BioMed Research International: Virology The latest articles from Hindawi © 2017 , Hindawi Limited . All rights reserved. Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells Wed, 30 Aug 2017 00:00:00 +0000 Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity. Olga Koval, Galina Kochneva, Anastasiya Tkachenko, Olga Troitskaya, Galina Sivolobova, Antonina Grazhdantseva, Anna Nushtaeva, Elena Kuligina, and Vladimir Richter Copyright © 2017 Olga Koval et al. All rights reserved. A Novel Genetic Group of Bovine Hepacivirus in Archival Serum Samples from Brazilian Cattle Sun, 20 Aug 2017 09:39:17 +0000 Hepatitis C virus (HCV) (genus Hepacivirus; family Flaviviridae) is a major human pathogen causing persistent infection and hepatic injury. Recently, emerging HCV-like viruses were described infecting wild animals, such as bats and rodents, and domestic animals, including dogs, horses, and cattle. Using degenerate primers for detecting bovine pestiviruses in a 1996 survey three bovine serum samples showed a low identity with the genus Pestivirus of the Flaviviridae family. A virus could not be isolated in cell culture. The description of bovine hepaciviruses (BovHepV) in 2015 allowed us to retrospectively identify the sequences as BovHepV, with a 88.9% nucleotide identity. In a reconstructed phylogenetic tree, the Brazilian BovHepV samples grouped within the bovine HCV-like cluster in a separated terminal node that was more closely related to the putative bovine Hepacivirus common ancestor than to bovine hepaciviruses detected in Europe and Africa. Cláudio W. Canal, Matheus N. Weber, Samuel P. Cibulski, Mariana S. Silva, Daniela E. Puhl, Hanspeter Stalder, and Ernst Peterhans Copyright © 2017 Cláudio W. Canal et al. All rights reserved. Male Partners of Infertile Couples with Seminal Infections of Human Papillomavirus Have Impaired Fertility Parameters Tue, 01 Aug 2017 06:29:05 +0000 Several studies have addressed the impact of viral infections on male infertility. However, it is still unknown whether human papillomavirus (HPV) can alter seminal parameters. The aim of this study was to determine the prevalence of HPV in the semen of male partners of couples seeking fertility evaluation. Additionally, we assessed the possibility that HPV infections affect seminal parameters. A total of 229 semen samples were collected from men in the Sperm Analysis Section of São Camilo Laboratory of Maringá, Brazil, between October 2015 and March 2016. Basic seminal parameters were analyzed, and HPV was detected and genotyped by polymerase chain reaction. HPV DNA was detected in 16.6% of samples. Of these, 10.5% had single type HPV infections, 6.1% had multiple HPV infections, 5.7% had exclusively high-risk HPV, and 6.1% had exclusively low-risk HPV. Samples positive for single and multiple types of HPV were associated with abnormal viscosity, and samples positive for multiple HPV types were also associated with hypospermia, higher pH, and increased leukocyte numbers. These findings suggest that the male partners of infertile couples with seminal HPV infections may have prostate disturbances indicative of glandular dysfunction, which may influence fertility. Edilson Damke, Fábio A. Kurscheidt, Valério A. Balani, Karen I. Takeda, Mary M. T. Irie, Fabrícia Gimenes, and Marcia E. L. Consolaro Copyright © 2017 Edilson Damke et al. All rights reserved. The Prevalence and Replication Capacity of a Tibetan Dominant HBV Strain, C/D Recombinant Wed, 21 Jun 2017 06:04:09 +0000 This study aimed to estimate the distribution of hepatitis B virus (HBV) C/D recombinant in Han and Tibet patients with chronic hepatitis B (CHB) and then learn such strain’s replication capacity in vivo. A total of 331 serum samples were collected from Han outpatients from Sichuan Province and Tibetan outpatients from Tibet. Viral genotypes in these samples were identified. An HBV replicative plasmid of C/D recombinant was constructed with selected genome. Sequentially, HBV replicative mouse models were established and the replication capacity of the viral strain was studied in vivo. In the 314 Han patients, 66% (207) were infected by genotype B strain while 31% (96) were by genotype C strain. Only 1% (3) were by C/D recombinant. In the 17 Tibetan patients, 41% (7) were by genotype D and 35% (6) by C/D recombinant. A plasmid with 1.3 copies of C/D recombinant genome was constructed. And its replication intermediates were found at similar levels to that of genotype D strain. Thus, C/D recombinant, the dominant viral strain in Tibet, was rather rare in the genotype B predominated Han patients from Sichuan Province. And the C/D recombinant replicated at a similar level to viral strain of genotype D in vivo. Lingyao Du, Menghan Liu, Miao Liu, Taoyou Zhou, Xing Cheng, Cong Liu, and Hong Tang Copyright © 2017 Lingyao Du et al. All rights reserved. A Novel Capillary Electrophoresis-Based High-Throughput Multiplex Polymerase Chain Reaction System for the Simultaneous Detection of Nine Pathogens in Swine Sun, 11 Jun 2017 00:00:00 +0000 Here we aimed to develop a capillary electrophoresis-based high-throughput multiplex polymerase chain reaction (PCR) system for the simultaneous detection of nine pathogens in swine. Nine pairs of specific primers and a set of universal primers were designed; the multiplex PCR was established. The specificity and cross-reactivity of this assay were examined, and the detection limit was determined using serial 10-fold dilutions of plasmids containing the target sequences. The assay was further tested using 144 clinical samples. We found that the nine specific amplification peaks were observed, and the assay had a high degree of specificity, without nonspecific amplification. The simultaneous detection limit for the nine viruses reached 10000 copies μL−1 when all of the premixed viral targets were present. Seventy-seven of the clinical samples tested positive for at least one of the viruses; the principal viral infections in the clinical samples were porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus. This approach has much potential for further development of high-throughput detection tools for the diagnosis of diseases in animals. Xu-long Wu, Lu Xiao, Hua Lin, Miao Yang, Shi-jie Chen, Wei An, Yin Wang, Xue-ping Yao, Ze-xiao Yang, and Zi-zhong Tang Copyright © 2017 Xu-long Wu et al. All rights reserved. Serum Dried Samples to Detect Dengue Antibodies: A Field Study Mon, 29 May 2017 08:43:57 +0000 Background. Dried blood and serum samples are useful resources for detecting antiviral antibodies. The conditions for elution of the sample need to be optimized for each disease. Dengue is a widespread disease in Mexico which requires continuous surveillance. In this study, we standardized and validated a protocol for the specific detection of dengue antibodies from dried serum spots (DSSs). Methods. Paired serum and DSS samples from 66 suspected cases of dengue were collected in a clinic in Veracruz, Mexico. Samples were sent to our laboratory, where the conditions for optimal elution of DSSs were established. The presence of anti-dengue antibodies was determined in the paired samples. Results. DSS elution conditions were standardized as follows: 1 h at 4°C in 200 µl of DNase-, RNase-, and protease-free PBS (1x). The optimal volume of DSS eluate to be used in the IgG assay was 40 µl. Sensitivity of 94%, specificity of 93.3%, and kappa concordance of 0.87 were obtained when comparing the antidengue reactivity between DSSs and serum samples. Conclusion. DSS samples are useful for detecting anti-dengue IgG antibodies in the field. Angelica Maldonado-Rodríguez, Othon Rojas-Montes, Guillermo Vazquez-Rosales, Adolfo Chavez-Negrete, Magdalena Rojas-Uribe, Araceli Posadas-Mondragon, Leopoldo Aguilar-Faisal, Ana Maria Cevallos, Beatriz Xoconostle-Cazares, and Rosalia Lira Copyright © 2017 Angelica Maldonado-Rodríguez et al. All rights reserved. A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray Wed, 24 May 2017 09:53:53 +0000 The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented. Ariel Vina-Rodriguez, Konrad Sachse, Ute Ziegler, Serafeim C. Chaintoutis, Markus Keller, Martin H. Groschup, and Martin Eiden Copyright © 2017 Ariel Vina-Rodriguez et al. All rights reserved. Infections Caused by HRSV A ON1 Are Predominant among Hospitalized Infants with Bronchiolitis in São Paulo City Wed, 24 May 2017 00:00:00 +0000 Human respiratory syncytial virus is the main cause of respiratory infections in infants. Several HRSV genotypes have been described. Goals. To describe the main genotypes that caused infections in São Paulo (2013–2015) and to analyze their clinical/epidemiological features. Methods. 94 infants (0–6 months) with bronchiolitis were studied. Clinical/epidemiological information was collected; a search for 16 viruses in nasopharyngeal secretion (PCR-real-time and conventional, sequencing, and phylogenetic analyses) was performed. Results. The mean age was 2.4 m; 48% were male. The mean length of hospital stay was 4.4 d (14% in the Intensive Care Unit). The positive rate of respiratory virus was 98.9%; 73 cases (77.6%) were HRSV (76,7% HRSVA). HRSVA formed three clusters: ON1 (), NA1 (), and NA2 (). All HRSVB were found to cluster in the BA genotype (BA9-; BA10-). Clinical analyses showed no significant differences between the genotype AON1 and other genotypes. Conclusion. This study showed a high rate of HRSV detection in bronchiolitis. HRSVA ON1, which has recently been described in other countries and has not been identified in previous studies in the southeast region of Brazil, was predominant. The clinical characteristics of the infants that were infected with AON1 were similar to infants with infections by other genotypes. Sandra E. Vieira, Luciano M. Thomazelli, Milena de Paulis, Angela E. Ferronato, Daniele B. Oliveira, Marina Baquerizo Martinez, and Edison L. Durigon Copyright © 2017 Sandra E. Vieira et al. All rights reserved. Ribonuclease from Bacillus Acts as an Antiviral Agent against Negative- and Positive-Sense Single Stranded Human Respiratory RNA Viruses Thu, 04 May 2017 00:00:00 +0000 Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses. Raihan Shah Mahmud, Christin Müller, Yulia Romanova, Ahmed Mostafa, Vera Ulyanova, Stephan Pleschka, and Olga Ilinskaya Copyright © 2017 Raihan Shah Mahmud et al. All rights reserved. Advancements in Developing Strategies for Sterilizing and Functional HIV Cures Wed, 26 Apr 2017 08:51:35 +0000 Combined antiretroviral therapy (cART) has been successful in prolonging lifespan and reducing mortality of patients infected with human immunodeficiency virus (HIV). However, the eradication of latent HIV reservoirs remains a challenge for curing HIV infection (HIV cure) because of HIV latency in primary memory CD4+ T cells. Currently, two types of HIV cures are in development: a “sterilizing cure” and a “functional cure.” A sterilizing cure refers to the complete elimination of replication-competent proviruses in the body, while a functional cure refers to the long-term control of HIV replication without treatment. Based on these concepts, significant progress has been made in different areas. This review focuses on recent advancements and future prospects for HIV cures. Wei Xu, Haoyang Li, Qian Wang, Chen Hua, Hanzhen Zhang, Weihua Li, Shibo Jiang, and Lu Lu Copyright © 2017 Wei Xu et al. All rights reserved. TDF Monotherapy Is Effective Regardless of Prior Nucleos(t)ide Analogue Treatment in Chronic Hepatitis B Patients in China Wed, 12 Apr 2017 09:30:33 +0000 Background/Aims. Many patients had to transfer to tenofovir disoproxil fumarate (TDF) if there is other nucleos(t)ide analogue (NA) resistance. We aimed to investigate antiviral effects of TDF monotherapy between NA-naive and NA-experienced chronic hepatitis B (CHB) patients in China. Methods. A total of 102 NA-naive and NA-experienced CHB patients with TDF monotherapy (300 mg/day) were retrospectively analyzed for useful parameters up to 72 weeks. Results. There were 36 and 66 patients with matched HBV DNA baseline level in NA-naïve and NA-experienced group, respectively. There were no significant differences between NA-naïve and NA-experienced groups in HBV DNA levels (all ) and HBV DNA undetectable rates (all ) at all time points. At the end of follow-up, HBV DNA undetectable rates in NA-naïve and NA-experienced group were 96.2% (25/26) and 91.8% (45/49), respectively (). Baseline HBV DNA level was the only independent predictor for HBV DNA negative time (). In addition, 27.8% (5/18) and 11.4% (4/35) achieved HBeAg seroconversion at the end of the follow-up, respectively (). Conclusions. TDF monotherapy was effective regardless of prior NA experienced. Baseline HBV DNA was a key predictive factor for HBV DNA negative time in TDF monotherapy. Mingxing Huang, Guoli Lin, Hong Shi, Yuankai Wu, Yusheng Jie, Zhe Zhu, and Yutian Chong Copyright © 2017 Mingxing Huang et al. All rights reserved. Cooccurrences of Putative Endogenous Retrovirus-Associated Diseases Thu, 23 Feb 2017 09:14:43 +0000 At least 8% of the human genome is composed of endogenous retrovirus (ERV) sequences. ERVs play a role in placental morphogenesis and can sometimes protect the host against exogenous viruses. On the other hand, ERV reactivation has been found to be associated with different diseases, for example, multiple sclerosis (MS), schizophrenia, type 1 diabetes mellitus (T1D), or amyotrophic lateral sclerosis (ALS). Little is known about the cooccurrence of these diseases. If all these diseases are caused by ERV, antiretroviral therapy should perhaps also show some effects in the other diseases. Here, we summarize literature demonstrating that some ERV-associated diseases seem to appear together more often than expected, for example, MS and ALS, MS and T1D, MS and schizophrenia, or ALS and T1D. In contrast, some ERV-associated diseases seem to appear together less frequently than expected, for example, schizophrenia and T1D. Besides, some reports demonstrate amelioration of MS, ALS, or schizophrenia under antiretroviral therapy in human immunodeficiency virus-infected patients. If such results could be confirmed in larger studies, alternative therapy strategies for ERV-associated diseases like MS and schizophrenia might be possible. Christine Brütting, Alexander Emmer, Malte E. Kornhuber, and Martin S. Staege Copyright © 2017 Christine Brütting et al. All rights reserved. Genetic Analysis of Two Chicken Infectious Anemia Virus Variants-Related Gyrovirus in Stray Mice and Dogs: The First Report in China, 2015 Thu, 23 Feb 2017 08:23:36 +0000 Chicken infectious anemia virus (CIAV) causes acute viral infection in chickens worldwide. It can infect chickens of all ages, but the disease is seen only in young chickens and is characterized by hemorrhagic lesions in the muscles, atrophic changes in the lymphoid organs, aplastic bone marrow, and immunosuppression causing increased mortality. Previous studies have demonstrated that CIAV can be isolated from blood specimens of humans and fecal samples of stray cats. In the present study, two variants of CIAV were isolated from fecal samples of mice (CIAV-Mouse) and stray dogs (CIAV-Dog), respectively. The genome of the two CIAV variants was sequenced and the results of the recombination detection program suggested that the CIAV-Dog strain could be a recombinant viral strain generated from parental CIAV strains, AB119448 and GD-1-12, with high confidence. Particularly, these findings were obtained from the comparison of genetic diversity and the relationship of CIAV between different hosts. This is the first report indicating that there is a significant difference in the number of transcription factor binding sites in CIAV noncoding regions from different hosts. Further studies are required to investigate the large geographic distribution of CIAV and monitor the variants, host range, and associated diseases. Lichun Fang, Yang Li, Yixin Wang, Jiayuan Fu, Shuai Cui, Xiaohan Li, Shuang Chang, and Peng Zhao Copyright © 2017 Lichun Fang et al. All rights reserved. Recombinant Lactococcus lactis Expressing Haemagglutinin from a Polish Avian H5N1 Isolate and Its Immunological Effect in Preliminary Animal Trials Wed, 22 Feb 2017 07:31:55 +0000 Lactic acid bacteria (LAB) are Gram-positive, nonpathogenic microorganisms that are gaining much interest as antigen producers for development of live vaccine vectors. Heterologous proteins of different origin have been successfully expressed in various LAB species, including Lactococcus lactis. Recombinant L. lactis strains have been shown to induce specific local and systemic immune responses against various antigens. Our study aimed at constructing a L. lactis strain expressing haemagglutinin of a Polish avian H5H1 influenza isolate and examining its effect on animals. Expression of the cloned H5 gene was achieved using the nisin-controlled gene expression system. Detection of the intracellular H5 antigen produced in L. lactis was performed by Western blot analysis and confirmed using mass spectrometry. The potential of L. lactis recombinant cells to induce an immune response was examined by setting up preliminary immunization trials on chickens and mice. Obtained sera were tested for specific antibodies by ELISA assays. The results of these studies are a promising step toward developing a vaccine against the bird flu using Lactococcus lactis cells as bioreactors for efficient antigen production and delivery to the mucosal surface. Agnieszka K. Szczepankowska, Katarzyna Szatraj, Przemysław Sałański, Agnieszka Rózga, Roman K. Górecki, and Jacek K. Bardowski Copyright © 2017 Agnieszka K. Szczepankowska et al. All rights reserved. Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant Wed, 01 Feb 2017 08:15:12 +0000 Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the β-glucuronidase gene (GUS) at the 3′ end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21–24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane. Sehrish Akbar, Muhammad Tahir, Ming-Bo Wang, and Qing Liu Copyright © 2017 Sehrish Akbar et al. All rights reserved. Associations of Adenovirus Genotypes in Korean Acute Gastroenteritis Patients with Respiratory Symptoms and Intussusception Wed, 01 Feb 2017 00:00:00 +0000 Human adenoviruses (HAdVs) cause a wide range of diseases, including respiratory infections and gastroenteritis, and have more than 65 genotypes. To investigate the current genotypes of circulating HAdV strains, we performed molecular genotyping of HAdVs in the stool from patients with acute gastroenteritis and tried to determine their associations with clinical symptoms. From June 2014 to May 2016, 3,901 fecal samples were tested for an AdV antigen, and 254 samples (6.5%) yielded positive results. Genotyping using PCR and sequencing of the capsid hexon gene was performed for 236 AdV antigen-positive fecal specimens. HAdV-41, of species F, was the most prevalent genotype (60.6%), followed by HAdV-2 of species C (13.8%). Other genotypes, including HAdV-3, HAdV-1, HAdV-5, HAdV-6, HAdV-31, HAdV-40, HAdV-12, and HAdV-55, were also detected. Overall, 119 patients (50.4%) showed concomitant respiratory symptoms, and 32 patients (13.6%) were diagnosed with intussusception. HAdV-1 and HAdV-31 were significantly associated with intussusception (). Our results demonstrate the recent changes in trends of circulating AdV genotypes associated with gastroenteritis in Korea, which should be of value for improving the diagnosis and developing new detection, treatment, and prevention strategies for broad application in clinical laboratories. Jae-Seok Kim, Su Kyung Lee, Dae-Hyun Ko, Jungwon Hyun, Han-Sung Kim, Wonkeun Song, and Hyun Soo Kim Copyright © 2017 Jae-Seok Kim et al. All rights reserved. Multiple Viral Infection Detected from Influenza-Like Illness Cases in Indonesia Mon, 23 Jan 2017 00:00:00 +0000 Influenza is one of the common etiologies of the upper respiratory tract infection (URTI). However, influenza virus only contributes about 20 percent of influenza-like illness patients. The aim of the study is to investigate the other viral etiologies from ILI cases in Indonesia. Of the 334 samples, 266 samples (78%) were positive at least for one virus, including 107 (42%) cases of multiple infections. Influenza virus is the most detected virus. The most frequent combination of viruses identified was adenovirus and human rhinovirus. This recent study demonstrated high detection rate of several respiratory viruses from ILI cases in Indonesia. Further studies to determine the relationship between viruses and clinical features are needed to improve respiratory disease control program. Kindi Adam, Krisna Nur Andriana Pangesti, and Vivi Setiawaty Copyright © 2017 Kindi Adam et al. All rights reserved. Immunogenicity and Cross Protection in Mice Afforded by Pandemic H1N1 Live Attenuated Influenza Vaccine Containing Wild-Type Nucleoprotein Sun, 22 Jan 2017 10:06:30 +0000 Since conserved viral proteins of influenza virus, such as nucleoprotein (NP) and matrix 1 protein, are the main targets for virus-specific CD8+ cytotoxic T-lymphocytes (CTLs), we hypothesized that introduction of the NP gene of wild-type virus into the genome of vaccine reassortants could lead to better immunogenicity and afford better protection. This paper describes in vitro and in vivo preclinical studies of two new reassortants of pandemic H1N1 live attenuated influenza vaccine (LAIV) candidates. One had the hemagglutinin (HA) and neuraminidase (NA) genes from A/South Africa/3626/2013 H1N1 wild-type virus on the A/Leningrad/134/17/57 master donor virus backbone (6 : 2 formulation) while the second had the HA, NA, and NP genes of the wild-type virus on the same backbone (5 : 3 formulation). Although both LAIVs induced similar antibody immune responses, the 5 : 3 LAIV provoked greater production of virus-specific CTLs than the 6 : 2 variant. Furthermore, the 5 : 3 LAIV-induced CTLs had higher in vivo cytotoxic activity, compared to 6 : 2 LAIV. Finally, the 5 : 3 LAIV candidate afforded greater protection against infection and severe illness than the 6 : 2 LAIV. Inclusion in LAIV of the NP gene from wild-type influenza virus is a new approach to inducing cross-reactive cell-mediated immune responses and cross protection against pandemic influenza. Andrey Rekstin, Irina Isakova-Sivak, Galina Petukhova, Daniil Korenkov, Igor Losev, Tatiana Smolonogina, Tatiana Tretiak, Svetlana Donina, Svetlana Shcherbik, Tatiana Bousse, and Larisa Rudenko Copyright © 2017 Andrey Rekstin et al. All rights reserved. Hepatitis E Virus in Industrialized Countries: The Silent Threat Wed, 14 Dec 2016 06:10:14 +0000 Hepatitis E virus (HEV) is the main cause of acute viral hepatitis worldwide. Its presence in developing countries has been documented for decades. Developed countries were supposed to be virus-free and initially only imported cases were detected in those areas. However, sporadic and autochthonous cases of HEV infection have been identified and studies reveal that the virus is worldwide spread. Chronic hepatitis and multiple extrahepatic manifestations have also been associated with HEV. We review the data from European countries, where human, animal, and environmental data have been collected since the 90s. In Europe, autochthonous HEV strains were first detected in the late 90s and early 2000s. Since then, serological data have shown that the virus infects quite frequently the European population and that some species, such as pigs, wild boars, and deer, are reservoirs. HEV strains can be isolated from environmental samples and reach the food chain, as shown by the detection of the virus in mussels and in contaminated pork products as sausages or meat. All these data highlight the need of studies directed to control the sources of HEV to protect immunocompromised individuals that seem the weakest link of the HEV epidemiology in industrialized regions. Pilar Clemente-Casares, Carlota Ramos-Romero, Eugenio Ramirez-Gonzalez, and Antonio Mas Copyright © 2016 Pilar Clemente-Casares et al. All rights reserved. Previously Unidentified Single Nucleotide Polymorphisms in HIV/AIDS Cases Associate with Clinical Parameters and Disease Progression Mon, 05 Dec 2016 12:19:02 +0000 The genetic background of an individual plays an important role in the progression of HIV infection to AIDS. Identifying previously unknown or uncharacterized single nucleotide polymorphisms (SNPs) that associate with disease progression may reveal important therapeutic targets and provide a greater understanding of disease pathogenesis. In the present study, we employed ultra-high multiplex PCR on an Ion Torrent next-generation sequencing platform to sequence 23 innate immune genes from 94 individuals with HIV/AIDS. This data was used to identify potential associations of SNPs with clinical parameters and disease progression. SNPs that associated with an increased viral load were identified in the genes for the interleukin 15 receptor (IL15RA), toll-like receptor 7 (TLR7), tripartite motif-containing protein 5 (TRIM5), and two killer-cell immunoglobulin-like receptors (KIR2DL1 and KIR2DL3). Additionally, SNPs that associated with progression from HIV infection to AIDS were identified in two 2′-5′-oligoadenylate synthetase genes (OAS2 and OAS3). In contrast, other SNPs identified in OAS2 and OAS3 genes, as well as in the TRIM5 and KIR2DS4 genes, were associated with a slower progression of disease. Taken together, our data demonstrates the utility of ultra-high multiplex PCR in identifying polymorphisms of potential clinical significance and further,identifies SNPs that may play a role in HIV pathogenesis. Vladimir V. Anokhin, Liliia B. Bakhteeva, Gulshat R. Khasanova, Svetlana F. Khaiboullina, Ekaterina V. Martynova, Richard L. Tillett, Karen A. Schlauch, Vincent C. Lombardi, and Albert A. Rizvanov Copyright © 2016 Vladimir V. Anokhin et al. All rights reserved. Human Bocavirus in Korean Children with Gastroenteritis and Respiratory Tract Infections Sun, 20 Nov 2016 08:38:29 +0000 Human bocaviruses (HBoVs) are suggested to be etiologic agents of childhood respiratory and gastrointestinal infections. There are four main recognized genotypes of HBoVs (HBoV1–4); the HBoV-1 genotype is considered to be the primary etiologic agent in respiratory infections, whereas the HBoV2–4 genotypes have been mainly associated with gastrointestinal infections. The aim of the present study was to determine the distribution of HBoV genotypes in children with respiratory or gastrointestinal infections in a hospital in Korea. A total of 662 nasopharyngeal swabs (NPSs) and 155 fecal specimens were collected from children aged 5 years or less. Polymerase chain reaction (PCR) was conducted to detect the NS1 HBoV gene. The VP1 gene of HBoV was further amplified in samples that were positive for the NS1 gene. The PCR products of VP1 gene amplification were genotyped by sequence analysis. HBoV was detected in 69 (14.5%) of 662 NPSs and in 10 (6.5%) of 155 fecal specimens. Thirty-three isolates from NPSs and five isolates from fecal specimens were genotyped, and all 38 sequenced isolates were identified as the HBoV-1 genotype. HBoV-1 is the most prevalent genotype in children with respiratory or gastrointestinal HBoV infections in a hospital in Korea. Eun Jin Lee, Han-Sung Kim, Hyun Soo Kim, Jae-Seok Kim, Wonkeun Song, MiYoung Kim, Young Kyung Lee, and Hee Jung Kang Copyright © 2016 Eun Jin Lee et al. All rights reserved. A Review of Management of Inflammation in the HIV Population Tue, 27 Sep 2016 12:23:20 +0000 Advancements in antiretroviral therapy have drastically increased the life expectancy for those infected with HIV. Today, a new subgroup of older patients with long-term controlled HIV exists, and its populace is continuously mounting. Therefore, it is essential to understand the enduring effects of chronic suppressed HIV infection in order to further improve HIV management in these patients. This paper will examine the role of HIV in chronic inflammation and immune dysfunction, the dynamic interaction that exists between comorbidity and HIV, and the potential consequences of long-term antiretroviral therapy in an effort to provide the best management options for the virally suppressed HIV patient. Jihad Slim and Christopher F. Saling Copyright © 2016 Jihad Slim and Christopher F. Saling. All rights reserved. Perilla Oil Supplementation Ameliorates High-Fat/High-Cholesterol Diet Induced Nonalcoholic Fatty Liver Disease in Rats via Enhanced Fecal Cholesterol and Bile Acid Excretion Wed, 24 Aug 2016 15:10:24 +0000 Recent experimental studies and clinical trials have shown that hepatic cholesterol metabolic disorders are closely related to the development of nonalcoholic fatty liver disease (NAFLD). The main goal of this study was to investigate the efficacy of the perilla oil rich in alpha-linolenic acid (ALA) against NASH and gain a deep insight into its potential mechanisms. Rats were fed a high-fat/high-cholesterol diet (HFD) supplement with perilla oil (POH) for 16 weeks. Routine blood biochemical tests and histological staining illustrated that the perilla oil administration improved HFD-induced hyperlipidemia, reduced hepatic steatosis, and inhibited hepatic inflammatory infiltration and fibrosis. Perilla oil also increased fecal bile acid and cholesterol excretion. Hepatic RNA-Seq analysis found that the long time perilla oil supplement notably modified the gene expression involved in cholesterol metabolism. Our results implicate that, after long-term high level dietary cholesterol feeding, rat liver endogenous synthesis of cholesterol and cholesterol-rich low density lipoprotein uptake was significantly inhibited, and perilla oil did not modulate expression of genes responsible for cholesterol synthesis but did increase cholesterol removed from hepatocytes by conversion to bile acids and increased fecal cholesterol excretion. Ting Chen, Fahu Yuan, Hualin Wang, Yu Tian, Lei He, Yang Shao, Na Li, and Zhiguo Liu Copyright © 2016 Ting Chen et al. All rights reserved. Detection of Peste des Petits Ruminants Viral RNA in Fecal Samples of Goats after an Outbreak in Punjab Province of Pakistan: A Longitudinal Study Mon, 15 Aug 2016 09:45:32 +0000 Peste des petits ruminants (PPR) is a highly contagious viral disease of domestic and wild small ruminants and thus has serious socioeconomic implications. In Pakistan, during the year 2012-2013, estimated losses due to PPR were worth Rs. 31.51 billions. Close contact between infected and susceptible animals is an important route of transmission of PPR. Therefore, carrier animals play an important role in unnoticed transmission of PPR. The objective of the study was to investigate the detection of PPR virus in goats recovered from PPR. A suspected PPR outbreak was investigated and confirmed as PPR after analysing appropriate samples collected from infected animals using rRT-PCR. A longitudinal study was conducted over the period of 16 weeks to ascertain the detection of PPR virus (PPRV) in faecal samples of recovered goats. Ninety-six (96) faecal samples from each sampling were collected at 4, 8, 12, and 16 weeks after the outbreak. Faecal samples were analysed using rRT-PCR. Of 96 from each sampling a total of 46, 37, 29, and 25 samples were positive for PPR viral genome at 4, 8, 12, and 16 weeks, respectively, after recovery. Attempts were made for the isolation of PPR virus on Vero cells, but results were negative. These results indicated the detection of PPR viral RNA up to 16 weeks after infection. Therefore, these results may help in the future epidemiology of PPR virus shedding and possible role as source of silent infection for healthy animals especially when there is no history of any outbreak in nearby flock or area. Riasat Wasee Ullah, Aamer Bin Zahur, Asma Latif, Javid Iqbal Dasti, Hamid Irshad, Muhammad Afzal, Tahir Rasheed, Adnan Rashid Malik, and Zafar-ul-Ahsan Qureshi Copyright © 2016 Riasat Wasee Ullah et al. All rights reserved. Vaccination with Astragalus and Ginseng Polysaccharides Improves Immune Response of Chickens against H5N1 Avian Influenza Virus Mon, 15 Aug 2016 07:29:00 +0000 To determine the effect of astragalus and ginseng polysaccharides (APS, GPS) on immune response and improvement of H5N1 vaccine, 360-day-old broilers were randomly divided into 8 groups of 45 chicks, comprising APS groups (1–3); GPS groups (4–6); vaccine group (7); and blank control (8) (without polysaccharide and vaccine). From day 12 after hatch groups 1–3 were given APS and groups 4–6 with GPS both at 100, 200, and 400 (mg/kg), respectively. At day 15 after hatch, groups 1–7 were vaccinated with 0.3 mL H5N1 vaccine subcutaneously; daily weight gain (DWG) and serum Ig antibody (by HI-test) were measured on 3, 7, 14, and 28 days after vaccination. Serum antibody titers and expression of cytokines (IL-2, IL-10, I FN-γ, and TNF) were determined by ELISA and RT-PCR. Results revealed that all the polysaccharide groups were numerically increased in antibody levels and the expression of cytokines was significant () in the APS and GPS groups compared to corresponding vaccine group and blank control. DWG was higher () in 400 mg/kg APS groups than control groups. Thus oral supplements of GPS and APS have shown their potential in the improvement of immune response and could be used as adjuvant in a formulation of H5N1 vaccine. Auwalu Yusuf Abdullahi, Sanpha Kallon, Xingang Yu, Yongliang Zhang, and Guoqing Li Copyright © 2016 Auwalu Yusuf Abdullahi et al. All rights reserved. Development of Primer Pairs from Molecular Typing of Rabies Virus Variants Present in Mexico Wed, 03 Aug 2016 08:29:54 +0000 Nucleoprotein (N) gene from rabies virus (RABV) is a useful sequence target for variant studies. Several specific RABV variants have been characterized in different mammalian hosts such as skunk, dog, and bats by using anti-nucleocapsid monoclonal antibodies (MAbs) via indirect fluorescent antibody (IFA) test, a technique not available in many laboratories in Mexico. In the present study, a total of 158 sequences of N gene from RABV were used to design eight pairs of primers (four external and four internal primers), for typing four different RABV variants (dog, skunk, vampire bat, and nonhematophagous bat) which are most common in Mexico. The results indicate that the primer and the typing variant from the brain samples, submitted to nested and/or real-time PCR, are in agreement in all four singleplex reactions, and the designed primer pairs are an alternative for use in specific variant RABV typing. Fernando Bastida-González, Dolores G. Ramírez-Hernández, Erika Chavira-Suárez, Eleazar Lara-Padilla, and Paola Zárate-Segura Copyright © 2016 Fernando Bastida-González et al. All rights reserved. Virological and Immunological Status of the People Living with HIV/AIDS Undergoing ART Treatment in Nepal Thu, 28 Jul 2016 14:01:50 +0000 Antiretroviral therapy (ART) has increased the life span of the people living with HIV (PLHIV), but their virological and immunological outcomes are not well documented in Nepal. The study was conducted at a tertiary care center including 826 HIV-1 seropositive individuals undergoing ART for at least six months. Plasma viral load (HIV-1 RNA) was detected by Real Time PCR and CD4+ T-lymphocyte (CD4+) counts were estimated by flow cytometry. The mean CD4+ count of patients was 501 (95% CI = 325–579) cells/cumm, but about 35% of patients had CD4+ T cell counts below 350 cells/cumm. With increasing age, average CD4+ count was found to be decreasing (). Of the total cases, 82 (9.92%) were found to have virological failure (viral load: >1000 copies/ml). Tenofovir/Lamivudine/Efavirenz (TDF/3TC/EFV), the frequently used ART regimen in Nepal, showed virological failure in 11.34% and immunological failure in 37.17% of patients. Virological failure rate was higher among children < 15 years (14.5%) (); however, no association was observed between ART outcomes and gender or route of transmission. The study suggests there are still some chances of virological and immunological failures despite the success of highly active ART (HAART). Chet Raj Ojha, Geeta Shakya, and Shyam Prakash Dumre Copyright © 2016 Chet Raj Ojha et al. All rights reserved. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication Thu, 21 Jul 2016 08:19:24 +0000 Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i.) but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy. Gefei Wang, Rui Li, Zhiwu Jiang, Liming Gu, Yanxia Chen, Jianping Dai, and Kangsheng Li Copyright © 2016 Gefei Wang et al. All rights reserved. The HPV16 E7 Oncoprotein Disrupts Dendritic Cell Function and Induces the Systemic Expansion of CD11b+Gr1+ Cells in a Transgenic Mouse Model Mon, 11 Jul 2016 14:19:57 +0000 Objective. The aim of this study was to analyze the effects of the HPV16 E7 oncoprotein on dendritic cells (DCs) and CD11b+Gr1+ cells using the K14E7 transgenic mouse model. Materials and Methods. The morphology of DCs was analyzed in male mouse skin on epidermal sheets using immunofluorescence and confocal microscopy. Flow cytometry was used to determine the percentages of DCs and CD11b+Gr1+ cells in different tissues and to evaluate the migration of DCs. Results. In the K14E7 mouse model, the morphology of Langerhans cells and the migratory activity of dendritic cells were abnormal. An increase in CD11b+Gr1+ cells was observed in the blood and skin of K14E7 mice, and molecules related to CD11b+Gr1+ chemoattraction (MCP1 and S100A9) were upregulated. Conclusions. These data suggest that the HPV16 E7 oncoprotein impairs the function and morphology of DCs and induces the systemic accumulation of CD11b+Gr1+ cells. Gabriela Damian-Morales, Nicolás Serafín-Higuera, Mario Adán Moreno-Eutimio, Enoc M. Cortés-Malagón, José Bonilla-Delgado, Genaro Rodríguez-Uribe, Rodolfo Ocadiz-Delgado, Paul F. Lambert, and Patricio Gariglio Copyright © 2016 Gabriela Damian-Morales et al. All rights reserved. Role of Autophagy and Apoptosis in the Postinfluenza Bacterial Pneumonia Wed, 08 Jun 2016 10:57:02 +0000 The risk of influenza A virus (IAV) is more likely caused by secondary bacterial infections. During the past decades, a great amount of studies have been conducted on increased morbidity from secondary bacterial infections following influenza and provide an increasing number of explanations for the mechanisms underlying the infections. In this paper, we first review the recent research progress that IAV infection increased susceptibility to bacterial infection. We then propose an assumption that autophagy and apoptosis manipulation are beneficial to antagonize post-IAV bacterial infection and discuss the clinical significance. Zhen Qin, Yuan Yang, Hongren Wang, Jun Luo, Xiaojun Huang, Jiangzhou You, Baoning Wang, and Mingyuan Li Copyright © 2016 Zhen Qin et al. All rights reserved. Genomic Analysis of the Chicken Infectious Anemia Virus in a Specific Pathogen-Free Chicken Population in China Thu, 19 May 2016 16:32:42 +0000 The antibody to chicken infectious anemia virus (CIAV) was positive in a specific pathogen-free (SPF) chicken population by ELISA test in our previous inspection, indicating a possible infection with CIAV. In this study, blood samples collected from the SPF chickens were used to isolate CIAV by inoculating into MSB1 cells and PCR amplification. A CIAV strain (SD1403) was isolated and successfully identified. Three overlapping genomic fragments were obtained by PCR amplification and sequencing. The full genome sequence of the SD1403 strain was obtained by aligning the sequences. The genome of the SD1403 strain was 2293 bp with a nucleotide identity of 94.8% to 98.5% when compared with 30 referred CIAV strains. The viral proteins VP2 and VP3 were highly conserved, but VP1 was not relatively conserved. Both amino acids 139 and 144 of VP1 were glutamine, which was in accord with the low pathogenic characteristics. In this study, we first reported that CIAV exists in Chinese SPF chicken populations and may be an important reason why attenuated vaccine can be contaminated with CIAV. Yang Li, Yixin Wang, Lichun Fang, Jiayuan Fu, Shuai Cui, Yingjie Zhao, Zhizhong Cui, Shuang Chang, and Peng Zhao Copyright © 2016 Yang Li et al. All rights reserved. Evaluation of Risk Factors for Peste des Petits Ruminants Virus in Sheep and Goats at the Wildlife-Livestock Interface in Punjab Province, Pakistan Sun, 15 May 2016 08:02:07 +0000 Peste des petits ruminants virus (PPRV) is causing infectious disease with high morbidity and mortality rate in domestic and wild small ruminants of Pakistan with valuable economical losses. The present study was carried out to investigate risk factors of PPRV in domestic small ruminants which were present in the vicinity of wildlife parks. A total of 265 sera samples (27 wild ruminants and 238 domesticated small ruminants) from apparently healthy animals from two different wildlife parks were collected and analysed for PPRV antibodies. Also, 20 nasal swabs from domestic small ruminants showing respiratory signs were collected to check for presence of PPRV antigen. Competitive ELISA revealed highest proportions of anti-PPRV antibodies in domestic small ruminants around the Wildlife Park at Lahore (35%) as compared to Faisalabad (13%), with no existence of PPRV antibodies in tested serum of wild ruminants at these parks. Higher seropositivity was observed in females (25.6%) than in males (5.1%) and in goats (34.5%) compared to sheep (11.2%). The results of N-gene based RT-PCR highlight the absence of PPRV due to lack of current PPR outbreak in the region during study period. Even though grazing was not a significant risk factor, there is still a possibility of wildlife-livestock interactions for feed and water reservoirs, resulting in spillover of PPR to wildlife. Keeping in view the high seropositivity and risk of PPR, vaccination should be adopted to avoid circulation of PPRV among wild and domestic small ruminants (sheep and goats). Aziz-ul-Rahman, Muhammad Abubakar, Muhammad Hidayat Rasool, Shumaila Manzoor, Muhammad Saqalein, Muhammad Rizwan, Muhammad Munir, Qurban Ali, and Jonas Johansson Wensman Copyright © 2016 Aziz-ul-Rahman et al. All rights reserved. Serum Biochemistry of Lumpy Skin Disease Virus-Infected Cattle Thu, 12 May 2016 07:57:04 +0000 Lumpy skin disease is an economically important poxvirus disease of cattle. Vaccination is the main method of control but sporadic outbreaks have been reported in Turkey. This study was carried out to determine the changes in serum biochemical values of cattle naturally infected with lumpy skin disease virus (LSDV). For this study, blood samples in EDTA, serum samples, and nodular skin lesions were obtained from clinically infected animals () whereas blood samples in EDTA and serum samples were collected from healthy animals (). A quantitative real-time PCR method was used to detect Capripoxvirus (CaPV) DNA in clinical samples. A real-time PCR high-resolution melt assay was performed to genotype CaPVs. Serum cardiac, hepatic, and renal damage markers and lipid metabolism products were measured by autoanalyzer. LSDV nucleic acid was detected in all samples which were obtained from clinically infected cattle. The results of serum biochemical analysis showed that aspartate aminotransferase, alkaline phosphatase, total protein, and creatinine concentrations were markedly increased in serum from infected animals. However, there were no significant differences in the other biochemical parameters evaluated. The results of the current study suggest that liver and kidney failures occur during LSDV infection. These findings may help in developing effective treatment strategies in LSDV infection. Murat Şevik, Oğuzhan Avci, Müge Doğan, and Ömer Barış İnce Copyright © 2016 Murat Şevik et al. All rights reserved. Feline Coronavirus 3c Protein: A Candidate for a Virulence Marker? Tue, 03 May 2016 13:28:44 +0000 Feline infectious peritonitis virus (FIPV) is highly virulent and responsible for the highly fatal disease feline infectious peritonitis (FIP), whereas feline enteric coronavirus (FECV) is widespread among the feline population and typically causes asymptomatic infections. Some candidates for genetic markers capable of differentiating these two pathotypes of a unique virus (feline coronavirus) have been proposed by several studies. In the present survey, in order to search for markers that can differentiate FECV and FIPV, several clones of the 3a–c, E, and M genes were sequenced from samples obtained from cats with or without FIP. All genes showed genetic diversity and suggested the presence of FCoV mutant spectrum capable of producing a virulent pathotype in an individual-specific way. In addition, all the feline coronavirus FIPV strains demonstrated a truncated 3c protein, and the 3c gene was the only observed pathotypic marker for FCoVs, showing that 3c gene is a candidate marker for the distinction between the two pathotypes when the mutant spectrum is taken into account. A. S. Hora, P. O. Tonietti, S. A. Taniwaki, K. M. Asano, P. Maiorka, L. J. Richtzenhain, and P. E. Brandão Copyright © 2016 A. S. Hora et al. All rights reserved. Genetic Characterization of a Novel Mutant of Peste Des Petits Ruminants Virus Isolated from Capra ibex in China during 2015 Sun, 21 Feb 2016 08:37:02 +0000 Peste des petits ruminants virus (PPRV) is the causative agent of peste des petits ruminants (PPR). The spread of PPR often causes severe economic losses. Therefore, special attention should be paid to the surveillance of PPR emergence, spread, and geographic distribution. Here we describe a novel mutant of PPRV China/XJBZ/2015 that was isolated from Capra ibex in Xinjiang province in China 2015. The sequence analysis and phylogenetic assessment indicate that China/XJBZ/2015 belongs to lineage IV, being closely related to China/XJYL/2013 strain. Interestingly, the V protein sequence of China/XJBZ/2015 showed lower homology with other Chinese PPRVs isolated during 2013 to 2014 (94%~95%), whereas it shared 100% identity with three Tibet strains isolated in China 2007. The 3′ UTR, V gene, and C gene were determined to be highly variable. Besides, 29 PPR genomic sequences available in GenBank were analyzed in this study. It is the first time to use PPRV genomic sequences to classify the different lineages which confirmed the lineage clustering of PPRVs using N gene 255 bp fragments and F gene 322 bp fragments. In conclusion, our findings indicate that the PPRVs continue to evolve in China, and some new mutations have emerged. Zixiang Zhu, Xiaocui Zhang, Gulizhati Adili, Jiong Huang, Xiaoli Du, Xiangle Zhang, Pengfei Li, Xueguang Zheng, Xiangtao Liu, Haixue Zheng, and Qinghong Xue Copyright © 2016 Zixiang Zhu et al. All rights reserved. Mutagenic Potential ofBos taurus Papillomavirus Type 1 E6 Recombinant Protein: First Description Wed, 09 Dec 2015 11:37:55 +0000 Bovine papillomavirus (BPV) is considered a useful model to study HPV oncogenic process. BPV interacts with the host chromatin, resulting in DNA damage, which is attributed to E5, E6, and E7 viral oncoproteins activity. However, the oncogenic mechanisms of BPV E6 oncoprotein per se remain unknown. This study aimed to evaluate the mutagenic potential of Bos taurus papillomavirus type 1 (BPV-1) E6 recombinant oncoprotein by the cytokinesis-block micronucleus assay (CBMNA) and comet assay (CA). Peripheral blood samples of five calves were collected. Samples were subjected to molecular diagnosis, which did not reveal presence of BPV sequences. Samples were treated with 1 μg/mL of BPV-1 E6 oncoprotein and 50 μg/mL of cyclophosphamide (positive control). Negative controls were not submitted to any treatment. The samples were submitted to the CBMNA and CA. The results showed that BPV E6 oncoprotein induces clastogenesis per se, which is indicative of genomic instability. These results allowed better understanding the mechanism of cancer promotion associated with the BPV E6 oncoprotein and revealed that this oncoprotein can induce carcinogenesis per se. E6 recombinant oncoprotein has been suggested as a possible vaccine candidate. Results pointed out that BPV E6 recombinant oncoprotein modifications are required to use it as vaccine. Rodrigo Pinheiro Araldi, Jacqueline Mazzuchelli-de-Souza, Diego Grando Modolo, Edislane Barreiros de Souza, Thatiana Corrêa de Melo, Diva Denelle Spadacci-Morena, Roberta Fiusa Magnelli, Márcio Augusto Caldas Rocha de Carvalho, Paulo Luis de Sá Júnior, Rodrigo Franco de Carvalho, Willy Beçak, and Rita de Cassia Stocco Copyright © 2015 Rodrigo Pinheiro Araldi et al. All rights reserved. Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients Tue, 08 Dec 2015 07:41:10 +0000 Monitoring antiretroviral therapy using measurements of viral load (VL) and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS) samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM) in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6%) of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings. Juan Pablo Rodriguez-Auad, Othon Rojas-Montes, Angelica Maldonado-Rodriguez, Ma. Teresa Alvarez-Muñoz, Onofre Muñoz, Rocio Torres-Ibarra, Guillermo Vazquez-Rosales, and Rosalia Lira Copyright © 2015 Juan Pablo Rodriguez-Auad et al. All rights reserved. Corrigendum to “Animal Models for the Study of Rodent-Borne Hemorrhagic Fever Viruses: Arenaviruses and Hantaviruses” Mon, 26 Oct 2015 08:31:04 +0000 Joseph W. Golden, Christopher D. Hammerbeck, Eric M. Mucker, and Rebecca L. Brocato Copyright © 2015 Joseph W. Golden et al. All rights reserved. Construction of a gE-Deleted Pseudorabies Virus and Its Efficacy to the New-Emerging Variant PRV Challenge in the Form of Killed Vaccine Thu, 17 Sep 2015 12:36:38 +0000 The new-emerging PRV variants plague the vaccinated pigs and caused huge economic loss to local pig industry in China since 2011. The current commercial PRV vaccines cannot provide complete protection as the new-emerging PRV variants are antigenically different from the classical viruses. It is urgent to develop more safe and effective PRV vaccines based on the current circulating field isolates. In this study, a gE gene-deleted PRV based on the PRV HN1201, a representative PRV variant, was generated and the efficacy was tested on 3-week-old pigs in the form of killed vaccine. After fatal PRV HN1201 challenge, all vaccinated pigs survived without showing any clinical symptoms, but all unvaccinated pigs exhibited pseudorabies-specific respiratory and neurological signs with 100% mortality rate within 6 days after infection. The vaccinated pigs developed high level of gB and neutralizing antibodies after vaccination which may correlate to the protection provided by vaccine. Therefore, this gE gene-deleted PRV could be a promising vaccine candidate for the control of currently epidemic pseudorabies in China. Tongyan Wang, Yan Xiao, Qingyuan Yang, Yuzhou Wang, Zhe Sun, Chaoling Zhang, Shijun Yan, Juan Wang, Linghua Guo, He Yan, Zhiyu Gao, Lilin Wang, Xiangdong Li, Feifei Tan, and Kegong Tian Copyright © 2015 Tongyan Wang et al. All rights reserved. A High-Performance Multiplex Immunoassay for Serodiagnosis of Flavivirus-Associated Neurological Diseases in Horses Thu, 17 Sep 2015 11:54:23 +0000 West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) are flaviviruses responsible for severe neuroinvasive infections in humans and horses. The confirmation of flavivirus infections is mostly based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs). These tests suffer from poor specificity, mainly due to antigenic cross-reactivity among flavivirus members. Robust diagnosis therefore needs to be validated through virus neutralisation tests (VNTs) which are time-consuming and require BSL3 facilities. The flavivirus envelope (E) glycoprotein ectodomain is composed of three domains (D) named DI, DII, and DIII, with EDIII containing virus-specific epitopes. In order to improve the serological differentiation of flavivirus infections, the recombinant soluble ectodomain of WNV E (WNV.sE) and EDIIIs (rEDIIIs) of WNV, JEV, and TBEV were synthesised using the Drosophila S2 expression system. Purified antigens were covalently bonded to fluorescent beads. The microspheres coupled to WNV.sE or rEDIIIs were assayed with about 300 equine immune sera from natural and experimental flavivirus infections and 172 nonimmune equine sera as negative controls. rEDIII-coupled microspheres captured specific antibodies against WNV, TBEV, or JEV in positive horse sera. This innovative multiplex immunoassay is a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases. Cécile Beck, Philippe Desprès, Sylvie Paulous, Jessica Vanhomwegen, Steeve Lowenski, Norbert Nowotny, Benoit Durand, Annabelle Garnier, Sandra Blaise-Boisseau, Edouard Guitton, Takashi Yamanaka, Stéphan Zientara, and Sylvie Lecollinet Copyright © 2015 Cécile Beck et al. All rights reserved. Evaluation of Novel Multiplex Antibody Kit for Human Immunodeficiency Virus 1/2 and Hepatitis C Virus Using Sol-Gel Based Microarray Thu, 17 Sep 2015 11:49:36 +0000 Background. Microarrays enable high-throughput screening (HTS) of disease-related molecules, including important signaling proteins/peptides and small molecules that are in low abundance. In this study, we developed a multiplex blood bank screening platform, referred to as the Hi3-1 assay, for simultaneous detection of human immunodeficiency virus 1/2 (HIV 1/2) and hepatitis C virus (HCV). Methods. The Hi3-1 assay was tested using four panels (Panel 1, patient samples; Panel 2, seroconversion samples; Panel 3, performance samples; and Panel 4, purchased positive control samples), and the results were collected by the Department of Laboratory Medicine, Korea University Medical College, Republic of Korea. The present study compares the sensitivity of the multiplex detection platform for both HIV and HCV using a sol-gel based microarray, which was based on a reference test (Architect HIV Ag/Ab Combo and Architect anti-HCV assays), in Korean patients. Results. The sensitivity of the multiplex detection platform for both HIV and HCV was 100%, and the specificity was 99.96% for HIV and 99.76% for HCV, which is equivalent to that of the reference test. Conclusion. We have successfully applied a novel screening technology to multiplex HIV and HCV diagnoses in a blood bank screening test. Seung Gyu Yun, Jin Woo Jang, Jong Han Lee, Chae Seung Lim, Jinhong Kim, Yeona Ki, Minjoung Jo, and Soyoun Kim Copyright © 2015 Seung Gyu Yun et al. All rights reserved. Sporadic Creutzfeldt-Jakob Disease: Prion Pathology in Medulla Oblongata—Possible Routes of Infection and Host Susceptibility Thu, 17 Sep 2015 10:32:48 +0000 Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disorder, is characterized by remarkable phenotypic variability, which is influenced by the conformation of the pathologic prion protein and the methionine/valine polymorphic codon 129 of the prion protein gene. While the etiology of sCJD remains unknown, it has been hypothesized that environmental exposure to prions might occur through conjunctival/mucosal contact, oral ingestion, inhalation, or simultaneous involvement of the olfactory and enteric systems. We studied 21 subjects with definite sCJD to assess neuropathological involvement of the dorsal motor nucleus of the vagus and other medullary nuclei and to evaluate possible associations with codon 129 genotype and prion protein conformation. The present data show that prion protein deposition was detected in medullary nuclei of distinct sCJD subtypes, either valine homozygous or heterozygous at codon 129. These findings suggest that an “environmental exposure” might occur, supporting the hypothesis that external sources of contamination could contribute to sCJD in susceptible hosts. Furthermore, these novel data could shed the light on possible causes of sCJD through a “triple match” hypothesis that identify environmental exposure, host genotype, and direct exposure of specific anatomical regions as possible pathogenetic factors. Diego Iacono, Sergio Ferrari, Matteo Gelati, Gianluigi Zanusso, Sara Mariotto, and Salvatore Monaco Copyright © 2015 Diego Iacono et al. All rights reserved. Molecular Epidemiology of Human Norovirus in Korea in 2013 Wed, 02 Sep 2015 09:36:14 +0000 Norovirus is a major cause of acute gastroenteritis. The molecular epidemiology of norovirus exhibits temporal and geographical fluctuations, and new variants of the GII.4 genotype emerge every 2-3 years to cause global epidemics of acute gastroenteritis. We investigated GI and GII genotypes of human norovirus strains isolated from patients with acute gastroenteritis in Korea in 2013. Norovirus antigen test was performed on 2,980 fecal specimens from January to December 2013. RNA was extracted from norovirus antigen-positive fecal suspensions, and the norovirus capsid (VP1) and polymerase (RdRp) genes were characterized by RT-PCR and sequencing. Of the 230 genotyped strains, GII.4 (77.3%) was the most frequently observed capsid genotype, followed by GII.3 (6.1%) and GII.13 (3.9%). A norovirus GII.4 variant, GII.Pe/GII.4 Sydney 2012, was the most frequently found polymerase/capsid genotype (65.7%), followed by GII.P17/GII.17 (2.1%) and GII.P21/GII.3 (2.1%). Phylogenetic, similarity, and capsid epitope analyses of GII.Pe/GII.4 Sydney 2012 strains were performed. We concluded that the norovirus GII.4 variant, GII.Pe/GII.4 Sydney 2012, was the main cause of norovirus-related gastroenteritis in Korea in 2013. Jae-Seok Kim, Hyun Soo Kim, Jungwon Hyun, Han-Sung Kim, and Wonkeun Song Copyright © 2015 Jae-Seok Kim et al. All rights reserved. Epidemiological Scenario of Dengue in Brazil Sun, 30 Aug 2015 13:39:32 +0000 Dengue is the most important reemerging mosquito-borne viral disease worldwide. It is caused by any of four Dengue virus types or serotypes (DENV-1 to DENV-4) and is transmitted by mosquitoes from the genus Aedes. Ecological changes have favored the geographic expansion of the vector and, since the dengue pandemic in the Asian and Pacific regions, the infection became widely distributed worldwide, reaching Brazil in 1845. The incidence of dengue in Brazil has been frequently high, and the number of cases in the country has at some point in time represented up to 60% of the dengue reported cases worldwide. This review addresses vector distribution, dengue outbreaks, circulating serotypes and genotypes, and prevention approaches being utilized in Brazil. Rafaelle C. G. Fares, Katia P. R. Souza, Germán Añez, and Maria Rios Copyright © 2015 Rafaelle C. G. Fares et al. All rights reserved. Comparison of Monkeypox Virus Clade Kinetics and Pathology within the Prairie Dog Animal Model Using a Serial Sacrifice Study Design Mon, 24 Aug 2015 06:35:33 +0000 Monkeypox virus (MPXV) infection of the prairie dog is valuable to studying systemic orthopoxvirus disease. To further characterize differences in MPXV clade pathogenesis, groups of prairie dogs were intranasally infected ( p.f.u.) with Congo Basin (CB) or West African (WA) MPXV, and 28 tissues were harvested on days 2, 4, 6, 9, 12, 17, and 24 postinfection. Samples were evaluated for the presence of virus and gross and microscopic lesions. Virus was recovered from nasal mucosa, oropharyngeal lymph nodes, and spleen earlier in CB challenged animals (day 4) than WA challenged animals (day 6). For both groups, primary viremia (indicated by viral DNA) was seen on days 6–9 through day 17. CB MPXV spread more rapidly, accumulated to greater levels, and caused greater morbidity in animals compared to WA MPXV. Histopathology and immunohistochemistry (IHC) findings, however, were similar. Two animals that succumbed to disease demonstrated abundant viral antigen in all organs tested, except for brain. Dual-IHC staining of select liver and spleen sections showed that apoptotic cells (identified by TUNEL) tended to colocalize with poxvirus antigen. Interestingly splenocytes were labelled positive for apoptosis more often than hepatocytes in both MPXV groups. These findings allow for further characterization of differences between MPXV clade pathogenesis, including identifying sites that are important during early viral replication and cellular response to viral infection. Christina L. Hutson, Darin S. Carroll, Nadia Gallardo-Romero, Clifton Drew, Sherif R. Zaki, Tamas Nagy, Christine Hughes, Victoria A. Olson, Jeanine Sanders, Nishi Patel, Scott K. Smith, M. Shannon Keckler, Kevin Karem, and Inger K. Damon Copyright © 2015 Christina L. Hutson et al. All rights reserved. Genetic Diversity of Human Adenovirus in Children with Acute Gastroenteritis, Albania, 2013–2015 Mon, 03 Aug 2015 09:46:15 +0000 The objectives of the present study were to assess the occurrence of human adenoviruses (HAdVs) in paediatric patients with gastroenteritis in Albania and to characterize HAdV strains. Faecal specimens from children admitted with acute gastroenteritis to the Paediatric Hospital in Tirana were screened for HAdV, using broad-range primers targeting the hexon gene, in combination with species-specific primers targeting the fiber gene. Phylogenetic analysis was then performed to assess the genetic relationships among the different sequences and between the sequences of the samples and those of the prototype strains. Adenovirus DNA was detected in 33/142 samples (23.2%); 14 belonged to species F (13 HAdV-41 and 1 HAdV-40), 13 to species C (1 HAdV-1, 8 HAdV-2, and 4 HAdV-5), 5 to species B (HAdV-3), and 1 to species A (HAdV-12). Rotavirus coinfection was present in 9/33 (27.2%) positive samples. In the remaining 24 positive samples (12 enteric—F species; 12 nonenteric—A, B, or C species), HAdVs were detected as unique viral pathogens, suggesting that HAdV may be an important cause of diarrhoea in children requiring hospitalization. This is the first study investigating the presence of human adenoviruses (species A–G) as etiologic agents of viral gastroenteritis in children in Albania. G. La Rosa, S. Della Libera, S. Petricca, M. Iaconelli, D. Donia, P. Saccucci, F. Cenko, G. Xhelilaj, and M. Divizia Copyright © 2015 G. La Rosa et al. All rights reserved. Challenges and Strategies of Laboratory Diagnosis for Newly Emerging Influenza Viruses in Taiwan: A Decade after SARS Tue, 28 Jul 2015 12:55:47 +0000 Since the first case of severe acute respiratory syndrome (SARS) in Taiwan was identified in March 2003, viral respiratory infections, in particular the influenza virus, have become a national public health concern. Taiwan would face a serious threat of public health problems if another SARS epidemic overlapped with a flu outbreak. After SARS, the Taiwan Centers for Disease Control accelerated and strengthened domestic research on influenza and expanded the exchange of information with international counterparts. The capacity of influenza A to cross species barriers presents a potential threat to human health. Given the mutations of avian flu viruses such as H7N9, H6N1, and H10N8, all countries, including Taiwan, must equip themselves to face a possible epidemic or pandemic. Such preparedness requires global collaboration. Jih-Hui Lin and Ho-Sheng Wu Copyright © 2015 Jih-Hui Lin and Ho-Sheng Wu. All rights reserved. Animal Models for the Study of Rodent-Borne Hemorrhagic Fever Viruses: Arenaviruses and Hantaviruses Tue, 21 Jul 2015 12:34:56 +0000 Human pathogenic hantaviruses and arenaviruses are maintained in nature by persistent infection of rodent carrier populations. Several members of these virus groups can cause significant disease in humans that is generically termed viral hemorrhagic fever (HF) and is characterized as a febrile illness with an increased propensity to cause acute inflammation. Human interaction with rodent carrier populations leads to infection. Arenaviruses are also viewed as potential biological weapons threat agents. There is an increased interest in studying these viruses in animal models to gain a deeper understating not only of viral pathogenesis, but also for the evaluation of medical countermeasures (MCM) to mitigate disease threats. In this review, we examine current knowledge regarding animal models employed in the study of these viruses. We include analysis of infection models in natural reservoirs and also discuss the impact of strain heterogeneity on the susceptibility of animals to infection. This information should provide a comprehensive reference for those interested in the study of arenaviruses and hantaviruses not only for MCM development but also in the study of viral pathogenesis and the biology of these viruses in their natural reservoirs. Joseph W. Golden, Christopher D. Hammerbeck, Eric M. Mucker, and Rebecca L. Brocato Copyright © 2015 Joseph W. Golden et al. All rights reserved. A One-Step Real-Time RT-PCR Assay for the Detection and Quantitation of Sugarcane Streak Mosaic Virus Mon, 22 Jun 2015 09:57:10 +0000 Sugarcane mosaic disease is caused by the Sugarcane streak mosaic virus (SCSMV; genus Poacevirus, family Potyviridae) which is common in some Asian countries. Here, we established a protocol of a one-step real-time quantitative reverse transcription PCR (real-time qRT-PCR) using the TaqMan probe for the detection of SCSMV in sugarcane. Primers and probes were designed within the conserved region of the SCSMV coat protein (CP) gene sequences. Standard single-stranded RNA (ssRNA) generated by PCR-based gene transcripts of recombinant pGEM-CP plasmid in vitro and total RNA extracted from SCSMV-infected sugarcane were used as templates of qRT-PCR. We further performed a sensitivity assay to show that the detection limit of the assay was 100 copies of ssRNA and 2 pg of total RNA with good reproducibility. The values obtained were approximately 100-fold more sensitive than those of the conventional RT-PCR. A higher incidence (68.6%) of SCSMV infection was detected by qRT-PCR than that (48.6%) with conventional RT-PCR in samples showing mosaic symptoms. SCSMV-free samples were verified by infection with Sugarcane mosaic virus (SCMV) or Sorghum mosaic virus (SrMV) or a combination of both. The developed qRT-PCR assay may become an alternative molecular tool for an economical, rapid, and efficient detection and quantification of SCSMV. Wei-Lin Fu, Sheng-Ren Sun, Hua-Ying Fu, Ru-Kai Chen, Jin-Wei Su, and San-Ji Gao Copyright © 2015 Wei-Lin Fu et al. All rights reserved. Cyclophilin A Interacts with Viral VP4 and Inhibits the Replication of Infectious Bursal Disease Virus Sun, 24 May 2015 12:14:27 +0000 Nonstructural protein VP4, a serine protease of infectious bursal disease virus (IBDV) that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins VP2, VP4, and VP3, is essential to the replication of IBDV. However, the interacting partners of VP4 in host cells and the effects of the interaction on the IBDV lifecycle remain incompletely elucidated. In this study, using the yeast two-hybrid system, the putative VP4-interacting partner cyclophilin A (CypA) was obtained from a chicken embryo fibroblast (CEF) expression library. CypA was further confirmed to interact with VP4 of IBDV using co-immunoprecipitation (CO-IP), GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV. Taken together, our findings indicate that the host cell protein CypA interacts with viral VP4 and inhibits the replication of IBDV. Nian Wang, Lizhou Zhang, Yuming Chen, Zhen Lu, Li Gao, Yongqiang Wang, Yulong Gao, Honglei Gao, Hongyu Cui, Kai Li, Changjun Liu, Yanping Zhang, Xiaole Qi, and Xiaomei Wang Copyright © 2015 Nian Wang et al. All rights reserved. Outbreak Control and Clinical, Pathological, and Epidemiological Aspects and Molecular Characterization of a Bovine Herpesvirus Type 5 on a Feedlot Farm in São Paulo State Thu, 21 May 2015 07:42:27 +0000 This paper describes the control, epidemiological, pathological, and molecular aspects of an outbreak of meningoencephalitis in calves due to bovine herpesvirus 5 at a feedlot with 540 animals in São Paulo State, Brazil. The introduction of new animals and contact between the resident animals and the introduced ones were most likely responsible for virus transmission. Bovine herpesvirus 1 vaccine was used, resulting in the efficacy of the outbreak control, although two bovine herpesvirus 1 positive animals, vaccinated and revaccinated, presented meningoencephalitis, thereby characterizing vaccinal failure. Jane Megid, Acácia Ferreira Vicente, Camila Michele Appolinario, Susan Dora Allendorf, Mateus de Souza Ribeiro Mioni, Thaís Gasparini Baraldi, Adriana Cortez, Marcos Bryan Heinemann, Clovis Reinaldo Silva Fonseca, Vanessa Cristina Pelícia, Bruna Leticia Devidé Ribeiro, Liria Hiromi Okuda, and Edviges Maristela Pituco Copyright © 2015 Jane Megid et al. All rights reserved. Analysis of Coinfections with A/H1N1 Strain Variants among Pigs in Poland by Multitemperature Single-Strand Conformational Polymorphism Wed, 15 Apr 2015 11:46:50 +0000 Monitoring and control of infections are key parts of surveillance systems and epidemiological risk prevention. In the case of influenza A viruses (IAVs), which show high variability, a wide range of hosts, and a potential of reassortment between different strains, it is essential to study not only people, but also animals living in the immediate surroundings. If understated, the animals might become a source of newly formed infectious strains with a pandemic potential. Special attention should be focused on pigs, because of the receptors specific for virus strains originating from different species, localized in their respiratory tract. Pigs are prone to mixed infections and may constitute a reservoir of potentially dangerous IAV strains resulting from genetic reassortment. It has been reported that a quadruple reassortant, A(H1N1)pdm09, can be easily transmitted from humans to pigs and serve as a donor of genetic segments for new strains capable of infecting humans. Therefore, it is highly desirable to develop a simple, cost-effective, and rapid method for evaluation of IAV genetic variability. We describe a method based on multitemperature single-strand conformational polymorphism (MSSCP), using a fragment of the hemagglutinin (HA) gene, for detection of coinfections and differentiation of genetic variants of the virus, difficult to identify by conventional diagnostic. Krzysztof Lepek, Beata Pajak, Lukasz Rabalski, Kinga Urbaniak, Krzysztof Kucharczyk, Iwona Markowska-Daniel, and Boguslaw Szewczyk Copyright © 2015 Krzysztof Lepek et al. All rights reserved. Proteolytic Disassembly of Viral Outer Capsid Proteins Is Crucial for Reovirus-Mediated Type-I Interferon Induction in Both Reovirus-Susceptible and Reovirus-Refractory Tumor Cells Thu, 19 Mar 2015 13:42:55 +0000 Oncolytic reovirus induces innate immune responses, which contribute to the antitumor activity of reovirus, following in vivo application. Reovirus-induced innate immune responses have been relatively well characterized in immune cells and mouse embryonic fibroblasts cells; however, the mechanisms and profiles of reovirus-induced innate immune responses in human tumor cells have not been well understood. In particular, differences in reovirus-induced innate immune responses between reovirus-susceptible and reovirus-refractory tumor cells remain unknown, although the intracellular trafficking of reovirus differs between these tumor cells. In this study, we examined reovirus-induced upregulation of interferon- (IFN-) β and of the proapoptotic gene, Noxa, in reovirus-susceptible and -refractory tumor cells. IFN-β and Noxa were significantly induced by reovirus via the IFN-β promoter stimulator-1 (IPS-1) signaling in both types of tumor cells. Inhibition of cathepsins B and L, which are important for disassembly of reovirus outer capsid proteins and escape into cytoplasm, largely suppressed reovirus-induced upregulation of IFN-β and Noxa expression in not only reovirus-susceptible but also reovirus-refractory tumor cells. These results indicated that in both reovirus-susceptible and reovirus-refractory tumor cells, disassembly of the outer capsid proteins by cathepsins and the escape into the cytoplasm were crucial steps for reovirus-induced innate immunity. Yuki Katayama, Yuichi Terasawa, Masashi Tachibana, Hiroyuki Mizuguchi, and Fuminori Sakurai Copyright © 2015 Yuki Katayama et al. All rights reserved. Current and Emerging Cell Culture Manufacturing Technologies for Influenza Vaccines Sun, 01 Mar 2015 12:55:33 +0000 Annually, influenza virus infects millions of people worldwide. Vaccination programs against seasonal influenza infections require the production of hundreds of million doses within a very short period of time. The influenza vaccine is currently produced using a technology developed in the 1940s that relies on replicating the virus in embryonated hens’ eggs. The monovalent viral preparation is inactivated and purified before being formulated in trivalent or tetravalent influenza vaccines. The production process has depended on a continuous supply of eggs. In the case of pandemic outbreaks, this mode of production might be problematic because of a possible drastic reduction in the egg supply and the low flexibility of the manufacturing process resulting in a lack of supply of the required vaccine doses in a timely fashion. Novel production systems using mammalian or insect cell cultures have emerged to overcome the limitations of the egg-based production system. These industrially well-established production systems have been primarily selected for a faster and more flexible response to pandemic threats. Here, we review the most important cell culture manufacturing processes that have been developed in recent years for mass production of influenza vaccines. Ernest Milián and Amine A. Kamen Copyright © 2015 Ernest Milián and Amine A. Kamen. All rights reserved. Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1 Sat, 28 Feb 2015 07:49:41 +0000 Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. Dinja Oosterhoff, Gerard van de Weerd, Gerco van Eikenhorst, Tanja D. de Gruijl, Leo A. van der Pol, and Wilfried A. M. Bakker Copyright © 2015 Dinja Oosterhoff et al. All rights reserved. Cross-Platform Evaluation of Commercial Real-Time SYBR Green RT-PCR Kits for Sensitive and Rapid Detection of European Bat Lyssavirus Type 1 Mon, 16 Feb 2015 14:36:07 +0000 This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCR kits using real-time PCR assays. Two real-time thermocyclers showing different throughput capacities were used. The analysed performance evaluation criteria included the generation of standard curve, reaction efficiency, analytical sensitivity, intra- and interassay repeatability as well as the costs and the practicability of kits, and thermocycling times. We found that the optimised one-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used, while no difference was detected in reaction efficiency, R2 values, and intra- and interreproducibility between the two methods. The limit of detection at the 95% confidence level varied between 15 to 981 copies/µL and 41 to 171 for one-step kits and two-step kits, respectively. Of the ten kits tested, the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95% of confidence of 20 and 22 copies/µL on the thermocyclers Rotor gene Q MDx and MX3005P, respectively. The study demonstrated the pivotal influence of the thermocycler on PCR performance for the detection of rabies RNA, as well as that of the master mixes. Evelyne Picard-Meyer, Carine Peytavin de Garam, Jean Luc Schereffer, Clotilde Marchal, Emmanuelle Robardet, and Florence Cliquet Copyright © 2015 Evelyne Picard-Meyer et al. All rights reserved. Molecular Dynamics, Monte Carlo Simulations, and Langevin Dynamics: A Computational Review Mon, 16 Feb 2015 13:01:14 +0000 Macromolecular structures, such as neuraminidases, hemagglutinins, and monoclonal antibodies, are not rigid entities. Rather, they are characterised by their flexibility, which is the result of the interaction and collective motion of their constituent atoms. This conformational diversity has a significant impact on their physicochemical and biological properties. Among these are their structural stability, the transport of ions through the M2 channel, drug resistance, macromolecular docking, binding energy, and rational epitope design. To assess these properties and to calculate the associated thermodynamical observables, the conformational space must be efficiently sampled and the dynamic of the constituent atoms must be simulated. This paper presents algorithms and techniques that address the abovementioned issues. To this end, a computational review of molecular dynamics, Monte Carlo simulations, Langevin dynamics, and free energy calculation is presented. The exposition is made from first principles to promote a better understanding of the potentialities, limitations, applications, and interrelations of these computational methods. Eric Paquet and Herna L. Viktor Copyright © 2015 Eric Paquet and Herna L. Viktor. All rights reserved. Prospects of HA-Based Universal Influenza Vaccine Sun, 15 Feb 2015 07:26:07 +0000 Current influenza vaccines afford substantial protection in humans by inducing strain-specific neutralizing antibodies (Abs). Most of these Abs target highly variable immunodominant epitopes in the globular domain of the viral hemagglutinin (HA). Therefore, current vaccines may not be able to induce heterosubtypic immunity against the divergent influenza subtypes. The identification of broadly neutralizing Abs (BnAbs) against influenza HA using recent technological advancements in antibody libraries, hybridoma, and isolation of single Ab-secreting plasma cells has increased the interest in developing a universal influenza vaccine as it could provide life-long protection. While these BnAbs can serve as a source for passive immunotherapy, their identification represents an important step towards the design of such a universal vaccine. This review describes the recent advances and approaches used in the development of universal influenza vaccine based on highly conserved HA regions identified by BnAbs. Anwar M. Hashem Copyright © 2015 Anwar M. Hashem. All rights reserved. Protection against Multiple Subtypes of Influenza Viruses by Virus-Like Particle Vaccines Based on a Hemagglutinin Conserved Epitope Thu, 12 Feb 2015 13:57:09 +0000 We selected the conserved sequence in the stalk region of influenza virus hemagglutinin (HA) trimmer, the long alpha helix (LAH), as the vaccine candidate sequence, and inserted it into the major immunodominant region (MIR) of hepatitis B virus core protein (HBc), and, by using the E. coli expression system, we prepared a recombinant protein vaccine LAH-HBc in the form of virus-like particles (VLP). Intranasal immunization of mice with this LAH-HBc VLP plus cholera toxin B subunit with 0.2% of cholera toxin (CTB*) adjuvant could effectively elicit humoral and cellular immune responses and protect mice against a lethal challenge of homologous influenza viruses (A/Puerto Rico/8/1934 (PR8) (H1N1)). In addition, passage of the immune sera containing specific antibodies to naïve mice rendered them resistant against a lethal homologous challenge. Immunization with LAH-HBc VLP vaccine plus CTB* adjuvant could also fully protect mice against a lethal challenge of the 2009 pandemic H1N1 influenza virus or the avian H9N2 virus and could partially protect mice against a lethal challenge of the avian H5N1 influenza virus. This study demonstrated that the LAH-HBc VLP vaccine based on a conserved sequence of the HA trimmer stalk region is a promising candidate vaccine for developing a universal influenza vaccine against multiple influenza viruses infections. Shaoheng Chen, Dan Zheng, Changgui Li, Wenjie Zhang, Wenting Xu, Xueying Liu, Fang Fang, and Ze Chen Copyright © 2015 Shaoheng Chen et al. All rights reserved. The Involvement of Microtubules and Actin during the Infection of Japanese Encephalitis Virus in Neuroblastoma Cell Line, IMR32 Sun, 01 Feb 2015 11:38:30 +0000 The role of the cytoskeleton, actin, and microtubules were examined during the process of Japanese encephalitis (JEV) infection in a human neuroblastoma cell line, IMR32. Cytochalasin D and nocodazole were used to depolymerise the cellular actin and microtubules, respectively, in order to study the effect of JEV infection in the cell. This study shows that depolymerisation of the actin cytoskeleton at early process of infection inhibits JEV infection in the cell; however infection was not inhibited when depolymerisation occurred at the later stage of infection. The microtubules, on the other hand, are required at 2 points in infection. The antigen production in the cells was inhibited when the infected cells were treated at time up to 2 hours after inoculation and there was no significant effect at later times, while the viable virus released continued to be affected until 10 hours after inoculation. In conclusion, infection of JEV in IMR32 cells required actin to facilitate early process in infection and the microtubular network is utilised as the transport system to the virus replication site and the release of mature virus. Magdline Sia Henry Sum Copyright © 2015 Magdline Sia Henry Sum. All rights reserved. Development of Enhanced Primer Sets for Detection of Norovirus Wed, 28 Jan 2015 07:23:40 +0000 Norovirus (NV) is a major viral pathogen that causes nonbacterial acute gastroenteritis and outbreaks of food-borne disease. The genotype of NV most frequently responsible for NV outbreaks is GII.4, which accounts for 60–80% of cases. Moreover, original and new NV variant types have been continuously emerging, and their emergence is related to the recent global increase in NV infection. In this study, we developed advanced primer sets (NKI-F/R/F2, NKII-F/R/R2) for the detection of NV, including the variant types. The new primer sets were compared with conventional primer sets (GI-F1/R1/F2, SRI-1/2/3, GII-F1/R1/F2, and SRII-1/2/3) to evaluate their efficiency when using clinical and environmental samples. Using reverse transcription polymerase chain reaction (RT-PCR) and seminested PCR, NV GI and GII were detected in 91.7% (NKI-F/R/F2), 89.3% (NKII-F/R/R2), 54.2% (GI-F1/R1/F2), 52.5% (GII-F1/R1/F2), 25.0% (SRI-1/2/3), and 32.2% (SRII-1/2/3) of clinical and environmental specimens. Therefore, our primer sets perform better than conventional primer sets in the detection of emerged types of NV and could be used in the future for epidemiological diagnosis of infection with the virus. Byoung-Hwa Kong, Sung-Geun Lee, Sang-Ha Han, Ji-Young Jin, Weon-Hwa Jheong, and Soon-Young Paik Copyright © 2015 Byoung-Hwa Kong et al. All rights reserved. Complete Nucleotide Sequence Analysis of the Norovirus GII.4 Sydney Variant in South Korea Mon, 19 Jan 2015 14:36:21 +0000 Norovirus is the primary cause of acute gastroenteritis in individuals of all ages. In Australia, a new strain of norovirus (GII.4) was identified in March 2012, and this strain has spread rapidly around the world. In August 2012, this new GII.4 strain was identified in patients in South Korea. Therefore, to examine the characteristics of the epidemic norovirus GII.4 2012 variant in South Korea, we conducted KM272334 full-length genomic analysis. The genome of the gg-12-08-04 strain consisted of 7,558 bp and contained three open reading frame (ORF) composites throughout the whole genome: ORF1 (5,100 bp), ORF2 (1,623 bp), and ORF3 (807 bp). Phylogenetic analyses showed that gg-12-08-04 belonged to the GII.4 Sydney 2012 variant, sharing 98.92% nucleotide similarity with this variant strain. According to SimPlot analysis, the gg-12-08-04 strain was a recombinant strain with breakpoint at the ORF1/2 junction between Osaka 2007 and Apeldoorn 2008 strains. This study is the first report of the complete sequence of the GII.4 Sydney 2012 strain in South Korea. Therefore, this may represent the standard sequence of the norovirus GII.4 2012 variant in South Korea and could therefore be useful for the development of norovirus vaccines. Ji-Sun Park, Sung-Geun Lee, Ji-Young Jin, Han-Gil Cho, Weon-Hwa Jheong, and Soon-Young Paik Copyright © 2015 Ji-Sun Park et al. All rights reserved. Epidemiological and Molecular Characteristics of the PB1-F2 Proteins in H7N9 Influenza Viruses, Jiangsu Wed, 14 Jan 2015 07:10:30 +0000 The recent sporadic infections of humans in China with previously unrecognized avian influenza A virus of the H7N9 subtype (A(H7N9)) have caused concern. The aim is to find out the epidemiological and molecular analysis of the PB1-F2 proteins in H7N9 influenza viruses, in Jiangsu province. Sequences were obtained from GISAID database. Data were analyzed by using Molecular Evolutionary Genetics Analysis software and Bayesian Markov chain Monte Carlo method. From March 1, 2013, to May 31, 2014, 53 patients were confirmed to be infected with the H7N9 virus; one was a retrospective case in Jiangsu province. 38 sequences of PB1 in H7N9 of Jiangsu were obtained from the GISAID online and were then divided into three lineages. Of these sequences, 4 sequences and 3 sequences encode an N-terminally truncated PB1-F2 (52aa)polypeptide and C-terminally truncated PB1-F2 (76aa) polypeptide, respectively. The remaining sequences encode a full-length PB1-F2 (90aa). We estimated a mean evolutionary rate of 3.053 × 10−3 subs/site/year (95% HPD: 2.021 × 10−3–4.051 × 10−3). The site-by-site analysis of selection pressure analysis revealed positively and negatively (12, 3), respectively, selected sites. Influenza A (H7N9) virus adapting into new host, PB1-F2 of H7N9, might be faced with higher selection pressures. Pingmin Wei, Wei Li, Hairong Zi, Michael Cunningham, Yan Guo, Yang Xuan, Taha Hussein Musa, and Pengfei Luo Copyright © 2015 Pingmin Wei et al. All rights reserved. Quantitative iTRAQ LC-MS/MS Proteomics Reveals the Proteome Profiles of DF-1 Cells after Infection with Subgroup J Avian Leukosis Virus Thu, 08 Jan 2015 07:14:55 +0000 Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that can induce various clinical tumors and has caused severe economic losses in China. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of ALV-J infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in DF-1 cells infected and mock-infected with ALV-J. A total of 75 cellular proteins were significantly changed, including 33 upregulated proteins and 42 downregulated proteins. The reliability of iTRAQ-LC MS/MS was confirmed via real-time PCR. Most of these proteins were related to the physiological functions of metabolic processes, biosynthetic processes, responses to stimuli, protein binding, signal transduction, cell cytoskeleton, and so forth. We also found some proteins that play important roles in apoptosis and oncogenicity. The differentially expressed proteins identified may provide valuable information to elucidate the pathogenesis of virus infection and virus-host interactions. Xiaofei Li, Qi Wang, Yanni Gao, Xiaole Qi, Yongqiang Wang, Honglei Gao, Yulong Gao, and Xiaomei Wang Copyright © 2015 Xiaofei Li et al. All rights reserved. Impact of Viral Activators and Epigenetic Regulators on HIV-1 LTRs Containing Naturally Occurring Single Nucleotide Polymorphisms Mon, 05 Jan 2015 13:34:21 +0000 Following human immunodeficiency virus type 1 (HIV-1) integration into host cell DNA, the viral promoter can become transcriptionally silent in the absence of appropriate signals and factors. HIV-1 gene expression is dependent on regulatory elements contained within the long terminal repeat (LTR) that drive the synthesis of viral RNAs and proteins through interaction with multiple host and viral factors. Previous studies identified single nucleotide polymorphisms (SNPs) within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III (3T, C-to-T change at position 3, and 5T, C-to-T change at position 5 of the binding site, respectively, when compared to the consensus B sequence) that are low affinity binding sites and correlate with more advanced stages of HIV-1 disease. Stably transfected cell lines containing the wild type, 3T, 5T, and 3T5T LTRs were developed utilizing bone marrow progenitor, T, and monocytic cell lines to explore the LTR phenotypes associated with these genotypic changes from an integrated chromatin-based microenvironment. Results suggest that in nonexpressing cell clones LTR-driven gene expression occurs in a SNP-specific manner in response to LTR activation or treatment with trichostatin A treatment, indicating a possible cell type and SNP-specific mechanism behind the epigenetic control of LTR activation. Sonia Shah, Vanessa Pirrone, Aikaterini Alexaki, Michael R. Nonnemacher, and Brian Wigdahl Copyright © 2015 Sonia Shah et al. All rights reserved. Cellular Levels of Oxidative Stress Affect the Response of Cervical Cancer Cells to Chemotherapeutic Agents Sun, 16 Nov 2014 08:26:03 +0000 Treatment of advanced and relapsed cervical cancer is frequently ineffective, due in large part to chemoresistance. To examine the pathways responsible, we employed the cervical carcinoma-derived SiHa and CaSki cells as cellular models of resistance and sensitivity, respectively, to treatment with chemotherapeutic agents, doxorubicin, and cisplatin. We compared the proteomic profiles of SiHa and CaSki cells and identified pathways with the potential to contribute to the differential response. We then extended these findings by comparing the expression level of genes involved in reactive oxygen species (ROS) metabolism through the use of a RT-PCR array. The analyses demonstrated that the resistant SiHa cells expressed higher levels of antioxidant enzymes. Decreasing or increasing oxidative stress led to protection or sensitization, respectively, in both cell lines, supporting the idea that cellular levels of oxidative stress affect responsiveness to treatment. Interestingly, doxorubicin and cisplatin induced different profiles of ROS, and these differences appear to contribute to the sensitivity to treatment displayed by cervical cancer cells. Overall, our findings demonstrate that cervical cancer cells display variable profiles with respect to their redox-generating and -adaptive systems, and that these different profiles have the potential to contribute to their responses to treatments with chemotherapy. Maria Filippova, Valery Filippov, Vonetta M. Williams, Kangling Zhang, Anatolii Kokoza, Svetlana Bashkirova, and Penelope Duerksen-Hughes Copyright © 2014 Maria Filippova et al. All rights reserved. Mechanisms of Action and Efficacy of Statins against Influenza Tue, 11 Nov 2014 13:40:24 +0000 The influenza virus (IV) is known to be a resistant virus with frequent mutations, causing severe respiratory diseases in the upper respiratory system. Public health concerns about clinical efficacy of all conventional drugs are ambiguous; therefore, finding additional therapeutic agents is critical to prevent and control influenza outbreaks. Influenza is associated with the induction of proinflammatory cytokines. Scientists have reported that anti-inflammatory drugs, with pleiotropic effects, reduce the burden of severe influenza diseases. Therefore, statins, which are cardioprotective drugs with anti-inflammatory and immunomodulatory effects, may help patients suffering from influenza virus (IV). This review delineates the potential use of statins as an alternative therapy in treating influenza related illness. Parvaneh Mehrbod, Abdul Rahman Omar, Mohd Hair-Bejo, Amin Haghani, and Aini Ideris Copyright © 2014 Parvaneh Mehrbod et al. All rights reserved. Rapid and Accurate Detection of Bacteriophage Activity against Escherichia coli O157:H7 by Propidium Monoazide Real-Time PCR Sun, 02 Nov 2014 12:02:26 +0000 Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol of E. coli require several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intact E. coli cells that survive phage exposure. Escherichia coli O157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cells E. coli O157:H7. Compared to PMA-qPCR, direct plating overestimated (P < 0.01) phage efficacy as cell surface-attached phage particles lysed E. coli O157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol of E. coli O157:H7. Hui Liu, Yan D. Niu, Jinquan Li, Kim Stanford, and Tim A. McAllister Copyright © 2014 Hui Liu et al. All rights reserved. Comparison of Structural Architecture of HCV NS3 Genotype 1 versus Pakistani Genotype 3a Tue, 21 Oct 2014 13:49:18 +0000 This study described the structural characterization of Pakistani HCV NS3 GT3a in parallel with genotypes 1a and 1b NS3. We investigated the role of amino acids and their interaction patterns in different HCV genotypes by crystallographic modeling. Different softwares were used to study the interaction pattern, for example, CLCBIO sequence viewer, MODELLER, NMRCLUST, ERRAT score, and MODELLER. Sixty models were produced and clustered into groups and the best model of PK-NCVI/Pk3a NS3 was selected and studied further to check the variability with other HCV NS3 genotypes. This study will help in future to understand the structural architecture of HCV genome variability and to further define the conserved targets for antiviral agents. Kaneez Fatima, Esam Azhar, Shilu Mathew, Ghazi Damanhouri, and Ishtiaq Qadri Copyright © 2014 Kaneez Fatima et al. All rights reserved. Delivery of Human EV71 Receptors by Adeno-Associated Virus Increases EV71 Infection-Induced Local Inflammation in Adult Mice Wed, 27 Aug 2014 07:54:27 +0000 Enterovirus71 (EV71) is now recognized as an emerging neurotropic virus in Asia and one major causative agent of hand-foot-mouth diseases (HFMD). However potential animal models for vaccine development are limited to young mice. In this study, we used an adeno-associated virus (AAV) vector to introduce the human EV71 receptors P-selectin glycoprotein ligand-1 (hPSGL1) or a scavenger receptor class-B member-2 (hSCARB2) into adult ICR mice to change their susceptibility to EV71 infection. Mice were administered AAV-hSCARB2 or AAV-hPSGL1 through intravenous and oral routes. After three weeks, expression of human SCARB2 and PSGL1 was detected in various organs. After infection with EV71, we found that the EV71 viral load in AAV-hSCARB2- or AAV-hPSGL1-transduced mice was higher than that of the control mice in both the brain and intestines. The presence of EV71 viral particles in tissues was confirmed using immunohistochemistry analysis. Moreover, inflammatory cytokines were induced in the brain and intestines of AAV-hSCARB2- or AAV-hPSGL1-transduced mice after EV71 infection but not in wild-type mice. However, neurological disease was not observed in these animals. Taken together, we successfully infected adult mice with live EV71 and induced local inflammation using an AAV delivery system. Hung-Bo Hsiao, Ai-Hsiang Chou, Su-I Lin, Shu-Pei Lien, Chia-Chyi Liu, Pele Chong, Chih-Yeh Chen, Mi-Hua Tao, and Shih-Jen Liu Copyright © 2014 Hung-Bo Hsiao et al. All rights reserved. Comment on “Clinical Profile and Outcome of Japanese Encephalitis in Children Admitted with Acute Encephalitis Syndrome” Wed, 27 Aug 2014 07:40:02 +0000 Girish Chandra Bhatt and Tanya Sharma Copyright © 2014 Girish Chandra Bhatt and Tanya Sharma. All rights reserved. The Sequential Tissue Distribution of Duck Tembusu Virus in Adult Ducks Mon, 18 Aug 2014 06:57:48 +0000 In 2010, a novel Tembusu virus (TMUV) that caused a severe decrease in the egg production of ducks was isolated in southeast China. Given the novelty of this duck pathogen, little information is available regarding its pathogenesis. Here, we systematically investigated the replication kinetics of TMUV PTD2010 in adult male and female ducks. We found that PTD2010 was detectable in most of the parenchymatous organs as well as the oviduct and intestinal tract from days 1 to 7 after inoculation. Viral titers were maintained at high levels for at least 9 days in the spleen, kidney, bursa of Fabricius, brain, and ovary. No virus was detected in any of these organs or tissues at 18 days after inoculation. PTD2010, thus, causes systemic infections in male and female ducks; its replication kinetics show similar patterns in most organs, with the exception of the ovaries and testes. Li Wu, Jinxiong Liu, Pucheng Chen, Yongping Jiang, Leilei Ding, Yuan Lin, Qimeng Li, Xijun He, Qiusheng Chen, and Hualan Chen Copyright © 2014 Li Wu et al. All rights reserved. Antiviral Action of Synthetic Stigmasterol Derivatives on Herpes Simplex Virus Replication in Nervous Cells In Vitro Thu, 24 Jul 2014 08:56:54 +0000 Polyfunctionalized stigmasterol derivatives, (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3β-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2), inhibit herpes simplex virus type 1 (HSV-1) replication and spreading in human epithelial cells derived from ocular tissues. Both compounds reduce the incidence and severity of lesions in a murine model of herpetic stromal keratitis when administered in different treatment modalities. Since encephalitis caused by HSV-1 is another immunopathology of viral origin, we evaluate here the antiviral effect of both compounds on HSV-1 infected nervous cell lines as well as their anti-inflammatory action. We found that both stigmasterol derivatives presented low cytotoxicity in the three nervous cell lines assayed. Regarding the antiviral activity, in all cases both compounds prevented HSV-1 multiplication when added after infection, as well as virus propagation. Additionally, both compounds were able to hinder interleukin-6 and Interferon-gamma secretion induced by HSV-1 infection in Neuro-2a cells. We conclude that compounds 1 and 2 have exerted a dual antiviral and anti-inflammatory effect in HSV-1 infected nervous cell lines, which makes them interesting molecules to be further studied. Erina Petrera, Analía G. Níttolo, and Laura E. Alché Copyright © 2014 Erina Petrera et al. All rights reserved. Progress in the Identification of Dengue Virus Entry/Fusion Inhibitors Thu, 24 Jul 2014 00:00:00 +0000 Dengue fever, a reemerging disease, is putting nearly 2.5 billion people at risk worldwide. The number of infections and the geographic extension of dengue fever infection have increased in the past decade. The disease is caused by the dengue virus, a flavivirus that uses mosquitos Aedes sp. as vectors. The disease has several clinical manifestations, from the mild cold-like illness to the more serious hemorrhagic dengue fever and dengue shock syndrome. Currently, there is no approved drug for the treatment of dengue disease or an effective vaccine to fight the virus. Therefore, the search for antivirals against dengue virus is an active field of research. As new possible receptors and biological pathways of the virus biology are discovered, new strategies are being undertaken to identify possible antiviral molecules. Several groups of researchers have targeted the initial step in the infection as a potential approach to interfere with the virus. The viral entry process is mediated by viral proteins and cellular receptor molecules that end up in the endocytosis of the virion, the fusion of both membranes, and the release of viral RNA in the cytoplasm. This review provides an overview of the targets and progress that has been made in the quest for dengue virus entry inhibitors. Carolina De La Guardia and Ricardo Lleonart Copyright © 2014 Carolina De La Guardia and Ricardo Lleonart. All rights reserved. Metabolic Variations, Antioxidant Potential, and Antiviral Activity of Different Extracts of Eugenia singampattiana (an Endangered Medicinal Plant Used by Kani Tribals, Tamil Nadu, India) Leaf Tue, 15 Jul 2014 08:27:49 +0000 Eugenia singampattiana is an endangered medicinal plant used by the Kani tribals of South India. The plant had been studied for its antioxidant, antitumor, antihyperlipidemic, and antidiabetic activity. But its primary and secondary metabolites profile and its antiviral properties were unknown, and so this study sought to identify this aspect in Eugenia singampattiana plant through different extraction methods along with their activities against porcine reproductive and respiratory syndrome virus (PRRSV). The GC-MS analysis revealed that 11 primary metabolites showed significant variations among the extracts. Except for fructose all other metabolites were high with water extract. Among 12 secondary metabolites showing variations, the levels of 4-hydroxy benzoic acid, caffeic acid, rutin, ferulic acid, coumaric acid, epigallocatechin gallate, quercetin, myricetin, and kaempferol were high with methanol extract. Since the flavonoid content of methanol extracts was high, the antioxidant potential, such as ABTS, and phosphomolybdenum activity increased. The plants antiviral activity against PRRSV was for the first time confirmed and the results revealed that methanol 25 µg and 75 to 100 µg in case of water extracts revealed antiviral activity. K. M. Maria John, Muniappan Ayyanar, Subbiah Jeeva, Murugesan Suresh, Gansukh Enkhtaivan, and Doo Hwan Kim Copyright © 2014 K. M. Maria John et al. All rights reserved. Molecular Epidemiology of Genogroup II Noroviruses Infection in Outpatients with Acute Gastroenteritis in Nanjing, China (2010–2013) Tue, 15 Jul 2014 08:27:04 +0000 Objective. Human noroviruses (NoVs) of genogroup II are the most common strains detected in sporadic cases of acute nonbacterial gastroenteritis in outpatients in Nanjing. To gain insight into the molecular epidemiology of GII strains, we analyzed 75 positive NoV cases from 2010 to 2013. Methods. The sporadic cases were detected by real-time PCR with specific primers and probes to human NoVs of genogroup I or II, human sapovirus, human rotavirus, human astrovirus, and human enteric adenovirus. Human NoVs of genogroup II were further studied by VP1 amplification (RT- PCR), cloning, sequencing, and phylogenetic analysis. Results. Rotavirus and human NoVs were more frequently detected in all the cases from 2010 to 2013. Human NoVs infection was more frequent since 2011 and more frequent than rotavirus infection after 2012. Out of the 75 NoV cases of genogroup II, there were 5 GII.6, 11 GII.3, and 59 GII.4. Of the 59 GII.4, 27 cases were previous GII.4.2006b strains that circulated between 2010 and 2012; while 32 cases were the newly emerging GII.4 strains GII.4.2012 from 2011 to 2013. Conclusion. Our data confirm other studies on the rapid emergence and displacement of highly virulent GII.4 strains. Zhang Hong-ying, Shi Li-min, Li Wei, Wang Xuan, Qiao Meng-kai, He Min, Wang Yan, and Xie Guo-xiang Copyright © 2014 Zhang Hong-ying et al. All rights reserved. Tick-Borne Encephalitis Virus Habitats in North East Germany: Reemergence of TBEV in Ticks after 15 Years of Inactivity Tue, 08 Jul 2014 00:00:00 +0000 The incidence of tick-borne encephalitis has risen in Europe since 1990 and the tick-borne encephalitis virus (TBEV) has been documented to be spreading into regions where it was not previously endemic. In Mecklenburg-West Pomerania, a federal state in Northern Germany, TBEV was not detectable in over 16,000 collected ticks between 1992 and 2004. Until 2004, the last human case of TBE in the region was reported in 1985. Following the occurrence of three autochthonous human cases of TBE after 2004, however, we collected ticks from the areas in which the infections were contracted. To increase the chance of detecting TBEV-RNA, some of the ticks were fed on mice. Using nested RT-PCR, we were able to confirm the presence of TBEV in ticks for the first time after 15 years. A phylogenetic analysis revealed a close relationship between the sequences we obtained and a TBEV sequence from Mecklenburg-East Pomerania published in 1992 and pointed to the reemergence of a natural focus of TBEV after years of low activity. Our results imply that natural foci of TBEV may either persist at low levels of activity for years or reemerge through the agency of migrating birds. Silvius Frimmel, Anja Krienke, Diana Riebold, Micha Loebermann, Martina Littmann, Karin Fiedler, Christine Klaus, Jochen Süss, and Emil Christian Reisinger Copyright © 2014 Silvius Frimmel et al. All rights reserved. Antagonizing Interferon-Mediated Immune Response by Porcine Reproductive and Respiratory Syndrome Virus Thu, 03 Jul 2014 00:00:00 +0000 Interferons (IFNs) are important components in innate immunity involved in the first line of defense to protect host against viral infection. Porcine reproductive and respiratory syndrome virus (PRRSV) leads to severe economic losses for swine industry since being first identified in early 1990s. PRRSV interplays with host IFN production and IFN-activated signaling, which may contribute to the delayed onset and low level of neutralizing antibodies, as well as weak cell-mediated immune response in infected pigs. PRRSV encodes several proteins that act as antagonists for the IFN signaling. In this review, we summarized the various strategies used by PRRSV to antagonize IFN production and thwart IFN-activated antiviral signaling, as well as the variable interference with IFN-mediated immune response by different PRRSV strains. Thorough understanding of the interaction between PRRSV and host innate immune response will facilitate elucidation of PRRSV pathogenesis and development of a better strategy to control PRRS. Rong Wang and Yan-Jin Zhang Copyright © 2014 Rong Wang and Yan-Jin Zhang. All rights reserved. A Complete Molecular Diagnostic Procedure for Applications in Surveillance and Subtyping of Avian Influenza Virus Thu, 26 Jun 2014 06:48:14 +0000 Introduction. The following complete molecular diagnostic procedure we developed, based on real-time quantitative PCR and traditional PCR, is effective for avian influenza surveillance, virus subtyping, and viral genome sequencing. Method. This study provides a specific and sensitive step-by-step procedure for efficient avian influenza identification of 16 hemagglutinin and 9 neuraminidase avian influenza subtypes. Result and Conclusion. This diagnostic procedure may prove exceedingly useful for virological and ecological advancements in global avian influenza research. Chun-Hsien Tseng, Hsiang-Jung Tsai, and Chung-Ming Chang Copyright © 2014 Chun-Hsien Tseng et al. All rights reserved. Seropositivity and Coinfection of Hepatitis B and C among Patients Seeking Hospital Care in Islamabad, Pakistan Tue, 24 Jun 2014 10:42:15 +0000 The undertaken study was conducted to find out the seroprevalence and coinfection of HBV and HCV infection among patients seeking hospital care. A total of 845 samples were received at tertiary care hospital of Islamabad and were screened for hepatitis B and C. The ELISA was used to detect antigen for HBV and antibodies for HCV in patient serum. Among 845 collected samples, 255 (30.1%) were seropositive for HBV and HCV. Out of 255 seropositive samples, 45 (5.3%) were positive for HBsAg while 199 (23.5%) were positive for anti-HCV. Among 255, 11 (1.3%) were seropositive for both HBsAg and anti-HCV (coinfection). Among the seropositive male, HBV was more prevalent (23.8%) while female patients had a high incidence of HCV (52.2%). Among the age group variable, HBV, HCV, and coinfection were found to be more common in the age groups of 21–30 (29%) and 30–40 (24%) years. The seropositivity for HBsAg was higher in unmarried individuals (31.2%) while anti-HCV was more prevalent in married individuals (84%). The present study provides the preliminary information about high HCV and HBV prevalence. Findings from the current study will be helpful for the better management and control of viral hepatitis among patients seeking hospital care. Jafar Khan, Mehwish Shafiq, Sameera Mushtaq, Sultan Ayaz, Riaz Ullah, Naser M. AbdEI-Salam, H. Fouad, and Mohammad Abdul Wasim Copyright © 2014 Jafar Khan et al. All rights reserved. Animal Arterivirus Infections Mon, 23 Jun 2014 05:52:53 +0000 Denis Archambault, Udeni B. R. Balasuriya, Raymond R. R. Rowland, Hanchun Yang, and Dongwan Yoo Copyright © 2014 Denis Archambault et al. All rights reserved. Sequence Variation in the E2-Binding Domain of HPV16 and Biological Function Evaluation in Tunisian Cervical Cancers Tue, 17 Jun 2014 06:29:16 +0000 HPV16 E2 variants have different effects on the transcriptional activity of the LCR. In this study, we examined the nucleotide and amino acid sequence variation within the HPV16 E2 gene and to correlate with disease progression. E2 gene disruption was detected by PCR amplification of the entire E2 gene using a single set of primers. Nucleotide variations were analyzed by bidirectional sequencing. mRNA expression patterns of E6 and E7 gene transcripts were evaluated by a reverse transcriptase-PCR method (RT-PCR). The detection of intact E2 genes was significantly higher among controls than cases (81.8% versus 37.5%, resp., ). Among the E subgroup, variation at position 3684 C>A results in the amino acid substitution T310K and was more common among the E2 undisrupted cases (7/9; 77.7%), compared to controls (2/9; 22.2%). In addition, specific sequence variations identified in the E2 ORF at positions 3684 C>A were associated with increased viral oncogenes E6-E7 production. Besides HPV16 E2 disruption, the 3684 C>A variation within undisrupted E2 genes could be involved in an alternative mechanism for deregulating the expression of the HPV16 E6 and E7 oncogenes and appears to be a major factor contributing to the development of cervical cancer in Tunisian women. Saloua Kahla, Lotfi Kochbati, Samia Hammami, Mohamed Badis Chanoufi, Mongi Maalej, and Ridha Oueslati Copyright © 2014 Saloua Kahla et al. All rights reserved. Genetic Characterization and Evolution of H1N1pdm09 after Circulation in a Swine Farm Mon, 16 Jun 2014 00:00:00 +0000 Following the emergence of the A(H1N1)pdm09 in humans, this novel influenza virus was reverse transmitted from infected people to swine population worldwide. In this study we investigated the molecular evolution of A(H1N1)pdm09 virus identified in pigs reared in a single herd. Nasal swabs taken from pigs showing respiratory distress were tested for influenza type A and A(H1N1)pdm09 by real-time RT-PCR assays. Virus isolation from positive samples was attempted by inoculation of nasal swabs samples into specific pathogen free embryonated chicken eggs (ECE) and complete genome sequencing was performed on virus strains after replication on ECE or from original swab sample. The molecular analysis of hemagglutinin (HA) showed, in four of the swine influenza viruses under study, a unique significant amino acid change, represented by a two-amino acid insertion at the HA receptor binding site. Phylogenetic analysis of HA, neuraminidase, and concatenated internal genes revealed a very similar topology, with viruses under study forming a separate cluster, branching outside the A(H1N1)pdm09 isolates recognized until 2014. The emergence of this new cluster of A(H1N1)pdm09 in swine raises further concerns about whether A(H1N1)pdm09 with new molecular characteristics will become established in pigs and potentially transmitted to humans. Arianna Boni, Gabriele Vaccari, Livia Di Trani, Guendalina Zaccaria, Giovanni Loris Alborali, Davide Lelli, Paolo Cordioli, and Ana Maria Moreno Copyright © 2014 Arianna Boni et al. All rights reserved. Influenza and Other Respiratory Viruses Involved in Severe Acute Respiratory Disease in Northern Italy during the Pandemic and Postpandemic Period (2009–2011) Thu, 12 Jun 2014 11:02:48 +0000 Since 2009 pandemic, international health authorities recommended monitoring severe and complicated cases of respiratory disease, that is, severe acute respiratory infection (SARI) and acute respiratory distress syndrome (ARDS). We evaluated the proportion of SARI/ARDS cases and deaths due to influenza A(H1N1)pdm09 infection and the impact of other respiratory viruses during pandemic and postpandemic period (2009–2011) in northern Italy; additionally we searched for unknown viruses in those cases for which diagnosis remained negative. 206 respiratory samples were collected from SARI/ARDS cases and analyzed by real-time RT-PCR/PCR to investigate influenza viruses and other common respiratory pathogens; also, a virus discovery technique (VIDISCA-454) was applied on those samples tested negative to all pathogens. Influenza A(H1N1)pdm09 virus was detected in 58.3% of specimens, with a case fatality rate of 11.3%. The impact of other respiratory viruses was 19.4%, and the most commonly detected viruses were human rhinovirus/enterovirus and influenza A(H3N2). VIDISCA-454 enabled the identification of one previously undiagnosed measles infection. Nearly 22% of SARI/ARDS cases did not obtain a definite diagnosis. In clinical practice, great efforts should be dedicated to improving the diagnosis of severe respiratory disease; the introduction of innovative molecular technologies, as VIDISCA-454, will certainly help in reducing such “diagnostic gap.” Elena Pariani, Marianna Martinelli, Marta Canuti, Seyed Mohammad Jazaeri Farsani, Bas B. Oude Munnink, Martin Deijs, Elisabetta Tanzi, Alessandro Zanetti, Lia van der Hoek, and Antonella Amendola Copyright © 2014 Elena Pariani et al. All rights reserved. Immunogenicity Studies of Bivalent Inactivated Virions of EV71/CVA16 Formulated with Submicron Emulsion Systems Wed, 11 Jun 2014 12:01:58 +0000 We assessed two strategies for preparing candidate vaccines against hand, foot, and mouth disease (HFMD) caused mainly by infections of enterovirus (EV) 71 and coxsackievirus (CV) A16. We firstly design and optimize the potency of adjuvant combinations of emulsion-based delivery systems, using EV71 candidate vaccine as a model. We then perform immunogenicity studies in mice of EV71/CVA16 antigen combinations formulated with PELC/CpG. A single dose of inactivated EV71 virion (0.2 μg) emulsified in submicron particles was found (i) to induce potent antigen-specific neutralizing antibody responses and (ii) consistently to elicit broad antibody responses against EV71 neutralization epitopes. A single dose immunogenicity study of bivalent activated EV71/CVA16 virion formulated with either Alum or PELC/CpG adjuvant showed that CVA16 antigen failed to elicit CVA16 neutralizing antibody responses and did not affect EV71-specific neutralizing antibody responses. A boosting dose of emulsified EV71/CVA16 bivalent vaccine candidate was found to be necessary to achieve high seroconversion of CVA16-specific neutralizing antibody responses. The current results are important for the design and development of prophylactic vaccines against HFMD and other emerging infectious diseases. Chih-Wei Lin, Chia-Chyi Liu, Tsung-Chun Lu, Shih-Jen Liu, Yen-Hung Chow, Pele Chong, and Ming-Hsi Huang Copyright © 2014 Chih-Wei Lin et al. All rights reserved. Discordant Correlation between Serological Assays Observed When Measuring Heterosubtypic Responses against Avian Influenza H5 and H7 Viruses in Unexposed Individuals Wed, 11 Jun 2014 11:41:30 +0000 The human population is constantly exposed to multiple influenza A subtypes due to zoonotic spillover and rapid viral evolution driven by intrinsic error-prone replication and immunological pressure. In this context, antibody responses directed against the HA protein are of importance since they have been shown to correlate with protective immunity. Serological techniques, detecting these responses, play a critical role for influenza surveillance, vaccine development, and assessment. As the recent human pandemics and avian influenza outbreaks have demonstrated, there is an urgent need to be better prepared to assess the contribution of the antibody response to protection against newly emerged viruses and to evaluate the extent of preexisting heterosubtypic immunity in populations. In this study, 68 serum samples collected from the Italian population between 1992 and 2007 were found to be positive for antibodies against H5N1 as determined by single radial hemolysis (SRH), but most were negative when evaluated using haemagglutination inhibition (HI) and microneutralisation (MN) assays. As a result of these discordant serological findings, the increased sensitivity of lentiviral pseudotypes was exploited in pseudotype-based neutralisation (pp-NT) assays and the results obtained provide further insight into the complex nature of humoral immunity against influenza A viruses. Eleonora Molesti, Francesca Ferrara, Giulia Lapini, Emanuele Montomoli, and Nigel Temperton Copyright © 2014 Eleonora Molesti et al. All rights reserved. Changes of Costimulatory Molecule CD28 on Circulating CD8+ T Cells Correlate with Disease Pathogenesis of Chronic Hepatitis B Tue, 10 Jun 2014 04:57:11 +0000 Costimulatory signals are critical for antiviral immunity. The aim of this study was to evaluate the change of costimulatory molecule CD28 on circulating CD8+ T cells in chronic hepatitis B patients (CHB). Seventy CHB patients and fifty-six healthy controls were included, and forty-eight CHB patients were recruited for 52 weeks of longitudinal investigation. The proportions of circulating CD8+CD28+ and CD8+CD28− subpopulations were determined by flow cytometry, and the CD8+CD28+/CD8+CD28− T cells ratio was calculated. Compared with the subpopulation in healthy controls, high proportions of CD8+CD28− subpopulation were observed in CHB patients. Similarly, the CD8+CD28+/CD8+CD28− T cells ratio was significantly decreased in CHB patients compared with healthy controls and correlated significantly with hepatitis B virus (HBV) loads. High proportions of CD8+CD28− subpopulation and low CD8+CD28+/CD8+CD28− T cells ratio were observed in hepatitis B e antigen- (HBeAg-) positive individuals as compared with that in HBeAg-negative subjects. A significant decrease in CD8+CD28− subpopulation, increase in CD8+CD28+ subpopulation, and CD8+CD28+/CD8+CD28− T cells ratio were seen in those patients who received efficient antiviral therapy. Thus, aberrant CD28 expression on circulating CD8+ T cells and the CD8+CD28+/CD8+CD28− T cells ratio reflect the dysregulation of T cell activation and are related to the pathogenesis of chronic HBV infection. Xuefen Li, Haishen Kong, Li Tian, Qiaoyun Zhu, Yiyin Wang, Yuejiao Dong, Qin Ni, and Yu Chen Copyright © 2014 Xuefen Li et al. All rights reserved. Protective Immunity Based on the Conserved Hemagglutinin Stalk Domain and Its Prospects for Universal Influenza Vaccine Development Sun, 25 May 2014 10:49:54 +0000 Influenza virus surface glycoprotein hemagglutinin (HA) is an excellent and chief target that elicits neutralizing antibodies during vaccination or natural infection. Its HA2 subunit (stem domain) is most conserved as compared to HA1 subunit (globular head domain). Current influenza vaccine relies on globular head domain that provides protection only against the homologous vaccine strains, rarely provides cross-protection against divergent strains, and needs to be updated annually. There is an urge for a truly universal vaccine that provides broad cross-protection against different subtype influenza A viruses along with influenza B viruses and need not be updated annually. Antibodies against the stem domain of hemagglutinin (HA) are able to neutralize a wide spectrum of influenza virus strains and subtypes. These stem-specific antibodies have great potential for the development of universal vaccine against influenza viruses. In this review, we have discussed the stem-specific cross-reactive antibodies and heterosubtypic protection provided by them. We have also discussed their epitope-based DNA vaccine and their future prospects in this scenario. Madhu Khanna, Sachin Sharma, Binod Kumar, and Roopali Rajput Copyright © 2014 Madhu Khanna et al. All rights reserved. Equine Arteritis Virus Does Not Induce Interferon Production in Equine Endothelial Cells: Identification of Nonstructural Protein 1 as a Main Interferon Antagonist Sun, 25 May 2014 09:26:28 +0000 The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-β was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-β mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-β promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response. Yun Young Go, Yanhua Li, Zhenhai Chen, Mingyuan Han, Dongwan Yoo, Ying Fang, and Udeni B. R. Balasuriya Copyright © 2014 Yun Young Go et al. All rights reserved. Antiviral Perspectives for Chikungunya Virus Thu, 15 May 2014 15:50:15 +0000 Chikungunya virus (CHIKV) is a mosquito-borne pathogen that has a major health impact in humans and causes acute febrile illness in humans accompanied by joint pains and, in many cases, persistent arthralgia lasting for weeks to years. CHIKV reemerged in 2005-2006 in several parts of the Indian Ocean islands and India after a gap of 32 years, causing millions of cases. The re-emergence of CHIKV has also resulted in numerous outbreaks in several countries in the eastern hemisphere, with a threat to further expand in the near future. However, there is no vaccine against CHIKV infection licensed for human use, and therapy for CHIKV infection is still mainly limited to supportive care as antiviral agents are yet in different stages of testing or development. In this review we explore the different perspectives for chikungunya treatment and the effectiveness of these treatment regimens and discuss the scope for future directions. Deepti Parashar and Sarah Cherian Copyright © 2014 Deepti Parashar and Sarah Cherian. All rights reserved. Growth and Replication of Infectious Bursal Disease Virus in the DF-1 Cell Line and Chicken Embryo Fibroblasts Wed, 14 May 2014 11:44:06 +0000 Infectious bursal disease virus (IBDV) causes a highly contagious disease in young chicks and leads to significant economic losses in the poultry industry. To determine a suitable cell line for IBDV infection, replication, and growth kinetics of the virus, DF-1 cells and chicken embryo fibroblasts (CEF) were used. The population doubling per day (Pd/D) was found to be higher in DF-1 as compared to CEF cells. A suitable time of infection (TOI) was established for increased production of virus and greater infectivity titers. The DF-1 and CEF cells were found to be susceptible to infection by producing marked cytopathic effects (CPEs), and the growth curves of IBDV in DF-1 and CEF cells were evaluated by infectivity assay using tissue culture infectious dose (TCID50). The cytopathic effects of the virus in DF-1 and CEF cells were found to be similar, but higher viral titers were detected in the DF-1 cells as compared to CEF. Thus the DF-1 cell line had a higher growth potential and infectivity, which will be of advantage in vaccine production. Kaliyaperumal Rekha, Chandran Sivasubramanian, Ill-Min Chung, and Muthu Thiruvengadam Copyright © 2014 Kaliyaperumal Rekha et al. All rights reserved. Induction of Antibodies and T Cell Responses by a Recombinant Influenza Virus Carrying an HIV-1 Tat Protein in Mice Wed, 14 May 2014 11:21:50 +0000 Recombinant influenza viruses hold promise as vectors for vaccines to prevent transmission of mucosal pathogens. In this study, we generated a recombinant WSN/Tat virus in which Tat protein lacking residues 51 to 59 of the basic domain was inserted into the N-terminus of the hemagglutinin (HA) of A/WSN/33 virus. The Tat insertion into the viral HA caused a 2-log reduction in viral titers in cell culture, compared with the parental A/WSN/33 virus, and severely affected virus replication in vivo. Nevertheless, Tat-specific antibodies and T cell responses were elicited upon a single intranasal immunization of BALB/c mice with WSN/Tat virus. Moreover, Tat-specific immune responses were also detected following vaccine administration via the vaginal route. These data provide further evidence that moderately large HIV antigens can be delivered by chimeric HA constructs and elicit specific immune responses, thus increasing the options for the potential use of recombinant influenza viruses, and their derivatives, for prophylactic and therapeutic vaccines. B. Garulli, G. Di Mario, M. G. Stillitano, D. Compagnoni, F. Titti, A. Cafaro, B. Ensoli, Y. Kawaoka, and M. R. Castrucci Copyright © 2014 B. Garulli et al. All rights reserved. Preparation of North American Type II PRRSV Infectious Clone Expressing Green Fluorescent Protein Thu, 08 May 2014 12:32:15 +0000 Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid by Spe I and Xho I sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesized in vitro and amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV. Liyue Wang, Kao Zhang, Hongyu Lin, Wenyan Li, Jiexia Wen, Jianlou Zhang, Yonghong Zhang, Xiujin Li, and Fei Zhong Copyright © 2014 Liyue Wang et al. All rights reserved. Quantitative Evaluation of the Severity of Acute Illness in Adult Patients with Tick-Borne Encephalitis Wed, 07 May 2014 12:24:05 +0000 The aim of the present study was to quantify the severity of acute illness in patients with tick-borne encephalitis and to ascertain this approach by comparing it to standard clinical assessment. We designed scoring system for quantification of the severity of acute illness in patients with tick-borne encephalitis. Certain number of points was allotted to the presence, intensity, and duration of individual symptoms/signs. According to the obtained score the disease was classified as mild, moderate, and severe. Tick-borne encephalitis was assessed clinically as mild when only signs/symptoms of meningeal involvement were found, moderate in case of monofocal neurological signs and/or mild to moderate signs/symptoms of central nervous system dysfunction, and severe in patients with multifocal neurological signs and/or symptoms of severe dysfunction of central nervous system. By designed scoring system 282 adult patients, 146 males and 136 females, average aged 52.2 ± 15.5 years (range 15–82 years), with confirmed tick-borne encephalitis, were prospectively assessed. In 279/282 (98.9%) patients the severity according to clinical assessment matched with the score ranges for mild, moderate, and severe disease. The proposed approach enables precise and straightforward appraisal of the severity of acute illness and could be useful for comparison of findings within/between study groups. Petra Bogovic, Mateja Logar, Tatjana Avsic-Zupanc, Franc Strle, and Stanka Lotric-Furlan Copyright © 2014 Petra Bogovic et al. All rights reserved. Taking Bacteriophage Therapy Seriously: A Moral Argument Tue, 29 Apr 2014 09:01:14 +0000 The excessive and improper use of antibiotics has led to an increasing incidence of bacterial resistance. In Europe the yearly number of infections caused by multidrug resistant bacteria is more than 400.000, each year resulting in 25.000 attributable deaths. Few new antibiotics are in the pipeline of the pharmaceutical industry. Early in the 20th century, bacteriophages were described as entities that can control bacterial populations. Although bacteriophage therapy was developed and practiced in Europe and the former Soviet republics, the use of bacteriophages in clinical setting was neglected in Western Europe since the introduction of traditional antibiotics. Given the worldwide antibiotic crisis there is now a growing interest in making bacteriophage therapy available for use in modern western medicine. Despite the growing interest, access to bacteriophage therapy remains highly problematic. In this paper, we argue that the current state of affairs is morally unacceptable and that all stakeholders (pharmaceutical industry, competent authorities, lawmakers, regulators, and politicians) have the moral duty and the shared responsibility towards making bacteriophage therapy urgently available for all patients in need. Gilbert Verbeken, Isabelle Huys, Jean-Paul Pirnay, Serge Jennes, Nina Chanishvili, Jacques Scheres, Andrzej Górski, Daniel De Vos, and Carl Ceulemans Copyright © 2014 Gilbert Verbeken et al. All rights reserved. Human Cytomegalovirus and Autoimmune Disease Tue, 29 Apr 2014 08:08:06 +0000 Human cytomegalovirus (HCMV) represents a prototypic pathogenic member of the β-subgroup of the herpesvirus family. A range of HCMV features like its lytic replication in multiple tissues, the lifelong persistence through periods of latency and intermitting reactivation, the extraordinary large proteome, and extensive manipulation of adaptive and innate immunity make HCMV a high profile candidate for involvement in autoimmune disorders. We surveyed the available literature for reports on HCMV association with onset or exacerbation of autoimmune disease. A causative linkage between HCMV and systemic lupus erythematosus (SLE), systemic sclerosis (SSc), diabetes mellitus type 1, and rheumatoid arthritis (RA) is suggested by the literature. However, a clear association of HCMV seroprevalence and disease could not be established, leaving the question open whether HCMV could play a coresponsible role for onset of disease. For convincing conclusions population-based prospective studies must be performed in the future. Specific immunopathogenic mechanisms by which HCMV could contribute to the course of autoimmune disease have been suggested, for example, molecular mimicry by UL94 in SSc and UL83/pp65 in SLE patients, as well as aggravation of joint inflammation by induction and expansion of CD4+/CD28− T-cells in RA patients. Further studies are needed to validate these findings and to lay the grounds for targeted therapeutic intervention. Anne Halenius and Hartmut Hengel Copyright © 2014 Anne Halenius and Hartmut Hengel. All rights reserved. Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India Sun, 27 Apr 2014 11:06:48 +0000 Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV). The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF. Aman Kamboj, Atul Kumar Pateriya, Anamika Mishra, Pradip Ranaware, Diwakar D. Kulkarni, and Ashwin Ashok Raut Copyright © 2014 Aman Kamboj et al. All rights reserved. Detection of Newcastle Disease Virus Minor Genetic Variants by Modified Single-Stranded Conformational Polymorphism Analysis Thu, 10 Apr 2014 00:00:00 +0000 Newcastle disease and Avian Influenza are considered to be the most dangerous fowl diseases which may cause huge economic losses. Newcastle disease is caused by the enveloped, and single-stranded RNA virus (NDV, APMV-1; belonging to Paramyxoviridae family), which can be further divided into sixteen different genotypes grouped into five pathotypes according to their pathogenicity. It has been reported that low pathogenic virus can greatly increase its pathogenicity even during a single passage. Additionally, due to the widespread use of live vaccines, a mixture of two or more different viruses in one sample can be detected. Hence, there is a great need for establishment of fast, inexpensive, sensitive, and relatively simple diagnostic method for multistrain and quasispecies detection of NDV infection. In this paper we describe a diagnostic method based on RT-PCR followed by a modified version of single-stranded conformational polymorphism analysis using short DNA fragments of gene encoding viral F protein. The method allows for rapid diagnosis of genetic variant emerging from previously stable population which may prevent the spread of the pathogenic viral variant. Lukasz Rabalski, Krzysztof Smietanka, Zenon Minta, and Boguslaw Szewczyk Copyright © 2014 Lukasz Rabalski et al. All rights reserved. The Regulation of Autophagy by Influenza A Virus Sun, 23 Mar 2014 06:32:47 +0000 Influenza A virus is a dreadful pathogen of animals and humans, causing widespread infection and severe morbidity and mortality. It is essential to characterize the influenza A virus-host interaction and develop efficient counter measures against the viral infection. Autophagy is known as a catabolic process for the recycling of the cytoplasmic macromolecules. Recently, it has been shown that autophagy is a critical mechanism underlying the interaction between influenza A virus and its host. Autophagy can be induced by the infection with influenza A virus, which is considered as a necessary process for the viral proliferation, including the accumulation of viral elements during the replication of influenza A virus. On the other hand, influenza A virus can inhibit the autophagic formation via interaction with the autophagy-related genes (Atg) and signaling pathways. In addition, autophagy is involved in the influenza virus-regulated cell deaths, leading to significant changes in host apoptosis. Interestingly, the high pathogenic strains of influenza A virus, such as H5N1, stimulate autophagic cell death and appear to interplay with the autophagy in distinct ways as compared with low pathogenic strains. This review discusses the regulation of autophagy, an influenza A virus driven process. Rong Zhang, Xiaojuan Chi, Song Wang, Baomin Qi, Xiaoqiang Yu, and Ji-Long Chen Copyright © 2014 Rong Zhang et al. All rights reserved. The Interplay of Reovirus with Autophagy Mon, 10 Mar 2014 16:18:00 +0000 Autophagy participates in multiple fundamental physiological processes, including survival, differentiation, development, and cellular homeostasis. It eliminates cytoplasmic protein aggregates and damaged organelles by triggering a series of events: sequestering the protein substrates into double-membrane vesicles, fusing the vesicles with lysosomes, and then degrading the autophagic contents. This degradation pathway is also involved in various disorders, for instance, cancers and infectious diseases. This paper provides an overview of modulation of autophagy in the course of reovirus infection and also the interplay of autophagy and reovirus. Hung-Chuan Chiu, Sarah Richart, Fong-Yuan Lin, Wei-Li Hsu, and Hung-Jen Liu Copyright © 2014 Hung-Chuan Chiu et al. All rights reserved. Commentary on the Regulation of Viral Proteins in Autophagy Process Mon, 10 Mar 2014 09:58:01 +0000 The ability to subvert intracellular antiviral defenses is necessary for virus to survive as its replication occurs only in the host cells. Viruses have to modulate cellular processes and antiviral mechanisms to their own advantage during the entire virus life cycle. Autophagy plays important roles in cell regulation. Its function is not only to catabolize aggregate proteins and damaged organelles for recycling but also to serve as innate immunity to remove intracellular pathogenic elements such as viruses. Nevertheless, some viruses have evolved to negatively regulate autophagy by inhibiting its formation. Even more, some viruses have employed autophagy to benefit their replication. To date, there are more and more growing evidences uncovering the functions of many viral proteins to regulate autophagy through different cellular pathways. In this review, we will discuss the relationship between viruses and autophagy and summarize the current knowledge on the functions of viral proteins contributing to affect autophagy process. Ching-Yuan Cheng, Pei-I Chi, and Hung-Jen Liu Copyright © 2014 Ching-Yuan Cheng et al. All rights reserved. Simple and Cost-Effective Restriction Endonuclease Analysis of Human Adenoviruses Sun, 09 Mar 2014 13:36:13 +0000 Restriction endonuclease analyses (REAs) constitute the only inexpensive molecular approach capable of typing and characterizing human adenovirus (HAdV) strains based on the entire genome. However, the application of this method is limited by the need for time-consuming and labor-intensive procedures. We herein developed a simple and cost-effective REA for assessing HAdV. The method consists of (1) simple and cost-effective DNA extraction, (2) fast restriction endonuclease (RE) digestion, and (3) speedy mini agarose gel electrophoresis. In this study, DNA was isolated according to the kit-based method and 21.0 to 28.0 μg of viral DNA was extracted from prototypes (HAdV-1, HAdV-3, HAdV-4, and HAdV-37) in each flask. The amount of DNA ranged from 11.4 to 57.0 μg among the HAdV-3 () isolates. The obtained viral DNA was found to be applicable to more than 10 types of REAs. Fast-cut restriction endonucleases (REs) were able to digest the DNA within 15 minutes, and restriction fragments were easily separated via horizontal mini agarose gel electrophoresis. The whole procedure for 10 samples can be completed within approximately six hours (the conventional method requires at least two days). These results show that our REA is potentially applicable in many laboratories in which HAdVs are isolated. Arun Kumar Adhikary, Nozomu Hanaoka, and Tsuguto Fujimoto Copyright © 2014 Arun Kumar Adhikary et al. All rights reserved. Differential Host Cell Gene Expression and Regulation of Cell Cycle Progression by Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Wed, 26 Feb 2014 12:25:31 +0000 Nonstructural protein 11 (nsp11) of porcine reproductive and respiratory syndrome virus (PRRSV) is a viral endoribonuclease with an unknown function. The regulation of cellular gene expression by nsp11 was examined by RNA microarrays using MARC-nsp11 cells constitutively expressing nsp11. In these cells, the interferon-β, interferon regulatory factor 3, and nuclear factor-κB activities were suppressed compared to those of parental cells, suggesting that nsp11 might serve as a viral interferon antagonist. Differential cellular transcriptome was examined using Affymetrix exon chips representing 28,536 transcripts, and after statistical analyses 66 cellular genes were shown to be upregulated and 104 genes were downregulated by nsp11. These genes were grouped into 5 major signaling pathways according to their functional relations: histone-related, cell cycle and DNA replication, mitogen activated protein kinase signaling, complement, and ubiquitin-proteasome pathways. Of these, the modulation of cell cycle by nsp11 was further investigated since many of the regulated genes fell in this particular pathway. Flow cytometry showed that nsp11 caused the delay of cell cycle progression at the S phase and the BrdU staining confirmed the cell cycle arrest in nsp11-expressing cells. The study provides insights into the understanding of specific cellular responses to nsp11 during PRRSV infection. Yan Sun, Dong Li, Sumanprava Giri, Supriya G. Prasanth, and Dongwan Yoo Copyright © 2014 Yan Sun et al. All rights reserved. Comparison of Host Immune Responses to Homologous and Heterologous Type II Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Challenge in Vaccinated and Unvaccinated Pigs Wed, 26 Feb 2014 08:42:50 +0000 Porcine reproductive and respiratory syndrome (PRRS) is a high-consequence animal disease with current vaccines providing limited protection from infection due to the high degree of genetic variation of field PRRS virus. Therefore, understanding host immune responses elicited by different PRRSV strains will facilitate the development of more effective vaccines. Using IngelVac modified live PRRSV vaccine (MLV), its parental strain VR-2332, and the heterologous KS-06-72109 strain (a Kansas isolate of PRRSV), we compared immune responses induced by vaccination and/or PRRSV infection. Our results showed that MLV can provide complete protection from homologous virus (VR-2332) and partial protection from heterologous (KS-06) challenge. The protection was associated with the levels of PRRSV neutralizing antibodies at the time of challenge, with vaccinated pigs having higher titers to VR-2332 compared to KS-06 strain. Challenge strain did not alter the cytokine expression profiles in the serum of vaccinated pigs or subpopulations of T cells. However, higher frequencies of IFN-γ-secreting PBMCs were generated from pigs challenged with heterologous PRRSV in a recall response when PBMCs were re-stimulated with PRRSV. Thus, this study indicates that serum neutralizing antibody titers are associated with PRRSV vaccination-induced protection against homologous and heterologous challenge. X. Li, A. Galliher-Beckley, L. Pappan, B. Trible, M. Kerrigan, A. Beck, R. Hesse, F. Blecha, J. C. Nietfeld, R. R. Rowland, and J. Shi Copyright © 2014 X. Li et al. All rights reserved. Genetic Diversity Analysis of Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses Emerging in Recent Years in China Tue, 25 Feb 2014 09:26:03 +0000 Porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by its extensive genetic diversity. Here we analyzed 101 sequences of NSP2 hypervariable region, 123 ORF3 sequences, and 118 ORF5 sequences from 128 PRRSV-positive clinical samples collected in different areas of China during 2008–early 2012. The results indicated that the amino acid identities of the three genes among these sequences were 87.6%–100%, 92.5%–100%, and 77%–100%, respectively. Meanwhile, 4 novel patterns of deletion and insertion in NSP2 region or GP5 were first found. The phylogenetic analysis on these 3 genes revealed that the Chinese PRRSV strains could be divided into three subgroups; majority of genes analyzed here were clustered in subgroup 3 with multiple branches; the strains with 30-aa deletion in NSP2-coding region were still the dominant virus in the field. Further phylogenetic analysis on four obtained complete genomic sequences showed that they were clustered into different branches with the Chinese corresponding representative strains. Our analyses suggest that the genetic diversity of genotype 2 PRRSV in the field displays a tendency of increasing in recent years in China, and the 30-aa deletion in NSP2-coding region should be no longer defined as the molecular marker of the Chinese HP-PRRSV. Lei Zhou, Xiaorong Yang, Yuan Tian, Shuoyan Yin, Gang Geng, Xinna Ge, Xin Guo, and Hanchun Yang Copyright © 2014 Lei Zhou et al. All rights reserved. Identification of a Chicken Anemia Virus Variant-Related Gyrovirus in Stray Cats in China, 2012 Thu, 13 Feb 2014 09:59:50 +0000 The chicken anemia virus (CAV), is a known member of the genus Gyrovirus and was first isolated from chickens in Japan in 1979. Some reports have also demonstrated that CAV can be identified in human stool specimens. In this study, a variant of CAV was detected using PCR with CAV-based primers in fecal samples of stray cats. The genome of CAV variant was sequenced and the results suggest that it could be a recombinant viral strain from parental CAV strains JQ690762 and AF311900. Recombination is an important evolutionary mechanism that contributes to genetic diversification. These findings indicate that CAV variant might have originated from CAV-infected chickens. The epidemiology and pathogenesis of this novel virus remains to be elucidated. This study underscores the importance of CAV surveillance and it presents the first evidence suggesting the possibility of CAV homologous recombination in cat. Xinheng Zhang, Yuanjia Liu, Jun Ji, Feng Chen, Baoli Sun, Chunyi Xue, Jingyun Ma, Yingzuo Bi, and Qingmei Xie Copyright © 2014 Xinheng Zhang et al. All rights reserved. A Novel Isolate with Deletion in GP3 Gene of Porcine Reproductive and Respiratory Syndrome Virus from Mid-Eastern China Wed, 12 Feb 2014 07:14:31 +0000 PRRSV strain SH1211 was isolated from the lung tissue of a piglet on a large-scale pig farm with approximately 30% morbidity and 50% mortality in mid-eastern China in 2012. The full-length genome of SH1211 was 15 313 nt in size, excluding the polyadenylated sequences, and shared 94.9% nucleotide sequence identity with the HP-PRRSV strain, JXA1. The GP2 and GP5 proteins of SH1211 shared only 91.5% and 85.1% amino acid sequence identities with those of the JXA1, respectively. A deletion at amino acid positions 68 and 69 was identified in the GP3 protein of SH1211, compared with the GP3 of Type-2 PRRSV isolates. A phylogenetic tree based on the nucleotide sequence of the complete genome showed that SH1211 is the most closely related to other HP-PRRSV strains isolated in China. However, phylogenetic analysis based on the GP2 and GP5 proteins showed that SH1211 is the most closely related to the QYYZ strain. A recombination analysis indicated that SH1211 might have been generated through recombination events between the JXA1 and QYYZ in which the GP2 and GP5 coding sequences were exchanged. Thus, SH1211 is a novel PRRSV strain with significant variation. Our analysis of SH1211 provides insight into the role of genetic variation in the antigenicity of PRRSVs in China. Baochao Fan, Hai Wang, Juan Bai, Lili Zhang, and Ping Jiang Copyright © 2014 Baochao Fan et al. All rights reserved. Development of a Reverse Transcription Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Subtype H7N9 Avian Influenza Virus Thu, 06 Feb 2014 00:00:00 +0000 A novel influenza A (H7N9) virus has emerged in China. To rapidly detect this virus from clinical samples, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7N9 virus. The minimum detection limit of the RT-LAMP assay was 0.01 PFU H7N9 virus, making this method 100-fold more sensitive to the detection of the H7N9 virus than conventional RT-PCR. The H7N9 virus RT-LAMP assays can efficiently detect different sources of H7N9 influenza virus RNA (from chickens, pigeons, the environment, and humans). No cross-reactive amplification with the RNA of other subtype influenza viruses or of other avian respiratory viruses was observed. The assays can effectively detect H7N9 influenza virus RNA in drinking water, soil, cloacal swab, and tracheal swab samples that were collected from live poultry markets, as well as human H7N9 virus, in less than 30 min. These results suggest that the H7N9 virus RT-LAMP assays were efficient, practical, and rapid diagnostic methods for the epidemiological surveillance and diagnosis of influenza A (H7N9) virus from different resource samples. Hongmei Bao, Yuhui Zhao, Yunhe Wang, Xiaolong Xu, Jianzhong Shi, Xianying Zeng, Xiurong Wang, and Hualan Chen Copyright © 2014 Hongmei Bao et al. All rights reserved. The Laboratory of Genetics and Physiology 2: Emerging Insights into the Controversial Functions of This RIG-I-Like Receptor Thu, 16 Jan 2014 06:46:38 +0000 The laboratory of genetics and physiology 2 (LGP2) is a key component of the RNA helicase family of retinoic acid-inducible gene 1- (RIG-I-) like receptors (RLRs) and is widely involved in viral RNA recognition and regulation during innate immune responses. Unlike RIG-I and melanoma differentiation-associated 5, both RLR members, LGP2 lacks the caspase-recruitment domain (CARD), which is required for recruiting and interacting with downstream signaling proteins to activate a cascade of downstream signaling events. The absence of the CARD results in divergent functional performance for LGP2 compared to these other RLR members. Both negative and positive regulatory roles have been reported for LGP2 in antiviral immune responses. It is currently unclear how the unusual properties of LGP2 mediate opposing roles. Future studies should elucidate the molecular mechanism(s) of LGP2 action. This minireview provides a brief overview of LGP2 structure and functions, with an expanded discussion on the regulation mechanisms in response to viral infection, hopefully stimulating insight into the divergent roles of LGP2 in the regulation of antiviral immune responses. Zixiang Zhu, Xiangle Zhang, Guoqing Wang, and Haixue Zheng Copyright © 2014 Zixiang Zhu et al. All rights reserved. Novel Bifunctional Single-Chain Variable Antibody Fragments to Enhance Virolysis by Complement: Generation and Proof-of-Concept Sun, 12 Jan 2014 09:45:28 +0000 When bound to the envelope of viruses, factor H (FH), a soluble regulator of complement activation, contributes to the protection against a potent immune defense mechanism, the complement-mediated lysis (CML). Thus, removing FH from the surface renders viruses, such as HIV, susceptible to CML. For a proof of concept, we developed a construct consisting of recombinant bifunctional single-chain variable fragment (scFv) based on a monoclonal antibody against Friend murine leukemia virus (F-MuLV) envelope protein gp70, which was coupled to specific binding domains (short consensus repeats 19-20; SCR1920) of FH. We used Pichia pastoris as expression system in common shake flasks and optimized expression in high density bench top fermentation. Specific binding of recombinant scFv was proven by flow cytometry. The recombinant scFv-SCR significantly enhanced CML of F-MuLV in vitro implying that FH binding to the viral surface was impaired by the scFv-SCR. This novel concept to enhance virolysis may provide a new approach for antiviral treatment. Georg Huber, Zoltán Bánki, Renate Kunert, and Heribert Stoiber Copyright © 2014 Georg Huber et al. All rights reserved. Potential Role of Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP2 in Apoptosis Inhibition Thu, 09 Jan 2014 09:50:04 +0000 Porcine reproductive and respiratory syndrome virus (PRRSV) is a serious threat to the pork industry, and its pathogenesis needs further investigations. To study the role of two structural proteins of PRRSV in virus-host cells interactions, two stable cell lines (MARC-2a and MARC-N) expressing GP2 and N proteins, respectively, were established. We induced apoptosis in these cells by treating them with staurosporine and found a significant reduction in the number of apoptotic cells in MARC-2a as compared to MARC-N and MARC-145 cells. In addition, we found significantly higher activities of transcriptional factors (NF-κB and AP-1) in both cell lines as compared to MARC-145 (parent cells). Overall, our data suggest that, although both stable cell lines activate NF-κB and AP-1, GP2 triggers the antiapoptotic process through an intermediate step that needs to be further investigated. Sujit Pujhari, Tayyba T. Baig, and Alexander N. Zakhartchouk Copyright © 2014 Sujit Pujhari et al. All rights reserved. Human Arthropod-Borne Viral Infections Sun, 29 Dec 2013 14:15:32 +0000 Aldo Manzin, Byron E. Martina, Ernest A. Gould, Patrizia Bagnarelli, and Vittorio Sambri Copyright © 2013 Aldo Manzin et al. All rights reserved. Seroprevalence of St. Louis Encephalitis Virus and West Nile Virus (Flavivirus, Flaviviridae) in Horses, Uruguay Sun, 29 Dec 2013 13:49:00 +0000 St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) belong to the Japanese encephalitis antigenic complex (Flavivirus genus, Flaviviridae family). They show antigenic close relationships and share many similarities in their ecology. Both are responsible for serious human diseases. The aim of this study was to investigate the presence of neutralizing antibodies to these viruses in horses from Uruguay. To do this, 425 horse sera were collected in 2007 and analyzed by plaque reduction neutralization tests. As a result, 205 sera (48.2%) were found positive for SLEV, with titers ranging between 10 and 80. Two sera remained inconclusive, since they showed low titers to WNV and SLEV (10 and 20), not allowing us to demonstrate activity of WNV in our territory. This is the first report of circulation of SLEV in horses in Uruguay. Analía Burgueño, Lorena Spinsanti, Luis Adrián Díaz, María Elisa Rivarola, Juan Arbiza, Marta Contigiani, and Adriana Delfraro Copyright © 2013 Analía Burgueño et al. All rights reserved. Clinical Profile and Outcome of Japanese Encephalitis in Children Admitted with Acute Encephalitis Syndrome Sun, 29 Dec 2013 13:19:40 +0000 Japanese encephalitis (JE) is an arthropod borne viral disease. Children are most commonly affected in Southeast Asian region showing symptoms of central nervous system with several complications and death. The clinical characteristics and outcomes in pediatric JE patients hospitalized with acute encephalitis syndrome (AES) are still poorly understood. A prospective study was conducted in pediatric ward of Assam Medical College Hospital to evaluate the clinical profile and outcome of JE in children. A total of 223 hospitalized AES cases were enrolled during March to December 2012. Serum and cerebro spinal fluids were tested for presence of JE specific IgM antibody. 67 (30%) were found to be JE positive. The most common presenting symptoms in JE patients were fever (100%), altered sensorium (83.58%), seizure (82.08%), headache (41.79%), and vomiting (29.85%). Signs of meningeal irritation were present in 55.22% of cases. Around 40.29%, JE patients had GCS ≤ 8. Among the JE patients, 14.7% died before discharge. The complete recoveries were observed in 63.9% of cases, while 21.3% had some sort of disability at the time of discharge. JE is still a major cause of AES in children in this part of India. These significant findings thus seek attentions of the global community to combat JE in children. Gitali Kakoti, Prafulla Dutta, Bishnu Ram Das, Jani Borah, and Jagadish Mahanta Copyright © 2013 Gitali Kakoti et al. All rights reserved. Construction and Immunogenicity of DNA Vaccines Encoding Fusion Protein of Porcine IFN-1 and GP5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Tue, 24 Dec 2013 13:43:13 +0000 Porcine reproductive and respiratory syndrome virus (PRRSV) has been mainly responsible for the catastrophic economic losses in pig industry worldwide. The commercial vaccines only provide a limited protection against PRRSV infection. Thus, the focus and direction is to develop safer and more effective vaccines in the research field of PRRS. The immune modulators are being considered to enhance the effectiveness of PRRSV vaccines. IFN-λ1 belongs to type III interferon, a new interferon family. IFN-λ1 is an important cytokine with multiple functions in innate and acquired immunity. In this study, porcine IFN-λ1 (PoIFN-λ1) was evaluated for its adjuvant effects on the immunity of a DNA vaccine carrying the GP5 gene of PRRSV. Groups of mice were immunized twice at 2-week interval with 100 μg of the plasmid DNA vaccine pcDNA3.1-SynORF5, pcDNA3.1-PoIFN-λ1-SynORF5, and the blank vector pcDNA3.1, respectively. The results showed that pcDNA3.1-PoIFN-λ1-SynORF5 can significantly enhance GP5-specific ELISA antibody, PRRSV-specific neutralizing antibody, IFN-γ level, and lymphocyte proliferation ratherthan the responses induced by pcDNA3.1-SynORF5. Therefore, type III interferon PoIFN-λ1 could enhance the immune responses of DNA vaccine of PRRSV, highlighting the potential value of PoIFN-λ1 as a molecular adjuvant in the prevention of PRRSV infection. Luping Du, Bin Li, Kongwang He, Haibin Zhang, Kehe Huang, and Shaobo Xiao Copyright © 2013 Luping Du et al. All rights reserved. The Usefulness of Clinical-Practice-Based Laboratory Data in Facilitating the Diagnosis of Dengue Illness Thu, 19 Dec 2013 12:58:42 +0000 Alertness to dengue and making a timely diagnosis is extremely important in the treatment of dengue and containment of dengue epidemics. We evaluated the complementary role of clinical-practice-based laboratory data in facilitating suspicion/diagnosis of dengue. One hundred overall dengue (57 dengue fever [DF] and 43 dengue hemorrhagic fever [DHF]) cases and another 100 nondengue cases (78 viral infections other than dengue, 6 bacterial sepsis, and 16 miscellaneous diseases) were analyzed. We separately compared individual laboratory variables (platelet count [PC] , prothrombin time [PT], activated partial thromboplastin time [APTT], alanine aminotransferase [ALT], and aspartate aminotransferase [AST]) and varied combined variables of DF and/or DHF cases with the corresponding ones of nondengue cases. The sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value (NPV) in the diagnosis of DF and/or DHF were measured based on these laboratory variables. While trade-off between sensitivity and specificity, and/or suboptimal PPV/NPV was found at measurements using these variables, prolonged APTT + normal PT + PC < 100 × 109 cells/L had a favorable sensitivity, specificity, PPV, and NPV in diagnosis of DF and/or DHF. In conclusion, these data suggested that prolonged APTT + normal PT + PC < 100 × 109 cells/L is useful in evaluating the likelihood of DF and/or DHF. Jien-Wei Liu, Ing-Kit Lee, Lin Wang, Rong-Fu Chen, and Kuender D. Yang Copyright © 2013 Jien-Wei Liu et al. All rights reserved. Biomarkers in Japanese Encephalitis: A Review Wed, 18 Dec 2013 18:26:51 +0000 JE is a flavivirus generated dreadful CNS disease which causes high mortality in various pediatric groups. JE disease is currently diagnosed by measuring the level of viral antigens and virus neutralization IgM antibodies in blood serum and CSF by ELISA. However, it is not possible to measure various disease-identifying molecules, structural and molecular changes occurred in tissues, and cells by using such routine methods. However, few important biomarkers such as cerebrospinal fluid, plasma, neuro-imaging, brain mapping, immunotyping, expression of nonstructural viral proteins, systematic mRNA profiling, DNA and protein microarrays, active caspase-3 activity, reactive oxygen species and reactive nitrogen species, levels of stress-associated signaling molecules, and proinflammatory cytokines could be used to confirm the disease at an earlier stage. These biomarkers may also help to diagnose mutant based environment specific alterations in JEV genotypes causing high pathogenesis and have immense future applications in diagnostics. There is an utmost need for the development of new more authentic, appropriate, and reliable physiological, immunological, biochemical, biophysical, molecular, and therapeutic biomarkers to confirm the disease well in time to start the clinical aid to the patients. Hence, the present review aims to discuss new emerging biomarkers that could facilitate more authentic and fast diagnosis of JE disease and its related disorders in the future. Ravi Kant Upadhyay Copyright © 2013 Ravi Kant Upadhyay. All rights reserved. Incidence of Japanese Encephalitis among Acute Encephalitis Syndrome Cases in West Bengal, India Mon, 11 Nov 2013 14:34:43 +0000 Background and Objectives. Japanese encephalitis (JE) is the most important cause of acute and epidemic viral encephalitis. Every year sporadic JE cases are reported from the various districts of West Bengal, indicating its endemicity in this state. JE vaccination programme has been undertaken by the State Health Department of West Bengal. This study was aimed at seeing the present scenario of JE among acute encephalitis syndrome (AES) cases in West Bengal. Materials and Methods. Blood and/or CSF samples were referred from suspected AES cases to the referral virology laboratory of the Calcutta School of Tropical Medicine from different hospitals of Kolkata. IgM antibody capture ELISA was performed on the CSF and serum samples by JE virus MAC ELISA kit supplied by the National Institute of Virology, Pune. Results. The present study reveals that 22.76% and 5% of the AES cases were positive for JE IgM in 2011 and 2012, respectively. JE is mainly prevalent in children and adolescents below 20 years of age with no gender predilection. Although the percentages of JE positive cases were high in 2011, it sharply decreased thereafter possibly due to better awareness programs, due to mass vaccination, or simply due to natural epidemiological niche periodicity due to herd immunity. Bhaswati Bandyopadhyay, Indrani Bhattacharyya, Srima Adhikary, Saiantani Mondal, Jayashree Konar, Nidhi Dawar, Asit Biswas, and Nemai Bhattacharya Copyright © 2013 Bhaswati Bandyopadhyay et al. All rights reserved. Proteomic Identification of Dengue Virus Binding Proteins in Aedes aegypti Mosquitoes and Aedes albopictus Cells Sun, 10 Nov 2013 09:24:10 +0000 The main vector of dengue in America is the mosquito Aedes aegypti, which is infected by dengue virus (DENV) through receptors of midgut epithelial cells. The envelope protein (E) of dengue virus binds to receptors present on the host cells through its domain III that has been primarily recognized to bind cell receptors. In order to identify potential receptors, proteins from mosquito midgut tissue and C6/36 cells were purified by affinity using columns with the recombinant E protein domain III (rE-DIII) or DENV particles bound covalently to Sepharose 4B to compare and evaluate their performance to bind proteins including putative receptors from female mosquitoes of Ae. aegypti. To determine their identity mass spectrometric analysis of purified proteins separated by polyacrylamide gel electrophoresis was performed. Our results indicate that both viral particles and rE-DIII bound proteins with the same apparent molecular weights of 57 and 67 kDa. In addition, viral particles bound high molecular weight proteins. Purified proteins identified were enolase, beta-adrenergic receptor kinase (beta-ARK), translation elongation factor EF-1 alpha/Tu, and cadherin. Maria de Lourdes Muñoz, Gustavo Limón-Camacho, Rosalinda Tovar, Alvaro Diaz-Badillo, Guillermo Mendoza-Hernández, and William C. Black IV Copyright © 2013 Maria de Lourdes Muñoz et al. All rights reserved. Hepatitis C Virus NS3 Inhibitors: Current and Future Perspectives Sun, 27 Oct 2013 17:30:14 +0000 Currently, hepatitis C virus (HCV) infection is considered a serious health-care problem all over the world. A good number of direct-acting antivirals (DAAs) against HCV infection are in clinical progress including NS3-4A protease inhibitors, RNA-dependent RNA polymerase inhibitors, and NS5A inhibitors as well as host targeted inhibitors. Two NS3-4A protease inhibitors (telaprevir and boceprevir) have been recently approved for the treatment of hepatitis C in combination with standard of care (pegylated interferon plus ribavirin). The new therapy has significantly improved sustained virologic response (SVR); however, the adverse effects associated with this therapy are still the main concern. In addition to the emergence of viral resistance, other targets must be continually developed. One such underdeveloped target is the helicase portion of the HCV NS3 protein. This review article summarizes our current understanding of HCV treatment, particularly with those of NS3 inhibitors. Kazi Abdus Salam and Nobuyoshi Akimitsu Copyright © 2013 Kazi Abdus Salam and Nobuyoshi Akimitsu. All rights reserved. N2 Gas Plasma Inactivates Influenza Virus by Inducing Changes in Viral Surface Morphology, Protein, and Genomic RNA Mon, 30 Sep 2013 08:30:43 +0000 We have recently treated with N2 gas plasma and achieved inactivation of bacteria. However, the effect of N2 gas plasma on viruses remains unclear. With the aim of developing this technique, we analyzed the virucidal effect of N2 gas plasma on influenza virus and its influence on the viral components. We treated influenza virus particles with inert N2 gas plasma (1.5 kpps; kilo pulses per second) produced by a short high-voltage pulse generated from a static induction thyristor power supply. A bioassay using chicken embryonated eggs demonstrated that N2 gas plasma inactivated influenza virus in allantoic fluid within 5 min. Immunochromatography, enzyme-linked immunosorbent assay, and Coomassie brilliant blue staining showed that N2 gas plasma treatment of influenza A and B viruses in nasal aspirates and allantoic fluids as well as purified influenza A and B viruses induced degradation of viral proteins including nucleoprotein. Analysis using the polymerase chain reaction suggested that N2 gas plasma treatment induced changes in the viral RNA genome. Scanning electron microscopy analysis showed that aggregation and fusion of influenza viruses were induced by N2 gas plasma treatment. We believe these biochemical changes may contribute to the inactivation of influenza viruses by N2 gas plasma. Akikazu Sakudo, Naohiro Shimizu, Yuichiro Imanishi, and Kazuyoshi Ikuta Copyright © 2013 Akikazu Sakudo et al. All rights reserved. Influenza Virus Specific CD8+ T Cells Exacerbate Infection Following High Dose Influenza Challenge of Aged Mice Thu, 26 Sep 2013 11:49:54 +0000 Influenza viruses cause severe illnesses and death, mainly in the aged population. Protection afforded by licensed vaccines through subtype-specific neutralizing antibodies is incomplete, especially when the vaccine antigens fail to closely match those of the circulating viral strains. Efforts are underway to generate a so-called universal influenza vaccine expressing conserved viral sequences that induce broad protection to multiple strains of influenza virus through the induction of CD8+ T cells. Here we assess the effect of a potent antiviral CD8+ T cell response on influenza virus infection of young and aged mice. Our results show that CD8+ T cell-inducing vaccines can provide some protection to young mice, but they exacerbate influenza virus-associated disease in aged mice, causing extensive lung pathology and death. E. M. Parzych, L. J. DiMenna, B. P. Latimer, J. C. Small, S. Kannan, B. Manson, M. O. Lasaro, E. J. Wherry, and H. C. Ertl Copyright © 2013 E. M. Parzych et al. All rights reserved. Dose- and Time-Dependent Apoptosis Induced by Avian H9N2 Influenza Virus in Human Cells Wed, 11 Sep 2013 12:09:45 +0000 To understand human response to avian H9N2 influenza, we investigated the effects of the viral infection on A549, HepG2, and HeLa cells at low and high MOIs. To identify virus-host interplay, expression of Mx and NP genes was measured in the cells supernatants. Cell viability and apoptosis were evaluated by MTT assay, DNA fragmentation, and florescent staining. The virus titration and NP gene transcript levels indicate lower susceptibility of HeLa cell to H9N2 replication than other cells. Although H9N2 did produce a faster CPE in HepG2, high dose of the virus induced apoptosis within early stage of A549 infection. The DNA laddering was enhanced in the cell correlated with increase in virus transcripts. The undetectable to different regulation levels of Mx gene were observed in response to H9N2 infection suggesting that an insufficient antiviral defense in the noncompetent-IFN HepG2 cell promotes efficient viral replication. These results showed that the permissivity of HepG2 for H9N2 is comparable with A549; however, liver cells are not target tissue respond to the infection. These data revealed that the H9N2 virus induced apoptosis signaling via mitochondrial pathway in human alveolar epithelial cells, indicating that the induction may be associated with a dose-dependent manner. Shahla Shahsavandi, Mohammad Majid Ebrahimi, Kaveh Sadeghi, Seyedeh Zahra Mosavi, and Ashraf Mohammadi Copyright © 2013 Shahla Shahsavandi et al. All rights reserved. High-Level Expression of Functionally Active Dengue-2 Non-Structural Antigen 1 Production in Escherichia coli Sun, 08 Sep 2013 18:02:15 +0000 Detection of nonstructural protein (NS1) is an important diagnostic marker during acute phase of dengue infection. Not only for diagnostic purpose, the protein had important role in vaccine design as well, as a candidate for studying virus assembly and maturation. Various researchers employed different expression systems and strategies for recombinant NS1 protein production. Attempts to express NS1 protein in prokaryotic and yeast expression system result in formation of insoluble protein which needs to undergo refolding to attain native structural and functional forms. Here, we report the production of soluble NS1 protein in E. coli by using appropriate vector and employing suitable culture conditions to maximize protein production. Proteins were purified using metal affinity chromatography. SDS-PAGE and western blot analysis reveal the native structure of NS1 protein. Solid phase ELISA using the recombinantly expressed antigen with positive and negative dengue samples showed that the expressed protein retains its antigenic and immunological properties. To our knowledge, this is the first report on the successful production of functionally active recombinant dengue-2 NS1 protein production without undergoing any in vitro posttranslational modification process. S. Gowri Sankar, K. J. Dhanajeyan, R. Paramasivan, V. Thenmozhi, B. K. Tyagi, and S. John Vennison Copyright © 2013 S. Gowri Sankar et al. All rights reserved. Soluble Form of Canine Transferrin Receptor Inhibits Canine Parvovirus Infection In Vitro and In Vivo Sun, 08 Sep 2013 15:15:31 +0000 Canine parvovirus (CPV) disease is an acute, highly infectious disease threatening the dog-raising industry. So far there are no effective therapeutic strategies to control this disease. Although the canine transferrin receptor (TfR) was identified as a receptor for CPV infection, whether extracellular domain of TfR (called soluble TfR (sTfR)) possesses anti-CPV activities remains elusive. Here, we used the recombinant sTfR prepared from HEK293T cells with codon-optimized gene structure to investigate its anti-CPV activity both in vitro and in vivo. Our results indicated that codon optimization could significantly improve sTfR expression in HEK293T cells. The prepared recombinant sTfR possessed a binding activity to both CPV and CPV VP2 capsid proteins and significantly inhibited CPV infection of cultured feline F81 cells and decreased the mortality of CPV-infected dogs, which indicates that the sTfR has the anti-CPV activity both in vitro and in vivo. Jiexia Wen, Sumin Pan, Shuang Liang, Zhenyu Zhong, Ying He, Hongyu Lin, Wenyan Li, Liyue Wang, Xiujin Li, and Fei Zhong Copyright © 2013 Jiexia Wen et al. All rights reserved. PDZ Domains and Viral Infection: Versatile Potentials of HPV-PDZ Interactions in relation to Malignancy Wed, 04 Sep 2013 11:59:13 +0000 Cervical cancer is caused by high-risk human papillomaviruses (HPVs), and a unique characteristic of these is a PDZ (SD-95/lg/O-1-)binding motif in their E6 proteins. Through this motif HPV E6 interacts with a variety of PDZ domain-containing proteins and targets them mainly for degradation. These E6-PDZ interactions exhibit extraordinarily different functions in relation to HPV-induced malignancy, depending upon various cellular contexts; for example, Dlg and Scrib show different distribution patterns from what is seen in normal epithelium, both in localization and in amount, and their loss may be a late-stage marker in malignant progression. Recent studies show that interactions with specific forms of the proteins may have oncogenic potential. In addition, it is interesting that PDZ proteins make a contribution to the stabilization of E6 and viral episomal maintenance during the course of HPV life cycle. Various posttranslational modifications also greatly affect their functions. Phosphorylation of hDlg and hScrib by certain kinases regulates several important signaling cascades, and E6-PDZ interactions themselves are regulated through PKA-dependent phosphorylation. Thus these interactions naturally have great potential for both predictive and therapeutic applications, and, with development of screening tools for identifying novel targets of their interactions, comprehensive spatiotemporal analysis is currently underway. Kazunori Nagasaka, Kei Kawana, Yutaka Osuga, and Tomoyuki Fujii Copyright © 2013 Kazunori Nagasaka et al. All rights reserved. Report on Influenza A and B Viruses: Their Coinfection in a Saudi Leukemia Patient Mon, 02 Sep 2013 13:57:58 +0000 Purpose. Influenza A and B viruses are the leading cause of respiratory infections in children worldwide, particularly in developing countries. There is a lack of data on coinfection of influenza A and B viruses circulating in Saudi Arabia. In this study, we aimed to identify the circulation of influenza viruses that contribute to respiratory tract infections in Saudi children. Methods. We collected 80 nasopharyngeal aspirates (NPAs) from hospitalized children with acute respiratory illness (ARI) at Riyadh during the period extended from October 2010 till April 2011. Samples were tested for the common respiratory viruses including influenza viruses by RT-PCR. Results. Overall, 6 samples were found positive for influenza A and/or B viruses. Among these positive clinical samples, only one collected sample from a female one-year-old immunocompromised child with leukemia showed a coinfection with influenza A and B viruses. In present study coinfection was confirmed by inoculation of the clinical specimen in specific pathogenfree embryonating chicken eggs and identification of the virus isolates by hemagglutination and one-step RT-PCR. Conclusion. This study opens the scene for studying the role of influenza virus’s coinfection in disease severity and virus evolution. Further studies are required to better understand the clinical importance of viral coinfection. Fahad N. Almajhdi and Ghazanfar Ali Copyright © 2013 Fahad N. Almajhdi and Ghazanfar Ali. All rights reserved. Increased Production of Interleukin-4, Interleukin-10, and Granulocyte-Macrophage Colony-Stimulating Factor by Type 2 Diabetes’ Mononuclear Cells Infected with Dengue Virus, but Not Increased Intracellular Viral Multiplication Mon, 02 Sep 2013 09:49:26 +0000 It has been reported that diabetes mellitus (DM) was an epidemiologically identified risk factor for development of dengue hemorrhagic fever (DHF)/severe dengue in dengue virus (DENV) affected patients, and T helper 2 (Th2) cytokines such as interleukin-4 (IL-4) and IL-10 each plays an important role in the immunopathogenesis of DHF in studies involving general population. To better understand the relationship between these epidemiological and immunological findings, we performed an in vitro study evaluating the sequential immunological reactions and viral load in the DENV infected mononuclear cells of adults with type 2 DM (T2DM group, ) and normal adults (control group, ). We found in the T2DM group significantly higher IL-4 level on the first and the third postinfection days, while higher IL-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were detected on the third postinfection day. No significant difference in DENV viral load between the cultured mononuclear cells from both groups was found on the first and third post-infection days. These data immunologically suggest that patients with T2DM are at higher risk for development of DHF/severe dengue and strengthen the previously epidemiologically identified role of DM being a predictive risk factor for progressing into DHF/severe dengue in DENV-affected patients. Ing-Kit Lee, Ching-Jung Hsieh, Rong-Fu Chen, Zih-Syuan Yang, Lin Wang, Chang-Mei Chen, Chiung-Fen Liu, Chung-Hao Huang, Chun-Yu Lin, Yen-Hsu Chen, Kuender D Yang, and Jien-Wei Liu Copyright © 2013 Ing-Kit Lee et al. All rights reserved. Molecular Mechanisms Involved in the Pathogenesis of Alphavirus-Induced Arthritis Wed, 28 Aug 2013 07:52:06 +0000 Arthritogenic alphaviruses, including Ross River virus (RRV), Chikungunya virus (CHIKV), Sindbis virus (SINV), Mayaro virus (MAYV), O'nyong-nyong virus (ONNV), and Barmah Forest virus (BFV), cause incapacitating and long lasting articular disease/myalgia. Outbreaks of viral arthritis and the global distribution of these diseases point to the emergence of arthritogenic alphaviruses as an important public health problem. This review discusses the molecular mechanisms involved in alphavirus-induced arthritis, exploring the recent data obtained with in vitro systems and in vivo studies using animal models and samples from patients. The factors associated to the extension and persistence of symptoms are highlighted, focusing on (a) virus replication in target cells, and tissues, including macrophages and muscle cells; (b) the inflammatory and immune responses with recruitment and activation of macrophage, NK cells and T lymphocytes to the lesion focus and the increase of inflammatory mediators levels; and (c) the persistence of virus or viral products in joint and muscle tissues. We also discuss the importance of the establishment of novel animal models to test new molecular targets and to develop more efficient and selective drugs to treat these diseases. Iranaia Assunção-Miranda, Christine Cruz-Oliveira, and Andrea T. Da Poian Copyright © 2013 Iranaia Assunção-Miranda et al. All rights reserved. Prevalence of Occult Hepatitis B Virus Infection in a Cohort of HIV-Positive Patients Resident in Sicily, Italy Mon, 26 Aug 2013 08:54:25 +0000 Occult hepatitis B virus (OBI) in HIV-infected groups is still debated, as well as the associated risk-factors and clinical significance. In this paper, we examined a total of 405 HBsAg-negative/HIV-infected patients enrolled from January 2007 to December 2009. Overall, the prevalence of OBI was 5.9% (95% confidence interval (CI95%): 3.8–8.7%); it was more frequently associated with “anti-HBc alone” serological marker (11.3%; adjusted odds ratio = 3.7, CI95%: 1.4–9.8), although it was also detected in the absence of any HBV serological marker (4.9%; CI95%: 2.3–9.1%). A low prevalence of anti-HCV-positive patients with OBI was found (3.1%; CI95%: 0.6–8.7%). HIV RNA plasma levels or other immunological/clinical characteristics were not significantly associated with OBI. All but one occult HBV infections were sustained by genotype D viral strains. OBI is relatively frequent in HIV-infected patients, although it does not seem to exert a relevant clinical impact. Viral genotypes in occult HBV infections reflect those circulating in the Mediterranean area. Fabio Tramuto, Carmelo Massimo Maida, Giuseppina M. E. Colomba, Paola Di Carlo, and Francesco Vitale Copyright © 2013 Fabio Tramuto et al. All rights reserved. Possible Future Monoclonal Antibody (mAb)-Based Therapy against Arbovirus Infections Thu, 22 Aug 2013 10:18:56 +0000 More than 150 arboviruses belonging to different families are known to infect humans, causing endemic infections as well as epidemic outbreaks. Effective vaccines to limit the occurrence of some of these infections have been licensed, while for the others several new immunogens are under development mostly for their improvements concerning safety and effectiveness profiles. On the other hand, specific and effective antiviral drugs are not yet available, posing an urgent medical need in particular for emergency cases. Neutralizing monoclonal antibodies (mAbs) have been demonstrated to be effective in the treatment of several infectious diseases as well as in preliminary in vitro and in vivo models of arbovirus-related infections. Given their specific antiviral activity as well-tolerated molecules with limited side effects, mAbs could represent a new therapeutic approach for the development of an effective treatment, as well as useful tools in the study of the host-virus interplay and in the development of more effective immunogens. However, before their use as candidate therapeutics, possible hurdles (e.g., Ab-dependent enhancement of infection, occurrence of viral escape variants) must be carefully evaluated. In this review are described the main arboviruses infecting humans and candidate mAbs to be possibly used in a future passive immunotherapy. Giuseppe Sautto, Nicasio Mancini, Giacomo Gorini, Massimo Clementi, and Roberto Burioni Copyright © 2013 Giuseppe Sautto et al. All rights reserved. Full-Length Genomic Sequence of Subgenotype IIIA Hepatitis A Virus Isolate in Republic of Korea Wed, 07 Aug 2013 13:09:05 +0000 Hepatitis A virus is known to cause acute hepatitis and has significant implications for public health throughout the world. In the Republic of Korea, the number of patients with hepatitis A virus infection has been increasing rapidly since 2006. In this study, the Kor-HAV-F strain was identified as subgenotype IIIA by RT-PCR, and its identity was confirmed by nucleotide sequencing and alignment analysis. Moreover, detailed phylogenetic analysis indicated that the Kor-HAV-F strain clustered into subgenotype IIIA, including strains isolated in Japan, Norway, and India. The entire amino acid sequence of the VP1 and 2A regions was compared with that of the reference strains isolated in various countries. We found 2 amino acid changes (T168A and L96P, resp.) in the VP1 and 2A regions, which had not been found in any other hepatitis A virus strain. To our knowledge, this study is the first to report the full-length sequence of a hepatitis A virus isolated in the Republic of Korea. Ah-Ra Lee, Sung-Geun Lee, Lae-Hyung Kang, Weon-Hwa Jheong, and Soon-Young Paik Copyright © 2013 Ah-Ra Lee et al. All rights reserved. Virus-Like Particles Containing the Tetrameric Ectodomain of Influenza Matrix Protein 2 and Flagellin Induce Heterosubtypic Protection in Mice Tue, 30 Jul 2013 08:10:10 +0000 The ectodomain of matrix protein 2 (M2e) is highly conserved among influenza A viruses and can be a promising candidate antigen for a broadly cross-protective vaccine. In this study, a tetrameric M2e (tM2e) and a truncated form of flagellin (tFliC) were coincorporated into virus-like particles (VLPs) to enhance its immunogenicity. Our data showed that the majority of M2e in VLPs was presented as tetramers by introducing a foreign tetramerization motif GCN4. Intranasal immunization with tM2e VLPs significantly enhanced the levels of serum IgG and IgG subclasses compared to soluble M2e (sM2e) in mice. tM2e VLPs also induced higher M2e-specific T-cell and mucosal antibody responses, conferring complete protection against homologous influenza virus infection. The immunogenicity of tM2e VLPs was further enhanced by coincorporation of the membrane-anchored tFliC (tM2e chimeric VLPs) or coadministration with tFliC VLPs as a mixture, but not the soluble flagellin, inducing strong humoral and cellular immune responses conferring cross-protection against lethal challenge with heterotypic influenza viruses. These results support the development of tM2e chimeric VLPs as universal vaccines and warrant further investigation. Li Wang, Ying-Chun Wang, Hao Feng, Tamanna Ahmed, Richard W. Compans, and Bao-Zhong Wang Copyright © 2013 Li Wang et al. All rights reserved. Understanding the Dengue Viruses and Progress towards Their Control Tue, 09 Jul 2013 10:01:00 +0000 Traditionally, the four dengue virus serotypes have been associated with fever, rash, and the more severe forms, haemorrhagic fever and shock syndrome. As our knowledge as well as understanding of these viruses increases, we now recognise not only that they are causing increasing numbers of human infections but also that they may cause neurological and other clinical complications, with sequelae or fatal consequences. In this review we attempt to highlight some of these features in the context of dengue virus pathogenesis. We also examine some of the efforts currently underway to control this “scourge” of the tropical and subtropical world. Rosmari Rodriguez-Roche and Ernest A. Gould Copyright © 2013 Rosmari Rodriguez-Roche and Ernest A. Gould. All rights reserved. Molecular Characterization of BK and JC Viruses Circulating among Potential Kidney Donors in Kuwait Tue, 09 Jul 2013 09:47:55 +0000 BK and JC polyomaviruses can be associated with nephropathy following renal transplantation. The aim of this study was to determine the prevalence, load, and genotypes of BK and JC viruses circulated in potential kidney donors in Kuwait. The detection of polyomavirus DNA was carried out in serum and urine samples of 165 potential kidney donors. Seventy (42%) individuals were tested positive for polyomavirus DNA, of whom 20 (12%) had detectable polyomavirus DNA in their serum samples, 40 (24%) in their urine samples, and 10 (6%) in both serum and urine samples. In the group of polyomavirus-positive patients, JC DNA could be detected in 78% of urine samples and 11% of serum samples, whereas BK DNA could be detected in 7% of urine samples and 3% of serum samples. The median polyomaviral load was low. The detected BK sequences in Kuwaiti adults formed new clusters sharing common ancestor with subgroups Ib1 and IVc, which are prevalent in Asia and Europe. Additionally, around half of the detected JCV sequences in Kuwaiti adults formed new clusters within the African subtype 3. Our results suggest high rate of polyomavirus shedding among healthy adults in Kuwait that can jeopardize their suitability for kidney donation. Wassim Chehadeh, Susan Silpi Kurien, and Mangalathillam Raman Nampoory Copyright © 2013 Wassim Chehadeh et al. All rights reserved. A Mouse Model of Interstitial Pneumonitis Induced by Murine Cytomegalovirus Infection after Allogeneic Skin Transplantation Tue, 02 Jul 2013 11:15:48 +0000 We investigated the effect of murine cytomegalovirus (MCMV) on interstitial pneumonia in transplant recipients in an experimental skin allograft model. Skin transplantation between C57BL/6J and BALB/c mice was performed in the presence or absence of cyclosporin A treatment. Flow cytometry showed that the number of CD4+ and CD8+ cells and the level of IFN-γ decreased significantly in the groups treated with cyclosporin A. We either mock-infected or infected the mice with MCMV by intranasal administration and monitored pathophysiological behavior and body weight. The infected mice were sacrificed at different days postinfection for histology, immunohistochemistry, and molecular biological evaluations. Interstitial pneumonitis was observed in positive control groups as well as in experimental group that received cyclosporin A, a skin transplant, and infected with the highest dose of virus (105 PFU). Transmission electronic microscopy demonstrated the presence of herpes virus particles. MCMV DNA and glycoprotein B were demonstrated in the epithelial cells of the lung tissue in those animals by in situ hybridization and immunohistochemistry, respectively. Our data demonstrated the establishment of a mouse model of interstitial pneumonitis via MCMV infection after allogeneic skin transplantation. Dequn Ni, Haiyang Yu, Wei Zhang, Lin Gan, Jiqiang Zhao, Mingli Wang, and Jason Chen Copyright © 2013 Dequn Ni et al. All rights reserved. Dengue in the United States of America: A Worsening Scenario? Thu, 20 Jun 2013 08:45:44 +0000 Dengue is a febrile illness caused by any of the four dengue virus types (DENV-1 to -4, genus Flavivirus, family Flaviviridae) mainly transmitted by the mosquito Aedes aegypti. DENV can be transmitted by blood transfusion. Dengue has been historically present in the continental United States (US), in the state of Hawaii, and in the US insular territories in the Caribbean and the Pacific. During the second half of the 20th century, most of the cases reported in the US were imported cases brought to the country by travelers. Since 2009, cases of autochthonous dengue have been recognized in the state of Florida after 75 years of absence, followed by intensification of transmission in endemic places including the US territories of US Virgin Islands and Puerto Rico, which experienced a large dengue epidemic in 2010. The widespread distribution of dengue mosquito vectors, deficient mosquito control measures and increased frequency of DENV-infected visitors to the US coming from dengue-endemic locations or places experiencing epidemics appear to be jointly responsible for the emergence and reemergence of dengue in the US and its territories. Germán Añez and Maria Rios Copyright © 2013 Germán Añez and Maria Rios. All rights reserved. Genetic Analysis of Clinical VZV Isolates Collected in China Reveals a More Homologous Profile Mon, 27 May 2013 09:44:03 +0000 Forty-four varicella-zoster virus (VZV) isolates from China were genotyped by using a scattered single nucleotide polymorphism (SNP) method, including open reading frames (ORFs) 1, 22, 31, 37, 60, 62, 67, and 68. Based on the analysis of the polymorphic markers in the 8 ORFs, all of the 44 isolates can be placed in genotype J defined by the SNP profiles in ORF22 or clade B defined by the SNP profiles in ORFs 31, 37, 60, 62, 67, and 68. The three consecutive nucleotide (CGG) in-frame insertions in ORF 1 were found in 8 (18.2%) isolates, which has not been described in VZV strains from any other part of the world. A novel synonymous A>G substitution in ORF60 was revealed in 4 (9.1%) of the isolates. In addition, a previously described three consecutive nucleotide (ATC) insertion in ORF 60 was found in all the Chinese isolates but not in the US isolate MLS. The results showed all the 44 strains that belong to genotype J/clade B with significantly high homogeneity, and phylogenetic analysis suggested that the 44 Chinese isolates consist of 4 clusters, but interstrain variations also exist. Overall, VZV isolates obtained in China showed significantly higher genetic homogeneity than isolates reported from other countries. Longfeng Jiang, Lin Gan, Jason Chen, and Mingli Wang Copyright © 2013 Longfeng Jiang et al. All rights reserved. Systemic Delivery of Tyrosine-Mutant AAV Vectors Results in Robust Transduction of Neurons in Adult Mice Mon, 20 May 2013 09:44:53 +0000 Recombinant adeno-associated virus (AAV) vectors are powerful tools for both basic neuroscience experiments and clinical gene therapies for neurological diseases. Intravascularly administered self-complementary AAV9 vectors can cross the blood-brain barrier. However, AAV9 vectors are of limited usefulness because they mainly transduce astrocytes in adult animal brains and have restrictions on foreign DNA package sizes. In this study, we show that intracardiac injections of tyrosine-mutant pseudotype AAV9/3 vectors resulted in extensive and widespread transgene expression in the brains and spinal cords of adult mice. Furthermore, the usage of neuron-specific promoters achieved selective transduction of neurons. These results suggest that tyrosine-mutant AAV9/3 vectors may be effective vehicles for delivery of therapeutic genes, including miRNAs, into the brain and for treating diseases that affect broad areas of the central nervous system. Asako Iida, Naomi Takino, Hitomi Miyauchi, Kuniko Shimazaki, and Shin-ichi Muramatsu Copyright © 2013 Asako Iida et al. All rights reserved. RNase P-Associated External Guide Sequence Effectively Reduces the Expression of Human CC-Chemokine Receptor 5 and Inhibits the Infection of Human Immunodeficiency Virus 1 Thu, 27 Dec 2012 14:03:04 +0000 External guide sequences (EGSs) represent a new class of RNA-based gene-targeting agents, consist of a sequence complementary to a target mRNA, and render the target RNA susceptible to degradation by ribonuclease P (RNase P). In this study, EGSs were constructed to target the mRNA encoding human CC-chemokine receptor 5 (CCR5), one of the primary coreceptors for HIV. An EGS RNA, C1, efficiently directed human RNase P to cleave the CCR5 mRNA sequence in vitro. A reduction of about 70% in the expression level of both CCR5 mRNA and protein and an inhibition of more than 50-fold in HIV (R5 strain Ba-L) p24 production were observed in cells that expressed C1. In comparison, a reduction of about 10% in the expression of CCR5 and viral growth was found in cells that either did not express the EGS or produced a “disabled” EGS which carried nucleotide mutations that precluded RNase P recognition. Furthermore, the same C1-expressing cells that were protected from R5 strain Ba-L retained susceptibility to X4 strain IIIB, which uses CXCR4 as the coreceptor instead of CCR5, suggesting that the RNase P-mediated cleavage induced by the EGS is specific for the target CCR5 but not the closely related CXCR4. Our results provide direct evidence that EGS RNAs against CCR5 are effective and specific in blocking HIV infection and growth. These results also demonstrate the feasibility to develop highly effective EGSs for anti-HIV therapy. Wenbo Zeng, Gia-Phong Vu, Yong Bai, Yuan-Chuan Chen, Phong Trang, Sangwei Lu, Gengfu Xiao, and Fenyong Liu Copyright © 2013 Wenbo Zeng et al. All rights reserved. In Silico Biology of H1N1: Molecular Modelling of Novel Receptors and Docking Studies of Inhibitors to Reveal New Insight in Flu Treatment Wed, 17 Oct 2012 09:03:53 +0000 Influenza is an infectious disease caused by RNA viruses of the family Orthomyxoviridae. The new influenza H1N1 viral stain has emerged by the genetic combination of genes from human, pig, and bird’s H1N1 virus. The influenza virus is roughly spherical and is enveloped by a lipid membrane. There are two glycoproteins in this lipid membrane; namely, hemagglutinin (HA) which helps in attachment of the viral strain on the host cell surface and neuraminidase (NA) that is responsible for initiation of viral infection. We have developed homology models of both Hemagglutinin and Neuraminidase receptors from H1N1 strains in eastern India. The docking studies of B-Sialic acid and O-Sialic acid in the optimized and energy-minimized homology models show important H-bonding interactions with ALA142, ASP230, GLN231, GLU232, and THR141. This information can be used for structure-based and pharmacophore-based new drug design. We have also calculated ADME properties (Human Oral Absorption (HOA) and % HOA) for Oseltamivir which have been subject of debate for long. Deepak Kumar Behera, Pabitra Mohan Behera, Laxmikanta Acharya, Anshuman Dixit, and Payodhar Padhi Copyright © 2012 Deepak Kumar Behera et al. All rights reserved. Sensitive HPV Genotyping Based on the Flow-Through Hybridization and Gene Chip Thu, 11 Oct 2012 16:14:49 +0000 Persistent infection of high-risk human papillomavirus (HPV) has been recognized as the direct cause of cervical carcinoma. Therefore, detection and genotyping of HPV are important to cervical-cancer screening. In this study, we have evaluated the efficacy of flow-through hybridization and gene chip (HybriMax) on HPV genotyping through comparison of the results with Hybrid Capture II (HC-II) and in situ hybridization (ISH). 591 women were classified into 6 groups according to their histological diagnoses. The overall accordance rate on 13 types of HPV genotypes between HybriMax and HC-II were 92.5% and 100% in the cancer group. The overall accordance was excellent with the Kappa index (KI) of 0.814. The value of KI in each group was 0.750 (normal cytological diagnosis), 0.781 (chronic cervicitis), 0.80 (condyloma acuminatum), 0.755 (cervical intraepithelial neoplasia (CIN) I), 0.723 (CIN II), and 0.547 (CIN III) (, good; , excellent). The 10 most common HPV subtype detected by HybriMax were 16, 52/58, 18, 33, 31, 81, 53, 68, and 66 in patients, and 16, 68, 18, 52, 58, 11, 53, 31/39, and 33 in normal controls. In conclusion, HybriMax is an efficient method for HPV genotyping and more suitable for clinical use. Pingping Tao, Weiping Zheng, Yungen Wang, and Mei-lu Bian Copyright © 2012 Pingping Tao et al. All rights reserved. Advances in Arbovirus Surveillance, Detection and Diagnosis Wed, 16 May 2012 14:09:20 +0000 Roy A. Hall, Bradley J. Blitvich, Cheryl A. Johansen, and Stuart D. Blacksell Copyright © 2012 Roy A. Hall et al. All rights reserved. Commercial Dengue Rapid Diagnostic Tests for Point-of-Care Application: Recent Evaluations and Future Needs? Thu, 10 May 2012 15:13:14 +0000 Dengue fever, dengue haemorrhagic fever, and dengue shock syndrome (DF/DHF/DSS) are tropical diseases that cause significant humanitarian and economic hardship. It is estimated that more than 2.5 billion people are at risk of infection and more than 100 countries have endemic dengue virus transmission. Laboratory tests are essential to provide an accurate diagnosis of dengue virus infection so that appropriate treatment and patient management may be administered. In many dengue endemic settings, laboratory diagnostic resources are limited and simple rapid diagnostic tests (RDTs) provide opportunities for point-of-care diagnosis. This paper addresses current issues relating to the application of commercial dengue RDTs for the diagnosis of acute dengue virus infection, recent diagnostic evaluations, and identifies future needs. Stuart D. Blacksell Copyright © 2012 Stuart D. Blacksell. All rights reserved. Approaches for the Development of Rapid Serological Assays for Surveillance and Diagnosis of Infections Caused by Zoonotic Flaviviruses of the Japanese Encephalitis Virus Serocomplex Wed, 18 Apr 2012 09:36:49 +0000 Flaviviruses are responsible for a number of important mosquito-borne diseases of man and animals globally. The short vireamic period in infected hosts means that serological assays are often the diagnostic method of choice. This paper will focus on the traditional methods to diagnose flaviviral infections as well as describing the modern rapid platforms and approaches for diagnostic antigen preparation. Jody Hobson-Peters Copyright © 2012 Jody Hobson-Peters. All rights reserved. Evolution of Mosquito-Based Arbovirus Surveillance Systems in Australia Sun, 11 Mar 2012 09:12:27 +0000 Control of arboviral disease is dependent on the sensitive and timely detection of elevated virus activity or the identification of emergent or exotic viruses. The emergence of Japanese encephalitis virus (JEV) in northern Australia revealed numerous problems with performing arbovirus surveillance in remote locations. A sentinel pig programme detected JEV activity, although there were a number of financial, logistical, diagnostic and ethical limitations. A system was developed which detected viral RNA in mosquitoes collected by solar or propane powered CO2-baited traps. However, this method was hampered by trap-component malfunction, microbial contamination and large mosquito numbers which overwhelmed diagnostic capabilities. A novel approach involves allowing mosquitoes within a box trap to probe a sugar-baited nucleic-acid preservation card that is processed for expectorated arboviruses. In a longitudinal field trial, both Ross River and Barmah Forest viruses were detected numerous times from multiple traps over different weeks. Further refinements, including the development of unpowered traps and use of yeast-generated CO2, could enhance the applicability of this system to remote locations. New diagnostic technology, such as next generation sequencing and biosensors, will increase the capacity for recognizing emergent or exotic viruses, while cloud computing platforms will facilitate rapid dissemination of data. Andrew F. van den Hurk, Sonja Hall-Mendelin, Cheryl A. Johansen, David Warrilow, and Scott A. Ritchie Copyright © 2012 Andrew F. van den Hurk et al. All rights reserved. Viral Bacterial Artificial Chromosomes: Generation, Mutagenesis, and Removal of Mini-F Sequences Thu, 23 Feb 2012 12:09:12 +0000 Maintenance and manipulation of large DNA and RNA virus genomes had presented an obstacle for virological research. BAC vectors provided a solution to both problems as they can harbor large DNA sequences and can efficiently be modified using well-established mutagenesis techniques in Escherichia coli. Numerous DNA virus genomes of herpesvirus and pox virus were cloned into mini-F vectors. In addition, several reverse genetic systems for RNA viruses such as members of Coronaviridae and Flaviviridae could be established based on BAC constructs. Transfection into susceptible eukaryotic cells of virus DNA cloned as a BAC allows reconstitution of recombinant viruses. In this paper, we provide an overview on the strategies that can be used for the generation of virus BAC vectors and also on systems that are currently available for various virus species. Furthermore, we address common mutagenesis techniques that allow modification of BACs from single-nucleotide substitutions to deletion of viral genes or insertion of foreign sequences. Finally, we review the reconstitution of viruses from BAC vectors and the removal of the bacterial sequences from the virus genome during this process. B. Karsten Tischer and Benedikt B. Kaufer Copyright © 2012 B. Karsten Tischer and Benedikt B. Kaufer. All rights reserved. Proximity of Residence to Bodies of Water and Risk for West Nile Virus Infection: A Case-Control Study in Houston, Texas Tue, 24 Jan 2012 10:15:28 +0000 West Nile virus (WNV), a mosquito-borne virus, has clinically affected hundreds of residents in the Houston metropolitan area since its introduction in 2002. This study aimed to determine if living within close proximity to a water source increases one’s odds of infection with WNV. We identified 356 eligible WNV-positive cases and 356 controls using a population proportionate to size model with US Census Bureau data. We found that living near slow moving water sources was statistically associated with increased odds for human infection, while living near moderate moving water systems was associated with decreased odds for human infection. Living near bayous lined with vegetation as opposed to concrete also showed increased risk of infection. The habitats of slow moving and vegetation lined water sources appear to favor the mosquito-human transmission cycle. These methods can be used by resource-limited health entities to identify high-risk areas for arboviral disease surveillance and efficient mosquito management initiatives. Melissa S. Nolan, Ana Zangeneh, Salma A. Khuwaja, Diana Martinez, Susan N. Rossmann, Victor Cardenas, and Kristy O. Murray Copyright © 2012 Melissa S. Nolan et al. All rights reserved. Rapid Molecular Detection Methods for Arboviruses of Livestock of Importance to Northern Europe Thu, 15 Dec 2011 10:29:35 +0000 Arthropod-borne viruses (arboviruses) have been responsible for some of the most explosive epidemics of emerging infectious diseases over the past decade. Their impact on both human and livestock populations has been dramatic. The early detection either through surveillance or diagnosis of virus will be a critical feature in responding and resolving the emergence of such epidemics in the future. Although some of the most important emerging arboviruses are human pathogens, this paper aims to highlight those diseases that primarily affect livestock, although many are zoonotic and some occasionally cause human mortality. This paper also highlights the molecular detection methods specific to each virus and identifies those emerging diseases for which a rapid detection methods are not yet developed. Nicholas Johnson, Katja Voller, L. Paul Phipps, Karen Mansfield, and Anthony R. Fooks Copyright © 2012 Nicholas Johnson et al. All rights reserved. A Novel System for Rapid and Cost-Effective Production of Detection and Diagnostic Reagents of West Nile Virus in Plants Sun, 04 Dec 2011 09:24:22 +0000 The threat of West Nile virus (WNV) epidemics necessitates the development of a technology platform that can produce reagents to support detection and diagnosis rapidly and inexpensively. A plant expression system is attractive for protein production due to its low-cost and high-scalability nature and its ability to make appropriate posttranslational modifications. Here, we investigated the feasibility of using plants to produce two WNV detection and diagnostic reagents to address the current cost and scalability issues. We demonstrated that WNV DIII antigen and E16 monoclonal antibody are rapidly produced at high levels in two plant species and are easily purified. Furthermore, they are effective in identifying WNV and in detecting human IgM response to WNV infection. E16 mAb does not cross-react with other flaviviruses, therefore, is valuable for improving diagnostic accuracy. This study provides a proof of principle for using plants as a robust and economical system to produce diagnostic reagents for arboviruses. Junyun He, Huafang Lai, Christopher Brock, and Qiang Chen Copyright © 2012 Junyun He et al. All rights reserved. Bacterial Artificial Chromosome Clones of Viruses Comprising the Towne Cytomegalovirus Vaccine Wed, 30 Nov 2011 09:15:03 +0000 Bacterial artificial chromosome (BAC) clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. Instead, genetic composition and in vitro growth properties have been inferred from studies of laboratory stocks that may not always accurately represent the viruses that comprise the vaccine. Here we describe the use of BAC cloning to define the genotypic and phenotypic properties of viruses from the Towne vaccine. Given the extensive safety history of the Towne vaccine, these BACs provide a logical starting point for the development of next-generation rationally engineered cytomegalovirus vaccines. Xiaohong Cui, Stuart P. Adler, Andrew J. Davison, Larry Smith, EL-Sayed E. Habib, and Michael A. McVoy Copyright © 2012 Xiaohong Cui et al. All rights reserved. Plasma Cell Cerebrospinal Fluid Pleocytosis Does Not Predict West Nile Virus Infection Wed, 02 Nov 2011 17:53:46 +0000 Purpose. Diagnosis of WNV (WNV) relies upon serologic testing which may take several days after the onset of clinical symptoms to turn positive. Anecdotal reports suggest the presence of plasma cells or plasmacytoid lymphocytes in the cerebrospinal fluid (CSF) may be an early indicator of WNV infection. Methods. The CSFs of 89 patients (12 with WNV, 12 with other viral illness {OVI}, and 65 with nonviral illness{NVI}) were compared for the presence of either plasma cells or plasmacytoid lymphocytes. Results. Plasma cells were rarely seen in any of the patients. Plasmacytoid lymphocytes were more commonly seen in WNV (58%) and OVI (50%) than NVI (11%). The differences were significant for WNV versus NVI, but not WNV versus OVI (𝑃<0.001 and 𝑃=0.58, resp.). Conclusions. A CSF pleocytosis with plasma cells or plasmacytoid lymphocytes was neither sensitive nor specific for the diagnosis of WNV infection. Michael Jordan, Avish Nagpal, William Newman, Paul A. Thompson, and Paul J. Carson Copyright © 2012 Michael Jordan et al. All rights reserved. Cervicovaginal Safety of the Formulated, Biguanide-Based Human Immunodeficiency Virus Type 1 (HIV-1) Inhibitor NB325 in a Murine Model Mon, 24 Oct 2011 13:10:11 +0000 Vaginal microbicides that reduce or eliminate the risk of HIV-1 sexual transmission must do so safely without adversely affecting the integrity of the cervicovaginal epithelium. The present studies were performed to assess the safety of the biguanide-based antiviral compound NB325 in a formulation suitable for topical application. Experiments were performed using a mouse model of cervicovaginal microbicide application, which was previously shown to be predictive of topical agent toxicity revealed in microbicide clinical trials. Mice were exposed vaginally to unformulated NB325 or NB325 formulated in the hydroxyethyl cellulose “universal placebo.” Following exposures to formulated 1% NB325 for 10 min to 24 h, the vaginal and cervical epithelia were generally intact, although some areas of minimal vaginal epithelial damage were noted. Although formulated NB325 appeared generally safe for application in these studies, the low but observable level of toxicity suggests the need for improvements in the compound and/or formulation. Karissa Lozenski, Tina Kish-Catalone, Vanessa Pirrone, Robert F. Rando, Mohamed Labib, Brian Wigdahl, and Fred C. Krebs Copyright © 2011 Karissa Lozenski et al. All rights reserved. Inequalities and Duality in Gene Coexpression Networks of HIV-1 Infection Revealed by the Combination of the Double-Connectivity Approach and the Gini's Method Thu, 29 Sep 2011 14:52:01 +0000 The symbiosis (Sym) and pathogenesis (Pat) is a duality problem of microbial infection, including HIV/AIDS. Statistical analysis of inequalities and duality in gene coexpression networks (GCNs) of HIV-1 infection may gain novel insights into AIDS. In this study, we focused on analysis of GCNs of uninfected subjects and HIV-1-infected patients at three different stages of viral infection based on data deposited in the GEO database of NCBI. The inequalities and duality in these GCNs were analyzed by the combination of the double-connectivity (DC) approach and the Gini's method. DC analysis reveals that there are significant differences between positive and negative connectivity in HIV-1 stage-specific GCNs. The inequality measures of negative connectivity and edge weight are changed more significantly than those of positive connectivity and edge weight in GCNs from the HIV-1 uninfected to the AIDS stages. With the permutation test method, we identified a set of genes with significant changes in the inequality and duality measure of edge weight. Functional analysis shows that these genes are highly enriched for the immune system, which plays an essential role in the Sym-Pat duality (SPD) of microbial infections. Understanding of the SPD problems of HIV-1 infection may provide novel intervention strategies for AIDS. Chuang Ma, Yanhong Zhou, and Sheng-He Huang Copyright © 2011 Chuang Ma et al. All rights reserved. Characterization of Porcine Endogenous Retrovirus Clones from the NIH Miniature Pig BAC Library Wed, 07 Sep 2011 11:56:29 +0000 Pigs have been considered as donors for xenotransplantation in the replacement of human organs and tissues. However, porcine endogenous retroviruses (PERVs) might transmit new infectious disease to humans during xenotransplantation. To investigate PERV integration sites, 45 PERV-positive BAC clones, including 12 PERV-A, 16 PERV-B, and 17 PERV-C clones, were identified from the NIH miniature pig BAC library. The analysis of 12 selected full-length sequences of PERVs, including the long terminal repeat (LTR) region, identified the expected of open reading frame length, an indicative of active PERV, in all five PERV-C clones and one of the four PERV-B clones. Premature stop codons were observed in only three PERV-A clones. Also, eleven PERV integration sites were mapped using a 5000-rad IMpRH panel. The map locations of PERV-C clones have not been reported before, thus they are novel PERV clones identified in this study. The results could provide basic information for the elimination of site-specific PERVs in selection of pigs for xenotransplantation. Seong-Lan Yu, Woo-Young Jung, Kie-Chul Jung, In-Cheol Cho, Hyun-Tae Lim, Dong-Il Jin, and Jun-Heon Lee Copyright © 2012 Seong-Lan Yu et al. All rights reserved. Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis Mon, 29 Aug 2011 10:56:24 +0000 In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W84S, P110S and V129L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM−1. Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection. Chen-Ji Huang, Hwei-Ling Peng, and Chih-Yu Cheng Copyright © 2011 Chen-Ji Huang et al. All rights reserved. Cloning and Expression of Highly Pathogenic Avian Influenza Virus Full-Length Nonstructural Gene in Pichia pastoris Thu, 24 Mar 2011 15:05:28 +0000 Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1). M. B. Abubakar, I. Aini, A. R. Omar, and M. Hair-Bejo Copyright © 2011 M. B. Abubakar et al. All rights reserved. Mutation of Herpesvirus Saimiri ORF51 Glycoprotein Specifically Targets Infectivity to Hepatocellular Carcinoma Cell Lines Thu, 09 Dec 2010 12:23:29 +0000 Herpesvirus saimiri (HVS) is a gamma herpesvirus with several properties that make it an amenable gene therapy vector; namely its large packaging capacity, its ability to persist as a nonintegrated episome, and its ability to infect numerous human cell types. We used RecA-mediated recombination to develop an HVS vector with a mutated virion protein. The heparan sulphate-binding region of HVS ORF51 was substituted for a peptide sequence which interacts with somatostatin receptors (SSTRs), overexpressed on hepatocellular carcinoma (HCC) cells. HVS mORF51 showed reduced infectivity in non-HCC human cell lines compared to wild-type virus. Strikingly, HVS mORF51 retained its ability to infect HCC cell lines efficiently. However, neutralisation assays suggest that HVS mORF51 has no enhanced binding to SSTRs. Therefore, mutation of the ORF51 glycoprotein has specifically targeted HVS to HCC cell lines by reducing the infectivity of other cell types; however, the mechanism for this targeting is unknown. Susan J. Turrell and Adrian Whitehouse Copyright © 2011 Susan J. Turrell and Adrian Whitehouse. All rights reserved. Pathogenicity of a Very Virulent Strain of Marek's Disease Herpesvirus Cloned as Infectious Bacterial Artificial Chromosomes Thu, 25 Nov 2010 14:02:25 +0000 Bacterial artificial chromosome (BAC) vectors containing the full-length genomes of several herpesviruses have been used widely as tools to enable functional studies of viral genes. Marek's disease viruses (MDVs) are highly oncogenic alphaherpesviruses that induce rapid-onset T-cell lymphomas in chickens. Oncogenic strains of MDV reconstituted from BAC clones have been used to examine the role of viral genes in inducing tumours. Past studies have demonstrated continuous increase in virulence of MDV strains. We have previously reported on the UK isolate C12/130 that showed increased virulence features including lymphoid organ atrophy and enhanced tropism for the central nervous system. Here we report the construction of the BAC clones (pC12/130) of this strain. Chickens were infected with viruses reconstituted from the pC12/130 clones along with the wild-type virus for the comparison of the pathogenic properties. Our studies show that BAC-derived viruses induced disease similar to the wild-type virus, though there were differences in the levels of pathogenicity between individual viruses. Generation of BAC clones that differ in the potential to induce cytolytic disease provide the opportunity to identify the molecular determinants of increased virulence by direct sequence analysis as well as by using reverse genetics approaches on the infectious BAC clones. Lorraine P. Smith, Lawrence J. Petherbridge, Susan J. Baigent, Jennifer Simpson, and Venugopal Nair Copyright © 2011 Lorraine P. Smith et al. All rights reserved. Expression and Purification of Z Protein from Junín Virus Tue, 29 Jun 2010 16:32:34 +0000 Arenaviridae comprises 23 recognized virus species with a bipartite ssRNA genome and an ambisense coding strategy. The virions are enveloped and include nonequimolar amounts of each genomic RNA species, designated L and S, coding for four ORFs (N, GPC, L, and Z). The arenavirus Junín (JUNV) is the etiological agent of Argentine Hemorrhagic Fever, an acute disease with high mortality rate. It has been proposed that Z is the functional counterpart of the matrix proteins found in other negative-stranded enveloped RNA viruses. Here we report the optimized expression of a synthetic gene of Z protein, using three expression systems (two bacterial and a baculoviral one). One of these recombinant proteins was used to generate antibodies. A bioinformatic analysis was made where Z was subdivided into three domains. The data presented contributes methodologies for Z recombinant production and provides the basis for the development of new experiments to test its function. S. E. Goñi, C. S. Borio, F. B. Romano, R. P. Rota, M. G. Pilloff, J. A. Iserte, M. A. Tortorici, B. I. Stephan, M. F. Bilen, P. D. Ghiringhelli, and M. E. Lozano Copyright © 2010 S. E. Goñi et al. All rights reserved. Proteomic Analysis of Pichindé virus Infection Identifies Differential Expression of Prothymosin- Tue, 08 Jun 2010 11:07:09 +0000 The arenaviruses include a number of important pathogens including Lassa virus and Junin virus. Presently, the only treatment is supportive care and the antiviral Ribavirin. In the event of an epidemic, patient triage may be required to more effectively manage resources; the development of prognostic biomarker signatures, correlating with disease severity, would allow rational triage. Using a pair of arenaviruses, which cause mild or severe disease, we analyzed extracts from infected cells using SELDI mass spectrometry to characterize potential biomarker profiles. EDGE analysis was used to analyze longitudinal expression differences. Extracts from infected guinea pigs revealed protein peaks which could discriminate between mild or severe infection and between times post-infection. Tandem mass-spectrometry identified several peaks, including the transcriptional regulator prothymosin-. Further investigation revealed differences in secretion of this peptide. These data show proof of concept that proteomic profiling of host markers could be used as prognostic markers of infectious disease. Gavin C. Bowick, Kizhake V. Soman, He Wang, Judith F. Aronson, Bruce A. Luxon, Lee O. Lomas, David G. Gorenstein, and Norbert K. Herzog Copyright © 2010 Gavin C. Bowick et al. All rights reserved. Detection of EBV, HBV, HCV, HIV-1, HTLV-I and -II, and SMRV in Human and Other Primate Cell Lines Thu, 29 Apr 2010 07:19:53 +0000 The high prevalence of contaminated cell cultures suggests that viral contaminations might be distributed among cultures. We investigated more than 460 primate cell lines for Epstein-Barr (EBV), hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus type 1 (HIV-1), human T-cell leukemia/lymphoma virus I and II (HTLV-I/-II), and squirrel monkey retrovirus (SMRV) infections for risk assessment. None of the cell lines were infected with HCV, HIV-1, or HTLV-I/-II. However, one cell line displayed reverse transcriptase activity. Thirty-nine cell lines harbored EBV DNA sequences. Studies on the lytic phase of EBV revealed that five cell lines produce EBV particles and six further cell lines produced EBV upon stimulation. One cell line contained an integrated HBV genome fragment but showed no virus production. Six cell lines were SMRV-infected. Newly established cell lines should be tested for EBV infections to detect B-lymphoblastoid cell lines (B-LCL). B-LCLs established with EBV from cell line B95-8 should be tested for SMRV infections. Cord C. Uphoff, Sabine A. Denkmann, Klaus G. Steube, and Hans G. Drexler Copyright © 2010 Cord C. Uphoff et al. All rights reserved. Characterization of Recombinant Dengue-2 Virus Derived from a Single Nucleotide Substitution in the 5′ Noncoding Region Mon, 22 Mar 2010 15:04:09 +0000 Variants of wild-type dengue serotype 2 (DEN-2) virus containing nucleotide substitutions at positions 14, 15, or 57 in the NCR were constructed by PCR-mediated site-directed mutagenesis. All three viruses containing a single point substitution demonstrated attenuation phenotype as evidenced by decreases replication and plaque size in cell culture assay. All three variants were less neurovirulent in newborn mice compared to the wild type. The mutants were immunogenic in adult mice immunogenicity and maintained stable replication characteristics following passage in mice. The variant viruses were competent for replication in Aedes aegypi mosquito vector, albeit at lower levels of infection and dissemination in the mosquito than the wild-type Den-2 16681 virus. Although all of the viruses, including the wild type, were found transmissible in mosquito life cycles, they were found subsequentially decreased in efficiency of infection, transmission, and dissemination rates along the mosquito generations and all of them remained genetically stable. Vijittra Leardkamolkarn, Wipawan Sirigulpanit, and Richard M. Kinney Copyright © 2010 Vijittra Leardkamolkarn et al. All rights reserved. The Adenovirus Type 3 Dodecahedron's RGD Loop Comprises an HSPG Binding Site That Influences Integrin Binding Thu, 04 Mar 2010 15:16:39 +0000 Human type 3 adenovirus dodecahedron (a virus like particle made of twelve penton bases) features the ability to enter cells through Heparan Sulphate Proteoglycans (HSPGs) and integrins interaction and is used as a versatile vector to deliver DNA or proteins. Cryo-EM reconstruction of the pseudoviral particle with Heparan Sulphate (HS) oligosaccharide shows an extradensity on the RGD loop. A set of mutants was designed to study the respective roles of the RGD sequence (RGE mutant) and of a basic sequence located just downstream. Results showed that the RGE mutant binding to the HS deficient CHO-2241 cells was abolished and unexpectedly, mutation of the basic sequence (KQKR to AQAS) dramatically decreased integrin recognition by the viral pseudoparticle. This basic sequence is thus involved in integrin docking, showing a close interplay between HSPGs and integrin receptors. E. Gout, G. Schoehn, D. Fenel, H. Lortat-Jacob, and P. Fender Copyright © 2010 E. Gout et al. All rights reserved. Oxidative Imbalance in HIV-1 Infected Patients Treated with Antiretroviral Therapy Mon, 26 Oct 2009 14:05:36 +0000 It is generally accepted that oxidative stress is involved in HIV infection. However, the role in oxidative balance of Highly Active Antiretroviral Therapy (HAART) is still debated. In our study we assessed serum oxidant and antioxidant levels in an HIV-1-infected population treated with HAART, and compared them with those of untreated HIV-1 patients and HIV-1-negative subjects. The study included 116 HIV-1-infected patients (86 HAART-treated and 30 untreated), and 46 HIV-negative controls. Serum oxidant levels were significantly higher in the HIV-1 treated group as compared to untreated and control groups. In addition, a decrease of serum total antioxidant status was observed in the HIV-1 treated group. To be noted is that patients who rigorously follow antiretroviral therapy (optimal HAART adherence) have significantly higher oxidative status than those who do not closely follow the therapy (poor HAART adherence). Analysis of variance revealed no significant further increase in oxidative status in HIV-1-infected patients taking antiretroviral and other drugs with the exception of psychiatric drugs (e.g. anxiolytics or antidepressants). Taken together, our results indicate that HAART may affect oxidative stress in HIV-1-infected patients and suggest that antiretroviral therapy plays an important role in the synergy of HIV infection and oxidative stress. Antonella Mandas, Eugenio Luigi Iorio, Maria Gabriella Congiu, Cinzia Balestrieri, Antonello Mereu, Daniela Cau, Sandra Dessì, and Nicoletta Curreli Copyright © 2009 Antonella Mandas et al. All rights reserved. HBX-Mediated Migration of HBV-Replicating HepG2 Cells: Insights on Development of Hepatocellular Carcinoma Wed, 16 Sep 2009 09:16:30 +0000 Hepatitus B virus (HBV) is a major cause of the development of hepatpcellular carcinoma (HCC). One of the significant characteristics of tumor progression is cell migration which is reflective of cytoskeletal dynamics. The Rho GTPases contribute to a multiple cellular processes, including the cellular cytoskeletal reorganization and motility. It has been found that some Rho GTPases have oncogenic activity and can promote cancer cell invasion. Here we discuss one of the Rho GTPases, Rac1 can be activated by HBV replication and such activation results in the high motility of HBV-replicating cells. The enhanced cell motility can be interestingly alleviated by the mutation at the sites of proline rich domain located in HBX. Our findings may provide new insights on the mechanism of HCC development associated with chronic HBV infection. Huixing Feng, Jianhua Zhang, Xi Li, and Wei Ning Chen Copyright © 2009 Huixing Feng et al. All rights reserved. Herpesvirus-Mediated Delivery of a Genetically Encoded Fluorescent Sensor to Canine Cardiomyocytes Tue, 14 Jul 2009 14:03:24 +0000 We report the development and application of a pseudorabies virus-based system for delivery of troponeon, a fluorescent sensor to adult canine cardiomyocytes. The efficacy of transduction was assessed by calculating the ratio of fluorescently labelled and nonlabelled cells in cell culture. Interaction of the virus vector with electrophysiological properties of cardiomyocytes was evaluated by the analysis of transient outward current (), kinetics of the intracellular transients, and cell shortening. Functionality of transferred troponeon was verified by FRET analysis. We demonstrated that the transfer efficiency of troponeon to cultured adult cardiac myocytes was virtually 100%. We showed that even after four days neither the amplitude nor the kinetics of the current was significantly changed and no major shifts occurred in parameters of transients. Furthermore, we demonstrated that infection of cardiomyocytes with the virus did not affect the morphology, viability, and physiological attributes of cells. János Prorok, Péter P. Kovács, Attila A. Kristóf, Norbert Nagy, Dóra Tombácz, Judit S. Tóth, Balázs Ördög, Norbert Jost, László Virág, Julius G. Papp, András Varró, András Tóth, and Zsolt Boldogkői Copyright © 2009 János Prorok et al. All rights reserved. Improved Coinfection with Amphotropic Pseudotyped Retroviral Vectors Mon, 25 May 2009 15:00:23 +0000 Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed. In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins (EGFP and dsRed2). Target cells were primary human fibroblasts (PHF) and 3T3 cells. Unconcentrated vector preparations produced a coinfection rate of 4% (defined as cells that are both red and green as a percentage of all cells infected). Optimized spinoculation, comprising centrifugation at 1200 g for 2 hours at , increased the coinfection rate to 10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to 25%. Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation, increased the coinfection rate to 35% (3T3) or 50% (PHF). Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors. Yuehong Wu, David W. Melton, Yong Zhang, and Peter J. Hornsby Copyright © 2009 Yuehong Wu et al. All rights reserved. Virus-Specific Read-Through Codon Preference Affects Infectivity of Chimeric Cucumber Green Mottle Mosaic Viruses Displaying a Dengue Virus Epitope Sun, 22 Mar 2009 13:54:40 +0000 A Cucumber green mottle mosaic virus (CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct. Pak-Guan Teoh, Aik-Seng Ooi, Sazaly AbuBakar, and Rofina Yasmin Othman Copyright © 2009 Pak-Guan Teoh et al. All rights reserved. A Sensitive and Rapid Assay for Investigating Vertical Transmission of Hepatitis B Virus via Male Germ Line Using EGFP Vector as Reporter Tue, 22 Jul 2008 00:00:00 +0000 Hepatitis B virus (HBV) constitutes a serious menace to man. DNA recombination and sequencing, interspecific in vitro fertilization, single-embryo PCR and RT-PCR were employed to establish a sensitive and rapid assay for exploring the vertical transmission of viruses via male germ line. Plasmid pIRES2-EGFP-HBs which expressed enhanced green fluorescent protein as reporter for the expression of hepatitis B virus S gene was successfully constructed and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize with zona-free hamster ova. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBs DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive control (PCR) indicating expression of pIRES2-EGFP-HBs, and not observed in the embryos without green fluorescence and negative controls (PCR and RT-PCR) indicating no pIRES2-EGFP-HBs in the cells. The advantages and application foreground of this assay for study on vertical transmission of viruses such as HCV, HIV, HPV, and SARS via germ line were discussed. Mohamed Morsi M. Ahmed, Tian-Hua Huang, and Qing-Dong Xie Copyright © 2008 Mohamed Morsi M. Ahmed et al. All rights reserved. A C-terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein Mon, 01 Jan 1900 00:00:00 +0000 Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressed in vitro or in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms. While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585–610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585–610 of the ORF2 protein might be critical for capsid biogenesis. Li Xiaofang, Mohammad Zafrullah, Faizan Ahmad, and Shahid Jameel Copyright © 2001 Hindawi Publishing Corporation. All rights reserved. Proteomic Profiling and Neurodegeneration in West-Nile-Virus-Infected Neurons Mon, 01 Jan 1900 00:00:00 +0000 West Nile virus, a mosquito-borne flavivirus, is a human, equine, and avian pathogen. High-resolution two-dimensional differential-gel electrophoresis (2D-DIGE) was used to characterize protein expression in primary rat neurons and to examine the proteomic profiling to understand the pathogenesis of West-Nile-associated meningoencephalitis. Three pH ranges, 3–10, 4–7, and 5–6, were used to analyze the protein spots. The proteins are labeled with fluorescent dyes Cy3 and Cy5 before being separated on the basis of charge and size respectively on a two-dimensional platform. About 55 proteins showed altered expression levels. These were then subsequently digested and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis using peptide mass fingerprinting and database searching. These cellular proteins could represent distinct roles during infection related to apoptosis. Our findings show that two-dimensional differential gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of proteins whose expression was altered due to West Nile virus infection. V. Dhingra, Q. Li, A. B. Allison, D. E. Stallknecht, and Z. F. Fu Copyright © 2005 Hindawi Publishing Corporation. All rights reserved.