BioMed Research International: Virology http://www.hindawi.com The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . 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 http://www.hindawi.com/journals/bmri/2014/878139/ 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 http://www.hindawi.com/journals/bmri/2014/767538/ 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 http://www.hindawi.com/journals/bmri/2014/703930/ 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 http://www.hindawi.com/journals/bmri/2014/947560/ 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 http://www.hindawi.com/journals/bmri/2014/825039/ 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 http://www.hindawi.com/journals/bmri/2014/726145/ 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 http://www.hindawi.com/journals/bmri/2014/620740/ 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 http://www.hindawi.com/journals/bmri/2014/308371/ 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 http://www.hindawi.com/journals/bmri/2014/315470/ 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 http://www.hindawi.com/journals/bmri/2014/653056/ 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 http://www.hindawi.com/journals/bmri/2014/516859/ 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 http://www.hindawi.com/journals/bmri/2014/303841/ 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 http://www.hindawi.com/journals/bmri/2014/639321/ 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 http://www.hindawi.com/journals/bmri/2014/598732/ 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 http://www.hindawi.com/journals/bmri/2014/241298/ 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 http://www.hindawi.com/journals/bmri/2014/670506/ 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 http://www.hindawi.com/journals/bmri/2014/231365/ 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 http://www.hindawi.com/journals/bmri/2014/423181/ 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 http://www.hindawi.com/journals/bmri/2014/546274/ 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 http://www.hindawi.com/journals/bmri/2014/420658/ 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 http://www.hindawi.com/journals/bmri/2014/631642/ 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 http://www.hindawi.com/journals/bmri/2014/494835/ 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 http://www.hindawi.com/journals/bmri/2014/904038/ 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 http://www.hindawi.com/journals/bmri/2014/368581/ 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 http://www.hindawi.com/journals/bmri/2014/841027/ 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 http://www.hindawi.com/journals/bmri/2014/621316/ 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 http://www.hindawi.com/journals/bmri/2014/472978/ 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 http://www.hindawi.com/journals/bmri/2014/496219/ 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 http://www.hindawi.com/journals/bmri/2014/632347/ 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 http://www.hindawi.com/journals/bmri/2014/498083/ 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 http://www.hindawi.com/journals/bmri/2014/483657/ 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 http://www.hindawi.com/journals/bmri/2014/962915/ 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 http://www.hindawi.com/journals/bmri/2014/363790/ 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 http://www.hindawi.com/journals/bmri/2014/430508/ 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 http://www.hindawi.com/journals/bmri/2014/416727/ 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 http://www.hindawi.com/journals/bmri/2014/748068/ 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 http://www.hindawi.com/journals/bmri/2014/313252/ 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 http://www.hindawi.com/journals/bmri/2014/306130/ 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 http://www.hindawi.com/journals/bmri/2014/525064/ 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 http://www.hindawi.com/journals/bmri/2014/960190/ 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 http://www.hindawi.com/journals/bmri/2014/971345/ 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 http://www.hindawi.com/journals/bmri/2014/160505/ 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 http://www.hindawi.com/journals/bmri/2013/608510/ 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 http://www.hindawi.com/journals/bmri/2013/582957/ 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 http://www.hindawi.com/journals/bmri/2013/152656/ 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 http://www.hindawi.com/journals/bmri/2013/318698/ 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 http://www.hindawi.com/journals/bmri/2013/198797/ 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 http://www.hindawi.com/journals/bmri/2013/591290/ 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 http://www.hindawi.com/journals/bmri/2013/896749/ 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 http://www.hindawi.com/journals/bmri/2013/875958/ 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 http://www.hindawi.com/journals/bmri/2013/467869/ 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 http://www.hindawi.com/journals/bmri/2013/694269/ 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 http://www.hindawi.com/journals/bmri/2013/876314/ 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 http://www.hindawi.com/journals/bmri/2013/524165/ 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 http://www.hindawi.com/journals/bmri/2013/343195/ 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 http://www.hindawi.com/journals/bmri/2013/172479/ 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 http://www.hindawi.com/journals/bmri/2013/369712/ 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 http://www.hindawi.com/journals/bmri/2013/290609/ 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 http://www.hindawi.com/journals/bmri/2013/965853/ 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 http://www.hindawi.com/journals/bmri/2013/973516/ 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 http://www.hindawi.com/journals/bmri/2013/859583/ 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 http://www.hindawi.com/journals/bmri/2013/838491/ 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 http://www.hindawi.com/journals/bmri/2013/426034/ 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 http://www.hindawi.com/journals/bmri/2013/686549/ 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 http://www.hindawi.com/journals/bmri/2013/690835/ 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 http://www.hindawi.com/journals/bmri/2013/683464/ 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 http://www.hindawi.com/journals/bmri/2013/341387/ 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 http://www.hindawi.com/journals/bmri/2013/678645/ 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 http://www.hindawi.com/journals/bmri/2013/681234/ 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 http://www.hindawi.com/journals/bmri/2013/974819/ 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 http://www.hindawi.com/journals/bmri/2013/509714/ 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 http://www.hindawi.com/journals/bmri/2012/714623/ 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 http://www.hindawi.com/journals/bmri/2012/938780/ 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 http://www.hindawi.com/journals/bmri/2012/512969/ 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 http://www.hindawi.com/journals/bmri/2012/151967/ 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 http://www.hindawi.com/journals/bmri/2012/379738/ 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 http://www.hindawi.com/journals/bmri/2012/325659/ 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 http://www.hindawi.com/journals/bmri/2012/472537/ 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 http://www.hindawi.com/journals/bmri/2012/159578/ 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 http://www.hindawi.com/journals/bmri/2012/719402/ 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 http://www.hindawi.com/journals/bmri/2012/106783/ 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 http://www.hindawi.com/journals/bmri/2012/428498/ 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 http://www.hindawi.com/journals/bmri/2012/697418/ 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 http://www.hindawi.com/journals/bmri/2011/941061/ 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 http://www.hindawi.com/journals/bmri/2011/926407/ 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 http://www.hindawi.com/journals/bmri/2012/482568/ 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 http://www.hindawi.com/journals/bmri/2011/359042/ 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 http://www.hindawi.com/journals/bmri/2011/414198/ 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 http://www.hindawi.com/journals/bmri/2011/785158/ 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 http://www.hindawi.com/journals/bmri/2011/412829/ 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 http://www.hindawi.com/journals/bmri/2010/970491/ 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 http://www.hindawi.com/journals/bmri/2010/956823/ 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 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 http://www.hindawi.com/journals/bmri/2010/904767/ 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 http://www.hindawi.com/journals/bmri/2010/934694/ Variants of wild-type dengue serotype 2 (DEN-2) virus containing nucleotide substitutions at positions 14, 15, or 57 in the 5 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 http://www.hindawi.com/journals/bmri/2010/541939/ 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 http://www.hindawi.com/journals/bmri/2009/749575/ 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 http://www.hindawi.com/journals/bmri/2009/930268/ 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 http://www.hindawi.com/journals/bmri/2009/361795/ 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 Boldogkoi Copyright © 2009 János Prorok et al. All rights reserved. Improved Coinfection with Amphotropic Pseudotyped Retroviral Vectors Mon, 25 May 2009 15:00:23 +0000 http://www.hindawi.com/journals/bmri/2009/901079/ 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 http://www.hindawi.com/journals/bmri/2009/781712/ 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 http://www.hindawi.com/journals/bmri/2008/495436/ 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. Proteomic Profiling and Neurodegeneration in West-Nile-Virus-Infected Neurons Mon, 01 Jan 1900 00:00:00 +0000 http://www.hindawi.com/journals/bmri/2005/420261/abs/ 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. 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 http://www.hindawi.com/journals/bmri/2001/804896/abs/ 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.