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Evidence-Based Complementary and Alternative Medicine
Volume 2013, Article ID 194976, 9 pages
http://dx.doi.org/10.1155/2013/194976
Research Article

Immunomodulatory Activity and Protective Effects of Polysaccharide from Eupatorium adenophorum Leaf Extract on Highly Pathogenic H5N1 Influenza Infection

1College of Biological Sciences and Biotechnology, Beijing Forestry University, 35 QingHua East Road, Beijing 100083, China
2College of Veterinary Medicine, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, China

Received 1 April 2013; Revised 12 August 2013; Accepted 20 August 2013

Academic Editor: Juliano Ferreira

Copyright © 2013 Yi Jin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

The development of novel broad-spectrum, antiviral agents against H5N1 infection is urgently needed. In this study, we evaluated the immunomodulatory activities and protective effect of Eupatorium adenophorum polysaccharide (EAP) against the highly pathogenic H5N1 subtype influenza virus. EAP treatment significantly increased the production of IL-6, TNF-α, and IFN-γ both in vivo and in vitro as measured by qPCR and ELISA. In a mouse infection model, intranasal administration of EAP at a dose of 25 mg/kg body weight prior to H5N1 viral challenge efficiently inhibited viral replication, decreased lung lesions, and increased survival rate. We further evaluated the innate immune recognition of EAP, as this process is regulated primarily Dectin-1 and mannose receptor (MR). These results indicate that EAP may have immunomodulatory properties and a potential prophylactic effect against H5N1 influenza infection. Our investigation suggests an alternative strategy for the development of novel antiinfluenza agents and benefits of E. adenophorum products.

1. Introduction

Highly pathogenic H5N1 subtype influenza virus can be transmitted directly from poultry to human and cause acute respiratory infections. Pandemic influenza virus H5N1 posed a worldwide threat to the public health because of rapid spread and high pathogenicity [1, 2]. The symptoms in animals or humans infected with H5N1 include fever, encephalitis, pneumonia, and severe acute respiratory syndrome (SARS) [3, 4]. The World Health Organization reported 622 human cases of highly pathogenic H5N1 influenza virus infection, including 371 deaths (a mortality rate >50%), from 2003 to 2013 (http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/index.html). Currently, the most effective preventive measure against the influenza virus is vaccination. Several antiinfluenza medications have been widely used, including zanamivir (Relenza) and oseltamivir (Tamiflu). Unfortunately, their benefits have been significantly restricted by drug-resistance and frequent antigenic mutation [5, 6]. Therefore, the development of novel antiinfluenza agents against the H5N1 subtype is very important.

The invasive plant Eupatorium adenophorum, native to Central America, has a strong ability to adapt to different environments all over the world. This plant first invaded southern Yunnan Province (China) in the 1940s from Burma and Vietnam, and quickly spread across southwestern China throughout the 1950s [7, 8]. Over the past 50 years, E. adenophorum has seriously impacted the ecological environment in China’s middle subtropical zones, including Yunnan, Guizhou, Sichuan, and Guangxi Provinces, by encroaching farmlands, pasture fields, and forests [7]. Manual, chemical, or biological control of E. adenophorum has hindered its comprehensive development and utilization for economic benefit. Many bioactive components isolated from E. adenophorum have shown antimicrobial activity and immunomodulating properties [9]. In a recent study, the anti-inflammatory properties of ethanolic leaf extract was evaluated [10]. However, there have been few reports addressing the bioactivity of E. adenophorum polysaccharide (EAP).

The immunomodulating properties and therapeutic potential of a large number of botanical polysaccharides have been reported [11]. Several polysaccharides from Cordyceps militaris, Portulaca oleracea, Gracilaria lemaneiformis, Gyrodinium impudium, and Panax ginseng have been described as efficacious antiinfluenza agents against H1N1 and H3N2 strains [1215]. In recent reports, polysaccharide-based adjuvants enhanced the immunogenicity and improved the protective efficacy of H5N1 vaccines in animal infection models [16, 17]. However, to our knowledge there have not been any reports regarding the treatment with EAP against highly pathogenic H5N1 influenza.

In the present study, we investigated the potential effect of EAP against H5N1 influenza infection in a mouse model. Immune enhancement effects and the innate immune recognition of EAP were also evaluated. Our results suggest the anti-H5N1 effects of EAP offer an alternative strategy for developing antiinfluenza agents and the utilization of E. adenophorum products.

2. Methods

2.1. Virus

The H5N1 influenza virus (A/bar-headed goose/Qinghai/1/2010) used in this study was isolated from Qinghai Lake in May 2010. This isolate is highly pathogenic in poultry, mouse, and Madin-Darby canine kidney (MDCK) cells. The virus was propagated in MDCK cells at 37°C for 48 h, and the viral supernatant was harvested, aliquoted, and stored at −80°C. Viral titers were determined by plaque assay as described previously [18].

2.2. Animal and Cells

8–10-week-old Female BALB/c mice were obtained from Vital River Laboratories (Beijing, China), and the original breeding pairs were purchased from Charles River (Beijing, China). Mice were raised in independent ventilated cages (IVC) and received pathogen-free food and water. Animal treatments were governed by the Regulations of Experimental Animals of Beijing Authority, and approved by the Animal Ethics Committee of the China Agriculture University.

The mouse leukemic monocyte macrophage Raw 264.7 cell line, human lung adenocarcinoma epithelial A549 cell line, and Madin-Darby canine kidney (MDCK) cell lines were provided by the Cell Resource Center of Peking Union Medical College. The cells were cultured and maintained according to the supplier’s recommendations.

2.3. EAP Preparation

E. adenophorum was collected from Yunnan province, China. The leaves were sliced and dried in shade. 100 g dried materials were powdered in a mixer and then filtered with 40 meshes. Leaf powder was extracted by ultrasonic treatment with 1000 mL of distilled water for 45 min. The supernatant was collected and the precipitate resuspended in 1000 mL of distilled water and again extracted by ultrasonic treatment for 30 min. The resulting supernatant was combined with that obtained from the first ultrasonic treatment. The final aqueous fraction was evaporated to dryness in a rotary evaporator. The residue obtained was dissolved in distilled water and kept frozen at 4°C.

The extract was centrifuged at 3000 g/min for 25 min and concentrated under 80°C for 8 h to prepare polysaccharide. The supernatant was then deproteinized using the Sevag method, and dialyzed against water for 48 h. The final liquid was mixed with three-fold volume of 95% ethanol (v/v) and centrifuged at 3000 g/min for 10 min. The precipitates were successively washed with absolute ethanol, ether, and dried under vacuum at 40°C to obtained the crude polysaccharide (yield = 1.2%). EAP content was determined by the phenol-H2SO4 method [19].

2.4. Effects of EAP on Raw 264.7 Cells and A549 Cells In Vitro

2.5 mL A549 and Raw 264.7 cells (4 × 105/mL) per well were plated in 6-well plates and cultured at 37°C under 5% CO2 for 24 h. Media was removed and 2.5 mL culture medium containing different concentrations of EAP (50, 100, 200 μg/mL) was added to each well. Controls were treated with phosphate-buffered saline (PBS). Cells were collected 36 h after treatment for RNA extraction and quantitative polymerase chain reaction (qPCR).

2.5. In Vivo Challenge Assay

Mice were administrated EAP at a dose of 5, 10, 25, or 50 mg/kg body weight, intranasally once daily for 5 days before the challenge. Control mice were administered PBS using the same schedule. Influenza virus stocks were diluted in PBS. Mice were anesthetized with Zotile (Virbac, France) intramuscularly at 15 mg/kg (body weight) and then infected intranasally with 120 plaque-forming units (PFU) of H5N1 influenza virus in 50 μL. The lung tissue of five mice per group was collected on day 0 before challenge for qPCR and ELISA. Lung tissue from another five mice on day 3 postinfection was collected for plaque assay and qPCR. Ten mice per group were observed for survival for 14 days and body weights recorded.

2.6. Plaque Assay

MDCK cells were cultured in DMEM (Hyclone Laboratories, Logan, UT, USA) containing 10% FBS (Hyclone Laboratories), 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, San Diego, CA, USA). Lung tissue supernatant was diluted 10-fold and added to a cell monolayer covered by semisolid agar containing 0.5 μg/mL of trypsin TPCK (Sigma-Aldrich, St. Louis, MO, USA). Plates were incubated at 37°C, 5% CO2 for 60–72 h and stained with 1% crystal violet.

2.7. RNA Extraction and qPCR

Total RNA from 1 × 106 cells or 10 mg lung tissue were prepared by Trizol (Invitrogen) according to the manufacturer’s instructions. DNaseI-treated RNA (0.2 μg) was reverse transcribed into cDNA using random primers. The expression of the hemagglutinin (HA) gene of H5N1 influenza virus was detected by qPCR using the Power SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA, USA). The following primers were used: forward primer, 5′-CGC AGT ATT CAG AAG AAG CAAGAC-3′; and reverse primer, 5′-TCC ATA AGG ATA GAC CAG CTA CCA-3′. The reaction was run on an ABI 7500 thermal cycler with an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 56°C for 30 s, and 72°C for 40 s. The copy number of the HA gene was calculated by 7500 software v2.0 (Applied Biosystems) using an HA-containing plasmid of known concentration as a standard.

Relative qPCR was performed for other eight genes: hβ-actin, h IL-6, h IFN-γ, and hTNF-α for A549 cells; mβ-actin, mTLR-2, mTLR-4, mDectin-1, mMR, mIL-6, mIFN-γ, and mTNF-α for Raw264.7 cells. The sequences of primers were shown in Table 1. The reaction was run with 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 52°C for 30 s, and extension at 72°C for 40 s. The fold change in gene expression was normalized to controls (naive mice) by using β-actin as an internal standard [20].

tab1
Table 1: The sequences of qPCR primers used in this study.
2.8. ELISA

IL-6, TNF-α, and IFN-γ levels in lung were tested with ELISA kits (Boster, Wuhan, China) according to the manufacturer’s protocol. One gram of lung tissue from each mouse was ground in 1 mL PBS and centrifuged for 20 min at 5000 rpm. The supernatants were collected and diluted 10-fold for ELISA.

2.9. Histopathological Analysis

Lung tissue was collected at day 3 postinfection and fixed by 4% neutral formalin at room temperature for 48 h. Serial tissue sections at 5-μm-thick were obtained after embedding in paraffin. Each slide was examined by light microscopy (Olympus CX31, Center Valley, PA, USA) after staining with hematoxylin and eosin (H & E) or Masson trichrome (GenMed Scientifics Inc. Wilmington, DE, USA) then examined.

2.10. Statistical Analysis

The statistical analysis was performed using one-way ANOVAs with SPSS 12.0 (SPSS Taiwan Corp., Taiwan), and was considered significant.

3. Results

3.1. Immunomodulatory Activities of EAP In Vitro

Many botanical polysaccharides exhibit an immunomodulatory effect [11]. To determine the immunomodulatory properties of EAP, we investigated the potential effect of the polysaccharides on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP (50, 100, 200 μg/mL) for 36 h. The mRNA levels of IL-6, TNF-α, and IFN-γ were detected by qPCR. Figure 1 shows the immunomodulatory activities of EAP in vitro. Various concentrations of EAP triggered a strong secretion of IL-6, TNF-α, and IFN-γ in a dose-dependent manner both in A549 cells (Figures 1(a)1(c)) and Raw264.7 cells (Figures 1(d)1(f)) compared with the PBS treatment group.

fig1
Figure 1: Effects of EAP on A549 and Raw264.7 cells. Cells were treated with various concentrations of EAP for 36 h. The mRNA levels of IL-6, TNF-α, and IFN-γ in A549 cells ((a)–(c)) and Raw264.7 cells ((d)–(f)) were determined by qPCR. *indicates when compared with the PBS group.
3.2. Antiinfluenza Effect of EAP in H5N1 Infected Mice

To test whether EAP could protect H5N1 infected mice, mice were treated with EAP at a dose of 5, 10, 25, or 50 mg/kg body weight intranasally once daily for 5 days prior to viral challenge with 120 PFU. Ten mice per group were monitored for 14 days for the survival rate. As shown in Figure 2(a), all mice receiving PBS died at day 11. Mice administrated 25 mg/kg EAP had a survival rate of 50% at day 14, which was significantly higher than those receiving PBS (by log rank analysis). EAP treatment of 10 mg/kg and 50 mg/kg also appeared to have a survival advantage, but not statistically significant. This result suggests that the protective effect of EAP against H5N1 infection requires a moderate dose. EAP treatment also alleviated weight loss in infected mice (Figure 2(b)).

194976.fig.002
Figure 2: Effect of EAP on H5N1 infected mice. (a) Ten mice per group were monitored for 14 days for survival rate. The body weights were recorded (b). Lung viral load at day 3 postinfection was determined by plaque assay (c) and qPCR (d). *indicates compared with the PBS group.

To determine the viral load in the lung of the infected mice, plaque assays and qPCR were performed. The pulmonary viral titers in the EAP (25 mg/kg) group were significantly lower than the titers in the mice that received PBS at day 3 postinfection (Figures 2(c) and 2(d)). These data clearly indicate that intranasal administration of EAP controls H5N1 viral replication and improves survival rates in a mouse model.

The protective effect of EAP against H5N1 virus is likely due to its immunomodulatory properties. To detect IL-6, TNF-α, and IFN-γ expression, lungs of five mice per group were collected at day 0 before infection and tested by qPCR and ELISA. The mRNA levels in the EAP group (25 mg/kg) were significantly higher than those in the PBS control (naive mice) (Figures 3(a)3(c)). Soluble cytokine levels at day 0 were measured by ELISA, and results were consistent with the qPCR results, even though IFN-γ production in the EAP group was not significantly higher than that of the PBS group ( ) (Figures 3(g)3(i)). These results suggest that EAP increases the IL-6, TNF-α, and IFN-γ production.

fig3
Figure 3: The expression of IL-6, TNF-α, and IFN-γ  in vivo. The expression level of IL-6, TNF-α, and IFN-γ in lung at day 0 was determined by qPCR ((a)–(c)) and ELISA ((g)–(i)). Day 3 postinfection levels were determined by qPCR ((d)–(f)). *indicates compared with the PBS group.

IL-6, TNF-α, and IFN-γ expression at day 3 postinfection was determined by qPCR. In contrast, TNF-α mRNA levels following EAP (25 mg/kg) treatment were significantly lower than those in the PBS group (Figure 3(e)), while IL-6 and IFN-γ expression were only slightly lower (not significant) (Figures 3(d) and 3(f)). These results may be explained by a higher viral load, and the more severe inflammatory response in PBS treated mice.

3.3. Histopathological Lesions in Lungs

Excessive inflammation can cause severe lung lesions during H5N1 influenza infection. To evaluate histopathological changes in the lungs of infected mice, tissues of each group at day 3 postinfection were examined. The lungs of PBS treated mice exhibited a severe inflammation response, characterized by interstitial edema, inflammatory cellular infiltration around small blood vessels, alveolar lumen flooded with edema fluid mixed with exfoliated alveolar epithelial cells, and a thickening of alveolar walls (Figures 4(c) and 4(d)). The lungs of EAP (25 mg/kg) treated mice exhibited milder lesions than those receiving PBS, characterized by signs of bronchopneumonia with interstitial edema, and inflammatory cell infiltration around small blood vessels (Figures 4(a) and 4(b)). Viral loads and inflammatory cytokine production in the lung were correlated; suggesting that EAP treatment reduces lung lesions in H5N1 infected mice.

194976.fig.004
Figure 4: The lung histopathology of H5N1 infected mice. Representative lung sections from EAP group ((a), (b)) and PBS group ((c), (d)) at day 3 postinfection were stained with H & E. The solid arrows indicate interstitial edema and inflammatory cellular infiltration around small blood vessels. The unshaded arrows indicate a dropout of mucous epithelium in bronchioles and inflammatory cellular infiltration.
3.4. Innate Immune Recognition of EAP

Polysaccharides derived from many plants enhance the secretion of cytokines and chemokines, such as TNF-α, IL-6, IL-8, and IL-12 [11]. This immunomodulatory effect is mediated mainly through recognition of polysaccharide polymers by several pattern recognition receptors (PRRs). To determine which receptor contributes directly to the innate immune recognition of EAP, Toll-like receptor 2 (TLR2), TLR4, Dectin-1, and mannose receptor (MR) were examined by qPCR both in vivo and in vitro. Mice were treated with EAP at a dose of 25 mg/kg body weight intranasally once daily for 5 days, with control mice receiving PBS. Lung total RNA was prepared for qPCR. The expression of Dectin-1 and MR in EAP treated mice was significantly elevated compared with controls, while expression of TLR2 and TLR4 were slightly higher, but not statistically significant (Figure 5(a)). In vitro assay showed similar trends. As shown in Figure 5(b), Raw264.7 cells were treated with 200 μg/mL EPA for 36 h before qPCR. Dectin-1 and MR levels were significantly higher, while expression of TLR2 and TLR4 did not change. These data suggest that EAP recognition occurred mainly via the Dectin-1 and MR pathway.

fig5
Figure 5: The expression of pattern recognition receptors. (a) Mice were treated with EAP (25 mg/kg body weight) intranasally once daily for 5 days while control mice received PBS. The TLR2, TLR4, Dectin-1, and MR mRNA levels in lung were detected by qPCR. (b) Raw264.7 cells were treated with 200 μg/mL EPA for 36 h, and then qPCR. *indicates compared with the PBS group.

4. Discussion

In this study, we evaluated the immunomodulatory activities and protective effect of EAP against H5N1 influenza infection in a mouse model. To our knowledge, these findings are the first to show the anti-H5N1 effect of EAP. Intranasal administration of EAP prior to H5N1 viral challenge improved survival rates of infected mice with a corresponding reduction of pulmonary viral load. The anti-H5N1 effect was very likely due to the innate immune recognition of EAP and the secretion of innate immune mediators (IL-6, TNF-α and IFN-γ) before infection. Furthermore, the effect of EAP on PRR expression (including TLR2, TLR4, Dectin-1, and MR) was determined both in vivo and in vitro. These results suggest that the innate immune recognition of EAP was dependent upon the activation of the Dectin-1 and MR pathways. Our data demonstrate the feasibility of using EAP as a novel immunomodulatory agent against influenza infection. Unfortunately, the sugar composition of EAP has not been characterized.

The emergence of new drug-resistant strains resulting from antigenic drift limits the therapeutic benefits of vaccination and antiviral agents in controlling influenza [6, 21, 22]. Thus, development of novel broad-spectrum antiinfluenza strategies is urgently needed. Most botanical polysaccharides are ideal candidates for novel immunomodulatory agents due to their nontoxic properties and fewer side effects compared with bacterially derived polysaccharides. A number of polysaccharides isolated from plant and fungi exhibit effective antiviral benefits against influenza A virus (including H1N1 and H3N2 subtypes) [1215]. The use of polysaccharides as immunomodulatory agent in anti-H5N1 studies is rare. In this paper, our data show the immunomodulatory activities of EAP both in vivo and in vitro. EAP treatment elevated the production of IL-6, TNF-α, and IFN-γ and provides a survival advantage in H5N1 infected mice. The survival rate following EAP pretreatment (25 mg/kg body weight) was significantly higher than in mice receiving PBS (50% to 0%).

In previous reports, high levels of proinflammatory cytokines and chemokines (including TNF-α, IL-6 and IFN-γ) were detected during H5N1 infection [23, 24]. This “cytokine storm” leads to the severe respiratory symptoms and host immune injury. Thus, H5N1-induced cytokine storms are hypothesized to be the main cause of mortality, and the use of anti-inflammatory agents may therefore provide a therapeutic effect [25, 26]. However, it is unclear whether the lack of proinflammatory cytokines (such as TNF-α and IL-6) facilitates viral clearance. Interestingly, knockout mice deficient in TNF-α, TNF-α receptor, IL-6, MIP-1α, and IL-1R or steroid-treated, wild-type mice did not have a survival advantage compared with wild-type mice following H5N1 influenza infection [27, 28]. Interestingly, prophylactic treatment of TLR3 agonist PolyICLC, which strongly upregulates cytokine production, provides protection against H1N1 and H5N1 infections [29, 30]. These conflicting studies may be explained in that the inflammatory response helps clear the virus, while aggravating host pathological damage. Elevated production of cytokines, such as IL-6, TNF-α, and IFN-γ are very important for viral clearance in the early stage of infection by activating the innate immune system. Once the viral infection has triggered a cytokine storm due to the high viral load, the inflammatory response causes severe pathological injury or even death. In this case, receiving an immunomodulator alone cannot help animal to survive [25]. This likely explains why immunomodulator treatment prior to viral infection results in a better survival rate [26, 30]. In our study, treatment of EAP shortly after infection or 24 h postinfection did not provide a survival advantage (data not show).

The antiinfluenza properties of IL-6, TNF-α, and IFN-γ have been discussed in many studies, despite their participation in cytokine storms triggered by influenza infection. IL-6 plays an important role in protecting against influenza A virus as it is required for viral clearance and essential for animal survival [31]. TNF-α has been reported to exert a defensive effect against influenza infection in vitro [32]. IFN-γ treatment in the early stages of influenza infection improves the survival rate in mouse models [33]. In addition, high levels of IFN-γ secretion stimulated by ginseng polysaccharides provide an antiinfluenza effect in vivo [12]. In this report, intranasal administration of EAP before H5N1 challenge elevates expression of IL-6, TNF-α, and IFN-γ compared with mice receiving PBS. The high levels of these mediators contribute to the viral clearance and antiviral response. Pulmonary viral titers following EAP treatment were lower at day 3 postinfection. In contrast, IL-6 and IFN-γ mRNA levels were slightly lower, while TNF-α production was significantly lower than that of PBS group. Regarding the excessive inflammation induced by H5N1 virus, massive secretion of mediators contributes to lung injury rather than an antiviral response. Therefore, the timing of EAP treatment as a prophylactic agent is very important.

The immunomodulatory activities of botanical polysaccharides are thought to be mediated by several PRRs [11]. In this study, we examined the mRNA levels of TLR2, TLR4, Dectin-1, and MR after EAP treatment. EAP was found to upregulate Dectin-1 and MR mRNA expressions significantly both in vivo and in vitro. Our hypothesis is that the innate immune recognition of EAP is driven mainly via a Dectin-1 and MR dependent pathway. Binding to these receptors, EAP may activate complex intracellular signaling pathways, and increase cytokine production, leading to an antiviral response. Thus, the protection against H5N1 by EAP treatment is less likely to cause drug resistance, and may represent a broad-spectrum antiinfluenza effect.

In conclusion, our study demonstrates that EAP leaf extract is a prophylactic and immune enhancement agent against H5N1 influenza virus infection. Treatment with EAP effectively inhibits H5N1 viral replication and improves animal survival. This approach offers an alternative strategy for antiinfluenza immunomodulatory agent development, and benefits the utilization of E. adenophorum products.

Authors’ Contribution

Yi Jin and Yuewei Zhang contributed equally to this work.

Acknowledgments

This work was supported by the Beijing Forestry University Young Scientist Fund (BLX006), the National Key Basic Research Program of China (2010CB530303), and the Fundamental Research Funds for the Central University (no. TD2012-03).

References

  1. H. Chen, G. J. D. Smith, K. S. Li et al., “Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 8, pp. 2845–2850, 2006. View at Publisher · View at Google Scholar · View at Scopus
  2. Y. Guan, L. L. M. Poon, C. Y. Cheung et al., “H5N1 influenza: a protean pandemic threat,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 21, pp. 8156–8161, 2004. View at Publisher · View at Google Scholar · View at Scopus
  3. T. Xu, J. Qiao, L. Zhao et al., “Acute respiratory distress syndrome induced by avian influenza A (H5N1) virus in mice,” American Journal of Respiratory and Critical Care Medicine, vol. 174, no. 9, pp. 1011–1017, 2006. View at Publisher · View at Google Scholar · View at Scopus
  4. W.-F. Ng, K.-F. To, W. W. L. Lam, T.-K. Ng, and K.-C. Lee, “The comparative pathology of severe acute respiratory syndrome and avian influenza A subtype H5N1—a review,” Human Pathology, vol. 37, no. 4, pp. 381–390, 2006. View at Publisher · View at Google Scholar · View at Scopus
  5. J. LeGoff, D. Rousset, G. Abou-Jaoude et al., “I223R mutation in influenza A(H1N1)pdm09 neuraminidase confers reduced susceptibility to oseltamivir and zanamivir and enhanced resistance with H275Y,” PLoS One, vol. 7, no. 8, Article ID e37095, 2012. View at Publisher · View at Google Scholar
  6. J. L. McKimm-Breschkin, P. W. Selleck, T. B. Usman, and M. A. Johnson, “Reduced sensitivity of influenza A (H5N1) to oseltamivir,” Emerging Infectious Diseases, vol. 13, no. 9, pp. 1354–1357, 2007. View at Google Scholar · View at Scopus
  7. R. Wang and Y.-Z. Wang, “Invasion dynamics and potential spread of the invasive alien plant species Ageratina adenophora (Asteraceae) in China,” Diversity and Distributions, vol. 12, no. 4, pp. 397–408, 2006. View at Publisher · View at Google Scholar · View at Scopus
  8. L. Zhu, O. J. Sun, W. Sang, Z. Li, and K. Ma, “Predicting the spatial distribution of an invasive plant species (Eupatorium adenophorum) in China,” Landscape Ecology, vol. 22, no. 8, pp. 1143–1154, 2007. View at Publisher · View at Google Scholar · View at Scopus
  9. M.-L. Zhang, M. Wu, J.-J. Zhang, D. Irwin, Y.-C. Gu, and Q.-W. Shi, “Chemical constituents of plants from the genus Eupatorium,” Chemistry and Biodiversity, vol. 5, no. 1, pp. 40–55, 2008. View at Publisher · View at Google Scholar · View at Scopus
  10. A. K. Chakravarty, T. Mazumder, and S. N. Chatterjee, “Anti-inflammatory potential of ethanolic leaf extract of Eupatorium adenophorum spreng. Through alteration in production of TNF-α, ROS and expression of certain genes,” Evidence-Based Complementary and Alternative Medicine, vol. 2011, Article ID 471074, 10 pages, 2011. View at Publisher · View at Google Scholar · View at Scopus
  11. I. A. Schepetkin and M. T. Quinn, “Botanical polysaccharides: macrophage immunomodulation and therapeutic potential,” International Immunopharmacology, vol. 6, no. 3, pp. 317–333, 2006. View at Publisher · View at Google Scholar · View at Scopus
  12. D.-G. Yoo, M.-C. Kim, M.-K. Park et al., “Protective effect of ginseng polysaccharides on influenza viral infection,” PLoS One, vol. 7, no. 3, Article ID e33678, 2012. View at Publisher · View at Google Scholar · View at Scopus
  13. M. Kim, J. H. Yim, S.-Y. Kim et al., “In vitro inhibition of influenza A virus infection by marine microalga-derived sulfated polysaccharide p-KG03,” Antiviral Research, vol. 93, no. 2, pp. 253–259, 2012. View at Publisher · View at Google Scholar · View at Scopus
  14. M.-Z. Chen, H.-G. Xie, L.-W. Yang, Z.-H. Liao, and J. Yu, “In vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis,” Virologica Sinica, vol. 25, no. 5, pp. 341–351, 2010. View at Publisher · View at Google Scholar · View at Scopus
  15. Y. Ohta, J.-B. Lee, K. Hayashi, A. Fujita, K. P. Dong, and T. Hayashi, “In vivo anti-influenza virus activity of an immunomodulatory acidic polysaccharide isolated from Cordyceps militaris grown on germinated soybeans,” Journal of Agricultural and Food Chemistry, vol. 55, no. 25, pp. 10194–10199, 2007. View at Publisher · View at Google Scholar · View at Scopus
  16. R. C. Layton, N. Petrovsky, A. P. Gigliotti et al., “Delta inulin polysaccharide adjuvant enhances the ability of split-virion H5N1 vaccine to protect against lethal challenge in ferrets,” Vaccine, vol. 29, no. 37, pp. 6242–6251, 2011. View at Publisher · View at Google Scholar · View at Scopus
  17. L. Guo, D. Wang, Y. Hu et al., “Adjuvanticity of compound polysaccharides on chickens against Newcastle disease and avian influenza vaccine,” International Journal of Biological Macromolecules, vol. 50, no. 3, pp. 512–517, 2012. View at Publisher · View at Google Scholar · View at Scopus
  18. Y. Wu, G. Zhang, Y. Li et al., “Inhibition of highly pathogenic avian H5N1 influenza virus replication by RNA oligonucleotides targeting NS1 gene,” Biochemical and Biophysical Research Communications, vol. 365, no. 2, pp. 369–374, 2008. View at Publisher · View at Google Scholar · View at Scopus
  19. M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith, “Colorimetric method for determination of sugars and related substances,” Analytical Chemistry, vol. 28, no. 3, pp. 350–356, 1956. View at Google Scholar · View at Scopus
  20. K. J. Livak and T. D. Schmittgen, “Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method,” Methods, vol. 25, no. 4, pp. 402–408, 2001. View at Publisher · View at Google Scholar · View at Scopus
  21. P. Palese, “Influenza: old and new threats,” Nature Medicine, vol. 10, no. 12, supplement, pp. S82–S87, 2004. View at Publisher · View at Google Scholar · View at Scopus
  22. C.-L. Cheung, J. M. Rayner, G. J. D. Smith et al., “Distribution of amantadine-resistant H5N1 avian influenza variants in Asia,” Journal of Infectious Diseases, vol. 193, no. 12, pp. 1626–1629, 2006. View at Publisher · View at Google Scholar · View at Scopus
  23. K. J. Szretter, S. Gangappa, X. Lu et al., “Role of host cytokine responses in the pathogenesis of avian H5N1 influenza viruses in mice,” Journal of Virology, vol. 81, no. 6, pp. 2736–2744, 2007. View at Publisher · View at Google Scholar · View at Scopus
  24. M. C. W. Chan, C. Y. Cheung, W. H. Chui et al., “Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells,” Respiratory Research, vol. 6, article 135, 2005. View at Publisher · View at Google Scholar · View at Scopus
  25. B.-J. Zheng, K.-W. Chan, Y.-P. Lin et al., “Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 23, pp. 8091–8096, 2008. View at Publisher · View at Google Scholar · View at Scopus
  26. Y. Jin, G. Zhang, Y. Hu et al., “Inhibition of highly pathogenic avian H5N1 influenza virus propagation by RNA oligonucleotides targeting the PB2 gene in combination with celecoxib,” Journal of Gene Medicine, vol. 13, no. 4, pp. 243–249, 2011. View at Publisher · View at Google Scholar · View at Scopus
  27. R. Salomon, E. Hoffmann, and R. G. Webster, “Inhibition of the cytokine response does not protect against lethal H5N1 influenza infection,” Proceedings of the National Academy of Sciences of the United States of America, vol. 104, no. 30, pp. 12479–12481, 2007. View at Publisher · View at Google Scholar · View at Scopus
  28. M. J. Carter, “A rationale for using steroids in the treatment of severe cases of H5N1 avian influenza,” Journal of Medical Microbiology, vol. 56, part 7, pp. 875–883, 2007. View at Publisher · View at Google Scholar · View at Scopus
  29. J. P. Wong, M. E. Christopher, S. Viswanathan et al., “Activation of toll-like receptor signaling pathway for protection against influenza virus infection,” Vaccine, vol. 27, no. 25-26, pp. 3481–3483, 2009. View at Publisher · View at Google Scholar · View at Scopus
  30. Y. Li, Y. Hu, Y. Jin et al., “Prophylactic, therapeutic and immune enhancement effect of liposome-encapsulated PolyICLC on highly pathogenic H5N1 influenza infection,” Journal of Gene Medicine, vol. 13, no. 1, pp. 60–72, 2011. View at Publisher · View at Google Scholar · View at Scopus
  31. O. Dienz, J. G. Rud, S. M. Eaton et al., “Essential role of IL-6 in protection against H1N1 influenza virus by promoting neutrophil survival in the lung,” Mucosal Immunology, vol. 5, no. 3, pp. 258–266, 2012. View at Publisher · View at Google Scholar · View at Scopus
  32. S. H. Seo and R. G. Webster, “Tumor necrosis factor alpha exerts powerful anti-influenza virus effects in lung epithelial cells,” Journal of Virology, vol. 76, no. 3, pp. 1071–1076, 2002. View at Google Scholar · View at Scopus
  33. I. D. Weiss, O. Wald, H. Wald et al., “IFN-γ treatment at early stages of influenza virus infection protects mice from death in a NK cell-dependent manner,” Journal of Interferon and Cytokine Research, vol. 30, no. 6, pp. 439–449, 2010. View at Publisher · View at Google Scholar · View at Scopus