Pathogenic Avian Influenza Virus Infection Research Articles

Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via upregulation of SOCS1 and SOCS3.

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species. Highly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Wide-spread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses after infection. Disease severity following avian influenza virus infection can be attributed in part to hyperinduction of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low-pathogenicity avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and downregulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combination with antiviral therapy may have important therapeutic benefits.

Subclinical highly pathogenic avian influenza virus infection among vaccinated chickens, China.

Subclinical infection of vaccinated chickens with a highly pathogenic avian influenza A(H5N2) virus was identified through routine surveillance in China. Investigation suggested that the virus has evolved into multiple genotypes. To better control transmission of the virus, we recommend a strengthened program of education, biosecurity, rapid diagnostics, surveillance, and elimination of infected poultry.

Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses.

Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0118-3) contains supplementary material, which is available to authorized users.

Human H7N9 and H5N1 influenza viruses differ in induction of cytokines and tissue tropism.

Since emerging in 2013, the avian-origin H7N9 influenza viruses have resulted in over 400 human infections, leading to 115 deaths to date. Although the epidemiology differs from human highly pathogenic avian H5N1 influenza virus infections, there is a similar rapid progression to acute respiratory distress syndrome. The aim of these studies was to compare the pathological and immunological characteristics of a panel of human H7N9 and H5N1 viruses in vitro and in vivo. Although there were similarities between particular H5N1 and H7N9 viruses, including association between lethal disease and spread to the alveolar spaces and kidney, there were also strain-specific differences. Both H5N1 and H7N9 viruses are capable of causing lethal infections, with mortality correlating most strongly with wider distribution of viral antigen in the lungs, rather than with traditional measures of virus titer and host responses. Strain-specific differences included hypercytokinemia in H5N1 infections that was not seen with the H7N9 infections regardless of lethality. Conversely, H7N9 viruses showed a greater tropism for respiratory epithelium covering nasal passages and nasopharynx-associated lymphoid tissue than H5N1 viruses, which may explain the enhanced transmission in ferret models. Overall, these studies highlight some distinctive properties of H5N1 and H7N9 viruses in different in vitro and in vivo models. The novel avian-origin H7N9 pandemic represents a serious threat to public health. The ability of H7N9 to cause serious lung pathology, leading in some cases to the development of acute respiratory distress syndrome, is of particular concern. Initial reports of H7N9 infection compared them to infections caused by highly pathogenic avian (HPAI) H5N1 viruses. Thus, it is of critical importance to understand the pathology and immunological response to infection with H7N9 compared to HPAI H5N1 viruses. We compared these responses in both in vitro and in vivo models, and found that H5N1 and H7N9 infections exhibit distinct pathological, immunological, and tissue tropism differences that could explain differences in clinical disease and viral transmission.

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections.

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

Protective efficacy of passive immunization with monoclonal antibodies in animal models of H5N1 highly pathogenic avian influenza virus infection.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.

Efficacy of repeated intravenous administration of peramivir against highly pathogenic avian influenza A (H5N1) virus in cynomolgus macaques.

Highly pathogenic avian influenza A (H5N1) viruses cause severe and often fatal disease in humans. We evaluated the efficacy of repeated intravenous dosing of the neuraminidase inhibitor peramivir against highly pathogenic avian influenza virus A/Vietnam/UT3040/2004 (H5N1) infection in cynomolgus macaques. Repeated dosing of peramivir (30 mg/kg/day once a day for 5 days) starting immediately after infection significantly reduced viral titers in the upper respiratory tract, body weight loss, and cytokine production and resulted in a significant body temperature reduction in infected macaques compared with that of macaques administered a vehicle (P < 0.05). Repeated administration of peramivir starting at 24 h after infection also resulted in a reduction in viral titers and a reduction in the period of virus detection in the upper respiratory tract, although the body temperature change was not statistically significant. The macaque model used in the present study demonstrated that inhibition of viral replication at an early time point after infection by repeated intravenous treatment with peramivir is critical for reduction of the production of cytokines, i.e., interleukin-6 (IL-6), tumor necrosis factor α, gamma interferon, monocyte chemotactic protein 1, and IL-12p40, resulting in amelioration of symptoms caused by highly pathogenic avian influenza virus infection.

Little evidence of subclinical avian influenza virus infections among rural villagers in Cambodia.

In 2008, 800 adults living within rural Kampong Cham Province, Cambodia were enrolled in a prospective cohort study of zoonotic influenza transmission. After enrollment, participants were contacted weekly for 24 months to identify acute influenza-like illnesses (ILI). Follow-up sera were collected at 12 and 24 months. A transmission substudy was also conducted among the family contacts of cohort members reporting ILI who were influenza A positive. Samples were assessed using serological or molecular techniques looking for evidence of infection with human and avian influenza viruses. Over 24 months, 438 ILI investigations among 284 cohort members were conducted. One cohort member was hospitalized with a H5N1 highly pathogenic avian influenza (HPAI) virus infection and withdrew from the study. Ninety-seven ILI cases (22.1%) were identified as influenza A virus infections by real-time RT-PCR; none yielded evidence for AIV. During the 2 years of follow-up, 21 participants (3.0%) had detectable antibody titers (≥1∶10) against the studied AIVs: 1 against an avian-like A/Migratory duck/Hong Kong/MPS180/2003(H4N6), 3 against an avian-like A/Teal/Hong Kong/w312/97(H6N1), 9 (3 of which had detectible antibody titers at both 12- and 24-month follow-up) against an avian-like A/Hong Kong/1073/1999(H9N2), 6 (1 detected at both 12- and 24-month follow-up) against an avian-like A/Duck/Memphis/546/74(H11N9), and 2 against an avian-like A/Duck/Alberta/60/76(H12N5). With the exception of the one hospitalized cohort member with H5N1 infection, no other symptomatic avian influenza infections were detected among the cohort. Serological evidence for subclinical infections was sparse with only one subject showing a 4-fold rise in microneutralization titer over time against AvH12N5. In summary, despite conducting this closely monitored cohort study in a region enzootic for H5N1 HPAI, we were unable to detect subclinical avian influenza infections, suggesting either that these infections are rare or that our assays are insensitive at detecting them.

H5-based DNA constructs derived from selected highly pathogenic H5N1 avian influenza virus induce high levels of humoral antibodies in Muscovy ducks against low pathogenic viruses.

BackgroundH5 low pathogenic avian influenza virus (LPAIV) infection in domestic ducks is a major problem in duck producing countries. Their silent circulation is an ongoing source of potential highly pathogenic or zoonotic emerging strains. To prevent such events, vaccination of domestic ducks might be attempted but remains challenging. Currently licensed vector vaccines derived from H5N1 HPAIV possess clade 0, clade 2.2 or clade 2.3.4 HA sequences: selection of the best HA candidate inducing the largest cross protection is a key issue. For this purpose, DNA immunization of specific pathogen free Muscovy ducks was performed using different synthetic codon optimized (opt) or native HA genes from H5N2 LPAIV and several H5N1 HPAIV clade 2.1, 2.2.1 and 2.3.4. Humoral cross-immunity was assessed 3 weeks after boost by hemagglutination inhibition (HI) and virus neutralization (VN) against three French H5 LPAIV antigens.FindingsVaccination with LP H5N2 HA induced the highest VN antibody titre against the homologous antigen; however, the corresponding HI titre was lower and comparable to HI titres obtained after immunization with opt HA derived from clades 2.3.4 or 2.1. Compared to the other HPAIV-derived constructs, vaccination with clade 2.3.4 opt HA consistently induced the highest antibody titres in HI and VN, when tested against all three H5 LPAIV antigens and H5N2 LPAIV, respectively: differences in titres against this last strain were statistically significant.ConclusionThe present study provides a standardized method to assess cross-immunity based on HA immunogenicity alone, and suggests that clade 2.3.4-derived recombinant vaccines might be the optimal candidates for further challenge testing to vaccinate domestic Muscovy ducks against H5 LPAIV.

Massive Mobilization of Dendritic Cells During Influenza A Virus Subtype H5N1 Infection of Nonhuman Primates

Highly pathogenic avian influenza virus infection is characterized by a marked inflammatory response, but the impact of infection on dendritic cells (DCs) is unknown. We show that influenza A virus subtype H5N1 infection rapidly and profoundly impacts DCs in cynomolgus macaques, increasing the number of blood myeloid and plasmacytoid DCs by 16- and 60-fold, respectively. Infection was associated with recruitment, activation, and apoptosis of DCs in lung-draining lymph nodes; granulocyte and macrophage infiltration in lungs was also detected, together with expression of CXCL10. This degree of DC mobilization is unprecedented in viral infection and suggests a potential role for DCs in the pathogenesis of highly pathogenic avian influenza virus.

Pathological and immunohistochemical findings of natural highly pathogenic avian influenza infection in tufted ducks during 2010-2011 outbreaks in Japan.

ABSTRACTIn the winter of 2010–2011, an outbreak of highly pathogenic avian influenza virus (HPAIV) infection occurred in wild and domestic birds in Japan. Tufted ducks were found dead in an urban area of Toyota City, Koriyama, Fukushima Prefecture. Two tufted ducks were examined histopathologically, immunohistochemically and molecularly. Gross findings included marked dark-red clotted blood in the pectoral muscles and multifocal hemorrhages on the serous membranes. Microscopically, non-suppurative meningoencephalitis, multifocal to coalescing pancreatic necrosis and severe pulmonary congestion were observed. HPAIV antigen was detected in the malacic areas, neuronal, glial and ependymal cells, pulmonary capillary endothelial cells and epithelium of pulmonary bronchioles, necrotic pancreatic acini and degenerated cardiac myocytes. The HPAIV isolate was genetically classified into clade 2.3.2.1 group A. The broad distribution of virus antigen in brain and pulmonary tissues associated with HPAIV spontaneous infection in tufted ducks might be useful in understanding its pathogenesis in nature.

Protection against H5N1 Highly Pathogenic Avian and Pandemic (H1N1) 2009 Influenza Virus Infection in Cynomolgus Monkeys by an Inactivated H5N1 Whole Particle Vaccine

H5N1 highly pathogenic avian influenza virus (HPAIV) infection has been reported in poultry and humans with expanding clade designations. Therefore, a vaccine that induces immunity against a broad spectrum of H5N1 viruses is preferable for pandemic preparedness. We established a second H5N1 vaccine candidate, A/duck/Hokkaido/Vac-3/2007 (Vac-3), in our virus library and examined the efficacy of inactivated whole particles of this strain against two clades of H5N1 HPAIV strains that caused severe morbidity in cynomolgus macaques. Virus propagation in vaccinated macaques infected with either of the H5N1 HPAIV strains was prevented compared with that in unvaccinated macaques. This vaccine also prevented propagation of a pandemic (H1N1) 2009 virus in macaques. In the vaccinated macaques, neutralization activity, which was mainly shown by anti-hemagglutinin antibody, against H5N1 HPAIVs in plasma was detected, but that against H1N1 virus was not detected. However, neuraminidase inhibition activity in plasma and T-lymphocyte responses in lymph nodes against H1N1 virus were detected. Therefore, cross-clade and heterosubtypic protective immunity in macaques consisted of humoral and cellular immunity induced by vaccination with Vac-3.

Innate immune response gene expression profiles in specific pathogen-free chickens infected with avian influenza virus subtype H9N2

In poultry species, H9N2 low pathogenic avian influenza (LPAI) virus infections frequently lead to low to moderate mortality rates and morbidity characterized by depression, respiratory disease symptoms, and decreases in egg production. Since current knowledge on the avian immune response to H9N2 infection is limited, we used microarray analysis to examine global changes in immune-related gene expression induced by H9N2 LPAI infection in specific pathogen-free chickens. The expression profiles of approximately 800 genes, including those that influence cell differentiation, transcription, transport, immune responses, and signal transduction, were altered by H9N2 infection. A complete chicken genome microarray analysis identified a strong innate antiviral host response after infection in spleens. In particular, a significant number of immune response genes, including interferon genes and related immune response genes, were upregulated following H9N2 infection. The increased transcription of 2′-5′-oligoadenylate synthetase and myxovirus resistance genes was confirmed by real-time RT-PCR. These results suggest that the host response generated against H9N2 infection may contribute to protective effects by manipulating innate immune responses.

CD8+Treg cells suppress CD8+T cell‐responses by IL‐10‐dependent mechanism during H5N1 influenza virus infection

Although Treg-cell-mediated suppression during infection or autoimmunity has been described, functions of Treg cells during highly pathogenic avian influenza virus infection remain poorly characterized. Here we found that in Foxp3-GFP transgenic mice, CD8+ Foxp3+ Treg cells, but not CD4+ Foxp3+ Treg cells, were remarkably induced during H5N1 infection. In addition to expressing CD25, the CD8+ Foxp3+ Treg cells showed a high level of GITR and produced IL-10. In an adoptive transfer model, CD8+ Treg cells suppressed CD8+ T-cell responses and promoted H5N1 virus infection, resulting in enhanced mortality and increased virus load in the lung. Furthermore, in vitro neutralization of IL-10 and studies with IL-10R-deficient mice in vitro and in vivo demonstrated an important role for IL-10 production in the capacity of CD8+ Treg cells to inhibit CD8+ T-cell responses. Our findings identify a previously unrecognized role of CD8+ Treg cells in the negative regulation of CD8+ T-cell responses and suggest that modulation of CD8+ Treg cells may be a therapeutic strategy to control H5N1 viral infection.

Effect of infectious bursal disease virus on pathogenicity of avian influenza virus subtype H9N2 in broiler chicks

In this experiment, pathogenesis of H9N2 avian influenza virus (AIV), experimentally infected with infectious bursal disease virus (IBDV) in broiler chicks was examined. Three groups of twenty were randomly selected. Day old chickens in group 1, were infected by 103 CID50 of IBDV intrabursaly, and in thirty days of age groups 1 and 2 were challenged with 106 EID50 H9N2, intranasaly-intraoculary. Chickens in group 3 remained as control (uninfected with neither IBDV or AIV). Tracheal and cloacal swabs, and tissue samples, were collected at 3, 7, and 11 days postinoculation (PI). Serum samples examined for antibodies against avian influenza virus (AIV) by hemagglutination inhibition test (HI). IBD caused lower H9N2 antibody level. IBDV infected chickens (g1) shed AI virus for a longer period than AIV infected birds (g2), from both trachea and cloac. IBDV was related with AIV in brain and liver. Isolation of AIV from trachea, conjunctiva, bursa and lung in IBDV infected group (1), prolonged till 11 days PI. Our study provides evidence that a previous history of IBDV infection in chickens may cause them to be more susceptible to H9N2 low pathogenic avian influenza (LPAI) virus infection and may alter its tissue tropism. Key words: Infectious bursal disease, avian influenza, virus shedding, broiler chicks.

Infection with influenza A viruses causes changes in promoter DNA methylation of inflammatory genes

Replication of influenza virus in the host cells results in production of immune mediators like cytokines. Excessive secretion of cytokines (hypercytokinemia) has been observed during highly pathogenic avian influenza virus (HPAI-H5N1) infections resulting in high fatality rates. The exact mechanism of hypercytokinemia during influenza virus infection is still not known completely. As promoter DNA methylation changes are linked with expression changes in genes, we intend to identify whether changes in promoter DNA methylation have any role in expression of cytokines during influenza A virus infection. A panel of 24 cytokine genes and genes known to be involved in inflammatory response were analyzed for their promoter DNA methylation changes during influenza A virus infections. Four different strains of influenza A viruses, viz. H5N1, H1N1, pandemic (2009) H1N1, and a vaccine strain of H5N1, were used for the study. We found seven of the total 24 inflammatory genes studied, showing significant changes in their promoter methylation levels in response to virus infection. These genes included proinflammatory cytokines CXCL14, CCL25, CXCL6, and interleukines IL13, IL17C, IL4R. The changes in DNA methylation levels varied across different strains of influenza viruses depending upon their virulence. Significant promoter hypomethylation in IL17C and IL13 genes was observed in cells infected with HPAI-H5N1 virus compared with other influenza viruses. This decrease in methylation was found to be positively correlating with the increased expression of these genes. Analysis of IL17C promoter region using bisulfite sequencing resulted in identification of a CpG site within Retinoid X receptor-alpha (RXR-α) transcription factor binding site undergoing demethylation specifically in H5N1-infected cells but not in other influenza-infected cells. Thus, the study could demonstrate that changes in promoter methylation in certain specific cytokine genes actually have a possible role in their expression changes during influenza A virus infection.

Simvastatin and oseltamivir combination therapy does not improve the effectiveness of oseltamivir alone following highly pathogenic avian H5N1 influenza virus infection in mice

Nonspecific anti-inflammatory drugs have been purported to reduce the burden of severe influenza disease. We demonstrate that, unlike oseltamivir administration, simvastatin administration did not reduce morbidity, mortality, or viral load of mice infected with H1N1 or H5N1 viruses. No added benefit to the efficacy of oseltamivir therapy was observed when mice were treated in combination with simvastatin. Modest reductions in lung cytokine production in H5N1 but not H1N1 virus-infected simvastatin-treated mice indicate a potential benefit for statin use in mitigating disease following severe virus infection.

Kinetic Characterization of PB1-F2-Mediated Immunopathology during Highly Pathogenic Avian H5N1 Influenza Virus Infection

The PB1-F2 protein encoded by influenza A viruses can contribute to virulence, a feature that is dependent of its sequence polymorphism. Whereas PB1-F2 from some H1N1 viruses were shown to exacerbate the inflammatory response within the airways, the contribution of PB1-F2 to highly pathogenic avian influenza virus (HPAIV) virulence in mammals remains poorly described. Using a H5N1 HPAIV strain isolated from duck and its PB1-F2 knocked-out mutant, we characterized the dynamics of PB1-F2-associated host response in a murine model of lethal pneumonia. The mean time of death was 10 days for the two viruses, allowing us to perform global transcriptomic analyses and detailed histological investigations of the infected lungs at multiple time points. At day 2 post-infection (pi), while no histopathological lesion was observed, PB1-F2 expression resulted in a significant inhibition of cellular pathways involved in macrophage activation and in a transcriptomic signature suggesting that it promotes damage to the epithelial barrier. At day 4 pi, the gene profile associated with PB1-F2 expression revealed dysfunctions in NK cells activity. At day 8 pi, PB1-F2 expression was strongly associated with increased transcription of genes encoding chemokines and cytokines implicated in the recruitment of granulocytes, as well as expression of a number of genes encoding enzymes expressed by neutrophils. These transcriptomic data were fully supported by the histopathological analysis of the mice lungs which evidenced more severe inflammatory lesions and enhanced recruitment of neutrophils in the context of PB1-F2 expression, and thus provided a functional corroboration to the insight obtained in this work. In summary, our study shows that PB1-F2 of H5N1 HPAIV markedly influences the expression of the host transcriptome in a different way than its H1N1 counterparts: H5N1 PB1-F2 first delays the initial immune response but increases the pulmonary inflammatory response during the late stages of infection.

Pathological study of archival lung tissues from five fatal cases of avian H5N1 influenza in Vietnam

Highly pathogenic avian H5N1 influenza virus (H5N1) infection in humans causes acute respiratory distress syndrome, leading to multiple organ failure. Five fatal cases of H5N1 infection in Vietnam were analyzed pathologically to reveal virus distribution, and local proinflammatory cytokine and chemokine expression profiles in formalin-fixed, paraffin-embedded lung tissues. Our main histopathological findings showed diffuse alveolar damage in the lungs. The infiltration of myeloperoxidase-positive and/or CD68 (clone KP-1)-positive neutrophils and monocytes/macrophages was remarkable in the alveolar septa and alveolar spaces. Immunohistochemistry revealed that H5N1 mainly infected alveolar epithelial cells and monocytes/macrophages in lungs. H5N1 replication was confirmed by detecting H5N1 mRNA in epithelial cells using in situ hybridization. Quantitation of H5N1 RNA using quantitative reverse transcription PCR assays revealed that the level of H5N1 RNA was increased in cases during early phases of the disease. We quantified the expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, regulated on activation normal T-cell expressed and secreted (commonly known as RANTES), and interferon-gamma-inducible protein of 10 kDa (IP-10) in formalin-fixed, paraffin-embedded lung sections. Their expression levels correlated with H5N1 RNA copy numbers detected in the same lung region. Double immunofluorescence staining revealed that TNF-α, IL-6, IL-8 and IP-10 were expressed in epithelial cells and/or monocytes/macrophages. In particular, IL-6 was also expressed in endothelial cells. The dissemination of H5N1 beyond respiratory organs was not confirmed in two cases examined in this study.

Goose Toll-like receptor 7 (TLR7), myeloid differentiation factor 88 (MyD88) and antiviral molecules involved in anti-H5N1 highly pathogenic avian influenza virus response

In mammals, Toll-like receptor 7 (TLR7) is an important membrane-bound receptor triggered by antiviral compounds and single-stranded RNA. It is implicated in the immune response to viruses such as influenza virus. It was not known whether geese, a natural host for avian influenza viruses, possess a homologue of mammalian TLR7 for recognizing avian influenza virus. In this study, we cloned the full-length of goose TLR7 and partial sequences of its adaptor protein, myeloid differentiation factor 88 (MyD88), some antiviral molecules such as RNA-dependent protein kinase (PKR) and 2′,5′-oligoadenylate synthetase (OAS). Goose TLR7 has a protein secondary structure identical to that of mammals, consisting of several leucine-rich domains, a transmembrane domain, and Toll/interleukin-1 receptor domain. To further understand whether the MyD88-dependent pathway of TLR7 is involved in the antiviral innate immune response against highly pathogenic avian influenza virus (HPAIV) infection in geese, we inoculated geese with an H5N1 HPAIV isolated from ducks in 2004. The virus, A/Duck/Guangdong/212/2004, replicated in various tissues resulting in 40% mortality. Quantitative real-time PCR analysis showed upregulation of mRNA transcripts for TLR7, MyD88, PKR and OAS in the lungs of geese at 1, 2 and 3 days post-inoculation. Therefore, the MyD88-dependent pathway of TLR7 was involved in the early stage of antiviral innate immune response in geese during H5N1 HPAIV infection.