Pathogenic Avian Influenza H5N1 Virus Research Articles

A neuraminidase activity-based microneutralization assay for evaluating antibody responses to influenza H5 and H7 vaccines.

Outbreaks of the highly pathogenic avian influenza H5N1 and H7N9 viruses have spurred an unprecedented research effort to develop antivirals and vaccines against influenza. Standardized methods for vaccine evaluation are critical for facilitating vaccine development. Compared with hemagglutination inhibition assays, mounting evidence suggest that microneutralization tests (MNTs) is a better choice for the evaluation of candidate pandemic influenza vaccines because they measure neutralizing antibody activity in cell cultures and are more sensitive in detecting H5 and H7. Here, we report a MNT measuring neuraminidase activity as the read-out (NA-MNT) for quantitative analysis of neutralizing antibodies against avian influenza viruses. Compared to the conventional microneutralization assay (ELISA-MNT), the NA-MNT is faster with lower intra- and inter-assay variations, while no difference in geometric mean titers was found between these two assays for the evaluation of H5N1 and H7N9 vaccines. These results suggest that NA-MNT is a reliable and high throughput method which could facilitate the development of candidate pandemic influenza vaccine.

Full sequence analysis of hemagglutinin and neuraminidase genes and proteins of highly pathogenic avian influenza H5N1 virus detected in Iran, 2015.

Over the last two decades, the highly pathogenic avian influenza H5N1 virus has gained a lot of attention due to its zoonotic and mutative nature. Iran is among the countries significantly affected by the virus as it hosts migratory birds during seasonal migration. In this study, the molecular characterizations of hemagglutinin (HA) and neuraminidase (NA) genes and proteins of H5N1 strain A/chicken/Iran/8/2015 detected in backyard poultry, Mazandaran province, were investigated. Phylogenetic analysis classified this virus as a member of subclade 2.3.2.1c, with the cleavage site motif of "PQRERRRK-R/GLF". HA carried a few mutations altering affinity to mammalian cells; however, the virus was categorized as avian. NA protein had the 20-amino acid deletion at aa position 49-69 similar to those isolated since 2000. Mutations of H253Y and H274Y contributing to antiviral resistance were present in NA. From this analysis, it can be concluded that the wild migratory birds flying from Western Asia to Eastern Africa are probably the main carriers of seasonal H5N1 in the country.

Deep sequencing of primary human lung epithelial cells challenged with H5N1 influenza virus reveals a proviral role for CEACAM1

Current prophylactic and therapeutic strategies targeting human influenza viruses include vaccines and antivirals. Given variable rates of vaccine efficacy and antiviral resistance, alternative strategies are urgently required to improve disease outcomes. Here we describe the use of HiSeq deep sequencing to analyze host gene expression in primary human alveolar epithelial type II cells infected with highly pathogenic avian influenza H5N1 virus. At 24 hours post-infection, 623 host genes were significantly upregulated, including the cell adhesion molecule CEACAM1. H5N1 virus infection stimulated significantly higher CEACAM1 protein expression when compared to influenza A PR8 (H1N1) virus, suggesting a key role for CEACAM1 in influenza virus pathogenicity. Furthermore, silencing of endogenous CEACAM1 resulted in reduced levels of proinflammatory cytokine/chemokine production, as well as reduced levels of virus replication following H5N1 infection. Our study provides evidence for the involvement of CEACAM1 in a clinically relevant model of H5N1 infection and may assist in the development of host-oriented antiviral strategies.

Complementary recognition of the receptor-binding site of highly pathogenic H5N1 influenza viruses by two human neutralizing antibodies

The highly pathogenic avian influenza virus H5N1 is a major threat to global public health and therefore a high-priority target of current vaccine development. The receptor-binding site (RBS) on the globular head of hemagglutinin (HA) in the viral envelope is one of the major target sites for antibody recognition against H5N1 and other influenza viruses. Here, we report the identification and characterization of a pair of human RBS–specific antibodies, designated FLD21.140 and AVFluIgG03, that are mutually complementary in their neutralizing activities against a diverse panel of H5N1 viruses. Crystallographic analysis and site-directed mutagenesis revealed that the two antibodies share a similar RBS-binding mode, and their individual specificities are governed by residues at positions 133a, 144, and 145. Specifically, FLD21.140 preferred Leu-133a/Lys-144/Ser-145, whereas AVFluIgG03 favored Ser-133a/Thr-144/Pro-145 residue triplets, both of which perfectly matched the most prevalent residues in viruses from epidemic-originating regions. Of note, according to an analysis of 3758 H5 HA sequences available in the Influenza Virus Database at the National Center for Biotechnology Information, the residues Leu-133a/Ser-133a and Ser-145/Pro-145 constituted more than 87.6 and 99.3% of all residues at these two positions, respectively. Taken together, our results provide a structural understanding for the neutralizing complementarity of these two antibodies and improve our understanding of the RBS-specific antibody response against H5N1 infection in humans.

Co-infection of highly pathogenic avian influenza and duck hepatitis viruses in Egyptian backyard and commercial ducks

Highly pathogenic avian influenza (HPAI) H5N1 virus poses a major challenge to the poultry industry and human health in Egypt. Twenty one households and eight duck farms in Sharkia Province, Egypt were investigated for the presence of avian influenza virus (AIV) and/or duck hepatitis virus 1 (DHV-1). Mortality rates among the investigated farms and yards were, 18.9% (69/365) of native ducks, 60.9% (25/41) of Pekin ducks, 60.2% (6306/10473) of Muscovy ducks and 44.9% (1353/3015) of Mallard ducks. The RT-PCR revealed the circulation of HPAI-H5N1 virus (81/104) among the examined birds with a high percentage in Muscovy (83.7%) and Pekin (83.4%) ducks. Interestingly, co-infection of HPAI and DHV-1 viruses in three ducklings with age of 4–19 days was detected. Severe neurological signs with high mortality were observed in ducklings as early as 4 days of age. Influenza virus antigen was detected in the neurons and glial cells of the brain, hepatocytes, and the intestinal submucosal plexus. Although, genetic characterization of H5N1 isolates revealed HPAIV of clade 2.2.1.2, such increased mortalities and neurological signs regardless of the duck age might imply the natural selection of HPAI in ducks. Crucial monitoring of the disease situation in ducks is essential for the implementation of an effective prevention and control program.

Highly Pathogenic H5N1 Influenza A Virus Spreads Efficiently in Human Primary Monocyte-Derived Macrophages and Dendritic Cells.

Influenza A viruses cause recurrent epidemics and occasional global pandemics. Wild birds are the natural reservoir of influenza A virus from where the virus can be transmitted to poultry or to mammals including humans. Mortality among humans in the highly pathogenic avian influenza H5N1 virus infection is even 60%. Despite intense research, there are still open questions in the pathogenicity of the H5N1 virus in humans. To characterize the H5N1 virus infection in human monocyte-derived macrophages (Mɸs) and dendritic cells (DCs), we used human isolates of highly pathogenic H5N1/2004 and H5N1/1997 and low pathogenic H7N9/2013 avian influenza viruses in comparison with a seasonal H3N2/1989 virus. We noticed that the H5N1 viruses have an overwhelming ability to replicate and spread in primary human immune cell cultures, and even the addition of trypsin did not equalize the infectivity of H7N9 or H3N2 viruses to the level seen with H5N1 virus. H5N1 virus stocks contained more often propagation-competent viruses than the H7N9 or H3N2 viruses. The data also showed that human DCs and Mɸs maintain 1,000- and 10,000-fold increase in the production of infectious H5N1 virus, respectively. Both analyzed highly pathogenic H5N1 viruses showed multi-cycle infection in primary human DCs and Mɸs, whereas the H3N2 and H7N9 viruses were incapable of spreading in immune cells. Interestingly, H5N1 virus was able to spread extremely efficiently despite the strong induction of antiviral interferon gene expression, which may in part explain the high pathogenicity of H5N1 virus infection in humans.

The PA-interacting host protein nucleolin acts as an antiviral factor during highly pathogenic H5N1 avian influenza virus infection

Polymerase acidic (PA) protein is a multifunctional regulator of influenza A virus (IAV) replication and pathogenesis. In a previous study, we reported that nucleolin (NCL) is a novel PA-interacting host protein. In this study, we further explored the role of NCL during highly pathogenic H5N1 avian influenza virus infection. We found that depletion of endogenous NCL in mammalian cells by siRNA targeting during H5N1 infection resulted in significantly increased viral polymerase activity, elevated viral mRNA, cRNA and vRNA synthesis, accelerated viral replication, and enhanced apoptosis and necrosis. Moreover, siRNA silencing of NCL significantly exacerbated the inflammatory response, resulting in increased secretion of IL-6, TNF-α, TNF-β, CCL-4, CCL-8, IFN-α, IFN-β and IFN-γ. Conversely, overexpression of NCL significantly decreased IAV replication. Collectively, these data show that NCL acts as a novel potential antiviral factor during H5N1 infection. Further studies exploring the antiviral mechanisms of NCL may accelerate the development of new anti-influenza drugs.

Recombinant Duck Interferon Gamma Inhibits H5N1 Influenza Virus Replication In Vitro and In Vivo.

The highly pathogenic H5N1 avian influenza virus (AIV) is widespread in waterfowl, causing enormous economic losses and posing a significant threat to public health. An increasing number of reagents have been identified to prevent the spread of influenza; however, there have been no reports on the anti-H5N1 effects of duck interferons, which exhibit antiviral activity against other viruses. Our aim was to investigate the antiviral effects of purified duck interferons. In this study, we successfully cloned and expressed duck interferon gamma (IFN-γ) in Escherichia coli. The antiviral effects of this recombinant duck IFN-γ (rDuIFN-γ) was assessed in vitro and in vivo. rDuIFN-γ displayed antiviral activity against vesicular stomatitis virus and AIV in duck embryo fibroblasts. Pretreating ducks with 3.4 × 104 U rDuIFN-γ also partially decreased mortality from 70% to 30% and delayed onset in 2-day-old Peking ducks. Virus titers in tissues and viral shedding decreased, and the expression of interferon-stimulated genes increased in brain and spleen in rDuIFN-γ-treated ducks. These results indicate that duck IFN-γ has the potential to inhibit viral replication in ducks.

Development of an Influenza Rapid Diagnostic Kit Specific for the H7 Subtype.

Since the spring of 2013, human infections with H7N9 viruses have been detected in China. Some of these viruses have become highly pathogenic. Highly and low pathogenic avian influenza H7N9 viruses are currently co-circulating with the seasonal influenza A viruses H3N2 and H1N1pdm09. Prompt identification and isolation of H7N9 patients is one measure to prevent the spread of H7N9 virus and help prevent a pandemic. The majority of commercially available point-of-care rapid influenza diagnostic kits can differentiate between influenza A and B viruses, but cannot distinguish between H7N9 viruses and seasonal influenza A viruses. Accordingly, we have developed a rapid diagnostic kit specific for the H7 subtype that is accessible, easy to use. Although the detection limit of this H7 kit is one-tenth lower than that of a commercially available rapid influenza A and B diagnostic kit of similar design, except for the specificity of the monoclonal antibodies used, this kit is highly specific, detecting only H7-subtype influenza viruses, including the recent highly pathogenic H7N9 viruses from humans, and does not show any non-specific reactions with other HA subtypes. This H7 kit will be of value for the early detection of H7N9-infected patients.

Characterization of pseudoparticles paired with hemagglutinin and neuraminidase from highly pathogenic H5N1 influenza and avian influenza A (H7N9) viruses

The reassortment of two highly pathogenic avian influenza (HPAI) H5N1 and H7N9 viruses presents a potential challenge to human health. The hemagglutinins (HAs) and neuraminidases (NAs) of these simultaneously circulating avian influenza viruses were evaluated using the pseudoparticle (pp) system. Native and mismatched virus pps were generated to investigate their biological characteristics. The HAs and NAs of the two viruses reassorted successfully to generate infectious viral particles. H7 was demonstrated to have the ability to reassort with NA from the H5N1 viruses, resulting in the generation of virions that were highly infectious to bronchial epithelial cells. Although the Anhui H5+Anhui N9 combination showed an moderate infectivity to the four cell lines, it was most sensitive to oseltamivir. The H7 in the pps was found to be predominantly HA0. Further, H5 in the pps primarily presented as HA1, owing to the particular mechanisms underlying its maturation. All NAs predominantly existed in monomer form. In our study, HAs/NAs, in all combinations, were functional and able to perform their corresponding function in the viral life cycle. Our data suggest that HAs/NAs from the (HPAI) H5N1 and H7N9 viruses are capable of assembly into infectious virions, posing a threat topublic health.

Characterization of highly pathogenic avian influenza H5N8 virus from Egyptian domestic waterfowl in 2017

ABSTRACTIn 2016, the highly pathogenic avian influenza (HPAI) H5N8 virus was detected in wild birds for the first time in Egypt. In the present study, we identified the HPAI virus H5N8 of clade 2.3.4.4 from domestic waterfowl in Egypt, suggesting its transmission to the domestic poultry from the migratory birds. Based on partial haemagglutinin gene sequence, this virus has a close genetic relationship with subtype H5N8 viruses circulating in Asia and Europe. Pathologically, H5N8 virus in hybrid duck induced nervous signs accompanied by encephalomalacia, haemorrhages, nonsuppurative encephalitis and nonsuppurative vasculitis. The granular layer of cerebellum showed multifocal areas of hydropic degeneration and the Purkinje cell neurons were necrotized or lost. Additionally, the lung, kidney and spleen were congested, and necrotizing pancreatitis was also observed. The co-circulation of both HPAI H5N1 and H5N8 subtypes with the low pathogenic avian influenza H9N2 subtype complicate the control of avian influenza in Egypt with the possibility of emergence of new reassortant viruses. Therefore, continuous monitoring with implementation of strict control measures is required.Research highlightsHPAI H5N8 virus clade 2.3.4.4 was detected in domestic ducks and geese in Egypt in 2017.Phylogenetically, the virus was closely related to HPAI H5N8 viruses identified in Asia and EuropeNonsuppurative encephalitis was widely observed in HPAI H5N8 virus-infected ducks.Degeneration of the cerebellar granular layer was found in most of the brain tissues examined.

Comparative analysis of MicroRNA expression in dog lungs infected with the H3N2 and H5N1 canine influenza viruses

MicroRNAs, a class of noncoding RNAs 18 to 23 nucleotides (nt) in length, play critical roles in a wide variety of biological processes. The objective of this study was to examine differences in microRNA expression profiles derived from the lungs of beagle dogs infected with the avian-origin H3N2 canine influenza virus (CIV) or the highly pathogenic avian influenza (HPAI) H5N1 virus (canine-origin isolation strain). After dogs were infected with H3N2 or H5N1, microRNA expression in the lungs was assessed using a deep-sequencing approach. To identify the roles of microRNAs in viral pathogenicity and the host immune response, microRNA target genes were predicted, and their functions were analyzed using bioinformatics software. A total of 229 microRNAs were upregulated in the H5N1 infection group compared with those in the H3N2 infection group, and 166 microRNAs were downregulated. MicroRNA target genes in the H5N1 group were more significantly involved in metabolic pathways, such as glycerolipid metabolism and glycerophospholipid metabolism, than those in the H3N2 group. The inhibition of metabolic pathways may lead to appetite loss, weight loss and weakened immunity. Moreover, miR-485, miR-144, miR-133b, miR-4859-5p, miR-6902-3p, miR-7638, miR-1307-3p and miR-1346 were significantly altered microRNAs that potentially led to the inhibition of innate immune pathways and the heightened pathogenicity of H5N1 compared with that of H3N2 in dogs. This study deepens our understanding of the complex relationships among microRNAs, the influenza virus-mediated immune response and immune injury in dogs.

Substitution of D701N in the PB2 protein could enhance the viral replication and pathogenicity of Eurasian avian-like H1N1 swine influenza viruses

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses (SIVs) have become predominant in pig populations in China and have recently been reported to have the most potential to raise the next pandemic in humans. The mutation D701N in the PB2 protein, which accounts for 31% of H1N1 SIVs, has previously been shown to contribute to the adaptation of the highly pathogenic H5N1 or H7N7 avian influenza viruses in mammals. However, little is known of the effects of this substitution on the EA H1N1 viruses. Herein, we investigated the contributions of 701N in the PB2 protein to an EA H1N1 SIV (A/Hunan/42443/2015(H1N1), HuN EA-H1N1), which had 701D in the PB2 protein. Our results found that viral polymerase activity, viral replication, and pathogenicity in mice were indeed enhanced due to the introduction of 701N into the PB2 protein, and the increased viral growth was partly mediated by the host factor importin-α7. Thus, substantial attention should be paid to the D701N mutation in pig populations.

Exploring contacts facilitating transmission of influenza A(H5N1) virus between poultry farms in West Java, Indonesia: A major role for backyard farms?

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been reported in Asia, including Indonesia since 2003. Although several risk factors related to the HPAIV outbreaks in poultry in Indonesia have been identified, little is known of the contact structure of farms of different poultry production types (backyard chickens, broilers, layers, and ducks). This study aims to quantify the contact rates associated with the movement of people, and movements of live birds and products and equipment that affect the risk of HPAIV H5N1 transmission between poultry farms in Indonesia. On 124 poultry farms in 6 districts in West Java, logbooks were distributed to record the movements of farmers/staff and visitors and their poultry contacts. Most movements in backyard chicken, commercial native chicken, broiler and duck farms were visits to and from other poultry farms, whilst in layer farms visits to and from poultry companies, visits to egg collection houses and visit from other poultry farms were most frequent. Over 75% of persons visiting backyard chicken and duck farms had previously visited other poultry farms on the same day. Visitors of backyard chicken farms had the highest average contact rate, either direct contact with poultry on other farms before the visits (1.35 contact/day) or contact during their visits in the farms (10.03 contact/day). These results suggest that backyard chicken farms are most at risk for transmission of HPAIV compared to farms of the other poultry production types. Since visits of farm-to-farm were high, backyard farms could also a potential source for HPAIV transmission to commercial poultry farms.

Comparative Epidemiology of Highly Pathogenic Avian Influenza Virus H5N1 and H5N6 in Vietnamese Live Bird Markets: Spatiotemporal Patterns of Distribution and Risk Factors.

Highly pathogenic avian influenza (HPAI) H5N1 virus has been circulating in Vietnam since 2003, whilst outbreaks of HPAI H5N6 virus are more recent, having only been reported since 2014. Although the spatial distribution of H5N1 outbreaks and risk factors for virus occurrence has been extensively studied, there have been no comparative studies for H5N6. Data collected through active surveillance of Vietnamese live bird markets (LBMs) between 2011 and 2015 were used to explore and compare the spatiotemporal distributions of H5N1- and H5N6-positive LBMs. Conditional autoregressive models were developed to quantify spatiotemporal associations between agroecological factors and the two HPAI strains using the same set of predictor variables. Unlike H5N1, which exhibited a strong north–south divide, with repeated occurrence in the extreme south of a cluster of high-risk provinces, H5N6 was homogeneously distributed throughout Vietnam. Similarly, different agroecological factors were associated with each strain. Sample collection in the months of January and February and higher average maximum temperature were associated with higher likelihood of H5N1-positive market-day status. The likelihood of market days being positive for H5N6 increased with decreased river density, and with successive Rounds of data collection. This study highlights marked differences in spatial patterns and risk factors for H5N1 and H5N6 in Vietnam, suggesting the need for tailored surveillance and control approaches.

Reassortant H5N1 Avian Influenza Virus Bearing PB2 Gene From a 2009 Pandemic H1N1 Exhibits Increased Pathogenicity in Mice.

Reassortment is a key driving force of the evolution and host adaptation of the influenza virus. A(H1N1)pdm2009 (pdm09), a novel H1N1 influenza viral subtype, caused a pandemic in 2009. The strain was established in pig herds and cocirculated with the highly pathogenic H5N1 avian influenza virus. The coexistence of pdm09 with H5N1 raises concerns that reassortment may cause the development of novel viral strains with unpredictable virulence. Given that the viral polymerase subunit PB2 is a determinant of host range and pathogenicity, and that the substantial amino acid differences in PB2 between pdm09 and H5N1, including positions 590/591 and 271, which are shown to play key roles in enhanced polymerase activity in mammalian host cells, we generated a reassortant virus containing PB2 derived from a pdm09 (A/Liaoning/1/2009, LN/09) to investigate if pdm09-derived PB2 can function in a heterologous avian virus isolate as an adaptive strategy, with H5N1 (A/duck/Hubei/hangmei01/2006, HM/06) as the backbone. We assessed the biological characteristics, including pathogenicity, replication, and polymerase activity, of the reassortant. Compared with HM/06 and LN/09, H5N1 hybrid virus containing PB2 from LN/09 exhibited significantly increased pathogenicity in mice and proliferation activity in mammalian cell lines, as well as markedly enhanced polymerase activity. Our results indicate that the coexistence of H5N1 and pdm09 may pose a great threat to public health through reassortment. Moreover, our results highlight the importance of monitoring the emergence of H5N1 reassortants containing pdm09-derived PB2.

Spatial clustering of pathology submissions during the initial introduction and spread of avian influenza H5N1 in poultry in Nigeria in 2006-2007.

Highly pathogenic avian influenza (HPAI) virus H5N1 spread throughout Nigeria between 2006 and 2007. Bird samples collected across the country were submitted through the free-of-charge (FOC) program to the National Veterinary Research Institute, Vom (NVRI-Vom) laboratory. The present article describes the spatial distributions and evaluated clustering of the FOC submissions from poultry farms at the global, local, and focal levels between 2006 and 2007 epidemic in Nigeria. Spatial statistics evaluating clustering of the FOC submissions were implemented using the Moran's I test, the purely spatial cluster analysis with the SaTScan Poisson model, and the Bithell's linear score test. A significant global clustering of the FOC submissions was observed. Significant local clusters of submissions were observed in the North-East, North-Central, and South-West zones. There was significant decline in FOC submissions with increasing distance from NVRI-Vom. These results indicated that the geographic area of influence of the FOC submission program in Nigeria was limited to regions closer to the diagnostic laboratory. This work provides a detailed insight into the surveillance activities during the HPAI outbreaks in Nigeria, and should assist policy-makers and field veterinarians to improve the effectiveness of national eradication plans in the face of any outbreak of animal diseases.

Potential Biological and Climatic Factors That Influence the Incidence and Persistence of Highly Pathogenic H5N1 Avian Influenza Virus in Egypt.

Highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 is endemic in poultry in Egypt where the highest number of human infections worldwide was reported. During the last 12 years the Egyptian A/H5N1 evolved into several genotypes. In 2007-2014 vaccinated poultry suffered from antigenic drift variants of clade 2.2.1.1 and in 2014/2015 an unprecedented upsurge of A/H5N1 clade 2.2.1.2 occurred in poultry and humans. Factors contributing to the endemicity or re-emergence of A/H5N1 in poultry in Egypt remain unclear. Here, three potential factors were studied: climatic factors (temperature, relative humidity, and wind speed), biological fitness in vitro, and pathogenicity in domestic Pekin and Muscovy ducks. Statistical analyses using negative binomial regression models indicated that ambient temperature in winter months influenced the spread of A/H5N1 in different geographic areas analyzed in this study. In vitro, at 4 and 56°C 2.2.1.1 and recent 2.2.1.2 viruses were more stable than other viruses used in this study. Further, Pekin ducks were more resistant than Muscovy ducks and the viruses were excreted for up to 2 weeks post-infection assuming a strong role as a reservoir. Taken together, ambient temperature in winter months potentially contributes to increasing outbreaks in some regions in Egypt. Heat stability of clade 2.2.1.1 and recent 2.2.1.2 viruses probably favors their persistence at elevated temperatures. Importantly, asymptomatically infected Pekin ducks may play an important role in the spread of avian and human-like A/H5N1 in Egypt. Therefore, control measures including targeted surveillance and culling of silently infected Pekin ducks should be considered.

Differences in Type I interferon response in human lung epithelial cells infected by highly pathogenic H5N1 and low pathogenic H11N1 avian influenza viruses.

Influenza A virus infection induces type I interferons (IFNs α/β) which activate host antiviral responses through a cascade of IFN signaling events. Herein, we compared highly pathogenic H5N1 and low pathogenic H11N1 avian influenza viruses isolated from India, for their replication kinetics and ability to induce IFN-β and interferon-stimulating genes (ISGs). The H5N1 virus showed a higher replication rate and induced less IFN-β and ISGs compared to the H11N1 virus when grown in the human lung epithelial A549 cells, reflecting the generation of differential innate immune responses during infection by these viruses. The non-structural 1 (NS1) protein, a major IFN-antagonist, known to help the virus in evading host innate immune response was compared from both the strains using bioinformatics tools. Analyses revealed differences in the composition of the NS1 proteins from the two strains that may have an impact on the modulation of the innate immune response. Intriguingly, H5N1 virus attenuated IFN-β response in a non-NS1 manner, suggesting the possible involvement of other viral proteins (PB2, PA, PB1/PB1-F2) of H5N1 in synergy with NS1. Preliminary analyses of the above proteins of the two strains by sequence comparison show differences in charged residues. The insight gained will be useful in designing experimental studies to elucidate a probable role of the polymerase protein(s) in association with NS1 in inhibiting the IFN signaling and understanding the molecular mechanism governing the difference.

A two dose immunization with an inactivated reassortant H5N2 virus protects chickens against lethal challenge with homologous 2.3.2.1 clade and heterologous 2.2 clade highly pathogenic avian influenza H5N1 viruses

The present study was aimed at generating a reassortant vaccine candidate virus with clade 2.3.2.1 Hemagglutinin (HA) and its evaluation in a challenge study for protection against homologous (2.3.2.1 clade) and heterologous (2.2 clade) highly pathogenic avian influenza (HPAI) H5N1 viruses. Plasmid-based reverse genetics technique was used to rescue a 5 + 3 reassortant H5N2 strain containing the modified HA of H5N1 (clade 2.3.2.1), the Neuraminidase (NA) of H9N2, the Matrix (M) of H5N1 and the internal genes of A/WSN/33 H1N1. In addition, another 6 + 2 reassortant virus containing modified HA from H5N1 (clade 2.3.2.1), the NA from H9N2 and the internal genes of A/WSN/33 H1N1 was also rescued. The 5 + 3 reassortant H5N2 virus could grow to a higher titer in both MDCK cells and chicken eggs compared to the 6 + 2 reassortant H5N2 virus. The vaccine containing the inactivated 5 + 3 reassortant H5N2 virus was used in a two-dose immunization regime which protected specific pathogen free (SPF) chickens against two repeated challenges with homologous 2.3.2.1 clade and heterologous 2.2 clade HPAI H5N1 viruses. The 5 + 3 reassortant H5N2 virus based on clade 2.3.2.1 generated in this study can be effective in protecting chickens in the case of an outbreak caused by antigenically different clade 2.2 HPAI H5N1 viruses and opens the way to explore its applicability as potential vaccine candidate especially in the Asian countries reporting these clades frequently. The study also indicates that sequential immunization can broaden protection level against antigenically diverse strains of H5N1 viruses.