Highly Pathogenic H5N1 Research Articles

Susceptibility of common dabbling and diving duck species to clade 2.3.2.1 H5N1 high pathogenicity avian influenza virus: an experimental infection study.

In the winter of 2010-2011, Japan experienced a large outbreak of infections caused by clade 2.3.2.1 H5N1 high pathogenicity avian influenza viruses (HPAIVs) in wild birds. Interestingly, many tufted ducks (Aythya fuligula), which are migratory diving ducks, succumbed to the infection, whereas only one infection case was reported in migratory dabbling duck species, the major natural hosts of the influenza A virus, during the outbreak. To assess whether the susceptibility of each duck species to HPAIVs was correlated with the number of cases, tufted duck and dabbling duck species (Eurasian wigeon, Mareca penelope; mallard, Anas platyrhynchos; Northern pintail, Anas acuta) were intranasally inoculated with A/Mandarin duck/Miyazaki/22M807-1/2011 (H5N1), an index clade 2.3.2.1 virus previously used for experimental infection studies in various bird species. All ducks observed for 10 days post-inoculation (dpi) mostly shed the virus via the oral route and survived. The tufted ducks shed a higher titer of the virus than the other dabbling duck species, and one of them showed apparent neurological symptoms after 7 dpi, which were accompanied by eye lesions. No clinical symptoms were observed in the dabbling ducks, although systemic infection and viremia were observed in some of them sacrificed at 3 dpi. These results suggest that the susceptibility of clade 2.3.2.1 HPAIVs might differ by duck species.

Inactivation of Avian Influenza Viruses by Chemical Disinfectants and the Influence of Faecal Matter

Inactivation of avian influenza viruses (AIVs) using chemical disinfectants is an indispensable biosecurity measure during outbreaks. The efficiency of disinfection process depends on disinfectant type and presence or absence of organic matter. It is also not clear whether AIV pathotype could influence its tolerability to a given disinfectant. Here, we assessed the efficiency of formalin and caustic soda against high (H5N1) and low (H9N2) pathogenic AIVs in the presence and absence of wet faeces. Our results showed that the effectiveness of both disinfectants depends on the target virus with the low pathogenic H9N2 virus being generally more susceptible to disinfection than the high pathogenic H5N1 virus. Wet faeces slightly influenced disinfection action in a virus- and contact time- dependent manner. This study highlights the importance of eliminating faecal matter before initiating a disinfection process and indicates the influence of disinfectant type and AIVs pathotype when selecting the appropriate disinfectant.

Virological, pathological, and glycovirological investigations of an Ezo red fox and a tanuki naturally infected with H5N1 high pathogenicity avian influenza viruses in Hokkaido, Japan

In winter/spring 2021–2022, high pathogenicity avian influenza viruses (HPAIVs) that are genetically closely related to each other were detected worldwide. In a public garden in Sapporo, Hokkaido, Japan, a crow die-off by HPAIV infection occurred from March 29 to May 18, 2022. During the event, H5N1 HPAIVs were isolated from an Ezo red fox (Vulpes vulpes schrencki) and a tanuki (Nyctereutes procyonoides albus) found in the same garden. The fox showed viral meningoencephalitis and moderate virus replication in the upper respiratory tract, whereas the tanuki showed viral conjunctivitis and secondary bacterial infection in the eyes accompanied with visceral larva migrans. Viruses isolated from the fox and the tanuki were genetically closely related to those isolated from crows in the same garden. Various α2-3 sialosides were found in the respiratory tracts of these canid mammals, consistent with HPAIV infections in these animals. This study highlighted the importance of monitoring HPAIV infections in wild carnivore mammals to detect the potential virus spreading in nature.

Detection of New H5N1 High Pathogenicity Avian Influenza Viruses in Winter 2021-2022 in the Far East, Which Are Genetically Close to Those in Europe.

Many high pathogenicity avian influenza (HPAI) cases in wild birds due to H5N1 HPAI virus (HPAIV) infection were reported in northern Japan in the winter of 2021–2022. To investigate the epidemiology of HPAIVs brought to Japan from surrounding areas, a genetic analysis of H5 HPAIVs isolated in northern Japan was performed, and the pathogenicity of the HPAIV in chickens was assessed by experimental infection. Based on the genetic analysis of the hemagglutinin gene, pathogenic viruses detected in northern Japan as well as one in Sakhalin, the eastern part of Russia, were classified into the same subgroup as viruses prevalent in Europe in the same season but distinct from those circulating in Asia in winter 2020–2021. High identities of all eight segment sequences of A/crow/Hokkaido/0103B065/2022 (H5N1) (Crow/Hok), the representative isolates in northern Japan in 2022, to European isolates in the same season could also certify the unlikeliness of causing gene reassortment between H5 HPAIVs and viruses locally circulating in Asia. According to intranasal challenge results in six-week-old chickens, 50% of the chicken-lethal dose of Crow/Hok was calculated as 104.5 times of the 50% egg-infectious dose. These results demonstrated that the currently prevalent H5 HPAIVs could spread widely from certain origins throughout the Eurasian continent, including Europe and the Far East, and implied a possibility that contagious viruses are gathered in lakes in the northern territory via bird migration. Active monitoring of wild birds at the global level is essential to estimate the geographical source and spread dynamics of HPAIVs.

Emergence of clade 2.3.4.4b novel reassortant H5N1 high pathogenicity avian influenza virus in South Korea during late 2021.

High pathogenicity H5N1 avian influenza viruses pose a threat to both animal and human health worldwide. In late 2020, outbreaks of H5 high pathogenicity avian influenza viruses belonging to clade 2.3.4.4b emerged in Europe, following on from outbreaks in East Asia in earlier years. However, very recent studies show that clade 2.3.4.4b H5N1, rather than 2.3.4.4b H5N8, has become predominant in wild birds and has infected poultry in several countries. In this study, we describe isolation of a novel H5N1 virus from a captured mandarin duck in South Korea, and another H5N1 virus from a quail farm. We performed genetic analysis of these two viruses to identify their origin and to determine their relationship with the clade 2.3.4.4b H5N1 viruses currently circulating in Europe. Based on our results, it is presumed that the novel H5N1 virus isolated in Korea originated from an unknown reassortant between clade 2.3.4.4b H5N8 viruses circulating from 2020 and other Eurasian viruses, with additional reassortment of genes and point mutations that discriminate them from the recently reported H5N1 virus in Europe.

Mathematical Modeling Finds Disparate Interferon Production Rates Drive Strain-Specific Immunodynamics during Deadly Influenza Infection.

The timing and magnitude of the immune response (i.e., the immunodynamics) associated with the early innate immune response to viral infection display distinct trends across influenza A virus subtypes in vivo. Evidence shows that the timing of the type-I interferon response and the overall magnitude of immune cell infiltration are both correlated with more severe outcomes. However, the mechanisms driving the distinct immunodynamics between infections of different virus strains (strain-specific immunodynamics) remain unclear. Here, computational modeling and strain-specific immunologic data are used to identify the immune interactions that differ in mice infected with low-pathogenic H1N1 or high-pathogenic H5N1 influenza viruses. Computational exploration of free parameters between strains suggests that the production rate of interferon is the major driver of strain-specific immune responses observed in vivo, and points towards the relationship between the viral load and lung epithelial interferon production as the main source of variance between infection outcomes. A greater understanding of the contributors to strain-specific immunodynamics can be utilized in future efforts aimed at treatment development to improve clinical outcomes of high-pathogenic viral strains.

Economic loss in poultry farms affected with highly pathogenic avian influenza A virus subtype H5N1 and low pathogenic avian influenza A virus subtype H9N2 in Chattogram, Bangladesh

Depending on the degree of pathogenicity in chickens, avian influenza virus (AIV) are divided into highly pathogenic (HPAI) or low pathogenic AI (LPAI) viruses. Typically, high morbidity accompanied by high and rapidly escalating unexplained mortality is associated with a HPAI virus. On the other hand, LPAI viruses normally cause only a mild or no clinical disease, but under certain circumstances such as with concomitant infection(s) may also cause high mortality. However, published literature seems to be absent in Bangladesh on the economic loss caused by them at the affected farm level. This study was aimed at assessing the economic losses caused by highly pathogenic avian influenza A virus subtype H5N1 (HPAI H5N1) and low pathogenic avian influenza A virus subtype H9N2 (LPAI H9N2) on poultry farms. Cloacal and oropharyngeal samples from chickens supplied from 262 farms between October 2017 and April 2019 were investigated by real-time reverse transcription polymerase chain reaction (rRT-PCR). Of them birds on 16, 15 and 12 farms were diagnosed positive with HPAI H5N1, LPAI H9N2 and with the both, respectively. For each of the categories of infection five farms were randomly chosen and owners or farm representatives were interviewed with a pretested questionnaire to assess the economic losses attributed to them. The results revealed that the farm-level economic loss was variable depending on the number of birds housed on the day of clinical illness and the type of birds (i.e. broiler, layer and Sonali) reared. On the other hand, the average per-bird loss for broiler, layer and Sonali chickens due to HPAI H5N1 infection alone was around BDT 26, BDT 182 and BDT 82, respectively. No Sonali farm was diagnosed positive with LPAI H9N2 or HPAI H5N2 plus LPAI H9N2, and therefore, not included in the study. The average per-bird loss attributed to HPAI H5N1 plus LPAI H9N2 was BDT46 in layer and BDT 47 in broiler farm. On the other hand, the average per-bird loss caused by LPAI H9N2 alone was BDT62 in broiler, much higher than layer in which the estimate was BDT 46.5. To avoid economic loss attributed to HPAI H5N1 and LPAI H9N2 proper biosecurity should be practiced to keep the birds on farms free from the introduction of the viruses

Genotyping and reassortment analysis of highly pathogenic avian influenza viruses H5N8 and H5N2 from Egypt reveals successive annual replacement of genotypes

Highly pathogenic (HP) H5N1, clade 2.2.1, and low pathogenic avian influenza (LPAI) H9N2 viruses, G1-B lineage, are endemic in poultry in Egypt and have co-circulated for almost a decade. Surprisingly, no inter-subtypic reassortment events have been reported from the field during that time. After the introduction of HPAIV H5N8, clade 2.3.4.4b, in Egyptian poultry in 2016, suddenly HP H5N2 reassortants with H9N2 viruses emerged. The current analyses focussed on studying 32 duck flocks, 4 broiler chicken flocks, and 1 turkey flock, suffering from respiratory manifestations with moderate to high mortality reared in two Egyptian governorates during 2019. Real-time RT-PCR substantiated the presence of HP H5N8 in 21 of the 37 investigated flocks with mixed infection of H9N2 in two of them. HP H5N1 was not detected. Full hemagglutinin (HA) sequencing of 10 samples with full-genome sequencing of three of them revealed presence of a single genotype. Very few substituting mutations in the HA protein were detected versus previous Egyptian HA sequences of that clade. Interestingly, amino acid substitutions in the Matrix (M2) and the Neuraminidase (NA) proteins associated with conferring both Amantadine and Oseltamivir resistance were present. Systematic reassortment analysis of all publicly available Egyptian whole genome sequences of HP H5N8 (n = 23), reassortant HP H5N2 (n = 2) and LP H9N2 (n = 53) viruses revealed presence of at least seven different genotypes of HPAI H5Nx viruses of clade 2.3.4.4b in Egypt since 2016. For H9N2 viruses, at least three genotypes were distinguishable. Heat mapping and tanglegram analyses suggested that several internal gene segments in both HP H5Nx and H9N2 viruses originated from avian influenza viruses circulating in wild bird species in Egypt. Based on the limited set of whole genome sequences available, annual replacement patterns of HP H5Nx genotypes emerged and suggested selective advantages of certain genotypes since 2016.

M1-like monocytes are a major immunological determinant of severity in previously healthy adults with life-threatening influenza.

In each influenza season, a distinct group of young, otherwise healthy individuals with no risk factors succumbs to life-threatening infection. To better understand the cause for this, we analyzed a broad range of immune responses in blood from a unique cohort of patients, comprising previously healthy individuals hospitalized with and without respiratory failure during one influenza season, and infected with one specific influenza A strain. This analysis was compared with similarly hospitalized influenza patients with known risk factors (total of n = 60 patients recruited). We found a sustained increase in a specific subset of proinflammatory monocytes, with high TNF-α expression and an M1-like phenotype (independent of viral titers), in these previously healthy patients with severe disease. The relationship between M1-like monocytes and immunopathology was strengthened using murine models of influenza, in which severe infection generated using different models (including the high-pathogenicity H5N1 strain) was also accompanied by high levels of circulating M1-like monocytes. Additionally, a raised M1/M2 macrophage ratio in the lungs was observed. These studies identify a specific subtype of monocytes as a modifiable immunological determinant of disease severity in this subgroup of severely ill, previously healthy patients, offering potential novel therapeutic avenues.

Emerging highly pathogenic H5 avian influenza viruses in France during winter 2015/16: phylogenetic analyses and markers for zoonotic potential

Several new highly pathogenic (HP) H5 avian influenza virus (AIV) have been detected in poultry farms from south-western France since November 2015, among which an HP H5N1. The zoonotic potential and origin of these AIVs immediately became matters of concern. One virus of each subtype H5N1 (150169a), H5N2 (150233) and H5N9 (150236) was characterised. All proved highly pathogenic for poultry as demonstrated molecularly by the presence of a polybasic cleavage site in their HA protein – with a sequence (HQRRKR/GLF) previously unknown among avian H5 HPAI viruses – or experimentally by the in vivo demonstration of an intravenous pathogenicity index of 2.9 for the H5N1 HP isolate. Phylogenetic analyses based on the full genomes obtained by NGS confirmed that the eight viral segments of the three isolates were all part of avian Eurasian phylogenetic lineage but differed from the Gs/Gd/1/96-like lineage. The study of the genetic characteristics at specific amino acid positions relevant for modulating the adaptation to and the virulence for mammals showed that presently, these viruses possess most molecular features characteristic of AIV and lack some major characteristics required for efficient respiratory transmission to or between humans. The three isolates are therefore predicted to have no significant pandemic potential.

Surveillance for highly pathogenic influenza A viruses in California during 2014–2015 provides insights into viral evolutionary pathways and the spatiotemporal extent of viruses in the Pacific Americas Flyway

We used surveillance data collected in California before, concurrent with, and subsequent to an outbreak of highly pathogenic (HP) clade 2.3.4.4 influenza A viruses (IAVs) in 2014–2015 to (i) evaluate IAV prevalence in waterfowl, (ii) assess the evidence for spill-over infections in marine mammals and (iii) genetically characterize low-pathogenic (LP) and HP IAVs to refine inference on the spatiotemporal extent of HP genome constellations and to evaluate possible evolutionary pathways. We screened samples from 1496 waterfowl and 1142 marine mammals collected from April 2014 to August 2015 and detected IAV RNA in 159 samples collected from birds (n=157) and pinnipeds (n=2). HP IAV RNA was identified in three samples originating from American wigeon (Anas americana). Genetic sequence data were generated for a clade 2.3.4.4 HP IAV-positive diagnostic sample and 57 LP IAV isolates. Phylogenetic analyses revealed that the HP IAV was a reassortant H5N8 virus with gene segments closely related to LP IAVs detected in mallards (Anas platyrhynchos) sampled in California and other IAVs detected in wild birds sampled within the Pacific Americas Flyway. In addition, our analysis provided support for common ancestry between LP IAVs recovered from waterfowl sampled in California and gene segments of reassortant HP H5N1 IAVs detected in British Columbia, Canada and Washington, USA. Our investigation provides evidence that waterfowl are likely to have played a role in the evolution of reassortant HP IAVs in the Pacific Americas Flyway during 2014–2015, whereas we did not find support for spill-over infections in potential pinniped hosts.Emerging Microbes & Infections (2017) 6, e80; doi:10.1038/emi.2017.66; published online 6 September 2017

No Evidence of On-farm Circulation of Avian Influenza H5 Subtype in Ca Mau Province, Southern Vietnam, March 2016 - January 2017.

Background:Subtype H5N1 avian influenza viruses, both high pathogenicity and low pathogenicity, have been enzootic in Vietnam since 2001. The viruses are readily identified at live bird markets, but virus prevalence on smallholder poultry is typically zero or very low. If the true direction of the viral transmission chain is farm to market, it is unknown why farm prevalence should be low when market prevalence is moderate to high.Methods:We established a cohort of 50 smallholder poultry farms in Ca Mau province in the Mekong Delta region of Vietnam. From March 2016 to January 2017, we collected naso-pharyngeal and cloacal samples from 156 ducks and 96 chickens. In addition, 126 environmental samples were collected. Samples were assayed for H5 subtype influenza by real-time RT-PCR.Results/Discussion: None of the 378 collected samples were positive for H5 influenza. This is likely to mean that circulation of subtype H5 influenza viruses was low in Ca Mau in 2016. Detection of avian influenza on smallholder poultry farms is necessary to determine the directionality and association between farm prevalence and market prevalence of avian influenza viruses. Larger farm-level studies should be planned as these will be critical for determining the presence and strength of this association.

Genome-wide gene expression pattern underlying differential host response to high or low pathogenic H5N1 avian influenza virus in ducks

The differences in the influenza viral pathogenesis observed between different pathogenic strains are associated with distinct properties of virus strains and the host immune responses. In order to determine the differences in the duck immune response against two different pathogenic strains, we studied genome-wide host immune gene response of ducks infected with A/duck/India/02CA10/2011 and A/duck/Tripura/103597/2008 H5N1 viruses using custom-designed microarray. A/duck/India/02CA10/2011 is highly pathogenic virus (HP) to ducks, whereas A/duck/Tripura/103597/2008 is a low pathogenic (LP) virus strain. Comparative lung tissue transcriptome analysis of differentially expressed genes revealed that 686 genes were commonly expressed, 880 and 1556 genes are expressed uniquely to infection with HP and LP virus, respectively. The up-regulation of chemokines (CCL4 and CXCR4) and IFN-stimulated genes (IFITM2, STAT3, TGFB1 and TGFB3) was observed in the lung tissues of ducks infected with HP virus. The up-regulation of other immune genes (IL17, OAS, SOCS3, MHC I and MHC II) was observed in both infection conditions. The expression of important antiviral immune genes MX, IFIT5, IFITM5, ISG12, β-defensins, RSAD2, EIF2AK2, TRIM23 and SLC16A3 was observed in LP virus infection, but not in HP virus infection. Several immune-related gene ontology terms and pathways activated by both the viruses were qualitatively similar but quantitatively different. Based on these findings, the differences in the host immune response might explain a part of the difference observed in the viral pathogenesis of high and low pathogenic influenza strains in ducks.

Model-based clustering with certainty estimation: implication for clade assignment of influenza viruses.

BackgroundClustering is a common technique used by molecular biologists to group homologous sequences and study evolution. There remain issues such as how to cluster molecular sequences accurately and in particular how to evaluate the certainty of clustering results.ResultsWe presented a model-based clustering method to analyze molecular sequences, described a subset bootstrap scheme to evaluate a certainty of the clusters, and showed an intuitive way using 3D visualization to examine clusters. We applied the above approach to analyze influenza viral hemagglutinin (HA) sequences. Nine clusters were estimated for high pathogenic H5N1 avian influenza, which agree with previous findings. The certainty for a given sequence that can be correctly assigned to a cluster was all 1.0 whereas the certainty for a given cluster was also very high (0.92–1.0), with an overall clustering certainty of 0.95. For influenza A H7 viruses, ten HA clusters were estimated and the vast majority of sequences could be assigned to a cluster with a certainty of more than 0.99. The certainties for clusters, however, varied from 0.40 to 0.98; such certainty variation is likely attributed to the heterogeneity of sequence data in different clusters. In both cases, the certainty values estimated using the subset bootstrap method are all higher than those calculated based upon the standard bootstrap method, suggesting our bootstrap scheme is applicable for the estimation of clustering certainty.ConclusionsWe formulated a clustering analysis approach with the estimation of certainties and 3D visualization of sequence data. We analysed 2 sets of influenza A HA sequences and the results indicate our approach was applicable for clustering analysis of influenza viral sequences.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1147-x) contains supplementary material, which is available to authorized users.

Protection Afforded by a Recombinant Turkey Herpesvirus-H5 Vaccine Against the 2014 European Highly Pathogenic H5N8 Avian Influenza Strain.

A highly pathogenic avian influenza (HPAI) H5N8 (clade 2.3.4.4) virus, circulating in Asia (South Korea, Japan, and southern China) since the beginning of 2014, reached the European continent in November 2014. Germany, the Netherlands, the United Kingdom, Italy, and Hungary confirmed H5N8 infection of poultry farms of different species and of several wild bird species. Unlike the Asian highly pathogenic (HP) H5N1, this HP H5N8 also went transatlantic and reached the American West Coast by the end of 2014, affecting wild birds as well as backyard and commercial poultry. This strain induces high mortality and morbidity in Galliformes, whereas wild birds seem only moderately affected. A recombinant turkey herpesvirus (rHVT) vector vaccine expressing the H5 gene of a clade 2.2 H5N1 strain (rHVT-H5) previously demonstrated a highly efficient clinical protection and reduced viral excretion against challenge with Asian HP H5N1 strains of various clades (2.2, 2.2.1, 2.2.1.1, 2.1.3, 2.1.3.2, and 2.3.2.1) and was made commercially available in various countries where the disease is endemic. To evaluate the protective efficacy of the rHVT-H5 vaccine against the first German H5N8 turkey isolate (H5N8 GE), a challenge experiment was set up in specific-pathogen-free (SPF) chickens, and the clinical and excretional protection was evaluated. SPF chickens were vaccinated subcutaneously at 1 day old and challenged oculonasally at 4 wk of age with two viral dosages, 10(5) and 10(6) 50% egg infective doses. Morbidity and mortality were monitored daily in unvaccinated and vaccinated groups, whereas viral shedding by oropharyngeal and cloacal routes was evaluated at 2, 5, 9, and 14 days postinoculation (dpi). Serologic monitoring after vaccination and challenge was also carried out. Despite its high antigenic divergence of the challenge H5N8 strain, a single rHVT-H5 vaccine administration at 1 day old resulted in a full clinical protection against challenge and a significant reduction of viral shedding in the vaccinated birds.

Application of FTA® Cards for detection and storage of avian influenza virus

Abstract Introduction: The aim of the study was to test the utility of Flinders Technology Associates filter paper (FTA® Cards) for molecular detection and storage of avian influenza virus (AIV). Material and Methods: There were two strains of AIV used in the study: low pathogenicity H7N1 and high pathogenicity H5N1 subtypes. Detection of viral material was conducted using molecular RT-PCR and rRT- PCR method. Results: The infectivity of LPAIV/H7N1 and HPAIV/H5N1 was completely inactivated within 1 h and 24 h after adsorption to FTA® Cards at room temperature, respectively. Viruses stored on FTA® Cards had detection limit approximately 1 log10 lower than live viruses. Viral RNA of both strains were detectable on the cards by rRT-PCR for a minimum of 150 d, irrespectively of storage temperatures (room temperature, -20ºC). RNA was also detected in all samples obtained from SPF chickens experimentally infected with HPAI/H5N1 on 3rd and 4th day post-infection (p.i.). Conclusion: FTA® Cards enable safe and effective alternative transport of samples for molecular diagnosis of AIV.

Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens

A synthetic hemagglutinin (HA) gene from the highly pathogenic avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1) (Indo/03) was expressed in aquatic plant Lemna minor (rLemna-HA). In Experiment 1, efficacy of rLemna-HA was tested on birds immunized with 0.2μg or 2.3μg HA and challenged with 106 mean chicken embryo infectious doses (EID50) of homologous virus strain. Both dosages of rLemna-HA conferred clinical protection and dramatically reduced viral shedding. Almost all the birds immunized with either dosage of rLemna-HA elicited HA antibody titers against Indo/03 antigen, suggesting an association between levels of anti-Indo/03 antibodies and protection. In Experiment 2, efficacy of rLemna-HA was tested on birds immunized with 0.9μg or 2.2μg HA and challenged with 106 EID50 of heterologous H5N1 virus strains A/chicken/Vietnam/NCVD-421/2010 (VN/10) or A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Birds challenged with VN/10 exhibited 100% survival regardless of immunization dosage, while birds challenged with PWT/06 had 50% and 30% mortality at 0.9μg HA and 2.2μg HA, respectively. For each challenge virus, viral shedding titers from 2.2μg HA vaccinated birds were significantly lower than those from 0.9μg HA vaccinated birds, and titers from both immunized groups were in turn significantly lower than those from sham vaccinated birds. Even if immunized birds elicited HA titers against the vaccine antigen Indo/03, only the groups challenged with VN/10 developed humoral immunity against the challenge antigen. None (rLemna-HA 0.9μg HA) and 40% (rLemna-HA 2.2μg HA) of the immunized birds challenged with PWT/06 elicited pre-challenge antibody titers, respectively. In conclusion, Lemna-expressed HA demonstrated complete protective immunity against homologous challenge and suboptimal protection against heterologous challenge, the latter being similar to results from inactivated whole virus vaccines. Transgenic duckweed-derived HA could be a good alternative for producing high quality antigen for an injectable vaccine against H5N1 HPAI viruses.

PB2 segment promotes high-pathogenicity of H5N1 avian influenza viruses in mice

H5N1 influenza viruses with high lethality are a continuing threat to humans and poultry. Recently, H5N1 high-pathogenicity avian influenza virus (HPAIV) has been shown to transmit through aerosols between ferrets in lab experiments by acquiring some mutation. This is another deeply aggravated threat of H5N1 HPAIV to humans. To further explore the molecular determinant of H5N1 HPAIV virulence in a mammalian model, we compared the virulence of A/Duck/Guangdong/212/2004 (DK212) and A/Quail/Guangdong/90/2004 (QL90). Though they were genetically similar, they had different pathogenicity in mice, as well as their 16 reassortants. The results indicated that a swap of the PB2 gene could dramatically decrease the virulence of rgDK212 in mice (1896-fold) but increase the virulence of rgQL90 in mice (60-fold). Furthermore, the polymerase activity assays showed that swapping PB2 genes between these two viruses significantly changed the activity of polymerase complexes in 293T cells. The mutation Ser715Asn in PB2 sharply attenuated the virulence of rgDK212 in mice (2710-fold). PB2 segment promotes high-pathogenicity of H5N1 avian influenza viruses in mice and 715 Ser in PB2 plays an important role in determining high virulence of DK212 in mice.

Impact of vaccination on infection with Vietnam H5N1 high pathogenicity avian influenza virus in hens and the eggs they lay

High pathogenicity avian influenza virus (HPAIV) infections in chickens negatively impact egg production and cause egg contamination. Previously, vaccination maintained egg production and reduced egg contamination when challenged with a North American H5N2 HPAIV. However, Asian H5N1 HPAIV infection has some characteristics of increased pathogenicity compared to other H5 HPAIV such as more rapid drop and complete cessation in egg production. Sham (vaccinated at 25 and 28 weeks of age), inactivated H5N1 Once (1X-H5-Vax; vaccinated at 28 weeks of age only) and inactivated H5N1 Twice (2X-H5-Vax; vaccinated at 25 and 28 weeks of age) vaccinated adult White Leghorn hens were challenged intranasally at 31 weeks of age with 6.1 log10 mean embryo infectious doses (EID50) of clade 2.3.2.1a H5N1 HPAIV (A/chicken/Vietnam/NCVD-675/2011) which was homologous to the inactivated vaccine. Sham-vaccinated layers experienced 100% mortality within 3 days post-challenge; laid soft and thin-shelled eggs; had recovery of virus from oral swabs and in 53% of the eggs from eggshell surface (35%), yolk (24%), and albumin (41%); and had very high titers of virus (average 7.91 log10 EID50/g) in all segments of the oviduct and ovary. By comparison, 1X- and 2X-H5-Vax challenged hens survived infection, laid similar number of eggs pre- and post-challenge, all eggs had normal egg shell quality, and had significantly fewer contaminated eggs with reduced virus quantity. The 2X-H5-Vax hens had significantly higher HI titers by the day of challenge (304 GMT) and at termination (512 GMT) than 1X-H5-Vax hens (45 GMT and 128 GMT). The current study demonstrated that AIV infections caused by clade 2.3.2.1a H5N1 variants can be effectively controlled by either double or single homologous vaccination.

Antibody titer has positive predictive value for vaccine protection against challenge with natural antigenic-drift variants of H5N1 high-pathogenicity avian influenza viruses from Indonesia.

Vaccines are used in integrated control strategies to protect poultry against H5N1 high-pathogenicity avian influenza (HPAI). H5N1 HPAI was first reported in Indonesia in 2003, and vaccination was initiated in 2004, but reports of vaccine failures began to emerge in mid-2005. This study investigated the role of Indonesian licensed vaccines, specific vaccine seed strains, and emerging variant field viruses as causes of vaccine failures. Eleven of 14 licensed vaccines contained the manufacturer's listed vaccine seed strains, but 3 vaccines contained a seed strain different from that listed on the label. Vaccines containing A/turkey/Wisconsin/1968 (WI/68), A/chicken/Mexico/28159-232/1994 (Mex/94), and A/turkey/England/N28/1973 seed strains had high serological potency in chickens (geometric mean hemagglutination inhibition [HI] titers, ≥ 1:169), but vaccines containing strain A/chicken/Guangdong/1/1996 generated by reverse genetics (rg; rgGD/96), A/chicken/Legok/2003 (Legok/03), A/chicken/Vietnam/C57/2004 generated by rg (rgVN/04), or A/chicken/Legok/2003 generated by rg (rgLegok/03) had lower serological potency (geometric mean HI titers, ≤ 1:95). In challenge studies, chickens immunized with any of the H5 avian influenza vaccines were protected against A/chicken/West Java/SMI-HAMD/2006 (SMI-HAMD/06) and were partially protected against A/chicken/Papua/TA5/2006 (Papua/06) but were not protected against A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Experimental inactivated vaccines made with PWT/06 HPAI virus or rg-generated PWT/06 low-pathogenicity avian influenza (LPAI) virus seed strains protected chickens from lethal challenge, as did a combination of a commercially available live fowl poxvirus vaccine expressing the H5 influenza virus gene and inactivated Legok/03 vaccine. These studies indicate that antigenic variants did emerge in Indonesia following widespread H5 avian influenza vaccine usage, and efficacious inactivated vaccines can be developed using antigenic variant wild-type viruses or rg-generated LPAI virus seed strains containing the hemagglutinin and neuraminidase genes of wild-type viruses. H5N1 high-pathogenicity avian influenza (HPAI) virus has become endemic in Indonesian poultry, and such poultry are the source of virus for birds and mammals, including humans. Vaccination has become a part of the poultry control strategy, but vaccine failures have occurred in the field. This study identified possible causes of vaccine failure, which included the use of an unlicensed virus seed strain and induction of low levels of protective antibody because of an insufficient quantity of vaccine antigen. However, the most important cause of vaccine failure was the appearance of drift variant field viruses that partially or completely overcame commercial vaccine-induced immunity. Furthermore, experimental vaccines using inactivated wild-type virus or reverse genetics-generated vaccines containing the hemagglutinin and neuraminidase genes of wild-type drift variant field viruses were protective. These studies indicate the need for surveillance to identify drift variant viruses in the field and update licensed vaccines when such variants appear.