H5N1 Highly Pathogenic Avian Influenza Virus Research Articles

Low Virulence and Lack of Airborne Transmission of the Dutch Highly Pathogenic Avian Influenza Virus H5N8 in Ferrets.

Highly pathogenic avian influenza (HPAI) H5N8 viruses that emerged in poultry in East Asia spread to Europe and North America by late 2014. Here we show that the European HPAI H5N8 viruses differ from the Korean and Japanese HPAI H5N8 viruses by several amino acids and that a Dutch HPAI H5N8 virus had low virulence and was not transmitted via the airborne route in ferrets. The virus did not cross-react with sera raised against pre-pandemic H5 vaccine strains. This data is useful for public health risk assessments.

Dryocrassin ABBA, a novel active substance for use against amantadine-resistant H5N1 avian influenza virus.

The occurrence of multi-drug resistant highly pathogenic avian influenza virus (HPAIV) strains highlights the urgent need for strategies for the prevention and control of avian influenza virus. The aim of our current study is to evaluate the antiviral activity of dryocrassin ABBA isolated from Rhizoma Dryopteridis Crassirhizomatis (RDC) against an amantadine-resistant H5N1 (A/Chicken/Hebei/706/2005) strain in a mouse model. Post inoculation with HPAIV H5N1 virus in mice, the survival rate was 87, 80, and 60% respectively in the 33, 18, and 12.5 mg/kg dryocrassin ABBA-treated groups. On the other hand, the survival rate was 53 and 20%, respectively in the amantadine-treated group and untreated group. Mice administered with dryocrassin ABBA or amantadine showed a significant weight increase compared to the untreated group. Moreover, 33 and 18 mg/kg dryocrassin ABBA have decreased lung index (P >0.05) and virus loads (P <0.01) compared to the untreated group on day 7. Also, on day 7 bronchoalveolar lavage fluid pro-inflammatory cytokines (IL-6, TNF-α, and IFN-γ) decreased significantly (P <0.01) while anti-inflammatory cytokines (IL-10 and MCP-1) were increased significantly (P <0.01) in the 33 and 18 mg/kg dryocrassin ABBA-treated groups compared to the amantadine group and the untreated group. Moreover, the concentrations of IL-12 in drug-treated groups were significantly (P < 0.01) lowered compared with the untreated group. Based on the above we conclude that orally administered dryocrassin ABBA provided mice protection against avian influenza virus H5N1 by inhibiting inflammation and reducing virus loads. Dryocrassin ABBA is a potential novel lead compound which had antiviral effects on amantadine-resistant avian influenza virus H5N1 infection.

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.

Genetic and antigenic characterization of H5 and H7 influenza viruses isolated from migratory water birds in Hokkaido, Japan and Mongolia from 2010 to 2014.

Migratory water birds are the natural reservoir of influenza A viruses. H5 and H7 influenza viruses are isolated over the world and also circulate among poultry in Asia. In 2010, two H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from fecal samples of water birds on the flyway of migration from Siberia, Russia to the south in Hokkaido, Japan. H7N9 viruses are sporadically isolated from humans and circulate in poultry in China. To monitor whether these viruses have spread in the wild bird population, we conducted virological surveillance of avian influenza in migratory water birds in autumn from 2010 to 2014. A total of 8103 fecal samples from migratory water birds were collected in Japan and Mongolia, and 350 influenza viruses including 13 H5 and 19 H7 influenza viruses were isolated. A phylogenetic analysis revealed that all isolates are genetically closely related to viruses circulating among wild water birds. The results of the antigenic analysis indicated that the antigenicity of viruses in wild water birds is highly stable despite their nucleotide sequence diversity but is distinct from that of HPAIVs recently isolated in Asia. The present results suggest that HPAIVs and Chinese H7N9 viruses were not predominantly circulating in migratory water birds; however, continued monitoring of H5 and H7 influenza viruses both in domestic and wild birds is recommended for the control of avian influenza.

Molecular cloning and immune responsive expression of LGP2 gene, a pivotal member of the RLR gene family from Muscovy duck Cairina moschata

Laboratory of genetics and physiology 2 (LGP2) is an important intracellular receptor that recognizes viral RNAs in innate immunity. In this study, a novel LGP2 cDNA was identified from the spleen of a Muscovy duck (Cairina moschata). The deduced amino acid sequence of Muscovy duck LGP2 (MdLGP2) consisted of 675 amino acid residues. The peptide contained two main structure domains: six important motifs, including a DExD/H box for RNA helicase activity in the RNA helicase region located at the N-terminal region, and two Zn2+-binding regions with an RNA-binding loop in the C-terminus regulatory domain (CTD). The MdLGP2 mRNA was ubiquitously expressed in the tested tissues, with high expression levels in glandular stomach, colon, ileum, crop, and caecum tissues, and low expression levels in the brain, skin, and heart. The mRNA expression of MdLGP2 was significantly upregulated in the brain, spleen, and lungs of ducks in the early stages of postinfection with H5N1 highly pathogenic avian influenza virus (HPAIV). These results suggested that MdLGP2 was involved in the early stages of antiviral innate immune response in ducks after infection with H5N1 HPAIV. However, whether it plays a positive or negative regulatory role in the host antiviral response requires further investigation.

Phylogenetic Analysis and Functional Characterization of the Influenza A H5N1 PB2 Gene.

Highly pathogenic avian influenza (HPAI) H5N1 viruses are endemic in poultry and cause continued inter-species transmission to human in Asia, such as China and Vietnam, leading to pandemic concerns and socio-economic challenges. Phylogenetic analysis of H5N1 viruses isolated from China and Vietnam during 2001-2012 showed that several geographically distinct sublineages have become established in these two countries. Subsequently, we reassigned HPAI H5N1 viruses into three distinct groups to reveal the intrasubtype reassortment. Apart from six reassortants detected here, we found that several viral strains showed signals for homologous recombination within PB2 and PB1 genes, suggestive of the fluidity of the H5N1 virus gene pool. Furthermore, sequenced-based analyses revealed that the viral polymerase displayed a higher level of genetic polymorphism but associated with lower substitution rate when compared with those of other gene segments. In addition, the selection pressure analysis indicated that purifying selection was predominant in eight genomic segments especially in the polymerase complex. However, the site-by-site analysis helped to detect 14 positively selected sites in the PB1, PA, HA, NA, MP and NS proteins. Despite the fact that PB2 protein of H5N1 viruses was highly conserved at the amino acid level, eleven adaptive mutations were still observed in the protein. Further comparative structural analysis of the K627E mutation indicated that there were no structural differences between the variants, which possessed either PB2-627E or PB2-627K. Transcriptomic analysis suggested the non-mitochondrial PB2 protein of H5N1 virus that forms a stable complex with the mitochondrial antiviral signalling protein (MAVS, also known as IPS-1, VISA or Cardif) can induce interferon-beta (IFN-β) expression, but the substitution (PB2-K627E) is not the sole determinant of the RIG-I-like receptors (RLR) signalling components induction in Calu-3 cells.

Susceptibility to and transmission of H5N1 and H7N1 highly pathogenic avian influenza viruses in bank voles (Myodes glareolus).

The study of influenza type A (IA) infections in wild mammals populations is a critical gap in our knowledge of how IA viruses evolve in novel hosts that could be in close contact with avian reservoir species and other wild animals. The aim of this study was to evaluate the susceptibility to infection, the nasal shedding and the transmissibility of the H7N1 and H5N1 highly pathogenic avian influenza (HPAI) viruses in the bank vole (Myodes glareolus), a wild rodent common throughout Europe and Asia. Two out of 24 H5N1-infected voles displayed evident respiratory distress, while H7N1-infected voles remained asymptomatic. Viable virus was isolated from nasal washes collected from animals infected with both HPAI viruses, and extra-pulmonary infection was confirmed in both experimental groups. Histopathological lesions were evident in the respiratory tract of infected animals, although immunohistochemistry positivity was only detected in lungs and trachea of two H7N1-infected voles. Both HPAI viruses were transmitted by direct contact, and seroconversion was confirmed in 50% and 12.5% of the asymptomatic sentinels in the H7N1 and H5N1 groups, respectively. Interestingly, viable virus was isolated from lungs and nasal washes collected from contact sentinels of both groups. The present study demonstrated that two non-rodent adapted HPAI viruses caused asymptomatic infection in bank voles, which shed high amounts of the viruses and were able to infect contact voles. Further investigations are needed to determine whether bank voles could be involved as silent hosts in the transmission of HPAI viruses to other mammals and domestic poultry.

Pathogenicity of Highly Pathogenic Avian Influenza Virus H5N1 in Naturally Infected Poultry in Egypt.

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been endemic in Egypt since 2006, and there is increasing concern for its potential to become highly transmissible among humans. Infection by HPAIV H5N1 has been described in experimentally challenged birds. However, the pathogenicity of the H5N1 isolated in Egypt has never been reported in naturally infected chickens and ducks. Here we report a 2013 outbreak of HPAIV H5N1 in commercial poultry farms and backyards in Sharkia Province, Egypt. The main symptoms were ecchymosis on the shanks and feet, cyanosis of the comb and wattles, subcutaneous edema of the head and neck for chickens, and nervous signs (torticollis) for ducks. Within 48-72 hrs of the onset of illness, the average mortality rates were 22.8-30% and 28.5-40% in vaccinated chickens and non-vaccinated ducks, respectively. Tissue samples of chickens and ducks were collected for analyses with cross-section immunohistochemistry and real-time RT-PCR for specific viral RNA transcripts. While viral RNA was detected in nearly all tissues and sera collected, viral nucleoprotein was detected almost ubiquitously in all tissues, including testis. Interestingly, viral antigen was also observed in endothelial cells of most organs in chickens, and clearly detected in the trachea and brain in particular. Viral nucleoprotein was also detected in mononuclear cells of various organs, especially pulmonary tissue. We performed phylogenetic analyses and compared the genomic sequences of the hemagglutinin (HA) and nonstructural proteins (NS) among the isolated viruses, the HPAIV circulated in Egypt in the past and currently, and some available vaccine strains. Further analysis of deduced amino acids of both HA and NS1 revealed that our isolates carried molecular determinants of HPAIV, including the multibasic amino acids (PQGERRRK/KR*GLF) in the cleavage site in HA and glutamate at position 92 (D92E) in NS1. This is the first report of the pathogenicity of the HPAIVH5N1 strain currently circulating in naturally infected poultry in Egypt, which may provide unique insights into the viral pathogenesis in HPAIV-infected chickens and ducks.

Detection prevalence of H5N1 avian influenza virus among stray cats in eastern China.

Since 1997, more and more cases of the infectious H5N1 avian influenza virus (AIV) in humans have been reported all over the world but the transmission of H5N1 avian influenza virus to stray cats has been little demonstrated. The objective of this pilot investigation was to determine the prevalence of H5N1 AIV antibodies in stray cats in eastern China where is the dominant enzootic H5N1 highly pathogenic avian influenza virus (HP AIV). A total of 1,020 nasal swab and 1,020 serum samples were collected and tested. Evidence of HPAI H5N1 virus antibodies was present in two of the 1,020 serum samples that were positive by HI assay and NT assay, respectively. The results imply little transmission and that the Clade 2.3.2 HPAIV H5N1 infections in poultry did not significantly affect the rural animal shelters or suburban environment in eastern China. In future studies, these results can be used as baseline seroepidemiological levels for H5N1 AIV among cats in China.

Highly Pathogenic Avian Influenza H5N1 in Mainland China.

Highly pathogenic avian influenza (HPAI) H5N1 has posed a significant threat to both humans and birds, and it has spanned large geographic areas and various ecological systems throughout Asia, Europe and Africa, but especially in mainland China. Great efforts in control and prevention of the disease, including universal vaccination campaigns in poultry and active serological and virological surveillance, have been undertaken in mainland China since the beginning of 2006. In this study, we aim to characterize the spatial and temporal patterns of HPAI H5N1, and identify influencing factors favoring the occurrence of HPAI H5N1 outbreaks in poultry in mainland China. Our study shows that HPAI H5N1 outbreaks took place sporadically after vaccination campaigns in poultry, and mostly occurred in the cold season. The positive tests in routine virological surveillance of HPAI H5N1 virus in chicken, duck, goose as well as environmental samples were mapped to display the potential risk distribution of the virus. Southern China had a higher positive rate than northern China, and positive samples were mostly detected from chickens in the north, while the majority were from duck in the south, and a negative correlation with monthly vaccination rates in domestic poultry was found (R = −0.19, p value = 0.005). Multivariate panel logistic regression identified vaccination rate, interaction between distance to the nearest city and national highway, interaction between distance to the nearest lake and wetland, and density of human population, as well as the autoregressive term in space and time as independent risk factors in the occurrence of HPAI H5N1 outbreaks, based on which a predicted risk map of the disease was derived. Our findings could provide new understanding of the distribution and transmission of HPAI H5N1 in mainland China and could be used to inform targeted surveillance and control efforts in both human and poultry populations to reduce the risk of future infections.

The Clinical Potential of Passive Immunization with Therapeutic Antibodies: Focus on Highly Pathogenic Avian Influenza Virus Infection

ABSTRACT The H5N1 highly pathogenic avian influenza (HPAI) virus has been reported to infect humans and posing a significant pandemic threat. Although neuraminidase inhibitors are currently available for the treatment of human influenza, alternative strategies need to be developed for the treatment of H5N1 HPAI virus infection in humans due to the appearance of drug-resistant viruses. Recently, passive immunization with virus-specific monoclonal antibodies has been tested for H5N1 HPAI virus infection in animal models, providing evidence that antibody therapy may be a promising option for prophylaxis or treatment of this infectious disease. Here we discuss potential benefits and limitations of antibody therapy in clinical cases of H5N1 virus infection in humans.

The East Jakarta Project: surveillance for highly pathogenic avian influenza A(H5N1) and seasonal influenza viruses in patients seeking care for respiratory disease, Jakarta, Indonesia, October 2011-September 2012.

Indonesia has reported the most human infections with highly pathogenic avian influenza (HPAI) A(H5N1) virus worldwide. We implemented enhanced surveillance in four outpatient clinics and six hospitals for HPAI H5N1 and seasonal influenza viruses in East Jakarta district to assess the public health impact of influenza in Indonesia. Epidemiological and clinical data were collected from outpatients with influenza-like illness (ILI) and hospitalized patients with severe acute respiratory infection (SARI); respiratory specimens were obtained for influenza testing by real-time reverse transcription-polymerase chain reaction. During October 2011-September 2012, 1131/3278 specimens from ILI cases (34·5%) and 276/1787 specimens from SARI cases (15·4%) tested positive for seasonal influenza viruses. The prevalence of influenza virus infections was highest during December-May and the proportion testing positive was 76% for ILI and 36% for SARI during their respective weeks of peak activity. No HPAI H5N1 virus infections were identified, including hundreds of ILI and SARI patients with recent poultry exposures, whereas seasonal influenza was an important contributor to acute respiratory disease in East Jakarta. Overall, 668 (47%) of influenza viruses were influenza B, 384 (27%) were A(H1N1)pdm09, and 359 (25%) were H3. While additional data over multiple years are needed, our findings suggest that seasonal influenza prevention efforts, including influenza vaccination, should target the months preceding the rainy season.

SiRNAs targeting PB2 and NP genes potentially inhibit replication of Highly Pathogenic H5N1 Avian Influenza Virus.

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is a threat to animal and public health worldwide. Till date, the H5N1 virus has claimed 402 human lives, with a mortality rate of 58 percent and has caused the death or culling of millions of poultry since 2003. In this study, we have designed three siRNAs (PB2-2235, PB2-479 and NP-865) targeting PB2 and NP genes of avian influenza virus and evaluated their potential, measured by hemagglutination (HA), plaque reduction and Real time RT-PCR assay, in inhibiting H5N1 virus (A/chicken/Navapur/7972/2006) replication in MDCK cells. The siRNAs caused 8- to 16-fold reduction in virus HA titers at 24 h after challenged with 100TCID50 of virus. Among these siRNAs, PB2-2235 offered the highest inhibition of virus replication with 16-fold reduction in virus HA titer, 80 percent reduction in viral plaque counts and 94 percent inhibition in expression of specific RNA at 24 h. The other two siRNAs had 68-73 percent and 87-88 percent reduction in viral plaque counts and RNA copy number, respectively. The effect of siRNA on H5N1 virus replication continued till 48h (maximum observation period). These findings suggest that PB2-2235 could efficiently inhibit HPAI H5N1 virus replication.

Efficient strategy for constructing duck enteritis virus-based live attenuated vaccine against homologous and heterologous H5N1 avian influenza virus and duck enteritis virus infection.

Duck is susceptible to many pathogens, such as duck hepatitis virus, duck enteritis virus (DEV), duck tembusu virus, H5N1 highly pathogenic avian influenza virus (HPAIV) in particular. With the significant role of duck in the evolution of H5N1 HPAIV, control and eradication of H5N1 HPAIV in duck through vaccine immunization is considered an effective method in minimizing the threat of a pandemic outbreak. Consequently, a practical strategy to construct a vaccine against these pathogens should be determined. In this study, the DEV was examined as a candidate vaccine vector to deliver the hemagglutinin (HA) gene of H5N1, and its potential as a polyvalent vaccine was evaluated. A modified mini-F vector was inserted into the gB and UL26 gene junction of the attenuated DEV vaccine strain C-KCE genome to generate an infectious bacterial artificial chromosome (BAC) of C-KCE (vBAC-C-KCE). The HA gene of A/duck/Hubei/xn/2007 (H5N1) was inserted into the C-KCE genome via the mating-assisted genetically integrated cloning (MAGIC) to generate the recombinant vector pBAC-C-KCE-HA. A bivalent vaccine C-KCE-HA was developed by eliminating the BAC backbone. Ducks immunized with C-KCE-HA induced both the cross-reactive antibodies and T cell response against H5. Moreover, C-KCE-HA-immunized ducks provided rapid and long-lasting protection against homologous and heterologous HPAIV H5N1 and DEV clinical signs, death, and primary viral replication. In conclusion, our BAC-C-KCE is a promising platform for developing a polyvalent live attenuated vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0174-3) contains supplementary material, which is available to authorized users.

Wild bird surveillance around outbreaks of highly pathogenic avian influenza A(H5N8) virus in the Netherlands, 2014, within the context of global flyways.

Highly pathogenic avian influenza (HPAI) A(H5N8) viruses that emerged in poultry in east Asia since 2010 spread to Europe and North America by late 2014. Despite detections in migrating birds, the role of free-living wild birds in the global dispersal of H5N8 virus is unclear. Here, wild bird sampling activities in response to the H5N8 virus outbreaks in poultry in the Netherlands are summarised along with a review on ring recoveries. HPAI H5N8 virus was detected exclusively in two samples from ducks of the Eurasian wigeon species, among 4,018 birds sampled within a three months period from mid-November 2014. The H5N8 viruses isolated from wild birds in the Netherlands were genetically closely related to and had the same gene constellation as H5N8 viruses detected elsewhere in Europe, in Asia and in North America, suggesting a common origin. Ring recoveries of migratory duck species from which H5N8 viruses have been isolated overall provide evidence for indirect migratory connections between East Asia and Western Europe and between East Asia and North America. This study is useful for better understanding the role of wild birds in the global epidemiology of H5N8 viruses. The need for sampling large numbers of wild birds for the detection of H5N8 virus and H5N8-virus-specific antibodies in a variety of species globally is highlighted, with specific emphasis in north-eastern Europe, Russia and northern China.

Pathological Evaluation of Natural Cases of a Highly Pathogenic Avian Influenza Virus, Subtype H5N8, in Broiler Breeders and Commercial Layers in South Korea.

Outbreaks of highly pathogenic avian influenza (HPAI) virus, subtype H5N8, were observed in two different flocks of local broiler breeder farms and a commercial layer farm in South Korea. Clinically, the cases were characterized by a gradual increase in mortality, slow transmission, and unrecognizable clinical signs of HPAI. Gross observations in both cases included hemorrhagic or necrotic lesions in internal organs, such as serosal and mucosal membranes, spleen, and pancreas. Both cases exhibited similar histopathologic lesions, including multifocal malacia in the brain and multifocal or diffuse necrosis in the spleen and pancreas. Immunohistochemical results indicated that neurons and glial cells in the brain, myocytes in the heart, acinar cells in the pancreas, and mononuclear phagocytic cells in several visceral organs were immunopositive for avian influenza viral antigen. To experimentally reproduce the low pathogenicity and the mortality observed in these two cases, 18 specific-pathogen-free chickens and 18 commercial layers were divided into an H5N8 virus-inoculated group and a contact-exposed group. The mortality of the chickens in the inoculation group was 50%-100%, whereas the mean time to death was delayed or death did not occur in the contact-exposed group. The distributions of the viral antigens and histopathologic lesions in the experimental study were similar to those observed in the field cases. These findings suggest that the H5N8 virus induces a different pattern of pathobiology, including slow transmission and low mortality, compared with that of other HPAI viruses. This is the first pathologic description of natural cases of H5N8 in South Korea, and it may be helpful in understanding the pathobiology of novel H5N8 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.

PA-X decreases the pathogenicity of highly pathogenic H5N1 influenza A virus in avian species by inhibiting virus replication and host response.

PA-X is a newly discovered protein that decreases the virulence of the 1918 H1N1 virus in a mouse model. However, the role of PA-X in the pathogenesis of highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype in avian species is totally unknown. By generating two PA-X-deficient viruses and evaluating their virulence in different animal models, we show here that PA-X diminishes the virulence of the HPAIV H5N1 strain A/Chicken/Jiangsu/k0402/2010 (CK10) in mice, chickens, and ducks. Expression of PA-X dampens polymerase activity and virus replication both in vitro and in vivo. Using microarray analysis, we found that PA-X blunts the global host response in chicken lungs, markedly downregulating genes associated with the inflammatory and cell death responses. Correspondingly, a decreased cytokine response was recapitulated in multiple organs of chickens and ducks infected with the wild-type virus relative to those infected with the PA-X-deficient virus. In addition, the PA-X protein exhibits antiapoptotic activity in chicken and duck embryo fibroblasts. Thus, our results demonstrated that PA-X acts as a negative virulence regulator and decreases virulence by inhibiting viral replication and the host innate immune response. Therefore, we here define the role of PA-X in the pathogenicity of H5N1 HPAIV, furthering our understanding of the intricate pathogenesis of influenza A virus. Influenza A virus (IAV) continues to pose a huge threat to global public health. Eight gene segments of the IAV genome encode as many as 17 proteins, including 8 main viral proteins and 9 accessory proteins. The presence of these accessory proteins may further complicate the pathogenesis of IAV. PA-X is a newly identified protein in segment 3 that acts to decrease the virulence of the 1918 H1N1 virus in mice by modulating host gene expression. Our study extends these functions of PA-X to H5N1 HPAIV. We demonstrated that loss of PA-X expression increases the virulence and replication of an H5N1 virus in mice and avian species and alters the host innate immune and cell death responses. Our report is the first to delineate the role of the novel PA-X protein in the pathogenesis of H5N1 viruses in avian species and promotes our understanding of H5N1 HPAIV.

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.