H5N1 Avian Influenza Virus Research Articles

The PB1 gene from H9N2 avian influenza virus showed high compatibility and increased mutation rate after reassorting with a human H1N1 influenza virus

BackgroundReassortment between human and avian influenza viruses (AIV) may result in novel viruses with new characteristics that may threaten human health when causing the next flu pandemic. A particular risk may be posed by avian influenza viruses of subtype H9N2 that are currently massively circulating in domestic poultry in Asia and have been shown to infect humans. In this study, we investigate the characteristics and compatibility of a human H1N1 virus with avian H9N2 derived genes.MethodsThe polymerase activity of the viral ribonucleoprotein (RNP) complex as combinations of polymerase-related gene segments derived from different reassortment events was tested in luciferase reporter assays. Reassortant viruses were generated by reverse genetics. Gene segments of the human WSN-H1N1 virus (A/WSN/1933) were replaced by gene segments of the avian A2093-H9N2 virus (A/chicken/Jiangsu/A2093/2011), which were both the Hemagglutinin (HA) and Neuraminidase (NA) gene segments in combination with one of the genes involved in the RNP complex (either PB2, PB1, PA or NP). The growth kinetics and virulence of reassortant viruses were tested on cell lines and mice. The reassortant viruses were then passaged for five generations in MDCK cells and mice lungs. The HA gene of progeny viruses from different passaging paths was analyzed using Next-Generation Sequencing (NGS).ResultsWe discovered that the avian PB1 gene of H9N2 increased the polymerase activity of the RNP complex in backbone of H1N1. Reassortant viruses were able to replicate in MDCK and DF1 cells and mice. Analysis of the NGS data showed a higher substitution rate for the PB1-reassortant virus. In particular, for the PB1-reassortant virus, increased virulence for mice was measured by increased body weight loss after infection in mice.ConclusionsThe higher polymerase activity and increased mutation frequency measured for the PB1-reassortant virus suggests that the avian PB1 gene of H9N2 may drive the evolution and adaptation of reassortant viruses to the human host. This study provides novel insights in the characteristics of viruses that may arise by reassortment of human and avian influenza viruses. Surveillance for infections with H9N2 viruses and the emergence of the reassortant viruses in humans is important for pandemic preparedness.

Hen raising helps chicks establish gut microbiota in their early life and improve microbiota stability after H9N2 challenge

BackgroundEarly gut microbial colonization is important for postnatal growth and immune development of the chicken. However, at present, commercial chickens are hatched and raised without adult hens, thus are cut off from the microbiota transfer between hens and chicks. In this study, we compared the gut microbiota composition between hen-reared and separately reared chicks, and its impact on the resistance to H9N2 avian influenza virus, with the motive of investigating the impact of this cutoff in microbiota transfer.ResultsWe used the 16SrRNA sequencing method to assess the composition of the gut microbiota in chicks represented by three hen-reared groups and one separately reared group. We found that the diversity of gut microbes in the chicks from the three hen-reared groups was more abundant than in the separately reared group, both at the phylum and genus levels. Our findings highlight the importance of early parental care in influencing the establishment of gut microbiota in the early life of chicks. SourceTracker analysis showed that the feather and cloaca microbiota of hens are the main sources of gut microbiota of chicks. After H9N2 exposure, the viral infection lasted longer in the separately reared chicks, with the viral titers in their oropharyngeal swabs being higher compared to the hen-reared chicks at day 5 post-infection. Interestingly, our results revealed that the gut microbiota of the hen-reared chicks was more stable after H9N2 infection in comparison to that of the separately reared chicks.ConclusionsMicrobiota transfer between the hens and their chicks promotes the establishment of a balanced and diverse microbiota in the early life of the chicks and improves microbiota stability after H9N2 challenge. These findings advance our understanding of the protective role of gut microbiota in the early life of chicks and should be instrumental in improving chick rearing in the commercial poultry industry.9rWXktHvFNPuqpjj52kMkpVideo

Detection of Highly Pathogenic Avian Influenza Virus H5N1 Clade 2.3.4.4b in Great Skuas: A Species of Conservation Concern in Great Britain.

The UK and Europe have seen successive outbreaks of highly pathogenic avian influenza across the 2020/21 and 2021/22 autumn/winter seasons. Understanding both the epidemiology and transmission of these viruses in different species is critical to aid mitigating measures where outbreaks cause extensive mortalities in both land- and waterfowl. Infection of different species can result in mild or asymptomatic outcomes, or acute infections that result in high morbidity and mortality levels. Definition of disease outcome in different species is of great importance to understanding the role different species play in the maintenance and transmission of these pathogens. Further, the infection of species that have conservation value is also important to recognise and characterise to understand the impact on what might be limited wild populations. Highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b has been detected in great skuas (Stercorarius skua) across different colonies on islands off the shore of Scotland, Great Britain during summer 2021. A large number of great skuas were observed as developing severe clinical disease and dying during the epizootic and mortalities were estimated to be high where monitored. Of eight skuas submitted for post-mortem examination, seven were confirmed as being infected with this virus using a range of diagnostic assays. Here we overview the outbreak event that occurred in this species, listed as species of conservation concern in Great Britain and outline the importance of this finding with respect to virus transmission and maintenance.

Molecular and Antigenic Characterization of Avian H9N2 Viruses in Southern China.

ABSTRACTThe H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in BJ94 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 × 10−3 substitutions/site/year, which was 1.5-fold faster than that of the average H9N2 HA rate (3.95 × 10−3 substitutions/site/year). Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses.IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry (especially in chicken in South Korea and China). Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 AIVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.

Expression and characterization of a recombinant broadly-reactive monoclonal antibody against group 1 and 2 influenza viruses

Production of broadly-reactive antibodies is critical for universal immunodiagnosis of rapidly-evolving influenza viruses. Most monoclonal antibodies (mAbs) are generated in mice using the hybridoma technology which involves labor- and time-consuming screening and low yield issues. In this study, a recombinant antibody based on a broadly-reactive mAb against the hemagglutinin (HA) stalk of H7N9 avian influenza virus was expressed in CHO cells and its biological characteristics, cross-reactivity and epitope recognition were identified. The variable genes of the parental antibody were amplified and cloned into the antibody-expressing plasmids containing the constant genes of murine IgG1. The recombinant antibody was expressed in high yield and purity in CHO cells and showed similar features to the parental antibody, including negative hemagglutination inhibition activity against H7N9 virus and high binding activity with the H7N9 HA protein. Notably, the recombinant antibody exhibited a broad reactivity with different influenza subtypes belonging to group 1 and group 2, which was associated with its recognition of a highly-conserved epitope in the stalk, as observed for the parental antibody. Our results suggest that cell-based antibody expression system can be utilized as an important alternative to the hybridoma technology for antibody production for influenza virus diagnostics.

The highly pathogenic H5N1 avian influenza virus induces the mitogen-activated protein kinase signaling pathway in the trachea of two Ri chicken lines.

ObjectiveThe highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the mitogen-activated protein kinase (MAPK) signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines.MethodsResistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, the tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing.ResultsA gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. A total of 1,794 DEGs were observed between control and H5N1-infected resistant Ri chickens, 432 DEGs between control and infected susceptible Ri chickens, and 1,202 DEGs between infected susceptible and infected resistant Ri chickens. The expression levels of MAPK signaling pathway-related genes (including MyD88, NF-κB, AP-1, c-fos, Jun, JunD, MAX, c-Myc), cytokines (IL-1β, IL-6, IL-8), type I interferons (IFN-α, IFN-β), and IFN-stimulated genes (Mx1, CCL19, OASL, and PRK) were higher in H5N1-infected than in non-infected resistant Ri chickens. MyD88, Jun, JunD, MAX, cytokines, chemokines, IFNs, and IFN-stimulated expressed genes were higher in resistant-infected than in susceptible-infected Ri chickens.ConclusionResistant Ri chickens showed higher antiviral activity compared to susceptible Ri chickens, and H5N1-infected resistant Ri chickens had immune responses and antiviral activity (cytokines, chemokines, interferons, and IFN-stimulated genes), which may have been induced through the MAPK signaling pathway in response to H5N1 infection.

Isolation and Identification of a Highly Pathogenic Avian Influenza H5N6 Virus from Migratory Waterfowl in Western Mongolia.

In April 2020, two Whooper Swans (Cygnus cygnus) and one Swan Goose (Anser cygnoides) were found dead at three different locations in western Mongolia. Virus isolation from organs taken from the carcasses and full genome sequencing revealed that all three birds were positive for highly pathogenic H5N6 avian influenza virus (HPAIV) belonging to subclade 2.3.4.4h. Confirming similar reports from central Mongolia and western China, these findings have important implications for the monitoring, control, and management of HPAIVs in wild bird and commercial poultry populations in Mongolia.

Poultry to Human Passport: Cross-species Transmission of Zoonotic H7N9 Avian Influenza Virus to Humans

Human infections with H7N9 avian influenza virus were first reported in the early spring of 2013, in the Yangtze-delta region of China. This virus subsequently caused five successive epidemic waves from 2013 to 2018 with highest reported cases in the last wave making this strain the most successful zoonosis influenza virus in humans in recent decades. No H7N9 human infections have been reported since 2019, probably because of the extensive vaccination of poultry. Although zoonoses of H7N9 and other subtypes of avian influenza viral infections remain rare, the virus could acquire sufficient mammalian adaptive mutations to allow it to cause a future influenza pandemic. Here, we summarize the main findings on viral and host factors affecting the interspecies transmission of the H7N9 avian influenza virus.

A H9N2 Human Case and Surveillance of Avian Influenza Viruses in Live Poultry Markets - Huizhou City, Guangdong Province, China, 2021.

What is already known about this topic?An increasing number of human infected avian influenza A (H9N2) cases have been reported. In 2021, 11 human infections with influenza A virus subtype H9N2 (A/H9N2) have been reported in China.What is added by this report?A new case of H9N2 that occurred in April 2021 in Huizhou City, Guangdong Province, China, was reported in this study. Epidemiological and laboratory information of the case and routine influenza surveillance data of avian influenza A were presented in this report.What are the implications for public health practice?The emergence of a human infected with Avian Influenza Virus H9N2 demonstrates that there is an urgent need to strengthen the surveillance of influenza-like illness and live poultry market.

Epizootic, Endemic and Pandemic Zoonotic Viral Infections

More than 60% human infectious diseases have zoonotic origin. Cross species transmission of pathogens is a continuous, dynamic process that occurs throughout the world, giving rise to epizootic (temporary, limited infection), endemic (on-going infection limited to a defined geographic region) and pandemic viral infections (infection spreading to every part of the world) like the current COVID-19 pandemic, which depends on the existing conditions on the ground. In Nov 2021, sudden mortality of numerous migrating demoiselle cranes was reported from their resting site near Jodhpur, Rajasthan. The symptomatic cranes became gradually weak and were unable to fly. They eventually fell dead which caused concern locally, given the current prevailing SARS-CoV-2 pandemic situation the world over. By the end of Dec 2021, the number of cranes with mortality and morbidity reduced, making it a temporary “epizootic infection”. Molecular diagnosis carried out at a specialized laboratory identified the etiological agent to be the highly pathogenic Avian Influenza Virus H5N1 (HPAIV), which has been responsible for morbidity of avian species from different parts of the world. There was no report of spreading the H5N1 AIV infection from the infected migratory cranes to nearby chicken farms or pig farms for now. In the absence of vaccines against the highly pathogenic H5N1 AIVs, and the inherent ability of influenza viruses, both avian AIV and human IAVs to constantly mutate its envelope gene or the surface antigens, resulting from the error-prone nature of the viral RNA Polymerase enzyme are the roadblocks for development of a universal, broad-spectrum influenza vaccine. Even when such a universal vaccine against H5N1 is available, vaccinating a large number of wild migratory cranes would be difficult. However, it is possible and indeed necessary to vaccinate chickens in poultry farms and pigs in farms that raise pigs for human consumption.

Immunogenicity and Cross-Protective Efficacy of Recombinant H5ha1 Protein of Clade 2.3.2.1a Highly Pathogenic H5n1 Avian Influenza Virus Expressed in E.Coli

Immunogenicity and Cross-Protective Efficacy of Recombinant H5ha1 Protein of Clade 2.3.2.1a Highly Pathogenic H5n1 Avian Influenza Virus Expressed in E.Coli

Molecular Detection of Avian Influenza Virus in Wild Birds in Morocco, 2016-2019.

Avian influenza (AI) is a zoonotic disease significant to both public and animal health, caused by influenza virus A, and affects domestic poultry, wild birds, and mammals including humans. Aquatic birds are considered the natural reservoir of this virus. In 2016, Morocco experienced the first occurrence of low pathogenic H9N2 avian influenza virus (AIV) in poultry; however, no cases were reported in wild birds. The present study aimed to monitor the presence of AIV in wild birds in Morocco in order to trace the possible sources of the viruses affecting poultry. Between 2016 and 2019, 967 samples obtained from 480 birds representing 56 different wild bird species, 20 families, and 8 orders, mostly from Charadriiformes, Anseriformes, Pelecaniformes, and Passeriformes, were collected from various wetlands and relevant ornithologic sites in Morocco. These field samples consisted of 374 cloacal swabs, 321 tracheal swabs, 54 fecal samples, and 218 organ pools including the trachea, lung, liver, spleen, heart, intestine, and brain. The samples were examined for the presence of AIV using TaqMan-based real-time reverse transcriptase-PCR (rRT-PCR) targeting the matrix gene, followed by further subtyping rRT-PCR tests targeting the H1-H16 genes. The AI matrix gene was detected in 18 out of 967 samples (1.86%); positive samples were detected in 17 birds belonging to 10 bird species: two redshanks (Tringa totanus), one little stint (Calidris minuta), one ruddy turnstone (Arenaria interpres), one common snipe (Gallinago gallinago), one common greenshank (Tringa nebularia), one black-winged stilt (Himantopus himantopus), two black-headed gulls (Chroicocephalus ridibundus), one slender-billed gull (Chroicocephalus genei), six cattle egrets (Bubulcus ibis), and one Eurasian coot (Fulica atra). AIV was detected in 2 wetlands and 1 ornithologic site (Sidi Moussa Oualidia Complex, Smir lagoon and El Jadida Coast) and the highest positivity was revealed in fresh fecal samples (11.1%), indicating the suitability of this matrix for wild bird surveillance. Our results highlight that waders, gulls, and cattle egrets are the most affected species and may represent a potential risk for AI introduction in the poultry sector in Morocco. Regular monitoring of wild birds in Morocco, focusing in particular in the areas and species identified in this study as a high risk of virus circulation, should be implemented to anticipate and prevent possible AIV spread.

Molecular Characterization of Avian Influenza H9N2 and H5N1 from Outbreak in a Backyard Layer Farm in Kaduna State, Nigeria

Avian influenza viruses (AIVs) have been reported in Nigeria since 2006, and the co-circulation of high-pathogenic avian influenza H5N1 and low-pathogenic avian influenza H9N2 subtypes in poultry has been reported. These Avian Influenza virus subtypes could lead to severe economic losses to the poultry farmers and may force the farmers out of the poultry production leading to unemployment for the staff of the farm. The co-infection with both highly pathogenic (H5N1) and low pathogenic (H9N2) could lead to possible re-assortment and the production of novel strain that can be highly pathogenic to chicken and may have the capacity to infect humans and be transmitted from human to humans which may lead to a pandemic of influenza. The clinical signs observed were somnolence, marginal cyanosis of the combs, ruffled feathers, matted vent and whitish/yellowish diarrhoea; while the post mortem examinations revealed dehydrated carcass, multiple foci of petechial haemorrhages on the abdominal fats, congested and necrotic-friable liver, edematous and congested lungs, congested spleen with multiple foci of necrosis, hemorrhagic and misshaped ovarian follicles, egg yolk peritonitis, swollen and hemorrhagic kidneys, erosion of the proventricular mucosa, congested trachea with mucoid exudates, and sinusitis. The disease was diagnosed based on the clinical signs, post mortem findings, competitive Enzyme-Linked Immunosorbent Assays (ELISAs) and clade-specific RT-qPCR, for the detection of co-circulating highly pathogenic avian influenza virus H5N1 and low pathogenic H9N2. The result of all sera (5/5) tested for Antibodies against Avian influenza Nucleoprotein produced positive results while 80 % of sera (4/5) tested positive for the H9 subtype of AIV. Antibodies against the H5 and H7 subtypes were not detected. The results of RRT-PCR and RRTPCR/ H5 were positive for both tracheal and cloacal samples. RRT-PCR/ H7 & H9 yielded negative results.

Seroprevalence and detection of H5N1 avian influenza virus in local chickens in Tabanan Regency, Bali, Indonesia

Aim: Avian Influenza (AI) is a zoonotic disease that causes death in poultry and humans. Monitoring the virus needs to be carried out continuously to prevent outbreaks of the disease. Seroprevalence and detection of H5N1 and H9N2 AI virus antigen were intended to monitor the presence of viruses in local chickens in Tabanan, a Regency of the Indonesian island Province of Bali. The research aims were to detect the presence of H5N1 AI virus, and to know the distribution of this virus in Tabanan Regency. Materials and Methods: Research located in six districts of Tabanan regency namely Baturiti, Penebel, Marga, Kediri, Tabanan, and Kerambitan. A total of 1,398 local chickens that never been vaccinated with AI were randomly sampled in this study. The samples collected were serum, cloacal and tracheal swabs. Serum samples were tested with hemagglutination inhibition (HI) assay. While samples of cloacal and tracheal swabs were isolated in 9-day-old germinated chicken eggs, followed by hemagglutination assay and RT-PCR test using H5N1 and H9N2 primers. Results: AI seroprevalence in local chickens in Tabanan Regency was 1% with the distribution in each district as follows; Penebel 1.6%, Kerambitan 1.2%, Marga 1%, while Tabanan, Kediri, and Baturiti 0.7% each. H5N1 AI virus was detected in 11 samples, i.e. five in Marga district and three in Penebel district, two in Kediri, and one in Tabanan, while the H9N2 AI virus was not detected. Conclusion: These results indicate that H5N1 AI virus may still circulate in local chickens in Tabanan Regency, Bali.

Spatiotemporal Associations and Molecular Evolution of Highly Pathogenic Avian Influenza A H7N9 Virus in China from 2017 to 2021.

Highly pathogenic (HP) H7N9 avian influenza virus (AIV) emerged in China in 2016. HP H7N9 AIV caused at least 33 human infections and has been circulating in poultry farms continuously since wave 5. The genetic divergence, geographic patterns, and hemagglutinin adaptive and parallel molecular evolution of HP H7N9 AIV in China since 2017 are still unclear. Here, 10 new strains of HP H7N9 AIVs from October 2019 to April 2021 were sequenced. We found that HP H7N9 was primarily circulating in Northern China, particularly in the provinces surrounding the Bohai Sea (Liaoning, Hebei, and Shandong) since wave 6. Of note, HP H7N9 AIV phylogenies exhibit a geographical structure compatible with high levels of local transmission after unidirectional rapid geographical expansion towards the north of China in 2017. In addition, we showed that two major subclades were continually expanding with the viral population size undergoing a sharp increase after 2018 with an obvious seasonal tendency. Notably, the hemagglutinin gene showed signs of parallel evolution and positive selection. Our research sheds light on the current epidemiology, evolution, and diversity of HP H7N9 AIV that can help prevent and control the spreading of HP H7N9 AIV.

Baculovirus-derived influenza virus-like particle confers complete protection against lethal H7N9 avian influenza virus challenge in chickens and mice

Currently, highly pathogenic avian influenza (HPAI) H7N9 viruses still pose a potential pandemic threat. Influenza virus-like particle (VLP) is one of the most promising vaccine strategies to complement traditional egg-dependent vaccines. Here, we generated a H7N9 VLP vaccine candidate by baculovirus expression system and evaluated its efficacy in chickens and mice. The H7N9 VLP was produced through co-infection of Sf9 insect cells with three recombinant baculoviruses expressing individual HA, NA and M1 gene of the HPAI H7N9 virus A/chicken/Guangdong/GD15/2016. Intramuscular immunization of the H7N9 VLP elicited robust antibody immune responses and conferred complete clinical protection against lethal H7N9 virus challenge both in chickens and mice. Meanwhile, H7N9 VLP significantly restrained virus shedding and dramatically alleviated pulmonary lesions caused by H7N9 virus infection in birds and mice. Interestingly, chicken antibodies induced by the H7N9 VLP also had a good cross-reactivity with H7N9 field strains isolated in different years. In addition, vaccination with the H7N9 VLP elicited high T cell immunity in mouse lung, evidenced by significantly upregulated expression of IL-2, IL-4 and IFN-γ. Furthermore, the H7N9 VLP significantly decreased the expression of some key inflammatory cytokines, such as IL6, RANTES and TNF-α in mouse lung, which may partially account for its contribution to alleviate lung pathology. Therefore, our study describes the good efficacy of the HA + NA + M1-containing H7N9 VLP both in chicken and mice models, highlighting the potential of VLP-based vaccine as a critical alternative of traditional egg-based vaccine for control of H7N9 influenza virus in both humans and poultry.

Development and application of a visual microarray for synchronously detecting H5N1, H7N9 and H9N2 avian influenza virus RNA

The aim of this study was to develop a microarray assay for the simultaneous detection of the H5, H7, H9, N1, N9 and N2 genes of the avian influenza virus (AIV) using a Nanogold-streptavidin and silver-stain-enhanced nucleic acid dot-blot hybridisation system. The conserved sequences of H5 genes from H5N1, H7 genes from H7N9, H9 genes from H9N2, N9 genes from H7N9 and N2 genes from H9N2 AIV were cloned, together with that of N1 obtained commercially, and were used as templates for generating the probes using biotin-labeled primers, which targeted the conserved regions of H5, H7, H9, N1, N9 and N2 genes, respectively. The oligonucleotide probes were diluted using the spotting buffer and ddH2O, and each probe was then spotted to each specific position on the microarray. The PCR products including biotin-labeled lambda, NP, H5, H7, H9, N1, N9 and N2 were mixed, 200 μL of which was then added to the microarray chamber after denaturing. Following a hybridization incubation at 45℃ for 120 min, the microarray was then incubated with nanogold-streptavidin about 4 μg/mL for 30 min. After the supplementary of 200 μL of silver buffer A and silver buffer B in the chamber, the hybridization results were assessed by direct visualization in the dark at room temperature.The microarray assay was optimized and its specificity, sensitivity and stability were evaluated. The optimal conditions comprised a probe concentration of 50 μmol/L, a hybridization temperature of 45℃ and a hybridization time of 2 h. The optimal concentration of nanogold-streptavidin was 4 μg/mL and the optimal staining time was 7 min. The results of specificity evaluation showed that no cross-binding of the probes with each other and no cross-hybridization with Newcastle disease virus, infectious bronchitis virus and infectious laryngotracheitis virus was observed. The optimized microarray assay was significantly more sensitivity than the reverse-transcription PCR assay. The microarray was available after storing at less 90 d at 4 ℃. The optimized microarray assay was validated on clinical specimens and the results showed that it had over 95.6 % correlation with reverse-transcription PCR method. Therefore, the microarray assay could be used for the high throughput detection of AIV infections due to H5N1, H7N9 and H9N2.

Mutations of 127, 183 and 212 residues on the HA globular head affect the antigenicity, replication and pathogenicity of H9N2 avian influenza virus.

H9N2 avian influenza virus (AIV), one of the predominant subtypes devastating the poultry industry, has been circulating widely in the poultry population and causing huge economic losses. In this study, two H9N2 viruses with similar genetic backgrounds but different antigenicity were isolated from a poultry farm, namely A/chicken/Jiangsu/75/2018 (JS/75) and A/chicken/Jiangsu/76/2018 (JS/76). Sequence analysis revealed that their surface genes differed in three amino acid residues (127, 183 and 212) on the head of hemagglutinin (HA). To explore the differences between the two viruses in their biological features, six recombinant viruses, including the wild-type or mutant HA and NA of JS/75 and JS/76 were generated with A/Puerto Rico/8/1934 (PR8) backbone via reverse genetics. The chicken challenge study and HI assay data indicated that r-76/PR8 showed the most obvious antigen escape due to 127 and 183 amino acid substitutions in HA gene. Further studies verified that the 127N site was glycosylated in JS/76 and its mutants. Receptor-binding assays showed that all the recombination viruses were prone to bind the human-like receptors, except for the mutants which glycosylated 127N was deleted. Growth kinetics and mice challenge experiments indicated that 127N-glycosylated viruses showed less replication in A549 cells and lower pathogenicity in mice compared with wild-type viruses. Therefore, the glycosylation site and two amino acid alternations in the HA globular head were responsible for the differences in antigenicity and pathogenicity between the two H9N2 isolates. This study is significant in the research of the antigenic variation and vaccine updates for the H9N2 AIV. Also, highlighted the critical functions of glycosylation in the influenza virus on the pathogenicity against mammals.

Single Dose of Bivalent H5 and H7 Influenza Virus-Like Particle Protects Chickens Against Highly Pathogenic H5N1 and H7N9 Avian Influenza Viruses.

Both H5N1 and H7N9 subtype avian influenza viruses cause enormous economic losses and pose considerable threats to public health. Bivalent vaccines against both two subtypes are more effective in control of H5N1 and H7N9 viruses in poultry and novel egg-independent vaccines are needed. Herein, H5 and H7 virus like particle (VLP) were generated in a baculovirus expression system and a bivalent H5+H7 VLP vaccine candidate was prepared by combining these two antigens. Single immunization of the bivalent VLP or commercial inactivated vaccines elicited effective antibody immune responses, including hemagglutination inhibition, virus neutralizing and HA-specific IgG antibodies. All vaccinated birds survived lethal challenge with highly pathogenic H5N1 and H7N9 viruses. Furthermore, the bivalent VLP significantly reduced viral shedding and virus replication in chickens, which was comparable to that observed for the commercial inactivated vaccine. However, the bivalent VLP was better than the commercial vaccine in terms of alleviating pulmonary lesions caused by H7N9 virus infection in chickens. Therefore, our study suggests that the bivalent H5+H7 VLP vaccine candidate can serve as a critical alternative for the traditional egg-based inactivated vaccines against H5N1 and H7N9 avian influenza virus infection in poultry.

Pathogen change of avian influenza virus in the live poultry market before and after vaccination of poultry in southern China

BackgroundThe fifth wave of H7N9 avian influenza virus caused a large number of human infections and a large number of poultry deaths in China. Since September 2017, mainland China has begun to vaccinate poultry with H5 + H7 avian influenza vaccine. We investigated the avian influenza virus infections in different types of live poultry markets and samples before and after genotype H5 + H7 vaccination in Nanchang, and analyzed the changes of the HA subtypes of AIVs.MethodsFrom 2016 to 2019, we monitored different live poultry markets and collected specimens, using real-time reverse transcription polymerase chain reaction (RT-PCR) technology to detect the nucleic acid of type A avian influenza virus in the samples. The H5, H7 and H9 subtypes of influenza viruses were further classified for the positive results. The χ2 test was used to compare the differences in the separation rates of different avian influenza subtypes.ResultsWe analyzed 5,196 samples collected before and after vaccination and found that the infection rate of AIV in wholesale market (21.73%) was lower than that in retail market (24.74%) (P < 0.05). Among all the samples, the positive rate of sewage samples (33.90%) was the highest (P < 0.001). After vaccination, the positive rate of H5 and H7 subtypes decreased, and the positive rate of H9 subtype and untypable HA type increased significantly (P < 0.001). The positive rates of H9 subtype in different types of LPMs and different types of samples increased significantly (P < 0.01), and the positive rates of untypable HA type increased significantly in all environmental samples (P < 0.05).ConclusionsSince vaccination, the positive rates of H5 and H7 subtypes have decreased, but the positive rates of H9 subtypes have increased to varying degrees in different testing locations and all samples. This results show that the government should establish more complete measures to achieve long-term control of the avian influenza virus.