H5N1 Avian Influenza Viruses Research Articles

The host tropism of current zoonotic H7N9 viruses depends mainly on an acid-labile hemagglutinin with a single amino acid mutation in the stalk region.

The incidence of human infection by zoonotic avian influenza viruses, especially H5N1 and H7N9 viruses, has increased. Current zoonotic H7N9 avian influenza viruses (identified since 2013) emerged during reassortment of viruses belonging to different subtypes. Despite analyses of their genetic background, we do not know why current H7N9 viruses are zoonotic. Therefore, there is a need to identify the factor(s) responsible for the extended host tropism that enables these viruses to infect humans as well as birds. To identify H7N9-specific amino acids that confer zoonotic properties on H7N9 viruses, we performed multiple alignment of the hemagglutinin (HA) amino acid sequences of A/Shanghai/1/2013 (H7N9) and A/duck/Zhejiang/12/2011(H7N3) (a putative, non- or less zoonotic HA donor to the zoonotic H7N9 virus). We also analyze the function of an H7N9 HA-specific amino acid with respect to HA acid stability, and evaluated the effect of acid stability on viral infectivity and virulence in a mouse model. HA2-116D, preserved in current zoonotic H7N9 viruses, was crucial for loss of HA acid stability. The acid-labile HA protein in H7 viruses played an important role in infection of human airway epithelial cells; HA2-116D contributed to infection and replication of H7 viruses. Finally, HA2-116D served as a H7 virulence factor in mice. These results suggest that acid-labile HA harboring HA2-116D confers zoonotic characteristics on H7N9 virus and that future novel zoonotic avian viruses could emerge from non-zoonotic H7 viruses via acquisition of mutations that remove HA acid stability.

Highly pathogenic avian influenza A virus subtype H5N1 (clade 2.3.4.4b) isolated from a natural protected area in Peru.

The panzootic caused by H5N1 avian influenza viruses is a high concern for wild birds' conservation and the study of spillover events into mammals. The near coding-complete genome of H5N1 clade 2.3.3.4b sequencing in the Miseq Illumina platform was performed from a bird located in Pantanos of Villa National Wildlife Refuge.

Bovine H5N1 influenza virus binds poorly to human-type sialic acid receptors.

Clade 2.3.4.4b highly pathogenic H5N1 avian influenza (HPAI) viruses started circulating widely in lactating dairy cattle in the United States at the end of 2023. Avian influenza viruses enter cells after binding to glycan receptors with terminally linked α2-3 sialic acid, whereas human influenza viruses typically bind to glycan receptors terminally linked α2-6 sialic acid in the upper respiratory tract. Here, we evaluated the receptor binding properties of hemagglutinin (HA) trimers from a clade 2.3.4.4b avian isolate (A/American Wigeon/South Carolina/22-000345-001/2021) and a cattle isolate (A/dairy cattle/Texas/24-008749-002-v/2024). Using two different methods, we found that both of the 2.3.4.4b H5s bound efficiently to glycan receptors with terminally linked α2-3 sialic acid with no detectable binding to glycan receptors with terminally linked α2-6 sialic acid. Our data suggest that clade 2.3.4.4b H5N1 viruses bind poorly to human receptors. It will be important to continue evaluating receptor binding properties of these viruses as they evolve in cattle.

Pathogenicity and transmissibility of bovine H5N1 influenza virus

Highly pathogenic H5N1 avian influenza (HPAI H5N1) viruses occasionally infect, but typically do not transmit, in mammals. In the spring of 2024, an unprecedented outbreak of HPAI H5N1 in bovine herds occurred in the USA, with virus spread within and between herds, infections in poultry and cats, and spillover into humans, collectively indicating an increased public health risk1–4. Here we characterize an HPAI H5N1 virus isolated from infected cow milk in mice and ferrets. Like other HPAI H5N1 viruses, the bovine H5N1 virus spread systemically, including to the mammary glands of both species, however, this tropism was also observed for an older HPAI H5N1 virus isolate. Bovine HPAI H5N1 virus bound to sialic acids expressed in human upper airways and inefficiently transmitted to exposed ferrets (one of four exposed ferrets seroconverted without virus detection). Bovine HPAI H5N1 virus thus possesses features that may facilitate infection and transmission in mammals.

Transmission dynamics of avian influenza viruses in Egyptian poultry markets

Live bird markets (LBMs) are considered hotspots for Avian Influenza Viruses (AIVs). In such markets, AIVs pose threats to both poultry and public health. Within LBMs, AIVs spread through various routes, including direct contact, environmental contamination, and aerosol transmission. Unique factors in Egyptian LBMs, such as the coexistence of wild and domestic birds, increase transmission risks between birds as well as spill-overs into exposed humans. Understanding the transmission dynamics of AIVs is vital for implementing effective control measures. We conducted a study in four Egyptian LBMs located in Mediterranean coast cities from November 2021 to March 2023. In this study we tested 3,971 samples from poultry, wild birds, and the environment, out of which 692 (17.4%) were positive for AIV. Poultry exhibited a higher prevalence (42.2%) than wild birds (34.4%). Environmental samples, including water (30.8%), surfaces (17.2%), and air (18.2%), also tested positive for AIV. Diverse AIV subtypes, including H5N1, H9N2, H5/H9 co-infection, and H5N8, were detected among avian species and the environment. Temporal analysis revealed fluctuating IAV positivity rates from November 2021 to March 2023. These results emphasize the importance of continuous surveillance, resource allocation, and multisectoral collaboration to protect poultry and human health, and prevent novel influenza strains’ emergence in Egyptian LBMs.

Genetic and molecular characterization of H9N2 avian influenza viruses in Yunnan Province, Southwestern China

The H9N2 subtype of the avian influenza virus (AIV) is widely prevalent in birds, threatening the poultry industry and providing genetic material for emerging human pathogens. The prevalence and genetic characteristics of H9N2 in Yunnan Province, China, are largely unknown. Samples were collected from live poultry markets (LPMs) and breeding farms in Yunnan Province. H9N2-positive samples were identified by polymerase chain reaction (PCR), with a high positivity rate of 42.86% in tissue samples. The positivity rate of swab samples in the LPMs in Kunming was 3.97% (17/564), but no AIV was detected in samples from poultry farms in Lijiang, Wenshan, and Yuxi. Evolutionary analysis and genotyping were performed for the 17 strains of isolated H9N2 virus. Phylogenetic analysis revealed that all H9N2 viral genes had 91.6%-100% nucleotide homology, belonged to the G57 genotype, and had high homology with H9N2 viruses isolated from Guangdong and Guangxi, suggesting that the H9N2 viruses in Yunnan Province may have been imported by chicks. Using a nucleotide divergence cutoff of 95%, we identified ten distinct H9N2 genotypes that continued to evolve. The surface genes of the H9N2 isolates displayed substantial genetic diversity, highlighting the genetic diversity and complexity of the H9N2-subtype AIVs in Yunnan. Molecular analysis demonstrated that all 17 strains of H9N2 isolates had mutations at H183N, Q226L, L31P, and I268V in hemagglutinin; S31N in matrix protein 2; and no replacements at positions 274 and 292 of the neuraminidase protein. Sixteen strains had the A558V mutation and one strain had the E627V mutation in polymerase basic protein 2. Analysis of these amino acid sites suggests that H9N2 influenza viruses in Yunnan continue to mutate and adapt to mammals and are sensitive to neuraminidase inhibitors but resistant to adamantanes. It is necessary to strengthen surveillance of AIV H9N2 subtypes in poultry and LPMs in Yunnan to further understand their genetic diversity.

Genetic diversity of H5N1 and H5N2 high pathogenicity avian influenza viruses isolated from poultry in Japan during the winter of 2022–2023

High pathogenicity avian influenza viruses (HPAIVs) of the H5N1 and H5N2 subtypes were responsible for 84 HPAI outbreaks on poultry premises in Japan during October 2022–April 2023. The number of outbreaks during the winter of 2022–2023 is the largest ever reported in Japan. In this study, we performed phylogenetic analyses using the full genetic sequences of HPAIVs isolated in Japan during 2022–2023 and those obtained from a public database to identify their genetic origin. Based on the hemagglutinin genes, these HPAIVs were classified into the G2 group of clade 2.3.4.4b, whose ancestors were H5 HPAIVs that circulated in Europe in late 2020, and were then further divided into three subgroups (G2b, G2d, and G2c). Approximately one-third of these viruses were classified into the G2b and G2d groups, which also included H5N1 HPAIVs detected in Japan during 2021–2022. In contrast, the remaining two-thirds were classified into the G2c group, which originated from H5N1 HPAIVs isolated in Asian countries and Russia during the winter of 2021–2022. Unlike the G2b and G2d viruses, the G2c viruses were first detected in Japan in the fall of 2022. Importantly, G2c viruses caused the largest number of outbreaks throughout Japan over the longest period during the season. Phylogenetic analyses using eight segment genes revealed that G2b, G2d, and G2c viruses were divided into 2, 4, and 11 genotypes, respectively, because they have various internal genes closely related to those of avian influenza viruses detected in wild birds in recent years in Asia, Russia, and North America, respectively. These results suggest that HPAIVs were disseminated among migratory birds, which may have generated numerous reassortant viruses with various gene constellations, resulting in a considerable number of outbreaks during the winter of 2022–2023.

Global antigenic landscape and vaccine recommendation strategy for low pathogenic avian influenza A (H9N2) viruses

The sustained circulation of H9N2 avian influenza viruses (AIVs) poses a significant threat for contributing to a new pandemic. Given the temporal and spatial uncertainty in theantigenicity of H9N2 AIVs, the immune protection efficiency of vaccines remains challenging. By developing an antigenicity prediction method for H9N2 AIVs, named PREDAC-H9, the global antigenic landscape of H9N2 AIVs was mapped. PREDAC-H9 utilizes the XGBoost model with 14 well-designed features. The XGBoost model was built and evaluated to predict the antigenic relationship between any two viruses with high values of 81.1%, 81.4%, 81.3%, 81.1%, and 89.4% in accuracy, precision, recall, F1 value, and area under curve (AUC), respectively. Then the antigenic correlation network (ACnet) was constructed based on the predicted antigenic relationship for H9N2 AIVs from 1966 to 2022, and ten major antigenic clusters were identified. Of these, four novel clusters were generated in China in the past decade, demonstrating the unique complex situation there. To help tackle this situation, we applied PREDAC-H9 to calculate the cluster-transition determining sites and screen out virus strains with thehigh cross-protective spectrum, thus providing aninsilico reference for vaccine recommendation. The proposed model will reduce the clinical monitoring workload and provide a useful tool for surveillance and control of H9N2 AIVs.

Hemagglutinin glycosylation pattern-specific effects: implications for the fitness of H9.4.2.5-branched H9N2 avian influenza viruses

ABSTRACT Since 2007, h9.4.2.5 has emerged as the most predominant branch of H9N2 avian influenza viruses (AIVs) that affects the majority of the global poultry population. The spread of this viral branch in vaccinated chicken flocks has not been considerably curbed despite numerous efforts. The evolutionary fitness of h9.4.2.5-branched AIVs must consequently be taken into consideration. The glycosylation modifications of hemagglutinin (HA) play a pivotal role in regulating the balance between receptor affinity and immune evasion for influenza viruses. Sequence alignment showed that five major HA glycosylation patterns have evolved over time in h9.4.2.5-branched AIVs. Here, we compared the adaptive phenotypes of five virus mutants with different HA glycosylation patterns. According to the results, the mutant with 6 N-linked glycans displayed the best acid and thermal stability and a better capacity for multiplication, although having a relatively lower receptor affinity than 7 glycans. The antigenic profile between the five mutants revealed a distinct antigenic distance, indicating that variations in glycosylation level have an impact on antigenic drift. These findings suggest that changes in the number of glycans on HA can not only modulate the receptor affinity and antigenicity of H9N2 AIVs, but also affect their stability and multiplication. These adaptive phenotypes may underlie the biological basis for the dominant strain switchover of h9.4.2.5-branched AIVs. Overall, our study provides a systematic insight into how changes in HA glycosylation patterns regulate the evolutionary fitness and epidemiological dominance drift of h9.4.2.5-branched H9N2 AIVs, which will be of great benefit for the glycosylation-dependent vaccine design.

Pathogenicity and transmission of novel highly pathogenic H7N2 variants originating from H7N9 avian influenza viruses in chickens

The H7 subtype avian influenza viruses are circulating widely worldwide, causing significant economic losses to the poultry industry and posing a serious threat to human health. In 2019, H7N2 and H7N9 co-circulated in Chinese poultry, yet the risk of H7N2 remained unclear. We isolated and sequenced four H7N2 viruses from chickens, revealing them as novel reassortants with H7N9-derived HA, M, NS genes and H9N2-derived PB2, PB1, PA,NP, NA genes. To further explore the key segment of pathogenicity, H7N2-H7N9NA and H7N2-H9N2HA single-substitution were constructed. Pathogenicity study showed H7N2 isolates to be highly pathogenic in chickens, with H7N2-H7N9NA slightly weaker than H7N2-Wild type. Transcriptomic analysis suggested that H7N9-derived HA genes primarily drove the high pathogenicity of H7N2 isolates, eliciting a strong inflammatory response. These findings underscored the increased threat posed by reassorted H7N2 viruses to chickens, emphasizing the necessity of long-term monitoring of H7 subtype avian influenza viruses.

Modelling the transmission dynamics of H9N2 avian influenza viruses in a live bird market

H9N2 avian influenza viruses (AIVs) are a major concern for the poultry sector and human health in countries where this subtype is endemic. By fitting a model simulating H9N2 AIV transmission to data from a field experiment, we characterise the epidemiology of the virus in a live bird market in Bangladesh. Many supplied birds arrive already exposed to H9N2 AIVs, resulting in many broiler chickens entering the market as infected, and many indigenous backyard chickens entering with pre-existing immunity. Most susceptible chickens become infected within one day spent at the market, owing to high levels of viral transmission within market and short latent periods, as brief as 5.3 hours. Although H9N2 AIV transmission can be substantially reduced under moderate levels of cleaning and disinfection, effective risk mitigation also requires a range of additional interventions targeting markets and other nodes along the poultry production and distribution network.

Evolutionary dynamics and comparative pathogenicity of clade 2.3.4.4b H5 subtype avian influenza viruses, China, 2021–2022

The recent concurrent emergence of H5N1, H5N6, and H5N8 avian influenza viruses (AIVs) has led to significant avian mortality globally. Since 2020, frequent human-animal interactions have been documented. To gain insight into the novel H5 subtype AIVs (i.e., H5N1, H5N6 and H5N8), we collected 6102 samples from various regions of China between January 2021 and September 2022, and identified 41 H5Nx strains. Comparative analyses on the evolution and biological properties of these isolates were conducted. Phylogenetic analysis revealed that the 41 H5Nx strains belonged to clade 2.3.4.4b, with 13 related to H5N1, 19 to H5N6, and 9 to H5N8. Analysis based on global 2.3.4.4b viruses showed that all the viruses described in this study were likely originated from H5N8, exhibiting a heterogeneous evolutionary history between H5N1 and H5N6 during 2015–2022 worldwide. H5N1 showed a higher rate of evolution in 2021–2022 and more sites under positive selection pressure in 2015–2022. The antigenic profiles of the novel H5N1 and H5N6 exhibited notable variations. Further hemagglutination inhibition assay suggested that some A(H5N1) viruses may be antigenically distinct from the circulating H5N6 and H5N8 strains. Mammalian challenge assays demonstrated that the H5N8 virus (21GD001_H5N8) displayed the highest pathogenicity in mice, followed by the H5N1 virus (B1557_H5N1) and then the H5N6 virus (220086_H5N6), suggesting a heterogeneous virulence profile of H5 AIVs in the mammalian hosts. Based on the above results, we speculate that A(H5N1) viruses have a higher risk of emergence in the future. Collectively, these findings unveil a new landscape of different evolutionary history and biological characteristics of novel H5 AIVs in clade 2.3.4.4b, contributing to a better understanding of designing more effective strategies for the prevention and control of novel H5 AIVs.

Molecular characterization of the whole genome of H9N2 avian influenza virus isolated from Egyptian poultry farms

H9N2 avian influenza viruses (AIVs) affect both poultry and humans on a global level, and they are especially prevalent in Egypt. In this study, we sequenced the entire genome of AIV H9N2 isolated from chickens in Egypt in 2021, using next-generation sequencing (NGS) technology. Phylogenetic analysis of the resulting sequences showed that the studied strain was generally monophyletic and grouped within the G1 sublineage of the Eurasian lineage. Four segments (polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acidic [PA], and non-structural [NS]) were related to Egyptian genotype II, while the nucleoprotein (NP), neuraminidase (NA), matrix (M), and haemagglutinin (HA) segments were related to Egyptian genotype I. Molecular analysis revealed that HA protein contained amino acid residues (191H and 234L) that suggested a predilection for attaching to human-like receptors. The antigenic sites of HA had two nonsynonymous mutations: V194I at antigenic site A and M40K at antigenic site B. Furthermore, the R403W and S372A mutations, which have been observed in H3N2 and H2N2 strains that caused human pandemics, were found in the NA protein of the detected strain. The internal proteins contained virulence markers: 504V in the PB2 protein, 622G, 436Y, 207K, and 677T in the PB1 protein, 127V, 550L, and 672L in PA protein, and 64F and 69P in the M protein. These results show that the detected strain had undergone intrasubtype reassortment. Furthermore, it contains changes in the viral proteins that make it more likely to be virulent, raising a question about the tendency of AIV H9N2 to become highly pathogenic in the future for both poultry and humans.

Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from 2020 to 2022

The H9N2 subtype of avian influenza virus (AIV) is widely prevalent in poultry and wild birds globally, and has become the predominant subtype circulating in poultry in China. The H9N2 AIV can directly or indirectly (by serving as a “donor virus”) infect humans, posing a significant threat to public health. Currently, there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province, central China. In this study, we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province, and genetic analysis revealed that these viruses belonged to 7 different genotypes. Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations, and exhibited diverse replication abilities within these birds. These viruses could replicate efficiently in the lungs of mice, with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys. At the cellular level, the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays, revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level. The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters. Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China. It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.

Genetics of H5N1 and H5N8 High-Pathogenicity Avian Influenza Viruses Isolated in Japan in Winter 2021-2022.

In winter 2021-2022, H5N1 and H5N8 high-pathogenicity avian influenza (HPAI) viruses (HPAIVs) caused serious outbreaks in Japan: 25 outbreaks of HPAI at poultry farms and 107 cases in wild birds or in the environment. Phylogenetic analyses divided H5 HPAIVs isolated in Japan in the winter of 2021-2022 into three groups-G2a, G2b, and G2d-which were disseminated at different locations and times. Full-genome sequencing analyses of these HPAIVs revealed a strong relationship of multiple genes between Japan and Siberia, suggesting that they arose from reassortment events with avian influenza viruses (AIVs) in Siberia. The results emphasize the complex of dissemination and reassortment events with the movement of migratory birds, and the importance of continual monitoring of AIVs in Japan and Siberia for early alerts to the intrusion of HPAIVs.

Evolutionary Events Promoted Polymerase Activity of H13N8 Avian Influenza Virus.

Wild birds are considered to be the natural reservoir hosts of avian influenza viruses (AIVs). Wild bird-origin AIVs may spill over into new hosts and overcome species barriers after evolutionary adaptation. H13N8 AIVs used to be considered primarily circulated in multispecies gulls but have recently been shown to possess cross-species infectivity. In this study, we analyzed the genetic changes that occurred in the process of the evolution of H13 AIVs. Phylogenetic analysis revealed that H13 AIVs underwent complex reassortment events. Based on the full genomic diversity, we divided H13 AIVs into 81 genotypes. Reassortment experiments indicated that basic polymerase 2 (PB2) and nucleoprotein (NP) genes of the H9N2 AIV significantly enhanced the polymerase activity of the H13N8 AIV. Using the replication-incompetent virus screening system, we identified two mutations, PB2-I76T and PB2-I559T, which could enhance the polymerase activity of the H13N8 AIV in mammalian cells. Notably, these mutations had been acquired by circulating H13N8 AIVs in 2015. These findings suggest that H13N8 AIVs are about to cross the host barrier. Occasional genetic reassortments with other AIVs and natural mutation events could promote this process. It is imperative to intensify monitoring efforts for H13N8 AIVs.

Genetic insights of H9N2 avian influenza viruses circulating in Mali and phylogeographic patterns in Northern and Western Africa.

Avian influenza viruses (AIVs) of the H9N2 subtype have become widespread in Western Africa since their first detection in 2017 in Burkina Faso. However, the genetic characteristics and diffusion patterns of the H9N2 virus remain poorly understood in Western Africa, mainly due to limited surveillance activities. In addition, Mali, a country considered to play an important role in the epidemiology of AIVs in the region, lacks more comprehensive data on the genetic characteristics of these viruses, especially the H9N2 subtype. To better understand the genetic characteristics and spatio-temporal dynamics of H9N2 virus within this region, we carried out a comprehensive genetic characterization of H9N2 viruses collected through active surveillance in live bird markets in Mali between 2021 and 2022. We also performed a continuous phylogeographic analysis to unravel the dispersal history of H9N2 lineages between Northern and Western Africa. The identified Malian H9N2 virus belonged to the G1 lineage, similar to viruses circulating in both Western and Northern Africa, and possessed multiple molecular markers associated with an increased potential for zoonotic transmission and virulence. Notably, some Malian strains carried the R-S-N-R motif at their cleavage site, mainly observed in H9N2 strains in Asia. Our continuous phylogeographic analysis revealed a single and significant long-distance lineage dispersal event of the H9N2 virus to Western Africa, likely to have originated from Morocco in 2015, shaping the westward diffusion of the H9N2 virus. Our study highlights the need for long-term surveillance of H9N2 viruses in poultry populations in Western Africa, which is crucial for a better understanding of virus evolution and effective management against potential zoonotic AIV strain emergence.

Spatiotemporal genotype replacement of H5N8 avian influenza viruses contributed to H5N1 emergence in 2021/2022 panzootic.

Since 2020, clade 2.3.4.4b highly pathogenic avian influenza H5N8 and H5N1 viruses have swept through continents, posing serious threats to the world. Through comprehensive analyses of epidemiological, genetic, and bird migration data, we found that the dominant genotype replacement of the H5N8 viruses in 2020 contributed to the H5N1 outbreak in the 2021/2022 wave. The 2020 outbreak of the H5N8 G1 genotype instead of the G0 genotype produced reassortment opportunities and led to the emergence of a new H5N1 virus with G1's HA and MP genes. Despite extensive reassortments in the 2021/2022 wave, the H5N1 virus retained the HA and MP genes, causing a significant outbreak in Europe and North America. Furtherly, through the wild bird migration flyways investigation, we found that the temporal-spatial coincidence between the outbreak of the H5N8 G1 virus and the bird autumn migration may have expanded the H5 viral spread, which may be one of the main drivers of the emergence of the 2020-2022 H5 panzootic.IMPORTANCESince 2020, highly pathogenic avian influenza (HPAI) H5 subtype variants of clade 2.3.4.4b have spread across continents, posing unprecedented threats globally. However, the factors promoting the genesis and spread of H5 HPAI viruses remain unclear. Here, we found that the spatiotemporal genotype replacement of H5N8 HPAI viruses contributed to the emergence of the H5N1 variant that caused the 2021/2022 panzootic, and the viral evolution in poultry of Egypt and surrounding area and autumn bird migration from the Russia-Kazakhstan region to Europe are important drivers of the emergence of the 2020-2022 H5 panzootic. These findings provide important targets for early warning and could help control the current and future HPAI epidemics.

One-Health Challenge in H9N2 Avian Influenza: Novel Human-Avian Reassortment Virus in Guangdong Province, China

China is one of the highest producers of poultry meat output in the world, with a large scale of chicken rearing. Statistically analyzed H9N2-subtype avian influenza viruses (AIVs) have become the dominant subtype in China’s live poultry market, with the highest detection rate. Although H9N2 AIV is of low pathogenicity and tends not to cause serious disease and high mortality in poultry, it poses a great challenge to the domestic poultry farming industry by causing a decrease in appetite, a decline in egg production, and deaths caused by mixed infections with another pathogenic microorganism. Moreover, novel influenza viruses (H7N9 and H3N8) infecting humans have emerged in China, and the H9N2 AIV provides all or part of the internal genes to the new recombinant viruses, posing a potential threat to public health and safety and human health. In this research, six H9N2 AIVs were isolated from feces or oropharyngeal swabs collected from live poultry markets and duck farms in Zhanjiang. After epidemiological investigations, phylogenetic analyses, and molecular characterization, we found that the ZJ81 strain was a chicken–human–mink recombinant virus, the ML3 strain was a chicken-human recombinant virus, and all six virus strains of the virus had a bias for the human receptor-binding site and a mutation that could cause an increase in virulence in mice. Therefore, surveillance and control of H9N2 AIV should be strengthened to provide data support for cross-species transmission of H9N2 AIV.

Bivalent Hemagglutinin Cleavage-Site Peptide Vaccines Protect Chickens from Lethal Infections with Highly Pathogenic H5N1 and H5N6 Avian Influenza Viruses.

Outbreaks of highly pathogenic avian influenza viruses cause huge economic losses to the poultry industry worldwide. Vaccines that can protect chickens from infections caused by various variants of highly pathogenic H5Nx avian influenza viruses are needed owing to the continuous emergence of new variants. We previously showed that vaccines containing the H5 cleavage-site peptide from clade 2.3.4.4. H5N6 avian influenza virus protects chickens from infection with homologous clade 2.3.4.4. H5N6 avian influenza virus, but not from infection with the heterologous clade 1 H5N1 avian influenza virus. Therefore, we developed bivalent peptide vaccines containing H5 cleavage sites of viruses from both clades to protect chickens from both H5N1 and H5N6 avian influenza viruses. Chickens were vaccinated with two doses of a combined peptide vaccine containing cleavage-site peptides from clade 1 and clade 2.3.4.4. highly pathogenic H5N1 and H5N6 avian influenza viruses and then challenged with both viruses. The infected chickens were monitored for survival and their tracheae and cloacae were sampled to check for viral shedding based on the median tissue culture infectious dose of 50 (log10TCID50/mL) in Madin-Darby canine kidney cells. Antibody production was induced at similar levels in the sera of chickens immunized with two doses of the combined peptide vaccines containing cleavage-site peptides from highly pathogenic H5N1 and H5N6 avian influenza viruses. The immunized chickens were protected from infection with both H5N1 and H5N6 avian influenza viruses without viral shedding in the tracheae and cloacae. Dual-peptide vaccines containing cleavage-site peptides of both clades can protect chickens from highly pathogenic avian influenza virus infections.