Subtype Avian Influenza Research Articles

Characterization of a reassortant H11N9 subtype avian influenza virus isolated from spot-billed duck in China

H11N9 viruses in wild birds might have provided the NA gene of human H7N9 virus in early 2013 in China, which evolved with highly pathogenic strains in 2017 and caused severe fatalities. To investigate the prevalence and evolution of the H11N9 influenza viruses, 16,781 samples were collected and analyzed during 2016–2020. As a result, a novel strain of influenza A (H11N9) virus with several characteristics that increase virulence was isolated. This strain had reduced pathogenicity in chicken and mice and was able to replicate in mice without prior adaptation. Phylogenetic analyses showed that it was a sextuple‐reassortant virus of H11N9, H3N8, H3N6, H7N9, H9N2, and H6N8 viruses present in China, similar to the H11N9 strains in Japan and Korea during the same period. This was the H11N9 strain isolated from China most recently, which add a record to viruses in wild birds. This study identified a new H11N9 reassortant in a wild bird with key mutation contributing to virulence. Therefore, comprehensive surveillance and enhanced biosecurity precautions are particularly important for the prediction and prevention of potential pandemics resulting from reassortant viruses with continuous evolution and expanding geographic distributions.

Protective efficacy of a bivalent H5 influenza vaccine candidate against both clades 2.3.2.1 and 2.3.4.4 high pathogenic avian influenza viruses in SPF chickens.

Worldwide, high pathogenic avian influenza viruses belonging to clades 2.3.4.4 and 2.3.2.1 have been circulating in both poultry and wild birds. Since 2018, Korea has built a national antigen bank to ensure preparedness in an emergency. In this study, we developed a bivalent vaccine candidate containing antigens derived from two reassortant KA435/2.3.2.1d and H35/2.3.4.4b strains for Korean national antigen bank. We evaluated its immunogenicity and protective efficacy in specific pathogen free chickens. The two vaccine strains, rgKA435-H9N2 PB2/2.3.2.1d and rgH35/2.3.4.4b, both of which were generated successfully by reverse genetics, were highly immunogenic (titres of haemagglutination inhibition: 8.3 and 8.4 log2, respectively) and showed good protective efficacy (100 and 147 50% protective dose, respectively) against lethal challenge with wild-type virus when delivered as a 1:1 mixture. Notably, the vaccine provided complete protection against viral shedding at a full dose (512 HAU) and a 1/10 dose (51.2 HAU), with no clinical signs, after challenge with H35/2.3.4.4b. The bivalent vaccine developed in this study may reduce the cost of vaccine production and could be used as a H5 subtype avian influenza vaccine candidate against two clades simultaneously.

Effect of avian influenza virus subtype H9N2 on the expression of complement-associated genes in chicken erythrocytes

ABSTRACT 1. The H9N2 subtype avian influenza virus can infect both chickens and humans. Previous studies have reported a role for erythrocytes in immunity. However, the role of H9N2 against chicken erythrocytes and the presence of complement-related genes in erythrocytes has not been studied. This research investigated the effect of H9N2 on complement-associated gene expression in chicken erythrocytes. 2. The expression of complement-associated genes (C1s, C1q, C2, C3, C3ar1, C4, C4a, C5, C5ar1, C7, CD93 and CFD) was detected by reverse transcription-polymerase chain reaction (RT-PCR). Quantitative Real-Time PCR (qRT-PCR) was used to analyse the differential expression of complement-associated genes in chicken erythrocytes at 0 h, 2 h, 6 h and 10 h after the interaction between H9N2 virus and chicken erythrocytes in vitro and 3, 7 and 14 d after H9N2 virus nasal infection of chicks. 3. Expression levels of C1q, C4, C1s, C2, C3, C5, C7 and CD93 were significantly up-regulated at 2 h and significantly down-regulated at 10 h. Gene expression levels of C1q, C3ar1, C4a, CFD and C5ar1 were seen to be different at each time point. The expression levels of C1q, C4, C1s, C2, C3, C5, C7, CFD, C3ar1, C4a and C5ar1 were significantly up-regulated at 7 d and the gene expression of levels of C3, CD93 and C5ar1 were seen to be different at each time point. 4. The results confirmed that all the complement-associated genes were expressed in chicken erythrocytes and showed the H9N2 virus interaction with chicken erythrocytes and subsequent regulation of chicken erythrocyte complement-associated genes expression. This study reported, for the first time, the relationship between H9N2 and complement system of chicken erythrocytes, which will provide a foundation for further research into the prevention and control of H9N2 infection.

Avian influenza H5N1 in a great white pelican (Pelecanus onocrotalus), Mauritania 2022.

In February 2022, mortalities among great white pelicans (Pelecanus onocrotalus) were reported in the Parc National de Diawling, southwestern Mauritania. Samples were collected and processed, indicating the presence of high pathogenicity avian influenza subtype H5N1. A nearly complete genome was generated for one sample, revealing a high similarity [> 99.5% (H5) nucleotide sequence identity] with Clade 2.3.4.4b H5N1 identified in Europe in 2022.

H9N2 avian influenza virus dispersal along Bangladeshi poultry trading networks.

Avian influenza virus subtype H9N2 is endemic in Bangladesh's poultry population. The subtype affects poultry production and poses a potential zoonotic risk. Insufficient understanding of how the poultry trading network shapes the dissemination of avian influenza viruses has hindered the design of targeted interventions to reduce their spread. Here, we use phylodynamic analyses of haemagglutinin sequences to investigate the spatial spread and dispersal patterns of H9N2 viruses in Bangladesh's poultry population, focusing on its two largest cities (Dhaka and Chattogram) and their poultry production and distribution networks. Our analyses suggest that H9N2 subtype avian influenza virus lineage movement occurs relatively less frequently between Bangladesh's two largest cities than within each city. H9N2 viruses detected in single markets are often more closely related to viruses from other markets in the same city than to each other, consistent with close epidemiological connectivity between markets. Our analyses also suggest that H9N2 viruses may spread more frequently between chickens of the three most commonly sold types (sunali-a cross-bred of Fayoumi hen and Rhode Island Red cock, deshi-local indigenous, and exotic broiler) in Dhaka than in Chattogram. Overall, this study improves our understanding of how Bangladesh's poultry trading system impacts avian influenza virus spread and should contribute to the design of tailored surveillance that accommodates local heterogeneity in virus dispersal patterns.

Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus.

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.

H10Nx avian influenza viruses detected in wild birds in China pose potential threat to mammals

H10 subtype avian influenza viruses (AIVs) have been isolated from wild and domestic avian species worldwide and have occasionally crossed the species barrier to mammalian hosts. Fatal human cases of H10N8 infections and the recent detection of human H10N3 infections have drawn widespread public attention. In this study, 25 H10Nx viruses were isolated from wild waterfowl in China during a long-term surveillance of AIVs. We conducted phylogenetic and phylogeographic studies of the hemagglutinin (HA) genes of global H10 viruses to determine the spatiotemporal patterns of spread and the roles of different hosts in viral transmission. We found the pattern of AIV transmission from wild birds to poultry to humans, and Anatidae have acted as the seeding population in the spread of the virus. Phylogenetic incongruence indicated complex reassortment events and our isolates were divided into eight genotypes (G1–8). We also found that the HA genes of the G8 viruses belonged to the North American lineage, indicating that intercontinental gene flow has occurred. Their receptor-binding specificity showed that the G1/4/5/6/7/8 viruses bind to both human-type α2,6-linked sialic acid receptors and avian-type α2,3-linked sialic acid receptors. Mouse studies indicated that the H10Nx isolates replicated efficiently in the respiratory system without preadaptation, but showed low pathogenicity in mice. The H10Nx isolates showed no (G2/4/7) or low pathogenicity (G1/3/5/6/8) in chickens, and the G6 and G8 viruses could be transmitted to chickens through direct contact. The asymptomatic shedding of these wild-bird-origin H10Nx isolates in chickens and their good adaptation in mice should increase the ease of their transmission to humans, and they therefore pose a threat to public health. Our findings demonstrate a further understanding of wild bird-origin H10 viruses and provide information for the continuous surveillance of H10 subtype viruses.

Characterization of an H7N9 Influenza Virus Isolated from Camels in Inner Mongolia, China.

The H7N9 subtype of influenza virus can infect birds and humans, causing great losses in the poultry industry and threatening public health worldwide. However, H7N9 infection in other mammals has not been reported yet. In the present study, one H7N9 subtype influenza virus, A/camel/Inner Mongolia/XL/2020 (XL), was isolated from the nasal swabs of camels in Inner Mongolia, China, in 2020. Sequence analyses revealed that the hemagglutinin cleavage site of the XL virus was ELPKGR/GLF, which is a low-pathogenicity molecular characteristic. The XL virus had similar mammalian adaptations to human-originated H7N9 viruses, such as the polymerase basic protein 2 (PB2) Glu-to-Lys mutation at position 627 (E627K) mutation, but differed from avian-originated H7N9 viruses. The XL virus showed a higher SA-α2,6-Gal receptor-binding affinity and better mammalian cell replication than the avian H7N9 virus. Moreover, the XL virus had weak pathogenicity in chickens, with an intravenous pathogenicity index of 0.01, and intermediate virulence in mice, with a median lethal dose of 4.8. The XL virus replicated well and caused clear infiltration of inflammatory cells and increased inflammatory cytokines in the lungs of mice. Our data constitute the first evidence that the low-pathogenicity H7N9 influenza virus can infect camels and therefore poses a high risk to public health. IMPORTANCE H5 subtype avian influenza viruses can cause serious diseases in poultry and wild birds. On rare occasions, viruses can cause cross-species transmission to mammalian species, including humans, pigs, horses, canines, seals, and minks. The H7N9 subtype of the influenza virus can also infect both birds and humans. However, viral infection in other mammalian species has not been reported yet. In this study, we found that the H7N9 virus could infect camels. Notably, the H7N9 virus from camels had mammalian adaption molecular markers, including altered receptor-binding activity on the hemagglutinin protein and an E627K mutation on the polymerase basic protein 2 protein. Our findings indicated that the potential risk of camel-origin H7N9 virus to public health is of great concern.

The health of influenza surveillance and pandemic preparedness in the wake of the COVID-19 pandemic.

The COVID-19 pandemic is the first to have emerged when Next Generation Sequencing was readily available and it has played the major role in following evolution of the causative agent, Severe Acute Respiratory Syndrome Coronavirus 2. Response to the pandemic was greatly facilitated though use of existing influenza surveillance networks: World Health Organization (WHO) Global Influenza Surveillance and Response System (GISRS), focussing largely on human influenza, and the OFFLU network of expertise on avian influenza established by the Food and Agricultural Organization of the United Nations (FAO) and the World Organization for Animal Health (WOAH). Data collection/deposition platforms associated with these networks, notably WHO's FluNet and the Global Initiative on Sharing All Influenza Data (GISAID) were/are being used intensely. Measures introduced to combat COVID-19 resulted in greatly decreased circulation of human seasonal influenza viruses for approximately 2 years, but circulation continued in the animal sector with an upsurge in the spread of highly pathogenic avian influenza subtype H5N1 with large numbers of wild bird deaths, culling of many poultry flocks and sporadic spill over into mammalian species, including humans, thereby increasing pandemic risk potential. While there are proposals/implementations to extend use of GISRS and GISAID to other infectious disease agents (e.g. Respiratory Syncytial Virus and Monkeypox), there is need to ensure that influenza surveillance is maintained and improved in both human and animal sectors in a sustainable manner to be truly prepared (early detection) for the next influenza pandemic.

Development and Evaluation of a Blocking Lateral Flow Assay Strip for Detection of Newcastle Disease Virus Antibodies.

Newcastle disease (ND) is an acute septicemic infectious disease caused by Newcastle disease virus (NDV). Considering that vaccination is currently the main modality for the prevention of ND, it is essential to assess the effectiveness of clinical immunization. In this study, we have developed a blocking lateral flow assay (bLFA) strip for the rapid detection of NDV antibodies using the monoclonal antibody 9C1 against haemagglutinin-neuraminidase (HN), which allows for the determination of an NDV-specific antibody titer within 10 min at room temperature. In addition, the bLFA strip has no cross-reactivity with the positive serum of other avian pathogens including avian influenza subtypes H5, H7, and H9, MD, IBD, IB, EDS, and avian adenovirus. The ability of the bLFA strip for detecting a neutralizing antibody was also estimated. The results showed that the chicken NDV hyperimmunized serum had a complete blocking (100%) titer of 11 log 2, and half-blocking titer of 13 log 2, which are 4 times less than and the same as that of the HI test (13 log 2), and 8 and 2 times less than that of the VN test (14 log 2), respectively. A total of 510 clinical samples were tested for NDV antibodies. The coincidence rate between the results of the bLFA strip and HI test was 97.65%. Therefore, it is an ideal alternative method for assessing the clinical immunity of ND vaccines in the field in real-time.

Genome sequence analysis of H7N3 subtype avian influenza virus originated from wild birds and its potential infectivity in mice.

In 2021, an H7N3 avian influenza virus (AIV) was isolated from a mallard in Tianhewan Yellow River National Wetland Park, Ningxia Hui Autonomous Region, China. Sequences analysis showed that this strain received its genes from H7, H6, H5, H3, and H1 AIVs of domestic poultry and wild birds in Asia and Europe. It was mild pathogenicity in mice. These results suggest the importance of continued surveillance of the H7N3 virus to better understand the ecology and evolution of the AIVs in poultry and wild birds and the potential threat to humans.

Evolution and mammalian adaptation of H3 and H10 subtype avian influenza viruses in wild birds in Yancheng Wetland of China

Wild birds play a critical role in avian influenza virus (AIV) ecology and some outbreaks of avian influenza in human originate from wild birds, suggesting that epidemiological surveillance and interspecies-transmission analysis of AIVs in wild birds are critical. Since 2019, we have performed sampling in Yancheng Wetland along the East Asian-Australasian Flyway. Totally, 2054 fecal swabs were collected and one H3N8, two H3N1, one H10N8, and three H10N1 were isolated. Three H3 gene of AIVs we isolated belonged to Eurasian lineage, but the four H10 gene clustered into North American lineage. What’s more, the H3 and the foreign H10 gene had generated novel reassortants in Yancheng wetland. Receptor binding assay indicated that nearly all strains, except D369/H10N1, presented a dual receptor-binding profile and bound to avian-type receptor preferentially. In animal experiment, all isolates could infect mice without prior adaptation and induce histopathological changes in mice lungs, moreover, all H3 subtype AIVs obviously triggered weight loss of mice. In addition to lung and turbinate, D322/H3N1, D338/H3N8, D211/H10N8 and D266/H10N1 could spread to brain and kidney or liver or spleen, showing a wider range of tissue tropism. Multiple mutants associated with mammalian adaptation were also detected in all isolates according to molecular analysis. These findings revealed that H3 and H10 AIVs circulating in wild birds in Yancheng Wetland underwent complex reassortment and increased mammalian adaptation, which highlighted the necessity to monitor the diverse reassortment of AIVs in wild birds and evaluate the risks of H3 and H10 viruses to human health.

Pathogenicity and pathogenesis of a recent highly pathogenic avian influenza subtype H5N8 in mule ducklings in Egypt.

In late 2017, an H5N8 highly pathogenic avian influenza (HPAI) virus, clade 2.3.4.4, was isolated from domestic ducks in Egypt, which was associated with high morbidity and low mortality. The pathogenicity increased due to the continuous circulation of virus in ducks. Thus, this study aimed to monitor the pathogenesis and pathogenicity of new H5N8 Avian influenza (AI) virus in mule ducklings. The lethal dose 50 (LD50) for this new local HPAI H5N8 isolate was calculated. Twenty ducklings were inoculated with 0.1 mL of dilution containing 10 LD50 HPAI per duck. The clinical signs and mortalities were recorded until 30 days post-infection (DPI) to confirm viral pathogenesis. Reverse transcription polymerase chain reaction was used to detect viral shedding from collected cloacal swabs after 3rd, 5th, 7th, 10th, 14th, 21st, and 30th DPI. The main histopathological lesions associated with the presence of HPAI virus were also recorded on the 3rd and 14th DPI. The result showed that the LD50 of the new HPAI H5N8 was 104 log10. Clinical signs were observed after 2nd DPI, but it was clinically severe on 3rd, 4th, and 5th DPI in the form of respiratory and gastric disorders, forming 90% of all diseased ducklings, whereas 30% of the infected ducks only showed nervous signs. The mortality rate peaked on 4th and 5th DPI with a cumulative mortality rate of 60% for the inoculated ducks, whereas no mortality was recorded after 6th DPI. Dead ducks showed typical postmortem lesions of AI disease. Necrosis and ecchymotic or petechial hemorrhages on the heart, pancreas, liver, and spleen were observed, whereas the lung showed pneumonia. With regard to viral shedding, infected ducklings shed the virus from its gut until 7th DPI, but the number of duck shedders gradually decreased until 14th DPI after viral shedding. The histopathological findings indicated that the spleen and thymus showed necrosis and hemorrhages, whereas the brain showed multifocal malacic foci and spread meningitis. Moreover, the lung had intrabronchial hyaline degeneration and fibrinous pneumonia on 3rd DPI. Furthermore, the liver showed multifocal necrotic foci and subcapsular hemorrhage, whereas the kidney showed remarkable tubular degeneration, mostly within the collecting tubules. Furthermore, the heart showed marked myocardiolysis of the cardiac muscle fibers. On 14th DPI, all histopathological lesions of the examined organs were restored to normal. The currently circulating HPAI H5N8 virus strain has high virulence, particularly for imported mule ducks that originated from non-vaccinated breeder ducks. Therefore, vaccination and quarantine measures must be applied on imported 1-day-old mule ducklings. Moreover, the pathogenesis must be reviewed and monitored for updating circulating AI strains caused by the continuous and rapid evolution of AI viruses.

Correlation of avian influenza-H9N2 with high mortality in broiler flocks in the southwest of Tripoli, Libya.

Low pathogenic H9N2 avian influenza (LPAI H9N2) caused by the influenza A virus which belongs to the family Orthomyxoviridae. It caused mild respiratory symptoms and a drop in egg production in poultry. Outbreaks of AI-H9N2 have occurred in poultry since the 1990s in many countries in USA, Europe, and Asia. Recently, outbreaks of H9N2 in commercial chicken were recorded in Morocco, Tunisia, Libya, and Egypt. Furthermore, numerous studies demonstrated that co-infection with AI H9N2 and other pathogens results in severe respiratory illness with high mortality in broiler chickens. Outbreaks of respiratory disease with variations in mortality rate were recorded in broiler flocks growing in the southwest of Tripoli in Libya. The present study was conducted to explain the variation of mortality rate on broiler flocks growing in the southwest area of Tripoli by detection of AI H9N2 antibodies and antigens. A total of 453 sera samples, 60 tracheal swabs, and 60 cloacal swabs were collected from unvaccinated broiler flocks against avian influenza. Specific avian influenza type A antibodies were detected by using the Elisa test, and specific AI-H9N2 antibodies were detected by using the HI test, whereas specific AI-H9N2 antigens were detected in tracheal and cloacal swabs by using One-Step RT-PCR (M gene) technique. Respiratory diseases with high variations in mortality rate were recorded in broiler flocks growing in the southwest of Tripoli in Libya; the broiler mortality rate in Twisha farms was higher than other farms (62.2% and 11%, respectively). Whereas avian influenza type A antibodies were detected at a high level in Twisha and other farms (95.2%, and 76.7%, respectively). The positive samples for AI type A were tested for AI H9N2 using the HI test. Interestingly the percentage of AI-H9N2 antibodies was quite similar in high and low mortality regions (53.4% and 46.8%, respectively). Additionally, AI-H9N2 antigens were detected only in tracheal swabs in Twisha farm 3, Al-Maamoura, and Ber Al-Tota districts. This study confirmed the endemic of AI- H9N2 in broiler flocks in the southwest of Tripoli-Libya. Also, it clarified that AI-H9N2 was not responsible for the high mortality rate by itself in broiler flocks. Moreover, this study supported the presence of other subtypes of avian influenza in the studied area.

Clade 2.3.4.4b H5N8 Subtype Avian Influenza Viruses Were Identified from the Common Crane Wintering in Yunnan Province, China.

The seasonal migration of wild aquatic birds plays a critical role in the maintenance, transmission, and incursion of the avian influenza virus (AIV). AIV surveillance was performed during 2020-2021 in two national nature reserves with abundant wild bird resources in Yunnan, China. Four H5N8 AIVs isolates from the common crane were identified by next-generation sequencing. Phylogenetic analysis demonstrated that all eight gene segments of these H5N8 AIVs belonged to clade 2.3.4.4b high-pathogenic AIV (HPAIV) and shared high nucleotide sequence similarity with the strains isolated in Hubei, China, and Siberia, Russia, in 2020-2021. The H5N8 HPAIVs from common cranes were characterized by both human and avian dual-receptor specificity in the hemagglutinin (HA) protein. Moreover, possessing the substitutions contributes to overcoming transmission barriers of mammalian hosts in polymerase basic 2 (PB2), polymerase basic protein 1 (PB1), and polymerase acid (PA), and exhibiting the long stalk in the neck region of the neuraminidase (NA) protein contributes to adaptation in wild birds. Monitoring AIVs in migratory birds, at stopover sites and in their primary habitats, i.e., breeding or wintering grounds, could provide insight into potential zoonosis caused by AIVs.

Pathogenicity and anti-infection immunity of animal H3N2 and H6N6 subtype influenza virus cross-species infection with tree shrews

Animal influenza viruses can spread across species and pose a fatal threat to human health due to the high pathogenicity and mortality. Animal models are crucial for studying cross-species infection and the pathogenesis of influenza viruses. Tupaia belangeri (tree shrew) has been emerging as an animal model for multiple human virus infections recently because of the close genetic relationship and phylogeny with humans. So far, tree shrew has been reported to be susceptible to human influenza virus subtype H1N1, avian influenza viruses subtype H9N2, subtype H5N1, and subtype H7N9. However, the pathogenicity, infection, and immunity of swine and land avian influenza viruses with low pathogenicity and the potential to jump to humans remain largely unexplored in the tree shrew model. Previously, our team has successfully isolated the newly emerging swine influenza virus subtype H3N2 (A/Swine/GX/NS2783/2010, SW2783) and avian influenza virus subtype H6N6 (A/CK/ZZ/346/2014, ZZ346). In this study, we observed the pathogenicity, immune characteristics, and cross-species infection potential ability of SW2783 and ZZ346 strains in tree shrew model with 50% tissue culture infective dose (TCID50), hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), real-time quantitative PCR (qRT-PCR) and other experimental methods. Both animal-borne influenza viruses had a strong ability on tissue infection in the turbinate and the trachea of tree shrews in vitro, in which SW2783 showed stronger replication ability than in ZZ346. SW2783 and ZZ346 both showed pathogenic ability with infected tree shrews model in vivo without prior adaptive culture, which mainly happened in the upper respiratory tract. However, the infection ability was weak, the clinical symptoms were mild, and the histopathological changes in the respiratory tract were relatively light. Furthermore, innate immune responses and adaptive immunity were observed in the tree shrew model after the infection of SW2783 and ZZ346 strains. We observed that the unadapted SW2783 and ZZ346 virus could transmit among tree shrews by direct contact. We also observed that SW2783 virus could transmit from tree shrews to guinea pigs. These results indicated that both animal-borne influenza viruses could induce similar pathogenicity and immune response to those caused by human-common influenza viruses. Tree shrews may be an excellent animal model for studying the interaction between the influenza virus and the host and the cross-species infection mechanism of the animal influenza virus.

A multiplex real-time RT-PCR assay for the detection of H1, H2 and H3 subtype avian influenza viruses.

Avian influenza viruses (AIVs) are influenza A viruses, of which subtypes H1, H2 and H3 are highly transmissible in poultry and have the risk of transmission to human as well. It is important to establish an accurate, sensitive and convenient means of virus detection. In this study, we developed a multiplex real-time RT-PCR assay based on conserved sequences of the virus hemagglutinin and matrix, and designed primers and probes for the simultaneous and rapid detection of AIV subtypes H1, H2 and H3. We used different subtypes of AIVs and other avian respiratory viruses for evaluation of the specificity of this method. The results showed good sensitivity, specificity and reproducibility. The detection limit was 10-100 copies per reaction. The method also achieved good concordance with the virus isolation method when compared to 81 poultry samples evaluated. It provides a new method for detecting mixed infections of AIVs.

Field efficacy of HVT-H9 vaccination after natural infection of H9N2 avian influenza virus in broilers

H9N2 subtype avian influenza virus (AIV) has been persistently circulating in China. It causes huge economic losses to the poultry industry and poses a great threat to public health. Previously, we constructed a turkey herpesvirus live vector vaccine candidate strain expressing an H9 gene, HVT-H9. Results showed that immunisation with HVT-H9 could provide good immunity in specific pathogen free (SPF) chickens. In this study, field-bred Arbour Acres plus (AA+) broilers were additionally immunised with HVT-H9 at one day of age. Then, broilers were naturally infected with H9N2 AIV. During the endemic period, death occurred in flocks without HVT-H9 immunisation and the mortality rate increased rapidly, forming a clear death wave. However, HVT-H9 vaccination prevented broiler mortality. Etiological tests and serological tests showed that broilers were positive for H9N2 AIV. Collectively, HVT-H9 immunisation provided good immunity for broilers in the field by inhibiting H9N2 virus infection and transmission.

An epidemiological overview of human infections with HxNy avian influenza in the Western Pacific Region, 2003-2022.

Avian influenza subtype A(HxNy) viruses are zoonotic and may occasionally infect humans through direct or indirect contact, resulting in mild to severe illness and death. Member States in the Western Pacific Region (WPR) communicate and notify the World Health Organization of any human cases of A(HxNy) through the International Health Regulations (IHR 2005) mechanism. This report includes all notifications in the WPR with illness onset dates from 1 November 2003 to 31 July 2022. During this period, there were 1972 human infections with nine different A(HxNy) subtypes notified in the WPR. Since the last report, an additional 134 human avian influenza infections were notified from 1 October 2017 to 31 July 2022. In recent years there has been a change in the primary subtypes and frequency of reports of human A(HxNy) in the region, with a reduction of A(H7N9) and A(H5N1), and conversely an increase of A(H5N6) and A(H9N2). Furthermore, three new subtypes A(H7N4), A(H10N3) and A(H3N8) notified from the People's Republic of China were the first ever recorded globally. The public health risk from known A(HxNy) viruses remains low as there is no evidence of person-to-person transmission. However, the observed changes in A(HxNy) trends reinforce the need for effective and rapid identification to mitigate the threat of a pandemic from avian influenza if person-to-person transmission were to occur.

PA-X protein of H9N2 subtype avian influenza virus suppresses the innate immunity of chicken bone marrow-derived dendritic cells

H9N2 subtype avian influenza (AI) is an infectious disease associated with immunosuppression in poultry. Here, the regulation function of PA-X protein was determined on the host innate immune response of H9N2-infected chicken bone marrow-derived DCs (chBM-DCs). Based on 2 mutated viruses expressing PA-X protein (rTX) or deficient PA-X protein (rTX-FS), and the established culture system of chBM-DCs, results showed PA-X protein inhibited viral replication in chBM-DCs but not in non-immune chicken cells (DF-1). Moreover, PA-X protein downregulated the expression of phenotypic markers (CD40, CD86, and MHCII) and proinflammatory cytokine (IL-12 and IL-1β) of chBM-DCs. The mixed lymphocyte reaction between chBM-DCs and chicken T cells showed PA-X protein significantly decreased H9N2-infected chBM-DCs to induce T cell proliferation, implying a suppression of the DC-induced downstream T cell response. Taken together, these findings indicated that PA-X protein is a key viral protein to help H9N2 subtype AIVs escape the innate immunity of chBM-DCs.