Highly Pathogenic Avian Influenza H5N8 Clade 2.3.4.4 Virus: Equivocal Pathogenicity and Implications for Surveillance Following Natural Infection in Breeder Ducks in the United Kingdom

Since early 2014, several outbreaks involving novel reassortant highly pathogenic avian influenza (HPAI) A(H5N8) viruses have been detected in poultry and wild bird species in Asia, Europe and North America. These viruses have been detected in apparently healthy and dead wild migratory birds, as well as in domestic chickens, turkeys, geese and ducks. In this study, we describe the pathology of an outbreak of H5N8 HPAIV in breeder ducks in the UK. A holding with approximately 6000 breeder ducks, aged approximately 60 weeks, showed a gradual reduction in egg production and increased mortality over a 7‐day period. Post‐mortem examination revealed frequent fibrinous peritonitis, with severely haemorrhagic ovarian follicles and occasional splenic and pancreatic necrosis and high incidence of mycotic granulomas in the air sacs and lung. Low‐to‐moderate levels of HPAI H5N8 virus were detected mainly in respiratory and digestive tract, with minor involvement of other organs. Although histopathological examination confirmed the gross pathology findings, intralesional viral antigen detection by immunohistochemistry was not observed. Immunolabelled cells were rarely only present in inflamed air sacs and serosa, usually superficial to granulomatous inflammation. Abundant bacterial microcolonies were observed in haemorrhagic ovaries and oviduct. The limited viral tissue distribution and presence of inter‐current fungal and bacterial infections suggest a minor role for HPAIV H5N8 in clinical disease in layer ducks.     

Comparative pathogenicity of H5N6 subtype highly pathogenic avian influenza viruses in chicken,Pekin duck and Muscovy duck

In Japan during the 2016–2017 winter season, clade 2.3.4.4 highly pathogenic avian influenza viruses (HPAIVs) of the H5N6 subtype caused 12 outbreaks in chicken and Muscovy duck farms. These viruses have been circulating in Vietnam and China since 2014. In this study, we evaluated the susceptibility of chicken, Pekin duck (Anas platyrhynchos domesticus) and Muscovy duck (Cairina moschata) to H5N6 HPAIVs that originated in Japan, Vietnam and China. The H5N6 HPAIVs examined in this study were highly lethal to chickens compared with their pathogenicity in Pekin duck and Muscovy duck. One of five chickens infected with A/Muscovy duck/Aomori/1‐3T/2016 (MusDk/Aomori) survived despite viral shedding, although all of the chickens infected with the other viruses died. The 50% chicken lethal dose differed among the Japanese strains that shared the same gene constellation indicating that gene constellation was not a major determinant of pathogenicity in chicken. MusDk/Aomori, A/chicken/Niigata/1‐1T/2016 (Ck/Niigata) and A/duck/Hyogo/1/2016 (Dk/Hyogo) infected all Muscovy ducks inoculated; Ck/Niigata killed 50% of the ducks it infected whereas the other two did not kill any ducks. A/chicken/Japan/AnimalQuarantine‐HE144/2016 (HE144) isolated from chicken meat that originated in China was highly pathogenic to Pekin duck: all of the ducks died within 3.75 days of inoculation. This study shows that the pathogenicity of the clade 2.3.4.4 H5N6 HPAIVs differs not only between hosts but also within the same host species.     

Epidemiological and Molecular Analysis of an Outbreak of Highly Pathogenic Avian Influenza H5N8 clade 2.3.4.4 in a German Zoo: Effective Disease Control with Minimal Culling

Outbreaks of highly pathogenic avian influenza A virus (HPAIV) subtype H5N8, clade 2.3.4.4, were first reported in January 2014 from South Korea. These viruses spread rapidly to Europe and the North American continent during autumn 2014 and caused, in Germany, five outbreaks in poultry holdings until February 2015. In addition, birds kept in a zoo in north‐eastern Germany were affected. Only a few individual white storks (Ciconia ciconia) showed clinical symptoms and eventually died in the course of the infection, although subsequent in‐depth diagnostic investigations showed that other birds kept in the same compound of the white storks were acutely positive for or had undergone asymptomatic infection with HPAIV H5N8. An exception from culling all of the 500 remaining zoo birds was granted by the competent authority. Restriction measures included grouping the zoo birds into eight epidemiological units in which 60 birds of each unit tested repeatedly negative for H5N8. Epidemiological and phylogenetical investigations revealed that the most likely source of introduction was direct or indirect contact with infected wild birds as the white storks had access to a small pond frequented by wild mallards and other aquatic wild birds during a period of 10 days in December 2014. Median network analysis showed that the zoo bird viruses segregated into a distinct cluster of clade 2.3.4.4 with closest ties to H5N8 isolates obtained from mute swans (Cygnus olor) in Sweden in April 2015. This case demonstrates that alternatives to culling exist to rescue valuable avifaunistic collections after incursions of HPAIV.     

Phylogenetic variations of highly pathogenic H5N6 avian influenza viruses isolated from wild birds in the Izumi plain,Japan, during the 2016–17 winter season

During the 2016–2017 winter season, we isolated 33 highly pathogenic avian influenza viruses (HPAIVs) of H5N6 subtype and three low pathogenic avian influenza viruses (LPAIVs) from debilitated or dead wild birds, duck faeces, and environmental water samples collected in the Izumi plain, an overwintering site for migratory birds in Japan. Genetic analyses of the H5N6 HPAIV isolates revealed previously unreported phylogenetic variations in the PB2, PB1, PA, and NS gene segments and allowed us to propose two novel genotypes for the contemporary H5N6 HPAIVs. In addition, analysis of the four gene segments identified close phylogenetic relationships between our three LPAIV isolates and the contemporary H5N6 HPAIV isolates. Our results implied the co‐circulation and co‐evolution of HPAIVs and LPAIVs within the same wild bird populations, thereby highlighting the importance of avian influenza surveillance targeting not only for HPAIVs but also for LPAIVs.     

A novel H7N3 reassortant originating from the zoonotic H7N9 highly pathogenic avian influenza viruses that has adapted to ducks

The first human case of zoonotic H7N9 avian influenza virus (AIV) infection was reported in March 2013 in China. This virus continues to circulate in poultry in China while mutating to highly pathogenic AIVs (HPAIVs). Through monitoring at airports in Japan, a novel H7N3 reassortant of the zoonotic H7N9 HPAIVs, A/duck/Japan/AQ‐HE30‐1/2018 (HE30‐1), was detected in a poultry meat product illegally brought by a passenger from China into Japan. We analysed the genetic, pathogenic and antigenic characteristics of HE30‐1 by comparing it with previous zoonotic H7N9 AIVs and their reassortants. Phylogenetic analysis of the entire HE30‐1 genomic sequence revealed that it comprised at least three different sources; the HA (H7), PB1, PA, NP, M and NS segments of HE30‐1 were directly derived from H7N9 AIVs, whereas the NA (N3) and PB2 segments of HE30‐1 were unrelated to zoonotic H7N9. Experimental infection revealed that HE30‐1 was lethal in chickens but not in domestic or mallard ducks. HE30‐1 was shed from and replicated in domestic and mallard ducks and chickens, whereas previous zoonotic H7N9 AIVs have not adapted well to ducks. This finding suggests the possibility that HE30‐1 may disseminate to remote area by wild bird migration once it establishes in wild bird population. A haemagglutination‐inhibition assay indicated that antigenic drift has occurred among the reassortants of zoonotic H7N9 AIVs; HE30‐1 showed similar antigenicity to some of those H7N9 AIVs, suggesting it might be prevented by the H5/H7 inactivated vaccine that was introduced in China in 2017. Our study reports the emergence of a new reassortant of zoonotic H7N9 AIVs with novel viral characteristics and warns of the challenge we still face to control the zoonotic H7N9 AIVs and their reassortants.     

Outbreaks of highly pathogenic avian influenza in zoo birds caused by HA clade 2.3.4.4 H5N6 subtype viruses in Japan in winter 2016

In late 2016, two zoos, one in northern Japan and the other in central Japan, experienced highly pathogenic avian influenza (HPAI) outbreaks, in which multiple zoo birds were infected with H5N6 subtype HPAI virus (HPAIV). Here, we report an overview of these HPAI outbreaks. HPAIV infections were confirmed by virus isolation in three black swans (Cygnus atratus) and three snowy owls (Bubo scandiacus) kept in the Omoriyama Zoo hospital. At Higashiyama Zoo and Botanical Gardens, following the death of a black swan at a zoo pond, nine waterfowl, including two black swans, four cackling geese (Branta hutchinsii leucopareia), two mallards (Anas platyrhynchos), and a wigeon (Anas penelope), died after HPAIV infection in isolation facilities. Based on the presence of H5‐specific antibodies in their sera, two surviving black swans and a surviving mallard at Higashiyama Zoo appeared to have HPAIV infection, although the virus was not isolated. The detectable levels of antibodies (≥10 HI) were maintained for at least 5–9 months, as determined by haemagglutinin inhibition test. Isolation of two H5N6 subtype HPAIVs from an open‐air pond where affected zoo birds were previously housed at Higashiyama Zoo strongly indicates that wild waterfowl associated with aquatic environments brought the virus to the zoo. The phylogenetic relationships of the 18 isolates indicated direct viral transmission among birds within each zoo. In both zoos, containment of suspected birds in isolation facilities might have allowed the virus spread among birds inside the facility. However, maintaining containment measures and strict sanitation procedures could facilitate successful physical containment and clearance of HPAIV in both zoos.     

Respiratory disease due to mixed viral infections in poultry flocks in Egypt between 2017 and 2018: Upsurge of highly pathogenic avian influenza virus subtype H5N8 since 2018

For several years, poultry production in Egypt has been suffering from co‐circulation of multiple respiratory viruses including highly pathogenic avian influenza virus (HPAIV) H5N1 (clade 2.2.1.2) and low pathogenic H9N2 (clade G1‐B). Incursion of HPAIV H5N8 (clade 2.3.4.4b) to Egypt in November 2016 via wild birds followed by spread into commercial poultry flocks further complicated the situation. Current analyses focussed on 39 poultry farms suffering from respiratory manifestation and high mortality in six Egyptian governorates during 2017–2018. Real‐time RT‐PCR (RT‐qPCR) substantiated the co‐presence of at least two respiratory virus species in more than 80% of the investigated flocks. The percentage of HPAIV H5N1‐positive holdings was fairly stable in 2017 (12.8%) and 2018 (10.2%), while the percentage of HPAIV H5N8‐positive holdings increased from 23% in 2017 to 66.6% during 2018. The proportion of H9N2‐positive samples was constantly high (2017:100% and 2018:63%), and H9N2 co‐circulated with HPAIV H5N8 in 22 out of 39 (56.8%) flocks. Analyses of 26 H5, 18 H9 and 4 N2 new sequences confirmed continuous genetic diversification. In silico analysis revealed numerous amino acid substitutions in the HA and NA proteins suggestive of increased adaptation to mammalian hosts and putative antigenic variation. For sensitive detection of H9N2 viruses by RT‐qPCR, an update of primers and probe sequences was crucial. Reasons for the relative increase of HPAIV H5N8 infections versus H5N1 remained unclear, but lack of suitable vaccines against clade 2.3.4.4b cannot be excluded. A reconsideration of surveillance and control measures should include updating of diagnostic tools and vaccination strategies.     

Isolation of highly pathogenic H5N6 avian influenza virus in Southern Vietnam with genetic similarity to those infecting humans in China

Since 2013, H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been responsible for outbreaks in poultry and wild birds around Asia. H5N6 HPAIV is also a public concern due to sporadic human infections being reported in China. In the current study, we isolated an H5N6 HPAIV strain (A/Muscovy duck/Long An/AI470/2018; AI470) from an outbreak at a Muscovy duck farm in Long An Province in Southern Vietnam in July 2018 and genetically characterized it. Basic Local Alignment Search Tool (BLAST) analysis revealed that the eight genomic segments of AI470 were most closely related (99.6%–99.9%) to A/common gull/Saratov/1676/2018 (H5N6), which was isolated in October 2018 in Russia. Furthermore, AI470 also shared 99.4%–99.9% homology with A/Guangxi/32797/2018, an H5N6 HPAIV strain that infected humans in China in 2018. Phylogenetic analyses of the entire genome showed that AI470 was directly derived from H5N6 HPAIVs that were in South China from 2015 to 2018 and clustered with four H5N6 HPAIV strains of human origin in South China from 2017 to 2018. This indicated that AI470 was introduced into Vietnam from China. In addition, molecular characteristics related to mammalian adaptation among the recent human H5N6 HPAIV viruses, except PB2 E627K, were shared by AI470. These findings are cause for concern since H5N6 HPAIV strains that possess a risk of human infection have crossed the Chinese border.     

Genetic relationship between poultry and wild bird viruses during the highly pathogenic avian influenza H5N6 epidemic in the Netherlands, 2017–2018

In the Netherlands, three commercial poultry farms and two hobby holdings were infected with highly pathogenic avian influenza (HPAI) H5N6 virus in the winter of 2017–2018. This H5N6 virus is a reassortant of HPAI H5N8 clade 2.3.4.4 group B viruses detected in Eurasia in 2016. H5N6 viruses were also detected in several dead wild birds during the winter. However, wild bird mortality was limited compared to the caused by the H5N8 group B virus in 2016–2017. H5N6 virus was not detected in wild birds after March, but in late summer infected wild birds were found again. In this study, the complete genome sequences of poultry and wild bird viruses were determined to study their genetic relationship. Genetic analysis showed that the outbreaks in poultry were not the result of farm‐to‐farm transmissions, but rather resulted from separate introductions from wild birds. Wild birds infected with viruses related to the first outbreak in poultry were found at short distances from the farm, within a short time frame. However, no wild bird viruses related to outbreaks 2 and 3 were detected. The H5N6 virus isolated in summer shares a common ancestor with the virus detected in outbreak 1. This suggests long‐term circulation of H5N6 virus in the local wild bird population. In addition, the pathogenicity of H5N6 virus in ducks was determined, and compared to that of H5N8 viruses detected in 2014 and 2016. A similar high pathogenicity was measured for H5N6 and H5N8 group B viruses, suggesting that biological or ecological factors in the wild bird population may have affected the mortality rates during the H5N6 epidemic. These observations suggest different infection dynamics for the H5N6 and H5N8 group B viruses in the wild bird population.     

Association of wild bird densities around poultry farms with the risk of highly pathogenic avian influenza virus subtype H5N8 outbreaks in the Netherlands, 2016

Highly pathogenic (HP) avian influenza viruses (AIV) can spread globally through migratory birds and cause massive outbreaks in commercial poultry. AIV outbreaks have been associated with proximity to waterbodies, presence of waterfowl or wild bird cases near poultry farms. In this study, we compared densities of selected HPAI high‐risk wild bird species around 7 locations (H farms) infected with HPAIV H5N8 in the Netherlands in 2016–2017 to densities around 21 non‐infected reference farms. Nine reference farms were in low‐lying water‐rich areas (R‐W) and 12 in higher non‐water‐rich areas (R‐NW). Average monthly numbers/km² of Eurasian wigeons, tufted ducks, Anatidae (ducks, geese and swans) and Laridae (gulls) were calculated between September and April in rings of 0–1, 1–3, 3–6 and 6–10 km around the farms. Linear mixed model analyses showed generally higher bird densities for H and R‐W compared to R‐NW farms between October and March. This was most striking for Eurasian wigeons, with in peak month December 105 (95% CI:17–642) and 40 (7–214) times higher densities around H and R‐W farms, respectively, compared to R‐NW farms. Increased densities around H farms for Eurasian wigeons and Anatidae were more pronounced for distances up to 10 km compared to 0–1 km that mostly consists of the farm yard, which is an unattractive habitat for waterfowl. This distance effect was not observed in gulls, nor in tufted ducks that live on large open waterbodies which are unlikely to be within 0–1 km of farms. This study provides insights into spatio‐temporal density dynamics of HPAI high‐risk birds around farms and their associations with poultry outbreaks. The outcomes indicate that knowledge of environmental and ecological drivers for wild bird presence and abundance may facilitate identification of priority areas for surveillance and biosecurity measures and decisions on establishments of poultry farms to reduce risk of HPAI outbreaks.     

First Case of Highly Pathogenic Avian Influenza in Poultry in Spain

In October 2009, highly pathogenic avian influenza virus (HPAIV) was isolated for the first time in poultry in Spain. Sequencing analysis revealed that it was an H7N7 HPAIV. The progenitors of H7 HPAIV strains involved in recent European poultry outbreaks were simultaneously circulating in wild birds. The infected Spanish farm is located close to a reservoir abundant in wild birds. Epidemiological investigation found no links to other poultry holdings and those located in the control area were negative for AIV. Previous spatial risk analyses had identified the area where the infected holding is located to beat high relative risk for the introduction and presence of H5N1 HPAIV by wild birds. We suggest a risk‐based surveillance scheme that targets smaller geographical units but maintains the number of wild birds being sampled, as early detection of potentially pathogenic AIV is crucial in preventing spread to poultry.     

Avian metapneumovirus subtype C in Wild Waterfowl in Ontario,Canada

Avian metapneumovirus (aMPV ) is an emerging poultry pathogen that has a significant economic impact on poultry production worldwide. The geographic range of the virus continues to expand, and wild birds have been implicated as reservoirs of aMPV that have the potential to spread the virus over long distances. Our objective was to determine the apparent prevalence of aMPV subtype C in wild waterfowl in Ontario, Canada. Wild waterfowl were captured in August and September, 2016 as part of routine migratory waterfowl population monitoring by the Ontario Ministry of Natural Resources and Forestry. Oropharyngeal and cloacal swabs were collected from each bird and placed together for aMPV testing using real‐time RT ‐PCR . A total of 374 live wild birds from 23 lakes were sampled and tested for aMPV . Among all ducks tested, 84 (22%) were positive for aMPV . The proportion of samples that tested positive ranged from 0% in ring‐necked ducks (Aythya collaris ) and green‐winged teal (Anas carolinensis ) to 44% (8 of 18) in American black ducks (A. rubripes ). Waterfowl positive for aMPV were found at 14 of 23 lakes in the study area and the percent positive at these 14 lakes ranged between 5% and 84%. Although subtype C aMPV has been detected in a variety of wild birds in North America, this is the first report of aMPV in wild ducks in Ontario, Canada. The high apparent prevalence, particularly in mallards and American black ducks (37 and 44%, respectively), suggests that these species may be important reservoirs of aMPV . Given the potential impact of aMPV on domestic poultry and the potential role of wild birds as reservoirs of the virus, further investigation of the geographic distribution, risk factors associated with aMPV carriage in wild waterfowl and potential role of other birds in the epidemiology of aMPV in Canada is warranted.     

Spatiotemporal evolutionary epidemiology of H5N1 highly pathogenic avian influenza in West Africa and Nigeria, 2006–2015

H5N1 highly pathogenic avian influenza virus (HPAIV) was first observed in Nigeria in early 2006 and has now spread to more than 17 African countries having severe economic and public health implications. Here, we explore the spatiotemporal patterns of viral dispersal both among West African countries and within Nigeria using sequence data from hemagglutinin (HA) gene region of the virus. Analyses were performed within a statistical Bayesian framework using phylodynamic models on data sets comprising of all publically available HA sequence data collected from seven West African countries and Egypt between 2006 and 2015. Our regional‐level analyses indicated that H5N1 in West Africa originated in Nigeria in three geopolitical regions, specifically north central and north‐east, where backyard poultry and wild birds are in frequent contact, as well as south‐west, a major commercial poultry area, then dispersed to West African countries. We inferred significant virus dispersal routes between Niger and Nigeria on one side and Burkina Faso, Ivory Coast, Ghana and Egypt on the other. Furthermore, south‐west Nigeria identified as a primary source for virus dispersal within Nigeria as well as to Niger in 2006 and 2008. Niger was an important epicentre for the virus spread into other West African countries in 2015. Egyptian introductions from West Africa were sporadic and resulted most likely from poultry trade with Nigeria rather than contact with infected wild birds. Our inferred viral dispersal routes reflected the large‐scale unrestricted movements of infected poultry in the region. Our study illustrates the ability of phylodynamic models to trace important HPAIV dispersal routes at a regional and national level. Our results have clear implications for the control and prevention of this pathogen across scales and will help improve molecular surveillance of transboundary HPAIVs.     

Poultry trading behaviours in Vietnamese live bird markets as risk factors for avian influenza infection in chickens

Vietnamese poultry are host to co‐circulating subtypes of avian influenza viruses, including H5N1 and H9N2, which pose a great risk to poultry productivity and to human health. AIVs circulate throughout the poultry trade network in Vietnam, with live bird markets being an integral component to this network. Traders at LBMs exhibit a variety of trading practices, which may influence the transmission of AIVs. We identified trading practices that impacted on AIV prevalence in chickens marketed in northern Vietnamese LBMs. We generated sequencing data for 31 H9N2 and two H5N6 viruses. Viruses isolated in the same LBM or from chickens sourced from the same province were genetically closer than viruses isolated in different LBMs or from chickens sourced in different provinces. The position of a vendor in the trading network impacted on their odds of having AIV‐infected chickens. Being a retailer and purchasing chickens from middlemen was associated with increased odds of infection, whereas odds decreased if vendors purchased chickens directly from large farms. Odds of infection were also higher for vendors having a greater volume of ducks unsold per day. These results indicate how the spread of AIVs is influenced by the structure of the live poultry trading network.     

Prevalence of H7N9 subtype avian influenza viruses in poultry in China, 2013–2018

H7N9 subtype avian influenza viruses (AIV) circulating in China over recent years have had an enormous impact on public health and economy. During the period between November 2016 and April 2017, an increase in human infections caused by these viruses was reported, with rapid emergence and spread of variants in China. Consequently, the government of China implemented a controversial vaccination strategy in September 2017. Here, we provide evidence of the prevalence of H7N9 AIVs in China based on systematic large‐scale surveillance in poultry during 2013–2018. Emerging variants were confirmed as highly pathogenic in chickens using the intravenous pathogenicity index (IVPI) test. The currently available vaccine provided complete protection against the H7N9 HPAIV challenge in chickens. The collective findings clearly indicate that the vaccination strategy implemented not only significantly decreases the prevalence of H7N9 AIVs in poultry but also effectively prevents human infection with H7N9 viruses.     

Experimental infection of highly pathogenic avian influenza viruses,Clade 2.3.4.4 H5N6 and H5N8, in Mandarin ducks from South Korea

Outbreaks of highly pathogenic avian influenza (HPAI ) have been reported worldwide. Wild waterfowl play a major role in the maintenance and transmission of HPAI . Highly pathogenic avian influenza subtype H5N6 and H5N8 viruses simultaneously emerged in South Korea. In this study, the comparative pathogenicity and infectivity of Clade 2.3.4.4 Group B H5N8 and Group C H5N6 viruses were evaluated in Mandarin duck (Aix galericulata ). None of the ducks infected with H5N6 or H5N8 viruses showed clinical signs or mortality. Serological assays revealed that the HA antigenicity of H5N8 and H5N6 viruses was similar to each other. Moreover, both the viruses did not replicate after cross‐challenging with H5N8 and H5N6 viruses, respectively, as the second infection. Although both the viruses replicated in most of the internal organs of the ducks, viral replication and shedding through cloaca were higher in H5N8‐infected ducks than in H5N6‐infected ducks. The findings of this study provide preliminary information to help estimate the risks involved in further evolution and dissemination of Clade 2.3.4.4 HPAI viruses among wild birds.     

Highly Pathogenic Avian Influenza H5N8 in Germany: Outbreak Investigations

Epidemiological outbreak investigations were conducted in highly pathogenic avian influenza virus of the subtype H5N8 (HPAIV H5N8)‐affected poultry holdings and a zoo to identify potential routes of entry of the pathogen via water, feedstuffs, animals, people, bedding material, other fomites (equipment, vehicles etc.) and the presence of wild birds near affected holdings. Indirect introduction of HPAIV H5N8 via material contaminated by infected wild bird seems the most reasonable explanation for the observed outbreak series in three commercial holdings in Mecklenburg‐Western Pomerania and Lower Saxony, while direct contact to infected wild birds may have led to outbreaks in a zoo in Rostock and in two small free‐range holdings in Anklam, Mecklenburg‐Western Pomerania.     

Characterization of avian influenza H5N3 reassortants isolated from migratory waterfowl and domestic ducks in China from 2015 to 2018

Wild and domestic aquatic birds are the natural reservoirs of avian influenza viruses (AIVs). All subtypes of AIVs, including 16 hemagglutinin (HA) and nine neuraminidase (NA), have been isolated from the waterfowls. The H5 viruses in wild birds display distinct biological differences from their highly pathogenic H5 counterparts. Here, we isolated seven H5N3 AIVs including three from wild birds and four from domestic ducks in China from 2015 to 2018. The isolation sites of all the seven viruses were located in the region of the East Asian‐Australasian Migratory Flyway. Phylogenetic analysis indicated that the surface genes of these viruses originated from the wild bird H5 HA subtype and the N3 Eurasian lineage. The internal genes of the seven H5N3 isolates are derived from the five gene donors isolated from the wild birds or ducks in Eastern‐Asia region. They were also divided into five genotypes according to their surface genes and internal gene combinations. Interestingly, two of the seven H5N3 viruses contributed their partial internal gene segments (PB1, M and NS) to the newly emerged H7N4 reassortants, which have caused first human H7N4 infection in China in 2018. Moreover, we found that the H5N3 virus used in this study react with the anti‐serum of the H5 subtype vaccine isolate (Re‐11 and Re‐12) and reacted well with the Re‐12 anti‐serum. Our findings suggest that worldwide intensive surveillance and the H5 vaccination (Re‐11 and Re‐12) in domestic ducks are needed to monitor the emergence of novel H5N3 reassortants in wild birds and domestic ducks and to prevent H5N3 viruses transmission from the apparently healthy wild birds and domestic ducks to chickens.     

Genetic characteristics,pathogenicity and transmission of H5N6 highly pathogenic avian influenza viruses in Southern China

Since 2014, H5 highly pathogenic avian influenza viruses (HPAIVs) from clade 2.3.4.4 have been persistently circulating in Southern China. This has caused huge losses in the poultry industry. In this study, we analysed the genetic characteristics of seven H5N6 HPAIVs of clade 2.3.4.4 that infected birds in Southern China in 2016. Phylogenetic analysis grouped the HA, PB2, PA, M and NS genes as MIX‐like, and the NA genes grouped into the Eurasian lineage. The PB1 genes of the GS24, GS25, CK46 and GS74 strains belonged to the VN 2014‐like group and the others were grouped as MIX‐like. The NP genes of GS24 and GS25 strains belonged to the ZJ‐like group, but the others were MIX‐like. Thus, these viruses came from different genotypes, and the GS24, GS25, CK46 and GS74 strains displayed genotype recombination. Additionally, our results showed that the mean death time of all chickens inoculated with 10⁵ EID₅₀ of CK46 or GS74 viruses was 3 and 3.38 days, respectively. The viruses replicated at high titers in all tested tissues of the inoculated chickens. They also replicated in all tested tissues of naive contact chickens, but their replication titers in some tissues were significantly different (p < 0.05). Thus, the viruses displayed high pathogenicity and variable transmission in chickens. Therefore, it is necessary to focus on the pathogenic variation and molecular evolution of H5N6 HPAIVs in order to prevent and control avian influenza in China.