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.     

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.     

Consecutive Natural Influenza A Virus Infections in Sentinel Mallards in the Evident Absence of Subtype‐Specific Hemagglutination Inhibiting Antibodies

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand‐raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT‐qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full‐length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian‐type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re‐introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein‐specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds.     

Detection of reassortant avian influenza A (H11N9) virus in wild birds in China

Human infectious avian influenza virus (AIV) H7N9 emerged in China in 2013. The N9 gene of H7N9, which has the ability to cause death in humans, originated from an H11N9 influenza strain circulating in wild birds. To investigate the frequency and distribution of the N9 gene of the H11N9 and H7N9 influenza virus circulating in wild birds between 2006 and 2015, 35,604 samples were collected and tested. No H7N9 but four strains of the H11N9 subtype AIV were isolated, and phylogenetic analyses showed that the four H11N9 viruses were intra‐subtype and inter‐subtype reassortant viruses. A sequence analysis revealed that all six internal genes of A/wild bird/Anhui/L306/2014 (H11N9) originated from an H9N2 AIV isolated in Korea. The H9N2 strain, which is an inner gene donor reassorted with other subtypes, is a potential threat to poultry and even humans. It is necessary to increase monitoring of the emergence and spread of H11N9 AIV in wild birds.     

Genetic characterization of an H13N2 low pathogenic avian influenza virus isolated from gulls in China

Low pathogenic avian influenza viruses circulate in wild birds but are occasionally transmitted to other species, including poultry, mammals and humans. To date, infections with low pathogenic avian influenza viruses of HA subtype 6, HA subtype 7, HA subtype 9 and HA subtype 10 among humans have been reported. However, the epidemiology, genetics and ecology of low pathogenic avian influenza viruses have not been fully understood thus far. Therefore, persistent surveillance of low pathogenic avian influenza virus infections in wild birds and other species is needed. Here, we found a low pathogenic avian influenza virus of the subtype H13N2 (abbreviated as WH42) in black‐tailed gulls in China. All gene sequences of this H13N2 virus were determined and used for subsequent analysis. Phylogenetic analysis of the HA gene and NA gene indicated that WH42 was derived from the Eurasian lineage. We analysed the timing of the reassortment events and found that WH42 was a reassortant whose genes were transferred from avian influenza viruses circulating in Asia, Europe and North America. Additionally, WH42 possessed several molecular markers associated with mammalian virulence and mammalian transmissibility. Interestingly, we also found low but detectable haemagglutination inhibition antibodies against H13N2 low pathogenic avian influenza virus in serum samples collected from chickens. Taken together, our findings show that the H13 virus may have been introduced into poultry and that sustainable surveillance in gulls and poultry is required.     

Complete genome sequence of a novel influenza A H1N2 virus circulating in swine from Central Bajio region,Mexico

The aim of this study was to perform the complete genome sequence of a swine influenza A H1N2 virus strain isolated from a pig in Guanajuato, México (A/swine/Mexico/GtoDMZC01/2014) and to report its seroprevalence in 86 counties at the Central Bajio zone. To understand the evolutionary dynamics of the isolate, we undertook a phylogenetic analysis of the eight gene segments. These data revealed that the isolated virus is a reassortant H1N2 subtype, as its genes are derived from human (HA, NP, PA) and swine (M, NA, PB1, PB2 and NS) influenza viruses. Pig serum samples were analysed by the hemagglutination inhibition test, using wild H1N2 and H3N2 strains (A/swine/México/Mex51/2010 [H3N2]) as antigen sources. Positive samples to the H1N2 subtype were processed using the field‐isolated H1N1 subtype (A/swine/México/Ver37/2010 [H1N1]). Seroprevalence to the H1N2 subtype was 26.74% in the sampled counties, being Jalisco the state with highest seroprevalence to this subtype (35.30%). The results herein reported demonstrate that this new, previously unregistered influenza virus subtype in México that shows internal genes from other swine viral subtypes isolated in the past 5 years, along with human virus‐originated genes, is widely distributed in this area of the country.     

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.     

Appearance of reassortant European avian‐origin H1 influenza A viruses of swine in Vietnam

Three subtypes—H1N1, H1N2 and H3N2—of influenza A viruses of swine (IAV s‐S) are currently endemic in swine worldwide, but there is considerable genotypic diversity among each subtype and limited geographical distribution. Through IAV s‐S monitoring in Vietnam, two H1N2 influenza A viruses were isolated from healthy pigs in Ba Ria‐Vung Tau Province, Southern Vietnam, on 2 December 2016. BLAST and phylogenetic analyses revealed that their HA and NA genes were derived from those of European avian‐like H1N2 IAV s‐S that contained avian‐origin H1 and human‐like N2 genes, and were particularly closely related to those of IAV s‐S circulating in the Netherlands, Germany or Denmark. In addition, the internal genes of these Vietnamese isolates were derived from human A(H1N1)pdm09 viruses, suggesting that the Vietnamese H1N2 IAV s‐S are reassortants between European H1N2 IAV s‐S and human A(H1N1)pdm09v. The appearance of European avian‐like H1N2 IAV s‐S in Vietnam marks their first transmission outside Europe. Our results and statistical analyses of the number of live pigs imported into Vietnam suggest that the European avian‐like H1N2 IAV s‐S may have been introduced into Vietnam with their hosts through international trade. These findings highlight the importance of quarantining imported pigs to impede the introduction of new IAV s‐S.     

Continued reassortment of avian H6 influenza viruses from Southern China, 2014–2016

H6 subtype avian influenza virus (AIV) was prevalent in poultry and could sporadically infect humans. Here, a total of 196 novel H6 AIVs isolated from poultry in eight provinces of China from 2014 to 2016 were phylogenetically characterized. Our analysis revealed that they could be divided into two clades in the Asian H6 HA lineage, A/wild duck/Shantou/2853/2003(H6N2) (ST2853‐like) (85.7%) and A/duck/Shantou/339/2000(H6N2) (ST339‐like) (14.3%), in which ST2853‐like strains predominate. These novel strains belonged to the H6N6 (n = 165, 84.2%), H6N2 (n = 30, 15.3%), and H6N3 (n = 1, 0.51%) subtypes, which could be classified into 36 genotypes including 12 novel genotypes described in this study. In particular, several strains possessed the V190 and S228 mutations in HA (H3 numbering), which is critical for human receptor binding and identical to the human‐derived strain A/Taiwan/2/2013(H6N1). Furthermore, 10.3% of the H6N6 isolates possessed the N6‐∆11b (59–69) deletion. In summary, we describe phylogenetic and molecular characterizations of H6 AIVs in southern China and highlight the constant prevalence of H6 AIVs in poultry as well as adaptation to mammalian hosts.     

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.     

A new reassortant clade 2.3.2.1a H5N1 highly pathogenic avian influenza virus causing recent outbreaks in ducks,geese, chickens and turkeys in Bangladesh

A total of 15 dead or sick birds from 13 clinical outbreaks of avian influenza in ducks, geese, chickens and turkeys in 2017 in Bangladesh were examined. The presence of H5N1 influenza A virus in the affected birds was detected by RT‐PCR. Phylogenetic analysis based on full‐length gene sequences of all eight gene segments revealed that these recent outbreaks were caused by a new reassortant of clade 2.3.2.1a H5N1 virus, which had been detected earlier in 2015 during surveillance in live bird markets (LBMs) and wet lands. This reassortant virus acquired PB2, PB1, PA, NP and NS genes from low pathogenic avian influenza viruses mostly of non‐H9N2 subtypes but retained HA, NA and M genes of the old clade 2.3.2.1a viruses. Nevertheless, the HA gene of these new viruses was 2.7% divergent from that of the old clade 2.3.2.1a viruses circulated in Bangladesh. Interestingly, similar reassortment events could be traced back in four 2.3.2.1a virus isolates of 2013 from backyard ducks. It suggests that this reassortant virus emerged in 2013, which took two years to be detected at a broader scale (i.e. in LBMs), another two years until it became widely spread in poultry and fully replaced the old viruses. Several mutations were detected in the recent Bangladeshi isolates, which are likely to influence possible phenotypic alterations such as increased mammalian adaptation, reduced susceptibility to antiviral agents and reduced host antiviral response.     

Evolutionary and Ecological Dynamics of Transboundary Disease Caused by H5N1 Virus in Southeast Asia

Southeast Asia has been the breeding ground for many emerging diseases in the past decade, and it is in this region that new genetic variants of HPAI H5N1 viruses have been emerging. Cross‐border movement of animals accelerates the spread of H5N1, and the changing environmental conditions also exert strong selective pressure on the viruses. The transboundary zoonotic diseases caused by H5N1 pose a serious and continual threat to global economy and public health. Here, we divided the H5N1 viruses isolated in Southeast Asia during 2003–2009 into four groups according to their phylogenetic relationships among HA gene sequences. Molecular evolution analysis suggests populations in expansion rather than a positive selection for group 2 and group 3, yet group 4 is under strong positive selection. Site 193 was found to be a potential glycosylation site and located in receptor‐binding domain. Note that site 193 tends to appear in avian isolates instead of human strains. Population dynamics analysis reveals that the effective population size of infections in Southeast Asia has undergone three obvious increases, and the results are consistent with the epidemiological analysis. Ecological and phylogeographical analyses show that agro‐ecological environments, migratory birds, domestic waterfowl, especially free‐ranging ducks, are crucial in the occurrence, maintenance and spread of H5N1 virus. The epidemiological links between Indonesia and Suphanburi observed suggest that viruses in Indonesia were originated from multiple introductions.     

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.     

Characteristics of the first H16N3 subtype influenza A viruses isolated in western China

The first documented avian influenza virus subtype H16N3 was isolated in 1975 and is currently detectable in many countries worldwide. However, the prevalence, biological characteristics and threat to humans of the avian influenza virus H16N3 subtype in China remain poorly understood. We performed avian influenza surveillance in major wild bird gatherings across the country from 2017 to 2019, resulting in the isolation of two H16N3 subtype influenza viruses. Phylogenetic analysis showed these viruses belong to the Eurasian lineage, and both viruses presented the characteristics of inter‐species reassortment. In addition, the two viruses exhibited limited growth capacity in MDCK and A549 cells. Receptor‐binding assays indicated that the two H16N3 viruses presented dual receptor‐binding profiles, being able to bind to both human and avian‐type receptors, where GBHG/NX/2/2018(H16N3) preferentially bound the avian‐type receptor, while GBHG/NX/1/2018(H16N3) showed greater binding to the human‐type receptor, even the mice virulence data showed the negative results. Segments from other species have been introduced into the H16N3 avian influenza virus, which may alter its pathogenicity and host tropism, potentially posing a threat to animal and human health in the future. Consequently, it is necessary to increase monitoring of the emergence and spread of avian influenza subtype H16N3 in wild birds.     

Emergence and adaptation of H3N2 canine influenza virus from avian influenza virus: An overlooked role of dogs in interspecies transmission

H3N2 canine influenza virus (CIV) originated from avian species and emerged in dogs in Asia around 2005 where it became enzootic before reaching the USA in 2015. To investigate the key aspects of the evolution of H3N2 CIV regarding its emergence and adaptation in the canine host, we conducted an extensive analysis of all publicly available H3N2 CIV sequences spanning a 10‐year period. We believe that H3N2 AIVs transferred to canines around 2002–2004. Furthermore, H3N2 CIVs could be divided into seven major clades with strong geographic clustering and some changed sites evidence of adaptive evolution. Most notably, the dN/dS of each H3N2 CIVs segment was higher than the correspondent of H3N2 AIVs and the U content of HA and NA was increasing over time, suggesting the idea that this avian‐origin virus may be gradually adapting to the host. Our results provide a framework to elucidate a general mechanism for emergence of novel influenza viruses.     

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.     

Domestic ducks play a major role in the maintenance and spread of H5N8 highly pathogenic avian influenza viruses in South Korea

The H5N8 highly pathogenic avian influenza viruses (HPAIVs) belonging to clade 2.3.4.4 spread from Eastern China to Korea in 2014 and caused outbreaks in domestic poultry until 2016. To understand how H5N8 HPAIVs spread at host species level in Korea during 2014–2016, a Bayesian phylogenetic analysis was used for ancestral state reconstruction and estimation of the host transition dynamics between wild waterfowl, domestic ducks and chickens. Our data support that H5N8 HPAIV most likely transmitted from wild waterfowl to domestic ducks, and then maintained in domestic ducks followed by dispersal of HPAIV from domestic ducks to chickens, suggesting domestic duck population plays a central role in the maintenance, amplification and spread of wild HPAIV to terrestrial poultry in Korea.     

Influenza A Virus in Backyard Pigs and Poultry in Rural Cambodia

Surveillance of influenza virus in humans and livestock is critical, given the worldwide public health threats and livestock production losses. Livestock farming involving close proximity between humans, pigs and poultry is often practised by smallholders in low‐income countries and is considered an important driver of influenza virus evolution. This study determined the prevalence and genetic characteristics of influenza A virus (IAV) in backyard pigs and poultry in Cambodia. A total of 751 animals were tested by matrix gene‐based rRT‐PCR, and influenza virus was detected in 1.5% of sampled pigs, 1.4% of chickens and 1.0% of ducks, but not in pigeons. Full‐length genome sequencing confirmed triple reassortant H3N2 in all IAV‐positive pigs and various low pathogenic avian influenza subtypes in poultry. Phylogenetic analysis of the swine influenza viruses revealed that these had haemagglutinin and neuraminidase genes originating from human H3N2 viruses previously isolated in South‐East Asia. Phylogenetic analysis also revealed that several of the avian influenza subtypes detected were closely related to internal viral genes from highly pathogenic H5N1 and H9N2 formerly sequenced in the region. High sequence homology was likewise found with influenza A viruses circulating in pigs, poultry and wild birds in China and Vietnam, suggesting transboundary introduction and cocirculation of the various influenza subtypes. In conclusion, highly pathogenic subtypes of influenza virus seem rare in backyard poultry, but virus reassortment, involving potentially zoonotic and pandemic subtypes, appears to occur frequently in smallholder pigs and poultry. Increased targeted surveillance and monitoring of influenza circulation on smallholdings would further improve understanding of the transmission dynamics and evolution of influenza viruses in humans, pigs and poultry in the Mekong subregion and could contribute to limit the influenza burden.