Experimental infection of H5N1 and H5N8 highly pathogenic avian influenza viruses in Northern Pintail (Anas acuta)

The wide geographic spread of Eurasian Goose/Guangdong lineage highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses by wild birds is of great concern. In December 2014, an H5N8 HPAI clade 2.3.4.4 Group A (2.3.4.4A) virus was introduced to North America. Long‐distance migratory wild aquatic birds between East Asia and North America, such as Northern Pintail (Anas acuta ), were strongly suspected of being a source of intercontinental transmission. In this study, we evaluated the pathogenicity, infectivity and transmissibility of an H5N8 HPAI clade 2.3.4.4A virus in Northern Pintails and compared the results to that of an H5N1 HPAI clade 2.3.2.1 virus. All of Northern Pintails infected with either H5N1 or H5N8 virus lacked clinical signs and mortality, but the H5N8 clade 2.3.4.4 virus was more efficient at replicating within and transmitting between Northern Pintails than the H5N1 clade 2.3.2.1 virus. The H5N8‐infected birds shed high titre of viruses from oropharynx and cloaca, which in the field supported virus transmission and spread. This study highlights the role of wild waterfowl in the intercontinental spread of some HPAI viruses. Migratory aquatic birds should be carefully monitored for the early detection of H5 clade 2.3.4.4 and other HPAI viruses.     

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.     

Amino acid substitutions in antigenic region B of hemagglutinin play a critical role in the antigenic drift of subclade 2.3.4.4 highly pathogenic H5NX influenza viruses

As one of the important control strategies for highly pathogenic avian influenza (HPAI) in China, vaccination has been implemented compulsively in poultry flocks since 2004. However, the emergence and dominance of the circulating antigenic variants require the update of vaccines periodically. In order to investigate the key molecular sites responsible for the antigenic drift, a total of 13 amino acid positions divergent between clade 2.3.4 H5 viruses and their descendent subclade 2.3.4.4 variants in or around the recognized antigenic epitopes A–E were initially identified through inspecting a comprehensive HA sequence alignment of the H5 subtype HPAI viruses. Subsequently, a panel of single‐site or multi‐site HA mutants was constructed by reverse genetics with two H5N1 viruses of S (clade 2.3.4) and QD1 (subclade 2.3.4.4) as the HA backbone to study their antigenic variations, respectively. The hemagglutination–inhibition assay revealed an evident impact of mutations at sites 88, 156, 205, 208, 239 and 289 to the HA antigenicity and highlighted that the amino acid substitutions located in the antigenic region B, especially the combined mutations at sites 205 and 208, were the major antigenic determinant which was also consistent with results from flow cytometry and antigenic mapping. Our findings provided more insights into the molecular mechanism of antigenic drift of the H5 subtype HPAI virus, which would be helpful for the selection of vaccine candidates and accordingly for the prevention and control of this devastating viral agent.     

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.     

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.     

Isolation and characterization of equine influenza virus (H3N8) from an equine influenza outbreak in Malaysia in 2015

Equine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. In this study, we carried out molecular characterization of Equine Influenza Virus (EIV) isolated from the Malaysian outbreak in 2015 by sequencing of the HA and NA gene segments using Sanger sequencing. The nucleotide and amino acid sequences of HA and NA were compared with representative Florida clade 1 and clade 2 strains using phylogenetic analysis. The Florida clade 1 viruses identified in this outbreak revealed numerous amino acid substitutions in the HA protein as compared to the current OIE vaccine strain recommendations and representative strains of circulating Florida sub‐lineage clade 1 and clade 2. Differences in HA included amino acids located within antigenic sites which could lead to reduced immune recognition of the outbreak strain and alter the effectiveness of vaccination against the outbreak strain. Detailed surveillance and genetic information sharing could allow genetic drift of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.     

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.     

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.     

Outbreaks of Clade 2.3.4.4 H5N8 highly pathogenic avian influenza in 2018 in the northern regions of South Africa were unrelated to those of 2017

Asian‐origin H5N8 highly pathogenic avian influenza (HPAI) viruses of the H5 Goose/Guangdong/96 lineage, clade 2.3.4.4 group B, reached South Africa by June 2017. By the end of that year, 5.4 million layers and broiler chickens died or were culled, with total losses in the poultry industry estimated at US$ 140 million, and thousands of exotic birds in zoological collections, endangered endemic species and backyard poultry and pet birds also perished. The 2017 H5N8 HPAI outbreaks were characterized by two distinct spatial clusters, each associated with specific reassortant viral genotypes. Genotypes 1, 2, 3 and 5 were restricted to the northern regions, spanning the provinces of Limpopo, Gauteng, North West, Mpumalanga, KwaZulu‐Natal and Free State. The second, much larger cluster of outbreaks was in the south, in the Western and Eastern Cape provinces, wherein 2017 and 2018 outbreaks were caused solely by genotype 4. The last confirmed case of H5N8 HPAI in the northern region in 2017 was in early October, and the viruses seemed to disappear over the summer. However, starting in mid‐February 2018, H5N8 HPAI outbreaks resurged in the north. Viruses from two of the eight outbreaks were sequenced, one from an outbreak in quails (Coturnix japonica) in the North West Province, and another from commercial pullets in the Gauteng province. Phylogenetic analysis identified the viruses as a distinct sixth genotype that was most likely a new introduction to South Africa in early 2018.     

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.     

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.     

Experimental infection of clade 1.1.2 (H5N1), clade 2.3.2.1c (H5N1) and clade 2.3.4.4 (H5N6) highly pathogenic avian influenza viruses in dogs

Since the emergence of highly pathogenic avian influenza (HPAI) H5N1 in Asia, the haemagglutinin (HA) gene of this virus lineage has continued to evolve in avian populations, and H5N1 lineage viruses now circulate concurrently worldwide. Dogs may act as an intermediate host, increasing the potential for zoonotic transmission of influenza viruses. Virus transmission and pathologic changes in HPAI clade 1.1.2 (H5N1)‐, 2.3.2.1c (H5N1)‐ and 2.3.4.4 (H5N6)‐infected dogs were investigated. Mild respiratory signs and antibody response were shown in dogs intranasally infected with the viruses. Lung histopathology showed lesions that were associated with moderate interstitial pneumonia in the infected dogs. In this study, HPAI H5N6 virus replication in dogs was demonstrated for the first time. Dogs have been suspected as a “mixing vessel” for reassortments between avian and human influenza viruses to occur. The replication of these three subtypes of the H5 lineage of HPAI viruses in dogs suggests that dogs could serve as intermediate hosts for avian–human influenza virus reassortment if they are also co‐infected with human influenza viruses.     

New Introduction of Clade 2.3.2.1 Avian Influenza Virus (H5N1) into Bangladesh

Since the first outbreak of highly pathogenic H5N1 avian inafluenza (HPAI) in Bangladesh in February 2007, a total of 519 disease events have been reported till 22 October 2011. Partial HA gene sequences of 11 selected H5N1 HPAI isolates of 2007 to 2011 were determined and subjected to phylogenetic analysis. The study revealed a recent introduction of clade 2.3.2 and 2.3.4 viruses into Bangladesh in 2011 in addition to clade 2.2 viruses that had been in circulation since 2007. Clade 2.3.2 virus isolates from Bangladesh are phylogenetically related to the newly designated clade 2.3.2.1 viruses, reported recently from Asia and Eastern Europe.     

Co‐circulation and characterization of HPAI‐H5N1 and LPAI‐H9N2 recovered from a duck farm,Yogyakarta, Indonesia

In July 2016, an avian influenza outbreak in duck farms in Yogyakarta province was reported to Disease Investigation Center (DIC), Wates, Indonesia, with approximately 1,000 ducks died or culled. In this study, two avian influenza (AI) virus subtypes, A/duck/Bantul/04161291‐OR/2016 (H5N1) and A/duck/Bantul/04161291‐OP/2016 (H9N2) isolated from ducks in the same farm during an AI outbreak in Bantul district, Yogyakarta province, were sequenced and characterized. Our results showed that H5N1 virus was closely related to the highly pathogenic AI (HPAI) H5N1 of clade 2.3.2.1c, while the H9N2 virus was clustered with LPAI viruses from China, Vietnam and Indonesia H9N2 (CVI lineage). Genetic analysis revealed virulence characteristics for both in avian and in mammalian species. In summary, co‐circulation of HPAI‐H5N1 of clade 2.3.2.1c and LPAI‐H9N2 was identified in a duck farm during an AI outbreak in Yogyakarta province, Indonesia. Our findings raise a concern of the potential risk of the viruses, which could increase viral transmission and/or threat to human health. Routine surveillance of avian influenza viruses should be continuously conducted to understand the dynamic and diversity of the viruses for influenza prevention and control in Indonesia and SEA region.     

Genetic Diversity and Phylogenetic Analysis of Highly Pathogenic Avian Influenza (HPAI) H5N1 Viruses Circulating in Bangladesh from 2007–2011

Highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in Bangladesh since its first isolation in February 2007. Phylogenetic analysis of the haemagglutinin (HA) gene of HPAI H5N1 viruses demonstrated that 25 Bangladeshi isolates including two human isolates from 2007–2011 along with some isolates from neighbouring Asian countries (India, Bhutan, Myanmar, Nepal, China and Vietnam) segregate into two distinct clades (2.2 and 2.3). There was clear evidence of introduction of clade 2.3.2 and 2.3.4 viruses in 2011 in addition to clade 2.2 viruses that had been in circulation in Bangladesh since 2007. The data clearly demonstrated the movement of H5N1 strains between Asian countries included in this study due to migration of wild birds and/or illegal movement of poultry across borders. Interestingly, the two human isolates were closely related to the clade 2.2 Bangladeshi chicken isolates indicating that they have originated from chickens. Furthermore, comparative amino acid sequence analysis revealed several substitutions (including 189R>K and 282I>V) in HA protein of some clade 2.2 Bangladeshi viruses including the human isolates, suggesting there was antigenic drift in clade 2.2.3 viruses that were circulating between 2008 and 2011. Overall, the data imply genetic diversity among circulating viruses and multiple introductions of H5N1 viruses with an increased risk of human infections in Bangladesh, and establishment of H5N1 virus in wild and domestic bird populations, which demands active surveillance.     

Limited pathogenicity and transmissibility of Korean highly pathogenic avian influenza H5N6 clade 2.3.4.4 in ferrets

The pathogenicity and transmissibility of a reassortant clade 2.3.4.4 avian influenza A (H5N6) virus were evaluated in ferrets. Virus excretion was detected in the upper respiratory tract, but the ferrets did not show any clinical signs of infection. Transmission did not occur between cohoused or respiratory droplet‐contact ferrets.     

Serological surveillance reveals patterns of exposure to H5 and H7 influenza A viruses in European poultry

Influenza A viruses of H5 and H7 subtype in poultry can circulate subclinically and subsequently mutate from low to high pathogenicity with potentially devastating economic and welfare consequences. European Union Member States undertake surveillance of commercial and backyard poultry for early detection and control of subclinical H5 and H7 influenza A infection. This surveillance has moved towards a risk‐based sampling approach in recent years; however, quantitative measures of relative risk associated with risk factors utilized in this approach are necessary for optimization. This study describes serosurveillance for H5 and H7 influenza A in domestic and commercial poultry undertaken in the European Union from 2004 to 2010, where a random sampling and thus representative approach to serosurveillance was undertaken. Using these representative data, this study measured relative risk of seropositivity across poultry categories and spatially across the EU. Data were analysed using multivariable logistic regression. Domestic waterfowl, game birds, fattening turkeys, ratites, backyard poultry and the ‘other’ poultry category holdings had relatively increased probability of H5 and/or H7 influenza A seropositivity, compared to laying‐hen holdings. Amongst laying‐hen holdings, free‐range rearing was associated with increased probability of H7 seropositivity. Spatial analyses detected ‘hotspots’ for H5 influenza A seropositivity in western France and England, and H7 influenza A seropositivity in Italy and Belgium, which may be explained by the demographics and distribution of poultry categories. Findings suggest certain poultry category holdings are at increased risk of subclinical H5 and/or H7 influenza A circulation, and free‐range rearing increases the likelihood of exposure to H7 influenza A. These findings may be used in further refining risk‐based surveillance strategies and prioritizing management strategies in influenza A outbreaks.     

Diagnosis and Control Measures of the 2010 Outbreak of Foot‐and‐Mouth Disease A Type in the Republic of Korea

In January 2010, foot‐and‐mouth disease (FMD) occurred for the first time in 8 years in Korea. The outbreaks were because of A serotype, different from the O type, which had occurred previously in 2000 and 2002. The FMD outbreaks were identified in seven farms, consisting of six cattle farms where viruses were detected and one deer farm where only FMDV antibody was detected. The seven farms were within 9.3 km of each other. All susceptible animals within 10 km radius of the outbreak farms were placed under movement restrictions for 3–11 weeks. No vaccination took place to facilitate the clinical observation of infected animals and virus detection. After clinical observations and serological tests within the control zones showed no evidence of FMD infection, the movement restrictions were lifted, followed by FMD‐free declaration (23 March) at 80 days after the first outbreak on 2 January. This communication describes the outbreak of FMD A serotype, and control measures applied to eradicate the disease in Korea.     

Isolation and genetic characterization of H13N8 low pathogenic avian influenza virus from migratory birds in eastern China

Low pathogenic avian influenza virus (LPAIV) is an important zoonotic pathogen. Migratory birds are the natural reservoir for all 16 haemagglutinin (HA) and nine neuraminidase (NA) subtypes of LPAIV. Surveillance of LPAIV in migratory waterfowl and poultry is important for animal and public health. An understanding of the ecology and epidemiology of LPAI viruses in their reservoirs is beneficial for routine surveillance projects. Here, we report the isolation of an H13N8 LPAIV from black‐tailed gulls in eastern China. Full genome sequences of this isolate were determined. Genetic analysis of the HA and NA segments of this isolate showed that this H13N8 LPAIV was derived from the Eurasian lineage. Additionally, we speculate that this H13N8 LPAIV was a reassortant between the North American and Eurasian lineages. Interestingly, we identified amino acid motifs responsible for increased virulence or transmission of influenza viruses in mammals. We also found weak but measurable haemagglutination inhibition antibody titers against H13N8 virus in serum samples collected from chickens. These results suggest that continued surveillance for LPAI viruses in migratory birds and poultry is required.