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.     

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.     

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.     

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.     

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.     

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.     

Shedding of clade 2.3.4.4 H5N8 and H5N2 highly pathogenic avian influenza viruses in peridomestic wild birds in the U.S.

European starlings (Sturnus vulgaris), house sparrows (Passer domesticus) and rock pigeons (Columba livia) are all wild birds commonly found in large numbers in and around human dwellings and domestic livestock operations. This study evaluated the susceptibility of these species to three strains of highly pathogenic avian influenza virus (HP AIV) clade 2.3.4.4 isolated in the U.S.. Experimental infection of European starlings and rock pigeons did not result in any overt signs attributable to AIV infection and no virus shedding was detected from the oral and cloacal routes. House sparrows shed by the oral route and exhibited limited mortality. Individuals from all three species seroconverted following infection. These data suggest that none of these birds are a likely potential bridge host for future HP AIV outbreaks but that their seroconversion may be a useful surveillance tool for detection of circulating H5 HP AIV.     

Emergence and spread of highly pathogenic avian influenza A(H5N8) in Europe in 2016‐2017

Circulation of highly pathogenic avian influenza (HPAI ) viruses poses a continuous threat to animal and public health. After the 2005–2006 H5N1 and the 2014–2015 H5N8 epidemics, another H5N8 is currently affecting Europe. Up to August 2017, 1,112 outbreaks in domestic and 955 in wild birds in 30 European countries have been reported, the largest epidemic by a HPAI virus in the continent. Here, the main epidemiological findings are described. While some similarities with previous HPAI virus epidemics were observed, for example in the pattern of emergence, significant differences were also patent, in particular the size and extent of the epidemic. Even though no human infections have been reported to date, the fact that A/H5N8 has affected so far 1,112 domestic holdings, increases the risk of exposure of humans and therefore represents a concern. Understanding the epidemiology of HPAI viruses is essential for the planning future surveillance and control activities.     

Genetic and pathogenic characteristics of clade 2.3.2.1c H5N1 highly pathogenic avian influenza viruses isolated from poultry outbreaks in Laos during 2015–2018

Since 2004, there have been multiple outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses in Laos. Here, we isolated H5N1 HPAI viruses from poultry outbreaks in Laos during 2015–2018 and investigated their genetic characteristics and pathogenicity in chickens. Phylogenetic analysis revealed that the isolates belonged to clade 2.3.2.1c and that they differed from previous Laos viruses with respect to genetic composition. In particular, the isolates were divided into two genotypes, each of which had a different NS segments. The results of possible migration analysis suggested a high likelihood that the Laos isolates were introduced from neighbouring countries, particularly Vietnam. The recent Laos isolate, A/Duck/Laos/NL‐1504599/2018, had an intravenous pathogenicity index score of 3.0 and showed a 50% chicken lethal dose of 10^2.5 EID₅₀/0.1 ml, indicating high pathogenicity. The isolated viruses exhibited no critical substitution in the markers associated with mammalian adaptation, but possess markers related to neuraminidase inhibitor resistance. These results emphasize the need for ongoing surveillance of circulating influenza virus in South‐East Asia, including Laos, to better prepare for and mitigate global spread of H5 HPAI.     

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.     

Characterization of H7N9 avian influenza viruses isolated from duck meat products

Avian influenza H7N9 viruses have caused five epidemic waves of human infections since the first human cases were reported in 2013. In 2016, the initial low pathogenic avian influenza (LPAI) H7N9 viruses became highly pathogenic, acquiring multi‐basic amino acids at the haemagglutinin cleavage site. These highly pathogenic avian influenza (HPAI) H7N9 viruses have been detected in poultry and humans in China, causing concerns of a serious threat to global public health. In Japan, both HPAI and LPAI H7N9 viruses were isolated from duck meat products carried illegally and relinquished voluntarily at the border by passengers on flights from China to Japan between 2016 and 2017. Some of the LPAI and HPAI H7N9 viruses detected at the border in Japan were characterized previously in chickens and ducks; however, their pathogenicity and replicative ability in mammals remain unknown. In this study, we assessed the biological features of two HPAI H7N9 virus isolates [A/duck/Japan/AQ‐HE29‐22/2017 (HE29‐22) and A/duck/Japan/AQ‐HE29‐52/2017 (HE29‐52); both of these viruses were isolated from duck meat at the border)] and an LPAI H7N9 virus isolate [A/duck/Japan/AQ‐HE28‐3/2016 (HE28‐3)] in mice and ferrets. In mice, HE29‐52 was more pathogenic than HE29‐22 and HE28‐3. In ferrets, the two HPAI virus isolates replicated more efficiently in the lower respiratory tract of the animals than did the LPAI virus isolate. Our results indicate that HPAI H7N9 viruses with the potential to cause severe diseases in mammals have been illegally introduced to Japan.     

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.     

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.     

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.     

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.     

Highly pathogenic avian influenza subtype H5Nx clade 2.3.4.4 outbreaks in Dutch poultry farms, 2014–2018: Clinical signs and mortality

In recent years, different subtypes of highly pathogenic avian influenza (HPAI) viruses caused outbreaks in several poultry types worldwide. Early detection of HPAI virus infection is crucial to reduce virus spread. Previously, the use of a mortality ratio threshold to expedite notification of suspicion in layer farms was proposed. The purpose of this study was to describe the clinical signs reported in the early stages of HPAI H5N8 and H5N6 outbreaks on chicken and Pekin duck farms between 2014 and 2018 in the Netherlands and compare them with the onset of an increased mortality ratio (MR). Data on daily mortality and clinical signs from nine egg‐producing chicken farms and seven Pekin duck farms infected with HPAI H5N8 (2014 and 2016) and H5N6 (2017–2018) in the Netherlands were analysed. In 12 out of 15 outbreaks for which a MR was available, MR increase preceded or coincided with the first observation of clinical signs by the farmer. In one chicken and two Pekin duck outbreaks, clinical signs were observed prior to MR increase. On all farms, veterinarians observed clinical signs of general disease. Nervous or locomotor signs were reported in all Pekin duck outbreaks, but only in two chicken outbreaks. Other clinical signs were observed less frequently in both chickens and Pekin ducks. Compared to veterinarians, farmers observed and reported clinical signs, especially respiratory and gastrointestinal signs, less frequently. This case series suggests that a MR with a set threshold could be an objective parameter to detect HPAI infection on chicken and Pekin duck farms at an early stage. Observation of clinical signs may provide additional indication for farmers and veterinarians for notifying a clinical suspicion of HPAI infection. Further assessment and validation of a MR threshold in Pekin ducks are important as it could serve as an important tool in HPAI surveillance programs.     

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.     

Risk factors associated with highly pathogenic avian influenza subtype H5N8 outbreaks on broiler duck farms in South Korea

Highly Pathogenic Avian Influenza (HPAI ) subtype H5N8 outbreaks occurred in poultry farms in South Korea in 2014 resulting in significant damage to the poultry industry. Between 2014 and 2016, the pandemic disease caused significant economic loss and social disruption. To evaluate the risk factors for HPAI infection in broiler duck farms, we conducted a retrospective case–control study on broiler duck farms. Forty‐three farms with confirmed laboratories on premises were selected as the case group, and 43 HPAI ‐negative farms were designated as the control group. Control farms were matched based on farm location and were within a 3‐km radius from the case premises. Spatial and environmental factors were characterized by site visit and plotted through a geographic information system (GIS ). Univariable and multivariable logistic regression models were developed to assess possible risk factors associated with HPAI broiler duck farm infection. Four final variables were identified as risk factors in a final multivariable logistic model: “Farms with ≥7 flocks” (odds ratio [OR ] = 6.99, 95% confidence interval [CI ] 1.34–37.04), “Farm owner with ≥15 years of raising poultry career” (OR  = 7.91, 95% CI 1.69–37.14), “Presence of any poultry farms located within 500 m of the farm” (OR  = 6.30, 95% CI 1.08–36.93) and “Not using a faecal removal service” (OR  = 27.78, 95% CI 3.89–198.80). This highlights that the HPAI H5N8 outbreaks in South Korea were associated with farm owner education, number of flocks and facilities and farm biosecurity. Awareness of these factors may help to reduce the spread of HPAI H5N8 across broiler duck farms in Korea during epidemics. Greater understanding of the risk factors for H5N8 may improve farm vulnerability to HPAI and other subtypes and help to establish policies to prevent re‐occurrence. These findings are relevant to global prevention recommendations and intervention protocols.