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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Biosecurity and Circulation of Influenza A (H5N1) Virus in Live‐Bird Markets in Bangladesh, 2012

Bangladesh has been considered as one of the five countries endemic with highly pathogenic avian influenza A subtype H5N1 (HPAI H5N1). Live‐bird markets (LBMs) in south Asian countries are believed to play important roles in the transmission of HPAI H5N1 and others due to its central location as a hub of the poultry trading. Food and Agriculture Organization (FAO) of the United Nations has been promoting improved biosecurity in LBMs in Bangladesh. In 2012, by enrolling 32 large LBMs: 10 with FAO interventions and 22 without assistance, we assessed the virus circulation in the selected LBMs by applying standard procedures to investigate market floors, poultry stall floors, poultry‐holding cases and slaughter areas and the overall biosecurity using a questionnaire‐based survey. Relative risk (RR) was examined to compare the prevalence of HPAI H5N1 in the intervened and non‐intervened LBMs. The measures practised in significantly more of the FAO‐intervened LBMs included keeping of slaughter remnants in a closed container; decontamination of poultry vehicles at market place; prevention of crows’ access to LBM, market/floor cleaning by market committee; wet cleaning; disinfection of floor/poultry stall after cleaning; and good supply of clean water at market (P < 0.05). Conversely, disposal of slaughter remnants elsewhere at market and dry cleaning were in operation in more of the FAO non‐intervened LBMs (P < 0.05). The RR for HPAI H5N1 in the intervened and non‐intervened LBMs was 1.1 (95% confidence interval 0.44–2.76), suggesting that the proportion positive of the virus in the two kinds of LBM did not vary significantly (P = 0.413). These observations suggest that the viruses are still maintained at the level of production in farms and circulating in LBMs in Bangladesh regardless of interventions, albeit at lower levels than in other endemic countries.     

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.     

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.     

Incidence of Highly Pathogenic Avian Influenza H5N1 in Nigeria, 2005–2008

Outbreaks of highly pathogenic avian influenza (HPAI) H5N1 occurred in Nigeria between December 2005 and July 2008. We describe temporal and spatial characteristics of these outbreaks at State and Local Government Area (LGA) levels. A total of 25 of 37 States (67.6%; Exact 95% CI: 50.2–82.0%) and 81 of 774 LGAs (10.5%; Exact 95% CI: 8.4–12.8%) were affected by HPAI outbreaks over the period from 2005 to 2008. The incidence risk of HPAI outbreak occurrence at the State level was 5.6% (0.7–18.7%) for 2005, 50.0% (30.7–69.4%) for 2006, 54.5% (29.9–80.3%) for 2007 and 0% for 2008. Only very few LGAs experienced HPAI outbreaks within the affected States. The incidence risk of HPAI outbreak occurrence on a LGA level was 0.3% (0.0–0.9%) for 2005, 6.6% (4.9–8.6%) for 2006, 4.2% (2.9–6.0%) for 2007 and 0% for 2008. The mean period between farmers noticing HPAI outbreaks and reporting them to veterinary authorities, and between reporting HPAI outbreaks and the depopulation of infected premises, was for both 4.5 days; both periods also had medians of 1 day. We have estimated the spatially smoothed incidence risk for the whole outbreak period and identified the existence of a large corridor in the western part of Nigeria and a smaller corridor in south‐eastern part, where the risk of HPAI occurrence was lower than in the rest of the country. The effect of HPAI control policies on the outbreaks patterns are discussed, as well as possible reasons why HPAI did not become endemic in Nigeria.     

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.     

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.