Antigenic, genetic, and pathogenic characterization of H5N1 highly pathogenic avian influenza viruses isolated from dead whooper swans (Cygnus cygnus) found in northern Japan in 2008

In April and May 2008, whooper swans (Cygnus cygnus) were found dead in Hokkaido in Japan. In this study, an adult whooper swan found dead beside Lake Saroma was pathologically examined and the identified H5N1 influenza virus isolates were genetically and antigenically analyzed. Pathological findings indicate that the swan died of severe congestive edema in the lungs. Phylogenetic analysis of the HA genes of the isolates revealed that they are the progeny viruses of isolates from poultry and wild birds in China, Russia, Korea, and Hong Kong. Antigenic analyses indicated that the viruses are distinguished from the H5N1 viruses isolated from wild birds and poultry before 2007. The chickens vaccinated with A/duck/Hokkaido/Vac-1/2004 (H5N1) survived for 14 days after challenge with A/whooper swan/Hokkaido/1/2008 (H5N1), although a small amount of the challenge virus was recovered from the tissues of the birds. These findings indicate that H5N1 highly pathogenic avian influenza viruses are circulating in wild birds in addition to domestic poultry in Asia and exhibit antigenic variation that may be due to vaccination.     

Surveillance and characterization of avian influenza viruses from migratory water birds in eastern Hokkaido, the northern part of Japan, 2009–2010

Avian influenza virus (AIV) surveillance was conducted around a small pond in Obihiro, eastern Hokkaido, Japan. Eleven AIVs were isolated from a total of 1,269 fecal samples of migratory wild birds collected during 2009 and 2010. The sample number covered approximately 60 % of the total number of birds observed during sampling periods. The subtypes of the isolates included H3N8 (4 isolates), H5N2 (3), H6N2 (2), H6N1 (1), and H11N2 (1). The H3N8 subtype was most prevalent as in the previous studies performed in Hokkaido. The three H5N2 isolates genetically characterized as low pathogenic AIV were closely related to the strains previously isolated from aquatic wild birds in Japan and also to the Korean strains isolated from aquatic birds in recent years. In Korea, H5N2 subtype virus has often been isolated from poultry and wild birds, as well as reassortant viruses generated from duck H5N2 viruses and chicken H9N2 virus, and avian-swine-like reassortant H5N2 viruses. Considering the previous chicken outbreaks caused by highly pathogenic H5N2 viruses, which affected many countries, it should be an important priority to continue, monitoring the evolution of H5N2 viruses circulating in the region.     

Molecular characterization of the glycoprotein genes of H5N1 influenza A viruses isolated in Israel and the Gaza Strip during 2006 outbreaks

Highly pathogenic H5N1 avian influenza A viruses (AIV) have caused outbreaks among domestic poultry and wild aquatic birds in many Asian, European, and African countries since 1997. In March 2006 an avian H5N1 influenza A virus was isolated from poultry in Israel. In the present study we molecularly characterized the hemagglutinin (HA) and neuraminidase (NA) genes of eleven H5N1 viruses isolated from domestic poultry in Israel and Gaza in March–April 2006. Phylogenetic analysis of the HA and NA genes showed that the Israeli and Gazian viruses were closely related to viruses isolated in Egypt in 2006.     

Genetic characterization and pathogenicity assessment of highly pathogenic H5N1 avian influenza viruses isolated from migratory wild birds in 2011, South Korea

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among wild birds and poultry has posed a potential threat to human public health. In the present study, we report the isolation of HPAI H5N1 viruses (A/Md/Korea/W401/11 and A/Md/Korea/W404/11) from fecal samples of migratory birds. Genetic and phlyogenetic analyses demonstrated that these viruses are genetically identical possessing gene segments from avian virus origin and showing highest sequence similarities (as high as 99.8%) to A/Ws/Hokkaido/4/11 and 2009-2010 Mongolian-like clade 2.3.2 isolates rather than previous Korean H5N1 viruses. Both viruses possess the polybasic motif (QRERRRK/R) in HA but other genes did not bear additional virulence markers. Pathogenicity of A/Md/Korea/W401/11 was assessed and compared with a 2006 clade 2.2 HPAI H5N1 migratory bird isolate (A/EM/Korea/W149/06) in chickens, ducks, mice and ferrets. Experimental infection in these hosts showed that both viruses have high pathogenic potential in chickens (2.3-3.0 LD(50)s) and mice (3.3-3.9 LD(50)s), but A/Md/Korea/W401/11 was less pathogenic in duck and ferret models. Despite recovery of both infection viruses in the upper respiratory tract, efficient ferret-to-ferret transmission was not observed. These data suggest that the 2011 Korean HPAI wild bird H5N1 virus could replicate in mammalian hosts without pre-adaptation but could not sustain subsequent infection. This study highlights the role of migratory birds in the perpetuation and spread of HPAI H5N1 viruses in Far-East Asia. With the changing pathobiology caused by H5N1 viruses among wild and poultry birds, continued surveillance of influenza viruses among migratory bird species remains crucial for effective monitoring of high-pathogenicity or pandemic influenza viruses.     

Evolutionary genetics of highly pathogenic H5N1 avian influenza viruses isolated from whooper swans in northern Japan in 2008

In April and May 2008, highly pathogenic avian influenza viruses subtype H5N1 were isolated from dead or moribund whooper swans in Aomori, Akita and Hokkaido prefectures in northern Japan. To trace the genetic lineage of the isolates, the nucleotide sequences of all eight genes were determined and phylogenetically analyzed. The Japanese strains were nearly identical to chicken viruses isolated in Russia in April 2008 and closely related to viruses isolated from dead wild birds in Hong Kong in 2007–2008. Their HA genes clustered in clade 2.3.2. On the other hand, NA and the other internal genes were closely related to those of clade 2.3.4 viruses (genotype V) whose NP genes originated from an HA clade 2.3.2 virus. In conclusion, the H5N1 viruses isolated in Japan, Russia and Hong Kong were derived from a common ancestor virus belonging to genotype V that was generated from genetic reassortment events between viruses of HA clades 2.3.2 and 2.3.4.     

Isolation and characterization of H6N1 and H9N2 avian influenza viruses from Ducks in Hanoi, Vietnam

We report the genetic characterization of low pathogenic avian influenza (LPAI) viruses isolated from domestic ducks in northern Vietnam in 2009. In total, 22 influenza A viruses consisting of 21 H6N1 subtypes and one H9N2 subtype were isolated from 1488 ducks collected in February, March, and April 2009, accounting the overall virus isolation rate for 1.5%. No H5N1 strain was isolated in this study. Phylogenetic analysis indicated that all the eight genes of the H6N1 and H9N2 subtypes analyzed in this study were similar to those isolated in Korea, southeast China and northern Japan, and wild birds which migrate along the coastal East Asian Flyway are estimated to transmit these viruses. There was no evidence that the H6N1 and H9N2 subtypes share the gene segments with H5N1 subtypes. However, it is important to monitor the prevalence and genetical backgrounds of LPAI viruses among poultry in an area where several different influenza A subtypes are in circulation.     

Isolation and molecular characterization of the influenza A/H5N1 viruses isolated during the outbreaks of avian influenza among birds in the European part of Russia in 2005: a virus strain with ozeltamivir-resistance mutation was found

The isolation and characterization of the influenza A/H5NI viruses isolated from hens that died during the outbreak of avian influenza in autumn 2005 in the Yandovka village (Tula oblast) and from a wild swan that died near the orifice of the Volga River in the zone of the Karalat Furrow were carried out. Molecular-biologic and phylogenetic analyses were performed with a view of determining possible geographical origin of strains, phylogenetic similarity of viruses and also estimating their pathogenicity, epidemic danger for people, and possible resistance to antiviral drugs. It was shown that the virus belonged to the high pathogenic variants that arose in China as a result of the reassortment of the viruses of the genotypes Z and V that circulated among poultry and wild birds. A number of molecular markers characterizing the high pathogenicity of the virus for gallinaceous birds and mammals were revealed, but the specific mutations in the hemagglutinin gene that promote the high rate of virus replication in a human organism and also the mutations of adaptation to it were not found. It was shown that the variants of the influenza A/H5N1 virus that circulated in this epizootic were sensitive to remantadine. The strain isolated from the wild swan had the mutation causing resistance to Tamiflu/ozeltamivir.     

The genetic and antigenic diversity of avian influenza viruses isolated from domestic ducks, muscovy ducks, and chickens in northern and southern Vietnam, 2010–2012

To estimate the prevalence of avian influenza virus infection in Vietnam, surveillance was conducted in domestic and wild birds from households, live-bird markets, slaughtering sites, and bird sanctuaries in Vietnam between October 2010 and October 2012. Of the 4,550 samples collected, 226 influenza A virus isolates were obtained from domestic ducks, muscovy ducks, and chickens. Of these, 25 and 22 H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from apparently healthy domestic ducks in live-bird markets and slaughtering sites in northern and southern Vietnam, respectively. The HA genes of H5 viruses isolated from birds in northern Vietnam phylogenetically belonged to the genetic clade 2.3.2.1 and those in southern Vietnam belonged to the genetic clade 1.1. In addition, 39 H3, 12 H4, 1 H5, 93 H6, 2 H7, 18 H9, 3 H10, and 11 H11 viruses were isolated. Phylogenetic and antigenic analyses of the H6 and H9 viruses revealed that they were closely related to the isolates obtained from domestic poultry in China. Phylogenetic analyses of internal gene segments of these isolates revealed that these viruses were circulating in both domestic and wild birds in Asia and reassortment events had occurred frequently. Therefore, it will be important to continue the surveillance and strict controls over the movement and trade of poultry and poultry products in order to eradicate H5N1 HPAIV from Asia.     

Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002.

Outbreaks of highly pathogenic H5N1 avian influenza have occurred in Hong Kong in chickens and other gallinaceous poultry in 1997, 2001, twice in 2002 and 2003. High mortality rates were seen in gallinaceous birds but not in domestic or wild waterfowl or other wild birds until late 2002 when highly pathogenic H5N1 avian influenza occurred in waterfowl (geese, ducks and swans), captive Greater Flamingo (Phoenicopterus ruber) and other wild birds (Little Egret Egretta garzetta) at two waterfowl parks and from two dead wild Grey Heron (Ardea cinerea) and a Black-headed Gull (Larus ridibundus) in Hong Kong. H5N1 avian influenza virus was also isolated from a dead feral pigeon (Columba livia) and a dead tree sparrow (Passer montanus) during the second outbreak. The first waterfowl outbreak was controlled by immediate strict quarantine and depopulation 1 week before the second outbreak commenced. Control measures implemented for the second outbreak included strict isolation, culling, increased sanitation and vaccination. Outbreaks in gallinaceous birds occurred in some live poultry markets concurrently with the second waterfowl outbreak, and infection on a chicken farm was detected 1 week after the second waterfowl park outbreak was detected, on the same day the second grey heron case was detected. Subsequent virus surveillance showed the outbreaks had been contained.     

Isolation of influenza A/H5N1 virus strains from poultry and wild birds in West Siberia during epizooty (July 2005) and their depositing to the state collection of viruses (August 8, 2005)

Six strains of influenza AH5N1 virus were isolated, by using PS and MDCK continuous cell lines from poultry and wild birds, which were collected on July 28, 2005 in the samples taken from 5 examines species of wild birds in the Novosibirsk region during the epizootic outbreak with a high mortality. The strains were identified by means of HIT, RT-PCR, and microchip-based techniques. Two strains, A/Grebe/Novosibirsk/29/05 (H5N1) and A/Duck/Novosibirsk/56/05 (H5N1), were deposited to the Russian State Collection of Viruses (Registration Nos. 2372 and 2371, respectively) with the priority date 08.08.2005. Positive results in RT-PCR for influenza A/H5N1 virus detec- tion were obtained in 100% of the samples from dead and sick poultry; 93% from the clinically healthy poultry kept together with sick one; positive results ranged from 0 to 36%. Sequencing established the identity of genetic characteristics of strains isolated for wild birds and poultry as well as their affiliation to high pathogenic avian influenza (HPAI). Phylogenetic analysis revealed a high homology of hemagglutining of West-Siberian strains and strains isoolated from bar-headed gooses (Eulabeia indica) on the Qinghai Lake (Western China) in the 2005 spring.     

Genetic characterization of low pathogenic H5N1 and co-circulating avian influenza viruses in wild mallards (Anas platyrhynchos) in Belgium, 2008

As part of a long-term wild bird monitoring programme, five different low pathogenic (LP) avian influenza viruses (AIVs) were isolated from wild mallards (subtypes H1N1, H4N6, H5N1, H5N3, and H10N7). A LP H5N1 and two co-circulating (same location, same time period) viruses were selected for full genome sequencing. An H1N1 (A/Anas platyrhynchos/Belgium/09-762/2008) and an H5N1 virus (A/Anas platyrhynchos/Belgium/09-762-P1/2008) were isolated on the same day in November 2008, then an H5N3 virus (A/Anas platyrhynchos/09-884/2008) 5 days later in December 2008. All genes of these co-circulating viruses shared common ancestors with recent (2001 to 2007) European wild waterfowl influenza viruses. The H5N1 virus shares genome segments with both the H1N1 (PB1, NA, M) and the H5N3 (PB2, HA) viruses, and all three viruses share the same NS sequence. A double infection with two different PA segments from H5N1 and from H5N3 could be observed for the H1N1 sample. The observed gene constellations resulted from multiple reassortment events between viruses circulating in wild birds in Eurasia. Several internal gene segments from these 2008 viruses and the N3 sequence from the H5N3 show homology with sequences from 2003 H7 outbreaks in Italy (LP) and the Netherlands (highly pathogenic). These data contribute to the growing sequence evidence of the dynamic nature of the avian influenza natural reservoir in Eurasia, and underline the importance of monitoring AIV in wild birds. Genetic information of potential hazard to commercial poultry continues to circulate in this reservoir, including H5 and H7 subtype viruses and genes related to previous AIV outbreaks.     

Characterization of the amantadine-resistant H5N1 highly pathogenic avian influenza variants isolated from quails in Southern China

Highly pathogenic H5N1 avian influenza viruses have spread in poultry and wild birds in Asia, Europe, and Africa since 2003. To evaluate the role of quails in the evolution of influenza A virus, we characterized three H5N1 viruses isolated from quails (QA viruses) in southern China. Phylogenetic analysis indicated that three QA viruses derived from the A/goose/Guangdong/1/96-like lineage and most closely related to HA clade 4 A/chicken/Hong Kong/31.4/02-like viruses. Molecular analysis suggested that QA viruses and clade 4 H5N1 viruses carried consistent residue signatures, such as the characteristic M2 Ser31Asn amantadine-resistance mutation, implying a common origin of these viruses. As revealed by viral pathogenicity tests, these QA viruses could replicate in intranasally infected mice, but were not lethal to them, showing low pathogenicity in mammals. However, they killed all intravenously inoculated chickens, showing high pathogenicity in poultry. Results from amantadine sensitivity tests of wild-type QA viruses and their reverse genetic viruses demonstrated that all QA viruses were resistant to amantadine, and the M2 Ser31Asn mutation was determined as the most likely cause of the increased amantadine-resistance of H5N1 QA viruses. Our study confirmed experimentally that the amino acid at residue 31 in the M2 protein plays a major role in determining the amantadine-resistance phenotype of H5N1 influenza viruses. Our findings provide further evidence that quails may play important roles in the evolution of influenza A viruses, which raises concerns over possible transmissions of H5N1 viruses among poultry, wild birds, and humans.     

Influenza virus subtypes in aquatic birds of eastern Germany

Summary We report the findings of a 12-year surveillance study (1977–89) of avian influenza A viruses in eastern Germany. Viruses were isolated directly from feral ducks (n=236) and other wild birds (n=89); from domestic ducks (n=735) living on a single farm; and from white Pekin ducks (n=193) used as sentinels for populations of wild aquatic birds; mainly sea birds. The efficiency of virus isolation was 9.9% overall, with considerable variability noted among species: 8.7% in wild ducks, 0.9% in other feral birds and 38% in Pekin ducks. Use of sentinel ducks in wild pelagic bird colonies improved virus detection rates fivefold, suggesting that this approach is advantageous in ecological studies. Among the 40 different combinations of hemagglutinin (HA) and neuraminidase (NA) subtypes we identified, H6N1 predominated (23.6% for all avian species), followed by H4N6 (11%). Among individual species, the frequency profiles favored H2N3 (20.8%) and H4N6 (20.3%) in feral ducks; H7N7 (22.3%), H4N6 (24.4%) and H2N3 (10.4%) in Pekin ducks used as sentinels; and H6N1 (34.8%) and H6N6 (15.1%) in domestic ducks maintained on a single farm. By relying on sentinel birds for serological assays, it was possible to trace an influenza season in feral swan populations, beginning in August and continuing through the winter months. Comparison of subtype distribution of influenza viruses for Europe and North American showed significant differences. This supports the fact of two geographically distinct gene pools of influenza viruses in birds connected with their distinct flyways of each hemisphere. The high frequency of isolation of H2 influenza viruses is of considerable interest to those interested in the recycling of this subtype in humans. Similarly the frequent isolation of H7N7 influenza viruses raises concern about reservoirs of potentially pathogenic influenza virus for domestic poultry. Our results confirm the existence of a vast reservoir of influenza A viruses in European aquatic birds, which possesses sufficient diversity to account for strains that infect lower animals and humans.This article is dedicated to the memory of Dr. Herbert Sinnecker who died in 1991 at the age of 61. He was the Director of the Institute of Viral Zoonosis in the former German Democratic Republic (GDR). It was Herbert Sinnecker's forsight and understanding of the need to resolve the origin of human and animal influenza pandemics that initiated the studies described in this article. He developed novel epidemiological and ecological methods that permitted definition of the influenza virus gene pool in central Europe. The unification of Germany made it possible to publish this article; otherwise, the studies encouraged and organized by H. Sinnecker would have been lost to the scientific community.     

Influenza viruses isolated from wild birds

Ninety-eight hemagglutinating agents were isolated from washings of cloaca and organs of 750 birds collected in southern and southeastern regions of the Kazakh SSR. Determinations of their type appurtenance allowed 36 agents to be classified into influenza A virus. Among them 4 strains had H1N1 surface antigens, 29 strains were Hav2 Nav5 and 3 strains had unidentified neuraminidase and Hav2. The data on the biological properties of influenza virus strains of both subtypes are presented.     

Genetic analysis of nonstructural genes (NS1 and NS2) of H9N2 and H5N1 viruses recently isolated in Israel

The avian influenza virus subtype H9N2 affects wild birds, domestic poultry, swine, and humans; it has circulated amongst domestic poultry in Israel during the last 6 years. The H5N1 virus was recorded in Israel for the first time in March 2006. Nonstructural (NS) genes and NS proteins are important in the life cycle of the avian influenza viruses. In the present study, NS genes of 21 examples of H9N2 and of two examples of H5N1 avian influenza viruses, isolated in Israel during 2000–2006, were completely sequenced and phylogenetically analyzed. All the H9N2 isolates fell into a single group that, in turn, was subdivided into three subgroups in accordance with the time of isolation; their NS1 and NS2 proteins possessed 230 and 121 amino acids, respectively. The NS1 protein of the H5N1 isolates had five amino acid deletions, which was typical of highly pathogenic H5N1 viruses isolated in various countries during 2005–2006. Comparative analysis showed that the NS proteins of the H9N2 Israeli isolates contained few amino acid sequences associated with high pathogenicity or human host specificity.     

Information of the Center for Ecology and Epidemiology of Influenza, D. I. Ivanovsky Research Institute of Virology, Russian Academy of Medical Sciences, on the results of the 2009-2010 influenza and acute respiratory viral infection epidemic season (at week 40 of 2009 to week 22 of 2010) in the world and Russia

The paper describes the specific features of the 2009-2010 epidemic season in Russia and the world, which are due to the wide spread of a new pandemic strain of influenza A(H1N1)v virus. There is an unusual early upsurge in the incidence of influenza and acute respiratory viral infection (ARVI) (in October-November 2009) with its peak at weeks 45 to 48 of the year with a succeeding reduction to the seasonal values by its end. The circulation of influenza B virus strains was recorded in February-April 2010, which was responsible for the higher epidemic thresholds of morbidity in a number of Russia's regions. A study of the antigenic properties of the strains defined their relationship to the reference strains A/California/07/2009 (H1N1)v and B/Brisbene/60/2008. There were strains with amino acid substitutions at position 222 of hemagglutinin in the population of pandemic influenza A(H1N1)v virus. The strains of the new pandemic influenza A(H1N1)v virus were resistant to remantadine and susceptible to oseltamivir, zanamivir, and arbidol. The influenza B virus strains were susceptible to oseltamivir, zanamivir, and arbidol. The proportion of pathogens of some ARVIs was as follows: parainfluenza viruses, 9.8%; adenoviruses, 5.5%; respiratory syncytial virus, 4.8%; and Mycoplasma pneumonia, 0.6%. There is evidence that there is a need for further monitoring of influenza viruses in Russia.     

Genetic characterization of highly pathogenic H5N1 avian influenza virus from live migratory birds in Bangladesh

Since the first outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Bangladesh in 2007, the virus has been circulating among domestic poultry causing severe economic losses. To investigate the presence of HPAIV H5N1 in migratory birds and their potential role in virus spread, 205 pools of fecal samples from live migratory birds were analyzed. Here, the first virus isolation and genome characterization of two HPAIV H5N1 isolates from migratory birds (A/migratory bird/Bangladesh/P18/2010 and A/migratory bird/Bangladesh/P29/2010)are described. Full-length amplification, sequencing, and a comprehensive phylogenetic analysis were performed for HA, NA, M, NS, NP, PA, PB1, and PB2 gene segments. The selected migratory bird isolates belong to clade 2.3.2.1 along with recent Bangladeshi isolates from chickens, ducks, and crows which grouped in the same cluster with contemporary South and South-East Asian isolates. The studied isolates were genetically similar to other H5N1 isolates from different species within the respective clade although some unique amino acid substitutions were observed among them. Migratory birds remain a real threat for spreading pathogenic avian influenza viruses across the continent and introduction of new strains into Bangladesh.     

Characterization of a highly pathogenic H5N1 avian influenza virus isolated from ducks in Eastern China in 2011

This study describes the characterization of seven H5N1 avian influenza viruses from domestic ducks in Eastern China in 2011. Phylogenetic analysis showed these viruses were closely related to an H5N1 virus circulating in wild birds in Hong Kong. Some characteristics of these viruses were similar to those of an H5N1 strain that circulated in China and Vietnam (2003-2004). The virulence of three isolates was examined in chickens and mice, and they were found to be highly pathogenic in chickens but showed low pathogenicity in mice. These results suggest that continued H5N1 surveillance in poultry should be used as an early warning system for avian influenza outbreaks.     

Characterization of An Avian Influenza Virus of Subtype H7N2 Isolated from Chickens in Northern China

An H7N2 avian influenza virus was isolated from chickens during routine surveillance in northern China in 2002. To understand the origin of this virus, we completely sequenced its genome. The PB1, PA, HA, and M genes of this virus were highly homologous with those of the wild bird virus A/Africa starling/Eng-Q/983/79 (H7N1). The NP and NS genes were closely related to those of two other wild bird viruses isolated 30 years ago. The closest relatives of the PB2 and NA genes of the virus were those of the A/swine/Germany/2/81 (H1NI) and A/Leningrad/134/57 (H2N2), respectively. Animal inoculation tests showed that the virus cannot replicate efficiently in chickens. However, after intranasal inoculation, the virus induced 20% weight loss and replicated well in the lungs of mice. The virus was also recovered from the hearts and brains of the mice. These results suggest that the influenza virus isolated in chickens in northern China in 2002 originated in wild birds and may pose a threat for both avian species and mammalian hosts.     

Investigating a crow die-off in January–February 2011 during the introduction of a new clade of highly pathogenic avian influenza virus H5N1 into Bangladesh

We investigated unusual crow mortality in Bangladesh during January-February 2011 at two sites. Crows of two species, Corvus splendens and C. macrorhynchos, were found sick and dead during the outbreaks. In selected crow roosts, morbidity was ~1 % and mortality was ~4 % during the investigation. Highly pathogenic avian influenza virus H5N1 clade 2.3.2.1 was isolated from dead crows. All isolates were closely related to A/duck/India/02CA10/2011 (H5N1) with 99.8 % and A/crow/Bangladesh/11rs1984-15/2011 (H5N1) virus with 99 % nucleotide sequence identity in their HA genes. The phylogenetic cluster of Bangladesh viruses suggested a common ancestor with viruses found in poultry from India, Myanmar and Nepal. Histopathological changes and immunohistochemistry staining in brain, pancreas, liver, heart, kidney, bursa of Fabricius, rectum, and cloaca were consistent with influenza virus infection. Through our limited investigation in domesticated birds near the crow roosts, we did not identify any samples that tested positive for influenza virus A/H5N1. However, environmental samples collected from live-bird markets near an outbreak site during the month of the outbreaks tested very weakly positive for influenza virus A/H5N1 in clade 2.3.2.1-specific rRT-PCR. Continuation of surveillance in wild and domestic birds may identify evolution of new avian influenza virus and associated public-health risks.