Susceptibility of herons (family: Ardeidae) to clade 2.3.2.1 H5N1 subtype high pathogenicity avian influenza virus.

The pathogenicity of the H5 subtype high pathogenicity avian influenza viruses (HPAIVs) in Ardeidae bird species has not been investigated yet, despite the increasing infections reported. Therefore, the present study aimed to examine the susceptibility of the Ardeidae species, which had already been reported to be susceptible to HPAIVs, to a clade 2.3.2.1 H5N1 HPAIV. Juvenile herons (four grey herons, one intermediate egret, two little egrets, and three black-crowned night herons) were intranasally inoculated with 106 50% egg infectious dose of the virus and observed for 10 days. Two of the four grey herons showed lethargy and conjunctivitis; among them, one died at 6 days post-inoculation (dpi). The viruses were transmitted to the other two cohoused naïve grey herons. Some little egrets and black-crowned night herons showing neurological disorders died at 4-5 dpi; these birds mainly shed the virus via the oral route. The viruses predominantly replicated in the brains of birds that died of infection. Seroconversion was observed in most surviving birds, except some black-crowned night herons. These results demonstrate that most Ardeidae species are susceptible to H5 HPAIVs, sometimes with lethal effects. Herons are mostly colonial and often share habitats with Anseriformes, natural hosts of influenza A viruses; therefore, the risks of cluster infection and contribution to viral dissemination should be continuously evaluated. RESEARCH HIGHLIGHTSClade 2.3.2.1 H5N1 HPAIV causes lethal infections in Ardeidae sp.Viruses are transmitted among grey herons.Some herons with HPAIV showed conjunctivitis or neurological symptoms.HPAIV systemically replicated in herons tissues.

Susceptibility of common family Anatidae bird species to clade 2.3.4.4e H5N6 high pathogenicity avian influenza virus: an experimental infection study.

BACKGROUND: There were large outbreaks of high pathogenicity avian influenza (HPAI) caused by clade 2.3.4.4e H5N6 viruses in the winter of 2016-2017 in Japan, which caused large numbers of deaths among several endangered bird species including cranes, raptors, and birds in Family Anatidae. In this study, susceptibility of common Anatidae to a clade 2.3.4.4e H5N6 HPAI virus was assessed to evaluate their potential to be a source of infection for other birds. Eurasian wigeons (Mareca penelope), mallards (Anas platyrhynchos), and Northern pintails (Anas acuta) were intranasally inoculated with 106, 104, or 102 50% egg infectious dose (EID50) of clade 2.3.4.4e A/teal/Tottori/1/2016 (H5N6). RESULTS: All birds survived for 10 days without showing any clinical signs of infection. Most ducks inoculated with ≥ 104 EID50 of virus seroconverted within 10 days post-inoculation (dpi). Virus was mainly shed via the oral route for a maximum of 10 days, followed by cloacal route in late phase of infection. Virus remained in the pancreas of some ducks at 10 dpi. Viremia was observed in some ducks euthanized at 3 dpi, and ≤ 106.3 EID50 of virus was recovered from systemic tissues and swab samples including eyeballs and conjunctival swabs. CONCLUSIONS: These results indicate that the subject duck species have a potential to be a source of infection of clade 2.3.4.4e HPAI virus to the environment and other birds sharing their habitats. Captive ducks should be reared under isolated or separated circumstances during the HPAI epidemic season to prevent infection and further viral dissemination.

Pathogenicity of clade 2.3.2.1 H5N1 highly pathogenic avian influenza virus in American kestrel (Falco sparverius).

Birds of prey, including endangered species, have been infected with H5 highly pathogenic avian influenza viruses (HPAIVs) in several countries. In this present study, we assessed the pathogenicity of the clade 2.3.2.1 H5N1 HPAIV in American kestrels (Falco sparverius) with a view to preventing future outbreaks in raptors. The kestrels were intranasally inoculated with the virus or fed the meat of chicks that had died from viral infection. Kestrels in both groups initially had reduced food intake, showed clinical signs such as depression and neurologic manifestations, and succumbed to the infection within 6 days. The kestrels primarily shed the virus orally from 1 day post-inoculation until death, with an average titre of 104.5-5.7 EID50/ml, which is comparable to the inoculum titre. The viruses replicated in almost all tested tissues; notably, the feather calamuses also contained infectious virions and/or viral genes. Pancreatic lesions were present in several infected birds, as shown in previous cases of HPAIV infection in raptors. These results indicate that kestrels are highly susceptible to infection by clade 2.3.2.1 H5 HPAIVs, which readily occurs through the consumption of infected bird carcasses. Early detection and removal of HPAIV infected carcasses in the field is essential for preventing outbreaks in raptors. RESEARCH HIGHLIGHTS Clade 2.3.2.1 H5 HPAIV caused lethal infection in American kestrels. Kestrels with the HPAIV showed neurologic signs and eye disorders. The HPAIV replicated in systemic tissues of kestrels, and was orally shed. The HPAIV was recovered from feather calamus of kestrels.

Pathogenicity of H5 highly pathogenic avian influenza virus in rooks (Corvus frugilegus).

Rooks (Corvus frugilegus) are considered migratory crows in Japan. Some rooks share a wintering site in the Izumi plain in Kagoshima Prefecture with hooded cranes (Grus monacha) and white-necked cranes (Grus vipio), which are designated as "endangered" in the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. Highly pathogenic avian influenza (HPAI), caused by H5 subtype viruses, has recently been reported in these crane species in Japan, in conjunction with a massive decrease in their population. In the present study, the pathogenicity of HPAI virus was assessed in rooks to evaluate the likelihood that they are a source of infections in other bird species. One of four rooks intranasally inoculated with A/mandarin duck/Miyazaki/22M807-1/2011 (H5N1) died at 10 days post-inoculation (d.p.i.). The other three rooks exhibited seroconversion but no clinical signs. All the rooks had shed virus by the oral route at <103 50% egg infectious dose/ml until 7 d.p.i. Virus was also recovered from multiple tissues of the rook that succumbed to the infection. These results suggest that rooks are susceptible to infection with H5 HPAI viruses, leading to prolonged viral shedding. The rooks shed the virus at low titres however, indicating that they are likely to function as transmission vectors in wintering bird flocks. The rooks exhibited clear antibody responses against the H5 HPAI virus, and thus serological surveillance of them in the field should be helpful for assessing viral pervasion into the habitats of crane species.

Characterization of Highly Pathogenic Avian Influenza Virus A(H5N6), Japan, November 2016.

Highly pathogenic avian influenza viruses (HPAIVs) A(H5N6) were concurrently introduced into several distant regions of Japan in November 2016. These viruses were classified into the genetic clade 2.3.4.4c and were genetically closely related to H5N6 HPAIVs recently isolated in South Korea and China. In addition, these HPAIVs showed further antigenic drift.

Characterization of clade 2.3.4.4 H5N8 highly pathogenic avian influenza viruses from wild birds possessing atypical hemagglutinin polybasic cleavage sites.

Since 2014, clade 2.3.4.4 H5 subtype highly pathogenic avian influenza viruses (HPAIVs) have been distributed worldwide. These viruses, which were reported to be highly virulent in chickens by intravenous inoculation, have a consensus HPAI motif PLRERRRKR at the HA cleavage site. However, two-clade 2.3.4.4 H5N8 viruses which we isolated from wild migratory birds in late 2014 in Japan possessed atypical HA cleavage sequences. A swan isolate, Tottori/C6, had a novel polybasic cleavage sequence, PLGERRRKR, and another isolate from a dead mandarin duck, Gifu/01, had a heterogeneous mixture of consensus PLRERRRKR and variant PLRERRRRKR sequences. The polybasic HA cleavage site is the prime virulence determinant of AIVs. Therefore, in the present study, we examined the pathogenicity of these H5N8 isolates in chickens by intravenous inoculation. When 106 EID50 of these viruses were intravenously inoculated into chickens, the mean death time associated with Tottori/C6 was substantially longer (>6.1 days) than that associated with Gifu/01 (2.5 days). These viruses had comparable abilities to replicate in tissue culture cells in the presence and absence of exogenous trypsin, but the growth of Tottori/C6 was hampered. These results indicate that the novel cleavage motif of Tottori/C6 did not directly affect the infectivity of the virus, but Tottori/C6 caused attenuated pathogenicity in chickens because of hampered replication efficiency. It is important to test for the emergence of diversified HPAIVs, because introduction of HPAIVs with a lower virulence like Tottori/C6 might hinder early detection of affected birds in poultry farms.

Amino acid substitution at position 44 of matrix protein 2 of an avirulent H5 avian influenza virus is crucial for acquiring the highly pathogenic phenotype in chickens.

The pathogenicity of highly pathogenic avian influenza (HPAI) viruses is dependent on multiple factors, but the sequence at the HA cleavage site plays the most important role. To better understand the mechanism of virulence of HPAI virus, an avirulent H5 avian influenza virus, A/teal/Tottori/150/02 (H5N3, teal/150), was passaged in respiratory organs of chickens to generate a virus with a highly pathogenic phenotype. After 12 consecutive passages, the virus (strain 12a) became highly pathogenic, with a 100 % mortality rate in chickens. Sequence analysis of the highly pathogenic variant revealed an amino acid change from aspartic acid (Asp) to asparagine (Asn) at position 44 of matrix protein 2 (M2). To investigate the role of M2 in the pathogenicity of HPAI virus, we generated reassortant viruses possessing a polybasic HA cleavage site and either Asp or Asn at position 44 of M2 using the highly pathogenic strain 12a and the avirulent strain 7a, which has Asp at position 44 of M2 derived from isolate teal/150, and we compared their pathogenicity in chickens. Experimental infections demonstrated that the pathogenicity of viruses possessing Asp in M2 was dramatically decreased, and the mortality rate of inoculated chickens was 0 %, in contrast to viruses with Asn, which showed 70 to 100 % mortality. Our findings indicate that M2 protein of the avirulent H5 avian influenza virus is important for acquiring high virulence and that Asn at position 44 of M2, in addition to the polybasic HA cleavage site, is crucial for high pathogenicity in chickens.

Susceptibility of wild passerines to subtype H5N1 highly pathogenic avian influenza viruses.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have spread throughout many areas of Asia, Europe and Africa, and numerous cases of HPAI outbreaks in domestic and wild birds have been reported. Although recent studies suggest that the dissemination of H5N1 viruses is closely linked to the migration of wild birds, information on the potential for viral infection in species other than poultry and waterfowl is relatively limited. To investigate the susceptibility of terrestrial wild birds to infection with H5N1 HPAI viruses, common reed buntings (Emberiza schoeniclus), pale thrushes (Turdus pallidus) and brown-eared bulbuls (Hypsipetes amaurotis) were infected with A/mountain hawk-eagle/Kumamoto/1/07(H5N1) and A/whooper swan/Aomori/1/08(H5N1). The results showed that common reed buntings and brown-eared bulbuls were severely affected by both virus strains (100% mortality). While pale thrushes did not exhibit any clinical signs, seroconversion was confirmed. In common reed buntings, intraspecies-transmission of A/whooper swan/Aomori/1/08 to contact birds was also confirmed. The findings show that three passerine species; common reed buntings, brown-eared bulbuls and pale thrushes are susceptible to infection by H5N1 HPAI viruses, which emphasizes that continued surveillance of species other than waterfowl is crucial for effective monitoring of H5N1 HPAI virus outbreaks.

A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice.

Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.

Novel Genotype of HA Clade 2.3.4.4b H5N8 Subtype High Pathogenicity Avian Influenza Virus Emerged at a Wintering Site of Migratory Birds in Japan, 2021/22 Winter.

Surveillance of avian influenza virus (AIV) was conducted in the 2021-2022 winter season at a wintering site of migratory Anatidae in Japan. An H5N8 subtype high pathogenicity AIV (HPAIV) with a unique gene constellation and four low pathogenicity AIVs (LPAIVs) were isolated from environmental samples. The genetic origin of the HPAIV (NK1201) was determined with whole-genome sequencing and phylogenetic analyses. Six of NK1201's eight genes were closely related to HA clade 2.3.4.4b H5N8 subtype HPAIVs, belonging to the G2a group, which was responsible for outbreaks in poultry farms in November 2021 in Japan. However, the remaining two genes, PB1 and NP, most closely matched those of the LPAIVs H7N7 and H1N8, which were isolated at the same place in the same 2021-2022 winter. No virus of the NK1201 genotype had been detected prior to the 2021-2022 winter, indicating that it emerged via genetic reassortment among HPAIV and LPAIVs, which were prevalent at the same wintering site. In addition, experimental infection in chickens indicated that NK1201 had slightly different infectivity compared to the reported infectivity of the representative G2a group H5N8 HPAIV, suggesting that the PB1 and NP genes derived from LPAIVs might have affected the pathogenicity of the virus in chickens. Our results directly demonstrate the emergence of a novel genotype of H5N8 HPAIV through gene reassortment at a wintering site. Analyses of AIVs at wintering sites can help to identify the emergence of novel HPAIVs, which pose risks to poultry, livestock, and humans.

Multiple infections with H5N8 subtype high pathogenicity avian influenza viruses in a feral mallard.

During the 2020-2021 winter, Eurasian countries experienced large outbreaks caused by the clade 2.3.4.4b H5N8 subtype high pathogenicity avian influenza viruses (HPAIVs) in the wild bird populations. At least seven gene constellations have been found in the causal HPAIVs. When and where the various HPAIVs emerged remains unclear. Here, we successfully cloned H5N8 HPAIVs with multiple gene constellations from a tracheal swab of a dead mallard found at its wintering site in Japan in January 2021. According to their phylogeny, the bird was most likely co-infected with the E2 and E3 genotype clade 2.3.4.4b HPAIVs. The result indicates that feral waterbirds can be infected with multiple HPAIVs, and shed an HPAIV with novel gene constellation in Southern wintering sites.

Genetic and antigenic analyses of H5N8 and H5N1 subtypes high pathogenicity avian influenza viruses isolated from wild birds and poultry farms in Japan in the winter of 2021-2022.

In the winter of 2021-2022, multiple subtypes (H5N8 and H5N1) of high pathogenicity avian influenza viruses (HPAIVs) were confirmed to be circulating simultaneously in Japan. Here, we phylogenetically and antigenically analyzed HPAIVs that were isolated from infected wild birds, an epidemiological investigation of affected poultry farms, and our own active surveillance study. H5 subtype hemagglutinin (HA) genes of 32 representative HPAIV isolates were classified into clade 2.3.4.4b lineage and subsequently divided into three groups (G2a, G2b, and G2d). All H5N8 HPAIVs were isolated in early winter and had HA genes belonging to the G2a group. H5N1 HPAIVs belong to the G2b and G2d groups. Although G2b viruses were widespread throughout the season, G2d viruses endemically circulated in Northeast Japan after January 2022. Deep sequence analysis showed that the four HPAIVs isolated at the beginning of winter had both N8 and N1 subtypes of neuraminidase genes. Environmental water-derived G2a HPAIV, A/water/Tottori/NK1201-2/2021 (H5N8), has unique polymerase basic protein 1 and nucleoprotein genes, similar to those of low pathogenicity avian influenza viruses (LPAIVs). These results indicate that multiple H5 HPAIVs and LPAIVs disseminated to Japan via transboundary winter migration of wild birds, and HPAIVs with novel gene constellations could emerge in these populations. Cross-neutralization test revealed that G2a H5N8 HPAIVs were antigenically distinct from a G2b H5N1 HPAIV, suggesting that antibody pressure in wild birds was involved in the transition of the HPAIV groups during the season.

Susceptibility of common dabbling and diving duck species to clade 2.3.2.1 H5N1 high pathogenicity avian influenza virus: an experimental infection study.

In the winter of 2010-2011, Japan experienced a large outbreak of infections caused by clade 2.3.2.1 H5N1 high pathogenicity avian influenza viruses (HPAIVs) in wild birds. Interestingly, many tufted ducks (Aythya fuligula), which are migratory diving ducks, succumbed to the infection, whereas only one infection case was reported in migratory dabbling duck species, the major natural hosts of the influenza A virus, during the outbreak. To assess whether the susceptibility of each duck species to HPAIVs was correlated with the number of cases, tufted duck and dabbling duck species (Eurasian wigeon, Mareca penelope; mallard, Anas platyrhynchos; Northern pintail, Anas acuta) were intranasally inoculated with A/Mandarin duck/Miyazaki/22M807-1/2011 (H5N1), an index clade 2.3.2.1 virus previously used for experimental infection studies in various bird species. All ducks observed for 10 days post-inoculation (dpi) mostly shed the virus via the oral route and survived. The tufted ducks shed a higher titer of the virus than the other dabbling duck species, and one of them showed apparent neurological symptoms after 7 dpi, which were accompanied by eye lesions. No clinical symptoms were observed in the dabbling ducks, although systemic infection and viremia were observed in some of them sacrificed at 3 dpi. These results suggest that the susceptibility of clade 2.3.2.1 HPAIVs might differ by duck species.

Reintroduction of H5N1 highly pathogenic avian influenza virus by migratory water birds, causing poultry outbreaks in the 2010-2011 winter season in Japan.

H5N1 highly pathogenic avian influenza virus (HPAIV) was reintroduced and caused outbreaks in chickens in the 2010-2011 winter season in Japan, which had been free from highly pathogenic avian influenza (HPAI) since 2007 when HPAI outbreaks occurred and were controlled. On 14 October 2010 at Lake Ohnuma, Wakkanai, the northernmost part of Hokkaido, Japan, H5N1 HPAIVs were isolated from faecal samples of ducks flying from their nesting lakes in Siberia. Since then, in Japan, H5N1 HPAIVs have been isolated from 63 wild birds in 17 prefectures and caused HPAI outbreaks in 24 chicken farms in nine prefectures by the end of March in 2011. Each of these isolates was genetically closely related to the HPAIV isolates at Lake Ohnuma, and those in China, Mongolia, Russia and Korea, belonging to genetic clade 2.3.2.1. In addition, these isolates were genetically classified into three groups, suggesting that the viruses were transmitted by migratory water birds through at least three different routes from their northern territory to Japan. These isolates were antigenic variants, which is consistent with selection in poultry under the immunological pressure induced by vaccination. To prevent the perpetuation of viruses in the lakes where water birds nest in summer in Siberia, prompt eradication of HPAIVs in poultry is urgently needed in Asian countries where HPAI has not been controlled.

Possible bidirectional human-swine and subsequent human-human transmission of influenza virus A(H1N1)/2009 in Japan.

In 2019, sows at a swine farm in Japan showed influenza-like illness (ILI) shortly after contact with an employee that exhibited ILI. Subsequently, a veterinarian became sick shortly after examining the sows and was diagnosed with influenza A virus (IAV) infection. Then, her family also contracted the infection. Subsequently, Pandemic A(H1N1)2009 viruses were isolated from all samples obtained from the sows, veterinarian and her family. Whole-genome analysis of the isolates confirmed that the viruses belonged to the same lineage (6B.1A) and the genome sequences obtained from all of the isolates were almost identical to each other. Furthermore, an epidemiological survey revealed no contact between veterinarians or their families and influenza patients prior to the onset of illness. These results strongly indicated a case of bidirectional infection between humans and sows. At the same time, we found a few unique mutations in the IAV genomes corresponding to the host species. The mutations that occurred in the virus after it was transferred from the farm worker to the sows were not observed in the humans infected from the sows, probably as a result of the mutations reverting to the original nucleotides. These results demonstrate that the bidirectional transmission of IAV is a potential risk for the next pandemic outbreak due to the emergence of new mutant strains.

Molecular changes associated with adaptation of equine influenza H3N8 virus in embryonated chicken eggs.

Embryonated chicken eggs (ECEs) are routinely used to isolate equine influenza virus. Propagation of the virus in ECEs results in selection of variants. In the present study, we determined nucleotide sequences of entire coding regions of parent A/equine/Tottori/1/07 (H3N8) and its derivatives that have different passage histories in ECE. After 12 passages, nucleotide sequence analysis predicted 3 amino acid substitutions in hemagglutinin (HA; 2 in HA1 and 1 in HA2). The two amino acid substitutions in HA1 were located in the vicinity of the cell receptor-binding site. Three other amino acid substitutions were predicted in internal proteins, 1 in the M1, 1 in the NP and 1 in the PA. This is the first report showing mutations in the internal protein genes of equine influenza virus associated with adaptation to ECE.

A sero-survey of subtype H3 influenza A virus infection in dogs and cats in Japan.

A sero-epidemiological survey of human and equine H3 influenza A virus infections in dogs and cats using the hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests was conducted. Serum samples were collected from 582 dogs and 237 cats in Japan during the periods 2002-2008 and 1997-2008, respectively. Although no HI antibodies against equine H3 virus were detected, 9 (3.8%) from cats and 12 (2.1%) from dogs were HI-positive against human H3 virus. Only one serum each from dogs and cats was NI-positive against N2 virus. These findings suggest that although equine H3 influenza virus infections have not been prevalent in companion animals, human H3N2 influenza A virus infections have occurred in dogs and cats in recent years in Japan.

Dynamics of invasion and dissemination of H5N6 highly pathogenic avian influenza viruses in 2016-2017 winter in Japan.

Large highly pathogenic avian influenza (HPAI) outbreaks caused by clade 2.3.4.4e H5N6 viruses occurred in Japan during the 2016-2017 winter. To date, several reports regarding these outbreaks have been published, however a comprehensive study including geographical and time course validations has not been performed. Herein, 58 Japanese HPAI virus (HPAIV) isolates from the 2016-2017 season were added for phylogenetic analyses and the antigenic relationships among the causal viruses were elucidated. The locations where HPAIVs were found in the early phase of the outbreaks were clustered into three regions. Genotypes C1, C5, and C6-8 HPAIVs were found in specific areas. Two strains had phylogenetically distinct hemagglutinin (HA) and non-structural (NS) genes from other previously identified strains, respectively. The estimated latest divergence date between the viral genotypes suggests that genetic reassortment occurred in bird populations before their winter migration to Japan. Antigenic differences in 2016-2017 HPAIVs were not observed, suggesting that antibody pressure in the birds did not contribute to the selection of HPAIV genotypes. In the late phase, the majority of HPAI cases in wild birds occurred south of the lake freezing line. At the end of the outbreak, HPAI re-occurred in East coast region, which may be due to the spring migration route of Anas bird species. These trends were similar to those observed in the 2010-2011 outbreaks, suggesting there is a typical pattern of seeding and dissemination of HPAIV in Japan.

A cluster epidemic of influenza A(H1N1)pdm09 virus infection in four captive cheetahs (Acinonyx jubatus).

In January 2019, four cheetahs (Acinonyx jubatus) kept at a Japanese zoo intermittently showed respiratory signs following the incidence of seasonal influenza in animal caregivers. Respiratory materials (saliva, sputum and food tray swabs) were non-invasively collected from the four cheetahs. Although we were unable to isolate the virus, the NP gene of influenza A virus was detected in three of the cheetahs but not in the fourth cheetah that had nearly recovered. From a food tray swab which tested weakly positive by a commercial influenza detection kit, we were able to obtain the whole-genome sequence of the influenza A virus. Analysis of the genome, A/cheetah/Kanagawa/2/2019(H1N1), revealed that the virus was closely related to influenza A(H1N1)pdm09 viruses isolated from humans in Japan in the 2018-2019 winter. Production of haemagglutinin inhibition (HI) antibodies (64-128 HI) against an A(H1N1)pdm09 virus in plasma samples confirmed infection of all four cheetahs. The animals continued to produce antibodies for at least 314 days after disease onset. These findings strongly suggest that reverse zoonotic transmission of A(H1N1)pdm09 virus occurred from human to cheetah and subsequently from cheetah to cheetah in the zoo. We also show that specimens can be safely and non-invasively collected from non-domesticated animals and used to investigate respiratory infectious diseases.

Susceptibility of two species of wild terrestrial birds to infection with a highly pathogenic avian influenza virus of H5N1 subtype.

The recent epidemic caused by H5N1 highly pathogenic avian influenza (HPAI) viruses has spread over many parts of Asia, Europe and Africa. Wild birds, particularly waterfowl, are considered to play a role in viral dissemination. However, detailed information on whether wild terrestrial birds act as carriers is currently unavailable. To investigate the susceptibility of terrestrial birds to HPAI viruses, two species of wild bird (great reed warbler and pale thrush) that are common in East Asia were infected with H5N1 HPAI virus. The results showed that both species were highly susceptible to the virus. The great reed warbler showed fatal infection with 100% mortality, but the pale thrush survived for longer periods (>8 days) with viral shedding. These findings suggest that there is variation in clinical outcome after infection of wild terrestrial birds, and that some bird species could become subclinical excretors of the H5N1 virus.