Susceptibility of chickens, quail, and pigeons to an H7N9 human influenza virus and subsequent egg-passaged strains.

H7N9 human influenza virus A/Anhui/1/2013 (Anhui2013) showed low pathogenicity in chickens, quail, and pigeons, with quail being the most susceptible among the species tested. IVPIE1-1, which was recovered from a dead chicken after intravenous inoculation of Anhui 2013, had broader tissue tropism in chickens than did the original inoculum, as well as amino acid substitutions in the polymerase acidic gene and neuraminidase gene segments, but its pathogenicity was not enhanced. Viruses obtained after passage of Anhui 2013 in 10- and 14-day-old embryonated eggs showed rapid accumulation of amino acid substitutions at the receptor-binding site of the hemagglutinin protein. Two strains obtained through egg passage, 10E4/14E17 and 10E4/10E13, replicated better in intranasally infected chickens than did the original Anhui 2013 strain, yet the new isolates showed low pathogenicity in chickens despite their amino acid substitutions. The increased virus replication in chickens of 10E4/14E17 and 10E4/10E13 was not correlated with temperature-sensitive replication, given that virus replication was suppressed at increased temperatures. The existence of highly susceptible hosts, such as quail, which permit asymptomatic infection, facilitates increased mutation of the virus through amino acid substitution at the receptor-binding site, and this might be one of the mechanisms underlying the prolonged circulation of H7N9 influenza virus.

Pathogenicity of H5N8 highly pathogenic avian influenza viruses isolated from a wild bird fecal specimen and a chicken in Japan in 2014.

Poultry outbreaks caused by H5N8 highly pathogenic avian influenza viruses (HPAIVs) occurred in Japan between December 2014 and January 2015. During the same period; H5N8 HPAIVs were isolated from wild birds and the environment in Japan. The hemagglutinin (HA) genes of these isolates were found to belong to clade 2.3.4.4 and three sub-groups were distinguishable within this clade. All of the Japanese isolates from poultry outbreaks belonged to the same sub-group; whereas wild bird isolates belonged to the other sub-groups. To examine whether the difference in pathogenicity to chickens between isolates of different HA sub-groups of clade 2.3.4.4 could explain why the Japanese poultry outbreaks were only caused by a particular sub-group; pathogenicities of A/chicken/Miyazaki/7/2014 (Miyazaki2014; sub-group C) and A/duck/Chiba/26-372-48/2014 (Chiba2014; sub-group A) to chickens were compared and it was found that the lethality of Miyazaki2014 in chickens was lower than that of Chiba2014; according to the 50% chicken lethal dose. This indicated that differences in pathogenicity may not explain why the Japanese poultry outbreaks only involved group C isolates.

Intracontinental and intercontinental dissemination of Asian H5 highly pathogenic avian influenza virus (clade 2.3.4.4) in the winter of 2014-2015.

Asian H5 highly pathogenic avian influenza viruses (HPAIVs) that possess the clade 2.3.4.4 HA gene have been identified in wild birds and poultry since late 2014 in both Europe and North America (N. America). Clade 2.3.4.4 H5 HPAIVs of the H5N8 subtype have been isolated in both regions, whereas reassortment viruses with NA N1 and N2 subtypes of the North American (N. American). avian lineage have only been identified in N. America. The HA genes of those isolates were closely related to genes of the HPAIVs that have caused massive outbreaks in poultry in Korea since January 2014. The outbreaks caused by those viruses and the genetic relatedness of their HA and NA genes are reviewed in this study. Although the illegal movement of poultry and poultry products cannot be ruled out as a cause of intercontinental and intracontinental dissemination of clade 2.3.4.4 H5 HPAIVs during the winter of 2014-2015, transmission of the viruses by infected migratory birds appears to be a more plausible mechanism for their dissemination. In particular, the involvement of migratory birds in HPAIV transmission between Asia and N. America is highly likely because of the reassortments between H5N8 HPAIV and the N. American lineage avian influenza viruses.

Characterization of an H5N8 influenza A virus isolated from chickens during an outbreak of severe avian influenza in Japan in April 2014.

A highly pathogenic avian influenza virus (HPAIV) of subtype H5N8, A/chicken/Kumamoto/1-7/2014, was isolated from a Japanese chicken farm during an outbreak in April 2014. Phylogenetic analysis revealed that this virus belonged to HA clade 2.3.4.4. All eight genomic segments showed high sequence similarity to those of the H5N8 subtype HPAIVs A/broiler duck/Korea/Buan2/2014 and A/baikal teal/Korea/Donglim3/2014, which were isolated in Korea in January 2014. Intranasal experimental infection of chickens and ducks with A/chicken/Kumamoto/1-7/2014 was performed to assess the pathogenicity of the virus in chickens and the potential for waterfowl to act as a virus reservoir and carrier. A high-titer virus challenge (10(6) EID50 per animal) was lethal in chickens, but they were unaffected by lower virus doses (10(2) EID50 or 10(4) EID50 per animal). Virus challenge at all doses examined was found to result in asymptomatic infection of ducks. An HI assay revealed that A/chicken/Kumamoto/1-7/2014 possessed relatively low cross-reactivity with H5 viruses belonging to clades other than clade 2.3.4.4. These results suggest that waterfowl may be able to spread the virus even if they possess antibodies resulting from a previous infection with H5 HPAIV that was antigenically distinguishable from viruses belonging to clade 2.3.4.4.

Ubiquitin-specific protease 2-69 in macrophages potentially modulates metainflammation.

Macrophages play a critical role in chronic inflammation and metabolic diseases. We identified a longer splice variant of ubiquitin specific protease (USP) 2-69 as a novel molecule that modulates pathways implicated in metabolic disorders. Expression levels of aP2/FABP4 and PAI-1/SERPINE1 genes were increased by 4- and 1.8-fold, respectively, after short hairpin RNA-mediated knockdown (KD) of the USP2 gene, and such expression was alleviated by overexpression of USP2-69 in human myeloid cell lines. Supernatants derived from USP2-KD cells induced IL6 (∼6-fold) and SAA3 (∼15-fold) in 3T3-L1 adipocytes to suggest the anti-inflammatory properties of USP2. In addition, we observed a 30% decrease in the number of macrophages in mesenteric adipose tissue derived from USP2-69 transgenic mice fed a high-fat diet for 14 wk compared with that in their C57BL/6 littermates (P<0.01), which was consistent with a ∼40% decrease in transcription of aP2 and PAI-1. The aP2 locus exhibited elevated chromatin accessibility (>2.1-fold), methylation of histone H3 lysine 4 (>4.5-fold), and acetylation of histone H4 (>2.5-fold) in USP2-KD cells. Transfection of isopeptidase-mutated USP2-69 did not alter chromatin conformation on the aP2 locus in USP2-KD cells. Our results suggest that USP2-69 suppresses meta-inflammatory molecules involved in the development of type-2 diabetes.

Reassortant swine influenza viruses isolated in Japan contain genes from pandemic A(H1N1) 2009.

In 2013, three reassortant swine influenza viruses (SIVs)-two H1N2 and one H3N2-were isolated from symptomatic pigs in Japan; each contained genes from the pandemic A(H1N1) 2009 virus and endemic SIVs. Phylogenetic analysis revealed that the two H1N2 viruses, A/swine/Gunma/1/2013 and A/swine/Ibaraki/1/2013, were reassortants that contain genes from the following three distinct lineages: (i) H1 and nucleoprotein (NP) genes derived from a classical swine H1 HA lineage uniquely circulating among Japanese SIVs; (ii) neuraminidase (NA) genes from human-like H1N2 swine viruses; and (iii) other genes from pandemic A(H1N1) 2009 viruses. The H3N2 virus, A/swine/Miyazaki/2/2013, comprised genes from two sources: (i) hemagglutinin (HA) and NA genes derived from human and human-like H3N2 swine viruses and (ii) other genes from pandemic A(H1N1) 2009 viruses. Phylogenetic analysis also indicated that each of the reassortants may have arisen independently in Japanese pigs. A/swine/Miyazaki/2/2013 were found to have strong antigenic reactivities with antisera generated for some seasonal human-lineage viruses isolated during or before 2003, whereas A/swine/Miyazaki/2/2013 reactivities with antisera against viruses isolated after 2004 were clearly weaker. In addition, antisera against some strains of seasonal human-lineage H1 viruses did not react with either A/swine/Gunma/1/2013 or A/swine/Ibaraki/1/2013. These findings indicate that emergence and spread of these reassortant SIVs is a potential public health risk.

Experimental West Nile virus infection in aigamo ducks, a cross between wild ducks (Anas platyrhynchos) and domestic ducks (Anas platyrhynchos var. domesticus).

Four 2-wk-old and four 4-wk-old aigamo ducks, a cross between wild and domestic ducks (Anas platyrhynchos and Anas platyrhynchos var. domesticus, respectively), were infected with the NY99 strain of West Nile virus (WNV) to investigate WNV's pathogenicity in aigamo ducks and the possibility that they could transmit WNV. In the group of infected 2-wk-old aigamo ducks (2w-infection group), all of the ducks ate and drank less and showed decreased activity, some showed ataxia, and one died. Meanwhile, the group of infected 4 wk olds (4w-infection group) showed no clinical signs during the experimental period. Viremia was observed in all of the ducks in both age groups. Peak viral titers in the three surviving members of the 2w-infection group were 10(3.7)-10(5.3) plaque-forming units (PFU)/ml serum; the peak was 10(7.1) PFU/ml serum in the 2w duck that died from the infection. Peak viral titers in the 4w-infection group were 10(4.1)-10(4.9) PFU/ml serum. Viral shedding in the oral and/or cloacal cavity was observed in all four members of the 2w-infection group and in three of the four members of the 4w-infection group. These results suggest that WNV-infected aigamo ducks can transmit WNV. Although aigamo ducks are reared in East Asia, where WNV is an exotic pathogen, the virus could be introduced and spread there in the future; thus it is important to take precautions against an introduction, and measures to prevent infection to aigamo duck operations should be prepared.

Antibody responses induced by experimental West Nile virus infection with or without previous immunization with inactivated Japanese encephalitis vaccine in horses.

A group of horses immunized with inactivated Japanese encephalitis (JE) vaccine (JE-Immune Group) and a group of non-immunized horses (Non-Immune Group) were infected with West Nile virus (WNV). After WNV infection, neutralizing (Nt) antibody (Ab) titers to WNV were higher than those to JE virus (JEV) in the Non-Immune Group, but the NtAb titers to JEV were higher than those to WNV during most of the post-challenge observation period in the JE-Immune Group. Immunoglobulin M (IgM) Abs to WNV tested positive in the Non-Immune Group but negative in the JE-Immune Group, except for in one horse. These results suggest that diagnosis of WNV infection in JE-immunized horses requires serological tests for NtAb and IgM titers to both WNV and JEV.

The inhibitory effect of a combination of imidacloprid and permethrin on blood feeding by mosquitoes in dogs raised under outdoor conditions.

Combinations of imidacloprid and permethrin were frequently used to control harmful arthropod of companion animals. The inhibitory effects on blood-feeding activity of mosquitoes in dogs raised under outdoor conditions were evaluated by using combination of 10% (w/v) of imidacloprid and 50% (w/v) of permethrin as spot-on form. Dogs in the treated group received the combination imidacloprid/permethrin spot-on. After treatment, dogs in the control and treated groups were kept separately from the evening (17:00) to the morning of the following day (09:00) in two different kennels installed outdoors to mimic realistic dog-raising conditions. Mosquitoes in the kennels were collected by light traps placed in the kennels and a sweep net to determine evidence of blood feeding, and for species identification. Mosquitoes were collected at Days 5, 3 and 1 before agent treatment, and the Day of treatment, and Days 3, 7, 14, 21, 28, 35 and 42 after treatment. The percentages of blood-fed mosquitoes measured at Days 0, 3, 21, 28 and 42 after treatment were statistically significantly lower (p<0.01) in the treated group than in the control group. The most commonly collected mosquito, Culex tritaeniorhynchus, revealed statistically significant lower percentages (p<0.01) of blood-fed mosquitoes in the treated group than in the control group at the Day of treatment, and Days 3, 7, 21, 28 and 42 after treatment. It appeared that the test agent was effective in inhibiting blood feeding by adult female mosquitoes, and the efficacy lasts for 42 days after treatment under outdoor conditions.

Transmission of an H5N8-Subtype Highly Pathogenic Avian Influenza Virus from Infected Hens to Laid Eggs.

We showed here that an H5N8-subtype highly pathogenic avian influenza virus (HPAIV) was transmitted to both the internal contents and shells of eggs laid by white leghorn hens experimentally infected with the virus. Seven of eight HPAIV-infected hens laid eggs until 4 days postinoculation (dpi). The mean number of eggs laid per head daily decreased significantly from 0.58 before inoculation to 0.18 after viral inoculation. The virus was detected in the eggs laid by three of the seven hens. Viral transmission was detectable beginning on 3 dpi, and virus titers in tracheal and cloacal swabs from the hens that laid the contaminated eggs exceeded 2.9 log10 EID50. The level of viral replication and its timing when virus replicates enough to be detected in oviduct after virus inoculation appear to be key factors in the transmission of H5N8 HPAIV from infected hens to laid eggs.

Specific Monoclonal Antibody Overcomes the Salmonella enterica Serovar Typhimurium's Adaptive Mechanisms of Intramacrophage Survival and Replication.

Salmonella-specific antibodies play an important role in host immunity; however, the mechanisms of Salmonella clearance by pathogen-specific antibodies remain to be completely elucidated since previous studies on antibody-mediated protection have yielded inconsistent results. These inconsistencies are at least partially attributable to the use of polyclonal antibodies against Salmonella antigens. Here, we developed a new monoclonal antibody (mAb)-449 and identified its related immunogen that protected BALB/c mice from infection with Salmonella enterica serovar Typhimurium. In addition, these data indicate that the mAb-449 immunogen is likely a major protective antigen. Using in vitro infection studies, we also analyzed the mechanism by which mAb-449 conferred host protection. Notably, macrophages infected with mAb-449-treated S. Typhimurium showed enhanced pathogen uptake compared to counterparts infected with control IgG-treated bacteria. Moreover, these macrophages produced elevated levels of pro-inflammatory cytokine TNFα and nitric oxide, indicating that mAb-449 enhanced macrophage activation. Finally, the number of intracellular bacteria in mAb-449-activated macrophages decreased considerably, while the opposite was found in IgG-treated controls. Based on these findings, we suggest that, although S. Typhimurium has the potential to survive and replicate within macrophages, host production of a specific antibody can effectively mediate macrophage activation for clearance of intracellular bacteria.

Surveillance of avian paramyxovirus serotype-1 in migratory waterfowls in Japan between 2011 and 2013.

To further understand the epidemiology of avian paramyxovirus serotype-1 (APMV-1) in migratory waterfowls in Japan, we conducted the surveillance of this virus from feces derived from the migratory waterfowls collected in 41 Japanese prefectures between October 2011 and March 2013. Six APMV-1 viruses were isolated from total 661 samples. All isolates were identified as the avirulent (lentogenic) type on the basis of intracerebral pathogenicity tests. Genetic analysis showed that these viruses possessed the deduced amino acid sequence of 112GKQGR-L117 or 112ERQER-L117 at the cleavage site of the F0 protein, which was identical to the motif in the avirulent type. Phylogenetic analysis based on the partial fusion protein gene classified these APMV-1 isolates into 2 major genetic groups. Four isolates were classified as class II genotype I, and they were genetically closely related to strains isolated in Asian countries, including Japan. In contrast, two isolates were classified as class I, and they were genetically closely related to strains mainly isolated in the U.S.A.

Phylogenetic analysis of avian paramyxovirus serotype-1 in pigeons in Japan.

To understand the epidemiology of Avian paramyxovirus serotype-1 (APMV-1) in pigeons in Japan, phylogenetic analysis was comprehensively conducted based on partial fusion protein gene using isolate from the surveillance of this virus with previously known Japanese pigeon strains. This surveillance was conducted using feces obtained from domestic pigeons collected in 40 prefectures throughout Japan from June 2011 to March 2013. From a total of 1,021 samples, a single virus (APMV1/pigeon/Japan/Kanagawa/2013: JP/Kanagawa-pg/2013) was isolated. All Japanese pigeon APMV-1 strains were clustered into a single genetic lineage, which was termed VIb/1 by phylogenetic analysis based on the F gene including the sequence of the cleavage site. These APMV-1 strains were further subdivided into four subgroups identified over 4 separate timeframes: 1984-1995 (group 1), 1995-2000 (group 2), 2001-2007 (group 3) and the novel subgroup isolated in 2013 (group 4). Each subgroup has specific amino acid motifs at a cleavage site of the F protein, namely, (112)GRQKR-F(117)(except for one strain), (112)RRKKR-F(117), (112)RRQKR-F(117) and (112)RRQKR-F(117), respectively. Our data suggest that Japanese APMV-1 strains from pigeons were diverse and reinforced the possibility that there were multiple introduction routes from foreign countries into Japan.

Canine mitochondrial uncoupling proteins: structure and mRNA expression of three isoforms in adult beagles.

Uncoupling proteins (UCPs) are members of the mitochondrial transporter family that dissipate the proton gradient as heat more than via ATP synthesis. In the present study, nucleotide and amino acid sequences of UCPs 1, 2 and 3 of a dog were determined, and their mRNA expression in various peripheral tissues was examined. The sequences were highly (76-97%) homologous to those of other species. Although lower homologies (60-74%) were found when compared among the three canine UCPs, their deduced amino acid sequences had some common domains, such as three mitochondrial carrier protein motifs, six transmembrane alpha-helix domains, and putative purine nucleotide binding domains. By Northern blot analyses, UCP1 mRNA was not detected in any tissues examined. UCP2 mRNA was expressed in most tissues, particularly abundantly in adipose tissue, spleen and lung. Two sizes of UCP3 mRNA were found exclusively in heart and skeletal muscle. These results suggest that canine UCPs have uncoupling activity, and are involved in the regulation of metabolic heat production and/or energy expenditure, as do those of other species.

Bacterial lipopolysaccharide induces mRNA expression of an IkappaB MAIL through toll-like receptor 4.

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IkappaB protein recently identified as a molecule appearing in immunocompetent organs after administration of bacterial lipopolysaccharide (LPS). Participation of Toll-like receptor (TLR) 4, which is a major form of LPS receptors, in the LPS-induced MAIL expression was investigated. When a human myelomonocytic cell line U937 was treated with phorbol 12-myristate 13-acetate for 3 days, the LPS-induced MAIL expression was much potentiated in parallel with an increase in TLR4 expression. The MAIL induction was attenuated when the cells were treated with a neutralizing antibody against TLR4. The in vivo induction of MAIL in the spleen was smaller in mice having a missense mutation of the Tlr4 gene than in normal control mice. These results collectively indicate that TLR4 contributes, at least in part, MAIL induction after LPS stimulation.

Simple differentiation of avirulent and virulent strains of avian paramyxovirus serotype-1 (Newcastle disease virus) by PCR and restriction endonuclease analysis in Japan.

To differentiate the avirulent from virulent strains of avian paramyxovirus serotype-1 (APMV-1, Newcastle disease virus), PCR and restriction endonuclease analysis (REA) was employed. Primer sequences were used to amplify a 766-base pair fragment that included the fusion protein cleavage site. REA of PCR products generated by Hin1 I and Apa I enabled the differentiation of avirulent field and vaccine strains from more virulent field strains of APMV-1 in Japan.

Genetics and infectivity of H5N1 highly pathogenic avian influenza viruses isolated from chickens and wild birds in Japan during 2010-11.

Outbreaks of H5N1 subtype highly pathogenic avian influenza virus (HPAIV) were recorded in chickens, domesticated birds and wild birds throughout Japan from November 2010 to March 2011. Genetic analysis of the Japanese isolates indicated that all gene segments, except the PA gene, were closely related to Japanese wild bird isolates in 2008 and belonged to clade 2.3.2.1 classified by the WHO/OIE/FAO H5N1 Evolution Working Group. Direct ancestors of the PA gene segment of all Japanese viruses analyzed in this study can be found in wild bird strains of several subtypes other than H5N1 isolated between 2007 and 2009. The PA gene of these wild bird isolates share a common ancestor with H5N1 HPAIVs belonging to clades 2.5, 7 and 9, indicating that wild birds were involved in the emergence of the current reassortant 2.3.2.1 viruses. To determine how viruses were maintained in the wild bird population, two isolates derived from chickens (A/chicken/Shimane/1/2010, Ck10 and A/chicken/Miyazaki/S4/2011, CkS411) and one from a wild bird (A/mandarin duck/Miyazaki/22M-765/2011, MandarinD11) were compared in their ability to infect and be transmitted to chickens. There was a significant difference in the survival of chickens that were infected with 10(6)EID(50) of CkS411 compared to those with MandarinD11 and the transmission efficiency of CkS411 was greater than the other viruses. The increased titer of CkS411 excreted from infected chickens contributed to the improved transmission rates. It was considered that reduced virus excretion and transmission of MandarinD11 could have been due to adaptation of the virus in wild birds.

Specific antibody responses to West Nile virus infections in horses preimmunized with inactivated Japanese encephalitis vaccine: evaluation of blocking enzyme-linked immunosorbent assay and complement-dependent cytotoxicity assay.

West Nile virus (WNV) and Japanese encephalitis (JE) virus are distributed separately in the world with some exceptions. There is a concern that WNV may invade into Asia where JE virus exists. On and after such invasion, any differential diagnosis could be complicated by serological crossreactivities. We previously demonstrated experimentally using horses infected with WNV that preimmunization with inactivated JE vaccine considerably affected the ability of neutralization tests and immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (ELISA) to diagnose WNV infection. Here, we investigated WNV-specific antibody responses in vaccinated horses using a blocking ELISA and complement-dependent cytotoxicity (CDC) assay to evaluate these two newly developed serodiagnostic methods for WNV infection. Sera previously collected from six experimentally infected horses were used: Three were vaccinated before the infection, whereas the other three remained unvaccinated. WNV-specific antibody responses were successfully detected in the vaccinated and unvaccinated horses using both new methods, except for one vaccinated horse in which responses were not induced, probably as a result of crossprotection induced by JE vaccination. Specific antibody responses were at earliest detected from days 9 to 10 postinfection in the blocking ELISA, whereas the CDC assay provided earlier detection (at days 7-8) in all horses. The time courses of antibody levels were similar between vaccinated and unvaccinated horses in either method, indicating no notable effect of vaccination on detection of specific antibody responses, as far as antibodies were induced. These results indicated that blocking ELISA, but preferably the CDC assay, can be useful for detecting WNV infection in JE-vaccinated horses.

Genetic characterization and susceptibility on poultry and mammal of H7N6 subtype avian influenza virus isolated in Japan in 2009.

From February to March 2009, six strains of H7N6 subtype avian influenza virus were isolated from quails in three farms in Aichi prefecture in Japan. The isolates were shown to be low pathogenic for chicken by the examination performed using the "Manual of Standards for Diagnostic Tests and Vaccines" by World organisation for Animal Health (OIE). The deduced amino acid sequence at the cleavage site was PE (I/Q/L) PKRR (nucleotide sequences were cct gaa (a/c) (t/a) a cc (a/g) aaa aga aga), suggesting persistence in domestic poultry for some time. The direct putative ancestor strain could not be elucidated by phylogenetic analysis of all genome segments of the quail isolates. Diverged date from a putative common ancestor in a non-rooted phylogenetic tree among quail viruses was estimated between March 2002 and July 2004. Three putative N-linked glycosylation sites resided in the vicinity of the receptor binding pocket of HA1 region. They are considered to decrease the reactivity of neutralizing antibody against the virus. Experiments for the infectivity and pathogenicity of a quail strain to poultry indicated that the quail isolate had higher infectivity to quails than chickens and ducks. Direct and dust-borne and/or droplet-borne transmissions among quail were proven in quails with and without direct contact with experimentally infected quails. The virus is seldom transmitted among chickens either directly or indirectly, and indirect transmission from infected quails to chickens was not observed. The pathogenicity of the quail strain for mammalian, pig and mouse was low, although it could replicate in those animals.

Experimental West Nile virus infection in jungle crows (Corvus macrorhynchos).

We experimentally infected jungle crows (Corvus macrorhynchos), which are representative corvids in East Asia, with West Nile virus (WNV) to study their susceptibility toward WNV infection. Six jungle crows were subcutaneously inoculated with 1,000 plaque-forming units (PFU) of the WNV NY99 strain. Within 7 days after inoculation, five of the six infected crows died, and peak viremias ranged from 10(6.5) to 10(10.9) PFU/mL serum. In addition, infected crows shed WNV in the oral cavity and cloaca, and the virus was widely disseminated in the organs of the crows. Based on these findings, we conclude that jungle crows are highly susceptible to WNV infection, and they could serve as amplifying hosts in the transmission of WNV. Although WNV has not been detected in East Asia, the virus could spread rapidly on introduction into this region because of the large number of potential amplifying hosts and vector mosquitoes that inhabit this region.