Different infection routes of avian influenza A (H5N1) virus in mice

The continuing outbreaks of avian influenza A H5N1 virus infection in Asia and Africa have caused worldwide concern because of the high mortality rates in poultry, suggesting its potential to become a pandemic influenza virus in humans. The transmission route of the virus among either the same species or different species is not yet clear. Broilers and BABL/c mice were inoculated with the H5N1 strain of influenza A virus isolated from birds. The animals were inoculated with 0.1 mL 10^6.83 TCID₅₀ of H5N1 virus oronasally, intraperitoneally and using eye drops. The viruses were examined by virological and pathological assays. In addition, to detect horizontal transmission, in each group, healthy chicks and mice were mixed with those infected. Viruses were detected in homogenates of the heart, liver, spleen, kidney and blood of the infected mice and chickens. Virus antigen was not detected in the spleen, kidney or gastrointestinal tract, but detected by Plaque Forming Unit (PFU) assay in the brain, liver and lung without degenerative change in these organs (in the group inoculated using eye drops. The detection results for mice inoculated using eye drops suggest that this virus might have a different tissue tropism from other influenza viruses mainly restricted to the respiratory tract in mice. All chicken samples tested positive for the virus, regardless of the method of inoculation. Avian influenza A H5N1 viruses are highly pathogenic to chickens, but its virulence in other animals is not yet known. To sum up, the results suggest that the virus replicates not only in different animal species but also through different routes of infection. In addition, the virus was detection not only in the respiratory tract but also in multiple extra‐respiratory tissues. This study demonstrates that H5N1 virus infection in mice can cause systemic disease and spread through potentially novel routes within and between mammalian hosts.     

Induction of a cross-reactive antibody response to influenza virus M2 antigen in pigs by using a Sendai virus vector

Protecting pigs from simultaneous infection with avian, swine, and human influenza viruses would be an effective strategy to prevent the emergence of reassortants with pandemic potential. M2 protein is a candidate antigen for so-called 'universal vaccines,' which confer cross-protection to different influenza viruses in a strain- and subtype-independent manner. We tested whether a recombinant F gene-deleted Sendai virus vector that contained an M2 gene derived from an H5N1 avian influenza virus (SeV/ΔF/H5N1M2) could induce a cross-reactive antibody response to the extracellular domain of M2 protein (M2e) in pigs. SeV/ΔF/H5N1M2 induced an antibody response to M2e when the vector was inoculated intramuscularly. The antibodies induced by SeV/ΔF/H5N1M2 cross-reacted with M2e derived from different avian, swine, and human influenza viruses. In mice, however, SeV/ΔF/H5N1M2 did not confer cross-protection to challenge with a heterologous H3N2 influenza virus. Our results confirm those of other groups indicating that antibodies to M2e do not mediate protection to influenza viruses in pigs.     

Different routes of inoculation impact infectivity and pathogenesis of H5N1 high pathogenicity avian influenza virus infection in chickens and domestic ducks

The H5N1 type A influenza viruses classified as Qinghai-like virus (clade 2.2) are a unique lineage of type A influenza viruses with the capacity to produce significant disease and mortality in gallinaceous and anseriform birds, including domestic and wild ducks. The objective of this study was to determine the susceptibility and pathogenesis of chickens and domestic ducks to A/Whooper Swan/Mongolia/224/05 (H5N1) high pathogenicity avian influenza (HPAI) virus when administered through respiratory or alimentary routes of exposure. The chickens and ducks were more susceptible to the H5N1 HPAI virus, as evidenced by low infectious and lethal viral doses, when exposed by intranasal as compared to alimentary routes of inoculation (intragastric or oral-fed infected chicken meat). In the alimentary exposure pathogenesis study, pathologic changes included hemorrhage, necrosis, and inflammation in association with virus detection. These changes were generally observed in most of the visceral organs of chickens, between 2 and 4 days postinoculation (DPI), and are similar to lesions and virus localization seen in birds in natural cases or in experimental studies using the intranasal route. Alimentary exposure to the virus caused systemic infection in the ducks, characterized by moderate lymphocytic encephalitis, necrotized hepatitis, and pancreatitis with a corresponding demonstration of virus within the lesions. In both chickens and ducks with alimentary exposure, lesions, virus, or both were first demonstrated in the upper alimentary tract on 1 DPI, suggesting that the alimentary tract was the initial site affected upon consumption of infected meat or on gavage of virus in liquid medium. However, as demonstrated in the infectivity study in chickens, alimentary infection required higher exposure doses to produce infection as compared to intranasal exposure in chickens. These data suggest that upper respiratory exposure to H5N1 HPAI virus in birds is more likely to result in virus infection and transmission than will consumption of infected meat, unless the latter contains high doses of virus, as found in cannibalized infected carcasses.     

核酸免疫制备抗猪流感病毒HA单克隆抗体

为制备H1N1猪流感病毒HA蛋白单克隆抗体(monoclonal antibody,McAb),本试验利用表达猪流感病毒A/Swine/Guangdong/2004(H1N1)毒株HA蛋白的表达质粒pVAX1-HA肌注股内肌免疫BALB/c小鼠,将其脾细胞与骨髓瘤细胞(SP2/0)进行融合;通过间接ELISA方法筛选和有限稀释法克隆,获得9株稳定分泌抗HA单克隆抗体的杂交瘤细胞。中和试验结果显示,单克隆抗体4D5株对H1N1流感病毒起中和作用,该单克隆抗体杂交瘤细胞培养上清的效价为1∶1024。这株单克隆抗体与哈尔滨兽医研究所国家重点实验室保存的H3N2流感病毒、H5N1流感病毒均不发生交叉反应,显示出了很好的H1特异性;间接免疫荧光试验结果显示,这株单克隆抗体能与H1N1流感病毒发生特异性反应。制备的特异性抗HA单克隆抗体为建立H1N1流感病毒免疫学检测方法和单链抗体抗病毒复制研究奠定了基础。     

Avian and swine influenza viruses: our current understanding of the zoonotic risk

The introduction of swine or avian influenza (AI) viruses in the human population can set the stage for a pandemic, and many fear that the Asian H5N1 AI virus will become the next pandemic virus. This article first compares the pathogenesis of avian, swine and human influenza viruses in their natural hosts. The major aim was to evaluate the zoonotic potential of swine and avian viruses, and the possible role of pigs in the transmission of AI viruses to humans. Cross-species transfers of swine and avian influenza to humans have been documented on several occasions, but all these viruses lacked the critical capacity to spread from human-to-human. The extreme virulence of H5N1 in humans has been associated with excessive virus replication in the lungs and a prolonged overproduction of cytokines by the host, but there remain many questions about the exact viral cell and tissue tropism. Though pigs are susceptible to several AI subtypes, including H5N1, there is clearly a serious barrier to infection of pigs with such viruses. AI viruses frequently undergo reassortment in pigs, but there is no proof for a role of pigs in the generation of the 1957 or 1968 pandemic reassortants, or in the transmission of H5N1 or other wholly avian viruses to humans. The major conclusion is that cross-species transmission of influenza viruses per se is insufficient to start a human influenza pandemic and that animal influenza viruses must undergo dramatic but largely unknown genetic changes to become established in the human population.     

Development of vaccine strains of H5 and H7 influenza viruses

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokkaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/ Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 viruses as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9 N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.     

PA-X基因的长度变化对H3N2犬流感病毒复制能力和致病性的影响

H3N2犬流感病毒(canine influenza virus, CIV)已在中国多地的犬群中流行,是禽流感跨宿主感染并形成新分支的近期案例。研究表明,PA-X基因与甲型流感病毒适应新宿主的能力相关,且其长度能够影响甲型流感病毒的复制及致病能力。为了解PA-X基因的长度变化对H3N2 CIV复制能力及致病力的影响,本研究利用H3N2 CIV的8质粒操作系统,拯救了三株重组H3N2 CIV毒株：PA-X基因表达大小为232个氨基酸多肽的亲本病毒CIV＿PA-X＿232;对PA编码区第191、192位氨基酸的密码子进行改造,PA-X基因不表达蛋白的重组病毒CIV＿PA-X＿Knock;对PA+1编码区第232位氨基酸进行突变,PA-X基因表达大小为252个氨基酸多肽的重组病毒CIV＿PA-X＿252。通过比较3株重组病毒的聚合酶活性,在MDCK细胞中的复制效率及对小鼠致病性的差异,来评价表达不同长度的PA-X基因对H3N2 CIV的影响。结果显示,CIV＿PA-X＿252和CIV＿PA-X＿Knock的聚合酶活性显著(P<0.05)高于CIV＿PA-X＿232,且CIV＿PA-X＿...     

Serological and virological surveillance of swine H1N1 and H3N2 influenza virus infection in two farms located in Hubei province, central China

Swine influenza viruses H1N1 and H3N2 have been reported in the swine population worldwide. From June 2008 to June 2009, we carried out serological and virological surveillance of swine influenza in the Hubei province in central China. The serological results indicated that antibodies to H1N1 swine influenza virus in the swine population were high with a 42.5% (204/480) positive rate, whereas antibodies to H3N2 swine influenza virus were low with a 7.9% (38/480) positive rate. Virological surveillance showed that only one sample from weanling pigs was positive by RT-PCR. Phylogenetic analysis of the hemagglutinin and neuraminidase genes revealed that the A/Sw/HB/S1/2009 isolate was closely related to avian-like H1N1 viruses and seemed to be derived from the European swine H1N1 viruses. In conclusion, H1N1 influenza viruses were more dominant in the pig population than H3N2 influenza viruses in central China, and infection with avian-like H1N1 viruses persistently emerged in the swine population in the area.     

Immunogenicity and efficacy of a recombinant adenovirus expressing hemagglutinin from the H5N1 subtype of swine influenza virus in mice

The H5N1 influenza viruses infect a range of avian species and have recently been isolated from humans and pigs. In this study we generated a replication-defective recombinant adenovirus (rAd-H5HA-EGFP) expressing the hemagglutinin (HA) gene of H5N1 A/Swine/Fujian/1/2001 (SW/FJ/1/01) and evaluated its immunogenicity and protective efficacy in BALB/c mice. The recombinant virus induced high levels of hemagglutination inhibition (HI) antibody at a median tissue culture infective dose of 10⁸ or 10⁷. Compared with mice in the control groups, the mice vaccinated with rAd-H5HA-EGFP did not show apparent weight loss after challenge with either the homologous SW/FJ/1/01 or the heterologous H5N1 A/Chicken/Hunan/77/2005 (CK/HuN/77/05). Replication of the challenge virus was partially or completely inhibited, and viruses were detected at significantly lower numbers in the organs of the vaccinated mice, all of which survived the challenge with CK/HuN/77/05, whereas most of the control mice did not. These results indicate that rAd-H5HA-EGFP can provide effective immune protection from highly pathogenic H5N1 viruses in mice and is therefore a promising new candidate vaccine against H5N1 influenza in animals.     

Pathogenicity of H5N1 influenza A viruses isolated in Vietnam between late 2003 and 2005

Since late 2003, highly pathogenic H5N1 influenza A viruses have spread among poultry and wild aquatic birds in Asian countries. Transmission of these viruses to humans can be lethal. Most human cases of infection with H5N1 viruses have occurred in Vietnam. Therefore, to understand the pathogenicity in mammals of these H5N1 viruses, we took viruses isolated from poultry (5 strains) and humans (2 strains) in Vietnam and tested their virulence in mice. The results showed that the H5N1 viruses from humans were pathogenic in mice and that one avian isolate was also pathogenic. These findings suggested that the H5N1 viruses circulating in poultry adapted during replication in humans or that strains pathogenic in mice were transmitted directly to humans.     

Pathogenicity in quails and mice of H5N1 highly pathogenic avian influenza viruses isolated from ducks

In our study, the pathogenicity of H5N1 influenza A viruses circulating in waterfowls in Southern China was investigated. Three H5N1 highly pathogenic avian influenza (HPAI) viruses isolated from ducks, A/Duck/Guangdong/383/2008(DK383), A/Duck/Guangdong/378/2008(DK378) and A/Duck/Guangdong/212/2004(DK212) were inoculated at 10(6) fifty-percent egg infectious doses (EID(50)) into ducks, quails and mice and showed varying levels of pathogenicity. In ducks, the mortality rates ranged from 0 to 60% and the mean death time (MDT) was 0-6.7 days post-inoculation (DPI). While the viruses were highly pathogenic in quails, resulting in 83.3-100% mortality and the MDT of 2.3-3 DPI, they were completely lethal in mice (100% mortality). The viruses replicated in many organs of ducks and quails and were found in the brain, and kidney, lung and spleen of the mice. Phylogenetic analysis revealed that DK383 and DK378 viruses of clade 2.3.2 belonged to genotype 11, while DK212 virus of clade 9 was genotype 3. Our study illustrated H5N1 influenza viruses within Clade 2.3.2 and 9 from duck in Southern China had very highly pathogenicity to Japanese quails and BALB/c mice, but viruses within Clade 2.3.2 had more highly lethality than those of clade 9 to Muscovy ducks. Therefore, they had posed a continued challenge for disease control and public health.     

Influenza virus infections in mammals

The natural reservoir of all known subtypes of influenza A viruses are aquatic birds, mainly of the orders Anseriformes and Charadriiformes in which the infection is asymptomatic and the viruses stay at an evolutionary equilibrium. However, mammals may occasionally contract influenza A virus infections from this pool. This article summarizes: (i) natural infections in mammals including pigs, horses, marine mammals, ferrets, minks; (ii) results from experimental infections in several animal models including mice, ferrets, primates, rats, minks, hamsters and (iii) evidence for the increased pathogenicity of the current influenza A H5N1/Asia viruses for mammals. Several reports have shown that a number of mammalian species, including pigs, cats, ferrets, minks, whales, seals and finally also man are susceptible to natural infection with influenza A viruses of purely avian genetic make up. Among the mammalian species naturally susceptible to avian influenza virus the pig and the cat might exert the greatest potential public health impact. Despite numerous studies in animal and cell culture models, the basis of the extended host spectrum and the unusual pathogenicity of the influenza A H5N1 viruses for mammals is only beginning to be unraveled. Recently, also the transmission of equine influenza A virus to greyhound racing dogs has been documented.     

An H9N2 influenza virus vaccine prepared from a non-pathogenic isolate from a migratory duck confers protective immunity in mice against challenge with an H9N2 virus isolated from a girl in Hong Kong

H9N2 influenza viruses circulate in wild birds and poultry in Eurasian countries, and have been isolated from pigs and humans in China. H9N2 viruses isolated from birds, pigs and humans have been classified into three sublineages based on antigenic and genetic features. Chicken antisera to H9N2 viruses of the Korean sublineage reacted with viruses of different sublineages by the hemagglutination-inhibition test. A test vaccine prepared from a non-pathogenic A/duck/Hokkaido/49/1998 (H9N2) strain of the Korean sublineage, obtained from our influenza virus library, induced immunity in mice to reduce the impact of disease caused by the challenge with A/Hong Kong/1073/1999 (H9N2), which is of a different sublineage. The present results indicate that an inactivated whole virus vaccine prepared from a non-pathogenic influenza virus from the library could be used as an emergency vaccine during the early stage of a pandemic caused by H9N2 infection.     

A serological survey of canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in dogs in China

Influenza viruses have been isolated from dogs in China; however, the extent of influenza infection among dogs is not yet clear. Here, we examined the seroprevalence of avian-origin canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in pet dogs in China during January 2012 to June 2013. The seropositivity rate of canine H3N2, H1N1/09 and human H3N2 were 3.5%, 1.5%, and 1.2%, respectively. Dogs aged 2–5 years were most commonly seropositive to canine H3N2 virus. It is worth noting that two serum samples were positive against both canine H3N2 and H1N1/09 viruses, suggesting the possibility of coinfection with both viruses. Our findings emphasize the necessity for continued surveillance of influenza viruses in dogs in China.     

Serologic surveillance of swine H1 and H3 and avian H5 and H9 influenza A virus infections in swine population in Korea

Influenza A is a respiratory disease common in the swine industry. Three subtypes, H1N1, H1N2 and H3N2 influenza A viruses, are currently co-circulating in swine populations in Korea. An outbreak of the highly pathogenic avian influenza H5N1 virus occurred in domestic bird farms in Korea during the winter season of 2003. Pigs can serve as hosts for avian influenza viruses, enabling passage of the virus to other mammals and recombination of mammalian and avian influenza viruses, which are more readily transmissible to humans. This study reports the current seroprevalence of swine H1 and H3 influenza in swine populations in Korea by hemagglutination inhibition (HI) assay. We also investigated whether avian H5 and H9 influenza transmission occurred in pigs from Korea using both the HI and neutralization (NT) tests. 51.2% (380/742) of serum samples tested were positive against the swine H1 virus and 43.7% (324/742) were positive against the swine H3 virus by HI assay. The incidence of seropositivity against both the swine H1 virus and the swine H3 virus was 25.3% (188/742). On the other hand, none of the samples tested showed seropositivity against either the avian H5 virus or the avian H9 virus by the HI and NT tests. Therefore, we report the high current seroprevalence and co-infectivity of swine H1 and H3 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 and H9 influenza transmission to Korean pigs.     

Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health?

Pandemic strains of influenza A virus might arise by genetic reassortment between viruses from different hosts. Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of 10 (A-J) genotypes in pigs in China by analyzing the eight genes of 28 swine H9N2 viruses isolated in China from 1998 to 2007. Swine H9N2 viruses in genotype A and B were completely derived from Y280-like and Shanghai/F/98-like viruses, respectively, which indicated avian-to-pig interspecies transmission of H9N2 viruses did exist in China. The other eight genotype (C-J) viruses might be double-reassortant viruses, in which six genotype (E-J) viruses possessed 1-4 H5-like gene segments indicating they were reassortants of H9 and H5 viruses. In conclusion, genetic diversity of H9N2 influenza viruses from pigs in China provides further evidence that avian to pig interspecies transmission of H9N2 viruses did occur and might result in the generation of new reassortant viruses by genetic reassortment with swine H1N1, H1N2 and H3N2 influenza viruses, therefore, these swine H9N2 influenza viruses might be a potential threat to human health and continuing to carry out swine influenza virus surveillance in China is of great significance.     

Avian influenza virus

Avian influenza viruses do not typically replicate efficiently in humans, indicating direct transmission of avian influenza virus to humans is unlikely. However, since 1997, several cases of human infections with different subtypes (H5N1, H7N7, and H9N2) of avian influenza viruses have been identified and raised the pandemic potential of avian influenza virus in humans. Although circumstantial evidence of human to human transmission exists, the novel avian-origin influenza viruses isolated from humans lack the ability to transmit efficiently from person-to-person. However, the on-going human infection with avian-origin H5N1 viruses increases the likelihood of the generation of human-adapted avian influenza virus with pandemic potential. Thus, a better understanding of the biological and genetic basis of host restriction of influenza viruses is a critical factor in determining whether the introduction of a novel influenza virus into the human population will result in a pandemic. In this article, we review current knowledge of type A influenza virus in which all avian influenza viruses are categorized.     

Studies on influenza viruses H10N4 and H10N7 of avian origin in mink

An influenza A virus, A/mink/Sweden/84 (H10N4), was isolated from farmed mink during an outbreak of respiratory disease, histopathologically characterised by severe interstitial pneumonia. The virus was shown to be of recent avian origin and closely related to concomitantly circulating avian influenza virus. Serological investigations were used to link the isolated virus to the herds involved in the disease outbreak. Experimental infection of adult mink with the virus isolate from the disease outbreak reproduced the disease signs and pathological lesions observed in the field cases. The mink influenza virus also induced an antibody response and spread between mink by contact. The same pathogenesis in mink was observed for two avian influenza viruses of the H10N4 subtype, circulating in the avian population. When mink were infected with the prototype avian H10 influenza virus, A/chicken/Germany/N/49, H10N7, the animals responded with antibody production and mild pulmonary lesions but neither disease signs nor contact infections were observed. Detailed studies, including demonstration of viral antigen in situ by immunohistochemistry, of the sequential development of pathological lesions in the mink airways after aerosol exposure to H10N4 or H10N7 revealed that the infections progress very similarly during the first 24h, but are distinctly different at later stages. The conclusion drawn is that A/mink/Sweden/84, but not A/chicken/Germany/N/49, produces a multiple-cycle replication in mink airways. Since the viral distribution and pathological lesions are very similar during the initial stages of infection we suggest that the two viruses differ in their abilities to replicate and spread within the mink tissues, but that their capacities for viral adherence and entry into mink epithelial cells are comparable.     

中国流行的2009甲型H1N1猪源流感病毒HA和NA基因的分子和遗传特征

目前流行的甲型H1N1流感病毒是一个复杂的基因重配病毒。对病毒的分子生物学研究,尤其是病毒囊膜蛋白血凝素（haemagglutini,HA）基因和神经氨酸酶（neuraminidase,NA）基因的研究,为控制和预防H1N1流感病毒具有重要的意义。本研究对中国流行的2009甲型H1N1猪源流感病毒的HA和NA基因与疫苗株A/California/07/2009（H1N1）,以及不同国家和地区的病毒株进行核苷酸和氨基酸序列分析。从NCBI的GenBank数据库下载所需要毒株的序列,采用Lasergene 6.0软件包中的EditSeq和MegAlign进行序列分析,进化树分析采用MEGA4.1软件。进化分析表明,中国流行的2009 H1N1流感病毒与疫苗株的核苷酸同源率分别在98.8%~99.7%和98.6%~99.6%之间;裂解位点处为I/VPSIQSR↓G,不具备高致病性流感病毒的特征;有1株NA抗性病毒。尽管与疫苗株相比,中国流行株2009甲型H1N1猪源流感病毒的HA和NA基因有部分突变,但这些突变并不是重要的。本研究首次详细分析了中国流行的2009甲型H1N1猪源流感病毒株与疫苗株的HA和NA基因的分子特征,对实时监测流感病毒HA和NA基因的变化具有重要意义。     

Pathogenicity of a Hong Kong-origin H5N1 highly pathogenic avian influenza virus for emus, geese, ducks, and pigeons

The H5N1 type A influenza viruses that emerged in Hong Kong in 1997 are a unique lineage of type A influenza viruses with the capacity to transmit directly from chickens to humans and produce significant disease and mortality in both of these hosts. The objective of this study was to ascertain the susceptibility of emus (Dramaius novaehollandiae), domestic geese (Anser anser domesticus), domestic ducks (Anas platyrhynchos), and pigeons (Columba livia) to intranasal (i.n.) inoculation with the A/chicken/Hong Kong/220/97 (H5N1) highly pathogenic avian influenza virus. No mortality occurred within 10 days postinoculation (DPI) in the four species investigated, and clinical disease, evident as neurologic dysfunction, was observed exclusively in emus and geese. Grossly, pancreatic mottling and splenomegaly were identified in these two species. In addition, the geese had cerebral malacia and thymic and bursal atrophy. Histologically, both the emus and geese developed pancreatitis, meningoencephalitis, and mild myocarditis. Influenza viral antigen was demonstrated in areas with histologic lesions up to 10 DPI in the geese. Virus was reisolated from oropharyngeal and cloacal swabs and from the lung, brain, and kidney of the emus and geese. Moderate splenomegaly was observed grossly in the ducks. Viral infection of the ducks was pneumotropic, as evidenced by mild inflammatory lesions in the respiratory tract and virus reisolation from oropharyngeal swabs and from a lung. Pigeons were resistant to HK/220 infection, lacking gross and histologic lesions, viral antigen, and reisolation of virus. These results imply that emus and geese are susceptible to i.n. inoculation with the HK/220 virus, whereas ducks and pigeons are more resistant. These latter two species probably played a minimal epidemiologic role in the perpetuation of the H5N1 Hong Kong-origin influenza viruses.