湖南省2006--2009年人感染高致病性禽流感病毒(H5N1)分子特征研究

目的 分析湖南省2006-2009年4例人感染高致病性禽流感病例感染病毒的可能来源、基因重配情况以及分子特征.方法 鸡胚分离核酸检测H5N1病毒阳性标本,获得高致病性H5N1病毒,对病毒进行全基因组序列测定,采用BLAST和MEGA4.0进行同源性比对、基因进化分析和各基因分子特征分析.结果 4株病毒的基因片段均为禽源,并未发现与人季节性流感病毒之间发生重配,且与当地禽类中分离的病毒高度同源.全基因组进化树分析显示,4株病毒在分支2.3.4中,2株为基因型V、2株为新的基因型.分子特征分析显示,4株病毒的血凝素(HA)分子裂解位点均为PLRERRKR/G,均出现A160T位点突变,神经氨酸酶(NA)分子49～68位均出现20个氨基酸(aa)缺失,非结构蛋白1(NSI)分子80～84位均出现5个aa的缺失.在HA分子大部分位点,4株病毒仍然表现出与禽类受体的亲和性,HN/1/09和HN/2/09出现可能使病毒对α-2,6连接的唾液酸人类受体的亲和性增强的T192I突变.HN/1/08的PB2基因出现增加小鼠致病力的D701N改变.耐药性基因片段分析显示,4株病毒对金刚烷胺和奥司他韦均敏感.结论 2006-2009年湖南省4株人感染高致病性禽流感病毒(H5N1)为禽源,但存在多种基因型,而且发生了部分位点的突变.

Abstract：

Objective To understand the possible origins,genetic re-assortment and molecular characterization of 4 highly pathogenic avian influenza A(H5N1)viruses isolated from humans in Hunan province,between 2006 and 2009,Methods H5N1 PCR test-positive specimens were inoculated in embryonated eggs while H5N1 virus was isolated and genomes sequenced.Genome homology and genetic molecular characterization were analyzed by BLAST and MEGA 4.0.Results All gene segments of the 4 viruses were avian in origin.No re-assortment was found between avian influenza A(H5N1)viruses and human seasonal influenza viruses.Virnses that isolated from domestic poultry shared high similarity with the 4 human viruses in gene homology.Data from the whole genome phylogenetic analysis showed that the 4 viruses were in clade 2.3.4,while 2 viruses belonged to genotype V,and another 2 were new genotypes.Results from molecular characterization showed that amino acid sequences of HA cleavage site of the 4 viruses were PLRERRKR/G.All 4 viruses had A160T mutation in HA,a 20 amino acid deletion in the neuraminidase(NA)stalk at position 49-68,and a 5 amino acid deletion in the non-structural protein 1(NS1).Most sites in the HA molecules showed that the viruses preferentially bound to avian influenza virus receptor.However,T192I mutation that might enhance the α2,6-linked sialic acid human influenza receptor binding had emerged in HN/1/09 and HN/2/09.D701N mutation of PB2 that increased the virulence in mice was found in HN/1/08.Analysis on drug resistance gene amino acid showed that all 4 viruses were sensitive to amantadine and oseltamivir.Conclusion Highly pathogenic avian influenza A(H5N1)viruses isolated from humans in Hunan province from 2006 to 2009 were avian in origin,and the 4 viruses belonged to different genotypes.Some mutations that related to virulence and receptor binding positions had emerged in some of the strains.     

Genetic and phylogenetic characterizations of a novel genotype of highly pathogenic avian influenza (HPAI) H5N8 viruses in 2016/2017 in South Korea

During the outbreaks of highly pathogenic avian influenza (HPAI) H5N6 viruses in 2016 in South Korea, novel H5N8 viruses were also isolated from migratory birds. Phylogenetic analysis revealed that the HA gene of these H5N8 viruses belonged to clade 2.3.4.4, similarly to recent H5Nx viruses, and originated from A/Brk/Korea/Gochang1/14(H5N8), a minor lineage of H5N8 that appeared in 2014 and then disappeared. At least four reassortment events occurred with different subtypes (H5N8, H7N7, H3N8 and H10N7) and a chicken challenge study revealed that they were classified as HPAI viruses according to OIE criteria.     

Development of Eurasian H7N7/PR8 high growth reassortant virus for clinical evaluation as an inactivated pandemic influenza vaccine

Avian-to-human transmission of the high pathogenicity (HP) H7N7 subtype avian influenza viruses in the Netherlands during 2003 caused zoonotic infections in 89 people, including a case of acute fatal respiratory distress syndrome. Public health emergency preparedness against H7N7 avian influenza viruses with pandemic potential includes the development of vaccine candidate viruses. In order to develop a high growth reassortant vaccine candidate virus, low pathogenicity (LP) A/mallard/Netherlands/12/2000 (H7N3) and A/mallard/Netherlands/2/2000 (H10N7) strains were selected as donors of the H7 haemagglutinin and N7 neuraminidase genes, respectively. The donor viruses exhibited high amino acid sequence homology with the surface glycoproteins of A/Netherlands/219/03 H7N7 virus (NL219), an isolate recovered from the fatal human case. Adhering to the seasonal influenza vaccine licensure regulations, we generated a H7N7/PR8 reassortant containing desired surface glycoprotein genes from the mallard viruses and internal genes of A/Puerto Rico/8/34 human vaccine strain (H1N1). Antigenic analysis revealed that the vaccine candidate virus confers broad antigenic cross-reactivity against contemporary Eurasian and the North American H7 subtype human isolates. Mice immunized with formalin inactivated (FI) H7N7/PR8 whole virus vaccine with or without aluminum hydroxide adjuvant conferred clinical protection from mortality and reduced pulmonary replication of the NL219 challenge virus. The FI H7N7/PR8 whole virus vaccine also afforded cross-protection in mice at the pulmonary level against antigenically distinct North American LP A/Canada/444/04 (H7N3) human isolate. The vaccine candidate virus satisfied the agricultural safety requirements for chickens, proved safe in mice, and has entered in phase-I human clinical trial in the United States.     

H7N3 live attenuated influenza vaccine has a potential to protect against new H7N9 avian influenza virus

After recent emergence of new avian influenza A(H7N9) viruses in humans many people and Governments are asking about H7 influenza vaccine which could provide cross-protection against new viruses, until H7N9 vaccine is prepared from a relevant strain. Here we scientifically justify that available H7N3 live attenuated influenza vaccine (LAIV) can be protective against H7N9 viruses due to the presence of conserved immune epitopes in its hemagglutinin. We used Immune Epitope Database analysis resource to predict B-cell and CTL epitopes distributed across H7N3 HA molecule and assessed their identity with new H7N9 viruses at near 70% and 60% of the epitopes, respectively. In addition, we tested serum samples of volunteers participated in phase I clinical trial of H7N3 LAIV for the presence of anti-H7N9 hemagglutination-inhibition and neutralizing antibodies and found seroconversions in 44.8% of vaccinated persons, which suggests the potential of H7N3 LAIV to protect against new H7N9 avian influenza viruses.     

上海市人群甲型流感病毒H1、H3、H5、H9抗体血清学监测

[目的 ]　了解上海市人群对甲 3型、甲 1型流感病毒和禽流感病毒H9、H5的抗体 ,探讨甲 3型、甲 1型流感流行的趋势和禽流感病毒H9、H5抗体存在情况。　 [方法 ]　应用常规血凝抑制试验微量半加敏法对上海市不同年龄组人群进行甲型流感抗体血清学监测。　 [结果 ]　对A/SH/1/98(H3N2 )毒株的抗体阳性率为 96.2 0 % ;对A/SH /7/99(H1N1)毒株的抗体阳性率为 72 .60 % ;对H9毒株的抗体阳性率为 7.5 6% ;对H5毒株的抗体阳性率仅 0 .75 %。　 [结论 ]　A/SH/1/98(H3N2 )类毒株已不可能在上海再度引起流行 ;A/SH/7/99(H1N1)类毒株今后在人群中 ,特别在 0～ 4岁年龄组中可能存在散在性发生或局限性小爆发 ;本市人群曾受到禽流感H 9病毒的感染 ,不能排除由本市禽类中的H9病毒感染及人的可能 ;受禽流感H5毒株感染只是个别现象。禽流感病毒在本市禽类中的感染状况及对人的影响需进一步研究     

Novel reassortant highly pathogenic H5N6 avian influenza viruses in poultry in China

We characterized two novel highly pathogenic H5N6 influenza viruses isolated from Chinese poultry in 2013. Genomic analysis showed that both isolates were reassortants, and derived their genes from H5 and H6 subtype viruses found in poultry in China. The virulence of the two isolates was examined in chickens and mice, and both isolates were found to be highly pathogenic in chickens and only moderately virulent for mice. Our results show that continued circulation of these viruses could endanger both avian species and humans.     

Evaluation of avian influenza virus isolated from ducks as a potential live vaccine candidate against novel H7N9 viruses

Recent outbreaks of a novel H7N9 avian influenza virus in humans in China raise pandemic concerns and underscore an urgent need to develop effective vaccines. Theoretically, live influenza vaccines are of multiple advantages over traditional inactivated influenza vaccines to be used in a pandemic, because they can be produced rapidly, safely, and inexpensively. However, studies on live vaccines against the novel H7N9 virus are limited. In this study, we evaluated a potential live influenza vaccine candidate using an H7N3 avian influenza virus isolated from ducks with controls of two recombinant viruses generated through reverse genetics. The potential candidate could be produced efficiently using chicken embryonated eggs, and is homogenous to the novel H7N9 virus in their viral hemagglutinin genes. The potential candidate is likely low pathogenic to birds and mammals, and likely sensitive to oseltamivir and amantadine, as suggested by its genomic sequences. Its low pathogenicity was further supported through inoculation in mice, chicken embryonated eggs and chickens. Specific antibodies elicited in mice were detectable at least during the period between day 14 and day 56 after intranasal administration of the candidate for one time. Titers of the specific antibodies increased significantly with a boost intranasal administration or a higher inoculation dose. The induced specific antibodies were of substantial cross-reactivity with the novel H7N9 virus. These primary but promising evaluation data suggest that the duck influenza virus could be used as a potential live vaccine candidate, favorably through a prime-boost route, to mitigate the severity of the possible pandemic caused by the newly emerging H7N9 virus, and is valuable to be further evaluated.     

Evidence for common ancestry among viruses isolated from wild birds in Beringia and highly pathogenic intercontinental reassortant H5N1 and H5N2 influenza A viruses

Highly pathogenic clade 2.3.4.4 H5N8, H5N2, and H5N1 influenza A viruses were first detected in wild, captive, and domestic birds in North America in November–December 2014. In this study, we used wild waterbird samples collected in Alaska prior to the initial detection of clade 2.3.4.4 H5 influenza A viruses in North America to assess the evidence for: (1) dispersal of highly pathogenic influenza A viruses from East Asia to North America by migratory birds via Alaska and (2) ancestral origins of clade 2.3.4.4 H5 reassortant viruses in Beringia. Although we did not detect highly pathogenic influenza A viruses in our sample collection from western Alaska, we did identify viruses that contained gene segments sharing recent common ancestry with intercontinental reassortant H5N2 and H5N1 viruses. Results of phylogenetic analyses and estimates for times of most recent common ancestry support migratory birds sampled in Beringia as maintaining viral diversity closely related to novel highly pathogenic influenza A virus genotypes detected in North America. Although our results do not elucidate the route by which highly pathogenic influenza A viruses were introduced into North America, genetic evidence is consistent with the hypothesized trans-Beringian route of introduction via migratory birds.     

Inferring the antigenic epitopes for highly pathogenic avian influenza H5N1 viruses

The evasion of influenza virus from host immune surveillance is mainly mediated through its surface protein hemagglutinin (HA), the main component of influenza vaccine. Thus, identification of influenza virus antigenic epitopes on HA can not only help us understand the molecular mechanisms of viral immune escape but also facilitate vaccine strain selection. Despite previous efforts, there is a lack of systematic definition of the antigenic epitopes for the highly pathogenic avian influenza (HPAI) H5N1 viruses. In this study, we infer the HA antigenic epitopes for H5N1 viruses by integrating the antigenic sites mapped from the HA of human influenza H3N2 viruses, the sites which were reported to be associated with immune escape in H5 viruses and the mutation hotspot sites identified in the evolutionary history of HPAI H5N1 viruses. We show that these inferred antigenic epitopes play significant roles in antigenic variation of HPAI H5N1 viruses. Based on inferred antigenic epitopes, we further develop a computational method to effectively predict antigenic variants for HPAI H5N1 viruses (available at http://biocloud.hnu.edu.cn/predict/html/index.html). Therefore, our work has not only inferred the antigenic epitopes for HPAI H5N1 viruses but also provided an effective computational method to assist vaccine recommendations for protection against the deadly bird flu.     

M2SR,a novel live influenza vaccine,protects mice and ferrets against highly pathogenic avian influenza

The emergence of highly pathogenic avian influenza H5N1 viruses has heightened global concern about the threat posed by pandemic influenza. To address the need for a highly effective universal influenza vaccine, we developed a novel M2-deficient single replication (M2SR) influenza vaccine virus and previously reported that it provided strong heterosubtypic protection against seasonal influenza viruses in mice. In the current study, we assessed M2SR induced protection against H5N1 influenza in mice and ferrets.Mice were intranasally inoculated with M2SR viruses containing the HA and NA from A/Vietnam/1203/2004 (M2SR H5N1) or A/California/07/2009 (M2SR H1N1). All M2SR vaccinated mice survived lethal challenge with influenza A/Vietnam/1203/2004 (H5N1), whereas 40% of mice vaccinated with recombinant H5 HA and none of the naïve controls survived. M2SR H5N1 provided sterile immunity, whereas low levels of virus were detected in the lungs of some M2SR H1N1 vaccinated mice. In contrast, recombinant H5 HA vaccinated mice and naïve controls showed systemic infection.M2SR H5N1 induced strong serum and mucosal antibody responses (IgG and IgA classes) against H5 HA, with high hemagglutination inhibition (HAI) titers. In contrast, while M2SR H1N1 elicited cross-reactive antibodies recognizing the H5 HA2 stalk region or the neuraminidase, no HAI activity against H5N1 virus was detected after M2SR H1N1 immunization.Both M2SR H5N1 and H1N1 also protected ferrets against lethal challenge with A/Vietnam/1203/2004. A prime–boost regimen provided optimal protection with no virus detected in the respiratory tract or brain after challenge. As in the mouse model, only the M2SR H5N1 vaccine induced HAI antibodies against the challenge virus in ferrets, while the M2SR H1N1 was able to provide protection without the induction of HAI antibodies.In summary, effective protection against highly pathogenic H5N1 influenza virus was provided by both homologous H5N1 M2SR and heterologous H1N1 M2SR demonstrating the cross-protective attributes of the M2SR platform.     

嘉兴市外环境禽流感病毒污染状况及职业暴露人群H5N6、H7N9和H9N2抗体水平调查

目的 了解嘉兴市外环境禽流感病毒分布情况及涉禽职业暴露人群禽流感病毒感染现状,为科学有效防控人感染禽流感疫情提供科学依据。 方法 实时荧光定量PCR检测2013年3月-2016年4月采集的嘉兴市3 115份外环境标本的A型流感病毒核酸,并对A型流感病毒核酸阳性的标本进一步进行H5、H7、H9亚型检测。采用马血红细胞凝集抑制试验检测2015年4月和2016年4月采集的嘉兴市140名涉禽职业暴露人群血清中的H5N6、H7N9和H9N2血凝素抗体。 结果 外环境标本中A型流感病毒核酸阳性率为18.11%,H5、H9、H7亚型阳性率分别为0.64%、3.21%、5.07%;同时,外环境标本中检出不同亚型混合污染标本74份,且H9/H7混合污染的阳性率达到1.86%。A型流感病毒及H7亚型阳性率在冬春季节出现高峰,且在城乡活禽市场的阳性率明显高于其他场所;宰杀或摆放禽肉案板表面和清洗禽类的污水标本中A型流感病毒及H7亚型的阳性率明显高于其他标本类型。140份人群血清标本共检出H9N2抗体阳性标本5份,阳性率为3.57%,未检出H5N6、H7N9抗体阳性标本。 结论 2013-2016年嘉兴市外环境中存在H5、H9、H7亚型污染,且涉禽职业暴露人群中少量的H9N2禽流感病毒无症状感染者,提示禽类接触、活禽交易市场暴露有感染禽流感病毒风险,因此应做好重点人群的健康教育及重点区域的禽流感病毒监测,防止人感染禽流感疫情的发生。     

An emerging avian influenza A virus H5N7 is a genetic reassortant of highly pathogenic genes

We full genome characterised the newly discovered avian influenza virus H5N7 subtype combination isolated from a stock of Danish game ducks to investigate the composition of the genome and possible features of high pathogenicity. It was found that the haemagglutinin and the acidic polymerase genes were closely related to a low pathogenic H5 strain (A/Duck/Denmark/65047/04 H5N2). The neuraminidase and the non-structural genes were closely related to the highly pathogenic H7N7 strains from The Netherlands 2003. The basic polymerase genes 1 and 2 were shared between the Danish H5N7 and H5N2 and the H7N7 from The Netherlands. The nucleoprotein and the matrix genes were closely related to H6 strains. Thus, the new H5N7 subtype share genes with H5, H7 and H6 subtypes and possesses internal genes originating from highly pathogenic strains. The findings emphasize the need for surveillance presumed low pathogenic avian influenza A viruses.     

Highly pathogenic avian influenza

Highly pathogenic (HP) avian influenza (AI) (HPAI) is an extremely contagious, multi-organ systemic disease of poultry leading to high mortality, and caused by some H5 and H7 subtypes of type A influenza virus, family Orthomyxoviridae. However, most AI virus strains are mildly pathogenic (MP) and produce either subclinical infections or respiratory and/or reproductive diseases in a variety of domestic and wild bird species. Highly pathogenic avian influenza is a List A disease of the Office International des Epizooties, while MPAI is neither a List A nor List B disease. Eighteen outbreaks of HPAI have been documented since the identification of AI virus as the cause of fowl plague in 1955. Mildly pathogenic avian influenza viruses are maintained in wild aquatic bird reservoirs, occasionally crossing over to domestic poultry and causing outbreaks of mild disease. Highly pathogenic avian influenza viruses do not have a recognised wild bird reservoir, but can occasionally be isolated from wild birds during outbreaks in domestic poultry. Highly pathogenic avian influenza viruses have been documented to arise from MPAI viruses through mutations in the haemagglutinin surface protein. Prevention of exposure to the virus and eradication are the accepted methods for dealing with HPAI. Control programmes, which imply allowing a low incidence of infection, are not an acceptable method for managing HPAI, but have been used during some outbreaks of MPAI. The components of a strategy to deal with MPAI or HPAI include surveillance and diagnosis, biosecurity, education, quarantine and depopulation. Vaccination has been used in some control and eradication programmes for AI.     

Prevalence and diversity of H9N2 avian influenza in chickens of Northern Vietnam, 2014

Despite their classification as low pathogenicity avian influenza viruses (LPAIV), A/H9N2 viruses cause significant losses in poultry in many countries throughout Asia, the Middle East and North Africa. To date, poultry surveillance in Vietnam has focused on detection of influenza H5 viruses, and there is limited understanding of influenza H9 epidemiology and transmission dynamics. We determined prevalence and diversity of influenza A viruses in chickens from live bird markets (LBM) of 7 northern Vietnamese provinces, using pooled oropharyngeal swabs collected from October to December 2014. Screening by real time RT-PCR revealed 1207/4900 (24.6%) of pooled swabs to be influenza A virus positive; overall prevalence estimates after accounting for pooling (5 swabs/pools) were 5.8% (CI 5.4–6.0). Subtyping was performed on 468 pooled swabs with M gene Ct < 26. No influenza H7 was detected; 422 (90.1%) were H9 positive; and 22 (4.7%) were H5 positive. There was no evidence was of interaction between H9 and H5 virus detection rates. We sequenced 17 whole genomes of A/H9N2, 2 of A/H5N6, and 11 partial genomes. All H9N2 viruses had internal genes that clustered with genotype 57 and were closely related to Chinese human isolates of A/H7N9 and A/H10N8. Using a nucleotide divergence cutoff of 98%, we identified 9 distinct H9 genotypes. Phylogenetic analysis suggested multiple introductions of H9 viruses to northern Vietnam rather than in-situ transmission. Further investigations of H9 prevalence and diversity in other regions of Vietnam are warranted to assess H9 endemicity elsewhere in the country.     

A subunit vaccine candidate derived from a classic H5N1 avian influenza virus in China protects fowls and BALB/c mice from lethal challenge

In recent years, numerous human infections with avian influenza viruses in Asia have raised the concern that the next influenza pandemic is imminent. The most effective way to combat human avian influenza is through vaccination of the public. In this study, we developed an influenza A recombinant protein (rH5HA) directed against the hemagglutinin (HA) of a classic H5N1 high pathogenic avian influenza virus isolated in South China in 1996. Following purification of the recombinant protein expressed from a baculovirus expression system, we evaluated the efficiency of rH5HA on specific pathogen free (SPF) chicken, commercial chicken, and in BALB/c mice in an infection-protection model. The results demonstrated that rH5HA induced antibody responses and provided full protection in both SPF chickens and commercial chickens. Protective immunity was generated within 2 weeks in chickens as young as 7-day post-hatch using a minimum amount of rH5HA protein (2 μg/bird/vaccination). The serum antibody generated from rH5HA immunization was protective and lasted more than 6 months. Our data also demonstrated that rH5HA immunization protected BALB/c mice from a lethal challenge with pathogenic avian influenza virus. These results suggested that vaccination with rH5HA could be a vaccine candidate for the control of H5N1 avian influenza in poultry, in mice, and potentially in other mammals including human.     

Phylogeographic analysis of H5N1 highly pathogenic avian influenza virus isolated in Cambodia from 2018 to 2019

Since 2004, several outbreaks of highly pathogenic avian influenza (HPAI) have been reported in Cambodia. Until 2013, all H5N1 viruses identified in Cambodia belonged to clade 1 and its subclades. H5N1 HPAI viruses belonging to clade 2.3.2.1c have been dominant since the beginning of 2014, with various genotypes (KH1-KH5) reported. Here, we isolated nine H5N1 HPAI viruses from domestic poultry farms and slaughterhouses in Cambodia during 2018–2019 and performed phylogenetic analysis of whole genome sequences. All isolates were classified as H5 clade 2.3.2.1c viruses and all harbored multi-basic amino acid sequences (PQRERRRKR/GLF) at the haemagglutinin (HA) cleavage site. Phylogenetic analysis revealed that the H5N1 isolates in this study belonged to the KH2 genotype, the dominant genotype in Cambodia in 2015. Phylogenetic analysis of the HA gene showed that the isolates were divided into two groups (A and B). The results of Bayesian discrete phylogeography analysis revealed that the viral migration pathways from Vietnam to Cambodia (Bayes factor value: 734,039.01; posterior probability: 1.00) and from Cambodia to Vietnam (Bayes factor value: 26,199.95; posterior probability: 1.00) were supported by high statistical values. These well-supported viral migrations between Vietnam and Cambodia demonstrate that viral transmission continued in both directions. Several factors may have contributed to this, including the free-grazing duck system and movement of poultry-related products. Thus, the results emphasize the need for an enhanced international surveillance program to better understand transboundary infection and evolution of H5N1 HPAI viruses, along with implementation of more stringent international trade controls on poultry and poultry products.     

Infection of mice with a human influenza A/H3N2 virus induces protective immunity against lethal infection with influenza A/H5N1 virus

The transmission of highly pathogenic avian influenza (HPAI) A viruses of the H5N1 subtype from poultry to man and the high case fatality rate fuels the fear for a pandemic outbreak caused by these viruses. However, prior infections with seasonal influenza A/H1N1 and A/H3N2 viruses induce heterosubtypic immunity that could afford a certain degree of protection against infection with the HPAI A/H5N1 viruses, which are distantly related to the human influenza A viruses. To assess the protective efficacy of such heterosubtypic immunity mice were infected with human influenza virus A/Hong Kong/2/68 (H3N2) 4 weeks prior to a lethal infection with HPAI virus A/Indonesia/5/05 (H5N1).     

Phylogenetic analysis of a novel H6N6 avian influenza virus isolated from a green peafowl in China and its pathogenic potential in mice

To explore the ecology of the H6 subtype avian influenza viruses in Hebei Province, China, a long-term surveillance was conducted in 2012 among domestic poultry and birds in a wildlife park. In this study, we report the characterization of a novel H6N6 avian influenza virus isolated from a healthy green peafowl in Qinghuangdao Wildlife Park in 2012. A phylogenetic analysis indicated that the isolated H6N6 strain has the same gene constellation as the ST3367-like strains, which are mainly distributed in southern and eastern China. A mouse experiment showed that the isolate replicated efficiently in the lungs and turbinates of infected mice without previous adaptation, resulting in locally thickened alveolar septa and interstitial pneumonia. Further studies of the H6 subtype viruses are required to clarify their evolutionary pattern in north China, which will benefit disease control and pandemic preparedness for novel viruses.     

2010-2012年南宁市职业暴露人群血清学和环境高致病性禽流感监测分析

目的了解南宁市职业暴露人群高致病性H5N1禽流感病毒的接触感染状况和南宁市及周边地区活禽市场、养殖厂等环境禽流感病毒H5、H7、H9亚型污染状况,为防治人禽流感提供科学依据。方法在2010-2012年采集从事家禽养殖、屠宰等人群血清标本用红细胞凝集抑制试验（HI）检测H5N1抗体;对在城乡活禽市场外环境采集的笼具涂抹、粪便和污水标本用荧光定量PCR法检测禽流感病毒FluA、HS、H7、H9核酸。结果共采集职业暴露人群80人份血清H5N1抗体均为阴性;环境标本175份禽流感病毒核酸监测阳性标本为47份,阳性率为26．86％。A型禽流感病毒核酸阳性标本分布于6个城区（县）的9个活禽农贸市场,各个季节采集的标本的阳性率有差异。3种类型的标本中,水的阳性率最高,其次是笼具涂抹标本,粪便的阳性率最低。结论禽流感H5亚型在南宁市职业暴露人群中未发现隐性感染;活禽市场环境普遍存在高致病性禽流感病毒的污染,H5和H9亚型是主要的病原体。