广东部分地区猪戊型肝炎的分子流行病学调查

为了解广东地区猪戊型肝炎的感染情况,本试验于2015年在广东不同猪场收集了2月龄内猪胆汁73份,RTnPCR检测、全基因组测序、同源性及进化分析结果显示:猪感染戊型肝炎的阳性率为6.8%(5/73),5个ORF2部分核苷酸序列同源性为87.2%~99.4%,属于基因4型,4b、4h亚型。对4b亚型中的1个阳性样品(GZHD)株进行全基因测序,其核苷酸序列与swGX40(EU676172)同源性最高为96.5%,与广东株SS19(JX855794)同源性为93.9%,与人源戊型肝炎病毒(HEV)株CVS-Sie10(LC042232)同源性为94.8%。结果表明:广东地区猪群间HEV流行毒株仍以基因4型为主,且为揭示戊型肝炎的跨种传播提供了新的分子生物学依据。     

四川地区猪源戊型肝炎病毒感染的分子流行病学调查

为了解四川地区猪源戊型肝炎病毒(hepatitis E virus, HEV)的感染情况,采集四川13个地区屠宰场的358份生猪组织样品,采用荧光RT-PCR进行HEV检测,对阳性样品进行开放阅读框(open reading frame, ORF)两部分序列测序、同源性及系统进化分析。结果显示:猪感染HEV的阳性率为4.75%,17个毒株ORF2部分序列之间同源率为85.4%～99.4%,均属于基因4型,其中4a亚型8株、4b亚型3株、4d亚型6株。与人、猪等不同宿主的HEV参考毒株比较,四川猪源HEV与我国主要流行的人源HEV遗传关系密切,其中10株与人源毒株同源率超过95%。结果表明:四川地区生猪HEV流行毒株以基因4型为主,具有较大的人畜共患风险,应有针对性、系统性地对其进行预防和控制。     

云南省部分猪场猪戊型肝炎的流行情况调查

为了解云南省猪群戊型肝炎感染情况,从云南省8个猪场采集166份新鲜猪粪便样品,用套式RT-PCR技术对戊型肝炎病毒(HEV)核酸进行检测,结果 21份样品阳性,阳性率为12.7%,感染率较高。通过PCR产物片段的测序分析,去除重复序列后得到11株基因序列。同源性分析结果表明,这11株毒株序列与选定的Gen Bank中基因1型、2型、3型和4型参考序列的核苷酸序列同源性分别为85.6%~87.8%,85.6%~87.8%,85.6%~95.6%和92.8%~98.9%,说明这些株毒株属于基因4型,与中国其他地区分离的毒株同源性最高,证实HEV的流行具有地理分布的特征。     

猪圆环病毒2型天津及周边地区分离株的遗传演化分析

为了解天津及周边地区猪圆环病毒2型（porcine circovirus 2,PCV2）的流行毒株遗传变异情况,本研究应用基因克隆技术和生物信息学分析软件对24株PCV2分离株全基因及ORF2基因分别进行克隆、测序、同源性比对和遗传进化分析。全基因组序列分析结果显示,24株PCV2分离株全基因组序列有14株为1 767 bp,10株为1 768 bp。用DNAStar软件对全基因组序列同源性比对表明,24株PCV2分离株与NCBI上的PCV2参考株的全基因序列同源性为93.6%~99.8%,24株分离株之间的全基因组序列差异很小,同源性为94.0%~99.9%。同时,用DNAStar对PCV2分离株的ORF2基因序列同源性分析表明,其与NCBI上的PCV2 ORF2基因参考序列的核苷酸序列同源性为87.3%~99.3%,24株分离株ORF2基因核苷酸序列同源性为89.0%~100.0%,3株分离株（10084F4、R14040及R14086）的核苷酸序列同源性为100.0%。对全基因和ORF2基因的遗传进化分析表明,24株分离株的系统进化树被分为2个大分支,其中23株分离毒株属于PCV2b亚型,1株属于PCV2a亚型,未分离出PCV2c亚型。综上所述,天津及周边地区PCV2的流行模式仍以PCV 2b亚型为主,本研究为该地区PCV2的流行变异情况研究提供了一定的理论依据。     

猪圆环病毒2型天津及周边地区分离株的遗传演化分析

为了解天津及周边地区猪圆环病毒2型(porcine circovirus 2,PCV2)的流行毒株遗传变异情况,本研究应用基因克隆技术和生物信息学分析软件对24株PCV2分离株全基因及ORF2基因分别进行克隆、测序、同源性比对和遗传进化分析。全基因组序列分析结果显示,24株PCV2分离株全基因组序列有14株为1 767bp,10株为1 768bp。用DNAStar软件对全基因组序列同源性比对表明,24株PCV2分离株与NCBI上的PCV2参考株的全基因序列同源性为93.6%~99.8%,24株分离株之间的全基因组序列差异很小,同源性为94.0%~99.9%。同时,用DNAStar对PCV2分离株的ORF2基因序列同源性分析表明,其与NCBI上的PCV2 ORF2基因参考序列的核苷酸序列同源性为87.3%~99.3%,24株分离株ORF2基因核苷酸序列同源性为89.0%~100.0%,3株分离株(10084F4、R14040及R14086)的核苷酸序列同源性为100.0%。对全基因和ORF2基因的遗传进化分析表明,24株分离株的系统进化树被分为2个大分支,其中23株分离毒株属于PCV2b亚型,1株属于PCV2a亚型,未分离出PCV2c亚型。综上所述,天津及周边地区PCV2的流行模式仍以PCV 2b亚型为主,本研究为该地区PCV2的流行变异情况研究提供了一定的理论依据。     

2014年-2016年广西地区猪繁殖与呼吸综合征病毒Nsp2和ORF5基因的遗传多样性分析

为了解广西地区猪繁殖与呼吸综合征病毒(PRRSV)流行毒株的遗传进化情况,对2014年-2016年来自广西各地的部分PRRSV阳性病料进行Nsp2和ORF5基因的扩增和测序分析.结果获得34个Nsp2基因序列和45个ORF5基因序列,均属于美洲型毒株.Nsp2基因间核苷酸序列的同源性为91.8％~100％,与PRRSV美洲型毒株VR-2332、CH-1a、JXA1及NADC30株核苷酸序列的同源性分别为81.3％~84.3％、88.9％~92.1％、94.3％~99.3％和73.5％~75.1％,而与PRRSV欧洲型毒株LV株核苷酸序列的同源性为51.5％~53.2％.ORF5基因间核苷酸序列的同源性为82.8％~100％,与PRRSV美洲型毒株VR-2332、CH-1a、JXA1及NADC30株核苷酸序列的同源性分别为83.7％~99.5％、85％~95％、83.8％~99.7％和83.2％~86.4％,而与PRRSV欧洲型毒株LV株核苷酸序列的同源性为62.4％~64.5％.基于Nsp2和ORF5基因推导的氨基酸序列绘制的遗传进化树中,广西地区的毒株主要分布在以JXA1为代表的Ⅳ亚群.表明当前广西PRRSV流行毒株以JXA1株为代表的高致病性美洲型毒株为主,各毒株Nsp2和ORF5基因序列存在一定的差异,尚未发现欧洲型毒株和美洲型NADC30类毒株.     

6株鸽源新城疫病毒广西分离株F基因的遗传演化分析

应用RT-PCR方法对分离自广西不同鸽场的6株鸽源新城疫病毒F基因进行扩增、序列测定和分析。结果显示:只有1株分离株F基因的ORF全长为1 659 bp,编码552个氨基酸,其他5株分离株F基因的ORF全长均为1 662 bp,编码553个氨基酸,6株分离株的F0蛋白裂解位点的序列均为112RRQKRF117,符合强毒株的序列结构特性;核苷酸同源性比较显示,6株分离株与NDV基因Ⅵ型中的Ⅵb亚型同源性最高,为91.3%~99.3%(与比利时分离株11(JX901124)的同源性最高),与其他基因Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅶ、Ⅷ、Ⅸ型毒株的同源性在81.3%~89.2%。与经典强毒株F48E9相比,核苷酸序列同源性为83.6%~84.8%,氨基酸序列同源性为89.2%~92.4%;与Ⅰ型疫苗株V4、I-2相比,核苷酸同源性分别为83.6%~85.1%和82.6%~84.2%,氨基酸序列同源性分别为88.1%~91.3%和87.7%~90.8%;与Ⅱ型疫苗株La Sota和B1相比,核苷酸同源性分别为81.7%~83.9%和82.0%~83.9%,氨基酸序列同源性分别为86.6%~89.9%和86.6%~89.9%。遗传进化分析显示,6株广西鸽源分离株与2011年比利时分离株11(JX901124)和中国毒株SDS、LLN713、BJP13、LJL404、P4的遗传关系最为接近,位于同一进化树分支上,属于基因Ⅵb亚型。     

东北边境地区野猪及放养杂交野猪戊型肝炎流行病学调查

为了解东北边境地区野猪及放养杂交野猪群体猪戊型肝炎病毒(HEV)感染情况,于2015—2018年在吉林省、黑龙江省的中朝、中俄边境和内蒙古自治区境内加格达奇周边地区采集6月龄以上杂交野猪血清、粪便或肛拭子样品共520份,采集野猪血清和粪便样品共248份。ELISA检测、RT-nPCR检测、全基因组测序、同源性及进化分析结果显示,杂交野猪和野猪感染HEV的血清抗体总阳性率为34.1%(136/399);核酸总阳性率为1.56%(12/771),12份核酸阳性样品均来自杂交野猪,病毒基因组ORF2部分核苷酸序列同源性为85.4%～100.0%,属于基因4型,4a、4b亚型。对4a亚型的1份阳性样品(LJG-18)进行病毒全基因组扩增测序,其核苷酸序列与日本的人源毒株JKO-ChiSai98C同源性最高,为94.9%,与吉林省猪源毒株Ch-S-1同源性为90.2%。结果表明:东北边境地区放养杂交野猪群具有较高的HEV血清抗体阳性率,HEV流行毒株以4a亚型为主。本试验针对我国野猪及放养杂交野猪群体开展猪戊型肝炎流行病学调查,为该病的流行情况提供了新的科学数据,对我国养猪业健康发展和公共卫生安全具有重要意义。     

广东地区新生仔猪先天性震颤猪群中PCV3感染情况的调查

对广东省不同地区新生仔猪先天性震颤猪群中PCV3的流行病学情况进行调查。采集的病料样品,通过常规PCR进行PCV3检测与全基因扩增,分析其全基因的遗传进化关系。将获得的16株PCV3毒株核酸序列与其他环状病毒参考毒株对全基因组和Cap基因进行遗传变异分析,结果显示,新生仔猪的先天性震颤猪群中PCV3样本总阳性率高达58.2%。16株PCV3毒株之间全基因核苷酸序列同源性为99.5%~100%,与美洲代表毒株PCV3-US/MO2015全基因核苷酸序列同源性在98.8%~99.1%之间;16株PCV3毒株之间Cap基因的核苷酸序列同源性为99.7%~100%,与国内外参考毒株的核苷酸同源性为99.1%~100%。遗传进化分析显示,16株PCV3毒株都属于PCV3a分支。PCV3在我国广东省新生仔猪先天性震颤猪群中广泛存在。     

2015-2016年北方某省猪繁殖与呼吸综合征病毒ORF5基因遗传变异分析

为了解北方某省猪繁殖与呼吸综合征病毒(PRRSV)流行毒株的遗传变异情况,本研究对2015-2016年省内各地163家养殖场的874份PRRS疑似病料进行RT-PCR检测,并对PRRSV阳性样品的ORF5基因进行测序及变异分析.结果显示,PRRSV阳性样品共209份,阳性率为23.9%.扩增阳性样品的ORF5基因序列,并选取44条有代表性的基因序列,遗传进化分析表明,北方某省PRRSV流行毒株为美洲型毒株,其ORF5基因核苷酸同源性为82.6%~99.5%,与VR-2332、CH-1a、HuN4和JXA1、CHsx1401和NADC30株的核苷酸同源性分别为83.5%~99.3%、85.1%~95.2%、83.5%~99.3%和82.4%~96.8%.基于ORF5基因核苷酸序列绘制的遗传进化树显示,目前北方某省流行的美洲型PRRSV可分为4个亚群,其中24株属于以CHsx1401和NADC30为代表的Ⅱ亚群,19株属于以HuN4和JXA1为代表的Ⅳ亚群,1株属于以VR2332为代表的Ⅰ亚群.各毒株ORF5基因序列之间存在一定差异,但无明显地域性.本研究结果可为北方某省PRRSV遗传进化研究和疫病防控提供参考.     

上海及新疆地区猪戊型肝炎病毒开放阅读码框架2序列的克隆与分析

通过已建立的动物戊型肝炎病毒（Hepatitis E virus,HEV）RT-nPCR方法,检测上海地区和新疆地区猪群样品。扩增HEV开放读码框架2（open reading frame 2,ORF2）保守区,产物为507 bp。经克隆、测序,获得20株上海株、3株新疆株序列,采用分子生物学软件与47株已知HEV进行同源性、进化关系分析。结果23株序列从同源性比对和进化关系分析均属HEVⅣ型,上海株间507 bp的核苷酸同源性为94.1%~100.0%,与上海猪源（DQ450072）、日本人源Japan1（AB369690）亲缘关系近;3株新疆株507 bp间的核苷酸同源性为86.1%~98.2%,与新疆猪源China SW1（AY594199）、中国人源T1株（AJ272108）亲缘关系近;上海株与新疆株间的核苷酸同源性为82.6%~100.0%,提示上海株与新疆株间存在差异。23株HEV在时间和地域关系分析,提示不同年份的新疆株存在差异,同年份不同猪场的新疆株进化关系存在差异,不同年份、不同区县的上海株进化关系也存在差异。     

Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb

Strains of hepatitis E virus (HEV) genotypes 1 and 4 have been detected on the Chinese mainland although there have been no previous reports of zoonotic genotype 3 HEV. In the present study, 65 swine fecal specimens were collected from five pig farms located in different Shanghai suburbs. RT-PCR and nested PCR were undertaken using partial nucleotide sequences of Open Reading Frame 2 (ORF2) of HEV to detect HEV RNA. Genetic analysis was based on alignments of an amplified 150-nt ORF2 sequence. RT-PCR revealed 15 HEV positive samples among 65-pig fecal specimens examined. Phylogenetic analysis of the amplified sequences indicated seven HEV strains belonged to genotype 3 and eight strains to genotype 4. This is the first time that genotype 3 hepatitis E virus has been identified on the Chinese mainland.     

Genotype 3 hepatitis E has been widespread in pig farms of Shanghai suburbs

Hepatitis E virus (HEV) genotype 3 was first identified in swine raised on a Shanghai suburban pig farm in late 2006. To accurately determine the prevalence of HEV infections among Shanghai pig farms, 426 pig fecal samples were collected from 37 pig farms located in all 10 Shanghai suburban districts and tested for the presence of HEV RNA using RT-PCR. Genetic analysis based on an amplified 150-bp ORF2 fragment revealed 111 samples to be HEV positive, and the prevalence of HEV infection within the different districts varied between 0 and 41.7%. Thirty-two samples were sequenced, and phylogenetic analysis indicated that 10 isolates belonged to HEV genotype 4 and were most closely related to 3 human and 2 swine HEV strains, all of which had originally been isolated from Asian countries including Japan and China. The remaining 22 isolates belonged to genotype 3 and were most closely related to a strain of swine HEV, US-SW, isolated from pigs in the United States. Our data indicated that genotype 3 HEV was widespread among suburban Shanghai pig farms although further study is required to determine the source and zoonotic nature of the virus.     

Prevalence and genetic characterization of Hepatitis E virus in paired samples of feces and serum from naturally infected pigs

This study describes the distribution of Hepatitis E virus (HEV) in a naturally infected swine population and the genetic relatedness of HEV strains on swine farms in Spain. Of fecal and serum samples collected from 131 pigs and manure-ditch samples collected from 17 farms, HEV was detected in 16%, 14%, and 59%, respectively, for an overall prevalence rate of 23%. The maximum prevalence rates for feces and serum were in pigs 5 to 12 wk old. A high prevalence of the virus in feces (18%) was observed in sows. Gene sequencing was performed on 6 strains from feces, serum, and manure ditch: the nucleotide identities varied from 81.5% to 99% when compared with those of other strains of genotype 3 isolated from swine. This is the first study in Europe to show the variation in virus distribution by age in feces and serum in a naturally infected swine population.     

Hepatitis E virus in swine and effluent samples from slaughterhouses in Brazil

Hepatitis E is an infectious disease which virus (HEV) is highly disseminated in swine herd populations. Sporadic acute human hepatitis E cases have been associated to genotype 3 and 4 strains of HEV also reported in swine populations of endemic and non-endemic areas. With the aim to evaluate the incidence of animals with current infection of HEV, 115 bile samples were collected from three slaughterhouses under inspection by Animal Sanitary Protection Agency of Rio de Janeiro, Brazil. In parallel, effluent samples were collected from six sewage pipe exit sites of two slaughterhouses. HEV RNA was detected in 11 out of 115 (9.6%) bile samples collected and three waste samples from one slaughterhouse. Viral loads observed for bile samples varied from 10(1)-10(5) genome copies/mL and for effluent samples mean load was 10(2) genome copies/mL. Sequencing and phylogenetic analysis classified samples within genotype 3 subtype 3b closely related to the sample obtained from the first reported autochthonous human case and samples from swine of commercial herds in Brazil. Our data demonstrates that although most animals achieve slaughter age (around 20 weeks old) already immune to HEV, a significant number of animals are with current infection at commercial age. Further studies should be addressed to consider risk analysis and possible evaluation of inspection regulations considering food safety measures regarding hepatitis E zoonotic aspect in Brazil.