2022年鄂西地区PRRSV ORF5基因遗传进化分析

为掌握鄂西地区PRRSV的流行及遗传变异情况,对2022年采自该地区8个县（市、区）的疑似感染发病猪血液、肺脏组织等样品共672份,进行PRRSV实时荧光RT-PCR检测,并对部分阳性样品的ORF5基因序列进行遗传进化分析。结果显示：检出PRRSV阳性样品33份,阳性检出率为5%;通过进一步分型鉴定,23份为HP-PRRSV阳性,占69.7%,10份为类NADC30（NADC30-like PRRSV）阳性,占30.3%;采用DNAstar软件分析4份阳性样品的GP5蛋白氨基酸序列,显示存在多个氨基酸突变,在诱骗表位（27~30 aa）出现29G→29C,在中和表位,HP-PRRSV毒株出现39A→39T,类NADC30毒株出现38A→38T。结果表明,鄂西地区存在高致病性和类NADC30两种PRRSV毒株流行,其GP5蛋白氨基酸序列已发生了多个变异。结果提示,应持续加强PRRSV分子流行病学监测,选择与流行毒株匹配的疫苗进行免疫。     

多重RT-PCR检测PRRSV和CSFV及PRRSV ORF5基因遗传变异分析

根据猪繁殖与呼吸综合征病毒(PRRSV)和猪瘟病毒(CSFV)的基因组序列设计了2对特异性引物,建立了同时检测PRRSV和CSFV的多重RT-PCR方法.2008年1月至2009年12月期间,采集山东各地71个不同规模猪场及农村散养户212份病料,采用建立的多重RT-PCR技术同时检测PRRSV和CSFV的感染状况.检测结果显示,病料中高致病性PRRSV占57.1%(121/212),CSFV占29.2%(62/212).应用RT-PCR方法扩增山东不同地区16株PRRSV的ORF5基因,并进行了序列分析.结果表明,16株PRRSV的ORF5基因核苷酸同源性为97.0%～100.0%;与参考变异毒株JXA1、传统毒株VR2332核苷酸同源性分别为98.0%～99.5%和86.4%～87.7%.推导的氨基酸序列分析表明,16株毒株GP5蛋白氨基酸不存在缺失,但存在位点突变.12株在非中和表位29位点与准种进化34位点发生了突变,分别由V→A和N→S;1株在非中和表位185位点发生突变,由A→V;这与2007年流行的PRRSV毒株JXA1、HUN等不同.     

2021—2022年我国部分地区猪繁殖与呼吸综合征病毒ORF5基因变异分析

为深入了解我国猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的流行和遗传变异情况，2021—2022年，本课题组在我国13个省份约50个养殖场收集了117份临床疑似猪繁殖与呼吸综合征患猪样品，针对PRRSV的ORF5基因进行了遗传变异分析。通过实时荧光定量PCR鉴定PRRSV阳性样品和基因型，应用RT-PCR扩增PRRSV ORF5和ORF7基因，将阳性样本进行测序并开展系统发育学分群和GP5氨基酸突变分析。结果显示，共检出PRRSV-2阳性样品48份(总体阳性率约41%),经测序共获取38条ORF5序列和10条ORF7序列。所有ORF5序列间核苷酸相似性为77.1%～99.8%,ORF7序列间核苷酸相似性为83.3%～98.7%。系统发育学分群显示，谱系1占比60%(29/48,28株NADC30-like毒株，1株NADC34-like毒株),谱系3占比25%(12/48),谱系5.1占比5%(2/48),谱系8.7占比10%(5/48)。GP5氨基酸分析表明，信号肽(aa 1—26)、中...     

2019—2021年我国部分地区PRRSV ORF5基因遗传变异分析

为了解2019—2022年我国南方地区猪繁殖与呼吸障碍综合征病毒（PRRSV）ORF5基因遗传变异情况,自广东、广西、湖南、江西4个省份的种猪场和育肥场,采集疑似PRRSV感染样品进行RT-PCR检测,并对部分阳性样品进行病毒分离培养和ORF5基因测序。结果显示：PRRSV总检出率为26.12%,其中育肥场场检出率（86.36%）高于种猪场（52.00%）;共成功分离到97株病毒并对其进行了ORF5测序分析,发现Sublineage 1.8占比最高（51.55%）,其次为Lineage 5(15.46%),Sublineage 1.5、Lineage 8、Lineage 3占比依次为13.40%、10.31%和9.28%;类NADC34毒株在2021年有3个省份（广东、广西和湖南）被首次检出,且当年检出率达13.40%。GP5蛋白分析结果显示,分离株中第13和151位氨基酸以及中和表位变异较多。对其中1株毒株进行重组分析发现,该毒株以类NADC30毒株为主要亲本,其多处基因组与JXA1和IA2014毒株发生了重组。结果表明：我国南方地区PRRSV流行较为普遍,尤其是在育肥猪群中;流行毒...     

猪繁殖与呼吸综合征病毒贵州流行株GP5基因的RLFP分析

为对贵州省某养猪场猪繁殖与呼吸综合征(PRRS)疑似病例进行确诊及分析其病原分子变异情况,采集该养猪场死亡猪病料样本进行猪繁殖与呼吸综合征病毒(PSSRV)的RT-PCR检测,并采用RLFP方法分析了现场流行毒株GP5基因的变异情况。结果表明,该猪场病猪样本中PRRSV的核酸检出率为100%,是由高致病性PRRSV变异毒株引起;RT-PCR扩增PRRSV贵州流行株GP5基因,经RLFP分析显示,流行株GP5基因呈1-3-3 PCR-RFLP模式;克隆测序后发现,贵州流行株GP5基因发生多个点突变,其中在第25、216、358、409和554位核苷酸的点突变,可导致一些酶切位点的丧失或显现。     

湖北省部分规模猪场猪繁殖与呼吸综合征流行病学调查

为探究猪繁殖与呼吸综合征病毒（PRRSV）在规模猪场的流行特征,2021年从湖北省7个规模猪场疑似PRRSV感染猪群以及所有种公猪群中,共收集样品3 324份,采用三重巢式RT-PCR法进行PRRSV病原学检测,分析不同养殖类型、不同生长阶段、不同季节及不同感染毒株的流行特点,并将阳性产物进行测序和遗传进化分析。结果显示：3 324份样品中共检测出PRRSV阳性样品323份,阳性检出率为9.72%（323/3 324）。育种场阳性检出率最低,为2.66%（20/752）;商品猪扩繁场与育肥场阳性检出率相近,分别为11.82%（169/1 430）与11.73%（134/1 142）;保育猪群阳性检出率最高,为17.23%（152/882）;其余依次为生长育肥猪、哺乳仔猪和母猪,阳性检出率分别为11.30%（121/1 071）、5.26%（30/570）和3.24%（20/618）;种公猪中未发现PRRSV感染。PRRSV一年四季均可流行,春、夏、秋、冬季的阳性检出率分别为18.16%（148/815）、6.94%（60/864）、6.10%（34/557）、7.44%（81/1 088）,春季阳性检出率显著高于其他季节（P＜0.05）,而夏、秋、冬季节间差异不显著（P≥0.05）。3种PRRSV毒株均有流行,类NADC30毒株、经典毒株和高致病性变异毒株的占比分别为49.54%、21.36%、29.10%,类NADC30毒株检出占比显著高于其他类型毒株（P＜0.05）。结果表明：PRRSV在所调查的7个规模猪场中流行普遍,尤其是商品猪扩繁场与育肥场;保育猪群PRRSV感染风险较高,种公猪感染风险较低,春季是高发季节,类NADC30毒株为优势流行毒株。结果提示,规模养殖场应根据PRRSV的流行特征,加强免疫和监测,有针对性地制定防控措施。     

2013年—2014年广西桂林市猪蓝耳病病毒流行病学调查

为了解2013年—2014年广西桂林市猪蓝耳病(PRRS)的流行情况,以及主要基因的变异情况,本研究应用RT-PCR的方法检测疑似猪蓝耳病病毒(PRRSV)样品35份,阳性为8份,阳性率22.9%。选择其中3个阳性的样品进行GP5和部分nsp2基因测序并分析其结果。GP5基因分析结果表明3株PRRSV的GP5基因与NCBI基因库中参考序列对应核苷酸的同源性为63.5%~99.8%,氨基酸同源性为57.1%~99.5%。通过GP5的遗传进化分析发现所获得的PRRSV为北美型,且与HP-PRRSV同属于一个小分支上;部分nsp2基因结果表明,与PRRSV原型毒株VR2332的核苷酸同源性在78%~78.8%之间,氨基酸同源性在69.1%~70.3%之间,与高致病性毒株PRRSV JXA1核苷酸同源性在96.3%~99.1%,氨基酸同源性在93.2%~97.6%。氨基酸的比对结果表明,本研究所获得的3株PRRSV部分nsp2基因均存在与HP-PRRSV一致的1+29个氨基酸的缺失,表明3株PRRSV毒株均为HP-PRRSV。     

华中地区猪繁殖与呼吸综合征病毒临床流行毒株Nsp2和GP5氨基酸的遗传变异分析

为了解当前猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)流行毒株的遗传变异情况,本研究对2013-2014年间来源于中国华中地区湖北、河南和湖南3省20个猪场65份疑似PRRSV感染的临床样品开展病毒分离工作,通过RT-PCR及间接免疫荧光试验进行鉴定,结果显示成功分离到4株PRRSV。通过测序获得它们的部分Nsp2氨基酸序列,经比对分析,确定分离毒株皆为变异毒株,并且与JXA1、TJ等高致病性毒株有相同的遗传特点,在Nsp2区域有30个氨基酸的不连续缺失。本研究将从临床样品中测序获得的23株PRRSV的GP5氨基酸序列与GenBank公布的有代表性的毒株序列进行遗传进化分析,结果显示所有毒株均为美洲型毒株,其中21株与高致病性变异毒株(JXA1、TJ和HUN4)同源性较高,同时在主要中和位点和N-糖基化位点存在部分氨基酸突变,另外两株与疫苗毒株RespPRRS MLV同源性较高。本试验结果表明,当前高致病性PRRSV依然是优势毒株,其GP5氨基酸突变频繁,不同地区流行毒株存在一定的遗传差异。     

猪繁殖与呼吸综合征病毒湖南分离株ORF5基因的遗传进化分析

为了解猪繁殖与呼吸综合征病毒（PRRSV）在湖南省地方猪保种场的感染情况,本研究在2019－2020年间从湖南省2个地方猪保种场采集287份全血样品。首先将血样混合成41份,采用RT-PCR或PCR法进行PRRSV病原检测,进一步通过高保真PCR扩增从PRRSV阳性样品中扩增PRRSV ORF5基因;测序后利用DNAStar软件分析获得的ORF5基因及其编码的GP5氨基酸与国内外不同PRRSV毒株的遗传进化关系;最后用PRRSV阳性血清接种Marc-145细胞,经盲传分离毒株,并用Reed-Muench法测定病毒滴度。结果显示,检测的41份混样中有3份PRRSV病原核酸呈阳性;从PRRSV阳性混样中单独扩增获得6条PRRSV ORF5基因序列,均属于PRRSV-2型的lineage 8分支,相似性为99.2%~99.8%;6条ORF5基因编码的GP5蛋白氨基酸序列在信号肽区域（第23位）、潜在的N-糖基化位点（第33位）和表位C （第59位）存在差异;PRRSV阳性血清接种Marc-145细胞盲传5代后出现明显的细胞病变,获得1株PRRSV毒株,命名为NX-1,病毒TCID₅₀为4×10⁵/mL。本研究表明,湖南省地方猪保种场存在PRRSV感染,感染的PRRSV属于PRRSV-2型的lineage 8,其GP5氨基酸序列存在的多处变异可能是造成疫苗免疫失败的原因之一,以上结果可为湖南省地方猪保种场的免疫防控提供一定参考。     

2株高致病性猪繁殖与呼吸综合征病毒分子特征分析

为进一步掌握黔西南州猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus,PRRSV）流行变异趋势,对2株高致病性PRRSV（HR-PRRSV）贵州分离株GZLPS、GZSB进行Nsp2与GP5基因克隆和测序,并分析毒株分子变异特征。结果显示,2株分离株Nsp2基因有90个核苷酸不连续缺失,不同位置的36个核苷酸发生突变;GP5基因存在不同位点置换。同源性比对与系统进化树分析显示,2株分离株Nsp2与GP5基因与HP-PRRSV JXA1株及贵州省2011年以前流行毒株的核苷酸同源性在96%以上,氨基酸同源性在95%以上,但在分子变异上存在差异。结果表明黔西南州PRRSV变异方式已呈多样性。     

Heartworm in dogs in Canada in 1989

In late November 1989, 1732 clinics and institutional veterinarians were sent a questionnaire to assess the status of Dirofilaria immitis, and 51.7% responded. Of 247,716 dogs tested, 394 had D. immitis microfilariae and 51 were amicrofilaremic for a total of 445 cases and heartworm prevalence of 0.17%. Most (408) of these dogs had no preventive medication and the prevalence among dogs tested and unprotected was 1.01%. That prevalence was considerably higher in endemic areas. Thirty-seven dogs with heartworm had preventive medication. Heartworm was most frequent in companion dogs over three years of age maintained outdoors in rural areas. About 75% of the cases had never left Canada, 26% had clinical signs and 125 were not treated.

Heartworm was reported from British Columbia, Manitoba, Ontario, Quebec, Nova Scotia and Newfoundland, but 383 cases were in Ontario. South-western Ontario was the primary focus of infection. There were 33 cases in Quebec and 24 in Manitoba, mainly found in and around Metropolitan Montreal and Winnipeg respectively.

     

Heartworm in dogs in Canada in 1987

In late November 1987, 1246 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1987, and 50% of them responded. Veterinarians reported that 165,428 dogs were blood tested for heartworm disease and 511 dogs were found with D. immitis microfilariae. Another 78 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1987 as 589 (0.35%).     

Heartworm in dogs in Canada in 1986

In late December 1986, 1224 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1986; 46% of them responded. Veterinarians reported that 150,989 dogs were blood-tested for microfilariae and 869 dogs were found with heartworm. Another 65 dogs were amicrofilaremic but diagnosed with heartworm disease and one was found with heartworm at necropsy to give the total number diagnosed in 1986 as 935 (0.62%).

Heartworm was reported from Manitoba, New Brunswick, Ontario and Quebec, but most (810) of the cases were from Ontario. South-western Ontario continued to be the primary focus of the infection in Canada. There were 103 cases reported from Quebec, mostly from and around Montreal, and 21 cases from Manitoba, from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 33% of the cases were observed with clinical signs of heartworm disease and 81% had a history of not having left Canada.

     

Heartworm in dogs in Canada in 1984

In late December 1984, 1853 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1984 and 35% of them responded. Veterinarians reported that 97,794 dogs were blood-tested to check for microfilariae and 1417 dogs (1.45% of those tested) were found with heartworm. Another 34 dogs were amicrofilaremic, but were diagnosed as having heartworm disease, to give the total number diagnosed in 1984 as 1451 (1.48%). Heartworm was reported from all provinces except Prince Edward Island and Newfoundland but most (1310) of the cases were in Ontario. In Quebec, 126 cases were reported mostly from west of Montreal.

Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 27% of the cases were observed with clinical signs of heartworm disease and 72% had a history of not having left Canada. Southwestern Ontario continued to be the primary focus of the infection.

     

Heartworm in dogs in Canada in 1983

In late December 1983, 2 800 veterinarians across Canada were sent a questionnaire in order to assess the status of heartworm disease in Canada in 1983 and 26% of them responded. Veterinarians reported that 59 504 dogs were blood-tested to check for microfilariae and 771 dogs (1.30% of those tested) were found with Dirofilaria immitis. Heartworm disease was diagnosed in all provinces except New Brunswick and Newfoundland but most (733) of the cases were in Ontario.

Heartworm disease was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 31% of the cases were observed with clinical signs of heartworm disease and 64% had a history of not having left Canada. Southwestern Ontario continues to be the focus of the infection and most of the dogs there had not left the province previously.

     

Heartworm in dogs in Canada in 1991

In late November 1991, 1883 clinics in Canada were sent a questionnaire to assess the status of Dirofilaria immitis in dogs in 1991 and there was a 60.0% response. There were 344,031 dogs tested for heart-worm (HW), 627 were found infected and the prevalence of HW infection was 0.18%. There were 417 dogs with HW in Ontario, 116 in Manitoba, 38 in Quebec, 53 in British Columbia, three in Alberta, and one in Nova Scotia. In British Columbia, all of the infected dogs but one were from the Okanagan valley which, as from 1991, is a new focus of infection in Canada. Most dogs with HW had not been on preventive medication in 1990, and the prevalence among dogs tested and unprotected was 0.59%. That prevalence was considerably higher in endemic areas. Companion dogs, over three years of age and maintained primarily outdoors in rural areas, were most frequently infected. One cat was diagnosed with D. immitis and 33 dogs had Dipetalonema reconditium.     

Heartworm in dogs in Canada in 1985

In late December 1985, 1485 institutional veterinarians and small and mixed animal clinics across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1985 and 44% of them responded. Veterinarians reported that 137,300 dogs were blood-tested to check for microfilariae and 1210 dogs were found with heartworm. Another 36 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number diagnosed in 1985 as 1247 (0.91%).

Heartworm was reported from all provinces except Prince Edward Island, Newfoundland and Saskatchewan but most (1126) of the cases were in Ontario. Southwestern Ontario continued to be the primary focus of the infection in Canada. From Quebec, 91 cases were reported mostly from and around Montreal. From Manitoba, 19 cases were reported from Winnipeg and surrounding areas. Heartworm was found most frequently in companion dogs over three years of age maintained mainly outdoors in rural areas. About 28% of the cases were observed with clinical signs of heartworm disease and 78% had a history of not having left Canada.