国外禽痘病毒疫苗自然重组株整合序列的研究

本文根据禽痘病毒（FPV）基因组中禽网状内皮组织增生症病毒（REV）常见整合区域的序列设计合成一对引物，对国外一株FPV疫苗进行了PCR扩增，结果扩增出一条1478bp的片段，经过测序和序列分析表明该片段为REV和FPV的整合序列，FPV序列中整合了大小为193bp的REV长末端重复序列，进一步比较显示该序列与国外REV毒株APC566、713和SNV的同源性分别为：99．6％、98.0％和87．3％，与我国REV分离株HA9901的同源性只有83．6％。同时该REV整合序列与国外已经测定的FPV毒株AJ581527、AY255633、AF198100、AF006064和AF246698比较时发现，这些毒株中含有的REV序列不仅完全一致，而且REV序列的插入位点也完全相同。这说明国外FPV疫苗中不仅含有REV整合序列，而且已经存在多年了。     

鸡痘病毒分离株及疫苗株整合网状内皮组织增生病病毒的基因分析

为了解山东地区鸡痘病毒(FPV)野毒流行株整合禽网状内皮组织增生病病毒(REV)前病毒的情况,同时比较FPV分离株和弱毒疫苗株整合REV前病毒序列的差异,本研究对山东不同地区的14个FPV分离株和国内外不同厂家的5个FPV疫苗株进行了REV前病毒整合区基因PCR扩增、核苷酸序列测定及基因分析.结果表明14个FPV分离株均整合了几乎全长的REV前病毒序列,这些序列与美国分离的FPV整合株AF246698的整合序列同源性最高,与REV标准株同源性较低;疫苗株均仅整合部分或完整的REV-LTR序列,但国产与进口疫苗株的整合序列不同,国产疫苗整合的LTR片段相对较小.研究表明所分离的山东FPV分离株均整合全长REV序列,疫苗株仅整合REV-LTR序列.然而FPV整合病毒株的致病特性、整合疫苗的安全性及对整合FPV野毒的免疫保护作用有待于进一步研究.     

整合禽网状内皮组织增殖病病毒基因的鸡痘病毒野毒株致病性研究

以分离保存的整合禽网状内皮组织增殖病病毒（REV）基因的鸡痘病毒（FPV）野毒株接种CEF细胞,间接免疫荧光试验检测REV,结果未检测到游离REV病毒粒子;以该毒株人工感染7日龄SPF雏鸡,同时设阴性对照,常规免疫新城瘦弱毒苗（ND）。接毒后每周采血动态检测NDV抗体、REV抗体、REV病毒血症;同时对试验鸡免疫器官指数、组织学变化动态观察,并对免疫器官的细胞凋亡动态检测。结果表明,整合REV基因的FPV感染后2周可引起鸡REV病毒血症,并产生REV抗体;导致中枢免疫器官发育分化不良,尤其对胸腺的影响最为明显,攻毒2周后,试验组与对照组相比,胸腺指数明显下降（P〈o．05）;免疫器官主要表现实质细胞数量少,正常结构发育不良,间质结缔组织增生而发生纤维化;细胞凋亡检测证实免疫器官的淋巴细胞和巨噬细胞均有明显的凋亡现象,细胞凋亡是引起免疫器官萎缩的主要原因。对新城疫疫苗免疫反应呈显著的抑制作用,攻毒3周后,试验组血清新城疫病毒抗体滴度显著低于对照组（P〈0．05）,其抗体滴度峰值低,维持时间短,下降速度快。本试验证实FPV整合REV前病毒基因组会明显改变FPV的致病性,表现某些REV的致病特征,使感染鸡REV抗体转为阳性并出现病毒血症,进一步证实REV可以通过基因组整合于FPV而进行传播的推测。     

禽痘病毒活疫苗中网状内皮组织增生病病毒的分离鉴定及其序列比较

为了从怀疑污染禽网状内皮组织增生病病毒（Reficuloendotheliosisvirus,REV）的禽痘病毒（Fcwlpoxvirus,FPV）活疫苗中分离病毒,将市场上购买的某些厂家的禽痘弱毒疫苗接种7日龄SPF雏鸡。在5d后采其抗凝血浆接种鸡胚成纤维细胞（chickenembryofibroblast,CEF）,在连续传2代后,用REV特异性单抗做间接免疫荧光试验,确认REV感染,命名为JS0809。提取感染细胞的DNA,采用PCR扩增env基因。测序分析表明,该毒株的env基因开放阅读框同大部分REV-毒株一致,为176lbp。JS0809的囊膜蛋白基因（envelopeproteingene,env）与国内已发表株的同源性高达96．0％-99．％。相对而言,它与早年分离到的毒株的同源性只有96％-97．3％,但与近几年的分离珠的同源性则均高达99．5％-99．8％。它与整合进FPV野毒株中的全基因组REV的env基因同源性很高为99．8％,而与整合进FPV疫苗株中全基因组REV的env基因同源性较低仅为97．3％。本研究证实了在我国生产的某些FPV疫苗中存在REV污染,所污染的REV的env基因更接近最近的流行野毒,这是国内第一次报道从禽痘弱毒疫苗中分离~qREV活毒。     

鸡痘病毒PCR检测方法的建立及临床应用

根据已发表的鸡痘病毒(fowlpox virus,FPV)4 b核心蛋白基因的核苷酸序列设计合成了(P3,P5)1对引物,通过对影响PCR扩增条件的优化,在不同的退火温度下,直接以病毒液为模板扩增出预期的1 107 bp的片段。特异性检测表明,两对引物对FPV102株、FPV282E4(北京)、大华农鸡痘疫苗毒株、重组鸡痘疫苗病毒及临床病料均能扩增出相应的特异性片段,而用鸡马立克氏病毒、传染性支气管炎病毒,鸽痘病毒及鸡胚成纤维细胞制备的模板进行PCR扩增均为阴性。敏感性检测表明,引物能检测0.195 ng/μL的模板量。用此方法检测50份临床样品,其中的阳性率为92%,用琼脂扩散试验方法检测的阳性率为70%。以上结果表明,该方法可以用于FPV临床样品的检测。     

1株基因Ⅶ型新城疫病毒的生物学特性研究及全基因组序列分析

为研究当前流行的新城疫病毒（Newcastle disease virus,NDV）基因型、致病性及其与传统新城疫病毒疫苗株（La Sota等）的核苷酸差异,试验从某发病鸡场病死鸡体内分离到1株疑似NDV毒株,经红细胞凝集试验（HA）和红细胞凝集抑制试验（HI）初步确定为鸡源NDV。参照GenBank公布的NDV F基因部分片段（登录号：JF950510.1）设计1对引物,通过RT-PCR技术扩增分离株的F基因并克隆、测序,测序结果与NCBI中NDV F基因序列进行比对,构建系统进化树并分析其基因型;通过测定鸡胚平均致死时间（MDT）、1日龄鸡脑内致病指数（ICPI）和6周龄鸡静脉致病指数（IVPI）判断病毒致病性;参照GenBank公布的NDV全基因组序列（登录号：JF950510.1）设计9对引物对分离株进行全基因组序列测定,并分析其基因组结构。结果表明,RT-PCR扩增得到F基因长约500 bp,基于F基因构建的系统进化树显示分离株为基因Ⅶ型NDV;MDT、ICPI和IVPI分别为52.8 h、1.675和2.46,表明分离株属于强毒株。全基因组序列分析显示,分离株全基因组全长15 192 bp,与传统La Sota株基因组相比,序列多出6个碱基,核苷酸序列同源性82.8%。本研究成功分离到1株基因Ⅶ型NDV强毒株,且与传统疫苗毒株La Sota的核苷酸序列同源性差异较大。     

区分犬瘟热病毒Asia-Ⅰ型野毒株及疫苗株的AS-PCR方法

犬瘟热是一种接触性传染病,可侵害免疫系统、呼吸系统、消化系统,甚至神经系统,导致全身性的病理变化,对宠物犬、毛皮动物等存在巨大威胁。目前常用的胶体金检测法不能有效区分疫苗免疫与动物自然感染。为建立一种高效准确的鉴别犬瘟热病毒野毒株和疫苗株的检测方法,本试验对从武汉地区收集已确诊犬瘟热的6只犬分离得到的野毒株以及3株广泛使用的CDV疫苗株进行全基因组测序,从氨基酸水平和碱基水平比对分析后,确定H基因为AS-PCR引物设计的靶基因。通过对H基因进行分型,发现武汉地区流行的CDV均为Asia-Ⅰ型,而疫苗株Y2为America-I型,疫苗株Y1和Y3均为America-Ⅱ型。对比179株Asia-Ⅰ型（6株野毒株样品+173株GenBank Asia-Ⅰ型）与3株疫苗株的CDV-H基因序列,采用AS-PCR技术（3'端错配）设计出1对能有效区分犬瘟热Asia-Ⅰ型野毒株和疫苗株的特异性引物,上游引物序列为5'-TTAAATGATAATGACATAGTG-3',下游引物序列为5'-CCTGGCAAGGCAAGA-3'。结果显示该引物有较强的特异性,6株样品野毒株均可扩增出长894 bp的片段,疫苗株不能扩增,且野毒株的H基因上存在9个较为规律的碱基（氨基酸）变异位点,而疫苗株在第277位氨基酸上均为天冬酰胺,这些变异可能导致N-糖基化位点的增加,从而对犬瘟热病毒疫苗株的毒力产生影响。本研究建立的AS-PCR方法能有效区分犬瘟热疫苗和Asia-Ⅰ型野毒株。     

鸡传染性法氏囊病病毒基因组A片段cDNA序列分析

用鸡胚成纤维细胞繁殖鸡传染性法氏囊病病毒（IBDV）天水毒株。用提纯的病毒颗粒提取基因组RNA。根据已报道的英国52／70株的序列设计引物，用反转录－聚合酶链式反应（RT－PCR）进行cDNA扩增，获得1558bp和1590bp两个部分重叠的片段。结果表明，所克隆片段为3099的IBDV大开放读框。经与已报道的多个毒株相应序列比较后发现，核苷酸同源性介于97.4%-99.8%之间，推导的氨基酸同源性介于98.1%-99.5%。     

禽网状内皮组织增生症病毒山东SD2101株的分离鉴定及全基因组序列和致病性分析

禽网状内皮组织增生症病毒（reticuloendotheliosis virus，REV）是一种常见的禽肿瘤性病毒，可诱发感染鸡群免疫抑制，给我国养禽业带来巨大经济损失。本试验运用DF-1细胞接种方法，在山东省某父母代白羽肉种鸡场鸡群中分离到1株REV，将其命名为SD2101株。为掌握SD2101株基因组遗传演化特征，设计合成6对引物，通过PCR扩增获得了SD2101株的前病毒全基因组序列，并与GenBank下载的参考毒株进行了序列比对和遗传进化分析。结果显示：SD2101株前病毒全基因组具有典型复制完整型逆转录病毒的基因组结构，与2011年分离自江苏省的鸡源REV野毒株HA1101同源性最高；遗传进化分析发现，SD2101株与大部分国内分离株处于同一进化支上。为了解SD2101株的致病性，将分离毒株接种于1日龄SPF鸡，通过体质量、免疫器官指数和灭活疫苗免疫应答等指标进行评价。结果显示，SD2101株对1日龄SPF鸡的生长和免疫功能具有明显的抑制作用。综上，与参考毒株相比，SD2101株基因组序列变异程度较小，具有一定致病作用，提示在生产中应加强对REV的关注和监测。本研究为REV的流行病学研究提供了信息资料。     

基因Ⅶ型鹅副粘病毒NA-1株全基因组的克隆及特性分析

将本实验室分离保存的NA-1株鹅副粘病毒（GPMV）经SPF鸡胚增殖，收集鸡胚尿囊液进行病毒纯化，提取病毒基因组RNA。参考GenBank已收录的GPMV ZJ1株基因组序列，设计了8对特异性引物，RT—PCR法分别特异性的扩增出病毒各基因片段，并将各目的基因片段回收纯化．克隆PGEMT载体．转化大肠杆菌DH5α，小提质粒选取阳性克隆酶切鉴定并进行序列测定．对测定结果进行拼接得到全长cDNA序列（GenBank登录号：DQ659677）。NA-1株核苷酸比新城疫病毒（NDV）核苷酸多6个碱基，位于1644～1645nt之间，符合NDV核苷酸“六碱基原则”。序列分析结果表明，GPMVNA-1株与各地代表株核苷酸同源性81．2％～91．3％。同时根据各毒株F基因开放阅读框前389个核苷酸序列绘制出系统发育进化树，结果表明GPMNNA-1株与SFO2和QY97病毒株同属于基因Ⅶ型，不同于基因Ⅸ型NDV的国家标准株F48E9和基因Ⅱ型的LaSota传统弱毒株。     

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 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.

     

Heartworm in dogs in Canada in 1988

In late November 1988, 1581 small and mixed animal clinics and institutional veterinarians across Canada were sent a questionnaire in order to assess the status of Dirofilaria immitis in Canada in 1988, and 46% of them responded. Veterinarians reported that 181,577 dogs were blood-tested for heartworm disease and 367 dogs were found with D. immitis microfilariae. Another 60 dogs were amicrofilaremic but diagnosed with heartworm disease to give the total number of cases diagnosed in 1988 as 441 (0.24%).     

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 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 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.