猪繁殖与呼吸综合征病毒ZB株的分离与鉴定

为了确定从高度疑似猪繁殖与呼吸综合征(PRRS)的猪内脏中分离得到的猪繁殖与呼吸综合征病毒(PRRSV)毒株的类型,试验进行了形态学观察、血清学分析、免疫学分析、RT-PCR扩增等一系列鉴定分析。结果表明:从疑似病料中分离获得的病毒可以使Marc-145细胞产生明显病变,而在Vero和PK-15细胞上不出现细胞病变,从而确定试验获得的病毒为PRRSV,命名为ZB株。     

水貂犬瘟热病毒LD-1株的分离与鉴定

取山东某貂场疑似犬瘟热病毒（canine distemper virus,CDV）感染的水貂肝脏等组织病料,通过RT-PCR检测呈CDV阳性,且无水貂细小病毒存在,将病料接种原代CEF细胞、传代系Vero细胞和DF1细胞3种细胞进行病毒分离,通过优化细胞培养条件,最终在Vero细胞上传代培养成功,出现露珠状典型细胞病变（CPE）。分离毒应用RT-PCR、PCR产物测序、抗体中和试验（SN）、间接免疫荧光试验（IFA）及病毒包涵体检查等多种方法进行了CDV的鉴定,结果显示CDV阳性,表明分离到的病毒为水貂CDV,并将其命名为CDV LD-1株。     

鸡肾型IBV的分离及其N基因的克隆与测序

通过病毒形态学观察、血凝试验、病毒干扰实验、动物回归实验等分离鉴定了1株鸡肾型传染性支气管炎病毒.利用RT-PCR技术对分离毒株的N基因进行了扩增,经克隆、序列测定和分析,证实分离株为肾型IBV.     

桂林老虎猫瘟热病毒的分离鉴定

我们在作猫细小病毒分子流行病学调查过程中,从桂林送棼的考虑粪便中分离出１株虎细小病毒,并对其进行了系统鉴定,经形态学理化学血清学交叉中和试验、动物感染试验与分子生物学,证明为一株猫瘟热病毒（猫泛白细胞减少症病毒）的强毒。     

鸡传染性支气管炎病毒宁夏株的分离与鉴定

为了研究宁夏地区流行的鸡传染性支气管炎病毒生物学特性及流行疾病的特点,试验对分离株进行了病毒血凝特性试验、病毒干扰试验、鸡胚致病性试验、动物回归试验,随后采用RT-PCR技术对病毒的S1蛋白基因进行扩增和序列分析,并将病毒在传代Vero、BHK-21细胞系上盲传6代,同时提取细胞基因组RNA,采用RT-PCR扩增S1蛋白基因。结果表明:分离的病毒为鸡传染性支气管炎病毒,不能在传代细胞上进行适应培养,该地区流行的病毒株与H120同源性极高。     

一株猪流行性腹泻病毒的分离鉴定及其致病性研究

应用Vero细胞对江苏省某猪场腹泻病料进行病毒分离,通过细胞病变观察、RT-PCR扩增、动物回归试验对病毒进行鉴定,通过半数致死量测定病毒的致病性。结果显示,成功分离到1株猪流行性腹泻病毒(PEDV),命名为PEDV JS14株。该毒株在Vero细胞上盲传至4代出现细胞病变,主要表现为细胞面粗糙、颗粒增多,细胞多核呈空斑样,细胞脱落等病变特征;分离株口服感染5头7日龄仔猪全部出现典型的临床症状,其中4头死亡。F6代培养物以不同滴度口服接种15头7日龄的仔猪,其半数致死量为10~(5.0)TCID_(50)/mL。结果表明,本次分离的PEDV为强毒株,为进一步的生物学特性研究奠定了基础。     

貉源犬瘟热病毒的分离鉴定

采集大庆某貉养殖场病貉的病料,处理后接种于非洲绿猴肾细胞(Vero)进行病毒分离。对分离毒株进行了中和试验、血凝试验、理化性质的鉴定,并用反转录-聚合酶链式反应(RT-PCR)检测感染细胞中的病毒核酸。病料接种 Vero 细胞 72 h 后产生明显的细胞病变(CPE);病毒分离株可以被犬瘟热病毒阳性血清中和,中和效价为 1∶25;分离株对氯仿、乙醚敏感,对酸和热抵抗力弱,可以凝集鸡红细胞,其凝集作用可被犬瘟热病毒阳性血清所抑制; RT-PCR检测病毒细胞培养液,扩增出的片段长287 bp,与预期设计的长度相同,经测序发现与MS01株的同源性为99%。结果表明,分离的病毒株为犬瘟热病毒,命名为CDV-DQ株。     

猪流行性腹泻病毒SDbz株的分离与鉴定

取病死仔猪十二指肠、空肠及肠内容物制成匀浆,应用套式RT-PCR检测呈现猪流行性腹泻病毒(PEDV)阳性,经处理将其接种到含终浓度50μg/mL胰酶的Vero细胞上进行病毒分离传代,盲传到10代左右开始出现局灶性细胞病变(CPE),20代以后特征性CPE稳定出现。将病毒Vero细胞培养物应用毒价测定及病毒中和试验、套式RT-PCR和间接免疫荧光进行检测鉴定,证明所分离到的病毒为PEDV,并将其命名为PEDVSDbz株。     

猪流行性腹泻病毒SDbz株的分离与鉴定

病料应用套式RT-PCR检测呈现PEDV阳性,经处理将其接种到含终质量浓度50 mg/L胰酶的Vero细胞上进行病毒分离传代,盲传到10代左右开始出现局灶性CPE,20代以后特征性CPE稳定出现.将病毒Vero细胞培养物应用毒价测定及病毒中和试验、套式RT-PCR和间接免疫荧光进行检测鉴定,证明所分离到的病毒为PEDV,并将其命名为PEDV SDbz株.     

一例猪伪狂犬病野毒感染的实验室诊断

为确诊衡阳市某猪场育肥猪发生的疑似猪伪狂犬病(PR)疫情,采集发病猪脾脏,运用PCR方法进行猪伪狂犬病病毒(PRV)核酸检测,利用非洲绿猴肾细胞(Vero)进行病毒分离培养,采用免疫过氧化物酶单层细胞染色法(IPMA)进行病毒血清学鉴定;用分离培养物进行家兔感染试验,观察临床症状和病例剖检变化,采用PCR检测各器官中的病毒分布情况。结果显示:病料接种Vero细胞培养3 d后,出现明显的细胞病变效应(CPE);分离培养的病毒与PRV阳性血清呈阳性反应;家兔感染后出现奇痒等神经症状并死亡,其肝、脾组织均为PRV gE核酸检测阳性。试验结果证实,分离到的病毒为PRV野毒,从而确诊了该疑似疫情。     

狐源犬瘟热病毒的分离及RT-PCR鉴定

为开展犬瘟热疫苗研究的前期工作,采用Vero细胞分离病毒的方法,选择保守基因N基因做RT-PCR鉴定,结果表明,该方法可成功分离出病毒并扩增出861 bp片段,与预计的片段大小一致。     

鸭坦布苏病毒江苏XZ株的分离和鉴定

从江苏徐州地区分离到的以引起樱桃谷鸭产蛋下降和死亡为特征的1株病毒,命名为XZ株。对该病毒进行电镜观察、血凝试验、ELD₅₀测定、RT-PCR扩增特异性目的基因、序列比对分析和动物回归试验。结果显示,分离毒株能致死鸭胚和鸡胚,电镜下观察到球形病毒粒子,不具有血凝性,对病料和接毒鸭胚尿囊液进行RT-PCR,均可扩增出基因片段,其核苷酸序列与坦布苏病毒奉贤株的相似性最高,为98.7%,与其他坦布苏病毒的毒株也具较高同源性,为86%~98%。用鸭胚分离毒株接种健康产蛋鸭,能复制出同样的疾病。结果表明分离病毒为鸭黄病毒属的坦布苏病毒。     

Isolation and identification of trout viruses in South Africa

A total of 3,257 samples of diseased rainbow trout were examined for the presence of viruses from January 1983 to December 1987. A virus closely related to the VR 299 serotype of infectious pancreatic necrosis virus was isolated from 13 cases. An additional 7,228 viscera samples from asymptomatic fish were collected during the same period and a similar virus was isolated from 2 sites. During the same period 2,892 ovarian fluid samples were collected and a similar virus was isolated from 1 site. A similar virus was also isolated from one consignment of imported trout ova. A total of 5,550 ova was examined during this period. The viruses were identified by various tests as being closely related to the VR299 serotype of infectious pancreatic necrosis virus. All these samples tested negative for infectious haematopoietic necrosis virus, viral haemorrhagic septicaemia virus and herpesvirus salmonis.     

猪细小病毒的分离与鉴定

本试验从福建省某猪场疑似猪细小病毒病死胎的淋巴结、肝脏中分离到1 株病毒。病料接种PK-15细胞36 h后出现了圆缩、集聚、脱落等细胞病变,猪细小病毒阳性血清能特异性地中和该分离病毒。根据已发表的细小病毒(PPV) VP2基因的序列设计并合成了一对引物,采用PCR方法可扩增531 bp DNA片段。测序结果表明,分离株VP2基因与NCBI公布的NADL-2株的同源性高达99.2％,证实分离的病毒株为猪细小病毒。为进一步开展该病毒致病机理、流行病学、诊断研究与疫苗免疫等奠定了基础。     

Isolation of bovine coronavirus from feces and nasal swabs of calves with diarrhea.

Fecal and nasal samples were collected from 180 calves with diarrhea and 36 clinically normal co-habitants, and tested for virus using HRT-18 cell cultures derived from human rectal adenocarcinoma. A cytopathic virus was isolated from 5 fecal and 56 nasal samples obtained from diarrheic calves. All calves in which the virus was isolated from diarrheic feces were positive for virus isolation from nasal swabs. The virus was also isolated from the nasal swabs of 10 clinically normal calves that were co-habitants with diarrheic calves. Because they were morphologically similar to coronavirus, agglutinated mouse erythrocytes and serologically identical with the Nebraska calf diarrhea coronavirus, new isolates were identified as bovine coronavirus. The demonstration of viral antigens in nasal epithelial cells by a direct immunofluorescence was in close agreement with the virus isolation in HRT-18 cell cultures. This is the first report on the isolation of bovine coronavirus from newborn calves with diarrhea in Japan. The evidence that the virus was frequently isolated from nasal swabs is of great interest for understanding the pathogenesis of bovine coronavirus infection.     

An epidemic of parapoxvirus infection among cattle: isolation and antibody survey.

A disease characterized by papules, nodules, vesicles and, rarely, pustules and ulcers on teats was seen among cattle on a farm in Chiba Prefecture, Japan. A virus was isolated by inoculation of fetal bovine lung cell cultures from a vesicle on a teat of an infected cow. The virus was subsequently passaged in fetal bovine lung and muscle cells in which it produced complete cytopathic changes. The virus was identified by physicochemical examinations and electromicroscopic observation as a parapoxvirus. A seroepidemiological survey was performed on antibody to the isolated virus by the agar gel immunodiffusion test. The isolated virus formed a precipitation line which cross reacted with other parapoxviruses isolated previously in Japan. The positive rate was more than 50% among cattle in the Kanto district. The positive rate increased with age. It was suggested that parapoxvirus infection might have already been prevalent among cattle in Japan.     

口蹄疫病毒灭活疫苗中VP1基因RNA抽提方法的建立

本试验提取疫苗种毒、BEI灭活病毒和白油乳化灭活疫苗病毒中的RNA,并就VP1基因进行了RT-PCR扩增、核酸序列测定和序列比对分析。结果显示,3种来源的VP1基因片段长度均为813 bp,与预期的目标相符,核苷酸序列显示3种方法处理的病毒VP1同源性为100%。说明灭活剂BEI和乳化工艺没有全部破坏口蹄疫病毒VP1基因核酸,为检测疫苗毒株的核酸序列建立了一种方法。     

番鸭呼肠病毒的分离与RT-PCR鉴定

从发病番鸭中分离到1株病毒,用该病毒接种番鸭胚,至第2代可引起鸭胚死亡,胚体出血,部分鸭胚肝脾有少量白点。分离毒经用RT-PCR方法进行检测,可与番鸭呼肠病毒标准株扩增出大小一致的特异条带,证实该分离毒为番鸭呼肠病毒。     

白鹭源新城疫病毒的分离与鉴定

从1只患病的白鹭的咽喉、泄殖腔棉拭子中分离到1株病毒,经血凝、血凝抑制试验和RT-PCR法鉴定为新城疫病毒。根据该毒株对鸡胚平均致死时间、鸡胚半数致死量、鸡胚半数感染量的测定和新城疫强弱毒鉴别的RT-PCR检测,表明该分离株为新城疫强毒株。