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。     

水貂阿留申病PCR检测方法的建立及应用

为了对水貂阿留申病毒(ADV)进行快速、准确地鉴定,试验根据基因库中ADV的NS1基因核苷酸序列,设计合成了一对特异性引物,经过反应条件的优化,建立了水貂阿留申病的PCR检测方法,对阳性病料进行特异性核酸扩增及产物测序,并对水貂肠炎细小病毒(MEV)、犬瘟热病毒(CDV)等病原核酸进行扩增检测该方法的特异性;用10倍系列稀释的ADV进行扩增检测敏感性;用该方法检测临床样品并与对流免疫电泳法进行了比较。结果表明:ADV阳性样品有明显目的条带(365 bp)出现,MEV、CDV均无特异条带出现,最低能检测到的病毒核酸量为2.53 ng/μL;临床样品检测表明PCR检出率为90%,对流免疫电泳法检出率为83%。     

一株水貂源伪狂犬病毒株的分离鉴定

从辽宁大连疑似水貂伪狂犬病发病死亡水貂脑、内脏中及饲喂的猪肝中分离到1株病毒并进行了鉴定。该分离毒株接种BHK-21细胞24 h后出现圆缩、聚集、脱落等典型的细胞病变(CPE);分离毒株能够被伪狂犬病病毒标准阳性血清中和;分离病毒接种家兔后,引起家兔出现奇痒等典型的伪狂犬病临床症状;同时根据Gen Bank公布的PRV的Tg ET基因设计引物并扩增出特异性的目的片段,扩增产物经过测序比较,表明扩增产物序列为猪伪狂犬病毒g E基因序列。以上结果表明,该病毒为伪狂犬病毒,依据来源确定为水貂源性伪狂犬病病毒株。     

一例水貂阿留申病毒和伪狂犬病毒混合感染的诊断

为确定河北衡水某水貂养殖场水貂大批量死亡的原因,笔者对死亡水貂进行解剖,采集其肾脏、脾脏等组织器官,进行阿留申病毒(ADV)、伪狂犬病毒(PRV)、犬瘟热病毒(CDV)和传染性肝炎病毒(CAV-Ⅰ/Ⅱ)检测。结果:阿留申病毒和伪狂犬病毒检测结果阳性,犬瘟热病毒和传染性肝炎病毒检测结果阴性,说明该养殖场的水貂感染了阿留申病毒和伪狂犬病病毒,应及时对畜舍进行消毒,隔离和淘汰阳性水貂。     

PRRSV云南株的分离与鉴定

从云南某猪场采取病料经RT-PCR检测为猪繁殖与呼吸综合征病毒(PRRS)阳性,将其处理后接种到猪肺泡巨噬细胞(PAM)和Marc-145细胞上。接种后3 d,猪肺巨噬细胞开始出现细胞病变(CPE);Marc-145细胞盲传3代后,细胞也出现了CPE。对出现CPE的细胞培养物,用RT-PCR法、间接免疫荧光试验进行检测,结果均为PRRSV阳性;细胞培养物经PEG初步浓缩后,接种到PRRSV阴性,PRV、CSFV抗体阳性的仔猪,15 d后猪只出现体温升高并伴有食欲减退,精神不振等症状。采血并用同上的RT-PCR法检测,结果为阳性;采血用同上的RT-PCR法检测,结果亦为阳性;用电镜对纯化后的病毒进行观察,可见有囊膜包裹的圆形病毒粒子,其直径约为50 nm;间接免疫荧光实验可见黄色荧光;TCID50测定其毒价为10-4.75/0.1 mL。该病毒对氯仿、紫外线、酸碱度变化(pH5.0或pH7.5)和热敏感;结果表明,已成功分离获得一株PRRSV云南地方流行毒株,命名为YN-1。     

三种饲料添加剂对阿留申病水貂血液常规指标的影响

为给抗水貂阿留申病(Aleutian disease of mink,ADM)的药物应用提供理论依据和进一步丰富水貂血液常规指标,试验研究了三种饲料添加剂对阿留申病水貂血液常规指标的影响,试验对常规饲养条件下的8～9月龄标准水貂母貂进行阿留申病毒(Aleutian disease virus,ADV)抗体检测,根据检测结果选取ADV阳性水貂(ADM水貂)238只、ADV阴性水貂(非ADM水貂)62只,分为抗申宝组、酵母多糖组、芪参散组和对照组,对照组只饲喂基础日粮。每组各随机选取3只(共24只)水貂分别在喂药前和喂药30 d后断趾采血,采用迈瑞BC-6600血细胞分析仪测定24项血液常规指标并比较。结果表明:饲喂三种饲料添加剂均可使ADM水貂的白细胞数(WBC)、血红蛋白含量(HGB)、血细胞比容(HCT)、血小板数(PLT)显著升高(P0.05)并达到正常水平,同时使红细胞数(RBC)和中性粒细胞数(NE)显著升高(P0.05);均可使非ADM水貂NE显著提高(P0.05);饲喂抗申宝还可使非ADM水貂的WBC、HGB、HCT、大血小板比率(P-LCR)、嗜酸细胞百分含量(EO%)和嗜碱细胞数(BA)显著升高(P0.05),饲喂芪参散后WBC、HGB、HCT、P-LCR、EO%显著升高(P0.05);未饲喂中草药的ADM水貂和非ADM水貂均出现淋巴细胞百分含量(LY%)显著下降(P0.05)。说明三种饲料添加剂均能增强水貂的生理机能,改善造血机能,均可调节ADM水貂的血常规指标,使其趋于正常;抗申宝对非ADM水貂血常规指标的改善相对最佳。     

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。     

水貂阿留申病免疫原性研究

本实验研究依据阿留申病毒(ADV)的特点,研制了四种不同类型的免疫生物制剂,并分别对临床健康的阿留申病对流免疫电泳(AD.CIEP)阴性水貂接种,攻毒后进行循环免疫复合物(CIC)和抗体(Ab)滴度检测,安死后检查病理组织学变化。本实验结果表明:四种不同类型的生物制剂,其免疫应答反应有显著不同;而同种类型免疫生物制剂,由于剂型、接种剂量不同,其免疫效果亦有明显差异。实验得出,白油佐剂ADV—G亚单位疫苗和白油佐剂(846)抗原的免疫效果好,是用于免疫预防水貂AD有希望的免疫生物制剂。     

水貂细小病毒性肠炎灭活疫苗保存期试验

对2~8℃条件下保存90,120,150,180,210,240,300,360 d的水貂细小病毒性肠炎灭活疫苗接种水貂进行免疫试验,结果表明:保存300 d的疫苗接种水貂30 d后血清中水貂肠炎细小病毒(MEV)HI抗体均在1∶32以上,免疫水貂强毒攻击均获得100%保护,确定水貂细小病毒细胞灭活疫苗保存期为300 d。该结果为水貂细小病毒细胞灭活疫苗运输和保存提供了理论依据。     

水貂犬瘟热病毒RC01株和细小病毒RC02株的分离鉴定及攻毒保护试验

取某水貂养殖厂疑似犬瘟热病毒和细小病毒混合感染病料,采用犬瘟热抗原快速检测试纸、HA和PCR、RTPCR法检测CDV和MEV。分别以抗CDV和抗MEV阳性血清中和后接种敏感细胞进行蚀斑纯化,成功分离一株细小病毒(MEV-RC02株)和犬瘟热病毒(CDV-RC01株)。将纯化后的MEV-RC01株和CDV-RC01株分别回归本动物,结果显示,分离株均具有较强的致病性。将水貂犬瘟热(CL08株)、病毒性肠炎(NA04株)二联活疫苗免疫健康非免貂后进行攻毒保护试验,结果表明,该二联疫苗对分离的MEV-RC02株和CDV-RC01株流行毒株具有很好的保护作用。     

Isolation and Identification of Local Aleutian Mink Virus Virulent Strain ADV-LN

Mink suspected infection aleutian mink disease virus (ADV) from mink breeding areas in Liaoning province were tested with CIEP method.The mink with antibody to ADV were selected and culled.Liver,spleen,kidney and mesenteric lymph node samples were taken for pathological examination and the viruses were observed under electron microscope.The grinded tissue fluid filter was added penicillin and treptomycin and inoculated into CRFK cells and passaged by 6 times for virus isolation.And cells cultures were identified as ADV by PCR.Then they were inoculated into healthy mink.Three days later,the mink showed clinical signs,which including the loss of appetite,anemia,hair dull,antifeedant and binge drinking.Some minks showed neurological symptoms,manifested symptoms of convulsions,cramps,staggering gait,ataxia,or hind limb paralysis and died.The virus strains isolated and identified were named as the ADV-LN.     

Characteristics of inapparent Aleutian disease virus infection in mink.

Inapparent of nonprogressive Aleutian disease virus (ADV) infection is a subclinical but persistent virus infection of mink. Mink with the inapparent type of ADV infection when subjected to stress did not develop the progessive form of the disease. However, when challenged with a large dose of the virus, these mink did develop progressive Aleutian disease indicating that they were not highly resistant to the virus. Sera of mink with either the progressive of the inapparent type of ADV infection did not neutralise the virus. The anti-ADV antibody activity in mink with inapparent type of ADV infection was in the IgG fraction of the serum the same as in mink with progressive Aleutian disease. These data indicate that the resistance of the mink with inapparent infection as compared to mink with progressive Aleutian disease was not due to a difference in the class of immunoglobulin response to the virus. However, mink with progressive Aleutian disease showed a greatly increased immunoglobulin response.     

水貂阿留申病毒结构蛋白与非结构蛋白的研究进展

水貂阿留申病毒(Aleutian mink disease virus,ADV)是一种主要侵染水貂的自主复制型细小病毒,是一种在水貂中广泛存在的重要病原体。病毒粒子的蛋白分为结构蛋白(VP1、VP2)和非结构蛋白(NS1、NS2)两类。VP1蛋白对病毒粒子产生感染性有重要作用;VP2蛋白是主要免疫功能区,能刺激机体产生中和抗体;NS1和NS2主要参与病毒的复制和基因的表达调节。文中对近年来国内外学者关于水貂阿留申病毒结构蛋白和非结构蛋白的研究情况进行归纳和总结。     

Detection of inapparent Aleutian disease virus infection in mink.

The normal serum gamma-globulin centration of mink from the Ontario Veterinary College field station was 13.2 +/- 2.6% of total serum proteins. Mink serum gamma-globulin concentrations above 21%, which represented 3 standard deviations above the normal mean, were considered to be hypergammaglobulinemic. About 39% of pastel mink infected naturally with Aleutin disease virus (ADV) exhibited an inapparent or nonprogressive infection. These nonprogressivley infected mink had serum gamma-globulin values below 21% andhad antibody titers less than 256 if tested by the couterimmunoelectrophoresis technique. Mink maintained inapparent infection for at least 10 months after infection with ADV. Neither gross nor histopathologic changes were present in the mink with inapparent ADV infection. The virus persisted in blood, mesenteric lymph nodes, kidney, liver, and spleen of mink with non-progressive infection, although the amount of virus present probably was small.     

Violet mink develop an acute disease after experimental infection with Aleutian disease virus (ADV) isolate ADV SL3

Six-Aleutian (aa)-genotype violet mink were infected intraperitoneally with the Aleutian Disease Virus (ADV) bone marrow derived isolate ADV SL3. All animals developed virus-specific antibodies and hypergammaglobulinaemia. Mortality during the fourteen week duration of the infection was 50%. The virus induced (histo)pathological lesions typical for Aleutian Disease. By immunohistochemical examination using a virus capsid-specific monoclonal antibody viral antigen was detected in lymph nodes, spleen, kidneys and once in hepatic Kupffer cells. By Southern blot and in situ hybridization studies with strand-specific RNA probes able to distinguish viral replicative forms from merely sequestered genomic DNA, ADV replication was detected in mesenteric lymph nodes and spleen. In one mink DNA replicative forms were also found in bone marrow cells or mononuclear cells of the peripheral blood, respectively. Only single-stranded viral DNA was detected in liver, kidney, gut and lung of infected animals. From Southern blot hybridization results a different, possibly organ-specific permissiveness of ADV in vivo is suggested.     

Immune complexes in Aleutian disease: demonstration of antibody on isolated virus

The specific binding of Staphylococcal protein A for mammalian immunoglobulin G was used to demonstrate IgG associated with Aleutian disease virus (ADV) when isolated from infected mink tissues. Protein A specifically bound to mink serum Ig with no reaction with other serum or tissue proteins. Protein A labeled with 131Iodine reacted with crude virus preparations but not with virus that had been purified by freon extraction to the point where it became reactive with antibody by counterimmunoelectrophoresis. Binding to purified ADV was restored when the purified virus was first reacted with antibody. Results of urea treatment indicated this as an alternative method for isolation of ADV free from antibody.     

Mink infected with aleutian disease virus have an elevated level of CD8-positive T-lymphocytes

Lymphocytes, monocytes, granulocytes, B-lymphocytes and CD8-positive T-lymphocytes of non-infected mink and mink infected with Aleutian disease virus (ADV) were measured by flow cytometry. The gammaglobulin levels of the sera were also measured. Besides development of hypergammaglobulinaemia in the infected mink, the most pronounced finding was that the number of CD8-positive lymphocytes doubled on average during development of Aleutian disease, while the number of B-lymphocytes did not change dramatically. The enhanced CD8 frequency was still apparent 6 months after initial ADV infection of the mink. The present experiments contribute to a better understanding of the immune deficiency stage seen in mink infected with ADV.     

水貂阿留申病病毒分子生物学研究进展

水貂阿留申病病毒是一种在水貂中广泛存在的重要病原体.该病毒属阿留申病毒属,主要编码4种蛋白(结构蛋白VP1、VP2和非结构蛋白NS1、NS2).VP1蛋白在协助病毒产生感染性方面起着重要作用;VP2蛋白是该病毒的主要免疫原性抗原,能体外中和病毒;NS1和NS2对病毒在宿主细胞中的复制起重要的调节作用.该病毒的分子生物学诊断技术主要有核酸杂交技术、PCR和基因芯片检测技术.     

Effect of a valine residue at codon 352 of the VP2 capsid protein on in vivo replication and pathogenesis of Aleutian disease parvovirus in mink

OBJECTIVE: To determine whether a group of 3 genetic differences in the nonstructural protein (NS1) or 1 genetic difference in the structural protein (VP2) of Aleutian disease parvovirus (ADV) is responsible for an increase in the in vivo replication and pathogenicity of G/U-8, a chimera of ADV-G (nonpathogenic) and ADV-Utah (pathogenic), compared with G/U-10. ANIMALS: 32 eight-month-old female sapphire mink (Mustela vison). PROCEDURE: Chimeric viruses were constructed, propagated in vitro, and used to inoculate mink. Antiviral antibody responses, presence of serum viral nucleic acid, and serum gamma globulin concentrations were monitored for 120 days following inoculation. Histologic examination of the liver, kidneys, spleen, and mesenteric lymph nodes was performed after necropsy. RESULTS: A chimera containing only the 3 amino acid substitutions in NS1 did not elicit measurable responses indicative of replication or pathogenicity in inoculated mink. Serum antiviral antibody responses, frequency of detection of viral nucleic acid in serum, gamma globulin response, and histologic changes in mink inoculated with chimeras containing a valine residue at codon 352 (352V) of VP2 capsid were increased, compared with values from mink inoculated with chimeric viruses that did not contain 352V. CONCLUSIONS AND CLINICAL RELEVANCE: A valine residue at codon 352 in the VP2 capsid protein of ADV affects in vivo viral replication and pathogenicity. This amino acid may be part of an incompletely defined pathogenic determinant of ADV. Further characterization of the pathogenic determinant may allow future development of focused preventive and therapeutic interventions for Aleutian disease of mink.