山羊地方性鼻内肿瘤病毒的gag基因克隆及分析

安徽省某山羊场内部分羊只发生羊鼻漏,面部一侧肿胀,进行病理剖检观察后,取面部皮下肿瘤组织研磨,提取RNA,用特异性引物通过RT-PCR方法扩增获得目的片段并测序。序列比对分析表明,获得的目的基因和山羊地方性鼻内肿瘤病毒的同源性最高,达99%。结果显示,此羊场的山羊感染了由山羊地方性鼻内肿瘤病毒引起的山羊地方性鼻内肿瘤。     

山羊地方性鼻内肿瘤病毒SC株gag基因分子克隆与原核表达

克隆山羊地方性鼻内肿瘤病毒(ENTV)的群抗原基因gag,并通过原核表达系统对gag进行表达研究。参照GenBank中收录的山羊地方性鼻内肿瘤病毒(ENTV-2)gag基因序列设计1对特异引物,以地方性鼻内腺癌(ENA)山羊鼻内分泌物中病毒RNA为模板,RT-PCR扩增获得目的片段,并将产物连接到pMD18-T载体获得阳性克隆(pMD18-gag)并测序。根据gag基因的ORF设计1对表达用引物进行亚克隆,并将ORF重组到pET-32,重组质粒转化至宿主菌Rostta中,用IPTG诱导表达,对表达的蛋白用SDS-PAGE分析,纯化表达产物,通过ELISA初步分析产物的反应原性。结果显示:克隆获得长2 249 bp的核酸序列(GenBank:HM104174),含有完整的ORF,与已报道的ENTV-2 gag基因的同源性高达87%,氨基酸水平上的同源性高达96.4%。表达产物大小约87 ku,与理论推理一致。采用ELISA方法检测表达产物未能与ENA阳性血清发生特异性反应。结果表明:gag基因在原核系统pET-32/Rostta中得到表达,表达产物与ENA阳性血清不具反应原性。     

山羊地方性鼻内肿瘤病毒和支原体混合感染山羊的诊断

<正>2016年10月福州市一山羊养殖户的山羊发病,经临床症状、剖检病变及实验室检验,确诊为羊地方性鼻内肿瘤病毒(Enzootic nasal tumor virus,ENTV)、绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)和丝状支原体山羊亚种(Mycoplasma mycoides subsp.capri,Mmc)混合感染引起的山羊呼吸     

山羊地方性鼻内肿瘤病毒Shaanxi株前病毒全基因组克隆及生物信息学分析

为研究山羊地方性鼻内肿瘤病毒(ENTV)Shaanxi株前病毒全基因组序列,本研究根据Gen Bank中收录的ENTV前病毒基因组序列设计5对引物,通过PCR方法从肿瘤组织中分段扩增获得了ENTV前病毒全基因组的5个片段,测序并分析。结果显示该病毒基因组全长7 275 bp,包含相互重叠的gag-pro-pol-env编码区及5'端非编码区;ENTV Shaanxi株基因组全序列与NC004994.2、AY197548.2、ENTV-SC株(HM104174.1)核苷酸同源性分别为89%、89%、99%。本研究为ENTV的分子生物学特性研究提供资料,为进一步研究其检测方法与致病机理奠定了基础。     

福清山羊地方性鼻内肿瘤病毒 绵羊肺炎支原体和溶血性曼氏杆菌混合感染的诊断病例

正2017年10月福清市某山羊养殖场发生了以流鼻涕、呼吸困难为主要特征的传染病,其中1～6月龄小羊病死率高达50%以上。通过临床症状、剖检病变和实验室检验,确诊为山羊地方性鼻内肿瘤病毒(Enzootic nasal tumor virus,ENTV)、绵羊肺炎支原体(Mycoplasma ovipneumoniae,Mo)和溶血性曼氏杆菌(Mannheimia haemolytica,Mh)混合感染引起的山羊呼吸道疾病,诊断过程报告如下。     

山羊地方性鼻内肿瘤病毒全基因组序列分析及其gag基因促肿瘤发生的机制研究

本研究旨在获得山羊地方性鼻内肿瘤病毒（enzootic nasal tumor virus 2,ENTV-2）全基因组序列并进行分析。从福建某羊场的山羊鼻内肿瘤组织取样,针对ENTV-2设计引物,通过RT-PCR方法从肿瘤组织中分段扩增出6个特异性产物,测序后利用生物学软件DNAStar进行拼接,获得长度为7 443 bp全基因组序列,将其命名为ENTV-2-FJ。ENTV-2-FJ基因组结构与其他逆转录病毒类似,具有5'-U5-gag-pro-pol-env-U3-3'典型结构,包含4个开放阅读框和侧翼非编码区以及末端重复序列。为制备兔抗ENTV-2 gag蛋白多克隆抗体,将特异性扩增的gag基因克隆至pET-21a （+）载体,构建重组质粒pET-21a-gag,鉴定后转化至BL21（DE3）细胞并经IPTG诱导成功表达分子量约为70 ku的融合蛋白。Western blot分析表明,制备的兔抗gag蛋白抗体能在患病山羊肿瘤组织和表达gag基因的细胞中特异性地检测出gag蛋白。为了深入研究ENTV-2-FJ致瘤机制,作者构建了过表达gag基因的K562细胞系,并进行裸鼠致瘤试验。结果显示, gag蛋白通过激活JAK2-STAT5信号通路促进肿瘤的生长。综上,本研究获得了ENTV-2-FJ全基因组序列并对其进行分析,制备并验证了gag蛋白的多克隆抗体,揭示了gag蛋白的作用机制,这些结果为阐明ENTV-2的致病机制提供了科学依据。     

山羊地方性鼻内肿瘤病毒gag蛋白的原核表达、纯化及多克隆抗体的制备

为获得纯化的山羊地方性鼻内肿瘤病毒(enzootic nasal tumor virus of goats,ENTV)gag蛋白和抗gag蛋白的多克隆抗体,根据GenBank已登录的ENTVgag基因序列,设计合成1对特异性引物,应用PCP扩增gag基因并连接于原核表达载体pET-28a(+)中,构建重组质粒pET-gag,经鉴定正确后转化大肠杆菌Rosetta(DE3)株进行诱导表达,并进行SDS-PAGE分析。重组菌经IPTG诱导后成功表达相对分子质量约为69 000的重组蛋白,重组蛋白经镍柱亲和层析纯化、尿素梯度透析复性后免疫小鼠制备多克隆抗体。Western blot试验表明,重组蛋白能与制得的多克隆抗体反应,而与正常小鼠血清和山羊地方性鼻内肿瘤患羊血清不反应。本试验成功获得了纯化的ENTV gag蛋白和小鼠抗gag蛋白的多克隆抗体,为进一步研究gag蛋白在ENTV致病过程中的作用提供了材料。     

四川一起山羊地方性鼻内肿瘤疫情的诊断

四川某地区山羊养殖基地发生一起特殊的肿瘤性疾病,通过流行病学及临床症状调查、尸体剖检、病理损伤的显微及超微结构观察和病原的核酸测序等一系列研究,将该病确诊为山羊地方性鼻内肿瘤(ENT)。本次疫病与国外报道的病例基本一致。这是国内首次报道ENT的病原,为该病的进一步研究奠定了基础。     

羊地方性鼻内腺瘤的研究进展

羊地方性鼻腺瘤(ENA)是绵羊和山羊的病毒性传染疾病,其特征在于鼻腔中的筛骨黏膜的肿瘤生长,已对羊养殖业造成严重的经济损失。为了对该病有更深入、全面的了解,本文主要从病原学、流行病学、临床症状和病理变化、诊断技术以及防治等方面对羊地方性鼻内腺瘤进行了综述。     

山羊鼻内腺瘤和腺癌的病理学研究

发生于内蒙古自治区某地的山羊鼻内肿瘤呈地方性流行，临床上病羊初期流出大量浆液性鼻液，随后逐渐出现呼吸困难和鼻塞音，肿瘤可侵犯周围组织引起眼球突出和头颅变形，最后死于窒息。１４例山羊鼻内肿瘤的病理学研究表明，肿瘤起源于筛骨迷路粘膜的腺体（包括浆液腺和粘液腺），组织学上为腺瘤，以后可转变为腺癌，并向周围组织作浸润性生长，未见转移。２例作透射电镜检查见瘤组织有异型性，瘤组织和瘤组织内未见病毒样颗粒。发生     

Development of a SYBR Green-based real-time quantitative polymerase chain reaction assay to detect enzootic nasal tumor virus in goats

Enzootic nasal adenocarcinoma is a contagious respiratory disease in goats that is caused by the enzootic nasal tumor virus 2 (ENTV-2). In order to increase the number of available detection methods for ENTV-2, we developed a SYBR Green real-time polymerase chain reaction (SGrPCR) assay that targets the gag gene of ENTV-2. The low limit of detection of the assay was 3.68 × 10¹ copies/μL, a hundredfold more sensitive than conventional PCR. The melt curve showed a single sharp melt peak at 83°C, which indicated that there was no non-specific amplification or primer dimer formation. The intra-assay and inter-assay coefficients of variation were 1.58% and 1.82%, respectively. There was no cross-reactivity with closely related goat viruses (i.e., orf virus, peste des petits ruminants virus, goatpox virus, foot-and-mouth disease virus) and endogenous retroviruses. In conclusion, the SGrPCR assay is specific for the gag gene of ENTV-2 and provides a rapid and sensitive approach for detecting ENTV-2 in clinical samples.     

Detection of enzootic nasal tumor virus (ENTV) in a sheep flock in southern Brazil

Enzootic nasal tumor (ENT) is a contagious neoplasm associated with enzootic nasal tumor virus (ENTV), which may induce disease in sheep (ENTV-1) and goats (ENTV-2). This study aimed to describe the occurrence of ENT in two Texel sheep (Ovis aries) from a 75-sheep flock, located in the city of Gravataí, southern Brazil. Animals used to be purchased from different origins, and no specific tests for disease monitoring or quarantine procedure were performed. Affected animals presented respiratory distress, anorexia with severe weight loss, and mucopurulent unilateral nasal discharge. Necropsy was performed in both animals and nasal cavity masses were observed. Histopathological analysis demonstrated an epithelial neoplasm compatible with nasal adenocarcinoma. PCR using a protocol that amplifies a 591 bp sequence of 5’LTR-gag region of ENTV1 was performed followed by DNA sequencing. Both samples were positive, and the sequences obtained presented highest identity (97%) with ENTV strain TN28 (GenBank accession number MH899613) detected in a Texel sheep from Scotland. This is the first report of ENTV-1 leading to enzootic nasal tumor in sheep in Latin America, which confirms the presence of the retrovirus in sheep flocks in the Brazilian territory.

     

山羊鼻内肿瘤病毒荧光定量PCR检测方法的建立及应用

为了解山羊鼻内肿瘤病毒(ENTV-2)的流行情况,本研究针对ENTV-2的env基因设计了1对特异性引物与探针,通过RT-PCR条件优化,成功建立了ENTV-2荧光RT-qPCR检测方法。该方法特异性高、灵敏性强(5.61 copies/μL)、重复性好(CV为0.09%~1.20%)。本研究建立的一步法荧光RT-qPCR方法为ENTV-2的临床检测以及流行病学调查提供技术支撑。     

Bronchioloalveolar carcinoma not related to jaagsiekte sheep retrovirus in a goat

A spontaneous lung tumor in a 5-year-old goat of the Murciano-Granadina breed is described in this paper. Clinical signs of cachexia and tachypnoea were evident, and a considerable amount of white mucous foamy fluid was discharged from the nostrils when the animal's head was lowered. A lung tumor with the characteristics of bronchioloalveolar carcinoma was detected during histopathologic examination. The tumor cells were positive for surfactant proteins C and B, confirming that alveolar type II cells were the origin of the neoplasia. Tumor samples were tested by polymerase chain reaction, immunoblotting, and immunohistochemistry for the presence of Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV), another retrovirus very closely related to JSRV, but all tests were negative. Therefore, this is the first reported case of spontaneous bronchioloalveolar carcinoma not related to JSRV or ENTV infection in a goat.     

奶山羊羔羊山羊痘的病理学、血清学及分子生物学诊断

目的 确定内蒙古某规模化奶山羊养殖场部分引进羔羊发病死亡的原因,为该场加强重点疫病免疫防控提供科学依据。方法 对出现典型临床症状的发病羊只进行病理解剖学观察,无菌采集肺脏病变样本,制备病理组织切片;无菌采集发病羊只的血液制备血清,采用高敏荧光技术检测血清中的山羊痘病毒抗体;无菌采集发病羊只的肺组织、鼻拭子、眼拭子,利用PCR技术检测山羊痘病毒P32基因,对测序获得的序列进行BLAST比对并进行同源性分析。结果 剖检可见病羊口腔、鼻腔、喉头、气管黏膜有圆形疹痘;肺部明显水肿,表面存在红色疹痘,切开后呈不透明、灰白色胶冻样;瘤胃、网胃、瓣胃、皱胃黏膜均出现疹痘。病理组织学检查可见肺泡红色深染,间质变宽,腔内存在脱落的上皮细胞,Ⅱ型肺泡上皮细胞增生,并化生为腺泡样状;支气管腔内存在大量的炎性细胞。发病山羊血清经高敏荧光技术检测,判定为山羊痘病毒抗体阳性。采集肺组织（GTPV-YP1）、鼻拭子（GTPV-YP2）、眼拭子（GTPV-YP3）样品各1份,3份样品经P32基因PCR检测均获得983 bp的扩增片段,与预期大小一致;GTPV-YP1、GTPV-YP2样品P32基因序列与山羊痘病毒中国分离株KC951854.1、Oman分离株 MN072621.1 P32基因的同源性为100%,GTPV-YP3与山羊痘病毒中国分离株EF514892.1、HM572329.1、JN596275.1、MG817382.1的同源性为99.0%。结论 经病理解剖学观察、病理组织学检查、血清学检测以及病原PCR鉴定,确定引起该场引进奶山羊羔羊发病的原因为山羊痘病毒感染。     

An immunofluorescence diagnostic test for feline viral rhinotracheitis.

Hyperimmune serum against feline viral rhinotracheitis was produced in a goat and conjugated with a fluorescent dye. Cell cultures infected with rhinotracheitis virus had positive immunofluorescence. Cell cultures infected with other feline viruses and herpesviruses of other species did not fluoresce. In cats experimentally infected with rhinotracheitis virus, the virus was isolated from nasal and conjunctival swabs 1 to 9 days after inoculation. Nasal smears stained with the conjugated antiserum fluoresced 1 to 9 days after inoculation when clinical disease was most apparent. Conjunctival smears had positive immunofluorescence 1 to 6 days, but not 9 days, after inoculation. On postinoculation day 23, rhinotracheitis virus was not isolated from nasal or conjunctival swabs and nasal and conjunctival smears did not fluoresce. Rhinotracheitis virus or feline calicivirus was isolated from naturally infected cats with upper respiratory tract disease. Nasal and conjunctival smears from rhinotracheitis virus-infected cats had positive immunofluorescence in all cast showing clinical illness. Smears from 1 clinically normal cat from which rhinotracheitis virus was isolated did not fluoresce. Nasal and conjunctival smears from calicivirus-infected cats did not fluoresce.     

Experimental peste des petits ruminants (goat plague) in goats and sheep.

In order to study the pathomorphology and immunohistochemistry of peste des petits ruminants, four goats and two sheep were inoculated intranasally with the Malig-Yemen strain of peste des petits ruminants virus. The animals developed fever, nasal discharge, oral erosions, cough and diarrhea. One goat and one sheep died and one moribund goat was killed. Three animals survived the infection. At necropsy, erosive stomatitis, pneumonia and gastroenteritis were found. Histopathologically the pneumonocytes and epithelial cells of the ileum had eosinophilic cytoplasmic and nuclear inclusions. By an indirect immunoperoxidase method, the nuclei and cytoplasm of the ileal epithelial cells of one goat contained positively (brown) stained antigen, which corresponded to viral nucleocapsids by electron microscopy. Virus appeared to be released through the microvilli of the epithelial cells. We also confirmed the formation of giant cells due to peste des petits ruminants virus.