Detection of hepatitis E virus in archived German wild boar serum samples

Hepatitis E is a rare human disease in Central Europe commonly imported from endemic regions. For autochthonous infections a zoonotic transmission from pigs, deer and wild boar is assumed. Using three different RT-PCR protocols, hepatitis E virus (HEV) RNA was detected in 10 out of 189 (5.3%) serum samples collected in 1995/1996 from wild boars in Germany. Sequence analysis indicates a close relationship with genotype 3 isolates of pigs and humans from the Netherlands and Japan. The results indicate that HEV is present in Germany since more than 10 years and that wild boar may function as a reservoir for HEV.     

猪圆环病毒2型检测技术研究进展

猪圆环病毒2型感染常引起不同生长阶段猪的免疫系统损伤,造成猪机体免疫功能不全和对疾病高度易感,给养猪业带来巨大损失,所以建立一种快速的检测技术对该病的防制至关重要,目前国内外许多学者对检测方法做了大量研究,文章对PCV2的检测方法作一介绍。     

Porcine circovirus 2-associated disease in Eurasian wild boar.

Porcine circovirus 2 (PCV2) was first identified in high-health herds of domestic swine and was associated with a debilitating disease called postweaning multisystemic wasting syndrome (PMWS). Most subsequent studies have indicated that PCV2 infects only swine but there is little information on porcids other than improved breeds of domestic swine. Multisystemic disease was reported in a group of Eurasian wild boars raised under free-range conditions. Affected young pigs had pneumonia and enteritis and were cachectic. Porcine circovirus 2 was identified in affected tissue by immunohistochemistry and in situ hybridization, and a PCV2-like virus was isolated from pooled organs. The open reading frame (ORF2) of the isolated PCV2 had a 98.7% homology with the ORF2 of a reference PCV2 isolate. These diagnostic data indicate that PCV2 can infect and cause disease in Sus scrofa subspecies other than domestic swine.     

安顺市猪Ⅱ型圆环病毒感的血清学调查

猪圆环病毒病是我地近年来新出现的一种危害较大的猪病,主要经呼吸道、消化道、精液及胎盘传染,引起断奶乳猪多系统衰竭综合症、猪皮炎与肾炎综合症、繁殖障碍、仔猪先天性震颤、增生性坏死性肺炎、肠炎等病症.     

我国猪圆环病毒病2型疫苗及相关联苗的研究进展

猪圆环病毒(PCV)能刺激机体产生中和性抗体,但该病毒体外增殖能力差,培养物病毒含量低,病毒接种细胞不产生病变等特性使传统的灭活疫苗、弱毒疫苗的生产受到了局限。通过对我国相关专利的分析,发现我国许多研究机构及学者在驯化和筛选适应PCV2生长的细胞系、构建重组病毒株、建立高密度培养技术等方面做了大量工作以期规避这些弊端,并开展了PCV2相关的二联苗、三联苗的研发工作,以有效预防PCV2及相关性疾病的发生。     

Genotyping of Mycoplasma hyopneumoniae in wild boar lung samples

Mycoplasma hyopneumoniae is the etiologic agent of enzootic pneumonia (EP), an important cause of disease-associated losses in swine production and a role of wild boar in recurrent infections can be supposed. Genotypes of M. hyopneumoniae from wild boar are unknown but could indicate its role as a potential reservoir. Therefore, 34 lung samples being PCR-positive for M. hyopneumoniae from wild boar from the Geneva region in Switzerland were assayed by genotyping using the p146 and multi-locus sequence typing (MLST) approaches and compared to data from outbreak cases from domestic swine in Switzerland. Successful genotyping was dependent on a sufficiently high concentration of M. hyopneumoniae DNA in the samples as assessed by different real-time PCR assays. The p146 genotyping was more successful with 24 samples (70.5%) being typeable whereas only 6 samples (17.6%) could be genotyped using the MLST approach. Variability of genotypes was high but identical types were found in geographically related animals. Genotypes from wild boar showed phylogenetic relatedness to those from domestic pigs but no matching types could be identified. Results show that direct genotyping from wild boar lung samples is possible and provides a promising approach to investigate future EP outbreak related samples from wild boar.     

猪圆环病毒2型感染的血清学分析

应用间接免疫荧光技术 ,测定了浙江省内 4 4个猪场 2 0 0 0～ 2 0 0 2年间的 2 0 39个血清样本 ,对不同地区、年龄和品种猪的血清学数据进行了系统分析。结果显示 ,浙江省 4 4个猪场均有猪圆环病毒 2型 (PCV2 )感染 ,猪场的感染率为 10 0 % ,全省猪的 PCV2感染的平均血清阳性率为 5 8.31% ,其中种猪感染的血清阳性率为 5 9.38% ,断奶后猪感染的血清阳性率为 5 6 .6 5 %。长白种猪感染的血清阳性率为 4 4 .83%、长白仔猪感染的血清阳性率为 6 4 .2 8% ,明显高于大约克和杜洛克种猪和仔猪的感染率。 PCV2感染的血清阳性率高的种猪和仔猪群 ,其 PRRSV和 Pr V的抗体阳性率低。这些结果说明 ,PCV2在猪群中的感染非常普遍 ,不同品种猪对 PCV2的易感性不同 ,PCV2的感染可降低猪群对疫苗的免疫应答     

2型猪圆环病毒研究进展

猪圆环病毒2型(Porcine circovirus type 2,PCV2)是一种基因组较小,但有较高进化速率的DNA病毒,其是影响养猪业经济发展的重要病原体。到目前为止,PCV2具有5种基因型-PCV2a到PCV2e,但对PCV2流行株的基因序列的检测发现,PCV2的基因型仍在持续的发展。自2012年以来,PCV2d基因型的圆环病毒在北美取代了以前主导的PCV2b基因型感染,同时在中国和韩国也有了类似的趋势。圆环病毒的新兴基因型,其毒力存在增强的可能性,基于PCV2a基因型制备的疫苗,是否对PCV2d基因型圆环病毒的感染具有保护作用,已引起各国学者的重视。本文通过对PCV2的生物学特性、抗体变异情况以及其基因进化情况进行综合的分析总结,从而为PCV2疫苗的开发与研制奠定理论基础。     

用PCR方法检测猪圆环病毒Ⅱ型

建立了一种能特异性地扩增猪圆环病毒 型 (porcine circovirus type 2 ,PCV- 2 )核酸片段的 PCR方法 ,从疑似断奶猪多系统衰竭综合征 (PMWS)的感染病例的淋巴结和脾脏中扩增出了预期长度的 PCV- 2 DNA片段 ,用限制性内切酶 Eco R 酶切鉴定了 PCR产物的特异性。 PCR产物的核苷酸序列分析表明 ,与已报道的 PCV- 2 (Gen Bank Ac-cession num ber AF0 2 72 17)相关区域的核苷酸序列同源性均大于 96 %。首次证实了我国猪群中存在 PCV- 2感染的PMWS。     

猪圆环病毒2型及其综合防控

猪圆环病毒2型(PCV2)可引起断奶仔猪多系统衰竭综合征(PMWS)、皮炎肾病综合征(PDNS)和母猪繁殖障碍等一系列猪圆环病毒相关疾病(PCVD),是当前危害养猪业的重要疫病之一,给我国养猪业造成严重的经济损失。另外,PCV2感染猪后还可以导致感染猪只产生免疫抑制,进而引起继发感染或混合感染,使疾病情况更为复杂。本文就PCV2的的病原学、流行病学、疫苗研究和综合防控等方面展开综述,以期为广大养户提供参考。     

Genome Sequence Analysis of Porcine Circovirus Type 2 Strains from Shanxi Province

In order to study genetic variation of porcine circovirus type 2 (PCV2) strains in Shanxi province, the genomic sequences of four PCV2 strains which were SXXZ1, SXXZ2, SXTY and SXJZ were isolated recently from some areas of Shanxi province, they were amplified, cloned and sequenced. The amplified PCV2 genomic sequences of these four strains were analyzed and compared with that of 34 published PCV2 stains by DNAStar and drawing phylogenetic tree. The results showed that the genomic sequences of SXJZ PCV2 strain was 1 768 bp, and the others were 1 767 bp, which accounted for 25% and 75%, respectively. The homologies of nucleotide sequences of the four strains were 95.9% to 100.0%, the homologies of nucleotide sequences of the four strains with the 34 isolates from different regions of the world PCV strain were 94.5% to 99.9%, and the homologies of nucleotide sequences of the four strains with the domestic vaccine strains were 95.6% to 99.8%. The phylogenetic tree analysis showed that SXXZ1, SXXZ2 and SXTY belonged to PCV-2b genotype, and SXJZ belonged to PCV-2e genotype. The evolutionary relationship among SXXZ1, SXXZ2 and the QY strain of Guangdong were closer, we also found a recent evolutionary relationship between SXTY and AUT5 of Austria, and a recent evolutionary relationship between SXJZ and FJ of Fujian. Besides the evolutionary relationship among SXXZ1, SXXZ2, SXTY and the DBN-SX07-2 of PCV2 vaccine of Shanxi province, a recent evolutionary relationship between SXJZ and LG of PCV2 vaccine were found. Thus PCV2 strains were popular based on PCV-2b in Shanxi province, also PCV-2e was isolated and existed. It laid a foundation for the molecular epidemiology, genetic variation, prevention and control of PCV2 in Shanxi province.     

猪圆环病毒2型山西株全基因组序列分析

为了解山西省猪圆环病毒2型（PCV2）的遗传变异情况,本试验采用PCR方法对山西省分离的4株猪圆环病毒流行株（SXXZ1株、SXXZ2株、SXTY株和SXJZ株）的全基因组进行了扩增、克隆和测序,并将其全基因组序列与国内外34株主要流行毒株进行核苷酸同源性与系统进化树分析。结果显示,4株PCV2山西流行株全基因组核酸序列全长SXJZ株为1 768 bp,其余均为1 767 bp,分别占25%和75%。4株毒株核苷酸同源性为95.9%~100.0%,与国内外34株参考株同源性为94.5%~99.9%,与国产疫苗株同源性为95.6%~99.8%;PCV2全基因组序列进化分析表明,本研究分离的SXXZ1株、SXXZ2株和SXTY株属于PCV-2b基因型,SXJZ株属于PCV-2e基因型,其中SXXZ1株和SXXZ2株与广东QY株的进化关系最近,SXTY与奥地利AUT5株的进化关系最近,SXJZ与福建FJ株的进化关系最近;而SXXZ1株、SXXZ2株和SXTY株与山西PCV2疫苗DBN-SX07-2株的进化关系较近,SXJZ株与国内PCV2疫苗LG株的进化关系较近。从而证实在山西省流行的PCV2毒株以PCV-2b为主,同时还分离出了PCV-2e亚型,表明山西省存在PCV-2e亚型毒株。本试验结果为山西省PCV2的分子流行病学、遗传变异及防控奠定了基础。     

圆环病毒2型人工感染猪的排毒情况及体内分布规律的初步研究

猪圆环病毒2型（PCV2）是引起断奶仔猪多系统衰竭综合症（PMWS）的重要病原,它给养猪业带来巨大的经济损失。本研究利用PCR及荧光定量PCR检测猪人工感染猪圆环病毒2型后的排毒及体内分布规律。结果表明,感染猪可以通过呼吸道和消化道进行排毒,而且病毒持续存在;PCV2病毒主要存在于病猪的淋巴结、肾脏、肺脏中,在肝脏和脾脏中也少量存在。     

表达猪圆环病毒2型Rep蛋白的重组杆状病毒的构建与鉴定

猪圆环病毒2型(PCV2)是近年来新发现的引起断奶仔猪多系统衰弱综合征的主要病原,含有两个主要的阅读框ORF1和ORF2,分别编码复制相关蛋白(Rep)和核衣壳蛋白(Cap)。为了探究ORF1基因结构及其与功能的关系,本研究利用Bac-to-Bac杆状病毒表达系统将PCV2的ORF1基因克隆到杆状病毒转移载体pFastBacTMDual中,获得重组转移载体pFBD-ORF1,再将其转化进含穿梭载体Bacmid的感受态细胞DH10Bac中,经蓝白菌落筛选获得含有ORF1基因的重组穿梭载体rBacmid-ORF1,经脂质体介导转染昆虫细胞sf9,获得重组杆状病毒rAc-Rep,SDS-PAGE和Western-blotting分析可见大小约为36ku的特异性条带,表明rAc-Rep在sf9中成功表达了PCV2Rep蛋白。     

猪圆环病毒2型CAP蛋白在flashBAC杆状病毒表达系统中的表达与鉴定

试验旨在通过真核表达系统表达猪圆环病毒2型（porcine circovirus type 2,PCV2） CAP蛋白。以PCV2 TZ0601株为模板,将PCV2 CAP全基因及CAP去除信号肽的基因编码序列克隆至pOET3载体上,酶切与测序鉴定正确后,将重组质粒pOET3-CAP及pOET3-CAP-X转染sf9昆虫细胞。采用flashBAC杆状病毒表达系统表达PCV2 CAP及去除信号肽的CAP蛋白,通过间接免疫荧光法、SDS-PAGE 和Western blotting鉴定目的蛋白的表达。结果表明,真核表达质粒pOET3-CAP及pOET3-CAP-X构建成功,目的基因在sf9昆虫细胞中高效表达,得到的蛋白经SDS-PAGE和Western blotting鉴定,在25~35 ku处有蛋白条带,表达的蛋白质可被PCV2阳性血清识别。试验结果为进一步制备PCV2亚单位疫苗及诊断抗原试剂盒的研发奠定了基础。     

贵州省猪圆环病毒Ⅱ型感染的PCR检测

为从病原学检测方面了解猪圆环病毒Ⅱ型在贵州的感染情况,采集贵州省9个集约化养猪场和养猪专业户断奶仔猪多系统衰竭综合征(PMW S)疑似发病猪组织病料137份,用针对猪圆环病毒Ⅱ型设计的特异性引物进行特异性目的基因片段的扩增,紫外光下观察,以420 bp处出现目的条带确定为阳性。结果表明,检出阳性病料77份,阳性率为56.2%,表明猪圆环病毒Ⅱ型感染在贵州省猪群中普遍存在。     

湘西山区猪2型圆环病毒血清学调查

2013年8月至2014年12月,从湖南湘西未接种任何疫苗(除猪瘟苗外)的山区小型猪场、农村传统饲养户以及部分野猪场采集了1 390份猪血清样品,通过酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA),血清学检测猪2型圆环病毒(Porcine circovirus type 2,PCV2)的血清抗体,对已确认为PCV2抗体阳性的血清进行猪蓝耳病毒(Porcine reproductive and respiratory syndrome virus,PRRSV),以及猪伪狂犬病毒(Pseudorabies virus,PRV)的血清抗体检测。检测结果显示PCV2血清抗体阳性率,小型猪场的为86%、农村传统饲养猪为78%、土猪场为69%、野猪场为25%。结果表明:湘西山区小型猪场、农村传统饲养户、部分野猪场的PCV2血清抗体阳性率不同,这种抗体阳性率的高低可能与饲养密度、饲养方式、品种和环境有关。本调查通过对湘西周边山区猪群的PCV2血清抗体阳性进行检测及统计分析,发现山区微小猪场及农户散养猪只具有较高的抗体阳性率,而抗体阳性率在一定程度上代表着PCV2的感染率。因此,加强PCV2免疫接种,将有利于防控PCV2相关性疾病的发生以及其他疾病继发的感染。     

Expression and Identification of Porcine Circovirus type 2 Capsid Protein Using flashBAC Baculovirus Expression System

The study was aimed to express CAP protein of porcine circovirus type 2 (PCV2) by eukaryotic expression system.The PCV2 TZ0601 strain was the template,the CAP protein with or without the signal peptide of PCV2 coding sequence were cloned into pOET3 vector.The constructed plasmid were confirmed by restriction enzyme digestion and DNA sequencing,then sf9 insect cells were transfected with recombinant plasmid pOET3-CAP and pOET3-CAP-X.The test was designed to express the CAP protein with or without the signal peptide of PCV2 by flashBAC baculovirus expression system.Expression of PCV2 gene were confirmed by indirect immunofluorescent assay (IFA),SDS-PAGE and Western blotting.The results showed that the eukaryotic expression plasmids of pOET3-CAP and pOET3-CAP-X were constructed successfully and the gene was highly expressed in sf9 insect cells.After expression,we could see our target band about 25 to 35 ku with SDS-PAGE and Western blotting.The immuno-reactivity of the protein was confirmed by antisera against PCV2.This would lay a foundation for a further study of PCV2 subunit vaccine and diagnostic antigen kit.     

猪圆环病毒2型诊断方法的研究进展

圆环病毒2型（PCV-2）是目前已知的最小的动物病毒,不仅是猪断奶后多系统衰弱综合征（PMWS）的病原,而且如猪皮炎肾病综合症（PDNS）、猪繁殖与呼吸综合征（PRRS）、仔猪先天震颤（CT）、增生性坏死性肺炎等疾病也与PCV-2有关,给养殖业带来了巨大的经济损失。作者从免疫学、分子生物学方面综述了当前国内外猪圆环病毒病常用的各种诊断方法的研究进展,便于我们今后的研究。     

一株2型猪圆环病毒的分离及其序列、致病性的初步分析

断奶仔猪多系统衰竭综合征(Postweaning multi-system ic wasting syndrome,PMWS)是由2型猪圆环病毒(Porcine circovirustype 2,PCV-2)引起的一种传染病。2006年,黑龙江省某猪场饲养的128头仔猪中,有80%的仔猪患有先天性震颤、吃奶困难,采集死亡与患病仔猪的病料经PCR鉴定,结果