Genetic Variation of Chinese PRRSV Strains Based on ORF5 Sequence

Thirteen isolates of porcine reproductive and respiratory syndrome virus (PRRSV) from different provinces of China were studied and compared with several PRRSV isolates from other countries. Phylogenetic analysis shows that all Chinese isolates of PRRSV in this study belong to the American genotype, except for one strain, B13, which clustered as a European genotype. Sequence analysis revealed that PRRSV Chinese isolates of the American genotype were highly similar in the ORF5 sequence and could be classified into two subclades. One contains PRRSV isolates that are more closely related to the American vaccine strain MLV Resp and its parent strain VR-2332, and the other contains ones only distantly related to them. Within the Chinese isolates slight genetic variation occurred, and some strains may originate directly from the vaccine virus.     

Immunogenicity of recombinant vaccinia virus vaccines co-expressing GP3/GP5 of European PRRSV and Cap protein of PCV2 in pigs

Applied Microbiology and Biotechnology - Porcine reproductive and respiratory syndrome (PRRS) is almost always caused by the North American strain of PRRS virus (PRRSV) in China; the European...     

Recombinant Encephalomyocarditis Viruses Elicit Neutralizing Antibodies against PRRSV and CSFV in Mice

Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of species and the infection can cause myocarditis and reproductive failure in pigs as well as febrile illness in human beings. In this study, we introduced the entire ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) or the neutralization epitope regions in the E2 gene of the classical swine fever virus (CSFV), into the genome of a stably attenuated EMCV strain, T1100I. The resultant viable recombinant viruses, CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2, respectively expressed partial PRRSV envelope protein GP5 or CSFV neutralization epitope A1A2 along with EMCV proteins. These heterologous proteins fused to the N-terminal of the nonstructural leader protein could be recognized by anti-GP5 or anti-E2 antibody. We also tested the immunogenicity of these fusion proteins by immunizing BALB/c mice with the recombinant viruses. The immunized animals elicited neutralizing antibodies against PRRSV and CSFV. Our results suggest that EMCV can be engineered as an expression vector and serve as a tool in the development of novel live vaccines in various animal species.     

表达猪圆环病毒2型Cap和猪繁殖与呼吸综合征病毒GP5融合蛋白重组腺病毒的构建和免疫原性测定

断奶后多系统衰弱综合征(PMWS)是目前规模化猪场常见的传染病,由PCV2感染引起。临床上PCV2与PRRSV混合感染较常见,死亡率和发病率很高,但目前尚无能有效预防该病的商品化疫苗。本研究利用基因克隆方法,构建融合表达PCV2-Cap蛋白与PRRSV-GP5蛋白的重组腺病毒rAd-Cap-GP5,经IPMA、IFA和Western blotting验证了Cap蛋白和GP5蛋白在该重组病毒中获得表达。将该重组腺病毒免疫小鼠测定其抗体、中和抗体,结果显示小鼠免疫后出现两种病毒的抗体和中和抗体,诱导小鼠产生明显的体液免疫应答,可以作为预防PCV2-PRRSV混合感染的基因工程疫苗候选毒株,为PCV2和PRRSV二联重组亚单位疫苗的研究奠定了基础。     

Andrographolide and Its Derivative Potassium Dehydrographolide Succinate Suppress PRRSV Replication in Primary and Established Cells via Differential Mechanisms of Action

Virologica Sinica - Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause significant economic loss worldwide and remains a serious threat to the pork industry. Currently,...     

The Novel PRRSV Strain HBap4-2018 with a Unique Recombinant Pattern Is Highly Pathogenic to Piglets

Virologica Sinica - Currently, various porcine reproductive and respiratory syndrome virus (PRRSV) variants emerged worldwide with different genetic characteristics and pathogenicity, increasing...     

猪传染性胸膜肺炎病料中PCV_2和PRRSV的PCR检测

应用PCR方法对从山东省不同地区采集的2 5 3份猪传染性胸膜肺炎肺脏和12 5份临床健康猪肺脏进行PCV_2和PRRSV的检测。结果显示,在传染性胸膜肺炎猪肺脏中,171份为PCV_2阳性,阳性率达6 7 5 % ;10 1份样品为PRRSV阳性,阳性率达4 0 % ;其中,6 8份样品同时检出PCV_2和PRRSV ,共感染阳性率达2 6 8%。而临床健康猪肺组织中,2 1份样品PCV_2检测结果为阳性,阳性率为16 8% ;12份样品PRRSV检测结果阳性,阳性率为9 6 % ,PCV_2和PRRSV共感染未检出。统计结果显示,传染性胸膜肺炎发病猪与临床健康猪PCV_2、PRRSV及PCV_2和PRRSV共感染的阳性率差异极显著,传染性胸膜肺炎发病猪的肺脏中PCV_2和PRRSV的检出率明显高于临床健康猪。上述检测结果提示,猪传染性胸膜肺炎的发生可能与PCV_2和PRRSV的感染和共感染有关     

Complete genome sequence of two novel Chinese virulent porcine reproductive and respiratory syndrome virus variants

A highly pathogenic strain of porcine reproductive and respiratory syndrome virus (PRRSV), characterized by a discontinuous 30-amino-acid deletion in its Nsp2-coding region, has been emerging in China since 2006. Here, we report the complete genomic sequence of two novel Chinese virulent PRRSV variants with additional NSP2-gene deletions, which will help us understand the molecular and evolutionary characteristics of PRRSV in Asia.     

MiR-506 inhibits PRRSV replication in MARC-145 cells via CD151

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases of swine, which is caused by PRRS virus (PRRSV). CD151, one of PRRSV entry mediators, determines the cell susceptibility for PRRSV. Emerging evidence indicates that the host microRNAs (miRNAs) play key roles in modulating virus infection and viral pathogenesis. In the present study, targeting porcine CD151 miRNAs were identified, and their function during PRRSV infection in MARC-145 cells was further verified. We found that miR-506 could directly target porcine CD151 3′-UTR mRNA by luciferase reporter assay. Overexpression of miR-506 significantly decreased CD151 expression at both mRNA and protein levels. Furthermore, overexpression of miR-506 reduced cellular PRRSV replication and virus release in MARC-145 cells. Our results suggested that miR-506 could inhibit PRRSV replication by directly targeting PRRSV receptor of CD151 in MARC-145 cells. However, the molecular mechanisms of miR-506 and its function in vivo need further investigation.     

从PRRSV BJ-4株基因组全长cDNA获得感染性病毒

对已构建的覆盖猪繁殖与呼吸综合征病毒(PRRSV)BJ-4全长cDNA的6个重组质粒进行测序,并对部分点突变进行定点回复突变,将突变片段顺次连接,获得了全长cDNA克隆pWSK-DCBA。通过体外转录获得病毒基因组RNA,将RNA与脂质体混合后直接转染MARC-145细胞,获得拯救病毒(rV68)。rV68能在MARC-145细胞上稳定传代,并可引起PRRSV特征性的细胞病变(CPE)。增殖动态分析表明,rV68在MARC-145细胞上的生长有所迟滞,达到最高滴度的培养时间比亲本病毒延迟12h,但滴毒无显著差异(P>0.05)。结果表明,构建的BJ-4全长cDNApWSK-DCBA具有感染性,为研究中国PRRSV的分子致病与免疫机制、新型疫苗等奠定了基础。     

表达猪繁殖与呼吸综合征病毒(PRRSV)GP5的重组伪狂犬病毒TK-/gG-/GP5+的构建及其生物学特性初步探讨

以伪狂犬病毒基因缺失标志疫苗株TK/gG-/LacZ 为亲本株,通过同源重组,构建了表达猪繁殖与呼吸综合征病毒(PRRSV)主要免疫原性蛋白GP5的重组伪狂犬病毒TK/gG-/GP5 .经PCR、Southern杂交、Western blot证实构建正确,并能表达GP5.同时,对重组病毒在PK-15细胞中的增殖特性进行了检测,结果与亲本株相比,增殖滴度无显著性差异.将重组病毒免疫BALB/c小鼠,2次免疫后4周,50%(3/6)小鼠产生了低水平的GP5特异性ELISA抗体,但中和抗体均小于1:4.     

我国猪繁殖与呼吸综合征病毒NT0901分离株分子特征

猪繁殖与呼吸综合征病毒(PRRSV)是目前引起国内外养猪业严重经济损失的重要病原之一,病毒基因和毒力变异较大。PRRSV NT0801株分离自我国发病猪群,毒力较强,但NSP2基因不存在高致病性PRRSV 30个氨基酸的缺失。为了进一步阐明该分离株的分子特征,本研究对该毒株全基因序列进行了测定和分析,结果该毒株基因组全长15 439 bp,其中包含29 nt Poly(A)。与高致病性PRRSV毒株JXA1比较,核酸序列同源性为96.7%,推导的GP3和GP5氨基酸序列同源性分别为97.2%和98.5%,但NSP2基因无30个氨基酸的缺失;与传统型毒株ch-1a比较,推导的GP3和GP5氨基酸序列同源性分别为92.9%和91.5%;基因进化树分析结果显示其介于高致病性和传统PRRSV毒株之间。与其它不同毒力PRRSV分离株基因序列比较,未发现明显重组信号。不同毒力毒株氨基酸残基比对分析结果显示,15个位点潜在毒力相关氨基酸残基中,该毒株有9个与高致病性PRRSV毒株一致,3个与高致病性PRRSV毒株不同,但与传统型和JXA1疫苗株相同,1个位点只与JXA1疫苗株相同,2个与其它毒株都不相同。表明该分离株与高致病性PRRSV密切相关,PRRSV流行毒株变异与基因突变有关,从而为该病毒毒力基因定位研究奠定了基础。     

Analysis of molecular variation in porcine reproductive and respiratory syndrome virus in China between 2007 and 2012

In the present study, 89 porcine reproductive and respiratory syndrome virus (PRRSV) isolates in China during 2007 to 2012 were randomly selected from the GenBank genetic sequence database. Evolutionary characteristics of these isolates were analyzed based on the sequences of non-structural protein 2 (Nsp2) and glycoprotein 5 (GP5). The genetic variations of the isolates were also compared with six representative strains. The results showed that a high degree of genetic diversity exists among the PRRSV population in China. Highly pathogenic PRRSV isolates, with a discontinuous deletion of a 30 amino acid residue in the Nsp2 region, remained the most dominant virus throughout 2007–2012 in China. Owing to the extensive use of representative vaccine strains, natural recombination events occurred between strains. Three isolates — HH08, DY, and YN-2011 — were more closely related to vaccine strains than the other isolates. Both YN-2011 and DY were the evolutionary products of recombination events between strains SP and CH-1R. The results of the present study provide useful information for the epidemiology of PRRSV as well as for vaccine development.     

三带喙库蚊体内猪繁殖与呼吸综合征病毒的分离与鉴定

【目的】调查猪场蚊虫是否能携带猪繁殖与呼吸综合征(PRRS)病毒。【方法】采集发生PRRS疫情的3个养猪场蚊虫样本,采用RT-PCR方法检测PRRS病毒核酸,取阳性蚊虫样本接种Marc-145细胞进行病毒的分离培养,以间接免疫荧光抗体技术和分子克隆技术进行病毒的鉴定。【结果】 养猪场内的蚊虫主要有三带喙库蚊Culex tritaeniorhychus、凶小库蚊Culex modestus、中华按蚊Anopheles sinensis和骚扰阿蚊Armigeres obturbans,其中三带喙库蚊占86.76%;以PRRS病毒N基因引物进行扩增,三带喙库蚊样本呈现阳性反应,而其他蚊种均为阴性。在蚊虫接种的Marc-145细胞中可见细胞融合和空泡形成等细胞病变效应;用抗PRRS病毒N蛋白抗体和羊抗猪IgG(H+L)-FITC进行间接免疫荧光染色,感染细胞呈现黄绿色荧光;以NSP2基因引物进行RT-PCR扩增、克隆与测序,发现库蚊源病毒与相应猪场猪源病毒中相应基因的序列具有较高同源性。【结论】 三带喙库蚊为猪舍优势蚊种,并能携带猪繁殖与呼吸综合征病毒。     

Effect of porcine reproductive and respiratory syndrome virus infection on the ovary and progesterone levels in third trimester pregnant sows

Porcine reproductive and respiratory syndrome virus (PRRSV) is a common cause of reproductive failure and abortion in swine. The mechanism of abortion is not fully defined, and the effect of the virus on luteal function has not been explored. In this study, we exposed late-term pregnant swine to varied doses of PRRSV strain NADC-8 and evaluated effects on ovarian function by serial determination of plasma progesterone levels and by microscopic evaluation of ovarian pathologic alterations combined with immunohistochemistry and in situ hybridization to detect PRRSV antigen. We identified no specific trend in plasma progesterone level associated with PRRSV infection status and no microscopic ovarian lesions. PRRSV antigen was not demonstrated in ovarian tissues by immunohistochemistry or in situ hybridization at necropsy 21 days postexposure. Based on these findings, it does not appear that either a direct or an indirect effect on luteal function contributes to PRRSV-induced abortion.     

PRRSV受体CD163的原核分段表达

为原核表达猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)受体CD163,本研究根据CD163基因序列(EU016226),通过生物信息学软件Scanprosite预测了CD163结构特征并进行分段。利用DNAstar软件设计合成3对特异引物,从猪肺泡巨噬细胞(porcine alveolar macrophages, PAM)中克隆CD163三个片段,经测序鉴定正确后,分别克隆于原核表达载体pET-28a(+),在IPTG诱导下,进行CD163重组蛋白的截短表达,经Western杂交检测证实表达的三段截短蛋白均具有良好的与抗原反应活性,从而为进一步研究CD163受体在病毒感染过程中的作用以及与其他受体之间的相互作用提供实验依据。     

The C/EBPβ-Dependent Induction of TFDP2 Facilitates Porcine Reproductive and Respiratory Syndrome Virus Proliferation

Virologica Sinica - Porcine reproductive and respiratory syndrome (PRRS) is an important infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), leading to...     

Complete Genome Sequence of a Novel Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Variant

Highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) emerged in China in 2006, and HP-PRRS virus (HP-PRRSV) has evolved continuously. Here, the complete genomic sequence of a novel HP-PRRSV field strain, JX, is reported. The present finding will contribute to further studies focusing on the evolutionary mechanism of PRRSV.