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     

猪圆环病毒2型及猪繁殖与呼吸综合症病毒的快速检测

According to the published genome sequences of Porcine circovirus type 2(PCV2)and Porcine reproductive and respiratory syndrome virus(PRRSV),primers were designed and PCR,RT-PCR were set up for the detection of PCV2 and PRRSV,respectively,With the established methods,38 clinical samples from the respiratory disease pigs were detected for the presence of PCV2 and PRRSV. The results demonstrated that 22 samples were positive for PCV2,27 samples were positive for PRRSV and among the above positive samples,18 samples were positive for both viruses,The data obtained in the present study indicated that PCV2 and PRRSV maybe play an important role in the course of the development of respiratiory diseases.     

Matrine exhibits antiviral activity in a PRRSV/PCV2 co-infected mouse model

BackgroundPRRSV and PCV2 co-infection is very common in swine industry which results in huge economic losses worldwide. Although vaccination is used to prevent viral diseases, immunosuppression induced by PRRSV and PCV2 leads to vaccine failure.PurposeOur previous results have demonstrated that Matrine possess antiviral activities against PRRSV/PCV2 co-infection in vitro. This study aims to establish a PRRSV/PCV2 co-infected KM mouse model and evaluate the antiviral activities of Matrine against PRRSV/PCV2 co-infection.Study designA total of 144 KM mice were randomly divided into six groups with 24 mice in each group, named as: normal control, PRRSV/PCV2 co-infected group (PRRSV/PCV2 group), Ribavirin treatment positive control (Ribavirin control) and Matrine treatment groups (Matrine 40 mg/kg, Matrine 20 mg/kg and Matrine 10 mg/kg).MethodsExcept normal control group, all mice in other five groups were inoculated with PRRSV, followed by PCV2 at 2 h later. At 7 days post-infection (dpi), mice in the treatment groups were intraperitoneally administered with various doses of Matrine and Ribavirin, twice a day for 5 consecutive days.ResultsPRRSV N and PCV2 CAP genes were detected by PCR in multiple tissues including heart, liver, spleen, lungs, kidneys, thymus and inguinal lymph nodes. The viral load of PCV2 was the highest in liver followed by thymus and spleen. Although PRRSV were detected in most of tissues, but the replication of PRRSV was not significantly increased, as shown by qPCR analysis. Comparing with PCV2 infection alone, PRRSV infection significantly elevated PCV2 replication and exacerbated PCV2 induced interstitial pneumonia. qPCR analysis demonstrated 40 mg/kg Matrine significantly attenuated PCV2 replication in liver and alleviated virus induced interstitial pneumonia, suggesting Matrine could directly inhibit virus replication. In addition, Matrine treatment enhanced peritoneal macrophages phagocytosis at 13 and 16 dpi, and 40 mg/kg of Matrine increased the proliferation activity of lymphocytes. Body weight gain was continuously promoted by administrating Matrine at 10 mg/kg.ConclusionMatrine possessed antiviral activities via inhibiting virus replication and regulating immune functions in mice co-infected by PRRSV/PCV2. These data provide new insight into controlling PRRSV and PCV2 infection and support further research for developing Matrine as a new possible veterinary medicine.     

猪圆环病毒2型、猪繁殖与呼吸综合征病毒及猪细小病毒混合感染的流行病学调查

根据GenBank上发表的PRRSVORF7、PPVVP2及PCV的基因组序列设计合成引物,建立了分别用于检测PRRSV、PPV和PCV的RT-PCR、PCR及复合PCR方法。应用建立的复合PCR方法对送检的127份病料进行了PCV的检测,对鉴定为PCV2阳性的67份病料再分别进行PRRSV和PPV的检测,以确定猪群中PCV2与PRRSV和,或PPV混合感染情况,结果表明,35份样品表现为PRRSV与PCV2混合感染,占样品总数的52．3％;18份样品表现为PCV2与PPV混合感染,占26．9％。另外,还有一定比例的三重感染,共5个样品,占7．5％。由此可见,猪群中PCV2与PRRSV及PPV混合感染比较普遍。     

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。     

高致病性猪繁殖与呼吸综合征病RT-PCR快速鉴别诊断方法的建立与临床应用

根据高致病性猪繁殖与呼吸综合征病毒(PRRSV)Nsp2基因的缺失信息,设计了3条特异性引物,以含高致病性PRRSV Nsp2基因的质粒pMDNSP2及普通PRRSV VR2332株RNA为模板,建立了快速诊断高致病性PRRSV RT-PCR方法.通过对临床组织病料的总RNA进行不同稀释倍数检测,结果表明该方法能从0.265 pg的总RNA中检测到PRRSV的基因,说明敏感性高.用该方法对猪瘟病毒(CSFV)、Ⅱ型圆环病毒(PCV-2)、伪狂犬病病毒(PRV),链球菌(Streptococcus)、副猪嗜血杆菌(Haemophilus parasuis)和大肠杆菌(Escherichia coli)同条件检测,结果都为阴性.进一步对36份疑似高致病性PRRSV临床组织病料细胞培养物、2株PRRSV商品活疫苗以及52个猪场所送检的184份临床样品进行了检测应用,结果36份疑似高致病性PRRSV临床组织病料的细胞培养物有5份样品为阳性且都为高致病性PRRSV,2株PRRSV商品活疫苗为普通PRRSV,52个猪场中有42个猪场(123份样品)呈阳性,其中只有1份为普通PRRSV.实验表明该方法能够准确地鉴别诊断高致病性PRRSV和普通PRRSV,且具有快速,敏感和特异的特点,具有临床实用性.     

Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2

Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n = 5), PRRSV inoculated (n = 5), PCV2 inoculated (n = 7), and PRRSV and PCV2 inoculated (n = 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.     

Porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication

An increasing number of studies demonstrate that autophagy, an intrinsic mechanism that can degrade cytoplasmic components, is involved in the infection processes of a variety of pathogens. It can be hijacked by various viruses to facilitate their replication. In this study, we found that PRRSV infection significantly increases the number of double- or single-membrane vesicles in the cytoplasm of host cells in ultrastructural analysis. Our results showed the LC3-I was converted into LC3-II after virus infection, suggesting the autophagy machinery was activated. We further used pharmacological agents and shRNAs to confirm that autophagy promoted the replication of PRRSV in host cells. Confocal microscopy analysis showed that PRRSV inhibited the fusion between autophagosomes and lysosomes, suggesting that PRRSV induced incomplete autophagy. This suppression caused the accumulation of autophagosomes which may serve as replication site to enhance PRRSV replication. It has been shown that NSP2 and NSP3 of arterivirus are two components of virus replication complex. We also found in our studies that NSP2 colocalized with LC3 in MARC-145 cells by performing confocal microscopy analysis and continuous density gradient centrifugation. Our studies presented here indicated that autophagy was activated during PRRSV infection and enhanced PRRSV replication in host cells by preventing autophagosome and lysosome fusion.     

Molecular characterization of a highly pathogenetic porcine reproductive and respiratory syndrome virus variant in Hubei, China

Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized as one of the most important pathogens of pigs throughout the world. In 2006, more than 10 provinces of China have experienced an epizootic outbreak of pig diseases characterized by high fever, reddened skin and high morbidity and mortality. From June 2006 to April 2007, we have investigated some clinical samples in Hubei province by RT-PCR and cloned several major genes, N, GP5 and NSP2 gene, shown in this study. Phylogenetic analysis of these genes revealed that the highly pathogenic PRRSV variant, ZB, was responsible for 2006 emergent outbreak of pig disease in Hubei province similar with those variants isolated from other provinces in China in 2006, and belongs to the NA-type PRRSV. In the PRRSV variants, the N and GP5 shear about 90% identity with prototypic ATCC VR-2332 and some typical NA-type Chinese isolates, except the 2850bp NSP2 gene (only shares 65% identity with ATCC VR-2332). But they all shear more than and 97% identity with other highly pathogenetic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and 2 deletions in the Nsp2 protein when compared with the previous isolates. Most of the variants found in 2006 epizootic outbreak of pig diseases in China were the farthest variants from the typical NA-type PRRSV in phylogenetic distance, and these diversities may be responsible for the differences in the pathogenicity observed between these variants and original Chinese PRRSV strains.     

Complete Genome Sequence of Two Variant Porcine Reproductive and Respiratory Syndrome Viruses Isolated from Vaccinated Piglets

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to affect the Chinese swine industry. Since 2006, variant PRRSV strains sharing two unique discontinuous deletions of 30 amino acids in the nonstructural protein Nsp2 have become dominant in Chinese swine herds and have caused huge economic losses to the swine industry in China. Here we report the complete genome sequence of two novel PRRSV variants isolated from vaccinated piglets with additional amino acid deletions in Nsp2.     

The origin and evolution of porcine reproductive and respiratory syndrome viruses

Porcine reproductive and respiratory syndrome viruses (PRRSV) are divided into North American and European types, which show about 40% difference in their amino acid sequences. The divergence time of these two types has been estimated to be about 1980 from epidemiological data. This suggested that PRRSV have evolved at a higher evolutionary rate (order of 10(-2)/site/year) compared with other RNA viruses of 10(-3) to 10(-5)/site/year. Here, to test the evolutionary history of PRRSV speculated by the epidemiological background, we estimated the divergence time and evolutionary rate of PRRSV with molecular evolutionary analysis. Estimated divergence time (1972-1988) corresponded well to that estimated by the epidemiological data, and the evolutionary rate (4.71-9.8) x 10(-2) of PRRSV was indeed the highest among RNA viruses so far reported. Furthermore, we inferred important sites for the adaptation in order to examine how PRRSV have adapted to swine since they emerged. The adaptive sites were located not only in the epitopes related to immunity but also in the transmembrane regions including a signal peptide. In particular, the adaptive sites in the transmembrane regions were considered to affect compatibility to the host cell membrane. We conclude that PRRSV were transmitted from another host species to swine in about 1980 and have adapted to swine by altering the transmembrane regions.     

Oxidative stress and Nrf2 in the pathophysiology of diabetic neuropathy: old perspective with a new angle

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically detrimental pig pathogen that causes significant losses for the pig industry. The immunostimulatory effects of hemagglutinating virus of Japan envelope (HVJ-E) in cancer therapy and the adjuvant efficacy of HVJ-E have been previously evaluated. The objective of this study was to investigate the adjuvant effects of HVJ-E on immunization with killed PRRSV vaccine, and to evaluate the protective effects of this immunization strategy against virulent PRRSV infection in piglets. Next, the PRRSV-specific antibody response, lymphocyte proliferation, PRRSV-specific IL-2, IL-10 and IFN-γ production, and the overall protection efficacy were evaluated to assess the immune responses of the piglets. The results showed that the piglets inoculated simultaneously with killed PRRSV vaccine and HVJ-E had a significantly stronger immune response than those inoculated with killed PRRSV vaccine alone. Our results suggest that HVJ-E could be employed as an effective adjuvant to enhance the humoral and cellular responses of piglets to PRRSV.     

Effects of Astragalus polysaccharide on immune responses of porcine PBMC stimulated with PRRSV or CSFV

Background

Astragalus polysaccharide (APS) has been used as an immunomodulator that can enhance immune responses, whereas the immunomodulatory effects of APS on porcine peripheral blood mononuclear cells (PBMCs) exposed to porcine reproductive and respiratory syndrome virus (PRRSV) and classical swine fever virus (CSFV) have not been investigated.

Methodology/Principal Findings

Porcine PBMCs were cultured in complete RPMI media in the presence of the R98-strain of PRRSV (5×10⁴ TCID₅₀/ml) or C-strain of CSFV (10³ TCID₅₀/ml) with or without APS. The expression of mRNA for CD28, cytotoxic T-lymphocyte antigen 4 (CTLA-4), transforming growth factor-β (TGF-β), interleukin 2 (IL-2) and IL-10 was assayed by TaqMan real-time RT-PCR. The expression of mRNA for CD28 and CTLA-4 increased at 24 h after stimulation of PBMCs with CSFV and the increased production of CTLA-4 was confirmed by western blot analysis, whereas the increases were inhibited by the addition of APS. In addition, APS alone upregulated IL-2 and TGF-β mRNA expression in PBMCs and the addition of APS had the capacity to prevent a further increase in IL-2 mRNA expression in PBMCs during CSFV or PRRSV infection, but had no effect on TGF-β mRNA expression. The production of tumor necrosis factor-alpha (TNF-α) increased at 12 h after stimulation with PRRSV or CSFV, but not with PRRSV plus APS or CSFV plus APS, whereas the addition of APS to PBMCs infected with PRRSV or CSFV promoted IL-10 mRNA expression.

Conclusions

We suggested that APS had immunomodulatory effects on cells exposed to PRRSV or CSFV. It might be that APS via different mechanisms affects the activities of immune cells during either PRRSV or CSFV infection. This possibility warrants further studies to evaluate whether APS would be an effective adjuvant in vaccines against PRRSV or CSFV.     

猪繁殖与呼吸综合征病毒非结构蛋白NSP2的原核表达及其单克隆抗体的研制

将猪繁殖与呼吸综合征病毒(PRRSV)S1株NSP2蛋白基因进行截短修饰 (tNSP2),克隆于pGEX-6P-1载体,转化大肠杆菌后用IPTG诱导表达。Western-blot结果表明,融合表达的GST-tNSP2蛋白能被PRRSV阳性血清特异性识别,大小约50kD。经GST柱提取纯化蛋白GST- tNSP2,免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞进行融合,获得2株能稳定分泌抗NSP2蛋白抗体的杂交瘤细胞株,将其命名为2B5、3H3。亚型鉴定结果均为IgG 1型,其轻链均为Κ链。间接免疫荧光试验证明,2B5和3H3均能与PRRSV S1毒株产生特异性反应,而不能与SY0608毒株反应。从而为PRRSV分离株的鉴定及NSP2功能研究奠定了重要基础。     

高致病性PRRSVNsp2基因RNA干扰对病毒复制的影响

旨在构建针对猪繁殖与呼吸综合征病毒(PRRSV)TJ株Nsp2基因的siRNA的表达载体,研究Nsp2基因的表达抑制对PRRSV复制的影响。设计并合成针对PRRSV TJ株Nsp2基因的3对优势小发卡RNA(shRNA),连接到pRNAT-U6.1/Neo,构建shRNA表达载体,将鉴定正确的载体质粒转染Marc-145细胞,6 h后接毒,每日观察CPE;在接毒PRRSV TJ株后不同时间点取上清样品,测定样品TCID50;并以β-actin为内参,RT-PCR对PRRSV Nsp2 mRNA进行半定量。结果表明,选取的3条Nsp2基因的优势siRNA均能够抑制PRRSV TJ株在Marc-145细胞上增殖,其中shRNA载体S-1和S-2抑制效果明显,与空载体相比较差异极显著。本研究构建的PRRSV Nsp2基因特异性shRNA载体能够有效抑制PRRSV的复制,可使病毒滴度TCID50最多降低103.38。     

Porcine reproductive and respiratory syndrome virus (PRRSV) infection spreads by cell-to-cell transfer in cultured MARC-145 cells, is dependent on an intact cytoskeleton, and is suppressed by drug-targeting of cell permissiveness to virus infection

Background

Porcine reproductive and respiratory syndrome virus (PRRSV) is the etiologic agent of PRRS, causing widespread chronic infections which are largely uncontrolled by currently available vaccines or other antiviral measures. Cultured monkey kidney (MARC-145) cells provide an important tool for the study of PRRSV replication. For the present study, flow cytometric and fluorescence antibody (FA) analyses of PRRSV infection of cultured MARC-145 cells were carried out in experiments designed to clarify viral dynamics and the mechanism of viral spread. The roles of viral permissiveness and the cytoskeleton in PRRSV infection and transmission were examined in conjunction with antiviral and cytotoxic drugs.

Results

Flow cytometric and FA analyses of PRRSV antigen expression revealed distinct primary and secondary phases of MARC-145 cell infection. PRRSV antigen was randomly expressed in a few percent of cells during the primary phase of infection (up to about 20–22 h p.i.), but the logarithmic infection phase (days 2–3 p.i.), was characterized by secondary spread to clusters of infected cells. The formation of secondary clusters of PRRSV-infected cells preceded the development of CPE in MARC-145 cells, and both primary and secondary PRRSV infection were inhibited by colchicine and cytochalasin D, demonstrating a critical role of the cytoskeleton in viral permissiveness as well as cell-to-cell transmission from a subpopulation of cells permissive for free virus to secondary targets. Cellular expression of actin also appeared to correlate with PRRSV resistance, suggesting a second role of the actin cytoskeleton as a potential barrier to cell-to-cell transmission. PRRSV infection and cell-to-cell transmission were efficiently suppressed by interferon-γ (IFN-γ), as well as the more-potent experimental antiviral agent AK-2.

Conclusion

The results demonstrate two distinct mechanisms of PRRSV infection: primary infection of a relatively small subpopulation of innately PRRSV-permissive cells, and secondary cell-to-cell transmission to contiguous cells which appear non-permissive to free virus. The results also indicate that an intact cytoskeleton is critical for PRRSV infection, and that viral permissiveness is a highly efficient drug target to control PRRSV infection. The data from this experimental system have important implications for the mechanisms of PRRSV persistence and pathology, as well as for a better understanding of arterivirus regulation.