Differential reactivity of a monoclonal antibody directed to the membrane protein of porcine reproductive and respiratory syndrome virus.

A monoclonal antibody (2C12) against the 19 kDa membrane (M) protein of a Canadian isolate of porcine reproductive and respiratory syndrome (PRRS) virus was produced. By indirect immunofluorescence (IIF) cytoplasmic fluorescence was observed in infected cells, but the pattern of fluorescence was generally different and intensity was weaker than that observed using the nucleocapsid protein-directed monoclonal antibody SDOW17. When tested by IIF towards a total of 26 PRRS virus isolates from Canada, 122 isolates from the US and 13 isolates from Europe the 2C12 MAb reacted with all the North American isolates tested including the VR-2332 isolate and the vaccine (RespPRRS) isolate. However no reactivity was observed towards the European isolates tested including the Lelystad virus. This reactivity pattern suggests that the epitope recognized by this MAb on the M protein of PRRS virus appears highly conserved among North American isolates but absent or weakly expressed on European isolates of PRRS virus.     

Antigenic variation and genotype of isolates of porcine reproductive and respiratory syndrome virus in Korea

A panel of three anti-glycoprotein 5 (gp5) protein monoclonal antibodies (mAbs) (15, 28 and 246) and three anti-nucleocapsid (N) protein mAbs (SDOW17, VO17 and EP147) was used to investigate, by an indirect fluorescent antibody test, the antigenic variations of 50 Korean isolates of porcine reproductive and respiratory syndrome virus (PRRSV), and compare them with a us ATCC vR2332-derived attenuated vaccine strain and the reference European Lelystad strain of PRRSV. A multiplex PCR assay for the differentiation of European and North American genotypes of PRRSV was used to determine the genotype of the 50 Korean isolates. Forty-six (92 per cent) of the 50 Korean isolates shared the epitopes recognised by the anti-N protein mAb SDOW17. No reactivity to the anti-gp5 and anti-N protein mAbs was observed with the other four isolates. Six distinct patterns could be identified on the basis of their reactivities with the anti-PRRSV mAbs. All 50 isolates were identified as North American genotypes by the differential PCR.     

Differentiation between porcine reproductive and respiratory syndrome virus isolates by restriction fragment length polymorphism of their ORFs 6 and 7 genes.

Three distinct antigenic profiles were identified by comparing the reactivities of 15 Canadian field isolates, the attenuated U.S. vaccine (Ingelvac MLV) strain and 2 European reference strains (Lelystad and Weybridge) of the porcine reproductive and respiratory syndrome virus (PRRSV) by indirect immunofluorescence with a set of 4 monoclonal antibodies to the nucleocapsid (N) protein and 2 other to the matrix (M) protein. In the present study, 9 Canadian isolates for which the sequences were determined appeared closely related to 2 U.S. reference strains (ATCC VR-2332 and ATCC VR-2385) with amino acid identities varying between 90 to 98% for the M and N proteins; substitutions in the nucleotide sequences were distributed randomly throughout the ORFs 6 and 7 genes, and most were 3rd base silent mutations. In comparison, more than 30% divergence was demonstrated with the Lelystad virus. Furthermore, differentiation between North American and European isolates, and between field isolates and the MLV strain could be achieved by cutting PCR-amplified products encompassing both ORFs 6 and 7 genes with 4 restriction endonucleases. When taken individually, BsaJI and AluI were the more appropriate restriction enzymes for distinguishing the vaccine strain from field isolates. The results obtained suggest that the restriction fragment length polymorphism of the genomic region covering the ORFs 6 and 7 genes may be a valuable tool to differentiate among PRRSV isolates.     

Performance of ELISA antigens prepared from 8 isolates of porcine reproductive and respiratory syndrome virus with homologous and heterologous antisera.

Porcine reproductive and respiratory syndrome virus (PRRSV) ELISA antigens of high quality were produced using 8 different isolates of PRRSV: the European Lelystad virus (LV), the U.S. MN-1b, 89-46448, 93-44927, and 93-24025B, and the Canadian LHVA-93-3, PA-8 and GH-6 virus isolates. The performance of each of these 8 antigens and a commercial PRRSV antibody test kit (Idexx's HerdChek) were measured against antisera raised in 5 groups of 6 piglets inoculated with either LV, MN-1b, 89-46448, 93-44927, or 93-24025B. Among the 8 isolates, the 89-46448 isolate produced the broadest spectrum of antigen and resulted in earlier detection of antibodies to various North American PRRSV isolates, followed by MN-1b as the 2nd best ELISA antigen for the detection of North American PRRSV antibodies. The GH-6 and PA-8 viral antigens exhibited restricted detection of PRRSV antibodies. The LV and 89-46448 combined antigens produced the best performance for the detection of antibodies against both European and North American antigenic types of PRRSV. Using 173 panel samples collected at 11 to 60 d after intranasal inoculation with 1 of the 5 PRRSV isolates, the sensitivities of the indirect ELISA used were 73.4%, 98.3%, 90.8%, 98.3%, 83.2%, 93.1%, 77.1%, 64.2%, 98.8% and 95.9% for LV, MN-1b, LHVA-93-3, 89-46448, 93-44927, 93-24025B, PA-8, GH-6 antigens, 89-46448-LV combined antigens and Idexx's PRRSV antibody test kit, respectively. All 8 antigens gave negative results with preinfection porcine sera (n = 30); high background or nonspecific reactions were not observed with the antigens.     

Effect of virus-specific antibodies on attachment, internalization and infection of porcine reproductive and respiratory syndrome virus in primary macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) induces respiratory distress in young pigs and reproductive failure in sows. In PRRSV infected pigs, virus persists for several weeks to several months. Although IPMA antibodies are detected from 7 days post inoculation (pi), virus neutralizing (VN) antibodies are commonly detected starting from 3 weeks pi with an SN test on Marc-145 cells. Since infection of Marc-145 cells is quite different compared to infection of macrophages, the in vivo target cell, the role of these VN antibodies in in vivo protection is questionable. In our study, we demonstrated that antibodies from pigs early in infection with PRRSV Lelystad virus (14 days pi) showed no neutralization in the SN test on Marc-145 cells, but partially reduced Lelystad virus infection of porcine alveolar macrophages. At 72 days pi, VN antibodies were detected by the SN test on Marc-145 cells, and these protected macrophages completely against Lelystad virus infection. In contrast, these VN antibodies only partially reduced porcine alveolar macrophage infection of a Belgian PRRSV isolate (homologous virus), and had no effect on infection of porcine alveolar macrophages with the American type VR-2332 strain (heterologous virus). Confocal analysis of Lelystad virus attachment and internalization in macrophages showed that antibodies blocked infection through both a reduction in virus attachment, and a reduction of PRRSV internalization. Western immunoblotting analysis revealed that sera from 14 days pi, which showed no neutralization in the SN test on Marc-145 cells but partially reduced Lelystad virus infection of macrophages, predominantly recognized the Lelystad virus N protein, and reacted faintly with the M envelope protein. Sera from 72 days pi, with VN antibodies that blocked infection of Marc-145 cells and PAM, reacted with the N protein and the two major envelope proteins M and GP5. Using the Belgian PRRSV isolate 94V360 an identical but less intense reactivity profile was obtained. VN sera also recognized the VR-2332 N and M protein, but not the GP5 protein.     

Genetics and geographical variation of porcine reproductive and respiratory syndrome virus (PRRSV) in Thailand

The Thai isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were obtained from the Chulalongkorn University-Veterinary Diagnostic Laboratory (CU-VDL). Virus isolation was confirmed by immunoperoxidase monolayer assay (IPMA) using SDOW-17. The virus genotype was determined using nested multiplex RT-PCR (nm RT-PCR) of ORF 1b. The nm RT-PCR was able to detect at least 10TCID50/ml of PRRSV. Of 137 Thai isolates, 66.42% belonged to the European (EU) genotype and 33.58% to the North American (US) genotype. ORF5 products of the eight US strains (00CS1, 01NP1, 01UD6, 02CB13, 02KK1, 02PB1, 02SP2 and 02SP3) and the six EU strains (01CB1, 01RB1, 02BR1, 02CB12, 02SB2 and 03RB1) were sequenced for genetic variation analysis. The US strains of the Thai isolates are clustered within the same group and are more closely related to the IAF-EXP91 from Canada (89-90% nucleotide identity), whereas the EU strains were very similar to the EU prototype, Lelystad virus (87-97.5% nucleotide identity). The ORF5 nucleotide identities within the US genotype tested in this study compared to the US prototype, VR-2332 varied from 83.7 to 85.2%, whereas 83.5-85.5% amino acid identities were found. Based on the phylogenetic tree, each pair of the Thai isolates (01NP1 and 02KK1, 00CS1 and 01UD6, and 01CB1 and 01RB1) was identical despite they were collected from different provinces. Therefore, there was no geographic influence on the spreading of PRRSV in Thailand. Interestingly, 02CB12 (EU genotype) shared over 99% similarity of the ORF5 nucleotide sequence and 98.6% of amino acid identity with the European vaccine, Porcillis (AF378819). However, modified live virus vaccines for PRRSV have not yet been used in the swine population in Thailand. The results suggested that both US and EU genotypes exist in Thailand, genetic variation does occur in both genotypes, and the sources of the viruses appear to be from Canada and Northern Europe, respectively. In addition, the spreading of PRRSV in Thailand might be due to introducing infected replacement pigs or infected semen into the farm.     

Immunohistochemical detection of porcine reproductive and respiratory syndrome virus using colloidal gold.

Two cytopathic agents were isolated on porcine alveolar macrophages following inoculation with homogenates of lung tissues from pigs showing respiratory problems. These isolates were identified as porcine reproductive and respiratory syndrome (PRRS) virus isolates by indirect immunofluorescence using a PRRS virus (PRRSV) specific monoclonal antibody (MAb) and were designated as LHVA-92-1 and LHVA-92-2. Immunogold electron microscopy using a porcine PRRS positive serum pool and protein A-gold resulted in an intense labelling of aggregates of viral particles. Dark specific cytoplasmic staining of porcine alveolar macrophages infected with both virus isolates could be observed by immunogold silver staining (IGSS) using the specific MAb. This method proved effective in detecting PRRSV antigens in several ethanol-fixed tissues of piglets intranasally inoculated with the supernatants of macrophages infected with each isolate. Immunogold silver staining was also successfully used for the detection of PRRSV antigens on sections of formalin-fixed paraffin-embedded lung tissues and on frozen sections of lungs. The present results indicate that colloidal gold may be useful for the identification and immunohistochemical detection of PRRSV in tissues.     

Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains six structural proteins the roles of which are not completely understood. In a preceding study, immunization with the dutch isolate I10 of PRRSV had led to the development of MAbs against four structural proteins [Wieczorek-Krohmer, M., 1994. Herstellung und Charakterisierung von monoklonalen Antik?rpern gegen das Virus des Porzinen Reproduktiven und Respiratorischen Syndroms (PRRSV). Inaugural-Dissertation, Ludwig-Maximilians-Universit?t, München] here finally identified by reaction with individual plasmid-expressed PRRSV proteins as products of ORFs 3 (GP3), 4 (GP4), 5 (GP5) and 7 (N). Surprisingly, the MAbs against GP5 revealed the presence of two antigenically distinct virus populations in the isolate I10, the population PRRSV-'PPV', isolated from plaques and the PRRSV-'EPV', gained by end point dilution. MAbs against GP3, GP4 and N reacted with both I10 populations as well as with natural PRRSV isolates. However, the anti-GP5 MAbs exclusively recognized PRRSV-'PPV'. In this study immunization of mice with both separated I10 populations confirmed that solely PRRSV-'PPV' possesses the property to induce an immune response ultimately leading to the establishment of MAbs against GP5. Whereas the 15 anti-GP5 MAbs (derived from four independent fusions) reacted exclusively with PRRSV-'PPV' of the isolate I10, anti-GP4 MAbs detected their target antigen on various isolates of European origin and were able to neutralize them. As indicated by competition assays and selection of neutralization-resistant virus mutants, all GP5 MAbs are directed against a single antigenic site on the ORF 5 protein. Both groups of neutralizing antibodies bound to the surface of purified virions demonstrating that the recognized epitopes represent surface structures of the virion envelope. However, anti-GP5 MAbs mediated the binding of more gold granules than anti-GP4 MAbs. Comparison of the neutralizing effect of anti-GP4 and anti-GP5 MAbs revealed the anti-GP5 MAbs as the more efficient antibodies. For the complete neutralization of about 100 ID50 of PRRSV-'PPV' anti-GP5 culture supernatant was effective up to a dilution of 1:1280 whereas the most effective anti-GP4 antibodies exhibited a comparable effect only up to 1:64. These results indicate that PRRSV GP5 in principle is a major target for neutralizing antibodies, as is found for other arteriviruses, but that in nature 'ORF 5 escape mutants' may develop as easily as in vitro.     

Genetic variation and phylogenetic analyses of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates

Swine herds in the US have experienced recent outbreaks of a severe form of porcine reproductive and respiratory syndrome (designated acute or atypical PRRS) characterized by abortion and high mortality in pregnant sows. Most of the affected herds had been vaccinated with modified live-vaccines (MLVs) against PRRS. To explore the possible mechanism of the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. The complete ORF5 gene of eight acute PRRSV isolates from herds experiencing acute PRRS outbreaks in Iowa and North Carolina was amplified and sequenced. Sequence analyses revealed that these acute PRRSV isolates shared 88-95% nucleotide and 88-96% amino acid sequence identities to each other, 87-97% nucleotide and 84-96% amino acid sequence identities with other North American PRRSV isolates and the MLVs. Most of the amino acid substitutions locate in the putative signal sequence and two short hypervariable regions at the amino terminus. The ORF5 gene sequence of the acute PRRSV isolate 98-37120-2 from a non-vaccinated swine herd in Iowa is very closely related to that of the RespPRRS MLV, with 97% nucleotide and 96% amino acid sequence identities. Phylogenetic analysis revealed that all eight acute PRRSV isolates are clustered within the North American genotype. Several minor branches that are not associated with geographic origins were also identified within the North American genotype. One acute PRRSV isolate (98-37120-2) is clustered with the RespPRRS MLV and several Danish isolates that were confirmed to be derived from the RespPRRS MLV. The ORF5 gene sequences of other seven acute isolates are more related to those of several earlier PRRSV isolates and the PrimePac MLV than to that of the RespPRRS MLV. Our results showed that the acute PRRSV isolates analyzed in this study differed from each other in ORF5 genes, although they all clustered within the North American genotype. The data from this study do not fully support the hypothesis that the emergence of acute PRRS is due to reversion of MLVs to a pathogenic phenotype, as only one of the eight acute isolates was shown to be very closely related to the RespPRRS MLV.     

一株类NADC30猪繁殖与呼吸综合征病毒的分离及遗传变异分析

为了解广西地区的猪繁殖与呼吸综合征病毒(PRRSV)的遗传变异及流行情况,本实验室从广西某发病猪场采集到的猪肺脏组织中检测到1株PRRSV,命名为GXNN1839,并对该毒株进行病毒的分离鉴定、GP5和Nsp2的测序分析.结果 显示:该毒株可在PAM细胞上分离增殖,有明显的细胞病变,通过IFA试验可以检测到细胞内PRR...     

用单克隆抗体鉴定猪繁殖与呼吸综合征病毒分离株

应用ＰＲＲＳＶ单克隆抗体,采用直接与间接免疫荧光抗体试验对分离获得的ＰＲＲＳＶ６个毒株进行了鉴定,结果所有分离毒株均能被单克隆抗体（ＳＤＯＷ１７、Ａ、Ｂ、Ｃ、Ｄ、Ｅ、Ｆ）所识别,呈现特异荧光,６个分离毒株均能与仅识别美洲型ＰＲＲＳＶ的单克隆抗体Ｆ反应,结果表明６个分离毒株均属于美洲型ＰＲＲＳＶ。利用微量细胞培养对分离毒株ＴＣＩＤ５０测定结果表明,６个分离毒株的ＴＣＩＤ５０分别为１０－７．５／０．１ｍｌ、１０－７．５／０．１ｍｌ、１０－７．５／０．１ｍｌ、１０－７．７５／０．１ｍｌ、１０－７．２５／０．１ｍｌ、１０－６．２５／０．１ｍｌ。病毒感染细胞的超薄切片电镜观察表明,在感染细胞浆内可见典型的ＰＲＲＳＶ病毒粒子,呈球形或椭圆形,直径约为６０ｎｍ左右,可见囊膜。     

Antigenic comparison of Lelystad virus and swine infertility and respiratory syndrome (SIRS) virus.

This study reports the antigenic relatedness of isolates of Lelystad virus collected in the Netherlands, Germany, and the United States. The binding of antibodies directed against these isolates was tested in a set of field sera collected during outbreaks of porcine epidemic abortion and respiratory syndrome in Europe and outbreaks of swine infertility and respiratory syndrome (SIRS) in North America. Two sets of sera from pigs experimentally infected with Lelystad virus or SIRS virus were also tested. Although all 7 isolates reacted with anti-Lelystad virus sera, antigenic variation was considerable. The 4 European isolates resembled each other closely, but differed from the American isolates, and the 3 American isolates differed antigenically from each other. To reliably diagnose Lelystad virus infection, a common antigen must first be identified.     

猪繁殖与呼吸综合征病毒强毒株HUB2株全基因组序列分析

从湖北省暴发猪"高热病"的猪场分离出1株猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV),并命名为HUB2株.根据GenBank上已发表的PRRSV全基因序列设计引物进行RTPCR扩增,获得PRRSV HUB2株全基因组cDNA序列.测序结果表明PRRSV HUB2株基因组全长15 320 bp(不包括PolyA尾).分析结果显示该毒株与PRRSV美洲型标准株(VR-2332)和欧洲型标准株(LV)全基因核苷酸同源性分别为89.6%和50.3%.说明HUB2属于美洲型毒株.与VR-2332相比,HUB2株非结构蛋白(Nsp2)存在2处不连续的缺失(共缺失30个氨基酸),其缺失位点位于推定氨基酸序列的第481位和532～560位.此次新出现的强毒株全基因组序列特性的揭示为科学防治猪高致病性蓝耳病奠定了理论基础.     

Epitope specificity of monoclonal antibodies to the N-protein of porcine reproductive and respiratory syndrome virus determined by ELISA with synthetic peptides

In an attempt to develop an alternate to ELISAs using recombinant N-proteins as antigen for the sero-diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infections of pigs I have measured the binding of nine anti-N-protein mAbs, which had been previously generated by various investigators, to overlapping peptides encompassing amino acids 19-70 of the N-proteins of the North American prototype (VR2332) and the European prototype (Lelystad virus, LV) of PRRSV. I also measured the binding of the mAbs to HerdChek ELISA plates coated with recombinant N-protein. All mAbs bound in an indirect ELISA to some of the peptides whether the mAbs had previously been reported to recognize continuous or discontinuous epitopes, but with different specificity and titer. Three mAbs bound with high titer to different linear epitopes located in amino acid segments 23-33, 31-50 and 43-56 and also with similar high titers to HerdChek plates. mAb SDOW17 bound with high titer to HerdChek plates but poorly to any of the peptides. In contrast, four mAbs bound with broad specificity to peptides containing an epitope(s) in amino acid segment 30-48, but poorly, or not at all, to HerdChek ELISA plates. Thus, this epitope is missing on the antigens of the HerdChek ELISA or is destroyed during immobilization of the antigens on the plate. A mAb to the N-protein of the closely related mouse arterivirus lactate dehydrogenase-elevating virus bound to the same epitope. Abs that bound with broad specificity to an epitope(s) in the 30-50 amino acid segment were also detected by the peptide ELISA in sera of 25 field sera that were sero-positive in the HerdChek ELISA, but also in sera of pigs from two out of three herds tested that were sero-negative by this test.     

Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies

Nineteen monoclonal antibodies (MAbs) with specificity for hog cholera virus (HCV) were prepared. They were used in an immune binding (peroxidase linked) assay to determine the reaction patterns of HCV isolates from Europe, Brazil, USA, Japan and Malaysia, as well as laboratory reference strains of the virus. A further panel of 17 MAbs raised against bovine virus diarrhoea virus (BVDV) was included in the study, together with 5 MAbs raised against a non-HCV pestivirus of porcine origin. All the MAbs were also tested against representative strains of BVDV and border disease virus. Six MAbs were HCV-specific, reacting with all isolates of HCV and none of the ruminant viruses. Among the other HCV MAbs geographical variation in reaction patterns was observed. There was evidence of antigenic distinction between recent European isolates, and archive material originally isolated more than 10 years ago.     

反转录-聚合酶链反应检测猪繁殖与呼吸综合征病毒的研究

本研究成功地建立了检测猪繁殖与呼吸综合征病毒（ＰＲＲＳＶ）的反转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术。根据ＰＲＲＳＶ两个标准毒株（美洲ＡＴＣＣ ＶＲ－２３３２株及欧洲ＬＶ株）膜蛋白和核衣壳蛋白的编码序列差异,自行设计合成了各自的引物,对两标准毒株及广州分离株（ＣＤＧ９５１２）进行扩增,获得了预期久为１ｋｂ的扩增片段。酶切鉴定,结果进一步主宰两标准毒株间基因差异显著,分离株与美洲株更为接近,百不同于