用LSAB免疫组化染色法检测猪生殖—呼吸道综合征病毒抗原？…

本研究在国内首次成功建立了辣根过氧化物酶标记的链霉亲和素－生物系（ＬＳＡＢ）免疫组化染色法检测猪生殖－呼吸道综合征病毒（ＰＲＲＳＶ）抗原。应用ＬＳＡＢ染色技术检测１２头人工感染ＰＲＲＳＶ美洲株（ＡＴＣＣ ＶＲ－２３３２）或国内分离株（Ｂ９６－４，Ｂ９６－５）的ＳＰＦ仔猪组织细胞内的ＰＲＲＳＶ抗原，阳性检出率为１００％。     

猪繁殖与呼吸综合征病毒SJ株(天津分离株)感染仔猪的病理学

选用10～15日龄健康仔猪20头，随机分为攻毒组（15头）与对照组（5头），攻毒组滴鼻接种猪繁殖与呼吸综合征病毒（PRRSV）SJ株，3mL／头；对照组滴鼻接种无PRRSV的细胞培养物，3mL／头。攻毒组的主要临床表现为眼睑水肿、打喷嚏、呼吸急促、嗜睡、体温略升高。攻毒组与对照组在试验开始后24h、7d、14d、21d、28d分别剖杀3头和1头仔猪，采集肺、脾、肾、扁桃体及各部位淋巴结作免疫组化及病理组织学观察；迅速采集肺组织，制作超薄切片，透射电镜观察。试验结果表明，PRRSV SJ株主要感染仔猪的肺脏与脾脏。免疫组化观察，攻毒后24h～7d，肺门淋巴结和扁桃体PRRSV抗原阳性反应最强；攻毒后14～28d，牌睦PRRSV抗原阳性反应最强。攻毒后14～28d，显微病变可见肺组织发生弥漫性、局灶性或间质性肺炎，肺泡隔因巨噬细胞、淋巴细胞及Ⅱ型上皮细胞增生而明显增厚。超微病变主要表现为，肺泡间隔中单核细胞增生，核孔消失，核内有异染色质聚集，Ⅱ型肺泡细胞高度变性等。     

寡核苷酸探针原位检测石蜡切片中PRRSV核酸

根据GenBank收录的美洲型猪繁殖与呼吸综合征病毒（PRRSV）ATCCVR-2332株ORF6和ORF7基因序列，用O1igo软件设计并合成大小为37bp的寡核苷酸探针，经生物素标记后，成功建立了原住检测石蜡组织切片中PRRSV核酸的方法。该探针能检测到56PgPRRSV核酸的RT—PCR产物DNA，能特异检测出PRRSV核酸及其PCR产物，而对猪瘟病毒（HCV）、猪细小病毒（PPV）、猪伪狂犬病病毒（PRV）、猪乙脑病毒（JEV）的核酸呈阴性反应。应用该方法检测PRRSVSC-1株人工感染的28日龄仔猪，在感染后7d即可在肺脏、肾脏、扁桃体、胸腺、肺门淋巴结、十二指肠和大脑检测到PRRSV核酸。该法可用于仔猪PRRSV感染的诊断和组织中核酸的定位及分布研究，也可用于甲醛固定组织的回顾性诊断。     

PRRSV间接免疫荧光检测法的建立和应用

以猪生殖与呼吸综合征病毒(PRRSV)ATCC VR-2332株人工单独感染3头3周龄仔猪，并用PRRSV ATCC VR-2332株和猪圆环病毒2型(PCV2)联合感染2头3周龄仔猪，每日检查试验猪体温、采食情况及临床表现，并用HerdChek IDExx试剂盒测其血清抗体。PRRSV PCV2联合感染猪分别在感染后1、3周剖杀，PRRSV单独感染猪和健康对照猪分别在感染后1、2和3周剖杀，对全部试验猪的肺组织进行灌洗处理以获得肺泡巨噬细胞(PAM)．用PAM进行抹片，同时用肺组织直接进行触片做间接免疫荧光(IIF)检测。结果表明，用所获得的3株单克隆抗体均检测到人工感染猪肺组织中的PRRSV抗原。另外，用IIF检测7头临床疑似PRRS病猪的PAM抹片。其中沭阳县的2头猪呈现阳性反应；对这2头猪的肺组织进行病毒分离。在盲传到第4代时Marc-145细胞上产生明显的细胞病变，进一步证实用所获得的3株单克隆抗体检测临床样品的结果准确而可靠，且获得的3株单克隆抗体均可通过IIF用于检测猪肺组织中的PRRSV抗原。     

PRRSV山西株ORF5和Nsp2基因序列分析

采用RT-PCR方法对2009—2011年山西省分离的5株猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus,PRRSV）的ORF5和Nsp2（2503～3269nt）基因进行克隆和测序,并对其基因序列和推导的氨基酸序列与国内外毒株进行了同源性分析。序列分析结果显示,5株分离株Nsp2基因与国内分离的PRRSV变异株（JXA1、HuN、HUN4、HUB1）的序列同源性最高,为96．8％～98．2％,且缺失位置一致,均存在2个位点30个氨基酸缺失;ORF5基因大小为603bp,编码200个氨基酸,第13、151位均为具有强毒特性的精氨酸（R）,137位为丝氨酸（S）,表明这5株均为野毒株,与国内分离的PRRSV变异株（JXA1、HuN、HUN4、HUB1）毒株的序列同源性最高,为96．5％～98．0％。结果表明,山西省内目前流行的PRRSV为Nsp2缺失30个氨基酸的变异毒株。     

间接免疫荧光试验（ⅡF）检测PRRSV方法的应用

用PRRSV ATCC VR-2332株人工单独感染了3头3周龄仔猪，并用PRRSV ATCC VR-2332株和猪圆环病毒2型(PCV2)联合感染了2头3周龄仔猪，同时设立了3头健康对照猪。攻毒后每日记录试验猪体温、采食、临床表现等，并用HerdChek IDEXX Kit测其血清抗体。PRRSV PCV2联合感染猪分别在感染后1周、3周剖杀，PRRSV单独感染猪和健康对照猪分别在感染后1周、2周和3周剖杀。对肺组织进行灌洗处理以获得肺泡巨噬细胞(PAM)，用PAM进行抹片，同时用肺组织直接进行触片作ⅡF检测。ⅡF试验结果表明，用所获得的3株单抗均检测到了人工感染猪肺组织中的PRRSV抗原。另外，用ⅡF检测临床疑似PRRS的7头猪的PAM抹片，其中沭阳2头猪呈现阳性反应。对这2头猪的肺组织进行病毒分离，结果在盲传到第四代时在Marc-145细胞上产生明显的细胞病变，进一步证实了所获得的3株单抗检测临床样品的准确性和可靠性。综合以上试验结果，证实了所获得的3株单抗均可通过ⅡF试验用于临床检测肺组织中的PRRSV抗原。     

检测伪狂犬病病毒双抗体夹心间接ELISA方法的建立与应用

以猪伪狂犬病病毒（PrV）Ea株特异性单克隆抗体576株为捕获抗体，纯化的抗PrV IgG为检测抗体，建立了检测PrV抗原的双抗体夹心ELISA方法。最佳反应条件为，单克隆抗体576株的包被浓度为12．2μg／mL，IgG的工作浓度为16．0μg／mL，对PrV的检测灵敏度达15．6ng（0．156μg／mL），与乙型脑炎病毒（JEV）、猪瘟病毒（HCV）、猪繁殖与呼吸障碍综合征病毒（PRRSV）、猪细小病毒（PPV）和猪流感病毒（SIV）等无交叉反应。批间和批内变异系数较小，分别为6．39％和4．1％。应用本方法对人工感染PrV的40日龄仔猪组织进行检测，肺和脑PrV抗原检出率最高，其次是脾、心、肝和肌肉。应用该方法和PrV对159份临床样本进行平行检测，发现二者的符合率可达到77．4％，比PCR敏感。实验结果表明：双抗体夹心间接ELISA方法具有敏感性高、特异性强和重复性好的特点，可广泛应用于动物组织中PrV的检测。     

一例猪繁殖与呼吸综合征醮实验室诊断

2011年10月,四川邛崃某猪场发生了一种以经产母猪流产、死产、产木乃伊胎、死胎及弱仔为主要特征的疾病。为确定病原,剖解2头病死仔猪,采集肺脏、脾脏组织,混合研磨提取组织总RNA,以实验室建立猪蓝耳病病毒（PRRSV）、猪瘟病毒（CSFV）核酸检测方法进行这2种病原的RT-PCR检测;采集小脑组织以酚氯仿法提取组织总DNA,进行伪狂犬病毒（PRV）PCR检测。结果显示CSFV与PRV均为阴性,PRRSV变异株和经典株均为阳性。诊断为猪蓝耳病变异株与经典株混合感染。     

猪繁殖与呼吸综合征病毒、猪圆环病毒2型与副猪嗜血杆菌混合感染的诊断及病原分析

为确定河南开封某猪场发生猪呼吸系统疾病综合征（PRDC）的病原,本研究无菌采集病死保育猪肺脏、心脏和脾脏等组织样品,进行细菌学检验和药敏试验,通过PCR/RT-PCR检测样品中猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）、猪流感病毒（SIV）、猪伪狂犬病病毒（PRV）、猪瘟病毒（CSFV）和猪肺炎支原体（Mhp）等病原,并对核酸阳性病毒性病原的抗原结构基因进行测序和遗传演化分析。结果表明,通过细菌分离培养、形态观察、卫星现象观察和16S rRNA基因鉴定,从病死保育猪体内分离鉴定出1株副猪嗜血杆菌（Hps）,药敏实验表明该菌株对对氨苄西林、阿莫西林克拉维酸、头孢噻呋和四环素几种药物敏感。核酸检测PRRSV和PCV2核酸阳性,分别命名为PRRSV/HN-2019和PCV2/HN11-2019;进一步对PRRSV/HN-2019和PCV2/HN11-2019的结构基因分析发现,PRRSV/HN-2019与与NADC30分支的毒株亲缘关系较近,属于NADC30-like毒株;PCV2/HN11-2019与PCV-2d分支的毒株亲缘关系较近,属于PCV-2d分支。综上所述,本研究确定该猪场存在PRRSV、PCV2和Hps的混合感染,为该猪场下一步的PRDC有效防控提供了参考依据。     

猪轮状病毒GZAS2020株与NX株VP7蛋白的结构预测和抗原表位分析

为了解猪轮状病毒（PoRV）VP7蛋白的生物信息学信息,运用Expasy-ProParam、YinOYang-1.2、TMHMM-2.0、ABCpred和IEDB等软件,预测分析PoRV临床分离株GZAS2020和疫苗株NX VP7蛋白的基本理化性质、结构功能和B细胞抗原表位差异。结果显示：两种VP7蛋白均属于稳定蛋白、亲水性蛋白、跨膜蛋白和非分泌性蛋白;分离株与疫苗株的O糖基化位点数分别为55和62,N糖基化位点数均为1,磷酸化位点数分别为29和30;亚细胞定位均位于质膜,均以α-螺旋和无规则卷曲为主要结构,延伸和β-转角贯穿其中;GZAS2020株的B细胞抗原表位位于67~72 aa（PYANSTT）、97~100 aa（KWTE）、176~179 aa（QQTD）、276~283 aa（TTAPQTER）,NX株位于96~99 aa（TKWK）、176~182 aa（QQTDEAN）、274~279 aa（DPTTTP）、310~317 aa（MSKRSRSL）,两者在97~100、176~179、276~279 aa处存在相同的B细胞抗原表位,但GZAS2020株在无规则卷曲位置（67~72 aa）存在独特的B细胞抗原表位。以上结果说明,疫苗株NX与贵州省流行株GZAS2020在主要抗原蛋白上存在差异。     

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.     

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.     

检测CSFV、JEV、PRRSV三种RNA病毒多重RT-PCR方法的建立

猪瘟病毒(CSFV)、流行性乙型脑炎病毒(JEV)和猪繁殖与呼吸综合征病毒(PRRSV)是引起严重的种猪繁殖障碍的病原,而且经常混合感染,及时准确诊断是防治的前提。根据GenBank发表序列选取3对引物建立检测CSFV、JEV和PRRSV病毒的多重RT-PCR方法,扩增产物分别为508 bp、380 bp、263 bp。经与IDEXX商品化的检测CSW抗原试剂盒比较,二者的符合率为96.7%;扩增JEV和PRRSV PCR产物分别经EcoR V和Sau3A I酶切得到预期的片段。建立的多重RT-PCR检测JEV、PRRSV和CSFV敏感度分别为12.5个TCID_(50)、10个TCID_(50)和10~(-3)ng总RNA。结果表明该多重RT-PCR方法具有很好的特异性和敏感性,可用于临床三种病毒核酸的检测。     

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.     

Effect of formalin fixation on the immunohistochemical detection of PRRS virus antigen in experimentally and naturally infected pigs.

The purpose of this study was to determine the effect of formalin fixation on the immunohistochemical detection of porcine reproductive and respiratory syndrome (PRRS) viral antigen in lungs of experimentally and naturally infected pigs. In separate trials, five 24-day-old pigs and six 10-day-old pigs were housed as separate groups in isolation and inoculated intranasally with 10(5.5) TCID50 of an isolate of PRRS virus (PRRSV; P129). The older and younger pigs were euthanatized at 7 and 10 days post inoculation (dpi), respectively. At necropsy, all pigs had gross and microscopic lung lesions typical of PRRS, and PRRSV was isolated from all pigs. To insure uniform fixation, lungs from each pig were cut into 1-cm-thick slices and immersed into 10% neutral-buffered formalin. After fixation in formalin for 8 hours or 1, 2, 3, 5, 6, 8, 10, and 15 days, 3 lung sections from some or all pigs were processed for histological examination using routine methods. Immunohistochemical staining for PRRSV antigen was positive at the following times (days unless otherwise stated) after fixation (percentage of pigs staining positive for PRRSV in parentheses): 8 hours (100); 1 (100); 2 (100); 3 (80); 5 (33); and 6, 8, 10, and 15 (0-all negative). To further evaluate the effects of formalin fixation on PRRSV immunodetection, 31 field cases of PRRS were selected for immunohistochemistry (IHC). Over a 3-month period, submitted cases were selected from the Purdue University Animal Disease Diagnostic Laboratory, W. Lafayette, Indiana, for IHC if 1) the clinical history included respiratory disease, 2) PRRSV was isolated from lung and/or serum from the submitted pigs or tissues, 3) at least 1 section of lung fixed in 10% neutral-buffered formalin was submitted for IHC, and 4) the duration of fixation could be accurately determined from the case history. Of the 31 PRRSV-infected pig cases meeting the selection criteria, 23 were fixed in formalin for 4 days or less. Twenty-one of these 23 (91%) were positive by IHC. Two of 8 cases fixed for greater than 4 days (25%) were positive by IHC. In practical terms, 1-day shipping of fixed samples to a laboratory followed by routine tissue processing within a laboratory should not adversely affect immunohistochemical detection of PRRS viral antigen. But a delay in shipping or processing of more than 2 days could reduce or prevent the detection of PRRS viral antigen by IHC.     

Identification of viral pathogens in aborted fetuses and stillborn piglets from cases of swine reproductive failure in Spain

The objective of the present study was to determine the presence of recognised abortifacient viruses such as porcine reproductive and respiratory virus (PRRSV), Aujeszky's disease virus (ADV), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2), in tissues from aborted fetuses and stillborn neonates in cases of late reproductive failure in swine. A total of 293 specimens (fetuses aborted in the last third of gestation and stillborn piglets) from 100 different cases of late-term abortions and premature farrowing from 15 different Spanish provinces were studied. PRRSV was detected in 9/100 cases by RT-PCR. Only 1/100 cases analysed (corresponding to a late-term aborted fetus with a negative PRRSV RT-PCR result) was positive for PCV2 by PCR. Neither ADV (monitored by viral isolation plus antigen detection) nor PPV (monitored by ELISA antigen capture test) infection was identified. The results suggest that PRRSV is one of the most important infectious agents, if not the most relevant one, associated with fetal infection leading to abortion or premature farrowing in Spain. Moreover, other viral pathogens such as ADV, PPV and PCV2 seem to have a minor impact on reproductive disease.     

重组N蛋白抗原检测PRRSV抗体ELISA的研究Ⅳ.与IDEXX ELISA试剂盒及Western blot的比较

利用重组N蛋白抗原建立了检测猪繁殖与呼吸综合征病毒（PRRSV）抗体的ELISA方法并组装成试剂盒，将试制的3批试剂盒分别与IDEXX生产的试剂盒及Western—blot进行了符合率试验。试验结果表明，所研制的试剂盒与Western blot的符合率达97．67％以上。对460份临床血清分别用自制的试剂盒和IDEXX公司生产的试剂盒进行检测，其中有37份不相符合，用Western blot对这37份血清进行验证，有35份血清检测结果与自制的试刺盒检测结果一致。由此表明，自行研制的试剂盒，其特异性和敏感性均能满足目前临床上该疫病的流行病学分析或免疫抗体检测。     

猪瘟病毒猪细小病毒猪繁殖与呼吸综合征病毒猪伪狂犬病病毒多重PCR方法的建立以及初步应用

猪瘟病毒、猪细小病毒、猪繁殖与呼吸综合征病毒及猪伪狂犬病病毒均能导致猪繁殖障碍,对养猪生产影响很大。本研究通过设计4对针对这4种病毒的特异引物建立了多重PCR方法,分别对其最佳反应条件、特异性及敏感性进行了测定,结果表明:其敏感性可达到CSFV 10 TCID50,PPV 10TCID50,PRRSV 1 TCID50,PRV 100 TCID50 CSFV 10TCID50,PPV 10TCID50,PRRSV 1 TCID50,PRV 100 TCID50。同时具有较好的特异性,对猪瘟病毒石门株、猪瘟病毒兔化弱毒株、PRV闽南A株、PPV弱毒疫苗株、PRRSV KY 35株及PRRSV B13株6个毒株均能扩增出相应的片段,而BVDV、BDV均未扩增出相应的片段。本方法的建立对于这4种病毒病的早期快速检测具有十分重要的意义。     

不同接毒方法对猪繁殖与呼吸综合征病毒增殖效果比较

通过对猪繁殖与呼吸综合征病毒（PRRSV）感染Marc-145细胞4种不同接毒方法的比较研究,筛选出一种在规模化生产中既能保证毒价,又能减少工艺流程,提高工作效率,降低污染几率及生产成本的接种方法。结果表明,与其他方法相比,同步接毒法效果最佳,不仅毒价最高,而且繁殖毒液时间缩短,减轻了工作量,节省生产成本。