Utilization of a rate enhancement hybridization buffer system for rapid in situ hybridization for the detection of porcine circovirus in cell culture and in tissues of pigs with postweaning multisystemic wasting syndrome.

A rapid in situ hybridization (ISH) technique for the detection of porcine circovirus (PCV) nucleic acid in cell culture and formalin-fixed paraffin-embedded tissues was developed. A fluorescein-labeled RNA probe was transcribed from a plasmid containing 530 bp of the ORF1 of a PCV isolated from a pig with postweaning multisystemic wasting syndrome (PMWS). Hybridization using standard hybridization buffer was performed at 42 C for 16 hours and was compared to hybridization using rate enhancement hybridization (REH) buffer at 67 C for 2 hours. Hybridization was detected with an alkaline phosphatase-conjugated antifluorescein antibody. In both cultured cells and tissues from pigs with PMWS, the signal intensity and number of labeled cells in sections hybridized with REH buffer were equal to those of sections hybridized with standard hybridization buffer. The total time required for ISH using the REH buffer is 7-8 hours, thus making this protocol suitable for application in routine PCV diagnosis.     

猪圆环病毒2型原位杂交检测方法的建立

参照GenBank发表的PCV2ORFl基因序列设计了1对引物,利用PCR地高辛探针合成的方法制备了长度为494bp的特异性探针,经检验具有良好的特异性和敏感性,可检测最低质粒DNA质量浓度为0．9728ug／L。用该探针建立了原位杂交组织切片检测方法,并用来检测PCV2感染猪的扁桃体和淋巴结组织,结果表明阳性信号主要存在于巨噬细胞胞浆中,信号强、背景良好,阴性对照无显色,说明该方法可作为PCV2实验室诊断和机理研究的一种有效检测方法。     

猪圆环病毒2型原位杂交检测技术的建立与应用

参照GenBank发表的猪圆环病毒2型（PCV2）ORF2基因序列设计引物，利用PCR扩增得到PCV2BF株341bp的核酸片段，用随机引物法制备出地高辛标记的核酸探针。制备的探针与PCV1、PRRSV、PPV、PRV等不发生反应，可检测的最低PCV2DNA含量为1．78Pg。对30份临床组织样本进行了检测，并与PCR比较，结果表明，阴性符合率为100％，阳性符合率为88．9％。应用原位杂交技术分析了PCV2在人工感染仔猪主要组织中的分布，结果表明，感染后3d，从仔猪的淋巴结、胸腺、肺脏、脾脏、鼻黏膜可检测到阳性信号，感染后21d，肝脏、肾脏、胰腺和回肠可检出阳性信号，至感染后42d，可从心脏、胃、脑检出阳性信号。在整个试验过程中会厌软骨、膀胱、皮肤、肌肉等组织均为阴性。本研究结果表明，建立的PCV2原位杂交技术具有良好的敏感性和特异性，可用于PCV2的实验室诊断和感染靶细胞的定位分析。     

Retrospective study on porcine circovirus type 2 infection in pigs from 1985 to 1997 in Spain

A retrospective survey was performed to detect lesions of Postweaning multisystemic wasting syndrome (PMWS) and nucleic acid of porcine circovirus type 2 (PCV2) in archived formalin-fixed, paraffin-embedded tissues from 189 pigs, and antibodies to this virus in sera of 388 pigs from the Spanish livestock between the years 1985 and 1997. PCV2 nucleic acid was detected by in situ hybridization (ISH) in tissues from 78 of 189 (41.3%) examined pigs. Variable amount of viral genome was detected in association with slight to severe microscopic lymphoid lesions consisting of lymphocyte depletion and histiocytic infiltration. The first positive case of PMWS with typical lesions and ISH positive corresponded to a pig necropsied in 1986. Two hundred and eighty-two of 388 (72.7%) sera were positive by immunoperoxidase monolayer assay. Serological and pathological data of the present study indicate that PCV2 was a enzootic infection in Spain since 1985, suggesting that the introduction of this virus in the livestock occurred previously.     

Differentiation of porcine circovirus 1 and 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome by in-situ hybridisation

Non-radioactive digoxigenin (DIG)-labelled probes that can differentiate porcine circovirus (PCV) 1 from PCV2 in formalin-fixed, paraffin-wax-embedded tissues by in-situ hybridisation were developed. A 349 base pair (bp) DNA fragment from open reading frame (ORF) 1 of PCV1 and a 481 bp DNA fragment from ORF2 of PCV2 generated by polymerase chain reaction (PCR) were used as PCV1 and PCV2 probes, respectively. A specific DIG-labelled PCV1 DNA probe did not hybridise with PCV2-infected PK-15 cells and vice versa. From the 40 field cases with postweaning multisystemic wasting syndrome tested by in-situ hybridisation, 30 (75 per cent) cases were PCV2-positive only and 10 (25 per cent) cases were positive for both PCV1 and PCV2. PCV1 and PCV2 DNAS were detected mainly in the macrophages of lymph nodes and spleens. Positive cells typically exhibited a dark brown to black reaction product mainly in the cytoplasm but also occasionally in the nucleus. In-situ hybridisation together with the differential probes developed in the present study represent an additional tool capable of differentiating of both types of PCV in formalin-fixed, paraffin-wax-embedded tissues.     

Double in situ hybridization for simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV).

A double in situ hybridization method for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV) genomes in the same tissue section was applied to lung tissues from 9 pigs in which PRRSV and PCV coinfection had been previously demonstrated. Paraffin-embedded tissue sections were simultaneously hybridized with a digoxigenin-labeled antisense RNA probe for PRRSV and a fluorescein-labeled antisense RNA probe for PCV, and hybridization was detected with anti-digoxigenin alkaline phosphatase/fast red and anti-fluorescein peroxidase/diaminobenzidine, respectively. PRRSV and PCV genomes were identified in the same pulmonary cell types as reported previously in all 9 pigs. In all pigs, PCV-positive cells outnumbered PRRSV-positive cells. A small proportion of alveolar macrophages contained both PRRSV and PCV genomes.     

Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome

Thirty-three pigs affected by porcine dermatitis and nephropathy syndrome, 30 from Spain and three from the USA, were investigated in order to detect porcine circovirus (PCV) in their tissues. A standard in situ hybridisation technique using a specific DNA 317-bp probe based on a well-conserved sequence of PCV (which recognises both PCV-1 and PCV-2) was applied to formalin-fixed, paraffin-embedded tissues. Twenty-eight of the 30 Spanish pigs and all three American pigs had PCV in at least one tissue. Viral nucleic acid was detected mainly in lymphoid organs, and especially the lymph nodes. The viral genome was also found, in order of decreasing quantity, in Peyer's patches, tonsil, lung, spleen, kidney, liver, and skin. Viral nucleic acid was located mainly within the cytoplasm of monocyte/macrophage lineage cells, including follicular dendritic cells, macrophages, histiocytes and Kupffer cells. No viral nucleic acid was found in damaged glomeruli or arteriolar walls. In frozen samples available from three Spanish pigs, the virus was identified as type 2 by using the polymerase chain reaction and restriction fragment length polymorphism. Most of the pigs from which serum was available were seropositive against porcine respiratory and reproductive syndrome virus (PRRSV), and PRRSV antigen was detected in the lung of two of the Spanish pigs. These results suggested that PCV is present in tissues of almost all pigs affected by PDNS, and PCV has to be considered as a possible agent involved in the pathogenesis of the syndrome.     

Characterization of interstitial nephritis in pigs with naturally occurring postweaning multisystemic wasting syndrome

Kidney samples with interstitial nephritis from 26 pigs affected by postweaning multisystemic wasting syndrome (PMWS) were selected. A histologic evaluation was carried out to describe the type of inflammation and its relationship with viral load, as assessed by in situ hybridization (ISH). Of 26 cases, 10 revealed a tubulointerstitial, lymphoplasmacytic nephritis, 11 an interstitial granulomatous nephritis, and 5 both types of inflammation (mixed type). In 4 cases of granulomatous inflammation, the pattern was not classically nodular, and a population of macrophages and lymphocytes was present (interstitial lymphohistiocytic nephritis). ISH confirmed the presence of porcine circovirus type 2 (PCV2) nucleic acid in all cases. The epithelium of the renal tubules was the most constantly ISH-positive structure. In tubulointerstitial nephritis, the higher the number of positive inflammatory cells, the more severe the inflammation. The ISH reaction was more heterogeneous and unpredictable in granulomatous nephritis, with some epithelioid and giant cells positive by ISH. To quantify macrophages distributed in the three patterns of nephritis, immunohistochemical methods using anti-major histocompatibility complex II (anti-MHC-II) and anti-lysozyme antibodies were undertaken, and semiquantitative evaluation was carried out. MHC-II was mainly expressed by lymphocytes in tubulointerstitial nephritis, but did not always stain macrophages in cases of granulomatous (including lymphohistiocytic) nephritis; the anti-lysozyme antibody revealed macrophages when present in tissues. The amount of PCV2 nucleic acid was not apparently associated with the pattern of inflammation (tubulointerstitial or granulomatous). PCV2 load seems to reflect the severity of the lymphoplasmacytic inflammation but not that of granulomatous and lympho histiocytic types.     

Study of the genetic variability of porcine circovirus type 2 detected in Serbia and Slovenia

Recent variants of porcine circovirus type 2 (PCV2) were obtained from tissues of domestic pigs with porcine circovirus associated disease and from randomly selected wild boar samples from Serbia and Slovenia. A 450-base-pair nucleotide sequence was obtained by PCR from the ORF2. The derived nucleotide and amino acid sequences were aligned and compared to the corresponding region of closely related PCV2 sequences determined in previous years and retrieved from the GenBank. The 30 Serbian and 17 Slovenian PCV2 sequences clustered into three previously determined genotypes (PCV2a: 7), (PCV2b: 38) and (PCV2d: 2). Three major variable regions, concerning 29 amino acid position substitutions within the ORF2, were observed, which further supports the segregation of the detected strains into three separate genotypes. This study indicates that PCV2b is the predominant genotype in Serbia and Slovenia and the detected PCV2 strains are closely related to those previously described in Europe and in other parts of the world.     

猪圆环病毒2型TaqMan实时荧光定量PCR检测方法的建立

本研究以猪圆环病毒2型(PCV2)核衣壳蛋白ORF2基因为目的基因,设计合成特异性引物和探针。PCR扩增得到目的基因,并克隆到pMD18-T载体,筛选得到标准阳性质粒。通过对荧光定量PCR反应条件的优化,建立了PCV2的TaqMan实时荧光定量PCR检测方法。试验结果表明,该方法特异性强,对猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)等猪常见病原的检测结果均为阴性;与普通PCR方法相比,其敏感性高出100倍,可达100拷贝/μL;对临床样品的检测证明,该方法可有效检测出淋巴结、肺脏等组织中的PCV2。     

Development and evaluation of an indirect in situ polymerase chain reaction for the detection of porcine circovirus type 2 in formalin-fixed and paraffin-embedded tissue specimens

Taking advantage of the high sensitivity of polymerase chain reaction (PCR) and the cell-localizing ability of in situ hybridization (ISH), an indirect in situ PCR (ISPCR) method was developed for detecting the distribution of porcine circovirus type 2 (PCV2) in formalin-fixed and paraffin-embedded inguinal lymph nodes obtained from clinically healthy PCV2-carrier pigs and postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Comparisons of the relative sensitivity of indirect ISPCR with other routinely used diagnostic methods for PCV2 indicated that nested PCR was the most sensitive method followed by indirect ISPCR, conventional PCR, ISH, and immunohistochemical (IHC) staining. Although indirect ISPCR, ISH, and IHC staining all revealed a similar signal distribution pattern of PCV2, using indirect ISPCR allowed specific amplification and detection of previously uneasily detected PCV2 signal than by routine ISH or IHC staining, particularly in those cells within the germinal center in clinically healthy PCV2-carrier pigs. Furthermore, six different PCV2 signal expression patterns in conjunction with the correlated lymphoid lesion stages were classified to describe the tissue morphological changes and viral infection. The result indicates that indirect ISPCR is a more effective, cell-based diagnostic tool with good specificity to detect limited PCV2 infection in formalin-fixed and paraffin-embedded tissue specimens and it would be a useful tool for further exploring the pathogenesis of PCV2 infection.     

Evaluation of commercial polyclonal- and monoclonal-antibody-based immunohistochemical tests for 2 genotypes of Porcine circovirus type 2 and comparison with in-situ hybridization assays

The objective of the present study was to evaluate polyclonal- and monoclonal-antibody-based immunohistochemical (IHC) tests for the detection of 2 genotypes of Porcine circovirus type 2 (PCV2), a and b, in formalin-fixed, paraffin-embedded lymph-node tissue from pigs with experimental or natural postweaning multisystemic wasting syndrome and to compare the IHC results with those of in-situ hybridization (ISH) assays. The ISH assays proved more sensitive than the IHC tests for the detection of PCV2a and PCV2b. According to these findings, polyclonal-antibody-based IHC testing is the most practical routine diagnostic method for the detection of PCV2 regardless of genotype because IHC testing is less technically complex than ISH testing. However, ISH assays are useful to differentiate between PCV2a and PCV2b in surveillance programs for the monitoring of PCV2 in swine herds.     

猪场母猪与仔猪猪圆环病毒2型感染情况调查

为了解目前母猪和仔猪猪圆环病毒2型(PCV2)感染情况,本试验对来自于15个猪场的85份母猪血清和29个猪场的55份仔猪可疑病料(脾脏、淋巴结、肾脏等),分别采用间接ELISA和PCR法进行PCV2抗体和病毒核酸检测。结果显示,在15个猪场中,1个猪场母猪抗体呈阴性,其他14个猪场母猪抗体均呈阳性,猪场PCV2阳性检出场为93.3%(14/15),85份母猪血清抗体总阳性率为75.3%(64/85)。29个猪场的仔猪,PCV2核酸检测阳性猪场为19个,猪场阳性率为65.5%(19/29),55份仔猪可疑病料病毒核酸检测总阳性率61.8%(34/55)。可见,母猪和仔猪感染PCV2相当普遍。对母猪群PCV2抗体阳性率及其仔猪群病毒核酸阳性率进行比较发现,母猪群PCV2抗体阳性率越高其仔猪群病毒核酸阳性率却越低。     

猪圆环病毒Ⅱ型广东分离株全基因组的克隆和序列分析

分离了9株猪圆环病毒Ⅱ型（PCV2）广东地方分离株，并进行了全基因组序列测定；对这9株PCV2广东分离毒株的ORF1和ORF2基因的序列分析表明ORF2的变异程度要比ORF1的变异程度大；对PCV2衣壳蛋白的氨基酸序列同源性比较发现了PCV2毒株间存在1个氨基酸变异程度较大的区域和2个氨基酸变异程度较小的区域，其中前两个区域与两个主要的免疫反应区域相对应；将这9个毒株的全基因组序列与GenBank上收录的18个PCV2毒株的全基因组序列基因进化树分析表明，这9个PCV2广东分离株彼此之间及与国内PCV2分离株、欧洲株之间更接近，而与韩国、中国台湾、日本和美洲毒株之间则稍远，因而在选PCV2疫苗免疫时，建议尽量使用国内生产的疫苗或自制组织灭活疫苗进行免疫。     

凉山州腹泻仔猪猪圆环病毒2型感染情况调查

为了解凉山州腹泻仔猪猪圆环病毒2型(PCV2)的感染情况,采用PCR方法对采自四川省凉山州西昌市和喜德县7个猪场85份腹泻仔猪粪便、病变组织等样品进行PCV2检测,并将扩增到的PCV2 ORF2基因片段进行测序和序列分析。结果显示,PCV2阳性样品有22份,阳性率为25.9%,阳性猪场占71.4%(5/7)。22个ORF2基因片段长度均为459 bp,核苷酸同源性为92.8%～100%,氨基酸同源性为93.5%～100%。构建的系统进化树显示22个测序序列分别处于2个分支:PCV2b和PCV2d,但未形成明显的地理分支。结果表明凉山州猪群中PCV2感染较为普遍,且以PCV2d亚型为主,并存在一定程度的遗传变异。     

Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica

Porcine circovirus (PCV) was recently divided into 2 antigenically distinct types that differ (65% amino acid identity) in the protein encoded by open reading frame 2 (ORF2). Porcine circovirus 1 is apparently non-pathogenic and, in contrast, PCV2 is associated with porcine multisystemic wasting syndrome (PMWS). Our objective was to determine the extent of exposure of normal pigs in Canada and Costa Rica to PCV2. Recombinant DNA techniques were used to produce an antigen from ORF2 of PCV2 that was suitable for the detection of antibody in swine sera. The presence of PCV2 nucleotide sequences was detected using polymerase chain reaction (PCR) techniques. Using these tests, specific antibody and nucleotide sequences were demonstrated in sera from a cohort of pigs during a PMWS outbreak. Antibody was detected in normal, healthy hogs slaughtered in Canada (82.4% of 386) and in Costa Rica (14.6% of 322). This is the first report indicating the presence of PCV2 in Latin America. More than 50% of these sera also contained PCV2 nucleotide sequence. Although these hogs were healthy when slaughtered, they were infected with PCV2 and may have previously been ill. The widespread occurrence of PCV2 in swine suggests that this virus is adapted to replication in porcine tissue.     

A comparison of immunohistochemistry and in situ hybridization for the detection of porcine circovirus type 2 in pigs

The aim of this study was to develop and to optimize an immunohistochemistry (IHC) method for PCV2 identification and to compare it with an in situ hybridization (ISH) technique. The results demonstrated that both ISH and IHC successfully detected PCV2 viral antigens or nucleic acid in the examined tissues. Most of the slides identified previously in ISH as PCV2-positive were also positive in IHC. In the case of nearly half of the slides the results of IHC examination revealed an increase in the intensity of staining. IHC presented higher sensitivity and specificity than ISH. No negative impact of the time of paraffin block storage on ISH detection results was observed. In addition, IHC results were easier to interpret due to better image quality after staining. Overall results confirmed IHC was a reliable and useful technique for PMWS diagnosis.     

ORF9基因缺失突变PCV2的构建与部分生物学特性

设计1对针对猪圆环病毒2型（PCV2）全基因组的特异性引物,从疑似断乳仔猪多系统衰竭综合征（PMWS）的病料中经PCR直接扩增出PCV2全基因组,再与载体pMD18-T Simple连接后构建重组质粒pMD18-T Simple-PCV2（命名为P-S-PCV2）。用ORF9特异的限制性内切酶Bpu10Ⅰ对P-S-PCV2进行酶切、补平连接反应,构建了ORF9基因缺失突变的重组质粒（命名为P-S-PCV2-J）。用SacⅡ对基因缺失突变的重组质粒进行酶切,获得的线性化基因突变PCV2基因组在体外进行自身环化,形成了相应缺失基因DNA（命名为PCV2-J）。用PCV2-J缺失突变株进行细胞转染、动物致病性和免疫原性、T淋巴细胞亚群的动态变化等部分生物学特性的研究。结果显示：PCV2-J转染IBRS-2细胞后,电镜观察可见病毒颗粒,PCR-RFLP检测有突变株生长;PCV2-J接种仔猪后无临床典型大体病变,PCR-RFLP检测淋巴结中有PCV2-J突变病毒的感染;PCV2-J免疫仔猪后的抗体水平在第2周开始上升,与对照组相比差异显著,CD3＋下降,与对照组无差异,CD4＋下降,第1周与对照组差异显著,CD8＋与对照组差异不显著。结果表明,ORF9基因缺失突变的PCV2仍具有复制感染能力,但免疫原性减弱。     

我国部分地区猪圆环病毒2型分离株的遗传变异分析

为了解我国猪圆环病毒2型(PCv2)流行毒株遗传变异情况,本研究对本实验室分离到的19株PCV2分离株通过病毒全基因组克隆和测序分析,将其分为2个大基因群和3个亚群,其基因组分别为1 766 nt、1 767 nt和l 768 nt,各占15.8%、73.7%和10.5%.以1 767 nt毒株为基准,其基因组第39或1 039位有1个碱基缺失后突变成1 766 nt毒株;第1 040位有1个碱基插入后突变成1 768 nt毒株.对19株病毒ORF2编码的Cap蛋白分析发现,有4株病毒编码基因发生了突变,出现了705 nt和708 nt两种突变型,使ORF2编码的Cap蛋白C末端分别有1和2个氨基酸增加.同时,本实验选用Acc I和Fba I内切酶对19株病毒基因组PCR产物进行了限制性片段长度多态性(PCR-RFLP)分析,其结果可作为流行毒株分型鉴别依据.