仔猪感染猪繁殖与呼吸综合征病毒的病毒定位研究

以猪繁殖与呼吸综合征病毒(Porcine reproduction and respiratory syndrome virus,PRRSV)天津分离株(PRRSV-SJ株)滴鼻感染10日龄～15日龄PRRSV阴性的健康仔猪,采用免疫组织化学法检测PRRSV在仔猪体内靶器官的动态分布,初步探讨PRRSV感染后在仔猪体内的病毒分布规律.结果表明,间接免疫组织化学法发现PRRSV-SJ株感染仔猪后1 d～28 d,均可在仔猪肺、淋巴结、扁桃体、气管、脾、肾等组织中检出PRRSV.感染后1 d～7 d,肺门淋巴结和扁桃体的病毒阳性细胞数量最多,阳性反应强.感染后14 d～28 d,脾脏的病毒阳性细胞数量多,阳性反应强.感染后1d,PRRSV就可在气管、肺门淋巴结和扁桃体检出.     

RNAscope原位杂交技术检测猪繁殖与呼吸综合征病毒RNA方法的建立

为了直观、高效、特异性地检测猪繁殖与呼吸综合征病毒(PRRSV)感染细胞和组织中PRRSV RNA,本研究根据HP-PRRSV Hu N4株ORF7基因序列设计8对28 bp~36 bp双"Z"结构的寡核苷酸探针(PRRSV-Nprobes),建立了一种新的PRRSV RNA原位杂交检测的方法。该方法能特异性的检测PRRSV感染猪肺泡巨噬细胞(PAMs)中PRRSV RNA,而对猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)、猪伪狂犬病毒(PRV)感染PAMs中的核酸呈现阴性。应用该方法检测PRRSV感染仔猪肺脏、腹股沟淋巴结和胸腺石蜡包埋组织切片中PRRSV RNA,在感染的腹股沟淋巴结中PRRSV RNA呈现集中分布,而PRRSV RNA在肺脏和胸腺中呈弥漫性分布。该方法可用于细胞和组织中核酸的定位及分布规律的研究,特别是用于病毒含量较少的潜伏感染的检测,为PRRSV实验室检测和致病机理研究奠定了良好的试验基础和技术支持。     

猪繁殖与呼吸综合征病毒和猪圆环病毒2型共感染对猪体内病毒动态及免疫反应的影响

将猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)单感染和共感染6周龄健康仔猪,采用实时定量PCR(Real-time PCR)对血清、组织(心、肝、肺、肾、胰、脾、胸腺、扁桃体、腹股沟淋巴结、肠系膜淋巴结)中的PRRSV和PCV2载量进行检测,分别采用间接ELISA和间接IFA对血清中的PRRSV和PCV2特异性抗体进行检测,采用MTT法对猪外周血淋巴细胞(PBLC)的增殖能力进行检测.结果,PRRSV/PCV2共感染组血清、组织中PRRSV的载量均显著高于PRRSV单感染组,PCV2的载量均显著高于PCV2单感染组;共感染组PRRSV和PCV2特异性抗体阳转的时间均晚于并且效价均显著低于PRRSV或PCV2单感染组;共感染组和单感染组PBLC的增殖能力均受到抑制,其严重程度依次为:PRRSV/PCV2组>PRRSV组>PCV2组.由此表明,PRRSV和PCV2在猪体内对彼此的增殖具有促进作用;PRRSV和PCV2共感染对猪的免疫抑制具有加重作用.     

华南地区猪圆环病毒和猪繁殖与呼吸综合征病毒检测分析

为了解华南地区猪圆环病毒及猪繁殖与呼吸综合征病毒的最新流行情况,采集了华南地区11个规模猪场各饲养阶段猪血清807份,用套式PCR(nPCR)检测猪圆环病毒1型(PCV-1)和猪圆环病毒2型(PCV-2);采集了若干猪场2010年1月至2011年8月期间有咳嗽、喘气、消瘦及疑似PDNS等临床症状的猪血清312份,以及无临床症状猪血清104份,以nPCR检测PCV-2,以一步法反转录PCR(RT-PCR)检测猪繁殖与呼吸综合征病毒。结果发现,所调查的11个规模猪场中只有5个检出PCV-1,所有猪场均检出PCV-2。PCV-2阳性率为30.61%,而PCV-1阳性率仅为4.21%;经产母猪和7周龄以上保育猪PCV阳性率最高;有临床症状的猪血清PCV-2阳性率为58.65%,PRRSV阳性率为37.82%,PCV-2阳性猪群中有39.89%的猪同时感染PRRSV;有症状猪群7月～9月的PCV-2感染率最高,而1月～3月最低;无临床症状猪血清PCV-2阳性率为27.9%,PRRSV阳性率为0.96%,PCV-2与PRRSV无混合感染。证明PCV尤其是PCV-2在华南地区仍广泛传播并流行,而且PCV-2与PRRSV混合感染致病情况较多。PCV-2的感染率与季节有一定的相关性,种猪带毒情况严重。     

猪繁殖与呼吸综合征病毒单克隆抗体的研制

以猪繁殖与呼吸综合征病毒(PRRSV)JYC株全病毒作为免疫原,免疫BALB/c小鼠,融合后经间接ELISA进行杂交瘤筛选,共获得4株能分泌针对PRRSV单克隆抗体(简称单抗)的杂交瘤细胞株,分别命名为2G7、6G7、7F2和7E8,其亚类均为IgG1;单抗腹水的间接ELISA效价分别为10×215、10×211、10×215、10×214;间接ELISA试验结果表明,4株单抗与实验室保存的江苏地区临床分离的12株PRRSV以及VR-2332均发生特异性反应,而与猪常见其他病毒如猪细小病毒(PPV)、猪伪狂犬病毒(PRV)、猪瘟病毒(HCV)、猪圆环病毒2(PCV2)均无反应性;间接荧光试验表明,4株单抗与Marc-145细胞感染的PRRSV均呈特异性的荧光反应。因此这些单抗均可用于ELISA方法以及间接荧光试验,从而为建立快速、敏感的PRRSV的抗原检测方法奠定了基础。     

用PCR法检测猪繁殖-呼吸综合征病毒和猪圆环病毒混合感染病例研究

根据已发表的猪繁殖-呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV-2)的核苷酸序列设计特异性引物，应用PCR方法对从福清市某养猪场病猪采集的淋巴结、肺、脾等器官组织病料进行检测，发现该猪场病猪病料的检测结果PRRSV和PCV-2同时为阳性，从而证实了PRRSV和PCV-2在该病猪体内混合感染。     

广西猪繁殖与呼吸综合征病毒感染状况调查

采用RT-PCR技术,对2004年1月至2005年4月期间,广西13个市104个疑似猪繁殖与呼吸综合征病毒(PRRSV)感染猪场,无菌采取231头病、死猪的组织病料(肺脏、淋巴结、脾脏)进行了病毒检测。同时,对鉴定为PRRSV阳性的组织病料和猪场进行了猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)和猪伪狂犬病毒(PRV)的检测,以确定猪群中PRRSV与PCV2、CSFV和PRV混合感染情况。结果从12个市的115份组织病料中检出PRRSV,病料的平均阳性率为49.78%(115/231),猪场的平均阳性率为61.54%(64/104),不同地市有一定的差异。PRRSV与PCV2、CSFV和/或PRV二重或多重混和感染的组织病料总数为53份,猪场总数为39个,混合感染的组织病料和猪场的总阳性率分别为22.94%(53/231)和37.50%(39/104)。混合感染的组织病料占PRRSV阳性组织病料的46.09%(53/115),混合感染的猪场占PRRSV阳性猪场的60.94%(39/64)。其中以PCV2和PRRSV混合感染的组织病料和猪场数最多。由此可见,PRRSV感染在广西猪场已普遍存在,与其他病毒混合感染现象逐渐趋向复杂化。     

断奶后多系统衰竭综合征病猪中存在沙门氏菌和猪繁殖与呼吸系统综合征病毒共感染

调查了曾经暴发沙门氏菌病的四个猪场的14头病猪,发现在这些猪的肿胀淋巴结中出现带有淋巴细胞排空的肉芽肿性炎症。应用免疫标记和PCR方法在病变部位检测到猪圆环病毒2(PCV2)抗原和PCV2DNA。此外,在这些病猪的肺脏中检测出猪繁殖与呼吸综合征病毒(PRRSV),分离到猪霍乱沙门氏菌。在9头沙门氏菌感染猪中,有5头为沙门氏菌、PMWS与PRRSV并发感染,其数量(55.6%)远远高于沙门氏菌与PMWS感染猪(22.2%)或沙门氏菌与PRRSV感染猪(22.2%)。     

猪繁殖与呼吸综合征病毒人工感染试验

为探讨单纯猪繁殖与呼吸综合征病毒(PRRSV)感染与猪"高热病"及猪群死亡率的关系,用经检测为PRRSV阳性的猪病料接种2头断奶仔猪,每日测定体温,将发病猪处死剖检,用PCR方法进行PRRSV检测和ELISA检测抗体,发现攻毒猪均迅速出现持续高热症状,无死亡趋向,剖检试验猪以肺脏损伤为主,无细菌的继发感染,PCR检测P...     

猪繁殖与呼吸综合征病毒与猪2型圆环病毒混合感染的诊断

猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)又称蓝耳病,由猪繁殖与呼吸综合征病毒(PRRSV)引起。该病可以引起妊娠母猪早产、流产、弱胎、死胎和木乃伊胎,种公猪性功能下降,育肥猪呼吸障碍甚至仔猪死亡。由于PRRSV可以引起淋巴细胞数量减少,导致机体免疫力下降,使感染猪容易引起其它细菌、病毒、支原体等的混合感染和继发感染。猪2型圆环病     

Effect of porcine circovirus type 2 (PCV2) vaccination on porcine reproductive and respiratory syndrome virus (PRRSV) and PCV2 coinfection

The objectives were to determine if PCV2 vaccination is effective in reducing disease and lesions associated with PRRSV and PCV2 coinfection and if there is a difference between intradermal (ID) and intramuscular (IM) route of PCV2 vaccination. Seventy-four, 21-day-old pigs were randomly allocated into one of six groups. On day 0, pigs were vaccinated with 2ml Suvaxyn((R)) PCV2 One Dose (Fort Dodge Animal Health, Inc.) by intramuscular (VAC-M-COINF) or intradermal (VAC-D-COINF) routes. On day 28, pigs were either singularly (PRRSV-only, PCV2-only) or coinfected (COINF) with PRRSV and PCV2. All pigs in all groups were necropsied on day 42. All vaccinated pigs seroconverted (IgM, IgG, and neutralizing antibodies) to PCV2 between 14 and 28 days post-vaccination. After challenge, all groups inoculated with PRRSV had reduced average daily gain compared to CONTROLS and PCV2-only (P<0.001). COINF pigs had significantly (P<0.05) reduced anti-PCV2-IgG antibody levels and neutralizing antibody levels compared to both vaccinated groups. COINF pigs had more severe lung lesions compared to VAC-M-COINF (P<0.05). COINF pigs had higher amounts of PCV2 DNA in serum samples and feces (P<0.05) and increased amounts of PCV2 in lymphoid tissues (P<0.05) compared to both vaccinated groups. In summary, PCV2 vaccination was effective at inducing a neutralizing antibody response and significantly reducing PCV2-associated lesions and PCV2 viremia in pigs coinfected with PCV2 and PRRSV. Differences between intradermal and intramuscular routes of vaccine administration were not observed.     

Epidemiology and horizontal transmission of porcine circovirus type 2 (PCV2)

Porcine circovirus type 2 (PCV2) is a small, non-enveloped, circular, single-stranded DNA virus of economic importance in the swine industry worldwide. Based on the sequence analyses of PCV2 strains, isolates can be divided into five subtypes (PCV2a-e). PCV2 is an ubiquitous virus based on serological and viremia data from countries worldwide. In addition, PCV2 DNA was discovered in archived samples prior to the first recognition of clinical disease. Recently, a worldwide shift in PCV2 subtype from PCV2a to PCV2b occurred. PCV2 DNA can be detected in fecal, nasal, oral and tonsillar swabs as well as in urine and feces from both naturally and experimentally infected pigs. PCV2 DNA can be detected early in the infectious process and persists for extended periods of time. The effectiveness of disinfectants for reducing PCV2 in vitro is variable and PCV2 is very stable in the pig environment. Limited data exist on the horizontal transmission of PCV2. Direct transmission of PCV2 between experimentally or naturally infected animals and na?ve animals has been documented and the incorporation of clinical or subclinically infected animals into a population represents a risk to the herd. Indirect transmission through the oral, aerosol or vaccine routes is likely a lesser risk for the transmission of PCV2 in most swine populations but may be worth evaluating in high heath herds. The objective of this review was to discuss data on the epidemiology and horizontal transmission of PCV2.     

猪圆环病毒2型ORF2/猪白介素2嵌合表达质粒在猪体内诱导的保护性免疫反应

为探讨猪圆环病毒2型(PCV2)ORF2/猪白介素-2(PoIL-2)嵌合重组表达质粒(rpcDNA3.1/PCV2-linker-PoIL-2)在猪体内的免疫效果和免疫保护效果进而研制高效PCV2核酸疫苗,将35只10日龄健康仔猪平均分成7组分别以rpcDNA3.1/PCV2-linker-PoIL-2重组表达质粒或PCV2ORF2基因原核表达的rCap蛋白进行免疫及免疫保护试验。共进行4次肌肉注射免疫,每次间隔2周;于第4次免疫后3周通过口腔和鼻腔途径感染PCV2细胞强毒。分别于第4次免疫后和攻毒后不同时间通过检测免疫猪血清抗体水平和外周血T淋巴细胞增殖活性、辅助性T细胞(Th)和细胞毒性T细胞(Tc)亚群的百分含量、排毒率和病毒血症阳性维持时间等指标以评价其免疫和免疫保护效果。结果表明,各免疫组猪均产生了抗PCV2特异性ELISA免疫抗体,但rCap蛋白免疫组猪抗体水平较低;rpcDNA3.1/PCV2-linker-PoIL-2质粒对猪体的免疫和免疫保护效果显著优于rpcDNA3.1/ORF2质粒;在rpcDNA3.1/PCV2-linker-PoIL-2质粒或rpcDNA3.1/ORF2质粒中添加rCap蛋白对重组质粒免疫及免疫保护效果无明显影响。因此,选取rpcDNA3.1/PCV2-linker-PoIL-2质粒为下一步研制PCV2核酸疫苗的主要成分。     

Reduction of porcine reproductive and respiratory syndrome virus (PRRSV) infection in swine alveolar macrophages by porcine circovirus 2 (PCV2)-induced interferon-alpha

Two common viral pathogens of swine, namely, porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV), were investigated in regard to their effects on monolayer cultures of swine alveolar macrophages (AMs). The purpose was to identify selected cellular changes and responses potentially associated with the clinical reactions of pigs infected with either or both of these viruses. Measurements included the (1) absolute and relative numbers of infected, viable, and apoptotic cells; (2) distribution of viral antigens; (3) levels of interferon-alpha (IFN-alpha) and tumor necrosis factor-alpha (TNF-alpha) produced and their association with the extent of virus-induced cytopathology. Four groups of AMs were studied, including mock-infected, PCV2 alone-infected (PCV2-A), PRRSV alone-infected (PRRSV-A), and PCV2 and PRRSV dually infected (PCV2/PRRSV) groups. The AMs of PCV2-A group had high antigen-containing rate without cell death. There was a marked increase in cell death and apoptosis in PRRSV-A group. However, a lower PRRSV-induced infectious rate, cell death, and apoptosis were seen in PCV2/PRRSV group. High levels of IFN-alpha production were detected in PCV2-infected groups, but not in mock-infected and PRRSV-A groups. The PRRSV-induced cytopathic effect (CPE) on MARC-145 cells or swine AMs was markedly reduced by pre-incubation of the cells with UV-treated or non-UV-treated supernatants of PCV2-infected AMs. In addition, the reduction in CPE was abolished when the supernatants of PCV2-infected AMs were pre-treated with a mouse anti-recombinant porcine IFN-alpha antibody. The results suggest that swine AMs were an important reservoir of PCV2; PCV2 infection reduced PRRSV infection and PRRSV-associated CPE in PCV2/PRRSV AMs; the reduction of PRRSV infection in AMs was mediated by IFN-alpha generated by PCV2 infection. The reduced PRRSV-associated CPE in AMs and increased pro-inflammatory cytokine production may lead to a more severe pneumonic lesion in those dually infected pigs.     

Expression of toll-like receptor 2 (TLR2) in porcine leukocyte subsets and tissues

Toll-like receptors (TLR) are a group of pattern recognition molecules that play a crucial role in innate immunity. TLR2 recognises a variety of microbial components leading to the development of inflammatory and immune responses. To characterise the expression and functional properties of porcine TLR2 (pTLR2), we have raised a panel of monoclonal antibodies (mAb) against this molecule. Mouse 3T3 cell transfectants expressing pTLR2 were used for immunisation of mice. The specificity of these antibodies was confirmed by their reactivity with CHO cells transfected with pTLR2 but not with pTLR4 or with non-transfected cells. Using one of these mAbs, named 1H11, pTLR2 was found on cells of the innate immune system, including monocytes, macrophages, and granulocytes, but not on peripheral blood lymphocytes. Staining of tissue sections showed that pTLR2 is also expressed on epithelial cells lining the tracheobronchial and intestinal tracts, bile ducts in the liver and renal tubules, and on the basal layer of the epidermis. This distribution is consistent with a surveillance function at entry sites, allowing for early detection of microbial invasion.     

Prevalence and association of porcine circovirus type 2 (PCV2), porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) in aborted fetuses, mummified fetuses, stillborn and nonviable neonatal piglets

Porcine circovirus type 2 (PCV2) seems to cause reproductive failure in sows not only in experimental studies. A retrospective study was made with a total of 252 aborted fetuses, mummified fetuses, stillborn and nonviable neonatal piglets to determine the presence of PCV2, porcine parvovirus (PPV) and porcine respiratory and reproductive syndrome virus (PRRSV) by PCR. PCV2 was found in all stages of gestation in 27.1 percent of samples examined. A statistically significant association could be shown between the detection of PCV2 and PRRSV. However, no significant association was seen between the detection of PCV2 and PPV and between PPV and PRRSV.     

猪圆环病毒2型细胞培养适应毒株的培育和鉴定

从临床表现为仔猪断奶后多系统衰竭综合征（PMWS）淋巴组织病料，经聚合酶链式反应（PCR）证实为猪圆环病毒2型（PCV2）感染，采用无污染的猪肾细胞系（PK15）分离培养，并连续传代培育成一株细胞培养适应毒，命名为PCV2／LG株。分离毒株经细胞培养，于第25代后毒价显著升高，于第35代毒价可达10^5.6TCID 50/mL。采用免疫过氧化物酶单层细胞染色法（IPMA）、免疫电镜技术、分子克隆及核酸序列分析等鉴定表明，分离株感染细胞后病毒抗原主要分布在细胞核及细胞质中；病毒感染的阳性细胞呈散在分布，阳性细胞数可达50％以上；免疫电镜观察到与PCV2特异抗体结合形成的病毒免疫复合物呈实心小颗粒样粒子团，病毒粒子直径约为17nm；病毒抗原基因组由1768个核苷酸组成，与GenBank登录的8个PCV2基因组序列同源性达96．2％以上。用2mL的病毒细胞培养物（10^5.6TCID 50/mL）接种30日龄PCV2抗体阴性仔猪3头，可引起典型PMWS临床症状。本研究为进一步开展该病毒的致病性、疫苗免疫、诊断及分子生物学等研究奠定了基础。     

Molecular diversity of Brazilian strains of porcine circovirus type 2 (PCV-2)

Porcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, eight pig tissue samples from two Brazilian states were analyzed using six PCR primer pairs amplifying a 1705-bp fragment of the PCV-2 genome. The NJ distance-based method was used for the phylogenetic analysis with the eight field strains herein, 15 GenBank sequences and using PCV-1 as an out-group. This yielded two major clusters (A and B) for this viral species, with the Brazilian strains segregating with European and Asian sequences. Nucleotide identity was 99.7 to 100% among the sequences. This information can be used in further studies of pathogenesis related to PCV-2 in Brazil.     

Experimental inoculation of porcine circoviruses type 1 (PCV1) and type 2 (PCV2) in rabbits and mice

The objective of this work was to investigate the susceptibility of rabbits and mice experimentally inoculated with porcine circoviruses type 1 (PCV1) and type 2 (PCV2) to infection and development of disease and/or lesions. Forty six New Zealand rabbits and 50 ICR-CDI mice were both divided into two groups comprising PCVI and PCV2 inoculated animals, and a third group inoculated with non-infected cell culture medium. Rabbits were inoculated intranasally while mice were inoculated intraperitoneally. Clinical signs and body weights were recorded at the start of the experiment and at necropsy. Animals were bled, euthanised and necropsied at days 0, 3, 7, 10, 14 and 20 post-inoculation and samples were collected for histopathological, serological, in situ hybridisation and PCR analysis. No clinical signs or gross and microscopic lesions compatible with PCV2 infections such as those seen in pigs were observed. No presence of PCV2 nucleic acid was detected in rabbits and mice by in situ hybridisation. Only one mouse inoculated with PCV1 seroconverted on day 20 P1. PCV1 and PCV2 genome was detected in serum by PCR in mice inoculated with each porcine circovirus, while rabbits were negative for both viral types. These studies indicated that porcine circoviruses did not cause any disease or microscopic lesions in inoculated rabbits and mice during the experimental period. However, intraperitoneally inoculated mice might have harboured PCV2 in circulation without evidence of viral replication.     

Genetic typing of porcine circovirus type 2 (PCV-2) isolates from Slovakia

Of 120 clinical specimens obtained from pigs bred on 28 PMWS-affected farms in Slovakia, porcine circovirus type 2 (PCV-2) was detected by single PCR in 77 samples. A short 224 bp fragment of ORF2 was used for preliminary grouping of isolates by phylogenetic analysis. Nucleotide sequences of the entire ORF2 region provided more precise genetic typing and segregation of preselected isolates (n = 10) into two known genotypes, PCV-2a (n = 1) and PCV-2b (n = 9). Complete genome sequencing of three selected isolates allowed their definitive grouping into genotype PCV-2b, cluster 1A or genotype PCV-2a, cluster 2D. No correlation between the mutations and the geographic origin of isolates was observed. Results confirmed that many PCV-2 isolates are genetically very stable since similar viruses circulate in Central and Western Europe.