Genetic analysis of ORF5 of recent Korean porcine reproductive and respiratory syndrome viruses (PRRSVs) in viremic sera collected from MLV-vaccinating or non-vaccinating farms

The 23 open reading frame (ORF) 5 sequences of Korean type II porcine reproductive and respiratory syndrome virus (PRRSV) were collected from viremic sera from the (modified live vaccine) MLV-vaccinating and non-vaccinating farms from 2007 to 2008. The samples were phylogenetically analyzed with previous ORF5 sequences, including type I Korean PRRSV, and previously reported or collected sequences from 1997 to 2008. A MN184-like subgroup of type II Korean PRRSV was newly identified in the viremic sera collected from 2007 to 2008. And of the type I PRRSVs, one subgroup had 87.2~88.9% similarity with the Lelystad virus, showing a close relationship with the 27~2003 strain of Spain. The maximum parsimony tree of type II PRRSV from 1997 to 2008 showed that they had evolved to four lineages, subgroups 1, 2, 3 and 4. Most of the recently collected type II PRRSVs belonged to subgroup 4 (48%). The region of three B-cell epitopes and two T-cell epitopes of ORF5 amino acids sequences was considerably different from the MLV in subgroups 3 and 4. In conclusion, the existence of type I PRRSV, which was genetically different from Lelystad virus (Prototype of type I PRRSV), and heterologous type II PRRSVs of viremic pigs detected even in the MLV-vaccinating farms indicated the need for new vaccine approaches for the control of PRRSV in Korea.     

Identification of genetically diverse sequences (ORF 5) of porcine reproductive and respiratory syndrome virus in a swine herd.

The ability of genetically diverse strains of porcine reproductive and respiratory syndrome virus (PRRSV) to coexist in a 1750-sow farm was assessed through the case study describing a chronically infected farm, and also by an animal experiment involving the use of swine bioassay. The case study employed a program of monitoring sera from suckling, nursery, and finishing pigs for the presence of PRRSV by polymerase chain reaction (PCR) and virus isolation (VI). The swine bioassay tested homogenates, consisting of lymphoid and pulmonary tissues, collected from 60 breeding animals from the same farm. The open reading frame (ORF) 5 portion of selected positive PRRSV detected from sera or tissues were nucleic acid sequenced and their phylogenies compared. The results indicated the presence of 3 genetically diverse groups, designated PRRSV-A, -B, and -C. Sequence heterology ranged from 5.8 to 11% between groups. Sequence homology ranged from 98.7 to 99.8% within groups. Swine bioassay verified the presence of PRRSV-A in 1 of 60 animals, and no evidence of strains B or C were detected. This paper indicates that based on the evaluation of ORF 5, genetically diverse strains of PRRSV appear to coexist, although the frequency and significance of this observation is not understood.     

PRRSV ZQ-GD-2010株的分离及ORF5和ORF7基因的序列分析

为了给研制基因工程疫苗选取毒株奠定基础,研究采集广东肇庆某猪场疑似患高热症猪的病料,通过RT-PCR检测、病毒分离传代,证实分离到1株猪繁殖与呼吸综合征病毒,命名为ZQ-GD-2010株,并对其ORF5和ORF7基因进行序列测定,绘制遗传进化树。结果表明:该毒株为美洲型,其ORF5和ORF7基因核苷酸与近年来我国分离到的美洲型毒株的相似性为96%～100%,而与欧洲型毒株LV株的相似性仅为58.9%～62.8%;其与2007—2009年国内分离株的遗传演化关系较近。     

2007-2010年河北省部分地区猪繁殖与呼吸综合征分子流行病学

为了弄清河北省秦皇岛、唐山、廊坊、邯郸等部分地区流行的猪繁殖与呼吸综合征病毒（PRRSV）的ORF5和Nsp2基因变异情况,对采自以上地区在2007—2010年期间的临床发病猪肺组织,提取其RNA,通过RT—PCR扩增样本中PRRSV的ORF5和Nsp2基因,对目的基因测序,并以VR-2332、CH—la、MLV、LV、HB-1、HB-2、JXA1、HUB1、HEB1等毒株为参考序列,进行序列分析和进化树分析。扩增出42个ORF5基因,14个Nsp2基因,通过完整的ORF5和部分Nsp2基因序列比较分析发现所有毒株均为美洲型,且大多数毒株与国内2006—2007年间分离的JXA1、HUB1、HEB1等高致病力毒株同源关系很近。42个ORF5基因编码的氨基酸序列分析表明GP5蛋白的糖基化位点数量和位置已发生变异,主要中和表位存在氨基酸变异（L／F^35→I^39）,与毒力相关的13位和151位点为强毒株的氨基酸特性（R^13、R^151）。14个Nsp2基因推导的氨基酸系列比较发现,1株PRRSV Nsp2基因未发生缺失突变,而剩余的13个毒株Nsp2基因均发生了氨基酸缺失,在481位和532～560位2处分别缺失1和29个氨基酸。系统发育进化树结果显示河北部分地区流行株分为2个小分支,1个毒株与HB-1聚为1支;河北流行株的大多数毒株在另一个分支,与高致病力毒株JXA1等聚为一支。本研究结果揭示了河北省部分地区流行的PRRSV ORF5和Nsp2基因的变异特征,丰富了河北省的PRRSV分子流行病学资料,提示要针对ORF5和Nsp2基因的变异采取PRRSV的防控措施。     

House fly vector for porcine circovirus 2b on commercial pig farms

We investigated the on-farm potential of common farm invertebrates to transmit porcine circovirus genotype 2 (PCV2) and other non-enveloped viruses. In 2007 (pre-PCV2 vaccination) and 2008 (post-PCV2 vaccination), invertebrate communities were trap-collected (8 trap-dates per year), counted and sorted into genus and species groups on 5 farm study sites within England. Total DNA was extracted from feces of representational cross-sections of pigs on each farm in each year and also from intact samples of Diptera flies (ca. 20 flies per trap) and dissected viscera of any cockroaches (ca. 5 per trap). Each DNA sample was tested for the presence of PCV2 DNA by separate PCRs for ORF1 and ORF2. Positive samples were sub-typed via DNA sequencing of PCR products. The pig-associated Diptera fly community was dominated by Musca domestica (house fly) in both years on all 5 farms; numerous Blatta orientalis cockroaches were only noted on 1 farm throughout. Specific PCV2b DNA elements were routinely detected (25-60% of samples) in weaner/nursery pig feces in 2007, but not in other age groups. Musca collected on 4 of the 5 farms in 2007 was also positive for PCV2b DNA elements. Comparison of ORF2 sequences indicated that ORF2 sequences indicating PCV2b genotype were identical in pigs and flies. Minor changes were noted in ORF1 sequences from different samples. Flies collected in the weaner/nursery area were most likely to be positive (22-50% of fly-trap samples). DNA extracted from all cockroaches (2007 and 2008) and all flies and pig feces in 2008 were also negative throughout. We suggest that Musca flies have the most likely on-farm potential to carry and transmit PCV2b due to their life cycle incorporating stages in close association with pigs and their habitat. Vaccination appeared to reduce environmental load of PCV2b.     

Molecular evolution of porcine reproductive and respiratory syndrome virus isolates from central China

To investigate the genetic diversity of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan Province of China, 61 ORF5 gene sequences, originating from Henan Province during 2003–2010, were subjected to amino acid variation and phylogenetic analysis. The analyzed PRRSV ORF5 sequences carried evidence of one unique recombination event. Phylogenetic analysis revealed that all Henan isolates belonged to type 2 genotype and were divided into two subgroups. The dominant isolates had shifted from subgroup 1 to subgroup 2 during 2003–2010. Amino acid variation analysis of the glycoprotein 5 revealed that Henan PRRSV strains tended to accumulate more substitutions within the N-terminus and hypervariable region. Selective pressure analysis revealed evidence that some ORF5 sites have likely evolved in response to immune pressure.     

A survey of porcine reproductive and respiratory syndrome among wild boar populations in Korea

No information is currently available on porcine reproductive and respiratory syndrome virus (PRRSV) infection in wild boars (Sus scrofa) in Korea. In this study, the status of PRRS in wild boars was investigated. Blood samples were collected from 267 wild boars from eight provinces in Korea. Four of the samples tested (1.5%) were positive for PRRSV antibodies and eight (3.0%) were positive for antigens. Of the virus-positive samples, three and five samples were typed as containing European (EU, type 1) or North American (NA, type 2) viruses, respectively. Two amplicons (one from type 1 and one from type 2) were used to analyze the PRRSV open reading frame 7 (ORF7) sequence. The nucleotide sequences of type 1 PRRSV ORF7 had identities between 96.1% and 98.4% with PRRSVs from domestic pigs in Korea. The sequences of type 2 PRRSV ORF7 had identities of 100% with the PRRSV strain VR-2332, which was prototypic North American strain. These results show that PRRSVs are present in wild boars in Korea, and effective PRRSV surveillance of the wild boar population might therefore be useful for disease control.     

Eradication of porcine reproductive and respiratory syndrome virus by serum inoculation of na?ve gilts

The acclimatization program included exposure to serum and recovery. A continuous flow unit (nursery to finishing) from the same farm was selected as a potential source of porcine reproductive and respiratory syndrome virus (PRRSV). Negative gilts were inoculated 5 d after arrival by intramuscular injection of serum from selected animals. There was a significant reduction in seroprevalence in the sow herd 1 y after implementation of the gilt inoculation program (P < 0.05). At that time, all of the tested nursery pigs were negative for PRRSV. The fully segregated finisher population had a significant reduction in the frequency of PRRSV positive animals (P < 0.05) by enzyme-linked immunosorbent assay (ELISA), with all animals testing negative by the end of the study. However a persistent seroconversion was observed in the partially segregated finisher pigs (P > 0.05). In conclusion, the gilt serum inoculation program achieved sow herd stabilization, as defined by the production of negative weaned pigs and this resulted in the eradication of PRRSV in the fully segregated flow.     

The diagnostic sensitivity of immunohistochemistry for the detection of porcine reproductive and respiratory syndrome virus in the lung of vaccinated and unvaccinated swine.

Immunohistochemistry (IHC) is used routinely to detect porcine reproductive and respiratory syndrome virus (PRRSV) in the lung of nursery and grow/finish pigs with respiratory disease and has been reported to be highly specific (100%) but only moderately sensitive (67%). When multiple sections of lung are examined from field cases of porcine pneumonia, it is common to detect PRRSV antigen in only 1 or 2 of the sections. This study was undertaken to determine the impact of the number of lung sections evaluated on the diagnostic sensitivity of IHC for the detection of PRRSV in vaccinated and unvaccinated swine. Five anterioventral sections of lung from animals experimentally challenged with PRRSV were evaluated on a single IHC slide. Utilizing a beta binomial model, observed results were used to calculate the probability of detecting PRRSV with IHC as a function of the number of lung sections assessed. Results demonstrate that the diagnostic sensitivity of PRRSV IHC is dependent on the number of lung sections examined. In unvaccinated pigs, a beta binomial model predicts that if a single lung section were evaluated, PRRSV would likely be confirmed in only 48% of infected animals, and at least 5 sections of anterioventral lung would need to be assessed to detect >90% of PRRSV-infected pigs. Vaccination resulted in significantly lower gross and microscopic lung lesion scores and significantly fewer antigen-positive cells. In vaccinated swine, the calculated probability of detecting a PRRSV-infected pig with IHC when a single lung section is evaluated was only 14%. If PRRSV is a primary concern, diagnosticians should collect at least 5 anterioventral sections of lung from each pig to be evaluated on a single IHC slide. This approach will diminish the number of false-negative results obtained with this method of antigen detection.     

Immune responses of swine following DNA immunization with plasmids encoding porcine reproductive and respiratory syndrome virus ORFs 5 and 7, and porcine IL-2 and IFNgamma

In the present study, five eukaryotic double-gene expression plasmids containing porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 and ORF7 genes combined with cDNAs encoding porcine IFNgamma and IL-2 were constructed for evaluation as PRRSV vaccine candidates. After immunization and viral challenge, two of three pigs immunized with pIRESorf5/IFNgamma, one of three pigs immunized with pIRESorf5/IL-2 and one of three pigs immunized with pIRESorf7/IL-2 were protected from lung lesions that were present in other vaccinated and control animals. Virus replication was reduced but not completely prevented in organs of the DNA-vaccinated animals as compared to controls. Therefore, the porcine cytokines IFNgamma and IL-2, delivered in combination with ORF5 or ORF7, may improve the immune efficacy of DNA vaccines against PRRSV.     

猪繁殖与呼吸综合征病毒与副猪嗜血杆菌混合感染的诊断

为确诊广东省阳江市某规模化猪场（存栏800头母猪）保育猪发病死亡的原因,本试验对从该发病猪场采集的3份肺脏、肝脏、脾脏临床样品进行细菌学检测及药敏试验,采用PCR/RT-PCR检测临床样品中猪伪狂犬病病毒（PRV）、猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）、猪圆环病毒2型（PCV2）、猪圆环病毒3型（PCV3）和猪肺炎支原体等病原。对特异性扩增的3株PRRSV的ORF5基因产物进行序列测定,与VR2332、HuN4、JXA1、CH-1a等代表毒株进行核苷酸序列同源性分析,并构建系统进化树。结果表明,试验分离鉴定出1株副猪嗜血杆菌（Hps）,对7种临床常用药如阿莫西林、头孢拉定等均有较强的敏感性。同源性比对结果表明,3株PRRSV （LJW1、LJW2和LJW3）ORF5基因核苷酸同源性为99.3%~99.8%,与欧洲型代表毒株Lelystad核苷酸同源性为64.0%~64.2%,与HP-PRRSV毒株JXA1、HuN4、CH-1a和TJ核苷酸同源性较高,分别为99.2%~99.5%、99.0%~99.3%、94.5%~94.9%和98.8%~99.2%;与中国河南和广西分离的HP-PRRSV毒株HeNzm1-16和GXLZ05-2015核苷酸同源性较高,分别为99.3%~99.7%和99.2%~99.7%,与美洲型经典疫苗株MLV、美洲型标准株NC、美洲型经典株VR2332核苷酸同源性较低,分别为88.5%~88.8%、85.2%~85.5%和82.3%~82.6%。PRRSV ORF5基因系统进化树分析表明,3株PRRSV均属于美洲型毒株,与国内HP-PRRSV代表毒株JXA1、HuN4和TJ等处于同一分支,亲缘关系较近。本研究揭示了该场保育猪发病病原,并从分子水平上明确了分离的3株PRRSV与不同代表毒株的亲缘关系,为弱毒疫苗的合理选择使用和综合防控PRRSV提供了参考依据。     

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。     

2005年-2010年我国部分地区PRRSV流行毒株的遗传变异分析

为了掌握高致病性猪繁殖与呼吸综合征病毒(PRRSV)的变异情况,揭示该病的发生规律,根据GenBank登录的PRRSV基因序列设计引物,采用RT-PCR法对2005年-2010年间送检的282份病料进行了PRRSV核酸检测,对其中9份阳性样品进行了ORF5～7基因片段扩增和测序,所得序列与GenBank下栽的PRRSV...     

Porcine reproductive and respiratory syndrome virus detection in Thailand during 2005–2010 in relation to clinical problems, pig types, regions, and seasons

The objectives of the present study were to determine the prevalence of porcine reproductive and respiratory syndrome virus (PRRSV) in Thailand between 2005 and 2010. The study was conducted by retrospectively investigating the detection of PRRSV from different pig types including boars, sows, piglets, nursery pigs, and fattening pigs from six regions of Thailand, i.e., the northern, eastern, northeastern, central, western, and southern parts. The data were obtained from cases submitted to the Chulalongkorn University Veterinary Diagnostic Laboratory for PRRSV detection between 2005 and 2010. Frequency analyses and generalized linear models were used to evaluate the prevalence of PRRSV in relation to various factors. In total, 2,273 tissues (n?=?636), semen (n?=?210) and serum (n?=?1,427) samples were included. PRRSV was detected in 32.6 % (740/2,273) of the pigs. The virus was found in 43.1 %, 15.7 %, and 30.3 % in the tissues, semen, and serum samples, respectively (P?<?0.001). The prevalence of PRRSV was highest in 2005 (43.6 %) and lowest in 2009 (23.6 %) (P?<?0.001). The prevalence of PRRSV was highest in nursery pigs (43.7 %) and lowest in boars (15.4 %) (P?<?0.001). The prevalence of PRRSV in the hot season (34.9 %) was higher than that found in the cool season (28.1 %, P?=?0.018) but did not differ significantly compared to rainy season (34.0 %, P?=?0.486). The strain of PRRSV isolated in the present study was genotype 2 (54.5 %), genotype 1 (31.0 %), and mixed genotypes (14.5 %). It can be concluded that PRRSV was detected in the tissue samples more frequently than the semen and serum samples. The prevalence of PRRSV was high in the nursery pigs. A high prevalence of PRRSV was found in the hot season, indicating that climatic factors may also contribute to the prevalence of PRRSV in Thailand. Of all the PRRSV detected, 31.0 %, 54.5 %, and 14.5 % belonged to genotype 1, genotype 2, and mixed genotypes, respectively.     

Genetic diversity analysis of the ORF5 gene in porcine reproductive and respiratory syndrome virus samples from South China

To understand the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in South China, we collected 231 clinical samples from pigs with suspected PRRSV infection in Guangdong between 2007 and 2009. We found that 74 of 231 samples were positive by RT-PCR. The PCR products of the ORF5 gene of 35 isolates from different farms were sequenced and their DNA sequences were compared to 23 other PRRSV isolates in the GenBank. We found that the nucleotide similarity among all South China isolates ranged from 87.6% to 100%, and all belonged to the North American genotype. Most of them were classified into subgenotype I, but the rest mapped to subgenotypes III, V or VI. Those in subgenotypes I and III were found to be highly variable in the primary neutralising epitope (PNE) with a specific amino acid mutation (F39/L39→I39), and a few isolates in subgenotypes I and III isolates also had a mutation at L41 (L41→S41). PRRSV isolates in subgenotypes III, V and VI had less potential glycosylation sites than those in subgenotype I. Our data contribute to the understanding of molecular variation of PRRSV in South China.     

Effects of challenge with a virulent genotype II strain of porcine reproductive and respiratory syndrome virus on piglets vaccinated with an attenuated genotype I strain vaccine

Porcine reproductive and respiratory syndrome virus (PRRSV) is endemic in most parts of Asia, where genotype I and II strains of diverse virulence may coexist. This study evaluated the outcome of infection with a highly virulent Asian genotype II PRRSV isolate in piglets vaccinated with a genotype I vaccine. Twenty-one 3-week-old piglets were divided in three groups: Pigs in group V (n=8) were vaccinated with an attenuated genotype I commercial PRRSV vaccine, while pigs in group U (n=8) and a control group (group C; n=5) were unvaccinated; 6 weeks later, pigs in groups V and U were challenged intranasally with a highly virulent strain of genotype II PRRSV (1×10(5) 50% tissue culture infectious doses/mL), while pigs in group C received a placebo. Over a period of 21 days after challenge, vaccinated pigs had significantly lower mortality (0/8 versus 2/8), fewer days of fever, a lower frequency of catarrhal bronchopneumonia, higher weight gains (13.4 versus 6.6 kg) and lower levels of viraemia compared to unvaccinated challenged pigs. Immunisation with a genotype I attenuated PRRSV vaccine provided partial protection against challenge with a highly virulent genotype II strain.     

猪繁殖与呼吸综合征病毒国内分离经典株与变异株的全基因组序列分析

通过分段设计引物,对猪繁殖与呼吸综合征病毒(PRRSV)LX、JX株基因组进行RT-PCR扩增,对各片段cDNA进行克隆和序列测定,拼接后获得全基因组序列。结果,PRRSV LX株全基因组序列长度为15 412 bp(不包括PolyA尾),PRRSV JX株全基因组序列长度为15 320 bp(不包括PolyA尾)。序列分析表明,JX株全基因组核苷酸序列与LX株、JXA1、VR2332、CH-1a、BJ-4、LV同源性分别为91.1%、98.6%、91.0%、94.6%、90.9%、61.7%;LX株全基因组核苷酸序列与JX株、JXA1、VR2332、CH-1a、BJ-4、LV同源性分别为91.1%、89.7%、99.7%、91.5%、99.7%、62.2%。对不同分离株的5′-UTR、Nsp2进行了序列比较,并根据5′-UTR、Nsp2、ORF5的基因序列和氨基酸序列,对国内外分离株进行了系统进化分析。根据5′-UTR核苷酸序列,可将PRRSV美洲型毒株分为4个亚群,LX株和JX株分别属于经典美洲型和"高热病"变异型。根据Nsp2氨基酸序列分析了不同分离株的分子进化关系,表明依据Nsp2序列美洲型分离株可初步划分为5个亚群,JX株独立于其他毒株,独自处于一个分支。依据ORF5序列也可将美洲型分离株划分为5个亚群。本研究为探讨PRRSV的分子进化奠定了基础。     

豫南地区规模化猪场猪繁殖与呼吸综合征病毒和猪圆环病毒2型抗体检测与分析

为掌握豫南地区规模化猪场中猪繁殖与呼吸综合征病毒（PRRSV）和猪圆环病毒2型（PCV2）疫苗免疫场猪群抗体水平和非疫苗免疫场猪群病原感染情况,试验利用ELISA方法对该地区猪群进行了PRRSV和PCV2抗体水平检测。结果表明,在疫苗免疫猪群中,PRRSV和PCV2抗体合格率分别为68.8%和58.7%,规模越大的猪场合格率越高,200~500头母猪规模场抗体阳性率最高（分别为76.8%和77.4%）,50头母猪以下场最低（分别为48.3%和44.2%）,种猪的合格率均最高,而育肥猪群合格率均最低。在非疫苗免疫猪群中,PRRSV和PCV2抗体阳性率分别为55.6%和65.3%,PRRSV抗体阳性率最低的是100~200头母猪规模场（46.5%）,最高的是50头母猪以下规模场（68.8%）,断奶仔猪和育肥猪群抗体阳性率均在71%以上;随猪场规模越大,PCV2抗体阳性率越低,200~500头母猪规模场抗体阳性率为41.4%,50~100头规模场和50头以下场的阳性率分别为78.9%和78.6%,种公、母猪的PCV2抗体阳性率最低（分别为37.5%和40.4%）,断奶仔猪和育肥猪抗体阳性率较高（分别为82.2%和79.5%）。本研究反映了豫南地区猪繁殖与呼吸综合征（porcine reproductive and respiratory syndrome,PRRS）和猪圆环病毒病（porcine circovirus disease,PCVD）疫苗免疫猪场的疫苗免疫效果和非疫苗免疫猪场病原感染的实际情况和规律,为该地区PRRS和PCVD防制工作提供了理论依据和指导。