Preclinical Detection of Porcine Circovirus Type 2 Infection Using an Ultrasensitive Nanoparticle DNA Probe-Based PCR Assay

Porcine circovirus type 2 (PCV2) has emerged as one of the most important pathogens affecting swine production globally. Preclinical identification of PCV2 is very important for effective prophylaxis of PCV2-associated diseases. In this study, we developed an ultrasensitive nanoparticle DNA probe-based PCR assay (UNDP-PCR) for PCV2 detection. Magnetic microparticles coated with PCV2 specific DNA probes were used to enrich PCV2 DNA from samples, then gold nanoparticles coated with PCV2 specific oligonucleotides were added to form a sandwich nucleic acid-complex. After the complex was formed, the oligonucleotides were released and characterized by PCR. This assay exhibited about 500-fold more sensitive than conventional PCR, with a detection limit of 2 copies of purified PCV2 genomic DNA and 10 viral copies of PCV2 in serum. The assay has a wide detection range for all of PCV2 genotypes with reliable reproducibility. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1, porcine parvovirus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus and classical swine fever virus. The positive detection rate of PCV2 specific UNDP-PCR in 40 preclinical field samples was 27.5%, which appeared greater than that by conventional and real-time PCR and appeared application potency in evaluation of the viral loads levels of preclinical infection samples. The UNDP-PCR assay reported here can reliably rule out false negative results from antibody-based assays, provide a nucleic acid extraction free, specific, ultrasensitive, economic and rapid diagnosis method for preclinical PCV2 infection in field, which may help prevent large-scale outbreaks.     

Detection of porcine circovirus type 1 in commercial porcine vaccines by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1. No cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. The analytical sensitivity of the LAMP for PCV1 DNA was 10 copies/μl in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction (nested PCR). Furthermore, 25 commercial swine vaccines were tested by both the LAMP and the nested PCR, and three of them were tested positive for PCV1 DNA. These results indicate that PCV1 DNA can be real-time detected by the LAMP; the method was highly specific, sensitive, and rapid for the detection of PCV1 DNA, particularly in commercial swine vaccines.     

Detection of porcine parvovirus using a taqman-based real-time pcr with primers and probe designed for the NS1 gene

A TaqMan-based real-time polymerase chain reaction (PCR) assay was devised for the detection of porcine parvovirus (PPV). Two primers and a TaqMan probe for the non-structural protein NS1 gene were designed. The detection limit was 1 x 102 DNA copies/μL, and the assay was linear in the range of 1 x 102 to 1 x 10? copies/μL. There was no cross-reaction with porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The assay was specific and reproducible. In 41 clinical samples, PPV was detected in 32 samples with the real-time PCR assay and in only 11 samples with a conventional PCR assay. The real-time assay using the TaqMan-system can therefore be practically used for studying the epidemiology and management of PPV.     

Complete Genome Sequence of Recombinant Porcine Circovirus Type 2 Strain SD-3

We report here the genome sequence of a recombinant porcine circovirus type 2 strain SD-3, isolated from a commercial swine farm with an outbreak of postweaning multisystemic wasting syndrome (PMWS) in pigs in Shandong Province of China. The complete circular genome of this isolate is 1,767 nucleotides in length. This recombinant isolate has the ORF1 regions from PCV2a viruses and ORF2 regions from PCV2b. The findings will help us to understand the molecular evolution of porcine circovirus type 2 and the relationship between porcine circovirus type 2 and disease.     

猪细小病毒LDR-PCR检测方法的建立和应用

为准确特异灵敏地检测猪细小病毒(PPV),建立一种新的LDR-PCR方法。首先在病毒的保守区内设计一对LDR探针,LDR探针两端各连有一段引物对应序列,以连接产物为模板进行PCR,琼脂糖凝胶电泳检测结果。以标准质粒为模板,通过对LDR反应的退火温度、连接酶浓度及探针浓度等反应条件进行优化,确定了LDR最佳的反应体系,并建立了LDR-PCR方法。结果表明,可以特异地检测PPV,与猪繁殖与呼吸障碍综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病毒(PRV)、猪圆环病毒(PCV)、猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)无交叉反应;最低检测限为102个拷贝。利用建立的方法对41例临床样本进行检测,14份样品PPV阳性,与普通PCR检测结果符合率为97.6%。     

An ELISA based on a truncated soluble ORF2 protein for the detection of PCV2 antibodies in domestic pigs

Postweaning multisystemie wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA)based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.     

The index herd with PMWS in Sweden: Presence of serum amyloid A,circovirus 2 viral load and antibody levels in healthy and PMWS-affected pigs

Background  

Postweaning Multisystemic Wasting Syndrome (PMWS) is an emerging disease in pigs of multifactorial origin, but associated to porcine circovirus type 2 (PCV2) infection. PMWS was first diagnosed in Sweden at a progeny test station that received pigs aged five weeks from 19 different nucleus herds on the day after weaning. The objective of this study was to examine, for the first time in an index outbreak of PMWS, the relationship between PCV2 virus, antibodies to PCV2 and serum amyloid a (SAA) in sequentially collected serum samples from pigs with and without signs of PMWS.     

Development of a loop-mediated isothermal amplification assay for porcine circovirus type 2

In this study,the loop-mediated isothermal amplification(LAMP)method was used to develop a rapid and simple detection system for porcine circovirus type 2(PCV2).According to the PCV2 sequences published in GenBank,multiple LAMP primers were designed targeting conserved sequences of PCV2.Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template,LAMP reactions in a PCV2 LAMP system was performed,the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye.The results showed highly-efficient and specific amplification in 30 min at 63℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng of nucleic acid,indicated that this assay was highly sensitive.The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter,showing the potential simplicity of interpretation of the assay results.The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples.In addition it offers higher specificity and sensitivity,shorter reaction times and simpler procedures than the currently available methods of PCV2 detection.It is therefore a promising tool for the effective and efficient detection of PCV2.     

Detection and genetic characterization of porcine circovirus 2 isolates from the first cases of postweaning multisystemic and wasting syndrome in wild boars in Greece

In 2002, postweaning multisystemic wasting syndrome (PMWS) was diagnosed in a European female wild boar (Sus scrofa), based on the detection of porcine circovirus 2 (PCV2) DNA in various organs, including the uterus, and on histopathologic lesions. This is the first detection of PCV2 DNA in the uterus of a wild boar. Three years later (2005), a wild boar < 6-8 mo of age was found moribund. It presented wasting and dyspnea and finally died. PCV2 DNA was detected in tissue samples, and histopathologic lesions consistent with PMWS were observed. Both wild boars were from neighboring hunting areas in central Greece. Two PCV2 strains from the wild boars were genetically characterized and compared to other reported PCV2 sequences from wild boars and domestic pigs. The PCV-2 sequences from the wild boars in this study were closely related to each other and were grouped with two isolates from wild boars from Hungary. The phylogenetic analysis revealed that the virus might be transmitted between hunting areas. In addition, PCV2 may spread from domestic pigs to wild boars and vice versa.     

一株家养野猪源猪圆环病毒2型的分离与鉴定

应用PCR方法从临诊疑似断奶仔猪多系统衰竭综合征(PMWS)家养野猪病例的淋巴结和脾脏中扩增出预期长度的猪圆环病毒2型(PCV2)的DNA片段,在Dulac细胞中进行分离和培养,扩增全基因组序列后进行同源性分析.结果显示,扩增产物与家猪源参考毒株的序列同源性均在98%以上,该病毒与家猪源病毒差异不大.     

Cellular adaptive immune response against porcine circovirus type 2 in subclinically infected pigs

Background  

Porcine circovirus type 2 (PCV2) is a dominant causative agent of postweaning multisystemic wasting syndrome (PMWS), a multifactorial disease complex with putative immunosuppressive characteristics. Little is known about adaptive PCV2-specific immune responses in infected pigs. Therefore, the T and B cell responses following PCV2 infection in 3-week old SPF piglets infected with PCV2 or PCV2 plus porcine parvovirus (PPV) were studied.     

Complete Genome Sequence of Porcine Circovirus 2b Strain Shandong

Shandong is a porcine circovirus 2b (PCV2b) strain that was isolated and purified from tissue samples from pigs with postweaning multisystemic wasting syndrome (PMWS) in the Shandong Province of China. Here, we report the complete genome sequence of strain Shandong, which may aid in understanding the molecular characteristics of this strain.     

Protective immunity conferred by porcine circovirus 2 ORF2‐based DNA vaccine in mice

Post‐weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP‐N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP‐Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP‐Cap, LG‐strain vaccine, pEGFP‐N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP‐Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN‐γ and IL‐10), and a small PCV2 viral load. The mice treated with the pEGFP‐Cap and LG‐strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP‐Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS.     

猪圆环病毒2型、猪繁殖与呼吸综合征病毒及猪细小病毒混合感染的流行病学调查

根据GenBank上发表的PRRSVORF7、PPVVP2及PCV的基因组序列设计合成引物,建立了分别用于检测PRRSV、PPV和PCV的RT-PCR、PCR及复合PCR方法。应用建立的复合PCR方法对送检的127份病料进行了PCV的检测,对鉴定为PCV2阳性的67份病料再分别进行PRRSV和PPV的检测,以确定猪群中PCV2与PRRSV和,或PPV混合感染情况,结果表明,35份样品表现为PRRSV与PCV2混合感染,占样品总数的52．3％;18份样品表现为PCV2与PPV混合感染,占26．9％。另外,还有一定比例的三重感染,共5个样品,占7．5％。由此可见,猪群中PCV2与PRRSV及PPV混合感染比较普遍。     

表达猪圆环病毒2型Cap和猪繁殖与呼吸综合征病毒GP5融合蛋白重组腺病毒的构建和免疫原性测定

断奶后多系统衰弱综合征(PMWS)是目前规模化猪场常见的传染病,由PCV2感染引起。临床上PCV2与PRRSV混合感染较常见,死亡率和发病率很高,但目前尚无能有效预防该病的商品化疫苗。本研究利用基因克隆方法,构建融合表达PCV2-Cap蛋白与PRRSV-GP5蛋白的重组腺病毒rAd-Cap-GP5,经IPMA、IFA和Western blotting验证了Cap蛋白和GP5蛋白在该重组病毒中获得表达。将该重组腺病毒免疫小鼠测定其抗体、中和抗体,结果显示小鼠免疫后出现两种病毒的抗体和中和抗体,诱导小鼠产生明显的体液免疫应答,可以作为预防PCV2-PRRSV混合感染的基因工程疫苗候选毒株,为PCV2和PRRSV二联重组亚单位疫苗的研究奠定了基础。     

Wild mink (Neovison vison) as sentinels in environmental monitoring

Superficial inguinal lymph nodes from 72 wild boars examined in a previous immunohistochemical (IHC) study on porcine circovirus type 2 (PCV2) were selected for a PCV2 polymerase chain reaction (PCR) analysis. Four of these lymph nodes were PCV2-IHC strongly positive with PMWS histological lesions (outcome 1), 6 weak to mild PCV2-IHC positive without PMWS histological lesions (outcome 2) and 62 PCV2-IHC negative. Considering IHC the gold standard for diagnosis, the aims of the study were to evaluate the suitability of the PCV2-DNA extraction from formalin-fixed and paraffin-embedded (FFPE) tissue and the sensitivity and specificity of PCR under two IHC interpretations criteria: (A) the sample was considered positive if the result was outcome 1; (B) the sample was considered positive if the result was outcome 1 or 2. Under (A) criteria, sensitivity and specificity of PCR were 100% and 89.7%, respectively; the Cohen's Kappa coefficient was 0.49. Under (B) criteria, sensitivity and specificity of PCR were 80.0% and 95.2%, respectively; the Cohen's Kappa coefficient was 0.72. The high Cohen's Kappa coefficient under the (B) interpretative criteria indicates good agreement between the two methods. In conclusion, 1) DNA extracted from FFPE specimens of wild boar is suitable for PCR and further represents a screening test for PCV2/PCVD (PCV2 Diseases) investigations in wild boar as well; 2) routine histological sampling can also be useful for PCV2 virological studies in wild boar.     

Prevalence of infection and genetic diversity of porcine circovirus type 2 (PCV2) in wild boar (Sus scrofa) in Poland

Porcine circovirus type 2 (PCV2) is a widespread, important pathogen of domestic swine and the causative agent of postweaning multisystemic wasting syndrome and other diseases and conditions referred to as "porcine circovirus diseases." Specific antibodies and DNA to PCV2 have also been detected in European wild boars and North American feral pigs. We collected 312 tonsil samples from wild boars shot in 13 of 16 districts of Poland, and tested them for PCV2 DNA using a real-time PCR. We detected PCV2 DNA in 75.6% of tested tonsils, and in particular, in 60% of samples from the 2006-07 season, and 91% from 2007-08. The phylogenetic analysis that included 12 PCV2 sequences from wild boars revealed that they belonged to two genetic clusters, PCV2b and PCV2a. We present data on prevalence of PCV2 in Polish wild boars and for the first time report the PCV2a genotype in Poland.     

Nucleotide Sequence of Porcine Circovirus Associated with Postweaning Multisystemic Wasting Syndrome in Pigs

This article describes the nucleotide sequence of a porcine circovirus (PCV) which possesses a high degree of association with postweaning multisystemic wasting syndrome (PMWS), a newly described disease of young pigs. The DNA sequence of this PMWS-associated PCV (pmws PCV) has 68% homology with that of a previously published nonpathogenic strain of PCV. The strains appear to be closely related yet distinct from one another.     

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed di...