Yonghou Jiang

Simultaneous detection of porcine circovirus type 2, classical swine fever virus, porcine parvovirus and porcine reproductive and respiratory syndrome virus in pigs by multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) was designed for the simultaneous detection of four viruses involved in reproductive and respiratory failure in pigs: porcine circovirus type 2 (PCV-2), porcine parvovirus (PPV), classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted fragment of the specific viruses. The sensitivity of the multiplex PCR using purified plasmid constructs containing the specific viral target fragments was 2.58x10(7), 2.64x10(5), 2.66x10(7) and 2.73x10(5) copies for PRRSV, PCV-2, CSFV and PPV, respectively. Using the multiplex PCR, co-infections with these four viruses were identified in 26/76 (34.2%) piglets born from sows with reproductive failure in China. This method is a rapid, sensitive and cost-effective diagnostic tool for the routine surveillance of viral infections in pigs.

ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of mRNAs in vitro

Porcine circovirus type 2 (PCV2) is the essential infectious agent of PCV associated disease (PCVAD). During previous in vitro studies, 11 RNAs and four viral proteins have been detected in PCV2-infected cells. Open reading frame (ORF) 4 is 180bp in length and has been identified at the transcription and the translation level. It overlaps completely with ORF3, which has a role in virus-induced apoptosis. In this study, start codon mutations (M1-PCV2) or in-frame termination mutations (M2-PCV2) were utilized to construct two ORF4-protein deficient viruses aiming to investigate its role in viral infection. The abilities of M1-PCV2 and M2-PCV2 to replicate, transcribe, express viral proteins, and to cause cellular apoptosis were evaluated. Viral DNA replication curves supported that the ORF4 protein is not essential for viral replication, but inhibits viral replication in the early stage of infection. Comparison of the expression level of ORF3 mRNA among wild-type and ORF4-deficient viruses in infected PK-15 cell demonstrated enhanced ORF3 transcription of both ORF4 mutants suggesting that the ORF4 protein may play an important role by restricting ORF3 transcription thereby preventing virus-induced apoptosis. This is further confirmed by the significantly higher caspase 3 and 8 activities in M1-PCV2 and M2-PCV2 compared to wild-type PCV2. Furthermore, the role of ORF4 in cell apoptosis and a possible interaction with the ORF1 associated Rep protein could perhaps explain the rapid viral growth in the early stage of infection and the higher expression level of ORF1 mRNA in ORF4 protein deficient PCV2 mutants.

Identification and characterization of two novel transcription units of porcine circovirus 2

Porcine circovirus type 2 (PCV2) infection is associated with porcine circovirus-associated diseases in pigs, which is a serious threat to the swine industry worldwide. To date, only three open reading frames (ORFs) within the PCV2 genome have been reported: ORF1 codes for two replicase proteins (Rep and Rep′), ORF2 for the structural protein (Cap), and ORF3 for a protein implicated in cellular apoptosis. In this study, based on transcription analysis of ORF3 mRNA, a potential ORF4 mRNA was detected and characterized by real-time RT-PCR and rapid amplification of cDNA ends analysis. The results indicate that the ORF4 gene is expressed at the level of transcription in the PCV2-infected cells. In addition, a novel ORF3 associated (ORF3′) mRNA was identified during virus replication in PK15 cells. Moreover, a 3′ poly(A) addition signal sequence (AUUAAA, nt 258–263) was found 10–30 nucleotides upstream of the cleavage site in the novel ORF4 mRNA in the complementary-strand of the PCV2 genome. Furthermore, alternate trans-splicing was identified in the ORF3′ mRNA between orientation diverse transcripts with typical GT-AG donor/acceptor junctions. Similar strategies as in this work can be applied to examine the transcription of other potential ORFs in PCV in the future.

Detection and genotyping of porcine circovirus in naturally infected pigs by oligo-microarray.

A rapid and reliable method for the identification of porcine circovirus (PCV) genotypes based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides (22-30 mer) immobilized on the surface of glass slides were selected to bind to the multiple target sites within the replication gene that are conserved among individual PCV genotypes. Cy5-labeled DNA targets were amplified in a PCR with primers common to both genotypes. The identification of PCV genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The utility and feasibility of oligonucleotide microarray hybridization was evaluated by testing standard and 87 clinical isolates. Analysis of the specimens showed that this microarray-based method is capable of unambiguous identification of both genotypes and fivefold more sensitive than gel electrophoresis. Our results indicated that the oligonucleotide array is useful for the identification and discrimination of PCV from clinical isolates and specimens in a clinical laboratory.

Development of an EvaGreen-based multiplex real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of six viral pathogens of porcine reproductive and respiratory disorder

Concurrent infection of pigs with two or more pathogens is common in pigs under intensive rearing conditions. Porcine circovirus type 2 (PCV2), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japanese encephalitis virus (JEV) and pseudorabies virus (PRV) are all associated with reproductive or respiratory disorders or both and can cause significant economic losses in pig production worldwide. An EvaGreen-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed in this study for simultaneous detection and differentiation of these six viruses in pigs. This method is able to detect and distinguish PCV2, PPV, PRRSV, CSFV, JEV and PRV with the limits of detection ranging from 100 to 500 copies/μL, high reproducibility, and intra-assay and inter-assay variation ranging from 0.11 to 3.20%. After validation, a total of 118 field samples were tested by the newly developed EG-mPCR. PCV2 was identified in 23%, PPV in 15%, PRRSV in 17% and PRV in 5% of the samples. Concurrent PCV2 and PRRSV infection was detected in 6.7%, PCV2 and PPV in 5% and PPV2 and PRRSV infection was detected in 5% of the cases. The agreement of the EG-mPCR and conventional PCR tests was 99.2%. This EG-mPCR will be a useful, rapid, reliable and cost-effective alternative for routine surveillance testing of viral infections in pigs.

A sensitive multiplex real-time PCR panel for rapid diagnosis of viruses associated with porcine respiratory and reproductive disorders

The objective of this study was to develop a multiplex real-time PCR panel using TaqMan probes for the detection and differentiation of porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus North American type (PRRSV-NA), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine parvovirus type 1 (PPV1) and Japanese encephalitis virus (JEV). Specific primer and probe combinations for PCV2, PRRSV, PRV, CSFV, PPV1 and JEV were selected within the conserved region of each viral genome. The multiplex real-time PCR panel which was run in two separate tubes was capable of specific detection of the six selected pig viruses, without cross-reactions with other non-targeted pig viruses. The detection limit of the assays was 10 copies/μL for PCV2, PRV, CSFV and PRRSV and 100 copies/μL for PPV and JEV. The two-tube multiplex real-time PCR panel showed 99.2% concordance with conventional PCR assays on 118 field samples. Overall, the multiplex real-time PCR panel provides a fast, specific, and sensitive diagnostic tool for detection of multiple viral pathogens in pigs and will be useful not only for diagnostics, or ecological, epidemiological and pathogenesis studies, but also for investigating host/virus or virus/virus interactions, particularly during coinfections.

A novel diagnostic platform based on multiplex ligase detection-PCR and microarray for simultaneous detection of swine viruses.

Simultaneous detection and identification of multiple pathogens is required in many diagnostic fields. In this study a novel method based on a multiplex ligase detection (LD)-polymerase chain reaction (PCR) and microarray (MLPM) is described to detect simultaneously several swine viruses involved in reproductive and/or respiratory problems. The multiplex diagnostic system was validated using standard plasmids, and clinical samples. Using this strategy as few as 10 copies of target plasmids were detected successfully. Each probe pair yielded specific positive signal only in its target site. In addition, when six target plasmids were present simultaneously sufficient robust signals were generated in their corresponding sites of six plasmid templates and no obvious signals were detected in non-target sites. Compared to real-time PCR, the MLPM showed specificities and sensitivities of 95.7-100% and 100% for 47 clinical samples tested, respectively. The results demonstrate that this novel assay is a specific, sensitive, and multiplex diagnostic method for detection of multiple pathogens and can also be adapted easily for diagnostic purposes.

Development and validation of a multiplex conventional PCR assay for simultaneous detection and grouping of porcine bocaviruses

Porcine bocavirus (PBoV), a newly described porcine parvovirus, has received attention because it can be commonly identified in clinically affected pigs including pigs with post-weaning multisystemic wasting syndrome (PWMS) and pigs with diarrhea. In recent years, novel PBoVs have been identified and were classified into three genogroups, but the ability to detect and classify these novel PBoVs is not comprehensive to date. In this study, a multiplex conventional PCR assay for simultaneous detection and grouping of PBoVs was developed by screening combinations of mixed primer pairs followed by optimization of the PCR conditions. This method exclusively amplifies targeted fragments of 531bp from the VP1 gene of PBoV G1, 291bp from the NP1 gene of PBoV G2, and 384bp from the NP1/VP1 gene of PBoV G3. The assay has a detection limit of 1.0×10(3)copies/μL for PBoV G1 4.5×10(3) for PBoV G2 and 3.8×10(3) for PBoV G3 based on testing mixed purified plasmid constructs containing the specific viral target fragments. The performance of the multiplex PCR assay was comparable to that of the single PCRs which used the same primer pairs. Using the newly established multiplex PCR assay, 227 field samples including faeces, serum and tissue samples from pigs were investigated. All three PBoV genogroups were detected in the clinical samples with a detection rate of 1.3%, 2.6% and 12.3%, respectively for PBoV G1, G2 and G3. Additionally, coinfections with two or more PBoV were detected in 1.7% of the samples investigated. These results indicate the multiplex PCR assay is specific, sensitive and rapid, and can be used for the detection and differentiation of single and multiple infections of the three PBoV genogroups in pigs.

Application of multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) mediated universal DNA microarray for detecting potato viruses in field samples

To facilitate the comprehensive and unbiased analysis of viral prevalence in a given biological setting, we have applied conventional PCR and multiplex reverse transcription-ligase detection reaction-polymerase chain reaction (MRLP) based universal microarray for the identification of potato virus infections in potato samples from tubers and leaves. With this MRLP based microarray (MRLPM) technique, total RNAs of 79 field samples were reverse transcribed by random primers, then subjected to a ligase detection reaction (LDR) and asymmetric labelling PCR as templates, finally, the MRLP amplicons were analyzed by zip-code microarray hybridization and subsequent scanning. The concordance rates between the MRLPM and the PCR results were 92.50 to 98.73% for ten potato viruses. In addition, the positive incidence obtained by the MRLPM was higher than that by PCR for corresponding viruses in field samples. In conclusion, the method was able to simultaneously detect ten potato viruses from samples with and without symptoms. Thus, this assay can be used as a rapid and sensitive technique for routine potato virus detection in field surveillance and in plant quarantine.