Wanzhe Yuan

Development of a TaqMan-based real-time PCR assay for the specific detection of porcine circovirus 3

Porcine circovirus 3 (PCV3) is a novel circovirus that was associated with porcine dermatitis and nephropathy syndrome, reproductive failure, and multisystemic inflammation. The objective of this study was to develop a rapid, simple, specific and sensitive TaqMan-based real-time PCR assay for PCV3 detection. Specific primers and probe were designed for the cap gene of PCV3 within the conserved region of viral genome. The assay was highly specific for PCV3, without cross-reactions with other non-targeted pig viruses. The detection limit of this assay was 102 copies. The assay had an efficiency of 95.7%, a regression squared value (R2) of 0.994 and showed a linear range of 102-107 copies PCV3 DNA per reaction. The assay was also very reproducible, with the intra- and inter-assay coefficient of variation less than 2.0%. For the 112 archived clinical samples collected from 2014 to March 2017, the PCV3 positive ratio was 12.5% (14/112) with the real-time PCR. The presence of the PCV3 dated back to at least 2014 in China and samples collected in 2017 had the highest PCV3 positive ratio (46.7%, 7/15). The real-time PCR assay could be used for detection of PCV3 in epidemiological and pathogenesis studies.

Development of a nanoparticle-assisted PCR assay for detection of porcine epidemic diarrhea virus

Abstract Porcine epidemic diarrhea virus (PEDV) is an important pig pathogen that can cause vomiting, diarrhea, and dehydration, leading to serious damage to the swine industry worldwide. In this study, a nanoparticle-assisted polymerase chain reaction (nanoPCR) assay targeting the N gene of PEDV was developed and the sensitivity and specificity were investigated. Under the optimized conditions for detection of PEDV RNA, the nanoPCR assay was 100-fold more sensitive than a conventional RT-PCR assay. The lower detection limit of the nanoPCR assay was 2.7×10−6 ng/μL of PEDV RNA and no cross-reaction was observed with other viruses. This is the first report to demonstrate the application of a nanoPCR assay for the detection of PEDV. The sensitive and specific nanoPCR assay developed in this study can be applied widely in clinical diagnosis and field surveillance of PEDV-infection.

Isolation and molecular analysis of porcine encephalomyocarditis virus strain BD2 from northern China.

Encephalomyocarditis virus (EMCV) can cause acute myocarditis in young pigs or reproductive failure in sows. In this study, an EMCV strain (BD2) was isolated from the suspected piglets with EMCV in China. It was identified by an indirect immunofluorescence assay and reverse-transcription polymerase chain reaction. The virus could reproduce on BHK-21 cells and reach a peak titer by 16 hpi with maximum titers of 10(8.22)TCID50/0.1 ml. Phylogenetic analyses of open reading frame and the VP3/VP1 genes using neighbor-joining method revealed that EMCV isolates fell into two clusters: groups I and II. The BD2 isolate belonged to group I, along with strains NJ08, HB1, BJC3, CBNU, K3, K11, BEL-2887A, GX0601, GXLC, pEC9, and PV21, whereas four strains (D variant, EMCV-B, EMCV-D, and PV2) belonged to group II.

Recombinase Polymerase Amplification Assay—A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection.

Reverse transcription recombinase polymerase amplification assay for the rapid detection of type 2 porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in pigs, and has tremendous negative economic impact on the swine industry worldwide. PRRSV is classified into the two distinct genotypes: type 1 and type 2, and most of the described PRRSV isolates in China are type 2. Rapid and sensitive detection of PRRSV is of great importance for the disease control and regional eradication programs. Recombinase polymerase amplification (RPA) has emerged as a novel isothermal amplification technology for the molecular diagnosis of infectious diseases. In this study, a fluorescence reverse transcription RPA (RT-RPA) assay was developed to detect the type 2 PRRSV using primers and exo probe specific for the viral nucleocapsid gene. The reaction was performed at 40°C within 20min. The RT-RPA assay could detect both the classical (C-PRRSV) and highly pathogenic PRRSV (HP-PRRSV), but there was no cross-reaction to other pathogens. Using the in vitro transcribed PRRSV RNA as template, the analytical sensitivity of RT-RPA was 690 copies. The assay performance was evaluated by testing 60 field samples and compared to real-time RT-PCR. The detection rate of RT-RPA was 86.6% (52/60), while the detection rate of real-time RT-PCR was 83.3% (50/60). This simple, rapid and reliable method could be potentially applied for rapid detection of PRRSV in point-of-care and rural areas.

Development of a TaqMan-based real-time reverse transcription polymerase chain reaction assay for the detection of encephalomyocarditis virus

Encephalomyocarditis virus (EMCV) is one of the major zoonosis pathogens and can cause acute myocarditis in young pigs or reproductive failure in sows. In this study, a TaqMan-based real-time reverse transcription polymerase chain reaction (RT-PCR) assay targeting 3D gene of EMCV was developed and their sensitivities and specificities were investigated. The results indicated that the standard curve had a wide dynamic range (10(1)-10(6) copies/μL) with a linear correlation (R(2)) of 0.996 between the cycle threshold (Ct) value and template concentration. The real-time RT-PCR assay is highly sensitive and able to detect 1.4×10(2) copies/μL of EMCV RNA, as no cross-reaction was observed with other viruses. These data suggested that the real-time RT-PCR assay developed in this study will be suitable for future surveillance and specific diagnosis of EMCV-infection.

Complete Genome Sequence of Porcine Circovirus Type 2 Strain HB-MC1.

ABSTRACT Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). Here, we report the complete genome sequence of PCV2 strain HB-MC1, which belongs to PCV2d.

Development of a taqman-based real-time PCR assay for the rapid and specific detection of novel duck- origin goose parvovirus

A real-time PCR assay was developed for specific detection of novel duck-origin goose parvovirus (N-GPV), the etiological agent of duck beak atrophy and dwarfism syndrome (BADS). The detection limit of the assay was 102 copies. The assay was useful in the prevention and control of BADS.

Effect of porcine circovirus type 2 (PCV2) on the function of splenic CD11c+ dendritic cells in mice

Porcine circovirus-associated disease (PCVAD) caused by porcine circovirus type 2 (PCV2) is an important disease in the global pig industry. Dendritic cells (DCs) are the primary immune cells capable of initiating adaptive immune responses as well as major target cells of PCV2. To determine whether PCV2 affects the immune functions of DCs, we evaluated the expression of endocytosis and co-stimulatory molecules on DCs (CD11c+) from PCV2-infected mouse spleen by flow cytometry (FCM). We also analyzed the main cytokines secreted by DCs (CD11c+) and activation of CD4+ and CD8+ T cells by DCs (CD11c+) through measurement of cytokine secretion, using ELISA. Compared with control mice, PCV2 did not affect the endocytic activity of DCs but it significantly enhanced TNF-α secretion and markedly decreased IFN-α secretion. Subsets of CD40+, MHCII+ CD40+ and CD137L+ CD86+ DCs did not increase obviously, but MHCII+ CD40- and CD137L- CD80+/CD86+ DCs increased significantly in PCV2-infected mouse spleen. Under the stimulation of DCs from PCV2-infected mouse, secretion of IFN-γ by CD4+ and CD8+ T cells and of IL-12 by CD8+ T cells was significantly lower than in control mice, while secretion of IL-4 by CD4+ T cells was remarkably higher. These results indicate that PCV2 modulates cytokine secretion and co-stimulatory molecule expression of DCs, and alters activation of CD4+ and CD8+ T cells by DCs. The immunomodulatory effects of PCV2 on DCs might be related to the host’s immune dysfunction and persistent infection with this virus.

Prevalence and genetic variation of porcine circovirus type 2 in Hebei, China from 2004 to 2014

Porcine circovirus type 2 (PCV2), the primary causative agent of porcine circovirus-associated disease (PCVAD), causes severe economic losses to the pig industry in China since 2002. To investigate the molecular epidemic characteristics and genetic evolution of PCV2, 12 PCV2 isolates obtained from different pig farms with various clinical symptoms of PCVAD in Hebei, China from 2004 to 2014 were sequenced and analyzed. The phylogenetic analysis showed that the 12 isolates were divided into two distinct genotypes, PCV2b (7/12) and PCV2d (5/12), based on the sequences of either viral complete genome or open reading frame 2 (ORF2). Of the 7 PCV2b strains, 5 were isolated from 2004 to 2008 while all PCV2d were isolated from 2009 to 2014. This exhibited that PCV2b isolates were the most common before 2009 and then PCV2d isolates became predominant and widely distributed in pig farms. Sequence comparisons among total isolates indicated that the nucleotide identity ranged from 95.5% to 100% for complete genome and 93.1%-100% for ORF2. Compared with seven PCV2b isolates, there were thirteen amino-acid substitutions in the ORF2 region and one additional amino-acid K at this region terminal for five PCV2d isolates. The results suggest that a higher genetic variation and a distinct genotype shift occurred among the PCV2 isolates collected from 2004 to 2014 in Hebei.