Development and application of a multiplex qPCR assay for the detection of duck circovirus, duck Tembusu virus, Muscovy duck reovirus, and new duck reovirus.

A multiplex qPCR assay was developed to simultaneously detect duck circovirus (DuCV), duck Tembusu virus (DTMUV), Muscovy duck reovirus (MDRV), and novel duck reovirus (NDRV), but it did not amplify other viruses, including duck virus enteritis (DVE), infectious bursal disease virus (IBDV), avian reovirus (ARV), H5 avian influenza virus (H5 AIV), H7 avian influenza virus (H7 AIV), H9 avian influenza virus (H9 AIV), Newcastle disease virus (NDV), and Muscovy duck parvovirus (MDPV), and the detection limit for DuCV, DTMUV, MDRV, and NDRV was 1.51 × 101 copies/µL. The intra- and interassay coefficients of variation were less than 1.54% in the repeatability test with standard plasmid concentrations of 1.51 × 107, 1.51 × 105, and 1.51 × 103 copies/µL. The developed multiple qPCR assay was used to examine 404 clinical samples to verify its practicability. The positivity rates for DuCV, DTMUV, MDRV, and NDRV were 26.0%, 9.9%, 4.0%, and 4.7%, respectively, and the mixed infection rates for DuCV + DTMUV, DuCV + MDRV, DuCV + NDRV, MDRV + NDRV, DTMUV + MDRV, and DTMUV + NDRV were 2.7%, 1.2%, 1.2%, 1.0%, 0.5%, and 0.7%, respectively.

[Sequencing and analysis of the complete genome of encephalomyocarditis virus strain GXLC isolated from swine].

The complete genome of encephalomyocarditis virus (EMCV)strain GXLC isolated from swine was sequenced and analyzed. Five overlapped gene fragments covering the entire open reading frame (ORF) were amplified by RT-PCR, and the 3'-untranslated region (UTR) and 5'-UTR were amplified by the 3'-rapid amplification of cDNA ends (RACE) and 5'-RACE method, respectively. The genome sequences of strain GXLC were obtained by assembling the sequences of RT-PCR-generated cDNA fragments. The length of the complete genome was 7 725 nucleotides (nt). The homology comparison and phylogenetic analysis of the nucleotide and deduced amino acid sequences between strain GXLC and other EMCV strains available in GenBank were performed. The results showed that the complete genome identity between GXLC strain and the strains from China, i.e. GX0601, GX0602, BJC3 and HB1 and the strains from other countries, i.e. CBNU, K3, K11, TEL-2887A, EMCV-R and PV21 was over 99%. The phylogenetic trees based on the complete genome, the structural protein or the non-structural protein gene sequences revealed that the tree topology was similar. All the EMCV strains could be divided into two groups: group I and group II, and group I could be subdivided into subgroup Ia and subgroup Ib. The strains from swine belonged to subgroup Ia or Ib, and the strains from mice belonged to subgroup Ia, while the strains from Sus scro fa belonged to group II. Strain GXLC, together with other EMCV isolates from China, belonged to subgroup Ia.

Cytokine mRNA expression profiles in peripheral blood mononuclear cells from piglets experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) significantly impact the swine industry worldwide. Co-infections with these viruses are common and several lines of evidence suggest that both PRRSV and PCV2 modify host immune responses that facilitate infection. This study examined cytokine mRNA expression profiles of peripheral blood mononuclear cells (PBMCs) from piglets experimentally co-infected with PRRSV and PCV2 to define the influence of co-infection on host immunity. PBMCs from infected and control piglets were stimulated with concanavalin A and the IL-2, IL-4, IL-6, IL-10, IL-12p40, IFN-gamma and TNF-alpha mRNA levels were determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR). PBMCs from PRRSV/PCV2 co-infected piglets had significantly reduced IL-2, IL-4, IL-6, IL-12p40 and IFN-gamma and significantly increased TNF-alpha mRNA levels compared to those of the piglets infected with either PRRSV or PCV2 alone. The IL-10 mRNA levels in all virus-infected groups were significantly up-regulated early during infection. These results suggested that co-infection synergistically suppresses T helper 1 (Th1)-type and Th2-type cytokine production by PBMCs, indicating that co-infection likely compromises cell-mediated and humoral immune responses resulting in increased severity of the diseases in piglets.