The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes.
Animals ; DNA Primers/genetics ; DNA, Mitochondrial/*genetics ; Gammaretrovirus/*genetics ; Polymerase Chain Reaction/*methods ; Proviruses/classification/*genetics ; Sensitivity and Specificity ; Sus scrofa/*genetics/*virology

Classical swine fever (CSF) is a contagious swine disease charactered by hemorrhagic fever and leukopenia,usually leading to substantial economic losses. To obtain a insight of leucopenia caused by CSFV infection, DNA microarray analyses of peripheral blood leucocytes (PBL) of the infected pigs was performed. Three health pigs were inoculated with a lethal dose of CSFV Shimen strain and their PBLs were isolated when the onset of typical clinical signs and then subjected to total RNA extraction followed by microarray analysis with Affymetrix Porcine Genome Array GeneChips. The results showed that the significant differences were observed in cellular apoptotic genes expression at 7 days post-infection (p. i.). The changes of the genes expression were confirmed by real time RT-PCR of some selected apoptosis-related genes. This study provided a valuable information for further investigating the molecular mechanism of apoptosis caused by CSFV infection.
Animals ; Apoptosis ; Cells, Cultured ; Classical Swine Fever ; genetics ; immunology ; virology ; Classical swine fever virus ; immunology ; physiology ; Gene Expression Profiling ; Leukocytes, Mononuclear ; cytology ; immunology ; virology ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; Sus scrofa

In early 2011, the serious outbreak of porcine pseudorabies virus (PRV) infection suddenly recurred in Henan and neighboring Provinces. To investigate the etiology of massive infection with PRV, 16 800 serum samples, 905 porcine epidemic diarrhea virus (PEDV) back-feeding tissues, and 56 PR gene deleted live vaccines were colleted from January 2011 to May 2013 to detect PRV field infection using a PRV gE antibody test kit. The gE and TK genes of 11 new epidemic PRV strains were sequenced by PCR, and their molecular characteristics were analyzed. Moreover, virus titer determination, protective test against PRV, and vaccine potency testing were performed. The results showed that the detection rate of PRV field infection-positive pig farms was 68.06%, and the overall positive rate of PRV field infection in serum was 38.47%; the positive rates in breeding sows, breeding boars, reserve pigs, and commercial pigs were 40.12%, 30.88%, 54.67%, and 26.52%, respectively. The new epidemic strains were in the same evolutionary branch and belonged to the virulent strain group. Compared with the classical PRV strain, the virulence of new epidemic strains changed a little. The length of gE gene was 1 787 bp, and the length of TK gene was 963 bp. The nucleotide homologies of gE and TK genes to Chinese reference strains were 98.2%-99.8% and 98.90%-99.6%, respectively, and the amino acid homologies were 97.1%-99.8% and 97.5%-99.4%, respectively. Commercial vaccine had a 100% protective effect against the new epidemic strains. The positive rate of PRV field infection was 0% in vaccine and 40.44% in back-feeding tissues. The results confirmed that PRV field infection rates were rising sharply among pigs in Henan and neighboring Provinces after 2011. The main virulence genes of new epidemic PRV strains did not change significantly over the years. PR gene deleted live vaccines had no PRV field infection and could completely resist the attack of new strains. The virus carriage of breeding boars and reserve pigs and the serious PRV field infection in PEDV back-feeding tissues were the main causative factors for massive infection with PRV and epidemic outbreak in Henan and neighboring Provinces from 2011 to 2013.
Amino Acid Sequence ; Animal Feed ; analysis ; virology ; Animals ; China ; epidemiology ; Epidemics ; Female ; Herpesvirus 1, Suid ; chemistry ; classification ; genetics ; isolation & purification ; Male ; Molecular Sequence Data ; Phylogeny ; Pseudorabies ; epidemiology ; virology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sus scrofa ; Swine ; Swine Diseases ; epidemiology ; virology ; Viral Proteins ; chemistry ; genetics