We designed two pairs of primers and their corresponding TaqMan probes according to gH, gE gene of PRV. By optimizing the probe's concentration, Mg2+ concentration, primers concentration and sample DNA extraction, real-time fluorescent quantitative PCR (FQ-PCR) which can quickly identity field virus and vaccine virus of PRV was established. According to our results, the dynamic range of the FQ-PCR assay is between 10 x 10(1) copies/microL and 10 x l0(8) copies/microL, and the detection limit of FQ-PCR is 1.0 x 10(1) copies/microL, which is 100 fold higher than that of conventional PCR. We detected 60 doubtful tissue samples using the FQ-PCR assay, serum neutralization and conventional PCR. In conclusion, the FQ-PCR method is rapid, sensitive, specific and accurate, and can be used to detect field strains of PRV rapidly. The closed-tube format of the assay minimized the risk of contamination of subsequent reaction and the assay can be performed in 2 h or less. Development of real-time quantitative PCR provides the basis for the early and rapid detection and analyzing quantitatively the infectious degree of PRV.
Animals ; Fluorescent Dyes ; Herpesvirus 1, Suid ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods ; Pseudorabies ; diagnosis ; prevention & control ; virology ; Pseudorabies Vaccines ; immunology ; isolation & purification ; Swine

The aim of the present study is to verify the functional and anatomical neural pathways which innervate the urinary bladder in the central nervous system of the rat. To identify the functional neural pathway, the urinary bladder was stimulated by infusing formalin for 2 h. Then, brain and spinal cord were dissected out and immunohistochemistry was done by using anti-c-fos antibody. Many c-fos immunoreactive (IR) neurons were identified in the telencephalic cortical areas and in several brainstem nuclei, which are known mostly to be related with urinary bladder. In the spinal cord, a number of c-fos IR neurons were found in the lamina I, IIo, dorsal gray commissure, sacral parasympathetic nucleus. To identify the anatomical neural pathway of the urinary bladder, Pseudorabies virus (PRV) was injected into the wall of urinary bladder and was identified with anti-PRV by using immunohistochemistry. Most PRV labeled neurons were found where c-fos IR neurons were identified and few of them were also in the areas where c-fos IR neurons were not found, e.g., prefrontal cortex, agranular insular cortex, and subfornical organ. In the spinal cord, PRV labeled cells were found all over the gray matter. The present study presents morphological evidence demonstrating the supraspinal areas are related with the neural control of the urinary bladder and most functional neural pathway of the urinary bladder is well consistent with the anatomical neural pathway except in some telencephalic cortical areas.
Animal ; Bladder/innervation* ; Central Nervous System/anatomy & histology* ; Female ; Herpesvirus 1, Suid/isolation & purification* ; Immunohistochemistry ; Neural Pathways/anatomy & histology ; Proto-Oncogene Proteins c-fos/analysis* ; Rats ; Rats, Sprague-Dawley

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

Our investigation was conducted in order to verify a recent severe epidemic at several swine farms in northern China that indicated a newly emerging disease. Evidence confirmed that the epidemic was caused by a virulent Pseudorabies virus infection in swine herds.
Animals ; China/epidemiology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Epidemics/*veterinary ; Herpesvirus 1, Suid/classification/*isolation & purification/*pathogenicity ; Pseudorabies/*epidemiology/mortality/pathology/virology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sequence Analysis, DNA/veterinary ; Swine ; Swine Diseases/*epidemiology/mortality/pathology/virology ; Vaccination/adverse effects/veterinary ; Virulence