Isolaiton And Identification Of A Mutated Swine Pseudorabies Virus And Its Biological Characteristics Analysis

Pseudorabies virus (PRV) is the pathogen of swine Pseudorabies. Currently, Pseudorabies iswidespread all over the world. It is one of the most serious infectious diseases that harms the pigindustry and causes huge economic losses to the world every year.Since2011, many pig farms that had been vaccinated the sows with the gE-deleted vaccine haveemerged a serious disease that appears to be pseudorabies symptoms. In these farms, the sows gavebirth to weak piglets, still birth and abortion and many newborn piglets had the neurological symptomsand died quickly. We perform this study to further understand the genetic variation of PRV. We isolatedone mutated strain of PRV from one pig farm of Jiangsu, and did the work of biological characteristicsanalysis, and then we amplified and sequenced the complete genome sequence and did its evolutionaryanalysis.1. The isolation and identification of a mutated swine Pseudorabies virusIn this study, we first detected the pathogen infection of the brain tissue samples from the deadpiglets that had the symptoms of Pseudorabies from one pig farm of Jiangsu. We first identified thepathogen as pseudorabies using PCR methods. The homogenated sample of brain tissue was subjectedto infecting Vero cells, classical cytopathogenic effects of herpesvirus lesions appeared post infection24hours. The infected cells showed positive reaction with antibodies against PRV in IFA. The titer of thisisolate propagated in Vero cells was up to107.43TCID50/mL. The growth curve shows that thereplication ability of the isolated virus is lower than that of PRV S isolate. We name the virus as JS-2012.The mice injected with the isolate and PRV S strain appeared unbearable itching and died finally. TheLD50of the isolate was lower than PRV S strain. This indicates that the virulence of JS-2012is differentfrom PRV S isolate.2. The complete genome sequencing and analysis of PRV JS-2012In this study, we harvested the supernatant of the infected cells and extracted the DNA as the PCRtemplate. We divided the whole genome into43segments and designed about200pair of primers to dothe PCR amplification. We joined the fragments together after successfully sequencing the fragments.Finally, we obtained the complete genome of JS-2012isolate. The length is143708bp. Then, we locatedthe genes by sequence comparison. We used the software to search the potential ORF and the poly (A)signals. We found that the location sites of the genes varied and we predicted there are73potentialORFs and35poly (A) signals. These ORFs can encode more than100viral proteins and there are sometranscripts existed.3. The genetic variation of main functional regions and its antigenic analysis of PRV JS-2012In this study, we analyzed the genetic variation of genes and its encoded proteins. It included thevariation analysis of the encoding genes and its proteins, the potential transmembrane regions,glycosylation sites and antigenic analysis. We found that the most variable glycosylation proteins are gE,gB, gC and gD. The nucleotide identity of gE gene and the isolates of2012is99.9%, and they belong to the same branch in the evolutionary tree. It has two characteristic insertions of three continuousnucleotides and thus has two aspartic acids insertion. gB gene varies greatly and have severalcharacteristic deletions. It belongs to the different branch comparing to the reference strains. gC genehas many point insertions and one region of discontinuous insertions and one hypervariable region. ThegD gene has the insertion of6continuous nucleotides. The membrane proteins, tegument proteins andnucleocapsid proteins all have different variations. The variations of nonstructural proteins mainlyfocused on the encoding genes which have the function of gene regulation, replication and packaging.The regulatory regions also have considerable variations, such as enhancers and replication origins. Theanalysis of these variable regions helps us to further understand the virulence variation and provide thebasic information of the newly genetic engineering vaccines development.