| Porcine pseudorabies(PR),caused by pseudorabies virus(PRV),is an important infectious disease.RRV is a member of the subfamily Alphaherpesvirinae and the family Herpesvindae.PRV be also known as suid herpesvirus 1(SuHV-1)or Aujeszky's Disease virus(ADV).Until now,only a single serotype of PRV is known.It is well known that vaccine immunization is an important mean for preventing and controlling the disease.In some countries,such as Germany,USA,PRV has been successfully eradicated from domestic pigs,but in recent years,there have been reported that it occurred on wild boar in the eradicated country.However,since late 2011,PR has reoccurred in several Bartha-k61-vaccinated pigs in swine farms across China.The disease is characterized by classical PR clinical symptoms,including high fever,respiratory distress,diarrhea,depression,anorexia and systemic neurological symptoms,which results in the high mortality in newborn piglets(up to 50%)and increased mobidity in growing and finishing pigs(10-30%).Sera samples positive for antibodies against PRV/gE(an indicator of field infection rather than vaccination,as all vaccines used in China are gE knock-out strains,including the Bartha-k61 strain)from the outbreak farms rapidly rose to 50%.The disease has spread to most areas in China and has caused huge economic losses.Furthermore,the PRV variant was found to be more virulent compared with classical PRVs in mice and in piglets of different ages.However,the mechanism of virulence enhancement of PRV variant is not clear.In this study,the biological characteristics of the variant strain,the whole genome sequencing of the PRV variant and the transcriptomics sequence of the PK-15 cells infected with PRV variant were studied.It may be helpful to understand the mechanism of virulence enhancement of PRV variant and it is also of great significance for the prevention and control of PR in our country.1.Isolation and identification of PRVIn the period from 2014 to 2016,five PRV strains were isolated and identified from the diseased pigs in Jiangsu and Shanghai,named as XJ5,CM,NT,HA and TX.The purified virus was confirmed by PCR,IF,A and electron microscopy observation.The sequence analysis of the gE,gC,gI,gD,gB and TK gene of five isolates,the sequence homology of the five isolates was very high between them.Based on the gE gene and gC gene to construct the genetic phylogenetic tree,the results showed that the five strains were close to PRV variant HeNl strain and JS-2012 strain,and all strains belonged to the same branch.The 50%lethal dose(LD50)of XJ5,CM,NT,HA and TX in mice model were 102 3TCID50,102.5TCID50,102.9TCID50,104.9TCID50 and 103.9TCID50,respectively.The results showed that although the genetic evolution distance was similar between them,there is a significant difference in virulence between each other.2.Bartha-k61 vaccine protects growing pigs against challenge with an emerging variant Pseudorabies virusSince late 2011,pseudorabies(PR)has resurfaced in many large pig farms,causing great economic loss for the swine industry in China.The PRV variant strain with high virulence and antigenic variation has been considered to be the main cause,and much attention has been focused on how to prevent and control the re-occurrence of this disease in China.In this study,two kinds of vaccination strategy were employed to evaluate the protective effects of Bartha-k61 vaccine against both variant PRV(XJ5)and classical PRV(Ra)strain challenge.Humoral immunity response,clinical signs,survival rate,body weight,virus shedding and pathology were assessed in commercial pigs.The results showed that Bartha-k61 vaccine,administered either once or twice,was effective against the PRV variant(XJ5)challenge,while no significant differences were observed between single and prime-boost vaccinated pigs.However,pigs vaccinated twice had better body weight gains than those vaccinated once,following challenge with the classical PRV strain(Ra)(p<0.01).Therefore,the Bartha-k61 vaccine appears to be an effective vaccine to control the spread of PRV variants in China in the absence of new powerful candidate vaccines specific to these PRV vatiant strains.3.Tissue distribution and pathogenicity assay of different pseudorabies virus on Balb/c micePrevious studies have confirmed that different strains have different virulence despite similar genetic evolution distances.First,the restriction fragment length polymorphism of BamHI digestion and the genetic evolution of gE gene and gC gene were analyzed by eight strains of pseudorabies virus including both variant and classical strains.The results showed that the genomes of 8 strains were different,and the genetic evolution belonged to different branches.Among them,variant strains including XJ5,CM,NT,HA and TX were closely related to the same branch,and classical strains JSZ and Ra was similar,belonging to the same small branch,Su strain was similar to foreign isolates.By comparing the one-step growth curve of eight viruses on PK-15 cells,the growth kinetics of 8 strains on PK-15 cells were similar.In order to verify whether the virulence differences were associated with the colonization and distribution of the virus in the tissue,Balb/c mice were inoculated through the nasal cavity to model natural infection means.The clinical symptoms,survival rate,mean survival time were recorded and assessed the virulence of different strain.The number of viral nucleic acid copies in different tissues were recorded and compared between the different strains.Balb/c mice nasal infection assay results showed that the replication of each virus was mainly in the lung and brain,followed by the heart and liver,spleen and kidney content of the least.And the virulence of HA strain was the weakest,consisted with its viral load in the tissue was also significantly lower than other viruses.This is consistent with the LD50 experimental results.This result also showed that the virulence of the virus was indeed associated with the ability of the virus to colonize and replicate within the body.4.Complete genome sequencing of PRV variant and classical strainPrevious studies have shown that the virulence of the variant strain(XJ5)and the classical strain(Ra)is quite different.In order to explore the cause of the difference between two strains in virulence,the complete genome sequences of two strains were determined by high-throughput sequencing.Sequencing with Illumina HiSeq2500,assembly and splicing were used to obtain a full length sequence without Gap.The genomic size of XJ5 and Ra were 141.955 kb and 143.227 kb,with corresponding G+C contents of 73.79%and 73.66%,respectively.Through the genetic prediction software analysis,the number of gene was 76 in the XJ5 genome and the total length of the coding gene was 106185 bp,and Ra genome included 83 genes,the total length of the coding gene was 105489 bp.Then about 40 kb belonged to the non-coding area.By repeated sequence prediction software analysis,it was found that there were a large number of repeats in the two genomes,including scattered in the repeat sequence and the tandem repeats,accounting for about 10%of the whole genome.At the same time,we used the Sanger sequencing method to verify the full sequence of six genes.The results showed that the sequencing results were reliable.Comparative genomic analysis showed that the sequence similarity of XJ5 strain to HeNl was 99.5%,and the sequence similarity to Ra was 98.81%.Through the analysis of coding genes,we found that the main variation of XJ5 strain was the mutation of US1,UL36 and IE180 compared with Ra strain.The genetic phylogenetic tree based on the complete genome was constructed,the results showed that the genetic evolution relationship of XJ5 strain was the closest to Ea strain.And the genetic evolution of Ra strain was close to that of HLJ8 strain.From the phylogenetic tree based on the complete genome,it shows that the PRV isolates were diverse in our country.5.Transcriptome of PK-15 Cells infected with RRV variant XJ5In order to screen related genes of PRV variant and its host interaction,XJ5 strain isolated in our laboratory was inoculated to PK-15 cell with an MOI=5,total RNAs were extracted from PRV infected PK-15 cells at 6 hours post-infection(hpi),12 hpi and mock.RNA-seq was performed using Illumina HiSeq2500.The results of sequencing showed that 1576 differentially expressed genes were screened at 6 hpi vs mock,and 944 genes were up-regulated and 632 genes were down-regulated.But,compared with mock group,only 84 differentially selected genes were screened for 12 hpi,and 78 genes were up-regulated,and 6 genes were down-regulated.Through the KEGG pathway analysis,the results showed differentially expressed genes screened 6 hpi group vs the mock were enriched to 258 signal pathways,including Metabolic pathways,TNF signaling pathway,Oxidative phosphorylation,Adherens junction,Parkinson's disease and so on.Differentially expressed genes screened 12 hpi group vs the mock group was enriched to 89 signal pathways through the KEGG pathway analysis,including Metabolic pathways,Oxidative phosphorylation,Parkinson's disease,Alzheimer's disease,Cardiac muscle contraction.In order to verify the reliability of RNA-seq data for transcriptome sequence,ten differential expression genes screened from 6 hpi group vs the mock were chosen and quantified by qRT-PCR.The variation tendency of these genes detected by qRT-PCR was consistent with that by RNA-seq analysis.These results provided important data support for the future study of the interaction between PRV variant and the host. |
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