Isolation,Identification And Comparative Genomics Analysis Of Porcine Pseudorabies Virus Strains

Pseudorabies?PR?,an infectious disease caused by pseudorabies virus?PRV?,caused high fever,diarrhea,depression,anorexia,mental and neurological disorders in pigs,also known as Aujeszky's Disease?AD?.PR is highly contagious disease and brings huge economic losses to the pig industry.Based on the experience of some European and American countries to eradication of pseudorabies in pigs successfully,China government introduced Bartha-K61 vaccine strain in the last century,and promoted and applicated this vaccine strain widely in China,and controlled the spread of pseudorabies in pig herds successfully.However,many large-scale immunized pig herds broke out pseudorabies,and gE antibodies turned from negative to positive since the end of 2011,which has seriously affected the pig industry in China.Most scholars believed that the national outbreak of pseudorabies may be related to factors such as viral virulence and genetic variation.The reason was not determined.In this study,four pseudorabies viruses that named as FJ01,FJ03,MS2018 and YK strains were successfully isolated from suspected PRV infected pigs in Sichuan and Fujian provinces,moreover,their titers were 10^-6.63,10^-7.08,10^-8.1 and 10^-7.18 TCID _50s/0.1 mL,respectively.The results of virulence test in Balb/c mice showed that LD₅₀ of the four viruses were 10^2.17,10^2.72,10^3.51 and 10^3.44 TCID_50s,respectively,and the virulence of FJ01was the strongest.Except YK strain,all three other viruses can cause severe itching symptoms in mice.The neutralization titer of YK strain was 1:512,which was significantly higher than that of FJ01,FJ03 and MS2018 strains and their neutralization titers were 1:64,1:16 and 1:32,respectively.The main immunogenic genes and virulence-related genes gB,gC,gE and TK of four PRV strains were amplified and sequenced,and their nucleotide and amino acid homologies were analyzed and the evolutionary trees were established.The results showed that the gB,gC,gE and TK genes of FJ01,FJ03 and MS2018 strains had higher homology with the mutants?TJ,HNX strains,etc.?isolated after 2011 at both nucleotide and amino acid levels.Compared with Fa strains,the homology of nucleotide and amino acid was 96.9%-100%and 98.9%-100%,respectively.In evolutionary relationship,although they belong to a large branch including Chinese strains,they belong to different evolutionary branches from Fa,which also indicated that the antigens of mutant strains may change at the gene level.The nucleotide homology and amino acid homology of the gB,gC,gE and TK genes of YK strain were 98.6%-99.7%and 97.8%-99.7%,respectively.YK was on the same branch as foreign strains in evolutionary relationship and had the closest affinity with Kaplan.According to the evolutionary relationship of antigenic genes and virulence of the isolated strains,we selected the strongest virulent FJ01 strain and YK which the main virulent strain were closer to the foreign strains for PacBio third-generation sequencing.The total length of PRV were 145465 bp and 142576 bp,respectively.The GC content were 73.53%and 73.64%,respectively.All of them encoded 70 genes.The genome structure of PRV was the same as that of PRV previously measured.It consisted of unique long region?U_L?,unique short region?U_S?,internal repeat sequence?IRs?and terminal repeat sequence?TRs?.Genome-wide alignment and coding gene homology analysis were performed between FJ01 and YK strains and reference sequences,respectively.The results showed that the nucleotide sequence of FJ01 genome had 96.36%-98.06%homology with the mutants isolated after 2011,and 96.36%-96.86%homology with the mutants isolated before 2011.YK strain had the highest homology with Kaplan strain,up to 97.72%,while the homology with other strains was lower than 95%.The differences between the two strains were mainly in the non-coding gene regions.YK strain has 100%homology with most of Kaplan genes,but there are dozens of amino acid deletions and insertions in UL36,UL47 and US1.Genome-wide evolutionary analysis showed that FJ01 was closer to the sub-branches of HNX and HNB,and YK and Kaplan were in the same branch.Therefore,YK strain was closer to foreign strains in the whole genome evolution level.It indirectly proved that the antigen of the strain was different from that of the domestic strain,which was also consistent with the results of serum neutralizing antibody test of the previous vaccine.The whole genomes of FJ01 and YK strains were recombined and analyzed by RDP4software.The recombinant signals were found in both strains.FJ01 recombined between12104 bp and 13552 bp,and the parents in this region were HLJ8 and TJ strains.Six recombination analysis algorithms supported the results.Between 66201 bp and 66735 bp,YK and Kaplan had the same parent strain,Becker,and recombined with the new variant HNX strain.Five algorithms supported this result.In addition,the recombination of immunogenic,virulence-related and homologous genes was analyzed.We found that YK was recombined in UL36 and UL47,respectively,and Becker and Ea were recombined.The phenomena of gene recombination were of great significance for studying the evolution and variation of PRV.In summary,four strains of PRV strain were isolated in this study,and their virulence to mice were determined.The complete genome sequence of two strains was detected,the genomic characteristics were analyzed,and the genetic recombination of the isolates was found.This will provided a reference for further research on porcine pseudorabies virus.