| Pseudorabies(PR) is characterized by fever, itching, acute encephalomyelitis and reproductive failure which caused by pseudorabies virus(PRV). It is harmful to pigs in various stages and causes severe economic losses to the pig industry in many countries. PR was eliminated from domestic pigs in the USA and some European countries after conducting national pseudorabies eradication program. PR was well controlled in some large-scale pig farms in China through using attenuated PRV vaccine and carrying out the national pseudorabies eradication program. However, since the October in 2011, PR broke out in many unvaccinated and vaccinated pig farms in more than 20 provinces in China. It brought great challenges for the prevention and control of PR in China. In order to the in-depth understanding of the pathogenesis of PR, the following works were carried out in this study.1. The epidemiological investigation of PRVA total of 1227 samples were collected from 386 pig farms in some provinces of China during January to November in 2012. The results showed that 13.73%(53/386) of the farms existed wild-type PRV strain infection and 12.47%(153/1227) of the samples were PRV-positive. Especially, more than 30% of samples collected in April, May and November in 2012 were found wild-type PRV-positive.The immunogenicity and virulence-related genes(g B, g C, g E, TK, RR1, and RR2 genes) from eight PRV-positive samples were amplified for sequencing, comparative analysis of phylogenetic trees. The results showed that the g B, g C and RR1 genes in positive samples had amino acid deletions and insertions compared with those of Bartha strain, indicating higher homology with the Ea strain than with the Bartha strain, and were all clustered into the same group with Ea strain, while different from Bartha strain. The wild-type PRV strains were genetically closely related to Ea strain and differed from Bartha strain.2. Isolation and identification of PRVThe homologized PRV-positive samples were isolated using PK-15 cells and identified by PCR, IFA and observation under transmission electron microscope(TEM). Virus-like spherical particles of PRV HNX, HNB, and Fa strains with envelope(approximately 150 nm in diameter) were visualized by negative-staining TEM. The growth characterization of the isolates was determined by measuring the 50% tissue culture infective dose(TCID50) and one-step growth curve. The obtained five PRV strains were named as HNX, HNB, HNQZ, HNQX, and HNZK. During the first to nine serial passages, the infectious titers of these strain ranged 10-5.0-10-6.4 TCID50/0.1m L. The growth kinetics showed no significant difference in the replication kinetics of PRV HNX and Fa strains.3. Pathogenicity of PRV strains in miceThe 50% lethal dose(LD50) of PRV HNX, HNB, Ea, and Fa strains in mice model were 102.0TCID50, 102.4TCID50, 102.0TCID50, and 100.7TCID50, respectively. The mice inoculated with PRV HNX or HNB strain at the same inoculated dose presented earlier and more serious onset of clinical signs, and more severe microglia, neuronal degeneration and necrosis, compared with those inoculated with PRV Ea strain.4. Pathogenicity of HNX strain in piglets with Bartha-derived maternal antibodyThe piglets with Bartha-derived maternal antibody were inoculated intramuscularly(i.m.) with 107 TCID50 of PRV HNX strain or Ea strain. The result showed that fever and itching symptom were observed at the injection site, the viruses were detected in nasal swabs and rectal swabs from piglets after i.m. with HNX strain. Low copy of PRV DNA was detected in the brain, lung, tonsil, spleen, liver, and kidney at the 28 th day in PRV HNX inoculated piglets. In PRV Ea inoculated piglets, no clinical symptoms were observed, the viruses were detected in nasal swabs and rectal swabs with 3 to 5 days post infection and low copy of PRV DNA was detected in the brain, lung, and tonsil at the 28 th day. The result indicateds that the PRV HNX strain was still pathogenic for piglets with PRV Bartha-induced maternal antibody. The PRV maternal antibody doesn’t provide piglets protection against fatal infection of PRV HNX strain and also can’t prevent PRV shedding.5. Pathogenicity of PRV HNX strain in piglets with immunized with Bartha vaccineThe PRV antibody free piglets were inoculated i.m with one dose of PRV Bartha-K61 strain vaccine or i.m. with PK-15 cells culture supernatant. Twenty-eight days after infection, they were challenged i.m. and intranasally with 107 TCID50 of PRV HNX strain. The result showed that the fever, lose of appetite, sneeze, dyspnea, depressed and other clinical symptoms were observed in all piglets. The viruses were detected in nasal swabs and rectal swabs for piglets and high copy of PRV DNA was detected in the brain, lung, tonsil, spleen, liver, and kidney at the 16 th day in all piglets. The pathological lesions and viral signals were observed in the tonsil and lung of all piglets at 16 days after HNX infection through hematoxylin and eosin staining and immunohistochemical assay. The neurological symptoms and 50% mortality were observed in naive piglets implying the high pathogenicity of HNX strain to both naive and Bartha-induced piglets. The vaccination with Bartha vaccine doesn’t prevent HNX strain shedding and colonization of PRV HNX in the brain, lung, tonsil, spleen, and kidney, thus it couldn’t provide pigs with effective protection against infection with PRV HNX strain.6. Complete genomic sequencing of PRV HNX, HNB, Fa, and Ea strainsThe complete genome sequences of HNX, Fa, HNB, and Ea strains were determined through next-generation sequencing(NGS) technology and Sanger sequencing. The genomic size of HNX, Fa, HNB, and Ea strains were 142294, 141930, 142255, and 142334 bp, with corresponding G+C contents of 73.56%, 73.67%, 73.61%, and 73.60%, and Gen Bank under accession numbers KM189912, KM189913, KM189914, and KU315430, respectively. The four strains consistently possessed 70 open reading frames and were consisted of two unique regions(UL and US), with the US region flanked by the internal and terminal repeat sequences(IRS and TRS), respectively.7. Comparative genomics analysis of PRVs complete genomeSequence comparison and phylogenetic tree analysis of PRV complete genome or genes were performed. The complete sequence of PRV HNX shared 98.1%, 96.7%, 96.4%, and 90.1% homology with HNB, Ea, Fa, and Bartha strains, respectively. The complete sequence of PRV Bartha shared 90.3% and 90.7% homology with HNX and HNB strains, respectively. The nucleotide sequences and amino acid sequences of all genes of PRV Bartha strain shared 91.1%-99.9% and 82.3%-99.7% homology with HNX, HNB, Ea, and Fa strains. The complete genome sequences were largely conserved between PRV Chinese and non-Chinese strains, whereas the main foci of divergence generally occurred in UL36 gene, US1 gene, and noncoding regions. The genome-wide phylogenetic analysis demonstrated that all tested PRV strains were clustered into two groups: the Chinese strain’ group and non-Chinese strain’ group. The Chinese strains were clustered into some different subgroups. On the basis of immunogenicity-related genes, the novel PRV strains and previous PRV strains were clustered into two different subgroups, on the basis of the virulence-related genes, the novel PRV strains and previous PRV strains were clustered in the same subgroup. The result indicates that the novel Chinese PRV strains have high homology with each other and are clustered into the same group, but distinct from Bartha, Becker, and Kaplan strains. The immunogenicity-related genes of the novel and previous Chinese PRV strains were clustered into two different subgroups, whereas the virulence-related genes in the same subgroup.8. Recombination possibility analysis among PRV complete genomes and genesSimilarity plots and bootscanning analysis of the complete genome or some genes of PRV HNX strain with HNB, Fa, Ea, TJ, ZJ01, He N1, JS-2012, Bartha, Kaplan, and Becker strains were performed. The result showed that no recombination events exist in the complete genome sequence, individual UL, IR/TR, and US region of the HNX strain and other new isolates. No recombination events exist in UL51, UL49.5, UL49, UL47, UL46, UL27, UL36, UL44, UL15, UL1, IE180, US1, and US8 genes of the HNX strain, other new isolate strains, previous Chinese strains, and non-Chinese strains. The result indicates that no recombination events exist among novel Chinese PRV strains as well as between new isolates and Bartha strain. |
PDF Full Text Request