Isolation, Identification And Sequence Analysis Of Pseudorabies Virus From Captive Wild Felids In China

Pseudorabies is caused by pseudorabies virus(PrV) infection with fever, itching and encephalomyelitis. The hosts of PRV are included pig, cattle, cat, rat, mink, fox, and rabbit. In recent years, the reports of Pseudorabies epidemic in China are increased dramatically. An outbreaks of Pseudorabies in pumas, jaguars, Korean leopard, ocelot occurred in 2012 in China. In these study, to confirm the outbreak, we performed virus isolation, identification, and preliminary investigation.From February 2012 to March 2012, two cougars, two jaguars, three ocelots, and three Korean leopard of a zoo of Beijing fell ill. The clinical manifestations included mental listlessness, Loss of appetite, strong thirstiness, and lastly dyspnea. All these animals died. We collected 40 samples including lung, nasal bone, intestine and contents, trachea, tonsils, kidney, liver, bladder, spleen to detect. PrV-specific primers(gBS/gBA, gDS/gDA) were used for PCR detect and the samples of lung, spleen, liver and kidney are PrV positive. According to the national standard GB/T 18641-2002 method, the homogenized tissue samples was used to inoculate to rabbits to evaluated the virulence of PrV. The inoculated rabbits showed characteristic manifestations of PRV including restlessness, and fear of people at post12 h of inculation, and died at post 30 h with convulsions. The virus was isolated by cultured in Vero cells and named BJ/Tiger/2012. The infectivity of BJ/Tiger/2012 were evaluated on MDCK, F81, and Vero cell lines. We found that the strain could induce CPE of rounding-up, shrinkage, and gatheringon F81 cell line within 48 h. CPE was also found in MDCK cell line with large-scale cell fusion, and formation of net-like structure. However, the virus induced cell fusion in Vero cell lines and shrinkage at post inoculation 72 h. after 4passages adapted on Vero, the CPE could be found at 48 h. the identify of biology, physical and chemical characterization revealed that BJ/Tiger/2012 doesnt agglutinate red blood cells of chicken, cat, guinea pigs, Kunming mouse, Balb/C mouse, or Amur tiger, and the virus can be inactivated by UV irradiation for 5min, or at 56℃ for 20 min.Total DNA of the fourth generation BJ/Tiger/2012 strain was isolated, and full-length gG, gE and the conserved region of gD genes were amplified by PCR. The 1540 bp, 1863 bp, and 767 bp PCR products were obtained respectively. Sequencing results revealed a 1500 bp ORF sequence for gG, a 1740 ORF sequence for gE, and a conserved 767 bp sequence for gD. Sequence of the gG gene showed a 98.9%~99.9 homology with that of PRV included in Genbank, and a 99.9% homology with the KC981239.1(Beijing) and KF711985.1(Hunan) sequences submitted by Chinese scholars in 2013; the gE gene showed homology of 97.8%~100% with the 16 stains of PRVs we submitted to NCBI; and the conserved region of gD showed similarities of 96.6%~100% with the 10 strains of PRVs submitted to NCBI by the United States, South Korea, and China.The domestic cats and canines were inoculated with BJ/Tiger/2012 virus. In intranasal infected group of cats, the animals started to show the symptoms at 42 hours post inoculation, including foamy salivation abundant, the body muscle rigidity, the cats suddenly died at 50 hours post infection. The samples of lungs, spleens, brains, intestines and kidneys of the infected cats were R positiveof PrV detected by PCR, while the samples of pancreas and skeletal muscles were negtive. The pathology analysis showed that increased alveolar macrophages,acute tubular mecrosis, skeletal muscle fracture. Meanwhile the immunohistochemistry analysis showed that the antigen of PrV were found in alveolar epithelial cells, renal tubular epithelial cell, mesenteric lymph node lymphocytes and a part of intestinal epithelial cells. The domestic canines, which were intranasally inoculated with BJ/Tiger/2012 vius, showed the clinical signs such as nervous disorders, respiratory disorders at 42 hours post infection, and died at 70 hours post infection. The organs of infected canines which were positive for the PrV, including lungs, spleens, brains, intestines and kidneys. The canines infected by hypodermic inoculated displayed the typical symptoms, including signs of severe pruritus, and died at 54 hours post infection. The lungs, spleens, brains, intestines kidneys and the inoculation domain were further confirmed with PrV PCR positive.To further investigate the epidemiology characterizations of PrV on Captive wild felids in China, totally 219 tissue and serum samples were collected from wild and economic animals since 2009, and were detected using PCR and gE-ELISA methods. The PCR results showed that 3 samples from 117 tissue samples were positive, collectedfrom a Zoo in Beijing, including two tigers and a wolf samples in 2013 and 2015, respectively. The ELISA results showed that 2 serum samples from 102 serum samples were PrV antibody positive. The twoserum samples were collected from a wolf from a Zoo in Beijing in 2012 and a tiger in a Zoo in Heilongjiang in 2009.We confirmed the lethal infection of captive wild feline with PrV by isolation and identification, animal challenge, and preliminary investigation. The results suggested that more attention should be paid on the control of the spreading of domestic infectious disease to wild animals.