| FPV(Feline panleukopenia virus) is hot fever which cats are most susceptible. Many feline animals are also susceptible to FPV. Due to the small size of the FPV, there were no reports about the virus. However, with the development of electron microscopes and techniques of viral isolation, the researchers isolated FPV from cells and tumor tissues. A French man, Verge discovered FPV and named it in 1939. In 1985, a FPV strain was first isolated from a natural infection case in China, which demonstrated that FPV existed in China. Reports confirmed that tigers, jaguars and lions which were infected by FPV in1986, which caused a great threat to the endangered wild animals in China. In 2008, a variant FPV strain(BJ-22) was isolated from monkey in China. The homology of sequence in VP2 strain is 98.75% and 98.15% similar to FPV and CPV. In a word, the host range of FPV has expanded from feline animals to non-human primates, which cause a great threat to public health.In this study, the physical and chemical charactarization of FPV strain HH-1/86 and analysis its whole genome sequence, On the basic of which the infectious clones of pFPV was built on. The F81 cells were transfected by liposome to rescue the virus rFPV, in order to provide a reverse genetics platform and reveal mechanism of transmission across species of FPV; a scientific basis of prevention and warnings about the cross transmission of FPV were provided.The result of the electronic microsocop show some visible three-dimensional symmetric virus particlcs with a diameter of about 20 nm. The tolerance to heat, acid and aether were detected. The virus was able to agglatinate 0.1% porcine red blood cells diluted at the pH of 5.8 ~ 6.8. The best pH for HA was 6.5. FPV strain HH-/86 has a full-genome of 5123 nt and contant 36.35% G+C. T he 3’ITR shows in a form of Y and obtain a length of 126 nt. On the other hand, 5’ITR is in an U form with the length of 198 nt. The method of over-lap PCR was used in the study, we use the fragment M1, M2, M3 as templates to clone the whole fragment as M1+M2+M3 in the middle of virus genome and identified it with gel electrophoresis. Then we use the infusion technology to insert the middle fragment into the PE plasmid which contain 3’ and 5’ ends. The 2981 nt A was mutant to G to form a new enzyme site Xho I to identify the recombinant virus after we rescue it. The full plasmid was identified by enzyme digestion to prove that it was successful constructed. The F81 cells were transected with Lip 3000 with the ratio of 7.5 μL Lip3000:3.5μg DNA. 72 hours after the infection, the rFPV0 was frozen and melted 3 times, then rFPV0 was passed to F81 at the volume ratio of 1:20. After rFPV1,the volume ratio for the passage was reduced to 1:100. The rFPV was passed to the fifteenth generation and was identified by the CPE, indirect fluorescence, growth curve, Western blot assay. The results indicted that there were no significance difference in the characteristics of virology and biological between the rFPV and its parent virus HH-1/86. This study provide the chance to examine the key amino acids for FPV to spread cross species and reveal the possible molecular mechanism of the high frequency cross species transmission of FPV. This would lay the foundation to prevent the FPV transmission cross species. |
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