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Isolation, Characterization and Phylogenetic Analysis of the Field Japanese Encephalitis Virus Isolates from Boars Farm in Henan Province

Author: WangXingTao
Tutor: LiuLi；LuoJun
School: Southwestern University
Course: Preventive Veterinary Medicine
Keywords: Pig Japanese encephalitis virus Isolation and identification of Phylogenetic tree
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 36
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Abstract


Epidemic of Japanese encephalitis (Japanese encephalitis, JE) is a mosquito from the Japanese encephalitis virus (Japanese encephalitis virus, JEV) (?) As the major media zoonotic natural foci disease. JEV can cause a person's nervous system disorders, neurotoxic to occur very serious consequences, sow pigs caused stillbirth, mummified, abortion, boar orchitis reproductive disorders. Although the mortality of the disease of pigs is not high, but can severely damage the economic interests of the pig industry is the most important reproductive disorders, one of the pig industry. Mosquitoes Japanese encephalitis popular media, the pig is the most important JEV proliferation of host and storage host. In this study, 19 cases of suspected Japanese encephalitis cases boar semen samples collected from the the Henan scale pig farm JEV Isolation and identification of molecular evolution analysis, aimed at in-depth study of Japanese encephalitis in Henan Province, the large-scale pig farm epidemiological situation, to provide a scientific basis for effective control pigs Japanese encephalitis pandemic. The study reference JEV have been reported in GenBank M E gene sequence homology analysis using DNAstar 5.0 software to identify conserved sequences, two pairs of specific primers for JEVE and M gene designed using Primer Premier 5.0 software. By RT-PCR on semen samples collected preliminary identification of suspected Japanese encephalitis cases the material extracted acquisition of total RNA as the template for the RT reaction, application design specific primers for PCR amplification JEVM, E gene, final two of JEV successfully isolated from 19 semen samples. Suckling mice challenged experiments show that the 3-day-old suckling mice by intracerebral inoculation with pig semen supernatant inoculated 72h after the onset rat stiff limbs, head to the side distortions typical neurological symptoms. The exchange that poison suckling mice brain tissue, RT-PCR amplification of JEV M and E genes result, the test rats two kinds of PCR products were consistent with the expected size were amplified and the positive control, negative control mice, there was no strip. The sequencing results showed that these two kinds of PCR products were JEVM or E gene, and before the sequencing results entirely consistent, which indicates that this experiment is successfully separated from the pig semen to 2 JEV strains, which were named as BSF.ZZ-1 and BSF.ZZ-3. Organizations with the BSF.ZZ-1 BSF.ZZ-3 challenged neonatal rat brain poison inoculated BHK-21 cells at 60 h after inoculation with indirect immunofluorescence anti JEVE monoclonal antibody identification. The results show that the two strains after infection of BHK-21 cells surpassed showed obvious specific staining within the cytoplasm of the majority of cells, whereas no fluorescence of the negative control cells, and further confirmed that the strains isolated in this experiment for JEV poison strains. New isolates were cultured in vitro in BHK-21 cells were sampled 72 h after infection, the virus titer determination. The results show that the viral titers were lower BSF.ZZ-1 and BSF.ZZ-3 of the 1st generation cell culture, respectively in 103.2 ± 02TCID50/mL and 101.6 ± 0.1 TCID50/mL then continues passaged virus volume gain a significant increase in the highest viral load of up to 104.5 ± 0.1 TCID50/mL and 104.2 ± 0.2 TCID50/mL. Finally, the newly isolated strains of JEV gene phylogenetic tree construction and analysis, and the use of foreign and domestic isolated SA14 and SA14-14-2, Ha-3, a total of 17, belonging to four different genetic subtypes M and E genes of JEV isolates JEV M gene and E genes were constructed phylogenetic tree for the reference sequence, Phylogenetic analysis of two newly isolated JEV. The results showed that 19 JEV strains were located in four different evolutionary branch, newly isolated BSF.ZZ-1 and BSF.ZZ-3 strain with domestic isolates SA14, SH-3, Ha-3, as well as vaccines strain SA14-14-2 M gene phylogenetic tree and E gene evolutionary tree branch are located in gene-III, and the new separation of these two strains of the vaccine strain SA14-14-2 genetic relationship.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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