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Development of a RT-nPCR Assay for Swine Hepatitis E Virus and Isolation of the SHEV-JX Strain

Author: ZhangXu
Tutor: DengShunZhou
School: Jiangxi Agricultural University,
Course: Preventive Veterinary Medicine
Keywords: Swine hepatitis E virus RT-nPCR Virus isolation 3'UTR
CLC: S858.28
Type: Master's thesis
Year: 2011
Downloads: 30
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Abstract


Swine hepatitis the (Swine Hepatitis E) is a zoonotic infectious disease caused by the the swine hepatitis virus (Swine Hepatitis E Virus, SHEV), predominantly latent infection in pigs. A growing number of studies have shown that swine hepatitis E virus and hepatitis E virus genome with high homology swine hepatitis E virus can directly cross-species infection, SHEV have important public health implications. Understanding of Jiangxi Province herd of swine hepatitis E infection and virus characteristics, the paper is the following. 1 according to the logged in GenBank the SHEV genomic sequence to design two pairs of degenerate primers, the establishment of a rapid detection of SHEV of RT-nPCR method, can be detected in a minimum content of 2.5 × 10-5NG SHEV RNA. Established method of bile SHEV as the the positive pig lungs, liver, spleen, kidney, and inguinal lymph nodes, blood, intestinal contents and feces SHEV detection and found that there are more than one organization organs SHEV. 270 copies of Jiangxi Province, parts of censorship 40-90 day-old pig bile SHEV testing, test results show that The SHEV the infection was seasonal changes: the first quarter of each year (1, 2, 3) and the fourth quarter (10,11,12) SHEV highest infection rate in the third quarter (7,8,9) the SHEV lower infection rates, second quarter (April, May and June) SHEV lowest rates of infection. Human lung adenocarcinoma cells (A549) as SHEV the host cell, isolated from pig bile samples get a swine hepatitis E virus, named after the isolates for SHEV-JX and its A549 cells cultural characteristics of a preliminary study. Bile samples were filtered to 0.22μm filter inoculated in A549 cells the Blind 3rd passage began to appear cell lesions (CPE), the cells refraction enhanced and gradually stretch to two, broken, cell growth state by dense arrangement gradually become loose arrangement to form a cross-linked structure, formed intracellular vacuoles off phenomenon. The 5-7 generation CPE is the most obvious 8th generation CPE began to weaken, and to 10 cells return to normal. The SHEV in 1-10 cells of the replication-specific RT-nPCR detection test results show that the proliferation of of the SHEV-JX strains in A549 cells can only be maintained for 7 generations. SHEV-JX strain RNA as a template, six degenerate primers designed SHEV genome sequences in GenBank have login amplified of the SHEV-JX genomic part sequence. The sequence length of 1629 bp, GenBank accession number: JN052926, including a partial sequence (1551 bp) of the SHEV ORF2 and 3'UTR sequences (78 bp). Sequence of swine hepatitis E virus nucleotide sequences in GenBank DNAstar software to compare and draw phylogenetic tree SHEV-JX strain homology with other swine hepatitis E virus strains from the results of 74.1 % -93.8%, the phylogenetic tree shows SHEV-JX strain of swine hepatitis E virus gene IV type. SHEV 3 `UTR, SHEV 3` UTR by two stem - loop structure, wherein the second stem - loop structure can be divided into two smaller stem - loop structure, the secondary structure of the poly (A) The length of the impact.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig
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