Getah virus strain and application thereof

A virus and strain technology, applied in the field of isolation and identification and application in inactivated vaccines, can solve problems such as non-availability, and achieve the effects of preventing epidemics and transmission, rapid preparation methods, and reducing economic losses.

Active Publication Date: 2021-12-03
CHINA ANIMAL HUSBANDRY IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no biological vaccine product available for the prevention and control of the disease. The prevention and control of the disease in domestic farms mainly

Method used

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  • Getah virus strain and application thereof
  • Getah virus strain and application thereof
  • Getah virus strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Isolation and identification of Getavirus GETV JXNC CGMCC No.22111

[0039] 1.1 Experimental materials

[0040] The disease samples used in the experiment came from a rabbit farm in Jiangxi Province, collected in October 2018; the pig kidney cell line (Porcine Kidney, PK-15) was purchased from ATCC, the American Culture Center.

[0041] 1.2 Primer design

[0042] Referring to the Getavirus genome sequence published in NCBI GenBank, a pair of specific primers P1-F and P1-R were designed with Primer Premier for the conserved region of gene nsP3 (Table 1). The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. .

[0043] Table 1 Primer information used in this study

[0044]

[0045]

[0046] NOTE: P2-R1 and P2-R2 primers are used for 5′ RACE by nested PCR, and P14-F primer is used for 3′ RACE.

[0047] 1.3 Collection and processing of disease materials

[0048] Collect the spleen of rabbits, cut it into pieces, make a 1:5 suspension w...

Embodiment 2

[0054] Example 2: Plaque purification and whole genome sequencing of Gatavirus

[0055] 2.1 Plaque purification of Gatavirus

[0056] T75cm 2 PK-15 cells that grow well in the cell culture flask and have a confluence of more than 95% are washed twice with PBS, digested with 2 mL of 0.25% trypsin (Gibco), and digested with 36 mL of 10% fetal bovine serum, 100 U / mL Penicillin and 100g / mL streptomycin were suspended in low-sugar DMEM medium to make a suspension, inoculated in a 6-well cell culture plate at 2 mL / well, and placed at 37°C, 5% CO 2 After culturing in an incubator for 12 hours, the cells grow into a monolayer, and the confluence is 95% at this time, and are washed twice with PBS for later use. Use serum-free low-sugar DMEM medium to make a 10-fold serial dilution of the 5th generation Getavirus solution in step 1.5 to 10 -1 ~10 -8 , the dilution 10 -3 ~10 -8 Add PK-15 monolayer cells in a volume of 1 mL respectively, and place at 37°C, 5% CO 2 Adsorb in the inc...

Embodiment 3

[0078] Embodiment 3: the mensuration of growth curve of Geta virus strain GETV JXNC virus

[0079] 3.1 Determination of virus titer

[0080] Inoculate the digested and blown PK-15 cell suspension into a 96-well cell culture plate and culture overnight. When the confluence of monolayer cells reaches 90%, take the cell culture of GETV JXNC strain cloned and purified 5 times in step 2.4 Carry out 10-fold dilution with cell maintenance solution, the dilution is from 10 -1 to 10 -10 ; Discard the cell culture medium in the 96-well cell culture plate, wash the PK-15 cells once with sterile PBS and discard, then add GETV JXNC strain virus dilutions of various dilutions to the 96-well plate, 100 μL per well, Each dilution was repeated 8 times, and each cell culture plate was set as a negative control group without virus inoculation; placed at 37°C, 5% CO 2 Incubator cultivation. Continuously observe for 4 days, observe and record the lesions every day. Calculate TCID according to...

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Abstract

The invention discloses a Getah virus strain and application thereof. The Getah virus GETV JXNC is obtained by being separated from the spleen of a domestic rabbit from Jiangxi Province and being subjected to passage and plaque purification, and the microbial preservation number of the GETV JXNC is CGMCC No.22111. The isolated strain can stably proliferate in passage cells to generate typical cytopathy. The separated GETV JXNC strain is separated from domestic rabbits for the first time in the world, is located in a gene group III through whole genome sequencing and evolutionary tree analysis, is close to the genetic relationship of the current swine Getah virus, has a good strain background, can be prepared into an inactivated vaccine and can be used as a virus seed for inspection, materials are provided for subsequent experimental research, and a material foundation is laid.

Description

technical field [0001] The invention relates to the field of pathogenic microorganisms, belongs to the field of veterinary biological products, and relates to the isolation and identification of a Getavirus strain and its application in inactivated vaccines. Background technique [0002] Getah virus (GETV) is a mosquito-borne virus that was first discovered in Malaysia in 1955. The GETV M1 strain was first isolated from mosquitoes in Hainan Province, my country in 1964. Disease caused by getavirus infection has become a growing threat to our nation's animals and public health. Getavirus is a single-stranded positive-sense RNA virus belonging to the genus Alphavirus of the Togaviridae family. Its genome contains a 5' non-transcoding region (5' UTR), a 3' UTR Two open reading frames (ORFs), two large polyproteins encoded by ORFs, are processed into four nonstructural proteins (nsP1, nsP2, nsP3 and nsP4) and five structural proteins (C, E3, E2, 6K and E1). According to the s...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/02C07K16/10A61K39/12A61P31/14C12R1/93
CPCC12N7/00C07K16/10A61K39/12A61P31/14C12N2770/36121C12N2770/36151C12N2770/36134A61K2039/5252A61K2039/552Y02A50/30
Inventor 李玲张艳宾张彤李鹏宇肖进张欣刘新月张国栋齐鹏
Owner CHINA ANIMAL HUSBANDRY IND
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