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Low virulent strain of pseudorabies virus variant and application of low virulent strain

A pseudorabies virus and mutant strain technology, which is applied in antiviral agents, medical preparations containing active ingredients, inactivation/attenuation, etc., can solve the problems of poor protection effect of mutant strains and achieve good safety results

Inactive Publication Date: 2017-01-04
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to solve the technical problem of PRV mutant strains appearing in China at present, but the existing commercialized PRV vaccines have poor protection effect on the mutant strains, and it is urgent to develop new PRV vaccines, and provides an attenuated strain of pseudorabies virus mutant strains. The strain has a good immune protection effect on both PRV variant strains and PRV classic virulent strains. PRV-negative piglets within 2 weeks of age did not show any clinical adverse symptoms, and it is safe and suitable as a vaccine candidate for the prevention and control of pseudorabies. strain

Method used

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  • Low virulent strain of pseudorabies virus variant and application of low virulent strain
  • Low virulent strain of pseudorabies virus variant and application of low virulent strain
  • Low virulent strain of pseudorabies virus variant and application of low virulent strain

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Effect test

Embodiment 1

[0040] The construction of embodiment 1 PRV JS-2012 high temperature passaging attenuated strain

[0041] Based on the pseudorabies mutant strain PRV JS-2012, the PRV JS-2012 strain was passaged on Vero cells by continuous high temperature at 40°C, and the passaged strain PRV JS-2012-F120 was obtained. The PRV JS-2012-F120 strain propagates on Vero cells, and its virus titer can reach 10 8.5 TCID 50 / 0.1mL, and the cytopathic morphology and plaque morphology caused by the virus on Vero cells were significantly different from its parent virus PRV JS-2012. It was verified by PCR that the gE and US2 genes of the PRV JS-2012-F120 strain were both deleted.

[0042] 1 method

[0043] 1.1 Primer design

[0044] According to the whole genome sequence of PRV JS-2012 strain, multiple primers were designed with Oligo 6.0 software (see Table 1), for gBU / gBD, gCU / gCD, gDU / gDD, gIU / gID, gEU / gED, US2U / US2D It is used to amplify partial or full-length sequences of pseudorabies virus gB, ...

Embodiment 2

[0063] Example 2 Deletion sequence analysis of PRV JS-2012-F120 viral genome

[0064]In embodiment 1, through PCR verification, it is found that the gE of PRV JS-2012-F120 virus has a deletion, and the US2 gene has a deletion. This embodiment designs a pair of primers (gE330up / US2down) for these two genes to amplify the parental virus PRV JS-2012 and The gE to us2 gene of the passage virus PRV JS-2012-F120 (see SEQ ID NO.14, SEQ ID NO.15), after sequencing, it was found that the 437th base in the CDS region of the gE gene of the passage virus was compared with the parent virus. The DNA sequence between the 396 bases in the CDS region of the US2 gene was continuously deleted, and a total of 2307 bases were deleted. Based on this deletion, a pair of primers (DF120up / DF120down) was designed to distinguish the PRV JS-2012-F120 virus from the parent virus PRV JS-2012.

[0065] 1 Materials and methods

[0066] 1.1 Primer design

[0067] According to the whole genome sequence of P...

Embodiment 3

[0080] Example 3 Pathogenicity experiment of PRV JS-2012 different generations of high-temperature passage virus to 14-day-old piglets

[0081] Select PRV JS-2012-F5, PRV JS-2012-F50, PRV JS-2012-F91, PRV JS-2012-F120 in Example 1, and inoculate 14-day-old PRV-negative piglets by nasal instillation to verify the attenuation of the virus Effect. It was found that the PRV JS-2012-F5 poison can kill 100% of the test pigs; the PRV JS-2012-F50 virus can make 100% of the pigs sick, but the lethal rate is 40%; the PRV JS-2012-F91 poison can make 80% of the pigs sick , but not lethal to pigs; PRV JS-2012-F120 is very safe for 14-day-old piglets. By analyzing the antibodies in the serum after challenge, the results showed that all the surviving test pigs could detect gB gene antibodies after the 7th day, and the gE antibodies were negative until the end of the experiment on the 21st day, indicating that PRV JS- 2012-F50, PRV JS-2012-F91, and PRVJS-2012-F120 virus gE genes were all de...

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Abstract

The invention discloses a low virulent strain of a pseudorabies virus variant. The low virulent strain is a low virulent strain with deleted genes of a pseudorabies virus variant PRVJS-2012. The deleted gene sequence includes an entire or partial DNA sequence of a CDS region of a gE gene in a genome of the PRVJS-2012 variant. The invention further discloses an application of the low virulent strain of the pseudorabies virus variant to prepare a vaccine for preventing or curing pseudorabies. The low virulent strain is good in immunoprotection effect on the PRV variant and a PRV classic virulent strain. A PRV-negative piglet in two weeks old is inoculated with the low virulent strain, and no bad clinic symptom appears. The low virulent strain is high in safety and suitable for being used as a vaccine candidate for pseudorabies.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an attenuated strain of a pseudorabies virus variant and an application thereof. Background technique [0002] Pseudorabies is a severe infectious disease caused by Pseudorabies virus (PRV), characterized by fever, itching, and encephalomyelitis. PRV can infect a variety of hosts, but pigs are the main natural host, reservoir and disseminator of the virus. Pigs of different ages can be infected, mainly causing abortion, stillbirth, mummified fetuses in pregnant sows, high mortality in suckling piglets, and infertility in breeding pigs. PRV belongs to the herpesviridae herpesvirus subfamily A, its genome is a linear double-stranded DNA, about 150kb in length, composed of a long unique region (UL), a short unique region (US) and repeat sequences on both sides of the US, encoding More than 70 kinds of proteins. Virions are composed of three layers: nucleocapsid, outer shell, and enve...

Claims

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Application Information

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IPC IPC(8): C12N7/04A61K35/763A61P31/22C12Q1/68C12Q1/70
Inventor 童光志童武梁超徐宏军高鹏郑浩李国新
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI