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H3N2 subtype canine influenza virus mouse adaptive virulent strain and application thereof

A canine influenza virus, H3N2 technology, applied in the field of microbial engineering, can solve problems such as difficulty in evaluating effects, adaptive mutant virus strains, etc., to achieve the effect of enhancing virulence

Active Publication Date: 2019-11-19
ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This provides an opportunity for the influenza virus to spread and further adapt in dogs, with serious potential public health risks
In addition, in the absence of secondary bacterial infection, the clinical symptoms caused by canine influenza virus in dogs are relatively mild, and it is difficult to evaluate the effect during the development of vaccines / drugs. Therefore, stable animal infection models and attack methods are currently available. It is especially important to use virulent strains
However, there have been no reports of adaptive mutation and lethality-enhanced strains of H3N2 CIVs obtained through continuous passage in mice.

Method used

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  • H3N2 subtype canine influenza virus mouse adaptive virulent strain and application thereof
  • H3N2 subtype canine influenza virus mouse adaptive virulent strain and application thereof
  • H3N2 subtype canine influenza virus mouse adaptive virulent strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Isolation identification and cultivation of embodiment 1 virus strain

[0047] Since 2016, H3N2 CIVs have caused nationwide epidemics in my country, and the epidemic strains in 2016 and the Asian epidemic strains in 2006 are different evolutionary branches. At present, the strain of this evolutionary branch is widely prevalent in my country. In 2017, the present invention detected a new type of H3N2 CIVs (QK32 for short) from the oral swab of a pet dog with clinical respiratory symptoms in Jilin City, and obtained the isolate from the allantoic fluid of chicken embryos. The separation method is as follows: vortex the oral swab of the dog with sterile PBS (pH: 7.2) for 30 seconds, put it in a centrifuge at 4°C at 4000r / min, and centrifuge for 5 minutes. Aspirate the supernatant and add penicillin and streptomycin with a final concentration of 1000IU, inoculate 9-day-old SPF embryos, place them in an incubator at 37°C for 72 hours, and after refrigerated at 4°C for 0.5h,...

Embodiment 2H3

[0061] The preparation of embodiment 2 H3N2 subtype canine influenza virus murine adaptation virulent strain

[0062] The present invention uses QK32 as the original virus strain, after several attempts to change the age of the inoculated BALB / c mice and the content of the inoculated virus, a preparation method is finally determined to obtain the mouse-adapted H3N2 subtype canine influenza virus that can cause death in mice. Virulent strain. The specific operation method is as follows:

[0063] Step 1: Inoculate 9g-10g BALB / c suckling mice with 25ul content of 106 / 100ulEID50 by bilateral intranasal method respectively. At 72 hours after inoculation, dissect the lung tissue and grind it in a tissue homogenizer after quick-freezing on dry ice , 8000×g for 5min, take the supernatant, put it again at 12000×g for 5min, take the supernatant tissue fluid and inoculate it into 9g~10g BALB / c suckling mice, each mouse is inoculated with 25ul of virus solution. The mouse lungs were con...

Embodiment 3

[0070] Embodiment 3 Identification of mouse-adapted virulent strains of the present invention

[0071] Take the 26th generation mouse lung tissue grinding solution in Example 2, use the method in Example 1 to extract viral nucleic acid, obtain cDNA through reverse transcription, and use the primer sequences and PCR reaction conditions in Example 1 for amplification. Sequence determination was performed by agarose gel electrophoresis and gel recovery. The full sequence of the non-coding region of the HA fragment of the mouse-adapted virulent strain is shown in SEQ ID NO.1, the full sequence of the non-coding region of its NA fragment is shown in SEQ ID NO.2, and the full sequence of the non-coding region of its NP fragment As shown in SEQ ID NO.3, the full sequence of the non-coding region of its PA fragment is shown in SEQ ID NO.4, the full sequence of the non-coding region of its PB1 fragment is shown in SEQ ID NO.5, and the non-coding region of its PB2 fragment The full seq...

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Abstract

The invention discloses a H3N2 subtype canine influenza virus mouse adaptive virulent strain and application thereof. The techniques of virus isolation, culture, infection of animals and the like areadopted, enhanced virulence and pathogenic lethal amino acid mutations and deletions are obtained through adaptation of mice, and the H3N2 subtype canine influenza virus mouse adaptive virulent strainis obtained; the adaptive strain can cause lethal infection in mice, and a powerful animal model is provided for evaluation and development of a vaccine, effectiveness evaluation of prevention and treatment drugs and the like, influenza virus infection and pathogenic and lethal mechanism research. In addition, due to the fact that the mouse adapts to the virulent strain, an HA gene is not mutated, the antigenicity is unchanged, and the strain can serve as an antigen for basic experimental research such as preparation and / or detection based on antibody levels. In conclusion, the adaptive virulent strain provides the powerful technical support for research and development of the H3N2 subtype canine influenza virus vaccine and research of screening, infection, virulence enhancing and other mechanisms of the high-efficiency prevention and / or treatment drugs.

Description

technical field [0001] The invention relates to a canine influenza virus strain, in particular to a H3N2 subtype canine influenza virus murine-adapted virulent strain and application thereof, belonging to the technical field of microbiological engineering. Background technique [0002] Influenza virus (IV) is an Orthomyxoviridae family with a diameter of 80-120 nm. Its structure is divided into three parts: nucleoprotein (NP), matrix protein (M) and envelope from inside to outside. Its genomic RNA consists of eight segments of single-stranded RNA, which are PB2, PB1, PA, HA, NP, NA, M, and NS genes. Currently, influenza A viruses have been found to have 18 HA subtypes and 11 NA subtypes, among which H1~H16HA subtypes and N1~N9NA subtypes are all derived from avian influenza, while H17N10 subtypes and H18N11 subtypes are only found in bat. At present, there are H3, H6, and H9 subtype influenza viruses that can cause canine infection. Among them, H3 subtype has been confirme...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A01K67/027G01N33/68C12R1/93
CPCA01K67/027A01K2207/20A01K2227/105A01K2267/0337C12N7/00C12N2760/16121C12N2760/16122G01N33/6854G01N2333/11
Inventor 高玉伟李元果张醒海金宁一王铁成孙伟洋石晶夏振强殷玉和胡鑫宇冯娜周博李乐天向海洋赵永坤杨松涛夏咸柱
Owner ACAD OF MILITARY SCI PLA CHINA ACAD OF MILITARY MEDICAL SCI INST OF MILITARY VETERINARY MEDICINE
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