CN113215113A

CN113215113A - Rabbit viral hemorrhagic disease virus type 2 and application thereof in preparation of inactivated vaccine

Info

Publication number: CN113215113A
Application number: CN202110594937.6A
Authority: CN
Inventors: 向王震; 王飞; 侯旭; 其他发明人请求不公开姓名
Original assignee: China Animal Husbandry Industry Co Ltd
Current assignee: China Animal Husbandry Industry Co Ltd
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2021-08-06
Also published as: CN113736752A

Abstract

The invention discloses a rabbit viral hemorrhagic disease virus type 2 and application thereof in preparation of inactivated vaccines. The strain is preserved in China general microbiological culture collection center with the preservation name of rabbit viral hemorrhagic disease virus CD2020 strain and the preservation number of: CGMCC NO. 21092. The strain has great difference in comparison with the sequence of the virus of viral hemorrhagic disease of domestic rabbit, wherein the homology of main antigen amino acid is only 88.4%. The current rabbit viral hemorrhagic disease vaccine has no effective protective effect on the strain. Therefore, the invention provides the oil emulsion inactivated vaccine prepared from the rabbit viral hemorrhagic disease virus CD2020 strain, and the vaccine has a good immune effect, has small side effect after rabbit immunization, high safety and no adverse effect on the external environment.

Description

Rabbit viral hemorrhagic disease virus type 2 and application thereof in preparation of inactivated vaccine

Technical Field

The invention relates to the technical field of veterinary infectious diseases, in particular to a rabbit viral hemorrhagic disease virus type 2 and application thereof in preparation of inactivated vaccines.

Background

Rabbit viral hemorrhagic disease type 2 virus (RHDV 2) can cause Rabbit hemorrhagic disease in Rabbit populations. The disease has high morbidity and mortality. The diseased rabbit group mainly presents pathological changes such as hepatosplenomegaly, lung hemorrhage and the like.

The virus was first detected in a farm in france in 2010. Compared with classical Rabbit viral hemorrhagic disease virus (RHDV), the strain and the age range of the infected host are wider, and even the disease and death of 11-day-old young rabbits can be caused. The RHDV2 virus appeared in recent years can break through the immune protection effect of the classical RHDV inactivated vaccine, and the vaccine for immunizing the traditional strain RHDV cannot generate good cross protection effect, and the virus forms wide epidemic in Italy, England, Spain, Germany, Norway, Australia and other countries.

21

days

5 and 21 in 2020, the livestock and veterinary bureau of rural agriculture issues an epidemic situation announcement, and rabbit hemorrhagic disease epidemic situation occurs in rabbit farms in Jintang county, metropolitan, Sichuan province, which causes 1500 rabbits to die, and the disease prevention and control center of the animal in Sichuan province confirms that the disease is rabbit hemorrhagic disease type 2. This means that RHDV2 type occurs for the first time in our country. No vaccine is available to prevent rabbit haemorrhage caused by RHDV 2. At present, the best preventive measure is to adopt a tissue inactivated vaccine for immunization.

Disclosure of Invention

The invention aims to solve the technical problem of providing RHDV2 causing rabbit viral hemorrhagic disease and application thereof in preparation of inactivated vaccines. The invention utilizes the RHDV2 type virus CD2020 to prepare a rabbit viral hemorrhagic disease oil emulsion inactivated vaccine (CD2020 strain) to prevent the prevalence of rabbit viral hemorrhagic disease caused by RHDV2 type virus infection. The vaccine has little side effect and good safety.

The purpose of the invention is realized by the following technical scheme:

the invention provides a rabbit viral hemorrhagic disease virus strain which is named as rabbit viral hemorrhagic disease virus CD 2020. The rabbit hemorrhagic disease virus CD2020 is a natural infectious virus separated from a suspected rabbit suffering from the rabbit hemorrhagic disease in a certain farm in Sichuan province, identified from the aspects of biological characteristics, specificity, immunogenicity and the like of a strain, and screened out the strain with strong toxicity and good immunogenicity. The rabbit hemorrhagic disease virus CD2020 has been stored in China general microbiological culture collection management center (address: China academy of sciences, institute of microbiology, No. 3, West Lu No.1, Beijing, Chaoyang, respectively) at 11 months and 16 days of 2020, and the storage number is CGMCC No. 21092.

The invention provides a separation method of rabbit viral hemorrhagic disease virus, which comprises the following steps:

A. aseptically collecting the livers of the dead rabbits, grinding while adding sterile normal saline in a proper container, centrifuging after freeze thawing, sucking supernatant, and filtering to obtain filtrate components;

B. and C, inoculating the tissue filtrate prepared in the step A to 50-60-day-old healthy non-immune rabbits, collecting liver tissues with typical pathological changes of the diseased and dead rabbits within 16-48h after inoculation, continuously subculturing the susceptible non-immune rabbits by using a toxic liver tissue suspension, and screening to obtain the rabbit viral hemorrhagic disease virus CD2020 strain.

Preferably, in the step a, the grinding is specifically performed by adding 10 times of sterilized normal saline; the centrifugation conditions were: centrifuging at 4000-8000 rpm for 15-30 min; the diameter of the filter hole of the filter is 0.45 um.

Preferably, in step B, the filtrate is inoculated into 1ml of healthy non-immunized rabbits, and injected intramuscularly in the legs.

The invention provides an application of the rabbit viral hemorrhagic disease virus in preparation of rabbit viral hemorrhagic disease oil emulsion inactivated vaccines.

The invention provides a preparation method of an oil emulsion inactivated vaccine for rabbit viral hemorrhagic disease type 2, which is characterized by comprising the following steps:

inoculating 50-60-day-old healthy non-immune rabbits with the rabbit hemorrhagic disease virus CD2020 strain as a seed virus, aseptically collecting liver tissues of the killed rabbits within 16-48h after inoculation, and removing viscera connective tissues;

adding sterile normal saline into liver tissue, fully grinding, and preparing into homogenate; homogenizing, freezing and thawing, adding double antibodies, centrifuging, taking supernatant, adding formaldehyde, and inactivating by a shaker to obtain inactivated tissue suspension;

and (3) mixing the inactivated tissue suspension obtained in the step (2) with a white oil adjuvant, and storing at 4 ℃ for later use.

Preferably, in the step (2), the grinding is carried out by adding 10 times of volume of sterilized normal saline; the number of freeze thawing times was 3.

Preferably, in the step (2), the double antibody is penicillin and streptomycin, and the addition amount of the penicillin and the streptomycin is 1000 IU/ml; the centrifugation conditions were 4000rpm, 30 min.

Preferably, in step (2), the final concentration of formaldehyde is 0.4%; the temperature of table inactivation is 37 ℃, and the inactivation time is 48-72 h.

The invention provides a rabbit viral hemorrhagic disease virus type 2 oil emulsion inactivated vaccine prepared according to the method.

Compared with the prior art, the invention has the following advantages:

the rabbit viral hemorrhagic disease virus CD2020 strain obtained by the invention has larger sequence comparison difference with the current domestic separated rabbit viral hemorrhagic disease virus, wherein the homology of main antigen protein is only 88.4 percent, the strain has larger harm to the infection of young rabbits, and the current inactivated vaccine for rabbit viral hemorrhagic disease can not provide effective protection for the infection of the strain.

The rabbit hemorrhagic disease virus CD2020 strain obtained by the invention can be stably passaged in a rabbit body, and the oil emulsion inactivated vaccine prepared according to the strain can provide good protection for the rabbit hemorrhagic disease virus. The rabbit viral hemorrhagic disease oil emulsion prepared by the invention is inactivated by a mature and stable preparation method, the process is simple, the operation is simple and convenient, and the prepared vaccine has good antigenicity and high safety.

Drawings

FIG. 1 is a genetic evolutionary tree analysis of the CD2020 strain and RHDV of the present invention;

FIG. 2 shows the result of RT-PCR amplification of CD2020 disease material in example 1; wherein: m is DL2000 Marker; 1. 2, 3 and 4 are pathological tissues; 5 is negative control;

FIG. 3 is a anatomical observation of the vaccine immunopotency test of example 3; wherein: FIG. 3A shows the lungs of the test group; FIG. 3B shows the liver of the experimental group; FIG. 3C shows lungs from control group; FIG. 3D is liver of control group;

FIG. 4 is a anatomical observation of the vaccine immunopotency test of example 3; wherein: FIG. 4A shows a first group of lungs; FIG. 4B is a liver of the first group; FIG. 4C is a second group of lungs; fig. 4D is a second group of livers.

FIG. 5 shows the results of amino acid homology alignment of the capsid proteins of the CD2020 strain and the conventional RHDV strain

Detailed Description

The methods in the following examples are conventional methods unless otherwise specified.

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit of the invention. All falling within the scope of the present invention.

Example 1 isolation and identification of Rabbit viral hemorrhagic disease Virus CD2020 Strain

1. Separation and identification of rabbit viral hemorrhagic disease virus CD2020 strain

Collecting dead rabbit liver, grinding with 10 times of sterilized normal saline, centrifuging at 8000rpm for 15min, collecting supernatant, filtering with 0.45 μm filter, collecting filtrate, and standing at-20 deg.C.

Designing a specific primer according to a VP60 nucleic acid sequence of RHDV published on GenBank, wherein an upstream primer P1: ATTGGGCCGGGTTTGGAAGTCAGAC, downstream primer P2: GCTCCAGTGGCAAAGCCCAACTCATAAG, amplifying a VP60 gene partial sequence fragment 722bp, wherein the sequence is shown as SEQ ID NO. 1. Extracting RNA of the collected diseased rabbit liver tissues by using a Trizol method, carrying out reverse transcription by using M-MLV (M-MLV) to obtain cDNA, and carrying out RT-PCR (reverse transcription-polymerase chain reaction) amplification by using the cDNA as a template under the reaction conditions of pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 1min45 s; 30 cycles, and finally 10min extension. The PCR product was subjected to 1% agarose gel electrophoresis. The result showed that a specific band appeared at 722 bp. Through Blast comparison, the homology of the amplified product fragment and VP60 of domestic separation classical RHDV is 79.8%, and the homology of the amplified product fragment and VP60 of RHDV2 published on GenBank is 96.7%, so that the strain is preliminarily judged to be rabbit viral hemorrhagic disease type 2.

2. Stability test and virulence identification

Selecting 5 healthy susceptible non-immune test rabbits, injecting the treated filtrate through leg muscle of the rabbit, wherein each filtrate is 1.0mL, and collecting liver disease materials of dead inoculated rabbits; the above test procedure was repeated 3 consecutive times. The results show that: the death time of 100% of the attacked rabbits is 18-48 h, which indicates that stable rabbit viral hemorrhagic disease virus type 2 is obtained and is named as CD2020 strain, the rabbit viral hemorrhagic disease virus type 2 is preserved in the China general microbiological culture collection center with the preservation name of SC strain of rabbit viral hemorrhagic disease virus type 2 and the preservation number of CGMCC No.21092 (preservation date: 11/16/2020, preservation unit address: institute of microbiology institute of China academy of sciences No.1, North Cheng Yang district, Beijing). The dead rabbit has nostril bleeding, and has tracheal circular bleeding, flush, serious lung bleeding and hepatomegaly through anatomical observation, which shows that the isolate has strong pathogenicity and is a virulent strain.

3. Subculture of rabbit viral hemorrhagic disease virus CD2020

Diluting the rabbit viral hemorrhagic disease virus CD2020 by 10 times of sterilized normal saline, and inoculating to healthy susceptible non-immune rabbits of 50-60 days old. Each leg was injected intramuscularly at 1 mL. And aseptically collecting dead rabbits within 24-48h after inoculation, aseptically collecting liver tissues with typical pathological changes, and removing connective tissues as virus seeds for later use.

Inoculating a strain CD2020 strain which is transmitted for 3-8 generations, adding physiological saline into liver pathological tissues of dead rabbits from which connective tissues are removed, grinding and filtering, wherein the ratio of the liver tissues to the physiological saline is 1:10 (W/V). Adding 1000IU/ml of penicillin and streptomycin, centrifuging at 4000rpm for 30min, sucking supernatant, and storing at-20 ℃.

Diluting the preserved rabbit liver tissue by 10 times, and diluting the rabbit liver tissue with a dilution ladderDegree of 10^-1～10^-9 Inoculating 5 healthy susceptible non-immune rabbits of 50-60 days old at each dilution, setting 5 control groups at an inoculation dose of 1 ml/each, continuously observing for 10 days after inoculation, recording death number of each gradient, and calculating LD according to Reed-Muench method₅₀The result showed that the titer of the strain was 10^7.32LD₅₀/ml。

Example 2 preparation of tissue suspension for Rabbit viral hemorrhagic disease

Diluting the rabbit viral hemorrhagic disease virus CD2020 strain by 10 times of normal saline, and inoculating to healthy susceptible non-immune rabbits of 50-60 days old. Injecting 1ml of the injection into each leg, selecting dead rabbits 24-48h after inoculation, aseptically collecting liver tissue with typical lesion, and removing connective tissue as virus seed.

Inoculating the virus seeds into healthy and susceptible non-immune rabbits after 3-8 generations, aseptically collecting the livers of the rabbits dead within 24-48h after inoculation, removing connective tissues, adding normal saline, grinding and filtering, wherein the ratio of the liver tissues to the sterilized normal saline is 1:10(W/V), and preparing into homogenate. Freezing and thawing for 3 times, adding 1000IU/ml each of penicillin and streptomycin, centrifuging at 4000rpm for 30min, and sucking supernatant to obtain tissue suspension. Adding formaldehyde solution into the suspension for inactivation, wherein the final concentration is 0.4%, and the inactivation conditions are as follows: inactivating in a shaker at 37 deg.C for 48-72 h. And (4) performing sterile test and inactivation test after inactivation is finished. After the sterility test and the inactivation test are qualified, the inactivated tissue suspension is obtained.

Example 3 vaccine evaluation test

The rabbit hemorrhagic disease virus CD2020 strain is a natural infection strain separated from a dead rabbit suspected of having the rabbit hemorrhagic disease in a certain rabbit farm in Sichuan by the inventor. The antigenic variation of the variant strain separated at this time and the classical strain is larger, which is probably the reason that the traditional inactivated vaccine for the viral hemorrhagic disease of the rabbit can not completely protect the rabbit group against the viral infection of the viral hemorrhagic disease type 2 of the rabbit.

Compared with the traditional RHDV reported in China before, the rabbit hemorrhagic disease virus CD2020 strain has larger sequence difference, and the amino acid homology of the main antigen gene has larger difference. The phylogenetic tree analysis shows that it is far from the conventional RHDV epidemic strain in genetic distance and is located in different clades (FIG. 5). The CD2020 strain is proved to be a variant RHDV isolate strain by showing that the CD2020 strain and some classical RHDVs in China are in different topological groups. The CD2020 strain has larger sequence comparison difference with the rabbit viral hemorrhagic disease virus separated in China at present, wherein the homology of main antigen protein is only 88.4%, and the strain has larger harm to the infection of young rabbits, and the test result further proves that the rabbit infected by the CD2020 strain cannot be completely protected by the existing commercial rabbit viral hemorrhagic disease vaccine. Therefore, the invention uses the CD2020 strain as vaccine seed virus to prepare an inactivated vaccine for preventing RHDV2 infection.

1. Preparation of inactivated vaccine

The inactivated tissue suspension prepared by adopting the 3 rd generation of virus strain CD2020 strain in the example 2 is mixed with the white oil adjuvant according to the proportion of 1:1 to obtain the inactivated vaccine, and the inactivated vaccine is stored at 4 ℃ for later use.

2. Vaccine safety test

15 SPF New Zealand rabbits were selected and randomized into three groups of 5 rabbits each. The first group was inoculated subcutaneously with 1mL of the previously described inactivated vaccine; the second group was vaccinated subcutaneously with 2mL of the inactivated vaccine described previously; the third group was a control group, and 1mL of sterilized saline was injected subcutaneously. Each group of test rabbits was kept in an isolator and continuously observed for 10 days. No adverse reaction is seen in the results, which shows that the inactivated vaccine has good safety.

3. Vaccine immunopotency assay

10 SPF New Zealand rabbits were purchased and randomly divided into two groups of 5 rabbits each. Each of the experimental groups was injected subcutaneously with 1.0ml of the aforementioned inactivated vaccine, and another 5 healthy New Zealand rabbits were used as controls. Two groups of new zealand rabbits were challenged 14 days after immunization, and each leg was injected intramuscularly with the lethal dose of venom prepared in example 1. Each group of experimental rabbits was kept in an isolator, and after 10 days of observation, all the New Zealand rabbits in the immune group survived and all the rabbits in the control group died. The vaccine had 100% protection against rabbit hemorrhagic disease type 2 and 100% mortality in the control (table 1). The cesarean examination is carried out on all experimental rabbits, and the visceral organs of the immunized rabbit have no obvious lesion (fig. 3A and B); the lungs of the control group had severe bleeding and the liver appeared yellowish (fig. 3C and D). Therefore, the rabbit viral hemorrhagic disease type 2 oil emulsion inactivated vaccine (CD2020 strain) has good immunoprotection.

New zealand rabbits of 15 SPF rating were selected and randomly divided into 3 groups of 5 rabbits each. The first group was injected subcutaneously with 1ml of the inactivated vaccine, the second group was inoculated with 1ml of a commercially available rabbit hemorrhagic disease virus tissue-inactivated vaccine, and the third group was used as a blank. The first and second groups were challenged 14 days after immunization, and each leg was intramuscularly injected with a lethal dose of venom. The experimental rabbits are placed in an isolation area for feeding, and after 10 days of observation, the first group of rabbits completely survive and the second group of rabbits completely attack. The first group of rabbit viscera has no obvious pathological changes by the observation of the experimental rabbit; the second group had lung bleeding and the liver appeared yellowish in color, as shown in fig. 4. Therefore, compared with the commercially available rabbit hemorrhagic disease virus tissue inactivated vaccine, the rabbit hemorrhagic disease oil emulsion inactivated vaccine (CD2020 strain) can protect rabbit groups against the invasion of the rabbit hemorrhagic disease virus type 2.

TABLE 1 protection against challenge with inactivated vaccine of CD2020 strain and commercially available vaccine

Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Zhongmu industries GmbH

<120> rabbit viral hemorrhagic disease virus type 2 and application thereof in preparation of inactivated vaccine

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attgggccag gtttggaagt tagacaattc cctcatgttg tcattgacgc tcgttcactc 60

gaaccagtca ccatcaccat gccggacttg cgccccaaca tgtaccaccc aacaggcaac 120

cctggcctcg ttcccacgtt ggtcctgagc gtttacaaca acctcatcaa cccatttggt 180

ggatccacga gcgcaatcca ggtcacggtg gaaacaaggc ccagtgagga ctttgagttt 240

gtgatgatcc gtgccccctc cagtaagacc gttgattcga tctcgcccgc agatctcctc 300

acaaccccag ttctcactgg ggttggcacc gataacagat ggaatggtga gatagttggg 360

ctgcaaccag tccccggtgg gttttctacg tgcaacagac actggaactt aaacggcagc 420

acatatggat ggtcaagccc gcggtttgcc gccattgacc acgacagagg caacgcaagt 480

ttccctggaa gcagttcgtc aaacgtgctt gagctttggt atgctagtgc cgggtctgca 540

gctgacaacc ccatctccca aattgctcca gatggtttcc ctgacatgtc atttgtaccc 600

ttcagcggta ccaccatccc taccgcaggg tgggtcgggt tcggtgggat ctggaacagc 660

agtaatggtg ccccctacgt cacgaccatg caggcttatg agttgggttt tgccactgga 720

gt 722

Claims

1. A strain of rabbit viral hemorrhagic disease virus is characterized in that: the name of the rabbit viral hemorrhagic disease virus strain is CD2020, and the preservation number in the China general microbiological culture Collection center is CGMCC No. 21092.

2. A vaccine for rabbit viral hemorrhagic disease type 2, the active ingredient of which is the inactivated rabbit viral hemorrhagic disease virus CD2020 of claim 1.

3. The vaccine of claim 2, further comprising a pharmaceutically acceptable adjuvant.

4. The use of the rabbit viral hemorrhagic disease virus CD2020 of claim 1 in the preparation of inactivated vaccine for rabbit viral hemorrhagic disease.

5. A preparation method of rabbit viral hemorrhagic disease type 2 oil emulsion inactivated vaccine comprises the following steps:

1) inoculating a susceptible non-immune rabbit of 50-60 days old by using the rabbit viral hemorrhagic disease virus strain as a seed virus according to claim 1, aseptically collecting the liver of the rabbit dead in 16-48h after inoculation, and removing the connective tissue of the liver;

2) fully grinding the liver without the connective tissue to prepare homogenate; freezing and thawing the ground homogenate, adding streptomycin, centrifuging, taking the supernatant, adding formaldehyde, and inactivating by a shaker to obtain an inactivated tissue suspension;

3) mixing the inactivated tissue suspension obtained in the step 2) with a white oil adjuvant to obtain the rabbit viral hemorrhagic disease virus type 2 oil emulsion inactivated vaccine, and storing at 4 ℃ for later use.

6. The method for preparing an inactivated oil emulsion vaccine for rabbit hemorrhagic disease 2 according to claim 5, wherein in the step 2), the specific steps are grinding by adding 5-10 times of volume of sterilized normal saline; the number of freeze thawing times was 3.

7. The method for preparing an inactivated oil emulsion vaccine of rabbit viral hemorrhagic disease virus type 2 according to claim 6, wherein the penicillin and streptomycin are added in an amount of 1000IU/ml each in step 2); the centrifugation conditions were: centrifuging at 4500r/min for 30 min.

8. The method for preparing inactivated oil emulsion vaccine of rabbit viral hemorrhagic disease virus type 2 according to claim 6, wherein in the step 2), the final inactivated concentration of formaldehyde is 0.4%; the inactivation temperature of the shaking table is 37 ℃, and the inactivation time is 48-72 h.

9. An inactivated rabbit viral hemorrhagic disease virus type 2 oil emulsion vaccine prepared by the method according to any one of claims 5 to 8.