CN113832113B

CN113832113B - Novel duck reovirus attenuated strain and application thereof

Info

Publication number: CN113832113B
Application number: CN202111076984.8A
Authority: CN
Inventors: 李泽君; 李雪松
Original assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Current assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Priority date: 2021-09-14
Filing date: 2021-09-14
Publication date: 2023-09-22
Anticipated expiration: 2041-09-14
Also published as: CN113832113A

Abstract

The invention discloses a novel duck reovirus attenuated strain which has sigma C structural protein with an amino acid sequence shown as SEQ ID NO. 1. The invention also discloses a vaccine comprising the novel duck reovirus attenuated strain. The invention also discloses application of the novel duck reovirus attenuated strain in preparation of a product for preventing novel duck reovirus diseases. The novel duck reovirus attenuated strain prepared by the method has the advantages that after 1-day-old healthy susceptible duckling is immunized, the protection rate of the novel duck reovirus attenuated strain on virulent attack reaches 100%, the novel duck reovirus attenuated strain has good safety and effectiveness, and the novel duck reovirus attenuated strain does not generate virulence back after artificial passage in the susceptible duckling, so that the novel duck reovirus attenuated strain has a very good application prospect in preventing and treating the novel duck reovirus of the duckling.

Description

Novel duck reovirus attenuated strain and application thereof

Technical Field

The invention belongs to the technical field of veterinary biological products, and particularly relates to a novel duck reovirus attenuated strain and application thereof as a novel duck reovirus live vaccine.

Background

The disease caused by the novel duck reovirus (Novel duck reovirus, NDRV) infection is a duck viral infectious disease which is mainly characterized by spleen irregular bleeding, swelling, necrosis and the like. The virus is separated from waterfowl such as geese, muscovy ducks, cherry valley ducks, sheldrake and the like, the NDRV can infect duckling of different varieties, the host range of infection is wider than that of typical Muscovy duck reovirus (Muscovy duck reovirus, MDRV), and the pathogenicity and clinical pathological changes caused by the two viruses are also quite different. Since 2017, the morbidity and mortality of novel duck reovirus diseases are gradually increased, and the novel duck reovirus diseases become one of important viral infectious diseases affecting the duck raising industry in China.

NDRV can infect and cause morbidity in muscovy ducks, semi-muscovy ducks, cherry valley ducks, sheldrake and geese, and the like. The virus has serious harm to duckling, the age of the muscovy duck and the half muscovy duck in the onset period is mainly 6-25 days, wherein the age of 7 days is the most, and the course of the disease is 5-7 days; the age of cherry valley ducks in the onset day is mainly 10-30 days. The incidence rate of the novel reovirus is 5-30%, and the death rate is 4-20%.

Vaccination is one of the keys to prevent novel duck reovirus disease. The current vaccines for preventing the disease comprise an inactivated vaccine and a attenuated live vaccine, wherein the inactivated vaccine has slower immunity generation time, usually needs multiple times of immunity to achieve better protection effect, and has weaker cell-mediated immunity, while the attenuated live vaccine has the effect of stimulating the organism to generate comprehensive immune response, has quicker immunity effect generation time and can generate enough protection effect once. Therefore, the novel duck reovirus attenuated live vaccine has more advantages in clinical application compared with the inactivated vaccine in the aspect of protecting duckling.

Disclosure of Invention

The invention aims to solve the technical problem that no novel duck reovirus attenuated live vaccine is clinically effective in preventing the novel duck reovirus disease, and provides a novel duck reovirus attenuated strain, and the novel duck reovirus live vaccine prepared by the attenuated strain can be used for effectively preventing the novel duck reovirus disease.

In order to solve the technical problems, the invention is realized by the following technical scheme:

the invention provides a novel duck reovirus attenuated strain, which has sigma C structural protein with an amino acid sequence shown as SEQ ID NO. 1.

Preferably, the attenuated strain of virus further comprises a sigma a structural protein having the amino acid sequence shown in SEQ ID No. 2.

Preferably, the attenuated strain of virus further comprises a sigma B structural protein having the amino acid sequence shown in SEQ ID NO. 3.

The invention provides a novel duck reovirus attenuated strain JS01-105P, the preservation number of which is CCTCC NO: v202168.

The invention also provides a nucleic acid encoding a structural protein or fragment thereof of:

a sigma C structural protein of the amino acid sequence shown in SEQ ID NO. 1;

or a sigma A structural protein of the amino acid sequence shown in SEQ ID NO. 2;

or a sigma B structural protein of the amino acid sequence shown in SEQ ID NO. 3.

Preferably, the nucleic acid is selected from:

the nucleotide sequence of the sigma C structural protein shown in SEQ ID NO. 4;

or the nucleotide sequence of the sigma A structural protein shown in SEQ ID NO. 5;

or the nucleotide sequence of the sigma B structural protein shown in SEQ ID NO. 6.

The invention also provides a vaccine containing the novel duck reovirus attenuated strain.

The vaccine has good safety and effectiveness to duckling, and the virus content of each feather is not less than 10 ^3.5 TCID ₅₀ 。

The invention also provides application of the novel duck reovirus attenuated strain in preparation of a product for preventing novel duck reovirus diseases.

The novel duck reovirus attenuated strain JS01-105P provided by the invention is used as a live vaccine for the novel duck reovirus disease, has good safety and effectiveness, and has a protection rate of 100% on virulent attack after 1 day of immunization on healthy and susceptible duckling, and has a very good application prospect on prevention and treatment of the novel duck reovirus disease of the duckling, wherein virulence return does not occur in the susceptible duckling after artificial passage.

Drawings

The invention will be described in further detail with reference to the drawings and the detailed description.

FIG. 1 is a diagram showing the survival rate of the NDRV JS01 strain virulent strain and the inoculated ducks with the virulent strain and the virulent strain after passage in example 2 of the invention;

FIG. 2 is a chart showing the virus titer of the viscera of 1 day old ducks inoculated with the NDRV JS01 strain virulent strain and the passaging strain of the invention in example 2;

FIG. 3 is a schematic diagram of the final product of the novel reovirus attenuated live vaccine of example 4 of the present invention.

The preferred novel duck reovirus attenuated strain is preserved in China center for type culture collection (CCTCC for short, address: university of Wuchang mountain and Wuhan collection in Wuhan, jua, hubei province) at the 1 st month of 2021, and the preservation number is CCTCC NO: v202168, which is classified and named as a novel duck reovirus attenuated strain JS01-105P.

Detailed Description

In order to develop a safe and effective novel duck reovirus attenuated live vaccine, the invention separates and identifies a novel duck reovirus attenuated strain JS01, and continuously passaging and purifying and culturing the novel duck reovirus attenuated strain JS01-105P on SPF chick embryos until 105 generations are obtained. The novel duck reovirus attenuated strain (JS 01-105P) is used as a live vaccine for immunizing 1-day-old healthy susceptible duckling, has good safety and effectiveness, and does not generate virulence return after artificial passage in the susceptible duckling, so that the novel duck reovirus attenuated strain has very good application prospect in the aspect of preventing and treating novel duck reovirus diseases of duckling

Example 1 cultivation and sequence analysis of novel attenuated reovirus vaccine strains

A novel duck reovirus is isolated and identified from cases causing spleen necrosis symptoms of duckling, named as a novel duck reovirus JS01 strain, and inoculated into 9-10-day-old SPF chick embryos by allantoic cavities for continuous culture in an incubator. Discarding dead chick embryo within 24 hours after inoculation, taking out the rest chick embryo once within 24 hours, taking out the dead chick embryo within 2-6 days, preserving at below-15 ℃, melting at room temperature, grinding, collecting supernatant, centrifuging at 3000 r/min for 10 minutes, and packaging the supernatant. Serial subculture was then carried out in the same manner to 100 generations, followed by purification by limiting dilution for 5 times to 105 generations. Full gene sequencing of JS01 and 105-generation viruses shows that compared with JS01, the 105-generation viruses are passaged to amino acid mutation sites of main structural proteins sigma C, sigma A and sigma B of the 105-generation viruses, and have 4 differences in total. Of the 5 amino acid mutations, the sigma C (sigma C) protein has 1 mutation point (T120P), the sigma A (sigma A) protein has 2 mutation points (T44P, E120G), and the sigma B (sigma B) protein has 1 mutation point (L260F). The JS01-105P strain structural protein sigma C has an amino acid sequence shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO. 4; the amino acid sequence of the structural protein sigma A is shown as SEQ ID NO.2, and the nucleotide sequence is shown as SEQ ID NO. 5; the amino acid sequence of the structural protein sigma B is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 6.

Example 2 evaluation of pathogenicity of NDRV JS01 strain passage toxin on 1 day old duckling

In order to evaluate the virus pathogenicity change conditions of the 5 th generation (5P), the 75 th generation (75P) and the 105 th generation (105P) of the NDRV JS01 strain, animal experiments are carried out after the purchased SPF duck embryos are hatched to 1 day old, 40 susceptible duckling are randomly divided into 4 groups, 10 duckling groups are respectively arranged in the 1 st group, the NDRV JS01-5P group, the 2nd group is the NDRV JS01-75P group, the 3 rd group is the NDRV JS01-105P group, and the 4 th group is the PBS control group. The 1 st to 3 rd groups of ducklings with 1 day age are subjected to leg intramuscular injection for virus passage, and the virus dosage is 10 ^5.0 TCID ₅₀ 0.1mL, each 0.1mL. Group 4 is a blank control group, and is injected with 0.1mLPBS buffer solution at the legs, continuously observed for 14 days, and observed whether the inoculated duck has clinical symptoms caused by viruses or whether the inoculated duck has local and systemic adverse reactions caused by inoculation.

Organ virus isolation assay: after the purchased SPF duck embryo is hatched to 1 day old, animal experiments are carried out, 40 duckling are randomly divided into 4 groups, 10 duckling groups are respectively, the 1 group is an NDRV JS01-5P generation virus group, the 2 group is an NDRV JS01-75P generation virus group, the 3 group is an NDRV JS01-105P generation virus group, and the 4 group is a PBS control group. Leg injection for passaging virus of 1 day-old duckling of groups 1-3, and virus dose of 10 ^5.0 TCID ₅₀ 0.1mL, each 0.1mL. Group 4 is a blank control group, and was injected with 0.1ml pbs buffer on each leg. 4 days after virus inoculation, all duckling are dissected and killed, and the dissected and the content of the virus in the viscera is measured, wherein the collected viscera comprise heart, liver, spleen, kidney, duodenum, brain, bursa of Fabricius and other viscera tissues.

Results:

clinical symptoms and mortality conditions within 14 days after infection of 1-day-old ducklings by different NDRV JS01 strains: after the muscle is inoculated with the NDRV JS01-5P strain to be virulent, two ducklings die within 14 days, the survival rate is 80%, the ducklings are not dead after the NDRV JS01-75P strain and the NDRV JS01-105P strain are inoculated, and the survival rate is 100% (shown in figure 1). The 3 days after the virus attack, the duckling of the inoculation group of the NDRV JS01-5P strain has symptoms of listlessness, reduced feed intake, diarrhea and the like; the young ducks of the NDRV JS01-75P strain inoculation group have diarrhea symptoms; NDRV JS01-105P vaccinations and PBS vaccinations had no adverse clinical symptoms.

Different NDRV JS01 strains are inoculated to ducklings of 1 day old after being subjected to virus passage, killing is carried out after 4 days, and pathological observation is shown as a figure 2, wherein the livers of the ducklings inoculated with NDRV JS01-5P show serious bleeding points, and the livers of the ducklings of 75P, 105P and the control group have no obvious change; the spleen of the NDRV JS01-5P inoculated duck has severe bleeding, swelling and necrosis, the spleen of the passaged virus 75P inoculated duck has bleeding, slight swelling and dead spots, and the spleen of the 105P and the spleen of the control duck have no obvious change. The NDRV JS01 strain is virulent and passaged to inoculate 1-day-old healthy susceptible duckling, the duckling is dissected and killed after 4 days, each group is used for taking heart, liver, spleen, lung, kidney, duodenum, bursa and brain of inoculated duckling to perform virus titration, NDRV sensitive LMH cells are used for titration, and the result of the viscera virus titration of the NDRV JS01-5P and passaged virus (75P and 105P) inoculated duckling is shown in figure 2. After the NDRV JS01-5P infects ducks, viruses can be detected in heart, liver, spleen, lung, kidney, duodenum, bursa of Fabricius and brain, and systemic infection is presented, wherein the content of the viruses in the lung, spleen, lung and kidney is highest; NDRV JS01-75P vaccinated ducks mainly infect spleen and bursa of Fabricius; the NDRV JS01 strain passaging virus 105P inoculated duck can only detect low-content virus in the spleen of part of ducks.

EXAMPLE 3 minimum immune dose study of novel reovirus JS01-105P strain

To determine the optimal immunization dose of NDRV JS01 strain attenuated strain JS-105P on 1-day-old ducklings: animal experiments were performed after incubating purchased SPF duck embryos to 1 day of age, and 40 healthy susceptible ducklings were randomly divided into 4 groups of 10 ducklings each. Group 1: leg muscle inoculation NDRV JS01-105P with dosage of 10 ^2.0 TCID ₅₀ Each 0.1mL; group 2: leg muscle inoculation NDRV JS01-105P with dosage of 10 ^2.5 TCID ₅₀ Each 0.1mL; group 3: leg muscle inoculation NDRV JS01-105P with dosage of 10 ^3.0 TCID ₅₀ Each 0.1mL; group 4: PBS control group, leg muscle inoculated with PBS, each 0.1mL. When duckling is 7 days old (i.e. 6 days after immunization), all 4 groups of duckling are subjected to virus elimination and muscle inoculation is carried out for 10 days ^5.0 TCID ₅₀ The NDRV JS01 is virulent, and on the 4 th day after virus attack, all ducks are subjected to section examination and virus titration of viscera. Section inspection display, 10 ^2.0 TCID ₅₀ After the dose immunization group attacks the toxin, 3/10 spleen still has a certain swelling and bleeding phenomenon. And 10 (10) ^2.5 TCID ₅₀ And 10 (V) ^3.0 TCID ₅₀ No obvious visceral lesions appear after the dose immunization of the group ducks attacks the toxin. Titration results of viruses in different organs show that after virus attack, the method is carried out at 10 ^3.0 TCID ₅₀ And 10 (V) ^2.5 TCID ₅₀ No virus could be detected in the viscera of the dose immunized duck, but 10 ^2.0 TCID ₅₀ Virus still exists in the spleen of 3/10 ducks of the dose immunization group. Description of the results above 10 ^2.5 TCID ₅₀ The NDRV strain JS01-105P can provide complete protection. Determination of minimum immunization dose of 10 for NDRV passage virus JS01-105P strain ^2.5 TCID ₅₀ 。

EXAMPLE 4 preparation and testing of novel reovirus attenuated live vaccines

In a sterile safety cabinet, adding the virus liquid amplified by NDRV JS01-105P, 110P and 115P for 3 generations into a freeze-drying protective agent in a ratio of 9:1, mixing uniformly, quantitatively split charging into sterile ampoule bottles, freeze-drying in a freeze dryer in vacuum, capping after the freeze drying, and preserving in a refrigerator below-15 ℃ to obtain a vaccine finished product shown in figure 3.

And (3) checking a vaccine finished product: the freeze-dried vaccine is subjected to character inspection, the character of the vaccine is spongy loose lumps, the vaccine is easy to separate from the wall of a vaccine bottle, and the vaccine is quickly dissolved after diluent is added; the sterility test, mycoplasma, exogenous virus, residual water and vacuum test are carried out according to the annex of the current Chinese animal pharmacopoeia. And (3) identification and inspection: epidemic disease to be preparedDiluting the virus maintenance solution for seedling to 100TCID ₅₀ 0.1ml, equal volume and NDRV hyperimmune serum inactivated at 56 ℃ for 30min (neutralization titer not less than 1:10), 96 well cell culture plates of LMH cells well grown were inoculated after neutralization at 37 ℃ for 1h, 10 wells per batch of vaccine, 200 ul/well, while normal control cells and virus control wells were established, 10 wells each. Placing at 37deg.C 5% CO ₂ Is cultured in a cell culture box for 7 days, and cytopathic conditions are observed and recorded. Normal maintenance fluid cell pores and neutralization pores should be free of cytopathic effects, and virus control pores should be cytopathic; the vaccine virus content was tested using LMH cells: randomly extracting one bottle of prepared vaccine per batch, diluting the vaccine with virus maintenance solution to 0.1ml containing 1 part according to the marked vaccine part, diluting with dilution solution by 10 times, and collecting 10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 4 dilutions, each followed by 4 replicates inoculated in 96 well plates of well grown LMH cells, 0.1mL per well, 37℃and 5% CO ₂ Culturing in a cell incubator for 7 days, observing and recording cytopathic effect, and calculating the virus content TCID according to Reed-Muench method ₅₀ The virus content of each feather should be more than or equal to 10 ^3.5 TCID ₅₀ 。

3 batches of laboratory test-prepared freeze-dried NDRV JS01-105P attenuated vaccine have good freeze-drying effect. And (3) the finished product inspection result shows that: the freeze-dried property is spongy loose agglomerate which is easy to separate from the wall of the vaccine bottle, and the diluent is added for quick dissolution; the sterility test, mycoplasma test and exogenous virus test are carried out according to the annex of the current Chinese animal pharmacopoeia, and 3 batches of vaccine have no bacteria, mycoplasma growth and exogenous virus pollution. The identification test results show that the virus control holes all generate cytopathy, and the neutralization holes and the maintenance liquid control holes in 3 batches of vaccine samples have no cytopathy.

Example 5 evaluation of safety of novel reovirus attenuated live vaccines

After the novel duck reovirus attenuated vaccine JS01-105P strain is inoculated into 1-day-old healthy susceptible duckling in single dose, single dose repetition and overdose, obvious adverse reactions are not seen at the inoculated part and the whole body, and the inoculated duckling is normal in feeding, drinking water, excretion and mental state and has no clinical reactions and pathological changes (see tables 1-3).

TABLE 1 safety test of novel reovirus attenuated vaccine JS01-105P strain single dose 1-inoculation of 1 day old duckling

TABLE 2 safety test of novel reovirus attenuated vaccine JS01-105P strain single dose repeated vaccination of 1 day old ducklings

TABLE 3 safety test of novel reovirus attenuated vaccine JS01-105P strain overdose inoculation of 1-day-old ducklings

EXAMPLE 6 evaluation of the effectiveness of novel attenuated live reovirus vaccines

Novel reovirus attenuated live vaccine JS01-105P strain is inoculated to 1-day-old duckling virus attack protection condition: (1) The clinical manifestations are that 1 day old duckling is immunized with 3 batches of NDRV JS01-105P strain vaccine, 7 days old duckling is subjected to virus attack, the vaccinated duckling group has no abnormal manifestation, and the contrast of the duckling group is characterized by reduced feed intake and diarrhea; (2) pathological changes: killing after 4 days of virus attack, checking pathological changes of immune ducks, wherein each viscera of duckling in each immune group has no obvious pathological changes, and spleen of duckling in the contrast virus attack group has different degrees of significance and necrosis, and other viscera have no obvious macroscopic lesions; (3) virus isolation: the animals were dissected 4 days after challenge, and heart, liver, spleen, lung, kidney, etc. organs of vaccinated ducklings were collected and virus isolated using LMH cells, which showed that no virus was isolated from 3 vaccine immunized ducklings, but no virus was isolated from the spleen of the control challenge group (table 4).

Table 4 effectiveness test of novel reovirus attenuated live vaccine JS01-105P strain

The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

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tcaggacttg tcgctgccca agaccctcct ccagctccac cgtaccaacc agcttcggga 360

cagtggtctg atctgcttca gtatcctcga tgggctaatc gtcaacgtga gttgcagtct 420

aaatatccta ttctgttgcg atctactttg ctttcagcta tgcgcgctgg accggtgttg 480

tacgttgaga cttggcccaa cacgatttct ggtcgacttg ctgactggtt catgtcgcag 540

tatgggaaca acttcgttga catgtgtgca cgattaactc agtcctgcat gaacatgcca 600

gttgagccgg atggtaatta tgaccagcag atgcgagcat taattagtct gtggctcctc 660

tcgtatattg gcgttgtgaa tcaatctaac actattaacg gcttctactt tgcttccaag 720

acgcgggggc aggctatgga taactggacg ctgttctatg ctaccaacac caatcgggtg 780

cagattactc agcgccactt tgcctacgtt tgcgctcgct cccccgactg gaatgtcgat 840

aagtcctgga tcagcgctgc caatttgact gccattgtca tggcttgccg ccagccgcca 900

gcctttgcca accagggggt aatcaaccag gcccagaatc gacctggatt ctcgatgaat 960

ggtggaacgc ctgtgcacga gctgaacatg ctgactaccg ctcaagcttg tatccagcag 1020

tgggtcgtag ctgatttgat ctcagcagct aagggtcagt ccatgacgca ggaggcgaat 1080

gacttctcga acctcatcca agccgatctt gggcggatca aagcgcaaga tgacgcgttg 1140

tacaaccaac aacctggtta cgctcgtcgc attaagccgt ttgccaacgg agattggact 1200

cctggaatga ctgcgcaagc attagctcta ctagccactt ttactgtcta g 1251

<210> 6

<211> 1104

<212> DNA

<213> novel Duck reovirus (Novel duck reovirus) attenuated strain JS01-105P

<400> 6

atggaggtgc gtgtgccaaa ctttcactcc tttatcgagg gtattactac tagttacttg 60

tgttctcctg cgagctggaa ttcgaagacg ttatgggata ttgaagaatt tcacacacct 120

gacgttatca gggtcggcaa tgcttattgt tgcactcagt gctgtggtgt tctgtactat 180

ggtgcccctc cctctgatgg aaactgtttt ccacatcaca agtgtcatca acagcaatat 240

cgtactgaga ctccgctcat gagatatatt aaggtgggtc gcactacaga gcaactgctt 300

gatcaatatg ccattgctct gcatgtcatt gcagattact atgacgaggc gagtaagcaa 360

cctcatgata tcgctgaaac tgagtcaatc gcaccatttg atatcgtaac caggactgaa 420

tctattcgca gtgaccgtgc cgttgacccg gaattctgga cttatccgtt agagagacga 480

ggatacgacg cgcgacatga gattgctaga gcgggttgga agatgatcga tgcttcatcg 540

cgaagtcaca ctcttcctga atgtctggtg tcaaatatgc tacatactag acatgtcttc 600

agccaaatgt tgaccacgac aaccatctat gatgtcgctg tcacgggtaa agctgttaca 660

ttcagcccga tggtagcaac catgccaact cgaggagatg gtgctgtggc tctgtcaaga 720

ggtaacttgg atcatgatgt cgaggactgt tggatggatg gttttgcatt ctcccccttc 780

atcggcggtg ttggcatcac tggtcaattt gagcgtggct cctgccataa ttttggacac 840

cccatgattg gaagcggtaa gaaggcttct cactaccgca atttattcat ggaatcctgg 900

cgtggatggt caaagtcgtg ctttacatgt gctgtaggga tggagcccgc ggagtgcgaa 960

tctaggctgc gaggccatgc cagaactatg ttcggacgtt ctcttccgga tatctgtgac 1020

ttcgaggaga ctacccacat tggccagtcg tccgcgccct taaagaaggc cacgaaattg 1080

tccttcctgg agtgtaggtg gtaa 1104

Claims

1. A novel duck reovirus attenuated strain with a preservation number of CCTCC NO: v202168.

2. A vaccine comprising the novel duck reovirus attenuated strain of claim 1.

3. The vaccine of claim 2, wherein the virus content per feather of the vaccine is not less than 10 ^3.5 TCID ₅₀ 。

4. Use of a novel duck reovirus attenuated strain of claim 1 in the preparation of a product for preventing a novel duck reovirus disease.