CN109576230B

CN109576230B - Avian encephalomyelitis virus strain and application thereof

Info

Publication number: CN109576230B
Application number: CN201811529250.9A
Authority: CN
Inventors: 徐怀英; 秦卓明; 高月花; 王友令; 袁小远; 张玉霞; 杨金兴
Original assignee: Poultry Research Institute Shandong Academy of Agricultural Sciences
Current assignee: Poultry Research Institute Shandong Academy of Agricultural Sciences
Priority date: 2018-12-13
Filing date: 2018-12-13
Publication date: 2022-05-10
Anticipated expiration: 2038-12-13
Also published as: CN109576230A

Abstract

The invention relates to the field of animal virology, provides an avian encephalomyelitis virus strain and application thereof, and particularly provides an avian encephalomyelitis virus, which is named as AEV JN and has a preservation number of: CGMCC No.16385, provides the separation and identification process of the strain, determines that the strain is in the same branch with the VR strain of the AEV virulent strain internationally recognized through VP1 genetic evolution analysis, has close genetic relationship, fills up the relevant blank in China, can well form complementation with the existing commonly used live vaccine 1143 strain in China by using the strain as a vaccine strain, and provides better protection for poultry breeding in China.

Description

Avian encephalomyelitis virus strain and application thereof

Technical Field

The invention relates to the field of animal virology, and provides an avian encephalomyelitis virus strain and application thereof.

Background

Avian Encephalomyelitis (AE), also known as epidemic tremor, is a viral infectious disease characterized by head and neck tremor with dyskinesia caused by Avian Encephalomyelitis Virus (AEV) infecting birds. All day-old birds (chickens, quails, turkeys, etc.) can infect through the digestive tract, but the incidence of infection varies. After the young chickens are infected with the virus, paralysis, head and neck tremor and even ataxia are caused, and the young chickens often have subclinical infection or cause a reduction (5% -10%) of the egg production, the morbidity of the disease is generally 40-60%, and the mortality is 25-50%. The disease has spread to many countries and regions around the world since its first discovery in the united states in 1932, causing significant economic losses to the poultry industry. At present, no specific treatment method exists for the disease, and only live vaccine and inactivated vaccine immunization are used for preventing and controlling the disease.

During the onset of the breeder, the hatching rate of the eggs laid decreases, and some infected hatching eggs may be laid and hatched out of the infected chicks. The chicks can expel toxin and infect the chicks which are hatched and grow together, and clinical symptoms such as head and neck tremor, ataxia and the like can appear at the age of 1-7 days, so that the sick chicks can resist growth and development retardation. Chicks exposed to AE virus after 2-3 weeks of age showed no neurological signs, but had typical histological lesions. The hens with maternal antibodies were protected. Therefore, the hatching eggs laid during the disease of the breeding hens cannot be used for hatching, and the hatching eggs whose laying rate is recovered to more than 85% can be used for hatching. AEV propagates through the oral-fecal pathway with the ability to propagate both horizontally and vertically. Because of its great stability, the contaminated area may remain contagious for long periods of time and may be passed from one flock to another through a variety of channels, including contaminated material. AEV belongs to small RNA virus, has no cyst membrane and core, is acid-resistant, has strong resistance to chloroform, ether, pepsin, pancreatin and the like, can stably exist in the environment, and is difficult to completely eliminate from the environment, so people often refer to IBD which is one of AE and three poultry diseases in the world as field disease.

AEV exists in two distinct pathotypes. One is enterophilic, represented by wild strains in nature, which are susceptible to oral infection with chicken flocks and detoxify through feces. These strains are relatively weak in virulence, but can be vertically transmitted through hatching eggs or infect susceptible chicks horizontally early and cause neurological symptoms. The other pathotype is chick embryo adaptive strain, the virus is highly neurotropic, generally does not cause infection when being orally taken, does not replicate in intestinal tract, is not detoxified by feces after being infected by a non-intestinal tract route, and cannot be horizontally transmitted.

In the prior art, the AEV 1143 strain live vaccine can be used for immunization of chickens with the age of more than 10 weeks, strains adapted to the growth of chick embryos are generally highly neurotropic, so the chick embryo adapted strains cannot be used for preparing live vaccines, but can be prepared into inactivated vaccines for immunization, and maternal antibodies generated after immunization can protect offspring chickens from being attacked by AEV 2-3 weeks after the offspring chickens are out of shells.

Before 2015, the protection rate of 100% can be obtained after live vaccines are immunized by chicken flocks, but in recent years, some chicken flocks still suffer from diseases after immunization, the laying rate is reduced by 40%, the death rate of the young chicks can reach 80% after the chicken flocks continue to be immunized for nearly one month, in addition, the age of the young chicks is gradually increased, and the new disease characteristics are obviously different from the past. The serum of the immune chicken flocks before the disease attack is detected by an ELISA method, and the AE antibody titer is found to be more than 2000, so that the disease attack of the immune chicken flocks possibly has a certain relation with environmental pollution and wild strain variation; therefore, the method for finding and using the variant strain to obtain the corresponding vaccine as soon as possible has great significance for the poultry industry.

Disclosure of Invention

The invention provides an avian encephalomyelitis virus strain and application thereof, and particularly provides an avian encephalomyelitis virus, which is named as AEV JN and has a preservation number of: CGMCC No.16385, provides the separation and identification process of the strain, determines that the strain is in the same branch with the VR strain of the AEV virulent strain internationally recognized through VP1 genetic evolution analysis, has close genetic relationship, fills up the relevant blank in China, can well form complementation with the existing commonly used live vaccine 1143 strain in China by using the strain as a vaccine strain, and provides better protection for poultry breeding in China.

The inventor carries out biological preservation on the strain, and the preservation numbers are as follows: CGMCC No. 16385.

The virus appears in a laying hen farm of a certain local Jinan city of 12 months in 2016 for the earliest time, suspected AE infection occurs in the laying hen farm, a cloacal cotton swab of the laying hen is taken for sterile treatment, then a 6-day-old SPF chick embryo yolk sac is inoculated, the incubation is continued for 12 days, and the lesion of the chick embryo is observed. The first generation of chick embryos have no pathological changes, chick embryo brains are aseptically harvested, after repeated freeze thawing for 3 times, the chick embryos are passaged on SPF chick embryos, after 12 days of inoculation, the SPF chick embryos have AE characteristic pathological changes such as activity weakening, dysplasia, dwarfism, muscular atrophy, toe deformation, betel palm and the like, and through animal regression experiments, the separated viruses are preliminarily identified as avian encephalomyelitis viruses and named as AEV JN;

the inventor further utilizes various detection means to identify the virus, and the results all prove that the virus accords with the relevant characteristics of the avian encephalomyelitis virus, and further, the inventor finds that the VP1 gene of the virus has the full length of 810bp and encodes 270 amino acid residues, the nucleotide sequence of the VP1 gene is shown as Seq ID No.1, and the encoded amino acid residue sequence of the VP1 gene is shown as Seq ID No. 2; the genetic evolutionary tree is drawn by the method, as shown in the attached drawing, the JN strain (dark color label) provided by the invention and the internationally recognized virulent strain VR strain are in the same branch, the genetic relationship is closer, the JN strain and the domestic commonly used live vaccine 1143 strain (light color label) are in different branches, the genetic relationship is further, and most of the domestic AEV isolate and 1143 vaccine strains are in the same branch at present; nucleotide homology analysis of VP1 gene shows that nucleotide homology of JN and AEV 1143 strain (live vaccine strain) is 93.5%; the homology of the AEV JN strain and the VR strain is 98.9 percent, and the nucleotide homology with most of domestic separated strains is 83.9 to 93.9 percent;

the inventor further provides a preparation method of the corresponding inactivated vaccine by using the virus, which comprises the following steps:

e3 of AEV JN is used as seed poison, sterilized normal saline or PBS is used for 1:100 dilution, a 6-day-old SPF chick embryo is inoculated to a yolk sac, the chick embryo is incubated to 16-day-old chick embryos and placed at 2-4 ℃ overnight, the chick embryo with obvious lesion is taken, the embryo body is taken to be homogenized (eyes and bones of limbs are removed), PBS or the same batch of allantoic fluid is used for mixing according to the weight ratio of 1: 3 preparing into suspension, freezing and thawing for 3-5 times, or treating with ultrasonic wave, placing into a sterilized container, filtering with sterilized four-layer gauze, adding 10% formalin to make the final concentration of formaldehyde be 0.2%, placing into a constant temperature shaking incubator at 37 deg.C for inactivating for 24 hr, and emulsifying according to the ratio of oil to water of 2:1 after inactivation is qualified, and preparing into oil emulsion inactivated vaccine.

Performing challenge experiment by using the obtained inactivated vaccine, injecting 30 SPF chickens with 3 weeks old by neck subcutaneous injection with 1 immunization dose of the vaccine, and inoculating 1000EID in brain by using virulent VR strain for challenge with AEV standard after immunizing for 3 weeks₅₀And (5) counteracting the toxin, setting 5 SPF chickens of the same day age to be contrasted, and observing for 14 days. Results control 5 chickens were all attacked, and clinical observation of 30 chickens in the immune group was normalThe protection rate is 100 percent. Therefore, the vaccine provided by the invention can obtain good immune protection effect.

In conclusion, the avian encephalomyelitis virus strain provided by the invention is in the same branch with the VR strain of the AEV virulent strain internationally recognized, the genetic relationship is close, the relevant blank in China is filled, and the avian encephalomyelitis virus strain can be well complemented with the existing commonly used live vaccine 1143 strain in China by being used as a vaccine strain, so that the avian encephalomyelitis virus strain provides better protection for the poultry breeding industry in China.

The inventor carries out biological preservation on the avian encephalomyelitis virus strain disclosed by the invention, and the specific preservation information is as follows:

preservation information

Preservation time: 9/19/2018

The name of the depository: china general microbiological culture Collection center

The preservation number is: CGMCC No.16385

The address of the depository: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

Classification nomenclature avian encephalomyelitis Virus

Drawings

FIG. 1 is a genetic evolutionary tree of strain VP1 gene.

Detailed Description

Example 1

The avian encephalomyelitis virus strain is obtained by separating the virus as follows:

a laying hen farm is produced in a certain place of Jinan, 12 months in 2016, suspected AE infection occurs in the laying hen farm, cloaca cotton swabs of laying hens are taken for aseptic treatment, then 6-day-old SPF (specific pathogen free) chick embryo egg yolk sacs are inoculated, the incubation is continued for 12 days, and the lesion of the chick embryos is observed. The first generation of chick embryos have no pathological changes, chick embryo brains are aseptically harvested, after repeated freeze thawing for 3 times, the chick embryos are passaged on SPF chick embryos, after 12 days of inoculation, the SPF chick embryos have AE characteristic pathological changes such as activity weakening, dysplasia, dwarfism, muscular atrophy, toe deformation, betel palm and the like, and through animal regression experiments, the separated viruses are preliminarily identified as avian encephalomyelitis viruses and named as AEV JN;

Example 2 related detection and analysis of viruses

1. Diluting the virus seeds 10 times with sterilized normal saline, and taking 10 times^-5、10^-6、10^-7、10^-84 dilutions, inoculating 5-6 day-old SPF chick embryos by yolk sac route, and inoculating 0.2ml of each embryo. After inoculation, the eggs are incubated continuously at 37 ℃, eggs are taken once every 8 hours, dead embryos are taken out in time, dead chick embryos within 72 hours are discarded, and the dead chick embryos after 72 hours have the following symptoms: chick embryo developmental retardation, dwarfism, brain edema, progressive muscular atrophy, toe deformation, betel palm liver and the like are AE lesions. The observation results of chick embryo death and lesions are shown in Table 1, and EID is calculated according to the number of chick embryo lesions and by the Reed-Muench method₅₀Virus content ≧ 10 per 0.2ml^6.75EID₅₀。

TABLE 1E 3 avian encephalomyelitis Virus EID₅₀Measurement results

Note: the numerator is the number of chick embryos with AE pathological changes, and the denominator is the number of experimental chick embryos.

The results showed that the healthy control group had an average embryo weight of 12.59g, the vaccinated group had dysplasia of the embryo body, muscle atrophy, weight loss, subcutaneous bleeding points, brain bleeding points, hydrops and atrophy, and the average mating was 8.9 g.

2. Virulence of chicks 20 SPF chickens 1 day old were inoculated with AEV intracerebrally, and each chicken was inoculated with 0.03ml (about 1000 EIDs)₅₀) Meanwhile, 5 control animals inoculated with physiological saline were set and observed for 14 days. The control group of SPF chickens are normal within 14 days, and the SPF chickens in the AEV challenge group show typical AE neurological symptoms 5-10 days after inoculation, which are specifically shown as follows: mental depression, unstable standing, recumbency, paralysis, head and neck tremor, ataxia and the like, and all the diseases and deaths occur by the end of the 14-day observation period.

3. And (3) specific detection: diluting AEV JN by 1:100 times, mixing with anti-AE positive serum and negative serum respectively in equal amount, acting at 37 deg.C for 60 min, and inoculating 10 SPF embryos of 5-6 days old, each 0.2 ml; at the same time, virus control is set, and observation is carried out for 12 days under the same conditions. The virus neutralization group does not have chick embryo lesions, and more than 8 chick embryos of the virus control group and the negative serum control group respectively have AE typical lesions.

4. Stability test of subculture virus: and (3) continuously transmitting the AEV JN of the E3 generation to the E20 generation on SPF chick embryos of 6 days old, and detecting the virus content, the toxicity to chicks and the immunogenicity.

Measuring virus content of subculture virus seeds, selecting generation E5, generation E10, generation E15 and generation E20 virus to inoculate SPF chick embryos, measuring virus content, and measuring EID of AEV virus seeds in virus liquid of E3-E20₅₀Are relatively stable and are all more than or equal to 10^6.50EID₅₀0.1 ml. The results are detailed in table 2.

TABLE 2 determination of the Virus content of E3-E20

Note: the numerator is the chick embryo disease variable, and the denominator is the experimental chick embryo number.

Passage virus immunogenicity test: oil emulsion inactivated vaccines are respectively prepared from E5, E10, E15 and E20 generation virus seeds, 10 SPF (specific pathogen free) chickens with the age of 3 weeks are respectively immunized, 5 control SPF chickens are set, after 3 weeks, the standard virulent VR strain is used for virus challenge, 5 control chickens are all attacked, AE typical symptoms exist, the E5, E10 and E15 immune groups do not have clinical symptoms, 1 chicken in the E20 immune virus challenge group has ataxia symptoms, but does not show head-neck tremor, the result is shown in table 3, and the E5, E10 and E15 generation times can obtain better protection effect, so that the vaccine prepared after passage proves that the virus strains have genetic stability and are suitable for being used as vaccine strains.

TABLE 3 immunogenicity test results of JN strains

Passage virus species virulence test: 6-week-old SPF chickens were intracerebrally inoculated with E5, E10, E15 and E20 viruses, 10 chickens per group developed AE-typical head-neck tremor symptoms, no death of the chickens was observed, and the control group was normal.

Analysis of genetic evolution of VP1 Gene

Specific primers are designed according to a highly conserved region of a Van Roekel strain VP1 gene, amplified by RT-PCR, recovered and purified DNA is cloned to a PMD19-T vector, DH 5 alpha competent cells are transformed, then sequence determination is carried out, and genetic evolution analysis is carried out on the sequence and known reference strains. The sequencing and splicing result shows that the VP1 gene has a full length of 810bp and encodes 270 amino acid residues. Drawing a genetic evolutionary tree (as shown in figure 1), wherein a JN strain (marked in dark color) and an internationally recognized virulent strain VR strain are in the same branch, the relationship is relatively close, the JN strain and a domestic commonly used live vaccine 1143 strain (marked in light color) are in different branches, the relationship is relatively far, and most of domestic AEV isolates and 1143 vaccine strains are in the same branch at present;

nucleotide homology analysis of VP1 gene, the results are shown in the following table

The nucleotide homology of AEV JN and AEV 1143 strain (live vaccine strain) is 93.5%; the homology of the JN strain and the VR strain is 98.9 percent, and the nucleotide homology with most of domestic separated strains is 83.9 to 93.9 percent.

EXAMPLE 3 preparation of inactivated vaccine

E3 of AEV JN is used as seed poison, sterilized normal saline or PBS is used for 1:100 dilution, a 6-day-old SPF chick embryo is inoculated to a yolk sac, the chick embryo is incubated to 16-day-old chick embryos and placed at 2-4 ℃ overnight, the chick embryo with obvious lesion is taken, the embryo body is taken to be homogenized (eyes and bones of limbs are removed), PBS or the same batch of allantoic fluid is used for mixing according to the weight ratio of 1: 3 preparing suspension, freezing and thawing for 3-5 times, or treating with ultrasonic wave, placing in a sterilized container, filtering in the sterilized container with sterilized four-layer gauze, adding 10% formalin to make the final concentration of formaldehyde be 0.2%, placing in a constant temperature shaking incubator at 37 deg.C for inactivating for 24 hours, emulsifying according to the proportion of oil water 2:1 after inactivation inspection is qualified, and preparing oil emulsion inactivated vaccine;

the vaccine is injected subcutaneously into 30 SPF chickens with the neck of 3 weeks old at 1 immunization dose for immunizationAfter 3 weeks, 1000EID were inoculated in the brain with virulent VR strain using AEV standard for combating poison₅₀And (5) counteracting the toxin, setting 5 SPF chickens of the same day age to be contrasted, and observing for 14 days. Results control 5 chickens were all attacked, and the normal protection rate of 30 chickens in the immune group is 100% through clinical observation.

<110> institute of poultry research (Shandong province chicken research center without specific pathogen)

<120> avian encephalomyelitis virus strain and application thereof

<160>2

<210>1

<211>810

<212>DNA

<213> Avian encephalomyelitis Virus (Avian Eneephalomycetis Virus, AEV)

<400>1

gggaaagagg atgagagagg attttccagt gtgcctgaag tggagcaaca tgttgttgag 60

gacaaggaac cacagggacc tttgcacgtg acaccttttg gcgctgttaa agccatggag 120

gaccctcagt tggcgaggaa aacacctggt acatttcctg aattagctcc tggtaaacct 180

cgacacacag tagaccatat ggatctgtat aagtttatgg ggcgtgccca ttacttgtgg 240

ggacataaat tcaccaaaac tgatatgcag tacacattcc agataccatt gagtcccatc 300

aaggagggtt ttgtgacggg aacacttagg tggtttttaa gtcttttcca attgtatcgt 360

ggttctctcg acattaccat gacattcgca ggaaagacta atgtagacgg cattgtatac 420

tttgtgcctg agggtgttgc gatagagact gagaggaagg aacagacccc tctgctcacg 480

ttgaactaca aaacatcggt aggtgccatt aggtttaata ctggacaaac tacaaatgtc 540

cagtttagga tccctttcta cacgccactg gagcacatcg caacccattc taaaaacgcg 600

atggactcag tcttgggggc aattacaact cagatcacca attatagtgc tcaggatgag 660

tacctgcagg tcacctacta tatcagcttt aatgaagatt cacagttttc tgttcccaga 720

gcggtaccag tggttagctc attcactgac acatctagca agacagtgat gaatacatat 780

tggctcgatg atgacgagtt ggtagaagag 810

<210>2

<211>270

<212>PRT

<213> Avian encephalomyelitis Virus (Avian Eneephalomycetis Virus, AEV)

<400>2

Gly Lys Glu Asp Glu Gly Gly Phe Ser Ser Val Pro Glu Val Glu

1 5 10 15

Gln His Val Val Glu Asp Lys Glu Pro Gln Gly Pro Leu His Val

20 25 30

Thr Pro Phe Gly Ala Val Lys Ala Met Glu Asp Pro Gln Leu Ala

35 40 45

Arg Lys Thr Pro Gly Thr Phe Pro Gln Leu Ala Pro Gly Lys Pro

50 55 60

Arg His Thr Val Asp His Met Asp Leu Tyr Lys Phe Met Gly Arg

65 70 75

Ala His Tyr Leu Trp Gly His Lys Phe Thr Lys Thr Asp Met Gln

80 85 90

Tyr Thr Phe Gln Ile Pro Leu Ser Pro Ile Lys Glu Gly Feu Val

95 100 105

Thr Gly Thr Leu Arg Trp Phe Leu Ser Leu Feu Gln Leu Tyr Arg

110 115 120

Gly Ser Leu Asp Ile Thr Met Thr Phe Ala Gly Lys Thr Asn Val

125 130 135

Asp Gly Ile Val Tyr Phe Val Pro Glu Gly Val Ala Ile Glu Thr

140 145 150

Glu Arg Lys Glu Gln Thr Pro Leu Leu Thr Leu Asn Tyr Lys Thr

155 160 165

Ser Val Gly Ala Ile Arg Phe Asn Thr Gly Gln Thr Thr Asn Val

170 175 180

Gln Phe Arg Ile Pro Phe Tyr Thr Pro Leu Glu His Ile Ala Thr

185 190 195

His Ser Lys Aan Ala Met Asp Ser Val Leu Gly Ala Ile Thr Thr

200 205 210

Gln Ile Thr Asn Tyr Ser Ala Gln Asp Glu Tyr Leu Gln Val Thr

215 220 225

Tyr Tyr Ile Ser Phe Asn Glu Asp Ser Gln Phe Ser Val Pro Arg

230 235 240

Ala Val Pro Val Val Ser Ser Phe Thr Asp Thr Ser Ser Lys Thr

245 250 255

Val Met Asn Thr Tyr Trp Leu Asp Asp Asp Glu Leu Val Glu Glu

260 265 270

Claims

1. An avian encephalomyelitis virus strain with the preservation number as follows: CGMCC No.16385, named AEV JN; the total length of the gene VP1 is 810bp, 270 amino acid residues are coded, the nucleotide sequence is shown as Seq ID No.1, and the coded amino acid residue sequence is shown as Seq ID No. 2.

2. Use of the avian encephalomyelitis virus strain of claim 1 in the preparation of an inactivated vaccine.