CN112501132A - Duck source goose parvovirus artificial attenuated strain and application thereof - Google Patents

Duck source goose parvovirus artificial attenuated strain and application thereof Download PDF

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CN112501132A
CN112501132A CN202010892082.0A CN202010892082A CN112501132A CN 112501132 A CN112501132 A CN 112501132A CN 202010892082 A CN202010892082 A CN 202010892082A CN 112501132 A CN112501132 A CN 112501132A
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张云
刘明
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Harbin Veterinary Research Institute of CAAS
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Abstract

The invention relates to a goose parvovirus attenuated strain GPV-ZYM with the preservation number of CGMCC No.15700 and application thereof as a vaccine for preventing duck or goose from infecting duck short beak syndrome and/or gosling plague.

Description

Duck source goose parvovirus artificial attenuated strain and application thereof
Technical Field
The invention belongs to the field of biology. In particular to an artificial attenuated strain of duck source goose parvovirus and application thereof.
Background
In recent years, the province of Fujian in China[1]Shandong province[2]Anhui province, Anhui province[3]Hefujian tea[4]The new goose parvovirus disease is epidemic in the provinces of the half muscovy ducks and the cherry valley ducks, the duck flock is infected with the disease, the disease is clinically manifested as hypoevolutism, beak atrophy, tongue outward extension and the like, and is commonly called as large (long) tongue disease or short beak-dwarfism syndrome and the like. The disease mostly attacks meat ducks of 10-30 days old, the morbidity is 15-100%, and the mortality is 3-10%. The weight of the sick duck is reduced by 20-30% compared with that of the healthy duck5]Those with severe weight are only 50% of normal weight[6]. At present, the disease incidence area is continuously enlarged, and huge economic loss is caused to the duck breeding industry in China.
The traditional goose parvovirus disease is also called gosling plague, is mainly characterized by the cellulose of the small intestine of a gosling and the embolic disease, and has the characteristics of quick transmission, high mortality rate and the like. GPV mainly infects goslings and young muscovy ducks within 30 days of age, the clinical manifestations are necrotic enteritis, the death rate is up to 80% -100%[7]. Although the pathogen of gosling plague and the pathogen of short beak-dwarfism syndrome belong to goose parvovirus in taxonomy, the homology of the gosling plague and the short beak-dwarfism syndrome is as high as 98.7%. However, the symptoms of the disease vary depending on the host infected with the disease. To date, no one has reported the pathogenesis of both viruses.
The whole genome sequence of GPV was first published in 1995 by Z.Z dori et al. GPV genome consists of about 5100 nucleotides, two ends of the genome are hairpin structures formed by folding palindromic sequences, and the genome contains two Open Reading Frames (ORF)[8]. The left ORF encodes two non-structural proteins NS1 and NS 2; the right ORF encodes three structural proteins VP1, VP2 and VP3, of which VP3 is the capsid protein, the major immunoprotective antigen, which induces the production of neutralizing antibodies. Since goose parvovirus belongs to DNA virus, its mutation is very slow.
The invention content is as follows:
the invention aims to provide a goose parvovirus strain GPV-ZYM which is preferably used as a vaccine for protecting ducks from infecting short beak syndrome of ducks and/or gosling plague of goslings (muscovy ducks). The strain is preserved in China general microbiological culture Collection center (China, Beijing) in 2018, 5 months and 16 days, and the preservation number is CGMCC No. 15700.
In particular, the present invention relates to the following aspects:
2. the goose parvovirus strain GPV-ZYM has the preservation number of CGMCC No. 15700.
The nucleotide sequence is shown as SEQ ID NO: 1 is shown.
3. The vaccine comprises the goose parvovirus strain GPV-ZYM in the item 1, and is applied to preparation of the vaccine for preventing duck or goose from infecting duck short beak syndrome and/or gosling plague.
4. Preparing duck short beak syndrome and/or gosling plagueThe vaccine method comprises the steps of inoculating the goose parvovirus attenuated strain GPV-ZYM in the item 1 into goose embryo fibroblast GEF, culturing at 37 ℃ until the cytopathy is more than 80%, collecting virus culture solution, wherein the virus content is TCID50Greater than or equal to 1040.1ml for formulating vaccines.
The invention relates to a method for preparing attenuated strain GPV-ZYM of goose parvovirus, which is prepared by carrying out continuous subculture on a novel duck source goose parvovirus strain which is clinically separated and causes short-Beak dwarfism syndrome (BADS) of duck through goose embryo fibroblast for 3 generations, then inoculating SPF golden-pig duck embryos, and repeating the continuous subculture for 120 generations alternately to obtain attenuated strain GPV-ZYM of goose parvovirus. The homology of the low virulent strain and other GPV strains in GenBank is 93.5-97.2%. The homology of the attenuated strain GPV-ZYM and the commercial attenuated vaccine GD strain (DQ665790) and the vaccine SYG61-41 strain (DQ299942) in China is respectively 92.8 percent and 93.0 percent on nucleotide, and the amino acid homology is respectively 95.3 percent and 95.9 percent. The low virulent strain GPV-ZYM is shown to be from an artificial low virulent strain rather than a vaccine strain. The 5' non-coding region of the strain carries the molecular markers GAACGAAC and GTTCGTTC which are unique to the GPV-ZYM low-virulent strain. Other GenBank strains and commercial vaccine strains do not have the molecular marker. The strain culture provides 100% protection for the short beak syndrome of cherry valley meat duck after being diluted by 1: 100 times; and also can provide 100% protection for gosling plague of gosling or muscovy duck. The gosling or the young muscovy duck of 1 day age only needs to be immunized once, and the gosling pestivirus infection and duck short beak syndrome of the gosling or the young muscovy duck can be effectively prevented by 100% in the whole course in 5 days. The live vaccine prepared by the method has the advantages of high efficiency, safety and low cost.
The invention aims to provide a preparation method of a novel duck source goose parvovirus cell attenuated live vaccine for protecting duck short beak syndrome and gosling and muscovy duck gosling plague, which is realized by the following technical scheme:
the invention uses the GPV-ZYM strain attenuated by the alternate continuous passage of the GEF and SPF duck embryos as the seed virus, and the live vaccine prepared by the GEF has the advantages of high efficiency, safety and low cost.
Description of the drawings:
FIG. 1: immunohistochemical detection of antigen distribution of goose parvovirus GPV-ZYM and virulent YG infected duckling small intestine (A) and liver (B).
The specific implementation mode is as follows:
the present invention is described in detail below by referring to examples. It will be understood by those of ordinary skill in the art that the following examples are for the purpose of illustrating the present invention only and should not be construed as limiting the present invention in any way. The scope of protection of the invention is defined by the appended claims.
Example 1 acquisition of an artificially attenuated novel attenuated Strain of goose parvovirus GPV-ZYM Strain
Suspected short-beak dwarfism syndrome of ducks is exploded in a certain cherry valley duck farm in Shandong in 2015, and the ducks which are inspected have obvious symptoms of short beaks, long tongues, emaciation and the like. The dead duck is seriously filled with blood, and the liver, the spleen, the kidney, the lung, the small intestine and the pancreas are seriously bleedings, even degenerated and necrotic. And grinding the disease material, inoculating the supernatant to duck embryos, and identifying the duck embryos as the novel goose parvovirus GPV.
Early studies showed that continuous alternate passage of GPV in Goose Embryo Fibroblast (GEF) and Duck Embryo Fibroblast (DEF) has an important relationship with attenuation of virulence[9-11]. With the continuous subculture of the virus in the cells, the host cells and the virus continuously interact, so that virulence related genes of the virus are modified and changed, and the virulence is weakened and even the pathogenic capability of the virus on host animals is lost.
We carry out continuous cross subculture on newly separated novel goose parvovirus GPV causing duck short beak-dwarfism syndrome in GEF and SPF golden duck embryos, and expect to obtain a low virulent strain capable of preventing duck short beak-dwarfism syndrome.
The specific process is as follows: the ZYM parent strain is subcultured for 3 generations, inoculated with SPF golden duck embryo (purchased from Harbin veterinary institute laboratory animal center), immediately harvested allantoic fluid after death of duck embryo, diluted by 1: 100 times, inoculated with GEF (purchased from New farm in Darriy district, Ha city) prepared from goose embryo, cultured for 6 days to obtain virus culture, and repeatedly frozen and thawed for three times and preserved at-70 ℃. After the GEF culture is diluted by 1: 100 times, the SPF golden duck embryos are continuously cultured for one generation, then the SPF golden duck embryos are continuously cultured for 3 generations in the GEF,and (3) diluting the last culture according to a ratio of 1: 100 under the same culture condition, inoculating SPF golden duck embryos, harvesting virus cultures, and repeatedly and alternately carrying out continuous passage for 120 times. The strain passaged 120 th generation was named GPV-ZYM. Calculation of cytotoxic TCID according to the Reed-Muench method50。
The 120 th generation strain (designated GPV-ZYM) has the following biological characteristics:
(1) the titer of the strain is as follows: higher potency in goose embryo fibroblast and can reach TCID50＞108.0/0.1ml；
(2) Biological information analysis: the homology between GPV-ZYM generation P120 and the structural protein (VP1) of the parent virus (the whole gene of the parent strain is shown as SEQ ID NO: 9 after sequencing, and the sequences of NS protein and VP1 protein are shown as SEQ ID NO: 10 and 11) is only 97.4%. The homology of the attenuated strain GPV-ZYM VP3 with the GD strain (DQ665790) and the SYG61-41 strain (DQ299942) of the commercial attenuated vaccine in China is 92.8 percent and 93.0 percent respectively, and the homology of the amino acid is 95.3 percent and 95.9 percent respectively. GPV-ZYM carries the unique molecular markers GAACGAAC and GTTCGTTC in the non-coding region.
(3) Safety: 1-2 days old healthy gosling and young muscovy duck without GPV maternal antibody respectively 10, each intramuscular injected with diluted 1: 1000 times of seed virus GPV-ZYM and goose parvovirus YG[12]0.2ml of stock solution, observing for 30 days, inoculating GPV-ZYM to the gosling and the young muscovy duck to be alive completely, and inoculating GPV virulent YG strains of the gosling and the young muscovy duck to be 80% dead.
(4) Immune protection:
protection of gosling plague: and (3) immunization group: selecting 10 goslings or Muscovy ducks which are 1 day old and have no GPV maternal antibodies, intramuscular injecting the minimum immune dose of 100 TCID50/0.2ml, and inoculating goose parvovirus virulent YG to the goslings or Muscovy ducks after 7 days; control group: 10 goslings or Muscovy ducks of 1 day old are respectively injected with PBS 0.2ml intramuscularly, and after 7 days, the goslings or Muscovy ducks of a control group are inoculated with the goose parvovirus virulent YG[12]As a result, the gosling or Muscovy duck immunized with GPV-ZYM is 100% protected, without disease and death after being challenged with the goose parvovirus YG.
Protection against short-beak dwarfism syndrome: is selected fromGPV maternal antibody 1 day old 20 cherry valley ducks, 10 intramuscular injections of the minimum immunization dose of 100 TCID50/0.2ml and 10 intramuscular injections of PBS 0.2ml, and after 7 days, inoculation of short-beak dwarf syndrome goose parvovirus virulent FJ-zz[4]0.2ml of stock solution, observing for 30 days, and obtaining 100% protection of GPV-ZYM post-immunization toxin counteracting FJ-zz group without short-beak dwarfism syndrome, wherein the weight is normally increased compared with a normal control group; and in the control group without immunity, after toxicity attack, 5 ducks have short beaks, 8 ducks grow slowly, and the weight average of the body is reduced by 15% compared with the weight average of the body of a normal duck.
(5) And culturing goose embryo fibroblast, and harvesting the virus when the cytopathic effect reaches over 75 percent.
(6) Weak seed stability test: the attenuated strain GPV-ZYM stock solution is injected into 1-day-old susceptible gosling, 1ml of the attenuated strain GPV-ZYM stock solution is injected into the gosling, 5 generations of the infected gosling are transmitted, the virus can be separated from the intestinal tract of the gosling after the gosling is passaged, but the attenuated strain has no pathological damage to the intestinal tract, has no influence on the growth and development of the gosling and has no difference (a diagram is not shown) with a control (no infection is carried out). Referring to FIGS. 1A and B, the left side of FIGS. 1A and 1B is virulent YG infection, and the right side is GPV-ZYM attenuated), so that the GPV-ZYM attenuated strain has no virulence reversion phenomenon, and the stability of the attenuated strain is good.
And (3) carrying out biological information analysis on the artificially attenuated novel goose parvovirus attenuated strain GPV-ZYM strain: extracting DNA of GPV-ZYM and parent virus as a template, and using the structural characteristics of the hairpin, wherein the single primer P1: 5-ACGCGCATGCCGCGCGGTCAG-3(SEQ ID NO: 12) was subjected to PCR amplification.
And (3) recovering the PCR product, linking the PCR product to a T vector, and sequencing by a gene company to obtain the nucleotide sequence of the GPV-ZYM virus. SEQ ID NO: 1 shows that the length of the full-length nucleotide sequence is 4740 bp; SEQ ID NO: 2 is a 5' non-coding region sequence; SEQ ID NO: 3 represents NS gene nucleotide sequence; SEQ ID NO: 4 represents the amino acid sequence of the NS protein; SEQ ID NO: 5 shows the sequence connecting the NS gene and the VP1 gene; SEQ ID NO: 6 is the nucleotide sequence of VP1 protein; SEQ ID NO: 7 shows the amino acid sequence of VP1 protein; SEQ ID NO: 8 shows the 3' non-coding sequence; SEQ ID NO: 9 is the full-length nucleotide sequence of the parental virus; SEQ ID NO: 10 is the amino acid sequence of its NS protein; SEQ ID NO: 11 shows the amino acid sequence of the VP1 protein.
After 120 generations of subculture adaptation, the replication capacity of the GPV-ZYM strain in the duck body is obviously reduced, and no clinical morbidity symptom exists. Replication was stable in GEF and virus titers were significantly increased (TCID50 ═ 10)8Per ml); the pathogenicity research result shows that after the ducklings of 1 day age are infected with GPV-ZYM, the spirit and the feed intake are normal, and the phenomena of death, weight loss and the like do not occur. After dissection, the appearance of each organ of the green duck infected by GPV-ZYM is normal, and pathological histological examination has no pathological change; immunohistochemical detection shows that the liver and enterovirus content of the duckling infected by the GPV-ZYM is obviously less than that of the duckling infected by parent virulent virus (or virulent YG or FJ-zz), which indicates that the replication capacity of the duckling in vivo is obviously reduced and the virus is completely weakened.
Immunohistochemical experiments were as follows:
the GPV-ZYM low virulent strain and the parent virus ZYM are respectively subjected to dissection examination on the third day after gosling infection, liver and small intestine are collected, the liver and small intestine are immediately fixed by 100mL/L neutral formalin solution after collection, a fixed tissue sample is dehydrated and embedded to prepare a tissue section, the tissue section is subjected to immunohistochemical GPV antigen detection, the monoclonal antibody is diluted by 1: 300 times, PBS is washed for three times, and then HRP-labeled anti-mouse secondary antibody is used for detection to observe the distribution condition of GP V in liver and intestinal organs.
Evolutionary analysis with other GenBank strains: sequence analysis by DNASTAR software shows that the GPV-ZYM strain and other GenBank GPV are in the same branch, and the nucleotide homology with GPV strain is 93.5-97.2%.
The homology of VP3 gene of GPV-ZYM strain and GD strain (DQ665790) and SYG61-41 strain (DQ299942) of commercial attenuated vaccine in China is 92.8% and 93.0% respectively, and the homology of amino acid is 95.3% and 95.9% respectively.
The preparation method of the artificially attenuated goose parvovirus attenuated strain GPV-ZYM strain applied to the novel vaccine of gosling plague and short-beak dwarfism syndrome comprises the following steps: GEF is inoculated to GPV-ZYM according to the ratio of 1: 1000, and when the cell lesion is cultured at 37 ℃ until the cell lesion is more than 80%, virus culture solution is collected. Viral content is in TCID50Greater than or equal to 1040.1ml, can be used for preparing vaccine.
The measures are as follows:
1. cell culture solution: DMEM with 3% fetal bovine serum.
2. Preparing goose embryo fibroblasts: taking 15-day-old goose embryos, disinfecting egg shells by iodine or alcohol, breaking the air chambers, and removing the egg shells along the edges of the air chambers. The chorion was penetrated with a small forceps, the neck of the embryo was clamped and the embryo was removed and placed in a dish. The head, limbs and internal organs were removed with an ophthalmic scissors, and then the embryo body was minced to form chyle. Repeatedly washing with DMEM for 3 times, placing into sterilized small green bottle, adding EDTA-pancreatin 3-4 times the volume, digesting in water bath at 37 deg.C for 30min, and shaking for 10 min. After digestion, the supernatant was discarded and washed 3 times with DMEM to remove pancreatin. Then, DMEM containing 10% serum was added, and the mixture was repeatedly blown by a gun about 100 times to blow out the digested tissue mass and disperse the cells. Filtering the liquid with 8 layers of gauze to remove impurities such as un-blown tissue blocks. Cell counting was performed with an erythrocyte counting plate, and the cells were diluted to 106one/mL. The cells were dispensed into cell culture flasks at 37 ℃ with 5% CO2Culturing under the condition.
3. Virus proliferation: the virus is diluted 1: 1000 and inoculated to primary fibroblast, and is cultured by rotating at 37 ℃ at the rotating speed of 10 r/h. And (5) observing cell lesions, and when more than 75% of cells are diseased, placing the bottle body at-20 ℃ for freezing storage.
4. And (3) virus content determination: mixing virus culture solution at a ratio of 1: 101；1∶102；……1∶106；1∶ 107Diluting, adding into a single-layer goose embryo fibroblast 96-well plate prepared in advance, culturing for 7 days with 5 wells per dilution degree, and calculating TCID 50. The potency should be greater than or equal to 105.5/ml。
5. And (4) safety inspection: selecting 10 healthy and susceptible young muscovy ducks or young geese with age of 1 day, injecting 10 times of vaccine into each muscle, observing for 30 days, and ensuring that the inoculated geese have no morbidity and normal appetite and spirit. And the growth and development are good, and all the plants survive.
6. And (3) testing the efficacy: 1) efficacy test for virulent gosling plague: 10 susceptible goslings or young muscovy ducks of one day old were injected intramuscularly with 1 feather vaccine, and the YGs were challenged simultaneously on days 5, 10, 20 and 30 after inoculation, together with 10 control groups. The result shows that the immune-inoculated goose and muscovy duck can resist the attack of strength, and the protection rate reaches 100 percent. While the goose in the control group died 100%, the Muscovy duck died 80%. 2) Efficacy experiments on short-beak dwarfism virus: intramuscular injection of 1 feather vaccine to 10 susceptible cherry valley ducks of one day age on 5 days, 10 days, 20 days and 30 days after inoculation, and simultaneous attack of short-beak dwarfism syndrome virus FJ-zz together with 10 control groups. The result shows that the immunized cherry valley duck can resist the attack of FJ-zz, and the protection rate reaches 100%. And 5 cherry valley ducks in the control group have short beaks, and the body weight of 8 ducks is reduced by 15%.
7. Weak seed stability test: the virus of the young goose can be separated from the intestinal tract after passage of the young goose, but has no pathological damage to the intestinal tract, no influence on the growth and development of the young goose and no difference with a control (the result is similar to a figure 1), so that the GPV-ZYM low virulent strain has no virulent reversion phenomenon and the low virulent strain has good stability.
A sequence table:
SEQ ID NO: 1: nucleotide sequence of GPV-ZYM strain
Figure BDA0002655935620000071
Figure BDA0002655935620000081
Figure BDA0002655935620000091
Figure BDA0002655935620000101
SEQ ID NO: 5' non-coding region sequence of 2 GPV-ZYM strain nucleotide
Figure BDA0002655935620000102
SEQ ID NO: NS gene sequence of 3 GPV-ZYM strain
Figure BDA0002655935620000103
Figure BDA0002655935620000111
SEQ ID NO: NS protein sequence of 4 GPV-ZYM strain
Figure BDA0002655935620000121
SEQ ID NO: 5 sequence linking NS Gene and VP Gene
Figure BDA0002655935620000122
SEQ ID NO: nucleotide sequence of VP1 encoding gene of 6 GPV-ZYM strain
Figure BDA0002655935620000123
Figure BDA0002655935620000131
Figure BDA0002655935620000141
SEQ ID NO: VP1 protein amino acid sequence of 7 GPV-ZYM strain
Figure BDA0002655935620000142
SEQ ID NO: 3' end non-coding sequence of 8 GPV-ZYM strain nucleotide
Figure BDA0002655935620000143
Figure BDA0002655935620000151
SEQ ID NO 9: ZYM virulent strain gene sequence (1-4692)
Figure BDA0002655935620000152
Figure BDA0002655935620000161
Figure BDA0002655935620000171
Figure BDA0002655935620000181
SEQ ID NO: NS protein amino acid sequence of 10 ZYM virulent strain
Figure BDA0002655935620000182
Figure BDA0002655935620000191
SEQ ID NO: VP1 protein amino acid sequence of 11 ZYM virulent strain
Figure BDA0002655935620000192
SEQ ID NO：12 P1∶5-ACGCGCATGCCGCGCGGTCAG-3
REFERENCES
[1]CHEN S，WANG S，CHENG x，et al.Isolation and characterization of a distinct duck-origin goose parvovirus causing an outbreak of duckling short beak and dologywarfism syndrome in China.Arch. Virol，2016,161(9)：2407-2416.
[2]YU K，MA X，SHENG Z，et al.Identification of Goose-Origin Parvovirus asa Cause of Newly Emerging Beak Atrophy and Dwarfism Syndrome in Ducklings.J.Clin.Microbiol，2016.
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[4] Liu rong chang, huang yu, lurong hui, fuqiu ling, wangchun and fuguanghua, cheng longfei, shaohua, cheng hong mei, short-beak dwarfism syndrome hemimuscovy duck etiology detection and histopathology characteristics, chinese veterinary medical report, 201838 (1): 5158.
[5] isolation and identification of novel Duck-derived parvovirus Anhui strain AH-D15 [ J ] Chinese preventive veterinary newspaper, 2016(07) ]: 528-531.
[6]CHEN H，DOU Y，TANG Y，et al.Isolation and Genomic Characterization of a Duck-Origin GPV-Related Parvovirus from Cherry Valley Ducklings in China.Plos One，2015，10(10)：e140284.
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[8]Zádori Z，Stefancsik R，Rauch T，Kisary J.Analysis of the complete nucleotide sequences of goose and muscovy duck parvoviruses indicates common ancestral origin with adeno-associated virus 2. Virology 1995.212：562-573.
[9]Kisary J，Derzsy D，Mészdros J.Attenuation of the goose parvovirus strain B laboratory and field trials of the attenuated mutant for vaccination agamst Derzsy′s disease.Avian Pathol，1978， 7(3)：397-406.
[10] Qina, liumin, feifei, etc., the culture and sequence analysis of goose parvovirus goose embryo fibroblast adapted virus strain, chinese prevention veterinary academy 2014, 36(11),
[11] the research on the pathogenicity change of the YG strain of goose parvovirus weakened by cell passage, 2016,47 (1): 135-140.
[12] Identification and distribution of the YG strain of the shore zhouwulin, li chen, liumin, zhangyun goose parvovirus in organs. 912917.
Figure IDA0002928457090000011
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Figure IDA0002928457090000051
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Figure IDA0002928457090000071
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Figure IDA0002928457090000091
Figure IDA0002928457090000101
Figure IDA0002928457090000111
Figure IDA0002928457090000121
Figure IDA0002928457090000131
Figure IDA0002928457090000141
Figure IDA0002928457090000151
Figure IDA0002928457090000161
Figure IDA0002928457090000171
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Figure IDA0002928457090000201
Figure IDA0002928457090000211
Figure IDA0002928457090000221
Figure IDA0002928457090000231

Claims (3)

1. The goose parvovirus attenuated strain GPV-ZYM has the preservation number of CGMCC No. 15700.
2. The goose parvovirus GPV-ZYM whole genome sequence is shown as SEQ ID NO: 1 is shown.
3. The use of the attenuated strain GPV-ZYM of goose parvovirus as claimed in claim 1 in preparing vaccine for preventing duck or goose from infecting duck beak syndrome and/or gosling plague.
CN202010892082.0A 2020-08-28 2020-08-28 Duck source goose parvovirus artificial attenuated strain and application thereof Pending CN112501132A (en)

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CN101880651A (en) * 2010-07-01 2010-11-10 福建省农业科学院畜牧兽医研究所 Preparation method of Muscovy duck parvo novel vaccines
CN103525771A (en) * 2013-10-18 2014-01-22 江苏省农业科学院 Goose parvovirus and applications thereof

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