CN112921006A - Goose astrovirus and application thereof - Google Patents

Abstract

The invention relates to a goose astrovirus N-GoAstV-190123 strain with strong toxicity and good immunity, which can generate higher neutralizing antibody after being prepared into an inactivated vaccine for immunity, can achieve the complete protection of goose groups, and has important economic value for the goose industry.

Description

Goose astrovirus and application thereof

The technical field is as follows:

the invention belongs to the technical field of biology, mainly relates to production of vaccines, and particularly relates to a goose astrovirus and application thereof.

Background art:

the astrovirus is a non-enveloped positive-strand RNA virus, can infect various animals such as human, dog, cat, pig, cow, sheep, chicken, duck and the like, is one of main pathogenic causes for animal diarrhea, and causes different diseases such as acute gastroenteritis, duck lethal hepatitis, chicken nephritis, goose visceral gout, mammalian encephalitis and the like. At present, astrovirus infection is widely prevalent worldwide and has become one of the important infectious diseases threatening human health and economic animal breeding. In recent years, a fatal infectious disease with gout as a main symptom is outbreaked in goose farms in Shandong, Jiangsu, Anhui and other areas. The disease mainly occurs to goslings of 5-20 days old, the goslings are infected with mental depression, anorexia, emaciation, quadriplegia and the like, serious people die, the death rate reaches 30 percent, and severe white urate precipitates appear in the liver, heart and kidney, wing-leg joints, coracoid parts and the like of the diseased goose through autopsy, so that serious economic loss is caused to the goose industry in China. Related cases have been reported by digital researchers, and the etiological agent causing the epidemic is goose astrovirus through research. Particularly, the gosling gout disease is rapidly spread throughout the country since 2017, huge economic loss is brought to the goose farming industry in China, and the development of the goose farming industry in China is seriously threatened.

Goose astrovirus belongs to RNA virus, and is characterized by severe lethal hepatitis, cholecystitis, nephritis and other diseases caused by diarrhea, arthritis and growth disturbance after goose astrovirus infection. The incidence of the goslings of 2-25 days old is high, the goslings within 15 days old are most susceptible, and the mortality rate is generally 15% -36% and can reach 50% at most. The avian astrovirus can have a plurality of hosts and can be spread horizontally, vertically and across species; meanwhile, the gene recombination of the avian astrovirus, the capability of adapting to a new host and the continuous discovery of a novel avian astrovirus virulent strain make the purification of the poultry group infected with the avian astrovirus extremely difficult. In addition, the strong resistance of the avian astrovirus to physical and chemical factors makes it very difficult to disinfect the environment polluted by the avian astrovirus. Currently, no specific drugs or vaccines are available on the market for avian astrovirus. The lack of suitable proliferation cell lines in vitro results in relatively few attenuated or inactivated vaccines capable of preventing and controlling the infection of the avian astrovirus, and even less commercial goose astrovirus vaccine products.

Research and development of various vaccines are reported in the prior art, for example, research and development of Rongxueliu and the like and verification of the immune effect of the goose astrovirus recombinant Capsid protein subunit vaccine, and research results show that the goose astrovirus recombinant Capsid protein subunit vaccine has a good immune effect and can effectively prevent infection of the goose astrovirus; patent document CN110699331A reports a preparation method and application of goose astrovirus attenuated virus, which adopts a method combining high-temperature treatment and low-temperature incubation to attenuate virus, and is used for preparing vaccine to effectively prevent goose gout caused by goose astrovirus, however, the research on goose astrovirus vaccine is still less, and based on the current continuous spread of goose astrovirus in farms, the economic loss caused by the continuous spread of goose astrovirus is more and more large, so there is an urgent need to develop safe and effective vaccine and yolk antibody and drug for preventing and treating goose astrovirus, so as to effectively control the epidemic situation of goose astrovirus and avoid the economic loss of aquaculture.

The invention content is as follows:

in order to solve the technical problems that the treatment means of goose astrovirus infection is limited and no effective vaccine exists in the prior art, the invention aims to provide a goose astrovirus which has the characteristics of good immunogenicity, high immune protection effect and easy culture and proliferation and is a proper strain for vaccine production.

In order to solve the technical problems, the invention adopts the following technical means:

goose astrovirus N-GoAstV-190123 strain, which is characterized in that the goose astrovirus is preserved in culture for patent procedures in China center for type culture Collection CCTCC, and the preservation numbers are as follows: CCTCC No: v202068, classified name is goose astrovirusGoose astrovirus(ii) a The preservation date is 2020, 10 months and 14 days; the preservation address is as follows: wuhan university Collection, Wuhan City, Hubei province.

The invention also requests to protect the application of the goose astrovirus N-GoAstV-190123 strain in preparing medicines for preventing and treating diseases caused by goose astrovirus infection.

Further, the medicament is a vaccine.

Further, the vaccine is an inactivated vaccine.

Furthermore, the inactivated vaccine is prepared from an adjuvant and inactivated goose astrovirus N-GoAstV-190123.

Further, the adjuvant is Montanide ISA 206 adjuvant.

Further, the inactivated vaccine is used for immunization by subcutaneous injection.

Based on the technical scheme, the invention has the following advantages and beneficial effects:

firstly, the goose astrovirus is obtained by separating from a goose field in the south-Yang region, and the goose astrovirus N-GoAstV-190123 with stronger purity and toxicity is obtained by a large number of subcultures, has higher lethality rate to goslings, has stronger toxicity after subculture and is transmitted to the 20 th generation, and the ELD of the culture₅₀Is 5 x 10^7.05Has strong multiplication capacity and is suitable for being used as a vaccine production strain.

Secondly, the goose astrovirus N-GoAstV-190123 is inactivated and then mixed with an adjuvant to prepare an inactivated vaccine, the inactivated vaccine can stimulate inoculated animals to generate neutralizing antibodies with higher concentration to prevent virus infection, and meanwhile, the protection rate of the inactivated vaccine to the goose astrovirus N-GoAstV-190123 and other strains after virus challenge reaches 100%, so that the inactivated vaccine prevents and controls the spread of the goose astrovirus in goose groups and has important significance for epidemic situation control.

In conclusion, the goose astrovirus N-GoAstV-190123 strain with strong toxicity and good immunity is obtained by separation, and the strain can generate higher neutralizing antibody after being prepared into an inactivated vaccine for immunity, can achieve complete protection on goose groups, and has important economic value for goose industry.

Description of the drawings:

FIG. 1. pathological material detection results: wherein 1, goose astrovirus (GoAstV); 2, Goose Parvovirus (GPV); goose Hemorrhagic Polyoma Virus (GHPV); 4, Duck Reovirus (DRV); tembusu virus (Tembusu).

FIG. 2 shows the detection results after the goose embryo is passed for 5 times: wherein 1, passage 5 subcultures; 2, passage 4 subcultures; 3, passage 3 subculture; 4, passage 2 subculture; passage 1 subculture 5.

FIG. 3 is a schematic representation of the results of animal regression experiments: a, a large amount of urate deposits exist in the kidney, and the ureter is full of urate; b, the liver and the heart are coated with urate samples and seriously exude; necrosis of renal tubular epithelial cells, cortical partial hemorrhage; d, glomerular cystectasia, degenerative necrosis of cortical tubular epithelial cells.

FIG. 4 results of measurement of neutralizing antibody titer.

The specific implementation mode is as follows:

example 1: isolation and identification of goose astrovirus

1. Collection of pathological material

The disease sample is collected from a farm for the disease attack in the south-yang area of Henan province, and the variety of the disease attack goose comprises the following components: thai goose and Langdian goose, the young goose died of illness is manifested by joint swelling, white free urate deposition, white urate deposition in heart, liver and kidney, etc. can be seen by caesarean section, and urate deposition is also found in some cases in subcutaneous part and gallbladder.

According to a method in a Zhangqing paper (Zhangqing. isolation and identification of new nephropathogenic goose astrovirus and attenuated strain breeding [ D ]. Chinese agriculture university, 2019.), virus genes such as Goose Parvovirus (GPV), Goose Hemorrhagic Polyoma Virus (GHPV), Duck Reovirus (DRV), Tembusu virus (Tembusu) and goose astrovirus (GoAstV) which possibly exist in a diseased gosling sample are detected, wherein GoAstV-SF is 5'-ATTCTTGGCTCGGTTGTC-3'; the GoAstV-SR was 5'-CCTGTGTTGCTCCTTCTC-3', and the amplified fragment size was 489 bp. The detection results are shown in fig. 1, wherein the detection results of Goose Parvovirus (GPV), Goose Hemorrhagic Polyomavirus (GHPV), Duck Reovirus (DRV) and Tembusu virus (Tembusu) are negative, and the detection result of goose astrovirus (GoAstV) is positive.

Taking a proper amount of livers and kidneys of the dead gosling which is detected to be positive by the goose astrovirus (GoAstV) aseptically, adding 5 times volume of physiological saline, shearing, fully homogenizing, repeatedly freezing and thawing for 5 times, centrifuging at 12000rpm for 10min, taking supernatant, filtering and sterilizing by a 0.22 mu m filter to obtain tissue homogenate supernatant, and carrying out virus separation operation.

2. Isolation of viruses

Inoculating 5 SPF goose embryos (0.5 mL/egg) with age of 8 days into the supernatant of the tissue homogenate by adopting a chorioallantoic membrane way, placing the supernatant into an incubator at 37 ℃ for continuous incubation, checking the inoculated goose embryos every day, observing the death condition of the goose embryos, and discarding the dead goose embryos within 24 hours. And if no death occurs, freezing and killing the goose embryo in a refrigerator at the temperature of-20 ℃ on the 7 th day after inoculation, opening the goose embryo by aseptic technique, grinding the liver, spleen, kidney and other organs of the goose embryo and the allantoic membrane of the goose embryo, taking the ground supernatant, inoculating the next generation of goose embryo, and continuously carrying out passage for 5 generations in the goose embryo according to the mode.

3. Identification and preservation of viruses

3.1 RT-PCT detection of goose-star Virus (GoAstV)

The detection result of the culture of each generation is shown in figure 2, the culture of each generation can amplify a 496bp specific band, which indicates that the virus can effectively proliferate in SPF goose embryos and is finally separated.

And finally obtaining two viruses through virus separation, wherein the two viruses are respectively named as goose astrovirus N-GoAstV-190123 and goose astrovirus N-GoAstV-190178, and RT-PCR product sequencing shows that the sequences of the two viruses have high homology with the reported goose astrovirus and both belong to the goose astrovirus, and the sequence homology of the two viruses is as high as 97.5%.

3.2 pathogenic median lethal dose (ELD)₅₀) Measurement of (2)

Continuously passaging the separated virus by SPF goose embryo until the 20 th generation, and performing 10-fold gradient dilution on the virus solution of the 1 st, 5 th, 10 th, 15 th and 20 th generation with sterilized normal saline for 8 groups of dilutions (10 groups of dilutions)^-1～10^-8) Inoculating 5 SPF goose embryos of 8 days old to each dilution according to the dose of 0.2 mL/chorioallantoic membrane via the chorioallantoic membrane, incubating in an incubator at 37 ℃, irradiating the embryos 1 time each day in the morning, at night, discarding dead goose embryos within 24h, continuously observing for 5 days, recording the death condition, and calculating half-embryo lethal dose ELD of the virus according to the death number and a Reed-Muench method₅₀. The specific results are shown in table 1 below:

TABLE 1 passage 1-5 Virus embryos half lethal ELD₅₀Measurement of (2)

Number of generations

Generation

1

5 th generation

Generation

10

Generation 15

Generation 20

N-GoAstV-190123

ELD50

5×104.00

5×105.25

5×106.30

5×107.00

5×107.05

N-GoAstV-190178

ELD50

5×103.50

5×104.55

5×105.45

5×105.50

5×105.50

Based on the results shown in Table 1, ELD of the 1 st passage culture of the goose astrovirus N-GoAstV-190123 isolated by the present invention was found₅₀Is 5 x 10^4.00The virulence gradually increased as the passage progressed, and when passed to passage 15, the ELD of the culture₅₀Is 5 x 10^7.00ELD of cultures when passed to passage 20₅₀Is 5 x 10^7.05. Shows that the virulence is basically stabilized at ELD by passage 15 by the same passage method₅₀Is 5 x 10^7.00Left and right. When the goose astrovirus N-GoAstV-190178 is passaged to 10 generations, the virulence is basically stabilized at ELD₅₀Is 5 x 10^5.45On the left and right, the tests show that the goose astrovirus N-GoAstV-190123 obtained by separation has stronger toxicity compared with the goose astrovirus N-GoAstV-190178.

3.2 animal regression test

The 50-feather 5-day-old goslings are randomly divided into two groups, and each group has 20 feathers. Wherein 10 feather of control group is injected with 0.2mL physiological saline per subcutaneous, 20 feather of regression test group is injected with 0.2mL virus diluent per subcutaneous₅₀Is 5 x 10^5.0/mL). The test gosling was observed under the same feeding conditions for 15 days, and the clinical symptoms and necropsy changes after infection were recorded.

After two days of challenge, the gosling starts to get ill, the death peak of the sick gosling is reached about 4 days after the virus of the challenge goose astrovirus N-GoAstV-190123 is attacked, the death rate of the gosling in a challenge test group can reach 65% when the gosling is about 20 days old, the death peak of the goose in a challenge test group is reached about 5 days after the virus of the challenge goose astrovirus N-GoAstV-190178 is attacked, and the death rate of the gosling in the challenge test group can reach 35% when the gosling is about 20 days old. The gosling in the challenge test group is clinically manifested as fatigue, anorexia, lassitude and limpid and white defecation. After the dead goslings are necropsied, a large amount of white urate deposits on the liver and the heart surface of the internal organs after two viruses are detoxified, and obvious grey-white urate deposits are also visible on the kidney surface and in the ureter. The fixed kidney tissue and HE staining result shows that the renal cortex is subjected to local hemorrhage on the serosa-proximal surface, the cortical renal tubular epithelial cells are denatured, and a large amount of necrosis is generated; the result of the specific test is shown in fig. 3, wherein the number of mesangial cells is reduced, the number of cortical tubular epithelial cells is denatured, and multiple necrosis is generated. The results show that the GoAstV separated by the invention shows typical symptoms of typical goose astrovirus infection after being attacked by virus, the attack is fast, the attack reaches the death peak only 4 days as soon as the GoAstV is attacked by virus, and the death rate reaches 65% after 15 days of attacking by virus. Wherein the goose astrovirus N-GoAstV-190123 has higher lethality rate than the goose astrovirus N-GoAstV-190178.

3.4 preservation of viruses

Through the tests, the invention obtains a highly pathogenic goose astrovirus N-GoAstV-190123, which is preserved in China Center for Type Culture Collection (CCTCC) for culture of patent programs, wherein the preservation numbers are as follows: CCTCC No: v202068, categorically designated as ansan-astrovirus (Goose astrovirus) (ii) a The preservation date is 2020, 10 months and 14 days; the preservation address is as follows: wuhan university Collection, Wuhan City, Hubei province.

Example 2: preparation of goose astrovirus inactivated vaccine

1. Preparation of inactivated vaccine

Step one, taking the 15 th-20 th generation goose astrovirus N-GoAstV-190123 virus seed to dilute the virus seed to ELD by using a diluent₅₀Is 5 x 10^5.00Inoculating 8-10 days old SPF goose embryos in 0.2mL per embryo through a chorioallantoic membrane way, and continuously incubating at 37 ℃ after inoculation without turning eggs;

step two, after SPF goose embryos are inoculated, eggs are laid for 3 times per day, the goose embryos which die before 24 hours are discarded, the goose embryos which die after 24 hours are taken out at any time till 72 hours, all live embryos are taken out, an air chamber is upright, and the eggs are cooled for 12 hours at 4 ℃;

taking out the goose embryos cooled for 12 hours, absorbing goose embryo liquid after surface disinfection, mixing the goose embryo liquid of every 20 goose embryos into a group, inspecting the goose embryos one by one while harvesting the embryo liquid, and discarding the goose embryos which are unclear in embryo liquid, rotten in fetus and suspected of being polluted;

step four, putting the harvested embryo liquid into a sterilization bottle, centrifuging the embryo liquid by a centrifuge, and mixing the embryo liquid and the sterilized embryo liquid in a storage tankFiltering with sterilized secondary prefilter column under aseptic condition, removing residues and other impurities from virus solution, sampling, determining toxicity value, and diluting with physiological saline to adjust ELD of embryo solution₅₀Is 5 x 10^5.00；

Accurately measuring formaldehyde, preparing 10% formaldehyde solution with water for injection, uniformly mixing, adding the formaldehyde solution into the goose embryo solution, stirring along with adding, fully mixing until the final concentration of the formaldehyde is 0.2%, pouring the goose embryo solution into a sterilized inactivation tank, inactivating at 37 ℃ for 24 hours while stirring, and sampling for sterile inspection and inactivation inspection after inactivating for 24 hours;

and seventhly, heating the inactivated virus liquid to 30 ℃, preserving heat, adding the Montanide ISA 206 adjuvant with the same volume into a double-layer jacketed container, keeping the temperature at 30 ℃, stirring at a low speed (200 r/min), gradually adding the inactivated virus liquid, increasing the stirring speed to 300r/min after the inactivated virus liquid is completely added, continuously stirring for 15 minutes at 30 ℃, cooling to below 10 ℃ under the condition of continuous high-speed stirring at 300r/min, stopping stirring, standing the emulsion for 24 hours at below 10 ℃, subpackaging, covering and sealing, and storing at about 4 ℃ for later use to obtain the goose astrovirus N-GoAstV-190123 inactivated vaccine.

Based on the same method, the goose astrovirus N-GoAstV-190178 inactivated vaccine is prepared.

2. Vaccine testing

2.1 physical Property test

Appearance: is a homogeneous emulsion.

The preparation formulation is as follows: the vaccine is water-in-oil-in-water type, a small amount of vaccine is absorbed by a liquid transfer gun and dropped on the surface of clean cold water, and the vaccine is dispersed in a cloud state.

Stability: the vaccine is sucked by 10ml and added into a centrifuge tube, and is centrifuged for 15 minutes at 3000r/min, and the water phase separated out from the tube bottom is not more than 0.5ml, which shows that the vaccine prepared by the invention has good stability.

And (4) sterile inspection: the test is carried out according to the appendix of the Chinese veterinary pharmacopoeia, and no bacteria grow in the results.

2.2 Security verification

2.2.1 goose embryo inoculation inspection:

inoculating 10 SPF goose embryos (0.5 mL inactivated vaccine/egg) of 5 days old by adopting a chorioallantoic membrane way, placing the inoculated goose embryos in an incubator at 37 ℃ for continuous incubation, checking the inoculated goose embryos every day, and observing the death condition of the goose embryos for 2 times per day, wherein the death condition of the goose embryos is continuously observed for 7 days, and the goose embryos do not die.

2.2.2 gosling inoculation test:

the method comprises the steps of selecting 35 feathers of a 10-day-old healthy gosling with GoAstV detection as negative, wherein 5 feathers are used as a control group, randomly dividing the remaining 30 feathers into a high dose group, a medium dose group and a low dose group, respectively carrying out vaccination according to the vaccination amounts of 2mL, 1mL and 0.5mL, feeding under the same condition, continuously observing for 28 days, wherein the gosling of a test group and the gosling of the control group are fed normally, the mental state is good, and the gosling does not die or suffer from epidemic diseases.

2.3 determination of the protective efficacy of the vaccine

2.3.1 neutralizing antibody Titers assay

Taking 15 feathers of healthy goslings with the age of 10-15 days and negative GoAstV nucleic acid detection and serum detection, randomly dividing the feathers into a control group and a test group, and numbering each feather separately, wherein the test group is injected with 1 mL/feather of vaccine subcutaneously, the control group is injected with an equal amount of physiological saline subcutaneously, the second inoculation is carried out after 14 days of inoculation, the boosting immunity is carried out, blood is collected after 3, 7, 14, 28, 60 and 90 days of first inoculation respectively, serum is separated, the neutralizing antibody titer of the goose astrovirus in the serum is measured, and the average neutralizing antibody titer of each group is calculated, wherein when the neutralizing antibody titer in the serum is less than or equal to 1:2, the average neutralizing antibody titer in the serum is recorded as 1log 2. The specific result is shown in the following figure 4, compared with the goose astrovirus N-GoAstV-190123 inactivated vaccine, the goose astrovirus N-GoAstV-190178 inactivated vaccine can generate a higher titer neutralizing antibody level, the neutralizing antibody concentration can reach more than 8log2 after 14 days and 28 days of first inoculation, the higher neutralizing antibody level can be still maintained after 60 days and 90 days of inoculation, and the goose astrovirus N-GoAstV-190123 inactivated vaccine has a certain preventive effect on viruses.

2.3.2 determination of protection against challenge

Collecting 900 feathers of 10-15 days old healthy gosling, and dividing into 3 groups, wherein group 1 adopts N-GoAstV-190123 inactivated vaccine for inoculationThe seeds (1 mL/feather of vaccine injected subcutaneously), group 2 was inoculated with N-GoAstV-190178 inactivated vaccine (1 mL/feather of vaccine injected subcutaneously), group 3 controls were injected subcutaneously with an equal amount of physiological saline, two vaccinations were performed at an interval of 14 days, each group was further divided into 3 groups at 28 days after the first immunization, and N-GoAstV-190123 virus dilutions (1 mL/feather of vaccine injected subcutaneously, EID) were used respectively₅₀Is 5 x 10^5.0mL), N-GoAstV-190178 virus dilution (1 mL/feather by subcutaneous injection, EID)₅₀Is 5 x 10^5.0mL), a control group injected with physiological saline is set, the survival rate after 14 days of challenge is calculated after 14 days of challenge, and the specific results are shown in table 2 below.

TABLE 2 challenge protection test results (survival,%)

	N-GoAstV-190123	N-GoAstV-190178	Physiological saline
				N-GoAstV-190123 inactivated vaccine	100%	100%	100%
N-GoAstV-190178 inactivated vaccine	85%	98%	100%
				Control group	48%	70%	100%

Based on the results in table 2, the protection rate of the gosling injected with the N-GoAstV-190123 inactivated vaccine is 100% after challenge, while the protection rate of the gosling injected with the N-GoAstV-190178 inactivated vaccine reaches 98% after challenge with the N-GoAstV-190178 virus diluent, and the survival rate of the gosling is only 85% after challenge with the N-GoAstV-190123 virus diluent with strong toxicity. The results show that the inactivated vaccine prepared from the N-GoAstV-190123 obtained by separation has better protection and better cross protection, and the strain is relatively suitable vaccine strain. Therefore, it is subjected to the preservation of cultures for patent procedures in the China Center for Type Culture Collection (CCTCC) under the preservation numbers: CCTCC No: v202068, categorically designated as ansan-astrovirus (Goose astrovirus) (ii) a The preservation date is 2020, 10 months and 14 days; the preservation address is as follows: wuhan university Collection, Wuhan City, Hubei province.

The above embodiments are merely detailed illustrations of the technical solutions of the present invention, and the conventional modifications made on the basis of the above embodiments are within the scope of the present invention.

Claims (7)

1. An anseriavirus strain N-GoAstV-190123, characterized in that the anseriavirus is preserved in China Center for Type Culture Collection (CCTCC) for patent programs, and the preservation numbers are as follows: CCTCC No: v202068, classified name is goose astrovirusGoose astrovirus(ii) a The preservation date is 2020, 10 months and 14 days; the preservation address is as follows: wuhan university Collection, Wuhan City, Hubei province.

2. The use of the goose astrovirus N-GoAstV-190123 strain of claim 1 in the preparation of medicaments for preventing and treating diseases caused by goose astrovirus infection.

3. The use according to claim 2, wherein the medicament is a vaccine.

4. The use according to claim 3, wherein the vaccine is an inactivated vaccine.

5. The use according to claim 4, wherein the inactivated vaccine is prepared from an adjuvant and inactivated goose astrovirus N-GoAstV-190123.

6. The use of claim 5, wherein the adjuvant is Montanide ISA 206 adjuvant.

7. The use according to claim 4, wherein the inactivated vaccine is administered by subcutaneous injection.

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