CN115287270B - Subtype B avian metapneumovirus (APV) subculture attenuated strain and application thereof - Google Patents
Subtype B avian metapneumovirus (APV) subculture attenuated strain and application thereof Download PDFInfo
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Abstract
The invention discloses a subtype B avian metapneumovirus (APV) subculture attenuated strain and application thereof. The attenuated strain LN16-A is obtained by continuously passaging in Vero cells and is named as LN16-A strain, and the accession number of the attenuated strain LN16-A is as follows: CGMCC NO.45261. The passage weakening strain LN16-A is used for immunizing SPF chickens of 3 weeks old and systematic immune protection evaluation is carried out, and the result shows that after LN16-A is used for immunizing organisms, high-level aMPV specific antibodies and neutralizing antibodies can be induced and generated, 100% immune protection can be provided for virulent attack, the immune protection effect is good, and pathological damage caused by virulent attack can be effectively prevented. The invention provides a new technical means for preventing and controlling the B subtype avian metapneumovirus disease and has important significance for the healthy development of poultry industry.
Description
Technical Field
The invention relates to a passage weakening strain and application thereof, in particular to a B subtype avian metapneumovirus passage weakening strain and application thereof, belonging to the technical field of biological medicines.
Background
Avian metapneumovirus (aMPV) is a member of the family Paramyxoviridae, pneumovirinae, metapneumovirus, a membrane-enveloped, non-segmented, single-negative-strand RNA virus with a genome spirally contained in a nucleoprotein shell. This virus, previously known as avian pneumovirus, is the causative agent of turkey rhinotracheitis. After infection of chicken, head swelling syndrome (SHS) and rapid decrease of egg production of chicken are caused, and nasal discharge, cough and opium are the main clinical symptoms. The virus is easy to be mixed with other pathogenic bacteria and viruses to infect, thereby aggravating the clinical symptoms of sick chickens, prolonging the disease process and causing more serious economic loss.
The aMPV was first isolated in south Africa in 1978 and was subsequently widely prevalent worldwide. The genome of aMPV has a total length of about 13.3 kb, contains 8 genes, and has the sequence of 3'-N-P-M-F-M2-SH-G-L-5'. Based on antigenic differences of the G genes, aMPV can be divided into four subtypes A, B, C, D. Subtype A viruses were first isolated in south Africa and the United kingdom, while subtype B viruses were first isolated in European continental countries including Hungary, germany and Italy. In recent years, subtype B viruses have developed into the main epidemic strain in china. Subtype C aMPV was first discovered in 1996 in turkeys in the United states and subsequently disseminated to Asia, and is currently reported separately in both Muscovy ducks in France and yellow-feathered broilers in China. The subtype C aMPV has wider host range, can infect mice besides birds, and is a potential zoonosis pathogen. Subtype D aMPV is currently reported only in france. In recent years, the popularity of aMPV in China is becoming more and more severe, and serious economic losses are caused. In 1998, shen Ruizhong and the like are separated and identified to obtain an aMPV strain in a certain broiler farm in Heilongjiang for the first time; in 2012, the YuanLing and the like investigate chicken flocks in Heilongjiang, jilin, changchun and Hebei, and the investigation result shows that the seropositivity of a plurality of chicken farms is as high as 100%; in addition, in 2020, shouztfu and the like find that the main prevalence of chicken flocks in China is B subtype aMPV, and a B, C subtype strain mixed infection phenomenon exists, so that the monitoring of the prevalence condition of aMPV in China needs to be further enhanced.
Although the disease is widespread in China, the research on the vaccine for the disease is less compared with other poultry diseases. At present, the combined use of the attenuated live vaccine and the inactivated vaccine is widely considered to be good for preventing the disease. The inventor of the invention develops an inactivated vaccine (LN 16-I strain) aiming at the disease in early stage, and the inactivated vaccine is proved to have good safety and immune protection effect. However, no related reports about attenuated vaccines exist in China. In the present invention, we attenuated the LN16 strain by serial passage in Vero cells. The LN16-A virus strain is finally selected as a candidate vaccine strain, the SPF chicken of 3 weeks old is immunized by the passage attenuated strain, and systematic immune protection evaluation is carried out, so that a new technical means is provided for prevention and control of the disease, and the method has important significance for healthy development of poultry industry.
Disclosure of Invention
The invention aims to provide a subtype B avian metapneumovirus (APV) subcultured attenuated strain and application thereof.
In order to achieve the purpose, the invention adopts the following technical means:
the invention relates to a subtype B avian metapneumovirus subculture attenuated strain which is named as LN16-A strain and classified named as subtype B avian metapneumovirus, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and is addressed to No. 3 of No. 1 Xilu Beijing of the sunward district in Beijing, and the microorganism research institute of the Chinese academy of sciences, wherein the accession numbers are as follows: CGMCC NO.45261, the preservation time is as follows: 8 and 29 months in 2022.
Furthermore, the invention also provides application of the subtype B avian metapneumovirus passage attenuated strain in preparation of a medicament for preventing diseases caused by infection of the subtype B avian metapneumovirus.
Preferably, the disease is turkey rhinotracheitis or chicken swollen head syndrome.
Wherein, preferably, the medicament is a B subtype avian metapneumovirus attenuated vaccine.
Furthermore, the invention also provides a B subtype avian metapneumovirus attenuated vaccine, and the effective component of the B subtype avian metapneumovirus attenuated vaccine comprises the B subtype avian metapneumovirus passage attenuated strain.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, aMPV/B LN16 virulent virus (LN 16-V) is attenuated through continuous passage in Vero cells, LN16-F35 attenuated strain is obtained as a candidate vaccine strain, named LN16-A, and the 3-week SPF chicken is immunized by the LN16-A attenuated strain and subjected to systemic immune protection evaluation, and the result shows that aMPV specific antibody can be induced and generated after the organism is immunized by the LN16-A strain. Serum is collected within 3 weeks after immunization, and the titer of neutralizing antibodies is determined, and the result shows that after the attenuated strain LN16-A is used for immunizing organisms, the organisms can be induced to generate the neutralizing antibodies with higher level. In order to determine the immune protection effect of the passage attenuated strain LN16-A on SPF (specific pathogen free) chickens, statistics is carried out on the morbidity of various groups of chickens after challenge, and the result shows that the passage attenuated strain LN16-A can provide 100% immune protection on strong virus challenge. The pathological results after challenge show that the chickens in the immune group have no obvious histopathological changes, which indicates that the passage weakening strain LN16-A can effectively prevent pathological damage caused by strong virus attack. The invention provides a basis for the prevention and control of the B subtype avian metapneumovirus disease and has important significance for the healthy development of poultry industry.
Drawings
FIG. 1 shows the pathogenicity of different progeny strains for SPF chickens;
FIG. 2 shows specific ELISA antibody levels in serum after LN16-A immunization;
FIG. 3 shows the level of neutralizing antibodies in serum after LN16-A immunization;
FIG. 4 is a statistic of disease incidence for each group after challenge;
FIG. 5 shows the pathological results after challenge.
Wherein, A is the natural layer of the turbinate bone of the control group of offensive toxin; b, attacking the control group of chicken turbinate epithelial cells; c, attacking the lower layer of the tracheal mucosa of the control group of chickens; d, immunizing chicken turbinate bone lamina propria; e, immunizing chicken turbinate epithelial cells; f, immunization chicken trachea submucosa.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. The examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1 preparation of a sub-type B avian metapneumovirus passaged attenuated strain and an immunopotency test thereof
1 materials and methods
1.1 Cell, virus and experimental animal
Vero cells are preserved in the laboratory; LN16-V is separated, identified and stored in the laboratory; SPF chickens at 3 weeks of age were purchased from the laboratory animal center of Harbin veterinary institute, national academy of agricultural sciences.
1.2 Primary reagents
The aMPV-specific ELISA antibody detection kit is purchased from IDEXX, USA; reverse transcriptase BioRT Master HiSensi cDNA First-Strand Synthesis Kit was purchased from Bori, hangzhou, inc.; fluorescent quantitative RT-PCR premixed enzyme Premix Premix Ex Taq was purchased from Dalibao Biotech.
1.3 Subculturing of LN16 Strain
Diluting parent strain LN16-V according to a certain titer, inoculating Vero cells, and adding 5% CO 2 Culturing in a 37 ℃ incubator, observing cytopathic effect, continuously passaging to more than 35 generations with 70% cytopathic effect time as virus collecting time, measuring the toxicity of 20 th generation (LN 16-F20 strain), 25 th generation (LN 16-F25 strain), 30 th generation (LN 16-F30 strain) and 35 th generation (LN 16-F35 strain) obtained by passaging Vero cells, and calculating the TCID of the virus according to the Reed-Muench method 50 。
1.4 Pathogenicity of different progeny strains
Taking 50 SPF chickens of 3 weeks old, randomly dividing into 5 groups (A-E groups), each group containing 10 chickens, and dividing the groups A-D into 20000 TCIDs 50 The single dose was inoculated by nasal drip with LN16-F20, LN16-F25, LN16-F30, andLN16-F35 passage 4 LN16 strains, group E immunized with equal amounts of PBS as a control. Clinical symptoms within 1-7 days after immunization were observed and the incidence of disease was counted for each group.
1.5 Immunopotentiality test
1.5.1 aMPV-specific antibody level detection
Collecting 26 SPF chickens of 3 weeks old, randomly dividing into 2 groups of 13 chickens, and immunizing and passaging attenuated strain LN16-A (500 TCID) by nasal drip route 50 One group), two groups of immune equivalent PBS as a control, collecting blood samples of 7d, 14d and 21d after each group of immune, separating serum after 1h at 37 ℃, detecting the antibody level of the serum according to the ELISA antibody detection kit instruction, and judging as positive antibody when the antibody titer is larger than 396.
1.5.2 Neutralizing antibody titer determination
Vero cells were cultured at 4X 10 5 In 96-well plates,/mL. Placing the collected 7d, 14d and 21d sera after immunization in 56 deg.C water bath for 30min to inactivate complement, filtering the sera with 0.22 μm filter, diluting by 2 times, and mixing the sera diluted by different times with LN16-V (2000 TCID) 50 /mL) was mixed in equal volume and incubated in an incubator at 37 ℃ for 1h, during which time the mixture was inverted one time. And inoculating 100 mu L of the incubated mixed solution into a full Vero cell 96-well plate, repeating 6 times for each sample, observing the cell state every day, counting the pathological change condition of each well after 7 days, and calculating the titer of the neutralizing antibody according to a Reed-Muench method.
1.5.3 Challenge protection test
Taking 26 SPF chickens of 3 weeks old, randomly dividing into 2 groups of 13, and immunizing and passaging attenuated strain LN16-A (500 TCID) by nasal drip route 50 /only), two groups immunized with equal amounts of PBS as control. After 21d, 5000TCID per chicken 50 The dosage of the composition is subjected to virus attack (LN 16-V nose drop), the clinical symptoms of all groups of chickens within 1-7d after virus attack are observed and recorded, and the virus attack protection rate is determined.
1.5.4 Histopathological observation
Collecting 11d of tissue of turbinate bone and trachea after challenge, fixing in 10% formalin solution, performing conventional treatment, and performing paraffin embedding section. After staining with hematoxylin and eosin, histopathological changes were observed under an optical microscope and the damage status of each group was recorded.
2. Results
2.1 The toxicity of different sub-strains
The LN16-V strain was serially passaged through Vero cells, and the cells began to have lesions after 72h of inoculation, which are shown as cells aggregating, shrinking and falling off, while the blank group of cells had no lesions. The toxin prices of 4 secondary LN16 strains of LN16-F20, LN16-F25, LN16-F30 and LN16-F35 are determined to be 10 in sequence 5.53 TCID 50 /mL，10 5.53 TCID 50 /mL，10 5.6 TCID 50 /mL，10 5.78 TCID 50 The results show that the LN16 strain gradually adapts to Vero cells during serial passage, and the virus titer also gradually increases.
2.2 Pathogenicity of different generation strains
The strain of different generations is given 20000TCID 50 After SPF chickens of 3 weeks old are inoculated with the dose of the single SPF chickens, the chickens inoculated with the LN16-F20 and LN16-F25 generation group have clinical symptoms of thick nasal discharge, head swing, depression and the like, and the morbidity is 40 percent and 20 percent respectively; the chicken inoculated with LN16-F30 and LN16-F35 generation strains have no clinical symptoms, the morbidity is 0 percent (figure 1), which shows that the toxicity is obviously reduced when the virus is transmitted to the F30, and the pathogenicity does not exist to the chicken, therefore, the 35 th generation strain (LN 16-F35) which has no pathogenicity to the chicken and has the highest toxicity price is taken as a candidate passage weakening strain, named as LN16-A strain, which is preserved in the general microbiological center of China Committee for culture Collection of microorganisms, and the preservation address is No. 3 of Xilu 1 of the North kingdom of the south China ministry of academic, the microbial institute of China, the preservation number is: CGMCC NO.45261, the preservation time is as follows: 8 and 29 months in 2022.
2.3 Immunopotential test
2.3.1 aMPV specific antibody levels
The determination of aMPV specific antibodies in serum 7-21d after immunization showed that the levels of antibodies in 21d after immunization gradually increased. At 7d, no aMPV-specific antibodies could be detected; at 14d, the average antibody titer is 2540, and the antibody positive rate is 90%; at 21d, the mean titer was 5831, the antibody positivity was 100%, and the blank antibody was consistently negative (fig. 2). The results show that the LN16-A strain can induce the generation of aMPV specific antibody after immunizing organisms.
2.3.2 Neutralizing antibody titer test results
Serum is collected within 3 weeks after immunization, and the neutralizing antibody titer is measured, and the result shows that the neutralizing antibody turns positive at 7d after immunization, the antibody positive rate is 100%, the antibody level gradually rises when the average antibody titer is 4.25log2 and 14d-21d, and the average titers are 5.25log2 and 7.33log2 respectively. This indicates that the body can be induced to produce higher level of neutralizing antibody after the attenuated strain LN16-A is immunized (FIG. 3).
2.3.3 Observation of symptoms
In order to determine the immune protection effect of LN16-A on SPF (specific pathogen free) chickens, statistics is carried out on the morbidity of chickens in each group after challenge, and the results show that the challenge control group starts to suffer from morbidity at the 3 rd moment, thick and turbid rhinorrhea is generated, and the morbidity reaches 100% at the 7 th moment; however, the immune group has no clinical symptoms all the time, the disease rate is 0 percent (figure 4), and the results show that the passage attenuated strain LN16-A can provide 100 percent of immune protection against virulent attack.
2.3.4 histopathological observations
11d after the challenge, 11 chickens were harvested from each group and killed, paraffin tissue sections were prepared from turbinates and trachea, and the sections were stained and placed under an optical microscope for observation of pathological changes. The results show that the chicken turbinate lamina propria and submucosa of the challenge control group can be infiltrated by a plurality of inflammatory cells (figure 5A), and mucous gland epithelial cells are degenerated (figure 5B); hemorrhage of submucosa in trachea and local inflammatory cell infiltration in lamina propria (fig. 5C); no obvious histopathological changes were observed in the immunized group of chickens (FIGS. 5D, 5E, 5F). The tests show that the passage weakening strain LN16-A can effectively prevent pathological damage caused by virulent attack.
Claims (5)
1.B subtype avian metapneumovirus subculture attenuated strain is named LN16-A strain and is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC NO.45261.
2. Use of the subtype B avian metapneumovirus passaged attenuated strain of claim 1 in the manufacture of a medicament for the prevention of disease caused by infection with subtype B avian metapneumovirus.
3. The use according to claim 2, wherein the disease is turkey rhinotracheitis or chicken swollen head syndrome.
4. The use of claim 2, wherein the medicament is a subtype B avian metapneumovirus attenuated vaccine.
5. An attenuated vaccine of subtype B avian metapneumovirus, characterized in that the effective component thereof comprises the attenuated strain of subtype B avian metapneumovirus according to claim 1.
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