Application of polyinosinic-polycytidylic acid combined water-in-oil adjuvant in preparation of animal vaccine or animal virus antiserum

Abstract

The invention provides application of a polyinosinic-polycytidylic acid combined water-in-oil adjuvant in preparation of animal vaccines or animal virus antiserum, and belongs to the technical field of animal virology. The invention provides application of a polyinosinic-polycytidylic acid combined water-in-oil adjuvant in preparation of animal vaccines or animal virus antiserum. According to the invention, the polyinosinic acid is used as the immunopotentiator and is matched with the water-in-oil adjuvant, so that the immune effect can be remarkably enhanced, and compared with a group without the polyinosinic acid, the titer of a neutralizing antibody of the prepared serum is increased by 3-4 times. The invention also provides a preparation method of the virus positive serum. According to the invention, the inactivated antigen prepared by the polyinosinic-polycytidylic acid combined with the water-in-oil adjuvant and the active virus antigen are used for alternative immunization, so that the titer of a neutralizing antibody can be improved by more than 10 times; the prepared virus positive serum can fully meet the requirements of product quality control on an inspection reagent in the aspects of titer and specificity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal virology, and particularly relates to application of polyinosinic-polycytidylic acid combined with water-in-oil adjuvant in preparation of animal vaccine or animal virus antiserum. BACKGROUND

[0002] Polyinosinic-polycytidylic acid (Poly I:C) is a kind of artificial double-stranded ribonucleic acid, is a kind of interferon inducer, has antiviral and immunoregulatory functions, is used as an antiviral and antitumor drug in human medicine, and is less used in the field of veterinary medicine. SUMMARY

[0003] The application aims to provide application of polyinosinic-polycytidylic acid combined with water-in-oil adjuvant in preparation of animal vaccine or animal virus antiserum, which can significantly enhance the immune effect of the animal vaccine or the animal virus antiserum.

[0004] The application provides application of polyinosinic-polycytidylic acid combined with water-in-oil adjuvant in preparation of animal vaccine or animal virus antiserum.

[0005] The application further provides an inactivated antigen, which comprises inactivated virus, polyinosinic-polycytidylic acid and water-in-oil adjuvant.

[0006] Preferably, the virus comprises duck plague virus.

[0007] The application further provides a preparation method of the inactivated antigen in the above scheme, comprising the following steps.

[0008] The inactivated virus and the polyinosinic-polycytidylic acid are mixed to obtain an aqueous phase mixture; the aqueous phase mixture and the water-in-oil adjuvant are mixed to perform emulsification, so as to obtain the inactivated antigen.

[0009] The application further provides a preparation method of virus positive serum, comprising the following steps.

[0010] The animal is immunized for the first time by using a live virus immunogen;

[0011] After the first immunization, the animal is immunized for the second time by using the live virus immunogen and the inactivated antigen;

[0012] After the second immunization, the animal is immunized for the third time by using the inactivated antigen;

[0013] After the third immunization, the animal is immunized for the fourth time by using the live virus immunogen;

[0014] After the fourth immunization, the serum of the animal is separated;

[0015] The inactivated antigen is the inactivated antigen in claim 2 or 3 or the inactivated antigen prepared by the preparation method in claim 4.

[0016] Preferably, the virus content of the live virus immunogen is ≥ 10 7.0 ELD 50 / 0.2ml.

[0017] Preferably, the immunization mode of the first immunization is intramuscular injection; 14 days after the first immunization, the second immunization is performed using a live virus immunogen and an inactivated antigen; during the second immunization, the immunization mode of the live virus immunogen comprises intramuscular injection and eye and nose drops; the immunization mode of the inactivated antigen comprises intramuscular injection; 21 days after the second immunization, the third immunization is performed using the inactivated antigen; during the third immunization, the immunization mode of the inactivated antigen comprises intramuscular injection; 28 days after the third immunization, the fourth immunization is performed using the live virus immunogen; during the fourth immunization, the immunization mode of the live virus immunogen comprises intramuscular injection and eye and nose drops; 21-28 days after the fourth immunization, the serum of the animal is isolated.

[0018] Preferably, the animal comprises an avian animal; the avian animal comprises a chicken; the age of the chicken at the first immunization is 21 days.

[0019] The application further provides a virus-positive serum prepared by the preparation method in the above-mentioned scheme, wherein the virus-positive serum does not contain antibodies against chicken infectious bronchitis virus, chicken Newcastle disease virus, avian adenovirus group I, avian adenovirus group III, avian influenza virus type A, chicken infectious laryngotracheitis virus, avian reovirus, chicken infectious bursal disease virus, avian reticuloendotheliosis virus, chicken Marek's disease virus, avian lymphoid leukosis virus, avian encephalomyelitis virus, chicken infectious anemia virus, muscovy duck parvovirus, duck hepatitis A virus type I, duck hepatitis A virus type III, duck Tembusu virus, and gosling plague virus.

[0020] Preferably, the application comprises at least one of the following aspects:

[0021] 1) preparing a product for detecting, identifying or diagnosing a virus;

[0022] 2) preparing a product for studying pathogenic and transmission mechanisms of a virus;

[0023] 3) preparing a product for evaluating or monitoring the quality of a live virus vaccine;

[0024] 4) preparing a virus-positive serum standard.

[0025] The application provides an application of poly I:C combined with a water-in-oil adjuvant in preparing an animal vaccine or an animal virus antiserum. The application uses poly I:C as an immune enhancer and combines the water-in-oil adjuvant, which can significantly enhance the immune effect. Compared with a group without poly I:C, the prepared serum has a neutralizing antibody titer that is 3-4 times higher.

[0026] The application also provides a preparation method of virus positive serum, and the inactivated antigen prepared by the polyriboinosinic acid-polyribocytidylic acid combined with water-in-oil adjuvant can improve the neutralizing antibody titer by more than 10 times through alternating immunization with live virus antigens, and the virus positive serum prepared can fully meet the needs of product quality control for test reagents in terms of titer and specificity. DETAILED DESCRIPTION

[0027] The application provides application of polyriboinosinic acid-polyribocytidylic acid combined with water-in-oil adjuvant in preparation of animal vaccines or animal virus antiserum.

[0028] The application also provides an inactivated antigen, comprising inactivated virus, polyriboinosinic acid-polyribocytidylic acid and water-in-oil adjuvant.

[0029] As an implementation form, the virus comprises duck plague virus.

[0030] The application also provides a preparation method of the inactivated antigen in the above scheme, comprising the following steps.

[0031] The inactivated virus and the polyriboinosinic acid-polyribocytidylic acid are mixed to obtain an aqueous phase mixture; the aqueous phase mixture and the water-in-oil adjuvant are mixed to perform emulsification, and an inactivated antigen is obtained.

[0032] As an implementation form, the inactivated virus is obtained by inactivating a live virus immunogen; the inactivation method is not particularly limited in the application, and a conventional method in the art can be used.

[0033] As an implementation form, the water-in-oil adjuvant comprises mineral oil.

[0034] As an implementation form, the volume ratio of the aqueous phase mixture to the water-in-oil adjuvant is 1:1.5.

[0035] As an implementation form, the inactivated antigen is prepared by emulsifying a mixture of virus inactivated liquid and polyriboinosinic acid-polyribocytidylic acid with water-in-oil adjuvant.

[0036] The application also provides a preparation method of virus positive serum, comprising the following steps.

[0037] A first immunization is performed on an animal by using a live virus immunogen;

[0038] A second immunization is performed after the first immunization by using the live virus immunogen and the inactivated antigen;

[0039] A third immunization is performed after the second immunization by using the inactivated antigen;

[0040] A fourth immunization is performed after the third immunization by using the live virus immunogen;

[0041] After the fourth immunization, the serum of the animal is separated;

[0042] The inactivated antigen is the inactivated antigen described in the above scheme or prepared by the preparation method.

[0043] The present application firstly uses a live virus immunogen to immunize the animal.

[0044] As an embodiment, the virus content of the live virus immunogen is ≥10 7.0 ELD 50 As an embodiment, the preparation method of the live virus immunogen comprises the following steps: inoculating a virus seed diluent into the chorioallantoic membrane of SPF chicken embryo, aseptically harvesting the chorioallantoic membrane, fetus and embryo liquid of the dead chicken embryo within 48-120 hours after inoculation and grinding to obtain a live virus immunogen; the dilution multiple is 50 times; the reagent used for dilution is sterile normal saline; the SPF chicken is 9-10-day-old SPF chicken; the inoculation amount of inoculation is 0.2 ml per embryo; the virus includes duck plague virus; the virus seed of the duck plague virus includes duck plague virus chicken embryo attenuated strain CVCC AV1211, which is preserved in the National Animal Germplasm Bank. The present application uses SPF chicken embryo to prepare the immunogen and uses SPF chicken as the immunized animal, which ensures the purity of the positive serum.

[0045] As an embodiment, the immunization mode of the first immunization is intramuscular injection; the intramuscular injection dose of the live virus immunogen is 1000 ELD 50 / chicken.

[0046] As an embodiment, the animal includes avian animals; the avian animals include chickens; the day-old of the chicken at the first immunization is 21 days.

[0047] As an embodiment, the virus positive serum is duck plague virus positive serum.

[0048] After the first immunization, the present application uses a live virus immunogen and an inactivated antigen to perform the second immunization.

[0049] As an embodiment, 14 days after the first immunization, a live virus immunogen and an inactivated antigen are used to perform the second immunization; during the second immunization, the immunization mode of the live virus immunogen includes intramuscular injection and eye and nose drops, the intramuscular injection dose of the live virus immunogen is 1000 ELD 50 / chicken, the eye and nose drop dose of the live virus immunogen is 0.1 ml per chicken; the immunization mode of the inactivated antigen includes intramuscular injection, and the intramuscular injection dose is 0.5 ml per chicken.

[0050] After the second immunization, the present application uses inactivated antigen for the third immunization.

[0051] As an embodiment, 21 days after the second immunization, the third immunization is carried out using inactivated antigen; during the third immunization, the immunization mode of the inactivated antigen includes intramuscular injection, and the dosage is 0.5 ml per SPF chicken.

[0052] After the third immunization, the present application uses live virus immunogen for the fourth immunization.

[0053] As an embodiment, 28 days after the third immunization, the fourth immunization is carried out using live virus immunogen; during the fourth immunization, the immunization mode of the live virus immunogen includes intramuscular injection and eye and nose drops, and the dosage is 10,000 ELD50 per SPF chicken, and 0.1 ml per eye and nose per SPF chicken.

[0054] The present application can increase the neutralizing antibody titer by more than 10 times through the alternate immunization of inactivated antigen and live virus antigen, and the prepared virus positive serum can fully meet the demand of product quality control on test reagents in terms of titer and specificity.

[0055] After the fourth immunization, the present application separates animal serum.

[0056] As an embodiment, 21-28 days after the fourth immunization, the animal serum is separated, and specifically, the heart blood is collected.

[0057] After the separation of the animal serum, the present application further includes a comprehensive identification of the serum to ensure the quality of the virus positive serum.

[0058] In the present application, the test includes the performance test, sterility test, mycoplasma test, foreign virus test, neutralization titer determination, specificity test, residual water determination, and vacuum degree determination. In addition to meeting the requirements of the national standard, the foreign virus test uses the chicken embryo method to detect the pathogen; the specificity test uses ELISA, AGP, SN or other suitable methods to detect the antibodies of chicken infectious bronchitis virus, chicken Newcastle disease virus, avian adenovirus group I, avian adenovirus group III, avian influenza virus type A, chicken infectious laryngotracheitis virus, avian reovirus, chicken infectious bursal disease virus, avian reticuloendotheliosis virus, chicken Marek's disease virus, avian lymphoid leukosis virus, avian encephalomyelitis virus, chicken infectious anemia virus, duck parvovirus, duck hepatitis A virus type I, duck hepatitis A virus type III, duck tembusu virus, gosling plague virus, and duck new reovirus. The qualified serum is mixed, subpackaged, and labeled with the serum name, batch number, specification, production date, etc., and stored below-15℃.

[0059] The preparation method of the application provides an innovative idea for preparing polyclonal antibodies of pathogenic agents with poor humoral immune effect.

[0060] The application further provides virus positive serum prepared by the preparation method in the above scheme, wherein the virus positive serum does not contain antibodies against chicken infectious bronchitis virus, chicken Newcastle disease virus, avian adenovirus group I, avian adenovirus group III, avian influenza virus type A, chicken infectious laryngotracheitis virus, avian reovirus, chicken infectious bursal disease virus, avian reticuloendotheliosis virus, chicken Marek's disease virus, avian lymphoid leukosis virus, avian encephalomyelitis virus, chicken infectious anemia virus, muscovy duck parvovirus, duck hepatitis A virus type I, duck hepatitis A virus type III, duck Tembusu virus, and small goose plague virus.

[0061] In the application, the virus positive serum is good in purity, does not contain other common avian pathogens, is good in specificity, does not contain antibodies against other common avian pathogens except for duck plague virus antibodies, is high in titer, is 4-5 times higher than serum prepared by a conventional method, and has a neutralization titer as high as 1:2048. The virus positive serum is mainly used for quality control of virus strains and live vaccines.

[0062] As an embodiment, the virus positive serum is duck plague virus positive serum; the method of the application is suitable for preparation of duck plague virus positive serum, and the prepared duck plague virus positive serum is high in titer against duck plague virus, single in composition, and good in purity; the duck plague virus positive serum can solve the problem that duck plague virus as a herpes virus is not easy to produce neutralizing antibodies in an animal body, and provides an innovative idea for preparation of high immune serum for animals.

[0063] The application further provides application of the virus positive serum in the above scheme, and the application comprises at least one of the following aspects:

[0064] 1) preparation of products for detecting, identifying or diagnosing viruses; 2) preparation of products for studying pathogenic and transmission mechanisms of viruses; 3) preparation of products for evaluating or monitoring quality of virus live vaccines; and 4) preparation of virus positive serum standard.

[0065] In the application, the virus positive serum is mainly used for quality control of veterinary biological products and related detection of viruses.

[0066] In order to further illustrate the application, the application of the poly I:C combined with a water-in-oil adjuvant in preparation of animal vaccines or animal virus antiserum is described in detail in the following examples, but they should not be understood as limiting the protection scope of the application.

[0067] Example 1

[0068] 1. Immunogen Preparation

[0069] 1) Preparation of live viral antigens

[0070] Dilute the virus seed 50 times with sterile saline and inoculate the chorioallantoic membrane of 9-10 day old SPF chicken embryos, 0.2 ml per embryo. Chicken embryos that die within 48-120 hours after inoculation should be cooled at 2-8℃ for 4-24 hours, and the chorioallantoic membrane, fetus, and embryonic fluid should be collected aseptically (if the fetus is corrupt, the embryonic fluid is turbid, or there is any suspected contamination, it should be discarded). Mix several of them into a group, place them in a sterile bottle, freeze them for later use or grind them. After grinding, filter through 8 layers of gauze, take the filtrate, add appropriate antibiotics, and then package it. After testing, the virus content is 10 7.5 ELD 50 / 0.2ml.

[0071] 2) Preparation of inactivated virus antigens

[0072] The above-mentioned live immunogen is added to formaldehyde solution at 0.15% of the total volume, shaken for 30 minutes, and inactivated for 7 days. During this period, shake once in the morning and afternoon every day for 20 minutes each time. After passing the inactivation test, the inactivated antigen is added to 0.5% (v / v) poly I-cell, mixed evenly, and emulsified at a ratio of 1:1.5 (aqueous phase: mineral oil, v / v) to prepare the inactivated antigen.

[0073] 2. The immunization procedure for serum preparation is:

[0074] For the first immunization, each SPF chicken is injected intramuscularly with 1000 ELD of live virus antigen. 50 .

[0075] The second booster vaccination is carried out 14 days after the first vaccination, with 1000 ELD of live virus antigen injected intramuscularly into each chicken. 50 The same dose is 0.1 ml for eye drops and nose drops. At the same time, each chicken is injected intramuscularly with 0.5 ml of inactivated virus antigen; 21 days after the second immunization, the third immunization is carried out, and each chicken is injected intramuscularly with 0.5 ml of inactivated virus antigen; 28 days after the third immunization, each chicken is injected intramuscularly with 10,000 ELD of live virus antigen. 50 21-28 days after the fourth immunization, blood was collected from all chicken hearts.

[0076] 3. Serological testing: The above-mentioned minimum sampling units shall be treated as independent samples for character testing, sterility testing, mycoplasma testing, exogenous virus testing, neutralization titer determination, specificity testing, residual moisture determination, and vacuum degree determination. In addition to meeting national standards, the exogenous virus test shall utilize the chicken embryo method to detect the pathogen. Specificity testing shall utilize ELISA, AGP, SN, or other appropriate methods to detect antibodies to infectious bronchitis virus, Newcastle disease virus, avian adenovirus group I, avian adenovirus group III, avian influenza A virus, infectious laryngotracheitis virus, avian reovirus, infectious bursal disease virus, avian reticuloendotheliosis virus, Marek's disease virus, avian lymphocytic leukosis virus, avian encephalomyelitis virus, infectious anemia virus, Muscovy duck parvovirus, duck hepatitis A virus type I, duck hepatitis A virus type III, duck Tembusu virus, gosling plague virus, and duck novel reovirus. All tests shall be negative. The serum that passes the inspection is mixed, packaged, labeled, and marked with the serum name, batch number, specifications, production date, etc., and stored below -15°C.

[0077] Comparative Example 1

[0078] The immunopotentiator poly I-C was omitted, and the rest was the same as in Example 1.

[0079] Comparative Example 2

[0080] Physiological saline was used instead of the inactivated duck plague virus antigen, 0.3% (V / V) poly I-C was added, mixed evenly, and emulsified at a ratio of 1:1.5 (aqueous phase: adjuvant, V / V) to prepare the product. The rest was the same as in Example 1.

[0081] Test Example 1

[0082] 1. Experimental Grouping and Immunization Treatment

[0083] Ten SPF chickens were immunized with the inactivated antigens prepared in Example 1 (immunized group 2), Comparative Example 1 (immunized group 1) and Comparative Example 2 (immunized group 3), respectively, with 0.5 ml per chicken for the first immunization. Booster immunizations were performed 14 and 35 days after the first immunization, and blood was collected 21 days after the last immunization to detect duck plague neutralizing antibodies.

[0084] 2. Serum Neutralizing Antibody Assay

[0085] At the end of the immunization period, serum from each chicken was collected and neutralizing antibody assayed according to the following method:

[0086] Serum was diluted twice in series with sterile saline, with dilutions ranging from 1:8 to 1:2566. Each dilution was mixed with 100 ELD 50 / 0.1 ml of the duck plague live virus antigen was mixed in equal amounts, neutralized for 1.5 h, and then inoculated on the chorioallantoic membrane of 9-10 day old chicken embryos. Five embryos were inoculated with 0.2 ml of each dilution, and five embryos were inoculated with 0.1 ml of serum without virus, virus, and physiological saline as controls. After 1 h at 37 °C, the embryos were incubated in a 37 °C incubator for 168 h. The embryos that died within 24 h were discarded. The neutralizing antibody titer was calculated according to the Reed-Muench method based on the dead embryos within 24-168 h. The geometric mean of the neutralizing antibody titer of each immunized group was calculated. The results (see Table 1) show that mineral oil can improve the immunization effect of inactivated antigen, but not significantly, while the addition of the immunopotentiator and the use of mineral oil together can greatly improve the immunization effect of inactivated antigen.

[0087] Table 1 Neutralizing antibody titers and geometric means of each group of experimental animals

[0088]

[0089] Test Example 2

[0090] 1. Preparation of immunogen The live virus antigen and inactivated antigen were prepared according to the antigen preparation method described in Example 1.

[0091] 2. The immunization effect of different immunization procedures was compared by grouping according to the following method, and immunizing 5 four-week-old SPF chickens in each group (see Table 2).

[0092] Table 2 Immunization effect of different immunization procedures

[0093]

[0094] 3. Antibody monitoring

[0095] During the immunization period, serum was collected every 14 days for antibody detection. When the neutralizing antibody titer was higher than 1:256

[0096] , the serum of all chickens was collected. The results show that the neutralizing antibody titers of the 5 chickens in group A were all higher than 1:1024, with the highest being 1:4087; the neutralizing antibody titers of the 5 chickens in group B ranged from 1:32 to 1:128; and the neutralizing antibody titers of the 5 chickens in group C ranged from 1:128 to 1:512. The results show that the use of inactivated antigen and live virus antigen for alternate immunization can obtain a higher level of neutralizing antibody.

[0097] Test Example 3 Preparation, detection, and application of duck plague positive serum

[0098] A batch of duck plague positive serum was prepared according to the immunization procedure described in Example 1 and was subjected to comprehensive examination. The results showed that the serum was a clear liquid, sterile, free of mycoplasma, free of exogenous virus contamination, free of duck plague virus contamination, with a neutralization titer of 1:2048, and free of antibodies against chicken infectious bronchitis virus, chicken Newcastle disease virus, avian adenovirus group I, avian adenovirus group III, avian influenza virus A, chicken infectious laryngotracheitis virus, avian reovirus, chicken infectious bursal disease virus, avian reticuloendotheliosis virus, chicken Marek's disease virus, avian lymphoid leukosis virus, avian encephalomyelitis virus, chicken infectious anemia virus, muscovy duck parvovirus, duck hepatitis A virus type I, duck hepatitis A virus type III, duck Tembusu virus, gosling plague virus, and duck new reovirus, with good specificity. The positive serum was used to conduct exogenous virus examination on three batches of duck plague live vaccine according to Appendix 3305 of the Third Volume of the Chinese Veterinary Pharmacopoeia, and no incomplete neutralization phenomenon occurred in all batches, and no cytotoxicity caused by the positive serum occurred.

[0099] Although the above examples have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and people can also obtain other embodiments according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. Application of poly I-C combined with water-in-oil adjuvant in the preparation of animal vaccines or animal virus antiserum.

2. An inactivated antigen, characterized in that Includes inactivated virus, poly Ionovirus and water-in-oil adjuvant.

3. The inactivated antigen according to claim 2, characterized in that The viruses include duck plague virus.

4. The method for preparing the inactivated antigen according to claim 2 or 3, characterized in that: The following steps are involved: The inactivated virus and poly I-C are mixed to obtain an aqueous phase mixture; the aqueous phase mixture is mixed with a water-in-oil adjuvant and emulsified to obtain an inactivated antigen.

5. A method for preparing virus-positive serum, characterized in that: The following steps are involved: Animals are first immunized with live viral immunogens; After the first immunization, a second immunization is performed using live virus immunogens and inactivated antigens; After the second immunization, a third immunization is performed using an inactivated antigen; After the third immunization, a fourth immunization is performed using a live virus immunogen; After the fourth immunization, the animal serum was isolated; The inactivated antigen is the inactivated antigen according to claim 2 or 3 or the inactivated antigen prepared by the preparation method according to claim 4.

6. The preparation method according to claim 5, characterized in that The virus content of the live virus immunogen is ≥10 7.0 ELD 50 / 0.2ml.

7. The preparation method according to claim 5, characterized in that The first immunization is by intramuscular injection; 14 days after the first immunization, a second immunization is performed using live virus immunogens and inactivated antigens; during the second immunization, the immunization methods of the live virus immunogen include intramuscular injection and eye drops and nasal drops; the immunization method of the inactivated antigen includes intramuscular injection; 21 days after the second immunization, a third immunization was performed using an inactivated antigen; During the third immunization, the inactivated antigen is administered by intramuscular injection. 28 days after the third immunization, a fourth immunization is performed using a live virus immunogen; during the fourth immunization, the immunization method of the live virus immunogen includes intramuscular injection and eye and nasal drops; 21 to 28 days after the fourth immunization, the animal serum was separated.

8. The preparation method according to claim 5, characterized in that The animals include poultry; the poultry include chickens; at the time of the first immunization, the chickens are 21 days old.

9. The virus-positive serum prepared by the method according to any one of claims 5 to 8, characterized in that: The virus-positive serum does not contain antibodies to infectious bronchitis virus, Newcastle disease virus, avian adenovirus group I, avian adenovirus group III, avian influenza A virus, infectious laryngotracheitis virus, avian reovirus, infectious bursal disease virus, avian reticuloendotheliosis virus, Marek's disease virus, avian lymphocytic leukosis virus, avian encephalomyelitis virus, infectious anemia virus, Muscovy duck parvovirus, duck hepatitis A virus type I, duck hepatitis A virus type III, duck Tembusu virus, gosling plague virus, and duck novel reovirus.

10. The use of the virus-positive serum according to claim 9, characterized in that: The application includes at least one of the following aspects: 1) Preparation of products for detecting, identifying or diagnosing viruses; 2) Preparation of products for research on viral pathogenicity and transmission mechanisms; 3) Prepare products for evaluating or monitoring the quality of live viral vaccines; 4) Prepare virus-positive serum standards.

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