Characterization of aphthovirus subtype Asia 1/2 recovered in India and development and potency testing of a highly concentrated purified vaccine

Transcription

1 Rev. sci. tech. Off. int. Epiz., 1983, 2 (4), Characterization of aphthovirus subtype Asia 1/2 recovered in India and development and potency testing of a highly concentrated purified vaccine A.C. GOEL and A. RAI* Summary : The aphthovirus subtype Asia 1/2 (strain India 21/80) recovered in India was further characterized by microneutralization, guinea pig protection and challenge tests in cattle. These tests confirmed it to differ from subtype Asia 1/1 and corroborated the results of microcomplement fixation tests. It multiplied to high titre in BHK21 cell culture. A PEG-concentrated vaccine was prepared using this strain, which when tested in cattle proved to be highly immunogenic and protective in a 1 ml dose. The studies also demonstrated that it is possible to readily adapt an indigenous strain and manufacture an effective vaccine in a short time to meet the need of an effective control of FMD in a given area. INTRODUCTION In India, aphthovirus (FMD virus) Asia 1/1 has been the prevalent subtype. However, we recently detected the occurrence of subtype Asia 1/2 during the period (6). We studied this virus by means of various tests, its growth potential in cell culture, immunogenicity and development of a polyethyleneglycol (PEG) concentrated vaccine for field use to control outbreaks due to Asia 1/2 virus. MATERIALS AND METHODS 1. Viruses. Aphthovirus Asia 1/2 (India 21/80) recovered recently in India (6) was used, with aphthovirus Asia 1/1 (vaccine strain) as the reference strain for comparison. * Central FMD Typing Laboratory, Indian Veterinary Research Institute, Mukteswar, (U.P.), India.

2 2. Guinea pig hyperimmune sera Antiserum against the viruses was prepared by the method of Davie (1). The reference guinea pig hyperimmune sera against aphthovirus Asia 1/1 and Asia 1/2 were received from the World Reference Laboratory, Animal Virus Research Institute, Pirbright, UK. 3. Microneutralization test for subtyping. The viruses were tested by two-dimensional microneutralization test and V values for antisera were calculated as described by Rweyemamu et al. (8). 4. Cross-protection test in guinea pigs. Guinea pigs that recovered after virus infection were challenged with homologous and heterologous viruses using 10 4 ID50 (50% infectious dose) of guinea pig-adapted virus in one hind foot by the intraplantar route. The generalization of infection to other foot pads and tip of the tongue was taken as evidence of generalized infection, and absence of lesions on these sites was taken as protection. 5. Adaptation of virus to BHK21 (clone 13) cell cultures. The aphthovirus Asia 1/2 (India 21/80) was passaged in BHK21 monolayer cell cultures for adaptation and cytopathic effect (CPE) was observed 18, 24, 36 and 48 hours after infection. 6. Purification and concentration of virus with chloroform and PEG The virus adapted to BHK21 cell culture in tubes was added to cell culture in a milk dilution bottle, which was then transferred to a Roux flask and finally a Provitsky flask. Virus of the 6th passage was used to infect the Provitsky flask culture, inducing a complete CPE within 18 hours after infection. The virus-infected cell culture fluid was clarified by centrifugation at 3000 rpm for 15 min and then treated with 2% chloroform (v/v) and again centrifuged at 3000 rpm for 30 min. The virus-containing fluid was harvested and the titre of complement-fixing units (CFU) was determined. Then polyethyleneglycol (PEG)-6000 was added at the rate of 6% (w/v) and the flask kept on a magnetic stirrer for 2 hours at 4 C, after which it was centrifuged at 3000 rpm for 30 minutes. The supernatant fluid was removed and the precipitate was dissolved in a maintenance medium at one-fifth of the original volume. It was again treated with 2% chloroform and centrifuged as above to spin down the debris. The supernatant fluid obtained was purified concentrated virus. The CFU in these preparations were determined.

3 Determination of complement-fixing units (CFU) in virus preparations. The virus suspensions were tested in two-fold dilutions by the microcomplement fixation test (7) and the highest dilution showing 50% fixation of the complement was taken as the end point titre containing 1 CFU. The dilution factor was multiplied by 40 to obtain CFU/ml. 8. Preparation of PEG vaccine. The virus preparation for vaccine manufacture contained 512 CFU/ml. Vaccine having a ph of 8.5 was prepared as follows: PEG-concentrated virus 100 ml Aluminium hydroxide gel ml Glycocol buffer, ph ml Phosphate buffer, ph ml Glycerine 1.25 ml Formaldehyde solution (0.6 ml formalin 9.4 ml distilled water) 1.25 ml Total volume 125 ml The vaccine thus prepared was stirred at 25 C for 48 hours and then kept at 4 C for 10 days. 9. Safety test of vaccine. The safety test was done by inoculating 0.1 ml of the prepared vaccine into BHK21 cell culture tubes, 0.05 ml into mice intramuscularly, 0.25 ml into guinea pigs by the intraplantar route, 0.5 ml into rabbits subcutaneously, and 1 ml into cattle by the intradermolingual (IDL) route. The animals were observed for development of lesions. 10. Potency test of vaccine. 1 ml of the vaccine was inoculated into 13 FMD-antibody-free cattle subcutaneously. 21 days after vaccination, serum samples were collected and then 8 animals were challenged by the India 21/80 (Asia 1/2) cattle virus containing 104 ID50, by the IDL route. The remaining 5 animals were challenged with the vaccine strain (Asia 1/1) virus as above. Unvaccinated controls were inoculated with each virus separately. The animals were examined during 10 days after challenge for development of primary and secondary lesions on tongue, gums, lips and feet. 11. Antibody assay. Serum from vaccinated cattle was tested by microneutralization test using homologous virus (Asia 1/2) as well as heterologous virus (Asia 1/1). A cons-

4 1052 tant dilution of serum (1/5) was tested against varying dilutions of virus. Virus titration without serum was done simultaneously to determine the virus titre. The serum neutralization index (SNI) was obtained by deducting the virus titre obtained in the presence of serum from that obtained in absence of serum. RESULTS The field strain India 21/80 (Asia 1/2) was passaged in BHK21 (clone 13) monolayer cell culture using infective tongue epithelium sample as the inoculum. Results obtained are shown in Table I. The virus readily adapted to BHK21 cell culture and produced a CPE 18 hours after infection. It reached a titre of TCID50/ml and contained 80 CFU/ml. The results of testing in both directions by a two-dimensional microneutralization test and the 'r' values obtained are shown in Table II. The values obtained were less than 0.25 in both directions. The cross-protection test in guinea pigs showed that there was no TABLE I Adaptation of aphthovirus India 21/80 in BHK21 cell culture by using infected cattle tongue epithelium as the inoculum Passage number CPE hours after infection = No CPE 1 = 25% of monolayer showing CPE 2 = 50% of monolayer showing CPE 3 = 75% of monolayer showing CPE 4 = > 75% of monolayer showing CPE TABLE II Subtyping of aphthovirus India 21/80 by two-dimensional microneutralization test using Asia 1/1 as the reference strain Antiserum Homologous Antiserum titre Heterologous r India 21/80 (Asia 1/2) Asia 1/1 1,

5 1053 protection against the heterologous virus (Table III). Cross-challenge of cattle vaccinated with India 21/80 (Asia 1/2) vaccine by homologous virus and heterologous virus (Asia 1/1) revealed protection against Asia 1/2 virus, but no protection against Asia 1/1 virus (Tables IV and V). Similarly, the SNI of vaccinated cattle was very high with homologous virus (Asia 1/2) and was very low with heterologous (Asia 1/1) virus (Table VI). TABLE III Cross-protection test in guinea pigs using India 21/80 (Asia 1/2) and Asia 1/1 aphthovirus subtypes Guinea pigs recovered after infection with Challenge virus No. protected/ No. challenged Protection rate India 21/80 (Asia 1/2) India 21/80 5/5 100% India 21/80 Asia 1/1 0/5 0% Asia 1/1 Asia 1/1 9/9 100% Asia 1/1 India 21/80 (Asia 1/2) 0/10 0% Control healthy India 21/80 (Asia 1/2) 0/5 guinea pigs Asia 1/1 0/5 TABLE IV Cross-challenge of cattle vaccinated with India 21/80 (Asia 1/2) vaccine after 21 days of vaccination by Asia 1/1 virus usine 10 4 ID cattle virus Observation of lesions number Primary Secondary T G L RF LF RH LH 1 2 Vaccinated Control 2 3 T = Tongue, G = Gum, L = Lip, RF = Right fore foot, LF = Left fore foot, RH = Right hind foot, LH = Left hind foot No. protected/no. challenged = 0/5 Percentage of protection = 0%

6 1054 TABLE V Potency testing of aphthovirus India 21/80 (Asia 1/2) PEG-concentrated vaccine given 1 ml subcutaneously and challenged 21 days after vaccination by IDL route using 10 4 ID50 cattle virus Vaccinated Control Cattle number 1 2 Primary Observation of lesions Secondary T G L RF LF RH LH c. o No. protected/no. challenged = 7/8 Percentage of protection = 87.5% The concentration of cell culture virus with 6% PEG-6000 was highly effective. The PEG-concentrated virus contained 2560 CFU/ml when dissolved in l/5th of original volume, while the original virus fluid contained 80 CFU/ml. The vaccine prepared from strain India 21/80 proved to be safe when tested in BHK21 cell culture, mice, guinea pigs, rabbits and cattle. The results of potency testing of the vaccine in cattle are shown in Table V. Only one of 8 animals developed a generalized reaction, and the remaining 7 animals were protected. The percentage of protection obtained was The SNI of serum from vaccinated cattle was very high (Table VI). Based on our experimental observations, we developed a scheme (Fig. 1) for use on occasions when a variant strain is encountered, in order to obtain as quickly as possible the information required to prepare a suitable vaccine. DISCUSSION We reported the occurrence of aphthovirus subtype Asia 1/2 in addition to subtype Asia 1/1 in India, the latter being in use as a vaccine strain (6). This

7 1055 TABLE VI Serum neutralization index (SNI) of 21 days after vaccination of cattle with aphthovirus India 21/80 (Asia 1/2) PEG-concentrated vaccine given 1 ml subcutaneously and tested against Asia 1/2 (homologous) and Asia 1/1 (heterologous) viruses by microneutralization test Log10 SNI of serum against aphthovirus subtypes Cattle number India 21/80 (Asia 1/2) Asia 1/ new subtype was responsible for severe outbreaks in vaccinated herds in this country. We screened several Asia 1/2 isolates by microcomplement fixation test and selected strain "India 21/80" as the Indian reference strain for Asia 1/2 because of its high growth potential in BHK21 cell culture and immunogenicity in guinea pigs. This virus adapted to BHK21 cell culture readily and reached high titres ( TCID 50 /ml), and thus would be highly suitable for largescale culture for vaccine production. Subtyping by the microneutralization test gave 'r' values in both directions of 0.13 and 0.06, well below the 0.25 required to demonstrate a difference between subtypes (5). Challenge tests in guinea pigs and cattle also showed that the two viruses belong to different subtypes. The SNI 21 days after vaccination of cattle with India 21/80 (Asia 1/2) and tested against India 21/80 as well as Asia 1/1 viruses revealed a high SNI against homologous virus, and a low SNI against Asia 1/1 virus. Consequently the vaccine would afford no protection against Asia 1/1. The virus grown in BHK21 cell culture and concentrated with 6% PEG-6000 was highly suitable for vaccine manufacture. While the original cell culture fluid contained 80 CFU/ml, the PEG-concentrated virus suspended in l/5th its original volume contained 2560 CFU/ml, and the virus had to be further diluted to contain 512 CFU/ml for use as a vaccine. Since PEG is considered highly suitable for purifying and concentrating aphthovirus (3), it would be advantageous to use this substance for large-scale virus concentration. The vaccine was highly immunogenic in cattle, since a 1 ml dose afforded 87.5% protection, and the SNI of vaccinated cattle was very high. This established the

8 1056 good quality and effectiveness of PEG-vaccine. Our experiments have demonstrated that the indigenous strain of aphthovirus can multiply to a high titre in BHK21 cell culture and can be advantageously used to produce an effective vaccine. Mowat et al. (4) reported similar observations in Nigerian cattle using vaccine produced from local strains. These observations are of great value for Field isolate ('Asia l' type) India 21/80 Unidirectional testing with Asia 1/1 antiserum 'r' 0.13 Passage in cattle \ Severe primary lesions 48 hours post infection Collected tongue epithelium for future use Secondary lesions 120 hours after infection Passage in BHK 21 cell culture using tongue epithelium material as inoculum Tubes (3 passages) \ Bottle (2 passages) Roux, flask Tested with Asia 1/1, Asia 1/2 subtype sera. Preliminarily characterized as Asia 1/2 Produce antiserum in guinea pigs Subtyping using homologous, Asia 1/1, Asia 1/2 subtype sera Provitsky flask Concentrated with PEG-6000 Field isolate identified as Asia 1/2 (Israel 3/63 strain) Further tests with virus e.g. complement fixing unit, 140 S content, immunogenicity Prepared vaccine after inactivation Tested in cattle (1 ml dose) and found effective FIG. 1 Virus ready for vaccine manufacture for field use Flow chart showing test procedure for detecting a variant strain of aphthovirus

9 1057 a country like India in which large areas have differing type and subtype distribution, requiring the selection of suitable vaccine strains and the production of effective vaccines for use in specific areas. A 1 ml dose of PEGconcentrated monovalent vaccine produced a high degree of immunity. Currently a 2.5 ml dose of monovalent vaccine is used in India, and a reduction in dose would be of great value since polyvalent vaccines formulated for field use would be more concentrated. It would enable PEG-concentrated virus stocks to be kept in liquid nitrogen containers, so that vaccines could be prepared when required. Recently it has been demonstrated that highly concentrated purified aphthoviruses can be stored in liquid nitrogen for several years without any loss of 140 S virus particles and the immunogenic protein VP1 (2). Thus there are sound reasons for this country to change to this system of virus concentration and storage, and to manufacture concentrated vaccines which are effective in a 1 ml dose, as demonstrated in the present study. The process of centrifugation can be replaced by filtration for commercial vaccine production (3). ACKNOWLEDGEMENTS The authors thank the Director, Indian Veterinary Research Institute and Indian Council of Agricultural Research, for providing facilities and financial assistance to carry out this work. * * * CARACTÉRISATION DU SOUS-TYPE APHTEUX ASIA 1/2 ISOLÉ EN INDE. MISE AU POINT ET CONTRÔLE D'ACTIVITÉ D'UN VACCIN PURIFIÉ HAU TEMENT CONCENTRÉ. A.C. Goel et A. Rai. Résumé : Les caratéristiques du sous-type Asia 1/2 (Inde 21/80) du virus aphteux isolé en Inde ont été étudiées par les méthodes de microneutralisation et de protection de cobaye, et par l'épreuve virulente sur bovins. Ces recherches ont confirmé que ce sous-type est différent de Asia 1/1, corroborant ainsi les résultats de la micro-méthode de fixation du complément. Il a été constaté que le virus se multipliait à un titre élevé en culture de cellules BHK21. Un vaccin concentré au polyéthylèneglycol (PEG) a été préparé à partir de cette souche. Les essais sur bovins ont révélé une immunogénicité élevée à la dose de 1 ml. Les études effectuées ont également mis en évidence la possibilité d'adapter facilement la souche autochtone et de produire rapidement un vaccin actif pour répondre aux besoins d'une prophylaxie efficace de la fièvre aphteuse dans une zone géographique donnée.

10 1058 CARACTERIZACIÓN DEL SUBTIPO AFTOSO ASIA 1/2 AISLADO EN LA INDIA. ELABORACIÓN Y CONTROL DE ACTIVIDAD DE UNA VACUNA PURI FICADA ALTAMENTE CONCENTRADA. A.C. Goel y A. Rai. Resumen : Se han estudiado las características del subtipo Asia 1/2 (India 21/80) del virus aftoso aislado en la India con los métodos de microneutralización y protección de cobayo, y con la prueba virulenta en bovinos. Confirmaron estas investigaciones que el aludido subtipo es distinto de Asia 1/1, corroborando asi los resultados del micrométodo de fijación del complemento. Se comprobó que se multiplicaba el virus con un elevado título en cultivo de células BHK21. Con esta cepa se preparó una vacuna concentrada con polietileno-glicol (PEG). Las pruebas en bovinos revelaron elevada inmunogenicidad con la dosis de 1 ml. Los estudios efectuados también evidenciaron la posibilidad de adaptar con facilidad la cepa autóctona y producir rápidamente une vacuna activa para hacer frente a las necesidades de un control eficiente de la fiebre aftosa en un área geográfica determinada. * * * REFERENCES 1. DAVIE J. (1964). A complement fixation technique for the quantitative measurement of antigenic differences between strains of the virus of foot and mouth disease. J. Hyg., 62, DUCHESNE M., GUERCHE J., LEGRAND B., PROTEAU M. and COLSON X. (1982). The use of highly concentrated purified (by a large scale method) and long term liquid nitrogen stored foot and mouth disease viruses for the preparation of vaccines: physicochemical quality controls and potency testing after storage. Dev. Biol. Stand., 50, LEI J.C. (1974). Application of polyethyleneglycol in the preparation of a concentrated, purified foot and mouth disease vaccine. Present status for research. Bull. Off. int. Epiz., 81, MOWAT G.N., PRINCE M.J., OWEN H. and TAYLOR W.P. (1975). Results of a small scale field trial in Nigerian cattle of foot and mouth disease vaccines produced from local virus strains. Bull. Off. int. Epiz., 83, PEREIRA H.G. (1977). Subtyping of foot and mouth disease virus. Dev. Biol. Stand., 35, RAI A. and GOEL A.C. (1982). Antigenic variation in FMD virus type Asia 1 strains recovered in India during Rev. sci. tech. Off. int. Epiz., 2 (1), RWEYEMAMUM.M., BOOTH J.C., PARRY N. and PAY T.W.F. (1977). Neutralization kinetics studies with type SAT 2 foot and mouth disease virus strains. II. Antigenic differentiation of vaccine strains. J. Hyg., 78, RWEYEMAMU M.M., BOOTH J.C., HEAD M. and PAY T.W.F. (1978). Microneutralization tests for serological typing and subtyping of foot and mouth disease virus strains. J. Hyg., 81,