Strain differentiation of foot and mouth disease virus type Asia 1 isolates of Indian origin

Transcription

1 Rev. sci. tech. Off. int. Epiz., 1989, 8 (3), Strain differentiation of foot and mouth disease virus type isolates of Indian origin L.M. BELWAL, V.A. SRINIVASAN and RAMA KANT * Summary: Antigenic variation among aphthovirus type isolates recovered from India was investigated by a two-dimensional microneutralisation test. Two vaccine strains and three field strains were employed for reference. Considerable antigenic variation was observed among the strains. While vaccine strain Asia 1 IND 8/79 exhibited a narrow spectrum, most strains could be related to Asia 1 63/72. A broad spectrum was observed for the strain WBN 117/85, which has since been recommended for incorporation in the quadrivalent vaccine. KEYWORDS: Antigenic variation - Aphthovirus - Foot and mouth disease - India. INTRODUCTION Foot and mouth disease (FMD) is endemic in India. Of the seven serotypes of virus, four (O, A, C, and ) are prevalent in the subcontinent (1). While type O accounts for most outbreaks (50-60%), type takes second place with 15-20%, followed by type A (10-15%) and type C (1-5%). While no significant antigenic variation has been encountered in type C isolates of Indian origin (unpublished findings), types O and A have been shown to exhibit moderate to significant antigenic variation (ll, 4). Some of these have also been blamed for breakdown of vaccine immunity (5). Evidence of antigenic variation in Indian strains of serotype was presented by Rai (6) who further reported the characterisation of a new subtype, /2 (7) on the basis of complement fixation tests. The present study was aimed at further elucidation of the antigenic variation among type strains of Indian origin, as well as at selection of an appropriate vaccine strain. Virus strains MATERIALS AND METHODS The vaccine strain India 8/79 was obtained from Wellcome FMD Laboratory, Pirbright, UK, as a suspension of baby hamster kidney (BHK 21 Cl. 13) * Indian Immunologicals, Lakdi-ka-pul, Hyderabad , India.

2 772 cells. Ten strains were obtained from the World Reference Laboratory for FMD at Pirbright as primary bovine kidney, primary bovine thyroid or porcine kidney (IBRS-2) cell culture isolates. Two strains ( HAH 17/86 and HAH 19/86) were obtained in cattle tongue epithelium from Haryana Agricultural University, Hissar. The vaccine strain 63/72 was obtained from Indian Veterinary Research Institute, Bangalore. The remaining twelve strains were isolated in our laboratory at Hyderabad. All the isolates were serially passaged in BHK cell lines until fully adapted. Details of virus isolates included in the study and their passage histories are given in Table I. Reference viruses and antisera Antisera prepared against vaccine strains India 8/79 and 63/72, and against field strains India 11/80, RAB 64/85 and WBN 117/85 were employed for reference. Anti-140S guinea pig serum against all the strains was prepared as described by Rweyemamu et al. (10). Briefly, 140S antigen inactivated with BEI (binary-ethyleneimine) and purified by sucrose density gradient was emulsified with Freund's incomplete adjuvant and inoculated intramuscularly into the hind leg of guinea pigs. A booster dose was administered 21 days later. Ten days after the second dose, the guinea pigs were bled and their serum pooled before use. Serum was obtained from cattle vaccinated with the vaccine viruses IND 8/79 and 63/72. After preliminary analysis with anti-140s guinea pig antisera, strain WBN 117/85 was selected as the vaccine strain, and serum was obtained from cattle vaccinated with it. A group of four to six seronegative steers maintained FMD-free at the Holding Farm of Indian Immunologicals was vaccinated with appropriate monovalent vaccine, repeated after 21 days. The animals were bled on the 35th day and their serum was pooled before use. Serum neutralisation test The strains were compared by cross neutralisation in the two-dimensional microneutralisation test described by Rweyemamu et al. (10). Antigenic diversity was determined in terms of 'r' values obtained as follows: r _ serum titre against heterologous virus serum titre against homologous virus The 'r' values were calculated from mean titres obtained from three replicate tests. The statistical significance of 'r' values was tested by using an estimated pooled variance of (9). The strains were differentiated at a 99% significance level, which requires a critical 'r' value > 0.24 for a strain to be declared homologous in a three replicate test. RESULTS AND DISCUSSION The antigenic relationships among Indian isolates of type FMD virus, expressed as 'r' values, are presented in Table II for anti-140s guinea pig serum and in Table III for serum from vaccinated cattle.

3 773 TABLE I Details of virus strains used in the study Status Source Passage history at the time of use ** c o Place < * CD Sta District Strain identification Regior strain Vaccine j j j Oi Di C i strain Vaccine IVRI RS1E/TB3S5B2 RS1E/TB5 B1E/TB6 BK/1E/TB7 RS2E/TB6 BT/IBK2E/TB6 BK3E/TB5 B7S4B2 BK2E/TB7 BK1E/TB6 ca CS Cî3 CC ca ca 1- - U-!_ ca ca ca ca ca ca OOOO ca ca ca s S 1_ larat harashl harashl harashl ij c+^ CU Used as issar issar I I WRL HAU, HAU, BK2E/TB6 E/TBK1B3 B2E/TB8 BK2E/TB6' BK2E/TB6 asthan asthan asthan ryana ryana cd ca ca ca ca 'ca <a ca aiocacxx Ahmedabad Ahmedabad Ahmedabad Baroda Kaira Kaira Kheda Pune Ahmednagar Bombay ovovocn ON Tf C^ CN un un oo oo oo oo oo oo oo un Tf m T}- cor-~ cn CN O ND ' ' NO Tf Q Q Q ^ zzzg é ü O S S Bikaner Boondi Jaipur Hissar Hissar un CN NO ND ND OO CO OO OO OO TÍ- un o\ ND Tf CQ ~., X X c tzc XX te strain Candida d d d BK2E/TB6S4B1 B1E/TB8 E/TBK3B3 BK2E/TB7 BT1B1E/TB7 st Bengal st Bengal ;am lam ssa Nadia Murshidabad Dibrugarh Kamrup Sambalpur Oí CU I-. Used as WRL WRL WRL WRI. ih BT1E/TB7 >h BT1B/TB5 BK1E/TB5 E/TBT2B5 BT1E/TB4 RT1F./TB3 CU CU -O T3 ca ca 3 3 dhra Pi dhra Pi nil Nac nil Nac 'nataka ala c ca ca ca cu < < H H i4 i Nalgonda Nizamabad Coimbatore Nilgiris Bellari Mallanuram un ON CN un oo oo oo oo NTNOÛTTICJ oo oo oo oo oo r- co CN NO O O oo TT Tt r- m < un oo NO Z Z Q Q Q Q TO TO TO ro ca ca ca ca ca ca TO TO TO TO TO o ro ro TO TO ro TO ro TO TO ro W7)(/)71l/ll/ll/ll711/l(/) Í/1 W C/1 (/) 1/1 co co co co co CO CO CO CO CO CO <<<<<<<<<< <<<<< <<<<< < <<<<< m India Westei ca Northern Indi n India Easter ca Southern Indi * Strains identified with prefix IND obtained from World Réf. Lab., Pirbright, U.K. (e.g. IND 8/79). Strains identified with prefix other than IND isolated and adapted at, Hyderabad (except the strain 63/72 which was obtained from Indian Vet. Res. Institute, Bangalore). ** BT = Bovine thyroid cell culture; BK = Bovine kidney cell culture; RS = Porcine kidney cell culture; B = Baby hamster kidney cell line (BHK 21 CI. 13); E/T = ether treated; S = BHK suspension cell culture; WRL = World Ref. Lab. for FMD, Pirbright, U.K.; IVRI = Indian Vet. Res. Institute; HAU = Haryana Agri. Univ., Hissar; = Indian Immunologicals Laboratory, Hyderabad.

4 774 TABLE II 'r' values obtained using anti-140s guinea pig antisera Antisera Viruses IND 8/79 63/72 WBN 117/85 IND 11/80 RAB 64/85 Gujarat IND 8/ * 0.09* 0.17* 0.04* IND 11/ * * IND 3/ * * 0.09* GUK 61/ * 0.13* * 0.04* GUB 104/ * GUA 25/ * * GUK 14/ * 0.14* * 0.02* Maharashtra 63/ * * 0.13* MAA 7/ * * MAB 142/ * * Rajasthan RAB 64/ * * IND 45/ * > >1.00 RAJ 1/ * * >1.00 West Bengal WBN 117/ * IND 68/ * * 0.63 Assam IND 47/ * IND 177/ * 0.51 > * Haryana HAH 17/ * * >1.00 HAH 19/ * 0.50 > * 0.07* Andhra Pradesh APN 32/ * * APN 6/ * 0.04* 0.06* 0.19* 0.02* Tamil Nadu IND 11/ * 0.16* 0.09* * IND 50/ * * 0.14* Karnataka IND 80/ * * 0.73 Kerala IND 48/ * * >1.00 Orissa ORS 8/ * * * 'r' significantly less than 1 at p =.01

5 775 TABLE III Serological interrelationship among type isolates of Indian origin (Based on 'r' values obtained with serum from vaccinated cattle) Antisera Viruses IND 8/79 63/72 WBN 117/85 WESTERN INDIA Gujarat Ahmedabad IND 8/ * 0.46 Ahmedabad IND 11/ Ahmedabad IND 25/ Baroda GUB 104/ * Kaira GUK 3/ Kaira GUK 61/ * 0.81 Kheda GUK 14/ * 0.10* 0.58 Maharashtra Ahmednagar MAA 7/ * 0.17* >1.00 Pune 63/ * * Bombay MAB 142/ * 0.10* 0.51 NORTHERN INDIA Rajasthan Bikaner RAB 64/ * Boondi IND 45/ * 0.23* 0.58 Jaipur RAJ 1/ * 0.13* 0.39 Haryana Hissar HAH 17/ * 0.20* 0.89 Hissar HAH 19/ * EASTERN INDIA West Bengal Nadia WBN 117/ * 0.17* 1.00 Murshidabad IND 48/ * Assam Dibrugarh IND 47/ * 0.19* 0.37 Kamrup ASK 177/ >1.00 Orissa Sambalpur ORS 8/ * SOUTHERN INDIA Andhra Pradesh Nalgonda APN 32/ * 0.21* 0.45 Nizamabad APN 6/ * 0.10* 0.23* Tamil Nadu Coimbatore IND 11/ * 0.42 Nilgiri IND 50/ * 0.13* 0.21* Karnataka Bellari IND 80/ * Kerala Malapuram IND 48/ * 0.33 >1.00 * 'r' significantly less than 1 at p = 0.01

6 776 The vaccine strain IND 8/79 exhibited a narrow spectrum with respect to V values obtained with anti-140s guinea pig serum. However, contemporary isolates from Gujarat, the state of origin of this strain, appeared homologous to Asia 1 IND 8/79, as did the strains WBN 117/85 from West Bengal and ORS 8/87. The Gujarat strains isolated after 1979, and those from other states, showed divergence from the vaccine strain IND 8/79 when guinea pig serum was used. The antigenic spectrum of IND 8/79 obtained with serum from vaccinated cattle was wider than that of anti-140s serum. Strains from Assam ( ASK 177/85) and Tamil Nadu ( IND 11/80) showed closer relationships to IND 8/79. A broad antigenic spectrum was exhibited by the vaccine strain 63/72 when anti-140s sera were employed. The 1979, 1984, 1987 Gujarat isolates and one isolate each from Andhra Pradesh, Tamil Nadu and Orissa appeared to be divergent. However, while higher 'r' values were obtained against divergent Gujarat isolates, the overall spectrum with serum from vaccinated cattle was narrower than that with anti-140s guinea pig serum. On the basis of 'r' values from anti-140s antiserum and bovine serum, most of the strains were related to WBN 117/85 (Tables II and III). Four isolates - IND 8/79, RAB 64/85, IND 11/80 and APN 6/87 - were found to be divergent with anti-140s guinea pig serum, while 63/72, Asia 1 APN 6/87 and IND 50/80 were significantly divergent (p < 0.01) with bovine serum. Thus, 23 strains out of 26 studied were observed to be nondivergent (not significantly different: r' < 1 at p = 0.01) from WBN 117/85 by virtue of serum neutralisation ratios obtained with serum from vaccinated cattle. As for the other two strains employed for reference, the Tamil Nadu strain (Asia 1 IND 11/80) presented a broader spectrum than the Rajasthan strain ( RAB 64/85). While 13 strains out of 26 studied showed divergence from Rajasthan strain, 18 strains were heterologous to the Tamil Nadu strain. Gujarat isolates appeared to be closer to IND 11/80 (Southern region), than to the strain RAB 64/85 which is from the Northwestern region of the country. However, strains from the state of Rajasthan gave 'r' values > 1, exhibiting homology with RAB 64/85 which originated in the same state. Evidence of antigenic variation among Indian type viruses based on complement fixation test results employing guinea pig hyperimmune serum against live FMD virus (5) has been presented in detail by Rai (6) and Rai and Goel (7). However, the preference for the serum neutralisation test using serum against inactivated and purified 140S antigen for strain differentiation is well documented (8) and endorsed by the Permanent Subcommittee on FMD of the International Association of Biological Standardisation (2). Results obtained in our laboratory show that cross neutralisation of field strains with serum from vaccinated cattle simulates the performance of candidate or vaccine strains, as expected from incorporation in the vaccine. Strain WBN 117/85 had the broadest antigenic spectrum. Except for the strain from Nizamabad (Andhra Pradesh, 1987), all appear to be closely related to this candidate vaccine strain. These studies show that there is considerable antigenic variation among Indian type strains, with some evidence of antigenic drift over the years. However,

7 777 uncontrolled livestock movements over vast territories might be responsible for antigenic divergence among strains which are contemporary in time and space. The strain WBN 117/85 exhibited a broad antigenic spectrum and has been recommended for incorporation in the quadrivalent vaccine. The antigenic relationship of field strains, originating from a particular region, to vaccine strain varies from homologous to divergent in nature. This observation indicates the necessity of continuous monitoring of field strains and incorporating more than one virus strain of a particular virus type in vaccine to control the disease, depending on the serological spectrum of candidate virus strains. ACKNOWLEDGMENTS This work was carried out under the project "Cataloguing and strain differentiation of FMD virus". Acknowledgments are due to the National Dairy Development Board, Anand (Gujarat), India, for providing financial assistance. The authors also wish to thank the Foot and Mouth Disease Virus Typing Centre of All India Coordinated Research Project at Haryana Agricultural University at Hissar, and the Indian Veterinary Research Institute, Bangalore, for providing the virus strains. * * DIFFÉRENCIATION ENTRE LES SOUCHES DE TYPE ASIA 1 DU VIRUS APHTEUX ISOLÉES EN INDE. - L.M. Belwal, V.A. Srinivasan et Rama Kant. Résumé : Les variations antigéniques entre les souches de type du virus aphteux isolées en Inde ont été étudiées à l'aide d'une microméthode de neutralisation bidimensionnelle. Deux souches vaccinales et trois souches sauvages ont été utilisées comme souches de référence. Des variations antigéniques considérables ont été constatées entre les souches. Tandis que la souche vaccinale IND 8/79 présentait un spectre antigénique étroit, la plupart des souches ont pu être apparentées à 63/72. Un spectre étendu a été observé pour la souche WBN 117/85, dont l'incorporation dans le vaccin quadrivalent a été depuis lors recommandée. MOTS-CLÉS : Aphthovirus - Fièvre aphteuse - Inde - Variations antigéniques. * * * DIFERENCIACIÓN ENTRE LAS CEPAS DE TIPO ASIA 1 DEL VIRUS AFTOSO AISLADAS EN INDIA. - L.M. Belwal, V.A. Srinivasan y Rama Kant. Resumen: Las variaciones antigénicas entre las cepas del tipo del virus aftoso aisladas en India se han estudiado mediante una microtécnica de neutralización bidimensional. Se utilizaron dos cepas vacunales y tres cepas salvajes como cepas de referencia, comprobándose considerables variaciones antigénicas entre ellas. En tanto que la cepa vacunal IND 8/79 presentaba un espectro antigénico estrecho, la mayoría de las cepas pudieron aparentarse

8 778 con 63/72. En la cepa WBN 117/85, se observó un espectro amplio, recomendándose su incorporación en la vacuna cuadrivalente. PALABRAS CLAVE: Aftovirus - Fiebre aftosa - India - Variaciones antigénicas. * * * REFERENCES 1. ANON. ( ). - Report of the Project Coordinator, All India Coordinated Research Project for Epidemiology of Foot and Mouth Disease. Indian Veterinary Research Institute, Izatnagar, U.P., India. 2. ANON. (1982). - Report on IABS foot and mouth disease meeting. 15 September 1982, Paris. IABS Biostandards, Geneva, Switzerland. 3. BELWAL L.M., PALANISAMY R., NAGAIAH K., KALANIDHI A.P., JAGANNATHA H.M., RAMANNA B.C. & SRINIVASAN V.A. (1986). - Serological study of foot and mouth disease virus type A in India. Rev. sci. tech. Off. int. Epiz., 5 (3), BELWAL L.M., KALANIDHI A.P., NAGAIAH K., RAMANNA B.C. & SRINIVASAN V.A. (1987). Immuno-serological studies on a foot and mouth disease virus type A strain involved in breakdown of vaccine immunity. Rev. sci. tech. Off. int. Epiz., 6 (1), DAVIE J. (1964). - A complement fixation technique for the quantitative measurement of antigenic differences between strains of foot and mouth disease. J. Hyg., Camb., 62, RAI A. (1980). Subtyping of foot and mouth disease virus isolates of type O and Asia 1 recovered from field outbreaks in India. Indian J. Anim. Sci., 50, RAI A. & GOEL A.C. (1983). - Antigenic variation in FMD virus type strains recovered in India during Rev. sci. tech. Off. int. Epiz., 2 (1), RWEYEMAMU M.M. (1984). - Antigenic variation in foot and mouth disease: studies based on virus neutralisation reaction. J. biol. Standard., 12, RWEYEMAMU M.M. & HINGLEY P.J. (1984). - Foot and mouth disease virus strain differentiation: analysis of serological data. J. biol. Standard., 12, RWEYEMAMU M.M., BOOTH J.C., HEAD M. & PAY T.W.F. (1978). - Microneutralisation test for serological typing and subtyping of foot and mouth disease virus strains. J. Hyg., Camb., 80, SRINIVASAN V.A., OULDRIDGE E.J., HEAD M. & RWEYEMAMU M.M. (1982). - A serological study of foot and mouth disease virus isolates. Rev. sci. tech. Off. int. Epiz., 2 (1),