WHO collaborative studies on enterovirus reference antisera

Bull. Org. mond. Sant| 1973, 48, 381-396 Bull. Wld Hith Org. WHO collaborative studies on enterovirus reference antisera * Fourth Report JOSEPH L. MELNICK & BETTYLEE HAMPIL on behalf of the participating laboratories This paper summarizes the results of the fourth part of a comprehensive programme undertaken by the WHO International Reference Centre for Enteroviruses and other laboratories for the testing of enterovirus equine antisera prepared for long-term use as reference antisera. The studies were designed to appraise the specificity of the immune serum ofhorses inoculated with prototype enteroviruses (coxsackievirus types A2, 4, 8, 10, 11, 14-16, 18-21, and 24, and echoviruses E21, 27, 30, 31, and 33). Tests for neutralizing antibody were performed against the homologous viruses and against available regional homotypic strains. Heterotypic tests were performed against reoviruses 1-3, adenoviruses 1-31, and the entire series of enteroviruses (with the exception of enterovirus 68). The homologous geometric mean titre of the S echovirus antisera ranged from 3 000 to 10 000; the titre of 1 coxsackievirus antiserum (A24) was only about 400, but the titres of the others ranged from 1 500 to 14 000. All corresponding preinoculation sera were negative. Heterotypic antibody of significant titre was found in 4 antisera: E31 serum against E5 virus, CA8 serum against CA3 virus, CAJ3 serum against CAM8 virus, and CAJS serum against CA2 virus. Information on other heterotypic antibody titres (where found) is recorded for guidance in the use of the sera. The results of homotypic tests with viruses isolatedby the collaborating laboratories, though limited in number, were satisfactory. Three previous studies made through the col- laborative efforts of a number of WHO Regional Reference Centres for Enteroviruses, WHO Virus Collaborating Laboratories, and other laboratories have provided pertinent information on 41 entero- virus equine antisera that were prepared for long- term use as research reference reagents. Data on the results of homologous, homotypic, and heterotypic tests by the participating laboratories were reported by Melnick & Hampil (1965, 1970) and Hampil & Melnick (1968). In the present study-the fourth part of the programme-laboratories in 10 countries examined an additional 19 enterovirus equine antisera.1 This report summarizes the antibody findings of the scientists in WHO and other labo- ratories throughout the world who participated in this study and collaborated in the writing of this report (the names and addresses of these scientists and laboratories are listed in the Annex, p. 395). The following abbreviations are used throughout the text and tables: P (poliovirus); CA (coxsackie- virus group A); CB (coxsackievirus group B); E (echovirus); Reo (reovirus); Ad (adenovirus); HI (haemagglutination-inhibition); NT (serum neu- tralization test); mouse NT (mouse in vivo neutraliza- tion test). Tissue culture (TC) cell systems are referred to as RhK (rhesus monkey kidney), GMK (vervet or green monkey kidney), Cyn K (cyno- * From the WHO International Reference Centre for Enteroviruses, Department of Virology and Epidemiology, Baylor College of Medicine, Houston, Tex., USA. 1 Information on how to obtain the reagents may be obtained from: Research Resources Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. 20014, USA; Virus Diseases, World Health Organisation, 1211 Geneva 27, Switzerland; the WHO International Reference Centre for Enteroviruses; or any WHO Regional Reference Centre for Enteroviruses. 3021 - 381 - Table 1. Summary of homologous, homotypic, and heterotypic tests a performed by 13 laboratories on 19 entero- virus equine sera b Viruses tested Laboratory homologous heterotypic NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNhomotypic Atlanta Mouse NT against CA2, 8, 10, 11, TC tests against El 2-20, 22-26 13-16, 18-20, 21, 24 using RhK cells and against E21 using HEL cells TC tests against E21 using HEL cells HI against Adl-31 and against E27, 30, 31, 33 using RhK cells Berkeley Mouse NT against CAl 5 Mouse NT against CAl, 3, 5, 6, 17, 19,22 TC tests against CA15, CA24, and TC tests against P1-3, CA7, 9, 16, CA24 antigenic variants using HEK CB1-6, El-9, 11-33, and entero- cells virus 68 using RhK cells; against CA3, 4, 8, 1 0, and 12 using HA cells; and against CA2, 11, 13-15, 18, 20, 20a, 20b, and 21 using HEK cells Copenhagen Mouse NT against CA2, 8, 10, 11, Mouse NT against CAI-22, 24 13-16, 18-20,21,24 Epsom Mouse NT against CA2, 8, 10, 11, Mouse NT against CAl -19, 21, 22, 13-16,18,19, 21, 24 24 TC tests against CA2, 8,10,11,13-16, TC tests against CA2, 3, 8, 10-18, 18, 20, 21, 24 using HA cells 20, 21, 24 using HA cells Houston Mouse NT against CA4, 19 TC tests against CA3, 4, 8, 10-18, 20, 21, 24 using HA or HEK cells TC tests against CA2, 8, 10, 11, 13-15, Mouse NT against CA3 18, 20, 21, 24, and E21 using HA or HEK cells; and against CA16, E27, 30, 31, 33 using RhK or GMK cells London TC tests against E27, 30, 31, 33 TC tests against El 2-20, 22-33 using RhK cells and against E21 using RhK cells; against E21 using using HA cells HA cells; and against E34 Mouse NT against CAI6 TC tests against E21 using HEL cells and against E30 and E31 using RhK cells Mouse NT against CAl 6 TC tests against E27, 30, 31 using RhK cells TC tests against CA14, 15, 18, and E21 using W1-38 cells; and against E27, 30, 33 using Cyn K or RhK cells using HEp-Z ceiis TC tests against P1, 2, 3, CA7, 9, CB2, 3, 4, 5, 6 and El-5, 6', 7-9, 11 using Cyn K or RhK cells Melbourne TC tests against E21 and E31 using HEL cells; against E27 using HeLa and HEL cells; and against E30 and 33 using HeLa cells Moscow TC tests against CAl 6, E27, 30, 31, 33 using RhK or GMK cells Ottawa TC tests against CAl 6, E21, 27, 30, 31, 33 using RhK cells Prague TC tests against CAl 6, E27, 30 using GMK cells; against E21 using HeLa cells; and against E31 using HEL cells Singapore TC tests against E27, 30, 31, 33 using Cyn K cells Tokyo TC tests against CAl 6, E21, 27, 30, 31, 33 using Cyn K cells Mouse NT against CAl 6 TC tests against P1-3 and CB1-6 using HEp-2 cells; against CA7 and 9 using Cyn K cells; against CAl 6 using HEL cells; against E1-3 using HeLa cells; against E4 using HEL cells; and against E5-9, 11 using HeLa cells TC tests against CAl 6, E20, 22-27, 29-33 using RhK or GMK cells TC tests against P1-3, CA7, 9, 16, CB1-6, E1-9, 11, 27, 30-33 using RhK cells E31 serum also tested against 2 field isolates of E5 virus TC tests against P1-3, CA7, 9, E1-3, 5-9, 11 using GMK cells, and against E4 using HEL cells TC tests against P1-3, CA7, 9,16, CBI-6, El-9, 11, 29 using Cyn K cells TC tests against El 2-27, 29-33 using Cyn K cells HI against Reo 1-3 TC tests against E21, 27 using HEL cells; against E30 using HEL or HeLa cells; against E31 using HEL cells; and against E33 using HeLa cells TC tests against CAl 6 and E31 using RhK cells TC tests against CAl 6, E21, 27 using Cyn K cells Mouse NT against CAl 6 Lyon a Hi = haemagglutination-inhibition; mouse NT = mouse in vivo test; TC = tissue culture; RhK = rhesus kidney; GMK = vervet or green monkey kidney; Cyn K = cynomolgus monkey kidney; HA = human amnion; HEK = human embryonic kidney; HEL = stable line of human embryonic lung other than WI-38. b The sera were: CA2, 4, 8, 10, 11, 13-16, 18-21, and 24; and E21, 27, 30, 31, and 33. In some cases, the heterotypic tests listed for a given laboratory were not done on all 19 sera; the tests performed appear in the appropriate summary tables. ENTEROVIRUS REFERENCE ANTISERA molgus monkey kidney), HA (human amnion- primary or secondary), and HEK (human embryonic kidney-primary or secondary). HEL refers to cell lines of human embryonic lung used by the reporting laboratory. PLAN OF STUDY AND METHODS The plan of study, the methods used in the serum neutralization tests, and the presentation of results were similar to those of earlier studies (Hampil & Melnick, 1968). Thirteen laboratories participated in the investiga- tion of the 19 antisera for homologous, homotypic, and heterotypic antibody. Three laboratories were new participants in the programme (Atlanta, Berke- ley, and Melbourne), and the methods used by two of these laboratories differed from those of the other laboratories in the following respects. Melbourne employed stable lines of cell cultures in the homolog- ous, homotypic, and heterotypic tests. Also, owing to the local quarantine regulations, the rehydrated sera were inactivated at 58.5°C for 1 h and stored frozen until used. (The other laboratories heated the sera at 56°C for 30 min before use.) The Atlanta laboratory, which had performed all the heterotypic tests against the adenoviruses in two previous studies, extended the testing in this study to include also serum neutralization tests against enteroviruses, using methods commonly employed in the labo- ratory. Furthermore, the reporting of titres differed slightly from that of the other laboratories. In tissue culture tests, the serum dilution that neutralized the challenge dose of virus in 2 out of 3 or 3 out of 3 tissue culture tubes inoculated with a given serum- virus mixture was reported as the end-point titre per 0.1 ml. In mouse neutralization tests, the serum dilu- tion protecting at least half of a litter of baby mice (unrandomized) was designated as the end-point titre per 0.05 ml. (The other laboratories reported titres of the sera as 50% end-point dilutions.) Table 1 summarizes the tests performed, the viruses used, and the host-cell systems used by each laboratory. MATERIALS Final vials of dried preinoculation and post- inoculation sera were supplied to the laboratories. The immune sera were from blood taken during the primary immunization of horses, and also after booster doses of antigen had been given. The sera were pooled, filtered, dispensed into appropriate containers, and freeze-dried. The 19 antisera were: CA2 (Fleetwood), CA4 (High Point), CA8 (Donovan), CAlO (Kowalik), CAl1 (Belgium-1), CA13 (Flores), CA14 (G-14), CA15 (G-9), CA16 (G-10), CA18 (G-13), CA19 (Dohi), CA20 (IH 35), CA21 (Kuykendall), CA24 (Joseph), E21 (Farina), E27 (Bacon), E30 (Bastianni), E31 (Caldwell), and E33 (Toluca-3). Live virus antigens concentrated 10 times by the magnesium aluminium carbonate method of Midulla et al. (1965) were used for immunizing the horses. The CA2, 8, 10, 11, 13, 16, and 20 and the E21, 27, and 31 virus fluids were collected and the con- centrated antigens were prepared in the laboratory of Dr S. S. Kalter (San Antonio). Virus fluids of CA14, 15, 18, 21, and 24 and E33 were collected in the laboratory of Dr H. A. Wenner (Kansas City) and shipped frozen to the Houston laboratory for preparation of the antigens. Dr Wenner's laboratory also provided a 10% mouse brain-torso suspension of CA19 virus for concentration. The Houston labo- ratory prepared the 10% mouse brain-torso sus- pension of CA4 virus as well as the concentrated antigen. Coxsackievirus types A2, 8, 10, 11, 1?, 14, 15, 18, 20, 21, and 24 and echovirus 21 were grown in secondary HA cell cultures. Coxsackievirus types A4 and 19 were propagated in 2-3-day-old mice. GMK cell cultures were used for the propagation of cox- sackievirus type A16 and echovirus 27; RhK cul- tures were used for echoviruses 30, 31, and 33. The concentrated antigens were shipped frozen to the laboratory of Dr R. W. Brown (Tuskegee Institute), where the horses were inoculated and bled according to methods described by Hampil et al. (1965). Prototype viruses were used in the homologous and heterotypic serum neutralization tests. The homotypic viruses were field isolates from the area of the laboratory performing the tests. RESULTS Tests on 14 coxsackievirus group A sera (CA2, 4, 8, 10, 11, 13-16, 18-21, and 24) Homologous tests. The results of tests for homolo- gous antibodies are shown in Table 2. All sera were subjected to the mouse NT; tests were also performed in TC in those instances in which the virus propa- gates in cell cultures. In general, there was fairly good agreement among the laboratories, with the 383 Table 2. Results of homologous serum neutralization tests on 14 coxsackievirus type A equine sera a Neutralizing antibody titre b Geometric mean titre Serum Laboratory mouse NT C TC test mouse NT TC test per 0.015-0.05 ml per 0.1 ml CA2 Atlanta Copenhagen Epsom Houston CA4 Copenhagen Epsom Houston CA8 Atlanta Copenhagen Epsom Houston CAl 0 Atlanta Copenhagen Epsom Houston CAl 1 Atlanta Copenhagen Epsom Houston CAI 3 Atlanta Copenhagen Epsom Houston CAI 4 Atlanta Copenhagen Epsom Houston Lyon CAl 5 Atlanta Berkeley Copenhagen Epsom Houston Lyon CAI 6 Atlanta Copenhagen Epsom Houston Moscow Ottawa Prague Tokyo CAl 8 Atlanta Copenhagen Epsom Houston Lyon CAI 9 Atlanta Copenhagen Epsom Houston CA20 Copenhagen Epsom Houston CA21 Copenhagen Epsom Houston CA24 Atlanta Berkeley Copenhagen Epsom Houston 2 560 (407)d 10 000 (20) 7 700 (316) 3 600 (8) 3 500 (56) 5 500 (53) 2 560 (100) 5 000 (10) 3 500 (178) 2 560 (56) 8 000 (40) 8 200 (100) 640 (100) 1 280 (32) 3 500 (42) >2 560 (320) >2 560 (40) 4 500 (133) >2 560 (234) 14 500 (200) 14 000 (316) 1 280 (239) 20 480 (80) 10 000 (8) 33 000 (73) 1 280 (100) 2 560 (158) 3 000 (42) 2 048 (100) 5 120 (316) 7 800 (120) 5 820 (137) 6 320 (195) 4100 (28) 7 100 (100) 7 300 (100) 2 560 (100) 8 000 (230) 1 500 (68) 1 600 (66) 7 100 (68) 2 000 (100) 10 000 (1 47) 32 000 (320) 9 600 (100) 640 (320) 5 000 (32) 5 700 (320) 2 400 (417) 1 800 (100) 2 000 (200) 2 330 (640) 4 240 (200) 4 000 (80) 1 024 (100) 2 560 (239) 3 600 (158) 10 000 (133) 10 000 (100) 800 (50) e 800 (230) 2 560 (100) 2 560 (1 6) 4 300 (73) 12 000 (42) 2 560 (40) 10 240 (63) 7 000 (56) 640 (100) 320 (200) 350 (100) 3 580 (56) 5 520 (60) 1 420 (51) 7 200 (100) 4 520 (152) 1 550 (67) >3 400 (200) 3 770 (82) >8 400 (251) 14 500 (167) 9 640 (57) 2120 (90) 2 580 (192) 2 300 (165) 4 520 (172) 4 120 (47) 2 560 (100) 20 000 (46) 2 560 (100) 16 000 (100) 256 (100) 640 (32) 620 (36) 7 150 (67) 8 450 (59) 415 (126) 6 400 (100) 406 (46) a Berkeley reported the preinoculation CAl 5 serum titre as 1: 20 by mouse NT test but as negative by TC test. The other laboratories reported all preinoculation sera as negative. b Houston tested 4 individual vials of dried serum. At least 2 separate tests were performed, usually 2 vials per test. Geometric mean titres are reported. Ottawa reported the geometric mean titre per 0.2 ml on CAI 6 serum. Epsom tested several sera more than once to obtain end-points. c Copenhagen and Epsom reported titres per 0.05 ml; Berkeley and Houston per 0.025 ml; Atlanta per 0.02 ml; and Tokyo per 0.015 ml. d The figures in parentheses indicate the LDso or TCDro values found in the test. e Pooled serum from 2 vials was also tested simultaneously with NIH reference monkey serum using a virus dose of 100 TCDso. The titre of the equine serum was 800; that of the monkey serum was 6 400-a value similar to that reported by Kamitsuka et al. (1965). ENTEROVIRUS REFERENCE ANTISERA exception of the tests performed on the CA18 and CA20 sera. One laboratory (Epsom) reported an unusually high titre for the CA18 serum when tested by both the mouse neutralization and TC methods. A second laboratory (Houston) reported significantly higher titres for the CA20 serum than were found by two other laboratories. In both cases, repeated tests by the laboratories concerned confirmed the original values assigned to the sera. The geo- metric mean titres of the 14 sera were calculated where feasible and the mean values assigned by the mouse NT and TC methods agreed surprisingly well. The quality of the CA24 serum was found to be poor: tests performed by 5 laboratories indicated that its titre was only about 1: 400. The Berkeley laboratory also found that the serum had little or no neutralizing activity against several antigenic variants of this immunotype (DN-19, Hu-39, and Pett) (Schmidt & Lennette, 1970). Heterotypic neutralization tests. The 14 coxsackie- virus A sera were tested for heterotypic antibody against the entire series of enteroviruses with the exception of enterovirus 68 (Fermon). The reports of positive heterotypic tests-i.e., sera with heterotypic titres of 1: 10 or above-are summarized in Tables 3, 4, 5, and 6. Tests against type A coxsackieviruses. No hetero- typic antibody to the 23 type A coxsackieviruses was found in the CA2, 4, 10, 18, and 24 immune sera subjected to the mouse NT (Table 3) These sera were also found to be negative in TC neutralization Table 3. Results of heterotypic tests a on 14 coxsackievirus type A equine sera b against coxsackievirus types Al -22 and 24 Antibody titre laboratory Serum Challenge virus Copenhagen Epsom Houston Berkeley mouse NT mouse NT TC test TC test mouse NT TC test per 0.05 ml per 0.05 ml per 0.1 ml per 0.1 ml per 0.05 ml per 0.1 ml CA8 CA3 320 (25)C 220 (56) 320 (100) 1 280 (1 000) CAll CAl5 100 (40) 40 (134) 0 0 CA17 60 (32) 14 (316) 20 (147) 0 CA24 40-100 (100) 0 20 (147) 0 CA13 CA17 >160 (32) 11 (316) 0 0 CA18 40-320 (158) 280 (133) 320 (68) 80 (32) CA24 20 (100) 0 0 0 CA14 CAl 15 (316) 0 CAll 15 (40) 14 (178) 10 (215) CA12 0 0 10 (100) CA15 CA2 >2 560 (80) 7 700 (316) 7 000 (100) 1 280 (100) 1 280 (320) CA5 20 (40) 0 CA8 80 (50) 0 0 CAl1 0 640 (133) 450 (46) 0 2 650 (320)d 40 (100) CA17 160 (32) 28 (316) 0 0 CA18 0 28 (100) 0 0 CA24 20(100) 28 (100) 0 0 CA16 CA12 0 0 10 (215) 0 CA19 CAl 10 (316) 0 0 CA20 CAll 80 (100) 34(133) 20 (250) 0 CA18 20 (158) 70(133) 20 (68) 0 CA21 CAll 40 (40) 0 0 a All tests were negative at a dilution of 1 :10 or lower except those shown in the table. In those cases where positive results were reported, negative tests where found are also shown. b No heterotypic antibody was found in the CA2, 4, 10, 18, and 24 immune sera. Copenhagen and Epsom performed mouse NT against CA viruses 1-22, and 24. Epsom also tested the sera in tissue culture against CA viruses 2-4, 7-18, 20, 21, and 24. Houston tested CA10 serum in tissue culture against CA viruses 2-4, 8, 11-15, 17, 18, and 24. c The figures in parentheses indicate the LD5o or TCDso values found in the test. d Preinoculation serum titre 1 40. 385 386 JOSEPH L. MELNICK & BETTYLEE HAMPIL Table 4. Results of cross-neutralization tests with sera and viruses CA3 and CA8; CAl 3 and CAl 8; and CA2 and CAl 5 Neutralizing antibody titre a Challenge CA3 serum CA8 serum CAI 3 serum CAI 8 serum CA2 serum CAI 5 serum virus mouse mouse mouse mouse mouse mouse NT TC NT TC NT TC NT TC NT TC NT TC CA3 2 000 290 950 CA8 80 16 3580 7 200 CA13 2 560- 4 770 <10 <10 4500 CA18 200 160 4 520 800- 10 000 CA2 5 820 6 320 7 000 7 000 CA15 <10 <10 9 640 2 580 a Data taken from Tables 2 and 3 and from the text. Geometric mean titres recorded wherever feasible. tests, except for tests on the CA18 serum against CA7 and CA9 viruses performed by one laboratory. The results of 3 other laboratories were negative (see Table 5A. However, as Table 3 shows, 9 sera (CA8, 11, 13-16, and 19-21) were found by one or more laboratories to contain heterotypic antibody against at least one coxsackievirus A. Positive results were obtained more frequently with the mouse NT than with the TC method of assay. Heterotypic antibody of significant titre was found in 3 sera: the CA8 serum against CA3 virus, the CA13 serum against CA18 virus, and the CA15 serum against CA2 virus. The results are recapitul- ated in Table 4 to show the two unidirectional neutralizations and the cross-neutralization of CA3 and CA8 sera and viruses.' The heterotypic titre of the CA15 serum against CA2 virus was found to be extraordinarily high-of the same order of mag- nitude as the homologous titre of the CA2 serum (see Table 2). Two laboratories also reported high levels of heterotypic antibody against CA 1I virus in the CA15 serum (Table 3), whereas two other laboratories reported negative results. Tests against viruses P1-3; CA7, 9, and 16; CBJ-6; and El-9, 11-27, and 29-33. The results of positive heterotypic tests against the enteroviruses that grow in monkey kidney tissue culture cells are recorded in Tables 5 and 6, except for those per- 1 CA3 equine serum, prepared after these studies had begun, was not available for testing by all laboratories. The results reported are those of Houston and Berkeley and are included for the information of laboratories using both sera. formed by one laboratory with echoviruses 7, 8, and 9. That laboratory (Lyon) reported heterotypic titres ranging from 1: 10 to 1: 80 in most of the immune sera as well as in the corresponding pre- inoculation sera. For brevity, these values do not appear in Table 6 whenever 3 other laboratories re- ported negative results. As shown in the tables, heterotypic antibodies, where found by a single laboratory, were of a low level, and similar tests performed by other laboratories were negative. Tests on echovirus sera (E21, 27, 30, 31, and 33) Homologous tests. The 5 echovirus sera were tested for homologous antibody titres by 10 laboratories. Table 7 shows the results and the TC cell systems employed in the tests. Three laboratories encountered difficulties in tests on the E27 serum and reported titres of less than 1: 100. Two of these laboratories (Ottawa and Tokyo) also used filtered virus suspensions, but failed to enhance the titre by the use of these sus- pensions. In contrast, two other laboratories (London and Lyon) reported homologous antibody titres of 1: 19 000 on the E27 serum. An exchange of information by Ottawa and Houston led to investiga- tions by both laboratories on the effect of filtration of the virus suspension on the results of the serum neutralization test. Ottawa used Houston seed virus and Houston used virus derived from the same source as the Ottawa seed. The results are sum- marized in Table 8. It appears that small differences in methods of filtration may affect monodispersion and that certain suspensions of prototype E27 virus ENTEROVIRUS REFERENCE ANTISERA Table 5. Summary results of heterotypic tests on 19 enterovirus immune sera (CA2, 4, 8, 10, 11, 13-16, 18-21, 24 and E21, 27, 30, 31, 33) against P1-3, CA7, 9, 16 and CB1-6 viruses a Antibody titre per 0.1 ml d Immune Heterotypic posiietst sserum b virus ye tests 6 negative tests laboratory titre CA2 CB3 CB4 CA8 CA7 CAI 0 CB3 CB4 CAl1t CB3 CB4 CA13 P1 P3 CA14 CA7 CAI 5 P2 CAl 6 CB4 CA1 8 CA7 CA9 CA20 CA7 E27 CA7 CB4 E30 CA9 CB3 CB4 E31 P1 CA9 CAl 6 E33 CB4 CB6 Lyon Lyon Lyon Lyon Lyon Lyon Lyon Lyon Ottawa Lyon Ottawa Lyon Lyon Ottawa Ottawa Lyon Lyon Lyon Lyon Lyon Lyon Lyon Ottawa Lyon Lyon Ottawa Lyon Ottawa Lyon Ottawa 10 10 10 10 10 20 10 70 71 70 20 40 30 24 17 20 10 10 25 10 10 10 11 10 20 10 10 10 30 14 Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Singapore Melbourne, Singapore Melbourne, Ottawa, Singapore Melbourne, Singapore Epsom, Melbourne, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Melbourne, Prague, Singapore Melbourne, Ottawa, Singapore Melbourne, Ottawa, Singapore Lyon, Melbourne, Prague, Singapore Melbourne, Ottawa, Prague, Singapore Melbourne, Prague, Singapore Melbourne, Ottawa, Singapore Lyon, Melbourne, Singapore a Results of neutralization of the challenge heterotypic virus at a serum dilution of 1 :10 or greater reported by 1 or more laboratories are shown; in such instances the laboratories reporting negative results are listed. Challenge doses of 32-320 TCD5o were used in the tests. For the cell cultures and viruses em- ployed by each laboratory, see Table 1. b All tests were negative on the CA4, CAI 6, CAI 9, CA21, CA24, and E21 sera (Berkeley, Lyon, Melbourne, Ottawa, Prague). c The challenge virus showing positive results is shown; tests against the other viruses were negative. It Ottawa reported geometric mean titres per 0.2 ml. e When positive results were found, Ottawa tested the corresponding preimmune serum, with negative results. Lyon did not test the preimmune sera. 387 388 JOSEPH L. MELNICK & BETTYLEE HAMPIL Table 6. Summary of results of positive echovirus heterotypic tests a reported by 8 laboratories b on 14 group A CA4 serum CA8 serum CAl 1 serum CAl 3 serum Laboratory against viruses: against virus: against viruses: against viruses: E4 E33 E4 E4 E18 E3 E4 E23 Atlanta 0 Lyon 1od lod lod 30d l0d Melbourne 0 0 0 0 0 Moscow 0 0 Ottawa 0 i of 0 0 0 0 Prague Singapore 0 0 0 0 0 Tokyo 0 0 i of Laboratory CAl 8 serum against viruses: CA20 serum against viruses:rEl E3 E4 E5 E8 E20 El E4 E6 E7 E8 El1 E21 Atlanta 0 0 Lyon l0d 80 d 20d 0 48 0 20 d lo0d 56 20 15d(13)e (17)e (11)e Melbourne 0 0 0 10f 20!f if 0 0 14f iof 0 Moscow 20 d Ottawa 10f 36 0 0 0 iof 0 0 0 0 0 13 f(28) e Prague Singapore 0 0 0 0 0 0 0 0 0 0 0 Tokyo 0 0 a Tests were made against echoviruses 1-9, 11-27, and 29-33. The viruses used by each laboratory are shown in Table 1. Challenge doses of 32-320 TCDco were employed. Neutralization of the challenge virus at a serum dilution of 1: 10 or greater by one or more labora- tories is shown; in these cases, all heterotypic tests, including those giving negative results, are listed. Berkeley tested CA23 serum against the entire series of echoviruses. Low heterotypic titres were reported against low test doses of El, 5, 7, 12, 17, 18, 21, and 29 viruses. b Lyon reported heterotypic antibody titres against echoviruses 7, 8, and 9 in most of the immune sera as well as in the corresponding preinoculation sera, with titres ranging from 1: 10 to 1: 80. For brevity, these results are omitted in instances where three other laboratories reported negative results (see text). contain small aggregates of virus particles that seriously affect the results of tests for neutralizing antibody titre (Wallis & Melnick, 1967).1 The titres assigned to the other 4 echovirus sera (E21, 30, 31, and 33) also varied widely. However, in most cases, there is less than a 4-fold difference between the individual titres reported and the calculated geometric mean titres. 1 We are indebted to Professor Craig Wallis for supplying the following details of the method used routinely in the Houston laboratory for the preparation of monodispersed enterovirus suspensions. A 25-mm microsyringe holder (Millipore Filter Corpora- tion, Bedford, Mass.) containing a Millipore membrane (average pore diameter: 0.05 ,um) is washed with 5 ml of 10% calf serum ultrafiltrate. The excess serum is removed by washing with 20 ml of saline buffered with 2-amino-2- (hydroxymethyl)-1,3-propanediol (" tris "). The residual fluids are then removed by repeatedly forcing air through the unit with a syringe. Before the syringe is reloaded with air, it is important to remove the barrel from the filter holder with the plunger intact. In this way, the membrane is not subjected to negative pressure, which may cause it to break or crack at the gasket screen. The virus suspension, previously clarified by centrifugation at 2 500 rev/min for 15 min, is diluted 1: 10 in distilled water for filtration; 5 ml of the 10% suspension is then filtered through the membrane. Gentle negative or positive pressure is exerted to permit filtration to proceed drop by drop for collection of the monodispersed virus suspension; the suspension is used reasonably soon after filtration so as to prevent reaggregation of the virus particles through prolonged standing. The serum ultrafiltrate is prepared as 10% fetal calf or calf serum in tris-buffered saline filtered successively through 0.22 pm, 0.1 ,um, and 0.05 pm Millipore membrane filters. Such a preparation, if stored in the refrigerator, is suitable for the pretreatment of filters for many months. ENTEROVIRUS REFERENCE ANTISERA coxsackie virus sera c CAl 4 serum CAl 5 serum CAl 6 serum against viruses: against viruses: against viruses: E4 E6 E7 El E3 E7 E8 Eli E12 E29 E32 El E3 E4 E7 l0d 15d l0d 0 30 lod 20d 10 33 20d (24) e (24) e (1 0) e 0 0 0 0 0 0 28f 0 0 0 0 0 10 0 0 0 10 18 0 0 0 0 0 iof 16 0 0 II f(12)e (12)e (1 0)e 0 0 0 14d 0 0 0 0 0 0 0 0 0 0 0 10f 40f 0 CA21 serum against viruses: CA24 serum against viruses: El E4 E5 E7 E8 ElI E32 El E2 E3 E4 E5 E7 E8 Ell E18 0 lod l0d 0 24 20 15d 0 0 0 l0d 0 47 32 15d (10)e (11)e (7)e (32)e 12f 0 10f 0 20f 0 17f 10f 0 0 10f 14f 160f 0 0 14f 0 0 iof 0 0 12f 0 0 0 0 36f 14f 0 0 0 0 0 0 0 0 0 0 0 0 0 10f 0 0 0 l0d c The 14 sera tested were: CA2, 4, 8, 10, 11, 13-16, 18-21, and 24. All tests were negative on CA2 and CAl 9 sera, except tests against E7, E8, and E9 viruses by Lyon (see footnote b). d Preinoculation serum not tested. e The figures in parentheses indicate the preinoculation serum titres. f Preinoculation serum reported either negative or less than 1 5. Heterotypic tests. The 5 echovirus sera were tested for heterotypic antibody against the entire series of enteroviruses including E34 (DN-19) virus. One laboratory (Copenhagen) tested all the sera by the mouse neutralization method against cox- sackieviruses types CA1-22 and 24. The tests were negative at the 1: 10 dilution of the sera except for 2 tests performed on the E21 serum. This serum neutralized CA12 virus at a dilution of 1: 40 and CA17 virus at a dilution of 1: 30. Tables 5 and 9 record the positive heterotypic titres found in tests against the prototype entero- viruses that are grown in monkey kidney cell cultures. Tests against viruses P1-3; CA7, 9, and 16; and CBJ-6. Positive heterotypic titres, when found by one laboratory in tests against these viruses (Table 5), were of a low order of neutralizing activity and diverged from negative heterotypic findings of several (usually three or more) other laboratories that performed similar tests. Tests against El-9, 11-27, and 29-33 viruses. Nine laboratories participated in the testing of the 5 echovirus sera for heterotypic antibody against these echoviruses. Both the positive and the negative results are given in Table 9, which also shows the viruses used by each laboratory. One laboratory (London) also tested the sera against E34 (DN-19) 389 JOSEPH L. MELNICK & BETTYLEE HAMPIL Table 7. Results of homologous tests on 5 echovirus equine sera (E21, E27, E30, E31, E33) a Serum antibody titre per 0.1 ml b Laboratory E21 E27 E30 E31 E33 Atlanta 640 (148) c 640 (144) 2 560 (320) 2 560 (100) 1 280 (32) Houston 2 000 (160) 2 000 (150) 5 500 (320) 9 600 (100) 20 000 (160) London 6 730 (100) 19 020 (180) 11 310 (1 000) 2 263 (180) 3 365 (320) Lyon 9 600 (317) 19 200 (41) 25 600 (67) 38 400 (67) Melbourne 1 000 (120) 2000 (47) 1 200 (132) 2450 (83) 16000 (41) Moscow 1 540 (71) 3400 (250) 1 200 (250) 3400 (35) Ottawa 6 730 (200) 41 (224)d 15 300 (112) 5660 (141) 18000 (68) 4 760 (500)6 Prague 1 200 (200) <200 (125) 2000 (200) 4000 (320) Singapore 4 742 (140)f 8 269 (260) 22 387 (57) 8 000 (316) 4000 (32) Tokyo 9400 (100) 51 (120)d 2025 (215) 14890 (320) 40960 (215) Geometric mean titre 2 990 (157) 3 590 (84)h 4 940 (222) 4 760 (165) 10 000 (96) a Serum neutralization tests on the E21 serum were performed in a variety of cell cultures: (1) stable lines of HEL cells (Atlanta, Lyon, Melbourne, Tokyo); (2) HA cells (Houston, London); (3) HeLa cells (Prague); (4) RhK cells (Ottawa); and (5) Cyn K cells (Tokyo). Melbourne used HeLa and HEL cell cultures for tests on E27 serum; HEL cells on E31 serum; and HeLa cells on E30 and E33 sera. The other laboratories employed monkey kidney cell cultures. b The results shown for Houston, Melbourne, Ottawa and, in some cases, Singapore and Tokyo are geometric mean titres of sera either reported or calculated by the authors. Ottawa reported titres per 0.2 ml. c The figures in parentheses indicate TCDso values found in the tests, or geometric mean TCDso values. d Unfiltered and filtered virus suspensions were employed. For further tests by Ottawa, see text and Table 8. 6 A filtered virus suspension was employed. f Geometric mean titre using NIH reference virus. Tests were read after 5 days' incubation. g Titre found with American Type Culture Collection virus; 7-day readings. h The values reported by Ottawa, Prague, and Tokyo were not used in the calculations. Table 8. Effect of filtration of certain virus suspensions of prototype echovirus 27 on the homologous antibody titre of the antiserum Laboratory Virus suspension Antibody titre TCD5o used Houston a unfiltered 355 400 filtered 2 445 400 Ottawa b unfiltered 81 170 filtered 427 148 a Houston reported similar results with the plaque count reduc- tion method. b Geometric mean titres of several tube neutralization tests are reported. virus, with negative results. Although it appears from the table that a higher incidence of heterotypic anti- body was found in the sera against the echoviruses with lower numbers (El-I 1), most of the positive tests were reported by a single laboratory. If the results from that laboratory are omitted from con- sideration, it will be seen that echovirus heterotypic antibody of low titre was found only infrequently. The exception is found in the tests on the E31 serum. Here, 5 laboratories reported heterotypic antibody against E5 virus with titres ranging from 1: 20 to 1: 400. A sixth laboratory (Houston) also tested this serum against ES virus with positive results (titre: approximately 1: 500). The preinocula- tion serum was negative. One laboratory (Ottawa) also tested the E31 serum against 2 field isolates of E5 virus, both of which were neutralized by the E31 serum at serum dilutions of approximately 1: 1 500. Heterotypic tests against adenoviruses and reoviruses The 19 sera were negative for heterotypic antibody against reoviruses 1-3 (Tokyo) and adenoviruses 1-31 (Atlanta). The HI method was employed for the tests. 390 ENTEROVIRUS REFERENCE ANTISERA 391 Homotypic tests As in previous studies, field isolates available in the participating laboratories were used as challenge viruses for either titration of the type- specific antisera or use of the sera at a given antibody unit level. Reports were received on 6 enterovirus types (CA16, E21, E27, E30, E31, and E33). Table 10 shows the results of each test on each isolate and the laboratory performing the test. The homologous titre assigned to the type-specific serum by the laboratory is also listed for comparison. Although the homotypic titres reported were often lower than the homologous titres of the type-specific serum, the usefulness of the sera was clearly demonstrated. COMMENTS With the completion of this collaborative study on 19 enterovirus equine antisera, the number of such antisera that have been examined for homologous, homotypic, and heterotypic antibodies now totals 60 and encompasses the entire series of recognized types of enterovirus with the exception of coxsackievirus types A3 and A17, echovirus 8, and enterovirus 68.1 In this study, as in the earlier investigations, the careful attention given to details in the tests is evi- dent from the excellence of the summaries of the results submitted by the participating laboratories. Repeat tests were performed frequently when indicated and a wealth of information is available for the guidance of laboratories using the sera. In general, the homologous titres assigned to the sera by the cooperating laboratories agreed well, with 3 exceptions. Three laboratories reported homologous titres of less than 1: 100 for the E27 serum, whereas 7 other laboratories found the serum to be of a useful titre, with titres ranging from a low of 1: 640 to a high of 1: 19 000. Presumably, certain prototype E27 virus suspensions contain an unusual number of aggregated virus particles and require special methods of filtration for the preparation of a monodispersed virus suspension (Wallis & Melnick, 1967). There was also a wide range in the titres assigned to the CAI 8 and CA20 sera when the serum neutralization tests were performed in tissue culture, although in both cases the lower values were of a useful titre (1: 800 and 1: 2 560, respectively). The results of tests performed on field isolates of 6 enteroviruses (CA16, E21, E27, E30, E31, and 1 Equine antisera against viruses CA3, CA17, and E8 have since been prepared and are now available. E33) demonstrated the usefulness of the type- specific equine antisera. In some cases, the homotypic titres were equivalent to the homologous titres, although most of the values were lower. It appears that it may prove difficult to identify some strains of E27 readily. No field isolates of the type A coxsackieviruses other than CA16 were available for study. An equally important facet of the programme is the abundance of information collected on the heterotypic properties of each of the 19 antisera investigated. All the sera were negative to reo- viruses 1-3 and adenoviruses 1-31 when tested by the haemagglutination-inhibition method. The entire series of enteroviruses, with the excep- tion of enterovirus 68, was also used in heterotypic tests, each serum being tested against a given entero- virus by at least two laboratories and more often three or four. Most of the tests were negative; low heterotypic titres (approximately 1: 10) where re- ported were usually not confirmed by other labo- ratories performing similar tests. However, in 4 sera there is confirmed evidence of the presence of hetero- typic antibody of significant titre against at least one enterovirus. These are: E31 serum against E5 virus; CA8 serum against CA3 virus; CA13 serum against CA18 virus; and CA15 serum against CA2 virus and possibly CAI1 and CA17 viruses. Heterotypic anti- bodies of low titres against CAll and CA18 were also reported to be present in the CA20 serum. These findings require careful consideration in the prepara- tion of combination serum pools, particularly at the 50 antibody unit level, although such pools are entirely feasible. Typing antisera may be used more effectively when combination pools are made (Lim & Benyesh- Melnick, 1960; Schmidt et al., 1961). The practic- ability of designing 8 combination pools containing 42 enterovirus equine sera (P1-3; CA7, 9, and 16; CB1-6; and E1-7, 9, 11-27, and 29-33), using each immunotype at the 50 antibody unit level, has recently been demonstrated (Schmidt et al., 1971). The pools were tested not only against the prototype strains but also against a number of field isolates of each available type. Sets of 8 dried combination serum pools of similar composition have been pre- pared for long-term storage and use (Melnick et al., 1973). A design for the preparation of satisfactory com- bination pools containing 19 coxsackievirus type A equine sera (CA1-6, 8, 10-15, and 17-22) is under consideration. 392 JOSEPH L. MIELICK & BETTYLEE HAMPIL Table 9. Summary of results reported by 9 laboratories a on heterotypic tests on 5 echovirus antisera (E21, 27, Antibody titre per 0.1 ml c Challenge E21 serum E27 serum E30 echovirus___________________________________________________________ A Lo Ly Me Mo 0 P S T A Lo Ly Me Mo 0 P S T A Lo Ly Me El E2 E3 E4 E5 E6 E7 E8 E9 Eli El12 El13 El14 El15 El 6 El17 El18 El19 E20 E21 E22 E23 E24 E25 E26 E27 E29 E30 E31 E32 E33 0 0 0 0 0 201 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0d 0 1 0ed 0 1 od 0 29Q 1 2e 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 12e 0 0 0 0 0 499 0 18i 11e 40e 0 20d 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 a The participating laboratories were: Atlanta (A), London (Lo), Lyon (Ly), Melbourne (Me), Moscow (Mo), Ottawa (0),Prague (P), Singapore (5), and Tokyo (T). b The results of the neutralization of the challenge virus at a serum dilution of 1 : 10 or greater reported by 1 or more laboratories are shown. Absence of neutralization at serum dilutions of less than 1:10O is shown as 0. Challenge doses of 32-320 TCDao were used. For cell cultures employed, see Table 1. c Ottawa reported geometric titres per 0.2 ml. The results of tests by Lyon against E7, E8, and E9 viruses are shown as geometric mean titres per 0.1 ml. d, Preinoculation serum not tested. e Preinoculation serum negative. ENTEROVIRUS REFERENCE ANTISERA 393 30, 31, and 33) against echoviruses 1-9, 11-27, and 29-33 bi Antibody titre per 0.1 mIC serum E31 serum E33 serum MO 0 P S T A Lo Ly Me Mo 0 P S T A Lo Ly Me Mo 0 P S T 20e 0 0 20d 28e 25e 10d 0 15d 0 25e 106 0 17e 0 0 20d 0 10e 0 0 0 0 0 0 0 0 12d 0 30d 0 0 1 6d 0 1 od o 0 0 0 0 0 0 1204 0 368 0 406 0 0 0 0 0 0 0 0 320d 166 4336 67f 20e 1 od 0 1oe 0 0 0 0 0 Iod 0 1060 0 0 0 0 0 0 14e 16d 0 579 0 28h 49d 0 306 0 0 0 0 16e 0 0 326 34e 186 0 0 37t 0 22e 14d 0 0 0 0 42J 0 0 0 0 60. 0 0 1oe 0 0 0 0 15d o0 00 0 10d 0 0 0 0 0 0 0 0 1060 0 0 0 0 0 0 0 0 0 20f 0 1od O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 I0O 0 0 10d 0 0 0 0 0 0 0 0 0 0 0 0 0 10e 0 14d 0 0 0 0 0 106 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20k 0 0 0 0 o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14e 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 281 28m 0 0 20e 13n 0 13e 0 0 0 146 0 f Preinoculation serum titre less than 1: 10. O Preinoculation serum titres of E21, E27, E30, and E31 sera against E7 virus were 1:21, 1:10. 1:30, and 1:40, respectively. h Preinoculation serum titre 1 :32. i Preinoculation serum titres of E21, E27, E30, and E33 sera against E8 virus were 1: 23, 1: 11, 1:11, and 1: 13, respectively. J Preinoculation serum titres of E21, E32, and E33 sera against E9 virus were 1:10, 1: 20, and 1:40, respectively. k Preinoculation serum titre 1: 20. I,mI Preinoculation serum titres 1 :14, 1 :40, and 1 :10, respectively. 2 JOSEPH L. MELNICK & BEITYLEE HAMPIL Table 10. Results of tests for homotypic serum antibody titre of CAl 6, E21, E27, E30, E31, and E33 sera virus isolate Laboratory N so.of Serum neutralizing antibody titre a Homologous titre reported b CAI 6 Atlanta c 16 neutralized by 20 antibody units in 1 280 (100) in mouse NT mouse NT (100) d Epsom 22 e geometric mean titre 537 in mouse NT 3 000 in mouse NT titre range (94-1 368) LDso range (32-215) Ottawa 1 380 (100) 4 240 (200) Tokyo 2 1 024 (20), 512 (50) 3! 256 (50), 64 (200), 64 (250) in TC 1 024 (100) in TC 147 (1 000), 512 (300), 512 (300) 2 048 (100) in mouse NT in mouse NT E21 Atlanta c 1 neutralized by 20 antibody units (100) 640 (148) Melbourne 3 345 (96), 4 000 (300), 749 (260) 1 000 (120) Tokyo 2 217 (69), 4551 (262) 9400 (100) E27 London 1 32000 (180) 19020 (180) Melbourne 1 180 (180) 2000 (47) Tokyo 1 32 (320) 51 (120) E30 Atlanta C 18 neutralized by 20 antibody units (100) 2 560 (320) (late break-throughs with 2 strains) Melbourne 4 150 (260), 3 350 (38), 1 410 (55), 1 200 (132) 592 (155) E31 Atlanta C 4 neutralized by 20 antibody units (100) 2 560 (100) London 1 13460 (56) 2263 (180) Melbourne 3 2 230 (110), 2 300 (55), 434 (177) 2 450 (83) Ottawa 2 160 (316) UF,96 170 (500) UF 5660 (141) UF 707 (316) F, 3 670 (316) F 4 760 (500) F E33 Melbourne 4 10500 (41), 8000 (75), 8000 (137), 16000 (40) 8 000 (750) a Antibody titres found per 0.1 ml in tissue culture tests (TC) or per 0.015-0.02 ml in mouse NT are shown for each field isolate. Where the results of 2 or more tests were reported,the calculated geometric mean titres are recorded. Ottawa reported geometric mean titres per 0.2 ml. b Values taken from Tables 2 and 7. c Serum dilution end-points were not reported. d The figures in parentheses indicate the TCD50 or LDso used in the test. 6 Isolated in the United Kingdom between 1961 and 1971. The results confirm earlier findings (unpublished data) that the CA16 virus circulating in the area since 1960 is a prime strain. f These 3 strains isolated from vesicles of patients with foot-and-mouth disease were tested by both the TC method and the mouse NT ° Unfiltered (UF) and filtered (F) virus suspensions were employed. ACKNOWLEDGEMENTS The participants in the various laboratories gratefully acknowledge the cooperation ofDr R. J. Byrne, Research Resources Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health. The preparation and testing of these antisera were supported by contracts PH 43-68-1044, PH 43-63-1177, PH 43-64- 529, PH 43-62-180, and NIH 69-89 from the Research Resources Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md., USA, and by grants from the World Health Organization. The sera were prepared as part of a collaborative programme involving Baylor College of Medicine (Dr J. L. Melnick and Dr B. Hampil), South- west Foundation for Research and Education (Dr S. S. Kalter), University of Kansas School of Medicine (Dr H. A. Wenner), and Tuskegee Institute (Dr R. W. Brown). 394 ENTEROVIRUS REFERENCE ANTISERA 395 RItSUME ETUDES COLLECTIVES DE L'OMS SUR LES SERUMS DE REFERENCE ANTI-ENTEROVIRUS: 40 RAPPORT Le pr6sent rapport expose l'essentiel des r6sultats obtenus au cours du 4e stade des 6tudes concert6es entre- prises par le Centre international OMS de r6f6rence pour les ent6rovirus ainsi que par un certain nombre de centres r6gionaux OMS de r6f6rence pour les virus et de labora- toires collaborateurs, dans le cadre d'un programme glo- bal d'essai de serums 6quins anti-ent6rovirus destin6s a servir de pr6parations de r6f6rence. On se proposait d'evaluer la specificit6 de 19 immun- s6rums de chevaux inocul6s par des souches prototypes d'ent6rovirus (coxsackievirus A 2, 4, 8, 10, 11, 13-16, 18-21 et 24; 6chovirus 21, 27, 30, 31 et 33). Des 6preuves de neu- tralisation ont ete pratiqu6es pour mettre en 6vidence les anticorps a l'6gard des virus homologues et des souches homotypiques dont disposait chaque laboratoire. On a egalement recherche les anticorps h6t6rotypiques pour les reovirus 1-3, les adenovirus 1-31 et 1'ensemble du groupe des enterovirus (a 1'exception de lent6rovirus 68). Pour les 5 serums anti-6chovirus, la moyenne geome- trique des titres homologues variait de 3000 a 10 000. Le titre du s6rum anti-coxsackievirus A24 n'6tait que de 400 alors que les titres des autres s6rums anti-coxsackievirus A s'6tageaient de 1500 a 14 000. Tous les s6rums t6moins pr6leves avant l'immunisation 6taient n6gatifs. Des titres 6leves d'anticorps het6rotypiques ont ;te deceles dans 4 s6rums: dans le serum anti--chovirus 31 pour 1'6cho- virus 5; dans le s6rum anti-coxsackievirus A13 pour le coxsackievirus A18; dans le serum anti-coxsackievirus A15 pour le coxsackievirus A2; dans le serum anti- coxsackievirus A8 pour le coxsackievirus A3. Les epreuves homotypiques pratiqu6es avec les isolats locaux ont per- mis de v6rifier l'utilit6 des antis6rums. REFERENCES Hampil, B. & Melnick, J. L. (1968) Bull. Wld Hlth Org., 38, 577-593 Hampil, B. et al. (1965) J. Immunol., 95, 895-908 Kamitsuka, P. S. et al. (1965) Amer. J. Epidem., 81, 283-306 Lim, K. A. & Benyesh-Melnick, M. (1960) J. Immunol., 84, 309-317 Melnick, J. L. & Hampil, B. (1965) Bull. Wld Hith Org., 33, 761-772 Melnick, J. L. & Hampil, B. (1970) Bull. Wld Hlth Org., 42, 847-863 Melnick, J. L. et al. (1973) Bull. Wld Hlth Org., 48, 263-268 Midulla, M. et al. (1965) J. Immunol., 95, 9-12 Schmidt, N. J. et al. (1961) J. Immunol., 87, 623-626 Schmidt, N. J. & Lennette, E. H. (1970) Amer. J. Epidem., 91, 99-109 Schmidt, N. J. et al. (1971) Bull. Wld Hlth Org., 45, 317-330 Wallis, C. & Melnick, J. L. (1967) J. Virol., 1, 478-488 Annex LIST OF PARTICIPATING LABORATORIES WHO Regional Reference Centre for Respiratory Virus Diseases other than Influenza Fairfield Hospital Melbourne, Victoria, Australia (Dr A. A. Ferris & Dr I. D. Gust) WHO Virus Collaborating Laboratory Laboratory of Hygiene Ottawa, Canada (Dr Fred P. Nagler) WHO Regional Reference Centre for Respiratory Virus Diseases other than Influenza Institute of Epidemiology and Microbiology Prague, Czechoslovakia (Dr 0. SobNslavskS & Dr D. Kopecka) WHO Regional Reference Centre for Enteroviruses State Serum Institute Copenhagen, Denmark (Dr Herdis von Magnus) JOSEPH L. MELNICK & BETTYLEE HAMPIL WHO Regional Reference Centre for Enteroviruses Virology Laboratory Lyon, France (Dr R. Sohier & Dr Odette G. Gaudin) WHO Regional Reference Centre for Enteroviruses National Institute of Health Tokyo, Japan (Dr Isamu Tagaya) WHO Regional Reference Centre for Enteroviruses Department of Bacteriology, University of Singapore Singapore (Dr Kok Ann Lim & Dr Liang Hin Lee) WHO Regional Reference Centre for Enteroviruses Institute for Poliomyelitis & Virus Encephalitis, Moscow, USSR. (Dr Marina Vorosilova) WHO Virus Collaborating Laboratory Central Public Health Laboratory London, England (Dr C. M. Patricia Bradstreet) Public Health Laboratory West Park Hospital Epsom, Surrey, England (Dr D. R. Gamble) WHO International Reference Centre for Enteroviruses Baylor College of Medicine Houston, Tex., USA (Dr Joseph L. Melnick & Dr Bettylee Hampil) WHO Regional Reference Centre for Enteroviruses Center for Disease Control Atlanta, Ga., USA (Dr M. H. Hatch & Dr J. C. Hierholzer) WHO Regional Reference Centre for Respiratory Virus Diseases other than Influenza Center for Disease Control Atlanta, Ga., USA (Dr W. R. Dowdle) Viral and Rickettsial Laboratory California State Department of Public Health Berkeley, Calif., USA (Dr Nathalie J. Schmidt) 396