Arbovirus neutralization tests with Peruvian sera in Vero cell cultures

Bull. Org. mond. Sante' 1972, 46, 451-455 Bull. Wld Hlth Org. Arbovirus neutralization tests with Peruvian sera in Vero cell cultures S. M. BUCKLEY,' J. L. DAVIS, 111,2 J. MADALENGOITIA,3 W. FLORES,3 & J. CASALS' Selected human serafrom Peru, previously examined by the haemagglutination-inhibition (HI) test with a number of arboviruses, were reexamined by neutralization tests carried out in Vero cell cultures. Results confirmed and extended the HI findings, indicating that the antibodies detected were evoked by Eastern equine encephalitis, Mayaro, Venezuelan equine encephalitis, Ilheus, St Louis encephalitis, yellow fever, Caraparu, and Guaroa viruses. Over 1 000 serum specimens collected from resi- dents of eastern Peru during March-July 1965 were subsequently examined with 27 different arbovirus antigens in haemagglutination-inhibition (HI) tests carried out at this laboratory by three of us (J.M., W.F., and J.C.). The results (to be reported) indi- cated widespread experience by the population sur- veyed with the following arboviruses (or closely related agents): Eastern equine encephalitis (EEE), Mayaro, Venezuelan equine encephalitis (VEE), Ilheus, St Louis encephalitis (SLE), yellow fever (YF), Caraparu, Murutucu, Guaroa, and Maguari. Occasional sera reacted with antigen for Bussuquara virus or for bat salivary gland virus. These findings provided the basis for selecting sera for reexamination by neutralization (N) tests carried out in Vero cell cultures with potentiating accessory factor (Morgan, 1945; Whitman, 1947; Taylor et al., 1955). This paper reports the N-test results obtained and compares them with the HI-test data. MATERIALS AND METHODS Sera The donors of the survey sera were apparently healthy males and females, 5-75 years of age, resi- dent in the half of Peru lying east of the eastern 1 Yale Arbovirus Research Unit, Department of Epide- miology and Public Health, Yale University School of Medicine, New Haven, Conn., USA, and The Rockefeller Foundation. 2Yale University School of Medicine. 3 University of San Marcos, Lima, Peru. foothills of the Andes. After collection the sera were stored in a freezer at -20°C. For the N tests, the specimens were inactivated at 56°C for 30 min immediately before use. Virus strains The following viruses were used: EEE (Ten- Broeck), Mayaro (Tr 4675), Una (BeAr 13136), VEE (Trinidad Donkey No. 1), Mucambo (BeAn 8), Pixuna (BeAr 35645), Ilheus (original), SLE (BeAr 23379), YF (Asibi), Caraparu (BeAn 3994), and Guaroa (CoH 35211). Virus stocks, representing the lyophilized infected fluid phase of either the first or the second passage of each virus in Vero cell cultures, were held in 1-ml ampoules in a freezer at -20°C until used. For tests, two ampoules of the given virus were rehydrated, pooled, and held at room temperature for 20 min prior to dilution. Cell cultures Stationary tube cultures of Vero cell monolayers in the fluid phase were used according to procedures already described (Buckley & Casals, 1970). Accessory factor A supply of normal rhesus monkey serum,' known to be free of virus antibodies and of virucidal and cytotoxic substances, was kept in a freezer at -65°C in screw-cap tubes (2-5 ml). 1 Kindly supplied by Dr Dorothy Horstmann, Depart- ment of Epidemiology and Public Health, Yale University School of Medicine. 2818 - 451- S. M. BUCKLEY ET AL. Diluent The diluent consisted of 0.75% bovine plasma albumin (Armour fraction V) in 0.05 M phosphate- buffered saline, pH 7.2. Virus titrations Two ampoules of the given virus were rehydrated, pooled, and held at room temperature for 20 min before being prepared as serial 10-fold dilutions with diluent. Each dilution was inoculated into three replicate Vero cell cultures, which were examined at intervals for the development of cytopathic effect (CPE). Virus titres were calculated by the method of Reed & Muench (1938) and are expressed as cytopathic dose50 (CPD50) index (Haldane, 1960) per millilitre. N tests In the qualitative N test, approximately 200 CPD,O of virus was mixed in equal volumes with an un- diluted, inactivated survey serum. To this serum- virus mixture was added an equal amount of acces- sory factor (AF, normal rhesus monkey serum), the latter thus making up 50% of the total mixture. After incubation for 1 hour at 37°C, the mixture was inoculated into two Vero cell cultures, 0.1 ml per tube. With each qualitative test, virus titrations were carried out on the day of test. Hyperimmune mouse sera for the respective viruses, as well as human sera without antibodies, served as additional con- trols. Tests were terminated when the expected end- point of the virus titration was obtained. The quantitative N test with AF was carried out as described above, with certain modifications. (1) Each survey serum was diluted serially twofold from 1: 2 to 1: 1024. (2) Each control hyperim- mune mouse serum was also diluted 1: 2 to 1: 1024, and each control human serum was diluted 1: 2. (3) As an additional control, the virus dose used, 200 CPD5o, was mixed in equal parts with diluent, to which mixture was then added an equal part ofAF (representing 50% of the total mixture); after incu- bation for 1 hour at 37°C, the mixture was imme- diately diluted serially 10-fold and each dilution was inoculated into 6-12 Vero cell cultures. The CPD50 titres obtained from these titrations accurately indi- cated the actual CPD5O used in the quantitative N test with AF. In addition, quantitative N tests were carried out in which AF was replaced throughout by inactivated normal rhesus monkey serum. Inactivation was per- formed by heating serum in 1-ml amounts for 30 min at 56°C immediately before the tests. Table 1. Results obtained with selected Peruvian sera by qualitative neutralization tests in Vero cell cultures with accessory factor, compared with Hi test survey results Virus Sera Sero - No. No. No. group Name tested protective positivein N test in Hi test A EEE 11 8 8 Mayaro 100 68 70 Mucambo 100 53 58 Pixuna 100 13 49 Una 84 54 41 VEE 100 64 65 B liheus 89 58 57 SLE 8 7 8 YF 84 52 54 C Caraparu 100 65 68 Bunyamwera Guaroa 100 68 70 RESULTS Qualitative N test In these tests, a serum was considered " protec- tive " if the serum-virus-AF mixture failed to induce CPE in the two Vero cell cultures inoculated. As shown in Table 1, there was good correlation be- tween the results of this test and the HI results with all viruses except Una and Pixuna. With Una virus, 54 of the 84 sera tested were protective, whereas only 41 of the 84 were HI-positive. With Pixuna, the respective ratios were 13/100 and 49/100. Quantitative N test In these tests, the serum neutralizing titre was expressed as the highest serum dilution giving pro- tection in one (50%) of the two Vero cell cultures inoculated. Table 2 shows the results obtained when 15 sur- vey sera, characterized by HI pattern as " anti- Mayaro ", were tested against Mayaro and Una viruses in tests performed with and without AF. In the presence of AF, each serum neutralized Mayaro virus to a higher titre than it did Una virus, the data thus confirming the HI findings. In the absence of AF, the N test titres were exceedingly low and it was impossible to say which virus was responsible for the antibody detected. Since the titres obtained with the remaining sera in the absence of AF were 452 ARBOVIRUS NEUTRALIZATION TESTS WITH PERUVIAN SERA Table 2. Quantitative neutralization tests, carried out with and without accessory factor, with Mayaro and Una viruses and 15 Peruvian sera characterized by HI pattern as - anti-Mayaro a N test titre HI titre Serum no. Accessory Mayaro Una Mayaro Una factor 258 Yes 256 <2 20 10 No 2 <2 552 Yes >32 8 20 10 No 4 6 821 Yes >32 12 20 10 No 2 4 647 Yes 512 6 40 10 No <2 3 692 Yes 384 3 40 10 No <2 <2 194 Yes 1 024 8 80 20 No 3 2 130 Yes 384 1 6 160 20 No 2 8 183 Yes 768 8 160 20 No 2 4 241 Yes 768 24 160 20 No 2 4 275 Yes >1 024 6 160 1 0 No 2 6 266 Yes >1 024 16 640 80 No 3 6 925 Yes 768 32 640 80 No 3 2 213 Yes >1 024 128 1 280 80 No 32 16 373 Yes >1 024 32 1 280 80 No 6 3 786 Yes 192 32 1 280 40 No 2 8 a50 CPDso of Mayaro virus and 40 CPD5o of Una virus were used in test (final dilution). All titres are expressed as the reciprocal of the serum dilution. Table 3. Neutralization tests, using accessory factor, carried out with 22 Peruvian sera characterized by HI pattern as ' anti-EEE ' or - anti-VEE - 8 ' anti-EEE ' sera: 14 ' anti-VEE - sera: HI titre a N test titres with N test titres with range EEE VEE Mucambo Pixuna 20-40 32-64 12-48 <2-8 <2-12 80-160 128-256 64-384 3-24 6-64 >160 >1 024 48-96 16-48 a HI titres with EEE and VEE viruses, respectively. All titres are expressed as the reciprocal of the serum dilution. also low, on the whole, the subsequent tables report only the data for N tests performed with AF. As shown in Table 3, for 14 sera characterized by HI pattern as " anti-EEE ", N test titres with EEE virus were consistently higher than the HI titres. For 14 sera characterized as " anti-VEE ", cross- N test results also confirmed the survey findings and indicated that VEE, rather than Mucambo or Pixuna virus, was responsible for the antibody detected. Table 4 summarizes the results of cross-N tests with Ilheus, SLE, and YF viruses and 36 sera char- acterized by HI pattern as either " anti-Ilheus ", " anti-SLE ", or " anti-YF ". With very few excep- tions, the data again confirmed the HI findings. For 15 survey sera characterized as " anti-Cara- paru ", N test titres with Caraparu virus perfectly matched HI titres (Table 5). N test results with 15 " anti-Guaroa " sera and Guaroa virus likewise con- firmed the HI results (Table 6), although in this instance some discrepancies in titre were observed. Table 4. Neutralization tests, using accessory factor, carried out with 36 Peruvian sera characterized by HI pattern as n anti-llheus ', - anti-SLE -, or - anti-yellow fever - 13 ' anti-llheus - sera: 8 - anti-SLE ' sera: 15 'anti-YF' sera: HI titre a N test titres with N test titres with N test titres with range liheus SLE YF liheus SLE YF liheus SLE YF 20-40 32-64 <2 <2-1 6 <2-4 24-48 2-3 <2-3 <2-6 4-64 80-160 32-128 <2 4-48 <2-3 48-128 8-16 2-6 <2-12 128-256 160 + 16-768 2-64 + 8-512 <2-2 <2-24 4-16 <2-192 <2-64 96-192 a HI titres with, respectively, llheus virus (' anti-llheus - sera), SLE virus ( anti-SLE n sera), and YF virus (' anti-YF ' sera). All titres are expressed as the reciprocal of the serum dilution. 453 S. M. BUCKLEY ET AL. Table 5. Neutralization tests, using accessory factor, carried out with Caraparu virus and 15 Peruvian sera characterized by HI pattern as "anti-Caraparu - a Serum no. N test titre HI titre 965 <2 1 0 555 8 10 122 16 10 52 32 + 20 524 32 + 20 957 32 + 20 513 64 40 106 64 + 40 173 64 + 40 635 128 80 1024 128 80 817 128 160 512 256 + 160 841 256 + 160 445 512 + 320 a All titres are expressed as the reciprocal of the serum dilution. DISCUSSION I he Vero cell monolayer (in fluid phase) technique described in this paper may be used as a simple qualitative N test or as a more cumbersome, but also more relevant, quantitative N test to confirm and Table 6. Neutralization tests, using accessory factor, carried out with Guaroa virus and 15 Peruvian sera characterized by HI pattern as - anti-Guaroa" a Serum no. N test titre HI titre 290 8 20 678 48 20 810 32 40 142 128 40 203 192 40 702 384 40 353 64 80 622 256 80 734 32 160 417 192 160 160 192 320 887 512 320 826 384 640 125 1 024 + 640 947 128 1 280 a All titres are expressed as the reciprocal of the serum dilution. extend serological patterns established in extensive HI test surveys. The comparative data presented in Tables 2-6 show that, for EEE, Mayaro, VEE, Ilheus, SLE, YF, Caraparu, and Guaroa viruses, the quantitative N test with AF is as sensitive as the HI test. ACKNOWLEDGEMENTS This investigation was supported by The Rockefeller Foundation and by the United States-Japan Cooperative Medical Science Program administered by the National Institute of Allergy and Infectious Diseases, National Insti- tutes of Health, US Department of Health, Education, and Welfare, under Grant No. l-R22-AI-08215-OlAl RESUME EPREUVES DE NEUTRALISATION D'ARBOVIRUS PRATIQUtES A L'AIDE DE SERUMS DE PERUVIENS EN CULTURES CELLULAIRES VERO Plus de 1000 serums preleves en 1965 chez des habitants de l'est du Perou avaient ete examin6s en epreuves d'inhibition de l'hemagglutination (IH) en presence d'une serie d'arbovirus. On avait releve un fort taux de positivite a l'egard des virus: encephalomy6lite equine de l'Est, Mayaro, encaphalomyelite equine du Venezuela, Ilheus, encephalite de St Louis, fievre jaune, Caraparu, Murutucu, Guaroa et Maguari. 454 ARBOVIRUS NEUTRALIZATION TESTS WITH PERUVIAN SERA 455 On a choisi un certain nombre de ces serums et on les a reexamines en epreuves de neutralisation (N) qualitatives et quantitatives pratiqu6es en cultures cellulaires Vero avec addition de facteur accessoire (serum de singe rhesus). En 6preuves qualitatives, on a note une correla- tion satisfaisante entre les resultats des epreuves IH et N, sauf en ce qui concerne les virus Una et Pixuna. Le test quantitatif s'est rev61e aussi sensible que le test IH et a permis de confirmer les donn6es foumies par ce dernier. REFERENCES Buckley, S. M. & Casals, J. (1970) Amer. J. trop. Med. Hyg., 19, 680-681 Haldane, J. B. S. (1960) Nature (Lond.), 187, 879 Morgan, I. M. (1945) J. Immunol., 50, 359-371 Reed, L. J. & Muench, H. (1938) Amer. J. Hyg., 27, 493-497 Taylor, R. M., Hurlbut, H. S., Work, T. H., Kingston, J. R. & Frothingham, T. E. (1955) Amer. J. trop. Med. Hyg., 4, 844-862 Whitman, L. (1947) J. Immunol., 56, 97-108