Eur. J. Epidemiol. 0392-2990 June 1986, p. 112-117 EUROPEAN JOURNAL OF EPIDEMIOLOGY Vol. 2, No. 2 PREVALENCE OF ANTIBODY TO HUMAN CORONAVIRUSES 229E, OC43 AND NEONATAL CALF DIARRHEA. CORONAVIRUS (NCDCV) IN PATIENTS OF NORTHERN ITALY P.M. CEREDA 1 L. PAGANI E. ROMERO Institute of Microbiology, University of Pavia, 27100 Pavia, Italy. Key words: Hnnaan coronaviruses - 229E - OC43 - Bovine coronavirus NCDCV. A seroepidemiological study for detection of antibody to hatman coronaviruses OC43, 229E, and neonatal calf diarrhea coronavirus (NCDCV), has been carried out using sera collected from hospitalized patients or healthy persons through routine laboratory tests in Northern Italy. Patients tested were children and adults with different pathological diseases. Antibody detection was performed by using an indirect immunoperoxidase staining technique (gor all viruses) and, in the case of OC43 and NCDCV, antibody detection was obtained even with a ,hemagglutination inhibition test and a plaque reduction neutralization assay. Results obtained show a significant difference in the prevalence of antibody to 229E betTeen children and adult group. Furthermore, a different tirer was observed, within the two groups, between patients affected by hematological diseases (leukemia) and patients w&h other diseases. Finally, our data seem to confirm previous studies reporting a very high prevalence of antibody to coronavirus OC43 but a less detectable seropositivity m coronavirus 229E. INTRODUCTION Human coronaviruses OC43 and 229E are well known as etiological agents of respiratory infec- tions generally affecting the upper respiratory tract and responsible for common colds during winter-spring seasons Isolation of these viruses is particularly difficult and, consequently, all epidemiological and reliable data about their diffusion are based upon serological studies performed using reference strains of cell culture adapted coronaviruses. To date, viruses are included in the coronavirus group for some properties (14) : i) the presence of one molecu!e of infectious ssRNA of MW ranging from 5.5xl06 and 6.1xl06 daltons with polyadenylated 3 terminus; 1 Corresponding author. ii) morphology showing pleomorphic enve- loped particles with diameter ranging from 80 to 160 nm and surface projections (spikes) pro.truding 12-24 nm from envelope; iii) common physicochemicai properties such as banding at 1.1.6.-1.23 g/cm 3 in sucrose gradients, disruption bv ether, chloroform, and detergents, removal of surface projections by bromelain and trypsin. Consequently, viruses could be included in the coronavirus group independently of their antigenic cross-reaction but, particu!arly, for their charac- teristic morphology (spikes), as revealed by electron microscopy. Human coronavirus OC43 and 229E are the prototypes of all coronaviruses affecting humans. No particular relatedness has been, to date, re- 112 Vol. 2. 1986 Antibody to coronavims 229E OC43 and NCDCV ported between these two viruses; furthermore, in vitro, 229E shows an easy growth in human lung fibroblast cell cultures with evident cytopathogenic effect; on the other hand, OC43 can grow in cell cultures (VERO cells or human fibroblast cell cultures) only after adaptation of virus strains cultured in suckling mouse brain. Despite their morphological and antigenic differences, these viruses are responsible, in humans, for common colds not easily distinguishable by symptoms or seasonality of appearence. Previous reports showed a high prevalence of antibody to human OC43 and 229E coronavirus in sera collected from patients of northern Italy (1, 2). Results were obtained using very complex tests such as Immune-Adherence Hemag- glutination (IAHA), in the case of 229E specific antibody detection, or not highly sensitive and reliable tests, as complement-fixation and hemag- glutination-inhibition, for OC43 antibody detec- tion (1, 2). Another interesting test was utilized for serodiagnosis of OC43 infection, based upon a microneutralization method, but, despite its high degree of specificity and sensitivity, its perfor- mance was economically very expensive (3). Furthermore, the presence of a non specific inhib- itor for OC43 and NCDCV coronavirus present in mammalian sera and in fetal calf serum ,(FGS) that can be removed by phospholipase C (PLC) treatment, prompted us to develop a new sensitive and specific hemagglutination inhibition test and a new plaque reduction assay (4). By the use of these new tests foc OC43 and by an indirect immunoperoxidase staining technique (IIP), for 229E, we carried out a seroepiderniologic study in order to verify previously reported data (2, 3). In addition, the strict antigenic relatedness between OC43 and NCDCV (5) suggested the investigation of the prevalence of a human immune response against this bovine virus. MATERIALS AND METHODS Viruses Coronavirus 229E was obtained from American Type Culture Collection (ATCC - Rockville, Md., USA) and adapted to grow in human embryo lung fibroblast cells (HELF). OC43 and NCDCV coronaviruses were cell culture adapted strains originally obtained from suckling mouse brain adapted virus supplied by Dr. H.S. Kaye (C.D.C., Respiratory Virology Unit, Atlanta, Ga., USA) and Dr. C.A. Mebus, (Dept. of Veterinary Science, University of Nebraska, Lin- coln, Neb., 68503). Adaptation to growth in HELF has been previously reported (5). Sela Three hundred and twenty nine sera were collected from hospitalized patients and healthy persons having routine laboratory tests in the University Hospital of Pavia, Italy. Sera from six age groups were studied for specific antibody to 229E, OC43 and NCDCV coro- naviruses. A number of sera were also collected from leukemic infants and children. Indirect immunoperoxidase saining technique (IIP) HELF cells were cultured in sterile microplates for tissue culture; when the monolayer was confluent, all cell wells were infected with 0.05 ml of a suspension of 22qE containing 100 infec- tious virus particles, incubated at 33C for 60 rain. To each well was then added 0.l ml of 199 medium containing 2% fetal calf serum. Infected cultures were incubated at 33C for 36 hours, washed 3 times with PBS Du!becco B and then fixed with absolute ethanol. Fixed preparations were immediately used for HP or stored at -70C until use. IIP for specific human antibody detection was performed as previously described for other viruses (5-8), except that the 3-amino -9-ethyl- carbazole-H202 colour developing system was used. Hemagglutination-inhibition test (HI) Antibody titer to OC43 and NCDCV was detected by HI following a reported procedure (10) and a suggested treatment of sera in order to avoid non-specific inhibitors of OC43 and NCDCV hemagglutination (9), except that the inactivation of phospholipase C (PLC) with heat treatment (56C for 60 rain) was used in substi- tution of phenanthroline treatment. Plaque reduction assay ( PRA). PRA for OC43 and NCDCV antibody detection was performed as previously described (4). Briefly, a titered virus suspension, known to contain 40-50 infectious units of virus, was mixed with an equal volume of serial twofold dilution of .PLC - treated sera diluted in serum - free 199 medium with addition of glutamine, penicillin, streptomycin and gentamycin. Mixtures were incubated at 33C for 60 rain and then inoculated in wells of microplate cell culture of HELF (0.05 rot/well). After adsorption at 33C for 60 rain, plaquing medium (medium 199 with fetal calf serum at the final concentration of 10% and 5% for OC43 and NCDCV, respective- ly), was added for 24 h and then cells were fixed with absolute ethanol and stained by the indirect immunoperoxidase staining technique described using mouse hyperimmune sera. 113 Cereda P.M. et al. Eur. J. Epidemiol. RESULTS As reported in Figure 1, antibody to 229E coronavirus was present in a high percentage of sera studied; its prevalence and the specific antibody titer increased with increasing age. Antibody titers in leukemic patients seemed to be consistently lower than in other patients (Fi- gure 1). The results obtained were not particularly different from those observed in previous reports (2, 11, 12), except for the fact that, because of the greater sensitivity of IIP technique used, antibody titers were greater. Furthermore, the IIP test was very easy to read, as shown in Figure 2, very simple to perform and can avoid some problems (i.e. false results and/or difficulties in interpreting data) frequently occurring using other conventional and tedious methods like comple- ment fixation and immune adherence hemagglu- tination. Finally, the IIP technique for 229E antibody detection had other advantages : it allowed 229 E titer 640 32C 16( 80 40 2 I0 10 OO 000o CO0 88888 OCX:K: 888 0 OO 0O0(3 OOOO COCO 0O0 CO 888 oolMm (30 O-6months 7 months 6-12years 13-30years 31-50years =51 years 5 years 33 66 70 66 48 46 Figure 1. - Reciprocal of antibody titer to 229E coronavirus as detected in different age groups by IIP. Antibody to 229E in sera from leukemic patients. O Antibody to 229E in non leukemic sera. Age Groups No. o.f sera Figure 2.- IIP for 229E antibody detection on inected human embryonic fibroblasts, 2A: positive human serum; 2B: negative serum. 114 Vol. 2, 1986 Antibody to coronavirus 229E OC43 and NCDCV OC 43 titer 64( 32( 16( Do AAAA 8 4C AAAAAAAA 0 _O OCXXX AAA AA 0 AAAA 8888 A A A AAA.A J 000 AA A AAAAA 000 AAAA iI AAAAA 0 AAAAA AAAA A AAAA AAAAA AAAAA AAA N AAA OCX: AAA cx:x zS o A J AAA :x)o 0-6 months 7 months 6-12years 14-30years 31-50 years 51 years Age Groups 5 years 33 66 70 66 48 46 No. of sera Figure 3. - Reciprocal of antibody titer to OC43 as detected in different age groups by HI and PRA tests. Antibody to OC43 in leukemic patients as detected by HI and PRA i. Antibody to OC43 in non leukemic patients as detected by HI 0 and PRA A. NCDV titer 64C 32 16( A 8O. 0(3 AA 40. 88888 a io. gig CK) A AAA& o0o AAAAA Ja SXD00OAAAAA AAA& 000C AA&AA ZAAA AAAIA 00 o A AAAA 00000AAAAA AAAAA A AAAAA xA ooe A oooo 8aeooo a g 88 oo8 AAA x2OAAAAA AAA A ooot AA 0-6 months 7 months 6-12 years 13-30years 31-50 years , Advances in experimental medicine and biology, V.ter Meulen, S. Siddel, and H. Wege E ds., Plenum Press., Vol. !42: 171-179. 14. Tyrrel D.A.J., Alexander D.J., Atmeida .I.D., Cunni- gham C.H., Easterday B.C., Garwes D.J., Hierholzer J.C., Kapikian A., MacNaughton M.R., Mc Intosh K. (1978): Coronaviridae: second report.- Intervi- rology 10: 321-328. 117