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 bet Teen children and adult group Furthermore a different tirer was observed within the two groups between patients affected by hematological diseases leukemia and patients w 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 N CDCV 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 Sel a 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 s aining 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 33 C 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 33 C 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 70 C 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 56 C 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 33 C for 60 rain and then inoculated in wells of microplate cell culture of HELF 0 05 rot well After adsorption at 33 C 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 in ected 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 A A A AAAAA 000 AAAA iI AAAAA 0 AAAAA AA AA A AAAA AAAAA AAAAA AAA N A AA 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 Z AAA AAAIA 00 o A AAAA 00000AAAAA AAAAA A AAAAA xA ooe A oooo 8aeooo a g 88 o o 8 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