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J gen Virol 1988 69 1725 1730 Printed in Great Britain Key words TGE virus porcine respiratory coronavirus antigenic relationship 1725 Antigenic Differentiation between Transmissible Gastroenteritis Virus of Swine and a Related Porcine Respiratory Coronavirus By P CALLEBAUT 1 I CORREA 2 M PENSAERT 1 G JIMI NEZ 2 AND L ENJUANES 2 1Laboratory of Virology Faculty of Veterinary Medicine State University of Gent Casinoplein 24 B 9000 Gent Belgium and 2Centro de Biologia Molecular CSIC UAM Facultad de Ciencias Universidad Aut6noma Canto Blanco 28049 Madrid Spain Accepted 25 March 1988 SUMMARY The antigenic relationship between a recently isolated porcine respiratory coronavirus TLM 83 and transmissible gastroenteritis TGE virus of swine was studied by neutralization immunoblotting and radioimmunoassay RIA using TGE virus specific monoclonal antibodies MAbs and polyclonal antibodies specific for both viruses A complete two way neutralization activity between the two viruses was found Immunoblotting revealed cross reactions between TLM 83 and TGE virus antigens at the level of the envelope protein El the nucleoprotein N and the peplomer protein E2 By virus neutralization assays and RIA with TGE virus specific MAbs the presence of similar epitopes in the E1 and N proteins and in the neutralization mediating antigenic site of the E2 protein were demonstrated E2 protein specific MAbs without neutralizing activity and reacting with antigenic sites B C and D previously defined failed to recognize TLM 83 These results indicated a close antigenic relationship and structural similarity between TLM 83 and TGE viruses and also suggested potential ways of differentiating between the two viruses Transmissible gastroenteritis TGE virus of swine is a member of the Coronaviridae family and was isolated for the first time in 1946 Doyle Kemeny et al 1975 The virus spreads by the oral faecal route it is not known whether the aerogenic route constitutes a significant mode of transmission Bohl 1981 The viral particle is composed of a non glycosylated nucleoprotein N and two glycoproteins one of which E2 is associated with the surface projections and the other El with the lipid envelope Garwes 4 d 3 2 1 I I I I 1 I I I 0 1 II 0 0 0 I I I I I I I I I 0 1 2 3 4 5 6 7 8 9 TGEV VN titre log2 Fig l Antigenic relationship between TLM 83 and TGE viruses examined by homologous and heterologous VN tests with sera from piglets experimentally infected with TLM 83 O and with TGE viruses 0 Titres are expressed as log2 of reciprocal endpoint dilution values immunoblotting analysis and by studying the reactivity with TGE virus specific monoclonal antibodies MAbs The results may provide the basis for a differential serological diagnosis The ability of virus neutralization VN tests to differentiate TGE and TLM 83 viruses was investigated by producing specific antisera to both viruses and comparing the homologous and heterologous antibody titres in cross VN assays The TLM 83 virus isolate was used at the ninth passage in cell culture It was cultivated in the swine kidney cell line PD5 grown as described by Horzinek et al 1982 Following inoculation cultures were incubated for 42 h at 37 C at that time the cells showed extensive cytopathic effect characterized by clustering of rounded up ceils and initial loss of infected ceils into the medium The virus was harvested by two cycles of freezing and thawing After clarifying an infectivity titre of 104 5 TCID5o ml was obtained The Purdue strain of TGE virus was the 114th passage in primary pig kidney PPK cells and for the neutralization assays was adapted to grow in PD5 cells by two passages It reached an infectivity titre of 105 5 TCIDs0 ml The porcine epidemic diarrhoea PED virus isolate CV777 and the haemagglutinating encephalomyelitis HE virus isolate VW 572 were used as the third pig passage and the 15th passage in PPK cells respectively produced as described earlier Debouck et al 1981 Pensaert the homologous blot with TLM 83 virus was too weak to give conclusive data The E2 N and E1 proteins of TGE virus had Mr values of 200000 48000 and 28000 respectively in agreement with values previously published Garwes Horzinek et al 1982 Laude et al 1986 TLM 83 proteins showed a pattern related to TGE virus proteins E2 from TLM 83 was demonstrated to have an Mr in the same range as that from TGE virus although some slight differences could not be excluded As common antigenic determinants appeared to be located on each of the structural polypeptides of TLM 83 and TGE viruses the degree of the antigenic relatedness was assessed by comparing the individual epitopes of the two viruses using a panel of TGE virus specific MAbs Both the binding assay and the MAbs which were used have been described previously Jim6nez et al 1986 Enjuanes et al 1987 Of these 20 nine and three MAbs were specific for the E2 N and E 1 proteins of TGE virus respectively we also used four MAbs to TGE virus for which the specificity at the protein level was unknown Fifteen E2 protein specific MAbs which were able to neutralize TGE virus and defined a single antigenic site were designated anti E2 A The five remaining non neutralizing E2 protein specific MAbs recognized three separate antigenic sites and were designated anti E2 B two MAbs anti E2 C two MAbs and anti E2 D one MAb The N and E1 protein specific MAbs used did not neutralize TGE virus In addition the neutralization of TGE and TLM 83 viruses by the anti E2 A MAbs was determined in PD5 cells as described above The results of these assays are shown in Fig 4 By radioimmunoassay RIA most MAbs recognized both TGE and TLM 83 viruses No differences between the epitopes located on the E1 and N polypeptides could be detected as all anti N and anti E1 MAbs bound to TLM 83 virus to a great extent Furthermore all E2 A specific MAbs were fully reactive with TLM 83 virus both in the binding and the neutralization assays indicating that the epitopes for the induction of neutralizing antibodies were indistinguishable However the non neutralizing anti E2 B anti E2 C and anti E2 D MAbs failed to recognize TLM 83 indicating that the antigenic sites in the E2 protein of TGE virus responsible for stimulating non neutralizing antibodies were modified or absent in the equivalent polypeptide of TLM 83 virus All MAbs with unknown specificity extensively bound to TLM 83 virus These results demonstrated that TGE and TLM 83 viruses were closely related and structurally similar as common antigenic determinants were found on E2 E1 and N proteins with porcine convalescent sera and with murine MAb preparations and it was found that these proteins were of similar M Therefore TLM 83 virus can be regarded as a TGE Virus variant which may have spontaneously emerged rather than as a new porcine coronavirus species The complete absence of serological cross reactivity of TLM 83 virus with the porcine coronaviruses HE and PED using immunofluorescent staining Pensaert et al 1981 and immunoblotting supports this conclusion Our results demonstrate that antigenic dissimilarities between TLM 83 and TGE viruses are associated with their respective peplomer proteins The differences may be exploited for developing a competitive ELISA for the differential detection of antibodies against each virus The lack of reactivity of site B C and D specific MAbs with TLM 83 is probably significant as these sites were present on three TGE virus clones of American PUR 54 Japanese SHI 56 and European BRE 79 origin that were collected over a period of 25 years Jim6nez et al 1986 Other isolates of the two viruses are currently being analysed in order to confirm the similarities and differences between them As a limited number of N and E1 protein specific MAbs have been used it cannot be ruled out that antigenic dissimilarities between TLM 83 and TGE viruses are also associated with these two proteins Further work is in progress to elucidate how the antigenic 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