【病毒外文文獻】1991 Antigenic analysis of feline coronaviruses with monoclonal antibodies (MAbs)_ Preparation of MAbs which discriminat

Veterinary Microbiology 28 1991 13 24 13 Elsevier Science Publishers B V Amsterdam Antigenic analysis of feline coronaviruses with monoclonal antibodies MAbs Preparation of MAbs which discriminate between FIPV strain 79 1146 and FECV strain 79 1683 T Hohdatsu a T Sasamoto b S Okada b and H Koyama b aDepartment of Veterinary Microbiology b Veterinary Infectious Diseases School of Veterinary Medicine and Animal Sciences Kitasato University Towada Shi Aomori Ken 034 Japan Accepted 5 December 1990 ABSTRACT Hohdatsu T Sasamoto T Okada S and Koyama H 1991 Antigenic analysis of feline coronavi ruses with monoclonal antibodies MAbs Preparation of MAbs which discriminate between FIPV strain 79 1146 and FECV strain 79 1683 Vet Microbiol 28 13 24 We prepared 31 monoclonal antibodies MAbs against either FIPV strain 79 1146 or FECV strain 79 1683 and tested them for reactivity with various coronaviruses by indirect fluorescent antibody assay IFA Sixteen MAbs which reacted with all of the 11 strains of feline coronaviruses also reacted with canine coronavirus CCV and transmissible gastroenteritis virus TGEV In many of them the polypeptide specificity was the recognition of transmembrane El protein of the virus We suc ceeded in obtaining MAbs which did not react with eight strains of FIPV Type I viruses showing cell associated growth but reacted with FIPV Type II 79 1146 KU 1 and or FECV Type II 79 1683 viruses showing non cell associated growth These MAbs also reacted with CCV or TGEV These MAbs recognized peplomer E2 glycoprotein and many antigenic differences were found in this E2 protein These results suggest that FIPV Type II and FECV Type II viruses are antigenically closer to TGEV or CCV than to FIPV Type I viruses Furthermore the MAb prepared in this study has enabled dis crimination between FIPV strain 79 1146 and FECV strain 79 1683 which was thought to be impos sible by the previous serological method INTRODUCTION For serological diagnosis of feline infectious peritonitis virus FIPV infec tion detection of antibody by indirect fluorescent antibody assay IFA is popular Pedersen 1976b Horzinek and Osterhaus 1979 Scott 1979 On the other hand feline enteric coronavirus FECV which antigenically cross reacts with FIPV and causes only mild enteritis without inducing FIP may 0378 1135 91 03 50 1991 Elsevier Science Publishers B V 14 T HOHDATSU E AL be present McKeirnan et al 1981 Pedersen et al 1981a Pedersen et al 1981b Pedersen et al 1984 Thus the serological diagnosis of FIP and the mechanisms of its onset become more complex Pedersen et al 1984a class ified the feline coronaviruses in terms of the disease types They divided FIPV into Types I and II according to the presence or absence of the induction of FIP ability of the viruses to proliferate in cell cultures and the antigenic re lationship with porcine and canine coronaviruses FECV has been divided into Types I and II in the same way Types I and II of FIPV in this classifica tion can be serologically discriminated by the neutralization test FIPV Type I and FECV Type II can also be distinguished by the neutralization test Cul tivation of FECV Type I in cells is not possible at present and its serological position remains unclear On the other hand even the neutralization test can not discriminate between FIPV Type II and FECV Type II It goes without saying that since all feline coronaviruses show cross reaction in IFA it is im possible to discern the types of virus infection There are many healthy but FIPV antibody positive cats living outdoors As long as FIPV Type II and FECV Type II cannot be distinguished serologically the clinical diagnostic significance of antibody detection in such cats is low In this study we attempted to distinguish between the 79 1146 strain class ified as FIPV Type II and the 79 1683 strain classified as FECV Type II by means of monoclonal antibodies MAbs At the same time we examined feline coronaviruses for antigenic differences by using the MAbs We also in vestigated the antigenic relationship between feline coronaviruses and canine and porcine coronaviruses MATERIALS AND METHODS Cell cultures Feline whole fetus cells fcwf 4 Crandell feline kidney cells CrFK and Swine kidney cells CPK were grown in Eagle s minimum es sential medium MEM containing 20 Leibovitz L 15 Medium L 15 10 fetal calf serum 100 units ml penicillin and 100 g ml streptomycin The maintenance medium was MEM containing 20 L 15 and antibiotics as above The cells were maintained in a humidified 5 CO2 incubator at 37 C Viruses The coronavirus isolates used in this study and their sources are shown in Table 1 Among these virus strains the authors isolated the strain KU 1 of FIPV from the liver cells of a kitten with the effusive form of FIP and the strain KU 2 of FIPV from the peritoneal cells of an adult cat also with effu sive FIP Among the FIPV strains used in the study strains UCD 1 NW 1 UCD 2 UCD 3 UCD 4 Black Yayoi and KU 2 show cell associated growth and are therefore regarded as Type I virus strains in the classification of Pedersen et al 1984a Moreover since strain KU 1 like strain 79 1146 MONOCLONAL ANTIBODIES FOR FELINE CORONAV1RUSES AND FIPV FECV DISCRIMINATION TABLE l Source or coronavirus isolates 15 Virus strain Source Reference FIPV M C Horzinek State University Utrecht The Netherlands N C Pedersen University of California Davis J K Yamamoto University of California Davis J K Yamamoto University of California Davis J K Yamamoto University of California Davis J K Yamamoto University of California Davis J K Yamamoto University of California Davxs M Hirano University of Iwate Japan Author et al Author et al FECV A J McKeirnan Washington State University Pullman TGEV National Institute of Animal Health of Japan National Institute of Animal Health of Japan CCV E Takahashi University of Tokyo Japan 79 1146 UCD 1 NW I UCD 2 UCD 3 UCD 4 Black Yayoi KU I KU 2 79 1683 TO 163 SH 1 71 Pedersen et al 1984b McKeirnan et al 1981 Pedersen et al 1981a Pedersen 1976a Pedersen et al 1981a Pedersen and Black 1983 Pedersen and Floyd 1985 Pedersen and Floyd 1985 Pedersen and Floyd 1985 Black 1982 Pedersen and Black 1983 Hayashi et al 1981 McKeirnan et al 1981 Pedersen et al 1984b Furuuchi et al 1975 Harada el al 1967 Binn et al 1975 grows well even in CrFK cells in a non cell associated manner it is considered to be a Type II virus strain FIPV and FECV TGEV and CCV were passaged two or three times in fcwf 4 cells CPK cells and CrFK cells respectively and were used for the study Preparation of virus antigen The antigen was prepared with the FIPV 79 1146 strain or FECV 79 1683 strain grown in fcwf 4 cell cultures Infectious culture fluid concentrated about tenfold by ammonium sulfate precipitation was layered onto a discontinuous sucrose density gradient 20 and 60 in an RPS 28 rotor Hitachi Koki Co Ltd Japan and centrifuged at 27 000 r p m for 2 h The virus bands formed were collected diluted in NTE buffer 0 1 M NaC1 0 01 M Tris HC1 pH 7 4 0 001 M EDTA and centrifuged at 80 000 g for 1 h The virus containing pellet was suspended in a 1 500 vol ume of NTE buffer 16 T HOHDATSU ET AL Production of antibody secreting hybrydomas BALB c mice about 5 weeks of age were inoculated intraperitoneally with a mixture of 50 g of the viral antigen prepared as above and 10 9 cells of pertussis adjuvant Four or six weeks later the mice received an intravenous booster dose of 50 g of viral antigen and spleen cells were obtained for fusing 3 d later The fusion was carried out by essentially the same method described by K Shler and Milstein 1975 Polyethyleneglycol 4000 Merck Germany was used as a fusing agent and the ratio of mouse spleen cells and mouse myeloma cells P 3 X 63 Ag8o6 5 3 was 10 1 The selective medium contained hypoxanthine 10 4 M aminopterin 4 l0 7 M and thymidine 1 6 10 5 M The fused cells at a concentration of 3 5 10 6 spleen cells per ml was dispensed in 100 A volumes into wells of 96 well flat bottomed microplates Corning Glass Works Corning NY and incubated at 37 C in a humid atmosphere contain ing 5 CO2 After incubation for 2 weeks the wells were examined and those which contained hybridoma cultures were tested for feline coronavirus spe cific antibody by an indirect immunofluorescence test see below The col onies in antibody positive wells were passaged in 24 well multiplates Com ing Glass Works Corning NY and incubated in medium containing hypoxanthine 10 4 M and thymidine 1 6 10 5 M The cells were then cloned by the soft agar method Determination of antibody class and subclass The supernatant fluids of anti body secreting hybridoma cultures were concentrated tenfold by 50 satu ration with ammonium sulfate and used for determination of antibody class and subclass by double diffusion in 1 agar gel containing 0 1 NAN3 Rab bit antisera against mouse immunoglobulins IgG 1 IgG 2a IgG 2b IgG 3 IgM and IgA and x and 2 chains Miles Laboratories U S A were placed in center wells and test samples were added to adjacent wells The plates were incubated overnight at room temperature in a humidified chamber Indirect fluorescent antibody assay Hybridoma culture supernatant fluid was added to acetone fixed infected monolayers incubated for 30 min at 37 C washed 3 times with phosphate buffered saline solution PBS and then stained with rabbit anti mouse IgG A M serum conjugated with fluorescein isothiocyanate FITC Miles Lab U S A After a further 30 min incuba tion at 37 C slides were washed in PBS Stained monolayers were mounted in buffered glycerol and examined using a fluorescence microscope Neutralization NT test Serial twofold dilution of the MAbs were mixed with an equal volume of a virus suspension diluted so as to contain approximately 200 TCIDso 0 1 ml The mixtures were incubated at 37 C for 60 min Each mixture was then inoculated into cell cultures in flat bottomed microplates and incubated in an atmosphere of 5 CO2 in air at 37 C for 6 d Two wells MONOCLONAL ANTIBODIES FOR FELINE CORONAVIRUSES AND FIPV FECV DISCRIMINATION 1 7 were employed for each antibody dilution The antibody titer was expressed as the reciprocal of the highest dilution of MAb that completely inhibited cytopathic effect in the test Western immunoblotting Viral antigen separated in polyacrylamide gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE were transferred to nitrocellulose sheets of 0 45 zm pore size The transfer was car ried out electrophoretically by the method adapted from Towbin et al 1979 in a Transfer Blot cell apparatus at 120 mA and 10 V for 14 h using transfer buffer consisting of 3 g 1 Tris pH 8 3 20 methanol and 43 2 g 1 1 gly cine The nitrocellulose sheets were then cut into strips and incubated at 37 C for 2 h in PBS containing 10 fetal calf serum The supernatant fluid of an tibody secreting hybridoma cultures was added in 1 ml volumes to individual strips and incubated at 37 C for 2 h The strips were then washed 3 times with PBS containing 0 05 Tween 20 and incubated at 37 C for 2 h with horseradish peroxidase conjugated rabbit antibody against mouse IgG A M Miles Lab U S A diluted 1 300 with PBS containing 10 fetal calf serum The strips were then washed and treated with substrate solution containing 0 05 g diaminobenzidine 50 1 of 30 H202 in 100 ml of 0 05 M Tris HC1 pH 7 2 When distinct bands appeared about 10 min later the reaction was stopped by pouring offthe substrate solution and rinsing with distilled water Determination of polypeptide specificity by enzyme linked immunosorbent as say ELISA The polypeptide specificity of the MAbs which could not be determined by western immunoblotting was determined according to the method of Fiscus and Teramoto 1987 Briefly the virus antigen described above was first disrupted with 1 Nonidet P 40 NP 40 This material was placed on a 15 50 linear sucrose density gradient containing 0 1 NP 40 and centrifuged at 80 000 g for 17 h After fractionation each fraction was diluted with NTE buffer and allowed to be absorbed by 96 well flat bot tomed Microelisa plates The MAbs against N E 1 and E2 proteins with poly peptide specificity clarified by western immunoblotting were delivered into the wells of each fraction and subjected to ELISA ELISA was performed according to the method of Hohdatsu et al 1987 Among the MAbs which recognize each protein fractions which reacted strongly with a single type of MAb were collected ELISA was performed with these fractions used as anti gens and the polypeptide specificity of the MAbs was determined RESULTS Isolation and characterization of monoclonal antibodies When FIPV strain 79 1146 was used as the immunogen 25 MAbs F2 1 F29 1 F16 4 F18 2 F19 1 F30 1 F34 1 F35 2 F36 1 F41 1 F51 1 F52 1 F70 2 F75 3 F15 18 T HOHDATSU ET AL 2 F24 1 F25 1 F46 4 F49 1 F69 3 F80 1 F6 3 F22 3 F23 2 F50 4 were obtained In addition with FECV strain 79 1683 as the immunogen 6 MAbs El 5 2 E19 1 E22 2 E6 2 E25 2 E 12 1 were obtained The polypeptide specificity Ig isotypes and NT activity to FIPV strain 79 1146 of these MAbs are shown in Table 2 For most MAbs western immunoblotting could deter TABLE 2 Polypeptide specificity Ig isotype and neutralization NT activity to FIPV strain 79 1146 of mon oclonal antibodies MAb no Polypeptide specificity lg isotype NT trier Blot ELISA F 2 1 N N IgG1 K 2 F 29 2 N N IgG 1 K 2 E 15 1 N N lgGl K 2 E 19 1 N N IgGl oK 2 E 22 2 N N igG 1 K 2 E 6 2 N N IgGl k 2 F 16 4 El El IgG3 K 2 F 18 2 El El IgGI K 2 F 19 I El El IgGI K 2 F 30 1 El E1 IgG3 K 2 F 34 1 El El IgG2a K 2 F 35 2 El E1 IgG3 K 2 F 36 1 El E1 IgG3 K 2 F41 1 E1 E1 IgGI K 2 F 51 1 El E1 lgG2a K 2 F 52 1 El E1 lgG2a K 2 F 70 2 E 1 E 1 lgG2a K 2 F 75 3 El E1 IgG2a K 2 F 15 2 E2 E2 IgG 1 K 2 F 24 1 E2 E2 IgG2a K 2 F 25 1 E2 E2 IgG2a K 2 F 46 4 E2 E2 IgG2a K 2 F 49 i E2 E2 lgG2a K 2 F 69 3 E2 E2 IgGl K 64 F 80 1 E2 E2 lgGl K 2 F 6 3 9 E2 IgGI K 2 F 22 3 E2 IgG2a K 2 F 23 2 9 E2 lgG2a K 2 F 50 4 E2 IgG2b K 2 E 25 2 E2 ND e lgG2b K 2 E 12 1 E2 ND IgM K 256 The polypeptide specificity of each of the MAbs was determined by its reactivity to each of the three major structural components of the F1PV virion either by immunoblotting of SDS PAGE or by ELISA For the ELISA the three structural components of FIPV were separated by sucrose gradient centrif ugation of detergent disrupted FIPV virions 2Not done MONOCLONAL ANTIBODIES FOR FELINE CORONAVIRUSES AND FIPV FECV DISCRIMINATION l 9 mine their polypeptide specificity However this method failed to determine the specificity of 4 MAbs F6 3 F22 3 F23 2 and F50 4 ELISA using NP 40 disrupted sucrose gradient purified viral polypeptide revealed that these MAbs recognise E2 protein Moreover western immunoblotting and ELISA using viral polypeptide yielded the same results with respect to the other MAbs Figure 1 shows examples of the western immunoblotting reaction of MAbs which recognize N E1 and E2 proteins As shown in Table 2 two F69 3 El2 1 of the 31 MAbs had NT activity to FIPV strain 79 1146 Reactivity of the monoclonal antibodies with the feline coronaviruses We ex amined the MAbs for reactivity with feline coronaviruses by IFA As shown in Table 3 the MAbs could be divided into six groups according to their reac tivity with 11 strains of feline coronavirus The 16 MAbs in Group I all re acted with the feline coronaviruses The two MAbs in Group II failed to react with two of the virus strains while the one MAb in Group III showed no reaction with four strains These MAbs were all found to recognize the N pro tein of the viruses MAbs in Group IV reacted with two strains of FIPV and the one strain of FECV while Group V MAbs reacted only with two strains of FIPV The two MAbs in Group VI did not react with any FIPV strain but a b c d et 94k 67k 43k 30k I Fig 1 Polypeptide specificity of monoclonal antibodies against FIPV 79 1146 strain in western immunoblotting SDS PAGE was performed on 10 gel under non reducing conditions before immunoblot a marker proteins b F 25 1 anti E2 c F 70 2 anti E1 d F 2 1 anti N e anti FIPV mouse serum TABLE 3 Reactivity of the monoclonal antibodies to the feline coronaviruses Group MAb no FIPV Type II Type I 79 1146 KU I UCD 1 NW I Yayoi KU 2 UCD 2 Black UCD 3 UCD 4 FECV Type lI 79 1683 Polypeptide specificity II III IV V1 F 2 1 N F16 4 E1 F18 2 El F19 1 El F30 1 El F34 1 El F35 2 E1 F36 1 E1 F41 1 El F51 1 El F52 1 E1 F70 2 El F75 3 E1 F80 1 E2 El5 1 N El9 1 N E 6 2 N E22 2 N F29 2 N F 50 4 E2 F 46 4 E2 F 69 3 E2 F15 2 E2 F25 1 E2 F 22 3 E2 F 23 2 E2 F 24 1 E2 F49 1 E2 F 6 3 E2 E 25 2 E2 E 12 1 E2 By means of indirect IF test with FITC conjugatcd rabbit anti mouse immunoglobulin on each virus infected fcwf 4 cell culture grown on coverslips The monoclonal antibodies were used undiluted cell culture fluids The minus sign indicates negative reactivity The polypeptide specificity of each of the MAbs was determined by its reactivity to each of the three major structural components of the FIPV virion either by immunoblotting of SDS PAGE or by ELISA MONOCLONAL ANTIBODIES FOR FELINE CORONAVIRUSES AND FIPV FECV DISCRIMINATION TABLE 4 Reactivity of the monoclonal antibodies to porcine and canine coronaviruses 21 Group MAb no CCV TGEV Polypeptide specificity 1 71 SH TO 163 111 IV F 2 1 N F16 4 El F18 2 El F19 1 El F30 1 E1 F34 1 El F35 2 E1 F36 1 E1 F41 1 El F51 1 El F52 2 El F70 1 El F75 3 El F 80 1 E2 E 15 1 N El9 1 N E 6 2 N E 22 2 N F 29 2 N F 50 4 E2 F 46 4 E2 F 69 3 E2 F 15 2 E2 V F 25 1 E2 F 22 3 E2 F 23 2 E2 F 24 1 E2 F 49 1 E2 F 6 3 E2 VI E 25 2 E2 E 12 1 E2 By means of indirect IF test with FITC conjugated rabbit anti mouse immunoglobulin on each vi rus infected CvFK or CPK cell culture grown on coverslips The monoclonal antibodies were used undiluted cell culture fluids The minus sign indicates negative reactivity The polypeptide specificity of each of the MAbs was determined by its reactivity to each of the three major structural components of the FIPV virion either by immunoblotting of SDS PAGE or by ELISA did react with the only strain of FECV All MAbs in Group IV V and VI were found to recognize E2 protein 22 T HOHDATSU ET AL Reactivity of the monoclonal antibodies with porcine and canine coronavi ruses We examined the MAbs for reactivity with porcine and canine corona viruses by IFA Table 4 shows the results All MAbs in Group I which reacted with all feline coronaviruses also reacted with CCV and TGEV MAbs in Groups II and III which did not react with UCD 2 UCD 3 UCD 4 and Black strains did not react with CCV and TGEV either However out of four MAbs in Group IV which reacted only with the 79 1146 and KU 1 strains of FIPV and strain 79 1683 of FECV three reacted with either CCV or TGEV More over among the MAbs in Group V which reacted only with the 79 1146 and KU 1 strains of FIPV two reacted with the SH strain of TGEV and 1 type reacted with the 1 71 strain of CCV Furthermore MAbs in Group VI which reacted with FECV alone reacted with the 1 71 strain of CCV DISCUSSION Thirty one MAbs were prepared by using strain 79 1146 classified as FIPV Type II and strain 79 1683 classified as FECV Type II as immunogens Ta ble 3 shows their reactivity with 11 strains of feline coronavirus All 16 MAbs in Group I reacted with feline coronaviruses Besides feline coronaviruses these MAbs reacted with the 1 71 strain of CCV and the SH and TO 163 strains of TGEV These results confirm the previous reports Pedersen et al 1978 Horzinek et al 1982 Pedersen et al 1984a that these virus strains are antigenically close to each other Concerning polypeptide specificity many MAbs in this Group I recognize E 1 protein Among these viruses many com mon epitopes seem to exist particularly in the E 1 protein Among eight virus strains with the characteristics of FIPV Type I reactivities of E6 2 and E22 2 in Group II and F29 2 in Group III with the MAbs were different and all of these MAbs recognized N protein Four MAbs in Group IV F50 4 F46 4 F69 3 and F 15 2 reacted with the 79 1146 and KU 1 strains of FIPV Type