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Vol 0123456789 1 3 Arch Virol DOI 10 1007 s00705 017 3489 8 ORIGINAL ARTICLE Antibody dependent enhancement ofuni00A0serotype II feline enteric coronavirus infection inuni00A0primary feline monocytes Tomomiuni00A0Takano 1 uni00A0 Mamikouni00A0Nakaguchi 1 uni00A0 Tomoyoshiuni00A0Doki 1 uni00A0 Tsutomuuni00A0Hohdatsu 1 uni00A0 Received 25 April 2017 Accepted 5 July 2017 Springer Verlag GmbH Austria 2017 Introduction Feline coronavirus FCoV is an enveloped positive strand RNA virus belonging to the family Coronaviridae subfam ily Coronavirinae and genus Alphacoronavirus 7 There are two serotypes of FCoV FCoV has been classified into types I and II based on the amino acid sequence of its spike S protein 11 18 Separate from these serotypes FCoV has been classified into two biotypes weakly pathogenic feline enteric coronavirus FECV avirulent FCoV and strongly pathogenic feline infectious peritonitis virus FIPV virulent FCoV 20 FIP is a lethal immune mediated infec tious disease of members of the family Felidae and there has been no established therapy for treatment It has been sug gested that FIPV is a mutant of FECV i e it is considered that after infecting a cat the open reading frame ORF 2 gene and ORF3c gene of FECV partially mutates in the cat s body and becomes virulent FCoV which is FIPV 21 The presence of FCoVs with a broad pathogenic spectrum in the field has also been proposed as another hypothesis 2 There are serotype I and II FECV and FIPV in FCoV Serotype II FCoVs are known to utilize feline aminopeptidase N fAPN as a virus receptor 6 8 There are 11 ORFs in the FCoV genome and of these ORF2 ORF4 ORF5 and ORF6 encode structural proteins 5 The S protein encoded by ORF2 plays an essential role in cell entry being involved in binding to the FCoV specific receptor on the host cell surface and fusion with the cell membrane 1 The S protein also plays an important role in antibody dependent enhancement ADE of FIPV infection 9 19 34 ADE is a phenomenon in which binding to an antibody makes host cell entry easier for the virus We pre viously reported that monoclonal antibodies mAbs to the S protein mediated ADE of FIPV infection 9 When ADE of FIPV infection occurs virus production in macrophages Abstract Feline coronavirus FCoV has been classified into two biotypes avirulent feline coronavirus feline enteric coronavirus FECV and virulent feline coronavirus feline infectious peritonitis virus FIPV In FIPV infection anti body dependent enhancement ADE has been reported and was shown to be associated with severe clinical disease On the other hand the potential role of ADE in FECV infection has not been examined In this study using laboratory strains of serotype II FIPV WSU 79 1146 FIPV 79 1146 and sero type II FECV WSU 79 1683 FECV 79 1683 we investi gated the relationship between ADE and induction of inflam matory cytokines which are pathogenesis related factors for each strain As with ADE of FIPV 79 1146 infection a mon oclonal antibody against the spike protein of FCoV mAb 6 4 2 enhanced FECV 79 1683 replication in U937 cells and primary feline monocytes However the ADE activity of FECV 79 1683 was lower than that of FIPV 79 1146 Moreover mRNA levels of inflammatory cytokines TNF uni03B1 IL 1uni03B2 and IL 6 significantly increased with ADE of FIPV 79 1146 infection in primary feline monocytes but FECV 79 1683 did not demonstrate an increase in these levels In conclusion infection of monocytes by FECV was enhanced by antibodies but the efficiency of infection was lower than that of FIPV Tsutomu Hohdatsu hohdatsu vmas kitasato u ac jp 1 Laboratory ofuni00A0Veterinary Infectious Disease School ofuni00A0Veterinary Medicine Kitasato University Towada Japan T uni00A0Takano et al 1 3 increases and production of inflammatory cytokines such as tumor necrosis factor alpha TNF uni03B1 interleukin IL 1uni03B2 and IL 6 is enhanced 27 30 31 Therefore ADE is closely associated with the severity of FIPV infection However no studies on the difference in pathogenicity between FIPV and FECV with regard to ADE of infection have been reported Since serotype I FCoV infection is dominant in the field 10 14 15 25 it is desirable to use serotype I FCoV in studies on FIP However isolation of serotype I FECV using cultured cell lines has not been successful and it is diffi cult to study ADE of infection in vitro Moreover an mAb inducing ADE activity of serotype I FCoV has not yet been prepared In this study using laboratory strains of serotype II FIPV WSU 79 1146 FIPV 79 1146 and serotype II FECV WSU 79 1683 FECV 79 1683 we investigated the relationship between ADE and induction of inflammatory cytokines which are pathogenesis related factors in each strain Materials anduni00A0methods Cell cultures anduni00A0viruses Felis catus whole fetus fcwf 4 cells kindly supplied by Dr M C Horzinek of the State University of Utrecht were grown in Eagle s minimum essential medium containing 50 L 15 medium 5 fetal calf serum FCS 100 U of pen icillin per mL and 100 uni03BCg of streptomycin per mL Cells of the human monocyte cell line U937 were cultured in RPMI 1640 medium containing 10 FCS and antibiotics Primary feline monocytes were maintained in RPMI 1640 growth medium supplemented with 10 FCS 100 U of penicillin per mL 100 uni03BCg of streptomycin per mL and 50 uni03BCM 2 mer captoethanol FIPV 79 1146 was kindly provided by Dr M C Horzinek FECV 79 1683 was kindly supplied by Dr A J McKeirnan of Washington State University These viruses were grown in fcwf 4 cells at 37 C with 5 CO 2 Antibodies mAb 6 4 2 IgG2a used in the present study recognizes the S protein of serotype II FCoV 9 It has been reported that mAb 6 4 2 exhibits neutralizing activity in fcwf 4 and CrFK cells but enhancing activity in primary feline mono cytes and macrophages depending on the reaction condi tions The mAb 6 4 2 was used at a dilution of 10 except in the experiment shown in Fig uni00A01A mAb R G 4 recogniz ing fAPN IgG1 and IgG1 mAb control recognizing feline interferon gamma prepared by our laboratory 8 were used Inoculation ofuni00A0U937 cells withuni00A0FCoV FIPV 79 1146 or FECV 79 1683 1 10 6 TCID 50 for both reacted with mAb 6 4 2 at 4 C for 1 h was added to the culture and adsorbed to the U937 cells 2 10 5 cells in tubes at 37 C with 5 CO 2 for 3 h The cells were washed three times with PBS and cultured in tubes at 37 C with 5 CO 2 for 48 h and the supernatants were collected The virus titer in the culture supernatant TCID 50 uni00A0was determined by theuni00A0method of Reed and Muench 22 withuni00A0fcwf 4uni00A0cells Inoculation ofuni00A0primary feline monocytes withuni00A0FCoV Primary feline monocytes were isolated from specific path ogen free SPF cats as described previously by Dewerchin et al 4 The blood sampling in SPF cats was performed in accordance with the Guidelines for Animal Experi ments of Kitasato University approval number 16 086 FIPV 79 1146 or FECV 79 1683 1 10 4 TCID 50 for both reacted with mAb 6 4 2 at 4 C for 1 h was added to the cul ture and adsorbed to the primary feline monocytes 2 10 5 cells in 24 well multi plates at 37 C with 5 CO 2 for 1 h The cells were washed three times with PBS and cultured in 24 well multi plates at 37 C with 5 CO 2 for 48 h and the supernatants and cells were collected The virus titer in the culture supernatant TCID 50 uni00A0was determined by theuni00A0method Fig 1 ADE of FECV infec tion in U937 cells and feline monocytes A U937 cells were infected with FCoV in the presence or absence of mAb 6 4 2 The results are shown as the mean SE n 5 B Feline monocytes were infected with FCoV in the presence or absence of mAb 6 4 2 The results are shown as the mean SE n 10 Black bar FIPV 79 1146 white bar FECV 79 1683 N D not detected Antibody dependent enhancement of coronavirus infection 1 3 of Reed and Muench 22 withuni00A0fcwf 4uni00A0cells Cells were used to measure the mRNA expression levels of inflammatory cytokines Effects ofuni00A0anti fAPN mAb inuni00A0primary feline monocytes infected withuni00A0FCoV Primary feline monocytes 2 10 5 cells were cultured in medium containing mAb R G 4 or IgG1 mAb control in 24 well multi plates at 4 C for 1 hour After washing with PBS FIPV 79 1146 or FECV 79 1683 1 x 10 4 TCID 50 for both with or without pretreatment with mAb 6 4 2 was added to the culture and allowed to adsorb to the cells at 37 C with 5 CO 2 for 1 h in the presence of mAbs After wash ing with PBS the cells were cultured in the medium and the culture supernatants were collected after 48 h The virus titer in the culture supernatant TCID 50 uni00A0was determined by theuni00A0method of Reed and Muench 22 with fcwf 4uni00A0cells Effects ofuni00A0lysosomotropic agents onuni00A0theuni00A0virus inuni00A0primary feline monocytes withuni00A0FCoV Primary feline monocytes 2 10 5 cells were cultured in medium containing chloroquine Wako Pure Chemical Industries Japan or ammonium chloride Wako Pure Chem ical Industries Japan in 24 well multi plates at 37 C with 5 CO 2 for 1 h After washing with PBS FIPV 79 1146 or FECV 79 1683 1 10 4 TCID 50 for both with or without pretreatment with mAb 6 4 2 was added to the culture and allowed to adsorb to the cells at 37 C with 5 CO 2 for 1 h in the presence of chloroquine or ammonium chloride After washing with PBS the cells were cultured in the medium containing chloroquine or ammonium chloride and the culture supernatants were collected after 48 h The virus titer in the culture supernatant TCID 50 uni00A0was determined by theuni00A0method of Reed and Muench 22 withuni00A0fcwf 4uni00A0cells To evaluate the cytotoxic effects of chloroquine and ammo nium chloride in primary feline monocytes cell viability was measured by the WST 8 assay as described before 29 RNA isolation anduni00A0cDNA preparation RNA isolation and cDNA preparation were performed employing the method of Takano et al 29 Determination ofuni00A0levels ofuni00A0feline GAPDH mRNA TNF uni03B1 mRNA IL 1uni03B2 mRNA anduni00A0IL 6 mRNA expression cDNA was amplified by PCR using specific primers for feline glyceraldehyde 3 phosphate dehydrogenase GAPDH mRNA TNF uni03B1 mRNA IL 1uni03B2 mRNA and IL 6 mRNA The primer sequences are shown in Tableuni00A01 PCR was performed using the method of Takano et al 32 The band density was quantified under appropriate UV exposure by video densi tometry using Image J software NIH USA TNF uni03B1 mRNA IL 1uni03B2 mRNA and IL 6 mRNA were semi quantitatively analyzed in terms of the relative density value to that of the mRNA for the housekeeping gene GAPDH Statistical analysis Data from two groups were analyzed by Student s t test and multiple groups were analyzed by one way ANOVA Results ADE ofuni00A0FECV infection inuni00A0U937 cells anduni00A0primary feline monocytes As U937 cells do not express fAPN FIPV alone does not infect U937 cells but it enters FIPV via the U937 cell sur face Fc receptor when anti FCoV S antibodies are present 12 31 therefore U937 cells were used as a prediction model to determine the mAb 6 4 2 concentrations required for ADE of FECV infection in vitro Fig uni00A01A FCoVs did not infect U937 cells in the absence of mAb 6 4 2 Several dilutions of mAb 6 4 2 were incubated with the virus The titer of FIPV 79 1146 was increased in the culture superna tant at several dilutions of mAb 6 4 2 1 1 7 7 10 2 3 9 Table 1 Sequences of PCR primers for feline GAPDH IL 1uni03B2 TNF uni03B1 and IL 6 Orientation Nucleotide sequence Location Length bp GAPDH Forward 5 AAT TCC ACG GCA CAG TCA AGG 3 158 178 97 Reverse 5 CAT TTG ATG TTG GCG GGA TC 3 235 254 IL 1uni03B2 Forward 5 CTG GTG CTG TCT GGC TCA TA 3 441 460 280 Reverse 5 TTC CCG TCT TTC ATC ACA CA 3 601 620 TNF uni03B1 Forward 5 TGG CCT GCA ACT AAT CAA CC 3 195 214 251 Reverse 5 GTG TGG AAG GAC ATC CTT GG 3 426 445 IL 6 Forward 5 GCA GAA AAC AAC CTG AAT CTT CCG 3 247 270 426 Reverse 5 GAG AAA GGA ATG CCC GTG AAC 3 601 620 T uni00A0Takano et al 1 3 10 2 TCID 50 mL 1 10 1 4 10 3 6 2 10 2 TCID 50 mL 1 100 8 3 10 2 8 2 10 1 TCID 50 mL In contrast to FIPV 79 1146 the titer of FECV 79 1683 did not increase at any dilution except 10 1 8 3 10 0 1 6 10 0 TCID 50 mL i e maximum ADE activity of FECV 79 1683 infec tion occurred at an mAb dilution of 1 10 We investigated the ADE activity of FECV infection in primary feline mono cytes Fig uni00A01B mAb 6 4 2 dilution of 1 10 enhanced the infection of monocytes with FIPV 79 1146 the median increase was 200 fold for the infection by virus alone virus alone 2 1 10 3 7 2 10 2 TCID 50 mL virus and mAb 6 4 2 6 1 10 5 1 6 10 5 TCID 50 mL On the other hand mAb 6 4 2 dilution of 1 10 slightly increased the infection of cells with FECV 79 1683 the median increase was tenfold virus alone 1 3 10 2 5 9 10 1 TCID 50 mL virus and mAb 6 4 2 1 5 10 3 9 9 10 2 TCID 50 mL i e the ADE activity of FECV 79 1683 was 10 to 20 fold lower than that of FIPV 79 1146 Involvement ofuni00A0theuni00A0virus receptor fAPN inuni00A0ADE ofuni00A0FECV infection inuni00A0primary feline monocytes The involvement of virus receptor fAPN in ADE of FECV infection in primary feline monocytes was investigated Fig uni00A02 When monocytes were allowed to react with mAb R G 4 beforehand and then inoculated with FECV 79 1683 alone the virus titer decreased significantly virus alone 1 5 10 2 5 2 10 1 TCID 50 mL virus and mAb R G 4 10 0 TCID 50 mL However when monocytes were allowed to react with mAb R G 4 beforehand and then inoculated with a mixture of FECV 79 1683 and mAb 6 4 2 the virus titer did not decrease as in cells without mAb R G 4 treatment virus and mAb 6 4 2 1 6 10 3 1 0 10 3 TCID 50 mL virus mAb 6 4 2 and mAb R G 4 5 6 10 2 3 6 10 2 TCID 50 mL Nearly identical results were obtained as in the case of FIPV 79 1146 Fig uni00A02 Effects ofuni00A0lysosomotropic agents onuni00A0ADE ofuni00A0FECV infection inuni00A0primary feline monocytes The ADE activity of FIPV is inhibited by the action of chlo roquine and ammonium chloride because the endosomal pH rises and inhibits the fusion of FIPV with the endosome membrane in cells treated with chloroquine and ammonium chloride Inhibition of FECV replication by treatment with chloroquine and ammonium chloride has been reported However the influence of chloroquine and ammonium chlo ride on ADE activity of FECV has not been reported Thus we investigated whether the ADE activity of FECV is inhib ited by chloroquine and ammonium chloride Primary feline monocytes were treated with chloroquine or ammonium chloride and the influence on viral replication after FECV infection was investigated The viability of primary feline monocytes after treatment with chloroquine and ammonium chloride was 98 7 1 6 mean SD and 101 2 3 6 mean SD respectively The virus titer was significantly higher in the culture supernatant of monocytes inoculated with a mixture of FECV 79 1683 and mAb 6 4 2 than in monocytes cultured with the virus alone virus alone 1 8 10 2 4 8 10 1 TCID 50 mL virus and mAb 6 4 2 1 5 10 3 4 9 10 2 TCID 50 mL However treatment with 20 uni03BCM chloroquine or 5 mM ammonium chloride significantly decreased the virus titer in the culture supernatant of cells infected with virus alone or a mixture of virus and mAb 0 TCID 50 mL in all cases of treatment with lysosomotropic agents Similar results were obtained with FIPV 79 1146 Fig uni00A03 Fig 2 Involvement of virus receptors fAPN in ADE in feline monocytes The results are shown as the mean SE n 10 Black bar FIPV 79 1146 white bar FECV 79 1683 N D not detected N S not significant Fig 3 Effects of lysosomotropic agents on ADE in feline monocytes The results are shown as the mean SE n 10 Black bar FIPV 79 1146 white bar FECV 79 1683 N D not detected Antibody dependent enhancement of coronavirus infection 1 3 Effect ofuni00A0ADE ofuni00A0FECV infection onuni00A0inflammatory cytokine mRNA levels inuni00A0primary feline monocytes Inflammatory cytokine production is enhanced when pri mary feline monocytes are inoculated with FIPV in the presence of anti FIPV S antibodies However it is not clear whether inflammatory cytokine production increases with ADE of FECV infection Thus we investigated the inflam matory cytokine mRNA levels in primary feline monocytes inoculated with a mixture of FECV 79 1683 and mAb 6 4 2 Fig uni00A04 The TNF uni03B1 IL 1uni03B2 and IL 6 mRNA levels were sig nificantly increased in monocytes inoculated with a mixture of FIPV 79 1146 and mAb 6 4 2 However these mRNA levels were not increased in monocytes inoculated with a mixture of FECV 79 1683 and mAb 6 4 2 Discussion FECV infects primary feline monocytes and macrophages but the infectivity of FECV for these cells is lower than that of FIPV 4 24 This difference has been suggested to be correlated with the pathogenicity of these viruses 24 FECV is assumed to cause almost no clinical symptoms because it does not readily infect primary feline monocytes or macrophages and it replicates only in local regions intes tinal epithelium 3 In contrast FIPV is considered to cause FIP because it readily infects primary feline monocytes and macrophages and circulates throughout the body However the conserved FCoV 7b gene is also detected in regions other than the intestine in some cases of FECV infection 13 16 and the reason for this has not yet been elucidated Mono cyte and macrophage infection by FIPV is potentiated in the presence of antibodies It was assumed that the efficiency of infection of monocytes and macrophages by FECV increases in the presence of antibodies as is observed with FIPV If this assumption is correct the mechanism of systemic cir culation of enterocyte tropic FECV may be explained Thus we investigated whether FECV has ADE activity A human histiocytic lymphoma cell line U937 was used to measure ADE of FIPV infection As U937 cells do not express fAPN FIPV alone does not infect U937 cells but it enters U937 cells via the cell surface Fc receptor when anti FCoV S antibodies are present 12 31 Furthermore as no feline monocyte macrophage cell line has been estab lished U937 cells are useful as a model to predict ADE activity of FCoV In this study FECV exhibited ADE activ ity in U937 cells but the activity level was lower than that of FIPV FIPV exhibited ADE activity in the presence of mAb 6 4 2 at several concentrations but FECV was only active at a tenfold dilution of mAb 6 4 2 i e the monocyte infection efficiency of FIPV was enhanced in the presence of the antibody but the increase in the efficiency of FECV infection in the presence of the antibody was only slight We also investigated the difference in the ADE activity of FIPV and FECV in primary feline monocytes The experiment was performed at an antibody concentration maximizing ADE activity of FECV based on the experimental results with U937 cells ADE activity in primary feline monocytes was confirmed for both FIPV and FECV but similar to the results of the experiment with U937 cells the ADE activity level of FECV was lower than that of FIPV Specifically the mean ADE activity level of FECV was approximately 1 20 of that of FIPV FIPV infected primary feline monocytes and macrophages expressing ADE release excess cytokines such as TNF uni03B1 and IL 6 as virus production increases 27 28 30 These cytokines cause a reduction in the number of lymphocytes and neutrophil activation aggravating the FIP pathology Therefore high level ADE activity is related to clinical aggravation to a serious state in FIPV infected cats Fig 4 Inflammatory cytokine mRNA expression levels in ADE in feline monocytes The results are shown as the mean SE n 10 Black bar FIPV 79 1146 white bar FECV 79 1683 N S not significant T uni00A0Takano et al 1 3 Therefore the low ADE activity of FECV may reflect the weak pathogenicity of FECV In primary feline monocytes the ADE activity level of FECV was lower than that of FIPV FIPV does not require the virus receptor to enter cells to induce ADE 31 but FECV is assumed to require both Fc and virus receptors therefore FECV enters cells