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Y Terada et al JVI Revised Ver 1 1 Establishment of a virulent full length cDNA clone for type I feline coronavirus 1 strain C3663 2 3 Yutaka Teradaa b Yudai Kurodab Shigeru Morikawac Yoshiharu Matsuurad Ken 4 Maedab c and Wataru Kamitania 5 6 aLaboratory of Clinical Research on Infectious Diseases Research Institute for 7 Microbial Diseases Osaka University Osaka Japan 8 bLaboratory of Veterinary Microbiology Joint Faculty of Veterinary Medicine 9 Yamaguchi University Yamaguchi Japan 10 cDepartment of Veterinary Science National Institute of Infectious Diseases Tokyo 11 Japan 12 dDepartment of Molecular Virology Research Institute for Microbial Diseases Osaka 13 University Osaka Japan 14 15 Running Head Infectious cDNA clone of virulent type I FCoV 16 17 18 Address correspondence to Wataru Kamitani DVM PhD 19 wakamita biken osaka u ac jp 20 Abstract word count 236 words 21 Text word count 9531 words 22 Number of pages 35 23 Number of figures 5 24 JVI Accepted Manuscript Posted Online 2 August 2019 J Virol doi 10 1128 JVI 01208 19 Copyright 2019 American Society for Microbiology All Rights Reserved on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 2 Abstract 25 Feline infectious peritonitis FIP is one of the most important infectious 26 diseases in cats and is caused by feline coronavirus FCoV Tissue culture adapted type 27 I FCoV shows reduced FIP induction in experimental infections which complicates the 28 understanding of FIP pathogenesis caused by type I FCoV We previously found that 29 the type I FCoV strain C3663 efficiently induces FIP in specific pathogen free cats 30 through the naturally infectious route In this study we employed a bacterial artificial 31 chromosome based reverse genetics system to gain more insights into FIP caused by the 32 C3633 strain We successfully generated recombinant virus rC3663 from Fcwf 4 cells 33 transfected with infectious cDNA that showed similar growth kinetics to the parental 34 virus Next we constructed a reporter C3663 virus carrying the nanoluciferase Nluc 35 gene to measure viral replication with high sensitivity The inhibitory effects of 36 different compounds against rC3663 Nluc could be measured within 24 h post infection 37 Furthermore we found that A72 cells derived from canine fibroblasts permit FCoV 38 replication without apparent cytopathic effects Thus our reporter virus is useful for 39 uncovering the infectivity of type I FCoV in different cell lines including 40 canine derived cells Surprisingly we uncovered aberrant viral RNA transcription of 41 rC3663 in A72 cells Overall we succeeded in obtaining infectious cDNA clones 42 derived from type I FCoV that retained its virulence Our recombinant FCoVs are 43 powerful tools for increasing our understanding of the viral life cycle and pathogenesis 44 of FIP inducing type I FCoV 45 46 Importance 47 Feline coronavirus FCoV is one of the most significant coronaviruses 48 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 3 because this virus induces feline infectious peritonitis FIP which is lethal disease in 49 cats Tissue culture adopted type I FCoV often loses pathogenicity which complicates 50 research on type I FCoV induced feline infectious peritonitis FIP Since we previously 51 found that the type I FCoV strain C3663 efficiently induces FIP in specific pathogen 52 free cats we established a reverse genetics system for the C3663 strain to obtain 53 recombinant viruses in the present study By using a reporter C3663 virus we were able 54 to examine the inhibitory effect of 68 compounds on C3663 replication in Fcwf 4 cells 55 and infectivity in a canine derived cell line Interestingly one canine cell line A72 56 permitted FCoV replication but with low efficiency and aberrant viral gene expression 57 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 4 Introduction 58 Coronaviruses CoVs are pathogens that infect a wide variety of animals 59 including humans and cause respiratory and enteric diseases 1 CoVs are enveloped 60 viruses possessing a large single stranded positive sense RNA 32 kb 2 are 61 classified as order Nidovirales family Coronaviridae and subfamily Coronavirinae 62 CoVs are further classified into four genera alpha beta gamma and delta 3 Feline 63 coronavirus belongs to alpha CoVs together with canine coronavirus porcine 64 transmissible gastroenteritis virus porcine epidemic diarrhea virus and human 65 coronavirus 229E and NL63 3 66 Feline CoV FCoV infections are distributed worldwide in domestic cats and 67 wild Felidae such as lions 4 5 and cheetahs 6 Based on their pathogenicity FCoVs 68 can be classified into two biotypes feline enteric CoV FECV and feline infectious 69 peritonitis virus FIPV FECV infections are asymptomatic or occasionally induce mild 70 intestinal inflammation in kittens 7 On the other hand FIPV infections induce the 71 more severe and immune mediated lethal disease feline infectious peritonitis FIP 8 72 9 73 FCoVs can also be further classified into two types types I and II based on 74 their antigenicity 10 11 Unlike type II FCoV type I FCoV infections occur 75 predominantly in felids worldwide 12 14 Furthermore their virological features differ 76 including growth characteristics in cell culture and receptor usage 7 15 Compared 77 with type I FCoV type II FCoV shows better growth kinetics and can more easily 78 induce FIP in specific pathogen free SPF cats Despite the fact that type II FCoV 79 infections occur with low frequency many researchers employ type II FCoVs to analyze 80 FIP pathogenesis Therefore a type I FCoV strain that can induce FIP is needed to fully 81 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 5 understand FIP pathogenesis 82 It has been proposed that type I FECV replicates and acquires mutations in its 83 viral genome in kittens and then the mutated FECV becomes a FIP associated virus 84 This hypothesis is known as the internal mutation theory 16 18 which is supported 85 by the proposal of virulent FIP markers Based on epidemiological studies spike S 86 and or open reading frame ORF 3c genes of type I FCoV are thought to be virulent 87 markers 18 20 However none of the proposed markers have been proven virulent 88 owing to the lack of feasible FIP cat models with type I FCoV It is difficult for most 89 type I FCoVs isolated from FIP cats to induce FIP in experimental settings using SPF 90 cats It is thought that adaptation of type I FCoV in tissue culture results in the loss of 91 pathogenicity 21 22 92 Recently we discovered a strain of type I FCoV C3663 isolated from FIP cats 93 23 that retained virulence despite adaptation in Fcwf 4 cells 9 Surprisingly three of 94 four SPF cats 75 developed FIP after infection with the C3663 strain 9 These 95 findings suggest that our C3663 strain is a candidate for analyzing FIP pathogenesis 96 induced by type I FCoV in experimental settings 97 In this study we constructed an infectious cDNA clone derived from the type I 98 FCoV C3663 strain by utilizing the bacterial artificial chromosome BAC system 99 Recombinant C3663 rC3663 virus was easily rescued from Fcwf 4 cells transfected 100 with BAC plasmids carrying the C3663 full length genome rC3663 showed similar 101 growth kinetics to the parental virus Furthermore we generated a recombinant virus 102 bearing the nanoluciferase Nluc gene in the ORF 3abc region This rC3663 Nluc 103 reporter virus was useful in investigating the inhibitory effects of compounds and 104 revealed the infectivity of type I FCoV in canine cells Interestingly the expression ratio 105 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 6 of subgenomic sg mRNA was different in canine derived A72 cells infected with 106 rC3663 virus suggesting that aberrant viral RNA transcription of the rC3663 virus 107 occurred in A72 cells 108 109 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 7 Results 110 Construction of BAC carrying the full length C3663 genome 111 The full genome sequence of type I FCoV strain C3663 was assembled into the 112 pBeloBAC11 vector to generate infectious cDNA clones under the control of a 113 cytomegalovirus CMV immediate early promoter Fig 1A To this end we separated 114 the genomic sequence of C3663 into 11 fragments and sequentially assembled them into 115 the BAC plasmid Fig 1A The vector backbone bears the CMV promoter followed by 116 the hepatitis delta virus HDV ribozyme and bovine growth hormone BGH 117 termination sequences Fig 1A the C3663 genomic sequence was cloned into the 118 pBeloBAC11 vector between the CMV promoter and the 25 nucleotide nt poly A 119 HDV ribozyme and BGH termination sequences Fig 1A The full length infectious 120 cDNA clone was designated pBAC FCoV C3663 and sequence analysis showed that it 121 possessed 25 nucleotide mutations compared with that of the C3663 reference sequence 122 Table 1 Of the 25 mutations 11 were synonymous and 14 were non synonymous 123 mutations Table 1 Two synonymous mutations at nt 9831 and nt 9834 were 124 introduced as the genetic marker which disrupts the EcoRI site EcoRI confirming 125 virus recovery from the cDNA clone Fig 1B and Table 1 126 127 Virus recovery by pBAC FCoV C3663 transfection 128 We produced rC3663 virus from Fcwf 4 cells which are highly susceptible to 129 FCoV infection by transfecting the cells with pBAC FCoV C3663 Small cytopathic 130 effects CPE were observed 2 days post transfection dpt which became larger by 3 131 dpt To determine rC3663 virus recovery we employed RT PCR on isolated RNA from 132 rC3663 and the parental strain We further confirmed the EcoRI genetic maker by 133 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 8 analyzing EcoRI digestion and Sanger sequencing Fig 1C and D Next we analyzed 134 the virological features of the rC3663 virus by comparing the growth kinetics of rC3663 135 and parental C3663 in Fcwf 4 cells after inoculating them with viruses at a multiplicity 136 of infection MOI of 0 01 The results showed that rC3663 growth kinetics were 137 similar to that of the parental C3663 Fig 1E Furthermore we compared viral RNA 138 replication in parental C3663 or rC3663 infected Fcwf 4 cells by northern blot analysis 139 Fig 1F and found that the amount of genomic g RNA and sg mRNAs in 140 rC3663 infected Fcwf 4 cells were similar to that of the parental C3663 infected cells 141 Fig 1F Taken together we were able to successfully generate infectious cDNA 142 clones derived from type I FCoV strain C3663 using the BAC system Our results 143 indicate that the recovered rC3663 virus possesses identical virological features as the 144 parental C3663 virus Fig 1E and F 145 146 Establishment of reporter rC3663 bearing the Nluc gene 147 In virology recombinant viruses carrying reporter genes GFP RFP or 148 luciferase provide many advantages for analyzing viral characteristics and screening 149 for therapeutic compounds 24 25 Thus we attempted to construct an infectious 150 cDNA clone of type I FCoV strain C3663 carrying an Nluc gene By following the 151 protocols of Tekes et al 26 we inserted the Nluc gene into pBAC FCoV C3663 in 152 place of the ORF 3abc gene to obtain pBAC FCoV C3663 Nluc Fig 2A The Nluc 153 gene replaced a region containing the start codon of ORF 3a to 71 nt upstream of the 154 ORF 3c stop codon to retain the transcription regulatory sequence TRS of the M gene 155 Fig 2A 156 To examine Nluc expression in Fcwf 4 cells infected with rC3663 Nluc virus 157 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 9 we inoculated Fcwf 4 cells with rC3663 Nluc at an MOI of 0 01 Infection with rC3663 158 was used as control After 24 48 and 72 h post infection hpi we found that Nluc 159 activity in rC3663 Nluc infected Fcwf 4 cells but not in rC3663 or mock infected 160 cells increased in a time dependent manner Fig 2B 161 We further investigated the viral growth of rC3663 Nluc in Fcwf 4 cells by 162 harvesting the supernatants of rC3663 Nluc or rC3663 infected Fcwf 4 cells at 24 48 163 and 72 hpi and then determining infectious titers of the supernatants by plaque assays 164 The production of infectious virus particles from rC3663 Nluc infected cells was 165 comparable to that of rC3663 infected cells Fig 2C As shown in Fig 2B and 2C the 166 increase in Nluc activity was significantly correlated with viral replication in 167 rC3663 Nluc infected cells Our data indicate that rC3663 carrying the Nluc reporter 168 gene is a powerful tool for investigating type I FCoV viral replication and production 169 170 Application of the rC3663 reporter virus in compound screening 171 Before applying the rC3663 reporter virus to compound screening we 172 determined sensitivity of the rC3663 Nluc virus to treatment with known inhibitors of 173 CoV replication cyclosporine A 27 28 and lopinavir 29 After adsorption of 174 rC3663 Nluc onto Fcwf 4 cells at an MOI of 0 01 the infected cells were treated with 175 various concentrations of cyclosporine A or lopinavir As shown in Fig 2D and 2E 176 both compounds inhibited luciferase activity in a dose dependent manner Furthermore 177 viral RNA levels in cyclosporine A or lopinavir treated cells were measured by 178 real time RT PCR Fig 2D and 2E Intracellular viral RNA levels were found reduced 179 in a dose dependent manner for both compounds and were correlated with luciferase 180 activity suggesting that detection sensitivity of luciferase expression in 181 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 10 rC3663 Nluc infected cells is comparable to that of viral RNA expression levels 182 Next to determine the usefulness of the rC3663 reporter virus for screening 183 antiviral compounds we utilized a commercially available library of 68 protease 184 inhibitors Fcwf 4 cells were inoculated with rC3663 Nluc MOI 0 01 and 10 M of 185 each protease inhibitor cyclosporine A and DMSO were used as positive and negative 186 control respectively The side effects of protease inhibitors were determined by MTT 187 assays Fig 2F Together with the MTT assay results 15 inhibitors were found to 188 exhibit more than 75 reduction in Nluc activity compared with that of the DMSO 189 control and without any accompanying cytotoxicity Fig 2F compound no 2 25 29 190 31 34 35 48 50 56 58 64 67 and 69 Indeed compound no 31 lopinavir 191 inhibited luciferase activity which is consistent with the results in Fig 2E Overall our 192 results support the suitability of rC3663 Nluc in compound screening 193 194 Identification of permissive cell lines for type I FCoV 195 In vitro propagation of type I FCoV is limited to a few cell lines including 196 Fcwf 4 cells AKD cells and CRFK cells because type I FCoV only shows a CPE in 197 such cell lines 23 30 31 Thus it is difficult to investigate the infectivity of type I 198 FCoV in cell lines derived from other animals such as dogs Nevertheless we explored 199 novel cell lines for propagation of type I FCoV by inoculating three canine derived cell 200 lines A72 canine fibroblasts MDCK canine kidney epithelial cells and DH82 201 canine macrophages with the rC3663 Nluc virus MOI 0 1 and investigated 202 infectivity by measuring Nluc activity Although a CPE was not observed for 203 rC3663 Nluc infected A72 cells Nluc activity was significantly high at 24 hpi and 204 increased in a time dependent manner Fig 3A and C On the other hand 205 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 11 rC3663 Nluc infected MDCK and DH82 cells did not exhibit detectable Nluc activity 206 Fig 3B 207 To determine viral RNA replication in A72 cells A72 MDCK DH82 and 208 Fcwf 4 cells were infected with rC3663 Nluc virus at an MOI of 0 01 followed by 209 real time RT PCR analysis of RNA extracted at 24 48 and 72 hpi Although the 210 amount of viral RNA in rC3663 Nluc infected A72 cells was lower than in Fcwf 4 cells 211 the amount of viral RNA in A72 cells but not in DH82 and MDCK cells was still 212 significantly high at 48 and 72 hpi Fig 3D These results indicate that A72 cells 213 permit replication of type I FCoV C3663 virus 214 Next we determined the production of infectious virus particles from 215 rC3663 Nluc infected A72 cells by collecting the culture supernatants at 24 48 and 72 216 hpi and measuring viral titers by plaque assays with Fcwf 4 cells Fig 3E The 217 supernatant infectious titers of Fcwf 4 cells reached 1 67 105 PFU mL at 72 hpi and 218 the amount of viral RNA determined by real time RT PCR increased in a 219 time dependent manner in Fcwf 4 cells Fig 3D and E As shown in Fig 3A and D 220 A72 cells support rC3663 virus replication but the production of infectious viruses was 221 lower compared with that of Fcwf 4 cells Meanwhile infectious viruses were not 222 produced by infected MDCK and DH82 cells Fig 3E These results indicate that A72 223 cells produce progeny viruses albeit with low efficiency while MDCK and DH82 224 cells are not permissive cell lines for type I FCoV 225 To further determine the low production of progeny virus by 226 rC3663 Nluc infected A72 cells we examined the propagation of rC3663 virus MOI 227 0 1 in A72 and Fcwf 4 cells by indirect immunofluorescence assays IFA using 228 confocal microscopy analysis Using an anti FCoV N monoclonal antibody N protein 229 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 12 expression in A72 cells was observed by IFA and exhibited small foci 48 hpi compared 230 with that of Fcwf 4 cells Fig 3F Therefore infectious particles were found generated 231 in A72 cells with a low efficiency of infection expansion by progeny particles to 232 neighboring cells We also examined N protein expression levels using immunoblotting 233 As expected N protein expression levels in rC3663 virus infected A72 cells were 234 significantly low Fig 3G Although the production of progeny virus and N protein 235 was low our results suggest that canine derived A72 cells are a permissive cell line for 236 type I FCoV infections without cytotoxic effects 237 238 MDCK cells do not permit viral replication during type I FCoV infection 239 As shown in Fig 3 and unlike A72 cells neither viral RNA replication nor 240 progeny virus production was observed in MDCK and DH82 cells infected with rC3663 241 virus These results led us to speculate that the entry receptor for type I FCoV is perhaps 242 not expressed in MDCK and DH82 cells or that viral RNA replication of type I FCoV is 243 not permitted in these cell lines Unfortunately the type I FCoV receptor remains 244 unknown Thus we examined viral replication levels in cells transfected with 245 pBAC FCoV C3663 Nluc As a negative control we generated 246 pBAC FCoV C3663 Nluc PolDead by mutating the active site of viral RNA dependent 247 RNA polymerase RdRp nsp12 from SDD to SAA Fig 4A and confirmed that virus 248 rescue did not occur in Fcwf 4 cells transfected with 249 pBAC FCoV C3663 Nluc PolDead because of disrupted RdRp activity data not 250 shown After transfecting MDCK cells with pBAC FCoV C3663 Nluc or 251 pBAC FCoV C3663 Nluc PolDead together with pcDNA3 1 fluc we determined 252 luciferase activity in cell lysates at 24 48 and 72 h p transfection Fig 4B the firefly 253 on August 5 2019 by guest http jvi asm org Downloaded from Y Terada et al JVI Revised Ver 1 13 luciferase reporter plasmid pcDNA3 1 fluc 32 was used as an internal control As 254 shown in Fig 4B Nluc expression levels in cells transfected with 255 pBAC FCoV C3663 Nluc were comparable to those of 256 pBAC FCoV C3663 Nluc PolDead Consistent with the luciferase assay results Fig 257 4B no progeny virus was produced in MDCK cell