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Advance Publication The Journal of Veterinary Medical Science Accepted Date 6 May 2018 J STAGE Advance Published Date 17 May 2018 1 Full Paper Virology 1 Mutation of the S and 3c genes in genomes of feline coronaviruses 2 3 Keisuke Oguma 1 Megumi Ohno 1 Mayuko Yoshida 1 2 and Hiroshi Sentsui 1 4 5 1 Laboratory of Veterinary Epizootiology Department of Veterinary Medicine College 6 of Bioresource Sciences Nihon University 1866 Kameino Fujisawa Kanagawa 252 7 0880 Japan 8 2 Present address Okinawa Zoo and Museum Foundation 5 7 1 Goya Okinawa City 9 Okinawa 904 0021 Japan 10 11 Corresponding author Oguma K Laboratory of Veterinary Epizootiology 12 Department of Veterinary Medicine Nihon University 1866 Kameino Fujisawa 13 Kanagawa 252 0880 Japan 14 E mail oguma keisuke nihon u ac jp 15 Tel Fax 81 466 84 3368 16 17 Running Head MUTATION OF FELINE CORONAVIRUS GENOME 18 19 2 ABSTRACT 20 Feline coronavirus FCoV is classified into two biotypes based on its pathogenicity 21 in cats a feline enteric coronavirus of low pathogenicity and a highly virulent feline 22 infectious peritonitis virus It has been suspected that FCoV alters its biotype via 23 mutations in the viral genome The S and 3c genes of FCoV have been considered the 24 candidates for viral pathogenicity conversion In the present study FCoVs were 25 analyzed for the frequency and location of mutations in the S and 3c genes from faecal 26 samples of cats in an animal shelter and the faeces effusions and tissues of cats that 27 were referred to veterinary hospitals Our results indicated that approximately 95 28 FCoVs in faeces did not carry mutations in the two genes However 80 FCoVs in 29 effusion samples exhibited mutations in the S and 3c genes with remainder displaying a 30 mutation in the S or 3c gene It was also suggested that mutational analysis of the 3c 31 gene could be useful for studying the horizontal transmission of FCoVs in multi cat 32 environments 33 34 KEYWORDS feline coronavirus multi cat environment mutation S gene 3c gene 35 36 3 INTRODUCTION 37 The genome of feline coronavirus FCoV a member of the Alphacoronavirus 1 38 species of the genus Alphacoronavirus comprises single stranded positive sense RNA 39 9 FCoV infection is prevalent in cats worldwide and is divided into two biotypes 40 feline enteric coronavirus FECV and feline infectious peritonitis virus FIPV The 41 former has low pathogenicity causing mild enteritis or unapparent infection and the 42 latter is highly virulent and lethal FIP is characterised by the accumulation of body 43 cavity effusions effusive or wet form and the formation of granulomatous lesions 44 affecting multiple organs non effusive or dry form 10 FIPVs are considered mutants 45 of FECVs 11 12 14 46 Although the viral genes responsible for biotype conversion have not been 47 completely elucidated the candidate genes have been identified The S gene encodes 48 spike protein on the viral membrane It was reported that 95 8 of 118 serotype I FIPVs 49 displayed missense mutations in codon 1 058 or 1 060 of the S gene whereas none of 50 the sample of 183 FECVs exhibited these mutations 6 The non synonymous 51 mutations in codons 1 058 and 1 060 substituted methionine to leucine M1 058L and 52 serine to alanine S1 060A respectively The 3c gene encoding an accessory viral 53 protein was also reported to be mutated in 60 100 of FIPVs resulting in the loss or 54 truncation of the 3c protein whereas most FECVs carried an intact 3c gene 3 55 5 8 11 13 14 Accordingly it was considered that mutation of the S gene 3c gene or 56 both was involved in the acquisition or augmentation of lethal pathogenicity in the 57 majorityof FIPV field strains In the present study we analyzed the S and 3c genes of 58 FCoVs detected in faecal materials effusion samples and tissues that were obtained 59 from cats in Japan to determine the frequency and location of the mutations An analysis 60 4 of the 3c gene suggested the horizontal infection of FCoVs which were detected in 61 effusions and tissues among several housemate cats in a multi cat environment 62 63 MATERIALS AND METHODS 64 Collection of clinical samples 65 Clinical specimens were obtained from 93 cats referred to private veterinary hospitals 66 in Japan for suspected FIP based on clinical symptoms including pyrexia vomiting 67 diarrhoea jaundice emaciation anaemia ascites pleural effusion ophthalmologic 68 abnormalities neurological signs and death Some animals displayed an enlargement of 69 abdominal organs that was noticed on palpation radiography or ultrasound The 70 samples of abdominal and pleural effusions whole blood serum rectal swabs faeces 71 and tissues were sent to our laboratory under refrigeration Tissues were obtained via 72 autopsy of four cats that had been kept by the same owner and referred to a veterinary 73 hospital The analyzed tissues included kidneys mesenteric lymph nodes a spleen and 74 an eye and its vitreous humor Whole blood samples were treated with 75 ethylenediaminetetraacetic acid as an anticoagulant 76 Faecal samples were collected from an animal shelter wherein each cat was housed 77 alone or with a few other cats per cage To prevent the redundant analysis of a cat when 78 2 cats were kept in a single cage only one faecal sample was taken 79 80 Nucleic acid extraction and complementary DNA synthesis 81 Total RNA samples were extracted from effusions supernatants of phosphate 82 buffered saline homogenised faecal and rectal swab samples serum plasma and a 83 vitreous humor sample from an eye using a QIAamp Viral RNA Mini Kit QIAGEN 84 5 Hilden Germany or ISOGEN LS reagent NIPPON GENE Tokyo Japan RNA 85 samples of whole blood were extracted using ISOGEN LS reagent In some cases 86 erythrocytes were lysed using 0 2 sodium chloride to isolate leukocytes and their 87 RNA was extracted using an RNeasy Mini Kit QIAGEN in combination with a QIA 88 shredder QIAGEN Tissues were homogenised in ISOGEN reagent NIPPON GENE 89 using a TissueRuptor with TissueRuptor disposable probes QIAGEN cDNAs were 90 synthesised using a PrimeScript 1st Strand cDNA Synthesis Kit Takara Bio Shiga 91 Japan All reagents and kits were used according to the manufacturers instructions 92 93 Amplification of the S and 3c genes by reverse transcription polymerase chain reaction 94 Reverse transcription polymerase chain reaction RT PCR was performed to amplify 95 the S and 3c genes using GoTaq Green Master Mix Promega Madison WI U S A 96 previously reported primers 1 5 6 and our designed primers Supplementary Table 1 97 The primers were used at a final concentration of 0 5 M The S gene fragments were 98 amplified to determine the FCoV serotype I or II in each animal Amplification of the 99 3c and S genes including codons 1 058 and 1 060 via first round PCR was performed 100 as follows initial denaturation at 94 C for 2 min 50 cycles of 94 C for 30 sec 50 C for 101 30 sec and 72 C for 45 sec and final extension at 72 C for 7 min In some cases the 3c 102 and S genes were amplified via nested RT PCR in which a second round reaction was 103 performed using the same PCR cycle parameters The S gene based serotyping was 104 carried out together with the 3c gene amplification under the same reaction protocol or 105 separately via single or nested RT PCR wherein the reaction protocol was the same 106 except for a shortened extension time of 20 sec The PCR products were 107 electrophoresed on a 2 agarose gel and amplified DNA fragments were retrieved 108 6 using the Wizard SV Gel and PCR Clean Up System Promega The extracted 109 product was directly sequenced using a BigDye Terminator v3 1 Cycle Sequencing Kit 110 on a genetic analyzer Applied Biosystems 3130 Thermo Fisher Scientific Waltham 111 MA U S A Some amplicons were cloned into a pCR2 1 TOPO vector using a 112 TOPO TA Cloning Kit Thermo Fisher Scientific and sequenced using M13 primers 113 or the primers used for RT PCR The obtained 3c gene sequences were analyzed to 114 determine the types and locations of mutations via comparisons with type I FECV 115 strains RM FJ938051 and UU19 HQ392470 and type II FIPV strain KUK H L 116 AB781789 none of which carry mutations resulting in the production of truncated 117 proteins GENETYX 13 Genetyx Corporation Tokyo Japan and BioEdit 7 1 3 0 7 118 software were used for sequence analysis All determined 3c gene sequences were 119 submitted to the DNA Data Bank of Japan Accession numbers are shown in 120 Supplementary Tables 2 and 3 121 122 RESULTS 123 Detection of FCoVs from clinical samples 124 Of the 53 samples obtained from 40 out of 93 cats that had been referred to animal 125 hospitals 55 3c gene sequences were obtained Supplementary Table 2 FCoV 126 serotypes I and II were detected in 38 and 2 cats respectively The ages of 39 animals 127 with FCoV positivity in any sample ranged from 2 months 17 years median 9 5 128 months and 30 animals were younger than two years old Cat 19 was of unknown age 129 Twenty cats were male 18 were female and the sex was not recorded for two animals 130 In stools collected from the animal shelter 3c genes were detected in 19 samples 131 Supplementary Table 3 132 7 133 Analysis of the S gene 134 Partial S gene fragments of FCoV including codons 1 058 and 1 060 were amplified 135 from the faeces of 19 cats from the animal shelter Codon mutations were not present in 136 all samples The FCoV S gene in faecal samples from 14 cats that had been referred to 137 animal hospitals was also examined Four of these cats were fed by a single owner and 138 the M1 058L or S1 060A mutation was detected in their faeces Half of the 14 hospital 139 cases presented with ascites or pleural effusion in which FCoV genomes were detected 140 Of the 30 ascites and pleural effusion samples that contained type I FCoVs M1 058L 141 and S1 060A mutations were discovered in 24 and 4 samples respectively and the 142 remaining 2 samples did not carry the mutations 143 Six tissue samples were obtained from four deceased cats that were 4 6 months of 144 age All tissues contained FCoVs that carried the M1 058L mutation 145 An FCoV that was detected in the blood sample from cat 55 had the M1 058L 146 mutation but because other samples were not taken this cat was not analyzed further 147 148 Analysis of the 3c gene 149 Previously reported information regarding the open reading frame ORF lengths of 150 the 3c gene was obtained from the National Center for Biotechnology Information 151 online database The majority of non truncated ORFs consisted of 714 nucleotides 152 coding 237 amino acids aa Some ORFs were longer because of one or more insertions 153 of several nucleotides Accordingly in the present study an intact 3c ORF was defined 154 as a sequence of at least 714 bases that did not contain a premature stop codon due to 155 any mutation type 156 8 The 3c ORF was 714 bases long in 18 out of 19 FCoV positive stool samples from 157 the animal shelter The ORF of the virus detected in the stool sample from cat S10 158 shelter cat was 711 bases due to a 3 base deletion spanning codons 23 24 resulting in 159 the deletion of 1 aa This mutation did not generate a premature stop codon Fig 1A 160 The 3c genes were also analyzed from the 14 FCoV containing faecal samples from cats 161 that had been referred to animal hospitals One faecal FCoV from cat 37 had a longer 162 intact 3c gene of 720 bases This sequence was genetically closest 96 3 in a BLAST 163 search to two intact 3c gene sequences of FIPV strains DSKUU48 GU053649 5 and 164 UU9 6 165 Ascites and pleural effusion samples containing FCoVs were taken from 32 cats in 166 animal hospitals Two ascites samples contained type II FCoVs with truncating 167 mutations in the ORF of 3c The other 30 samples were type I FCoVs Of these 26 168 samples carried truncating mutations in the 3c genes Fig 1B Some FCoVs were not 169 expected to express the 3c protein because of a mutation involving the start codon 170 All FCoVs identified in the six tissue samples of four cats contained a truncated ORF 171 in each 3c gene Fig 1C In a kidney and mesenteric lymph node from cat 80 two 172 FCoV variants were detected in each tissue in which the 3c ORFs were 712 and 684 173 bases respectively Both variants shared an identical two base deletion at codon 153 174 and one variant had an additional 28 base deletion located 46 bases downstream of the 175 two base deletion site An FCoV in blood of cat 55 had an intact 3c gene 176 The lengths of truncated 3c proteins expressed by FCoVs were predicted to range 177 from 3 to 235 aa corresponding to 1 3 99 2 of the length of the wild type protein 178 179 Mutation types leading to truncation or deletion of the 3c protein 180 9 Mutations that resulted in the production of truncated 3c proteins less than 237 aa or 181 complete protein loss were detected in 39 samples collected from 33 cats This included 182 one faecal sample from a cat housed in an animal shelter and clinical samples from 32 183 hospital referred cats Two deletions faeces from cat S10 and ascites from cat 75 and 184 one insertion faeces from cat 37 did not create premature stop codons The other 3c 185 genes amplified from 37 samples of 31 cats had mutations resulting in premature stop 186 codons or no protein expression because of a mutation that involved the start codon of 187 each sequence The most common mutation type that generated premature stop codons 188 was a frameshift resulting from a deletion or insertion 18 samples 48 6 from 16 189 cats Deletions accounted for the majority 17 of 18 samples of the frameshifts The 190 second most common cause of premature termination was a nonsense mutation 15 191 samples 40 5 from 11 cats A missense mutation at the start codon was found in 192 three samples 8 1 from three cats and an ATG codon next to the original start codon 193 in each sequence was out of frame in all three samples Deletion of a region including 194 the start codon was found in one sample 2 7 195 196 Relationship of the mutation of S and 3c genes 197 The relationship of S and 3c gene mutations in each sample type is indicated in Table 198 1 For FCoVs in 19 faecal samples that were obtained from the animal shelter no 199 viruses carried missense mutations at codons 1 058 and 1 060 of the S gene Only one 200 sample showed a deletion of three consecutive nucleotides in the 3c gene causing the 201 lack of one aa 202 In the four cats belonging to a single owner the faecal samples contained FCoVs 203 where the M1 058L mutation was found together with truncating mutations of the 3c 204 10 genes FCoVs in the other ten hospital samples did not carry mutations in the S and 3c 205 genes 206 In the effusion samples type I FCoVs had mutations in both the S and 3c genes in 24 207 of 30 samples A mutation at either codon 1 058 or 1 060 was present in 4 out of 30 208 samples The remaining two effusion samples carried only 3c gene truncating mutations 209 The present study detected two type II FCoVs in ascites samples both viruses carrying 210 truncating mutations in the 3c gene In the tissue samples of the four cats that belonged 211 to one owner all FCoVs in tissues had both the M1 058L mutation and 3c gene 212 truncating mutation 213 214 Sequence relationship among co habitants 215 Six cats included in this study were co habitants 80 81 82 85 87 and 88 that were 216 fed by a single owner Cats 80 82 were 4 month old littermates that died within a 217 month of disease onset and were autopsied Cat 87 died approximately 2 months later 218 and was also autopsied Consequently 14 samples including faecal samples and rectal 219 swabs from the six cats were analyzed and some identical and closely related mutations 220 were identified Fig 2 The ORF homology among the samples ranged from 95 66 221 99 86 222 A 714 base consensus sequence generated from these samples was identical to the 3c 223 gene of an FCoV in a rectal swab from cat 80 Deletion of the second and third 224 nucleotides at codon 153 was found in FCoVs detected in the lymph node and kidney 225 tissues of cat 80 An identical deletion was shared in a virus detected in ascites from cat 226 85 A frameshift caused by this deletion resulted in the generation of a premature stop 227 codon The kidney and lymph node samples of cat 80 demonstrated another virus 228 11 variant featuring a 28 base deletion located 45 bases downstream of the two nucleotide 229 deletion site An FCoV in a rectal swab from cat 85 had a closely related 29 base 230 deletion at the same position in the 3c ORF 231 A nonsense mutation at codon 210 was identified in the rectal swab and vitreous 232 humor sample from cat 82 The same mutation was detected in viruses in a rectal swab 233 ascites and kidney samples from cat 87 The homology of the sequences between the 234 rectal swab from cat 80 and samples from cats 82 and 87 ranged from 99 44 99 86 235 Another nonsense mutation at codon 205 was found in the spleen and lymph node 236 samples of cat 81 A two nucleotide deletion at codon 123 was detected in an FCoV 237 isolated from the faeces of cat 88 238 Cats 7 1 and 7 2 were 3 month old kittens that were housed together Both cats 239 displayed the accumulation of ascites and pleural effusion over the same period and the 240 effusion samples were obtained from the hospital on the same day The sequence 241 homology of the samples was 99 44 but premature stop codons were caused by a 242 deletion and frameshift in cat 7 1 and a nonsense mutation in cat 7 2 243 244 DISCUSSION 245 A previous investigation had determined that 96 2 FIPVs causing wet form FIP had 246 either an M1 058L 89 9 or S1 060A 6 3 mutation in the S gene 6 Our present 247 study revealed that type I FCoVs in ascites and pleural effusion samples had the 248 M1 058L and S1 060A missense mutations at a rate of 80 0 and 13 3 respectively 249 Because histopathological examinations of the cats were not performed the biotypes of 250 FCoVs analyzed in this study could not be determined Therefore the relationship 251 between the biotypes and gene mutations was not analyzed However it is considered 252 12 that approximately 90 FCoVs in effusion samples have one of the S gene mutations 253 On the contrary neither M1 058L nor S1 060A mutations were found in FCoVs in any 254 of the 19 faecal samples from shelter cats some of which had soft stools indicating 255 enteritis A previous report indicated that none of the FECVs in rectal swabs carried a 256 mutation at codon 1 058 or 1 060 6 Accordingly it is suggested that majority FCoVs 257 in faeces of clinically healthy cats and cats with only mild enteritis carry the S genes 258 without any of these mutations The M1 058L mutation was also detected in faeces and 259 tissues of four young diseased cats that had died The biotype of the viruses was 260 unknown but faecal FCoVs with the M1 058L or S1 060A mutation would require 261 experimental infection for pathogenicity determination even when FIP was confirmed 262 via histopathological examination 263 Previous studies have identified truncating mutations of the 3c gene in the genomes 264 of 60 of FIPVs whereas most FECVs carried intact 3c genes Although the precise 265 molecular function of the 3c protein is unknown it has been reported to play an 266 essential role in FECV replication in the intestines 2 13 Our present study determined 267 that 87 5 FCoVs in body cavity effusions which included type I and II viruses and all 268 type I FCoVs in six tissues collected from four cats carried 3c gene mutations resulting 269 in the truncation or loss of 3c protein However such mutations were not detected in the 270 majority of faecal FCoVs of shelter cats that did not exhibit any clinical symptoms 271 except for soft stools This finding is similar to previously published data 3 6 272 Therefore it is considered that in addition to the M1 058L and S1 060A mutations in 273 the S gene a truncating mutation of the 3c gene is another