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METHODOLOGY ARTICLE Molecular epidemiological of ciently replicates in macrophages monocytes and can against FCoV is rarely of diagnostic value in FIP Thus Tanaka et al BMC Veterinary Research 2015 11 57 DOI 10 1186 s12917 015 0372 2 Institute and we have been able to readily obtain nu cleotide sequences of the FCoV genome With regard Life Science University 1 7 1 Kyounan Musashino Tokyo 180 8602 Japan Full list of author information is available at the end of the article lead to FIP which is a highly lethal systemic granuloma tous disease 2 FIPV exists in two serotypes based on virus neutralizing antibodies type I and type II 1 3 Serotype I virus has a distinctive spike protein while the spike protein of serotype II is a recombinant protein be tween feline and canine enteric coronaviruses 4 Type I Histopathological examination of infected tissues is needed for the aetiological diagnosis of FIP 10 11 RT PCR plus nested PCR and real time PCR tech niques have allowed the detection or genotyping of FCoV 3 12 15 However they have not been fully ree valuated whether they can tolerate the genetic diversity of FCoV In recent years whole genome sequences of clinical strains of FCoV have been registered in the NCBI database by Rottier et al at the J Craig Venter Correspondence ytanaka nvlu ac jp Equal contributors 1 Department of Veterinary Hygiene Veterinary School Nippon Veterinary thus a universal method for heteropopulations of FCoV variants in clinical specimens is desirable Results To develop an RT PCR method for detection and genotyping of FCoV we performed comparative genome analysis using WGSs of 32 FCoV 7 CCoV and 5 TGEV strains obtained from NCBI As the PCR target we focused on the nsp14 coding region which is highly conserved and phylogenetically informative and developed a PCR method targeting nsp14 partial sequences Among 103 ascites 45 pleural effusion and 214 blood specimens from clinically ill cats we could detect FCoV in 55 53 4 14 31 1 and 19 8 9 specimens using the present method Direct sequencing of PCR products and phylogenetic analysis allowed discrimination between type I and II FCoV serotypes Our nsp14 amino acid sequence typing nsp14 aa ST showed that the FCoV clone with sequence type ST 42 which was the most predominant genotype of WGS strains was prevalent in domestic cats in Japan Conclusions Our nsp14 PCR scheme will contribute to virus detection epidemiology and ecology of FCoV strains Keywords Feline coronavirus Epidemiology Phylogenetic study Background Feline coronavirus FCoV can be classified into two bio types namely low virulent feline enteric coronavirus FECV and highly virulent feline infectious peritonitis virus FIPV 1 Clinical appearance of FECV if any is FECVs FIPVs predominate throughout the world but type II strains appear to be more adaptable to tissue cul ture However type I strains are more likely to cause clinical FIP signs 3 FCoV is also common in healthy cats worldwide less than 10 of FCoV seropositive cats develop FIP 5 9 Therefore measuring antibody levels coronavirus strains in Japan targeting nsp14 gene Yoshikazu Tanaka 1 Takashi Sasaki 1 Ryo Matsuda 1 Yosuke Abstract Background Feline infectious peritonitis is a fatal disease FCoV For detecting or genotyping of FCoV some RT PCR previously However referring to the whole genome sequences 2015 Tanaka et al licensee BioMed Central Commons Attribution License http creativecommons reproduction in any medium provided the original Dedication waiver http creativecommons org publicdomain zero 1 0 unless otherwise stated Open Access study of feline using RT PCR Uematsu 2 and Tomohiro Yamaguchi 2 cats caused by infection with feline coronavirus plus nested PCR techniques have been reported WGSs registered at NCBI there are no detection This is an Open Access article distributed under the terms of the Creative org licenses by 4 0 which permits unrestricted use distribution and work is properly credited The Creative Commons Public Domain applies to the data made available in this article ants in FCoV strains primers were designed by mul imens 103 ascites 45 pleural effusion 214 blood 9 Tanaka et al BMC Veterinary Research 2015 11 57 Page 2 of 9 to genome information there have been no reports on PCR based methods that can tolerate the genetic diver sity in whole genome sequenced FCoV strains Thus a detection method for all variants of FCoV is highly desirable In the present study in order to construct a universal method for FCoV variants we performed a genome wide analysis of FCoV and developed an RT PCR method for detecting FCoV in clinical specimens Consequently direct sequencing of PCR products and phylogenetic analysis allowed discrimination between type I and II serotypes of FCoV Using this method we investigated the population genetics of FCoV strains from diseased cats in Japan Methods Bioinformatics for FCoV CCoV and TGEV As shown in Additional file 1 thirty two FCoV strains seven canine coronavirus CCoV strains four transmis sible gastroenteritis coronavirus TGEV strains a por cine respiratory coronavirus PRCV strain and a Mink coronavirus MiCoV strain were used for comparative genome analysis All sequences were obtained from the NCBI database Gene searches and annotation were car ried out by GeneMarkS http opal biology gatech edu genemarks cgi 16 RAST annotation server http rast nmpdr org 17 and the blast based method MAFFT v7 037 was used for multiple sequence alignment 18 and Aminosan v1 0 2011 was performed for amino acid substitution model selection 19 Phylogenetic inference using maximum likelihood ML and bootstrapping was performed using MEGA v5 05 20 In order to perform a genome wide comparison among type I and II FCoV CCoV and TGEV strains we used the Genotyping tool at NCBI http www ncbi nlm nih gov projects genotyping formpage cgi which helps identify genotype and recombinant sequences using the blast based method 21 Default values were used 300 for window and 100 for increment Cell culture and virus Felis catus whole fetus 4 fcwf 4 American Type Culture Collection VA USA cells were maintained in Dulbecco s modified Eagle s medium D MEM Sigma Aldrich Tokyo Japan supplemented with 10 fetal bovine serum JRH Nissui Tokyo Japan We purified FCoV using linear sucrose gradient ultracentrifugation from FCoV 79 1146 strains a gift from Tsutomu Hodatsu Kitasato University Japan propagated in fcwf 4 cells RNA extraction and reverse transcription PCR Isogen LS Nippon Gene Toyama Japan was used for RNA preparation from clinical specimens whole blood cerebral fluid and 1 pericardial effusion which were ob tained in the examination of clinically ill cats for the presence of FCoV were used in the present study To detect FCoV in clinical specimens we performed RT PCR using a random primer plus a single PCR targeted nsp14 constructed in the present study To differentiate type I FCoV from type II FCoV or CCoV genotypes dir tiple alignments of nucleotide sequences of the nsp14 genes in all whole genome sequenced FCoV closely related subspecies CCoV and TGEV strains The pri mer set nsp14 F 5 GTGATGCTATCATGACTAG 3 and nsp14 R 5 CACCATTACAACCTTCTAA 3 was used The expected size of PCR products was 417 bp The reaction mixture for PCR consisted of 4 uni03BCl of cDNA in a total volume of 25 uni03BClcomposedof1 UofExTaq Takara Bio 10 pmol of each primer 0 2 mM deoxynucleoside triphosphate mixture and 1 re action buffer Takara Bio Reaction mixtures were thermally cycled once at 95 C for 2 min 40 times at 95 C for 30 s 48 C for 35 s and 72 C for 45 s and then once at 72 C for 5 min Using 6 uni03BClofPCRsam ple DNA fragments were analyzed by electrophoresis in 1 Tris acetate EDTA on a 1 agarose gel stained with ethidium bromide In addition these PCR products were directly sequenced using a Big Dye terminator version 3 1 cycle sequencing kit Applied Biosystems Tokyo Japan with an ABI Prism 3100 genetic analyzer Applied Biosystems Total RNA was extracted from the fcwf 4 cells in fected with FCoV strain 79 1146 and was reverse transcribed into cDNA Viral cDNA was quantified using a real time PCR method as reported previously 22 Using cDNA samples of known copy numbers we evaluated the detection limit of our PCR method target ing nsp14 Study population in molecular epidemiological study of FCoV strains from clinically ill cats in Japan In the period between 2007 and 2014 in Japan 372 spec pleural fluid ascites pericardial effusion and cerebral fluid and fcwf 4 cells infected with FCoV 79 1146 strain according to the manufacturer s protocol Total RNA was reverse transcribed using the PrimeScript RT PCR kit Perfect Real Time Takara Bio Shiga Japan as reported previously 22 Construction of PCR method for detection and genotyping of FCoV strains In order to construct a PCR method that detects vari ect sequencing of their PCR products and phylogenetic analysis were carried out strains by specimen Genome wide comparison between FCoV strains and nucleocapsid gene as reported previously 25 26 The Tanaka et al BMC Veterinary Research 2015 11 57 Page 3 of 9 recombination site varied slightly from one strain to an other In the phylogenetic tree based on concatenated sequences of nsp13 nsp14 nsp15 nsp16 and spike which are protein coding regions located within recom binant sequences all type II FCoV strains were clustered into a clade belonging to canine coronavirus strains closely related subspecies CCoV and TGEV In order to compare the genome structures between type I FCoV and type II FCoV and the closely related subspecies CCoV TGEV and PRCV we carried out a blast based genome wide comparison After performing genotyping genomic regions of 300 bp in each viral strain were color coded according to scores 0 to 300 based on nucleotide similarities against FCoV strain UU9 Figure 1 In the genome structures of all strains with type II FCoV genotype events of large scale recom bination were found in a locus that stretches for about 12 000 bp from the first half of nsp12 to upstream of the The diversity and evenness of ST distribution by spe cimen type were calculated using Simpson sdiversity index 1 andPielou s evenness index J 23 24 These parameters have generally been used for compar isons of biodiversity between geographically or eco logically separated environments Both values range from 0 no diversity or evenness to 1 extreme diver sity or evenness Results In order to determine the significance of differences in detection rate between ascites pleural effusion and blood specimens relative risk and odds ratio were calcu lated using MedCalc software http www medcalc org index php nsp14 amino acid sequence typing nsp14 aa ST and comparison of genetic diversity in FCoV strains between different specimens In order to devise a genotyping method for FCoV we assigned an ST number to all distinct nsp14 partial amino acid sequences 135 aa found in all FCoV strains which were sequenced in the present study and previ ously registered in the NCBI database We compared the population genetic structures of FCoV strains in Japan with whole genome sequenced strains from 6 dif ferent countries Comparison of diversity and evenness indexes of FCoV data not shown Among the alpha coronavirus 1 subspecies to which FCoV CCoV TGEV and PRCV belonged the coding regions from nsp14 to nsp16 were the most highly conserved Figure 1 PCR method targeting nsp14 for detection of FCoV strains from clinical specimens Basedonagenome widecomparisonusingtheGeno typing tool at NCBI we focused on protein coding se quences within nsp14 to nsp16 as a candidate PCR target The sequence region was suited to universal de tection of variants because of its highly conserved se quence Among the primer sets designed in nucleotide sequences conserved in all FCoV CCoV TGEV and PRCV strains we determined the best primer set nsp14 F and nsp14 R which allowed amplification of 417 bp of the nsp14 partial sequences Consequently this PCR is universally applicable to all alpha coronavirus 1 subspecies We were able to detect FCoV strains from ascites pleural effusion pericardial effusion and blood samples using the present method Few nonspecific bands were found in agarose gel electrophoresis Figure 2 Specific amplification of target sequences against all PCR prod ucts was confirmed by sequencing analysis To evaluate the detection limit the present method was applied to cDNA samples of known copy number using PCR The detection limit was 4 59 10 2 copies mL Sequence based differentiation of type I FCoV from type II FCoV or CCoV The nsp14 coding region was located on the recombin ation hotspot in type II FCoV and sequencing analysis of the region permitted discrimination between type I and II serotypes The nucleotide identity of the nsp14 partial sequence among FCoV CCoV TGEV and PRCV strains ranged from 89 9 to 100 By phylogenetic ana lysis based on nsp14 partial nucleotide sequences 406 bp we were able to successfully distinguish the type I FCoV genotype from type II FCoV CCoV or TGEV Figure 3 Unfortunately our method had no discrim inating power for differentiating between type II FCoV and CCoV genotypes Comparison of FCoV detection rate from ascites pleural effusion and blood in cats Using the PCR and direct sequencing method targeting nsp14 in the present study we surveyed FCoV strains from clinically ill cats on which FIP was suspected but not confirmed by a complete clinico pathological or pathological workup in veterinary hospitals in Japan In peritoneal and pleural fluid samples FCoV was detected in 55 of 103 53 4 and 14 of 45 31 1 respectively All of these exhibited the type I FCoV genotype In a com parison of positivity rate of FCoV between peritoneal and pleural effusion samples relative risk and odds ratio were Tanaka et al BMC Veterinary Research 2015 11 57 Page 4 of 9 1 7164 and 2 5372 95 confidence interval 1 21 5 32 z statistic 2 465 P 0 0137 respectively and we found sta tistically significant differences between the two On the other hand positive results on nsp14 PCR from 214 blood samples 9 cerebral fluid samples and 1 pericardial effusion sample from 224 clinically ill cats were seen in 19 8 9 0 0 and 1 100 respectively Only one of the blood samples exhibited type II FCoV or CCoV genotype Population genetics of FCoV strains in Japan by nsp14 amino acid sequence typing nsp14 aa ST Using nsp 14 amino acid sequence typing nsp14 aa ST which was an allelic analysis based on 135 amino acid residue of nsp14 we identified fifty three unique STs among the 89 FCoV positive samples in this study and 39 whole genome sequenced FCoV and CCoVstrains regis tered in the NCBI database Additional file 2 27 29 The Figure 1 Genome wide comparison of FCoV CCoV TGEV and PRCV strains bp of strain UU9 were compared with 43 whole genome sequenced strains a red yellow green gradient with green being the top score 300 and red corresponding region in FCoV strain UU9 assigned ST numbers of nsp14 aa ST in whole genome sequenced strains are shown in Additional file 1 As shown in Figure 4 our nsp14 aa ST indicated that the most predominant genotype among domestic cats in Japan was ST42 24 of 89 27 0 which was also found most frequently in whole genome sequenced strains followed by ST8 n 10 and ST25 n 7 and the top three STs accounted for 46 1 of total FCoV positive samples Among ascites specimens in the present study ST42 was the most frequent genotype n 18 followed by ST8 n 5 and ST25 n 5 Of pleural effusion and blood samples multiple strains of ST42 n 4 ST8 n 2 and ST8 n 3 ST25 n 2 ST42 n 2 were identified respectively We were unable to find any cor relations between types of specimen and STs As shown in Table 1 populations of FCoV strains from blood specimens showed high values for both diversity and evenness indexes which indicates an almost random registered at NCBI database Genomic regions of every 300 using BLAST Genotyping tool 21 The blast score were visualized in being the bottom score 0 indicating nucleotide similarity against the Tanaka et al BMC Veterinary Research 2015 11 57 Page 5 of 9 pattern The lowest values for both diversity and evenness indexes in ascites suggested the presence of ascites tropic clones In addition to FCoV detection rate population structures of FCoV strains between ascites and pleural effu Figure 2 Agarose gel electrophoresis of nsp14 PCR amplification products from clinical specimens Lane 1 pleural fluid lane 2 ascites lane 3 pericardial effusion lane 4 FCoV positive whole blood by aqPCRmethod lane5 FCoV negative whole blood by a qPCR method lane 6 fcwf 4 cells infected with FCoV strain 79 1146 M W M molecular weight marker 100 bp ladder is loaded on the agarose gel sion specimens also exhibited different trends Discussion In previous epidemiological studies nested PCR tech niques targeting 3 UTR and spike sequences have been widely used for viral detection and discrimination be tween type I and II serotypes respectively 3 12 30 36 However previous methods are time consuming and nonspecific amplification often occurs in the course of nested PCR Our nsp14 PCR method allowed detection of FCoV using a single PCR and little nonspecific ampli fication occurred during PCR in various clinical speci mens suggesting that the primer set exhibited high specificity to only viral genome Consequently our method allows to ascertain accurately the presence of FCoV in any type of specimen without confirmation by sequencing of PCR products Recently Soma et al re ported that positivity of FCoV was 44 1 377 of 854 in ascites from cats suspected of wet FIP in Japan using the 3 UTR nested PCR method 36 Although a simple comparison was not clear because of different study populations detection sensitivity of FCoV in ascites specimens of our nsp14 single PCR was comparable or better P 0 0746 compared with the study using the nested PCR method There have been some reports on the quasispecies in FCoV 31 37 41 Battilani et al reported that quasispecies composition is correlated with the seriousness of clinical form and lesions in the organs 37 Therefore detection of a population that consists of a complex of heterogeneous variants is necessary in the diagnosis of FCoV infection Consequently this may have resulted in improved sensitivity for FCoV detection by the present PCR targeting nsp14 which is a highly conserved region among alpha coronavirus1 subspecies The present method is the most reliable PCR method that can tolerate the genetic diversity of FCoV Using the scheme of nsp14 aa ST we found that a clone of FCoV ST42 is prevalent in domestic cats in Japan It is likely that the ST42 FCoV clone is also en demic worldwide as it accounts for most of the whole genome sequenced strains registered at NCBI The glo bal distribution of ST8 and ST25 FCoV clones which are second and third most common STs after ST42 in Japan remains to be clarified Further epidemiological study is thus needed Aggregated distribution of specific STs in FCoV strains from ascites was in contrast to that of blood specimens that formed an almost random pattern suggesting that a tropic ascites clone is present among FCoV populations A previous study also reported differences of quasispe cies compositions of FCoV in ORF7b and N region be tween organs 38 Interestingly diversity and evenness of FCoV strains from pleural effusion were similar to those of blood but not ascites With regard to both viral detection rate and population structures different trends between ascites and pleural effusion specimens suggest that pathology of FCoV relevant pleural effusion or pleural FIP have markedly different characteristics from those of FCoV relevant ascites or peritoneal FIP Previ ous studies reported that genomic alteration or variabil ity of viral population during infection could affect the organ specificity severity and immunological escape 37 39 41 Although an onset of FCoV infection or FIP highly depends on host factors FCoV also might exhibit the pathogenic