key: cord-0912963-3um507y0 authors: Minami, Shohei; Kuroda, Yudai; Terada, Yutaka; Yonemitsu, Kenzo; Van Nguyen, Dung; Kuwata, Ryusei; Shimoda, Hiroshi; Takano, Ai; Maeda, Ken title: Detection of novel ferret coronaviruses and evidence of recombination among ferret coronaviruses date: 2016-07-01 journal: Virus Genes DOI: 10.1007/s11262-016-1365-3 sha: 9bacabad197baf592cc66d688701917ca036064b doc_id: 912963 cord_uid: 3um507y0 In an epidemiological study of ferret coronaviruses (FRCoVs), novel FRCoV strains (Saitama-1 and Aichi-1) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of partial RNA-dependent RNA polymerase (RdRp) genes. Phylogenetic analysis indicated that these strains belonged to different clusters from other FRCoV strains. Next, the nucleotide sequence of the 3′-terminal region of Saitama-1 (8271 bases) strain was determined and compared with those of the other FRCoVs, indicating that the Saitama-1 strain differed from the previously reported MSU-1 and MSU-2 strains in the regions encoding spike (S) protein, nucleocapsid, and open reading frame 7b. Furthermore, the results of SimPlot analysis indicated that FRCoV (MSU-2 strain) emerged via a recombination event of S protein between the MSU-1 and Saitama-1 strains. This mechanism is similar to that responsible for the emergence of type II feline coronavirus. This information will be useful for understanding the pathogenesis of FRCoV in ferrets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11262-016-1365-3) contains supplementary material, which is available to authorized users. (FRSCV) [2, 3, 6] . The characteristic clinical sign of FIPlike disease is large palpable intra-abdominal masses, like dry-type FIP [2, 3, 6] . In addition, there are two genotypes of FRCoV based on differences in the spike (S) gene. This suggests that genotype I is associated with FIP-like disease and genotype II is associated with ECE [18] . However, we previously showed that there was no significant relationship between the genotypes of FRCoV and disease in Japan [14] . In addition, genotype I FRCoV was also detected in numerous asymptomatic ferrets in the Netherlands [9] . The relationship between FRCoV genotype and clinical symptoms thus remains unclear. Reverse transcription-polymerase chain reaction (RT-PCR) was used for epidemiological study of FRCoV infection. FRCoV gene detection in 56-61 % of ferrets in Japan and the Netherlands has been reported [9, 14] . Recently, an enzyme-linked immunosorbent assay (ELISA) using recombinant nucleocapsid (N) protein of FRCoV had been established, indicating that many ferrets possess antibodies against FRCoV [7] . However, FRCoV has not yet been isolated. In animal hospitals throughout Japan, 201 fecal samples were collected from domestic ferrets between Aug 1, 2012 and Dec 8, 2015 and were examined by RT-PCR. RNA was extracted from fecal samples using QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) and RT-PCR was performed using a QIAGEN OneStep RT-PCR Kit (QIA-GEN, Hilden, Germany). For specific and sensitive detection of FRCoV, FRCoV-specific primers, FRCoV RdRp-F1 (5 0 -GTT GGT TGC TGC ACA CAT AG-3 0 ) and FRCoV RdRp-R1 (5 0 -GGA GAA GTG CTT ACG CAA ATA-3 0 ) were used [14] . Coronavirus (CoV)-consensus primers, IN-6 (5 0 -GGT TGG GAC TAT CCT AAG TGT GA-3 0 ) and IN-7 (5 0 -CCA TCA TCA GAT AGA ATC ATC ATA-3 0 ), were also used for the detection of coronaviruses [8] . Both primer pairs target partial RNA-dependent RNA polymerase (RdRp) gene. The results showed that 126 ferrets (62.7 %) in Japan were positive (Supplementary Table S1 ). Significant differences were statistically analyzed using Chi-squared and Fisher's exact probability tests. P values of .05 were considered to be statistically significant. The ratio of detection of FRCoV between diarrhea (75.9 %) and other symptoms (53.4 %) was significantly different. In particular, the difference was marked in ferrets aged more than 3 years, suggesting that FRCoV is the primary pathogen or exacerbating factor for diarrhea in ferrets. In addition, most ferrets aged less than 1 year (81.6 %) were infected with FRCoV, regardless of their clinical symptoms, suggesting that most ferrets are infected with FRCoV soon after birth. Importantly, some strains, Saitama-1 strain from ferret no. 22 and Aichi-1 strain from ferret no. 160, were detected by RT-PCR using CoV-consensus primers, but not FRCoV-specific primers, even though the latter can detect FRCoV with more sensitivity than CoV-consensus primers [14] . The nucleotide sequences of partial RdRp genes were determined using a BigDye Terminator Ver. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. All sequences were deposited to the DNA Data Bank of Japan (DDBJ). A phylogenetic tree was constructed using the program MrBayes Ver. 3.2.2 [11] for MrModeltest analysis with a general time reversible (GTR) or WAG substitution matrices [15] . All trees were graphically represented using FigTree Ver. 1.4.2 [10] . The phylogenetic tree based on the partial RdRp genes showed that the FRCoV Saitama-1 and Aichi-1 strains belonged to a different cluster from other FRCoV strains (Fig. 1a) . In order to determine the 3 0 -terminal region of the Saitama-1 genome, further sequence analysis was performed. QIAGEN OneStep RT-PCR Kit (QIAGEN, Hilden, Germany) and TaKaRa RNA LA PCR TM Kit (AMV) Ver. 1.1 (TaKaRa, Shiga, Japan) were used to amplify each fragment of the Saitama-1 and Aichi-1 strains using the primer pairs listed in Supplementary Table S2 The nucleotide sequence from the S gene to poly A was compared among FRECV MSU-2, FRSCV MSU-1, and Saitama-1 strain using SimPlot Ver. 3.5.1 [5] . SimPlot analysis showed that the Saitama-1 strain was different in some regions, including the N, 3x-like, and ORF7b genes, when compared with other strains (data not shown). Surprisingly, the 3 0 -terminal in two-thirds of S genes in the MSU-2 strain was similar to that in the Saitama-1 strain, but one-third of S genes in the MSU-2 strain was similar to that in the MSU-1 strain (Fig. 2a) . Alignment of S proteins among Saitama-1, MSU-1, and MSU-2 strains also indicated that the MSU-2 strain was similar to the MSU-1 strain in the N-terminal one-third of S protein and to the Saitama-1 strain in the C-terminal two-thirds of the protein Virus Genes (2016) 52:858-862 859 (Fig. 2b) . In addition, a low similarity in nucleotide sequences between the MSU-1 and MSU-2 strains and the Saitama-1 strain was observed in the N, 3x-like, and ORF7b genes, but the E and M genes were highly conserved among all strains (data not shown). Phylogenetic analysis was performed based on the Nterminal one-third of the S protein (a.a. 1-438 of Saitama-1 S protein) and the C-terminal two-thirds of the protein (a.a. 439-1435 of Saitama-1 S protein), indicating that the MSU-2 strain was more similar to the MSU-1 strain than the Saitama-1 strain in the N-terminal onethird of the S protein (Fig. 1b) and was more similar to the Saitama-1 strain than the MSU-1 strain in the Cterminal two-thirds (Fig. 1c) . Furthermore, a phylogenetic tree based on the N protein indicated that Saitama-1 and Aichi-1 strains differed more from the other FRCoVs than from mink coronaviruses (Fig. 1d) . These results indicate that the Saitama-1 strain possesses different S, N, 3x-like, and ORF7 genes than other FRCoVs, and that the E and M genes are conserved among FRCoVs. Therefore, we consider the Saitama-1 and Aichi-1 strains to comprise a novel FRCoV genotype. In addition, these results indicate that the MSU-2 strain emerged by recombination of the S protein between the MSU-1 and Saitama-1 strains. These recombination events often occurred among CoVs, resulting in the diversity of CoV genomes. Especially, the recombination of S protein caused the cross-species transmission or change of pathogenesis of SARS-CoV [4] , porcine transmissible gastroenteritis virus (TGEV), canine coronavirus (CCoV) type II [1] , and feline coronavirus (FCoV) [13] . Novel CoVs must emerge and drastically evolve by these recombination events. Further investigation will thus be required to determine the evolution of CoV including FRCoV. In conclusion, novel FRCoV strains (Saitama-1 and Aichi-1) were detected in Japan. These FRCoVs appear to have emerged by recombination events among other FRCoVs. This information will be useful for understanding the pathogenesis of FRCoV in ferrets. Recombinant canine coronaviruses related to transmissible gastroenteritis virus of swine are circulating in dogs Clinicopathologic features of a systemic coronavirusassociated disease resembling feline infectious peritonitis in the domestic ferret (Mustela putorius) Systemic coronavirus-associated disease resembling feline infectious peritonitis in ferrets in the UK Recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination Detection of feline infectious peritonitis virus-like antigen in ferrets Establishment of serological test to detect antibody against ferret coronavirus Identification of a novel coronavirus in bats Enteric coronavirus in ferrets, the Netherlands FigTree v1.4.2 computer program and documentation distributed by the author MrBayes 3: Bayesian phylogenetic inference under mixed models Coronaviruses: structure and genome expression Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses Genetic characterization of coronaviruses from domestic ferrets A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach Coronavirus-associated epizootic catarrhal enteritis in ferrets Molecular characterization of a novel coronavirus associated with epizootic catarrhal enteritis (ECE) in ferrets Comparative sequence analysis of the distal one-third of the genomes of a systemic and an enteric ferret coronavirus SimPlot analysis was carried out using the nucleotide sequence of the Saitama-1 strain from the S gene to poly A (8263 bases), the MSU-1 strain (8025 bases), and the MSU-2 strain (8309 bases). Green and red lines indicate Saitama-1 and MSU-1, respectively. b Alignments of S proteins of Saitama-1, MSU-1, and MSU-2 Acknowledgments This study was supported by JSPS KAKENHI Grant Number 15H04599.