key: cord-0746255-j6hcp6zx authors: Li, Chunqiu; Liu, Qiujin; Kong, Fanzhi; Guo, Donghua; Zhai, Junjun; Su, Mingjun; Sun, Dongbo title: Circulation and genetic diversity of Feline coronavirus type I and II from clinically healthy and FIP‐suspected cats in China date: 2018-12-05 journal: Transbound Emerg Dis DOI: 10.1111/tbed.13081 sha: d5e1a2285e027d161fb7098f665b63f382087b17 doc_id: 746255 cord_uid: j6hcp6zx Feline infectious peritonitis (FIP) is a fatal infectious disease of wild and domestic cats, and the occurrence of FIP is frequently reported in China. To trace the evolution of type I and II feline coronavirus in China, 115 samples of ascetic fluid from FIP‐suspected cats and 54 fecal samples from clinically healthy cats were collected from veterinary hospitals in China. The presence of FCoV in the samples was detected by RT‐PCR targeting the 6b gene. The results revealed that a total of 126 (74.6%, 126/169) samples were positive for FCoV: 75.7% (87/115) of the FIP‐suspected samples were positive for FCoV, and 72.2% (39/54) of the clinically healthy samples were positive for FCoV. Of the 126 FCoV‐positive samples, 95 partial S genes were successfully sequenced. The partial S gene‐based genotyping indicated that type I FCoV and type II FCoV accounted for 95.8% (91/95) and 4.2% (4/95), respectively. The partial S gene‐based phylogenetic analyses showed that the 91 type I FCoV strains exhibited genetic diversity; the four type II FCoV strains exhibited a close relationship with type II FCoV strains from Taiwan. Three type I FCoV strains, HLJ/HRB/2016/10, HLJ/HRB/2016/11 and HLJ/HRB/2016/13, formed one potential new clade in the nearly complete genome‐based phylogenetic trees. Further analysis revealed that FCoV infection appeared to be significantly correlated with a multi‐cat environment (p < 0.01) and with age (p < 0.01). The S gene of the three type I FCoV strains identified in China, BJ/2017/27, BJ/2018/22 and XM/2018/04, exhibited a six nucleotide deletion (C(4035) AGCTC (4040)). Our data provide evidence that type I and type II FCoV strains co‐circulate in the FIP‐affected cats in China. Type I FCoV strains exhibited high prevalence and genetic diversity in both FIP‐affected cats and clinically healthy cats, and a multi‐cat environment and age (<6 months) were significantly associated with FCoV infection. species Alphacoronavirus 1, the genus Alphacoronavirus, the subfamily Coronavirinae, the family Coronaviridae, and the order Nidovirales. FCoVs are single-stranded positive sense RNA viruses with an approximately 29 kb nonsegmented genome, which consisting of 11 open reading frames (ORFs) (Myrrha et al., 2011) . FCoVs are classified into two serotypes, type I and type II FCoVs, according to their serological properties; they are also separated into two biotypes, FECV and FIPV, based on pathogenicity. These two biotypes, FECV and FIPV, exist in both serotypes I and II (Tekes & Thiel, 2016) . Both type I and type II FCoVs can cause FIP. FIP remains one of the most frequently fatal infectious feline diseases for which there are no effective therapies. America, reaching 80%-95%, while type II FCoVs are reportedly less common in the field (Benetka et al., 2004; Kummrow et al., 2005) . However, type II FCoV infection has predominantly been observed in various Asian countries, reaching 25% (Amer et al., 2012; Sharif et al., 2010) . Double homologous recombination between type I FCoV and CCoV leads to the emergence of type II FCoV Haijema, Rottier, & de Groot, 2007; Herrewegh, Smeenk, Horzinek, Rottier, & de Groot, 1998; Lin, Chang, Su, & Chueh, 2013; Lorusso et al., 2008; Terada et al., 2014) . These data indicate that the causative agent of FIP exhibits immense complexity in different countries and areas. The occurrence of FCoV is frequently reported in China and attracts extensive concern due to its high mortality rate. At present, little information is available on the molecular epidemiology of FCoV in China. In this study, molecular epidemiological investigation of FCoV was carried out in China from November 2015 to January 2018. Partial S genes and nearly complete genomes were used to analyze the genetic evolution of the FCoV strains identified. The aim of the study was to provide insights into the epidemiology and genetic diversity of the FCoV strains circulating in China. The resulting data will provide valuable information for prevention and control of FCoV infections. The current study was approved by the Animal Experiments Committee of the Heilongjiang Bayi Agricultural University (registration protocol 01/2015). The field study did not involve endangered or protected species. No specific permissions were required for location of samples because the samples were collected from public areas or non-protected areas. The sampling and data publication were approved by the cats' owners. In total, 169 samples were collected from veterinary hospitals in China from November 2015 to January 2018 using commercial virus sampling tubes (YOCON Biological Technology Co. Ltd. Beijing, China) with a volume of 3.5 ml. The 169 samples were sourced from Beijing (n = 83), Harbin of Heilongjiang province (n = 17), Daqing of Heilongjiang province (n = 28), Qiqihar of Heilongjiang province (n = 2), Dalian of Liaoning province (n = 4), Chengdu of Sichuan province (n = 14), Huzhou of Zhejiang province (n = 3), Haining of Zhejiang province (n = 4), Xining of Qinghai province (n = 6), and Xiamen of Fujian province (n = 8). For all samples, the cat's age, breed, gender, and collection date were recorded. Of the 169 samples, 54 fecal samples were obtained from clinically healthy cats and 115 samples of ascetic fluid were obtained from FIP-suspected cats. Clinical diagnosis was made for the diseased cats mainly as described by Addie et al. (2009) . The FIP-suspected cats were detected using routine blood parameters, blood biochemical parameters, ultrasonography, and the Rivalta test. The clinical signs shown by the FIP-suspected cats were thoracic effusion, inappetence, anorexia, weight loss, lethargy, icterus, fever, diarrhea, leukocytosis, decrease in lymphocytes, an albumin: globulin ratio <0.8, and ascitic fluid were positive on the Rivalta test. All samples were stored at −80°C. Feline coronavirus infection was detected by RT-PCR targeting the 6b gene. For amplification of the 6b gene of FCoV, RNA extraction and cDNA synthesis were carried out according to the protocol described by Wang et al. (2016) . Random primers (six nucleotides) were used for synthesis of the first-strand cDNA. The PCR amplification of the 6b gene was carried out according to the protocol described by Herrewegh et al. (1995) . In order to differentiate the type I and type II FCoV strains identified in our study, the partial S gene was amplified by RT-PCR using the primers described by Lin et al. (2009) . The purified PCR products of the partial S genes were directly subjected to Sanger sequencing. All nucleotide sequences generated in our study were submitted to GenBank. Sequence analysis was performed using the EditSeq tool in Lasergene DNASTAR ™ 5.06 software (DNASTAR Inc., Madison, WI). Multiple sequence alignments were performed using the Multiple Sequence Alignment tool of DNAMAN 6.0 software (Lynnon BioSoft, Point-Claire, Quebec, Canada). In order to obtain the entire genome of the identified FCoV strains, the cDNAs of 50 selected samples were sequenced by Illumina nextgeneration sequencing (Shanghai Biozeron Biotechnology Co., Ltd, Shanghai, China). Briefly, RNA extraction and cDNA synthesis for the 50 samples were carried out according to the protocol described by Wang et al. (2016) using random primers (six nucleotides) and an oligo dT18 primer. The double-stranded DNA was synthesized by second strand cDNA synthesis kit (Beyotime Biotechnology, Shanghai, China). Subsequently, 150 ng of cDNA was used to construct a library according to the manufacturer's instructions (TruSeq Nano DNA HT Library Prep Kit), and loaded on to a HiSeq × ten for sequencing. Raw sequence reads were trimmed to remove the reads of the adaptor, duplicate reads and host genomic sequences. The trimmed reads were assembled using SOAPdenovo v2.04 (http://soap.genomics.org.cn/), and the assembled genomes were corrected using GapCloser v1.12 software. Viral genes were predicted using the software GeneMarkS (http://topaz.gatech.edu/GeneMark/genemarks.cgi), and annotation of the predicted viruses was carried out through the nonredundant protein database (NR) using BLASTp. In this study, the nearly complete genomes of four FCoV strains were obtained, according to the porcine coronavirus PEDV described by Marthaler et al. (2013) . After genome assembly, the single genomic gap of the four FCoV strains was closed using standard Sanger sequencing technology. A similarity plots analysis of the genomes of the FCoV strains identified in our study was performed by the sliding window method as implemented in the SimPlot, v.3.5.1 package (Lole et al., 1999) . For the phylogenetic analysis, the partial S genes and nearly complete genome sequences of the FCoV strains were retrieved from GenBank. To construct phylogenetic trees, multiple alignments of all target sequences were carried out using the Clustal X program version 1.83 (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) . Furthermore, the phylogenetic trees of all target sequences were generated from the Clustal X-generated alignments by MEGA 6.06 software using the neighbor-joining method (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013) . Neighbor-joining phylogenetic trees were built with the p-distance model, 1,000 bootstrap replicates, and otherwise default parameters in MEGA 6.06 software. Phylogenetic trees were pruned and re-rooted by using the Interactive Tree Of Life (iTOL) software version 4.2.3 (https://itol.embl.de/) which is an online tool for displaying the circular tree and annotation (Letunic & Bork, 2007) . For genotyping of the FCoV strains identified in our study, the partial S gene was amplified by RT-PCR. Of the 126 FCoV positive samples, a total of 95 partial S genes were successfully sequenced. Detailed information on the 95 FCoV-positive samples is shown in FCoV positive samples, the positive rate of FCoV in the FIP-suspected cats was 75.7% (87/115), which was higher than in Korea (19.3%) and Turkey (37.3%) Tekelioglu et al., 2015) . The FCoV positive rate of cats from south China (80.0%) was higher than that of north China (73.1%). Several studies have reported that age, breed, sex, and a multi-cat environment are associated with FCoV infection and development of FIP (Addie et al., 2009; Bell, Malik, & Norris, 2006; Sharif et al., 2009; Worthing et al., 2012) . In the present study, FCoV infection was significantly associated with living environment and age, which is in line with previous studies (Addie et al., 2009; Drechsler, Alcaraz, Bossong, Collisson, & Diniz, 2011; Tekelioglu et al., 2015) ; the positive rate of FCoV in a multi- cat environment was higher than that in single cat households. These data suggest that a multi-cat environment confers a high risk for FCoV infection and development of FIP in China. In our study, the prevalence of FCoV was not significantly associated with sex, which is in agreement with several studies (Bell et al., 2006; Sharif et al., 2009; Taharaguchi, Soma, & Hara, 2012) . On the contrary, several studies have shown that FCoV infection appeared to be significantly correlated with the male sex (Pesteanu-Somogyi, Radzai, & Pressler, 2006; Worthing et al., 2012) . However, Tekelioglu et al. (2015) reported that the seroprevalence of FCoV was not associated with sex. In the current study, FCoV infection was also not associated with the breed of cat, which is in line with a study in Turkey (Tekelioglu et al., 2015) . In contrast, several studies have reported that FCoV infection exhibited a significant association with breed: purebred cats appear to be more susceptible to FCoV infection (Foley & Pedersen, 1996; Kiss, Kecskeméti, Tanyi, Klingeborn, & Belák, 2000) . In Australia, FCoV infection occurred at a significantly higher prevalence in British Shorthairs, Cornish Rex and Burmese cats than in Siamese, Persian, Domestic Shorthairs, and Bengal cats (Bell et al., 2006) . In Malaysia, the FCoV infection rate in Persian purebred cats (96.0%) was higher than that in the mixedbreed cats (70.0%) (Sharif et al., 2009) . In Japan, the prevalence of antibodies against FCoV in purebred cats (66.7%) was higher than that in random breeds (31.2%) (Taharaguchi et al., 2012) . In our study, FCoV infection was significantly associated with young cats (aged <6 months). Other studies have reported that cats of ages ranging from 3 to 11 months exhibit a higher FCoV prevalence than those of other ages (Bell et al., 2006; Pedersen, 2009; Taharaguchi et al., 2012) . However, FCoV infection in Australia and Malaysia was reported not to be associated with age (Bell et al., 2006; Sharif et al., 2009 CCoV leads to the emergence of type II FCoV Haijema et al., 2007; Herrewegh et al., 1998; Lin et al., 2013; Lorusso et al., 2008; Terada et al., 2014 The current study is the first to reveal the circulation and genetic diversity of type I and II FCoVs from clinically healthy and FIP-suspected cats in China. The type I and type II FCoV strains co-circulate in the FIP-affected cats in China. Type I FCoV strains exhibited high prevalence and genetic diversity in both FIP-affected and clinically healthy cats. 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