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1、Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan Takehisa Soma a,*, Tsuyoshi Ohinatab, Hiroshi Ishiic, Toshikazu Takahashid, Satoshi Taharaguchie, Motonobu Hara e aVeterinary Diagnostic Laboratory, Marupi Lifetech Co. Ltd., 103 Fushiocho, Ikeda, Osaka 563-0011, Japan b A
2、nimal Health, Pfi zer Japan Inc., 3-22-7 Yoyogi, Shibuya, Tokyo 151-8589, Japan cSukagawa Animal Hospital, 44-11 Morijuku, Sukagawa, Fukushima 962-0011, Japan dTakahashi Animal Hospital, 1785-1 Shimotsuruma, Yamato, Kanagawa 242-0001, Japan eDepartment of Microbiology, School of Veterinary Medicine,
3、 Azabu University, 1-17-71 Fuchinobe, Sagamihara, Kanagawa 229-8501, Japan a r t i c l ei n f o Article history: Received 27 August 2009 Accepted 23 May 2010 Keywords: Canine coronavirus Canine parvovirus Genotype Japan Prevalence PCR a b s t r a c t To clarify the prevalence of canine coronavirus (
4、CCoV) infection in Japan, faecal samples from 109 dogs with diarrhoea were examined for CCoV RNA together with canine parvovirus type 2 (CPV-2) DNA. The detection rates of CCoV and CPV-2 for dogs aged less than 1 year were 66.3% and 43.8%, while those for dogs aged 1 year or older were 6.9% and 10.3
5、%, respectively, which were signifi cantly different (p 0.0001 and p = 0.0003, respectively), indicating not CPV-2 but CCoV is an important diarrhoea-causing organism in juvenile dogs. Among the CCoV-positive dogs, 65.5% and 72.7% showed to be positive for CCoV types I and II, respectively, and simu
6、ltaneous detection rate of both types was high at 40.0%. Furthermore, trans- missible gastroenteritis virus (TGEV)-like CCoV RNA was detected from 8 dogs. These fi ndings indicate that CCoV type I and TGEV-like CCoV are already circulating in Japan, though no reports have been pre- sented to date. ?
7、 2010 Elsevier Ltd. All rights reserved. Canine coronavirus (CCoV) is a positive stranded RNA virus, and has been classifi ed as a Group 1 coronavirus along with feline coro- navirus (FCoV), transmissible gastroenteritis virus (TGEV), and hu- man coronavirus 229E (Pratelli, 2006). CCoV infection occ
8、urs mainly by oral ingestion of the virus in excreted faeces, and the major symptoms are digestive conditions such as diarrhoea and vomiting. Juvenile dogs and dogs under stress tend to be affected more frequently. Furthermore, the symptoms become more severe in dogs co-infected with canine parvovir
9、us type 2 (CPV-2) (Appel, 1988). CCoV was initially isolated from military dogs with diarrhoea in 1971 (Pratelli, 2006). The virus has spread extensively throughout the world and in Japan it was fi rst isolated in 1982 (Yasoshima et al., 1983). Thereafter, evidence of CCoV infections in Japan has be
10、en confi rmed by serological and genetic studies (Bandai et al., 1999; Mochizuki et al., 1987, 2001; Soma et al., 2001). CCoV has long been recognized as a single genotype. However, Pratelli et al. (2002b, 2003) reported two genotypes of CCoV; atypical CCoV, designated as CCoV type I with a spike-pr
11、otein gene similar to that of FCoV, and the well-known typical CCoV, designated as CCoV type II. It has been confi rmed that CCoV type I is in circula- tion in Europe and China (Benetka et al., 2006; Decaro et al., 2010; Pratelli et al., 2003; Stavisky et al., 2010; Yesilbag et al., 2004; Wang et al
12、., 2006). Furthermore, Wesley (1999) reported a novel CCoV type II named TGEV-like CCoV, which possesses an S gene similar to that of TGEV, and this virus also appears to be cir- culating in dogs in Europe and the USA (Decaro et al., 2009, 2010; Elia et al., 2010; Erles and Brownlie, 2009). It is hi
13、ghly likely that CCoV type I and TGEV-like CCoV are already in circulation in Japan, though no reports have been presented to date. In the present study, for the purpose of clarifying the present state of CCoV infec- tion in Japan, faecal samples of dogs with diarrhoea were examined for CCoV RNA and
14、 detected CCoV strains were examined for geno- type. In addition, CPV-2 DNA was investigated and its detection rate was compared with that of CCoV RNA. Rectal swab samples were collected from a total of 109 pri- vately owned (not kennelled) family pet dogs with diarrhoea which visited veterinary cli
15、nics in Japan in the period from Sep- tember 2007 to December 2009. None had received CCoV live vac- cine and CPV-2 vaccine derived from CPV-2 new type, the antigenic variants referred to as types 2a, 2b and 2c, though some dogs had received the vaccine from the original CPV-2 (old type). The swab s
16、amples were soaked in a small amount of sterile saline and imme- diately preserved in a frozen state. Viral RNA was extracted from the swab samples using QIAamp Viral RNA Mini Kit (Qiagen, Tokyo, Japan) according to the manufacturers instruction. RT-PCR for detecting CCoV RNA was performed using QIAGEN OneStep RT- PCR Kit (Qiagen, Tokyo, Japan). About 2.5lL of the extracted RNA was added to the 25lL reaction mixtures containing 5lL of 5? 0034-5288/$ - see front matter ? 2010 Elsevier Ltd. All
