Although this and the previous report (Hohdatsu et al., 1992) were different in methods and samples, it might suggest that type II FCoV contamination has been decreasing in Japanese domestic cats in recent years. In conclusion, our newly established VN test can specifically detect and distinguish between types of FCoV infection. positive-stranded Rebaudioside C RNA viruses. Antigenically and genetically, coronaviruses are currently grouped into at least four groups. FCoV belongs to group I coronavirus which includes canine coronavirus (CCoV), transmissible gastroenteritis computer virus (TGEV), porcine respiratory coronavirus and human coronavirus 229E (Wege et al., 1982). Two pathotypes of FCoVs are also known: one is feline enteric coronavirus (FECV) which causes from asymptomatic contamination to severe enteritis and another is usually feline infectious peritonitis computer virus (FIPV) that causes fatal immune-mediated disease FIP (Pedersen, 1987, Pedersen et al., 1981). It was exhibited that FIPV arises inside of cats by mutation from infected FECV (Vennema et al., 1998). However, it is difficult to distinguish between FECV and FIPV by serological and genetical methods (Boyle et al., 1984, Fiscus and Teramoto, 1987, Pedersen et al., 1984). Both FECV and FIPV are divided into two types I and II, based on their neutralization reactivity with spike (S) protein-specific mAbs (Fiscus and Rebaudioside C Teramoto, 1987, Hohdatsu et al., 1991b, Hohdatsu et al., 1992) and sequence analysis of the S protein gene (Motokawa et al., 1995, Motokawa et al., 1996). In addition to this serological property, some other biological characteristics to each type have been described. While FCoV type I grows poorly in cell culture, type II can grow well in many different cell lines (Pedersen et al., 1984). Furthermore, type II FCoV shows close antigenic and genetic relationship to CCoV and TGEV, suggesting that FCoV type II arose from double recombination between FCoV type I and CCoV (Herrewegh et al., 1998) FCoV type II strains are more frequently utilized than type I strains for experiments because of higher efficient growth. For serological survey of FCoV contamination, antibody has been detected by indirect fluorescent antibody assay (IFA) or enzyme-linked immunosorbent assay (ELISA) using FCoV (Pedersen, 1976, Scott, 1979, Horzinek and Osterhaus, 1979, Ishida et al., 1987). Alternatively CCoV or TGEV are also used as a target antigen because Mouse monoclonal to FBLN5 of serological close relations to FCoV. However, discrimination ability (specificity) of these methods using heterologous antigens seems to be low since they detect also cross-reactive antibodies between types I and II, such as antibody to nucleocapsid (N) protein. In this study, we developed a plaque-reduction neutralization test (PRNT) to serologically distinguish FCoV type I and II infections in cats, and we applied it for field cases. 2.?Materials and methods 2.1. Cell cultures Feline whole fetus cells (fcwf-4 cells), which are Rebaudioside C fetal feline lung cells having characteristics of macrophages (Jacobse-Geels and Horzinek, 1983) were produced in Dulbecco’s altered Eagle’s medium (DMEM) made up of 10% fetal calf serum (FCS), 100?U/ml penicillin and 100?g/ml streptomycin. The cells were maintained in a humidified 5% CO2 incubator at 37?C. 2.2. Viruses FIPV type I strains C3663 and Yayoi and FIPV type II strains KUK-H/L and M91-267 were used in this study. KUK-H strain was isolated from the spleen sample of effusive form FIP case in 1987 by using CRFK cells, and KUK-H/L was plaque-purified from the KUK-H strain (Mochizuki et al., 1997). M91-267 strain was isolated from the spleen sample taken at the postmortem examination of effusive form FIP case in 1991(Mochizuki et al., 1997). These two strains were classified into type II by using FCoV type-specific mAbs, kindly provided by Dr. T. Hohdatsu (Hohdatsu et al., 1991a, Hohdatsu et al., 1991b). C3663 strain was isolated from a cat with an effusive form of FIP in 1994 and used Rebaudioside C as a reference FCoV type I Rebaudioside C (Mochizuki et al., 1997). In addition, it was genetically confirmed that C3663 belongs to type I FCoV and M91-267 and KUK-H/L do to type II (manuscript in preparation). Yayoi strain was isolated from liver homogenate from non-effusive form of FIP by serial passage in suckling mouse brain and then in fcwf-4 cells and have been used as Japanese prototype.