2 MATERIALS AND METHODS
The swab samples of eye, nose and anal from 1,288 clinically diseased cats, tissues from 17 died cats and ascites from 21 cats were collected from Harbin, Shenyang, Hohhot, Tangshan, Tianjin, Beijing, Langfang, Shijiazhuang, Qingdao, Zhengzhou, Luoyang, Hefei, Chongqing, Taizhou, Guangzhou, Haikou in China from 2016 to 2019 (Figure 1). And 316 blood samples were collected from Tianjin, Beijing, Qingdao, Zhengzhou. The study was approved by the Animal Care and Ethics Committee of National Research Center for Veterinary Medicine and conventional animal welfare regulations and standards were taken into account.
The primers for PCR test of FIPV, FeLV, FCV, FHV-1, FPV, FIV were shown in Table 1. The viral nucleotide was extracted using the viral nucleic acid extraction kit II (Geneaid, Taiwan, China). The nucleotide samples of FIPV, FeLV, FCV and FIV were used as templates in the detection of one-step RT-PCR (TransGen Biotech, China), and the nucleotide samples of FHV-1 and FPV were detected using Premix Taq (Ex Taq Version 2.0 plus dye) (TakaRa, Japan).
Prior to further testing, the blood samples of cats were centrifuged (3000 ×g, 15 min) and inactivated (56℃, 30 min). For neutralization assay, two-fold diluted sera with the range of 1:2 to 1:4,096 were added in 96-well microtiter plates and preincubated with the virus (102 TCID50/well) for 1 h before the addition of F81 cells (2.5×104/well). The plates were then incubated at 37℃ in a humidified atmosphere of 5% CO2for 4-5 days. The titer was calculated based on cytopathic effect by the Reed-Muench method. For haemagglutination inhibition (HI) assay of FPV, the serum was two-fold diluted from 1:2 to 1:4,096 and mixed at the equal volume of 0.025 ml with FPV (8 HA unit per sample), and then incubated at 37℃ for 30 min before the equal volume of 1% suspensions of pig red blood cells were added. HI titer was defined as the last dilution that shows completely HI effect after the incubation at 4℃ for 90 min.