The Transmissible Gastroenteritis Virus Isolation And Partial Characteristic Identification

Transmissible Gastroenteritis virus of swine(TGEV) belongs to the family of Coronaviridae, genus, which is the main pathogen of viral diarrhea in swine. Transmissible gastroenteritis(TGE) caused by TGEV results in a series of clinical symptoms such as severe diarrhea, vomiting and dehydration. TGE is characterized by acute onset, wide range of prevalence as well as co-infection with other pathogens which are responsible for the high rates of morbidity and death, bringing serious losses to the swine husbandry. The research of isolation and identification of TGEV is be of great significance to the basic virology study, the preparation of related biologicals as well as the prevention of TGE.In this study, the TGEV potentially contaminated faeces samples were chosen as the source of virus. The proliferative status in the porcine primary testicular cell, ST cell line and PK15 cell line were explored.The virus passaged about 25 rounds in the three corresponding cell was subjected to the RTPCR experiments. The specific viral bands were found during the test which indicated that TGEV were able to proliferate in the given cells.By comparing the viral titer, the one presented in ST cell line was close to the others. However the CPE appeared earlier in ST cell line. Thus, the ST cell was chosen as the optimal one for the cultivation of TGEV.When being adapt to the ST cell line, the virus was examined by RT-PCR experiments in which the specific primers of TGEV, PEDV and PRV were applied, respectively. The results showed that a potential co-infection of TGEV, PEDV and PRV had taken place.The plaque cloning technology was used for the purification of the TGEV in this study. By screening the conditions for the formation of TGEV plaque, we found the optimal condition depicted in the following sequence. The concentration of agarose and neutral red were 1.6% and 0.02%, respectively. The temperature of the nutrient fluid is about 30℃. The thickness of culture-agarose is in 3mm. The plaque purification was carried on until the plagues were in the same size and shape. Meanwhile, the PCR results of each plague presented TGEV-positive. The initially purified virus was cultured in ST cell continuously and verified to proliferate on ST cell steadily through RT-PCR experiments.The indirect immunofluorescence test showed TGEV positive. Microscopic observation showed that the virus particle processing the typical coronary structure was about 150 nm in diameter, when dispersing in the cytoplasm about 60-100 nm in diameter.A series of physicochemical properties of TGEV were explored. The results showed that TGEV was sensitive to heat, the PH value and some organic reagents. The virus could be eliminated in 60℃ water bath for 30 min. The viral titer decreased dramatically when TGEV was placed in both acidic(PH = 3) and basic(PH = 9) environments. The virus also exhibited sensitivity to some organic reagents such as diethyl ether and chloroform.The purified TEGV was applied to the animal experiment. The piglet was found diarrhea, vomiting, weight losing and dehydrating after 30 hours of oral infection of TEGV. The infected individual became sluggish in motion then spastic and ultimately died in 8 days. Faeces collection was developed during the specific periods of time after infection for the RT-PCR experiment. The PCR results indicated that viral shedding began after 72 hours of infection. During the autopsy, the intestines were found inflated and full of fluid, and the small intestinal villi of which became short or even detached.The completion of isolation and purification of TGEV in this communication may lay a foundation for the future epidemiological studies, laboratory diagnosis as well as the production of vaccine.