Low Serum Acclimation Of Marc-145 Cells And Evaluation Of Autophagy Characteristics After Infection Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome is a highly lethal infectious disease.Since its outbreak in China in 2006,it has continued to cause huge economic impact on China's pig industry.As a country that promotes mandatory vaccine immunization,the demand for PRRS vaccine in China is very large.In order to optimize the production process of PRRS vaccine and reduce the production cost,our study explored the low serum culture process of Marc-145 cells,and explored the replication of RPRSV and autophagy after infected with PRRSV under low serum conditions.Acclimation of Marc-145 cells with low serum culture by stepwise serum reduction,with serum addition as a variable?10%,8%,6%,4%,2%,1%,0%?.After 18 consecutive passages,it was found that the minimum serum addition of Marc-145 cells was 1%.By measuring the cell viability and calculating the cell multiplication ratio,it was found that the Marc-145 cells in the 1%serum addition amount were in a good state,and thus the Marc-145 cell line which was stably passaged with a serum addition amount of 1%was successfully obtained.To investigate the replication of PRRSV in Marc-145 cells under low serum conditions,the virus replication was determined by real-time fluorescent quantitative PCR;and the TCID₅₀ of PRRSV was determined.The autophagy in Marc-145cells infected with PRRSV was observed by electron microscopy.The autophagy of cells in different culture conditions was preliminarily determined.The transcription levels of key autophagy-related genes Beclin1,ATG5 and ATG12 were determined by real-time PCR,and the level of autophagy was explored.We found that Marc-145 cells in low serum cultures were maintained longer after infected with PRRSV;the virus content was higher,and the replication peak was about 12 h earlier than in conventional serum culture,but the virus titer did not change much.At 36h after infected with PRRSV,obvious autophagosomes were observed in Marc-145 cells under different culture conditions,indicating that autophagy occurred in the cells after infection with PRRSV.At the transcriptional level of Beclin1,ATG5 and ATG12 autophagy genes,the transcription levels of autophagy-related genes were increased under low serum conditions,suggesting that autophagy enhanced after PRRSV infection in low serum environment.Copying is beneficial.In this study,the culture conditions of Marc-145 cells were successfully explored,and the low serum culture of cells was realized.The culture characteristics and autophagy of PRRSV under low serum conditions were elucidated.It provided reference and ideas for the production and development of PRRSV vaccine and related research on PRRSV and autophagy.