Abstract:

Objective To explore the replication of rotavirus SA11 strain in vivo and in vitro after adding Enterococcus faecalis （E. faecalis），and to provide the evidence for clarifying the effect of E. faecalis on rotavirus infection. Methods In in vitro experiment， 10⁸－10¹² CFU ·mL^－1E. faecalis were diluted with 10 times concentration gradient and co-cultured with Caco-2 cells for 12， 18， 24， 30，and 48 h，and control group （Caco-2 cells infacted with rotavius without the addttion of E. faecalis） was set up at the same time. The survival rates ofCaco-2 cellsin various groups after treatment of different concentrations of E. faecalis were determined by CCK-8 method.One hour after the Caco-2 cells were treated with rotavirus with multiplicity of infection （MIO） value of 0.1，they were cultured with 10⁸－10¹² CFU·mL^－1E.faecalis for 18 h. The rotavirus VP6 gene copy numbers of Caco-2 cells in various groups were detected by real-time fluorescence quantitative PCR（RT-qPCR） method and the rotavirus titers of Caco-2 cells in various groups were detected by immunofluorescence focus method.In in vivo experniment， 18 litters of 4－5 d old SPF Kunming suckling mice were randomly divided into control group， antibiotic treatment group and E. faecalis transplantation group. The suckling mice in antibiotic treatment group and E. faecalis transplantation group were gavaged with quadruple antibiotics for 3 d to deplete their intestinal flora， and the suckling mice in three groups received 10⁷ FFU·mL^－1 rotavirus 100 μL by gavage daily for 8 d， and the suckling mice in E. faecalis transplantation group were given 10¹⁰ CFU·mL^－1E. faecalis 100 μL at the same time.The feces were collected on the 2nd，4th，6th，and 8th days after virus infection， and the copy numbers of rotavirus in feces were measured by RT-qPCR method. Results In in vitro experiment，the CCK-8 method results showed that compared with control group，the survival rates of Caco-2 cells in various groups in 48 h of treatment of different concentrations of E. faecalis were increased（P<0.05）， indicating that E. faecalis was non-toxic to the Caco-2 cells within this concentration range.The RT-qPCR results showed that compared with control group，the number of rotavirus gene copies in the Caco-2 cells in 10¹⁰， 10¹¹，and 10¹² CFU·mL^－1E. faecalis groups weredecreased by 75.8%， 99.9%， and 99.9%，respectively（P<0.05）. The results of immunofluorescence focus detection showed that compared with control group，the rotavirus titer in 10⁸，10¹⁰，and 10¹² CFU·mL^－1E. faecalis groups were decreased by 13%，77%， and 100%， respectively （P<0.05）.In in vivo experiment， compared with control group， the copy number of fecal virus gene of the suckling mice in E. faecalis transplantation group was significantly decreased（P<0.05）. Conclusion In in vitro cell model and rotavirus infection model of suckling mice， E. faecalis can inhibit the replication of rotavirus.

Key words: Enterococcus faecalis, Rotavirus, Flora transplantation, Probiotics