Effect Of Intestinal Flora On Rotavirus Infection In Vivo And Inhibition Of Rotavirus Replication In Vivo And In Vitro By Enterococcus Faecalis

Objective1.To clarify the role of intestinal flora during rotavirus infection,and to analyze the effects of intestinal flora depletion and fecal microbiota transplantation(FMT)on rotavirus replication,so as to provide theoretical basis for clinical adjuvant treatment of rotavirus infection.2.To explore the effect of Enterococcus faecalis on the replication of rotavirus in vivo and in vitro,and to clarify the important role of E.faecalis in rotavirus infection.Methods1.(1)40 litters of 3-5-day-old SPF BALB/c suckling mice.About 15 rats from each litter were randomly divided into 8 groups: rotavirus group(RV),control group(Control),antibiotic treatment group(AB),antibiotic treatment control group(AB-Control),self-fecal group transplantation group(self-FMT),self-fecal group transplantation control group(self-FMT-Control),infant fecal group transplantation group(h-FMT),infant fecal group transplantation control group(hmi FMT-Control).The suckling mice in the RV group were fed with 100 μL 107 FFU/m L SA11 strain rotavirus for 10 days when they were 10 days old,and the suckling mice in the Control group were given 100 μL PBS for 10 days.The suckling mice in the AB and AB-Control groups were fed with 100 μL of four antibiotics for 16 days at the age of 3-5 days,the suckling mice in the AB group were fed with 100 μL 107 FFU/m L rotavirus for 10 days at the age of 10 days,and the suckling mice in the AB-Control group were given 100 μL PBS for 10 days at the age of 10 days.The suckling mice in the self-FMT,self-FMTControl,h-FMT and h-FMT-Control groups were given 100 μL of quadruple antibiotics for 3-5 days and then stopped.The self-FMT and self-FMT-Control groups were fed with 100 μL fecal bacteria suspension per suckling mouse for 3 days,and the h-FMT and h-FMT-Control groups were given 100 μL fecal bacteria suspension per suckling mouse for 3 days.Each suckling mouse in self-FMT and h-FMT groups was intragastrically fed with 100 μL 107 FFU/m L rotavirus for 10 days,and each suckling mouse in self-FMT-Control and h-FMT-Control groups was intragastrically fed with 100 μL PBS for 10 days.The feces and jejunal tissues of suckling mice infected with rotavirus on the 2nd,4th,6th,8th and 10 th day were collected and treated.(2)the copy number of rotavirus VP6 gene in the feces of suckling mice was detected by real-time fluorescence quantitative PCR,and the rotavirus titer in the feces of suckling mice was detected by immunofluorescence focal method.(3)the expressions of pro-inflammatory and anti-inflammatory cytokines IL-1 β,IL-10,TGF-β,TNF-α and intestinal barrier related proteins F11 R,Claudin-4,TJP-1 and Occludin in jejunum were detected by real-time fluorescence quantitative PCR.2.(1)A total of 80 4-week-old SPF BALB/c mice were randomly divided into 8 groups: rotavirus group(RV),control group(Control),antibiotic treatment group(AB),antibiotic treatment control group(AB-Control),self-fecal group transplantation group(self-FMT),self-fecal group transplantation control group(self-FMT-Control),infant fecal group transplantation group(h-FMT),infant fecal group transplantation control group(h-FMT-Control).Mice in RV group were given 500 μL 107 FFU/m L SA11 rotavirus for 1 week,mice in Control group were given 500 μL PBS for 1 week,mice in AB and AB-Control groups were given 500 μL antibiotics for 5 weeks,mice in AB group were given 500 μL 107 FFU/m L SA11 rotavirus for 1 week after 4 weeks of antibiotic administration,and mice in AB-Control group were given 500 μL PBS for 1 week after 4 weeks of antibiotic administration.The mice in the self-FMT,self-FMT-Control,h-FMT and h-FMT-Control groups were given 500 μL of four antibiotics for 3 weeks and then stopped.The mice in the self-FMT and self-FMT-Control groups were given 500 μL of fecal microflora suspension in each mouse for 1 week,and the treated infant fecal bacterial suspension in the h-FMT and h-FMT-Control groups were given intragastric administration of 500 μL in each mouse for 1 week.The mice in the self-FMT and h-FMT groups were given 500 μL rotavirus of SA11 strain for 1 week,and the mice in the self-FMT-Control and h-FMT-Control groups were given 500 μL PBS for 1 week.The feces and jejunal tissues of mice in each group were collected and treated on the 7th day after rotavirus infection.(2)the copy number of rotavirus VP6 gene in the feces of mice in each group was detected by real-time fluorescence quantitative PCR,and the rotavirus titer in the feces of mice in each group was detected by immunofluorescence focus method.(3)the expressions of pro-inflammatory and anti-inflammatory cytokines IL-1 β,IL-10,TGF-β,TNF-α and intestinal barrier related proteins F11 R,Claudin-4,TJP-1 and Occludin in jejunum were detected by real-time fluorescence quantitative PCR.3.E.faecalis affected rotavirus replication in vitro: E.faecalis was diluted with 10 times concentration gradient of 1 × 108 ~ 1 × 1012 CFU/m L and co-cultured with Caco-2 cells for 12 h,18 h,24 h,30 h and 48 h.The toxicity of E.faecalis to Caco-2 was determined by CCK-8 test,and the effects of different concentrations of E.faecalis on the expression of Claudin-1 and Occludin in Caco-2 cells were detected by Western blotting.Caco-2 cells were incubated with 1 × 108 ~ 1 × 1012 CFU/m L E.faecalis for 18 hours after the rotavirus was treated with rotavirus with a MOI value of 0.1.Rotavirus VP6 gene copies were detected by RT-q PCR and rotavirus titers were detected by immunofluorescence focus method.4.E.faecalis affected rotavirus replication in vitro: 18 litters of 4-5-day-old SPF BALB/c suckling mice were randomly divided into three groups: control group (Control),antibiotic treatment group(AB)and E.faecalis transplantation group(EF).Ab group and EF group were fed with quadruple antibiotics for 3 days to deplete their intestinal flora,then EF group stopped quadruple antibiotic administration.The suckling mice of three groups began to receive 100 μL of 107 FFU/m L rotavirus per day for 8 days,while those of EF group were given 100 μL of 1010 CFU/m L E.faecalis at the same time.Feces were collected every 2 days during virus infection,and the number of rotavirus copies in feces was measured by RT-q PCR.Results1.Intestinal microflora affects rotavirus replication in suckling mice and expression of intestinal related factors(1)The suckling mice in the RV group developed yellow watery diarrhea on the fourth day after rotavirus infection,while the suckling mice in the control group had no diarrhea.Mild diarrhea appeared in Ab,self-FMT and h-FMT groups on the fourth day,but there was no diarrhea in each control group.The feces of suckling rats treated with antibiotics were diluted with PBS and coated with drawing board after culture.There was almost no colony growth after antibiotic treatment,and large colonies were seen in each control group,indicating that the intestinal flora was depleted successfully.After fecal transplantation,a large area of colony growth could be seen in the feces of suckling mice,which was almost no different from that of self-FMT-Control group and h-FMT-Control group,indicating that the faeces were successfully transplanted into suckling mice.(2)Compared with RV group,the fecal rotavirus copy number of suckling mice in AB group decreased by 95% and 83% on the 8th and 10 th day of infection,respectively(P< 0.05).Compared with AB group,the number of virus gene copies in self-FMT group decreased by 95%,91%,63% and 59% respectively on the 4th,6th,8th and 10 th day after infection(P< 0.05).The number of viral gene copies in h-FMT group decreased by 93%,91%,83% and 79% on the 4th,6th,8th and 10 th day of infection,respectively(P< 0.01).The results of fecal rotavirus titer of suckling mice on the 4th day of infection showed that compared with RV group,the fecal rotavirus titer of AB group decreased by 48%(P< 0.01),and compared with AB group,the rotavirus titer of self-FMT group and h-FMT group decreased by 83% and 87%,respectively(P< 0.01).The above results suggest that antibiotic treatment can reduce rotavirus replication in suckling mice,and fecal transplantation can further reduce rotavirus replication.The above results showed that the intestinal antibiotic treatment reduced rotavirus replication in suckling mice,and the rotavirus replication efficiency decreased significantly after the intestinal flora of suckling mice was restored by fecal group transplantation.(3)The expression of inflammatory factors was detected.Compared with RV group,the expression of IL-1β decreased in AB self-FMT and h-FMT groups,and increased 19% in AB group(P< 0.05);TNF-α expression increased in self-FMT and h-FMT groups;IL-10 expression decreased in AB,self-FMT and h-FMT groups;TGF-β expression decreased 33% in h-FMT group(P< 0.05);TGF-β expression increased in ABand self-FMT groups.(4)Detection of intestinal barrier related protein expression.Compared with RV group,the expression of F11 R in AB,self-FMT and h-FMT groups decreased by 49%,24% and 32% respectively(P< 0.05),and the expression of Claudin-4 in AB,self-FMT and h-FMT groups decreased by 52%,32% and 19%,respectively(P< 0.05).TJP-1 expression decreased in AB,self-FMT and h-FMT groups,and Occludin expression decreased by 33%,29% and 34% in AB,self-FMT and h-FMT groups respectively(P< 0.05).2.Effects of intestinal flora on rotavirus replication and intestinal related factor expression in mice(1)No diarrhea was observed in each group after rotavirus infection,and the feces of mice were basically the same as those of the control group,which were black solid slender particles.After antibiotic treatment,the feces of suckling mice in each group were diluted with PBS and coated with drawing board,and there was basically no colony growth in the control group,indicating that the intestinal flora was depleted successfully.After fecal transplantation,a large area of colony growth could be seen in the feces of suckling mice in each group,which was almost no difference compared with the control group,indicating that the feces were successfully transplanted into mice.(2)Compared with RV,the number of rotavirus gene copies in the feces of AB group increased by 86% on the 7th day after infection(P< 0.01),and decreased by 33% in self-FMT group(P< 0.05),26% in h-FMT group compared with AB group(P< 0.05).The results of virus titer on the 7th day showed that compared with RV group,the fecal rotavirus titer of SA11 strain in AB group increased by 106%(P< 0.01).Compared with AB group,self-FMT group and h-FMT group decreased by 53.5% and 53% respectively(P< 0.01).The above results suggest that antibiotic treatment can promote rotavirus replication in adult mice,and fecal transplantation can reduce rotavirus replication.The above results showed that intestinal antibiotic treatment could promote rotavirus replication in adult mice,while fecal group transplantation could reduce rotavirus replication after recovering intestinal flora.(3)The expression of inflammatory factors was detected.Compared with RV group,IL-1 β expression increased in AB,self-FMT and h-FMT groups,TNF-α expression increased in AB group and h-FMT group,TNF-α expression decreased in self-FMT group,IL-10 expression increased by 210% in AB group(P< 0.05),IL-10 expression increased in self-FMT and h-FMT groups,TGF-β expression decreased in AB and self-FMT groups,and TGF-β expression increased in h-FMT group.(4)The expression of intestinal barrier protein was detected.Compared with RV group,the expression of F11 R,Claudin-4,TJP-1 and Occludin in AB group increased by 465%,4765%,621% and 2283% respectively(P< 0.01),and Claudin-4 in self-FMT and h-FMT group increased by 94% and 159%,respectively(P< 0.05).F11 R expression decreased by 51% and 43% in self-FMT and h-FMT group(P< 0.05),and TJP-1 expression decreased by 57% and 38%,respectively(P< 0.01).The expression of decreased in Occludin.3.Enterococcus faecalis inhibits rotavirus replication in vivo and in vitro(1)In vitro experiment,compared with the control group,the concentration of E.faecalis less than 1×1012 CFU/m L within 48 hours had no effect on cell survival rate(P< 0.05),indicating that E.faecalis was non-toxic to Caco-2 within this concentration range.1×108 CFU/m L E.faecalis increased the expression of Claudin-1 and Occludin protein in Caco-2 by 323%(P< 0.01)and 62.5%(P< 0.01),respectively.When the concentration of E.faecalis was 1×1010,1×1011,1×1012 CFU/m L,the copy number of rotavirus gene decreased by 75.8% 99.9% 99.9% compared with the positive control group(P< 0.05).When the concentration of E.faecalis was 1×108,1×1010 and 1×1012 CFU/m L,the rotavirus titer decreased by 13%,77% and 100% respectively compared with the positive control(P< 0.05).(2)In the in vivo experiment,there was no colony growth on the fecal plates of suckling mice in different treatment groups,LB plate and anaerobic plate in antibiotic treatment group,indicating that the intestinal flora of suckling mice was depleted successfully by antibiotic consumption;after intragastric administration of E.faecalis,there were more gray-white,protruding and smooth colonies in the area of LB coating plate,indicating that E.faecalis colonized in the intestinal tract of suckling mice in this group.The number of rotavirus copies in the feces of suckling mice in the three treatment groups decreased with the increase of infection days.Compared with the control group,the fecal rotavirus copies of suckling mice infected with EF for 2,4,6 and 8 days decreased by 98%,92%,81% and 81%,respectively(P< 0.01).The results showed that Enterococcus faecalis inhibited the replication of rotavirus in suckling mice.Conclusions1.The models of rotavirus infection in suckling mice and mice were successfully established,and the models of intestinal flora depletion and rotavirus infection after fecal group transplantation were successfully established.2.Intestinal flora depletion of suckling mice inhibited rotavirus replication,and fecal group transplantation could further inhibit rotavirus replication.The depletion of intestinal flora in adult mice promotes rotavirus replication in vivo,while the reconstruction of intestinal flora after fecal transplantation can alleviate rotavirus replication in vivo.3.The difference of rotavirus replication between suckling mice and mice may be due to the difference of species composition of intestinal flora,and it may also be related to the expression of intestinal inflammatory factors IL-1 β,IL-10,TGF-β,TNF-α and intestinal barrier related factors F11 R,Claudin-4,TJP-1 and Occludin.4.E.faecalis can inhibit rotavirus replication in vitro when the concentration of E.faecalis is more than 1×108 CFU/m L.E.faecalis can reduce rotavirus replication in suckling mice by oral administration.