Study On Changes Of Intestinal Flora Of Tree Shrews Infected With Persistent Occult HBV

Objective:To investigate the effects of persistent occult HBV infection on intestinal flora of tree shrews,and explore the mechanism of occult HBV infection from the perspective of intestinal flora.Methods:The newborn tree shrews were subcutaneously inoculated with C-type HBV DNA 7.53 × 108 IU/ml virus venomous 300ul via bilateral thighs on the first and third day after birth respectively,and the control group was not given any intervention.Serum HBV specific markers（HBsAg,HBsAb,HBeAg,HBeAb,HBcAb）and HBV DNA copy numbers were dynamically observed.From the 12th week,tree shrews were anesthetized regularly and liver biopsy was performed.The changes of HBV DNA load in liver tissue were persistently observed by PCR TaqMan probe method and the histopathological changes of tree shrews were dynamically observed by HE staining.At the 72th week,after the liver biopsy of tree shrews,the total RNA was extracted by Trizol method,and the mRNA relative levels of TLR4,TNF-α,IFN-γ,IL-10 and IL-6 in the liver tissues of the two groups were observed by qRT-PCR.Ten tree shrews meeting the criteria for persistent occult HBV infection were selected as the occult HBV infection group（OC group,n=10）and eight tree shrews without intervention as the normal control group（NC group,n=8）macthed with gender and ages.Feces of tree shrews in both groups were collected at 72th week,total DNA was extracted,16S rDNA V3+V4 region was amplified by PCR,and the amplified products were sequenced to observe the differences in intestinal microflora of tree shrews between the two groups.Results:1.The animal model of persistent occult HBV infection was successfully established by subcutanically injecting 106-107 IU/ml HBV DNA venom into the newborn tree shrews.Serum HBV specific markers expression patterns in OC group were diverse,with HBsAg（-）/HBcAb（+）being the most common.Serum HBV DNA load showed intermittent low level fluctuation,most of which were less than 200IU/mL,and it could also increase rapidly spontaneously accompanied by HBsAg（-）/HBsAg（+）serological conversion,analogous to HBV reactivation.In the liver tissue,HBV DNA copy number of tree shrews with persistent occult HBV infection was consistently positive,mostly fluctuating at the level of 103 IU/ml-104 IU/ml,and in a few tree shrews,HBV DNA in the liver tissue fluctuated at the level of 102 IU/ml-103 IU/ml.In pathology,the liver tissue of tree shrews with persistent occult HBV infection was characterized by persistent damage,mainly diffuse or scattered hepatocyte hydrodegeneration,without or with slightly inflammation.The extent of the damage were independent of the copy number of the duration of HBV infection.2.At the level of cytokines,the mRNA relative expression levels of cytokines TLR4,IFN-γ,IL-10 and IL-6 in the OC group were significantly lower than those in the NC group（TLR4:0.22±0.10 vs 1.00±0.24,P<0.001;IFN-γ:0.42±0.25 vs 1.00±0.40,P<0.05;IL-10:0.29±0.22 vs 1.00±0.36,P<0.01;IL-6:0.24±0.14 vs 1.00±0.68,P<0.05）,and there was no significant difference in the relative expression level of TNF-α mRNA between OC group and NC group（1.96±1.24 vs 1.00±0.44,P>0.05）.3.In gut,compared with NC group,there was no significant difference in a diversity（P>0.05）,while the β diversity was significantly decreased（P<0.01）in intestinal flora diversity of the tree shrews in OC group.In species composition,the intestinal flora of tree srews in both two groups was mainly composed of Firmicutes,Proteobacteria,Bacteroidetes,Actinobacteria,Cyanobacteria,etc.and the relative abundance of the intestinal flora in tree shrews was 99.72%and 99.84%in OC group and NC group,respectively.In class level,the relative abundance（%）of Clostridia in intestinal tract of tree shrews in OC group was significantly higher than that in NC group（1.35±0.804 vs 0.223±0.183,P<0.01）.Similarly,the relative abundance（%）of Clostridiales in intestinal tract of tree shrews in OC group was significantly higher than that in NC group in order level（1.35±0.804 vs 0.223±0.183,P<0.01）.While in family level,the relative abundance（%）of Moraxellaceae in intestinal tract of tree shrews in OC group was significantly lower than that in NC group in order level（0.05±0.053 vs 0.13±0.056,P<0.01）.In genus level,relative abundance of Escherichia-Shigella and Psychrobacter in OC group were both significantly lower than those in tree shrews of NC group（1.34±1.10 vs 4.39±3.40;0.0359±0.042 vs 0.099±0.023,both P<0.05）.And in species level,the relative abundance（%）of Bacteroidetes DSM 18228=JCM 13818 in OC group was significantly lower than that in NC group（0.07±0.082 vs 0.62±0.66,P<0.05）.LEfSE analysis showed that the different intestinal microorganism between OC group and NC group was mainly composed of Firmicutes,Bacteroidetes and Metobacteria,including Clostridia（LDA=3.75,P=0.001）,Clostridiales（LDA=3.72,P=0.0059）,Ruminococcaceae（LDA=3.42,P=0.016）,Lachnoclostridium（LD A=2.74,P=0.0076）,Bacteroides_Fr agilis(LDA=3.07,P=0.037,Prevotella₇（LDA=3.27,P=0.00042）,Muribaculacea e（LD A=2.58,P=0.041）,Sucinivibrionaceae（LD A=2.42,P=0.034）,Snodgrassella（LDA=2.1 3,P=0.042）,Snodgrassella_Alvi（LD A=2.13,P=0.024）,Klebsiella（LDA=3.00,P=0.033）,etc,which may be biomarkers for potential persistent occult HBV infection.Conclusion:1.Tree shrew is a good animal model of persistent occult HBV infection,and its potential mechanism may be related to immune tolerance.2.Persistent occult HBV infection affects changes in intestinal flora of tree shrews,and intestinal flora may be involved in mediating specific immune responses to occult HBV infection,the specific mechanism still needs to be further studied.