Comparative genomic analysis of genogroup 1 and genogroup 2 rotaviruses circulating in seven US cities, 2014-2016.

For over a decade, the New Vaccine Surveillance Network (NVSN) has conducted active rotavirus (RVA) strain surveillance in the USA. The evolution of RVA in the post-vaccine introduction era and the possible effects of vaccine pressure on contemporary circulating strains in the USA are still under investigation. Here, we report the whole-gene characterization (eleven ORFs) for 157 RVA strains collected at seven NVSN sites during the 2014 through 2016 seasons. The sequenced strains included 52 G1P[8], 47 G12P[8], 18 G9P[8], 24 G2P[4], 5 G3P[6], as well as 7 vaccine strains, a single mixed strain (G9G12P[8]), and 3 less common strains. The majority of the single and mixed strains possessed a Wa-like backbone with consensus genotype constellation of G1/G3/G9/G12-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1, while the G2P[4], G3P[6], and G2P[8] strains displayed a DS-1-like genetic backbone with consensus constellation of G2/G3-P[4]/P[6]/P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Two intergenogroup reassortant G1P[8] strains were detected that appear to be progenies of reassortment events between Wa-like G1P[8] and DS-1-like G2P[4] strains. Two Rotarix® vaccine (RV1) and two RV5 derived (vd) reassortant strains were detected. Phylogenetic and similarity matrices analysis revealed 2-11 sub-genotypic allelic clusters among the genes of Wa- and DS-1-like strains. Most study strains clustered into previously defined alleles. Amino acid (AA) substitutions occurring in the neutralization epitopes of the VP7 and VP4 proteins characterized in this study were mostly neutral in nature, suggesting that these RVA proteins were possibly under strong negative or purifying selection in order to maintain competent and actual functionality, but fourteen radical (AA changes that occur between groups) AA substitutions were noted that may allow RVA strains to gain a selective advantage through immune escape. The tracking of RVA strains at the sub-genotypic allele constellation level will enhance our understanding of RVA evolution under vaccine pressure, help identify possible mechanisms of immune escape, and provide valuable information for formulation of future RVA vaccines.

Whole gene analysis of a genotype G29P[6] human rotavirus strain identified in Central African Republic.

OBJECTIVE: Rotavirus A (RVA) remains the main causative agent of gastroenteritis in young children and the young of many mammalian and avian species. In this study we describe a RVA strain detected from a 6-month-old child from Central African Republic (CAR). RESULTS: We report the 11 open reading frame sequences of a G29-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H2 rotavirus strain, RVA/Human-wt/CAR/CAR91/2014/G29P[6]. Nine genes (VP1-VP3, VP6, NSP1-NSP5) shared 90-100% sequence similarities with genogroup 2 rotaviruses. Phylogenetically, backbone genes, except for VP3 and NSP4 genes, were linked with cognate gene sequences of human DS-1-like genogroup 2, hence their genetic origin. The VP3 and NSP4 genes, clustered genetically with both human and animal strains, an indication genetic reassortment human and animal RVA strains has taken place. The VP7 gene shared nucleotide (93-94%) and amino acid (95.5-96.7%) identities with Kenyan and Belgian human G29 strains, as well as to buffalo G29 strain from South Africa, while the VP4 gene most closely resembled P[6]-lineage I strains from Africa and Bangladesh (97%).

Metagenomic sequencing generates the whole genomes of porcine rotavirus A, C, and H from the United States.

The genus Rotavirus comprises eight species, designated A to H, and two recently identified tentative species I in dogs and J in bats. Species Rotavirus A, B, C and H (RVA, RVB, RVC and RVH) have been detected in humans and animals. While human and animal RVA are well characterized and defined, complete porcine genome sequences in the GenBank are limited compared to human strains. Here, we used a metagenomic approach to sequence the 11 segments of RVA, RVC and RVH strains from piglets in the United States (US) and explore the evolutionary relations of these RV species. Metagenomics identified Astroviridae, Picornaviridae, Caliciviridae, Coronoviridae in samples MN9.65 and OK5.68 while Picobirnaviridae and Arteriviridae were only identified in sample OK5.68. Whole genome sequencing and phylogenetic analyses identified multiple genotypes with the RVA of strain MN9.65 and OK5.68, with the genome constellation of G5/G9-P[7]/P[13]-I5/I5- R1/R1-C1-M1-A8-N1-T7-E1/E1-H1 and G5/G9-P[6]/P[7]-I5-R1/R1-C1-M1-A8-N1-T1/T7-E1/E1-H1, respectively. The RVA strains had a complex evolutionary relationship with other mammalian strains. The RVC strain OK5.68 had a genome constellation of G9-P[6]-I1-R1-C5-M6-A5-N1-T1-E1-H1, and shared an evolutionary relationship with porcine strains from the US. The RVH strains MN9.65 and OK5.68 had the genome constellation of G5-P1-I1-R1-C1-M1-A5-N1-T1-E4-H1 and G5-P1-I1-R1-C1-M1-A5-N1-T1-E1-H1, indicating multiple RVH genome constellations are circulating in the US. These findings allow us to understand the complexity of the enteric virome, develop improved screening methods for RVC and RVH strains, facilitate expanded rotavirus surveillance in pigs, and increase our understanding of the origin and evolution of rotavirus species.

Distribution of rotavirus genotypes in the postvaccine introduction era in Ashaiman, Greater Accra Region, Ghana, 2014-2016.

Group A Rotaviruses (RVAs) are the most important etiological agents of acute gastroenteritis (AGE) in children less than 5 years of age. Mortality resulting from RVA gastroenteritis is higher in developing countries than in developed ones, causing a huge public health burden in global regions like Africa and South-East Asia. This study reports RVA genotypes detected in Ashaiman, Greater Accra Region, Ghana, in the postvaccine introduction era for the period 2014-2016. Stool samples were collected from children less than 5 years of age who visited Ashaiman Polyclinic with AGE from November 2014 to May 2015 and from December 2015 to June 2016. The samples were tested by enzyme immunoassay (EIA), and one-step multiplex reverse transcription polymerase chain reaction was performed on the EIA positive samples for gel-based binomial genotyping. Of the 369 stool samples collected from children with AGE, 145 (39%) tested positive by EIA. Five VP7 (G1, G3, G9, G10, and G12) and three VP4 (P[4], P[6] and P[8]) genotypes were detected. Eight G/P combinations were identified of which, G3P[6], G12P[8], G1P[8], and G9P[4] were the most prevalent and responsible for 93 (68%) of the AGE cases, and seven mixed-types were detected which represented 8% of the RVA cases. High prevalence, diversity, and mixed-types of RVAs were detected from Ashaiman with the emergence of unusual genotypes.

Whole-gene analysis of inter-genogroup reassortant rotaviruses from the Dominican Republic: Emergence of equine-like G3 strains and evidence of their reassortment with locally-circulating strains.

Inter-genogroup reassortant group A rotavirus (RVA) strains possessing a G3 VP7 gene of putative equine origin (EQL-G3) have been detected in humans since 2013. Here we report detection of EQL-G3P[8] RVA strains from the Dominican Republic collected in 2014-16. Whole-gene analysis of RVA in stool specimens revealed 16 EQL-G3P[8] strains, 3 of which appear to have acquired an N1 NSP1 gene from locally-circulating G9P[8] strains and a novel G2P[8] reassortant possessing 7 EQL-G3-associated genes and 3 genes from a locally-circulating G2P[4] strain. Phylogenetic/genetic analyses of VP7 gene sequences revealed nine G3 lineages (I-IX) with newly-assigned lineage IX encompassing all reported human EQL-G3 strains along with the ancestral equine strain. VP1 and NSP2 gene phylogenies suggest that EQL-G3P[8] strains were introduced into the Dominican Republic from Thailand. The emergence of EQL-G3P[8] strains in the Dominican Republic and their reassortment with locally-circulating RVA could have implications for current vaccination strategies.

Gut microbial composition can differentially regulate bile acid synthesis in humanized mice.

We previously reported that alcohol drinkers with and without cirrhosis showed a significant increase in fecal bile acid secretion compared to nondrinkers. We hypothesized this may be due to activation by alcohol of hepatic cyclic adenosine monophosphate responsive element-binding protein 3-like protein 3 (CREBH), which induces cholesterol 7α-hydroxylase (Cyp7a1). Alternatively, the gut microbiota composition in the absence of alcohol might increase bile acid synthesis by up-regulating Cyp7a1. To test this hypothesis, we humanized germ-free (GF) mice with stool from healthy human subjects (Ctrl-Hum), human subjects with cirrhosis (Cirr-Hum), and human subjects with cirrhosis and active alcoholism (Alc-Hum). All animals were fed a normal chow diet, and none demonstrated cirrhosis. Both hepatic Cyp7a1 and sterol 12α-hydroxylase (Cyp8b1) messenger RNA (mRNA) levels were significantly induced in the Alc-Hum and Ctrl-Hum mice but not in the Cirr-Hum mice or GF mice. Liver bile acid concentration was correspondingly increased in the Alc-Hum mice despite fibroblast growth factor 15, fibroblast growth receptor 4, and small heterodimer partner mRNA levels being significantly induced in the large bowel and liver of the Ctrl-Hum mice and Alc-Hum mice but not in the Cirr-Hum mice or GF mice. This suggests that the normal pathways of Cyp7a1 repression were activated in the Alc-Hum mice and Ctrl-Hum mice. CREBH mRNA was significantly induced only in the Ctrl-Hum mice and Alc-Hum mice, possibly indicating that the gut microbiota up-regulate CREBH and induce bile acid synthesis genes. Analysis of stool bile acids showed that the microbiota of the Cirr-Hum and Alc-Hum mice had a greater ability to deconjugate and 7α-dehydroxylate primary bile acids compared to the microbiota of the Cirr-Hum mice. 16S ribosomal RNA gene sequencing of the gut microbiota showed that the relative abundance of taxa that 7-α dehydroxylate primary bile acids was higher in the Ctrl-Hum and Alc-Hum groups. Conclusion: The composition of gut microbiota influences the regulation of the rate-limiting enzymes in bile acid synthesis in the liver. (Hepatology Communications 2017;1:61-70).

Gut microbiome and liver disease.

Gut microbiota changes are important in determining the occurrence and progression of chronic liver disease related to alcohol, nonalcoholic fatty liver disease, and cirrhosis. Specifically, the systemic inflammation, endotoxemia, and the vasodilation that leads to complications such as spontaneous bacterial peritonitis and hepatic encephalopathy could be related to the gut milieu. Given the poor prognosis of these events, their prevention and early management are essential. Microbiota may be an essential component of the gut milieu that can impact these clinical events, and the study of their composition and function in a culture-independent manner could help understand the prognosis. Recent human and animal studies have shown that the relative abundance and the functional changes of microbiota in the stool, colonic mucosa, and saliva have varying consequences on the presence and prognosis of chronic liver disease and cirrhosis. The impact of therapies on the microbiota is slowly being understood and will likely lead to a more targeted approach to gut microbiota modification in chronic liver disease and cirrhosis.

Rifaximin Exerts Beneficial Effects Independent of its Ability to Alter Microbiota Composition.

OBJECTIVES: Rifaximin has clinical benefits in minimal hepatic encephalopathy (MHE) but the mechanism of action is unclear. The antibiotic-dependent and -independent effects of rifaximin need to be elucidated in the setting of MHE-associated microbiota. To assess the action of rifaximin on intestinal barrier, inflammatory milieu and ammonia generation independent of microbiota using rifaximin. METHODS: Four germ-free (GF) mice groups were used (1) GF, (2) GF+rifaximin, (3) Humanized with stools from an MHE patient, and (4) Humanized+rifaximin. Mice were followed for 30 days while rifaximin was administered in chow at 100 mg/kg from days 16-30. We tested for ammonia generation (small-intestinal glutaminase, serum ammonia, and cecal glutamine/amino-acid moieties), systemic inflammation (serum IL-1ß, IL-6), intestinal barrier (FITC-dextran, large-/small-intestinal expression of IL-1ß, IL-6, MCP-1, e-cadherin and zonulin) along with microbiota composition (colonic and fecal multi-tagged sequencing) and function (endotoxemia, fecal bile acid deconjugation and de-hydroxylation). RESULTS: All mice survived until day 30. In the GF setting, rifaximin decreased intestinal ammonia generation (lower serum ammonia, increased small-intestinal glutaminase, and cecal glutamine content) without changing inflammation or intestinal barrier function. Humanized microbiota increased systemic/intestinal inflammation and endotoxemia without hyperammonemia. Rifaximin therapy significantly ameliorated these inflammatory cytokines. Rifaximin also favorably impacted microbiota function (reduced endotoxin and decreased deconjugation and formation of potentially toxic secondary bile acids), but not microbial composition in humanized mice. CONCLUSIONS: Rifaximin beneficially alters intestinal ammonia generation by regulating intestinal glutaminase expression independent of gut microbiota. MHE-associated fecal colonization results in intestinal and systemic inflammation in GF mice, which is also ameliorated with rifaximin.

Gut microbiota drive the development of neuroinflammatory response in cirrhosis in mice.

UNLABELLED: The mechanisms behind the development of hepatic encephalopathy (HE) are unclear, although hyperammonemia and systemic inflammation through gut dysbiosis have been proposed. The aim of this work was to define the individual contribution of hyperammonemia and systemic inflammation on neuroinflammation in cirrhosis using germ-free (GF) and conventional mice. GF and conventional C57BL/6 mice were made cirrhotic using CCl4 gavage. These were compared to their noncirrhotic counterparts. Intestinal microbiota, systemic and neuroinflammation (including microglial and glial activation), serum ammonia, intestinal glutaminase activity, and cecal glutamine content were compared between groups. GF cirrhotic mice developed similar cirrhotic changes to conventional mice after 4 extra weeks (16 vs. 12 weeks) of CCl4 gavage. GF cirrhotic mice exhibited higher ammonia, compared to GF controls, but this was not associated with systemic or neuroinflammation. Ammonia was generated through increased small intestinal glutaminase activity with concomitantly reduced intestinal glutamine levels. However, conventional cirrhotic mice had intestinal dysbiosis as well as systemic inflammation, associated with increased serum ammonia, compared to conventional controls. This was associated with neuroinflammation and glial/microglial activation. Correlation network analysis in conventional mice showed significant linkages between systemic/neuroinflammation, intestinal microbiota, and ammonia. Specifically beneficial, autochthonous taxa were negatively linked with brain and systemic inflammation, ammonia, and with Staphylococcaceae, Lactobacillaceae, and Streptococcaceae. Enterobacteriaceae were positively linked with serum inflammatory cytokines. CONCLUSION: Gut microbiota changes drive development of neuroinflammatory and systemic inflammatory responses in cirrhotic animals. (Hepatology 2016;64:1232-1248).

Impaired Gut-Liver-Brain Axis in Patients with Cirrhosis.

Cirrhosis is associated with brain dysfunction known as hepatic encephalopathy (HE). The mechanisms behind HE are unclear although hyperammonemia and systemic inflammation through gut dysbiosis have been proposed. We aimed to define the individual contribution of specific gut bacterial taxa towards astrocytic and neuronal changes in brain function using multi-modal MRI in patients with cirrhosis. 187 subjects (40 controls, 147 cirrhotic; 87 with HE) underwent systemic inflammatory assessment, cognitive testing, stool microbiota analysis and brain MRI analysis. MR spectroscopy (MRS) changes of increased Glutamate/glutamine, reduced myo-inositol and choline are hyperammonemia-associated astrocytic changes, while diffusion tensor imaging (DTI) demonstrates changes in neuronal integrity and edema. Linkages between cognition, MRI parameters and gut microbiota were compared between groups. We found that HE patients had a significantly worse cognitive performance, systemic inflammation, dysbiosis and hyperammonemia compared to controls and cirrhotics without HE. Specific microbial families (autochthonous taxa negatively and Enterobacteriaceae positively) correlated with MR spectroscopy and hyperammonemia-associated astrocytic changes. On the other hand Porphyromonadaceae, were only correlated with neuronal changes on DTI without linkages with ammonia. We conclude that specific gut microbial taxa are related to neuronal and astrocytic consequences of cirrhosis-associated brain dysfunction.

Changes in the Intestinal Microbiome and Alcoholic and Nonalcoholic Liver Diseases: Causes or Effects?

The prevalence of fatty liver diseases is increasing rapidly worldwide; after treatment of hepatitis C virus infection becomes more widespread, fatty liver diseases are likely to become the most prevalent liver disorders. Although fatty liver diseases are associated with alcohol, obesity, and the metabolic syndrome, their mechanisms of pathogenesis are not clear. The development and progression of fatty liver, alcoholic, and nonalcoholic liver disease (NAFLD) all appear to be influenced by the composition of the microbiota. The intestinal microbiota have been shown to affect precirrhotic and cirrhotic stages of liver diseases, which could lead to new strategies for their diagnosis, treatment, and study. We review differences and similarities in the cirrhotic and precirrhotic stages of NAFLD and alcoholic liver disease. Differences have been observed in these stages of alcohol-associated disease in patients who continue to drink compared with those who stop, with respect to the composition and function of the intestinal microbiota and intestinal integrity. NAFLD and the intestinal microbiota also differ between patients with and without diabetes. We also discuss the potential of microbial therapy for patients with NAFLD and ALD.

Gut Microbiota Alterations can predict Hospitalizations in Cirrhosis Independent of Diabetes Mellitus.

Diabetes (DM) is prevalent in cirrhosis and may modulate the risk of hospitalization through gut dysbiosis. We aimed to define the role of gut microbiota on 90-day hospitalizations and of concomitant DM on microbiota. Cirrhotic outpatients with/without DM underwent stool and sigmoid mucosal microbial analysis and were followed for 90 days. Microbial composition was compared between those with/without DM, and those who were hospitalized/not. Regression/ROC analyses for hospitalizations were performed using clinical and microbial features. 278 cirrhotics [39% hepatic encephalopathy (HE), 31%DM] underwent stool while 72 underwent mucosal analyses. Ultimately, 94 were hospitalized and they had higher MELD, proton pump inhibitor (PPI) use and HE without difference in DM. Stool/mucosal microbiota were significantly altered in those who were hospitalized (UNIFRAC p < = 1.0e-02). Specifically, lower stool Bacteroidaceae, Clostridiales XIV, Lachnospiraceae, Ruminococcacae and higher Enterococcaceae and Enterobacteriaceae were seen in hospitalized patients. Concomitant DM impacted microbiota UNIFRAC (stool, p = 0.003, mucosa, p = 0.04) with higher stool Bacteroidaceae and lower Ruminococcaeae. Stool Bacteroidaceaeae and Clostridiales XIV predicted 90-day hospitalizations independent of clinical predictors (MELD, HE, PPI). Stool and colonic mucosal microbiome are altered in cirrhotics who get hospitalized with independent prediction using stool Bacteroidaceae and Clostridiales XIV. Concomitant DM distinctly impacts gut microbiota without affecting hospitalizations.

Salivary microbiota reflects changes in gut microbiota in cirrhosis with hepatic encephalopathy.

UNLABELLED: Altered gut microbiome is associated with systemic inflammation and cirrhosis decompensation. However, the correlation of the oral microbiome with inflammation in cirrhosis is unclear. Our aim was to evaluate the oral microbiome in cirrhosis and compare with stool microbiome. Outpatients with cirrhosis (with/without hepatic encephalopathy [HE]) and controls underwent stool/saliva microbiome analysis (for composition and function) and also systemic inflammatory evaluation. Ninety-day liver-related hospitalizations were recorded. Salivary inflammation was studied using T helper 1 cytokines/secretory immunoglobulin A (IgA), histatins and lysozyme in a subsequent group. A total of 102 patients with cirrhosis (43 previous HE) and 32 age-matched controls were included. On principal component analysis (PCA), stool and saliva microbiome clustered far apart, showing differences between sites as a whole. In salivary microbiome, with previous HE, relative abundance of autochthonous families decreased whereas potentially pathogenic ones (Enterobacteriaceae, Enterococcaceae) increased in saliva. Endotoxin-related predicted functions were significantly higher in cirrhotic saliva. In stool microbiome, relative autochthonous taxa abundance reduced in previous HE, along with increased Enterobacteriaceae and Enterococcaceae. Cirrhotic stool microbiota demonstrated a significantly higher correlation with systemic inflammation, compared to saliva microbiota, on correlation networks. Thirty-eight patients were hospitalized within 90 days. Their salivary dysbiosis was significantly worse and predicted this outcome independent of cirrhosis severity. Salivary inflammation was studied in an additional 86 age-matched subjects (43 controls/43 patients with cirrhosis); significantly higher interleukin (IL)-6/IL-1ß, secretory IgA, and lower lysozyme, and histatins 1 and 5 were found in patients with cirrhosis, compared to controls. CONCLUSIONS: Dysbiosis, represented by reduction in autochthonous bacteria, is present in both saliva and stool in patients with cirrhosis, compared to controls. Patients with cirrhosis have impaired salivary defenses and worse inflammation. Salivary dysbiosis was greater in patients with cirrhosis who developed 90-day hospitalizations. These findings could represent a global mucosal-immune interface change in cirrhosis.

Systems biology analysis of omeprazole therapy in cirrhosis demonstrates significant shifts in gut microbiota composition and function.

Proton pump inhibitors (PPI) have been associated with infectious complications in cirrhosis, but their impact on distal gut microbiota composition and function is unclear. We aimed to evaluate changes in stool microbiota composition and function in patients with cirrhosis and healthy controls after omeprazole therapy. Both 15 compensated cirrhotic patients and 15 age-matched controls underwent serum gastrin measurement, stool microbiota profiling with multitagged pyrosequencing, and urinary metabolic profiling with NMR spectroscopy to assess microbial cometabolites before/after a 14-day course of 40 mg/day omeprazole under constant diet conditions. Results before (pre) and after PPI were compared in both groups, compared with baseline by systems biology techniques. Adherence was >95% without changes in diet or MELD (model for end-stage liver disease) score during the study. Serum gastrin concentrations significantly increased after PPI in cirrhosis (pre 38.3 ± 35.8 vs. 115.6 ± 79.3 pg/ml P < 0.0001) and controls (pre 29.9 ± 14.5 vs. 116.0 ± 74.0 pg/ml, P = 0.001). A significant microbiota change was seen in both controls and cirrhosis after omeprazole (QIIME P < 0.0001). Relative Streptococcaceae abundance, normally abundant in saliva, significantly increased postomeprazole in controls (1 vs. 5%) and cirrhosis (0 vs. 9%) and was correlated with serum gastrin levels (r = 0.4, P = 0.005). We found significantly reduced hippurate in cirrhosis vs. controls both pre- and postomeprazole and increased lactate in both groups post vs. preomeprazole, whereas dimethylamine (DMA) decreased in cirrhosis only. On correlation network analysis, significant changes in linkages of bacteria with metabolites (hippurate/DMA/lactate) were found postomeprazole, compared with pre-PPI in cirrhosis patients. In conclusion, omeprazole is associated with a microbiota shift and functional change in the distal gut in patients with compensated cirrhosis that could set the stage for bacterial overgrowth.