SARS-CoV-2 mitochondrial metabolic and epigenomic reprogramming in COVID-19.

To determine the effects of SARS-CoV-2 infection on cellular metabolism, we conducted an exhaustive survey of the cellular metabolic pathways modulated by SARS-CoV-2 infection and confirmed their importance for SARS-CoV-2 propagation by cataloging the effects of specific pathway inhibitors. This revealed that SARS-CoV-2 strongly inhibits mitochondrial oxidative phosphorylation (OXPHOS) resulting in increased mitochondrial reactive oxygen species (mROS) production. The elevated mROS stabilizes HIF-1α which redirects carbon molecules from mitochondrial oxidation through glycolysis and the pentose phosphate pathway (PPP) to provide substrates for viral biogenesis. mROS also induces the release of mitochondrial DNA (mtDNA) which activates innate immunity. The restructuring of cellular energy metabolism is mediated in part by SARS-CoV-2 Orf8 and Orf10 whose expression restructures nuclear DNA (nDNA) and mtDNA OXPHOS gene expression. These viral proteins likely alter the epigenome, either by directly altering histone modifications or by modulating mitochondrial metabolite substrates of epigenome modification enzymes, potentially silencing OXPHOS gene expression and contributing to long-COVID.

Prolonged Shedding of SARS-CoV-2 in Feces of COVID-19 Positive Patients: Trends in Genomic Variation in First and Second Wave.

The main route of the transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are through respiratory pathways and close contact of human-to-human. While information about other modes of transmission is comparatively less, some published literature supporting the likelihood of a fecal-oral mode of transmission has been accumulating. The diagnosis of SARS-COV-2 infected cases is based on the real-time reverse transcription-PCR (RT-PCR). The fecal excretion of SARS-COV-2 has been reported frequently, however, the role of fecal viral load with the severity of disease is not yet clear. Our study focused on the investigation of SARS-CoV-2 shedding in the fecal samples of patients with coronavirus disease 2019 (COVID-19). A total of 280 RT-PCR-positive patients were enrolled, among them 15.4% had gastrointestinal (GI) symptoms. It was shown that 62% of the patients were positive for SARS-CoV-2 RNA in fecal specimens. This positivity was not related to the presence of GI symptoms and the severity of disease. The next generation sequencing [NGS] of SARS-CoV-2 from fecal samples of patients was performed to analyze mutational variations. Findings from this study not only emphasized the potential presence of SARS-CoV-2 in feces, but also its continuing mutational changes and its possible role in fecal-oral transmission.

Full genome based sequence and structural characterization of an unusual group A rotavirus G12P[11] isolated from neonates in Pune, western India.

Studies conducted at neonatal intensive care units in Pune, western India, suggested early exposure to rotaviruses and predominance of unusual human-bovine-like G12P[11] strains. The whole genome sequencing and phylogenetic analyses of a naturally attenuated, culture adapted neonatal strain, (NIV-1740121) revealed multiple-gene reassortment events, containing ROTAVAC® vaccine strain, 116E-like VP4, VP6, NSP3, NSP5 genes, VP7 gene of G12 origin and VP3 gene of porcine ancestry in a human Wa-like backbone. Analysis of 3D structure modeling of the VP7 and VP4 proteins with respect to 116E suggested amino acid variations in the major neutralizing epitopes of VP7, contributed to a modified charge density. Visualization of receptor-glycan interaction structures of NIV-1740121 and 116E VP8* showed type I glycan binds with a similar conformation at the same active site as represented in the available crystal structure of G10P[11] VP8*. The study adds to the knowledge of age restricted tropism of P[11] strains in neonates.

Clinical and genetic characteristics of unusual G12P[11] rotavirus strains recovered from neonates: A study from Pune, Western India.

Rotavirus infections in neonates are generally nosocomial, and differ from pediatric infections both clinically and epidemiologically. These infections are predominantly asymptomatic and often associated with unusual strains. Globally, so far limited data is available on rotavirus infections in neonates admitted at Neonatal Intensive Care Unit. The aim of the present study is to determine the prevalence of rotavirus among neonates and to study their genetic characteristics. Stool specimens (n = 701) collected from neonates (n = 621) admitted during April 2016 to March 2018 mainly for prematurity, low birth weight and associated respiratory distress syndrome from two hospitals from Pune were tested for rotavirus, genotyped and representative strains were sequenced for the genes encoding outer capsid proteins, VP7 and VP4. Rotavirus was detected in 24.31% neonates. Majority of rotavirus infected neonates (98.68%) were asymptomatic. Peak rotavirus antigen detection (91.38%) occurred during the first 2 weeks of admission. Low, very low and normal birth weight neonates with gestational age ≥28 weeks had significantly higher rotavirus infection than those with extreme low birth weight with gestational age <28 weeks. Rotaviral infections occurred almost evenly throughout the year without an apparent peak in colder months. Predominance of unusual G12P[11] strains (97.1%) was observed. Phylogenetic analysis of the partial VP7 coding gene revealed all G12 strains clustered in lineage III and shared 96.94%-100% (nucleotide) and 96.26%-100% (amino acid) identities among themselves, and 95.69%-98.98% (nucleotide) and 94.77%-98.98% (amino acid) with other lineage III G12 strains respectively. Similarly VP4 partial gene sequences of P[11] study strains shared 97.5%-100% (nucleotide and amino acid) identities among themselves and highest 93.34%-94.53% (nucleotide) and 93.57%-94.64% (amino acid) identity with vaccine strain 116E, G9P[11]. The study highlights high frequency of unusual G12P[11] strains among neonates for the first time in western India and reaffirms limited strain diversity in this population. The knowledge of neonatal strains is important for estimating the efficacies of rotavirus vaccines.

Evaluation of different genomic regions of Rotavirus A for development of real time PCR.

The nucleotide alignment of all 11 genes of human Rotavirus A (RVA) strains revealed suitability of NSP2, NSP3 and VP6 genes for the development of real time PCR (qRT-PCR). Evaluation of qRT-PCR assays using known rotavirus ELISA positive and negative fecal specimens showed non-overlapping ranges of Mean ±3SD cycle threshold (Ct) values for NSP3 and VP6 based assays. Using serial dilutions of purified RVA, high sensitivity of VP6 qRT-PCR assay (1.95 × 10-5 pg/µL of RNA) was recorded as compared to NSP2 and NSP3 qRT-PCR assays (1.95 × 10-4 pg/µL of RNA). Further, evaluation of the VP6 qRT-PCR assay involving 266 fecal specimens and frequency polygon analysis of the data indicated cut-off value of 35 for Ct with high sensitivity (126/131, 96%) and specificity (12/12, 100%). This VP6 qRT-PCR assay will be a useful diagnostic tool to evaluate clinical presentations in rotaviral gastroenteritis under different conditions such as breast feeding and administration of rotavirus vaccines.

Identification of group B rotavirus as an etiological agent in the gastroenteritis outbreak in Maharashtra, India.

Acute gastroenteritis outbreak occurred at Pargaon, Maharashtra, India in 1789 cases with an attack rate of 32.5% between November to December 2015. The stool specimens (n = 32) were investigated for different enteric viral agents using conventional methods. Transmission electron microscopy and RNA polyacrylamide gel electrophoresis respectively identified morphologically distinct rotavirus particles in 28% and RNA migration pattern of Group B Rotavirus (GBR) in 72% of the specimens. Reverse transcription polymerase chain reaction and nucleotide sequencing confirmed presence of GBR in 97% of the samples analyzed. The predominance of GBR infections and absence or insignificant presence of other agents confirmed GBR as an etiological agent of the gastroenteritis outbreak occurred in Maharashtra, India.

Changing trends in circulating rotavirus strains in Pune, western India in 2009-2012: emergence of a rare G9P[4] rotavirus strain.

BACKGROUND: A vast diversity in rotaviruses at inter- and intra-genotypic level underscores the need for monitoring of circulating rotavirus strains. The aim of this study was to update the data on rotavirus disease and strains for the period from January 2009 to December 2012 in Pune, western India which has been one of the sites of the Indian Rotavirus Strain Surveillance Network since November 2005. METHODS: Children aged <5 years admitted for acute gastroenteritis in three different hospitals from Pune city were included in the study. The stool specimens were collected and tested for rotavirus antigen by a commercial enzyme immunoassay. The rotavirus strains were genotyped by multiplex reverse transcription polymerase chain reaction. RESULTS: During the study period, we found 35.1% of 685 stool specimens contained rotavirus antigen. Frequency of rotavirus detection was greatest (58.5%) among children aged 7-12 months. The G1P[8] (31.4%), G2P[4] (20.2%) and G9P[8] (11.8%) strains were the most common types. We noted predominance of G1P[8] strains (39.6%-46.1%) in all the years of study except 2009 wherein G9P[8] strains scored highest level (15.3%). Subsequent to this, we identified G9P[8] strains at the second highest position in 2010, their sudden decline and rise in G9P[4] strains in 2011-2012. We detected G12 strains in combination with P[6] and P[8] at variable rates (0-10.2%) and highest level (27.1%) of mixed rotavirus infections in 2009 as compared to 2010-2012 (0-3.8%). CONCLUSION: The study highlights the huge burden of rotavirus disease and changing profile of circulating rotavirus strains displaying emergence of G9P[4] reassortant strains in Pune, western India and emphasizes the need to analyze the entire genomic constellation of rotavirus strains for better evaluation of the impact of rotavirus.

Isolation and characterization of dually reactive strains of group a rotavirus from hospitalized children.

Seven rotavirus strains dually reactive to serotype G1- and G2-specific monoclonal antibodies (MAbs) from hospitalized children with rotavirus diarrhea were culture adapted. Six strains were neutralized with G1 antiserum to a higher titer than that of G2, and one was neutralized with G1 and G2 antisera to equal titers. Of these, four strains were also neutralized with G6 antiserum. Five strains with short RNA pattern could not be serotyped, and the remaining two strains with long RNA pattern were serotyped as G1 strains. In addition, two strains showing dual reactivity to G2 and G4 MAbs and one G2-like strain from a nontypeable specimen were isolated. The dual reactivity of the isolates could not be attributed to mixed infections.