MFGM-enriched whey displays antiviral activity against common pediatric viruses in vitro.

Background: Among the most common mucosal viral infections in infants are rotavirus, one of the main causes of severe gastroenteritis in infants and children up to 5 years, and respiratory syncytial virus (RSV), one of the leading causes of lower respiratory tract infections. Both human milk and bovine milk derived factors may provide protection against mucosal viral infections. More recently, a similar activity of milk derived proteins was suggested for SARS-CoV-2. The goal of the current study was to test antiviral activity of the bovine milkfat globule membrane (MFGM) against rotavirus, RSV and SARS-CoV-2 and to further characterize MFGM-enriched whey to identify which components in MFGM-enriched whey may contribute to the inhibitory activity. Methods: The effects of MFGM-enriched whey, its whey protein isolate counterpart (WPI, obtained from the same production process) and a conventional whey protein concentrate (WPC) on rotavirus (strains Wa and SA114F), RSV (strain RSV-A2) and SARS-CoV-2 (Alpha variant) infectivity were determined using MA104 cells, human alveolar basal epithelial (A549) cells and monkey kidney (Vero E6) cells, respectively. The compounds were characterized in detail by LC-MS/MS and 31P-NMR to determine protein and phospholipid composition, respectively. Results: Relative to its WPI counterpart, MFGM-enriched whey demonstrated a dose-dependent inhibition for both rotavirus and RSV whereas for SARS-CoV-2 inhibition was only observed at the highest concentration tested. Label-free quantification (LFQ) and intensity based absolute quantification (iBAQ) of identified proteins revealed a clear difference between MFGM-enriched whey and its controls including enrichment of known MFGM proteins and non-MFGM proteins that are enriched simultaneously, some of which have previously been demonstrated to display anti-viral activity. Although not completely absent from other whey protein preparations, MFGM-enriched whey had the highest specific and total phospholipid levels. Conclusion: MFGM-enriched whey displayed antiviral activity against multiple viruses of clinical importance. This study provides insights into the active components in MFGM-enriched whey and may contribute to previous clinical observations with MFGM-enriched formula demonstrating reduced respiratory and gastrointestinal infections in formula fed infants.

Select gut microbiota impede rotavirus vaccine efficacy.

BACKGROUND& AIMS: The protection provided by rotavirus (RV) vaccines is highly heterogeneous amongst individuals. We hypothesized that microbiota composition might influence RV vaccine efficacy. METHODS: First, we examined the potential of segmented filamentous bacteria (SFB) colonization to influence RV vaccine efficacy in mice. Next, we probed the Influence of human microbiomes on RV vaccination via administering mice fecal microbial transplants (FMT) from children with robust or minimal RV vaccine responsiveness. Post-FMT, mice were subjected to RV vaccination followed by RV challenge. RESULTS: SFB colonization induced a phenotype that was reminiscent of RV vaccine failure, i.e. failure to generate RV antigens and, consequently, anti-RV antibodies following RV vaccination resulting in proneness to RV challenge after SFB levels diminished. FMT from children to mice recapitulated donor vaccination phenotype. Specifically, mice receiving FMT from high-responsive vaccinees copiously shed RV antigens and robustly generated anti-RV antibodies following RV vaccination. Concomitantly, such mice were impervious to RV challenge. In contrast, mice receiving FMT from children who had not responded to RV vaccination exhibited only modest responses to RV vaccination and, concomitantly, remained prone to RV challenge. Microbiome analysis ruled out a role for SFB but suggested involvement of Clostridium perfringens. Oral administration of cultured C. perfringens to gnotobiotic mice partially recapitulated the RV vaccine non-responder phenotype. Analysis of published microbiome data found C. perfringens abundance in children modestly associated with RV vaccine failure. CONCLUSION: Microbiota composition influences RV vaccine efficacy with C. perfringens being one, perhaps of many, potential contributing taxa.

Infant and adult human intestinal enteroids are morphologically and functionally distinct.

Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell, and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex vivo model to advance studies of infant-specific diseases and drug discovery for this population. IMPORTANCE: Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens.

Gut-resident C. perfringens impedes rotavirus vaccine efficacy.

Background & Aims: The extent to which live orally-administered rotavirus (RV) vaccines elicit protective immunity is highly heterogeneous. We hypothesized microbiota composition might influence vaccine efficacy. Methods: We tested this concept by examining extent to which colonizing mice with segmented filamentous bacteria (SFB) influenced RV vaccine efficacy.Influence of human microbiomes on RV vaccination was studied via administering germ-free mice fecal microbial transplants (FMT) from children with robust or minimal RV vaccine responsiveness. Post-FMT, mice were subjected to vaccination and challenge doses of RV. Results: SFB administration resulted in a phenotype reminiscent of RV vaccine failure, i.e. minimal generation of RV antigens and, consequently, lack of anti-RV antibodies resulting in proneness to RV challenge once SFB levels diminished. Transplant of microbiomes from children to mice recapitulated donor vaccination phenotype. Specifically, mice receiving FMT from high-responding children exhibited high levels of fecal RV antigen shedding and RV antibodies in response to RV vaccination and, concomitantly, were impervious to RV challenge. In contrast, mice receiving FMT from children who had not responded to RV vaccination exhibited only modest responses to RV challenge and, accordingly, remained prone to RV challenge. Microbiome analysis ruled out a role for SFB but suggested that RV vaccine failure might involve Clostridium perfringens . Oral administration of cultured C. perfringens to gnotobiotic mice partially recapitulated the RV vaccine non-responder phenotype. Analysis of previously-reported microbiome data found C. perfringens abundance in children associated with RV vaccine failure. Conclusion: Microbiota composition influences RV vaccine virus infection and, consequently, protective immunity. C. perfringens may be one, perhaps of many, bacterial species harbored in the intestine of RV-vaccine non-responders that influences RV vaccine outcomes.

Divergent responses of human intestinal organoid monolayers using commercial in vitro cytotoxicity assays.

In vitro models, such as primary cells and continuous cell lines routinely used for evaluating drug candidates, have limitations in their translational relevance to human diseases. Organotypic cultures are increasingly being used to assess therapeutics for various cancers and infectious diseases. Monitoring drug cytotoxicity in cell cultures is crucial in drug development, and several commercially available kits for cytotoxicity assessment offer distinct advantages and limitations. Given the complexity of organoid cultures, including donor-driven variability, we investigated drug-treated, tissue stem cell-derived human intestinal organoid responses with commonly used cell cytotoxicity assay kits. Using seven different compounds, we compared the cytotoxicity assay performance of two different leaky membrane-based and two metabolism-based assays. Significant variability was seen in reported viability outcomes across assays and organoid lines. High baseline activity of lactate dehydrogenase (LDH) in four human intestinal organoid lines required modification of the standard LDH assay protocol. Additionally, the LDH assay reported unique resilience to damage in a genetically-modified line contrasting results compared to other assays. This study highlights factors that can impact the measurement of cell cytotoxicity in intestinal organoid models, which are emerging as valuable new tools for research and pre-clinical drug testing and suggest the need for using multiple assay types to ensure reliable cytotoxicity assessment.

GI.1 Norovirus Neutralizing Antibody Levels Are Correlated with GI.1 Histo-blood Group Antigen-Blocking Antibody Levels.

BACKGROUND: The in vitro cultivation of human noroviruses allows a comparison of antibody levels measured in neutralization and histoblood group antigen (HBGA)-blocking assays. METHODS: Serum samples collected during the evaluation of an investigational norovirus vaccine (HIL-214 [formerly TAK-214]) were assayed for neutralizing antibody levels against the vaccine's prototype Norwalk virus/GI.1 (P1) virus strain. Results were compared to those previously determined using HBGA-blocking assays. RESULTS: Neutralizing antibody seroresponses were observed in 83% of 24 vaccinated adults, and antibody levels were highly correlated (r=0.81, P<0.001) with those measured by HBGA-blocking. CONCLUSIONS: GI.1-specific HBGA-blocking antibodies are a surrogate for neutralization of GI.1 norovirus.

2'-Fucosyllactose Inhibits Human Norovirus Replication in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Currently, there are no targeted antivirals for the treatment of HuNoV infection. Histo-blood group antigens (HBGAs) on the intestinal epithelium are cellular attachment factors for HuNoVs; molecules that block the binding of HuNoVs to HBGAs thus have the potential to be developed as antivirals. Human milk oligosaccharides (HMOs) are glycans in human milk with structures analogous to HBGAs. HMOs have been shown to act as decoy receptors to prevent the attachment of multiple enteric pathogens to host cells. Previous X-ray crystallography studies have demonstrated the binding of HMO 2'-fucosyllactose (2'FL) in the same pocket as HBGAs for some HuNoV strains. We evaluated the effect of 2'FL on the replication of a globally dominant GII.4 Sydney [P16] HuNoV strain using human intestinal enteroids (HIEs) from adults and children. A significant reduction in GII.4 Sydney [P16] replication was seen in duodenal and jejunal HIEs from multiple adult donors, all segments of the small intestine from an adult organ donor and in two pediatric duodenal HIEs. However, 2'FL did not inhibit HuNoV replication in two infant jejunal HIEs that had significantly lower expression of α1-2-fucosylated glycans. 2'FL can be synthesized in large scale, and safety and tolerance have been assessed previously. Our data suggest that 2'FL has the potential to be developed as a therapeutic for HuNoV gastroenteritis.

Advancements in Human Norovirus Cultivation in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are a significant cause of both epidemic and sporadic acute gastroenteritis worldwide. The lack of a reproducible culture system for HuNoVs was a major obstacle in studying virus replication and pathogenesis for almost a half-century. This barrier was overcome with our successful cultivation of multiple HuNoV strains in human intestinal enteroids (HIEs), which has significantly advanced HuNoV research. We previously optimized culture media conditions and generated genetically-modified HIE cultures to enhance HuNoV replication in HIEs. Building upon these achievements, we now present additional advancements to this culture system, which involve testing different media, unique HIE lines, and additional virus strains. HuNoV infectivity was evaluated and compared in new HIE models, including HIEs generated from different intestinal segments of individual adult organ donors, HIEs made from human embryonic stem cell-derived human intestinal organoids that were transplanted into mice (H9tHIEs), genetically-engineered (J4 FUT2 knock-in [ KI ], J2 STAT1 knock-out [ KO ]) HIEs, as well as HIEs derived from a patient with common variable immunodeficiency (CVID) and from infants. Our findings reveal that small intestinal HIEs, but not colonoids, from adults, H9tHIEs, HIEs from a CVID patient, and HIEs from infants support HuNoV replication with segment and strain-specific differences in viral infection. J4 FUT2-KI HIEs exhibit the highest susceptibility to HuNoV infection, allowing the cultivation of a broader range of GI and GII HuNoV strains than previously reported. Overall, these results contribute to a deeper understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research. Importance: Human noroviruses (HuNoVs) are very contagious and cause significant acute gastroenteritis globally, but studying them has been hindered by the lack of a reproducible culture system for nearly 50 years. This barrier was overcome by successfully cultivating multiple HuNoV strains in human intestinal enteroids (HIEs), advancing HuNoV research. We previously optimized culture conditions and developed genetically modified HIEs to enhance HuNoV replication. In this study, we tested different media, unique HIE lines, and additional virus strains, evaluating HuNoV infectivity in new HIE models. These models include HIEs from various intestinal segments of adult donors, human embryonic stem cell-derived HIEs transplanted into mice (H9tHIEs), genetically-engineered HIEs (J4 FUT2 knock-in [ KI ], J2 STAT1 knock-out [ KO ]), HIEs from a common variable immunodeficiency (CVID) patient, and from infants. Our findings show that adult small intestinal HIEs, H9tHIEs, CVID patient HIEs, and infant HIEs support HuNoV replication with segment and strain-specific differences. J4 FUT2-KI HIEs exhibited the highest susceptibility, allowing cultivation of a broader range of HuNoV strains. These results enhance the understanding of HuNoVs and highlight the transformative potential of HIE cultures in HuNoV research.

Analyzing efficiency of a lentiviral shRNA knockdown system in human enteroids using western blot and flow cytometry.

Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes.

Human milk cream alters intestinal microbiome of preterm infants: a prospective cohort study.

BACKGROUND: In very low birth weight (VLBW) infants, human milk cream added to standard human milk fortification is used to improve growth. This study aimed to evaluate the impact of cream supplement on the intestinal microbiome of VLBW infants. METHODS: Whole genome shotgun sequencing was performed on stool (n = 57) collected from a cohort of 23 infants weighing 500-1250 grams (control = 12, cream = 11). Both groups received an exclusive human milk diet (mother's own milk, donor human milk, and donor human milk-derived fortifier) with the cream group receiving an additional 2 kcal/oz cream at 100 mL/kg/day of fortified feeds and then 4 kcal/oz if poor growth. RESULTS: While there were no significant differences in alpha diversity, infants receiving cream significantly differed from infants in the control group in beta diversity. Cream group samples had significantly higher prevalence of Proteobacteria and significantly lower Firmicutes compared to control group. Klebsiella species dominated the microbiota of cream-exposed infants, along with bacterial pathways involved in lipid metabolism and metabolism of cofactors and amino acids. CONCLUSIONS: Cream supplementation significantly altered composition of the intestinal microbiome of VLBW infants to favor increased prevalence of Proteobacteria and functional gene content associated with these bacteria. IMPACT: We report changes to the intestinal microbiome associated with administration of human milk cream; a novel supplement used to improve growth rates of preterm very low birth weight infants. Since little is known about the impact of cream on intestinal microbiota composition of very low birth weight infants, our study provides valuable insight on the effects of diet on the microbiome of this population. Dietary supplements administered to preterm infants in neonatal intensive care units have the potential to influence the intestinal microbiome composition which may affect overall health status of the infant.

Infant and Adult Human Intestinal Enteroids are Morphologically and Functionally Distinct.

Background & Aims: Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. Methods: We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We validated differences in key pathways through functional studies and determined if these cultures recapitulate known features of the infant intestinal epithelium. Results: RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. Conclusions: HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex-vivo model to advance studies of infant-specific diseases and drug discovery for this population.

A Bivalent Human Norovirus Vaccine Induces Homotypic and Heterotypic Neutralizing Antibodies.

A GII.2 outbreak in an efficacy study of a bivalent virus-like particle (VLP) norovirus vaccine, TAK-214, in healthy US adults provided an opportunity to examine GII.4 homotypic vs. GII.2 heterotypic responses to vaccination and infection. Three serological assays (VLP-binding, histoblood group antigen-blocking, and neutralizing) were performed for each genotype. Results were highly correlated within a genotype but not between genotypes. Although the vaccine provided protection from GII.2-associated disease, little GII.2-specific neutralization occurred after vaccination. Choice of antibody assay can affect assessments of human norovirus vaccine immunogenicity.

Standardization of an antiviral pipeline for human norovirus in human intestinal enteroids demonstrates nitazoxanide has no to weak antiviral activity.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis. In immunocompetent hosts, symptoms usually resolve within 3 days; however, in immunocompromised persons, HuNoV infection can become persistent, debilitating, and sometimes life-threatening. There are no licensed therapeutics for HuNoV due to a near half-century delay in its cultivation. Treatment for chronic HuNoV infection in immunosuppressed patients anecdotally includes nitazoxanide, a broad-spectrum antimicrobial licensed for treatment of parasite-induced gastroenteritis. Despite its off-label use for chronic HuNoV infection, nitazoxanide has not been clearly demonstrated to be an effective treatment. In this study, we standardized a pipeline for antiviral testing using multiple human small intestinal enteroid lines representing different intestinal segments and evaluated whether nitazoxanide inhibits replication of five HuNoV strains in vitro. Nitazoxanide did not exhibit high selective antiviral activity against any HuNoV strain tested, indicating it is not an effective antiviral for HuNoV infection. Human intestinal enteroids are further demonstrated as a model to serve as a preclinical platform to test antivirals against HuNoVs to treat gastrointestinal disease. Abstr.

Report of the Assay Guidance Workshop on 3-Dimensional Tissue Models for Antiviral Drug Development.

The National Center for Advancing Translational Sciences (NCATS) Assay Guidance Manual (AGM) Workshop on 3D Tissue Models for Antiviral Drug Development, held virtually on 7-8 June 2022, provided comprehensive coverage of critical concepts intended to help scientists establish robust, reproducible, and scalable 3D tissue models to study viruses with pandemic potential. This workshop was organized by NCATS, the National Institute of Allergy and Infectious Diseases, and the Bill and Melinda Gates Foundation. During the workshop, scientific experts from academia, industry, and government provided an overview of 3D tissue models' utility and limitations, use of existing 3D tissue models for antiviral drug development, practical advice, best practices, and case studies about the application of available 3D tissue models to infectious disease modeling. This report includes a summary of each workshop session as well as a discussion of perspectives and challenges related to the use of 3D tissues in antiviral drug discovery.

Human intestinal organoids as models to study enteric bacteria and viruses.

Laboratory studies of host-microbe interactions have historically been carried out using transformed cell lines and animal models. Although much has been learned from these models, recent advances in the development of multicellular, physiologically active, human intestinal organoid (HIO) cultures are allowing unprecedented discoveries of host-microbe interactions. Here, we review recent literature using HIOs as models to investigate the pathogenesis of clinically important enteric bacteria and viruses and study commensal intestinal microbes. We also discuss limitations of current HIO culture systems and how technical advances and innovative engineering approaches are providing new directions to improve the model. The studies discussed here highlight the potential of HIOs for studying microbial pathogenesis, host-microbe interactions, and for preclinical development of therapeutics and vaccines.

A Standardized Antiviral Pipeline for Human Norovirus in Human Intestinal Enteroids Demonstrates No Antiviral Activity of Nitazoxanide.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis. In immunocompetent hosts, symptoms usually resolve within three days; however, in immunocompromised persons, HuNoV infection can become persistent, debilitating, and sometimes life-threatening. There are no licensed therapeutics for HuNoV due to a near half-century delay in its cultivation. Treatment for chronic HuNoV infection in immunosuppressed patients anecdotally includes nitazoxanide, a broad-spectrum antimicrobial licensed for treatment of parasite-induced gastroenteritis. Despite its off-label use for chronic HuNoV infection, nitazoxanide has not been clearly demonstrated to be an effective treatment. In this study, we established a standardized pipeline for antiviral testing using multiple human small intestinal enteroid (HIE) lines representing different intestinal segments and evaluated whether nitazoxanide inhibits replication of 5 HuNoV strains in vitro . Nitazoxanide did not exhibit high selective antiviral activity against any HuNoV strains tested, indicating it is not an effective antiviral for norovirus infection. HIEs are further demonstrated as a model to serve as a pre-clinical platform to test antivirals against human noroviruses to treat gastrointestinal disease.

Norovirus Protease Structure and Antivirals Development.

Human norovirus (HuNoV) infection is a global health and economic burden. Currently, there are no licensed HuNoV vaccines or antiviral drugs available. The protease encoded by the HuNoV genome plays a critical role in virus replication by cleaving the polyprotein and is an excellent target for developing small-molecule inhibitors. The current strategy for developing HuNoV protease inhibitors is by targeting the enzyme's active site and designing inhibitors that bind to the substrate-binding pockets located near the active site. However, subtle differential conformational flexibility in response to the different substrates in the polyprotein and structural differences in the active site and substrate-binding pockets across different genogroups, hamper the development of effective broad-spectrum inhibitors. A comparative analysis of the available HuNoV protease structures may provide valuable insight for identifying novel strategies for the design and development of such inhibitors. The goal of this review is to provide such analysis together with an overview of the current status of the design and development of HuNoV protease inhibitors.

Glycan Recognition in Human Norovirus Infections.

Recognition of cell-surface glycans is an important step in the attachment of several viruses to susceptible host cells. The molecular basis of glycan interactions and their functional consequences are well studied for human norovirus (HuNoV), an important gastrointestinal pathogen. Histo-blood group antigens (HBGAs), a family of fucosylated carbohydrate structures that are present on the cell surface, are utilized by HuNoVs to initially bind to cells. In this review, we describe the discovery of HBGAs as genetic susceptibility factors for HuNoV infection and review biochemical and structural studies investigating HuNoV binding to different HBGA glycans. Recently, human intestinal enteroids (HIEs) were developed as a laboratory cultivation system for HuNoV. We review how the use of this novel culture system has confirmed that fucosylated HBGAs are necessary and sufficient for infection by several HuNoV strains, describe mechanisms of antibody-mediated neutralization of infection that involve blocking of HuNoV binding to HBGAs, and discuss the potential for using the HIE model to answer unresolved questions on viral interactions with HBGAs and other glycans.

Persistence of G10P[11] neonatal rotavirus infections in southern India.

BACKGROUND: Neonatal rotavirus infections are predominantly caused by distinct genotypes restricted to this age-group and are mostly asymptomatic. METHOD: Stool samples from neonates admitted for >48 h in neonatal intensive care units (NICUs) in Vellore (2014-2015) and Chennai (2015-2016) in southern India, and from neonates born at hospitals in Vellore but not admitted to NICUs (2015-2016) were tested for rotavirus by ELISA and genotyped by hemi-nested RT-PCR. RESULTS: Of 791 neonates, 150 and 336 were recruited from Vellore and Chennai NICUs, and 305 were born in five hospitals in Vellore. Positivity rates in the three settings were 49.3% (74/150), 29.5% (99/336) and 54% (164/305), respectively. G10P[11] was the commonly identified genotype in 87.8% (65/74), 94.9% (94/99) and 98.2% (161/164) of the neonates in Vellore and Chennai NICUs, and those born at Vellore hospitals, respectively. Neonates delivered by lower segment cesarian section (LSCS) at Vellore hospitals, not admitted to NICUs, had a significantly higher odds of acquiring rotavirus infection compared to those delivered vaginally [p = 0.002, OR = 2.4 (1.4-4.3)]. CONCLUSIONS: This report demonstrates the persistence of G10P[11] strain in Vellore and Chennai, indicating widespread neonatal G10P[11] strain in southern India and their persistence over two decades, leading to interesting questions about strain stability.