Sasirekha Ramani

Replication of human noroviruses in stem cell–derived human enteroids

The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the leading cause of gastroenteritis worldwide. We report the successful cultivation of multiple HuNoV strains in enterocytes in stem cell–derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for strain-dependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.

Determination of the 50% Human Infectious Dose for Norwalk Virus

BACKGROUND  Noroviruses are the most common cause of gastroenteritis in the United States. An understanding of the infectious dose of these viruses is important for risk assessment studies. METHODS  Healthy adults were enrolled in a randomized, double-blind, placebo-controlled evaluation of different dosages of Norwalk virus. Eligible subjects were monitored for clinical gastroenteritis, and infection status was determined. The presence of virus in vomitus was also assessed. RESULTS  Fifty-seven persons were enrolled; 8 received placebo and an additional 8 persons were considered to be nonsusceptible on the basis of being secretor negative. Twenty-one persons were infected (all blood group O or A), and 67% of those infected developed viral gastroenteritis. The 50% human infectious dose was calculated to be 3.3 reverse transcription polymerase chain reaction units (approximately 1320 genomic equivalents [gEq]) for secretor-positive blood group O or A persons and 7.0 (approximately 2800 gEq) for all secretor-positive persons. The time of illness onset was inversely correlated with inoculum dose. The maximal concentration of virus shedding was higher for persons with gastroenteritis. Norwalk virus was identified in 15 of 27 (56%) vomitus samples at a median concentration of 41 000 gEq/mL. CONCLUSIONS  The 50% human infectious dose measured is higher than previous estimates and similar to that of other RNA viruses. Clinical Trials Registration NCT00138476.

Protective Effect of Natural Rotavirus Infection in an Indian Birth Cohort

BACKGROUND More than 500,000 deaths are attributed to rotavirus gastroenteritis annually worldwide, with the highest mortality in India. Two successive, naturally occurring rotavirus infections have been shown to confer complete protection against moderate or severe gastroenteritis during subsequent infections in a birth cohort in Mexico. We studied the protective effect of rotavirus infection on subsequent infection and disease in a birth cohort in India (where the efficacy of oral vaccines in general has been lower than expected). METHODS We recruited children at birth in urban slums in Vellore; they were followed for 3 years after birth, with home visits twice weekly. Stool samples were collected every 2 weeks, as well as on alternate days during diarrheal episodes, and were tested by means of enzyme-linked immunosorbent assay and polymerase-chain-reaction assay. Serum samples were obtained every 6 months and evaluated for seroconversion, defined as an increase in the IgG antibody level by a factor of 4 or in the IgA antibody level by a factor of 3. RESULTS Of 452 recruited children, 373 completed 3 years of follow-up. Rotavirus infection generally occurred early in life, with 56% of children infected by 6 months of age. Levels of reinfection were high, with only approximately 30% of all infections identified being primary. Protection against moderate or severe disease increased with the order of infection but was only 79% after three infections. With G1P[8], the most common viral strain, there was no evidence of homotypic protection. CONCLUSIONS Early infection and frequent reinfection in a locale with high viral diversity resulted in lower protection than has been reported elsewhere, providing a possible explanation why rotavirus vaccines have had lower-than-expected efficacy in Asia and Africa. (Funded by the Wellcome Trust.).

Comparative Study of the Epidemiology of Rotavirus in Children from a Community-Based Birth Cohort and a Hospital in South India

ABSTRACT Rotavirus gastroenteritis is the major cause of severe dehydrating diarrhea in children worldwide. This study compares rotavirus diarrhea in 351 children in a community-based cohort and 343 children admitted to a hospital during the same period. Clinical information and fecal specimens were obtained during diarrheal episodes. Fecal samples were screened for VP6 antigen, and the positive samples were G and P typed by reverse transcription-PCR. Rotavirus was detected in 82/1,152 (7.1%) episodes of diarrhea in the community and 94/343 (27.4%) cases in the hospital. The median age of affected children (7.5 versus 10.5 months) and the mean severity of symptoms (Vesikari score, 7.6 ± 3.4 versus 11 ± 2.5) were lower in the community. A larger proportion of children in the community were breast-fed than were children admitted to the hospital (73% versus 34.8%). In the community, the genotypes identified in symptomatic patients, in order of frequency, were G1 (36.5%), G10 (17.1%), G2 (15.9%), and G9 (7.3%) and mixed infections (7.3%). The most common G-P combinations were G1P[8], G2P[4], G1P[4], and G10P[11]. The distribution of G types from hospitalized children was G1 (46.8%), G9 (19.1%), G2 (8.5%), G10 (1.1%), and 4.3% mixed infections. The most common G-P combinations were G1P[8] and G9P[8]. This study documents significant genetic heterogeneity of rotaviruses in the community and the hospital. G10P[11] strains resembling a vaccine candidate strain caused disease in the community, indicating the need for careful epidemiological studies as well as safety studies for the vaccine candidates.

Multilocus Genotyping of Cryptosporidium sp. Isolates from Human Immunodeficiency Virus-Infected Individuals in South India

ABSTRACT This study characterized cryptosporidial infections in 48 human immunodeficiency virus-infected individuals in India by multilocus genotyping. Cryptosporidium hominis, C. parvum, C. felis, C. muris, and C. meleagridis were identified. Cpgp40/15 PCR-restriction fragment length polymorphism identified six subgenotypes. Cryptosporidial diarrhea was associated with decreased CD4 counts, below 200 (P = 0.009), but not high viral loads.

Epidemiology of human noroviruses and updates on vaccine development.

Purpose of review Noroviruses (NoVs) are the most common cause of epidemic and sporadic cases of acute gastroenteritis worldwide. This review summarizes recent NoV disease burden estimates, epidemiology findings and provides an update on virus-like particle (VLP) vaccine studies. Recent findings NoVs are the leading cause of food-borne gastroenteritis and are replacing rotavirus as the predominant gastrointestinal pathogen in pediatric populations. Genogroup II, genotype 4 NoVs (GII.4) remain the dominant genotype worldwide. Increased NoV activity reported in late 2012 was associated with the emergence of a new GII.4 variant called Sydney 2012. New studies suggest that human NoVs can bind a larger range of histoblood group antigens, a susceptibility factor for NoV illness, thus expanding the susceptible population pool for infection. Intranasal immunization with a monovalent GI NoV VLP vaccine showed proof-of-concept efficacy. Studies using intramuscular immunization with a bivalent formulation including GII.4 VLPs are now underway. Summary The importance of NoVs as a major gastrointestinal pathogen underscores the need for well tolerated and effective vaccines. Results of VLP vaccine trials appear promising. However, the rapid evolution of NoV genotypes through antigenic drift and changing glycan specificities provide new challenges to epidemiology studies and vaccine trials.

Disease and economic burden of rotavirus diarrhoea in India

We used published and unpublished studies and national statistics to estimate the number of deaths, hospitalizations, and outpatient visits due to rotavirus diarrhoea and the associated national economic burden of disease in India. Annually in India, rotavirus diarrhoea causes an estimated 122,000-153,000 deaths, 457,000-884,000 hospitalizations, and 2 million outpatient visits in children <5 years of age. India spends Rs 2.0-3.4 billion (US

Viruses causing childhood diarrhoea in the developing world

41-72 million) annually in medical costs to treat rotavirus diarrhoea. The use of specific interventions against rotavirus, such as newly available vaccines, would help prevent much of this large disease and economic burden.

Closing the diarrhoea diagnostic gap in Indian children by the application of molecular techniques.

Purpose of review Acute gastroenteritis is one of the leading causes of morbidity and mortality in children in the developing world. With improvements in hygiene and sanitation, the burden of disease due to bacterial and parasitic infections has decreased and an increasing proportion of diarrhoea hospitalizations are attributed to viruses. This review focuses on enteric viruses and their role in childhood diarrhoea in the developing world. Recent findings With the use of sensitive molecular techniques, it is evident that a significant proportion of childhood diarrhoea is attributable to enteric viruses, with at least one viral agent in nearly 43% of samples from childhood diarrhoea in developing countries. Rotaviruses remain the most common pathogens in children, followed by noroviruses in almost all countries. There is increasing evidence that both rotaviruses and caliciviruses spread beyond the gut in a large proportion of infections. Summary The review highlights the importance of viral agents of gastroenteritis in developing countries. Wider use of molecular techniques is resulting in rapid identification of new or emerging strains and in the detection of extra-intestinal spread. There is a need to better understand susceptibility and immune response to these agents to be able to design suitable interventions.

Human milk contains novel glycans that are potential decoy receptors for neonatal rotaviruses

A large proportion of diarrhoeal illnesses in children in developing countries are ascribed to an unknown aetiology because the only available methods, such as microscopy and culture, have low sensitivity. This study was aimed at decreasing the diagnostic gap in diarrhoeal disease by the application of molecular techniques. Faecal samples from 158 children with and 99 children without diarrhoea in a hospital in South India were tested for enteric pathogens using conventional diagnostic methods (culture, microscopy and enzyme immunoassays) and molecular methods (six PCR-based assays). The additional use of molecular techniques increased identification to at least one aetiological agent in 76.5 % of diarrhoeal specimens, compared with 40.5 % using conventional methods. Rotavirus (43.3 %), enteropathogenic Escherichia coli (15.8 %), norovirus (15.8 %) and Cryptosporidium spp. (15.2 %) are currently the most common causes of diarrhoea in hospitalized children in Vellore, in contrast to a study conducted two decades earlier in the same hospital, where bacterial pathogens such as Shigella spp., Campylobacter spp. and enterotoxigenic E. coli were more prevalent. Molecular techniques significantly increased the detection rates of pathogens in children with diarrhoea, but a more intensive study, testing for a wider range of infectious agents and including more information on non-infectious causes of diarrhoea, is required to close the diagnostic gap in diarrhoeal disease.