Sue E. Crawford

Norwalk virus shedding after experimental human infection.

Noroviruses are shed in feces up to 8 weeks after infection.

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.

The 3′ End of Norwalk Virus mRNA Contains Determinants That Regulate the Expression and Stability of the Viral Capsid Protein VP1: a Novel Function for the VP2 Protein

ABSTRACT Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. The capsid protein VP1 is synthesized from a subgenomic RNA that contains two open reading frames (ORFs), ORF2 and ORF3, and the 3′ untranslated region (UTR). ORF2 and ORF3 code for the capsid protein (VP1) and a small structural basic protein (VP2), respectively. We discovered that the yields of virus-like particles (VLPs) composed of VP1 are significantly reduced when this protein is expressed from ORF2 alone. To determine how the 3′ terminus of the NV subgenomic RNA regulates VP1 expression, we compared VP1 expression levels by using recombinant baculovirus constructs containing different 3′ elements. High VP1 levels were detected by using a recombinant baculovirus that contained ORF2, ORF3, and the 3′UTR (ORF2+3+3′UTR). In contrast, expression of VP1 from constructs that lacked the 3′UTR (ORF2+3), ORF3 (ORF2+3′UTR), or both (ORF2 alone) was highly reduced. Elimination of VP2 synthesis from the subgenomic RNA by mutation resulted in VP1 levels similar to those obtained with the ORF2 construct alone, suggesting a cis role for VP2 in upregulation of VP1 expression levels. Comparisons of the kinetics of RNA and capsid protein expression levels by using constructs with or without ORF3 or the 3′UTR revealed that the 3′UTR increased the levels of VP1 RNA, whereas the presence of VP2 resulted in increased levels of VP1. Furthermore, VP2 increased VP1 stability and protected VP1 from disassembly and protease degradation. The increase in VP1 expression levels caused by the presence of VP2 in cis was also observed in mammalian cells.

Cell attachment protein VP8* of a human rotavirus specifically interacts with A-type histo-blood group antigen

As with many other viruses, the initial cell attachment of rotaviruses, which are the major causative agent of infantile gastroenteritis, is mediated by interactions with specific cellular glycans. The distally located VP8* domain of the rotavirus spike protein VP4 (ref. 5) mediates such interactions. The existing paradigm is that ‘sialidase-sensitive’ animal rotavirus strains bind to glycans with terminal sialic acid (Sia), whereas ‘sialidase-insensitive’ human rotavirus strains bind to glycans with internal Sia such as GM1 (ref. 3). Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies, it is not yet known how VP8* of human rotaviruses interacts with Sia and whether their cell attachment necessarily involves sialoglycans. Here we show that VP8* of a human rotavirus strain specifically recognizes A-type histo-blood group antigen (HBGA) using a glycan array screen comprised of 511 glycans, and that virus infectivity in HT-29 cells is abrogated by anti-A-type antibodies as well as significantly enhanced in Chinese hamster ovary cells genetically modified to express the A-type HBGA, providing a novel paradigm for initial cell attachment of human rotavirus. HBGAs are genetically determined glycoconjugates present in mucosal secretions, epithelia and on red blood cells, and are recognized as susceptibility and cell attachment factors for gastric pathogens like Helicobacter pylori and noroviruses. Our crystallographic studies show that the A-type HBGA binds to the human rotavirus VP8* at the same location as the Sia in the VP8* of animal rotavirus, and suggest how subtle changes within the same structural framework allow for such receptor switching. These results raise the possibility that host susceptibility to specific human rotavirus strains and pathogenesis are influenced by genetically controlled expression of different HBGAs among the world’s population.

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.

Norwalk Virus RNA Is Infectious in Mammalian Cells

ABSTRACT Human noroviruses are positive-sense RNA viruses and are the leading cause of epidemic acute viral gastroenteritis in developed countries. The absence of an in vitro cell culture model for human norovirus infection has limited the development of effective antivirals and vaccines. Human histo-blood group antigens have been regarded as receptors for norovirus infection, and expression of the α(1,2) fucosyltransferase gene (FUT2) responsible for the secretor phenotype is required for susceptibility to Norwalk virus (NV) infection. We report for the first time that transfection of NV RNA, isolated from stool samples from human volunteers, into human hepatoma Huh-7 cells leads to viral replication, with expression of viral antigens, RNA replication, and release of viral particles into the medium. Prior treatment of the RNA with proteinase K completely abolishes RNA infectivity, suggesting a key role of an RNA-protein complex. Although overexpression of the human FUT2 gene enhances virus binding to cells, it is not sufficient to allow a complete viral infection, and viral spread from NV-transfected cells to naïve cells does not occur. Finally, no differences in NV RNA replication are observed between Huh-7 and Huh-7.5.1 cells, which contain an inactivating mutation in retinoic acid-inducible gene I (RIG-I), suggesting that the RIG-I pathway does not play a role in limiting NV replication. Our results strongly suggest that the block(s) to NV replication in vitro is at the stage of receptor and/or coreceptor binding and/or uncoating, either because cells lack some specific factor or activation of cellular antiviral responses independent of RIG-I inhibits virus replication.

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.).

Immunogenicity and protective efficacy of rotavirus 2/6-virus-like particles produced by a dual baculovirus expression vector and administered intramuscularly, intranasally, or orally to mice.

Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. Rotavirus VLPs composed of simian SA11 strain VP2 and VP6 proteins (homologous 2/6-VLPs) were produced by cloning the rotavirus simian SA11 genes 2 and 6 into a single baculovirus transfer vector (pAcAB4). The overall yield of homologous 2/6-VLPs produced with the dual recombinant baculovirus was at least 30-fold higher than that of VLPs composed of bovine RF strain VP2 and simian SA11 strain VP6 (heterologous 2/6-VLPs), produced with single recombinant baculoviruses. Adult mice were immunized intramuscularly twice with various doses of homologous or heterologous 2/6-VLPs in QS-21, orally with or without cholera toxin (CT), or intranasally with mutant Escherichia coli heat-labile enterotoxin (LT-R192G). Both homologous and heterologous 2/6-VLPs were immunogenic and induced protection from challenge, with those administered parenterally or intranasally affording the highest mean protection from challenge. The 2/6-VLPs did not induce serum neutralizing antibody (N-Ab) responses, but these VLPs primed for a broad heterotypic N-Ab response, which was elicited after rotavirus challenge. Heterotypic N-Ab responses were not observed in 2/6-VLP vaccinated mice that were > or =94% protected from challenge. After challenge, control mice immunized with adjuvant alone developed only homotypic serum N-Ab responses. Similar results were obtained after challenge of rabbits immunized parenterally or intranasally with heterologous 2/6-VLPs. These results suggest that 2/6-VLPs prime the immune system to enhance the production of heterotypic N-Ab responses, but the induction of heterotypic N-Abs requires that virus replication occurs after challenge. The use of 2/6-VLPs expressed from a single recombinant baculovirus simplifies production and would reduce the cost of a VLP-based vaccine.

Rotavirus Antigenemia in Children Is Associated with Viremia

Background Antigenemia is commonly detected in rotavirus-infected children. Although rotavirus RNA has been detected in serum, definitive proof of rotavirus viremia has not been shown. We aimed to analyze a defined patient population to determine if infectious virus could be detected in sera from children with rotavirus antigenemia. Methods and Findings Serum samples obtained upon hospitalization from children with gastroenteritis (57 stool rotavirus-positive and 41 rotavirus-negative), children with diagnosed bronchiolitis of known (n = 58) or unknown (n = 17) viral etiology, children with noninfectious, nonchronic conditions (n = 17), and healthy adults (n = 28) were tested for rotavirus antigen by enzyme immunoassay (EIA). Results of serum antigen testing were assessed for association with clinical and immunological attributes of the children. Rotavirus antigenemia was detected in 90% (51/57) of children with rotavirus-positive stools, in 89% (8/9) of children without diarrhea but with rotavirus-positive stools, in 12% (2/17) of children with bronchiolitis of unknown etiology without gastroenteritis, and in 12% (5/41) of children with gastroenteritis but with rotavirus-negative stools. Antigenemia was not detected in sera from children with noninfectious nonchronic conditions, children with bronchiolitis of known etiology and no gastroenteritis, or healthy adults. Neither age nor timing of serum collection within eight days after onset of gastroenteritis significantly affected levels of antigenemia, and there was no correlation between antigenemia and viral genotype. However, there was a negative correlation between serum rotavirus antigen and acute rotavirus-specific serum IgA (r = −0.44, p = 0.025) and IgG (r = −0.40, p = 0.01) titers. We examined 11 antigen-positive and nine antigen-negative sera for infectious virus after three blind serial passages in HT-29 cells using immunofluorescence staining for rotavirus structural and nonstructural proteins. Infectious virus was detected in 11/11 (100%) sera from serum antigen-positive children and in two out of nine (22%) sera samples from antigen-negative children (p = 0.002). Conclusions Most children infected with rotavirus are viremic. The presence of viremia is directly related to the detection of antigenemia and is independent of the presence of diarrhea. Antigenemia load is inversely related to the titer of antirotavirus antibody in the serum. The finding of infectious rotavirus in the blood suggests extraintestinal involvement in rotavirus pathogenesis; however, the impact of rotavirus viremia on clinical manifestations of infection is unknown.

Rotavirus Viremia and Extraintestinal Viral Infection in the Neonatal Rat Model

ABSTRACT Rotaviruses infect mature, differentiated enterocytes of the small intestine and, by an unknown mechanism, escape the gastrointestinal tract and cause viremia. The neonatal rat model of rotavirus infection was used to determine the kinetics of viremia, spread, and pathology of rotavirus in extraintestinal organs. Five-day-old rat pups were inoculated intragastrically with an animal (RRV) or human (HAL1166) rotavirus or phosphate-buffered saline. Blood was collected from a subset of rat pups, and following perfusion to remove residual blood, organs were removed and homogenized to analyze rotavirus-specific antigen by enzyme-linked immunosorbent assay and infectious rotavirus by fluorescent focus assay or fixed in formalin for histology and immunohistochemistry. Viremia was detected following rotavirus infection with RRV and HAL1166. The RRV 50% antigenemia dose was 1.8 × 103 PFU, and the 50% diarrhea dose was 7.7 × 105 PFU, indicating that infection and viremia occurred in the absence of diarrhea and that detecting rotavirus antigen in the blood was a more sensitive measure of infection than diarrhea. Rotavirus antigens and infectious virus were detected in multiple organs (stomach, intestines, liver, lungs, spleen, kidneys, pancreas, thymus, and bladder). Histopathological changes due to rotavirus infection included acute inflammation of the portal tract and bile duct, microsteatosis, necrosis, and inflammatory cell infiltrates in the parenchymas of the liver and lungs. Colocalization of structural and nonstructural proteins with histopathology in the liver and lungs indicated that the histological changes observed were due to rotavirus infection and replication. Replicating rotavirus was also detected in macrophages in the lungs and blood vessels, indicating a possible mechanism of rotavirus dissemination. Extraintestinal infectious rotavirus, but not diarrhea, was observed in the presence of passively or actively acquired rotavirus-specific antibody. These findings alter the previously accepted concept of rotavirus pathogenesis to include not only gastroenteritis but also viremia, and they indicate that rotavirus could cause a broad array of systemic diseases in a number of different organs.