Hepatitis A: detection by immune electron microscopy of a viruslike antigen associated with acute illness.

Spherical 27-nanometer particles were visualized in stools obtained from hepatitis A patients in the acute phase of the disease. The particle was serologically specific for this disease, and every hepatitis A patient tested demonstrated a serologic response to this antigen. The findings suggest that it is the etiologic agent of hepatitis A.

Enzyme-linked immunosorbent assay for identification of rotaviruses from different animal species.

Rotaviruses cause gastroenteritis in man and a wide variety of animal species. They cross-react in many immunologic tests and have a similar appearance by electron microscopy, making differentiation among them difficult. Rotaviruses derived from different host species were distinguished by postinfection serum blocking virus activity in an enzyme-linked immunosorbent assay (ELISA). Thirty-three rotavirus isolates from children living in three different parts of the world could not be differentiated by this technique, but they were distinct from four strains recovered from calves, and a series of strains isolated from piglets, foals, monkeys, and infant mice. The four bovine strains were similar, but they could be differentiated from the other animal strains, each of which exhibited a distinct pattern when tested by the ELISA blocking technique.

Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus.

The possibility of immunizing human infants against rotaviruses, which cause severe dehydrating diarrheal disease, may depend on the use of a related rotavirus, derived from another animal species, as a source of antigen. To test the feasibility of this approach, calves were infected in utero with a bovine rotavirus and challenged with bovine or human type 2 rotavirus shortly after birth. Infection in utero with bovine rotavirus induced resistance to diarrheal disease caused by the human virus as well as the homologous bovine virus. These data suggest that the bovine virus is sufficiently related antigenically to the human type 2 virus to warrant further evaluation of the former as a source of vaccine.

Reoviruslike agent in stools: association with infantile diarrhea and development of serologic tests.

Reoviruslike particles were visualized by electron microscopy in stool filtrates prepared from stools of infants and young children with severe acute gastroenteritis. Patients who had such particles in their stools and whose paired acute and convalescent serums were tested developed an antibody response to the reoviruslike agent, which was measured by immune electron microscopy and by complement fixation. The reoviruslike agent was antigenically related to the epizootic diarrhea of infant mice virus and the Nebraska calf diarrhea virus.

Human rotavirus type 2: cultivation in vitro.

A strain of type 2 human rotavirus (Wa) was grown to relatively high titer through 14 passages in primary cultures of African green monkey kidney (AGMK) cells. This passage series was initiated with virus that had been passaged 11 times serially in newborn gnotobiotic piglets. In contrast, virus present in the stool of patient Wa as well as virus from the first, second, or third passage in piglets could not be propagated successfully in African green monkey kidney cells. Prior to each passage in cell culture, the virus was treated with trypsin and the inoculated cultures were centrifuged at low speed. Cultivation of a type 2 human rotavirus should aid attempts to characterize this virus and to develop a means of immunoprophylaxis for a serious diarrheal disease of human infants.

Rotavirus vaccine development for the prevention of severe diarrhea in infants and young children.

Rotaviruses were first detected 20 years ago and emerged rapidly as the single most important recognized etiological agents of severe diarrhea among infants and children under 2 years in both developed and developing countries. They are estimated to cause over 870,000 deaths annually in developing countries. This review highlights recent approaches to the development of a rotavirus vaccine.

Immunity to rotaviruses.

A potent, multivalent, serotype-specific RV vaccine and improved tests for measuring vaccine potency would help eliminate the necessity to pretest for vaccine efficacy in every country selected for its deployment. Until then, the need will continue for vaccine trials in various countries because the pathogenesis and epidemiology of RV and RV serotypes differ between and within countries. Although RV vaccinology is complex, it has forged ahead of our knowledge of RV immunopathogenesis and epidemiology.

Safety and efficacy of high-dose rhesus-human reassortant rotavirus vaccines--report of the National Multicenter Trial. United States Rotavirus Vaccine Efficacy Group.

OBJECTIVE: Rotavirus is a leading cause of morbidity and mortality from dehydrating gastroenteritis in infants and young children worldwide. Virtually every child is infected by age 4 years, justifying universal childhood immunization when a safe and effective vaccine is available. We report the results of a multicenter, placebo-controlled field trial in the United States of monovalent serotype 1 and tetravalent (TV) rhesus-human reassortant rotavirus vaccines (RRVs). DESIGN: In this randomized, double-blind trial, 1278 healthy infants ages 5 to 25 weeks received three oral doses of RRV serotype 1, RRV-TV, or a placebo at approximately 2, 4, and 6 months of age. Vaccines contained 4 x 10(5) plaque-forming units of virus. Gastroenteritis episodes were monitored, and severity was graded throughout one rotavirus season. Two stool specimens per episode were tested for rotavirus. RESULTS: The incidence of reactions did not differ among treatment groups during the 5-day, postvaccination safety surveillance period for any of the three doses. Both vaccines significantly reduced the incidence of rotavirus gastroenteritis. Vaccination was most protective against serious rotavirus illness; RRV-TV prevented 49% of rotavirus episodes, 80% of very severe episodes, and 100% of dehydrating rotavirus illness. Reduction of rotavirus disease by RRV-TV resulted in significantly fewer total episodes of gastroenteritis of all causes and an 82% reduction in all cases of dehydrating diarrhea. CONCLUSION: RRV-TV is highly protective against very severe, dehydrating rotavirus gastroenteritis.

A rotavirus vaccine for prevention of severe diarrhoea of infants and young children: development, utilization and withdrawal.

The importance of rotaviruses (RVs) as the single most important cause of severe diarrhoea of infants and young children is well recognized. At NIH, we developed a quadrivalent (tetravalent [TV]) vaccine to protect against the four epidemiologically important RV serotypes. It is comprised of live attenuated rhesus RV (RRV), a VP7 serotype G3 strain (the 'Jennerian' approach), and three reassortant RVs, each containing 10 RRV genes and one human RV gene that codes for the major outer protein, VP7, that determines serotype G1, G2 or G4 specificity (the 'modified Jennerian' approach). The vaccine was safe and effective against severe diarrhoea in a major prelicensure collaborative effort of phase III trials. In February 1998 and again in June 1998, the Advisory Committee on Immunization Practices (ACIP) recommended routine immunization with three oral doses at 2, 4 and 6 months of age. The tetravalent vaccine (RotaShield) was licensed in the USA by the FDA in August 1998. In July 1999, after about 1.5 million doses had been given, the CDC recommended suspending administration of the vaccine because post-licensure surveillance of adverse events had suggested an association with intussusception. After further investigation by CDC, in October 1999, the ACIP withdrew its recommendation concluding that '...intussusception occurs with significantly increased frequency in the first 1-2 weeks after vaccination with RRV-TV, particularly following the first dose'. The implications of these developments from a practical, epidemiological, analytical and ethical perspective are discussed.

Systemic and mucosal immune responses to rhesus rotavirus vaccine MMU 18006.

Thirty-four children 3 to 20 months of age ingested either 10(5), 10(4) or 10(3) plaque-forming units of rhesus rotavirus vaccine, MMU 18006, which possesses human rotavirus serotype 3 neutralization antigen. Immune responses were evaluated by a plaque reduction neutralization (PRN) assay to rotavirus serotypes 1, 2 and 3 and by a serum IgG, IgM and IgA and fecal IgA class-specific enzyme-linked immunosorbent assay. Homotypic PRN antibody seroconversions to serotype 3 rotavirus were detected in 31 of 34 children (91%), whereas rises in heterotypic PRN antibody to human rotavirus serotypes 1 or 2 were found in only 3 of 21 (14%) (p less than 0.00000001). Thirty of the 34 vaccinated children (88%) had at least one class of rotavirus-specific serum antibody detected by enzyme-linked immunosorbent assay. A rotavirus-specific IgA coproantibody response was seen in 11 of 16 children (69%) following vaccination. Two children who had no evidence of PRN antibody to serotype 3 after vaccination had evidence of both a fecal and a serum rotavirus-specific IgA response, suggesting that in these children the response to the vaccine was primarily mucosal. These data show that orally administered rhesus rotavirus vaccine MMU 18006 elicits local intestinal immunity but produces primarily a homotypic serum neutralization response as measured by plaque reduction neutralization assays.

Evaluation of the M37 human rotavirus vaccine in 2- to 6-month-old infants.

Human rotavirus strain M37, isolated from an asymptomatic neonate, was evaluated as a live oral vaccine in a double-blinded, placebo-controlled trial involving 282 infants ages 2 to 6 months. Either 10(4) or 10(5) plaque-forming units (PFU) of the M37 vaccine were tested in 102 and 39 infants, respectively. The vaccine was well-tolerated; fever on Days 1 to 7 after vaccination was recorded in 12 and 18% of infants receiving 10(4) and 10(5) PFU of the M37 vaccine, respectively, compared with 6% of those receiving placebo; none of the vaccinees developed diarrhea. A rotavirus IgA enzyme-linked immunosorbent assay serum antibody response was detected in 47 and 76% of the infants receiving the 10(4) and 10(5) PFU vaccines, respectively. No clinical protection against rotavirus diarrhea was observed in the group vaccinated with the 10(4) PFU dose; the number of infants vaccinated with 10(5) PFU was too small for evaluation of vaccine efficacy. The M37 vaccine in a titer of 10(4) PFU was found to be inadequate; the 10(5) PFU dose was more immunogenic than the lower dose and warrants further study for clinical efficacy.

Protective efficacy against serotype 1 rotavirus diarrhea by live oral rhesus-human reassortant rotavirus vaccines with human rotavirus VP7 serotype 1 or 2 specificity.

Rhesus-human rotavirus (RV) reassortant vaccine strains D x RRV or DS 1 x RRV with VP7 serotype 1 or 2 specificity were evaluated for safety, immunogenicity and protective efficacy in a double blind placebo-controlled three cell trial involving 359 infants ages 2 to 5 months. The titer of the D x RRV vaccine was 10(4) and that of the DS 1 x RRV vaccine was 10(5) plaque-forming units/1-ml dose. The vaccines were acceptably reactogenic, each inducing a transient febrile response in fewer than one-third of the vaccinees. Seroconversion by RV enzyme-linked immunosorbent assay IgA antibody was detected in 61 and 75% of the vaccinees receiving a single dose of the serotype 1 or 2 reassortant vaccine, respectively. Efficacy against RV diarrhea was evaluated in two successive epidemic seasons; RV serotype 1 was prevalent in both. Clinical efficacy was observed with both vaccines and was associated with seroconversion after vaccination; considering only such vaccinees both vaccines showed equal efficacy. The overall rates of protection for the two vaccines combined against clinical RV disease in children with seroconversion after vaccination were 92 and 59% in the first and second RV epidemic seasons, respectively. Protection against asymptomatic RV infection, as measured by serologic responses, was 59% in the first season and nil in the second season. It is concluded that each of the reassortant RV vaccines was effective in inducing protection against symptomatic RV disease associated with RV serotype 1.

Reactogenicity and antigenicity of rhesus rotavirus vaccine (MMU-18006) in newborn infants in Venezuela.

The reactogenicity and antigenicity of the rhesus rotavirus vaccine, strain MMU18006, developed at the Laboratory of Infectious Diseases (National Institute of Allergy and Infectious Diseases, National Institutes of Health) were examined in a double blind, placebo-controlled study of 40 newborn infants in Caracas, Venezuela. The children were observed for the first few days after birth in the hospital nursery and by home visits for 10 days after vaccination to detect any adverse reactions. No reactions could be attributed to the vaccine. Serologic responses to the vaccine were evaluated in paired sera obtained at birth (cord blood) and 4 weeks after vaccination. Serologic responses to the vaccine were not observed by complement fixation, neutralization or a rhesus rotavirus VP7 epitope-specific competition assay. However, such responses were found in 9 of 14 tested infants by an immunoglobulin A-specific enzyme-linked immunosorbent assay. Seventeen of the 20 vaccinees also shed rhesus rotavirus vaccine in stool during the postvaccination period.

Immunogenicity and reactogenicity of lowered doses of rhesus rotavirus vaccine strain MMU 18006 in young children.

Rhesus rotavirus oral vaccine strain MMU 18006 at a dose of 10(5) plaque-forming units (PFU), a 1:10 dilution of the original undiluted vaccine, is highly immunogenic in young children. Fevers have occurred, however, on Days 3 and 4 following vaccination. This study was conducted to determine whether febrile reactions could be eliminated and immunogenicity maintained by (1) giving smaller doses of vaccine or (2) vaccinating younger infants. Thirty-one children between 3 and 11 months of age received, in a randomized, double blind manner, either 10(4) PFU of vaccine virus, 10(3) PFU of vaccine virus or placebo. All recipients of the 10(4) PFU dose had a seroresponse; however, some degree of immunogenicity was lost with the smaller dose (10(3) PFU). Fevers were observed in recipients of both of the lowered doses of vaccine but the febrile reactions were related to the age of the vaccinee. No infant younger than 5 months of age experienced a temperature elevation, whereas the majority of children older than 5 months had fevers. Our data suggest that the lack of reaction in the younger infants correlates with the presence of prevaccination neutralizing antibody, presumably transplacentally acquired. We conclude that the rhesus rotavirus oral vaccine at a dose of 10(4) PFU is immunogenic and appears to be safe in young infants.

Longitudinal study of rotavirus infection and gastroenteritis in families served by a pediatric medical practice: clinical and epidemiologic observations.

During 29 months of prospective longitudinal study of diarrhea in the home, human rotaviruses (HRVs) infected one or more members in 51% of 65 families, 35 of 126 children (28%) and 16 of 124 adults (13%). Within the 33 affected families, 57% of 62 children and 25% of 65 adults were infected. HRV gastroenteritis peaked at 40/100 person years at ages 12 to 23 months and decreased to 5 episodes/100 person years in adults. Among 25 children 0 through 36 months of age who had HRV infection, 88% were symptomatic. Of the 22 children with symptomatic HRV infection, 1 required hospitalization and 8 were seen by their physician for supportive care. HRVs were found in 12% of 216 stools obtained during gastrointestinal illness, but in only 0.2% of 1238 non-illness stools tested. HRV infections were noted as early as October and as late as April. Of 33 families who were studied for 2 seasons, at least 1 individual in each of 3 families experienced HRV infections in both years, but only one, an adult, shed virus and had symptoms in both seasons.

Simultaneous administration of rhesus rotavirus vaccine and oral poliovirus vaccine: immunogenicity and reactogenicity.

Rotavirus vaccine could be administered most efficiently if it were incorporated into routine childhood immunizations and did not interfere with the immune response to the other vaccines, principally oral poliovirus vaccine (OPV). We conducted a placebo-controlled randomized trial giving oral rhesus rotavirus vaccine (RRV) (strain MMU 18006) alone and together with a child's first dose of OPV and diphtheria-tetanus toxoids-pertussis to examine the possible interaction of these vaccines. A total of 102 infants 2 to 3 months of age were randomized into 3 groups to receive (1) RRV with OPV, (2) placebo with OPV and (3) RRV 2 weeks after OPV. All infants were given diphtheria-tetanus toxoids-pertussis. Serum samples were collected at the time of OPV immunization and 3 to 5 weeks later. Three to 5 weeks after OPV immunization 60% of infants had a 4-fold rise in neutralization titer to at least one of the three poliovirus serotypes. The rate of antibody response to poliovirus did not differ by RRV groups but a lower rate was correlated with a shorter interval (3 vs. 5 weeks) between OPV vaccination and antibody measurement. Fifty-six percent of infants had a 4-fold rise of IgA and 62% had a 4-fold rise of neutralizing antibody to RRV; this rise did not differ according to time of OPV immunization. RRV was not associated with side effects and may be safely given with OPV to infants 2 to 3 months of age.

Rotaviruses detected by reverse transcription polymerase chain reaction in acute gastroenteritis during a trial of rhesus-human reassortant rotavirus tetravalent vaccine: implications for vaccine efficacy analysis.

BACKGROUND: Rotaviruses are routinely diagnosed by detection of rotavirus antigen in stools using an enzyme immunoassay (EIA). A sensitive method, like reverse transcription polymerase chain reaction (RT-PCR), may reveal more rotaviruses, but the clinical significance of such findings is not well established. OBJECTIVES: To study whether RT-PCR can detect more episodes of rotavirus-associated gastroenteritis than EIA and to determine how rotavirus RT-PCR findings might change efficacy analysis of a rotavirus vaccine trial, in which the outcome measure was rotavirus gastroenteritis diagnosis with EIA. STUDY DESIGN: We applied RT-PCR for detection of rotaviruses in gastroenteritis episodes encountered in an efficacy trial of rhesus-human reassortant rotavirus tetravalent (RRV-TV) vaccine, in a total of 2398 infants. During a follow-up, covering two rotavirus epidemic seasons, 256 cases of rotavirus associated gastroenteritis were detected by EIA; 226 were in the primary efficacy analysis period that included children who had received three doses of vaccine or placebo. RESULTS: With RT-PCR, 84 (33%) more cases of rotavirus gastroenteritis were diagnosed than with EIA, 65 of these were in the primary efficacy analysis period. Clinically, cases of rotavirus gastroenteritis diagnosed by RT-PCR were much milder (median severity score 6 on a 20-point scale) than those diagnosed by EIA (median score 11), P < 0.0001. RT-PCR revealed proportionally more G2 and G4 rotaviruses than EIA. G1 rotaviruses detected by RT-PCR were almost equally divided between RRV-TV (25) vaccine and placebo (28) groups, whereas an apparent vaccine protective effect was seen in the distribution of G2 (one in the RRV-TV and eight in the placebo group) and G4 rotaviruses (six in the RRV-TV and 14 in the placebo group). CONCLUSION: RT-PCR is a useful tool in the diagnosis of rotavirus gastroenteritis, particularly for cases associated with other than the epidemiologically dominant G-type. Application of RT-PCR contributes to the overall appraisal of performance of rotavirus vaccine.

A serological survey of St Lucia.

A serological survey of a random sample of 541 of the population of St Lucia was undertaken. The prevalence of antibodies to dengue, herpes virus, VZ, rotavirus, rubella and syphilis is described and compared with other communities.

Overview of viral gastroenteritis.

Diarrheal illnesses in humans have been recognized since antiquity. Such illnesses continue to take a great toll of lives, with a disproportionately high mortality in infants and young children in developing countries. Bacteriologic and parasitologic advances made during the past century led to the discovery of the etiology of some of the diarrheal illnesses, but the etiology of the major portion remained unknown. It was assumed that viruses caused most of these illnesses because: (i) bacteria were recovered from only a small proportion of episodes, and (ii) bacteria-free filtrates were found to induce gastroenteritis in adult volunteer studies. However, an etiologic agent could not be recovered despite the "golden age" of virology in the 1950's and 1960's when tissue culture technology enabled the discovery of numerous cultivatable enteric viruses, none of which emerged as an important etiologic agent of gastroenteritis. The discoveries of the Norwalk virus in 1972, and of rotaviruses in 1973, both without the benefit of in vitro tissue culture systems, ushered in a new era in the study of the etiology of viral gastroenteritis. The Norwalk virus was found to be an important cause of non-bacterial epidemic gastroenteritis in adults and older children, and rotaviruses were shown to be the single most important etiologic agents of severe diarrheal illnesses of infants and young children in both developed and developing countries. With the major advances in the study of rotaviruses, there is a high degree of optimism that in the not-too-distant future, a rotavirus vaccine will be available. In addition, the recent molecular biologic advances in the study of the Norwalk and Norwalk-like viruses, now firmly established as caliviviruses, represent a major new horizon in the study of these viruses.

Classification of rotavirus VP4 and VP7 serotypes.

Rotaviruses, members of the Reoviridae family, are major etiologic agents of acute nonbacterial gastroenteritis of the young in a wide variety of mammalian and avian species, including humans. The need for effective immunoprophylaxis against rotaviral gastroenteritis has stimulated interest in the biochemical, molecular, genetic, and clinical aspects of these agents with the aim of developing safe and effective vaccines. Because neutralizing antibodies appear to play an important role in protection against many viral diseases, rotavirus antigens that induce neutralizing antibodies have played a central role in research and development of a rotavirus vaccine. The VP7 glycoprotein and VP4 spike protein that constitute the outer capsid of a complete rotavirus particle have been shown to be independent neutralization antigens. Since type specificity of the outer capsid proteins of a rotavirus appears to play an important role in protection against disease in experimental animal models, continued efforts have been made for classification and typing of neutralization specificities on the VP7 or VP4 capsid protein. Based on a criterion of > 20-fold differences between the homologous and heterologous reciprocal neutralizing antibody titers, fourteen VP7 (G) serotypes have been established. Studies are underway to characterize and classify the VP4 (P) serotypes among the strains that exhibit the fourteen different G serotypes. Attempts to classify the VP4 serotypes based on the same criterion (i.e., > 20-fold antibody differences) that is applied to classification of VP7 serotypes are in progress. This standard of > 20-fold antibody differences can be applied with hyperimmune serum raised to a reassortant possessing the VP4 encoding gene (and an unrelated VP7 encoding gene). Genotypes can provide leads towards classification but the serotype of a strain should be based on neutralization.