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Approaches to immunization of infants and young children against gastroenteritis due to rotaviruses.

Recent studies have shown that in developed countries rotaviruses are the single most important etiologic agents of acute gastroenteritis that requires hospitalization of infants and young children. Although deaths from gastroenteritis are, in general, infrequent in the developed countries, an effective rotavirus vaccine would clearly be of benefit to reduce the heavy toll of morbidity from gastroenteritis due to rotavirus. In the developing countries the impact of diarrheal diseases is staggering. It was recently estimated that in Asia, Africa, and Latin-America during a one-year period there would be 3.5 billion cases of diarrhea and 5-10 million deaths associated with diarrhea; in addition, diarrhea was ranked first in freqency in the categories of disease and mortality. In the developing countries rotaviruses are known to cause diarrhea, but their relative role in this high mortality rate is not yet known. epidemiologic data indicate that development of an effective rotavirus vaccine would reduce morbidity, and they suggest that a vaccine would also reduce a portion of the mortality from diarrheal disease. The prospects and approaches for the development of an effective rotavirus vaccine are presented. The recent successful propagation of rotavirus type 2 in cell culture represents an important step in this regard. In addition, the antigenic relation between human and animal strains offers another possible approach. The need for a live attenuated vaccine is indicated by the prime role played by local intestinal immunity in resistance to rotavirus disease.

Animals↗

Rotavirus in travelers' diarrhea: study of an adult student population in Mexico.

The role of rotavirus in adult diarrhea was evaluated in 165 students attending a Mexican university. Students were divided into three groups: newly arrived summer students from the United Sttes, regular students from the United States, and Mexican and Venezuelan students. Ninety-one students with diarrhea and 74 corresponding, matched, asymptomatic control students were included in the study. The frequency of rotavirus in stools was determined by electron microscopy with use of the pseudoreplica technique. Twenty-five percent of those who were ill and 12% of the controls had rotavirus in their stools. A significantly (P less than 0.05) greater number of newly arrived United States summer students with diarrhea had rotavirus in their stool than did matched controls (26% vs. 3%). There was no significant difference in rate of recovery of bacterial pathogens from rotavirus-positive and rotavirus-negative stools (52% vs. 53%) from students with diarrhea. Although significantly more rotavirus was identified from ill American summer students than from controls, the role of rotavirus as a cause of diarrhea in these students could not be established in all cases since bacterial pathogens were also commonly found in stool.

Diarrhea↗

Epidemiology of rotaviruses in a cohort of 45 Guatamalan Mayan Indian children observed from birth to the age of three years.

A cohort of 45 children was observed from birth to three years of age in their natural ecosystem to determine patterns of infection, morbidity, and growth. Data from enzyme-linked immunosorbent assay analysis for rotavirus of 5,891 extracts (kept frozen since 1964-1969) of weekly fecal specimens were compared against growth, morbidity, and specimen data files, permitting a retrospective description of the epidemiology of rotavirus infection in the cohort. Rotavirus infections were uncommon in the first months of life in intensively breast-fed infants. Infection increased with age to reach a maximal rate in the six- to 18-month age period. While there was a high incidence of diarrhea in the cohort, rotavirus was associated with only 10% of such episodes. The incidence of rotavirus infection was 1.2 episodes per child-year, and the incidence of rotavirus-associated diarrhea was 0.8 episodes per child-year. Serious outbreaks of rotavirus generally occurred from September through December, with as many as one-half of the children becoming infected. Repeated rotavirus infection was a common phenomenon.

Aging↗

Epidemiology of rotavirus infection and gastroenteritis in prospectively monitored Argentine families with young children.

A prospective study was carried out in 49 families of newborns in Avellaneda District, Argentina, recruited when the mother was in the last trimester of pregnancy and followed until the newborn was aged two years, to study the epidemiology of rotavirus infection under natural conditions. Weekly surveillance of all family members for diarrhea and rotavirus-related diarrhea was carried out from May 1983 to July 1986. Serum samples were also collected every six months to study the incidence of rotavirus infection in all family members. The peak of diarrhea incidence was observed in children aged less than two years (2.1 cases per person-year). Rotavirus infection was a common rather than a sporadic phenomenon (0.63 cases per person-year, on average) in all age groups, and at least 61% of rotavirus infections were reinfections. In contrast, the peak of rotavirus infection in seronegative persons (0.58 cases per person-year) and the peak of rotavirus-related diarrhea incidence (0.25 cases per person-year) were found during the first year of life. These results showed that infants aged less than one year are the most appropriate candidates for a rotavirus vaccine in the Avellaneda population. However, improvements in sanitary conditions and personal hygiene and education of the high-risk population should be considered the primary means of reducing infectious diarrhea in Argentina.

Adolescent↗

Randomized, placebo-controlled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea.

BACKGROUND: Hyperimmunized bovine colostrum containing antibodies has been shown to be effective in the treatment of rotavirus diarrhea. Antibodies derived from eggs of immunized hens may be a less expensive and more practical alternative. In this study, children with proven rotavirus diarrhea were treated with immunoglobulin extracted from eggs of chicken immunized with human rotavirus strains. METHODS: In a randomized, double-blind study, 79 children with known rotavirus diarrhea were assigned to receive either 10 g hyperimmune egg yolk (HEY) daily in four equally divided doses for 4 days (HEY group) or a similar preparation obtained from nonimmunized chicken (placebo group). The daily stool frequency and amount, oral rehydration solution iORS) intake, and presence of rotavirus in the stool were monitored for 4 days. RESULTS: In the HEY-treated group, there was significant reduction in stool output (in grams per kilogram per day; HEY vs. placebo; 87+/-59 vs. 120+/-75, P = 0.03), and significant reduction of ORS intake (in milliliters per kilogram per day) (HEY vs. placebo; 84+/-46 vs. 122+/-72, P = 0.008) on day 1 and clearance of virus on day 4 (HEY vs. placebo; 73% vs. 46%, P = 0.02). There was, however, no difference in diarrheal duration between the groups. CONCLUSIONS: Treatment with HEY against four human rotavirus strains resulted in modest improvement of diarrhea associated with earlier clearance of rotavirus from stools. These results indicate an encouraging role of HEY in the treatment of rotavirus-induced diarrhea in children. Further studies are needed to optimize the dose and neutralization titer and thus improve the efficacy of egg yolk immunoglobulin IgY derived from immunized hens.

Animals↗

Comparative sequence analysis of the genomic segment 6 of four rotaviruses each with a different subgroup specificity.

The nucleotide sequences of the genes that code for the major inner capsid protein, VP6, of the human rotavirus strain 1076 (subgroup I), porcine rotavirus Gottfried (subgroup II), equine rotavirus strain H-2 (non-I/II) and equine rotavirus strain FI-14 (both subgroups I and II) have been determined. The sixth segment positive-stranded RNA encodes a protein of 397 amino acids in all strains with the exception of strain H-2 in which it encodes a protein of 399 amino acids. Alignment of amino acid sequences of the VP6 protein of strain FI-14 and subgroup II rotaviruses (Wa and Gottfried) indicates a high degree of homology (94%), while homology between strain FI-14 and subgroup I rotaviruses (SA-11, RF and 1076) was somewhat less (90 to 92%). On the other hand a high degree of conservation of amino acid sequence (95 to 97%) was observed between the H-2 strain and subgroup I rotaviruses. Five regions that may contribute to subgroup epitopes were identified. Region A (amino acids 45, 56) and region C (amino acids 114, 120) may contribute to subgroup I epitopes and regions B (amino acids 83, 86, 89, 92), D (amino acids 312 or 314, 317 or 319) and E (amino acids 341 or 343, 350 or 352) may contribute to subgroup II epitopes. When analysed using the Western blot technique monoclonal antibodies specific for VP6 epitopes shared by all rotaviruses were observed to react with both monomeric and trimeric forms of VP6, while monoclonal antibodies specific for a subgroup I or II epitope reacted only with the trimeric form of VP6. This observation and the sequence analyses suggest that subgroup antigenic specificity is determined by conformational epitopes produced by the folding of VP6 or the interaction between VP6 monomers.

Amino Acid Sequence↗

Identification of four VP4 serological types (P serotypes) of bovine rotavirus using viral reassortants.

A series of five reassortant viruses each containing the VP4 gene of a distinct bovine rotavirus and the VP7 gene of human rotavirus strain ST3 was prepared, and antisera to these were produced in rabbits. In neutralization tests, these antisera allowed the differentiation of the five original strains (from three different VP7 or G serotypes) into three or possibly four VP4 or P serotypes. All of a further seven bovine rotavirus strains adapted to cell culture were successfully typed by these antisera. There was a degree of cross-reaction between antiserum to the fourth bovine rotavirus P serotype and the predominant human rotavirus serotype. However, antisera raised in guinea-pigs to recombinant VP4 from this serotype showed the bovine serotype to be distinct. There was no significant serological relationship between these four bovine rotavirus P serotypes and previously described P serotypes from rotaviruses isolated from man and non-bovine animals. The predominant bovine rotavirus VP7 serotypes G6 and G10 tended to have distinct P serotypes also.

Animals↗

Gastroenteritis in suckling mice caused by human rotavirus can be prevented with egg yolk immunoglobulin (IgY) and treated with a protein-bound polysaccharide preparation (PSK).

Oral inoculation of human rotavirus MO strain (serotype 3) into 5-day-old BALB/c mice cause gastroenteritis characterized by diarrhea. Clinical symptoms, histopathological changes in the small intestine, and the detection of rotavirus antigen in enterocytes were all characteristic of rotavirus-induced gastroenteritis. Using this small animal model, passive protection of suckling mice against human rotavirus infection was achieved with the use of immunoglobulin (IgY) from the yolks of eggs of rotavirus-immunized hens. When IgY against a rotavirus strain homotypic to the challenge virus (MO strain) was administered in the mice, complete protection against rotavirus infection was achieved. On the other hand, with oral administration of IgY against a heterotypic strain (serotype 1, Wa strain), a lower protective effect was nevertheless obtained. The four different strains of human rotavirus (Wa, KUN, MO, and ST3) were inactivated in vitro by treatment with PSK, a protein-bound polysaccharide preparation, in a dose-dependent manner. Oral administration of 2.5 mg of PSK caused a therapeutic effect on experimentally MO-infected suckling mice. The antiviral effect of PSK was indicated by the reduction of the duration of diarrhea.

Animals↗

Burden of severe rotavirus disease in Australia.

AIM: To analyse the epidemiology of coded rotavirus hospitalisations in Australia from 1993 to 2002, with a view to understanding the pre-vaccination burden of severe disease in Australia. This study also seeks to determine the burden of rotavirus-related mortality. METHODS: Hospitalisation data from the Australian Institute of Health and Welfare for the period 1993-2002 were analysed. Rotavirus-related mortality data from the National Mortality Database were also analysed for the period 1990-2002. RESULTS: There were an average of 4260 patients hospitalised for rotavirus each year. The majority of rotavirus hospitalisations occurred in those under the age of 5 years, with the highest rate being in those aged 6-12 months (618.4 per 100,000). The Northern Territory was the most distinct region, with exceptionally high rates of admission (148.9 per 100,000 total population), younger age of admission and longer lengths of stay. Mapping of rotavirus hospitalisation in Australia showed well-defined areas of high hospitalisation rates. Thirteen rotavirus-related deaths occurred during 1990-2002. CONCLUSION: Rotavirus infection causes considerable morbidity and mortality in Australia. A vaccination programme would need to be completed by 6 months of age to have maximal impact.

Adolescent↗

Acute gastroenteritis in children attending day-care centres with special reference to rotavirus infections. I. Aetiology and epidemiologic aspects.

Acute gastroenteritis (GE) among 214 children (aged 6 months-7 years) attending day-care centres (DDCs) in the Copenhagen County was studied during a 12-month period. A total of 197 cases of GE was observed in 109 children (i.e. 51% of the participants). The aetiology was as follows: rotavirus (n = 48) (24%), pathogenic bacteria (n = 11) (6%), Giardia lamblia (n = 3) (2%), while the aetiology of 68% remains unknown. The pathogenic bacteria included Yersinia enterocolitica, thermophilic Campylobacter, Clostridium difficile (+/- toxin) and enteropathogenic E. coli. In 4% of the GE the infections were multiple and Cryptosporidium was seen in one of these cases. The rate of GE declined with age from 1.35 GE per child per year (age group 1.0- less than 2.0 years) to 0.36 (6.0- less than 8.0 years). Serum sampled at the start of the study period showed that the frequency of detectable rotavirus IgG increased with age from 48% in the 6 months- less than 1.0 year group to 96% in the 4.0- less than 7.0 year group. The highest rates of rotavirus GE occurred from January to April (i.e. the rotavirus season). Moreover, rotavirus GE was almost absent after the age of 4. Hence, the rates of rotavirus GE per rotavirus season per child were 0.80 (age group 6 months-less than 1.0 year), 0.32 (1.0-less than 2.0), 0.14 (2.0-less than 3.0), 0.16 (3.0-less than 4.0), 0.06 (4.0-less than 5.0) and 0.04 (5.0-less than 6.0). Only 2 out of the 48 rotavirus GE were reinfections.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

Comparison of clinical characteristics between astrovirus and rotavirus infections diagnosed in 1997 to 2002 in Hungary.

AIM: To determine the severity and clinical characteristics of human astrovirus (HAstV) infections among hospitalized children and compare them with children infected by rotavirus. METHODS: Retrospective, case-control study of astrovirus-infected and rotavirus-infected children. Astroviruses were detected in stool samples by enzyme immunoassay and/or reverse transcriptase-polymerase chain reaction. All stool samples were tested for rotavirus and bacterial pathogens, and all negative samples were further tested for human astrovirus. Children with astrovirus-positive stool samples and complete clinical data were included in this study. RESULTS: Astrovirus was detected in 29 (1.8%) children, and 63 rotavirus-infected children were included as controls. Astrovirus-infected children had shorter duration of diarrhea than rotavirus-infected children (median 4 and 6 d, respectively; p<0.05), and 79% of the astrovirus infections were associated with a short duration of vomiting (median 1 and 4 d, respectively; p<0.0001). Rotavirus-infected children had longer hospitalization (p<0.050) than astrovirus-infected children. CONCLUSION: HAstV-infected children had similar symptoms to those occurring in rotavirus infection. However, astrovirus-infected patients had a significantly shorter duration of diarrhea and vomiting, and they required a shorter hospitalization. On the basis of the clinical data and severity scores, children with rotavirus infection had more severe illness.

Astroviridae Infections↗

Comparison of methods for rotavirus detection in water and results of a survey of Jerusalem wastewater.

Methods for the detection of viable rotaviruses and rotavirus antigen in water were developed and compared. The methods included laboratory-developed enzyme-linked immunosorbent assays (ELISAs) with chromogenic and luminescent substrates, commercial Rotazyme and Enzygnost ELISAs, and an indirect immunofluorescent assay. Of the methods tested, the immunofluorescent assay and the Enzygnost ELISA were the most sensitive for the simian rotavirus SA-11. All of the methods were positive for human rotavirus from clinical specimens. Seeded SA-11 rotavirus was concentrated from water by absorption to and elution from Zeta Plus filters followed by organic flocculation. Interference with the assays by components of the wastewater concentrates was minimal for the ELISAs, although the undiluted organic flocs were cytotoxic for the immunofluorescent assay. A survey of Jerusalem wastewater was carried out over the course of 1 year, and samples were assayed for rotaviruses and enteroviruses. Although enteroviruses were found in almost all of the samples, all samples were negative for rotaviruses. The concentration of rotaviruses in the wastewater was thus below the detection limit of the method used.

Antigens, Viral↗

Rotavirus virus-like particles as surrogates in environmental persistence and inactivation studies.

Virus-like particles (VLPs) with the full-length VP2 and VP6 rotavirus capsid proteins, produced in the baculovirus expression system, have been evaluated as surrogates of human rotavirus in different environmental scenarios. Green fluorescent protein-labeled VLPs (GFP-VLPs) and particles enclosing a heterologous RNA (pseudoviruses), whose stability may be monitored by flow cytometry and antigen capture reverse transcription-PCR, respectively, were used. After 1 month in seawater at 20 degrees C, no significant differences were observed between the behaviors of GFP-VLPs and of infectious rotavirus, whereas pseudovirus particles showed a higher decay rate. In the presence of 1 mg of free chlorine (FC)/liter both tracers persisted longer in freshwater at 20 degrees C than infectious viruses, whereas in the presence of 0.2 mg of FC/liter no differences were observed between tracers and infectious rotavirus at short contact times. However, from 30 min of contact with FC onward, the decay of infectious rotavirus was higher than that of recombinant particles. The predicted Ct value for a 90% reduction of GFP-VLPs or pseudoviruses induces a 99.99% inactivation of infectious rotavirus. Both tracers were more resistant to UV light irradiation than infectious rotavirus in fresh and marine water. The effect of UV exposure was more pronounced on pseudovirus than in GFP-VLPs. In all types of water, the UV dose to induce a 90% reduction of pseudovirus ensures a 99.99% inactivation of infectious rotavirus. Recombinant virus surrogates open new possibilities for the systematic validation of virus removal practices in actual field situations where pathogenic agents cannot be introduced.

Flow Cytometry↗

Heterogeneity of VP4 neutralization epitopes among serotype P1A human rotavirus strains.

We have used serotype-specific VP4 and VP7 neutralizing monoclonal antibodies (Nt-MAbs), as well as subgroup (SG)-specific MAbs, to characterize by enzyme immunoassay rotavirus strains isolated from diarrheic infants in the city of Monterrey, Mexico, from July 1993 to March 1994. Of a total of 465 children studied, 140 were rotavirus positive, including 3 patients infected with non-group A rotaviruses. The SG and VP7 (G) serotype specificities could be determined for 118 (84%) of the 140 rotavirus-positive stool specimens; 4 rotavirus strains were serotype G1 and SGII; 1 strain was serotype G2 and SGI+II; 112 strains were serotype G3 and SGII; 1 strain was serotype G3 and SGI; and none of the strains was serotype G4. Fifty-eight specimens, representing the 13 different group A rotavirus electropherotypes detected, were chosen for VP4 (P) serotyping. Of these, 48 (83%) strains reacted with the P1A serotype-specific Nt-MAb 1A10. None of the strains reacted with the serotype P2-specific Nt-MAbs tested. Not all viruses that reacted with Nt-MAb 1A10 were recognized by Nt-MAbs 2A3 and 2G1, which also recognize P1A strains, indicating heterogeneity of neutralization epitopes among serotype P1A human rotaviruses. This heterogeneity could be relevant for the specificity of the VP4-mediated neutralizing antibody immune response and indicates the need for antigenic characterization, in addition to genomic typing, of the VP4 proteins of circulating human rotavirus field strains.

Antibodies, Viral↗

Shedding of rotavirus in feces of sows before and after farrowing.

The purpose of this study was to determine whether sows shed rotavirus near the time of farrowing. Twelve sows purchased from a common source and seropositive for rotavirus were housed in isolation in farrowing crates from 5 days before to 3 weeks after farrowing. Fecal samples were collected at 3- to 4-day intervals and examined for the presence of rotavirus by direct electron microscopy. Samples were also treated with pancreatin and inoculated onto monkey kidney cells. Rotaviral antigens were detected by a direct immunofluorescence technique, and selected positive cultures were examined by immunoelectron microscopy. Rotavirus was detected in the feces of 5 of 12 sows as early as 5 days before to 2 weeks after farrowing. Diarrhea related to rotavirus developed in 4 of 12 litters. Two of these four litters were farrowed by sows which shed rotavirus at 7 and 10 and 14 days after farrowing. The results of this study indicate that sows immune to rotavirus can shed virus in their feces at a time when piglets are particularly susceptible to infection and that adult swine are of primary importance in the epidemiology of rotavirus as initiators of infection.

Animals↗

Heterogeneity of Raft-type membrane microdomains associated with VP4, the rotavirus spike protein, in Caco-2 and MA 104 cells.

Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-beta-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release.

Animals↗

RNA-binding proteins of bovine rotavirus.

Two major bovine rotavirus proteins have RNA-binding activity as shown by an RNA overlay-protein blot assay. Of the six proteins in purified virions, only one showed RNA-binding activity. This 92,000-molecular-weight (92K) protein was present in both single- and double-shelled particles. Its RNA-binding activity was blocked by preincubation with monospecific antibody to VP2. Thus, the 92K RNA-binding protein in rotavirus virions is VP2, the second most abundant protein in single-shelled particles. In infected cell extracts, numerous cellular RNA-binding proteins and two virus-specific RNA-binding proteins were detected, VP2 and a 31K nonstructural (NS31) protein. VP2 bound single-stranded RNA in preference to double-stranded RNA, whereas NS31 bound both single- and double-stranded RNA equally well. Binding did not appear to be nucleotide sequence specific, because RNA from uninfected cells and an unrelated RNA virus bound to VP2 and to NS31 as did rotavirus RNA. This technique showed that both cellular and rotavirus RNA-binding proteins also bound DNA. VP2 interacted with rotavirus RNA over a broad pH range, with an optimum at pH 6.4 to 6.8, and at NaCl concentrations between 0 and 100 mM. The RNA-binding activity of NS31 exhibited similar pH and NaCl dependency. Sequence-specific nucleic acid binding could be detected by this method. When labeled synthetic oligodeoxyribonucleotides corresponding to the 3' and 5' plus-sense terminal sequences of rotavirus gene segments were used as probes, the 3' synthetic oligodeoxyribonucleotide bound to one 48K protein in control and infected cells. This suggests that there may be a specific functional interaction between the 48K cellular protein and this 3'-terminal noncoding region of the rotavirus genome or mRNA. These data show that the RNA overlay-protein blot assay is a useful test to identify some cellular and viral proteins with RNA-binding activity. For bovine rotavirus, the evidence suggests that, of all the virus-specific proteins, VP2 and NS31 are most likely to interact with RNA during transcription and replication or virus assembly or both.

Animals↗

Relative concentrations of serum neutralizing antibody to VP3 and VP7 proteins in adults infected with a human rotavirus.

Two outer capsid rotavirus proteins, VP3 and VP7, have been found to elicit neutralizing-antibody production, but the immunogenicity of these proteins during human rotavirus infection has not been determined. The relative amounts of serum neutralizing antibody against the VP3 and VP7 proteins of the CJN strain of human rotavirus were, therefore, determined in adult subjects before and after infection with this virus. Reassortant strains of rotavirus that contained the CJN gene segment for only one of these two neutralization proteins were isolated and used for this study. The geometric mean titer of serum neutralizing antibody to a reassortant virus (CJN-M) that contained VP7 of CJN and VP3 of another human rotavirus was 12.7 times less than that of antibody to CJN before infection and 20.3 times less after infection. This indicated that most neutralizing antibody was against the VP3 rather than the VP7 protein of CJN. This result was confirmed with other reassortants between CJN and animal rotavirus strains (EDIM and rhesus rotavirus). These findings suggest that VP3 is the primary immunogen that stimulates neutralizing antibody during at least some rotavirus infections of humans.

Animals↗