Genetic relatedness of VP1 genes of Australian and Taiwanese rotavirus isolates.

Gene 1 (which encodes the viral RNA-dependent RNA polymerase, VP1) of an atypical human reassortant rotavirus strain, E210 (serotype G2P1B), is unrelated to genes 1 of standard human rotaviruses. To ascertain the origin of this gene, we determined a partial sequence and found that it exhibited greatest identity to gene 1 of a Taiwanese isolate, TE83, which is representative of G2 strains that caused an epidemic of gastroenteritis in 1993. Limited sequence identity to genes 1 of standard human and animal viruses was observed. This was confirmed by phylogenetic analysis. However, hybridization analysis using an E210 gene 1-specific probe indicated that a related gene was found among other Australian G2 isolates and in a Japanese strain isolated in the 1970s.

Rotavirus encephalopathy: pathogenesis reviewed.

Two cases of rotavirus gastroenteritis associated with neurological involvement, one with encephalitis (defined by abnormal neurological signs, cerebrospinal fluid (CSF) pleocytosis and detection of rotavirus genomic nucleic acid in the CSF) and one with a non-inflammatory encephalopathy (defined by abnormal neurological signs, an entirely normal CSF and detection of rotavirus genomic nucleic acid in the CSF), are presented and used as a basis to review and explore potential pathogenetic mechanisms, including direct viral replication within neurons and indirect effects of the newly described rotavirus 'enterotoxin'.

Epidemiological patterns of rotaviruses causing severe gastroenteritis in young children throughout Australia from 1993 to 1996.

Rotavirus strains that caused severe diarrhea in 4,634 (2,533 male) children aged less than 5 years and admitted to major hospitals in eight centers throughout Australia from 1993 to 1996 were subject to antigenic and genetic analyses. The G serotypes of rotaviruses were identified in 81.9% (3,793 of 4,634) children. They included 67.8% (from 3,143 children) serotype G1 isolates (containing 46 electropherotypes), 11.5% (from 531 children) serotype G2 isolates (27 electropherotypes), 0.8% (from 39 children) serotype G3 isolates (8 electropherotypes), and 1.6% (from 76 children) serotype G4 isolates (9 electropherotypes). G6 (two strains) and G8 (two strains) isolates were identified during the same period. G1 serotypes were predominant in all centers, with intermittent epidemics of G2 serotypes and sporadic detection of G3 and G4 strains. With the exception of two strains (typed as G1P2A[6] and G2P2A[6]) all serotype G1, G3, and G4 strains were P1A[8] and all serotype G2 strains were P1B[4]. Two contrasting epidemiological patterns were identified. In all temperate climates rotavirus incidence peaked during the colder months. The genetic complexity of strains (as judged by electropherotype) was greatest in centers with large populations. Identical electropherotypes appeared each winter in more than one center, apparently indicating the spread of some strains both from west to east and from east to west. Centers caring for children in small aboriginal communities showed unpredictable rotavirus peaks unrelated to climate, with widespread dissemination of a few rotavirus strains over distances of more than 1,000 km. Data from continued comprehensive etiological studies of genetic and antigenic variations in rotaviruses that cause severe disease in young children will serve as baseline data for the study of the effect of vaccination on the incidence of severe rotavirus disease and on the emergence of new strains.

Comparison of enzyme immunoassay, PCR, and type-specific cDNA probe techniques for identification of group A rotavirus gene 4 types (P types).

This study was designed to evaluate three techniques most commonly used to identify the VP4 (P) types of human group A fecal rotaviruses. The techniques included PCR with nested primers and hybridization with PCR-generated probes (to determine the P genotypes). The results obtained by these genetic techniques were evaluated against those obtained by an enzyme immunoassay (EIA) incorporating neutralizing monoclonal antibodies (N-MAbs) reacting with three major human P serotypes (serotypes P1A, P1B, and P2A). The P types of the rotaviruses present in 102 fecal specimens were determined under code by each of the three assays. The specificity of each assay was evaluated against a "gold standard" putative P type (P serotype and genotype) deduced from knowledge of the VP7 (G) type and the origin of the fecal specimen. Overall comparison of the results showed respective sensitivities and specificities of 92 and 92% for reverse transcription-PCR, 80 and 99% for hybridization, and 73 and 91% for EIA with N-MAbs. The hybridization assay retained high sensitivity with specimens stored for > or = 10 years. Hybridization assays with nonradioactive probes are relatively inexpensive and are suited for use in developing countries. In summary, both genetic assays showed high sensitivities and specificities in assigning a P type to human fecal rotavirus strains. Further evaluation of the EIA with N-MAbs is required, together with incorporation of new N-MAbs for the detection of the additional P types detected in developing countries.

Sequence of the VP7 gene of an atypical human rotavirus: evidence for genetic and antigenic drift.

The nucleotide sequence of the gene encoding the outer capsid glycoprotein, VP7, isolated from a reassortant human rotavirus, M3014, was determined. The deduced amino acid sequence exhibited significant identity to the VP7 from a standard strain belonging to serotype G4, although the antigenic regions of the M3014 VP7 resembled sequences from both serotype G4 and G9 viruses. However, reactivity with G4 or G9 serotype-specific monoclonal antibodies was not observed. We suggest that the M3014 VP7 was derived from sequential mutation of a G4-like progenitor gene resulting in a protein with novel antigenic properties.

Serum, fecal, and breast milk rotavirus antibodies as indices of infection in mother-infant pairs.

Sixty-eight mother-infant pairs were followed for 12-17 months after birth. Rotavirus infections in children were detected by EIA of weekly fecal antigen and anti-rotavirus IgA levels, by EIA of anti-rotavirus IgG in sera at birth, 6, or 12-17 months of age, and by anti-rotavirus EIA IgA and neutralizing antibody (NA) in monthly samples of maternal breast milk. Primary rotavirus infection was detected in 26 children (in 15 [58%] by fecal excretion, 12 [46%] by IgG seroconversion, and 22 [85%] by elevations of IgA anti-rotavirus antibodies [IgA coproconversion] in consecutive fecal specimens). Rotavirus "challenge" was detected by rises in levels of NA in breast milk in 9 (47%) of 19 mothers, including 5 (26%) from pairs in which there was no other evidence of rotavirus infection. Reinfections were detected in 2 children by rotavirus excretion and in 4 by coproconversion. IgA coproconversion is the most sensitive technique for detection of symptomatic and asymptomatic rotavirus infection in young children.

Multiple-gene rotavirus reassortants responsible for an outbreak of gastroenteritis in central and northern Australia.

Two rotavirus strains, E210 and E212, implicated in epidemics of gastroenteritis in children in central and northern Australia during 1993-1994, exhibited the unusual combination of a 'short' RNA electrophoretic pattern and subgroup II specificity. The outer capsid protein VP7 was found by PCR typing and sequence analysis to be related to that of serotype G2 viruses. Both strains displayed a novel pattern of reactivity to G2-specific monoclonal antibodies that correlated with sequence variation in the antigenic regions of VP7. The VP4 serotype of E210 and E212 was determined as P1B in an enzyme immunoassay, consistent with other G2 viruses. Analysis of the VP6 gene indicated significant identity (98-99%) with other human subgroup II viruses. Northern hybridization analysis of E210 RNA using total genome probes derived from the prototype strains RV4 and RV5 indicated that E210 was derived from multiple gene reassortment between rotaviruses belonging to different genetic types.

Amino acids involved in distinguishing between monotypes of rotavirus G serotypes 2 and 4.

Neutralizing monoclonal antibodies (N-MAbs) to serotype G2 and G4 rotaviruses were used to study intraserotypic variation by selection and characterization of N-MAb-resistant antigenic variants and reaction of N-MAbs with prototype rotavirus strains. Two G2-specific N-MAbs reacted with G2 rotaviruses S2, DS-1, RV-5 and RV-6 but not with 1076. Sequence analysis of the gene encoding VP7 of 1076 virus showed that the differences in amino acid sequence between 1076 virus and the other G2 strains at position 147, 213 and 217 correlated with the loss of N-MAb reactivity. Rotavirus variant mutation mapping data suggested that the amino acid difference at position 213 was likely to be of greatest importance. Rotavirus 1076 was defined as monotype b within G2 strains, whereas S2, DS-1, RV-5 and RV-6 belong to monotype a. The molecular basis for G4 subtypes/monotypes was also studied. The monotype G4b N-MAb 3A3 selected an antigenic variant with an amino acid mutation at position 96, whereas variants of the G4a-reactive N-MAb ST-3:1 showed a mutation at position 94, which produced a new, utilized glycosylation site. Neutralization by N-MAb ST-3:1 was also affected by amino acid changes at position 96. Reactions with these N-MAbs show that serotype G2 viruses can be divided into monotypes and confirm the observation that serotype G4 rotaviruses can be subdivided into subtypes/monotypes a and b. The G2 monotypes relate to differences at particular amino acids within antigenic region C and possibly region B, whereas antigenic region A is most important for G4 monotype differentiation.

Rotavirus antigenicity is affected by the genetic context and glycosylation of VP7.

Rotavirus variants resistant to neutralization were selected using monoclonal antibodies (N-MAbs) raised to VP7 of rotavirus G types 2, 3, and 6. Their neutralization resistance patterns and deduced VP7 amino acid sequences were obtained. Variants selected by two G2-specific N-MAbs from the homologous parent virus RV-5 showed single amino acid (aa) mutations in the antigenic A region. However, variants selected from reassortant virus RV-5 x SA11 (all genes from SA11 virus except that encoding VP7, which was from RV-5 virus) fell into two neutralization resistance groups. The first group showed identical mutations to the variants selected from RV-5 virus. The second group showed antigenic C region mutations, either alone or in combination with a mutation at aa 69. Variants selected from G3 parent viruses glycosylated at position 238 had a mutation at aa 96 in the A region, otherwise a C-region mutation at 211 was selected. Mutations at amino acid positions 94 or 96 were selected by monoclonal antibodies specific for each of the three serotypes. G3-specific monoclonal antibodies also selected mutations at position 148 and the new position of 264. This latter mutation resulted in substitution of aspartic acid for glycine and was located in a highly conserved and hydrophobic region of VP7. A G2-specific N-MAb selected variants with a mutation at aa 190 producing a new, utilized glycosylation site which we propose to be in new antigenic site E. The positions of mutations in antigenic variants and their antigenicity were determined by parental background genes and VP7 glycosylation.

Rotavirus serotypes causing severe acute diarrhea in young children in six Australian cities, 1989 to 1992.

Rotavirus VP7 serotypes were identified in stools from 72.9% (1,302/1,784) of hospitalized Australian children in six cities (1989 to 1992) and comprised 1,088 (83.6%) serotype G1 isolates, 84 (6.4%) serotype G2 isolates, 64 (4.9%) serotype G3 isolates, 49 (3.8%) serotype G4 isolates, and 17 (1.3%) isolates of mixed serotypes. The most densely populated cities yielded the greatest diversity of serotypes.

Characteristics and location of cross-reactive and serotype-specific neutralization sites on VP7 of human G type 9 rotaviruses.

The neutralization antigens of human rotavirus VP7 were studied by producing eight neutralizing monoclonal antibodies to G type 9 rotaviruses F45 and WI61 and selecting antigenic variants resistant to neutralization by these monoclonal antibodies. Neutralization resistance patterns and sequence analysis of the antigenic variants indicated the presence of overlapping serotype-specific and serotype cross-reactive epitopes in antigenic region A, and one distinct type-specific epitope. Cross-reactive monoclonal antibodies were more tolerant of amino acid sequence change than type-specific monoclonal antibodies. The existence of a new antigenic region, F, including amino acids 235 to 242 was confirmed. This region contained a cross-reactive epitope not detectable in the presence of glycosylation at amino acid 238. This glycosylation also affected neutralization by a cross-reactive monoclonal antibody directed to antigenic region C. Antigenic regions A, B, C, and F all contain epitopes shared between G types, of which at least two (C and F) are affected by glycosylation.

Measurement of rotavirus-neutralizing coproantibody in children by fluorescent focus reduction assay.

A fluorescent focus reduction assay suitable for the measurement of rotavirus-neutralizing antibodies in the feces of children was developed. Of 408 stools tested, 7% showed false-positive neutralization, and the number of rotavirus serotypes neutralized by a fecal extract was proportional to the levels of antirotaviral immunoglobulin A in the extract.

Comparison of rotavirus immunoglobulin A coproconversion with other indices of rotavirus infection in a longitudinal study in childhood.

In order to determine the sensitivity and reliability of antirotaviral fecal immunoglobulin A (IgA) as an indicator of rotavirus reinfection, the antibody responses to rotavirus of 44 infants with severe rotavirus gastroenteritis recruited on admission to a hospital were studied. Feces were collected daily during hospitalization and weekly thereafter, and sera were obtained every 4 months, for 6 to 32 months (median, 17 months). Antirotaviral IgG, IgA, and IgM were measured by enzyme immunoassay in all samples. Rotavirus antigen, rotavirus-neutralizing antibody, and total IgA were measured in feces. The results showed that use of an IgA index (ratio of specific IgA to total IgA) was unnecessary to identify copro-IgA conversion to rotavirus. The other markers of rotavirus infection tested showed a high level of predictive accuracy of coproconversion in rotavirus-neutralizing antibody. Copro-IgM, serum IgM, and virus in feces were insensitive measures of neutralizing antibody coproconversion. Seroconversion in IgG or IgA was detected in 46% of neutralizing coproconversions. The most sensitive marker, present in 92% of neutralizing coproconversions, was antirotaviral fecal IgA conversion. This correlation of fecal IgA with fecal neutralizing antibody suggests that coproconversions in IgA represent true elevations in antirotaviral IgA with neutralizing capacity. A coproconversion in IgA appears to indicate genuine rotavirus infection. Copro-IgA conversions in feces collected weekly are likely to be more sensitive markers of rotavirus reinfection than are seroconversion and virus detection combined in epidemiological studies of acute diarrhea in children and in rotavirus vaccine trials.