Characterisation and phylogenetic analysis of the VP7 proteins of serotype G6 and G8 human rotaviruses.

Serotype G6 and G8 rotaviruses are rarely found in man and may have originated in animals. Human serotype G6 and G8 rotaviruses, isolated from hospitalised children at various locations in Australia, were characterised. Deduced amino acid sequences of the major neutralising antigen, V7, showed significant identity to the cognate proteins of prototype human and bovine G6 and G8 viruses, respectively, and the strains reacted with G6 and G8 serotype-specific neutralising monoclonal antibodies, respectively, in an enzyme immunoassay. The VP4 type was determined as P[14] for all strains tested. Phylogenetic analysis of these and other human and bovine VP7 sequences suggested that a single inter-species transmission event, possibly from cattle, may have led to the emergence of G6 viruses in man. In contrast, the exchange of genes between human and bovine G8 viruses may have occurred onmore than one occasion, or these genes may have originated in a different host.

Homotypic and heterotypic serum neutralizing antibody response to rotavirus proteins following natural primary infection and reinfection in children.

Worldwide trials of rotavirus vaccines are currently in progress, but the basis of cross-reactive immunity between rotavirus serotypes is yet to be elucidated. The involvement of the outer capsid proteins, VP7 and VP4, in the production of cross-reactive neutralizing antibody (N-Ab) is unclear, and may be important for the success of animal rotavirus-based candidate vaccines that lack a VP4 of human rotavirus origin. In this study, VP7- and VP4-specific N-Ab was assayed in sera from children experiencing primary (27 children) and/or secondary (14 children) rotavirus infections using human-animal reassortant strains. These reassortants contained genes encoding the major G- and P-types found in human infection, including G1, 2, 3, and 4; or P1A[8], 1B[4], and 2[6]. After primary infection, the N-Ab response to VP7 was generally serotype-specific, whereas the response to VP4 was heterotypic. After reinfection (with the same or different serotypes) there was a significant increase (P=0.0313) in the number of VP7 serotypes seroconverted against with no broadening of cross-reactivity to VP4. Increases in homotypic N-Ab titer, following both primary and secondary infection, were greater against VP7 than VP4, with the seroconversion against VP7 being significantly greater upon reinfection than following primary infection (P=0.0280). In summary, heterotypic N-Ab produced following primary infection appears to be primarily against VP4. However, upon reinfection, VP7 becomes increasingly immunodominant both in terms of cross-reactive N-Ab production and increases in N-Ab titer.

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.

Production of reassortant viruses containing human rotavirus VP4 and SA11 VP7 for measuring neutralizing antibody following natural infection.

The outer capsid proteins VP4 and VP7 of group A rotaviruses are both targets of neutralizing antibody produced following natural infection in humans. Of interest is the relative importance and immunodominance of each protein in the generation of a protective immune response. In order to measure neutralizing antibody responses to VP4 and VP7 separately, reassortants bearing VP4 of each of the major human rotavirus P types with VP7 of SA11 origin were successfully produced by neutralizing monoclonal antibody selection. The resulting reassortants, together with reassortants representing each of the major VP7 types, were antigenically characterized with serotype-specific neutralizing monoclonal antibodies and hyperimmune sera. The neutralization proteins of human rotavirus origin were found to be unaffected antigenically by reassortment. The abilities of these reassortants to discriminate between VP4 and VP7 immune responses were evaluated with postinfection sera collected from three patients infected with either a P1A[8],G1, a P1B[4],G2, or a P1A[8],G4 rotavirus strain. The reassortants were shown to be capable of separating the neutralizing antibody responses to VP4 and VP7, with each patient showing a different immune response with respect to VP4 or VP7 immunodominance. These reassortants can now be applied to analyses of individual immune responses to VP4 and VP7 proteins after primary rotavirus infections and reinfections in humans.

Use of non-radioactive probes for VP4 typing of human rotaviruses.

The rotavirus outer capsid proteins VP4 and VP7 determine the P- and G-serotypes, respectively, of the virus. Three types of VP4 protein are commonly found in human rotaviruses (P4, P6 and P8) which are encoded by distinct VP4 gene alleles. We developed a non-radioactive Northern hybridization method for the P-genotyping of rotavirus field isolates. Double-stranded RNA was isolated from faecal specimens of rotavirus positive patients. Digoxigenin (DIG)-labelled cDNA probes derived from the VP4 gene of the standard strains RV5 (P4), ST3 (P6) and RV4 (P8) were used to discriminate between the different alleles. Although the P4 probe exhibited cross-reactivity with some P8 samples, the P6 and P8 probes were found to be type-specific. In addition, the probes did not react with standard strains representative of other defined human and animal rotavirus P-types. Use of these probes on viral RNA of faecal origin allowed approximately 70% of samples to be assigned a P-type. This method complements PCR- and EIA-based P-typing methods, is relatively inexpensive and is readily applicable to large numbers of samples, thus proving useful for epidemiological studies.

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.