Different Isoforms of HPV-16 E7 Protein are Present in Cytoplasm and Nucleus.

The E7 protein of high risk HPV types has been found with different molecular weights, mainly because of phosphorylation, an event that changes protein charge and mobility in SDS-PAGE. Distribution of E7 protein in the cellular compartments has also been subject of debate as some groups report the protein in nucleus and others in cytoplasm. The different subcellular distribution and molecular weights reported for the E7 protein suggest the presence of isoforms. We examined this possibility by using several antibodies that recognize different epitopes on the HPV-16 E7 protein. We showed that E7 is processed in 3 isoforms with different molecular weights and isoelectric points (IEP), and described as E7a1 (17.5 kDa, IEP 4.68), E7a (17 kDa, IEP 6.18) and E7b (16 kDa, IEP 6.96). The immunofluorescense results also showed that E7 is distributed into different compartments (ER, Golgi and nucleus), which suggest the presence of other posttranslational modifications, besides phosphorylation.

Post-translational regulation of rotavirus protein NSP1 expression in mammalian cells.

The nonstructural rotavirus protein NSP1 binds specifically to viral mRNAs and to interferon regulatory factor 3 (IRF3), inducing IRF3 degradation through a proteasome-dependent pathway. By using a vaccinia virus expression system in mammalian cells, we found that the yield of NSP1 was 8- and 13-fold lower than the viral proteins VP2 or NSP3, respectively; while in the presence of proteasome inhibitors such difference could be reduced to 2- to 2.5-fold, respectively. The susceptibility of NSP1 to proteasome degradation was fully reversed in a dose-dependent manner by transfection with the full complement of 11 molecules of translation-competent rotavirus mRNAs, but this effect was abrogated by the protein synthesis inhibitor cycloheximide. These results demonstrate that NSP1 is degraded through a proteasome-dependent pathway, and viral proteins, alone or in combination with viral mRNAs, interfere with such degradation.

[Prognosis of rotavirus diarrhea]. / Pronóstico de la diarrea por rotavirus.

OBJECTIVE: To compare the severity of rotavirus diarrhea (RV) and non-rotavirus diarrhea. MATERIAL AND METHODS: Between October 1994 and March 1995, a cross-sectional study was performed in 520 infants with acute diarrhea, at seven primary care level centers in five states of Mexico. Diagnosis of RV was done through immunoenzymatic assay or electrophoresis. Central tendency measures were used for data analysis. Results were presented as means and standard deviations, or median and variation. RESULTS: RV was isolated from 264 children; most of them were males aged 6 months to 1 year. Differences in clinical manifestations were statistically significant between the rotavirus-positive group and the rotavirus-negative group, in the following variables: median number of stools/24 hours; frequency of vomiting; temperature > 38 degrees C; dehydration; and clinical severity scoring. CONCLUSIONS: These results showed a poorer prognosis and a higher severity of rotavirus diarrhea, as compared to non-rotavirus diarrhea in infants.

Antigenic and genomic diversity of human rotavirus VP4 in two consecutive epidemic seasons in Mexico.

In the present investigation we characterized the antigenic diversity of the VP4 and VP7 proteins in 309 and 261 human rotavirus strains isolated during two consecutive epidemic seasons, respectively, in three different regions of Mexico. G3 was found to be the prevalent VP7 serotype during the first year, being superseded by serotype G1 strains during the second season. To antigenically characterize the VP4 protein of the strains isolated, we used five neutralizing monoclonal antibodies (MAbs) which showed specificity for VP4 serotypes P1A, P1B, and P2 in earlier studies. Eight different patterns of reactivity with these MAbs were found, and the prevalence of three of these patterns varied from one season to the next. The P genotype of a subset of 52 samples was determined by PCR. Among the strains characterized as genotype P[4] and P[8] there were three and five different VP4 MAb reactivity patterns, respectively, indicating that the diversity of neutralization epitopes in VP4 is greater than that previously appreciated by the genomic typing methods.

Serotype specificity of the neutralizing-antibody response induced by the individual surface proteins of rotavirus in natural infections of young children.

The relative contribution of the rotavirus surface proteins, VP4 and VP7, to the induction of homotypic as well as heterotypic neutralizing antibodies (NtAbs) in natural infections was studied. The NtAb titers of paired sera from 70 infants with serologically defined primary rotavirus infections were determined with a panel of rotavirus reassortants having one surface protein from a human rotavirus (serotypes G1 to G4 for VP7 and P1A and P1B for VP4) and the other surface protein from a heterologous animal rotavirus strain. A subset of 37 children were evaluated for epitope-specific antibodies to the two proteins by an epitope-blocking assay. The infants were found to seroconvert more frequently to VP4 than to VP7 by both methods, although the titers of the seroconverters were higher to VP7 than to VP4. Both proteins induced homotypic as well as heterotypic NtAbs. G1 VP7 frequently induced a response to both G1 and G3 VP7s, while G3 VP7 and P1A VP4 induced mostly homotypic responses.

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.

Identification of two independent neutralization domains on the VP4 trypsin cleavage products VP5* and VP8* of human rotavirus ST3.

The antigenic structure of the VP4 protein of human rotavirus (HRV) strains Wa and ST3 was studied by using a panel of Wa- and ST3-derived VP4-specific neutralizing monoclonal antibodies (NMAbs) and NMAb-resistant variants. The VP4-coding genes from three Wa and three ST3 variants were sequenced. For Wa VP4, one homotypic and one heterotypic neutralization site, at amino acids 458 and 392, respectively, were identified. For ST3 VP4, three neutralization sites were found at amino acids 72, 217, and 385 that are either homotypic or associated with limited cross-reactivity. Cross-neutralization assays using several pairs of NMAbs and resistant variants showed that Wa VP4 has at least one large neutralization domain on its larger trypsin cleavage product, VP5*, consisting of several operationally related epitopes. VP4 of ST3 has at least two neutralization domains, one located on VP5* that is operationally related to the large neutralization domains on VP5* from HRVs Wa and KU, as well as an independent neutralization domain on VP8*, the smaller trypsin cleavage product of VP4.

[Correlation between serotype and electrophoretype of rotaviruses isolated in 2 Mexican populations]. / Correlación entre serotipo y electroferotipo de rotavirus aislados en dos poblaciones de México.

In order to study the correlation between the serotype specificity and the genomic RNA electrophoretic pattern (electropherotype) of human rotavirus (HRV) strains, we analyzed the electropherotypes of 54 HRV that had been collected during a four year study in Mexico, and whose serotypes had been previously determined. We detected 17 different electropherotypes, four in association with serotype G1, two with serotype G2, six with serotype G3, and five with serotype G4. There were no viruses with the same electropherotype having a different serotype. The variations in RNA electrophoretic migration were greater between viruses belonging to different serotypes than between viruses of the same serotype. It is of note that the relative separation of RNA segments 7 and 9 remained constant among viruses of the same serotype. Electropherotyping might have a serotype predictive value for rotavirus specimens lacking the virion outer capsid.

Serologic analysis of human rotavirus serotypes P1A and P2 by using monoclonal antibodies.

Three human rotavirus (HRV) VP4 serotypes and one subtype have been described on the basis of a fourfold or an eightfold-or-greater difference in neutralization titer when tested with hyperimmune antisera to recombinant VP4 or VP8* (serotypes P1A, P1B, P2, and P3). To start to analyze the antigenic basis underlying serotype specificity, we produced a library of 13 VP4-specific neutralizing monoclonal antibodies (NMAbs) to two HRVs, the serotype P1A strain Wa and the serotype P2 strain ST3, and characterized the reactivity of these NMAbs with a panel of serotypically diverse HRV strains by neutralization assay and enzyme-linked immunosorbent assay (ELISA). We characterized the serotypic specificity of the NMAbs by using a fourfold or an eightfold-or-greater difference in titer against the homologous (i.e., immunogen) and heterologous strains as a criterion for serotype. Some ST3-derived NMAbs reacted specifically with serotype P2 HRVs by ELISA and/or neutralization assay, while some Wa-derived NMAbs reacted specifically by ELISA and/or neutralization assay with some or all serotype P1A HRVs. Other Wa- and ST3-derived NMAbs reacted with some or all serotype P1A and P2 HRV strains by neutralization assay and ELISA. Most NMAbs did not react with serotype P1B or P3 strains. In previous studies, three distinct operationally defined epitopes have been identified on VP4 by examining the reactivity patterns of selected antigenic variants of HRV strain KU. At least one of the NMAbs described here recognizes an epitope unrelated to these previously identified epitopes, since it neutralized both KU and its variants.

Humoral immune responses to VP4 and its cleavage products VP5* and VP8* in infants vaccinated with rhesus rotavirus.

The humoral immune response to rhesus rotavirus (RRV) VP4 and its cleavage products VP5* and VP8* was determined in paired serum samples from 44 infants vaccinated with RRV or human rotavirus-RRV reassortants and 5 placebo recipients. Our aim was to try to measure the response to those regions of VP4 most closely related to protection. An enzyme-linked immunosorbent assay (ELISA) was used to measure the immunoglobulin G immune response to baculovirus-expressed full-length RRV VP4, full-length VP8*, and the amino-terminal polypeptide of VP5* called VP5*(1) (amino acids 248 to 474). The two antigenic regions of VP4 selected for study, VP5*(1) and VP8*, have previously been shown to contain most of the cross-reactive and strain-specific neutralization epitopes, respectively, while the remaining carboxy-terminal half of VP5* (amino acids 475 to 776) has not been clearly associated with neutralization. All three recombinant proteins were antigenically conserved, since they reacted with a library of neutralizing monoclonal antibodies directed at VP4. There was a high percentage of seroresponders to VP4 (61%) or to VP8* (52%), but fewer infants seroresponded to VP5*(1) (11%). In addition, infants responding to VP5*(1) had considerably lower titers than to VP4 or VP8*. Immune response to VP4 correlated strongly with the responses detected by the plaque reduction neutralization assay but did not correlate with the responses detected by the ELISA to whole RRV. These data imply that the VP5*(1) region is less immunogenic than the VP8* region of VP4 in infants immunized with RRV or RRV reassortants. The low immunogenicity of VP5* might adversely affect the efficacy of RRV vaccine candidates.

Diversity of rotavirus serotypes in Mexican infants with gastroenteritis.

One hundred thirty-two stool specimens from infants with rotavirus gastroenteritis hospitalized in two Mexican cities (Mexico City and Mérida) were examined by serotype- and subgroup-specific enzyme immunoassays. Among them, 38 (29%) were serotype 1, 15 (11%) were serotype 2, 13 (10%) were serotype 3, 22 (17%) were serotype 4, none was serotype 5 or 6, and 44 (33%) could not be serotyped. By subgrouping, 121 specimens were characterized as follows: 24 (18%) were subgroup 1, 97 (74%) were subgroup 2, and none had both subgroup specificities. While serotype 1 rotavirus predominated in the Mexico City area for 4 consecutive years (1984 to 1987), serotype 4 predominated in Mérida during the single epidemic season studied (1985). These data demonstrate that all four primary human rotavirus serotypes circulated in Mexico, with serotype 1 being the most prevalent. The seroneutralization responses of 14 of the 22 patients infected with serotype 4 strains had been previously studied. Of these 14 infants, 11 appeared to have primary infections, as indicated by absence of neutralizing antibodies in the acute-phase sera and their young age (8 months on average) at the time of illness. Seven patients seroresponded to serotypes 1 and 4; two seroresponded to serotypes 1, 3, and 4; three seroresponded to serotype 1; and two had low-level seroresponses to serotype 3 or 4. These data indicate that heterotypic neutralizing antibody responses occur frequently following infection with serotype 4 rotaviruses.

Lack of cosegregation of the subgroup II antigens on genes 2 and 6 in porcine rotaviruses.

The rotavirus subgroup I and II specificities associated with gene 2 and 6 products (vp2 and vp6, respectively) were shown not to cosegregate in a number of porcine rotavirus strains. The porcine OSU rotavirus strain and OSU-vp7-like strains were all found to possess a subgroup II-specific region on vp2 and a subgroup I-specific region on vp6. Of interest is the observation that the subgroup II-specific epitope on vp2 appears to be present only in human and porcine rotavirus strains, suggesting a possible human-pig ancestral lineage for gene 2.

Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to the heterotypic neutralization domain of VP7 and the VP8 fragment of VP4.

A murine model was used to determine whether neutralizing monoclonal antibodies (MAbs) with heterotypic specificity directed to VP7 (MAb 57-8) or to the VP8 fragment of VP4 (MAb M14) passively protect mice against challenge with various strains of rotavirus. (The gene 4 product, an outer capsid protein, has traditionally been called VP3. It has been proposed, however, that the rotavirus gene 4 product be named VP4. The gene 3 product, a core protein, has been identified recently and named VP3 [M. Liu, P. A. Offit, and M. K. Estes, Virology 163:28-32, 1988]). Suckling mice orally inoculated with MAb 57-8 did not develop diarrhea when challenged with virulent serotype 3, 4, or 6 rotaviruses, while those inoculated with MAb M14 were passively protected from challenge with serotype 3 or 6 rotaviruses, as predicted by in vitro neutralization tests. These MAbs, however, did not protect mice from infection when the mice were challenged with rotaviruses of other serotypes. We conclude that specific neutralization epitopes on each surface protein are capable of mediating protection against one or several rotavirus serotypes.

Prevalent patterns of serotype-specific seroconversion in Mexican children infected with rotavirus.

The level of neutralizing antibodies to rotaviruses belonging to serotypes 1, 3, and 4 was determined in acute- and convalescent-phase sera from 36 Mexican children with rotaviral diarrhea. Most of the infants who seroconverted fell into one of the following three patterns: single seroconversion to serotype 1; seroconversion to serotypes 1 and 4; or seroconversion to all three serotypes tested. The heterotypic neutralizing antibody responses to rotavirus infections are discussed.