Search

Aim: We aimed to determine the functionality of rotavirus NSP3 dimers. Materials & methods: We expressed rhesus rotavirus NSP3 and determined the kinetics of host cell translation inhibition and the levels of accumulated dimerization intermediates and dimers. Results: We observed a linear kinetics of host cell translation inhibition, which correlated well with the sum of the dimerization intermediates and dimers. Treatment with 17-dimethylaminoethylamino-17-demethoxygeldanamycin reduced the accumulation of NSP3 dimers and potentiated host cell translation inhibition. Conclusion: Our results show that NSP3 dimer formation does not correlate with host cell translation inhibition and suggest that both NSP3 dimers and dimerization intermediates are functional and inhibit host cell translation.

El estudio de los mecanismos utilizados por diversos virus para controlar la traducción de la célula hospedera y favorecer la expresión de proteínas virales ha sido muy instructivo para conocer mecanismos celulares fundamentales que regulan la traducción. La proteína NSP3 de los rotavirus de la especie A (RVA) es capaz de inhibir la traducción de los RNAm celulares como consecuencia de su unión al factor de iniciación de la traducción eIF4G y de estimular la traducción de los RNAm virales como consecuencia de su unión a la secuencia UGACC de su extremo 3’. Se ha propuesto que NSP3 inhibe la traducción por interferir con la circularización de los extremos 5’-3’ de los RNAm celulares mediada por eIF4E-eIF4G-PABP (proteína de unión a poli-A), y simultáneamente se propone que NSP3 estimula la traducción de los RNAm del RVA circularizándolos de manera análoga a la que realiza PABP en los RNAm celulares. Sin embargo, la importancia de la dimerización de NSP3 asistida por la chaperona HSP90 en su función inhibitoria de la traducción celular se desconoce. Recientemente, se exploró la importancia de los intermediarios de la dimerización de NSP3 sobre su función inhibitoria de la traducción, y se encontró que mutaciones puntuales en la región coiled-coil afectan la formación de los dímeros y conservan parcialmente su función inhibitoria de la traducción celular. Además, se detectó que los dímeros de NSP3 se degradan con mayor rapidez que los intermediarios de la dimerización. Estos datos demuestran que la función de NSP3 se adquiere previamente a la aparición de los dímeros, y sugiere que la susceptibilidad al proteasoma de las distintas formas oligoméricas de NSP3 son relevantes en el establecimiento de la función inhibitoria de la traducción celular.

A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.

Electron microscopy is routinely used to identify viral infections in protozoan parasites. These viruses have been described as non-enveloped and icosahedral structures with a diameter of 30-60 nm. Most of them are classified within the non-segmented dsRNA Totiviridae family. We observed virus-like particles (VLPs) through transmission electron microscopy in the cytoplasm of Trypanosoma cruzi epimastigotes grown in cultures. Clusters of electrodense enveloped VLPs having a diameter of 48 nm were also observed. These clusters appear to have been released from distended Golgi cisternae. Furthermore, a paracrystalline array of electrodense, non-enveloped VLPs (with a diameter of 32 nm) were found in distended Golgi cisternae or as smaller clusters at a distance from the RE or Golgi. We cannot rule out that the 48 nm enveloped VLPs belong to the ssRNA Flaviviridae family because they are within its size range. The localization of enveloped VLPs is consistent with the replication strategy of these viruses that transit through the Golgi to be released at the cell surface. Due to the size and shape of the 32 nm non-enveloped VLPs, we propose that they belong to the dsRNA Totiviridae family. This is the first description of cytoplasmic enveloped and non-enveloped VLPs in T. cruzi epimastigotes.

Overproduction of pro-inflammatory cytokines and chemokines is frequently associated with severe clinical manifestations in patients infected with influenza A/H1N1 virus. Micro-RNAs (miRNAs) are highly conserved small non-coding RNA molecules that post-transcriptionally regulate gene expression and are potential biomarkers and therapeutic targets in different inflammatory conditions.We studied the circulating and miRNA profiles in critically ill A/H1N1 patients, A/H1N1 patients with milder disease, asymptomatic housemates and healthy controls. Cytokine, chemokine and growth factors that were potential targets of differentially expressed miRNAs were assessed. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and interactome analysis of these miRNAs were also performed.Critically ill patients exhibited a significant over-expression of circulating miR-150 (p0.005) when compared to patients with milder disease. miR-29c, miR-145 and miR-22 were differentially expressed in patients with severe A/H1N1 disease whereas miR-210, miR-126 and miR-222 were downregulated in individuals exposed to the A/H1N1 virus. Significant correlations (p0.05) between circulating levels of miR-150 with IL-1ra, IL-2, IL-6, CXCL8, IFN-γ, CXCL10 and G-CSF were detected, particularly in critically ill patients.The up-regulation of miR-150 is associated with poorer outcomes of A/H1N1 infection. The differential expression of miRNAs related with immune processes in severe A/H1N1 disease supports the potential role of these miRNAs as biomarkers of disease progression.

Chronic infections by hepatitis C virus (HCV) are a major cause of cirrhosis and hepatic cancer. The replication of HCV involves translation and proteolytic processing of polyproteins. The HCV single-stranded RNA encodes a single polyprotein of C/E1/E2/p7/NS2/NS3/NS4A/NS4B/NS5A/NS5B. The structural proteins, C, E1, E2, and p7, arise from the viral polyprotein by host proteases. Cleavage at the non-structural NS2/NS3 junction is performed by the NS2 protease. NS3 forms a complex with NS4A to cleave the rest of the viral polyprotein. The central 12-amino-acid sequence of NS4A, 21-GSVVIVGRIILS-32 (NS4Awt) is a determinant to enhance the NS3 protease activity at the NS5A/5B junction. We found that, from 13 blood donors infected with HCV, one sample showed five amino acid changes in the NS4A central region at V23I, I25C, I30S, L31T, and S32L, and another sample showed three changes at V23I, I25C, and I30V in this region. The other 11 samples showed the NS4Awt sequence. The effect of such amino acid variations on the NS3 proteolytic activity was evaluated in vitro using the central 12-amino-acid NS4Awt sequence with specific changes joined to NS3, and NS5A/5B as a substrate. Our results indicate that the amino acid changes of NS4A at V23I and I25C do not enhance the protease activity of NS3, whereas the amino acid changes at I30S, L31T, and S32L, as well as the NS4Awt sequence, enhance NS3 activity. Our results confirm that protease cofactor, encoded in NS4A, is of major regulatory relevance for the replication cycles of HCV.

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.

Rotavirus replication and virus assembly take place in electrodense spherical structures known as viroplasms whose main components are the viral proteins NSP2 and NSP5. The viroplasms are produced since early times after infection and seem to grow by stepwise addition of viral proteins and by fusion, however, the mechanism of viropIasms formation is unknown. In this study we found that the viroplasms surface colocalized with microtubules, and seem to be caged by a microtubule network. Moreover inhibition of microtubule assembly with nocodazole interfered with viroplasms growth in rotavirus infected cells. We searched for a physical link between viroplasms and microtubules by co-immunoprecipitation assays, and we found that the proteins NSP2 and NSP5 were co-immunoprecipitated with anti-tubulin in rotavirus infected cells and also when they were transiently co-expressed or individually expressed. These results indicate that a functional microtubule network is needed for viroplasm growth presumably due to the association of viroplasms with microtubules via NSP2 and NSP5.

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.

Se estableció la técnica de transcripción reversa-reacción en cadena de la polimerasa (RT-PCR) para la detección del virus de influenza aviar en tejidos de aves infectadas experimentalmente, incluidos en parafina, lo cual se correlacionó con las lesiones histológicas. Se inocularon por vía intravenosa ocho equilotes de diez pollos Arbor Acres de cinco semanas de edad, con 101 a 108 dosis letales de pollo del 50% (DLP50) del virus de influenza aviar A/Ck/Querétaro/20/95 (H5N2). Todas las aves se sometieron a evaluación histopatológica. Se ensayaron por RT-PCR tres tipos de tejidos incluidos en parafina (un ave por dosis): pulmón, encéfalo y riñón. Las principales lesiones histológicas observadas en las aves infectadas incluyeron necrosis del tejido linfoide (en pulmón, bazo, bolsa de Fabricio y tonsilas cecales), vasculitis, necrosis tubular aguda, encefalitis no supurativa y necrosis multifocal en los órganos examinados. Las principales diferencias con otras cepas altamente patogénicas ?peste de las aves (H7N7), Ck/Scot/59 (H5N1), Tern/SA (H5N3), Ty/Ont/66 (H5N9) y Ck/Penn/83 (H5N2)? fueron: mayor frecuencia de daño vascular, nefropatogenicidad, necrosis en islotes pancreáticos y necrosis difusa del tejido linfoide. Estos hallazgos le confieren a la cepa bajo estudio características propias distintivas. Se detectó virus de influenza en pulmón con la técnica de RT-PCR, independientemente de la dosis, y en encéfalo y riñón en siete de las ocho dosis empleadas, lo cual indica que el virus se replica en estos tejidos; los cambios patológicos observados pueden atribuirse a la replicación viral, sin excluir la participación de otros mecanismos de daño celular.

This report is of a community-based case control study to assess whether the severity of acute diarrhea by rotavirus (RV) in young children is associated with a particular VP7 (G) or VP4 (P) RV serotype. Five hundred twenty children younger than 2 years of age with diarrhea lasting less than 3 days were age and gender matched with 520 children with no diarrhea. The G and P serotypes were determined with specific monoclonal antibodies, and the VP4 serotype specificity in a subgroup was confirmed by genotyping. Infection with a G3 serotype led to a higher risk of diarrhea than infection with a G1 serotype. Infection with a G3-nontypeable-P serotype was associated with more severe gastroenteritis than infection with a G3 (or G1) P1A[8] serotype. A child with diarrhea-associated dehydration was almost five times more likely to be infected with a G3-nontypeable-P serotype than a child without dehydration ( P < 0.001). Moreover, the two predominant monotypes within serotype P1A[8] had significantly different clinical manifestations. In this study, the severity of RV-associated diarrhea was related to different P serotypes rather than to G serotypes. The relationship between serotype and clinical outcomes seems to be complex and to vary among different geographic areas.

A recombinant vaccinia virus encoding rotavirus protein NSP3 driven by an internal ribosome entry site (IRES) from the encephalomyocarditis (EMC) virus was able to abate protein synthesis in BSC1 cells by 25-fold, with as much as 30% of the remaining protein synthesis being NSP3. Hence NSP3 shuts off host cell protein synthesis down to the level seen during rotavirus infection but is unable to prevent translation from EMC IRES-driven genes. This effect was abolished by deletions in the eIF4G-binding (aa 274–313) and the dimerization (aa 150–206) but not the viral mRNA-binding (aa 83–149) domains, supporting that NSP3 functions in vivo as a dimer. Binding of eIF4G by NSP3 has been implicated in interfering with mRNA 5′-3′ circularization, hence such circularization is essential for translation in mammalian cells.

To compare the severity of rotavirus diarrhea (RV) and non-rotavirus diarrhea.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.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; temperature38 degrees C; dehydration; and clinical severity scoring.These results showed a poorer prognosis and a higher severity of rotavirus diarrhea, as compared to non-rotavirus diarrhea in infants.

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°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. The English version of this paper is available at: http://www.insp.mx/salud/ index.html

Group A rotaviruses are currently recognizedas the most important cause of acute gastroenteri-tis among both infants and young children world-wide, accounting for about one million deaths peryear in developing countries (I DeZoysa et al. 1985Bull WHO 63: 569-583, NR Blacklow & HBGreenberg 1991 N Engl J Med 325: 252-264).Rotaviruses are members of the familyReoviridae and their genome consists of 11 dis-crete segments of double-stranded (ds) RNA sur-rounded by a triple-layered shell virion (MK Estes& J Cohen 1989 Microbiol Rev 53 : 410-449). Theusual RNA electrophoretic profile of group Arotaviruses displays four different size classes con-taining RNA segments 1 to 4, 5 and 6, 7 to 9 and10 and 11. Those rotavirus strains with genomicpatterns not matching this basic configuration maybe regarded as either non-group A rotaviruses orgroup A rotaviruses with rearranged genome (EMendez et al. 1992 Arch Virol 125: 331-338). Inthis context, avian-like rotavirus strains have beenrecognized which display five segments in the firstdsRNA class (H Brussow et al. 1992 J ClinMicrobiol 30: 67-73, RHP Gusmao et al. 1994 JDiarrhoeal Dis Res 12: 129-132).Each of the 11 RNA segments codes for a singlepolypeptide, with the exception of gene 11 whichcodes for two polypeptides. Three of the virusstructural proteins (VP4, gene 4; VP6, gene 6; andVP7, genes 7, 8 or 9) induce antibodies that areused for classification of rotaviruses on serologi-cal basis (Estes & Cohen loc. cit.). VP6 forms themiddle shell of the virus and induces cross-reac-tive group- and subgroup-specific, non-neutraliz-ing antibodies. On the other hand, the two surfaceproteins VP7 and VP4 independently induce type-specific neutralizing antibodies that form the basisfor the current classification of group A rotavirusesinto G (VP7) and P (VP4) serotypes (Estes C [P8], G3;[P8], G4; and P[4], G2 (JR Gentsch et al. 1992 JClin Microbiol 30 : 1365-1373, A Steele et al. 1993J Clin Microbiol 31: 1735-1740). Rotavirus strainscharacterized as being [P8], G1and [P4], G2 seemto be the most common worldwide (BK Das et al.1994 J Clin Microbiol 32 : 1820-1822, NBG Rasoolet al. 1993 Arch Virol 133: 275-282).Although limited, the available data in Brazilconcerning the occurrence of rotavirus G and Pgenotypes indicate that the predominant strains areessentially the same as those most commonly foundworldwide. Thus, [P8], G1 and [P4], G2 accountfor more than 50% of isolates (MCS Timenetskyet al. 1994 J Clin Microbiol 32: 2622-2624, VGouvea et al. 1994 J Clin Microbiol 32 : 1408-1409,JPG Leite et al. 1996 Arch Virol 141 : 2353-2364).In these studies, the detection of mixed infectionsis an indicator that in vivo reassortments may oc-cur, as previously reported in northern Brazil (JDPMascarenhas et al. 1988 Virus Res 14: 235-240).Of interest, these investigators have shown thatrotavirus genotype [P8], G5 accounts for a signifi

H5N2 strains of low-pathogenicity avian influenza virus (LPAIV) have been circulating for at least 17 years in some Mexican chicken farms. We measured the rate and duration of viral excretion from Pekin ducks that were experimentally inoculated with an H5N2 LPAIV that causes death in embryonated chicken eggs (A/chicken/Mexico/2007). Leghorn chickens were used as susceptible host controls. The degree of viral excretion was evaluated with real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) using samples from oropharyngeal and cloacal swabs. We observed prolonged excretion from both species of birds lasting for at least 21 days. Prolonged excretion of LPAIV A/chicken/ Mexico/2007 is atypical.

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.

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.

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.

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.

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.

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.

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.

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.