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C F Arias

Publications and source records attributed to C F Arias.

At least 37 records · Page 2Linked to original sources
PubMed1998

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 G3 was found to be the prevalent VP7 serotype during the first year, being superseded by serotype G1 strains during the second season.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.

Antibodies, Monoclonal↗
PubMed1997

Identification of a T-helper cell epitope on the rotavirus VP6 protein.

In this work, we have studied the T-helper (Th)-cell response against rotavirus, in a mouse model. Adult BALB/c mice were inoculated parenterally with porcine rotavirus YM, and the Th-cell response from spleen cells against the virus and two overlapping fragments of the major capsid protein VP6 (VP6(1-192) and VP6(171-397)) were evaluated in vitro. The Th cells recognized the YM virus and the two protein fragments, suggesting that there are at least two Th-cell epitopes on the VP6 molecule. To study the specificity of Th cells against VP6 at the clonal level, we established two Th-cell hybridomas cross-reactive for the VP6 protein of rotavirus strains YM and SA11. Both hybridomas recognized the VP6(171-397) polypeptide, and a synthetic peptide comprising the amino acids 289 to 302 (RLSFQLVRPPNMTP) of YM VP6 in the context of the major histocompatibility complex class II IEd molecule. The Th-cell hybridomas recognized rotavirus VP6 in a highly cross-reactive fashion, since they could be stimulated by eight different strains of rotavirus, including the murine rotavirus EDIM, that represent five G serotypes and at least two subgroups. The amino acid sequence of the VP6 epitope is highly conserved in most group A rotavirus strains sequenced so far. On the other hand, it was found that Th cells specific for the VP6 epitope may constitute an important proportion of the total polyclonal Th-cell response against rotavirus YM in spleen cells. These results demonstrate that VP6 can be a target for highly cross-reactive Th cells.

Animals↗
PubMed1997

Rotaviruses induce an early membrane permeabilization of MA104 cells and do not require a low intracellular Ca2+ concentration to initiate their replication cycle.

In this work, we found that rotavirus infection induces an early membrane permeabilization of MA104 cells and promotes the coentry of toxins, such as alpha-sarcin, into the cell. This cell permeability was shown to depend on infectious virus and was also shown to be virus dose dependent, with 10 infectious particles per cell being sufficient to achieve maximum permeability; transient, lasting no more than 15 min after virus entry and probably occurring concomitantly with virus penetration; and specific, since cells that are poorly permissive for rotavirus were not permeabilized. The rotavirus-mediated coentry of toxins was not blocked by the endocytosis inhibitors dansylcadaverine and cytochalasin D or by the vacuolar proton-ATPase inhibitor bafilomycin A1, suggesting that neither endocytocis nor an intraendosomal acidic pH or a proton gradient is required for permeabilization of the cells. Compounds that raise the intracellular concentration of calcium ([Ca2+]i) by different mechanisms, such as the calcium ionophores A23187 and ionomycin and the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, did not block the coentry of alpha-sarcin or affect the onset of viral protein synthesis, suggesting that a low [Ca2+]i is not essential for the initial steps of the virus life cycle. Since the entry of alpha-sarcin correlates with virus penetration in all parameters tested, the assay for permeabilization to toxins might be a useful tool for studying and characterizing the route of entry and the mechanism used by rotaviruses to traverse the cell membrane and initiate a productive replication cycle.

Abrin↗
PubMed1997

Functional and structural analysis of the sialic acid-binding domain of rotaviruses.

The infectivity of most animal rotaviruses is dependent on the interaction of the virus spike protein VP4 with a sialic acid (SA)-containing cell receptor, and the SA-binding domain of this protein has been mapped between amino acids 93 and 208 of its trypsin cleavage fragment VP8. To identify which residues in this region are essential for the SA-binding activity, we performed alanine mutagenesis of the rotavirus RRV VP8 expressed in bacteria as a fusion polypeptide with glutathione S-transferase. Tyrosines were primarily targeted since tyrosine has been involved in the interaction of other viral hemagglutinins with SA. Of the 15 substitutions carried out, 10 abolished the SA-dependent hemagglutination activity of the protein, as well as its ability to bind to glycophorin A in a solid-phase assay. However, only alanine substitutions for tyrosines 155 and 188 and for serine 190 did not affect the overall conformation of the protein, as judged by their interaction with a panel of conformationally sensitive neutralizing VP8 monoclonal antibodies (MAbs). These findings suggest that these three amino acids play an essential role in the SA-binding activity of the protein, presumably by interacting directly with the SA molecule. The predicted secondary structure of VP8 suggests that it is organized as 11 beta-strands separated by loops; in this model, Tyr-155 maps to loop 7 while Tyr-188 and Ser-190 map to loop 9. The close proximity of these two loops is also supported by previous results from competition experiments with neutralizing MAbs directed at RRV VP8.

Alanine↗
PubMed1997

[Workshop on Molecular Epidemiology of Viral Diseases].

A workshop on viral epidemiology was held on September 29, 1995 at the Medical School of the Universidad Nacional Autónoma de Mexico. The aim of this workshop was to promote interaction among scientists working in viral epidemiology. Eighteen scientists from ten institutions presented their experiences and work. General aspects of the epidemiology of meaningful viral diseases in the country were discussed, and lectures presented on the rota, polio, respiratory syncytial, dengue, papiloma, rabies, VIH and hepatitis viruses.

Adult↗
PubMed1996

Interactions between the two surface proteins of rotavirus may alter the receptor-binding specificity of the virus.

The infection of target cells by most animal rotavirus strains requires the presence of sialic acids (SAs) on the cell surface. We recently isolated variants from simian rotavirus RRV whose infectivity is no longer dependent on SAs and showed that the mutant phenotype segregates with the gene coding for VP4, one of the two surface proteins of rotaviruses (the other one being VP7). The nucleotide sequence of the VP4 gene of four independently isolated variants showed three amino acid changes, at positions 37 (Leu to Pro), 187 (Lys to Arg), and 267 (Tyr to Cys), in all mutant VP4 proteins compared with RRV VP4. The characterization of revertant viruses from two independent mutants showed that the arginine residue at position 187 changed back to lysine, indicating that this amino acid is involved in the determination of the mutant phenotype. Surprisingly, sequence analysis of reassortant virus DS1XRRV, which depends on SAs to infect the cell, showed that its VP4 gene is identical to the VP4 gene of the variants. Since the only difference between DS1XRRV and the RRV variants is the parental origin of the VP7 gene (human rotavirus DS1 in the reassortant), these findings suggest that the receptor-binding specificity of rotaviruses, via VP4, may be influenced by the associated VP7 protein.

Animals↗
PubMed1996

Trypsin activation pathway of rotavirus infectivity.

The infectivity of rotaviruses is increased by and most probably is dependent on trypsin treatment of the virus. This proteolytic treatment specifically cleaves VP4, the protein that forms the spikes on the surface of the virions, to polypeptides VP5 and VP8. This cleavage has been reported to occur in rotavirus SA114fM at two conserved, closely spaced arginine residues located at VP4 amino acids 241 and 247. In this work, we have characterized the VP4 cleavage products of rotavirus SA114S generated by in vitro treatment of the virus with increasing concentrations of trypsin and with proteases AspN and alpha-chymotrypsin. The VP8 and VP5 polypeptides were analyzed by gel electrophoresis and by Western blotting (immunoblotting) with antibodies raised to synthetic peptides that mimic the terminal regions of VP4 generated by the trypsin cleavage. It was shown that in addition to arginine residues 241 and 247, VP4 is cleaved at arginine residue 231. These three sites were found to have different susceptibilities to trypsin, Arg-241 > Arg-231 > Arg-247, with the enhancement of infectivity correlating with cleavage at Arg-247 rather than at Arg-231 or Arg-241. Proteases AspN and alpha-chymotrypsin cleaved VP4 at Asp-242 and Tyr-246, respectively, with no significant enhancement of infectivity, although this enhancement could be achieved by further treatment of the virus with trypsin. The VP4 end products of trypsin treatment were a homogeneous VP8 polypeptide comprising VP4 amino acids 1 to 231 and a heterogeneous VP5, which is formed by two polypeptide species (present at a ratio of approximately 1:5) as a result of cleavage at either Arg-241 or Arg-247. A pathway for the trypsin activation of rotavirus infectivity is proposed.

Amino Acid Sequence↗
PubMed1995

The Salmonella ompC gene: structure and use as a carrier for heterologous sequences.

The Salmonella typhi (St) ompC gene codes for a major outer membrane protein (OMP) that is highly expressed in both low and high osmolarity. By hybridization studies with the entire gene or with segments thereof, ompC was found to be highly conserved within 11 different Salmonella serotypes, with the exception of S. arizonae. The study included several St isolates from Mexico and Indonesia. Variation was only detected in two (e and f) of the seven regions previously found to vary between St and E. coli ompC. Chimeric OmpC proteins, carrying a rotavirus VP4 capsid protein epitope, are well recognized by a specific monoclonal antibody (mAb) against this epitope, either in OMP preparations (by enzyme-linked immunosorbent assay; ELISA) or intact cells (by ELISA and immunogold-labelling), indicating that regions c and f are oriented towards the cell surface and are probably exposed. As has been shown before for other regulated OMP, this experimental approach could be useful for the presentation of heterologous epitopes in order to gain knowledge about porin topology, for testing the effect of altered porin surface epitopes on bacterial physiology, or else, in the development of multivalent vaccines.

Amino Acid Sequence↗
PubMed1995

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.

Antibodies, Monoclonal↗
PubMed1995

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↗
PubMed1995

Mapping the hemagglutination domain of rotaviruses.

Most strains of animal rotaviruses are able to agglutinate erythrocytes, and the surface protein VP4 is the virus hemagglutinin. To map the hemagglutination domain on VP4 while preserving the conformation of the protein, we constructed full-length chimeras between the VP4 genes of hemagglutinating (YM) and nonhemagglutinating (KU) rotavirus strains. The parental and chimeric genes were expressed in insect cells, and the recombinant VP4 proteins were evaluated for their capacity to agglutinate human type O erythrocytes. Three chimeric genes, encoding amino acids 1 to 208 (QKU), 93 to 208 (QC), and 93 to 776 (QYM) of the YM VP4 protein in a KU VP4 background, were constructed. YM VP4 and chimeras QKU and QC were shown to specifically hemagglutinate, indicating that the region between amino acids 93 and 208 of YM VP4 is sufficient to determine the hemagglutination activity of the protein.

Animals↗
PubMed1994

Mapping the subgroup epitopes of rotavirus protein VP6.

VP6, the most abundant protein of rotaviruses, contains epitopes that allow the classification of these viruses into four subgroups (SG), depending on the presence or absence of two epitopes called I and II. The subgroup-specific epitopes are conformational and appear to be present on trimeric but not monomeric VP6. We have identified on VP6 some of the amino acids that determine the reactivity of the subgroup-specific mAbs 255/60 and 631/9. A single amino acid mutation at positions 172 (Met to Ala) or 305 (Asn to Ala) was sufficient to change the subgroup specificity of the human rotavirus Wa VP6 protein from SGII to SGI/II, since either of these mutations allowed the protein to be recognized by the SGI mAb 255/60, while retaining its capacity to interact with the SGII mAb 631/9. In the case of the SGII epitope, the mutation of two contiguous amino acids (Ala305 Asn306 to Asn305 Ala306) in the porcine rotavirus YM VP6 protein (SGI) enabled the protein to be efficiently recognized by the SGII mAb 631/9, while causing the YM VP6 protein to lose its capacity to interact with mAb 255/60. These results suggest that both subgroup Abs interact with an antigenic domain in VP6 that is composed of at least two regions of the protein that, although distant in the linear sequence, might be in close proximity in the structured VP6 trimer.

Amino Acid Sequence↗
PubMed1994

Neutralizing antibody immune response in children with primary and secondary rotavirus infections.

We have characterized the neutralizing antibody immune response to six human rotavirus serotypes (G1 to G4, G8, and G9) in Brazilian children with primary and secondary rotavirus infections and correlated the response with the G serotype of the infecting rotavirus strain. Twenty-five children were studied: 17 had a single rotavirus infection, 4 were reinfected once, and 4 experienced three infections. Two of the reinfections were by non-group A rotaviruses. Among the 25 primary infections, we observed homotypic as well as heterotypic responses; the serotype G1 viruses, which accounted for 13 of these infections, induced mostly a homotypic response, while infections by serotype G2 and G4 viruses induced, in addition to the homotypic, a heterotypic response directed primarily to serotype G1. Two of the primary infections induced heterotypic antibodies to 69M, a serotype G8 virus that by RNA electrophoresis analysis was found not to circulate in the population during the time of the study. The specificity of the neutralizing antibody immune response induced by a virus of a given serotype was the same in primary as well as secondary infections. These results indicate that the heterotypic immune response induced in a primary rotavirus infection is an intrinsic property of the virus strain, and although there seem to be general patterns of serotype-specific seroconversion, these may vary from serotype to serotype and from strain to strain within a serotype.

Antibodies, Viral↗
PubMed1993

Immunological characterization of a rotavirus-neutralizing epitope fused to the cholera toxin B subunit.

A highly conserved neutralizing epitope from the surface protein VP4 (amino acids 296-313) of human rotaviruses was genetically fused to the B subunit of cholera toxin (CTB). Synthetic oligodeoxyribonucleotides encoding the VP4 peptide were inserted between the 3' end of the DNA that codes for the leader peptide, and the 5' end of the gene encoding mature CTB. The hybrid protein synthesized in Escherichia coli was found to maintain the ability of CTB to pentamerize, and to adhere to its cell receptor, the GM1 ganglioside. The chimera was efficiently recognized by a monoclonal antibody (mAb) directed at CTB and by a virus-neutralizing mAb against the VP4 peptide. The hybrid polypeptide was shown to induce high titers of serum antibodies (Ab) against CTB and the synthetic VP4 peptide following subcutaneous immunization; paradoxically, however, the Ab obtained did not recognize the virus by an enzyme-linked immunosorbent assay method, nor had detectable neutralizing activity. Potential implications of these results for future design and evaluation of fusion proteins as immunogens are discussed.

Amino Acid Sequence↗
PubMed1993

Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain.

Flavivirus genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. The N-termini of several nonstructural proteins are produced by cleavage at dibasic sites by a two-component viral proteinase consisting of NS2B and NS3. NS3 contains a trypsin-like serine proteinase domain at its N-terminus, whereas the function of NS2B in proteolysis is yet to be determined. We have used an NS3-specific antiserum, under nondenaturing conditions, to demonstrate that NS2B and NS3 form a complex both in vitro and in vivo. The N-terminal 184 residues of NS3 are sufficient to form the complex with NS2B. The complex forms efficiently when the NS2B and NS3 are translated from two different mRNAs as well as when NS2B and NS3 are translated as a polyprotein from the same mRNA. A chimeric complex can be formed between yellow fever NS2B and a chimeric yellow fever-dengue 2 NS3. Using anti-NS3 antisera, we also found that a 50-kDa fragment of NS3, consisting of the N-terminal approximately 460 residues, is produced in infected mammalian cells. This fragment is not produced in infected mosquito cells, but will form in Triton X-100 lysates of mosquito cells. The cleavage of NS3 to form this fragment is catalyzed by the NS3 proteinase itself and proteolysis requires NS2B. Examination of the amino acid sequence of NS3 reveals a potential conserved cleavage site that resembles other sites cleaved by the NS3/NS2B proteinase; this site occurs within a conserved RNA helicase sequence motif. The importance of this alternatively processed form of NS3 and its role in the replication cycle of dengue virus remain to be determined.

Aedes↗
PubMed1993

Sequence analysis of rotavirus YM VP6 and NS28 proteins.

We have determined the nucleotide sequence of genes 6 and 10 of porcine rotavirus YM. When the amino acid sequences of VP6 and NS28, the protein products of genes 6 and 10 respectively, were compared with other published sequences it was evident that the proteins of human rotavirus Wa have the highest degree of identity with rotavirus YM. This is in contrast with the observation that when other proteins of these two strains have been compared they have been found to be among the most distantly related pairs of rotavirus strains. This observation is in accordance with the proposed receptor-ligand interaction between NS28 and VP6 during virus morphogenesis, and suggests a specificity in the interaction between these two proteins. In addition, when rotavirus YM VP6, which belongs to subgroup I, was compared with the VP6 proteins of rotavirus strains having different subgroup specificities, it was found to be more closely related to subgroup II rather than subgroup I proteins. This finding allowed us to identify five potential amino acids on VP6 that may contribute to determining the subgroup antigens.

Amino Acid Sequence↗
PubMed1993

Binding to sialic acids is not an essential step for the entry of animal rotaviruses to epithelial cells in culture.

The infection of target cells by animal rotaviruses requires the presence of sialic acids on the cell surface. Treatment of the cells with neuraminidases or incubation of the viruses with some sialoglycoproteins, such as glycophorin A, greatly reduces virus binding, with the consequent reduction of viral infectivity. In this work, we report the isolation of animal rotavirus variants whose infectivity is no longer dependent on the presence of sialic acids on the cell surface. In addition, although these variants bind to glycophorin A as efficiently as the wild-type virus, this interaction no longer inhibit viral infectivity. These observations indicate that the initial interaction of the mutants with the cell occurs at a site different from the sialic acid-binding site located on VP8, the smaller trypsin cleavage product of VP4. Reassortant analysis showed that the mutant phenotype segregates with the VP4 gene. Neutralizing monoclonal antibodies directed to VP4 and VP7 were tested for their ability to neutralize the variants. Antibodies to VP7 and VP5, the larger trypsin cleavage product of VP4, neutralized the mutants as efficiently as the wild-type virus. In contrast, although antibodies to VP8 were able to bind to the mutants, they showed little or no neutralizing activity. The implications of these findings in rotavirus attachment to and penetration of epithelial cells in culture are discussed.

Antibodies, Monoclonal↗