Modeling the Transport of Human Rotavirus and Norovirus in Standardized and in Natural Soil Matrix-Water Systems.

We modeled Group A Rotavirus (RVA) and Norovirus genogroup II (GII NoV) transport experiments in standardized (crystal quartz sand and deionized water with adjusted pH and ionic strength) and natural soil matrix-water systems (MWS). On the one hand, in the standardized MWS, Rotavirus and Norovirus showed very similar breakthrough curves (BTCs), showing a removal rate of 2 and 1.7 log10, respectively. From the numerical modeling of the experiment, transport parameters of the same order of magnitude were obtained for both viruses. On the other hand, in the natural MWS, the two viruses show very different BTCs. The Norovirus transport model showed significant changes; BTC showed a removal rate of 4 log10, while Rotavirus showed a removal rate of 2.6 log10 similar to the 2 log10 observed on the standardized MWS. One possible explanation for this differential behavior is the difference in the isoelectric point value of these two viruses and the increase of the ionic strength on the natural MWS.

Rotavirus VP6 protein as a bio-electrochemical scaffold: Molecular dynamics and experimental electrochemistry.

This paper reports a theoretical and experimental investigation on the recombinant protein rotavirus VP6 as a bioelectrochemical interface. Our motivation arises from the highly active zones of VP6 which can interact with biological structures and metals, as well as its useful features such as self-assembly, polymorphism, and active surface charge. A molecular simulation study was performed to analyze the charge transfer properties of theVP6 trimer under an applied electric field. The electrostatic properties were evaluated via the nonlinear second-order Poisson-Boltzmann equation, using finite element methods based on parameter discretization and calculation of solute/solvent interaction forces, which account for mean-field screening effects. The electrochemical study validated the theoretical predictions for VP6 in their different assemblies (trimers and nanotubes) when they are used as electrodes in 10 mM K3[Fe(CN)6], 1 M KCl. Applying a potential sweep promotes charge transfer, facilitates redox activity of the ferricyanide ion. Furthermore, protein assemblies decreased electrode electrical resistance and enabled gold particle electrodeposition on the protein VP6. These results suggest that VP6 is a promising conductive biomaterial that promotes charge transfer of redox probes and could be used as a new scaffold to create bio-electrochemical interfaces.

Detection of a novel equine-like G3 rotavirus associated with acute gastroenteritis in Brazil.

Genotype G3P[8] of rotavirus A (RVA) is detected worldwide, usually associated with Wa-like constellation and exhibiting a long RNA migration pattern. More recently, a novel inter-genogroup, G3P[8] reassortant variant with a short electropherotype, has emerged in Asia, Oceania and Europe, denoting an overall potential of unusual rotavirus strains. During a RVA surveillance in Brazil, G3P[8] strains were found displaying a short electropherotype pattern, which had not been detected before in this region. This study aims to characterize the complete genome of 10 G3P[8] strains detected in the northern region of Brazil. All G3P[8] samples were subjected to partial sequencing, and the whole-genome phylogenetic analysis demonstrated that all strains possessed I2-R2-C2-M2-A2-N1-T2-E2-H2 genotype background, representing reassortants with an equine-like G3 VP7 and amino acid changes in VP4 and VP7 antigenic regions as compared to vaccine strains. Phylogenetic analysis demonstrated high nucleotide identity in almost all RNA segments of G3P[8] DS-1 samples detected in Asia, Oceania and Europe as well as G3P[4] strains in Japan. This study reports a novel, equine-like G3P[8] strain circulating in Brazil and isolated from children hospitalized for severe gastroenteritis, and highlights the complex dynamics of RVA molecular epidemiology. Our findings point to a novel RVA strain emerging in this region, and studies should be done to detect whether this may represent a challenge to current vaccine strategies.

Detection of emerging rotavirus G12P[8] in Sonora, México.

Rotavirus is the most common cause of gastroenteritis in children up to five years of age worldwide. The aim of the present study was to analyze the genotypes of rotavirus strains isolated from children with gastroenteritis, after the introduction of the rotavirus vaccine in México. Rotavirus was detected in 14/100 (14%) fecal samples from children with gastroenteritis, using a commercial test kit. The viral genome was purified from these samples and used as a template in RT-PCR amplification of the VP4 and VP7 genes, followed by gene cloning and sequencing. Among the rotavirus strains, 4/14 (28.5%) were characterized as G12P[8], 2/14 (14.3%), as G12P (not typed), and 3/14 (21.42%) as G (not typed) P[8]. Phylogenetic analysis of the VP7 gene showed that G12 genotypes clustered in lineage III. Phylogenetic analysis revealed that VP4 genotype P[8] sequences clustered in lineage V, whereas other P[8] sequences previously reported in Mexico (2005-2008) clustered in different lineages. Rotavirus genotype G12 is currently recognized as a globally emerging rotavirus. To our knowledge, this is the first report of this emerging rotavirus strain G12P[8] in México. Ongoing surveillance is recommended to monitor the distribution of rotavirus genotypes and to continually reassess the suitability of currently available rotavirus vaccines.

Rotavirus vaccine stability in the aquatic environment.

AIMS: To evaluate the thermal and length of stability of the Rotaviruses (RV) vaccine (RotaTeq) in the aquatic environment. METHODS AND RESULTS: Surface freshwater, brackish and drinking water were spiked with RV vaccine strain and stored at 22 and 4°C. The virus infectivity and genome persistence were evaluated by plaque assay and RT-qPCR, respectively, up to 180 days. Infectious RV vaccine particles showed to be less stable in the brackish water matrix than in surface and drinking water either at 22 or 4°C. The estimated T90 values obtained by the linear regression model were 18, 55 and 59 days, respectively for brackish, surface and drinking water stored at 22°C and 68, 154 and 240 days at 4°C. As expected, the genome persistence showed to be less affected by length and temperature of storage in all the matrices evaluated. CONCLUSIONS: The evidence of high stability of the RV vaccine in water matrices reinforces the importance for surveillance of RV vaccines strains in the environment regarding the potential occurrence of unexpected infections and virus genomic reassortments. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of reassortants and the shedding of the live attenuated vaccine strains after vaccination can compromise the vaccine safety by introducing new viral variants in the environment.

Rotavirus genotypes and the indigenous children of Brazilian midwest in the vaccine era, 2008-2012: Footprints of animal genome.

World group A rotavirus (RVA) surveillance data provides useful estimates of the disease burden, however, indigenous population might require special consideration. The aim of this study was to describe the results of G- and P-types from Brazilian native children ≤ 3 years. Furthermore, selected strains have been analyzed for the VP7, VP6, VP4, and NSP4 encoding genes in order to gain insight into genetic variability of Brazilian strains. A total of 149 samples, collected during 2008-2012, were tested for RVA using ELISA and PAGE, following by RT-PCR and sequencing. RVA infection was detected in 8.7% of samples (13/149). Genotype G2P[4] was detected in 2008 and 2010, G8P[6] in 2009, and G3P[8] in 2011. The phylogenetic analysis of the VP7 and VP4 genes grouped the Brazilian G2P[4] and G3P[8] strains within the lineages currently circulating in humans worldwide. However, the phylogenetic analysis of the VP6 and NSP4 from the Brazilian G2P[4] strains, and the VP7 and NSP4 from the Brazilian G3P[8] strains suggest a distant common ancestor with different animal strains (bovine, caprine, and porcine). The epidemiological and genetic information obtained in the present study is expected to provide an updated understanding of RVA genotypes circulating in the native infant population, and to formulate policies for the use of RVA vaccines in indigenous Brazilian people. Moreover, these results highlight the great diversity of human RVA strains circulating in Brazil, and an in-depth surveillance of human and animal RVA will lead to a better understanding of the complex dynamics of RVA evolution.

Rotavirus genotypes and the indigenous children of Brazilian midwest in the vaccine era, 2008-2012: footprints of animal genome

World group A rotavirus (RVA) surveillance data provides useful estimates of the disease burden, however, indigenous population might require special consideration. The aim of this study was to describe the results of G­ and P­types from Brazilian native children ≤3 years. Furthermore, selected strains have been analyzed for the VP7, VP6, VP4, and NSP4 encoding genes in order to gain insight into genetic variability of Brazilian strains. A total of 149 samples, collected during 2008­2012, were tested for RVA using ELISA and PAGE, following by RT­PCR and sequencing. RVA infection was detected in 8.7% of samples (13/149). Genotype G2P[4] was detected in 2008 and 2010, G8P[6] in 2009, and G3P[8] in 2011. The phylogenetic analysis of the VP7 and VP4 genes grouped the Brazilian G2P[4] and G3P[8] strains within the lineages currently circulating in humans worldwide. However, the phylogenetic analysis of the VP6 and NSP4 from the Brazilian G2P[4] strains, and the VP7 and NSP4 from the Brazilian G3P[8] strains suggest a distant common ancestor with different animal strains (bovine, caprine, and porcine). The epidemiological and genetic information obtained in the present study is expected to provide an updated understanding of RVA genotypes circulating in the native infant population, and to formulate policies for the use of RVA vaccines in indigenous Brazilian people. Moreover, these results highlight the great diversity of human RVA strains circulating in Brazil, and an in­depth surveillance of human and animal RVA will lead to a better understanding of the complex dynamics of RVA evolution

Analysis of rotavirus non-structural protein NSP5 by mass spectrometry reveals a complex phosphorylation pattern.

Genomic replication and partial assembly of Rotavirus takes place in cytoplasmic viral structures called viroplasms. NSP5 is a viral phosphoprotein localized in viroplasms and its expression is imperative for viral cycle progress. During infection three isoforms of NSP5 can be observed by SDS-PAGE (26, 28 and 33-35kDa) and previous reports suggested that they differ in their phosphorylation patterns. In this study we obtained NSP5 from infected cells and by mass spectrometry we were able to identify nine phosphorylation sites. We detected that in all the isoforms the same residues can be found either phosphorylated or unmodified. Quantitative analysis showed that the 28kDa isoform has a higher phosphorylation level than the 26kDa isoform suggesting that migration properties depend on the total number of phosphorylated residues. Moreover, we identified two not previously described modifications for this protein: an N-acetylation in Serine-2 and an intramolecular disulfide bond in a highly conserved motif, CXXC which is located between two charged alpha-helix motifs.

Brucella spp. lumazine synthase as a bovine rotavirus antigen delivery system.

Brucella spp. lumazine synthase (BLS) is a highly immunogenic decameric protein. It has been previously evaluated as a carrier to increase the immunogenicity of peptides fused to its N-termini. VP8 is a sialic acid binding domain of rotavirus external capsid protein VP4, which is involved in virus adhesion to host cells. In this work, the C486 bovine rotavirus (BRV) VP8 core protein (VP8d) was fused to the structure of BLS with the aim to produce an enhancement of the immune response against BRV VP8 and to evaluate the possible use of this antigen for vaccine development. The feasibility of using BLS as an antigen delivery system of polypeptides larger in size than those previously tested was also evaluated. Groups of female mice were immunized with BLS-VP8d fusion protein, VP8d or an equimolar mixture of purified VP8d and BLS (BLS+VP8d). Dams immunized with BLS-VP8 induced 97.5-100% protection against homologous challenge with C486 BRV; while pups born to dams immunized either with VP8d or BLS+VP8d presented a significant lower level of protection. The neutralizing antibody pattern was also significantly different among these experimental groups, and in concordance with challenge experiment. Overall, these results demonstrate that the BLS-VP8d chimeric protein is properly folded and stable, and that the BLS scaffold is a potent antigen delivery system that enhances the antibody response against BRV and elicits complete homotypic passive protection in a suckling mouse model.

The peptide-binding and ATPase domains of recombinant hsc70 are required to interact with rotavirus and reduce its infectivity.

The heat shock cognate protein hsc70 has been implicated as a postattachment cell receptor for rotaviruses. Here we show that hsc70 interacts specifically with rotaviruses through its peptide-binding domain, since a recombinant full-length hsc70 protein and its peptide-binding domain, but not its ATPase domain, bound triple-layered particles in a solid-phase assay, and known ligands of hsc70 competed this binding. The peptide ligands of hsc70 were also shown to block rotavirus infectivity when added to cells before virus infection, suggesting that hsc70 on the surface of MA104 cells also interacts with the virus through its peptide-binding domain and that this interaction is important for virus entry. When purified infectious virus was incubated with soluble hsc70 in the presence of the cochaperone hsp40 and ATP and then pelleted through a sucrose cushion, the recovered virus had lost 60% of its infectivity, even though hsc70 was not detected in the pellet fraction. The hsc70-treated virus showed slightly different reactivities with monoclonal antibodies and was more susceptible to heat and basic pHs than the untreated virus, suggesting that hsc70 induces a subtle conformational change in the virus that results in a reduction of its infectivity. The relevance of the ATPase activity of hsc70 for reducing virus infectivity was demonstrated by the finding that in the presence of a nonhydrolyzable analogue of ATP, virus infectivity was not affected, and a mutant protein lacking ATPase activity failed to reduce virus infection. Altogether, these results suggest that during cell infection, the interaction of the virus with hsc70 on the surface of MA104 cells results in a conformational change of virus particles that facilitates their entry into the cell cytoplasm.

Prevalence of antibodies to dengue virus, hepadnavirus and rotavirus in non-human primates

The aim of this study was to determine the prevalence of several viral antibodies in non-human primates from two zoological gardens from Venezuela. Only two out of 66 sera were positive for antibodies to dengue virus by hemagglutination inhibition. Six out of 62 sera exhibited antibodies against Hepatitis B virus (HBV) core antigen. Viral DNA was detected by nested PCR in one positive serum and in three negative without serological evidence of HBV infection. Sequence analysis showed the circulation of HBV genotype F, predominant in Venezuela. Antibodies against rotavirus antigens were found in 87 percent (20/23) of Old World primates and in 50 percent (13/26) of New World primates. Both the prevalence of antibodies and the mean O.D. value by ELISA were significantly lower in New World primate sera. These results suggest that non-human primates do not seem to represent an important reservoir for dengue virus infection, highly endemic in Venezuela. Anthropozoonotic infection of HBV seems to occur among primates not only from the Old but also from the New World, reinforcing the importance of vaccination of species at risk. This study also suggests a lower frequency of infection by rotavirus of non-human primates from the New World, compared to primates from the Old World.

En este estudio se determinó la prevalencia de anticuerpos contra varios virus en primates no humanos de dos parques zoológicos de Venezuela. Sólo dos de 66 sueros fueron positivos, por inhibición de la hemaglutinación, para anticuerpos contra virus dengue. Seis de 62 sueros presentaron anticuerpos contra la cápside del virus de la hepatitis B virus (VHB). Se detectó el ADN viral, mediante PCR en dos rondas, en uno de éstos y en tres sueros sin evidencia serológica de infección por VHB. El análisis de la secuencia mostró la circulación del VHB genotipo F, predominante en Venezuela. Un 87 por ciento (20/23) de los sueros de primates del Viejo Mundo y un 50 (13/26) de los del Nuevo Mundo mostraron anticuerpos contra antígenos de rotavirus. Tanto la prevalencia de anticuerpos como los valores promedio de D.O. por ELISA fueron significativamente menores en sueros de primates del Nuevo Mundo. Los primates no humanos no parecen jugar un papel importante como reservorio de la infección por virus dengue, altamente prevalente en el país. La infección por cepas humanas del VHB en primates sugiere infección antroponótica y la importancia de vacunar las especies a riesgo. Los resultados sugieren igualmente una menor frecuencia de infección por rotavirus en primates del Nuevo Mundo.

Evaluation of serum antibody responses against the rotavirus nonstructural protein NSP4 in children after rhesus rotavirus tetravalent vaccination or natural infection.

The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S-transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly (P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of < or = 200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant (P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate.

WHO informal consultation on quality, safety and efficacy specifications for live attenuated rotavirus vaccines Mexico City, Mexico, 8-9 February 2005.

Rotavirus vaccines are at an advanced stage of development but there are as yet no WHO recommendations on production and quality control to provide regulatory guidance. A meeting of experts was convened by WHO and PAHO/AMRO to review the scientific basis for production and quality control of rotavirus vaccines, and to discuss specific measures to assure the safety and efficacy of rotavirus vaccines. The meeting was attended by 25 experts from 14 countries, drawn from academia, public health, national regulatory authorities and vaccine producers. It was agreed that existing guidance for other live virus vaccines provides a very good basis for product characterization, especially for source materials and control of production. The basis for attenuation of current vaccines or vaccine candidates is not known but, at least for the vaccines based on the Jennerian approach of using animal (bovine) rotaviruses, is likely to be multigenic. The risk of intussusception in humans is influenced by genetic background and age. Recent analyzes of large vaccine safety trials found that certain strains of vaccine virus were not associated with intussusception, although in these trials the first dose of vaccine was not administered to children over 3 months of age. Since age is a risk factor for intussusception, this may suggest that early delivery of the first dose of vaccine is desirable. However, maternal antibodies may mitigate against early delivery of the first vaccine dose. Factors which could affect vaccine efficacy or safety include strain diversity, malnutrition, other enteric infections, parasitic infection or immune suppression. It was concluded that data from clinical trials conducted in one part of the world would not necessarily be predictive of vaccine efficacy in other places. It was agreed that in nonclinical evaluations there was a need to use oral dosing for toxicity studies and, because rotavirus is non-neurovirulent, that there was no need for an animal neurovirulence test. For clinical evaluations there was a need for a standard definition of severe disease as the clinical endpoint, a need for studies in diverse geographical regions, and the need to control for possible interference by factors such as other oral vaccinations, intercurrent infections and breast feeding. There is no accepted correlate of protection. Secretory IgA is the most satisfactory laboratory parameter currently available and for a period after vaccination much of the specific serum IgA is of this type, so that serum IgA levels can act as measure of seroconversion. The need for standardization of the potency assay for release of vaccine was identified, as was a need to develop guidance on standardized approaches to post-marketing surveillance for rotavirus vaccines.

Characterization of neuraminidase-resistant mutants derived from rotavirus porcine strain OSU.

Infection by some rotavirus strains requires the presence of sialic acid on the cell surface, its infectivity being reduced in cells treated with neuraminidase. A neuraminidase treatment-resistant mutant was isolated from the porcine rotavirus strain OSU. In reassortant strains, the neuraminidase-resistant phenotype segregated with the gene coding for VP4. The mutant retained its capacity to bind to sialic acid. The VP4 sequence of the mutant differed from that of the parental OSU strain in an Asp-to-Asn substitution at position 100. Neutralization escape mutants selected from an OSU neuraminidase-sensitive clone by monoclonal antibodies that failed to recognize the neuraminidase-resistant mutant strain carried the same mutation at position 100 and were also neuraminidase resistant. Neuraminidase sensitivity was restored when the mutation at position 100 was compensated for by a second mutation (Gln to Arg) at position 125. Molecular mechanics simulations suggest that the neuraminidase-resistant phenotype associated with mutation of OSU residue 100 from Asp to Asn reflects the conformational changes of the sialic acid cleft that accompany sialic acid binding.

The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6.

Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalytic kinase activity, and is present in infected cells as various isoforms having molecular masses of 26, 28 and 30-34 kDa. We have previously shown that NSP5 forms oligomers and interacts with NSP6 in yeast cells. Here we have mapped the domains of NSP5 responsible for these associations. Deletion mutants of the rotavirus YM NSP5 were constructed and assayed for their ability to interact with full-length NSP5 and NSP6 using the yeast two-hybrid assay. The homomultimerization domain was mapped to the 20 C-terminal aa of the protein, which have a predicted alpha-helical structure. A deletion mutant lacking the 10 C-terminal aa (DeltaC10) failed to multimerize both in yeast cells and in an in vitro affinity assay. When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30-34 kDa isoforms). In contrast, the DeltaC10 mutant produced forms equivalent to the 26 and 28 kDa species, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein. The interaction domain with NSP6 was found to be present in the 35 C-terminal aa of NSP5, overlapping the multimerization domain of the protein, and suggesting that NSP6 might have a regulatory role in the self-association of NSP5. NSP6 was also found to interact with wild-type NSP5, but not with its mutant DeltaC10, in cells transiently transfected with plasmids encoding these proteins, confirming the relevance of the 10 C-terminal aa for the formation of the heterocomplex.

Rotavirus vaccines and vaccination in Latin America.

Worldwide, rotaviruses account for more than 125 million cases of infantile gastroenteritis and nearly 1 million deaths per year, mainly in developing countries. Rather than other control measures, vaccination is most likely to have a major impact on rotavirus disease incidence. The peak incidence of rotavirus diarrhea occurs between 6 and 24 months of age. In developing countries, however, cases are not uncommon among children younger than 6 months. G serotypes 1 to 4 are responsible for most disease, but there are indications that in Brazil that G type 5 is of emerging epidemiological importance. Both homotypic and heterotypic responses are elicited during natural rotavirus infection, and the immunological response at the intestinal mucosal surface is probably the more consistent predictor of clinical immunity. With the primary objective of protecting children against life-threatening dehydrating diarrhea, many approaches to rotavirus vaccine development have been attempted. One vaccine, the tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV), was given licensing approval in the United States of America, introduced to the market, and later withdrawn. A number of studies have found better efficacy of RRV-TV in developed countries than in developing ones. Field trials with a 4 x 10(4) plaque-forming units (PFU) preparation of RRV-TV have been carried out in two countries in Latin America, Brazil and Peru. Those trials yielded protective efficacy rates against all rotavirus diarrhea ranging from 18% to 35%. Data from a large catchment trial in Venezuela with a higher RRV-TV dose, of 4 x 10(5) PFU/dose, indicated an efficacy rate of 48% against all rotavirus diarrhea and 88% against severe rotavirus diarrhea. It appears that breast-feeding does not compromise the efficacy of RRV-TV if three doses of the vaccine are administered. Similarly, possible interference of oral poliovirus vaccine with the "take" of the rotavirus vaccine can be overcome by giving three doses of the rotavirus vaccine or by using a higher-titer formulation of it. Wild enteroviruses, however, may cause primary rotavirus vaccine failure in developing countries. Studies in Peru with RRV-TV have shown a trend towards higher vaccine efficacy rates against "pure" (rotavirus-only) diarrheal episodes. Economic analyses made in the United States indicate that a vaccine that costs less than US$ 9 per dose would lead to a net savings in medical costs. To date, however, cost-benefit studies have not been done in developing countries. In the future, it is possible that some Latin American countries might adapt their polio production facilities to the preparation of rotavirus vaccines for human use. A year after RRV-TV was licensed for vaccination of infants in the United States, the occurrence of intussusception as an adverse event led to the vaccine's withdrawal from the market. The implications of that action, particularly for Latin America, will be addressed in this article, including the need to explore alternative rotavirus candidate vaccines, particularly through the conduct of parallel clinical trials in both developed and developing countries.

Humoral and cell-mediated immune responses in humans to the NSP4 enterotoxin of rotavirus.

Rotavirus nonstructural protein NSP4 has recently been suggested to function as a viral enterotoxin and play a role in the pathophysiological mechanism whereby rotaviruses induce diarrhea. The ability of rotavirus NSP4 to stimulate a humoral immune response was examined in naturally infected children and adults, rotavirus vaccinated children, as well as a cellular immune response in adults. In this study, 10 of 10 naturally infected children and 9 of 10 rotavirus-vaccinated children showed a weak humoral IgG immune response to recombinant NSP4 (rNSP4) and/or a synthetic peptide corresponding to residues 114-134 of NSP4. Modest serum IgG antibody responses were detected in 20 of 20 adults. A cellular immune response to rNSP4 and/or NSP4(114-134) were detected in 8 of 10 adults measured either as a T-cell proliferative response (7 of 10), an increased production of IL-2 (6 of 10), or increased production of interferon-gamma (8 of 10). These results indicate that NSP4 induces a humoral immune response in humans and show for the first time that NSP4 stimulates a cellular immune response, possibly including cytolytic T-cells.

Identification of two lineages (WA-like and F45-like) within the major rotavirus genotype P[8].

The fourth gene of a porcine (S8) and eight human rotavirus isolates possessing the major human VP4 specificity (P1A serotype and/or P[8] genotype) were partially sequenced and compared to other available P[8] sequences from rotaviruses types G1, G3, G5 and G9 specificities which had been originally recovered from children with diarrhea in Japan, Brazil and the USA. Brazilian rotavirus S8 represented the single known porcine rotavirus with this P specificity. Phylogenetic analysis revealed two lineages or subgenotypes within P[8] strains: the F45-like P subgenotype comprised most of the strains, including all the human G5 isolates analyzed, whereas the Wa- or S8-like subgenotype consisted of only a human isolate obtained in the same geographic region as S8 and an American strain with atypical RNA profile besides the prototypes Wa and S8 viruses. A conserved basic amino acid residue at position 131 in VP4 seemed characteristic of the F45-like P[8] subgenotype.

Comparative amino acid sequence analysis of the major outer capsid protein (VP7) of porcine rotaviruses with G3 and G5 serotype specificities isolated in Venezuela and Argentina.

Seven porcine group A rotavirus strains isolated in Venezuela were shown to be antigenically related to serotype G3 (five strains) or to serotype G5 (two strains), whereas two strains isolated in Argentina were classified as serotype G5. The serological classification of eight of these strains was confirmed by sequence analysis of the gene encoding the VP7 glycoprotein. A high degree of homology was observed among strains belonging to the same G serotype, although some variations in the serotype-specific regions were detected among different strains. Comparison with the published VP7 amino acid sequences of serotype G3 indicated that most porcine rotavirus strains are more closely related to each other and to human rotavirus strains than to rotavirus strains isolated from other species. Amino acid sequence comparison among serotype G5 porcine strains revealed that Venezuelan porcine isolates were more closely related to the American strain OSU, while the Argentinian strains had a higher similarity to the Australian strain TRF-41. This report confirms the worldwide distribution of these G serotypes among the porcine population.

VP6 from porcine rotavirus strain CN86: amino acid sequence divergence with conservation of subgroup II specificity.

A porcine rotavirus strain, CN86, originally isolated from rotavirus-infected piglets in Argentina, has been shown to possess unique characteristics. It was the first animal strain described to be antigenically related to human serotype G1 and the standard counterpart of another porcine strain showing rearrangement of genome segment 11. Owing to these features, molecular characterization of this virus seemed relevant. The gene encoding the major inner capsid protein, VP6, was cloned and its nucleotide sequence was determined. Comparative analysis of the deduced amino acid sequence of CN86 VP6 with those representing the four different subgroups showed that it is more closely related to subgroup II human Wa and porcine Gottfried strains, albeit to a lesser extent than they are to each other. Despite exhibiting sequence divergence, CN86 VP6 has 12 out of the 14 residues expected to be conserved in strains bearing subgroup II specificity. Interestingly, CN86 VP6 shows a high degree of homology with VP6 of porcine strain YM rotavirus which, although being closely related to subgroup II strains, has been serologically characterized as subgroup I. Subgroup II reactivity of CN86 strain, predicted by sequence analysis, was confirmed by ELISA with subgroup-specific monoclonal antibodies. Taken together, our results provide evidence for the existence of a human-pig lineage for rotavirus gene 6.