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PubMed1998

Rotavirus-specific proteins are detected in murine macrophages in both intestinal and extraintestinal lymphoid tissues.

Rotaviruses replicate in mature, villous epithelial cells of the mammalian small intestine. Although rotavirus has not been detected in plasma of infants with rotavirus-induced gastroenteritis, rotavirus particles and rotavirus genomic RNA have been detected in extraintestinal sites (e.g. cerebrospinal fluid). Using a murine rotavirus strain well adapted to growth in the small intestines of suckling mice, we found that macrophages (and to a lesser extent B cells) in gut-associated lymphoid tissue contained rotavirus-specific proteins, and that these antigen-containing cells travelled to sites distant to the intestine.

Age Factors↗
PubMed1994

Identification of a new VP4 serotype of human rotaviruses.

A new VP4 gene allele was identified by sequencing of two rotavirus recovered from infants with diarrhea. Strains HAL1166 (a VP7 serotype 8 human rotavirus from Finland) and PA169 (a VP7 serotype 6 human rotavirus from Italy) present 22-43% nucleotide sequence divergence and 14-45% amino acid divergence in the VP4 gene with rotaviruses belonging to various VP4 gene alleles previously reported. Cross-neutralization analysis between PA169 and rotaviruses belonging to the other recognized VP4 (P) serotypes had previously shown that none of them was similar to PA169. Hyperimmune serum prepared against a reassortant with the VP4 gene of strain PA169 and the VP7 gene of serotype 2 strain DS1 failed to neutralize previously recognized human rotavirus VP4 serotypes, indicating that PA169 represents a new VP4 serotype specificity. In addition, one of four neutralizing monoclonal antibodies directed to the VP4 of PA169 was specific for this virus, while three others had very limited cross-reactivity with rotaviruses possessing different VP4s.

Amino Acid Sequence↗
PubMed1994

Molecular characterization of the outer capsid spike protein (VP4) gene from human group C rotavirus.

A cDNA copy of the third genomic RNA segment of a noncultivatable human group C rotavirus (Bristol) was generated by single primer amplification. Human group C rotavirus genome segment 3 contains 2283 bp and encodes the VP4 gene with an open reading frame of 2232 nucleotides (744 amino acids) starting at nucleotide 21 and terminating at nucleotide 2251. PCR primers designed from the 5' and 3' terminal sequences of the C/Bristol VP4 gene were used to amplify the corresponding VP4 gene of a human group C rotavirus from Belém, Brazil. Nucleotide sequence comparisons of the Bristol and Belém VP4 genes revealed 45 differences of which only 6 were predicted to give amino acid changes. Alignment of the two human VP4 sequences with the prototype porcine group C/Cowden rotavirus VP4 showed only 71.2% nucleotide sequence identity. Protein sequence alignments showed that the human group C rotavirus VP4 sequences were 8 amino acids longer than the porcine VP4 sequence with an insertion of 6 amino acids, 252NSKLGD257 adjacent to the proposed proteolytic cleavage region (amino acids 231-250). The large overall number of differences between the human and porcine VP4 sequences strongly suggested that the porcine C/Cowden isolate may belong to a different group C rotavirus P "serotype." In contrast the very close similarity of the VP4 sequences of the UK and Brazilian group C rotaviruses support the hypothesis that these human isolates originate from a recent common ancestor.

Amino Acid Sequence↗
PubMed1996

Rotavirus stimulates IL-8 secretion from cultured epithelial cells.

Rotavirus is the most important cause of severe gastroenteritis in children worldwide. We have investigated cytokine responses to rotavirus infection of cultured intestinal epithelial cells. Interleukin 8 (IL-8) is a chemotactic and cell-activating cytokine that is synthesized by epithelial cells and induced in response to bacterial enteric pathogens. Rotavirus inoculation increased IL-8 mRNA levels in cultured intestinal epithelial cells within 2 hr of infection. IL-8 secretion increased 10(2)- to 10(3)-fold by 8 hr postinfection. Secretion of TNF alpha or IL-1 beta, cytokines which themselves increase IL-8 secretion, was not induced by rotavirus, nor was that of TNF alpha, IFN alpha, IFN gamma, or IL-6. Neutralizing antibodies to TNF alpha or IL-1 alpha/beta did not affect the IL-8 response. Secretion of IL-8 was dependent on an intact viral capsid, as single-shell particles were inert. Neutralizing monoclonal antibodies (vp7-specific) that do not block cell attachment did block rotavirus stimulation of IL-8 secretion, indicating that attachment to the cell surface is not a sufficient stimulus to induce IL-8. Genetically inactivated rotavirus was also effective for IL-8 induction, indicating that viral replication was not required. These data suggest that epithelial cytokine IL-8 may be an important mediator of the host response to viral gastroenteritis pathogens such as rotavirus.

Animals↗
PubMed1999

Rotavirus-specific cytotoxic T lymphocytes recognize overlapping epitopes in the amino-terminal region of the VP7 glycoprotein.

Rotavirus-specific cytotoxic T lymphocytes (CTL) play an important role in the resolution of rotavirus infection. The outer capsid glycoprotein, VP7, elicits a class I MHC-restricted CTL response. Vaccinia virus recombinants expressing the VP7 genes from simian rotavirus SA11 (serotype G3) and from the RF strain of bovine rotavirus (serotype G6) were used to analyze the CTL activity to this antigen in BALB/c (H-2(d)) and C57BL/6 (H-2(b)) mice neonatally infected with homologous and heterologous rotaviruses. A vaccinia virus recombinant expressing the first amino-terminal 88 amino acids of VP7 was constructed and used to search for cross-reactive CTL against this region of the protein. By using synthetic Kb, Db, and Kd motif-fitting peptides two overlapping CTL epitopes have been identified located in the first hydrophobic domain (H1) of VP7. Splenocytes obtained from rotavirus SA11-infected C57BL/6 mice induced the strongest CTL response against target cells sensitized with a peptide containing a Kb-restricted CTL epitope (amino acids 8-16). A second Kd-restricted epitope (residues 5-13) was recognized by splenocytes derived from rotavirus-infected BALB/c mice. These findings reveal the existence of CTL epitopes in the H1 signal sequence of the VP7 glycoprotein that coexist with a CTL epitope (residues 31-40) previously described within the H2 region.

Animals↗
PubMed1991

Molecular evidence for naturally occurring single VP7 gene substitution reassortant between human rotaviruses belonging to two different genogroups.

Twenty four stool rotaviruses that comprised 22 distinct electropherotypes were selected for genome analysis from the collection of diarrheal specimens obtained over an eight-year period. These 22 electropherotypes were found in 46% of the total electropherotypes identified during the previous studies and represented 328 (64%) of rotavirus specimens in the collection. When genomic RNAs from these stool rotaviruses were hybridized to the 32P-labeled transcription probes prepared from prototypes representing three human rotavirus genogroups, Wa, DS-1, and AU-1, any one of the isolates showed a high degree of homology only with one of the three probes, which data confirmed and extended our previous observation on the existence of three distinct genogroups among human rotaviruses. Two stool rotaviruses which had an unusual combination of serotype (G1), subgroup (I) and RNA pattern (an identical short electropherotype), however, yielded the hybridization pattern indicative of an intergenogroupic single VP7 gene substitution reassortant. When they were cell culture adapted and analyzed by RNA-RNA hybridization, molecular evidence was obtained indicating that their VP7 gene derived from viruses belonging to the Wa genogroup whereas the remaining 10 genes hybridized with viruses belonging to the DS-1 genogroup. Interestingly, these natural reassortants emerged in the midst of the rotavirus season in which G1 strains predominated.

Antigens, Viral↗
PubMed1982

Isolation and characterization of a canine rotavirus.

Canine rotavirus particles were visualized by direct electron microscopy in the feces from a clinically normal dog. The virus was subsequently propagated in cell cultures; it was characterized and compared with rotaviruses from other species. Replication of the virus in cell culture was found to be less dependent upon trypsin than that of human, bovine and porcine rotaviruses. Reproducible, sharp-edged plaques of various sizes were produced by the canine rotavirus in an established cell line of fetal rhesus monkey kidney, MA 104, under overlays of carboxymethyl cellulose or agarose. Intracytoplasmic inclusion bodies of different sizes and shapes were produced in infected MA 104 cells. By plaque reduction neutralization assay, a two-way antigenic relationship was found between the canine (CU-1) and simian (rhesus MMU 18006 and SA-11) rotaviruses. The canine rotavirus had a one-way antigenic relationship with feline (Taka), bovine (NCDV), and porcine (OSU) rotaviruses.

Animals↗
PubMed1982

Antigenic relationships between rotaviruses from different species as studied by neutralization and immunofluorescence.

Antigenic relationships between the Wa, Hochi, Ito, Hosokawa and Nemoto strains of human rotavirus, the SA-11 strain of simian rotavirus, the Lincoln strain of bovine rotavirus, the OSU strain of porcine rotavirus and the R-2 strain of lapine rotavirus, which were all established in cell culture, were studied by neutralization (NT) and immunofluorescence (IF) using guinea pig antisera against these strains. The strains could not be distinguished by indirect IF staining of infected cells because of marked cross reactions. On the other hand, the NT test clearly distinguished these strains, although there was a one-way antigenic relationship between some of them. The five strains of human rotavirus were clearly distinguished from each other by NT test. These human strains exhibited much higher homologous titers than heterologous titers, the former being at least eight-fold higher than the latter. The presence of at least five serotypes of human rotavirus was indicated.

Animals↗
PubMed1998

Serotypic characterization of outer capsid spike protein VP4 of vervet monkey rotavirus SA11 strain.

The vervet monkey rotavirus SA11, a prototype strain of group A rotaviruses, has been shown to possess VP7 serotype 3 specificity but its neutralization specificity with regard to the other outer capsid protein VP4 has not been elucidated. We thus determined its VP4 specificity by two-way cross-neutralization with guinea pig antiserum prepared with a single gene substitution reassortant that had only the VP4-encoding gene from the simian rotavirus SA11 strain and remaining ten genes from human rotavirus DS-1 strain (G serotype 2). The SA11 VP4 was related antigenically in a one-way fashion to rhesus monkey rotavirus MMU18006 VP4 (a P5B strain) and marginally to human and canine rotavirus VP4s with P serotype 5A specificity. In addition, the SA11 VP4 was shown to be distinct antigenically from those of other known P serotypes (1-4, and 6-11) as well as those of uncharacterized equine, lapine, and avian rotavirus strains. The SA11 VP4 is thus proposed for classification as a P5B serotype.

Animals↗
PubMed2001

Group C rotavirus NSP4 induces diarrhea in neonatal mice.

Nonstructural glycoprotein NSP4 of group A rotavirus induces diarrhea in neonatal mice by functioning as an enterotoxin. Previously, our laboratory reported that the structural features of group A and group C rotavirus NSP4 proteins are well conserved despite a lack of sequence homology between group A and group C rotavirus NSP4 proteins [Horie Y, et al., Arch Virol (1997) 142: 1865-1872]. To test whether group C rotavirus NSP4 has an enterotoxigenic activity, we expressed in Escherichia coli the carboxy two-thirds (corresponding to amino acid residues 55-150) of the NSP4 protein derived from group C rotavirus strain Ehime 9301. This truncated NSP4 protein was able to induce diarrhea in 5-day-old CD-1 mice when administered intraperitoneally. Thus, group C rotavirus NSP4 acts as an enterotoxin like group A rotavirus NSP4.

Animal Population Groups↗
PubMed1991

Outer capsid glycoprotein vp7 is recognized by cross-reactive, rotavirus-specific, cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) generated in mice orally inoculated with rotaviruses lyse target cells infected with different rotavirus serotypes (cross-reactive CTLs). Using vaccinia virus recombinants expressing individual rotavirus proteins from two different rotavirus serotypes, we found that cross-reactive CTLs recognize target cells expressing outer capsid protein vp7 better than those expressing outer capsid protein vp4 or inner capsid protein vp6. These findings may be relevant to vaccine strategies which include immunization with reassortant rotaviruses or viral or bacterial vectors expressing individual rotavirus proteins. The region or regions of vp7 which are antigenically conserved among different rotavirus serotypes and recognized by cross-reactive CTLs remain to be determined.

Animals↗
PubMed1991

Comparative growth of different rotavirus strains in differentiated cells (MA104, HepG2, and CaCo-2).

The production of viral antigen after infection of MA104, HepG2 (derived from human liver), and CaCo-2 (derived from human colon) cells with various cultivatable human and animal rotavirus strains was compared using immunofluorescence tests. All rotavirus strains examined expressed antigen in CaCo-2 cells and MA104 cells, but only some virus strains, namely, SA11-Cl3 (simian), RRV (simian), CU-1 (canine), and Ty1 (turkey), produced antigen in numbers of infected HepG2 cells comparable to infections in MA104 and CaCo-2 cells. Fl-14 (equine), OSU (porcine), NCDV (bovine), and Ch2 (chicken) strains were found to infect moderate numbers of HepG2 cells. Most human rotaviruses (representing viruses in serotypes 1, 2, 3, 4, 8, and 9), a simian rotavirus variant (SA11-4F), lapine (Ala, C-11 and R-2) viruses and porcine (Gottfried) virus infections resulted either in no detectable antigen or antigen synthesis in a low percentage of HepG2 cells. Human rotavirus isolates obtained from the stool specimens of an immunocompromised child with rotavirus antigen in his liver showed two different patterns of replication in HepG2 cells. Examination of the replication of a subset of viruses in the liver and intestinal tissues of orally infected suckling mice showed the CU-1 and Ty1 strains replicated well, while the OSU and human rotavirus strains did not. These results indicate that growth restriction in HepG2 cells is not serotype-specific, and growth of a virus in HepG2 cells does not necessarily correlate with the hepatotropic potential of a virus strain. Factors that may influence these differences of virus infectivity in HepG2 cells are discussed.

Animals↗
PubMed1991

Human rotavirus strain 69M has a unique VP4 as determined by amino acid sequence analysis.

The group A rotavirus strain 69M has recently been classified as a new rotavirus serotype, G8, based on the antigenic specificity of its VP7 outer capsid glycoprotein. The fourth gene of 69M, which encodes the other outer capsid protein VP4, was sequenced. The gene is 2362 nucleotides in length and contains one long open reading frame beginning at the 10th nucleotide from the 5' end and terminating with a stop codon 22 bases from the 3' end. The encoded VP4 contains 776 amino acids. Comparative analysis of the deduced amino acid sequence with other rotavirus strains demonstrated that the VP4 of strain 69M shares 68.9 to 74.5% amino acid identity with the VP4 from strains known to represent four human rotavirus VP4 genetic alleles and 74.9 to 86.2% identity with VP4 from various animal rotaviruses. Northern blot hybridization with either radiolabeled ssRNA transcripts from 69M or cloned 69M gene 4 cDNA as probes indicated that the fourth gene of strain 69M is genetically distinct from any group A rotavirus gene 4 described thus far. These data suggest that at least one additional antigenic specificity of VP4 exists in circulating human group A rotavirus strains.

Amino Acid Sequence↗
PubMed1992

Amino acid sequence analysis of bovine rotavirus B223 reveals a unique outer capsid protein VP4 and confirms a third bovine VP4 type.

The nucleotide and deduced amino acid sequence of the gene 4 of bovine rotavirus strain B223 is described. The open reading frame is predicted to encode a VP4 of 772 amino acids, shorter than described for any other rotavirus strain sequenced to date. B223 VP4 shows 70 to 73% similarity to other rotavirus VP4 proteins, demonstrating the presence of a unique VP4 type, and confirming a third VP4 allele in the bovine rotavirus population. Multiple sequence alignment with several other rotavirus strains created gaps in the sequence to account for a shorter VP4. The alignment shows a two contiguous amino acid deletions within the trypsin cleavage region of B223 VP4. Comparisons of two regions flanking the trypsin cleavage site, (aa 224 to 235, and aa 257 to 271) which show high homologies between strains, demonstrate that the region 5' to the trypsin cut site has a low homology (66%) to other rotavirus strains, although the region 3' to the trypsin cleavage site shows high homologies (86 to 93%) with other rotavirus strains. The lack of a conserved proline residue within the 5' flanking region suggests a possible altered local conformation of this site in B223 VP4. A second gap inserted into the VP4 of B223 on multiple sequence alignment is a three contiguous amino acid deletion at position 613-615 in the VP5* subunit. Previously defined biologic properties of this strain in relation to the determination of the amino acid composition of VP4 are discussed.

Alleles↗
PubMed1984

An improved enzyme-linked immunosorbent assay for the detection of rotavirus in faeces of neonates.

A direct enzyme-linked immunosorbent assay (EIA) for the detection of rotavirus in neonatal stools was developed. Rabbit antiserum against SA 11 rotavirus was incorporated as both coating and detector antibody, and rotavirus-negative rabbit serum was applied as a coating antibody control to eliminate false positive results. Pretreatment of stools with EDTA was found to increase both the sensitivity and specificity of the assay. This effect was greatest when 0.25 M EDTA (tetrasodium salt) was included in homogenized stool suspensions before the removal of solid debris by centrifugation. By electron microscopy, this EDTA pretreatment appeared to partly uncoat human rotavirus particles in faeces. Potentially suitable solid phase supports and horseradish peroxidase substrates were evaluated in the development of the assay. Screening of stool samples revealed that repeated freezing and thawing of stools eliminated positive EIA reactions. The SA 11 coating antibody compared favourably with a reference coating antiserum prepared against human faecal rotavirus strains. This EIA showed greater sensitivity for rotavirus detection than electron microscopy of stool concentrates prepared by ultracentrifugation, on testing 143 stools from 99 neonates and children. The assay has been applied successfully to detection of rotavirus in stools of neonates containing meconium, smaller amounts of viral antigen than in older children, and lacteal antirotaviral antibody. It is likely to be particularly useful for cross-infection studies in hospital wards and neonatal nurseries.

Animals↗
PubMed1983

Intestinal changes associated with rotavirus and enterotoxigenic Escherichia coli infection in calves.

Newborn calves inoculated with rotavirus, enterotoxigenic Escherichia coli (ETEC) serotype 020:K' x 106':K99:HNM, either alone or in combination, became depressed, anorectic, diarrhoeic and dehydrated. ETEC did not adhere to the intestine although there was extensive proliferation in the lumen. Only slight mucosal changes were induced by ETEC and the activity of membrane bound lactase remained normal. More severe mucosal damage and a decrease in lactase activity were found in newborn calves inoculated with either rotavirus or rotavirus and ETEC in combination. The most severe clinical illness was found in calves inoculated with both rotavirus and ETEC. Calves inoculated at 1 week of age with either rotavirus or ETEC remained clinically normal. Rotavirus infection produced slight mucosal changes and a reduction of lactase activity. In contrast, colostrum-fed or suckling calves up to 2 weeks old inoculated with both rotavirus and ETEC became clinically affected, showed severe mucosal damage and decreased lactase activity. There was no bacterial adhesion to the intestinal mucosa as observed by immunofluorescent labelling and light microscopy.

Animals↗
PubMed1990

Development of specific nucleic acid probes for the differentiation of porcine rotavirus serotypes.

A dot blot hybridization assay is described for the detection and differentiation of porcine rotavirus serotypes. Recombinant complementary DNA (cDNA) representing gene 9 (the gene encoding the neutralization antigens in VP7 glycoprotein) from OSU (porcine rotavirus serotype 1) and Gottfried (porcine rotavirus serotype 2) strains were used to determine the optimal hybridization conditions which allow specific detection of group A porcine rotaviruses. Probes were prepared by excision of the inserts from the recombinant plasmids and radiolabeling of cDNA with 32P by the random primer extension method. Probes were hybridized at various stringencies with viral RNA from different rotavirus serotypes bound to nylon membranes. Hybridization at low stringency (26% base pair mismatch for stable hybrid formation) had high sensitivity but low specificity. Hybridization at high stringency (16% base pair mismatch for stable hybrid formation) produced high specificity but decreased the sensitivity observed at low stringency. Probes were specific for rotavirus at both stringencies and did not hybridize with nucleic acids from other porcine viruses. Subgenomic gene 9 fragments were then tested to provide more specific probes. A 322 bp fragment from OSU gene 9 between nucleotides 382 and 704 and a 266 bp fragment from Gottfried gene 9 between nucleotides 230 and 496 were found to be specific as hybridization probes. These studies demonstrated the feasibility of the dot blot hybridization assay using subgenomic fragments of gene 9 to detect and differentiate serotypes of porcine rotavirus. Additional studies are warranted to further evaluate the sensitivity and the capability of these probes to detect porcine field isolates of the same serotype.

Animals↗
PubMed2001

Winter seasonality and rotavirus diarrhoea in adults.

We investigated the aetiological role of group A rotavirus in adults with acute diarrhoea in a 4-year prospective study. Of 683 patients with acute diarrhoea, 97 (14%) shed rotavirus as a sole agent, whereas six (5%) of 115 patients without diarrhoea shed rotavirus. Half of patients with rotavirus diarrhoea required admission to hospital. Unlike rotavirus diarrhoea in children, the occurrence of rotavirus-positive cases did not show a significant winter seasonality. Rotavirus infection should be included in the differential diagnosis of diarrhoeal diseases in adults.

Adult↗