Rotavirus acceleration of murine type 1 diabetes is associated with a T helper 1-dependent specific serum antibody response and virus effects in regional lymph nodes.

AIMS/HYPOTHESIS: Rotavirus infection in at-risk children correlates with production of serum autoantibodies indicative of type 1 diabetes progression. Oral infection with rhesus monkey rotavirus (RRV) accelerates diabetes onset in mice. This relates to their rotavirus-specific serum antibody titre and local pro-inflammatory cytokine induction without pancreatic infection. Our aim was to further investigate the roles of serum antibodies and viral extra-intestinal spread in diabetes acceleration by rotavirus. METHODS: Rotavirus-specific serum antibody production was detected by ELISA in diabetes-prone mice given either inactivated or low-dose RRV, in relation to their diabetes development. Serum anti-rotavirus antibody titres and infectious virus in lymph nodes were measured in mice given RRV or porcine rotavirus CRW-8. In lymph node cells, rotavirus antigen presence and immune activation were determined by flow cytometry, in conjunction with cytokine mRNA levels. RESULTS: Acceleration of diabetes by RRV required virus replication, which correlated with antibody presence. CRW-8 induced similar specific total immunoglobulin and IgA titres to those induced by RRV, but did not accelerate diabetes. RRV alone elicited specific serum IgG antibodies with a T helper (Th)1 bias, spread to regional lymph nodes and activated antigen-presenting cells at these sites. RRV increased Th1-specific cytokine expression in pancreatic lymph nodes. Diabetes onset was more rapid in the RRV-infected mice with the greater Th1 bias. CONCLUSIONS/INTERPRETATION: Acceleration of murine diabetes by rotavirus is virus strain-specific and associated with virus spread to regional lymph nodes, activation of antigen-presenting cells at these sites and induction of a Th1-dominated antibody and cytokine response.

Rotavirus-neutralizing antibodies inhibit virus binding to integrins alpha 2 beta 1 and alpha 4 beta 1.

Rotavirus outer capsid proteins VP5(*), VP8(*) and VP7 elicit neutralizing, protective antibodies. The alpha 2 beta 1 integrin is a cellular receptor for rotavirus that is bound by VP5(*). Some rotaviruses also recognize the alpha 4 beta 1 integrin. In this study, the effects of antibodies to rotavirus on virus binding to recombinant alpha 2 beta 1 and alpha 4 beta 1 expressed on K562 cells were determined. All neutralizing monoclonal antibodies to VP5(*) tested (YO-2C2, 2G4, 1A10) and two to VP7 (RV-3:2, RV-4:2) inhibited rotavirus binding to alpha 2 beta 1. Rotavirus binding to alpha 4 beta 1 was reduced by 2G4 and neutralizing antibody F45:2, directed to VP7. However, a neutralizing antibody to VP8(*) (RV-5:2) and one to VP7 (RV-3:1) did not affect rotavirus binding to these integrins. Virus-cell binding was unaffected by non-neutralizing antibody RVA to the rotavirus inner capsid protein VP6. The attachment of human rotavirus strain Wa to these integrins was inhibited by infection sera with neutralizing activity collected from two children hospitalised with severe rotavirus gastroenteritis. A negative reference serum did not affect rotavirus-cell attachment. As the binding of rotaviruses to alpha 2 beta 1 and alpha 4 beta 1 is inhibited by neutralizing antibodies to VP5(*) and VP7, and serum from children with rotavirus disease, rotavirus recognition of these integrins may be important for host infection.

Phorbol dibutyrate-induced megakaryocytic differentiation increases susceptibility of K562 cells to SA11 rotavirus infection.

The role of integrins previously implicated as rotavirus receptors in determining cellular susceptibility to SA11 rotavirus was studied, using phorbol dibutyrate (PDB) treatment of K562 cells to induce megakaryocytic differentiation. Expression of alpha2beta1 integrin was detected after 2 days in PDB, and peaked after PDB treatment for 4-7 days. SA11 titres were increased by 1.8- to 10.8-fold over untreated cells after PDB treatment for 2-7 days, and correlated with levels of alpha2beta1 integrin expression in PDB-treated K562 cells.

Association between rotavirus infection and pancreatic islet autoimmunity in children at risk of developing type 1 diabetes.

Pancreatic islet autoimmunity leading to type 1 diabetes could be triggered by viruses in genetically susceptible individuals. Rotavirus (RV), the most common cause of childhood gastroenteritis, contains peptide sequences highly similar to T-cell epitopes in the islet autoantigens GAD and tyrosine phosphatase IA-2 (IA-2), suggesting T-cells to RV could trigger islet autoimmunity by molecular mimicry. We therefore sought an association between RV infection and islet autoantibody markers in children at risk for diabetes who were followed from birth. There was a specific and highly significant association between RV seroconversion and increases in any of these antibodies: 86% of antibodies to IA-2, 62% to insulin, and 50% to GAD first appeared or increased with increases in RV IgG or IgA. RV infection may therefore trigger or exacerbate islet autoimmunity in genetically susceptible children.

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells.

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.

Comparison of enzyme immunoassay and reverse transcriptase PCR for identification of serotype G9 rotaviruses.

While only four globally important rotavirus G serotypes (1 to 4) have been documented, many studies suggest that serotype G9 viruses may be widely distributed and more important than previously recognized. We have evaluated 10 serotype G9 rotavirus-neutralizing monoclonal antibodies (MAbs) directed to VP7, which bound by direct enzyme immunoassay (EIA) to P1A[8], G9 rotaviruses F45, WI61, and AU32, for their ability to recognize the New Delhi G9 rotavirus 116E. Only one MAb (MAb F45:1) bound to P[11], G9 virus 116E to a high titer by EIA. This MAb was incorporated into an indirect EIA for G serotyping, which was validated with prototype cultivable human rotaviruses of G types 1 to 4 and 9. The EIA was compared with genotyping by reverse transcriptase PCR (RT-PCR) under code for the determination of the G types of rotaviruses obtained from neonates in New Delhi, India. The sensitivities of RT-PCR and EIA (after two additional freeze-thaw cycles) for the typing of G9 rotaviruses were 91 and 86%, respectively, for 24 culture-adapted rotavirus strains. The untypeable culture-adapted rotavirus samples also were unreactive with VP7 group antigen-reactive MAb 60. After two additional freeze-thaw cycles, only 26 of 42 (62%) of stools containing rotavirus typed as G9 by RT-PCR were positive for G9 rotavirus by EIA. Stools containing rotavirus untypeable by EIA contained significantly less MAb 60-reactive VP7 antigen (P = 0. 0001) than the stools containing typeable rotavirus. Thus, RT-PCR genotyping was the more sensitive method for determination of G9 type, but a serotype was readily determined in rotavirus samples containing MAb 60-reactive VP7 antigen by an EIA that incorporates MAb F45:1.

Longitudinal studies of neutralizing antibody responses to rotavirus in stools and sera of children following severe rotavirus gastroenteritis.

Rotavirus-neutralizing antibody responses in sera and stools of children hospitalized with rotavirus gastroenteritis and then monitored longitudinally were optimally detected by using local rotavirus strains. Stool responses were highest on days 5 to 8 after the onset of diarrhea. Longitudinal monitoring suggested that serum neutralizing antibody responses were a more useful measure of severely symptomatic rotavirus infection than stool responses but that stool antibody responses may be a useful measure of rotavirus immunity.

Detection and characterization of novel rotavirus strains in the United States.

We recently established a rotavirus strain surveillance system in the United States to monitor the prevalent G serotypes before and after the anticipated implementation of a vaccination program against rotavirus and to identify the emergence of uncommon strains. In this study, we examined 348 rotavirus strains obtained in 1996 to 1997 from children with diarrhea in 10 U.S. cities. Strains were characterized for P and G types, subgroups, and electropherotypes by using a combination of monoclonal antibody immunoassay, reverse transcription-PCR, and hybridization. The four strains most commonly found worldwide comprised 83% of the isolates (P[8]G1, 66.4%; P[4]G2, 8.3%; P[8]G3, 6.9%; P[8]G4, 1.4%), but 9.2% were unusual strains (P[6]G9, 5.5%; P[8]G9, 1.7%; P[6]G1, 1.4%; and P[4]G1 and P[8]G2, 0. 3% each). Strains not typeable for P or G type accounted for 5.5% of the total, while 2.3% of the strains had more than one G type (mixed infections). All P[6]G9 strains tested had short electropherotypes and subgroup I specificity and were detected in 4 of 10 cities, while P[8]G9 strains had long electropherotypes and subgroup II VP6 antigens. Both sequence analysis of the VP7 open reading frame (about 94 to 95% amino acid identity with the VP7 gene of G9 prototype strain WI61) and binding to a G9-specific monoclonal antibody strongly suggest that U.S. G9 strains belong to serotype G9. The high detection rates of unusual rotaviruses with G9 (7.2%) or P[6] (6.9%) specificity in multiple U.S. cities suggest the emergence of new strains or inadequate diagnosis in the past. The epidemiologic importance of these strains remains to be determined.

Attachment and growth of human rotaviruses RV-3 and S12/85 in Caco-2 cells depend on VP4.

Studies with human neonatal rotaviruses RV-3 and S12/85 and their reassortants showed that VP4 is a determinant of rotavirus attachment to and growth in Caco-2 cells. The binding of these viruses to MA104 and Caco-2 cells correlated with their growth ability. Virus sensitivity to trypsin and the VP4 fusion region may be implicated in these processes.

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.

Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells.

Rotavirus contains two outer capsid viral proteins, the spike protein VP4 and major capsid component VP7, both of which are implicated in cell entry. We show that VP4 and VP7 contain tripeptide sequences previously shown to act as recognition sites for integrins in extracellular matrix proteins. VP4 contains the alpha2beta1 integrin ligand site DGE. In VP7, the alphaxbeta2 integrin ligand site GPR and the alpha4beta1 integrin ligand site LDV are embedded in a novel disintegrin-like domain that also shows sequence similarity to fibronectin and the tie receptor tyrosine kinase. Microorganism sequence homology to these ligand motifs and to disintegrins has not been reported previously. In our experiments, peptides including these rotaviral tripeptides and mAbs directed to these integrins specifically blocked rotavirus infection of cells shown to express alpha2beta1 and beta2 integrins. Rotavirus VP4-mediated cell entry may involve the alpha2beta1 integrin, whereas VP7 appears to interact with alphaxbeta2 and alpha4beta1 integrins.

Multiple-gene rotavirus reassortants responsible for an outbreak of gastroenteritis in central and northern Australia.

Two rotavirus strains, E210 and E212, implicated in epidemics of gastroenteritis in children in central and northern Australia during 1993-1994, exhibited the unusual combination of a 'short' RNA electrophoretic pattern and subgroup II specificity. The outer capsid protein VP7 was found by PCR typing and sequence analysis to be related to that of serotype G2 viruses. Both strains displayed a novel pattern of reactivity to G2-specific monoclonal antibodies that correlated with sequence variation in the antigenic regions of VP7. The VP4 serotype of E210 and E212 was determined as P1B in an enzyme immunoassay, consistent with other G2 viruses. Analysis of the VP6 gene indicated significant identity (98-99%) with other human subgroup II viruses. Northern hybridization analysis of E210 RNA using total genome probes derived from the prototype strains RV4 and RV5 indicated that E210 was derived from multiple gene reassortment between rotaviruses belonging to different genetic types.

Amino acids involved in distinguishing between monotypes of rotavirus G serotypes 2 and 4.

Neutralizing monoclonal antibodies (N-MAbs) to serotype G2 and G4 rotaviruses were used to study intraserotypic variation by selection and characterization of N-MAb-resistant antigenic variants and reaction of N-MAbs with prototype rotavirus strains. Two G2-specific N-MAbs reacted with G2 rotaviruses S2, DS-1, RV-5 and RV-6 but not with 1076. Sequence analysis of the gene encoding VP7 of 1076 virus showed that the differences in amino acid sequence between 1076 virus and the other G2 strains at position 147, 213 and 217 correlated with the loss of N-MAb reactivity. Rotavirus variant mutation mapping data suggested that the amino acid difference at position 213 was likely to be of greatest importance. Rotavirus 1076 was defined as monotype b within G2 strains, whereas S2, DS-1, RV-5 and RV-6 belong to monotype a. The molecular basis for G4 subtypes/monotypes was also studied. The monotype G4b N-MAb 3A3 selected an antigenic variant with an amino acid mutation at position 96, whereas variants of the G4a-reactive N-MAb ST-3:1 showed a mutation at position 94, which produced a new, utilized glycosylation site. Neutralization by N-MAb ST-3:1 was also affected by amino acid changes at position 96. Reactions with these N-MAbs show that serotype G2 viruses can be divided into monotypes and confirm the observation that serotype G4 rotaviruses can be subdivided into subtypes/monotypes a and b. The G2 monotypes relate to differences at particular amino acids within antigenic region C and possibly region B, whereas antigenic region A is most important for G4 monotype differentiation.

Human rotavirus VP4 contains strain-specific, serotype-specific and cross-reactive neutralization sites.

The neutralization epitopes of human rotavirus VP4 were studied by using a panel of neutralization monoclonal antibodies previously shown to be strain-specific (RV-3:3), serotype-specific (RV-5:2, ST-3:3) or cross-reactive (F45:4). Antigenic variants of human rotaviruses RV-3, ST-3, RV-5 and F45 resistant to neutralization by the appropriate of VP4 specific monoclonal antibodies (RV-3:3, ST-3:3, RV-5:2 and F45:4 respectively) were selected. By nucleotide sequence analysis and single strand conformational polymorphism analysis of these variants, three sites of neutralization on VP5* and one site on VP8* were identified. At or near to the putative fusion region on VP5*, a strain-specific site (aa383), a serotype P1A-P2 cross-reactive site (aa392) and a serotype P2-specific site (aa397) were found. On VP8*, a serotype P1B-specific site at aa148 was detected. These results confirmed the importance of the putative fusion region in neutralization and have identified a new neutralization site in the hypervariable region of VP8* which is specific for serotype P1B human rotaviruses.

G3P2 rotaviruses causing diarrhoeal disease in neonates differ in VP4, VP7 and NSP4 sequence from G3P2 strains causing asymptomatic neonatal infection.

During longitudinal epidemiological studies of rotavirus infections in children in Melbourne, Australia human G3P2 rotavirus strains causing asymptomatic or symptomatic infections have been identified. Eleven strains (AS strains) associated with asymptomatic infection of newborn babies from 1974-1984, and five strains (S strains) associated with symptomatic infection of newborn babies (4) or a 22 week old infant (1) during 1980-1986 were studied. The entire nucleotide sequences of genes coding for VP4, VP7, NSP4 and VP6 were derived for representative AS and S strains. The nucleotide sequences of neutralization epitope regions present on the outer capsid proteins VP4 and VP7 (regions C and F) showed extensive conservation of nucleotide and deduced amino acid sequence in all strains. Minor variations were observed over the 12 year period in VP7 epitope regions A and B in some strains. Specific conserved amino acids differences between the asymptomatic and symptomatic strains were observed in the genes encoding VP4 at aa133 and 303 (asparagine or threonine) and 380 (serine or isoleucine), VP7 at aa27 (threonine or isoleucine), aa29 (isoleucine or threonine), aa42 (valine or alanine) and aa238 (asparagine or aspartic acid/serine) and NSP4 at aa135 (isoleucine or valine). No amino acid changes were identified in gene 6. The observed amino acid differences occurred in proteins that have been implicated in virulence, and correlate with differences in clinical symptoms of infants infected with these strains. These results permit speculation about the genetic basis for virulence of human strains.

VP4 and VP7 typing using monoclonal antibodies.

Both rotavirus outer capsid proteins, VP4 and VP7, elicit neutralizing antibodies. Neutralizing mouse monoclonal antibodies (N-MAbs) to VP7 are easily derived and have been used widely and successfully to serotype both stool-derived and culture-adapted rotaviruses by enzyme immunoassay (EIA). Generally, approximately 70% of rotaviruses in stool samples are typable by VP7 EIA, an inexpensive and practical method. Variations in antigenic regions between strains within human rotavirus serotypes 1, 2, 4, and 9 have been recorded. These have been termed monotypes because they are detected with N-MAbs. The molecular basis for monotypes has been determined by mapping mutations selected in N-MAb-resistant antigenic variants, and by sequence analysis of the gene encoding VP7 in newly recognized monotypes. Antigenic regions A, B and C in VP7 are involved. In order to detect all members of a particular VP7 serotype, it is necessary to type with a panel of N-MAbs specific for that serotype. N-MAbs to VP4 of human rotavirus are difficult to raise and few have proven suitable for VP4 serotyping by EIA. The specificity of the assay for each P type is highest when the VP7 serotype specificity of the capture antiserum is matched to the G type of the rotavirus in the test sample. The VP4 EIA gives similar typing rates to the VP7 typing EIA. N-MAbs directed to VP8, the smaller subunit of VP4 generated by proteolytic cleavage, are more likely to show serotype specificity. Some N-MAbs that select mutations in the putative fusion region of VP5, the larger subunit of VP4, show cross-reactivity with extracts of normal, uninfected MA 104 cells and with fetal bovine serum. These N-MAbs also give elevated EIA OD readings with rotavirus-positive, but previously non-reactive fecal samples which have been frozen and thawed repeatedly. Overall, VP8-reactive N-MAbs appear most suitable for VP4 typing by EIA.

Rotavirus antigenicity is affected by the genetic context and glycosylation of VP7.

Rotavirus variants resistant to neutralization were selected using monoclonal antibodies (N-MAbs) raised to VP7 of rotavirus G types 2, 3, and 6. Their neutralization resistance patterns and deduced VP7 amino acid sequences were obtained. Variants selected by two G2-specific N-MAbs from the homologous parent virus RV-5 showed single amino acid (aa) mutations in the antigenic A region. However, variants selected from reassortant virus RV-5 x SA11 (all genes from SA11 virus except that encoding VP7, which was from RV-5 virus) fell into two neutralization resistance groups. The first group showed identical mutations to the variants selected from RV-5 virus. The second group showed antigenic C region mutations, either alone or in combination with a mutation at aa 69. Variants selected from G3 parent viruses glycosylated at position 238 had a mutation at aa 96 in the A region, otherwise a C-region mutation at 211 was selected. Mutations at amino acid positions 94 or 96 were selected by monoclonal antibodies specific for each of the three serotypes. G3-specific monoclonal antibodies also selected mutations at position 148 and the new position of 264. This latter mutation resulted in substitution of aspartic acid for glycine and was located in a highly conserved and hydrophobic region of VP7. A G2-specific N-MAb selected variants with a mutation at aa 190 producing a new, utilized glycosylation site which we propose to be in new antigenic site E. The positions of mutations in antigenic variants and their antigenicity were determined by parental background genes and VP7 glycosylation.

Characteristics and location of cross-reactive and serotype-specific neutralization sites on VP7 of human G type 9 rotaviruses.

The neutralization antigens of human rotavirus VP7 were studied by producing eight neutralizing monoclonal antibodies to G type 9 rotaviruses F45 and WI61 and selecting antigenic variants resistant to neutralization by these monoclonal antibodies. Neutralization resistance patterns and sequence analysis of the antigenic variants indicated the presence of overlapping serotype-specific and serotype cross-reactive epitopes in antigenic region A, and one distinct type-specific epitope. Cross-reactive monoclonal antibodies were more tolerant of amino acid sequence change than type-specific monoclonal antibodies. The existence of a new antigenic region, F, including amino acids 235 to 242 was confirmed. This region contained a cross-reactive epitope not detectable in the presence of glycosylation at amino acid 238. This glycosylation also affected neutralization by a cross-reactive monoclonal antibody directed to antigenic region C. Antigenic regions A, B, C, and F all contain epitopes shared between G types, of which at least two (C and F) are affected by glycosylation.

Typing of human rotavirus VP4 by an enzyme immunoassay using monoclonal antibodies.

Two different neutralization specificities exist on the outer capsid of group A rotaviruses. At least seven VP7 (G) antigenic types are distinguishable among human rotaviruses. Four distinct antigenic (P) types of human rotavirus VP4 corresponding to separate rotavirus gene 4 groups have been described. The aim of this study was to identify P types in clinical specimens by developing an enzyme immunoassay, using P-type-specific neutralizing monoclonal antibodies (N-MAbs). Three N-MAbs primarily or solely recognizing each of P types 4, 6, and 8 and binding to VP4 or its subunit VP5* were derived. These N-MAbs served as detector antibodies in an enzyme immunoassay P-typing system similar to that in use for G typing. P-type specificity was highest when the G-type specificity of the capture antiserum was matched to the G type of the rotavirus in the test sample. The method correctly identified the P types of 13 well-characterized, cell culture-adapted human rotaviruses and was used to classify a further six strains. P typing of 118 rotavirus-positive stools gave results consistent with the P type inferred from the G type for 98 (83%) samples. Twelve (10%) of the stools showed no reaction with any N-MAb and eight (7%) samples were untypeable because of cross-reactivity between N-MAbs or high background readings. This P-typing enzyme immunoassay system is economical and amenable to large-scale use in epidemiological studies. Its use will facilitate assessment of the distribution of P types worldwide and of the role of VP4 in eliciting protective immune responses.