Molecular and immunologic characterization of group A bovine rotavirus field isolates with P8[11] spike protein

Bovine rotavirus strain B223 is the North American prototype for group A P8[11] serotype/genotype rotaviruses. Rotaviruses with this serotype/genotype are a prevalent cause of neonatal calf diarrhea and have also been isolated from asymptomatic human infants. On the basis of deduced amino acid sequence of the outer capsid viral protein 4 (VP4), strain B223 lacks a cysteine at position 318 that is conserved among all other rotavirus strains. It has been speculated that this may result in the loss of disulfide bond and a change in the structure of the VP4 that may affect the infectivity and antigenicity of the virus. This paper describes partial sequences of the VP4 gene (nucleotides 613 to 1016 coding for amino acid positions 202 to 335) of 16 bovine rotavirus field isolates with P8[11] that were obtained from calves in Nebraska and Indiana. All the isolates lack a cystein at position 203 and had a conserved valine residue at position 318, thus indicating that the prototype strain B223 is representative of group A rotaviruses with P8[11] serotype/genotype.     

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.     

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.     

Survey on the distribution of the gene 4 alleles of human rotaviruses by polymerase chain reaction

The presence of six gene 4 alleles (or VP4 genotypes) in human rotaviruses has been recognized. Using 16 representative cultivable human rotavirus strains, we confirmed the specificity of VP4 genotyping by polymerase chain reaction (PCR) using the nested oligonucleotides specific to each of the four representative gene 4 alleles. Using the PCR, we surveyed the gene 4 alleles of 199 human rotaviruses in stools collected in Japan and Thailand. Strains with the gene 4 allele, corresponding to P1A serotype, were shown to be the most prevalent, but two strains with P2 gene 4 allele and one strain with P3 gene 4 allele were detected in Thailand and in Japan, respectively.     

Electrophoretic typing of nosocomial rotavirus infection in a general paediatric unit showing the continual introduction of community strains

During 1989 stool specimens from hospitalised children with gastroenteritis at Ga-Rankuwa Hospital in South Africa were examined for the presence of rotaviruses. Overall 16% of the children were positive for rotavirus. However, 43% of the rotavirus positive patients were infected in the hospital. Further characterisation of the rotavirus strains was performed by electrophoresis of the RNA genome and hybridisation analysis of the VP7 and VP4 genes present. The strains associated with nosocomial infection were similar to those strains acquired in the community. The majority of the strains, both community- or hospital-acquired, were associated with a serotype 1 strain with a long electrophoretype and bearing the Wa-like VP4 gene. Three minor rotavirus strains with a long electrophoretype were also observed to be circulating bearing serotype 1 or 4 VP7 genes and the Wa-like VP4 gene. Interestingly, a serotype 4 strain bearing the M37-like VP4 gene was identified to occur almost exclusively in neonates although the gene was associated with diarrhoea in these cases. Two strains with differing short RNA electrophoretypes were also observed, members of which hybridised to VP7 serotype 2 and VP4 DS-1 type probes.     

Inhibition effect of shengma-gegen-tang on measles virus in Vero cells and human peripheral blood mononuclear cells

The nucleotide sequence of gene 6 encoding the rotavirus major capsid protein VP6 of EDIM strain (EW) was determined and compared to that of 20 previously reported strains with known subgroup specificities. Multiple alignments of amino acid sequences exhibited a high level of sequence conservation (87 to 99.2%). Site-specific mutagenesis experiments were undertaken to localize regions involved in subgroup specificity. Amino acid positions 305, 315, and a region 296-299 (or 301 for equine strain H-2) were identified as contributing to subgroup epitopes. A single amino acid mutation at position 305 or 315 was sufficient to change the subgroup specificity of EW VP6 protein from non I/II to subgroup I- or subgroup II-like, respectively. Mutation at these sites may be another important mechanism for subgroup variation, along with gene reassortment.     

Characterisation of G serotype dependent non-antibody inhibitors of rotavirus in normal mouse serum

Serotype specific (non-immunoglobulin) inhibitors of rotavirus have been identified in normal mouse serum obtained from BALB/c, CBA, and BL10 mice. Sialic acid was essential for the neutralising activity sera treated with the neuraminidase from Vibrio cholerae failed to neutralise rotavirus. G serotypes 4, 5, 7, 8, 9, and 10 were unaffected by the inhibitor(s) while G serotypes 1, 2, 6 and two G3 strains were neutralised to significant titres. Assessment of neutralisation of reassortants suggested that VP7 is the virus protein involved in the interaction although it remains possible that VP7 is influencing VP4 binding. Analysis of the sera by Western blot followed by virus overlay confirmed that binding is dependent on the presence of sialic acid. The human strain tested, Wa, bound to two (glyco) proteins (50 and 80 kDa) while the bovine strains tested, NCDV and UK bound to one (55 kDa) and two (36 and 55 kDa) proteins respectively. This indicates that while the bovine rotaviruses may bind to a common element, the human strain binds to clearly distinct proteins. We propose that these inhibitors interact with animal rotaviruses in a manner analogous to that by which they attach to target cells. The glycoprotein to which NCDV bound was purified and identified by N-terminal sequencing as murine alpha-1-anti-trypsin (MuAAT) and was confirmed to possess both neutralisation and anti-trypsin activity. Since MuAAT is known to possess only three N-linked glycans, identification and analysis of the actual virus-binding structure should now be possible.     

Adverse events and discomfort in studies on healthy subjects: the volunteer''s perspective. A survey conducted by the German Association for Applied Human Pharmacology

The aim of this work was to adapt the Western blot method to analyse the humoral response to proteins of rotavirus strains having various antigens. 10 serum samples from animals immunized with SA11, 18 serum samples from hospitalized children with rotavirus infections and 17 serum samples from healthy adults were examined for rotavirus antibodies using Western blot. Antibodies against all structural rotavirus proteins were detected. The examined sera reacted differently with rotavirus proteins depending on the strain used. The highest IgG reactivity was observed for Wa and SA11, while the lowest was noted for DS-1. At the same time a different level of cross-reactivity of human sera with specific proteins of antigenically varying rotaviruses was observed. The sera taken from adults showed a lower reactivity with proteins of antigens used compared with sera from children. The exception was VP6 of the Ito strain for which the reactivity of adult sera was statistically higher. Essential differences in the reactivity of children's and adult sera were found mainly in the case of VP2 and VP4 belonging to the SA11 strain and VP4 and VP7 of the Ito strain. From these investigations we can conclude that the Western blot method may be useful in assessing immune response caused by rotaviruses. The results of reactivity of some human sera with proteins of strains used indicates that the persons had previously been infected by antigenically varied rotaviruses.     

Baculovirus expression of gene 6 of the IDIR strain of group B rotavirus (GBR): coding assignment of the major inner capsid protein

The polypeptide product of gene 6 of the IDIR strain of group B rotavirus was synthesized by means of a baculovirus expression system in order to confirm the coding assignment of the gene and to develop reagents broadly reactive with heterologous strains of Group B rotaviruses (GBR). Earlier experiments indicated that IDIR virus gene 6 encoded the group-specific, major inner capsid protein, but direct confirmation of this coding assignment was not previously reported. The expression of IDIR virus gene 6 from baculovirus recombinants resulted in production of a protein with an apparent molecular weight equivalent to that deduced from the gene sequence (44 kDa). In addition, larger recombinant proteins were also observed, and these appeared to be consistent in size with oligomers of the primary VP6 product. The expressed protein reacted with antibody directed against the IDIR agent and other strains of GBR, but no reaction was observed with antibody directed against group A rotavirus. Serologic reactivity was also observed between the gene 6 product and a monoclonal antibody directed against the GBR group-specific antigen. Antibody directed against the recombinant gene 6 product specifically reacted with the IDIR virus major inner capsid protein in an immunoblot format. These experiments conclusively demonstrated that IDIR virus gene 6 encoded the major inner capsid protein and confirmed the presence of group B-specific antigenic epitopes on the protein.     

Attenuation of a human rotavirus vaccine candidate did not correlate with mutations in the NSP4 protein gene

The NSP4 protein of a simian rotavirus was reported to induce diarrhea following inoculation of mice. If NSP4 is responsible for rotavirus diarrhea in humans, attenuation of a human rotavirus may be reflected in concomitant mutations in the NSP4 gene. After 33 passages in cultured monkey kidney cells, a virulent human rotavirus (strain 89-12) was found to be attenuated in adults, children, and infants. Nucleotide sequence analysis of the NSP4 protein gene revealed only one base pair change between the virulent (unpassaged) and attenuated 89-12 viruses, which resulted from a substitution of alanine for threonine at amino acid 45 of the encoded NSP4 protein. Because both threonine and alanine have been found at position 45 of NSP4 in symptomatic and asymptomatic human rotaviruses, neither amino acid in this position could be established as a marker of virulence. Therefore, attenuation of rotavirus strain 89-12 appears to be unrelated to mutations in the NSP4 gene.     

Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability

A group A rotavirus isolated from a lamb with diarrhea in Qinhai province, China, was serially passaged in fetal calf kidney cells. In passage 96, rearrangements of RNA segments 5 and 6 of the viral genome were found. Here we report the nucleotide and predicted amino acid sequences of normal and rearranged RNA 6, coding for the major inner capsid protein VP6. In comparison with the normal gene (N6), the rearranged RNA 6 (R6) contained the normal open reading frame followed by a 473-nucleotide (nt) duplication of the gene beginning 23 nt after the termination codon. The duplicated region starts at nt 768 and runs through to the 3' end of the gene. In accordance with the nucleotide sequence of the rearranged RNA 6, a normal-length VP6 product was found in cells infected with the mutant. However, a single-amino-acid change from proline to glutamine at position 309 slightly affected the electrophoretic mobility of the VP6 monomer of the R6 mutant and reduced the stability of VP6 trimers on gels and at low pH values compared with the normal gene product. The degree of relatedness of VP6 of the Chinese lamb rotavirus Lp14 to those of other group A rotaviruses was determined.     

The transactivation domain AF-2 but not the DNA-binding domain of the estrogen receptor is required to inhibit differentiation of avian erythroid progenitors

Insight into the origin of human rotaviruses carrying the AU-1 VP4 allele was gained by examining their genomic RNA constellation using RNA-RNA hybridization and by sequencing the VP8* portion (nucleotides 1-750) of their gene 4. AU-1 like viruses isolated in Israel from children attending outpatient clinics were classified into three sub-genogroups based on RNA-RNA hybridization analysis: Subgenogroup 1 consists of two strains (Ro-5829 and Ro-5960) which belong to the AU-1 genogroup, since all their 11 segments hybridized to AU-1 segments. Subgenogroup 2 consists of one reassortant virus (Ro-5193) of which seven RNA segments hybridized to AU-1 segments and the remaining four segments hybridized to NCDV (bovine rotavirus). Subgenogroup 3 consists of four reassortant viruses (Ro-6460, Ro-6584, Ro-6784 and Ro-7044) which had a common genome constellation: only four of their RNA segments hybridized to AU-1 and the other seven segments hybridized to NCDV segments. Sequence analysis of the VP8* gene also revealed a three level pattern of homology with the AU-1 prototype and the local AU-1 like strains which was consistent with the overall genomic (RNA-RNA) constellation: Subgenogroup 1 had 98-98.1% homology with the AU-1 prototype; Subgenogroup 2 had 96.8% homology with the AU-1 prototype and 95.6-96.7% homology with Subgenogroup 1; Subgenogroup 3 had 95.3-95.6% homology with the prototype AU-1 and 93.4-94.3% homology with Subgenogroup 1. Possible evolutionary pathways are discussed.     

Homotypic and heterotypic serum neutralizing antibody response to rotavirus proteins following natural primary infection and reinfection in children

Worldwide trials of rotavirus vaccines are currently in progress, but the basis of cross-reactive immunity between rotavirus serotypes is yet to be elucidated. The involvement of the outer capsid proteins, VP7 and VP4, in the production of cross-reactive neutralizing antibody (N-Ab) is unclear, and may be important for the success of animal rotavirus-based candidate vaccines that lack a VP4 of human rotavirus origin. In this study, VP7- and VP4-specific N-Ab was assayed in sera from children experiencing primary (27 children) and/or secondary (14 children) rotavirus infections using human-animal reassortant strains. These reassortants contained genes encoding the major G- and P-types found in human infection, including G1, 2, 3, and 4; or P1A[8], 1B[4], and 2[6]. After primary infection, the N-Ab response to VP7 was generally serotype-specific, whereas the response to VP4 was heterotypic. After reinfection (with the same or different serotypes) there was a significant increase (P=0.0313) in the number of VP7 serotypes seroconverted against with no broadening of cross-reactivity to VP4. Increases in homotypic N-Ab titer, following both primary and secondary infection, were greater against VP7 than VP4, with the seroconversion against VP7 being significantly greater upon reinfection than following primary infection (P=0.0280). In summary, heterotypic N-Ab produced following primary infection appears to be primarily against VP4. However, upon reinfection, VP7 becomes increasingly immunodominant both in terms of cross-reactive N-Ab production and increases in N-Ab titer.     

Nigerian rotavirus serotype G8 could not be typed by PCR due to nucleotide mutation at the 3' end of the primer binding site

A rotavirus strain HMG89 from Nigeria with short electrophoretic pattern was typed G3 by PCR. A cDNA clone from the PCR product which hybridised in Northern blots to RNA segment 9 of the homologous Nigerian rotavirus strain HMG89 and laboratory reference strain 69M but not to other mammalian group A rotaviruses was sequenced. The VP7 gene 9 sequence is 1060 nucleotides long with two base deletions at positions 1034-1035. Sequence analysis of the primer (aAT8) used in the previous PCR serotyping assay revealed a mutation in one of the three nucleotide bases at the 3' end of the primer binding site accounting for our inability to serotype G8 strains in our samples. These findings demonstrate that PCR analysis can, albeit infrequently, lead to error in typing of rotaviruses due to small numbers of mutations in the primer binding region.     

G (VP7) serotype-dependent preferential VP7 gene selection detected in the genetic background of simian rotavirus SA11

We previously found the preferential selection of VP7 gene from a parent rotavirus strain SA11 with G serotype 3 (G3) in the sequential passages after mixed infection of simian rotavirus SA11 and SA11-human rotavirus single-VP7 gene-substitution reassortants with G1, G2, or G4 specificity. However, it has not been known whether or not VP7 genes derived from other strains with G3 specificity (G3-VP7 gene) are preferentially selected in the genetic background of SA11. To address this question, mixed infections followed by multiple passages were performed with a reassortant SA11-L2/KU-R1 (SKR1) (which possesses VP7 gene derived from G1 human rotavirus KU and other 10 genes of SA11 origin) and one of the five G3-rotaviruses, RRV, K9, YO, AK35, and S3. After the 10th passage, selection rates of SA11-L2/KU-R1 gene 9 (G1-VP7 gene) and gene 5 (NSP1 gene) reduced considerably (0 to 20.4%) in the clones obtained from all the coinfection experiments, while all or some of other segments were preferentially selected from SKR1 depending on the pairs of coinfection. When viral growth kinetics was examined, SKR1 exhibited better growth and reached a higher titer than any G3 viruses. Although the generated reassortants with VP7 gene and NSP1 gene derived from G3 viruses showed almost similar growth kinetics to that of SKR1 during the first 20 h of replication, the titers of these reassortants were higher than that of SKR1 after 36 h postinfection. The results obtained in this study suggested that G3-VP7 gene is functionally more adapted to the genetic background of SA11.     

Molecular basis of changes in biological properties of foot and mouth disease virus of subtype A22

Primary structure of capsid proteins and RNA polymerase of three closely related strains of foot and mouth disease virus (FMDV), subtype A22, differing by biological properties (the initial epitheliotropic strain A22 550 and its derivatives: thermoresistant myotropic A22 550/4 and thermosensitive attenuated A22 645) are compared by nucleic acid sequencing and analysis of the amino acid sequencing. The study revealed 1 substitute in VPI and 8 in RNA polymerase in the myotropic variant and 1 substitute in VP2, 2 in VP3, 13 in VP1, and 3 in RNA polymerase. Alteration of A22 550/4 tropism is probably due to a single substitution Gly 145-->Thr in the RGD site of capsid protein VP1. Analysis of the origin and biological properties of the attenuated strain A22 645 and the results of studies of the primary structure of proteins permit us to hypothesize that attenuation is polygenic, caused by adaptation to a heterologous host (continuous porcine cell culture), and can be expressed by changes in the structure of virus antireceptor providing its binding to cell receptors. Sites responsible for the reproduction of A22 FMDV at certain temperatures are presumably located in RNA polymerase.     

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.     

Capsid protein VP1 deletions in JC virus from two AIDS patients with progressive multifocal leukoencephalopathy

PCR on 52 cerebrospinal fluid (CSF) specimens and 33 brain biopsies obtained from HIV-1 positive patients utilized pairs of primers from both the early region (JTP) and late region (JLP). In these patients, in whom progressive multifocal leukoencephalopathy (PML) was suspected on the basis of clinical symptoms and magnetic resonance imaging (MRI) studies, eight CSFs (15%) and 14 brain biopsy specimens (42%) contained JCV DNA sequences. In two patients' samples, the CSFs were positive for JCV DNA in the VP1 region using the primer pair for the VP1 region (JLP), but the fragment amplified migrated more rapidly than the 129-bp product obtained from prototype JCV(Mad-1) or the fragment amplified from the antigenic variant of JCV known as Mad-11. These patients died 3-4 months after onset of progressive neurological symptoms. Cycle sequencing of the fragments revealed overlapping deletions of 24 and 27 nucleotides. These strains were of different genotypes, designated strain 107 and strain 206. Computer analysis of the VP1 amino acid sequence predicts that the eight or nine amino acid residue deletions represent a surface loop with a high antigenic index. These naturally occurring deletion mutants are the first examples of a phenomenon observed experimentally in the mouse polyoma virus capsid protein VP2.