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.     

Monitoring of rotavirus infection in a paediatric hospital by RNA electrophoresis

During the spring of 1987 and the autumn of 1988, stool specimens were collected from infants and young children in the paediatric unit at H. F. Verwoerd Hospital, Pretoria, and examined for the presence of rotaviruses to assess the potential for hospital-acquired infection in the paediatric wards. Stool samples were also collected from children admitted to the hospital for causes unrelated to gastro-enteritis to investigate the possible asymptomatic carriage of rotavirus in this population. Hospital-acquired rotavirus infection was determined in only 9% of cases. Very little asymptomatic carriage of the virus was identified. Electrophoretic analysis of the rotavirus strains showed that the majority of the infections (20 of 42) were associated with a particular strain with a long RNA profile, while 7 minor strains co-circulated (5 with a long electrophoretype and 2 with a short one). An apparent small outbreak of nosocomial infection with a single strain was observed to occur in one of the paediatric wards during the spring and early summer.     

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.     

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.     

Genetic and antigenic analysis of human rotavirus prevalent in Al-Taif, Saudi Arabia

The subgroup, serotype and electropherotype diversity of human rotavirus strains was investigated in Al-Taif, Saudi Arabia. Out of 349 faecal samples collected from diarrhoeic children, 150 (43 percent) tested rotavirus positive by a group-A specific enzyme-linked immunosorbent assay (ELISA). The majority (87 percent) of the infected children were below 2 years of age. Subgrouping and serotyping of rotaviruses with specific monoclonal antibodies showed that of the 150 rotavirus positive specimens, 17 percent belonged to subgroup I, 59 per cent belonged to subgroup II, and 24 percent were neither subgroup I nor subgroup II. The specimens were typed, as serotype 1 (43 percent), serotype 2 (5 percent), serotype 3 (11 percent), serotype 4 (10 percent) or mixed serotypes (3 percent). The remaining 41 (27 percent) specimens were untypeable. None of the serotypes showed association with a particular age group. An electrophoretic analysis of viral RNA revealed 11 distinct patterns (six long and five short). The majority, 78 percent were long patterns and 22 percent were short patterns. Analysis of the specimens for which subgroups, serotypes and electropherotypes were available indicated that a given RNA pattern does not correspond to a particular subgroup or serotype.     

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.     

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.     

Nucleotide sequence of the VP4-encoding gene of an unusual human rotavirus (HCR3)

Human rotavirus strain HCR3 was isolated from the stool of a clinically normal infant and identified as a serotype G3 rotavirus; however, it could not be grouped into any known human VP4 genetic groups by a polymerase chain reaction assay. The fourth gene of strain HCR3, which encodes the outer capsid protein VP4, was sequenced. This gene is 2362 nucleotides in length and contains one open reading frame capable of encoding a protein of 776 amino acids. The VP4 protein of strain HCR3 shared 67.5-73.5% amino acid identity with those of strains KU, RV-5, 1076, and K8, representing four human genetic groups, and relatively high homology (84.7%) with a fifth genetic group represented by strain 69M, whose VP4 shows more similarity to animal than to human strains. Strain HCR3 shared higher VP4 amino acid homology with various animal rotaviruses, ranging from 74.5 to 89.4%. These observations suggest that the VP4 outer capsid protein of strain HCR3 represents a new VP4 genetic group that is more closely related to animal rotaviruses than to human rotaviruses.     

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.     

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.     

Genetic variation in the VP7 gene of human rotavirus serotype 3 (G3 type) isolated in China and Japan

Sequence analysis of the VP7 gene was performed on twenty-one human isolates of serotype 3 related-rotavirus in China and Japan. The five Chinese isolates were found to be not similar to the 16 Japanese isolates and to SA11 (simian rotavirus). The Chinese isolates, especially CHW2 and CH-32, were different from the major serotype 3 human isolates. AU-1 and 02/92 which previously showed a wider spacing between RNA segments 10 and 11 by RNA polyacrylamide gel electrophoretic analysis, were more closely related to each other and could be differentiated from the other Chinese and Japanese isolates. For these reasons, serotype 3 viruses were considered to be intraserotypically more heterologous than serotype 1, 2 and 4 viruses.     

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.     

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.     

Managing pain after coronary artery bypass surgery

From June 1992 to May 1993, rotaviruses were detected by an immunoenzymatic assay in 159 (49.5%) of 321 children admitted to the hospital with acute diarrhea. Of the 159 cases ELISA positive, 80 samples were chosen at random to investigate subgroups and serotypes of group A human rotavirus. By the ELISA test 9 (11.3%) of the strains were subgroup I, 46 (57.5%) were subgroup II, and 25 (31.3%) could not be grouped. The serotype G1 was identified in 52 cases (65%), G2 in 11 cases (13.8%), G3 in 1 case (1.2%), and 7 cases (8.8%) showed more than one serotype. By electrophoretic analysis of viral RNA, 137 (42.7%) of the samples exhibited an RNA pattern. The long pattern (59.1%) prevailed over the short pattern (35.8%), and by coelectrophoresis 8 different electropherotypes were found throughout the period of study. These results illustrate the great variety of rotavirus strains in this region of the country.     

Particle-bombardment-mediated DNA vaccination with rotavirus VP4 or VP7 induces high levels of serum rotavirus IgG but fails to protect mice against challenge

We recently reported that epidermal immunization using the PowderJet particle delivery device with plasmid vector pcDNA1/EDIM6 encoding rotavirus VP6 of murine strain EDIM induced high levels of serum rotavirus IgG but failed to protect mice against EDIM infection (Choi, A. H., Knowlton, D. R., McNeal, M. M., and Ward, R. L. (1997) Virology 232, 129-138.). This was extended to determine whether pcDNA1/EDIM4 or pcDNA1/EDIM7, which encode either rotavirus VP4 or VP7, the rotavirus neutralization proteins, could also induce rotavirus-specific antibody responses and if these responses resulted in protection. Titers of rotavirus serum IgG increased with the first dose in mice immunized with pcDNA1/EDIM7, but little or no serum rotavirus IgG was detected in mice immunized with pcDNA1/EDIM4. In vitro assays with these plasmids in rabbit reticulocyte lysates showed that VP4 was expressed but the amount was considerably lower than VP6 or VP7. To improve expression of VP4 and induction of rotavirus-specific humoral responses, the coding region of VP4 was cloned into the high-expression plasmid WRG7054 as a fusion protein containing the 22-amino-acid secretory signal peptide of tissue plasminogen activator (tPA) at its N terminus. In vitro expression of tPA::VP4 was significantly higher than unmodified VP4, and mice inoculated with WRG7054/EDIM4 generated high titers of rotavirus IgG. The coding sequence of VP7 without the first 162 nucleotides was also cloned into WRG7054, but no difference was observed between titers of serum rotavirus IgG in mice immunized with this plasmid (WRG7054/EDIM7Delta1-162) and pcDNA1/EDIM7. The rotavirus-specific IgG titers in all immune sera were predominantly IgG1 indicating induction of Th 2-type responses. None of the mice immunized with any of the VP4 or VP7 plasmids developed serum or fecal rotavirus IgA or neutralizing antibody to EDIM. When immunized mice were challenged with EDIM virus, there was no significant reduction in viral shedding relative to unimmunized controls. Therefore epidermal immunization with VP4 or VP7 alone elicited rotavirus IgG responses but did not protect against homologous rotavirus challenge.     

Epidemiology of symptomatic human rotaviruses in Bangalore and Mysore, India, from 1988 to 1994 as determined by electropherotype, subgroup and serotype analysis

Epidemiology of symptomatic rotaviruses from Bangalore and Mysore in Southern India was investigated. While serotype G3 predominated throughout the 7-year study period from 1988 to 1994 in Bangalore, serotype G1 was more predominant than serotype G3 in Mysore during 1993 and 1994. Serotype G2 strains were either not detected or infrequently observed in both the cities. However, several strains with subgroup I and 'short' RNA pattern that exhibited high reactivity with typing MAbs specific for serotype 2 as well as other serotypes were detected throughout the period. Among the nonserotypeable strains from both cities, several exhibited dual subgroup (SGI + II) or subgroup I specificity and 'long' RNA pattern indicating their probable animal origin. Notably, a gradual, yet highly significant reduction in rotavirus gastroenteritis, from 45.3% in 1988 to 1.8% during 1994, was observed in Bangalore in stark contrast to the consistently high (about 34%) incidence of asymptomatic infections among neonates by I321-like G10P11 type strains during the same period. Moreover, I321-like asymptomatic strains were not detected in children with diarrhea.     

A minor prevalent strain in a severe outbreak of foal diarrhea associated with serotype 3 rotavirus

An epizootic of foal diarrhea due to serotype 3 rotavirus (RV) was observed in 89 of 168 cases (53%) during the period from March to July in 1987. A total of 51 strains of RV were isolated from the 62 diarrheal feces examined, and one isolate (CH-3) showed a unique electropherotype of viral RNA which differed from the others that widely prevailed on this farm. No positive reaction was observed between strain CH-3 and each of the antisera against serotypes 1 to 12 of human and animal RV in neutralization tests. However, dsRNAs of the CH-3 virus were hybridized with a probe prepared from a strain of equine serotype 3 RV.     

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.