Simian Rotavirus SA11 Research Articles

Expression of the rotavirus SA11 protein VP7 in the simple eukaryote Dictyostelium discoideum.

The outer capsid protein of rotavirus, VP7, is a major neutralization antigen and is considered a necessary component of any subunit vaccine developed against rotavirus infection. For this reason, significant effort has been directed towards producing recombinant VP7 that maintains the antigenic characteristics of the native molecule. Using a relatively new expression system, the simple eukaryote Dictyostelium discoideum, we have cloned the portion of simian rotavirus SA11 genome segment 9, encoding the mature VP7 protein, downstream of a native D. discoideum secretion signal sequence in a high-copy-number extrachromosomal vector. The majority of the recombinant VP7 expressed by transformants was intracellular and was detected by Western immunoblot following gel electrophoresis as two or three bands with an apparent molecular mass of 35.5 to 37.5 kDa. A small amount of VP7 having an apparent molecular mass of 37.5 kDa was secreted. Both the intracellular VP7 and the secreted VP7 were N glycosylated and sensitive to endoglycosidase H digestion. Under nonreducing electrophoresis conditions, over half the intracellular VP7 migrated as a monomer while the remainder migrated with an apparent molecular mass approximately twice that of the monomeric form. In an enzyme-linked immunosorbent assay, intracellular VP7 reacted with both nonneutralizing and neutralizing antibodies. The monoclonal antibody recognition pattern paralleled that found with VP7 expressed in either vaccinia virus or herpes simplex virus type 1 and confirms that D. discoideum-expressed VP7 is able to form the major neutralization domains present on viral VP7. Because D. discoideum cells are easy and cheap to grow, this expression system provides a valuable alternative for the large-scale production of recombinant VP7 protein.

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.

Genotypic diversity of reassortants between simian rotavirus SA11 and human rotaviruses having different antigenic specificities and RNA patterns

A total of 200 clones of reassortants were generated in vitro between simian rotavirus SA11 (SA11-L2 strain) and each of five human rotavirus (HRV) strains with different antigenic specificities and RNA patterns. When culture fluid coinfected with parent viruses was subjected to repeated selection with anti-HRV-VP4 and anti-SA11-VP7 neutralizing monoclonal antibodies, all yielded progenies were antigenic mosaic reassortants possessing VP4 and VP7 derived from SA11 and HRV, respectively. In genomic analysis of viral RNA by PAGE, two and five different genotypes were identified in 40 clones, generated between SA11 and HRV strain Hochi (G serotype 4, G4) and between SA11 and KU (G1), respectively. In contrast, 11 and 17 genotypes were found in progenies between SA11 and subgroup I G2 HRV strain HN126 or DS-1, respectively. Although SA11 genes, except for the VP7 gene, were predominant in most reassortants, gene 5 was preferentially selected from HRV. Incorporation of HRV segments 1, 3 and 10 into progenies was more frequent in SA11 x HN126 and SA11 x DS-1 reassortants than in other reassortants. These results indicated that the extent of diversity of reassortant genotypes differs between the two groups of the parental HRV strain; one has subgroup I specificity and a short RNA pattern (DS-1 genogroup), while the other has subgroup II specificity and a long RNA pattern (Wa genogroup).

Detection of viruses in environmental samples: Suitability of commercial rotavirus and adenovirus test kits

Commercially marketed kits are now available for rapid viral assay of clinical specimens. This study was conducted to determine the suitability of these kits for use in environmental testing. Eight rotavirus kits and one enteric adenovirus kit were screened for sensitivity using simian rotavirus SA11, human rotavirus Wa, and adenovirus 41. The most sensitive rotavirus kit and the adenovirus kit were selected for further evaluation using virus-seeded and unseeded sewage samples. The selected rotavirus kit proved capable of detecting virus at the 10 1 PFU/ml level. The enteric adenovirus kit was similarly sensitive, detecting virus at the 10 1 TCID 50/ml level. Neither kit was adversely affected by the presence of sewage. Kit assay revealed 3 of 30 unseeded sewage samples to be positive for rotavirus. Adenovirus positive samples were not detected among the 30 samples. These results were confirmed using electron microscopy. It was concluded that sensitive commercial kits could provide a reasonable alternative to cell culture for the presumptive testing of environmental samples.

Monoclonal antipeptide antibodies recognize epitopes upon VP4 and VP7 of simian rotavirus SA11 in infected MA104 cells.

To study morphogenetic events of rotavirus SA11-infected MA104 cells with strictly defined reagents we produced monoclonal antibodies against synthetic peptides from both outer capsid proteins VP4 (aa residues 228-241: QNTRNIVPVSIVSR) and VP7 (aa residues 319-326: SAAFYYRV) of simian rotavirus SA11. Two of the selected monoclonal antibodies proved to be reactive with determinants of SA11-infected MA104 rhesus monkey kidney cells, with purified SA11 as well as with the particular peptides used for immunization. The anti-VP4 antibody had a demonstrable neutralizing titer of 200 (50% focus reduction) whereas the anti-VP7 MuMAb revealed no detectable neutralizing activity. In peptide-inhibition experiments, the corresponding peptide inhibited its MuMAb whereas the noncorresponding peptide had no effect on antibody binding to intracellular viral antigen. Localization of VP7 was preceded by VP4 as shown by immunofluorescence microscopy.

Expression of the gene coding for the major outer capsid protein of SA-11 rotavirus in a baculovirus system

The gene coding for the major outer capsid protein (VP7) of simian rotavirus SA-11 has been expressed in a baculovirus-insect cell system. The resulting protein is 35 kDa and is primarily associated with the endoplasmic reticulum. Neutralizing SA-11 polyclonal antiserum and VP7 monospecific antiserum reacted specifically with the expressed gene product. Antiserum derived against the recombinant VP7 protein neutralized SA-11 rotavirus infectivity in a fluorescent focus assay.

A Comparison of Methods for the Isolation of a Wide Range of Viruses from Shellfish

Four methods were compared for the isolation of a wide range of enteric viruses from shellfish. The comparison was aimed at finding a practical and sensitive method to be used routinely. Three of the methods (A, B and C) were based on viral adsorption and desorption using buffers at different pH values. A fourth method (D) consisted of a single homogenization step with no pH adjustments. High recoveries for polio 1 were shown with the first three methods A-C, namely 81%, 79% and 77% respectively. Recoveries for reo 1 (Lang strain) and the simian rotavirus SA11 were not satisfactory using methods A-C. By contrast, method D gave high recoveries for reo and SA11 virus (31% and 68%, respectively). A modified method D generally yielded higher recoveries as indicated by a 91%, 33% and 35% recovery for polio 1, reo and SA11 viruses respectively. Comparison of method A and modified method D for the detection of viruses from environmental shellfish samples indicated that the two methods were of equal sensitivity.

Protection of agammaglobulinemic piglets from porcine rotavirus infection by antibody against simian rotavirus SA-11.

Rotavirus, a double-stranded RNA virus, has been implicated as a diarrhea-provoking agent in a variety of animal species. Several previous reports have shown that immunization with a single serotype may result in increased in vitro neutralization titers against serotypes not represented in the immunogen. This study was undertaken to determine whether antibody from cows immunized against simian rotavirus strain SA-11 (which is alien to pigs) could protect neonatal piglets from infection with a North Carolina isolate of porcine rotavirus. Accordingly, cows were immunized with SA-11 and an immunoglobulin G (IgG)-rich fraction was isolated from their colostrum. An IgG-rich fraction was similarly isolated from colostrum of nonimmunized cows. At equal concentrations, IgG from SA-11-immunized cows had two- to fourfold higher neutralization titers to seven of eight test strains of rotavirus, including SA-11 (serotype 3); human rotavirus serotypes 1, 3, and 4; North Carolina porcine rotavirus (serotype undetermined); Ohio State porcine rotavirus (serotype 5); and bovine rotavirus (serotype 6). The IgG-rich fractions were fed as dietary supplements to agammaglobulinemic piglets infected with the North Carolina porcine rotavirus. IgG from the SA-11-immunized cows was about eightfold more effective in protecting piglets than was IgG from nonimmunized cows.

Detection of Rotavirus in South African Waters: A Comparison of a Cytoimmunolabelling Technique with Commercially Available Immunoassays

A cytoimmunolabelling technique was compared with commercially available immunoassays for the detection of the simian rotavirus SA11 and human rotavirus in various environmental samples. The technique is based on labelling MA104 cells with antibody conjugated with an enzyme for the detection of rotavirus. Water samples were concentrated by ultrafiltration and inoculated on trypsin-treated MA104 cells. After 18 h incubation, evidence of viral replication was determined by immunolabelling of viral antigen using antibodies tagged with horseradish peroxidase. The cytoimmunolabelling method was shown to be 105 times more sensitive for the detection of rotavirus than commercially available ELISA and latex agglutination kits. The cytoimmunolabelling technique was also shown to be successful for the detection of human rotavirus in river and marine waters and wastewater effluents.

Preparation and characterization of a neutralizing monoclonal antibody directed to VP4 of rotavirus strain K8 which has unique VP4 neutralization epitopes

For selecting the neutralizing monoclonal antibodies (N-MAbs) directed to VP4 of rotavirus strain K8, which has unique VP4 neutralization epitopes, we prepared several reassortant viruses by mixed infection of two different strains K8 (serotype 1) and P (serotype 3) in vitro: three reassortant clones having VP4 of K8 and VP7 of P and four clones having VP4 of P and VP7 of K8. By using these reassortants in screening hybridomas, a N-MAb (K8-2C12) directed to strain K8-specific VP4 was obtained. The MAb K8-2C12 neutralized only K8 when tested against numerous strains of different serotypes, while in enzyme-linked immunosorbent assay this MAb reacted also with simian rotavirus SA11 (serotype 3), bovine rotavirus NCDV (serotype 6), and human rotavirus (HRV) strain 69M (serotype 8). Neutralization-resistant mutants of K8 were selected by the K8-2C12 antibody and VP4 amino acid sequences of the mutants were determined. Single amino acid substitution was detected in the three mutant clones at position 394, which is included in the major cross-reactive neutralization region identified in other rotaviruses.

Evidence for equimolar synthesis of double-strand RNA and minus-strand RNA in rotavirus-infected cells

The genome of the rotaviruses consists of eleven segments of double-strand RNA (dsRNA). Each segment is replicated asymmetrically with viral plus-strand RNA, i.e. messenger (m)RNA, serving as the template for the synthesis of minus-strand RNA to produce dsRNA. To examine the relative frequency of replication of each of the eleven genome segments, MA104 cells were infected with low (3rd) and high (12th) passage stocks of simian rotavirus SA11. The total cytoplasmic RNA of the infected cell was radiolabeled either by maintaining the infected cells in the presence [ 3H]uridine prior to harvest or by 3'-endlabeling the purified RNA with [ 32P]pCp and T4 RNA ligase. The RNA was then analyzed for the presence of 3H- and 32P-labeled dsRNA by electrophoresis on 10% polyacrylamide gels. Total cytoplasmic RNA from infected cells was also 3'-end-labeled with [ 32P]pCp and T4 RNA ligase and examined for the presence of minus-strand RNA by electrophoresis on low pH agarose-urea gels. Bands representing dsRNAs and minus-strand RNAs on autoradiographs of the gels were analyzed for intensity by densitometry. The results showed that the eleven segments of viral dsRNA were present in equimolar concentrations in cells either infected with low or high passage stocks of virus. Like intracellular dsRNAs, full-length minus-strand RNAs were also present in equimolar concentration in cells either infected with low or high passage rotavirus. These data indicate that, despite the non-equimolar levels of viral mRNAs in the cell, the eleven genome segments of rotavirus are replicated with equal frequencies in vivo.

Rotavirus RNA replication: VP2, but not VP6, is necessary for viral replicase activity

Temperature-sensitive mutants of simian rotavirus SA11 were previously developed and organized into 10 of a possible 11 recombination groups on the basis of genome reassortment studies. Two of these mutants, tsF and tsG, map to genes encoding VP2 (segment 2) and VP6 (segment 6), respectively. To gain insight into the role of these proteins in genome replication, MA104 cells were infected with tsF or tsG and then maintained at permissive temperature (31 degrees C) until 9 h postinfection, when some cells were shifted to nonpermissive temperature (39 degrees C). Subviral particles (SVPs) were recovered from the infected cells at 10.5 and 12 h postinfection and assayed for associated replicase activity in a cell-free system shown previously to support rotavirus genome replication in vitro. The results showed that the level of replicase activity associated with tsF SVPs from cells shifted to nonpermissive temperature was ca. 20-fold less than that associated with tsF SVPs from cells maintained at permissive temperature. In contrast, the level of replicase activity associated with tsG SVPs from cells maintained at nonpermissive temperature was only slightly less (twofold or less) than that associated with tsG SVPs from cells maintained at permissive temperature. Analysis of the structure of replicase particles from tsG-infected cells shifted to nonpermissive temperature showed that they were similar in size and density to virion-derived core particles and contained the major core protein VP2 but lacked the major inner shell protein VP6. Taken together, these data indicate that VP2, but not VP6, is an essential component of enzymatically active replicase particles.

Intracellular RNA synthesis directed by temperature-sensitive mutants of simian rotavirus SA11

The kinetics of intracellular synthesis of single-stranded (ss) RNA and double-stranded (ds) RNA directed by prototype temperature-sensitive (ts) mutants representing the 10 mutant groups of rotavirus SA11 were examined. Cells were infected with individual mutants or wild type under one-step growth conditions and maintained at permissive temperature (31°) or nonpermissive temperature (39°). At various times postinfection, infected cells were pulse-labeled, ssRNA and dsRNA were purified, RNA species were resolved by electrophoresis and autoradiography, and RNA synthesis was quantitated by computer-assisted densitometry. The mutants representing all groups synthesized significantly less ssRNA and dsRNA at both 31° and 39°, when compared to wild type. When the ratio of synthesis at 39° 31° was determined for ssRNA and dsRNA of each mutant, three RNA synthesis phenotypes were evident. The tsB(339), tsC(606), and tsE(1400) mutants synthesized both ssRNA and dsRNA in a temperature-dependent manner. The group G mutant, tsG(2130), synthesized ssRNA in temperature-independent fashion but was temperature-dependent for the synthesis of dsRNA. The remaining mutants, tsA(778), tsD(975), tsF(2124), tsH(2384), tsI(2403), and tsJ(2131), synthesized both ssRNA and dsRNA in a temperature-independent fashion. The RNA synthesis phenotypes of the ts mutants are discussed in terms of what is known of the function(s) of the protein species to which is lesions have been assigned.

Neutralization epitopes on rotavirus SA11 4fM outer capsid proteins

The VP7 and VP4 genes of seven antigenic mutants of simian rotavirus SA11 4fM (serotype 3) selected after 39 passages in the presence of SA11 4fM hyperimmune antiserum, were sequenced. Nucleotide sequence analysis indicated the following. (i) Twice as many amino acid substitutions occurred in the VP7 protein than in VP4, which has a molecular weight twice that of VP7. (ii) Most amino acid changes that occurred clustered in six variable regions of VP7 and in two variable regions of VP4; these variable regions may represent immunodominant epitopes. (iii) Most amino acid substitutions that occurred in VP7 and VP4 of these mutants were also observed in antigenic mutants selected with neutralizing monoclonal antibodies (NMAbs); however, some amino acid substitutions occurred that were not selected for NMAbs. (iv) On VP7, some of the neutralization epitopes appeared to be interrelated because amino acid substitution in one site affected binding of specific NMAbs to other sites, while other neutralization epitopes on VP7 appeared to be independent, in that amino acid substitution in one site did not affect the binding of NMAbs to another distant site.

Polypeptide composition of rotavirus empty capsids and their possible use as a subunit vaccine

Two types of empty capsid particles that differed with respect to the presence of the two outer shell proteins were isolated from MA-104 cells infected with bovine rotavirus V1005. Three previously uncharacterized polypeptides, I, II, and III, migrating between VP2 and VP6, were detected in empty capsids but not in single- and double-shelled rotavirus particles. Peptide mapping revealed that all three proteins were related to VP2. Polypeptides I, II, and III could be generated by in vitro trypsin digestion of empty capsids not exposed to trypsin in the infection medium. Labeled polypeptides appeared in empty capsids before they were detected in intracellular single- or double-shelled rotavirus particles. Empty capsids were also observed in MA-104 cells infected with bovine rotaviruses UK and NCDV, simian rotavirus SA11, and human rotavirus KU. VP7-containing empty capsid is the minimal subunit vaccine for cows; we failed to induce a substantial neutralizing antibody increase with VP7 purified under denaturating or nondenaturating conditions or with synthetic peptides corresponding to two regions of VP7.

Completion of the genomic sequence of the simian rotavirus SA11: nucleotide sequences of segments 1, 2, and 3

The nucleotide sequences for gene segments 1, 2, and 3 of the simian rotavirus SA11 genome, coding for the structural polypeptides VP1, VP2, and VP3, respectively, have been determined. Comparison of the VP1 and VP2 amino acid sequences with those determined for other strains indicates that certain features of these proteins are conserved. The possible functions of the viral polypeptides VP1, VP2, and VP3 are discussed in the light of enzyme functions known to be present in the rotavirus particle. The complete sequence of the entire SA11 genome, which consists of 11 segments of dsRNA totaling 18,555 nucleotides, has now been determined. This is the first complete sequence available for a rotavirus genome. Each genome segment appears to code for only one primary product; there are no significant, alternative open reading frames which are conserved between strains. Relevant data for each genome segment are tabulated.

Superinfecting rotaviruses are not excluded from genetic interactions during asynchronous mixed infections in vitro

Asynchronous infections of MA104 cells with temperature-sensitive ( ts) mutants of simian rotavirus SA11 were performed to determine if the superinfecting ts mutant could contribute to the formation of reassortant progeny. Significant yields of ts + reassortant progeny were obtained in crosses where infection with the first and second ts mutant was separated by as much as 24 hr, indicating that superinfecting viruses were not excluded from participation in genetic interactions. The practical and theoretical implications of this result are discussed.

A recombinant vaccinia virus expressing the major capsid protein of Simian rotavirus-induced anti-rotavirus antibodies

cDNA molecules encoding the major structural protein (VP6) of the Simian rotavirus SA11 were inserted under the control of the vaccinia virus 7.5 kDa promoter into the thymidine kinase gene. Synthesis of VP6 was demonstrated by immunoprecipitation of recombinant virus-infected cell. Mice inoculated via several routes with this recombinant vaccinia produce high titers of antirotavirus antibodies lacking neutralizing activity.

Conservation of a potential metal binding motif despite extensive sequence diversity in the rotavirus nonstructural protein NS53

The nucleotide sequence for the simian rotavirus SA11 gene segment 5 has been determined. The gene is 1611 nucleotides in length and contains a single open reading frame of 1485 nucleotides. The segment codes for the nonstructural protein NS53 which is predicted to be a polypeptide of 495 amino acids with a molecular weight of 58,484. When compared to the sequence for bovine RF gene segment 5 there are homologies of only 49 and 36% at the nucleotide and amino acid levels, respectively. This is in marked contrast to the situation with other rotavirus nonstructural proteins which are highly conserved between isolates. Nevertheless, there is a conserved region between amino acids 37–81 which contains a generalized motif for a metal binding domain. All eight cysteine and two histidine residues in this short sequence are conserved between the simian and bovine NS53 proteins. The conservation of this domain despite extensive sequence diversity in the remainder of the protein suggests that this region is functionally important.

Viruses with bisegmented double-stranded rna in pig faeces

Viruses similar to the bisegmented double-stranded (ds) RNA picobirnaviruses described in human faeces and the intestinal contents of Oryzomys nigripes rats and guinea pigs were isolated from the faeces of pigs taken from several areas in the state of Sao Paulo, Brazil. Samples were collected from 912 pigs of several breeds, aged nine to 61 days, and assayed by polyacrylamide gel electrophoresis with silver staining and a combined enzyme immunoassay for rotavirus and adenovirus, using the simian rotavirus SA11 as control. Electrophoretic profiles resembling the bisegmented dsRNA viruses were detected in 106 pigs with 15.3 per cent occurring in animals with diarrhoea compared to 9.6 per cent in animals without diarrhoea.