Nucleotide sequence of the simian rotavirus SA11 genome segment 3.
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Animals↗
Biomedical subjects
M K Estes
Publications and source records attributed to M K Estes.
Biological and immunological characterization of a simian rotavirus SA11 variant with an altered genome segment 4.
We have studied a variant virus isolated from a stock of SA11 virus (H. G. Pereira, R. S. Azeredo, A. M. Fialho, and M. N. P. Vidal, 1984, J. Gen. Virol. 65, 815-818). This virus, designated 4F, was initially identified by its faster electrophoretic mobility for genome segment 4. The variant was analyzed to determine if the altered electrophoretic mobility of genome segment 4 could be correlated with phenotypic changes. Comparison of our standard laboratory SA11 virus (clone 3) with the 4F variant showed the following: (i) The 4F variant possesses a viral hemagglutinin (VP4) with a higher apparent molecular weight than clone 3. (ii) The 4F variant produces large plaques when assayed in vitro, as compared to clone 3. (iii) The 4F variant produces plaques in the absence of proteolytic enzymes, whereas clone 3 does not. (iv) The 4F variant reacts with serotype-specific neutralizing monoclonal antibodies to VP7, but fails to react with several neutralizing anti-VP4 monoclonal antibodies generated to SA11 clone 3. (v) The 4F variant grows to a higher titer and is more stable than clone 3. (vi) The 4F variant produces a VP4 that appears to be more susceptible to cleavage by trypsin than is the VP4 of clone 3. Further analyses with the 4F variant may lead to an understanding of the molecular basis for these altered phenotypes that appear to be related, at least in part, to the product of genome segment 4.
Antigens, Viral↗
Nucleotide sequence of bovine rotavirus gene 1 and expression of the gene product in baculovirus.
The nucleotide sequence of the gene that encodes for the structural viral protein VP1 of bovine rotavirus (RF strain) has been determined. The sequence data indicate that segment 1 contains 3302 bp and is A + T rich (65.3%). The positive strand of segment 1 contains a single open reading frame that extends 1088 codons and possesses 5'- and 3'-terminal untranslated regions of 18 and 20 bp, respectively. The first AUG conforms to the Kozak consensus sequence and if utilized, would yield a protein having a calculated molecular weight of 124,847, very close to the apparent molecular weight of VP1 (M.W. 125,000). The deduced amino acid sequence presents significant similarities with RNA-dependent RNA polymerase of several RNA viruses. VP1 was also synthesized in baculovirus using two transfer vecors: pAC461 and pVL941. Following infection of Sf9 cells with a recombinant baculovirus, a full-length nonfusion protein was synthesised which shares properties with authentic VP1 made in monkey kidney cells. The level of VP1 synthesis was about 10-fold higher when the baculovirus recombinant was derived from the pVL941 transfer vector. In that case, VP1 was expressed in yields approximately equivalent to 10% of the cellular protein. The recombinant protein was immunoprecipitated by hyperimmune serum raised against purified rotavirus. It also was immunogenic; a hyperimmune serum made in guinea pigs reacted with VP1 using immunoprecipitation and Western blot. This serum did not possess neutralization activity.
Amino Acid Sequence↗
Rotavirus gene detection with biotinylated single-stranded RNA probes.
Biotinylated single-stranded RNA probes from two of the eleven genome segments of the simian rotavirus SA11 were synthesized from cloned DNA and used in dot-blot and Northern-blot hybridization assays. Different types of membranes and conditions to prepare and use synthetic non-radioactive transcript probes were evaluated to obtain optimal test results. Nytran membranes showed the highest sensitivity and lowest backgrounds for hybridization with biotinylated RNA probes. When a gene 6 single-stranded biotinylated probe was used in a dot-blot format, test sensitivity was 0.1 ng for detection of homologous RNA and 0.4-1.5 micrograms for detection of RNA from heterologous rotavirus strains. When used in Northern blots, detection with this gene 6 probe required 1 ng of total SA11RNA or 50 ng of heterologous RNA to be applied to the gels for transfer. Simultaneous hybridization with probes from two different genes on one membrane showed a detection level similar to that seen with single probes alone. The advantages of using biotinylated single-stranded RNA probes to detect or characterize the genes of viruses with double-stranded RNA genomes are shown.
Biotin↗
Phenotypes of rotavirus reassortants depend upon the recipient genetic background.
We have previously characterized the biological and immunological properties of a simian rotavirus SA11 variant (4F) with an altered genome segment 4. The SA11-4F variant formed large plaques in the presence of protease, formed small clear plaques in the absence of protease, and grew to high titer in the presence of protease when compared to our standard wild type (SA11 clone 3). To determine the genome segment of the rotavirus SA11 variant 4F that encoded the unique protease-associated phenotypes of the variant, reassortants were generated that segregated the outer capsid genes of 4F onto a genetic background derived from either the bovine rotavirus B223 or our standard SA11 wild type (clone 3), both of which have contrasting protease-associated phenotypes. The parental and reassortant viruses were examined to determine which genes from the 4F variant encoded the ability (i) to form large plaques in the presence of protease, (ii) to form small clear plaques in the absence of exogenous protease, and (iii) to grow to significantly higher titer in the presence of protease. These phenotypes could be transferred to a clone 3 genetic background by a single genome segment from the 4F variant segment 4. However, in the 4F/B223 reassortants a different and unexpected situation was found. On a B223 genetic background the same phenotypes segregated with a combination of a minimum of two 4F genome segments, segments 4 and 9. These results indicate that the recipient genetic background onto which the genes of a donor rotavirus are reassorted can affect the phenotypes conferred by the presence of the donor segments. Thus, the results of segregation mapping experiments using reassortant viruses should be interpreted with caution.
Cell Line↗
Human calicivirus-associated diarrhea in children attending day care centers.
We investigated human calicivirus (HCV)-associated diarrhea in children attending day care centers by using stool specimens collected in 1981-1983. We used a screening enzyme-linked immunosorbent assay (ELISA) derived from reagents prepared against the Sapporo strain of HCV and confirmed positive results with a blocking ELISA and immunosorbent electron microscopy. HCV was detected in 11 (2.9%) of 375 diarrheal stools and in none of 86 stools from asymptomatic contacts. This incidence rate was half that noted for rotaviruses and higher than that noted for Campylobacter, Salmonella, and Shigella in the original study. HCV was found in stool specimens from children in nine day care centers; HCV-associated diarrhea was sporadic, occurred with greater frequency in young children, and had a summer-fall predominance. Our results indicate that HCV is an important cause of diarrhea in day care centers and that frozen stool samples can yield epidemiological data on HCV infection.
Arizona↗
Seroepidemiology of adult diarrhea rotavirus in China, 1977 to 1987.
In 1982, large outbreaks of diarrhea that were caused by group B adult diarrhea rotavirus (ADRV) occurred throughout the People's Republic of China. Until 1982, group B rotavirus had never been associated with disease in humans. To determine whether ADRV was a new virus introduced in 1982 or had been present before that time, we examined antibody titers of ADRV in gamma globulin (pooled immunoglobulin) pools that were prepared during 1977 to 1987 in four cities in the People's Republic of China (Shanghai, Lanzhou, Wuhan, and Chandu). ADRV antibodies were assayed by using a blocking enzyme-linked immunosorbent assay. Antibodies were present in most Chinese gamma globulins tested, including those collected in Shanghai before the 1982 epidemic, and absent from American reference pools. The highest titers of antibody to ADRV (3,200) were found in gamma globulins collected in 1983 in Shanghai just after the epidemic, and these were fourfold higher than titers present in the preceding years. The quality of the gamma globulins stored for up to 12 years was tested by measuring levels of immunoglobulin G to group A rotavirus; these were equally high in gamma globulin pools prepared in the United States and in all samples from the People's Republic of China. Serum samples from patients from an outbreak of ADRV had elevated titers to ADRV 3 and 16 months after the onset of symptoms. These findings, as well as other epidemiologic findings on ADRV, suggest that the organism is an important and continuing cause of diarrhea in the People's Republic of China, was present before the first epidemic in 1982, and represents a risk to surrounding populations in Asia.
Adult↗
Serological characterization of bovine rotaviruses isolated from dairy and beef herds in Argentina.
Bovine rotaviruses isolated from beef and dairy herds in Argentina were serotyped by the immunoperoxidase focus reduction assay as previously described (G. Gerna, M. Battaglia, G. Milenesi, N. Passarani, E. Percivalle, and E. Cattaneo, Infect. Immun. 43:722-729, 1984). Three strains from beef herds were related to the UK and NCDV bovine rotavirus strains defined as serotype 6 (Y. Hoshino, R. G. Wyatt, H. B. Greenberg, J. Flores, and A. Z. Kapikian, J. Infect. Dis. 149:694-702, 1984). Two other strains from dairy herds were classified as bovine viruses related to the bovine B223 strain reported by Woode and co-workers (G. N. Woode, N. E. Kelso, T. F. Simpson, S. K. Gaul, L. E. Evans, and L. Babiuk, J. Clin. Microbiol. 18:358-364, 1983) in the United States. A serotyping antibody-capture enzyme-linked immunoassay to detect serotype 6 rotavirus using a serotype 6-specific monoclonal antibody was developed and evaluated for strain characterization. Characterization of 72 group A rotavirus-positive fecal samples from beef herds and 43 fecal samples from dairy herds showed a predominance of serotype 6 rotavirus in beef herds but both serotype 6 and non-serotype 6 rotaviruses in dairy herds. Analysis of genomic double-stranded RNA by polyacrylamide gel electrophoresis showed that when outbreaks were caused by one serotype only a single electropherotype was present in all samples.
Animals↗
Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay.
Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus.
Animals↗
In situ hybridization for quantitative assay of infectious hepatitis A virus.
A method of in situ hybridization using single-stranded RNA probes of opposite polarity for quantitative enumeration of hepatitis A virus (HAV) in infected cells has been developed. Kinetic experiments showed that foci of infected cells appeared as early as day 2 postinfection. The absence of foci in cells examined immediately after virus adsorption indicated that foci detected subsequently were related to viral replication. Foci were detected by hybridization with RNA probes complementary to HAV genomic RNA but not with RNA probes identical to HAV genomic RNA. The number of foci observed was linearly related to the HAV dose inoculated. Focus formation was reduced when a virus inoculum was pretreated with guinea pig anti-HAV hyperimmune serum but not when it was pretreated with preimmune serum. The high resolution of hybridization signals and relative rapidity of the test indicated that this technique will be useful for measuring serum neutralizing antibodies and for quantitative assay of infectious HAV.
Animals↗
Receptor activity of rotavirus nonstructural glycoprotein NS28.
Rotavirus morphogenesis involves the budding of subviral particles through the rough endoplasmic reticulum (RER) membrane of infected cells. During this process, particles acquire the outer capsid proteins and a transient envelope. Previous immunocytochemical and biochemical studies have suggested that a rotavirus nonstructural glycoprotein, NS28, encoded by genome segment 10, is a transmembrane RER protein and that about 10,000 Mr of its carboxy terminus is exposed on the cytoplasmic side of the RER. We have used in vitro binding experiments to examine whether NS28 serves as a receptor that binds subviral particles and mediates the budding process. Specific binding was observed between purified simian rotavirus SA11 single-shelled particles and RER membranes from SA11-infected monkey kidney cells and from SA11 gene 10 baculovirus recombinant-infected insect cells. Membranes from insect cells synthesizing VP1, VP4, NS53, VP6, VP7, or NS26 did not possess binding activity. Comparison of the binding of single-shelled particles to microsomes from infected monkey kidney cells and from insect cells indicated that a membrane-associated component(s) from SA11-infected monkey kidney cells interfered with binding. Direct evidence showing the interaction of NS28 and its nonglycosylated 20,000-Mr precursor expressed in rabbit reticulocyte lysates and single-shelled particles was obtained by cosedimentation of preformed receptor-ligand complexes through sucrose gradients. The domain on NS28 responsible for binding also was characterized. Reduced binding of single-shelled particles to membranes was seen with membranes treated with (i) a monoclonal antibody previously shown to interact with the C terminus of NS28, (ii) proteases known to cleave the C terminus of NS28, and (iii) the Enzymobead reagent. VP6 on single-shelled particles was suggested to interact with NS28 because (i) a monoclonal antibody to the subgroup I epitope on VP6 reduced particle binding, (ii) a purified polyclonal antiserum raised against recombinant baculovirus-produced VP6 reduced ligand binding, and (iii) a monoclonal antibody to a conserved epitope on VP6 augmented ligand binding. These experimental data provide support for the hypothesized receptor role of NS28 before the budding stage of rotavirus morphogenesis.
Animals↗
Purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins.
Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus that has been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins present in the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SA11. EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of three polypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-beta-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particles removed the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. The remaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis when probed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant and was strongly immunoreactive with human sera, animal sera raised against ADRV and against other group B animal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovine enteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This 47-kDa inner capsid polypeptide contains a common group B antigen and is similar to the VP6 of the group A rotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid that seems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations on the basis of similarities to the control preparation of SA11, including a 136-kDa polypeptide designated VP1, a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and several proteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivity on Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses are antigenically quite distinct.
Adult↗
Rotavirus SA11 genome segment 11 protein is a nonstructural phosphoprotein.
We investigated properties of the rotavirus genome segment 11 protein. A rotavirus SA11 genome segment 11 cDNA which contains the entire coding region was sequenced and inserted into the baculovirus transfer vector pVL941. Recombinants containing gene 11 cDNA were selected, and the gene 11 product expressed in Spodoptera frugiperda cells infected with these recombinants was inoculated into guinea pigs to produce hyperimmune antiserum. Characterization of the antiserum showed that it recognized a primary translation product with a molecular weight of 26,000 (26K protein) in recombinant-infected insect cells, in SA11-infected monkey kidney cells, and in cell-free translation reactions programmed with SA11 mRNA. A modified 28K product was also detected but only in SA11-infected monkey kidney cells. The 26K 28K proteins were shown to be phosphorylated in infected monkey kidney cells, and the 26K protein was phosphorylated in insect cells. We were unable to identify what type of modification caused the molecular weight shift to 28,000 in infected monkey kidney cells. Large amounts of the gene 11 product were detected by immunofluorescence in discrete foci in the cytoplasm of infected monkey kidney cells. Viruses of all known serotypes were also detected by immunofluorescence by using hyperimmune antiserum to the SA11 gene 11 product. The antiserum reacted with particle-depleted cytosol fractions but did not react with purified virus particles by immunoprecipitation or immunoblotting; it also did not neutralize virus infectivity in plaque reduction neutralization assays. Therefore, we conclude that the primary gene 11 product is a nonstructural phosphoprotein which we designated NS26.
Animals↗
Rotavirus gene structure and function.
Knowledge of the structure and function of the genes and proteins of the rotaviruses has expanded rapidly. Information obtained in the last 5 years has revealed unexpected and unique molecular properties of rotavirus proteins of general interest to virologists, biochemists, and cell biologists. Rotaviruses share some features of replication with reoviruses, yet antigenic and molecular properties of the outer capsid proteins, VP4 (a protein whose cleavage is required for infectivity, possibly by mediating fusion with the cell membrane) and VP7 (a glycoprotein), show more similarities with those of other viruses such as the orthomyxoviruses, paramyxoviruses, and alphaviruses. Rotavirus morphogenesis is a unique process, during which immature subviral particles bud through the membrane of the endoplasmic reticulum (ER). During this process, transiently enveloped particles form, the outer capsid proteins are assembled onto particles, and mature particles accumulate in the lumen of the ER. Two ER-specific viral glycoproteins are involved in virus maturation, and these glycoproteins have been shown to be useful models for studying protein targeting and retention in the ER and for studying mechanisms of virus budding. New ideas and approaches to understanding how each gene functions to replicate and assemble the segmented viral genome have emerged from knowledge of the primary structure of rotavirus genes and their proteins and from knowledge of the properties of domains on individual proteins. Localization of type-specific and cross-reactive neutralizing epitopes on the outer capsid proteins is becoming increasingly useful in dissecting the protective immune response, including evaluation of vaccine trials, with the practical possibility of enhancing the production of new, more effective vaccines. Finally, future analyses with recently characterized immunologic and gene probes and new animal models can be expected to provide a basic understanding of what regulates the primary interactions of these viruses with the gastrointestinal tract and the subsequent responses of infected hosts.
Amino Acid Sequence↗
Molecular characterization of three rabbit rotavirus strains.
We report biochemical (RNA and protein patterns and gene-coding assignments) and serologic (serotype and subgroup) properties of three strains of rabbit rotaviruses--Ala C11 and R2. The RNA electropherotypes were a standard "short" pattern for R2, an unusual "short" pattern for Ala, and an unusual "long" pattern for C11. Serologic studies indicated that these viruses were all group A serotype 3 rotaviruses. In addition, the Ala and C11 viruses were found to possess subgroup I specificity, whereas the R2 virus possessed subgroup II specificity. In contrast to their distinctive RNA patterns, the polypeptide patterns of the rabbit viruses were similar to those of SA11. To identify cognate genes and determine gene-coding assignments for the rabbit rotaviruses, cDNA probes of individual SA11 genes were hybridized to rabbit rotaviral genomic RNA segments that had been separated by polyacrylamide gel electrophoresis and transferred to filters (Northern blots). The order of genome segments 7-11 for each of the rabbit rotaviruses was unique and differed from that of SA11 genes. These differences were possibly due to rearrangements of the RNA sequences within these individual genome segments. Sequence analysis of the individual RNA segments will confirm whether genome rearrangements are the molecular basis for these novel migration patterns.
Animals↗
Molecular cloning and sequence determination of the fusion protein gene of human parainfluenza virus type 1.
Undegraded mRNA transcripts were isolated from human parainfluenza virus type 1 (hPIV-1)-infected LLC-MK2 cells and their size was determined through denaturing agarose electrophoresis. The two predominantly represented mRNA species (1.65 and 1.87 kb) are similar in size to other paramyxoviral mRNAs that encode their respective glycoproteins. The cDNA transcripts corresponding to these two mRNAs were used to construct two size-restricted cDNA libraries. A cDNA clone, containing a 1.87-kb insert, was identified as encoding the hPIV-1 fusion protein by positively hybridizing with a synthetic oligonucleotide mix whose sequence was derived from the conserved sequences of other paramyxoviral F0 genes. The nucleotide sequence of the cDNA insert was determined and found to contain a single, large open reading frame encoding a putative protein of 60,795 Da consisting of 556 amino acids. Comparison of the amino acid sequence with the fusion proteins of other paramyxoviruses enabled the identification of the highly conserved amino acids of the F1 N-terminus. In addition, the positions of the hydrophobic signal and transmembrane regions, cysteine, and proline residues are all conserved. These analyses confirm that the cDNA sequence is that of the F0 protein. The 5' end of the fusion protein mRNA was determined by primer extension to lie 155 bases beyond the 5' end of the cDNA insert.
Amino Acid Sequence↗
Identification of the simian rotavirus SA11 genome segment 3 product.
Previous studies on rotavirus gene-coding assignments failed to clearly identify the simian rotavirus SA11 genome segment 3 protein product. This question was reexamined by using new conditions of electrophoresis with improved resolution of proteins in the high-molecular-weight range. Our results showed that the SA11 genome segment 3 codes for a protein with an apparent mol wt of 88,000. This protein normally comigrates with the protein product of genome segment 4. The gene 3 protein was located in viral core particles by comparing the electrophoretic patterns of [35S]methionine-labeled viral polypeptides from infected cells and from purified double-shelled, single-shelled, and core particles. To confirm the identity of the gene 3 product, we also studied two reassortant viruses in which genome segment 3 was reassorted from each of two parental virus strains (SA11 and NCDV). The gene 3 and gene 4 products of these viruses were identified by (i) their separation by two different polyacrylamide gel systems, (ii) their location in distinct particle types, (iii) their differential sensitivity to trypsin digestion, and (iv) their distinctive protease peptide maps. We propose that the genome segment 3 product be called VP3 and that the gene 4 product be named VP4 from now on.
Animals↗
Topography of the simian rotavirus nonstructural glycoprotein (NS28) in the endoplasmic reticulum membrane.
The simian rotavirus SA11 genome segment 10 codes for a nonstructural glycoprotein, NS28, that has been hypothesized to be involved in budding of viral particles into the endoplasmic reticulum (ER) membrane. Previous studies had suggested that NS28 is an integral membrane protein of the ER, possibly a transmembrane protein. We have examined the topography of NS28 inserted in microsomal membranes following cell-free translation of genome segment 10 transcripts. These transcripts were obtained either by hybrid selection of mRNA synthesized by the endogenous viral RNA polymerase or by in vitro transcription of genome segment 10 cDNA using SP6 polymerase. Full-length and truncated gene 10 transcripts were translated in a cell-free system supplemented with dog pancreatic microsomes. The existence of a cytoplasmic domain of the translation product was demonstrated by protease protection experiments. An 18,000 (18K) mol wt glycosylated polypeptide was protected from digestion with proteinase K and trypsin, whereas chymotrypsin digestion yielded a 23K mol wt glycosylated polypeptide. Correlation of these biochemical data with the known sequence of NS28 suggests that a 10K mol wt hydrophilic, carboxy-terminal fragment (from amino acid number 86 to amino acid number 175) of this glycoprotein is exposed on the cytoplasmic side of the ER membrane. A model of how NS28 folds in the ER membrane is proposed.
Capsid↗