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The Lang strain of reovirus serotype 1 and the Dearing strain of reovirus serotype 3 differ in their sensitivities to beta interferon.

Replication of the Dearing strain of reovirus serotype 3 in mouse L cells was decreased 17- to 100-fold when a saturating dose of beta interferon (1,000 IU/ml) was used. Replication of the Lang strain of reovirus serotype 1 was inhibited only two- to threefold under similar conditions. It therefore appears that closely related strains of reovirus differ in their sensitivities to beta interferon treatment of mouse L cells.

Animals↗

Sequential partially overlapping gene arrangement in the tricistronic S1 genome segments of avian reovirus and Nelson Bay reovirus: implications for translation initiation.

Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the final sigmaC viral cell attachment protein initiates over 600 nucleotides distal from the 5' end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3'-proximal ORF present on the NBV S1 genome segment also encodes a final sigmaC homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the final sigmaC coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and final sigmaC gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream final sigmaC ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal final sigmaC start site.

Amino Acid Sequence↗

In vitro and in vivo characterization of avian reoviruses. I. Pathogenicity and antigenic relatedness of several avian reovirus isolates.

Pathogenicity, pathogenesis, and antigenic relatedness of four avian reovirus isolates obtained from commercially reared broilers were investigated. Chickens of various ages were inoculated both orally and intratracheally with reovirus. Based on disease signs, mortality, weight depression, tissue lesions, invasiveness, and viral persistence in chickens inoculated at 1 day of age, the isolates were classified as being of low, intermediate, or high pathogenicity. The low-pathogenicity isolate (2177) did not cause mortality, weight depression, or clinical disease. The isolate of intermediate pathogenicity (2035) produced low mortality rates (8%), some weight reduction by 7 weeks postinoculation, and microscopic lesions in the intestine and gastrocnemius tendons. The pathogenic isolates, 2408 and 1733, caused severe clinical disease characterized by stunting, feathering abnormalities, mortality as high as 84%, and microscopic lesions in the liver, intestine, pancreas, and/or gastrocnemius tendon. Highly pathogenic isolates also persisted longer in tissues of infected birds and elicited a more prompt and prolonged antibody response. Birds inoculated at 1 day or 1 week of age were more susceptible to reovirus-induced disease than birds inoculated at 2 weeks, suggesting an age-associated resistance. All isolates produced mortality with equal frequency in embryos. The isolates characterized were found to be antigenically similar based on cross-neutralization and cross-protection studies.

Age Factors↗

In vitro and in vivo characterization of avian reoviruses. II. Clinical evaluation of chickens infected with two avian reovirus pathotypes.

The effect of two avian reovirus isolates (2408 and 1733) on digestion and nutrient metabolism in infected chickens was assessed by an in vitro absorption assay and clinical blood chemistry analysis. Birds of various ages were inoculated orally and intratracheally with reovirus and sampled periodically for the respective assays. Transitory malabsorption was observed in the duodenum of birds infected with reovirus 2408. Conversely, increased absorption was detected in the ileum of these same birds. Clinical blood chemistry analyses of birds infected with both isolates revealed that severely affected birds had abnormally elevated plasma total protein, plasma albumin, and calcium levels. Decreases were found in percent bone ash and, due to abnormally high globulin levels, in albumin:globulin (A:G) ratios. A significant (P less than 0.05) correlation between body weights and total protein, albumin, A:G ratio, and bone ash was found in infected birds. The most pronounced metabolic and physiologic changes occurred in the severely affected birds, and, in general, pathogenicity of the isolates was reflected by the degree of metabolic change.

Animals↗

Cell receptors for the mammalian reovirus. I. Syngeneic monoclonal anti-idiotypic antibody identifies a cell surface receptor for reovirus.

We have prepared and characterized a syngeneic monoclonal anti-idiotype directed against the idiotype (Id3) of the anti-reovirus type 3 hemagglutinin B cell hybridoma 9BG5. We have shown this monoclonal antibody to be anti-idiotypic in a series of inhibition studies with purified sigma 1 (hemagglutinin) protein and with absorption studies on the idiotypic R1.1 cell line. That the idiotype Id 3 defined by the monoclonal anti-idiotype is distinct from H-2 was demonstrated by its presence on an idiotype-expressing H-2 negative cell line (R1.E). Binding of the anti-idiotype is relatively tunicamycin resistant and pronase/trypsin sensitive, which suggests that the recognition site of the anti-idiotypic receptor is poorly glycosylated. The presence of idiotype-like receptors on reovirus type 3 susceptible neurons may have important implications for the presence of related gene families between neural and lymphoid tissue. The implications of recognition by neuronal cells of an anti-idiotypic monoclonal antibody directed against the reovirus 3 attachment receptor for the pathogenesis of viral-induced auto-immunity are discussed.

Animals↗

Biosynthesis of reovirus-specified polypeptides: identification of regions of the bicistronic reovirus S1 mRNA that affect the efficiency of translation in animal cells.

The reovirus S1 gene cDNA was systematically altered by site-directed mutagenesis in an attempt to identify regions important in determining the relative efficiency of translation of the two open reading frames of the bicistronic S1 mRNA. The synthesis of the minor capsid protein sigma 1 encoded by ORF1, extending from AUG14 to UGA1424, and the synthesis of the nonstructural protein sigma 1NS encoded by ORF2, extending from AUG75 to UAG432, were examined in transfected COS cells. Deletion of the 5'-untranslated region upstream of ORF1 AUG14 did not significantly affect either the relative amount, or the ratio, of sigma 1 and sigma 1NS synthesized. Creation of a strong context ORF1 initiation site by substitution of 5'-untranslated region nucleotides flanking AUG14 likewise did not affect sigma 1 synthesis, but sigma 1NS synthesis from ORF2 was decreased about twofold relative to wild-type S1 mRNA. The amount of sigma 1NS synthesis was increased less than twofold either by elimination of the ORF1 AUG14 initiation codon or by termination of sigma 1 synthesis shortly after initiation from AUG14. No sigma 1 synthesis was detected when the ORF1 AUG14 was mutated to a UUG14 codon. When ribosomes which initiated translation at AUG14 were frameshifted at the next codon after AUG14, elongation occurred with comparable efficiency in the sigma 1NS ORF2 and in sigma 1 ORF1. No sigma 1NS synthesis was detected when the ORF2 AUG75 was mutated to an CUG75 codon, and this mutation did not affect the amount of sigma 1 synthesis. sigma 1NS synthesis was not affected by truncation of the 3'-untranslated region or premature termination of sigma 1 synthesis shortly after the ORF2 UAG432. However, truncation of the ORF2 5'-untranslated region at either 6 or 36 nt following the ORF1 AUG14 significantly increased the efficiency of sigma 1NS synthesis. These results indicate that the region of the S1 mRNA immediately downstream of the sigma 1 ORF1 initiation codon AUG14 but well upstream of the sigma 1NS ORF2 initiation codon AUG75 plays a major role in determining the relative efficiency of synthesis of sigma 1 and sigma 1NS from the reovirus bicistronic s1 mRNA.

2-Aminopurine↗

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain bicistronic s1 mRNA which encodes the minor capsid polypeptide sigma 1a and the nonstructural polypeptide sigma 1bNS.

Human reovirus serotype 1 Lang strain s1 mRNA, which encodes the minor capsid cell attachment protein sigma 1a and the nonstructural protein sigma 1bNS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. The Lang strain s1 mRNA is 1462 nucleotides in length and possesses two open reading frames. The first begins at nt 14 and has a coding capacity of 418 amino acids, sufficient to account for sigma 1a; the second begins at nt 75 and has a coding capacity of 119 amino acids, sufficient to account for sigma 1bNS. Comparison of the Lang serotype s1 sequence derived from cDNA clones of s1 mRNA with the Lang S1 sequence derived from cDNA clones of the S1 dsRNA genome segment definitively establishes that reovirus plus-strand mRNA is structurally equivalent to the plus-strand of the dsRNA genome segment.

Base Sequence↗

Biosynthesis of reovirus-specified polypeptides. Multiplication rate but not yield of reovirus serotypes 1 and 3 correlates with the level of virus-mediated inhibition of cellular protein synthesis.

Kinetic analysis of mouse L fibroblast cells infected with reovirus revealed that serotypes 1 (Lang strain) and 3 (Dearing strain) differ significantly from each other in terms of their rates of multiplication and their effects on cellular protein synthesis. Serotype 1 did not significantly affect the synthesis of cellular polypeptides in monolayer cultures of L cells at late times after infection when virus-specific protein synthesis was at a maximum. By contrast, under identical culture conditions, serotype 3 essentially completely inhibited the synthesis of cellular polypeptides at late times when viral protein synthesis was at a maximum. The kinetics of virus-specific polypeptide synthesis and the production of infectious progeny were considerably slower for the serotype 1 Lang strain as compared to the serotype 3 Dearing strain, both at 30 and 37 degrees. However, the relative pattern of viral polypeptide synthesis and the final yield of infectious progeny did not differ significantly between serotypes 1 and 3. For both serotypes, the maximum yield of infectious progeny was obtained shortly after the maximum rate of viral polypeptide synthesis was reached. These results suggest that the rate of multiplication, but not the final yield, of reovirus serotypes 1 and 3 is related to the extent of virus-mediated inhibition of cellular protein synthesis.

Animals↗

Biosynthesis of reovirus-specified polypeptides. Efficiency of expression of cDNAs of the reovirus S1 and S4 genes in transfected animal cells differs at the level of translation.

Full-length cDNAs of the reovirus serotype 1 Lang strain S1 and S4 genes were cloned in Escherichia coli using bacteriophage M13 and expressed in monkey COS cells under the control of the SV40 late promoter using the eukaryotic expression vector pJC119. The s1-encoded sigma 1 and s4-encoded sigma 3 gene products were expressed in transfected COS cells and were indistinguishable from the authentic sigma 1 and sigma 3 polypeptides synthesized in reovirion-infected COS cells. The relative translational efficiencies of the s1 and s4 mRNAs in transfected COS cells were similar to the efficiencies observed in virion-infected cells; the s4 mRNA was translated approximately five times more efficiently than the s1 mRNA. Our results suggest that the differential translation of the reovirus s1 and s4 mRNAs in vivo may be attributed to intrinsic structural properties of the individual mRNAs and is independent of competition with other viral mRNAs.

Animals↗

Biosynthesis of reovirus-specified polypeptides: ribosome pausing during the translation of reovirus S1 mRNA.

The steady-state distribution of translating ribosomes on the reovirus serotype 1 Lang strain polycistronic s1 mRNA and the monocistronic s4 mRNA was mapped using a sensitive primer extension assay and cDNA clones of the S1 and S4 genes. The distribution of translating ribosomes on the reovirus s1 and s4 mRNAs was not uniform. Major positions of ribosome pausing were detected in both rabbit reticulocyte and wheat germ cell-free protein synthesizing systems programmed with wild-type and site-directed mutant mRNAs. Two of the ribosome pause sites represent initiation and termination, rate-limiting steps of translation. For the polycistronic s1 mRNA, analysis of mutants in which either the sigma 1 ORF1 initiation codon or the sigma 1NS ORF2 initiation codon was eliminated by site-directed mutagenesis unequivocally established the identity of the specific ribosome pauses with specific ORF translational events. Ribosomes were far less uniformly distributed along the overlapping ORF regions of the polycistronic s1 mRNA than they were along the ORF of the monocistronic s4 mRNA. Furthermore, the rate-limiting initiation event at the sigma 1NS ORF2 AUG led to ribosome stacking and elongation arrest in the sigma 1 ORF1. These results begin to provide a conceptual framework for the dynamics of translation of complex as compared to simple viral mRNAs and suggest that ribosome activity may vary at multiple discrete regions on an mRNA.

Amino Acid Sequence↗

Reovirus genome segment assortment into progeny genomes studied by the use of monoclonal antibodies directed against reovirus proteins.

Using a panel of monoclonal antibodies (MABs) against reovirus proteins, we have identified proteins that associate with reovirus messenger RNA molecules prior to the generation of progeny double-stranded (ds) genome segments and proteins that are components of the structures within which progeny ds genome segments are generated. The following conclusions can be drawn from the results obtained. (1) Three proteins rapidly become associated with mRNA molecules to form single-stranded RNA-containing complexes (ssRCCs): the nonstructural protein microNS, the nonstructural protein sigma NS, and protein sigma 3. (2) Analysis of populations of ssRCCs in density gradients and by sequential exposure to various MABs indicates that some ssRCCs contain only microNS, others microNS and sigma NS or sigma 3, and others all three proteins. Each ssRCC contains one RNA molecule and, depending on the size of the RNA, 10-30 protein molecules. (3) The relative proportions of the individual RNA species in the ssRCC populations reflect the composition of the total mRNA population present in infected cells (which differs substantially from equimolarity). (4) RCCs that contain dsRNA, which become detectable as early as 4 hr after infection, contain not only microNS, sigma NS, and sigma 3, but also lambda 2. (5) The relative proportions of the 10 genome segments in dsRCCs are equimolar. This suggests that genome segment assortment into progeny genomes is linked to the transcription of plus strands into minus strands.

Animals↗

Genetic variation during lytic reovirus infection: high-passage stocks of wild-type reovirus contain temperature-sensitive mutants.

Wild-type clones of reovirus serotypes 1 (Lang), 2 (Jones), and 3 (Dearing) were serially passaged in L cells at a high multiplicity of infection, and the virus population was examined at passage levels 2, 5, and 11 for the presence of temperature-sensitive (ts) mutants. By passage 11 all three serotypes contained ts mutants that were not present in the original wild-type stock. ts mutants representing three mutant groups were identified. The majority of these mutants were in group G. Our results show that high-passage stocks of reovirus consist of a genetically heterogeneous population.

Genes, Viral↗

Detection and identification of avian, duck, and goose reoviruses by RT-PCR: goose and duck reoviruses are part of the same genogroup in the genus Orthoreovirus.

A reverse transcription-polymerase chain reaction (RT-PCR) procedure for the detection of avian, duck, and goose reovirus (ARV, DRV, and GRV) RNA from cell culture supernatant and clinical samples was established. Based on multiple sequence alignment, a pair of degenerate primers was selected and synthesized. The amplified, cloned, and sequenced 598-base-pair products from the sigmaA-encoding gene fragment from 16 isolates (ranging over 30 years) indicated that the primer regions were well conserved. The sensitivity of this method was determined to be 10(-2) PFU. The specificity of the RT-PCR method was determined by testing specimens containing avian influenza A viruses, Newcastle disease virus, and infectious bronchitis virus, all of which yielded negative results with no discernible background. The efficiency of the system for detection of ARV, DRV, and GRV directly in 71/83 clinical samples was confirmed. The nucleotide sequence analysis indicated that DRV and GRV isolated from China in different locales and years were closely related, showing 97.4-100% homology to each other, but with only 86.7-88.5% identity to DRV 89026. The nucleotide and amino acid sequence identities in the amplified sigmaA-encoding gene were 74.2-78.4% and 86.9-92.0%, respectively, between duck/goose and chicken species. Phylogenetic analysis indicated that GRV and DRV aggregated into the same specified genogroup within subgroup II of the genus Orthoreovirus and are more closely related to ARV than to Nelson Bay virus. Overall, this study developed a sensitive and specific technique for the identification ARV, DRV, and GRV, and sequencing analysis has enhanced our understanding of the evolutionary relationship between ARV, DRV, and GRV.

Animals↗

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s4 mRNA which encodes the major capsid surface polypeptide sigma 3.

Serotype 1 Lang strain s4 mRNA, which encodes the major capsid surface polypeptide sigma 3 of reovirions, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence for s4 mRNA has been determined from cDNA clones. The Lang strain s4 mRNA is 1196 nucleotides in length and possesses an open reading frame with a coding capacity of 365 amino acids, sufficient to account for a sigma 3 polypeptide of 41,212 daltons. Comparison of the serotype 1 (Lang) s4 sequence with the serotype 3 (Dearing) s4 sequence reveals 94% homology at the nucleotide level; the predicted sigma 3 polypeptides of the Lang and Dearing strains display 96% homology at the amino acid level. Two third base C codons (leu:CUC and ser:AGC) are used about one-tenth as frequently in the reovirus s4 mRNAs as compared to mammalian cellular mRNAs.

Amino Acid Sequence↗

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s3 mRNA which encodes the nonstructural RNA-binding protein sigma NS.

Human reovirus serotype 1 Lang strain s3 mRNA, which encodes the nonstructural RNA-binding polypeptide sigma NS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s3 mRNA is 1198 nucleotides in length and possesses an open reading frame with a coding capacity of 366 amino acids, sufficient to account for a sigma NS polypeptide of 41,179 daltons. Comparison of the serotype 1 (Lang) s3 sequence with the serotype 3 (Dearing) s3 sequence reveals 86.8 percent homology at the nucleotide level. The predicted sigma NS polypeptides of the Lang and Dearing strains display 97 percent homology at the amino acid level.

Amino Acid Sequence↗

Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s2 mRNA which encodes the virion core polypeptide sigma 2.

Human reovirus serotype 1 Lang strain s2 mRNA, which encodes the virion inner capsid core polypeptide sigma 2, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s2 mRNA is 1331 nucleotides in length and possesses an open reading frame with a coding capacity of 335 amino acids, sufficient to account for a sigma 2 polypeptide of 37,682 daltons. Comparison of the serotype 1 Lang s2 sequence derived from cDNA clones of s2 mRNA with the serotype 3 Dearing S2 sequence derived from cDNA clones of the S2 dsRNA genome segment reveals 86 percent homology at the nucleotide level. The predicted sigma 2 polypeptides of the Lang and Dearing strains display 98 percent homology at the amino acid level. Of 147 silent nt differences in the translated region, 136 were in the third base position of codons.

Amino Acid Sequence↗

Development and characterization of monoclonal antibodies against avian reovirus sigma C protein and their application in detection of avian reovirus isolates.

Avian reovirus (ARV) is a non-enveloped virus with a segmented double-stranded RNA genome surrounded by a double icosahedral capsid shell. ARVs are associated with viral arthritis, immunosuppression, and enteric diseases in poultry. The sigma C protein was involved in induction of apoptosis and neutralization antibody. In the present study, sigma C-His protein was expressed in Sf9 insect cells and purified by immobilized metal affinity chromatography. Eight monoclonal antibodies (mAbs) against sigma C-His and three mAbs against His were screened from hybridoma cells produced by fusion of splenocytes from immunized mice with NS1 myeloma cells. Among the eight mAbs against sigma C protein, all belonged to the IgG isotype except three for IgM. It was discovered that all anti-His mAbs were mixtures of IgG and IgM isotypes. mAbs reacted with sigma C-His protein in a conformation-independent manner based on dot blot and western blotting assays. The competitive binding assay indicated that all mAbs recognized the same epitope on sigma C protein that was conserved in different isolates. Compared with the commercial anti-ARV S1133 polyclonal antibody, mAb (D15) had universal reactivity to all serotypes or genotypes of ARVs tested. This monoclonal antibody may therefore be useful for the development of an antigen-capture enzyme-linked immunosorbent assay for rapid detection of field isolates.

Animals↗

Immunological response to infection with human reovirus-like agent: measurement of anti-human reovirus-like agent immunoglobulin G and M levels by the method of enzyme-linked immunosorbent assay.

The report describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the human reovirus-like agent of infantile gastroenteritis (HRVLA). This ELISA system proved to be four times as sensitive as the standard anti-HRVLA fluorescent-antibody assay and ten times as sensitive as the standard anti-HRVLA complement fixation assay. In addition, the ELISA was capable of determining immunoglobulin G (IgG) and IgM subclasses of anti-HRVLA antibody using a single dilution os serum. With this assay, it was discovered that 11 of 21 infected children had anti-HRVLA IgM in their acute sera before the appearance of anti-HRVLA IgG. ELISA is a useful tool in the evaluation of immunological response to HRVLA infection.

Adult↗