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R S Baric

Publications and source records attributed to R S Baric.

At least 55 records · Page 3Linked to original sources
PubMed1992

An experimental model for dilated cardiomyopathy after rabbit coronavirus infection.

A rabbit model for coronavirus-induced dilated cardiomyopathy is described. Acute rabbit coronavirus infection results in virus-induced myocarditis and congestive heart failure. Of the survivors of rabbit coronavirus infection, 41% had increased heart weight and heart weight-to-body weight ratios, biventricular dilation, myocyte hypertrophy, myocardial fibrosis, and myocarditis consistent with the development of dilated cardiomyopathy. These changes were also seen in the remaining 59% of the survivors, except that the degree of myocyte hypertrophy was reduced and only right ventricular dilation was present. In most survivors, myocarditis was usually mild (1-5 foci/transverse section), but in some cases it was severe (> 20 foci/transverse section). Interstitial and replacement fibrosis was more pronounced in the papillary muscles. These data suggest that rabbit coronavirus infection may progress to dilated cardiomyopathy.

Animals↗
PubMed1992

Detection of Norwalk virus in stool specimens by reverse transcriptase-polymerase chain reaction and nonradioactive oligoprobes.

A reverse transcriptase (RT)-polymerase chain reaction (PCR)-oligoprobe (OP), or RT-PCR-OP, method was developed for the detection of the Norwalk virus, which causes acute, epidemic gastroenteritis, in stool specimens. The Norwalk virus genome regions encoding the following two proteins were amplified by RT-PCR: the RNA polymerase (260-bp product) and a putative immunogenic protein (224-bp product). The resulting DNA fragments (amplicons) were hybridized to a digoxigenin-labeled internal OP specific to each amplicon. The detection limit of Norwalk virus, as determined by the endpoint of RT-PCR amplification for serially diluted, positive stool specimens, was similar to the actual virion titer as estimated by electron microscopy and at least 100-fold greater than the titer determined by radioimmunoassay (RIA). The RT-PCR-OP assay was specific for Norwalk virus and negative for other enteric viruses, including human and animal caliciviruses, hepatitis E virus, Snow Mountain agent, astroviruses, 16 human enteroviruses, and 5 human rotaviruses. Components of fecal specimens that interfere with RT-PCR were removed successfully by Sephadex G-200 gel chromatography. Of 20 stool specimens from human volunteers that were positive for Norwalk virus by RIA, a specific RT-PCR-OP result was obtained in 95% (19 of 20) of the samples by using the immunogenic protein primers and 75% (15 of 20) by using the polymerase primers. Twenty-six stool specimens from asymptomatic children and adults were negative by the Norwalk virus RT-PCR-OP. RT-PCR-OP detected Norwalk virus in the 4 of 21 coded fecal specimens that were also positive by enzyme immunoassay. Two samples that were positive by RIA or enzyme immunoassay were negative by RT-PCR, perhaps because viral RNA was not present or RT-PCR inhibitors were not adequately removed.

Adult↗
PubMed1991

Detection of hepatitis A virus and other enteroviruses in water by ssRNA probes.

Sensitive and specific methods are needed to detect hepatitis A virus (HAV) and other human enteroviruses in environmental samples such as drinking water and foods. Clones of cDNA encoding the 5'-most 1 kb of the HAV and coxsackievirus B3 (CB3) genomes were subcloned into T7/SP6 RNA transcription vectors. In vitro transcribed RNA from the T7 promoter detected their respective HAV or CB3 genomic RNA. Conversely, SP6 transcripts detected viral negative-stranded RNA but not the genome. When both ssRNA probes were tested at high temperature (65 degrees C), they did not hybridize with intracellular RNAs from 6 primate cell cultures used for isolation of HAV and other enteroviruses. The HAV probe did not hybridize with 13 different enteroviruses but detected as little as 500-1000 infectious units of the 7 strains of HAV tested. Conversely, the CB3 probe showed strong homology with all 13 enteroviruses tested but not HAV. The probes were used to detect HAV and other enteroviruses in water samples after virus amplification in cell culture. HAV was detected in water samples obtained during a waterborne hepatitis outbreak using the ssRNA probe. These samples were negative for HAV by direct solid phase radioimmunoassay and were not positive by immunoassays of inoculated cell cultures until several weeks of propagation. The CB3 ssRNA probe detected enteroviruses in samples of surface water and drinking water that were negative for cytopathic effects in inoculated cell cultures.

Animals↗
PubMed1990

Genetics of mouse hepatitis virus transcription: identification of cistrons which may function in positive and negative strand RNA synthesis.

A panel of 26 temperature-sensitive mutants of MHV-A59 were selected by mutagenesis with either 5-fluorouracil or 5-azacytidine. Complementation analysis revealed the presence of one RNA+ and five RNA- complementation groups. None of the RNA- complementation groups transcribed detectable levels of positive- or negative-stranded RNA at the restrictive temperature. Temperature shift experiments after the onset of mRNA synthesis revealed at least two classes of RNA- mutants. RNA- complementation groups A, B, D, and E were blocked in the ability to release infectious virus and transcribe mRNA and genome, while group C mutants continued to release infectious virus and transcribe both mRNA and genome. Temperature shift experiments at different times postinfection suggest that the group C mutants encode a function required early in viral transcription which affects the overall rate of positive strand synthesis. Analysis of steady state levels of negative strand RNA after the shift indicate that the group C mutants were probably blocked in the ability to synthesize additional minus strand RNA under conditions in which the group E mutants continued low levels of minus strand synthesis. These data suggest that at least four cistrons may be required for positive strand synthesis while the group C cistron functions during minus strand synthesis.

Animals↗
PubMed1990

Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups.

MHV-A59 temperature-sensitive mutants, representing one RNA+ and five RNA- complementation groups, were isolated and characterized by genetic recombination techniques. Maximum recombination frequencies occurred under multiplicities of infection greater than 10 each in which 99.99% of the cells were co-infected. Recombination frequencies between different ts mutants increased steadily during infection and peaked late in the virus growth cycle. These data suggest that recombination is a late event in the virus replication cycle. Recombination frequencies were also found to range from 63 to 20,000 times higher than the sum of the spontaneous reversion frequencies of each ts mutant used in the cross. Utilizing standard genetic recombination techniques, the five RNA- complementation groups of MHV-A59 were arranged into an additive, linear, genetic map located at the 5' end of the genome in the 23-kb polymerase region. These data indicate that at least five distinct functions are encoded in the MHV polymerase region which function in virus transcription. Moreover, using well-characterized ts mutants the recombination frequency for the entire 32-kb MHV genome was found to approach 25% or more. This is the highest recombination frequency described for a nonsegmented, linear, plus-polarity RNA virus.

Animals↗
PubMed1988

Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription.

The interaction of the mouse hepatitis virus (MHV) nucleocapsid protein (N) and viral RNA was examined. Monoclonal antibody specific for N protein coimmunoprecipitated MHV genomic RNA as well as all six MHV subgenomic mRNAs found in MHV-infected cells. In contrast, monoclonal antibodies to the MHV E2 or E1 envelope glycoproteins, an anti-I-A monoclonal antibody, and serum samples from lupus patients did not immunoprecipitate the MHV mRNAs. Moreover, the anti-N monoclonal antibody did not coimmunoprecipitate vesicular stomatitis virus RNA or host cell RNA under conditions which immunoprecipitated all MHV RNAs. These data suggest a specific interaction between the N protein and the virus-specific mRNAs. Both the membrane-bound and cytosolic small MHV leader-specific RNAs of greater than 65 nucleotides long were immunoprecipitated only by anti-N monoclonal antibody. These data suggest that an N binding site is present within the leader RNA sequences at a site at least 65 nucleotides from the 5' end of genomic RNA and all six subgenomic mRNAs. The larger leader-containing RNAs originating from mRNA 1 and mRNA 6, as well as the MHV negative-stranded RNA, were also immunoprecipitated by the anti-N monoclonal antibody. These data indicate that the MHV N protein is associated with MHV-specific RNAs and RNA intermediates and may play an important functional role during MHV transcription and replication.

Antibodies, Monoclonal↗
PubMed1988

Specific interaction between coronavirus leader RNA and nucleocapsid protein.

Northwestern blot analysis in the presence of competitor RNA was used to examine the interaction between the mouse hepatitis virus (MHV) nucleocapsid protein (N) and virus-specific RNAs. Our accompanying article demonstrates that anti-N monoclonal antibodies immunoprecipitated all seven MHV-specific RNAs as well as the small leader-containing RNAs from infected cells. In this article we report that a Northwestern blotting protocol using radiolabeled viral RNAs in the presence of host cell competitor RNA can be used to demonstrate a high-affinity interaction between the MHV N protein and the virus-specific RNAs. Further, RNA probes prepared by in vitro transcription were used to define the sequences that participate in such high-affinity binding. A specific interaction occurs between the N protein and sequences contained with the leader RNA which is conserved at the 5' end of all MHV RNAs. We have further defined the binding sites to the area of nucleotides 56 to 65 at the 3' end of the leader RNA and suggest that this interaction may play an important role in the discontinuous nonprocessive RNA transcriptional process unique to coronaviruses.

Base Sequence↗
PubMed1987

Analysis of intracellular small RNAs of mouse hepatitis virus: evidence for discontinuous transcription.

We have previously shown the presence of multiple small leader-containing RNA species in mouse hepatitis virus (MHV)-infected cells. In this paper, we have analyzed the origin, structure, and mechanism of synthesis of these small RNAs. Using cDNA probes specific for leader RNA and genes A, D, and F, we demonstrate that subsets of these small RNAs were derived from the various viral genes. These subsets have discrete and reproducible sizes, varying with the gene from which they are derived. The size of each subset correlates with regions of secondary structure, whose free energy ranges from -1.6 to -77.1 kcal/mol, in each of the mRNAs examined. In addition, identical subsets were detected on the replicative intermediate (RI) RNA, suggesting that they represent functional transcriptional intermediates. The biological significance of these small RNAs is further supported by the detection of leader-containing RNAs of 47, 50, and 57 nucleotides in length, which correspond to the crossover sites in two MHV recombinant viruses. These data, coupled with the high frequency of RNA recombination during MHV infection, suggest that the viral polymerase may pause in or around regions of secondary structure, thereby generating pools of free leader-containing RNA intermediates which can reassociate with the template, acting as primers for the synthesis of full-length or recombinant RNAs. These data suggest that MHV transcription uses a discontinuous and nonprocessive mechanism in which RNA polymerase allows the partial RNA products to be dissociated from the template temporarily during the process of transcription.

DNA↗
PubMed1985

Characterization of leader-related small RNAs in coronavirus-infected cells: further evidence for leader-primed mechanism of transcription.

Mouse hepatitis virus (MHV), a murine coronavirus, replicates in the cytoplasm and synthesizes 7 viral mRNAs containing an identical stretch of leader RNA sequences at the 5'-end of each RNA. The leader-coding sequences at the 5'-end of genomic RNA are at least 72 nucleotides in length and are joined to the viral mRNAs by a unique mechanism. Utilizing a leader-specific cDNA probe, we have detected several free leader RNA species ranging from 70 to 82 nucleotides in length. The predominant leader RNA was approximately 75 nucleotides. In addition, larger distinct leader-containing RNAs were also detected ranging from 130 to 250 nucleotides in length. The 70-82-nucleotide leader-related RNAs were present in both the cytosol and membrane fractions of infected cells. They were also detected only in the small RNA fractions but not associated with the replicative-intermediate RNA. These data suggest that the leader RNAs were associated with the membrane-bound transcription complex but at least part of them were dissociated from the RNA template. We have also identified a temperature-sensitive mutant, which synthesizes only leader RNA but not mRNAs at nonpermissive temperature, indicating that leader RNA synthesis is distinct from the transcription of mRNAs. These data support the leader-primed mechanism for coronavirus transcription and suggest that one or more free leader RNAs are used as primers of mRNA synthesis.

Animals↗
PubMed1985

Recombination between nonsegmented RNA genomes of murine coronaviruses.

We have isolated a recombinant virus between the A59 and JHM strains of mouse hepatitis virus, which contain a single species of nonsegmented RNA genome. This recombinant was derived by mixed infection of DBT cells with temperature-sensitive mutants of A59 and JHM at nonpermissive temperature. Viruses recovered at this temperature were screened by oligonucleotide fingerprinting of their genomic RNAs. One recombinant virus, B1, was found to contain mostly A59-derived sequences, but the 3 kilobases at the 5' end of the genomic RNA was derived from JHM. Thus, the crossover point in the B1 genome is located within gene A, which codes for the viral RNA polymerases. The study of the intracellular RNA species of B1 virus revealed that probably all of the virus-specific subgenomic mRNA species contained the body sequences of strain A59 but the leader sequences of JHM. This result indicates that the JHM leader RNA, which differs from the A59 leader RNA, could be fused to the mRNAs of a different virus strain during RNA transcription. Furthermore, B1 virus-infected cells contain an additional subgenomic mRNA species which is transcribed from a new initiation site within gene C, suggesting that the leader RNA could determine the site of initiation for coronavirus mRNAs. These data represent a first report of RNA recombination between viruses, other than picornaviruses, which contain nonsegmented RNA genomes.

Base Sequence↗
PubMed1984

Sindbis virus mutants selected for rapid growth in cell culture display attenuated virulence in animals.

Mutants of Sindbis virus were selected for rapid growth in baby hamster kidney (BHK) cell cultures and screened for attenuation of virulence in suckling mice. Comparisons among independently isolated virulent and attenuated strains, as well as a classical reversion analysis, showed that accelerated penetration of BHK cells was correlated with attenuation in vivo. Both phenotypic changes resulted from a reorganization of virion structure as detected by monoclonal antibodies. These results suggest that mutants selected for rapid growth in cell culture may be useful as attenuated vaccines and for studies of the molecular basis of virus pathogenesis.

Animals↗
PubMed1984

Studies on the mechanism of RNA synthesis of a murine coronavirus.

The mechanism of viral RNA replication in mouse hepatitis virus (MHV)-infected cells was studied by oligonucleotide mapping of every mRNA. We discovered that an oligonucleotide, No. 10, was localized at the 5'-end of every mRNA, and was not colinear with the sequences of the virion genomic RNA. This result indicates that all of the mRNAs contain a leader sequence which is joined to the body sequences of the mRNAs. We have also studied the structure of the replicative intermediate (RI) RNA in the MHV-infected cells. This RI RNA consists of a single species corresponding to the MHV genomic RNA. No subgenomic RI structures were detected. Furthermore, the nascent RNA chains in the RI structure contained the leader sequences, suggesting that the leader RNA was not added to the mRNA post- transcriptionally , but rather, it was probably synthesized independently and then used as a primer for the synthesis of mRNAs. We have also shown that the poly (A) sequences in the MHV genome were transcribed from the poly (U) sequences present in the negative-strand template. The RNA polymerases involved in the MHV RNA synthesis were also characterized. The early polymerase synthesizes a single negative-stranded, full-length RNA. The late polymerases could be separated into two activities, one synthesizing positive-stranded genomic RNA, and the other synthesizing genomic as well as subgenomic RNAs. Thus, the replication and transcription functions of MHV could probably be separated. A plausible model of MHV replication is presented.

Animals↗
PubMed1984

Characterization of leader RNA sequences on the virion and mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus.

Mouse hepatitis virus, which replicates in cytoplasm, contains leader RNA sequences at the 5' end of the virus-specific mRNAs. We have sequenced this leader RNA by synthesizing cDNA from a synthetic oligodeoxyribonucleotide primer (15-mer) that is complementary to the sequences at the junction site between the leader and body sequences of the mRNAs. The leader sequences on each mRNA have exactly the same size, which span approximately equal to 70 nucleotides. Leader cDNA fragments obtained from several mRNA species were sequenced and found to be identical. Computer analysis of the leader RNA sequences shows that they share extensive sequence homology with the long-terminal-repeat region of several mammalian sarcoma viruses, suggesting possible common functions. This is a novel case of spliced leader sequences in the mRNAs of a cytoplasmic virus. An identical leader sequence is also present at the 5' end of the virion genomic RNA. The leader RNA is thus probably encoded by the virion genomic RNA template and is fused to the different body sequences of the various mRNAs. Since conventional RNA splicing is not involved, a novel mechanism for fusing two noncontiguous RNA segments in the cytoplasm must be utilized during viral transcription. Several minor cDNA bands longer than the leader were also synthesized, suggesting the possible presence of partially homologous sequences in other parts of the genome RNA.

Base Sequence↗
PubMed1983

Requirement for host transcription in the replication of Sindbis virus.

Host cell involvement in Sindbis virus (SB) replication was examined in cells which had been treated with either actinomycin D (AMD) or alpha-amanitin (alpha-A). Treatment with these inhibitors of host transcription before infection reduced the ability of cells to support SB growth by 1 to 2 orders of magnitude, while having little or no effect on the replication of vesicular stomatitis virus. SB replication was sensitive to alpha-A in wild-type Chinese hamster ovary (CHO) cells but was resistant to alpha-A in CHOama-1 cells, a line which contains an alpha-A-resistant RNA polymerase II. A mutant of SB, SBamr, was isolated by mutagenesis followed by selection in cells which had been treated with AMD. SBamr grew normally not only in cells treated with AMD but also in alpha-A-treated cells. Our results suggest (i) that the synthesis of cellular mRNA (and presumably protein) is required for replication of SB, (ii) that prior treatment with either drug affects the same aspect of SB replication, and (iii) that mutations in the SB genome allow the virus to overcome the effect of inhibitors of host transcription.

Amanitins↗
PubMed1983

Presence of leader sequences in the mRNA of mouse hepatitis virus.

To determine the structure and the mechanism of synthesis of mouse hepatitis virus mRNA, the map positions of the large RNase T1-resistant oligonucleotides of the seven mouse hepatitis virus strain A59 intracellular mRNA species were studied. We found that all but one of the oligonucleotides were mapped at the positions within each mRNA consistent with the nested-set, stairlike structure of mouse hepatitis virus mRNA (Lai et al., J. Virol. 39:823-834). However, one oligonucleotide, 10, was mapped near the 5' ends of every mRNA and virion genomic RNA. In other words, oligonucleotide 10 and, therefore, the sequences around the 5' ends of the mRNAs are not colinear with the genomic sequences. Because this oligonucleotide is present only once in the genomic RNA, this result indicates that oligonucleotide 10 is not transcribed from multiple sites on the genomic template, but rather represents a leader RNA sequence which is joined to the body sequences of the different mRNAs during mRNA transcription. This provides the most direct evidence thus far for the presence of leader sequences in the mRNAs of mouse hepatitis virus, which is a cytoplasmic virus. Several possible mechanisms of RNA synthesis are discussed.

Base Sequence↗