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PubMed2002

Outbreak of jaundice and hemorrhagic fever in the Southeast of Brazil in 2001: detection and molecular characterization of yellow fever virus.

Between January and March 2001, an outbreak of jaundice and hemorrhagic fever occurred in the state of Minas Gerais, Southeast region of Brazil, in which a mortality rate of 53% was reported. Seroconversion, virus isolation, histopathological and immunohistochemical findings, and reverse transcription-polymerase chain reaction (RT-PCR) identified yellow fever virus (YFV) as the etiological agent responsible for the outbreak. Partial nucleotide sequence analysis from a fragment of the YFV genome spanning parts of nonstructural (NS) 5 gene and 3' noncoding region (3' UTR) showed that the YFV involved in this outbreak belongs to South American genotype I and differs from the Brazilian virus identified in 1996.

Amino Acid Sequence↗
PubMed1993

Use of genomic probes to detect hepatitis A virus and enterovirus RNAs in wild shellfish and relationship of viral contamination to bacterial contamination.

Genomic probes were used to investigate hepatitis A virus (HAV) and enterovirus RNAs in two types of shellfish from natural beds (Atlantic coast, France). After elution concentration, nucleic acid extracted by proteinase K and purified by phenol-chloroform and ethanol precipitation was assayed by dot blot hybridization. The probes used were a specific HAV probe corresponding to the 3' end (3D polymerase coding region) and an enterovirus probe corresponding to the 5' noncoding region. The method was first tested under experimental conditions by using virus-spiked shellfish before being applied under field conditions. Our results show that shellfish were highly contaminated: enterovirus and HAV RNAs were found in 63 and 67%, respectively, of samples examined with the riboprobes. On the same site, viral (HAV and enterovirus) RNAs were found in a larger fraction of cockles than mussels. Statistical tests of dependence showed no relationship between viral contamination and bacterial contamination (evaluated by fecal coliform counts).

Animals↗
PubMed1999

An attenuated variant of the GDVII strain of Theiler's virus does not persist and does not infect the white matter of the central nervous system.

The DA strain of Theiler's virus causes a persistent and demyelinating infection of the white matter of spinal cord, whereas the GDVII strain causes a fatal gray-matter encephalomyelitis. Studies with recombinant viruses showed that this difference in phenotype is controlled mainly by the capsid. However, conflicting results regarding the existence of determinants of persistence in the capsid of the GDVII strain have been published. Here we show that a GDVII virus whose neurovirulence has been attenuated by an insertion in the 5' noncoding region does not persist in the central nervous systems of mice. Furthermore, this virus infects the gray matter efficiently, but not the white matter. These results confirm the absence of determinants of persistence in the GDVII capsid. They suggest that the DA capsid controls persistence by allowing the virus to infect cells in the white matter of the spinal cord.

Animals↗
PubMed2000

[Sequence analysis of phosphoprotein genes of respiratory syncytial virus field strains isolated in China].

OBJECTIVE: To get information about phosphoprotein (P) genes of RSV field strains isolated in China. METHODS: The complete nucleotide sequences of the P protein genes from six respiratory syncytial virus strains were determined with cDNA clones which had been amplified by RT-PCR and cloned into PTZ18R plasmid vector and compared with those of previously published counterparts of A2 and CH18537 strains. RESULTS: The data indicated that there had high identities (>95%) at nucleotide and amino acid levels among the strains within the same subgroup; there were more divergence among strains (<85%) from different subgroups at nucleotide and amino acid levels of P genes, especially in the 3'-noncoding regions and the divergent domains. The analysis of hydrophobicity indicated that there had difference from 50 to 100 amino acid of P proteins between A and B subtypes. CONCLUSIONS: The analysis of the P genes confirm that there existed variability among RSV field strains and strains A2 and CH18537. The data described above revealed the characteristics of the P genes of RSV field strains isolated in China.

Base Sequence↗
PubMed1993

[Typing on 5'-terminal noncoding region of hepatitis C virus genome with restrict endonuclease].

We reported typing of the 5'-terminal noncoding region of HepCV genome in 219 patients with hepatitis C using restrict endonuclease. These patients came from different areas of China. The infective percentage of HCV type II was 83%, type III 14%, and mixed type II and III (II/III) 2%. It is indicated that type III and II/III. The constituent rate of HCV subgenome type in different areas was not significantly different.

China↗
PubMedDate not supplied

[3'terminal nucleotide sequence of barley stripe mosaic virus RNA].

Barley stripe mosaic virus (BSMV) is a representative of the hordeiviruses which has a functionally fragmented RNA genom. 3'-terminal nucleotide sequence of the noncoding region of BSMV RNA 2 has been determined. This region contains the internal olygo(A) sequence (n = 21) and the tyrosine accepting tRNA-like structure at 3'-extreme end. tRNA-like sequence can fold into the two distinct secondary structure which strikingly similar to such a structures which has been proposed recently for cucumo- and bromoviruses.

Base Sequence↗
PubMed1981

Uninfected cell polymerase efficiently transcribes early but not late herpes simplex virus type 1 mRNA.

The sequences of the DNAs encoding the 5' ends of one early and one late herpes simplex virus type 1 mRNA were analyzed, and the 5' ends of these mRNA species were precisely located. Neither mRNA species is spliced and the noncoding strand of the DNA contains recognizable T-A-T-A and C-A-T boxes upstream from their respective 5' ends. The early mRNA was efficiently transcribed by a commercially available uninfected cell lysate system, but the late mRNA was not. This difference between early and late mRNAs appears to be general in this virus.

Base Sequence↗
PubMed2000

The 5' repeat elements of the mouse Xist gene inhibit the transcription of X-linked genes.

X chromosome inactivation in mammals requires the Xist gene, which is exclusively expressed from the inactive X chromosome (Xi). The large heterogeneous Xist nuclear RNA colocalizes with Xi, most likely through nuclear protein interactions. The 5' region of the Xist RNA contains a series of well-conserved tandem repeats known to bind heteronuclear proteins in vitro and to enhance human XIST transcription. We show in an in vitro system that the conserved repeat element located in the 5' region of the mouse Xist gene (Xcr) represses three X-linked genes but has no effect on the autosomal genes Aprt, Ins, and the viral SV40 gene. The repression effect is not mediated by the conserved core sequence (Ccs) of Xcr, but requires the presence of the complete Xcr. This Xcr effect on X-linked genes suggests that Xcr transcript recognizes the genes to be silenced and is involved in the spreading of X inactivation.

Animals↗
PubMed1992

Translational enhancement of the poliovirus 5' noncoding region mediated by virus-encoded polypeptide 2A.

Genetic and biochemical studies have revealed that the 5' noncoding region of poliovirus mediates translation of the viral mRNA by an unusual mechanism involving entry of ribosomes in internal sequences of mRNA molecules. We have found that mRNAs bearing the 5' noncoding region of poliovirus were translated at an enhanced rate in poliovirus-infected mammalian cells at a time when translation of cellular mRNAs was not yet inhibited. This translational enhancement of the polioviral 5' noncoding region was mediated by the expression of virus-encoded polypeptide 2A. This indicates that 2A is a multifunctional protein involved directly or indirectly in the activation of viral mRNA translation, in addition to its known roles in viral polyprotein processing and in inhibition of cellular protein synthesis. Thus, 2A represents an activator of translation of a viral mRNA that is translated by an internal ribosome binding mechanism. A likely consequence of this role of 2A is the efficient translation of viral mRNAs early in the infectious cycle, when host cell mRNAs can still compete with viral mRNAs for the host cell translation apparatus.

Animals↗
PubMed1991

Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae.

We report the introduction of functional RNA molecules into yeast spheroplasts. Plasmids containing the firefly luciferase coding region were transcribed to yield RNAs suitable for introduction into yeast cells and direct assay of their translation products. The 5' noncoding regions of the RNAs were derived either from the 5' noncoding regions of firefly luciferase, poliovirus, or yeast virus-like-particle (VLP) L-A or M1 RNAs. Capped and non-capped mRNAs were made by T7 RNA polymerase-directed transcription and introduced into yeast spheroplasts. The peak time of luciferase transient expression from introduced RNAs was 2-4 h after their introduction. In contrast, transient expression of luciferase from a non-replicative, luciferase-encoding plasmid introduced into the cells was maximal at 16 h. For capped mRNAs, luciferase activity increased linearly with transcript amount for both yeast and human (HeLa) cells. Although non-capped luciferase mRNAs were expressed more efficiently following introduction into yeast than into HeLa cells, the 5' noncoding sequences from yeast double-stranded (ds)RNA VLP RNAs conferred no greater apparent cap-independence than non-VLP RNA sequences in this transient expression assay. The RNA transient expression system will allow the study of translation of capped and non-capped RNAs in yeast cells and of the replicative cycle of yeast virus-like RNA genomes.

Base Sequence↗
PubMed1995

Studies on neurovirulence in poliovirus-sensitive transgenic mice and cynomolgus monkeys for the different temperature-sensitive viruses derived from the Sabin type 3 virus.

We have studied methods for testing the neurovirulence of live poliovaccine viruses by intraspinal inoculation into mice carrying the human poliovirus receptor gene (Tg mice). A comparison of the neurovirulence of Sabin type 3 vaccine virus and related viruses using the 50% paralysis dose determined after intraspinal inoculation into the Tg mice as an index revealed a close correlation between the results of the paralysis dose in Tg mice, the neurovirulence expressed by the histopathological lesions core in monkeys, and the temperature sensitivity of the viruses. The results of experiments in the Tg mice also showed a good correlation with the number of mutations at position 472 from U to C in the 5' noncoding region in the genomes of the viruses tested. These results strongly suggest that the neurovirulence test for oral poliomyelitis vaccine using the Tg mice is an excellent method and may be used in place of the test using monkeys.

Animals↗
PubMed2003

Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA.

Viroids are small noncoding and infectious RNAs that replicate autonomously and move systemically throughout an infected plant. The RNAs of the family Pospiviroidae contain a central conserved region (CCR) that has long been thought to be involved in replication. Here, we report that the CCR of Potato spindle tuber viroid (PSTVd) also plays a role in pathogenicity. A U257A change in the CCR converted the intermediate strain PSTVd(Int) to a lethal strain that caused severe growth stunting and premature death of infected plants. PSTVd with nucleotide U257 changed to C or G did not cause such symptoms. The pathogenic effect of the U257A substitution was abolished by a C259U substitution in the same RNA. Analyses of the pathogenic effects of the U257A substitution in three other PSTVd variants established A257 as a new pathogenicity determinant that functions independently and synergistically with the classic pathogenicity domain. The U257A substitution did not alter PSTVd secondary structure, replication levels, or tissue tropism. The stunted growth of PSTVd(Int)U257A-infected tomato plants resulted from restricted cell expansion but not cell division or differentiation. This was correlated positively with the downregulated expression of an expansin gene, LeExp2. Our results demonstrate that specific nucleotides in a noncoding, pathogenic RNA have a profound effect in altering distinct cellular responses, which then lead to well-defined alterations in plant growth and developmental patterns. The feasibility of correlating viroid RNA sequence/structure with the altered expression of specific host genes, cellular processes, and developmental patterns makes viroid infection a valuable system in which to investigate host factors for symptom expression and perhaps also to characterize the mechanisms of RNA regulation of gene expression in plants.

Base Sequence↗
PubMed1994

Nucleotide sequence of the 3'-terminal region of the genome confirms that pea mosaic virus is a strain of bean yellow mosaic potyvirus.

The 1,035 nucleotides at the 3'end of the I strain of pea mosaic potyvirus (PMV-I) genomic RNA, encoding the coat protein, have been cloned and sequenced. A comparison of the derived coat protein sequence with those of the bean yellow mosaic virus (BYMV) strains, CS, S, D and GDD, indicates that PMV-I is a strain of BYMV. Sequence comparisons and hybridisation studies using the 3'-noncoding region support this classification. The nucleotide and protein sequence data also suggest that PMV-I and BYMV-CS form one subset of BYMV strains while the other three strains form another.

Amino Acid Sequence↗
PubMed1991

The polyadenylation signal of influenza virus RNA involves a stretch of uridines followed by the RNA duplex of the panhandle structure.

Appropriate RNAs are transcribed and amplified and proteins are expressed after transfection into cells of in vitro-reconstituted RNA-protein complexes and infection with influenza virus as the helper. This system permits us to study the signals involved in transcription of influenza virus RNAs. For the analysis we used a plasmid-derived RNA containing the reporter gene for chloramphenicol acetyltransferase (CAT) flanked by the noncoding sequences of the NS RNA segment of influenza A/WSN/33 virus. Mutations were then introduced into both the 5' and 3' ends, and the resulting RNAs were studied to determine their transcription in vitro and their CAT expression activity in the RNA-protein transfection system. The results reveal that a stretch of uninterrupted uridines at the 5' end of the negative-strand RNA is essential for mRNA synthesis. Also, a double-stranded RNA "panhandle" structure generated by the 5'- and 3'-terminal nucleotides appears to be required for polyadenylation, since opening up of these base pairs diminished mRNA synthesis and eliminated expression of CAT activity by the mutant RNAs. Finally, it was shown that this double-stranded RNA structural requirement is not sequence specific, since a synthetic GC clamp can replace the virus-coded RNA duplex. The data suggest that the viral RNA polymerase adds poly(A) by a slippage (stuttering) mechanism which occurs when it hits the double-stranded RNA barrier next to the stretch of uridines.

Base Composition↗
PubMed1997

Secondary structure of the 5'-noncoding region of border disease virus genome RNAs.

The computer predicted secondary structures from the 5'-NC genome region of four border disease virus (BDV) strains collected from sheep in England and Scotland over a period 1976-1986 were prepared. The FOLD program from GCG sequence analysis software package was used for the analysis of a 243 bp RNA fragment. Two typical shapes of secondary structures were observed which contained multiple imperfect stem-loop motifs. The shape of those structures exactly fit with the grouping of BDV strains to two phylogenetic groups. Secondary structures are typical only of BDV strains and they are different from the structures prepared for NADL (BVDV) and Alfort (CSFV) strains.

Animals↗
PubMed1994

Increase in the relative fitness of a plant virus RNA associated with its recombinant nature.

A recombination event at the 3' end of RNA 3 in a pseudorecombinant virus (C1C2T3) having RNAs 1 and 2 from TrK7-cucumber mosaic virus (CMV) and RNA 3 from P-tomato aspermy virus (TAV) was detected by ribonuclease protection assay (RPA). Sequence analysis of the 3' end of RNA 3 of C1C2T3 and of its parental CMV and TAV strains showed that RNA 3 of C1C2T3 had a hybrid nature in which most of the 3' noncoding region (3' ncr), including the whole 3' terminal tRNA-like structure, was derived from CMV RNA 2 by recombination. Recombination may have occurred in two steps. The first one would have caused the duplication of a large region 5' to the 3' end tRNA-like structure, derived from TAV and from CMV. The second one would have eliminated the TAV-derived sequences in this duplicated region. Competition experiments in tobacco plants showed that in a context of RNA 1 + 2 from CMV, RNA 3 from TAV is outcompleted by RNA 3 of CMV, but the recombinant TAV RNA 3 with the 3' end of CMV outcompetes both TAV and CMV RNA 3. This shows an increase in relative fitness associated with the recombinant nature of RNA 3. Our results document the potential importance of RNA-RNA recombination in the determination of the genetic structure of RNA viral populations.

Base Sequence↗
PubMed2002

Quasispecies heterogeneity and constraints on the evolution of the 5' noncoding region of hepatitis C virus (HCV): relationship with HCV resistance to interferon-alpha therapy.

Hepatitis C virus (HCV) polyprotein translation depends on direct internal entry of the 40S ribosomal subunit mediated by an internal ribosome entry segment (IRES) located in the 5' noncoding (5'NC) region of the viral genome. HCV is genetically heterogeneous and is characterized by the existence of a quasispecies distribution of the virus population within a single infected individual. Cloning and sequencing strategies were used to characterize 5'NC quasispecies genetically. Similar to coding regions, the HCV 5'NC region was distributed as a quasispecies, but it appeared to be subjected to stronger conservatory constraints than other regions of the HCV genome, probably due to the need for structural (and functional) conservation of the IRES. Indeed, most of the quasispecies substitutions were in unpaired regions of the IRES or clustered such that base-pairing was maintained, whereas only 21% were expected to result in a loss of base-pairing. Quasispecies-related structural changes could be predicted in the core cruciform of IRES domain III composed of the RNA helices which extend from the four-way junction JIIIabc, mostly in minor variants, but sometimes in major ones. The results presented here suggest the simultaneous presence in infected patients of a mixture of genetically distinct but closely related IRES sequences that may have different structures. No significant genetic changes of 5'NC quasispecies were observed after interferon-alpha treatment, except in patients with mixed genotype infection who cleared one of the infecting strains during therapy, suggesting that the quasispecies distribution of IRES sequences does not play a role in HCV resistance to interferon-alpha therapy. In contrast, the overall quasispecies distribution of HCV genomes (including IRES sequences) might participate in regulation of hepatic and extrahepatic HCV replication.

5' Untranslated Regions↗
PubMed1990

Polymerase errors accumulating during natural evolution of the glycoprotein gene of vesicular stomatitis virus Indiana serotype isolates.

We report the entire glycoprotein (G) gene nucleotide sequences of 26 vesicular stomatitis virus Indiana serotype (VSV IND) type 1 isolates from North and Central America. These sequences are also compared with partial G gene sequences of VSV IND type 2 (Cocal) and type 3 (Alagoas) viruses and the complete G gene sequences of the more distantly related VSV New Jersey (NJ) and Chandipura viruses. Phylogenetic analysis of the G gene sequences by maximum parsimony revealed four major lineages or subtypes within the classical VSV IND (type 1) viruses, each with a distinct geographic distribution. A high degree of VSV genetic diversity was found in Central America, with several virus subtypes of both VSV IND and NJ serotypes existing in this mainly enzootic disease region. Nineteen percent sequence variation but no deletions or insertions were evident within the 5' noncoding and the coding regions of the VSV IND type 1 G genes. In addition to numerous base substitutions, the 3' noncoding regions of these viruses also contained numerous base insertions and deletions. This resulted in striking variation in G gene sizes, with gene lengths ranging from 1,652 to 1,868 nucleotides. As the VSV IND type 1 subtypes have diverged from the common ancestor with the NJ subtypes, their G mRNAs have accumulated more 3' noncoding sequence inserts, ranging up to 303 nucleotides in length. These primarily consist of an imprecise reiteration of the sequence UUUUUAA, apparently generated by a unique polymerase stuttering error. Analysis of the deduced amino acid sequence differences among VSV IND type 1 viruses revealed numerous substitutions within defined antigenic epitopes, suggesting that immune selection may play a role in the evolution of these viruses.

Amino Acid Sequence↗