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PubMed1993

Rapid identification of flavivirus using the polymerase chain reaction.

A rapid and accurate detection and identification system was developed for flaviviruses that makes use of reverse transcription-polymerase chain reaction (RT-PCR). A primer pair (YF-1 and YF-3), which corresponds to the highly conserved sequence at 3' noncoding region among flaviviruses, was useful for identification of mosquito-borne flaviviruses. Nine sets of species specific primer pairs were also selected to identify and distinguish species, i.e., yellow fever, West Nile, Murray Valley encephalitis, Japanese encephalitis, St. Louis encephalitis and dengue type 1 to 4 viruses. This method required only two hours for completion using infected culture fluid, thus facilitating rapid identification of mosquito-borne flavivirus species.

Animals↗
PubMed1998

Hepatitis C virus population dynamics in human lymphocytes and hepatocytes infected in vitro.

We previously found two cell lines (MT-2 and PH5CH) that were susceptible to hepatitis C virus (HCV) infection. Analysis of the infectivity of sera from HCV-positive blood donors for MT-2 and PH5CH cells suggested the cell tropism of HCV. To investigate further the cell tropism of HCV, the dynamics of HCV populations during culture were examined using three MT-2 clones and three PH5CH clones, infected with inoculum 1B-2. To type HCV populations in these infected cells, the HCV hypervariable region 1 (HVR1) in these cloned cells was characterized by sequence analysis and HpaII digestion analysis, which could distinguish three major HVR1 types (I, II and III) derived from the inoculum 1B-2. It was found that genomes containing HVR1 type I became predominant in MT-2 clones, and genomes containing HVR1 type II became predominant in PH5CH clones during culture after inoculation. These results suggest that inoculum 1B-2 contains both lymphotropic and hepatotropic HCV species, which can be distinguished by HVR1 type. To search for cell type-specific sequences in regions other than HVR1, three HCV cDNA clones (3.4 kb of the 5' noncoding region to the nonstructural 2 region) containing HVR1 type I obtained from HCV-infected MT-2C cells, and three HCV cDNA clones containing HVR1 type II obtained from HCV-infected PH5CH7 cells were sequenced. Following a comparison of the sequences, 11 amino acids were identified as candidates for determinants of the cell tropism of HCV.

Amino Acid Sequence↗
PubMed2002

Prevalence of GB virus C (hepatitis G virus) and risk factors for infection in São Paulo, Brazil.

The prevalence of GB virus C (GBV-C) varies widely throughout the world. A cross-sectional study was conducted in the city of São Paulo, Brazil, to estimate the prevalence of GBV-C infection and to identify associated risk factors, using a large sampling of the general population rather than blood donors or an illness-related group of subjects. GBV-C RNA was detected by reverse-transcriptase polymerase chain reaction using primers directed to the 5' noncoding region (NCR) and nonstructural 5A region (NS5A) in serum samples from 1,039 healthy individuals 2 years of age or more. Fifty-two individuals were positive for both sets of primers and one was positive for NS5A only (prevalence of GBV-C infection, 5.1%; 95%CI, 3.9-6.7%). No child under 5 years of age was found positive. Among subjects aged 5 years or more, the prevalence of infection increased consistently with age, up to 30-39 years (8.3%), and decreased from then on. The number of sexual partners in the last 3 years (2 or more: OR, 2.6; 95%CI, 1.3-5.5) and history of contact with blood-sucking insects (OR, 2.5; 95%CI 1.2-5.4) were independently associated with GBV-C infection. In conclusion, the prevalence of GBV-C infection is high in São Paulo. In addition to parenteral transmission, another route, e.g. sexual or vertical, may be involved.

Adolescent↗
PubMed2002

Athila4 of Arabidopsis and Calypso of soybean define a lineage of endogenous plant retroviruses.

The Athila retroelements of Arabidopsis thaliana encode a putative envelope gene, suggesting that they are infectious retroviruses. Because most insertions are highly degenerate, we undertook a comprehensive analysis of the A. thaliana genome sequence to discern their conserved features. One family (Athila4) was identified whose members are largely intact and share >94% nucleotide identity. As a basis for comparison, related elements (the Calypso elements) were characterized from soybean. Consensus Calypso and Athila4 elements are 12-14 kb in length and have long terminal repeats of 1.3-1.8 kb. Gag and Pol are encoded on a single open reading frame (ORF) of 1801 (Calypso) and 1911 (Athila4) amino acids. Following the Gag-Pol ORF are noncoding regions of ~0.7 and 2 kb, which, respectively, flank the env-like gene. The env-like ORF begins with a putative splice acceptor site and encodes a protein with a predicted central transmembrane domain, similar to retroviral env genes. RNA of Athila elements was detected in an A. thaliana strain with decreased DNA methylation (ddm1). Additionally, a PCR survey identified related reverse transcriptases in diverse angiosperm genomes. Their ubiquitous nature and the potential for horizontal transfer by infection implicates these endogenous retroviruses as important vehicles for plant genome evolution.

Amino Acid Sequence↗
PubMed1996

Type 2 poliovirus recombinants isolated from vaccine-associated cases and from healthy contacts in Brazil.

In a previous study (Friedrich et al., 1995b) P2/Sabin-derived strains isloated in Brazil from vaccine-associated paralytic poliomyelitis (VAPP) cases and from healthy contacts were analyzed for the presence of mutations at nucleotide (nt) 481 in the 5'-noncoding region (5'NCR) and at the codon of amino acid (aa) 143 of the capsid protein VP1, that are known to increase neurovirulence. In the present study a part of the 3Dpol-coding region of these strains was sequenced (3Dpol seq.) with the aim to find recombinant strains. In the 3Dpol seq., four out of ten strains isolated from VAPP cases turned out to be recombinants: one had 3Dpol seq. from the P1/Sabin strain, while the second had a part of 3Dpol seq. both from the P2/Sabin and P1/Sabin strains; the third and fourth recombinants had 3Dpol seq. from non-vaccine strains. The strains isolated from healthy contacts of the two VAPP cases, from which type 2 vaccine/non-vaccine recombinant strains were isolated, also consisted from recombinant genomes with the same nt sequences as those of the isolates from VAPP cases, confirming the transmission of P2/Sabin-derived recombinants. Comparison of the aa sequence of the viral RNA polymerase of the P2/Sabin strain with the predicted aa sequences of these recombinants in 3Dopl seq. demonstrated that an aa 69 (Asp-->Glu)) substitution was observed in most of the recombinant genomes, while an aa 113 (Thr-->Ser) substitution was observed in all the recombinant genomes. The possibility that the genomic recombination increased the neurovirulence of these strains cannot be excluded.

Amino Acid Sequence↗
PubMed1992

Intracellular modifications induced by poliovirus reduce the requirement for structural motifs in the 5' noncoding region of the genome involved in internal initiation of protein synthesis.

A series of genetic deletions based partly on two RNA secondary structure models (M. A. Skinner, V. R. Racaniello, G. Dunn, J. Cooper, P. D. Minor, and J. W. Almond, J. Mol. Biol. 207:379-392, 1989; E. V. Pilipenko, V. M. Blinov, L. I. Romanova, A. N. Sinyakov, S. V. Maslova, and V. I. Agol, Virology 168:201-209, 1989) was made in the cDNA encoding the 5' noncoding region (5' NCR) of the poliovirus genome in order to study the sequences that direct the internal entry of ribosomes. The modified cDNAs were placed between two open reading frames in a single transcriptional unit and used to transfect cells in culture. Internal entry of ribosomes was detected by measuring translation from the second open reading frame in the bicistronic mRNA. When assayed alone, a large proportion of the poliovirus 5' NCR superstructure including several well-defined stem-loops was required for ribosome entry and efficient translation. However, in cells cotransfected with a complete infectious poliovirus cDNA, the requirement for the stem-loops in this large superstructure was reduced. The results suggest that virus infection modifies the cellular translational machinery, so that shortened forms of the 5' NCR are sufficient for cap-independent translation, and that the internal entry of ribosomes occurs by two distinct modes during the virus replication cycle.

DNA Mutational Analysis↗
PubMed1992

Glucocorticoid-dependent transformation by human papillomavirus type 16 E7 coding and 3' noncoding sequences.

The establishment of transformation of primary rodent cells by human papillomavirus (HPV) type 16 DNA requires glucocorticoid hormones (Pater et al., Nature 335, 832-835, 1988). Here we provide evidence by mutational analysis that, in the context of the hormone-regulated HPV 16 promoter/enhancer, the only protein coding sequences of HPV 16 required are those of the E7 gene. Moreover, additional sequences adjacent to the 3' end of E7 coding sequences are also essential for the establishment of the transformed phenotype. Splice donor sites, especially an E7 ORF 3' proximal one, are implicated for this cis-acting function, since specific deletion mutations of these splice sites greatly or completely reduced the frequency of transformation and the level of E7 RNA.

Animals↗
PubMed1992

Mutagenesis of a hexanucleotide sequence conserved in potexvirus RNAs.

Biologically active in vitro transcripts were synthesized from a cloned cDNA of a defective RNA (D RNA) of clover yellow mosaic virus (CYMV) and were used to determine if a hexanucleotide motif (5'-ACUUAA) conserved in the 3' noncoding region of potexvirus RNAs is essential for accumulation of progeny D RNA in planta. Deletion of rearrangement of the entire hexanucleotide sequence in the D RNA resulted in no detectable accumulation of progeny D RNA when coinoculated with helper CYMV RNA. Single-base substitutions of the four central nucleotides of the hexanucleotide sequence revealed that viable D RNAs can tolerate single residue changes at positions 3 and 5 only. These results suggest that the hexamer motif is involved in the process of D RNA propagation. The hexanucleotide sequence was also identified in the negative strand of potexvirus RNAs in the regions proposed to represent subgenomic RNA (sgRNA) promoters. In addition, the hexamer motif is present in similar regions in carlavirus RNAs. The conservation of this hexanucleotide (in orientation and position) in potexvirus and carlavirus RNAs, which serve as templates for full-length negative-strand synthesis and sgRNA production, strongly suggests that it plays a functional role in the synthesis of viral RNAs. Taken together, our data support our previous proposal (Bancroft et al., 1991. J. Gen. Virol. 72, 2173-2181) that the hexanucleotide sequence acts as a cis element involved in synthesis of full-length negative-sense viral RNA and further implicate the sequence in a similar role in production of positive-sense sgRNA.

Capsid↗
PubMed1997

Serological determination of hepatitis C virus genotype: comparison with a standardized genotyping assay.

In patients with chronic hepatitis C, determination of hepatitis C virus (HCV) genotype could be routinely run in the future to tailor treatment schedules. The suitabilities of two versions of a serological, so-called serotyping assay (Murex HCV Serotyping Assay version 1-3 [SA1-3] and Murex HCV Serotyping Assay version 1-6 [SA1-6]; Murex Diagnostics Ltd.), based on the detection of genotype-specific antibodies directed to epitopes encoded by the NS4 region of the genome, for the routine determination of HCV genotypes were studied. The results were compared with those of a molecular biology-based genotyping method (HCV Line Probe Assay [INNO-LiPA HCV]; Innogenetics S.A.), based on hybridization of PCR products onto genotype-specific probes designed in the 5' noncoding region of the genome, obtained with pretreatment serum samples from 88 patients with chronic hepatitis C eligible for interferon therapy. Definitive genotyping was performed by sequence analysis of three regions of the viral genome in all samples with discrepant typing results found among at least two of the three assays studied. In all instances, sequence analysis confirmed the result of the INNO-LiPA HCV test. The sensitivity of SA1-3 was 75% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 92% of the samples typeable by SA1-3. The sensitivity of SA1-6 was 89% relative to the results obtained by the genotyping assay. The results were concordant with those of genotyping for 94% of the samples typeable by SA1-6. Overall, SA1-6 had increased sensitivity relative to SA1-3 but remained less sensitive than the genotyping assay on the basis of PCR amplification of HCV RNA. Cross-reactivities between different HCV genotypes could be responsible for the mistyping of 8 (SA1-3) and 6% (SA1-6) of the samples. Subtyping of 1a and 1b is still not possible with the existing peptides, but discriminating between subtypes may not be necessary for routine use.

Female↗
PubMed1990

Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region.

The poliovirus mutant 5NC-11 has a 4-base insertion at position 70 within the 5' untranslated region and is deficient in RNA synthesis. Revertants from 5NC-11 were isolated, showing a partial recovery of wild-type levels of RNA synthesis. The 5' noncoding region of those revertants contained the mutation intact; mix-and-match experiments with the cDNA from these revertants revealed that a restricted region within the 3C gene was the site of the suppressing mutations in the revertants. The suppressors were point mutations, confirmed by introducing them into the 3C gene by site-directed mutagenesis. Although complementation studies indicated that the suppressors were cis active, we believe that protein changes rather than RNA sequence alterations are responsible for the suppression because RNA changes that did not alter protein sequence had no effect, whereas various protein alterations were suppressive. The results therefore imply that protein 3C interacts with the 5' end of the RNA and may play a role in RNA replication.

Amino Acid Sequence↗
PubMed1994

HCV RNA is present in the menstrual blood of women with chronic hepatitis C infection.

OBJECTIVES: To further determine potential routes of sexual transmission of hepatitis C virus (HCV), we examined the menstrual blood of women chronically infected with this virus. METHODS: Ten premenopausal women with documented HCV infection were studied. All patients were anti-HCV positive by ELISA-II and positive for HCV RNA by polymerase chain reaction. Eight patients acquired their infection via intravenous drug abuse, one patient through blood transfusion, and one patient was a health care worker. Liver biopsies showed evidence of chronic hepatitis in all patients. Menstrual blood was collected on the first day of menses utilizing a sterile 15-ml conical centrifuge tube. Total RNA was isolated from serum by the one-step guanidinium method. Reverse transcriptase polymerase chain reaction was performed with "nested" primers from the 5' noncoding region of the HCV genome. All samples were run twice, and negative controls were run with each sample. Three anti-HCV negative volunteers served as controls. RESULTS: HCV RNA was present in the menstrual blood of all chronically infected patients tested. All controls were negative for menstrual blood HCV RNA. CONCLUSIONS: 1) HCV RNA is routinely present in the menstrual blood of women chronically infected with this virus. 2) Knowledge of the presence of HCV RNA in menstrual blood should help facilitate appropriate guidelines for the sexual counseling of patients with chronic HCV infection.

Adult↗
PubMed1999

Sequence and insertion sites of murine melanoma-associated retrovirus.

We previously showed that B16 melanoma cells produce ecotropic melanoma-associated retrovirus (MelARV) which encodes a melanoma-associated antigen recognized by MM2-9B6 monoclonal antibody. The biological significance of MelARV in melanoma formation remains unknown. We found that infection of normal melanocytes with MelARV resulted in malignant transformation. It is likely that MelARV emerged from the defective Emv-2 provirus, a single copy of ecotropic provirus existing in the genome of C57BL/6 mice. In the present study, we cloned and sequenced the full-length MelARV genome and its insertion sites and we completed sequencing of the Emv-2 provirus. Our data show that MelARV has a typical full-length retroviral genome with high homology (98.54%) to Emv-2, indicating a close relationship between both viruses. MelARV probably emerged as a result of recombination between Emv-2 and an endogenous nonecotropic provirus. Some observed differences in the gag and pol regions of MelARV might account for the restoration of productivity and infectivity of a novel retrovirus that somatically emerged during melanoma formation. MelARV does not contain any oncogene and therefore might induce transformation by insertional mutagenesis. We sequenced two insertion sites of MelARV. The first insertion site represents the 3' coding region of the c-maf proto-oncogene at 67.0 centimorgans (cM) on chromosome 8. The c-maf proto-oncogene encodes a basic leucine zipper protein homologous to c-fos and c-jun. Insertion of MelARV in BL6 melanoma cells resulted in the up-regulation of c-maf. It is noteworthy that the Emv-2 provirus is also inserted into a noncoding region at 61.0 cM on the same chromosome 8. The second insertion site is the 3' noncoding region of the DNA polymerase gamma (PolG) gene on chromosome 7. The expression of PolG was not affected by the MelARV insertion. Further investigation of the biological significance of MelARV in melanoma formation is being undertaken.

Animals↗
PubMed1988

The adenovirus tripartite leader may eliminate the requirement for cap-binding protein complex during translation initiation.

The adenovirus tripartite leader is a 200-nucleotide 5' noncoding region that is found on all late viral mRNAs. This segment is required for preferential translation of viral mRNAs at late times during infection. Most tripartite leader-containing mRNAs appear to exhibit little if any requirement for intact cap-binding protein complex, a property previously established only for uncapped poliovirus mRNAs and capped mRNAs with minimal secondary structure. The tripartite leader also permits the translation of mRNAs in poliovirus-infected cells in the apparent absence of active cap-binding protein complex and does not require any adenovirus gene products for this activity. The preferential translation of viral late mRNAs may involve this unusual property.

Adenoviruses, Human↗
PubMed1991

Frameshifting in gene 10 of bacteriophage T7.

Gene 10 of bacteriophage T7, which encodes the most abundant capsid protein, has two products: a major product, 10A (36 kDa), and a minor product, 10B (41 kDa). 10B is produced by frameshifting into the -1 frame near the end of the 10A coding frame and is incorporated into the capsid. The frameshift occurs at a frequency of about 10% and is conserved in bacteriophage T3. This study shows that sequences important to frameshifting include the originally proposed frameshift site, consisting of overlapping phenylalanine codons and the 3' noncoding region that includes the transcriptional terminator over 200 bases downstream of the frameshift site. The frameshift occurs at the overlapping phenylalanine codons as determined from peptide sequencing data. Complementation studies show that there is only a very weak phenotype associated with phage infections in which there is no 10A frameshifting. Capsids from such infections are devoid of 10B and are as stable as wild-type capsids.

Amino Acid Sequence↗
PubMed1996

Genotype determination of hepatitis C virus from northern India: identification of a new subtype.

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the non-structural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India.

Amino Acid Sequence↗
PubMed1996

Reverse transcription-PCR detection of hepatitis G virus.

Hepatitis G virus (HGV) was recently identified as a new member of the Flaviviridae, but its clinical significance is still unclear. Since no immunoassay for the diagnosis of HGV is available, we developed a sensitive reverse transcription-PCR (RT-PCR) assay to facilitate the detection of the viral genome by mass screening in the clinical laboratory. Sequences within the 5'-noncoding region and within the putative NS5a region are independently amplified in the presence of digoxigenin-11-dUTP and are detected by hybridization with biotinylated capture probes binding to a streptavidin-coated matrix. Semiquantitative Enzymun-Test DNA detection via chemiluminescence can be performed either in a microtiter plate format or on fully automated ES 300 machines. We were able to detect at least 8 x 10(2) genome equivalents per ml of serum using both primer pairs. HGV was shown to be present in 43 of 130 (33%) serum samples from intravenous drug abusers with a high risk of parenteral exposure. However, only two of the patients were positive when the NS5a primers only were used, and only one patient was positive when only the 5'-noncoding region primers were used, demonstrating the increased sensitivity of HGV detection with two sets of primers. Among these patients, there was no obvious correlation with other viral infections like hepatitis B virus, hepatitis C virus, or human immunodeficiency virus. Within a blood donor panel, 3 of 92 (3%) samples were found to be HGV positive, suggesting that donated blood may need to be screened for HGV.

Base Sequence↗
PubMed1991

Identification of two determinants that attenuate vaccine-related type 2 poliovirus.

The poliovirus P2/P712 strain is an attenuated virus that is closely related to the type 2 Sabin vaccine strain. By using a mouse model for poliomyelitis, sequences responsible for attenuation of the P2/P712 strain were previously mapped to the 5' noncoding region of the genome and a central region encoding VP1, 2Apro, 2B, and part of 2C. To identify specific determinants that attenuate the P2/P712 strain, recombinants between this virus and the mouse-adapted P2/Lansing were constructed and their neurovirulence in mice was determined. By using this approach, the attenuation determinant in the central region was mapped to capsid protein VP1. Candidate attenuating sequences in VP1 and the 5' noncoding region were identified by comparing the P2/P712 sequence with that of vaccine-associated isolate P2/P117, and the P2/117 sequences were introduced into the P2/Lansing-P2/P712 recombinants by site-directed mutagenesis. Results of neurovirulence assays in mice indicate that an A at nucleotide 481 in the 5' noncoding region and isoleucine (Ile) at position 143 of capsid protein VP1 are the major determinants of attenuation of P2/P712. These determinants also attenuated neurovirulence in transgenic mice expressing human poliovirus receptors, a new model for poliomyelitis in which virulent viruses are not host restricted. These results demonstrate that A-481 and Ile-143 are general determinants of attenuation.

Animals↗
PubMed2007

Sequence determination and functional analysis of the Akabane virus (family Bunyaviridae) L RNA segment.

Akabane virus (AKAV) causes epizootic congenital deformities in cattle, sheep, and goats. Due to the lack of a complete genome sequence, the molecular biological properties of this virus are not known. We have cloned and sequenced the functional large (L) RNA segment of AKAV, and shown that it has polymerase activity using a minireplicon system with RNA polymerase I. The complete L RNA segment is 6868 nucleotides long and encodes an L protein of 2251 amino acids, which functions as an RNA-dependent RNA polymerase. A minireplicon reporter plasmid was constructed by flanking either the firefly luciferase or the green fluorescent protein gene in the antisense orientation with the 5'- and 3'-terminal noncoding regions of the small RNA segment. HmLu-1 cells were transfected with the reporter plasmid, and the L protein and nucleoprotein (N protein) expression plasmids. The reporter activity was upregulated in a dose-dependent manner with increasing concentration of either the L or N protein expression plasmid. Furthermore, the reporter activity could be downregulated by the AKAV NSs protein as well as by other orthobunyaviruses. These results show that the AKAV minireplicon system is a powerful tool for studying transcription and for rescuing infectious viruses from cloned cDNAs.

Animals↗