bluuebunny/crossref_metadata_2025_split huggingface dataset:
Viral RNA Mutations Are Region Specific and Increased by Ribavirin in a Full-Length Hepatitis C Virus Replication System
Authors: Contreras Ana Maria , Hiasa Yoichi , He Wenping , Terella Adam , Schmidt Emmett V. , Chung Raymond T. 
Published: October 2002

ABSTRACT

High rates of genetic variation ensure the survival of RNA viruses. Although this variation is thought to result from error-prone replication, RNA viruses must also maintain highly conserved genomic segments. A balance between conserved and variable viral elements is especially important in order for viruses to avoid “error catastrophe.” Ribavirin has been shown to induce error catastrophe in other RNA viruses. We therefore used a novel hepatitis C virus (HCV) replication system to determine relative mutation frequencies in variable and conserved regions of the HCV genome, and we further evaluated these frequencies in response to ribavirin. We sequenced the 5′ untranslated region (5′ UTR) and the core, E2 HVR-1, NS5A, and NS5B regions of replicating HCV RNA isolated from cells transfected with a T7 polymerase-driven full-length HCV cDNA plasmid containing a
cis
-acting hepatitis delta virus ribozyme to control 3′ cleavage. We found quasispecies in the E2 HVR-1 and NS5B regions of untreated replicating viral RNAs but not in conserved 5′ UTR, core, or NS5A regions, demonstrating that important
cis
elements regulate mutation rates within specific viral segments. Neither T7-driven replication nor sequencing artifacts produced these nucleotide substitutions in control experiments. Ribavirin broadly increased error generation, especially in otherwise invariant regions, indicating that it acts as an HCV RNA mutagen in vivo. Similar results were obtained in hepatocyte-derived cell lines. These results demonstrate the potential utility of our system for the study of intrinsic factors regulating genetic variation in HCV. Our results further suggest that ribavirin acts clinically by promoting nonviable HCV RNA mutation rates. Finally, the latter result suggests that our replication model may be useful for identifying agents capable of driving replicating virus into error catastrophe.
Involvement of the Leader Sequence in Sendai Virus Pathogenesis Revealed by Recovery of a Pathogenic Field Isolate from cDNA
Authors: Fujii Yutaka , Sakaguchi Takemasa , Kiyotani Katsuhiro , Huang Cheng , Fukuhara Noriko , Egi Yoshiko , Yoshida Tetsuya 
Published: October 2002

ABSTRACT

We previously demonstrated that a systematic passage of a pathogenic field isolate of Sendai virus (SeV), the Hamamatsu strain, in embryonated eggs caused attenuation of virulence to mice, and we isolated viral clones of distinct virulence (K. Kiyotani et al. Arch. Virol. 146:893-908, 2001). One of the clones, E15cl2, which was obtained from the virus at the 15th egg passage of E0, the parental Hamamatsu clone for egg passage, had 165-fold-attenuated virulence to mice and possessed only four mutations in the entire 15,384-base genome: in an antigenomic sense, U to A at position 20 (U
20
A) and U to A at position 24 (U
24
A) in the leader sequence, the promoter for transcription and replication, and A to G at position 9346 (silent) and A to U at position 12174 (Ser to Cys) in the L gene. To examine the possibility that leader mutations affect virus pathogenesis, we recovered live viruses from cDNA derived from the Hamamatsu strain. A mutant virus possessing either a mutation of U
20
A or U
24
A in the leader sequence showed a slightly lower pathogenicity than that of the parental virus, whereas a double mutant virus possessing both of the mutations showed 25-fold-attenuated virulence, accompanying a significantly lower virus replication in the mouse lung. Replications of the leader mutant viruses were also impaired in a primary culture of mouse pulmonary epithelial cells but not in chicken embryo fibroblasts. These findings suggest that leader mutations of SeV affect virus pathogenesis by altering virus replication in a host-dependent manner.
Modification of Asn374 of nsP1 Suppresses a Sindbis Virus nsP4 Minus-Strand Polymerase Mutant
Authors: Fata Cori L. , Sawicki Stanley G. , Sawicki Dorothea L. 
Published: October 2002

ABSTRACT

Our recent study (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8632-8640, 2002) found minus-strand synthesis to be temperature sensitive in vertebrate and invertebrate cells when the Arg183 residue of the Sindbis virus nsP4 polymerase was changed to Ser, Ala, or Lys. Here we report the results of studies identifying an interacting partner of the region of the viral polymerase containing Arg183 that suppresses the Ser183 codon mutation. Large-plaque revertants were observed readily following growth of the nsP4 Ser183 mutant at 40°C. Fifteen revertants were characterized, and all had a mutation in the Asn374 codon of nsP1 that changed it to either a His or an Ile codon. When combined with nsP4 Ser183, substitution of either His374 or Ile374 for Asn374 restored wild-type growth in chicken embryo fibroblast (CEF) cells at 40°C. In
Aedes albopictus
cells at 34.5°C, neither nsP1 substitution suppressed the nsP4 Ser183 defect in minus-strand synthesis. This argued that the nsP4 Arg183 residue itself is needed for minus-strand replicase assembly or function in the mosquito environment. The nsP1 His374 suppressor when combined with the wild-type nsP4 gave greater than wild-type levels of viral RNA synthesis in CEF cells at 40°C (∼140%) and in
Aedes
cells at 34.5°C (200%). Virus producing nsP1 His374 and wild-type nsP4 Arg183 made more minus strands during the early period of infection and before minus-strand synthesis ceased at about 4 h postinfection. Shirako et al. (Y. Shirako, E. G. Strauss, and J. H. Strauss, Virology 276:148-160, 2000) identified amino acid substitutions in nsP1 and nsP4 that suppressed mutations that changed the N-terminal Tyr of nsP4. The nsP4 N-terminal mutants were defective also in minus-strand synthesis. Our study implicates an interaction between another conserved nsP1 region and an internal region, predicted to be in the finger domain, of nsP4 for the formation or activity of the minus-strand polymerase. Finally, the observation that a single point mutation in nsP1 results in minus-strand synthesis at greater than wild-type levels supports the concept that the wild-type nsP sequences are evolutionary compromises.
A Single Amino Acid Mutation in the PA Subunit of the Influenza Virus RNA Polymerase Inhibits Endonucleolytic Cleavage of Capped RNAs
Authors: Fodor Ervin , Crow Mandy , Mingay Louise J. , Deng Tao , Sharps Jane , Fechter Pierre , Brownlee George G. 
Published: October 2002

ABSTRACT
The influenza A virus RNA-dependent RNA polymerase consists of three subunits—PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA→cRNA→vRNA), but its transcriptional activity (vRNA→mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNA-binding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.
Nucleoside Analog Resistance Caused by Insertions in the Fingers of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Involves ATP-Mediated Excision
Authors: Boyer Paul L. , Sarafianos Stefan G. , Arnold Edward , Hughes Stephen H. 
Published: October 2002

ABSTRACT
Although anti-human immunodeficiency virus type 1 (HIV-1) therapy has prolonged the lives of patients, drug resistance is a significant problem. Of particular concern are mutations that cause cross-resistance to a particular class of drugs. Among the mutations that cause resistance to several nucleoside analogs are the insertion of amino acids in the fingers subdomain of HIV-1 reverse transcriptase (RT) at positions 69 and 70. These insertions are usually associated with changes in the flanking amino acids and with a change to F or Y at position 215. We have proposed that the T215F/Y mutation makes the binding of ATP to HIV-1 RT more effective, which increases the excision of 3-azido-3′-deoxythymidine-5′-monophosphate (AZTMP) in vitro and increases zidovudine (AZT) resistance in vivo. Although the mechanism of AZT resistance involves enhanced excision, resistance to 3TC involves a block to incorporation of the analog. We measured the effects of fingers insertion mutations on the misincorporation and excision of several nucleoside analogs. RT variants with the amino acid insertions in the fingers and T215Y have a decreased level of misincorporation of ddATP and 3TCTP. These mutants also have the ability to excise AZTMP by ATP-dependent pyrophosphorylysis. However, unlike the classic AZT resistance mutations (M41L/D67N/K70R/T215Y or F/K219E or Q), the combination of the amino acid insertions in the fingers and the T215Y mutation allows efficient excision of ddTMP and d4TMP, even when relatively high levels of deoxynucleoside triphosphates are present in the reaction. Although the dideoxynucleoside analogs of other nucleosides were excised more slowly than AZTMP, ddTMP, and d4TMP, the mutants with the fingers insertion and T215Y excised all of the nucleoside analogs that were tested more efficiently than wild-type RT or a mutant RT carrying the classical AZT resistance mutations. In the ternary complex (RT/template-primer/dNTP), the presence of the bound dNTP prevents the end of the primer from gaining access to the nucleotide binding site (N site) where excision occurs. Gel shift analysis showed that the amino acid insertions in the fingers destabilized the ternary complex compared to wild-type HIV-1 RT. If the ternary complex is unstable, the end of the primer can gain access to the N site and excision can occur. This could explain the enhanced excision of the nucleoside analogs.
In Vivo Gene Transfer Using a Nonprimate Lentiviral Vector Pseudotyped with Ross River Virus Glycoproteins
Authors: Kang Yubin , Stein Colleen S. , Heth Jason A. , Sinn Patrick L. , Penisten Andrea K. , Staber Patrick D. , Ratliff Kenneth L. , Shen Hong , Barker Carrie K. , Martins Inês , Sharkey C. Matthew , Sanders David Avram , McCray Paul B. , Davidson Beverly L. 
Published: October 2002

ABSTRACT

Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5 × 10
8
TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.
Persistence of Wild-Type Virus and Lack of Temporal Structure in the Latent Reservoir for Human Immunodeficiency Virus Type 1 in Pediatric Patients with Extensive Antiretroviral Exposure
Authors: Ruff Christian T. , Ray Stuart C. , Kwon Patricia , Zinn Rebekah , Pendleton Amanda , Hutton Nancy , Ashworth Roxann , Gange Stephen , Quinn Thomas C. , Siliciano Robert F. , Persaud Deborah 
Published: October 2002

ABSTRACT

Although highly active antiretroviral therapy (HAART) for human immunodeficiency virus type 1 (HIV-1) infection can reduce levels of HIV-1 RNA in plasma to below the limit of detection, replication-competent forms of the virus persist in all infected individuals. One form of persistence involves a stable reservoir of latent but potentially infectious virus that resides in resting memory CD4
+
T cells. The mechanisms involved in maintaining this latent reservoir are incompletely understood. In the present study, we examined the dynamic characteristics of this reservoir in a cohort of children who developed drug-resistant HIV-1 as a result of extensive exposure to inadequately suppressive one- or two-drug regimens prior to the advent of HAART. We have previously shown that drug-resistant viruses selected by nonsuppressive pre-HAART regimens can enter and persist in this reservoir. We have extended these findings here by demonstrating that archival wild-type HIV-1 persists in this reservoir despite the fact that in these patients drug-resistant mutants have been favored by the selective conditions for many years. Phylogenetic analysis of replication-competent viruses persisting in resting CD4
+
T cells revealed a striking lack of temporal structure in the sense that isolates obtained at later time points did not show greater sequence divergence than isolates from earlier time points. The persistence of drug-sensitive virus and the lack of temporal structure in the latent reservoir provide genetic evidence for the idea that HIV-1 can persist in a latent form free of selective pressure from antiretroviral drugs in long-lived resting memory CD4
+
T cells. Although there may be other mechanisms for viral persistence, this stable pool of latently infected cells is of significant concern because of its potential to serve as a lasting source of replication-competent viruses, including the infecting wild-type form and all drug-resistant variants that have arisen subsequently.
Molecular Evolution of Hepatitis A Virus: a New Classification Based on the Complete VP1 Protein
Authors: Costa-Mattioli Mauro , Cristina Juan , Romero Héctor , Perez-Bercof Raoul , Casane Didier , Colina Rodney , Garcia Laura , Vega Ines , Glikman Graciela , Romanowsky Victor , Castello Alejandro , Nicand Elisabeth , Gassin Michelle , Billaudel Sylviane , Ferré Virginie 
Published: October 2002

ABSTRACT

Hepatitis A virus
(HAV) is a positive-stranded RNA virus in the genus
Hepatovirus
in the family
Picornaviridae
. So far, analysis of the genetic variability of HAV has been based on two discrete regions, the VP1/2A junction and the VP1 N terminus. In this report, we determined the nucleotide and deduced amino acid sequences of the complete VP1 gene of 81 strains from France, Kosovo, Mexico, Argentina, Chile, and Uruguay and compared them with the sequences of seven strains of HAV isolated elsewhere. Overall strain variation in the complete VP1 gene was found to be as high as 23.7% at the nucleotide level and 10.5% at the amino acid level. Different phylogenetic methods revealed that HAV sequences form five distinct and well-supported genetic lineages. Within these lineages, HAV sequences clustered by geographical origin only for European strains. The analysis of the complete VP1 gene allowed insight into the mode of evolution of HAV and revealed the emergence of a novel variant with a 15-amino-acid deletion located on the VP1 region where neutralization escape mutations were found. This could be the first antigenic variant of HAV so far identified.
The Core of Bluetongue Virus Binds Double-Stranded RNA
Authors: Diprose J. M. , Grimes J. M. , Sutton G. C. , Burroughs J. N. , Meyer A. , Maan S. , Mertens P. P. C. , Stuart D. I. 
Published: October 2002

ABSTRACT
Double-stranded RNA (dsRNA) viruses conceal their genome from the host to avoid triggering unfavorable cellular responses. The crystal structure of the core of one such virus, bluetongue virus, reveals an outer surface festooned with dsRNA. This may represent a deliberate strategy to sequester dsRNA released from damaged particles to prevent host cell shutoff.
The Human Cytomegalovirus Ribonucleotide Reductase Homolog UL45 Is Dispensable for Growth in Endothelial Cells, as Determined by a BAC-Cloned Clinical Isolate of Human Cytomegalovirus with Preserved Wild-Type Characteristics
Authors: Hahn Gabriele , Khan Hanna , Baldanti Fausto , Koszinowski Ulrich H. , Revello M. Grazia , Gerna Giuseppe 
Published: October 2002

ABSTRACT

An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in
Escherichia coli
, and the ribonucleotide reductase homolog UL45 was deleted. Reconstituted virus RVFIX and RVΔUL45 grew equally well in human fibroblasts and human endothelial cells. Thus, UL45 is dispensable for growth of HCMV in both cell types.
A comparison of physiological responses and rating of perceived exertion in two modes of aerobic exercise in men and women over 50 years of age
Authors: Grant S , Corbett K , Todd K , Davies C , Aitchison T , Mutrie N , Byrne J , Henderson E , Dargie H J 
Published: October 2002

Objectives: To compare the physiological responses and ratings of perceived exertion to aerobic dance and walking sessions completed at a self selected pace.Methods: Six women and six men with a sample mean (SD) age of 68 (7) years completed aerobic dance and walking sessions in random order. A treadmill test was performed by each subject from which peak oxygen uptake (V̇o2) and maximum heart rates (HRmax) were determined. During the aerobic dance and walking sessions, heart rate and V̇o2were measured continuously throughout. Rate of perceived exertion (RPE) was measured every three minutes throughout the session.Results: The sample means (SD) for %peak V̇o2were 67 (17)% for the aerobic dance sessions and 52 (10)% for the walking sessions, and the %HRmax sample means (SD) were 74 (12)% for the aerobic dance sessions and 60 (8)% for walking sessions. The sample mean (SD) RPE for the aerobic dance sessions was 11 (2), and for the walking sessions it was 10 (2).Conclusions: %peak V̇o2, %HRmax, and RPE were significantly higher for aerobic dance than for walking. However, both the aerobic dance and walking sessions were of adequate intensity to improve aerobic fitness in most subjects. Further investigation into the relation between RPE and %peak V̇o2in a field setting over representative exercise time periods would be useful.
Older driver involvements in police reported crashes and fatal crashes: trends and projections
Authors: Lyman S , Ferguson S A , Braver E R , Williams A F 
Published: October 2002

Objectives:Older drivers have become a larger part of the driving population and will continue to do so as the baby boomers reach retirement age. The purpose of this study was to identify the potential effects of this population increase on highway safety.Methods:Driver involvement rates for all police reported crashes were calculated per capita, per licensed driver, and per vehicle-mile of travel for 1990 and 1995. Also, driver involvement rates for fatal crashes were calculated for 1983, 1990, and 1995. Based on current crash rates per licensed driver and estimates of the future number of licensed drivers, projections of crashes involving drivers aged 65 and older were made for years 2010, 2020, and 2030.Results:Driver crash involvement rates per capita decreased with age, but fatal involvement rates per capita increased starting at age 70. The same pattern existed for involvement rates per licensed driver. For both all crashes and fatal crashes, involvement rates per mile driven increased appreciably at age 70. Using projections of population growth, it was estimated that for all ages there will be a 34% increase in the number of drivers involved in police reported crashes and a 39% increase in the number involved in fatal crashes between 1999 and 2030. In contrast, among older drivers, police reported crash involvements are expected to increase by 178% and fatal involvements may increase by 155% by 2030. Drivers aged 65 and older will account for more than half of the total increase in fatal crashes and about 40% of the expected increase in all crash involvements; they are expected to account for as much as 25% of total driver fatalities in 2030, compared with 14% presently.Conclusions:By most measures, older drivers are at less risk of being involved in police reported crashes but at higher risk of being in fatal crashes. Although any projections of future crash counts have inherent uncertainty, there is strong evidence that older drivers will make up a substantially larger proportion of drivers involved in fatal crashes by 2030 because of future increases in the proportion of the population aged 65 and older, and trends toward increased licensure rates and higher annual mileage among older persons. Countermeasures to reduce the anticipated death toll among older drivers should address the increased susceptibility to injury of older vehicle occupants in crashes.
Participation in maternal serum screening following screen positive results in a previous pregnancy
Authors: Rausch D.N. , Lambert-Messerlian G.M. , Canick J.A. 
Published: October 2002

Objective To determine whether women who have had a positive serum screening result in one pregnancy have a lower rate of participation in screening in their next pregnancy. Setting The Women and Infants Hospital triple marker screening programme. Methods Pregnancy and screening information was collected from laboratory and hospital databases to compare subsequent screening participation in women who were screen negative and screen positive for risk of Down's syndrome (DS) or neural tube defect (NTD) pregnancy. Results In an age matched comparison, 108 women who had a previous screen positive result were significantly less likely than 108 women who were screen negative to participate in maternal serum screening in their next pregnancy. When examined according to type of screen positive result, the effect was significant for both those who were screen positive for DS and those who were screen positive for NTD. The degree of risk in screen positive women did not significantly affect their uptake of screening in the next pregnancy. Conclusions Anxiety related to a screen positive result probably causes decreased participation in maternal serum screening in the next pregnancy. Reducing the screen positive rate in prenatal serum screening would alleviate maternal anxiety and would probably lead to more stable participation.