bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Altering the Transcription Termination Signals of Respiratory Syncytial Virus on Viral Gene Expression and Growth In Vitro and In Vivo
Authors: Tran Kim C. , Collins Peter L. , Teng Michael N. 
Published: December 2003

ABSTRACT
Nonsegmented negative-sense RNA viruses (mononegaviruses) control viral gene expression largely through a transcription gradient such that promoter-proximal genes are transcribed more abundantly than downstream genes. For some paramyxoviruses, naturally occurring differences in the levels of efficiency of transcription termination by various gene end (GE) signals provide an additional level of regulation of gene expression. The first two genes (NS1 and NS2) of respiratory syncytial virus (RSV) are particularly inefficient in termination. We investigated whether altering the termination efficiency (TE) of these two genes in infectious recombinant virus would affect transcription of promoter-proximal and promoter-distal genes, production of viral proteins, and viral replication in cell culture and in the respiratory tract of mice. Recombinant RSVs were constructed with mutations that increased or decreased the TE of the NS1 GE signal, increased that of the NS2 GE signal, or increased that of both signals. Increasing the TE of either or both GE signals resulted in decreased production of the related polycistronic readthrough mRNAs, which normally arise due to the failure of the viral polymerase to recognize the GE signal. This was accompanied by a small increase in the levels of monocistronic NS1 and NS2 mRNAs. Conversely, decreasing the TE of the NS1 GE increased the production of readthrough mRNAs concomitant with a decrease of monocistronic NS1 and NS2 mRNA levels. These changes were reflected in the levels of NS1 and NS2 protein. All of the mutant viruses displayed growth kinetics and virus yields similar to wild-type recombinant RSV (rA2) in both HEp-2 and Vero cells. In addition, all mutants grew similarly to rA2 in the upper- and lower-respiratory tract of BALB/c mice, though some of the mutants displayed slightly decreased replication. These data suggest that the natural inefficiencies of transcription termination by the NS1 and NS2 GE signals do not play important roles in controlling the magnitude of RSV gene expression or the efficiency of virus replication. Furthermore, while changes in the TE of a GE signal clearly can affect the transcription of its gene as well as that of the one immediately downstream, these changes did not have a significant effect on the overall transcriptional gradient.
Nucleocapsid-RNA Interactions Are Essential to Structural Stability but Not to Assembly of Retroviruses
Authors: Wang Shainn-Wei , Noonan Kristin , Aldovini Anna 
Published: December 2003

ABSTRACT
The process of RNA incorporation into nascent virions is thought to be critical for efficient retroviral particle assembly and production. Here we show that human immunodeficiency virus type 1 mutant particles (which are highly unstable and break down soon after release from the cell) lacking nucleocapsid (NC) core protein-mediated RNA incorporation are produced efficiently and can be recovered at the normal density when viral protease function is abolished. These results demonstrate that RNA binding by Gag is not necessary for retroviral particle assembly. Rather, the RNA interaction with NC is critical for retroviral particle structural stability subsequent to release from the membrane and protease-mediated Gag cleavage. Thus, the NC-RNA interaction, and not simply the presence of RNA, provides the virus with a structural function that is critical for stable retroviral particle architecture.
Dual Stimulation of Epstein-Barr Virus (EBV)-Specific CD4+- and CD8+-T-Cell Responses by a Chimeric Antigen Construct: Potential Therapeutic Vaccine for EBV-Positive Nasopharyngeal Carcinoma
Authors: Taylor G. S. , Haigh T. A. , Gudgeon N. H. , Phelps R. J. , Lee S. P. , Steven N. M. , Rickinson A. B. 
Published: December 2003

ABSTRACTVirus-associated malignancies are potential targets for immunotherapeutic vaccines aiming to stimulate T-cell responses against viral antigens expressed in tumor cells. Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma, a high-incidence tumor in southern China, expresses a limited set of EBV proteins, including the nuclear antigen EBNA1, an abundant source of HLA class II-restricted CD4+T-cell epitopes, and the latent membrane protein LMP2, a source of subdominant CD8+T-cell epitopes presented by HLA class I alleles common in the Chinese population. We used appropriately modified gene sequences from a Chinese EBV strain to generate a modified vaccinia virus Ankara recombinant, MVA-EL, expressing the CD4 epitope-rich C-terminal domain of EBNA1 fused to full-length LMP2. The endogenously expressed fusion protein EL is efficiently processed via the HLA class I pathway, and MVA-EL-infected dendritic cells selectively reactivate LMP2-specific CD8+memory T-cell responses from immune donors in vitro. Surprisingly, endogenously expressed EL also directly accesses the HLA class II presentation pathway and, unlike endogenously expressed EBNA1 itself, efficiently reactivates CD4+memory T-cell responses in vitro. This unscheduled access to the HLA class II pathway is coincident with EL-mediated redirection of the EBNA1 domain from its native nuclear location to dense cytoplasmic patches. Given its immunogenicity to both CD4+and CD8+T cells, MVA-EL has potential as a therapeutic vaccine in the context of nasopharyngeal carcinoma.
Gamma Interferon-Mediated Inflammation Is Associated with Lack of Protection from Intravaginal Simian Immunodeficiency Virus SIVmac239 Challenge in Simian-Human Immunodeficiency Virus 89.6-Immunized Rhesus Macaques
Authors: Abel Kristina , La Franco-Scheuch Lisa , Rourke Tracy , Ma Zhong-Min , de Silva Veronique , Fallert Beth , Beckett Laurel , Reinhart Todd A. , Miller Christopher J. 
Published: December 2003

ABSTRACTAlthough gamma interferon (IFN-γ) is a key mediator of antiviral defenses, it is also a mediator of inflammation. As inflammation can drive lentiviral replication, we sought to determine the relationship between IFN-γ-related host immune responses and challenge virus replication in lymphoid tissues of simian-human immunodeficiency virus 89.6 (SHIV89.6)-vaccinated and unvaccinated rhesus macaques 6 months after challenge with simian immunodeficiency virus SIVmac239. Vaccinated-protected monkeys had low tissue viral RNA (vRNA) levels, vaccinated-unprotected animals had moderate tissue vRNA levels, and unvaccinated animals had high tissue vRNA levels. The long-term challenge outcome in vaccinated monkeys was correlated with the relative balance between SIV-specific IFN-γ T-cell responses and nonspecific IFN-γ-driven inflammation. Vaccinated-protected monkeys had slightly increased tissue IFN-γ mRNA levels and a high frequency of IFN-γ-secreting T cells responding to in vitro SIVgag peptide stimulation; thus, it is likely that they could develop effective anti-SIV cytotoxic T lymphocytes in vivo. In contrast, both high tissue IFN-γ mRNA levels and strong in vitro SIV-specific IFN-γ T-cell responses were detected in lymphoid tissues of vaccinated-unprotected monkeys. Unvaccinated monkeys had increased tissue IFN-γ mRNA levels but weak in vitro anti-SIV IFN-γ T-cell responses. In addition, in lymphoid tissues of vaccinated-unprotected and unvaccinated monkeys, the increased IFN-γ mRNA levels were associated with increased Mig/CXCL9, IP-10/CXCL10, and CXCR3 mRNA levels, suggesting that increased Mig/CXCL9 and IP-10/CXCL10 expression resulted in recruitment of CXCR3+activated T cells. Thus, IFN-γ-driven inflammation promotes SIV replication in vaccinated-unprotected and unvaccinated monkeys. Unlike all unvaccinated monkeys, most monkeys vaccinated with SHIV89.6 did not develop IFN-γ-driven inflammation, but they did develop effective antiviral CD8+-T-cell responses.
Potential Selection of LMP1 Variants in Nasopharyngeal Carcinoma
Authors: Edwards Rachel H. , Sitki-Green Diane , Moore Dominic T. , Raab-Traub Nancy 
Published: December 2003

ABSTRACTSeven distinct sequence variants of the Epstein-Barr virus latent membrane protein 1 (LMP1) have been identified by distinguishing amino acid changes in the carboxy-terminal domain. In this study the transmembrane domains are shown to segregate identically with the distinct carboxy-terminal amino acid sequences. Since strains of LMP1 have been shown to differ in abundance between blood and throat washes, nasopharyngeal carcinomas (NPCs) from areas of endemicity and nonendemicity with matching blood were analyzed by using a heteroduplex tracking assay to distinguish LMP1 variants. Striking differences were found between the compartments with the Ch1 strain prevalent in the NPCs from areas of endemicity and nonendemicity and the B958 strain prevalent in the blood of the endemic samples, whereas multiple strains of LMP1 were prevalent in the blood of the nonendemic samples. The possible selection against the B958 strain appearing in the tumor was highly significant (P< 0.0001). Sequence analysis of the full-length LMP1 variants revealed changes in many of the known and computer-predicted HLA-restricted epitopes with changes in key positions in multiple, potential epitopes for the specific HLA of the patients. These amino acid substitutions at key positions in the LMP1 epitopes may result in a reduced cytotoxic-T-lymphocyte response. These data indicate that strains with specific variants of LMP1 are more likely to be found in NPC. The predominance of specific LMP1 variants in NPC could reflect differences in the biologic or molecular properties of the distinct forms of LMP1 or possible immune selection.
PEGylation of a Vesicular Stomatitis Virus G Pseudotyped Lentivirus Vector Prevents Inactivation in Serum
Authors: Croyle Maria A. , Callahan Shellie M. , Auricchio Alberto , Schumer Gregg , Linse Klause D. , Wilson James M. , Brunner Lane J. , Kobinger Gary P. 
Published: December 2003

ABSTRACT

One disadvantage of vesicular stomatitis virus G (VSV-G) pseudotyped lentivirus vectors for clinical application is inactivation of the vector by human serum complement. To prevent this, monomethoxypoly(ethylene) glycol was conjugated to a VSV-G-human immunodeficiency virus vector expressing
Escherichia coli
beta-galactosidase. The modification did not affect transduction efficiency in vitro and protected the vector from inactivation in complement-active human and mouse sera. Blood from mice dosed intravenously with either the unmodified or the PEGylated virus particles was assayed for active vector by a limiting-dilution assay to evaluate transduction efficiency and for p24, an indicator of the total number of virus particles present. PEGylation extended the circulation half-life of active vector by a factor of 5 and reduced the rate of vector inactivation in the serum by a factor of 1,000. Pharmacokinetic profiles for the total number of virus particles present in the circulation were unaffected by PEGylation. Modification of the vector with poly(ethylene) glycol significantly enhanced transduction efficiency in the bone marrow and in the spleen 14 days after systemic administration of the virus. These results, in concert with the pharmacokinetic profiles, indicate that PEGylation does protect the virus from inactivation in the serum and, as a result, improves the transduction efficiency of VSV-G pseudotyped lentivirus vectors in susceptible organs in vivo.
Small-Molecule Inhibition of Human Immunodeficiency Virus Type 1 Replication by Specific Targeting of the Final Step of Virion Maturation
Authors: Zhou Jing , Yuan Xiong , Dismuke David , Forshey Brett M. , Lundquist Christopher , Lee Kuo-Hsiung , Aiken Christopher , Chen Chin Ho 
Published: December 2003

ABSTRACT

Despite the effectiveness of currently available human immunodeficiency virus type 1 (HIV-1) therapies, a continuing need exists for new drugs to treat HIV-1 infection. We investigated the mechanism by which 3-
O
-{3′,3′-dimethylsuccinyl}-betulinic acid (DSB) inhibits HIV-1 replication. DSB functions at a late stage of the virus life cycle but does not inhibit the HIV-1 protease in vitro or interfere with virus assembly or release. DSB specifically delays the cleavage of Gag between the capsid (CA) and p2, resulting in delayed formation of the mature viral core and reduced HIV-1 infectivity. Replication of simian immunodeficiency virus (SIV) was resistant to DSB; however, a chimeric SIV carrying CA-p2 sequences from HIV-1 was inhibited by the drug, indicating that susceptibility to DSB maps to the CA-p2 region of the HIV-1 Gag protein. A single point mutation at the CA-p2 cleavage site of HIV-1 conferred strong resistance to DSB, confirming the target of the drug. HIV-1 strains that are resistant to a variety of protease inhibitors were sensitive to DSB. These findings indicate that DSB specifically protects the CA-p2 cleavage site from processing by the viral protease during virion maturation, thereby revealing a novel mechanism for pharmacologic inhibition of HIV-1 replication.
Persistent Infection with Ebola Virus under Conditions of Partial Immunity
Authors: Gupta Manisha , Mahanty Siddhartha , Greer Patricia , Towner Jonathan S. , Shieh Wun-Ju , Zaki Sherif R. , Ahmed Rafi , Rollin Pierre E. 
Published: December 2003

ABSTRACT
Ebola hemorrhagic fever in humans is associated with high mortality; however, some infected hosts clear the virus and recover. The mechanisms by which this occurs and the correlates of protective immunity are not well defined. Using a mouse model, we determined the role of the immune system in clearance of and protection against Ebola virus. All CD8 T-cell-deficient mice succumbed to subcutaneous infection and had high viral antigen titers in tissues, whereas mice deficient in B cells or CD4 T cells cleared infection and survived, suggesting that CD8 T cells, independent of CD4 T cells and antibodies, are critical to protection against subcutaneous Ebola virus infection. B-cell-deficient mice that survived the primary subcutaneous infection (vaccinated mice) transiently depleted or not depleted of CD4 T cells also survived lethal intraperitoneal rechallenge for ≥25 days. However, all vaccinated B-cell-deficient mice depleted of CD8 T cells had high viral antigen titers in tissues following intraperitoneal rechallenge and died within 6 days, suggesting that memory CD8 T cells by themselves can protect mice from early death. Surprisingly, vaccinated B-cell-deficient mice, after initially clearing the infection, were found to have viral antigens in tissues later (day 120 to 150 post-intraperitoneal infection). Furthermore, following intraperitoneal rechallenge, vaccinated B-cell-deficient mice that were transiently depleted of CD4 T cells had high levels of viral antigen in tissues earlier (days 50 to 70) than vaccinated undepleted mice. This demonstrates that under certain immunodeficiency conditions, Ebola virus can persist and that loss of primed CD4 T cells accelerates the course of persistent infections. These data show that CD8 T cells play an important role in protection against acute disease, while both CD4 T cells and antibodies are required for long-term protection, and they provide evidence of persistent infection by Ebola virus suggesting that under certain conditions of immunodeficiency a host can harbor virus for prolonged periods, potentially acting as a reservoir.
Continued Production of Drug-Sensitive Human Immunodeficiency Virus Type 1 in Children on Combination Antiretroviral Therapy Who Have Undetectable Viral Loads
Authors: Persaud Deborah , Siberry George K. , Ahonkhai Aima , Kajdas Joleen , Monie Daphne , Hutton Nancy , Watson Douglas C. , Quinn Thomas C. , Ray Stuart C. , Siliciano Robert F. 
Published: December 2003

ABSTRACT

Highly active antiretroviral therapy (HAART) can suppress plasma human immunodeficiency virus type 1 (HIV-1) levels to below the detection limit of ultrasensitive clinical assays. However, HIV-1 persists in cellular reservoirs, and in adults, persistent low-level viremia is detected with more sensitive assays. The nature of this viremia is poorly understood, and it is unclear whether viremia persists in children on HAART, particularly those who start therapy shortly after birth. We therefore developed a reverse transcriptase PCR (RT-PCR) assay that allows genotyping of HIV-1 protease even when viremia is present at levels as low as 5 copies of HIV-1 RNA/ml. We demonstrated that viremia persists in children with plasma virus levels below the limit of detection of clinical assays. Viremia was detected even in children who began HAART in early infancy and maintained such strong suppression of viremia that HIV-1-specific antibody responses were absent or minimal. The low-level plasma virus lacked protease inhibitor resistance mutations despite the frequent use of nelfinavir, which has a low mutational barrier to resistance. Protease sequences resembled those of viruses in the latent reservoir in resting CD4
+
T cells. Thus, in most children on HAART with clinically undetectable viremia, there is continued virus production without evolution of resistance in the protease gene.
Sequence Motifs Involved in the Regulation of Discontinuous Coronavirus Subgenomic RNA Synthesis
Authors: Zúñiga Sonia , Sola Isabel , Alonso Sara , Enjuanes Luis 
Published: December 2003

ABSTRACT

Coronavirus transcription leads to the synthesis of a nested set of mRNAs with a leader sequence derived from the 5′ end of the genome. The mRNAs are produced by a discontinuous transcription in which the leader is linked to the mRNA coding sequences. This process is regulated by transcription-regulating sequences (TRSs) preceding each mRNA, including a highly conserved core sequence (CS) with high identity to sequences present in the virus genome and at the 3′ end of the leader (TRS-L). The role of TRSs was analyzed by reverse genetics using a full-length infectious coronavirus cDNA and site-directed mutagenesis of the CS. The canonical CS-B was nonessential for the generation of subgenomic mRNAs (sgmRNAs), but its presence led to transcription levels at least 10
3
-fold higher than those in its absence. The data obtained are compatible with a transcription mechanism including three steps: (i) formation of 5′-3′ complexes in the genomic RNA, (ii) base-pairing scanning of the nascent negative RNA strand by the TRS-L, and (iii) template switching during synthesis of the negative strand to complete the negative sgRNA. This template switch takes place after copying the CS sequence and was predicted in silico based on high base-pairing score between the nascent negative RNA strand and the TRS-L and minimum Δ
G
.
Production of Novel Ebola Virus-Like Particles from cDNAs: an Alternative to Ebola Virus Generation by Reverse Genetics
Authors: Watanabe Shinji , Watanabe Tokiko , Noda Takeshi , Takada Ayato , Feldmann Heinz , Jasenosky Luke D. , Kawaoka Yoshihiro 
Published: December 2003

ABSTRACT

We established a plasmid-based system for generating infectious Ebola virus-like particles (VLPs), which contain an Ebola virus-like minigenome consisting of a negative-sense copy of the green fluorescent protein gene. This system produced nearly 10
3
infectious particles per ml of supernatant, equivalent to the titer of Ebola virus generated by a reverse genetics system. Interestingly, infectious Ebola VLPs were generated, even without expression of VP24. Transmission and scanning electron microscopic analyses showed that the morphology of the Ebola VLPs was indistinguishable from that of authentic Ebola virus. Thus, this system allows us to study Ebola virus entry, replication, and assembly without biosafety level 4 containment. Furthermore, it may be useful in vaccine production against this highly pathogenic agent.
Are mobile speed cameras effective? A controlled before and after study
Authors: Christie S M , Lyons R A , Dunstan F D , Jones S J 
Published: December 2003

Objective:To identify the most appropriate metric to determine the effectiveness of mobile speed cameras in reducing road traffic related injuries.Design:Controlled before and after study which compares two methods for examining the local effectiveness of mobile speed cameras—a circular zone around the camera and a route based method to define exposure at various distances from sites.Setting:South Wales, UK.Subjects:Persons injured by road traffic before and after intervention.Intervention:Use of mobile speed cameras at 101 sites.Main outcome measures:Rate ratio of injurious crashes at intervention and control sites.Results:Camera sites had lower than expected numbers of injurious crashes up to 300 metres using circles and up to 500 metres using routes. Routes methods indicated a larger effect than the circles method except in the 100 metres nearest sites. A 500 metre route method was used to investigate the effect within strata of time after intervention, time of day, speed limit, and type of road user injured. The number of injurious crashes after intervention was substantially reduced (rate ratio 0.49, 95% confidence interval 0.42 to 0.57) and sustained throughout two years after intervention. Significant decreases occurred in daytime and night time, on roads with speed limits of 30 and 60–70 miles/hour and for crashes that injured pedestrians, motorcycle users, and car occupants.Conclusions:The route based method is the better method of measure effectiveness at distances up to 500 metres. This method demonstrates a 51% reduction in injurious crashes.
Helmet rental practices at United States ski areas: a national survey
Authors: Clingenpeel J M , Marshall S W 
Published: December 2003

Objectives:Studies have shown that head injuries are the leading cause of death on ski slopes. Statistics on helmet rental practices at ski areas across the United States have never been reported. This study sought to determine the prevalence of United States ski areas offering helmet rental during the 2002–03 ski season. Secondarily it sought to analyze the relationships of geographic region and size of ski area with helmet rental availability and to gather information on ski helmet rental cost to the consumer.Methods and setting:A stratified cross sectional telephone survey of a sample of 331 United States ski area onsite rental shops during February 2003.Results:Altogether 50% of ski areas offered helmet rental with significant variation in the prevalence of helmet rental among ski areas of differing regions and sizes (p<0.01). A majority of Northeastern (57%), Western (63%), and Rocky Mountain (71%) ski areas rented helmets, whereas a minority of Southern (37%) and Midwestern (23%) ski areas rented helmets. Twenty five percent of the smallest ski areas (⩽50 acres) rented helmets compared with 74% of the largest ski areas (⩾501 acres).Conclusions:United States ski area helmet rental practices vary by region of the country and ski area size. Winter sports participants interested in wearing protective headgear should be aware of the helmet rental practices at the ski area in which they plan to visit and consider helmet purchase if they visit mainly smaller ski areas or areas within the South or Midwest.