3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy.

Cytoplasmic polyhedrosis virus (CPV) is unique within the Reoviridae family in having a turreted single-layer capsid contained within polyhedrin inclusion bodies, yet being fully capable of cell entry and endogenous RNA transcription. Biochemical data have shown that the amino-terminal 79 residues of the CPV turret protein (TP) is sufficient to bring CPV or engineered proteins into the polyhedrin matrix for micro-encapsulation. Here we report the three-dimensional structure of CPV at 3.88 A resolution using single-particle cryo-electron microscopy. Our map clearly shows the turns and deep grooves of alpha-helices, the strand separation in beta-sheets, and densities for loops and many bulky side chains; thus permitting atomic model-building effort from cryo-electron microscopy maps. We observed a helix-to-beta-hairpin conformational change between the two conformational states of the capsid shell protein in the region directly interacting with genomic RNA. We have also discovered a messenger RNA release hole coupled with the mRNA capping machinery unique to CPV. Furthermore, we have identified the polyhedrin-binding domain, a structure that has potential in nanobiotechnology applications.

Sexual Health Education from the Perspective of School Staff: Implications for Adoption and Implementation of Effective Programs in Middle School

Introduction: US teens are having sex early; however, the vast majority of schools do not implement evidence-based sexual health education (SHE) programs that could delay sexual behavior and/or reduce risky behavior. This study examines middle school staff’s knowledge, attitudes, barriers, self-efficacy, and perceived support (psychosocial factors known to influence SHE program adoption and implementation). Methods: Professional school staff from 33 southeast Texas middle schools completed an internet or paper-based survey. Prevalence estimates for psychosocial variables were computed for the total sample. Chi-square and t-test analyses examined variation by demographic factors. Results: Almost 70% of participants were female, 37% white, 42% black, 16% Hispanic; 20% administrators, 15% nurses/counselors, 31% non-physical education/non-health teachers, 28% physical education/health teachers; mean age = 42.78 years (SD = 10.9). Over 90% favored middle school SHE, and over 75% reported awareness of available SHE curricula or policies. More than 60% expressed confidence for discussing SHE. Staff perceived varying levels of administrator (28%-56%) support for SHE and varying levels of support for comprehensive sex education from outside stakeholders (e.g., parents, community leaders) (42%-85%). Overall, results were more favorable for physical education/health teachers, nurses/counselors, and administrators (when compared to non-physical education/non-health teachers) and individuals with experience teaching SHE. Few significant differences were observed by other demographic factors. Conclusions: Overall, study results were extremely positive, which may reflect a high level of readiness among school staff for adopting and implementing effective middle school SHE programs. Study results highlight the importance of several key action items for schools.

Mapping T-cell epitopes of the rubella virus structural proteins

Rubella virus (RV) typically causes a mild childhood illness, but complications can result from both viral and immune-mediated pathogenesis. RV can persist in the presence of neutralizing antibodies, suggesting that cell-mediated immune responses may be necessary for viral clearance. However, the molecular determinants recognized by RV-specific T-cells have not been identified. Using recombinant proteins which express the entire RV structural open reading frame in proliferation assays with lymphocytes of RV-immune individuals, domains which elicit major histocompatibility complex class II-restricted helper T-cells were identified. Synthetic peptides representing these domains were used to define specific epitopes. Two immunodominant domains were mapped to the capsid protein sequence C$\sb1$-C$\sb{29}$ and the E1 glycoprotein sequence E1$\sb{202}$-E1$\sb{283}.$ RV-specific MHC class I-restricted cytotoxic T lymphocytes (CTLs) were identified using a chromium-release assay with infected fibroblasts as target cells. An infectious Sindbis virus vector expressing each of the RV structural proteins identified the capsid, E2 and E1 proteins as targets of CTLs. Specific CTL epitopes were mapped within the previously identified immunodominant domains. This study identified domains of the RV structural proteins that may be beneficial for development of a synthetic vaccine, and provides normative data on RV-specific T-cell responses that should enhance our ability to understand RV persistence and associated complications. ^

{\it In vivo\/} induction of cytotoxic T lymphocytes against human immunodeficiency virus type 1 by synthetic viral peptides

Cytotoxic T lymphocytes (CTLs) play an important role in the suppression of initial viremia after acute infection with the human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS). Most HIV-infected individuals attain a high titer of anti-HIV antibodies within weeks of infection; however this antibody-mediated immune response appears not to be protective. In addition, anti-HIV antibodies can be detrimental to the immune response to HIV through enhancement of infection and participating in autoimmune reactions as a result of HIV protein mimicry of self antigens. Thus induction and maintenance of a strong HIV-specific CTL immune response in the absence of anti-HIV antibodies has been proposed to be the most effective means of controlling of HIV infection. Immunization with synthetic peptides representing HIV-specific CTL epitopes provides a way to induce specific CTL responses, while avoiding stimulation of anti-HIV antibody. This dissertation examines the capacity of synthetic peptides from the V3 loop region of the gp120 envelope protein from several different strain of HIV-1 to induce HIV-specific, MHC-restricted CD8$\sp+$ CTL response in vivo in a mouse model. Seven synthetic peptides representative of sequences found throughout North America, Europe, and Central Africa have been shown to prime CTLs in vivo. In the case of the MN strain of HIV-1, a 13 amino acid sequence defining the epitope is most efficient for optimal induction of specific CTL, whereas eight to nine amino acid sequences that could define the epitope were not immunogenic. In addition, synthesis of peptides with specific amino acid substitutions that are important for either MHC binding or T cell receptor recognition resulted in peptides that exhibited increased immunogenicity and induced CTLs that displayed altered specificity. V3 loop peptides from HIV-1 MN, SC, and Z321 induced a CTL population that was broadly cross-reactive against strains of HIV-1 found throughout the world. This research confirms the potential efficacy of using synthetic peptides for in vivo immunization to induce HIV-specific CTL-mediated responses and provides a basis for further research into development of synthetic peptide-based vaccines. ^

Phenotypic and genotypic analysis of mouse hepatitis virus (JHM) complementation group A temperature-sensitive mutants

Temperature sensitive (ts) mutant viruses have helped elucidate replication processes in many viral systems. Several panels of replication-defective ts mutants in which viral RNA synthesis is abolished at the nonpermissive temperature (RNA$\sp{-})$ have been isolated for Mouse Hepatitis Virus, MHV (Robb et al., 1979; Koolen et al., 1983; Martin et al., 1988; Schaad et al., 1990). However, no one had investigated genetic or phenotypic relationships between these different mutant panels. In order to determine how the panel of MHV-JHM RNA$\sp{-}$ ts mutants (Robb et al., 1979) were genetically related to other described MHV RNA$\sp{-}$ ts mutants, the MHV-JHM mutants were tested for complementation with representatives from two different sets of MHV-A59 ts mutants (Koolen et al., 1983; Schaad et al., 1990). The three ts mutant panels together were found to comprise eight genetically distinct complementation groups. Of these eight complementation groups, three complementation classes are unique to their particular mutant panel; genetically equivalent mutants were not observed within the other two mutant panels. Two complementation groups were common to all three mutant panels. The three remaining complementation groups overlapped two of the three mutant sets. Mutants MHV-JHM tsA204 and MHV-A59 ts261 were shown to be within one of these overlapping complementation groups. The phenotype of the MHV-JHM mutants within this complementation class has been previously characterized (Leibowitz et al., 1982; Leibowitz et al, 1990). When these mutants were grown at the permissive temperature, then shifted up to the nonpermissive temperature at the start of RNA synthesis, genome-length RNA and leader RNA fragments accumulated, but no subgenomic mRNA was synthesized. MHV-A59 ts261 produced leader RNA fragments identical to those observed with MHV-JHM tsA204. Thus, these two MHV RNA$\sp{-}$ ts mutants that were genetically equivalent by complementation testing were phenotypically similar as well. Recombination frequencies obtained from crosses of MHV-A59 ts261 with several of the gene 1 MHV-A59 mutants indicated that the causal mutation(s) of MHV-A59 ts261 was located near the overlapping junction of ORF1a and ORF1b, in the 3$\sp\prime$ end of ORF1a, or the 5$\sp\prime$ end of ORF1b. Sequence analysis of this junction and 1400 nucleotides into the 5$\sp\prime$ end of ORF1b of MHV-A59 ts261 revealed one nucleotide change from the wildtype MHV-A59. This substitution at nucleotide 13,598 (A to G) was a silent mutation in the ORF1a reading frame, but resulted in an amino acid change in ORF1b gene product (I to V). This amino acid change would be expressed only in the readthrough translation product produced upon successful ribosome frameshifting. A revertant of MHV-A59 ts261 (R2) also retained this guanidine residue, but had a second substitution at nucleotide 14,475 in ORF1b. This mutation results in the substitution of valine for an isoleucine.^ The data presented here suggest that the mutation in MHV-A59 ts261 (nucleotide 13,598) would be responsible for the MHV-JHM complementation group A phenotype. A second-site reversion at nucleotide 14,475 may correct this defect in the revertant. Sequencing of gene 1 immediately upstream of nucleotide 13,296 and downstream of nucleotide 15,010 must be conducted to test this hypothesis. ^

A case-control study of hepatitis C virus and its interaction with hepatitis B virus on the development of hepatocellular carcinoma in the USA

Objective. The aim of this study was to assess the independent risk of hepatitis C virus (HCV) infection in the development of hepatocellular carcinoma (HCC). The independent risk of hepatitis B virus (HBV), its interaction with hepatitis C virus and the association with other risk factors were examined.^ Methods. A hospital-based case-control study was conducted between January 1994 and December 1995. We enrolled 115 pathologically confirmed HCC patients and 230 nonliver cancer controls, who were matched by age ($\pm$5 years), gender, and year of diagnosis. Both cases and controls were recruited from The University of Texas M. D. Anderson Cancer Center at Houston. The risk factors were collected through personal interviews and blood samples were tested for HCV and HBV markers. Univariate and multivariate analyses were performed through conditional logistic regression.^ The prevalence of anti-HCV positive is 25.2% in HCC cases compared to 3.0% in controls. The univariate analysis showed that anti-HCV, HBsAg, alcohol drinking and cigarette smoking were significantly associated with HCC, however, family history of cancer, occupational chemical exposure, and use of oral contraceptive were not. Multivariate analysis revealed a matched odds ratio (OR) of 10.1 (95% CI 3.7-27.4) for anti-HCV, and an OR of 11.9 (95% CI 2.5-57.5) for HBsAg. However, dual infection of HCV and HBV had only a thirteen times increase in the risk of HCC, OR = 13.9 (95% CI 1.3-150.6). The estimated population attributable risk percent was 23.4% for HCV, 12.6% for HBV, and 5.3% for both viruses. Ever alcohol drinkers was positively associated with HCC, especially among daily drinkers, matched OR was 5.7 (95% CI 2.1-15.6). However, there was no significant increase in the risk of HCC among smokers as compared to nonsmokers. The mean age of HCC patients was significantly younger among the HBV(+) group and among the HCV(+)/HBV(+) group, when compared to the group of HCC patients with no viral markers. The association between past histories of blood transfusion, acupuncture, tattoo and IVDU was highly significant among the HCV(+) group and the HBV(+)/HCV(+) group, as compared to HCC patients with no viral markers. Forty percent of the HCC patients were pathologically or clinically diagnosed with liver cirrhosis. Anti-HCV(+) (OR = 3.6 95% CI 1.5-8.9) and alcohol drinking (OR = 2.7 95% CI 1.1-6.7), but not HBsAg, are the major risk factors for liver cirrhosis in HCC patients.^ Conclusion. Both hepatitis B virus and hepatitis C virus were independent risk factors for HCC. There was not enough evidence to determine the interaction between both viruses. Only daily alcoholic drinkers showed increasing risk for HCC development, as compared to nondrinkers. ^

Establishing occupational and environmental health design requirements for Lunar and Mars settlements

In a study of Lunar and Mars settlement concepts, an analysis was made of fundamental design assumptions in five technical areas against a model list of occupational and environmental health concerns. The technical areas included the proposed science projects to be supported, habitat and construction issues, closed ecosystem issues, the "MMM" issues--mining, material-processing, and manufacturing, and the human elements of physiology, behavior and mission approach. Four major lessons were learned. First it is possible to relate public health concerns to complex technological development in a proactive design mode, which has the potential for long-term cost savings. Second, it became very apparent that prior to committing any nation or international group to spending the billions to start and complete a lunar settlement, over the next century, that a significantly different approach must be taken from those previously proposed, to solve the closed ecosystem and "MMM" problems. Third, it also appears that the health concerns and technology issues to be addressed for human exploration into space are fundamentally those to be solved for human habitation of the earth (as a closed ecosystem) in the 21st century. Finally, it is proposed that ecosystem design modeling must develop new tools, based on probabilistic models as a step up from closed circuit models. ^

The role of substance abuse treatment in preventing human immunodeficiency virus transmission among injecting drug users

This exploratory study assesses the utility of substance abuse treatment as a strategy for preventing human immunodeficiency virus (HIV) transmission among injecting drug users (IDUs). Data analyzed in this study were collected in San Antonio, TX, 1989 through 1995 using both qualitative and quantitative methods. Qualitative data included ethnographic interviews with 234 active IDUs; quantitative data included baseline risk assessments and HIV screening plus interviews follow-up interviews administered approximately six months later to 823 IDUs participating in a Federally-funded AIDS community outreach demonstration project.^ Findings that have particularly important implications for substance abuse treatment as an HIV prevention strategy for IDUs are listed below. (1) IDUs who wanted treatment were significantly more likely to be daily heroin users. (2) IDUs who want treatment were significantly more likely to have been to treatment previously. (3) IDUs who wanted treatment at baseline reported significantly higher levels of HIV risk than IDUs who did not want treatment. (4) IDUs who went to treatment between their baseline and follow-up interviews reported significantly higher levels of HIV risk at baseline than IDUs who did not go to treatment. (5) IDUs who went to treatment between their baseline and follow-up interviews reported significantly greater decreases in injection-related HIV risk behaviors. (6) IDUs who went to treatment reported significantly greater decreases in sexual HIV risk behaviors than IDUs who did not go to treatment.^ This study also noted a number of factors that may limit the effectiveness of substance abuse treatment in reducing HIV risk among IDUs. Findings suggest that the impact of methadone maintenance on HIV risk behaviors among opioid dependent IDUs may be limited by the negative manner in which it is perceived by IDUs as well as other elements of society. One consequence of the negative perception of methadone maintenance held by many elements of society may be an unwillingness to provide public funding for an adequate number of methadone maintenance slots. Thus many IDUs who would be willing to enter methadone maintenance are unable to enter it and many IDUs who do enter it are forced to drop out prematurely. ^

MOUSE MAMMARY TUMOR VIRUS EPITOPES EXPRESSED ON TUMOR SPECIFIC TRANSPLANTATION ANTIGENS OF CHEMICALLY INDUCED C3H/HEJ SARCOMAS (TSTA, FIBROSARCOMA, CARCINOGENESIS, MMTV, METHYLCHOLANTHRONE)

This dissertation addressed the hypothesis that the unique tumor specific transplantation antigens (TSTA) of chemically induced sarcomas express epitopes encoded by endogenous viral genes. TSTA from two 3-methylcholanthrene-induced, C3H/HeJ fibrosarcomas (MCA-F and MCA-D) were serologically assessed for viral epitopes in an enzyme-linked immunospecific assay (ELISA) and by immunoaffinity chromatography. Initial evidence with an anti-TSTA antiserum suggested that TSTA were associated with mouse mammary tumor virus (MMTV) peptides, but not peptides from murine leukemia virus (MuLV). TSTA extracted from MCA-F, was assessed with specific anti viral antibodies at three levels of purification for its association with MuLV peptides (gp 70 and p 15E) and MMTV peptides (gp52, gp36 and p27). The results demonstrate that purified preparations enriched for TSTA activity are devoid of MuLV epitopes, but enriched for a subset of MMTV epitopes. Immunoaffinity supports constructed with anti-MMTV antibodies retained TSTA from partially purified MCA-F or MCA-D extracts. Immunoaffinity chromatography with antibodies against individual MMTV peptides demonstrated that the MCA-F TSTA was specifically retained by anti-gp36 and anti-p27 supports, but not by anti-gp52 supports nor a support made with bovine serum albumin. Analysis of the affinity purified TSTA preparations by HPGPC and SDS-PAGE revealed only a few components. Application of the anti-gp36 and anti-p27 retained materials to HPGPC and subsequent in vivo analysis demonstrated that the TSTA migrated in a low and a high molecular weight region. These results suggest that TSTA specificity in C3H/HeJ mice, results from MMTV recombinant proteins. ^

EPSTEIN-BARR VIRUS TRANSFORMATION OF B LYMPHOCYTE SUBPOPULATIONS

Epstein-Barr virus is a herpes virus distinguished by its remarkable specificity for the B lymphocyte of humans and certain other primates. Although the transformation process is very efficient, is has become clear that only a fraction of B lymphocytes is susceptible. Therefore the question may be raised if transformation is related to B cell stage of activation. B cells were purified from peripheral blood mononuclear cells by the removal of monocytes using elutriation and sheep red blood cell rosetting to remove T cells. Retesting B cells were purified using discontinuous Percoll gradients. Activation of resting cells for 24 hours with anti-mu or Staphylococcus aureus Cowan I (SAC) resulted in transition of susceptible cells into the G(,1) phase of the cell cycle as shown by an increase in cell size, an increase in uridine incorporation and an increase in sensitivity to B cell growth factor (BCGF). Entry into S phase was achieved by extending the period of activation to 48-96 hr as shown by an increase in thymidine incorporation. By this criterion, SAC activated cells entered S phase on day 2 and anti-mu treated cells on day 3. Control (G(,0)) cells and cells activated for varying lengths of time (G(,1), G(,1) plus S) were exposed to EBV and plated in a limiting dilution assay to determine the frequency of EBV-transformable cells. Control cells and cells activated for 24 hr had a precursor frequency of 1% to 2%. With continued activation, however, precursor frequency decreased as a function of the duration of activation. The decrease in frequency of transformable cells correlated with the entry of the population into S phase. The transformation frequency in the SAC-treated population was reduced twenty-fold on day 4, whereas in the anti-mu treated population it was reduced ten-fold. Treating cells with BCGF in conjunction with low concentrations of anti-mu decreased the transformation frequency to levels lower than anti-mu alone, further suggesting that entry into S phase is accompanied by a reduction in transformability. These results indicate that resting B cells are highly susceptible to transformation and that with in vitro activation into the cell cycle B cells become progressively insensitive to EBV. ^

„Es war, als hätte das Virus mich geschwängert.“ Vertextungsformen in Aids-Autobiographien

Die vorliegende Untersuchung der Vertextung von Aids in Autobiografien fokussiert die Frage, welcher Darstellungsstrategien diese sich bedienen und welche Funktionen sie in den westlichen Kulturen übernehmen. Vier Autobiografien werden exemplarisch mit Hilfe der Systemtheorie und der Diskursanalyse analysiert und auf folgende Leitfragen hin untersucht: Sind die AutorInnen an Aids erkrankt oder nicht? Welche Lebenszeit steht ihnen zur Verfügung? Sind sie professionelle Schriftsteller oder Laien? Welche Rolle spielt ihr Geschlecht? Welche Werte werden wie vermittelt? Wird Akzeptabilität geschaffen? Wie wird mit den Grenzen des Akzeptablen umgegangen? Wie wird die Konstruktion und Destruktion des schreibenden Subjektes angesichts der Krankheitserfahrung verhandelt? Das untersuchte Material umfasst ein Spektrum, das • das schnelle Sterben an Aids, das lange Leben mit Aids sowie das Leben als HIV-Negativer in Gegenwart von Aids zeigt. • von gesellschaftlich orientierter Bewältigung der Krankheitserfahrung über individuelle Bewältigung bis hin zur Verweigerung der gesellschaftlichen Integration reicht. • den unterschiedlichen Einsatz von Metaphern bei der Sinngebung und der Vertextung von Körpererfahrung aufzeigt: Sterben als Geburt (Normalisierungsrhetorik), Sterben als Holocaust (Eskalationsrhetorik), Krankheitserfahrung als Generator immer neuer, überbordender Sprachbilder.

Distribution of human immunodeficiency virus (HIV) in the CNS of children with severe HIV encephalomyelopathy

The presence and distribution of human immunodeficiency virus (HIV) were examined in the CNS of two children with severe HIV encephalitis and myelitis. Using polymerase chain reaction-mediated DNA amplification and subsequent Southern analysis, proviral HIV gag sequences were identified in brain tissue of both patients. In situ hybridization using antisense oligonucleotide probes revealed abundant HIV gag and env/nef RNAs selectively in areas with histopathological evidence for HIV-induced tissue damage. The spinal cord of one patient exhibited a striking subpial accumulation of HIV RNAs strongly suggestive of a liquorigenic spread of the infection. HIV RNAs were typically associated with cells of the monocyte/macrophage lineage, as shown by a combined immunohistochemical and in situ hybridization procedure. The present study supports the view that the pattern and distribution of HIV-induced brain lesions is largely determined by the extent of focal HIV replication within the CNS.

A Model Study of the Circulation in the Pearl River Estuary (PRE) and Its Adjacent Coastal Waters: 1. Simulations and Comparison with Observations

The Princeton Ocean Model is used to study the circulation features in the Pearl River Estuary and their responses to tide, river discharge, wind, and heat flux in the winter dry and summer wet seasons. The model has an orthogonal curvilinear grid in the horizontal plane with variable spacing from 0.5 km in the estuary to 1 km on the shelf and 15 sigma levels in the vertical direction. The initial conditions and the subtidal open boundary forcing are obtained from an associated larger-scale model of the northern South China Sea. Buoyancy forcing uses the climatological monthly heat fluxes and river discharges, and both the climatological monthly wind and the realistic wind are used in the sensitivity experiments. The tidal forcing is represented by sinusoidal functions with the observed amplitudes and phases. In this paper, the simulated tide is first examined. The simulated seasonal distributions of the salinity, as well as the temporal variations of the salinity and velocity over a tidal cycle are described and then compared with the in situ survey data from July 1999 and January 2000. The model successfully reproduces the main hydrodynamic processes, such as the stratification, mixing, frontal dynamics, summer upwelling, two-layer gravitational circulation, etc., and the distributions of hydrodynamic parameters in the Pearl River Estuary and coastal waters for both the winter and the summer season.

Efficacy of lead-in silibinin and subsequent triple therapy in difficult-to-treat HIV/hepatitis C virus-coinfected patients

OBJECTIVES: The efficacy of current hepatitis C virus (HCV) triple therapy, including a protease inhibitor, is limited in HIV/HCV-coinfected patients with advanced liver fibrosis and nonresponse to previous peginterferon-ribavirin. These patients have a low chance (only 30%) of achieving a sustained virological response (SVR) during triple therapy and cannot wait for next-generation anti-HCV drugs. In a pilot study, we investigated the efficacy of a lead-in therapy with silibinin before triple therapy in difficult-to-treat patients. METHODS: Inclusion criteria were HIV/HCV coinfection with advanced liver fibrosis and documented failure of previous peginterferon-ribavirin treatment. Intervention was lead-in therapy with intravenous silibinin 20 mg/kg/day for 14 days. Subsequently, peginterferon-ribavirin combined with telaprevir was initiated for 12 weeks, followed by peginterferon-ribavirin dual therapy until week 48 after initiation of triple therapy. The outcome measurements were HCV RNA after silibinin lead-in, at weeks 2, 4 and 12 of triple therapy, and SVR at week 24 after the end of treatment. RESULTS: We examined six HIV/HCV-coinfected patients (four infected with genotype 1a). All had fibrosis grade METAVIR ≥F3 and were on fully suppressive antiretroviral therapy. Mean HCV RNA decline after silibinin therapy was 2.6 log10 IU/mL (range 2-3 log10 IU/mL). Five of the six patients were virologically suppressed at weeks 2 and 4, and all six at week 12 of triple therapy. One experienced a viral breakthrough thereafter. Four of five patients (80%) showed an SVR 24. One patient had an SVR 12 but has not yet reached week 24. CONCLUSIONS: A lead-in with silibinin before triple therapy is highly effective and increases the probability of HCV treatment success in difficult-to-treat HIV/HCV-coinfected patients with advanced liver fibrosis and previous failure of peginterferon-ribavirin.