Project description:The abundance of HSV mRNAs was determines over 16h of infection in wt (KOS) and n199 (ICP22 mutant) infected cells. ICP22 is an immediate early gene of HSV that affects gene expression Overall design: 2 X 10E6 MRC5 cells were infected at an MOI of 10 PFU/cell. mRNA was isolated at different times post infection using Ambion RNaqueous-4PCR kit and quantified using an Agilent Eukaryotic RNA Nano kit and a 2100 bioanalyzer. Individual samples each with unit barcodes were quantified using an Agilent DNA 7500 chip an a 2100 bioanalyzer. Pooled multiplexed samples were sequenced at the Tufts University Genomics facility. The demulitplexed sequence read files were mapped to the HSV strain KOS genome and quantified using CLC Genomics Workbench.
Project description:During lytic herpes simplex virus (HSV) infections, the half-lives of host and viral mRNAs are regulated by the HSV virion host shutoff (Vhs) protein (UL41). The sequences of the UL41 polypeptides of HSV type 1 (HSV-1) strain KOS and HSV-2 strain 333 are 87% identical. In spite of this similarity, HSV-2 strains generally shut off the host more rapidly and completely than HSV-1 strains. To examine type-specific differences in Vhs function, we compared the Vhs activities of UL41 alleles from HSV-1(KOS) and HSV-2(333) by assaying the ability of a transfected UL41 allele to inhibit expression of a cotransfected reporter gene. Both HSV-1 and HSV-2 alleles inhibited reporter gene expression over a range of vhs DNA concentrations. However, 40-fold less of the HSV-2 allele was required to yield the same level of inhibition as HSV-1, indicating that it is significantly more potent. Examination of chimeric UL41 alleles containing various combinations of HSV-1 and HSV-2 sequences identified three regions of the 333 polypeptide which increase the activity of KOS when substituted for the corresponding amino acids of the KOS protein. These are separated by two regions which have no effect on KOS activity, even though they contain 43 of the 74 amino acid differences between the parental alleles. In addition, alleles encoding a full-length KOS polypeptide with a 32-amino-acid N-terminal extension retain considerable activity. The results begin to identify which amino acid differences are responsible for type-specific differences in Vhs activity.
Project description:We found that the germline transcription factor double homeobox 4 (DUX4) is upregulated upon infection with wild-type herpes simplex virus-1 (HSV-1). The goal of this experiment was to compare the cellular transcriptome of HEK293T cells that were infected with HSV-1 (KOS strain), or transfected with a plasmid encoding human DUX4. Overall design: HEK293T cells remained either untreated (mock), were infected with wild-type HSV-1 (KOS strain) for 18 h, or were transiently transfected with a plasmid encoding untagged human DUX4 (pmkg-DUX4) for 18 h, followed by extraction of total RNA and RNAsequencing.
Project description:Herpes simplex virus type 2 (HSV-2) is the most frequent cause of genital ulcer disease worldwide and has been associated with increased risk for HIV-1 acquisition and transmission. We conducted a cross-sectional analysis of risk factors for HSV-2 infection among HIV-1 uninfected partners, whose partners were co-infected with HIV-1 and HSV-2.Between November 2004 and April 2007, 3408 HIV-discordant couples, in which the HIV-1 infected partners were HSV-2 seropositive with CD4 250 cells/mm3 or greater, were enrolled in an HSV-2 suppression trial to prevent HIV-1 transmission at 14 sites in 7 African countries. Clinical & behavioral data, HSV-2 and HIV-1 testing were conducted at enrolment. Univariate and multivariate Poisson regression analyses were performed separately, by gender of the HIV-1 infected partner.Among 3354 HIV-1 uninfected participants, 32% were female and overall 71% were HSV-2 seropositive. Among couples with female HIV-1 infected partners, HIV-1 plasma RNA [aPR 1.03; 95% CI: 0.99 to 1.06; p = 0.11] and CD4 count [aPR 1.00; 95% CI: 0.98 to 1.01; p = 0.48] in the HSV-2/HIV-1 dually infected female and circumcision in the HIV-1 uninfected male partner [aPR 0.94; 95% CI: 0.88 to 1.00; p = 0.06] were not associated with reduced risk of HSV-2 seropositivity, after adjusting for other factors.In this cross-sectional analysis of African HIV-1 serodiscordant heterosexual couples with prevalent HSV-2 infection in the HIV-1 infected partner, HIV-1 plasma RNA and CD4 count in the dually-infected partner and male circumcision in the HIV-1 uninfected partner were not associated with HSV-2 concordance.ClinicalTrials.gov NCT00194519.
Project description:Suppressive herpes simplex virus (HSV) therapy can decrease plasma, cervical, and rectal HIV-1 levels in HIV-1/HSV-2 co-infected persons. We evaluated the effect of HSV-2 suppression on seminal HIV-1 levels.Twenty antiretroviral therapy (ART)-naive HIV-1/HSV-2 men who have sex with men (MSM) in Lima, Peru, with CD4 >200 cells/microl randomly received valacyclovir 500 mg twice daily or placebo for 8 weeks, then the alternative regimen for 8 weeks after a 2-week washout. Peripheral blood and semen specimens were collected weekly. Anogenital swab specimens for HSV DNA were self-collected daily and during clinic visits.HIV-1 RNA was quantified in seminal and blood plasma by TaqMan real-time polymerase chain reaction (RT-PCR) or Roche Amplicor Monitor assays. HSV and seminal cytomegalovirus (CMV) were quantified by RT-PCR. Linear mixed models examined differences within participants by treatment arm.Median CD4 cell count of participants was 424 cells/microl. HIV-1 was detected in 71% of 231 semen specimens. HSV was detected from 29 and 4.4% of swabs on placebo and valacyclovir, respectively (P < 0.001). Valacyclovir significantly reduced the proportion of days with detectable seminal HIV-1 (63% during valacyclovir vs. 78% during placebo; P = 0.04). Seminal HIV-1 quantity was 0.25 log10 copies/ml lower [95% confidence interval (CI) -0.40 to -0.10; P = 0.001] during the valacyclovir arm compared with placebo, a 44% reduction. CD4 cell count (P = 0.32) and seminal cellular CMV quantity (P = 0.68) did not predict seminal plasma HIV-1 level.Suppressive valacyclovir reduced seminal HIV-1 levels in HIV-1/HSV-2 co-infected MSM not receiving ART. The significance of this finding will be evaluated in a trial with HIV-1 transmission as the outcome.
Project description:The aim of this experiment was to determine whether viral double-stranded RNA from HSV-1 infected cells were detectable in non infected cells Overall design: Vero cells were infected with mCherry-tagged HSV-1 virus and sorted for mCherry in order to deliniate between infected and uninfected cells. Stranded RNAseq was subsequently performed in order to identify overlapping HSV-1 RNA derived from both strands of the viral genome which correspond to double-stranded RNA.
Project description:Herpes simplex viruses (HSV) are responsible for a broad variety of human diseases, including cold sores, ocular herpes and herpetic encephalitis, leading to a great burden worldwide. However, the role for sex in herpes infection is controversial and unclear due to the complexity of factors involving in the formation of HSV diseases. Herein, we ask whether there is a sexual dimorphism based on the chromosomal rather than hormonal differences in the primary skin cells during low exposure of HSV. Profiling of male and female transcriptional programs reveals that cytosolic sensing pathway is induced to a greater degree in female cells, correlated with higher yields of infectious virions in male counterparts. Female skin cells distinctively reactivate Xist, a critical component of X inactivation, to silence the expression of the transcriptional repressor on X chromosome, which thereby maintains higher viperin level and further explains the different growth phenotypes between two sexes. Collectively, we propose a model that HSV-1 triggers a sex-specific regulation of antiviral response in the cytosolic sensing signaling via the control of Xist. Overall design: We report transcriptomic data from both male and female HSV-1-infected mouse skin fibroblasts to nvestigate sex differences in HSV-1 infection
Project description:Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 ICP0- mutant virus, HSV-2 0?NLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0?NLS vaccine. Western blot analyses indicated the live HSV-2 0?NLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0?NLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2's thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0?NLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine's capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine.
Project description:BACKGROUND:Following acute infection, Herpes Simplex virus-1 (HSV-1) establishes lifelong latency and recurrent reactivation in the sensory neurons of trigeminal ganglia (TG). Infected tree shrew differs from mouse and show characteristics similar to human infection. A detailed transcriptomic analysis of the tree shrew model could provide mechanistic insights into HSV-1 infection in humans. METHODS:We sequenced the transcriptome of infected TGs from tree shrews and mice, and 4 human donors, then examined viral genes expression up to 58?days in infected TGs from mouse and tree shrew, and compare the latency data with that in human TGs. RESULTS:Here, we found that all HSV-1 genes could be detected in mouse TGs during acute infection, but 22 viral genes necessary for viral transcription, replication and viral maturation were not expressed in tree shrew TGs during this stage. Importantly, during latency, we found that LAT could be detected both in mouse and tree shrew, but the latter also has an ICP0 transcript signal absent in mouse but present in human samples. Importantly, we observed that infected human and tree shrew TGs have a more similar LAT region transcription peak. More importantly, we observed that HSV-1 spontaneously reactivates from latently infected tree shrews with relatively high efficiency. CONCLUSIONS:These results represent the first longitudinal transcriptomic characterization of HSV-1 infection in during acute, latency and recurrent phases, and revealed that tree shrew infection has important similar features with human infection.