Multidimensional analysis of Gammaherpesvirus RNA expression reveals unexpected heterogeneity of gene expression.

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi's sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level

Sexually transmitted infections, benign prostatic hyperplasia and lower urinary tract symptom-related outcomes: results from the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.

ObjectiveTo examine whether a history of sexually transmitted infections (STIs) or positive STI serology is associated with prevalent and incident benign prostatic hyperplasia (BPH)/lower urinary tract symptoms (LUTS)-related outcomes in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial.MethodsSelf-reported history of STIs (gonorrhoea, syphilis) was ascertained at baseline, and serological evidence of STIs (Chlamydia trachomatis, Trichomonas vaginalis, human papillomavirus (HPV)-16, HPV-18, herpes simplex virus type 2, human herpesvirus type 8 and cytomegalovirus) was detected in baseline serum specimens. We used data collected on the baseline questionnaire, as well as results from the baseline prostate-specific antigen (PSA) test and digital rectal examination (DRE), to define prevalent BPH/LUTS-related outcomes as evidence of LUTS (self-reported diagnosis of an enlarged prostate/BPH, BPH surgery or nocturia [waking ≥2 times/night to urinate]) and evidence of prostate enlargement (PSA > 1.4 ng/mL or prostate volume ≥30 mL) in men without prostate cancer. We created a similar definition of incident BPH using data from the follow-up questionnaire completed 5-13 years after enrolment (self-reported diagnosis of an enlarged prostate/BPH or nocturia), data on finasteride use during follow-up, and results from the follow-up PSA tests and DREs. We used Poisson regression with robust variance estimation to calculate prevalence ratios (PRs) in our cross-sectional analysis of self-reported (n = 32 900) and serologically detected STIs (n = 1 143) with prevalent BPH/LUTS, and risk ratios in our prospective analysis of self-reported STIs with incident BPH/LUTS (n = 5 226).ResultsGenerally null results were observed for associations of a self-reported history of STIs and positive STI serologies with prevalent and incident BPH/LUTS-related outcomes, with the possible exception of T. vaginalis infection. This STI was positively associated with prevalent nocturia (PR 1.36, 95% confidence interval (CI) 1.18-1.65), prevalent large prostate volume (PR 1.21 95% CI 1.02-1.43), and any prevalent BPH/LUTS (PR 1.32 95% CI 1.09-1.61); too few men had information on both STI serologies and incident BPH/LUTS to investigate the associations between T. vaginalis infection and incident BPH/LUTS-related outcomes.ConclusionsOur findings do not support associations of several known STIs with BPH/LUTS-related outcomes, although T. vaginalis infection may warrant further study