HIV-1 viral blips are associated with repeated and increasingly high levels of cell-associated HIV-1 RNA transcriptional activity.

OBJECTIVE: Some HIV+ patients, virally suppressed on ART, show occasional 'blips' of detectable HIV-1 plasma RNA. We used a new highly sensitive assay of cell-associated HIV-1 RNA to measure transcriptional activity in PBMCs and production of infectious virus from the viral reservoir, in patients with and without 'blips'. DESIGN/METHODS: RNA and DNA extracted from cells in 6 ml of peripheral blood, from suppressed patients with one to two 'blip' episodes over the past 2 years of ART (n = 55), or no 'blips' (n = 52), were assayed for HIV-1 RNA transcripts and proviral DNA targeting the highly conserved 'R' region of the LTR. Follow-up samples were also collected. Purified CD4+ T cells were cultured with anti-CD3/CD28/CD2 T-cell activator to amplify transcription and measure replication competent virus. RESULTS: HIV-1 RNA transcripts ranged from 1.3 to 5415 copies/106 white blood cells. 'Blip' patients had significantly higher levels vs. without blips (median 192 vs. 49; P = 0.0007), which correlated with: higher levels of inducible transcripts after activation in vitro, sustained higher HIV-1 transcription levels in follow-up samples along with increasing HIV-1 DNA in some, and production of replication-competent HIV-1. CONCLUSION: Viral 'blips' are significant reflecting higher transcriptional activity from the reservoir and contribute to the reservoir over time. This sensitive assay can be used in monitoring the size and activity of the HIV-1 reservoir and will be useful in HIV-1 cure strategies.

Comparative proteomic analysis of two pathogenic Tritrichomonas foetus genotypes: there is more to the proteome than meets the eye.

Certain clinical isolates of Tritrichomonas foetus infect the urogenital tract of cattle while others infect the gastrointestinal tract of cats. Previous studies have identified subtle genetic differences between these isolates with the term "genotype" adopted to reflect host origin. The aim of this work was to seek evidence of host-specific adaptation and to clarify the relationship between T. foetus genotypes. To do this we characterised the proteomes of both genotypes using two-dimensional gel electrophoresis (2DE) coupled with LC-MS/MS. Our comparative analysis of the data revealed that both genotypes exhibited largely similar proteoform profiles; however differentiation was possible with 24 spots identified as having a four-fold or greater change. Deeper analysis using 2DE zymography and protease-specific fluorogenic substrates revealed marked differences in cysteine protease (CP) expression profiles between the two genotypes. These variances in CP activities could also account for the pathogenic and histopathological differences previously observed between T. foetus genotypes in cross-infection studies. Our findings highlight the importance of CPs as major determinants of parasite virulence and provide a foundation for future host-parasite interaction studies, with direct implications for the development of vaccines or drugs targeting T. foetus.