Elucidation of the events responsible for HIV-1 and SIV mucosal transmission in the vagina, cervix and rectum, including a precise definition of virus - host cell - innate/adaptive immune interactions that precede the establishment of viremia and viral latency, could be instrumental in the design and development of effective vaccines. In recent years, our laboratories have taken significant strides toward addressing these objectives by developing a theoretical and experimental framework with which to identify transmitted/founder HIV-1 and SIV genomes responsible for virus transmission, and in parallel, developing in situ hybridization and quantitative PCR methods for detecting HIV-1 and SIV sequences in relevant mucosal and lymphoreticular tissues. However, these complementary molecular assays have not previously been used together in a systematic manner to analyze SIV transmission. Virology Core D will, for the first time, combine these technologically advanced approaches to study mucosal infections by SIVmac251, SIVmac239 and SHIV{SF162P3} with and without vaccine intervention in support of Projects 1-5. The overall objective of the Virology Core is thus to provide state-of-the-art molecular tools, experience and service to consortium investigators that will enable a spatial, temporal, genetic and immunocytohistological understanding of mucosal SIV transmission and subsequent virus dissemination in naive animals and to define sites and mechanisms of interception of this process in animals that are actively or passively vaccinated using different vaccine modalities.
Specific aims are: (i) to provide support to the five projects for quantitative analysis of viral RNA and DNA using regular and ultra-sensitive nested qRT-PCR and PCR methods applied to cell-free and cell-associated blood, biopsy and necropsy specimens;(ii) to provide advanced sequencing approaches including single genome amplification (SGA) - direct amplicon sequencing and next generation Life Technologies 454 pyrosequencing and Pacific Biosciences SMRT sequencing to assess transmission bottlenecks and post-transmission viral spread and evolution;and (iii) to provide in situ hybridization-laser capture microdissection and SGA sequencing for recovery and analysis of viral sequences from infected cells and cell foci, especially at early times after inoculation, in order to assess spatiotemporal patterns of early virus amplification and spread. The results will provide novel insight into pathways and mechanisms of SIV transmission and vaccine prevention.
We have developed a comprehensive suite of virologic assays that will be used to analyze early virus-host events in mucosal, submucosal, regional and systemic tissues. The Virology Core will provide sensitive and specific assays to reliably detect and quantify limited numbers of rare SIV events in a large background of challenging analyte and in so doing contribute importantly to the objectives of this consortium.
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