Impact of Intrahost Diversity on Disease Severity and Genetic Diversity in Primary and Secondary Infection

Justification/Summary

RNA viruses exhibit a high degree of sequence variation not just between isolates as addressed above, but also among viruses within an individual.  This in part is due to the viral RNA polymerase and its inability to proofread.  As a result, viral infections within an individual exist as a population of closely related sequences that are called quasispecies.  The role of quasispecies in disease pathogenesis, transmission, and viral evolution is suspected to possibly be important for RNA viruses.   Recent studies indicate that intra-host genetic diversity may be important for pathogenesis and tropism in mammalian hosts.              

Of particular interest are the comparisons between individuals with different disease outcomes and multiple time points within an individual infection (e.g., first acute sample and sample at defervescence). Sequence generated for this project will characterize  the intrahost diversity found in patients with severe versus non-severe, and primary versus secondary infection.  These data will help to define Dengue diversity within a host and allow an assessment of the capacity of present isolation techniques from serum to capture actual viral diversity.  Generation of this data will assist in project design and sequencing strategy for this project and future viral initiatives.

People

Principal External Collaborator

Eva Harris - Division of Infectious Diseases, School of Public Health, University of California, Berkeley, CA

Funding

Funding for this sequencing project was provided by the National Institute of Allergy and Infectious Diseases.

Cohort Description

Hospital - This Nicaraguan hospital-based study has been ongoing since 1998, and in its current form since 2005. Children, 6 months to 14 years of age, suspected of Dengue who present to the Nicaraguan National Pediatric Reference Hospital, the Hospital Infantil Manuel de Jesus Rivera, are invited to participate in the study, which follows children (as inpatients and outpatients) daily during the acute phase until discharge and again at convalescence for clinical symptoms and signs, fluid management, and collection of biological samples (serum, plasma, PBMCs, DNA). These samples are used for clinical management (complete blood count, blood chemistry) and dengue serology, virology, and molecular biology for both diagnostic and research purposes. The aims of the study are to improve Dengue case management and to better understand the pathogenesis of severe dengue using tools such as full-length genome sequencing, microarray, studies of antibody repertoire, and the like. As in the PDCS, ICT tools and clinical mononitoring have been widely implemented to ensure quality control and high-quality clinical data and biological samples.

Mouse Adapted - Human clinical isolates have been serially passaged in mosquito C6/36 cells and inoculated into mice to select for virus mutants that are adapted to grow in mice. Virus in mouse sera is collected, passaged in C6/36 cells and re-inoculated into mice repeatedly. The process is continued until a phenotype of increased pathogenesis/lethality in the mice is achieved.

PDVI-NI - The Pediatric Dengue Cohort Study (PDCS) follows 3,800 children in District II of Managua, Nicaragua and has been ongoing since August of 2004. Healthy blood samples are collected every July, and during the year, children are followed for all medical consults (including all febrile illnesses) at the Health Center Socrates Flores Vivas (HCSFV). Suspected dengue cases are followed clinically at the HCSFV and at the study hospital (Hospital Infantil Manuel de Jesus Rivera) when necessary, and acute and convalescent samples undergo serological, virological, and molecular biological work-up for dengue diagnosis and isolation of dengue virus at the National Virology Laboratory in the Centro Nacional de Diagnostico y Referencia at the Ministry of Health. Information and communication technologies (ICT) have been integrated into all levels of the study to ensure the highest level of sample and data quality. The aims of the study are to investigate the natural history of dengue transmission (clinical manifestations, viral sequence), collect biological samples for immunological studies and vaccine safety research, and prepare the site for potential clinical trials of tetravalent dengue vaccines.

Experimental RNA Elements - We have recently identified coding-region elements that regulate the viral genome in both mammalian and mosquito cells, including the conserved capsid-coding region 1 (CCR1).  CCR1 is a small 18 nucleotide region contained within a stable stem loop structure, and has been implicated in regulating translation and a yet unidentified role in the viral life cycle in mammalian cells, though its effects are more pronounced in mosquito cells.  When these viruses are passaged from different cell lines we have observed revertant viral sequences in the region of interest. We will be investigating whether any of these sequence changes are biologically relevant and whether they extend to other positions of the genome, which would implicate a secondary RNA element is required for CCR1 function.