Phase 3 (2007 Deadline)
Hepatitis C Virus Management in Pakistan
Charles M. Rice, The Rockefeller University, New York
Ishtiaq Qadri, (formerly) National University of Sciences and Technology, Islamabad
Pakistani Funding (HEC): $ 300,000
US Funding (State): $ 250,000
Project Dates on US Side: July 1, 2008 - June 30, 2012
Project Overview
The overall objective of this grant is to broaden the understanding and management of the Hepatitis C virus (HCV) pandemic in Pakistan. One of the initial goals is to develop HCV models that better represent the genotype 3 virus that predominates in Pakistan. The majority of tools used to study HCV biology represent viruses from either genotype 1 or 2. It is clear from these studies that there will be genotype-specific treatment effects; however, it remains to be delineated what these effects will be for genotype 3. This research team has been working towards better describing the HCV variants circulating in Pakistan and developing tools to specifically characterize genotype 3 virus.
Major Results
- Provided training to 18 students and researchers, including facilitating the visit and training of a female Pakistani scientist in the US partner’s lab
- Organized the 1st International HCV Management Symposium, held at NUST February 10-11, 2011
- Established the Diagnostic Lab at the NUST Center of Virology and Immunology
- Developed HCV intergenotypic chimeras, robust in vitro serine protease FRET-based assay, HCV NS3 helicase Assay, and an ELISA-based in vitro infectivity assay for HCV, with a patent application submitted to the Intellectual Property office at NUST
- Developed and tested HCV pseudotype particles (HCVpp) for HCVpp production and infectivity
- Published seven papers resulting from the project
- Obtained additional funding from the Pakistani Higher Education Commission and the Centre National de la Recherche Scientifique
Quarterly Update
In the second quarter of 2011, Dr. Mohsan Saeed, a Pakistani postdoctoral fellow who recently completed his PhD in Japan, began his visit to Dr. Rice’s lab. He cataloged the clinical samples Dr. Rice had received from Dr. Qadri and did RNA load measurements on those reported by the Pakistani researchers to have the highest titers. Selected samples were subjected to further analyses, including (1) troubleshooting plasma samples with various anticoagulants to optimize conditions for infection of primary liver cells and (2) testing of Pakistani isolates for cell culture infectivity. The research team expects to create a battery of functional systems for Pakistani genotype 3a isolates that can be used to evaluate new antiviral therapies for this prevalent genotype in the next few months. High on the list of priorities will be to create a genotype 3a replicon, which would be the workhorse for compound screening and evaluation. Dr. Saeed’s experience in accomplishing this for a non-Pakistani genotype 3a isolate in the latter part of his PhD thesis work should be a great asset as he works with Dr. Rice and his colleagues on this new research effort. To accomplish the remaining project objectives, Dr. Rice has received a one year no-cost extension on his grant through June 30, 2012.
Progress Report Summaries
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2011 Download full report
2010 Show summary || Hide summary || Download full report
A new shipping method was successfully used to ship Fatima’s samples and reagents back to Pakistan, and the next batch of Pakistani patient samples to the U.S. was successfully received in 2010 with another shipment en route.
During the final quarter of 2010, Kimberly Ritola, who was a postdoctoral fellow heading this project in the Rice lab, left for a position as a Senior Reviewer with the FDA. No visits to Pakistan were scheduled for any Rice lab members due to the current security situation. In October 2010, Dr. Qadri visited Rockefeller University to meet with the Rice lab members and had a one-on-one meeting with Dr. Rice. The meeting focused on identifying and overcoming any roadblocks to completing the project objectives.
One option, which was discussed with the NAS staff, was to support and recruit a Pakistani postdoctoral fellow to continue the work in Rice lab. Drs. Rice and Qadri agreed to earmark the remaining funds of the U.S. portion of the grant for an outstanding candidate, Mohsan Saeed, who is currently finishing his PhD in one of the top HCV laboratories in Japan. The project team is currently finalizing the logistic and visa issues associated with Dr. Saeed’s arrival at the Rockefeller University in the spring of 2011.
2009 Show summary || Hide summary || Download full report
Kaneez Fatima, a graduate student from the National University of Sciences and Technology (NUST) in Islamabad, Pakistan, finished a six-month training period in the Rice lab in December 2009. Kaneez continues to work on determining consensus sequences for specific HCV genes of clinical interest such as the NS3-4A protease (a target for antiviral therapy). Following PCR amplification of the NS3-4A region from multiple patient samples, she is assembling a consensus NS3-4A sequence. The consensus sequence will be cloned into a protein expression vector and the activity of the purified genotype 3 protease protein assessed against HCV specific protease inhibitors currently being developed. While accumulating enough sequences to create an accurate consensus sequence, individual patient NS3-4A clonal sequences were used to optimize the protein purification steps and the protease activity assays. To date, the cloning and protein purification steps have been worked out as well as a FRET based assay to measure protease activity. Work will now focus on generating more NS3-4A sequences to examine both the range of protease activities and inhibitor sensitivity within the Pakistani population.
Robust models allowing for the study of steps within the HCV life cycle were limited to genotypes 1 and 2. Cell lines stably expressing HCV replicons can recapitulate the virus replication and protein production steps but fail to make infectious virus. A model in which virus production and entry can be studied exists only for genotype 2 (HCVcc). Multiple approaches are being used to replicate these models for genotype 3 viruses. Chimeras using the genotype 2 HCVcc virus as a backbone and selected genes (such as the NS3-4A protease gene discussed above) from Pakistani patients were in the cloning phase. Additionally, a genotype 3 replicon cell line was created using the complete HCV sequence from individual Pakistani patients and eventually from a consensus sequence. These projects were very sequence intensive and may require selection steps to find a complete genome that will be capable of replicating in culture. Kaneez Fatima presented her projects for discussion at the Rice lab meeting before she left to continue her work in Pakistan.
Work by the Rice lab group focused on trying to isolate infectious virus from Pakistani patient samples. Patient samples obtained from Pakistan were directly screened for the presence of HCV capable of replicating in culture. This approach has been historically technically difficult. Initial entry and protein production has been seen with a few samples; however, no sustained replication has been detected. One major limitation for this sub-project has been the state of samples received from Pakistan. Packages initially received have were thawed up arrival, compromising the integrity of any infectious virus that might have been be present.
2008 Show summary || Hide summary || Download full report
(Will be available soon)
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