Transcript Virology Core

Einstein-Montefiore CFAR
Virology Core
Director: Dr. Ganjam V. Kalpana, Ph.D.
Overarching Goal
• To provide CFAR Investigators with the appropriate
support, training and access for the most relevant
and cutting-edge approaches to rapidly and
efficiently support their virology research programs.
Virology Core Aims
• Providing the Infrastructure, reagents, assays and technologies to
enable basic and translational researchers to Investigate HIV
Infection, pathogenesis and therapy;
• Performing cellular and molecular assays characterizing HIVlnfection to support clinical and translational investigators engaged
in patient-based studies of HIV/AIDS patients; and
• Providing training to investigators In a wide range of cellular and
molecular assays used in the study of HIV pathogenesis.
• Maintaining a central repository of molecular clones of infectious
HIV, viral isolates, reporter HIV clones and HIV reporter cells lines as
well as HIV-based expression vectors.
Virology Core Training Programs
 Measurement of p24 antigen by ELISA
 Co-culture, quantification and sequence analysis of HIV
from PBMCs
 Determining the viral titers, infectious units (i.u.) and
m.o.i. of HIV using reporter cell lines
 Real-time PCR for quantification of HIV RNA and
cellular gene expression
 Application of advanced virological techniques to
analyze all stages of HIV-1 replication-including entry,
reverse transcription, nuclear localization, integration,
transcription, assembly and particle production.
Virological Core Services
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HIV culture and isolation
HIV p24 antigen capture assay
Repository of titered primary HIV isolates
HIV-1 neutralization assays
Quantitative HIV-1 DNA and RNA measurements
HIV and cellular RNA quantification by real-time qPCR
Determination of viral tropism.
Reporter virus entry assay.
HIV sequence analysis.
Patient sample processing and storage.
Immunosorting of control and HIV-infected cellular
subpopulations.
Measurement of serum HCV antibody and plasma HCV virus
levels.
Molecular Biological Core Services
• Routine
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Custom vector and plasmid design and construction
Provision and expression of reporter and full-length molecular
clones of viruses of many clades
Site-directed mutagenesis
Large-scale expression of recombinant HIV, human or other
pathogen proteins
• Advanced
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Lentiviral vector construction and preparation of virus particles
RNA interference analysis
Genetic and biochemical systems to study protein-protein
interactions
Chromatin immunoprecipitation (ChIP) techniques
FRET-based BLam assay (beta-Lactamase based assay)
Heteroduplex mobility assay (HMA)
Expansion of Core Activities and
Innovation
• The core (in collaboration with ATRP program)
purchased a new Applied Biosystem qPCR
machine to facilitate the demand for use of this
assay in studying HIV-1 infection.
• Core is developing methods to carry out
microRNA analysis in HIV-1 infected samples.
• In collaboration with Epigenomics and Genomics
Center, the core is planning to optimize the
methods for RNA/microRNA-seq, ChIP-seq and
Affymatrix techniques. Consultation for the
developing and carrying out these techniques will
be provided for CFAR users.
Expansion of Core Activities and
Innovation
In collaboration with Bronx WHIS (PI: Dr. Anastos) and the Einstein’s Microbicide
program (PI: Dr. Herold), CFAR has purchased an Abbott m2000 machine
M200sp
Sample preperation
station
M2000rt
RT-PCR
machine
Expansion of Core Activities and
Innovation
• Abbot m2000 Provides automation for sample preparation and real time
PCR.
• Ideal for using a variety of clinical samples and allows the use of bar
coded tubes, creates an efficient workflow, minimizes errors and
contamination.
• Barcoded Laboratory Tubes, Precision Pipetting-no manual mixing or
manipulation, Open Mode-Flexible protocol for various sample types and
volumes
• Efficient Sample Extraction--Flexible throughput options of 24 to 96
samples—allows isolation of nucleic acids, RNA or DNA from variety of
samples (serum, plasma) based on the magnetic bead method.
• Samples are prepared automatically and loaded onto 96 well plates and
sealed for transfer to PCR machine.
Expansion of Core Activities and
Innovation --- continued
• Abbot m2000 currently being used for HIV-1 viral
load assays (range 40 to 10,000,000 copies/mL).
• Can be used for other clinical applications.
Other viral loads—HCV, HBV
Detection of cellular transcripts-biomarkers, depending
on the availability of the kit to use in the Abbott
machine.
Expansion of Core Activities and
Innovation --- continued
Technical Innovations—Alpha Technology
AlphaLISA-instead of p24 ELISA
Various enzymatic- Kinase Assays
Protein-protein interactions
A reduced number of assay steps---no
washing steps.
Automated---good for adopting to HTS
Versatile—measure large biomolecules
(up to 2000 kDa) or
weak interactions (mM).
Proposed Activities for
Next Project Period
• The Core will expand its sequencing activities for
molecular characterization of clinical isolates
• Expansion of virological support for WIHS studies
• Expansion of support to carry out epigenetic
(methylation and ChIP assays) and microRNA
analysis during HIV-1 pathogenesis
• Expand service to include support of protein
production
Accomplishments
• Over the past 5 years, we have contributed to data in
31 publications
• In the past year we have supported studies by 32
NlH-funded investigators.
• We have expanded the Core services In response to
the needs of Investigators including developing
highly cost-effective lab-developed assays.