Venkatakrishna Shyamala has extensive experience in molecular diagnostics and research and development. She has held leadership roles at several companies developing molecular diagnostic tests and was involved in the development, validation, and implementation of alternative nucleic acid tests for HCV and WNV screening at Chiron Corporation. She has also consulted for Novartis Diagnostics and contributed to establishing standards for molecular diagnostic methods through her work with CLSI.

1. Accomplishments (Selected)
Venkatakrishna Shyamala, PhD
•Consultant: Novartis Dx- Scientific Affairs;
Document writer - Molecular Diagnostics
•Innovative Biosensors, Inc.
•Senior VP, Research and Development
• Digene Corporation
•Director, Research and Development
• Chiron Corporation
•Associate Director, Research
• Univ of California, Berkeley
•Visiting Scientist
V. Shyamala 1

2. Accomplishments (Selected)
Venkatakrishna Shyamala, PhD
• Consultant: Scientific Affairs
Novartis Vaccines & Diagnostics
• Monitoring Procleix ® Blood Screening
Assay Testing and other NAT Trials
•Assist Asia-Pacific region through
Seminars, Conference Presentations,
Workshops, and Publication Support
V. Shyamala 2

3. Accomplishments (Selected)
Venkatakrishna Shyamala, PhD
• Document writer - Molecular Diagnostics
Clinical & Laboratory Standards Institute
Reviewer , Consensus Committee on Molecular Methods
Contributor:
MM06: Quantitative Molecular Methods for Infectious Diseases
MM19: Establishing Molecular Testing in Clinical Laboratories
MM01-A3: Diagnostic Molecular Methods for Genetic Diseases
MM09: Nucleic acid sequencing methods in Diagnostic
Laboratory Medicine
MM22: Microarrays for Diagnosis and Monitoring of Infectious
Diseases
POCT14: Point-of-Care Testing for Infectious Diseases
V. Shyamala 3
•

4. Molecular Methods and Platforms for Infectious Diseases Testing
A review of FDA Approved and Cleared Assays*
R. Emmadi, J. B. Boonyaratanakornkit, R. Selvarangan, V. Shyamala,
B. L. Zimmer, L. Williams, B. Bryant, T. Schutzbank, M. M.
Schoonmaker, J. A. AmosWilson, L. Hall, P. Pancholi and K. Bernard
J. Mol. Diagnostics 13, Nov. (2011)
•The performance of various assays as described in public forums,
Product Inserts and publications are reviewed
•The challenges, limitations, testing and indications related to
implementation of various assays in a clinical laboratory setting is
summarized
•The categories of diseases include Sexually Transmitted Diseases -
HPV, CT-GC, HIV-1, and HSV; Hospital Acquired Infections - MRSA,
VRE, C. difficile, Respiratory tract and CNS infections, MTB, and CNS
viral infections, other infections such as HCV, HBV, GBS, Culture tests
for fungal and bacterial identification.
*A by-product of CLSI MM19 document
V. Shyamala 4

5. Accomplishments (Selected)
Venkatakrishna Shyamala, PhD
•Consultant: Novartis Dx- Scientific Affairs;
Document writer - Molecular Diagnostics
•Innovative Biosensors, Inc.
•Senior VP, Research and Development
• Digene Corporation
•Director, Research and Development
• Chiron Corporation
•Associate Director, Research
• Univ of California, Berkeley
•Visiting Scientist
V. Shyamala 5

6. Innovative Biosensors, Inc.,
http://www.innovativebiosensors.com/VP%20R&D_July2007.pdf
Management Team
IBI's management has extensive experience in the
commercialization of innovative technologies in the
pharmaceutical, research and diagnostic markets.
V. Shyamala, Ph.D. — Senior Vice President of Research
and Development
Dr. Venkatakrishna Shyamala has extensive experience in
commercializing NAT technologies, primer/probe designs and assay
development, cDNA library screening, molecular cloning, sequencing
and expression. She has also participated in IND and BLA
preparations and submissions with broad project and people
management experience. Dr. Shyamala has been cited as inventor on
a dozen patents and published nearly fifty peer reviewed articles in
leading industry journals. She has a PhD in Biochemistry and
Molecular Biology.
V. Shyamala 6

7. V. Shyamala
Senior Vice President Research and Development
Accomplishments
http://www.innovativebiosensors.
com/pressreleases_pathogen_detection.html
•Innovative Biosensors Inc. Expands Scientific Advisory Board —
August 5, 2008
•Innovative Biosensors Inc. Raises $11.5 Million — Series B
Financing Combines Equity and Debt Capital — May 19, 2008
•Innovative Biosensors Inc. and ATCC® Partner to Develop High-
speed Test for Avian Flu — April 9, 2008
•Innovative Biosensors, Inc. and the University of Maryland
Receive Funding for Handheld Biosensor Development —
February 25, 2008
V. Shyamala 7

8. V. Shyamala
Senior Vice President Research and Development
Accomplishments
•NSF Grant Reviewer – Ohio State Interdisciplinary Grant —
October 2007
•AACC Oakridge Conference poster — A Rapid, Sensitive,
Specific Assay for the Detection of Chlamydia trachomatis By F.
Benahmed, J. Simpson, N. Chakraborty, I. Mielzynska, K. Modarress,
T. Hazel and V. Shyamala — April 2008
•Completion of MRSA CANARYTM Assay Feasibility — Feb 2008
•Pre-IDE Presentation — April 2008
•Alpha Clinical trials — Univ MD Shock Trauma Center; Indiana
University — July 2008
•Three Provisional Patents — Feb - April 2008
V. Shyamala 8

9. Accomplishments (Selected)
Venkatakrishna Shyamala, PhD
•Consultant: Novartis Dx- Scientific Affairs;
Document writer - Molecular Diagnostics
•Innovative Biosensors, Inc.
•Senior VP, Research and Development
• Digene Corporation
•Director, Research and Development
• Chiron Corporation
•Associate Director, Research
• Univ of California, Berkeley
•Visiting Scientist
V. Shyamala 9

10. V. Shyamala
Director, Research and Development
Accomplishments
•Evaluate comparator assays for Chlamydia trachomatis of Roche
Amplicor, Gen-Probe Aptima, Abbott m2000, with in-house HC2,
helicase-isothermal, whole genome amplification and real-time
PCR technologies
•Optimize nucleic acid extraction with silica from large sample
volume for increased Diagnostic Sensitivity
•Five Invention Disclosures — Dec 2007 - July 2008
V. Shyamala 10

11. Accomplishments (Selected)
Venkatakrishna Shyamala, PhD
•Consultant: Novartis Dx- Scientific Affairs;
Document writer - Molecular Diagnostics
•Innovative Biosensors, Inc.
•Senior VP, Research and Development
• Digene Corporation
•Director, Research and Development
• Chiron Corporation
•Associate Director, Research
• Univ of California, Berkeley
•Visiting Scientist
V. Shyamala 11

12. Alternative NAT HCV assay- Development, Validation , Implementation
Screen for Procleix ® HIV-HCV Clinical positives
S. Nguyen, P. Arcangel, D. Madriaga, David Chien, V. Shyamala, B.
Phelps, BSRI members
Chiron Corporation, Blood Systems Research Institute
•Development of an Alternative HCV Assay. June 2000
•Alternate Technology: Target amplification by PCR
•Nucleic acid isolation: Organon-Teknika NucliSens reagent
•Amp-Det: Roche COBAS Amplicor
•Validation July-2000
•BSRI- reference lab Aug-Sep 2000
•Timeline BLA filing Oct 2000
•FDA Approval Feb 2002
V. Shyamala 12

13. Conference presentations: Hepatitis C virus RNA assay
VIIth European congress of the ISBT meeting, 2001,15th-18th July, Paris, France
1. V. Shyamala, D. Chien, S. Nguyen, N. Lagwinski, D. Madriaga, P. Carmichael,
B. Phelps. J. Heitman, D. Hirschkorn, L. Tobler, and M. Busch. Development
and Evaluation of Alternative NAT assay: a highly sensitive RT-PCR based
diagnostic assay for HCV RNA.
54th Annual AABB meeting, 2001, 13th-17th Oct. San Antonio, TX.
2. L. H. Tobler, J. M. Vargo, K. M. Smith, D. Hirschkorn, J. Heitman, C. Degula, V.
Shyamala, D. Chien, B. Phelps, L. Mimms, M.P.Busch. Sensitivity and Specificity
of an HCV supplemental NAT assay. Transfusion, 41, 83.
AACC meeting, 2000, 16th-18th Nov, Anaheim, CA.
3. Shyamala, D. Madriaga, D. Chien and B. Phelps. A highly sensitive,
Transcription mediated amplification (TMA) and Polymerase chain reaction (PCR)
dual amplification assay for the detection of Hepatitis C viral RNA molecules.
V. Shyamala 13

14. Alternative NAT WNV assay- Development, Validation , Implementation
Screen for Procleix ® WNV Clinical positives
D. Madriaga, J. Cottrell, P. Arcangel, David Chien, V. Shyamala, B.
Phelps, BRTL members
Chiron Corporation, Bayer Research and Testing Labs
•Development of an Alternative WNV Assay Nov 2002 – Feb 2003
•Alternate Technology: Target amplification by PCR
•Nucleic acid isolation: Specific target capture technology
•Amp-Det: PCR- TaqMan technology
•Validation March 2003
•BRTL- Implement April 2003
•Timeline IND filing May 2003
•Blood Screening 2003-2004 WNV season
V. Shyamala 14

15. Analytical and Clinical sensitivity of West Nile virus RNA screening
and Confirmatory assays
M Busch, L Tobler, J Saldahna, S Caglioti, V Shyamala, J Linnen, J
Gallarda, B Phelps, R Smith, S Kleinman
Blood Systems Research Institute , the Canadian blood Services, Blood
Systems Laboratory, Chiron Corporation, Gen-Probe, Inc., Roche Molecular
Systems, the National Genetics Institute, the National Microbiology Lab-
Canada, Univ of British Columbia
Transfusion 45, 492 - 499 (2005)
•Coded samples, the First generation Chiron WNV assay implemented at
Bayer Reference and Testing Labs had a 95% LoD of 33 Cps/mL and the
improved Chiron assay had a 95% LoD of 6.4 Cps/mL
LoD Copies/mL Neat Minipools
Assay G-P Roche NGI Bayer G-P Chiron G-P G-P Roche
(Chiron) 1:4 1:16 1:6
Study A B C D E F G H I
Code
95% LoD 15 125 26 33 6.4 6.4 55 184 1336
50% LoD 3.4 29 6.1 7.7 1.5 1.5 13 43 309
V. Shyamala 15

16. Detection of West Nile Virus RNA and antibody in frozen plasma
components from a voluntary market withdrawal during the 2002 peak
epidemic
L Tobler, C Bianco, S A Glynn, G B Schriber, B J Dille, H E Prince, R S
Lanciotti, J M Linnen, J Gallarda, V Shyamala, D Smith, S H
Kleinman, and M P Busch for the REDS study Group
Blood Systems Research Institute, America’s Blood Centers, Westat, Abbott
Laboratories, Focus Technologies, The Centers for Disease Control and
Prevention, , Blood, Gen-Probe, Inc., Roche Molecular Systems, Chiron
Corporation, UCSF
Transfusion 45, 480 - 486 (2005) F
o
•During the year 2002 in the WNV peak epidemic regions 60,000
plasma units were voluntarily withdrawn from the market
• Of this 1468 were retrospectively screened by immunoassays (Focus
Technologies, Abbott Labs) and RNA detection assays (Gen-Probe
Inc., and Roche Molecular Systems)
•RNA screening yielded one positive sample that was negative by the
immunoassays
•RNA quantitation by Target-capture RT PCR (Chiron Corporation)
suggested 440 cps/mL in the positive unit
V. Shyamala 16

17. Screening the Blood supply for West Nile virus RNA by Nucleic Acid
Amplification testing
M Busch, S Caglioti, G Robertson, J McAuley, L Tobler, H Kamel, J
Linnen, V Shyamala, P Tomasulo, S Kleinman
Blood Systems Research Institute, Dept. Of Laboratory Medicine, Blood
Systems Laboratory, Blood Systems, Gen-Probe, Inc., Chiron Corporation,
Univ of British Columbia
New Eng J Med 353, 460-467 (2005)
ms Foundation
•For the early part of 2003 WNV RNA screening assay was
performed by minipool (16 samples) testing
•For the regions with highest reactivity in minipool screening,
retrospective individual screening was performed
•Individual unit testing yielded additional positives that was IgM-
negative
•Recommend Staged Minipool to Individual testing during the year
V. Shyamala 17

18. Conference presentations: West Nile virus RNA assay
58th Annual AABB meeting, 2005, Seattle, WA
1. J. Cline, C. Deza, R. Cory, A. Garcia, M. Lewis, A. Broulik, M. Deras, V. Shyamala, S. Pichuantes,
C. Giachetti, J. M. Linnen. Stability of WNV Viral RNA from tissue culture and blood donor samples in
stored blood. Transfusion 45, 149.
2. L. H. Tobler, V. Shyamala, J. Saldhana, C. Cameron, R. Lanciotti, R. Smith, I. Walsh, B. Munneke,
B. H. Phelps, D. Chien, M. P. Busch. West Nile virus (WNV) viral load comparison study. Transfusion
45, 151.
3. S. Nguyen, V. Shyamala, H. Huang, D. Madriaga, M. Badgett, J. Hedges, C. WalkerPeach, D.
Chien, B. Phelps, S. Pichuantes. Cloning of West Nile virus internal control and nucleotide fragments
spanning the full-length viral genome for production of stable RNA standards. Transfusion 45, 151.
2005 National conference on West Nile Virus, San Jose, CA
4. . S. Cagliotti, G. F. Robertson, J. McAuley, S. H. Kleinman, L. H. Tobler, H. Kamel, J. M. Linnen, V.
Shyamala, P. A. Tomasulo, M. P. Busch. Screening the blood supply for West Nile Virus RNA by
nucleic acid amplification testing.
57th Annual AABB meeting, 2004, Baltimore, MD
5. V. Shyamala, D. Madriaga, S. Pichuantes, B. Jaitner, D. Chien and B. Phelps. Performance
characteristics of the Validated and Improved qualitative and quantitative Target-Capture PCR WNV
NAT assays. Transfusion 44, 140.
6. M. Busch, L. Tobler, J. McAuley, J. Linnen, V. Shyamala, G. Robertson, D. Wright, S. Kleinman, S.
Cagliotti. West Nile Virus RNA dynamics and antibody evolution based on follow-up of viremic blood
donors. Transfusion 44, 2.
7. J. Cline, M. Lewis, W. Wu, S. Miller, A. Broulik, J. Savage, V. Shyamala, M. Cass, C. Giachetti, J.M.
Linnen. Gen-Probe Alternative WNV assay: A TMA-based confirmatory assay for West Nile Virus.
Transfusion 44, 138.
V. Shyamala 18

19. Conference presentations: West Nile virus RNA assay (Cont.)
28th congress of the ISBT meeting, 2004, Edinburgh, UK.
8. V. Shyamala, S. Pichunates, B. Jaitner, D. Madriaga, P. Arcangel, J. Cottrell, S. Nguyen, H.
Huang, A. Medina-Selby, D. Coit, D. Chien B. Phelps. Performance characteristics of the qualitative
and quantitative Target-Capture PCR WNV NAT assay. Vox Sanguinis, 87, 26.
9. L. H. Tobler, H. Prince, G. Hafner, B. Dille, R. A. Gutierrez, W. Andrews, C. Harrington, V.
Shyamala, J. McAuley, V. Winkelman, S. Cagliotti, M. P. Busch. Relative performance of four West
Nile Virus antibody assays in viremic blood donor specimens. Vox Sanguinis, 87, 65.
56th Annual AABB meeting, 2003, San Diego, CA
10. V. Shyamala, S. Pichuantes, B. Jaitner, D. Madriaga, P. Arcangel, J. Cottrell, S. Nguyen, H.
Huang, A. Medina-Selby, D. Coit, C. McCoin, D. Chien, B. Phelps. Detection and Quantitation of West
Nile Virus RNA by the Alternative NAT WNV Assay. Transfusion. 43, 128.
11. B. Jaitner, V. Shyamala, S. Nguyen, H. Huang, Y-L Fong, D. Chien, B. Phelps, S. Pichuantes.
Propagation, quantitation, and inactivation of West Nile Virus to support nucleic acid and IgM assay
development. Transfusion. 43, 128.
12. V. Shyamala, P. Arcangel, D. Madriaga, J. Cottrell, J. Linnen, D. Chien, B. Phelps. Compatibility
of ProcleixR- West Nile Virus (WNV) assay in various anticoagulants. Transfusion. 43, 129.
10th EPFA/NIBSC workshop & SOGAT meeting, 2003, Langen, Germany
13. V. Shyamala, S. Pichuantes, B. Jaitner, D. Madriaga, P. Arcangel, J. Cottrell, S. Nguyen, H.
Huang, A. Medina-Selby, D. Coit, C. McCoin, D. Chien, B. Phelps. Use of quantitative NAT assay to
correlate West Nile Virus titration bioassay (pfu/ml) with genomic copy numbers (geq/mL).
V. Shyamala 19

20. Alternative NAT HBV assay- Development, Validation , Implementation
Screen for Procleix ® Ultrio Clinical positives
J. Cottrell, P. Arcangel, D. Madriaga, David Chien, V. Shyamala, B.
Phelps, BRTL members
Chiron Corporation, Bayer Research and Testing Labs
Development of Target-Capture PCR HBV DNA Alternative
Assay for ProcleixR Ultrio Clinical Trials 2001
Technology: Target-Capture PCR
Nucleic acid isolation: Specific Target-Capture
Amplification-Detection: TaqMan assay
Agreement with BRTL as the Reference lab Aug-Sep 2002
BLA filing Oct 2004
FDA Approval 2006-2008
Used to Support TUV submission for Ultrio, Tigris-Ultrio,
RAS, FEP
V. Shyamala 20

21. Assessment of the Target-Capture PCR Hepatitis B Virus (HBV) DNA
Quantitative Assay and Comparison with Commercial HBV DNA
Quantitative Assays
V. Shyamala, P. Arcangel, J. Cottrell, D. Coit, A. Medina-Selby, C.
McCoin, D. Madriaga, D. Chien and B. Phelps
Chiron Corporation
J. Clin. Microbiol. 42, 5199 - 5204 (2004)
•The performance of the Target-Capture HBV assay was
demonstrated with Reference panels and Standards
•The range of Quantitation was 10-50 IU/mL
•The accuracy of quantitation of several concentrations of serially
diluted WHO standard was between 100-142% and of the QCMD 2003
six member panel was in the 74-140% range
•The comparative commercial assays included Roche Amplicor,
National Genetics Institute SuperQuant, Bayer Quantiplex version 2.0,
and Digene Hybrid Capture assay
•The Target-Capture HBV assay was more sensitive, accurate, high-
throughput, rapid, and reproducible.
V. Shyamala 21

22. ProcleixTM Ultrio Registration and Market Trials
V. Shyamala

23. External quality assessment for the detection of blood-borne viruses
in plasma by nucleic acid amplification technology: the first human
immunodeficiency virus and hepatitis B virus studies (HIV EQA/1 and
HBV EQA/1) and the fifth hepatitis C virus study (HCV EQA/5).
G. Pisani, K. Cristiano, J. Saldanha, M. Wirz, G. M. Bisso, C. Mele, G.
Gentili and the EQA Participants.
Department of Infectious, parasitic and immune-mediated Diseases, Rome,
Italy, Canadian Blood services, Ottawa, Canada
Vox Sanguinis 87, 91- 95 (2004)
• Sixteen laboratories received HBV EQA/1 coded panel of two HBV
concentrations
• All qualitative assays detected both members
•The Chiron Target-Capture PCR assay (Lab 46) assigned a mean
titer of 1506 IU/mL and 104 IU/mL to the 1000 IU/mL and 100 IU/mL
samples respectively.
V. Shyamala 23

24. The risk of hepatitis B virus infection by transfusion in Kumasi, Ghana
J.-P. Allain, D. Candotti, K. Soldan, F. Sarkodie, B. Phelps, C.
Giachetti, V. Shyamala, F. Yeboah, M. Anokwa, S. Owusu-Ofori, and
O. Opare-Sem
Division of Transfusion Medicine, Cambridge, UK, Departments of Medicine
and Biochemistry, Komfo Anokye Teaching Hospital, Kumasi, Ghana, Chiron
Corporation, Gen-Probe, Inc.
Blood 101, 2419-2425 (2003)
• In Africa more than 50% of blood donors and recipients are HBV
positive through natural exposure
•There are currently no HBV screening programs
•Relative merits of various antigen screening methods such as Particle
agglutination, dipstick, and EIA assays were compared
•The risk of HBV transmission was predicted by screening HBsAg
negative donors and a group of potential blood recipients for HBV DNA
(0.05% DNA positivity)
•The risk of transmission for <10 years old ranged between 1:11 and
1:326 for unscreened vs.EIA screened. This risk decreased four fold
in adults due to natural exposure to HBV.
V. Shyamala 24
•

25. Conference presentations: Hepatitis B virus DNA assay
59th Annual AABB meeting, 2006, Miami Beach, FL
1. Y.-L. Fong, D. Madriaga, V. Shyamala, J. Cottrell, R. Lewis, L. Eudey, G. Crutcher, N. Lelie, A.
Heaton, B. Phelps, D. Chien. Evaluation of Analytical Sensitivity of Chiron Target Capture HBV DNA
Assay for HBV Detection, and Comparison with NGI SuperQuantTM HBV DNA Assay. Transfusion 46,
96.
57th Annual AABB meeting, 2004, Baltimore, MD
2. V. Shyamala, P. Arcangel, J. Cottrell, D. Coit, A. Medina-Selby, C. McCoin, D. Chien, B.Phelps.
Performance characteristics of the qualitative and quantitative Target-Capture PCR HBV NAT assay.
Transfusion 44, 85.
56th Annual AABB meeting, 2003, San Diego, CA
3. V. Shyamala, J.Cottrell, P. Archangel, D. Coit, A. Medina-Selby, C. McCoin, J. Turczyn, D. Chien and
B. Phelps. Validation Of Alternative NAT HBV Assay: A Highly Sensitive PCR Based Assay For HBV
DNA. Transfusion. 43, 125.
27th congress of the ISBT meeting, 2002, Vancouver, Canada.
4. V. Shyamala, P. Arcangel, J. Cottrell, J. Linnen, C. Giachetti, D. Chien, B. Phelps. Performance
characteristics of hepatitis B virus DNA confirmatory assay for ProcleixR triplex assay. Vox Sanguinis,
83, 183.
5. J. Linnen, A. Umali, A. Broulik, D. Kolk, J. Dockter, S. McDonough, V. Shyamala, J. Cottrell, P.
Arcangel, L. Mimms and C. Giachetti. Effect of donor mini-pool size on closure of the HBV detection
window: A comparison of Triplex TMA to surface antigen detection. Vox Sanguinis, 83, 42.
54th Annual AABB meeting, 2001, San Antonio, TX.
6. J. Linnen, M. Ho-Sing-Lloy, M. Miyano, D. Kolk, A. Menez, A. Vaughn, E. Peterson, V. Shyamala, P.
Arcangel, D. Chien, B. Phelps. Performance of the TMA Triplex Assay which simultaneously detects
HIV-1, HCV and HBV nucleic acid. Transfusion, 41, 82.
V. Shyamala 25

26. Prevalence and quantitation of Parvovirus B19 DNA levels in blood
donors using a sensitive PCR screening assay
S. Kleinman, S. Glynn, T.-H. Lee, L. Tobler, L. Montalvo, D. Todd, J.
Kiss, V. Shyamala, M. Busch
Westat, Blood Systems Research Institute, The Institute for Transfusion
Medicine, Chiron Corporation; National Heart, Lung, and Blood Institute
Retrovirus Epidemiology Donor Study (REDS-II)
Transfusion 47, 1756- 1764 (2007)
•A retrospective study was conducted with 5020 plasma samples for
Parvo B19 DNA detection by Target Capture-PCR assay
•The Assay performance was 50% LoD at 1.6 IU/mL and 95% LoD at
16.5 IU/mL
•B19 DNA prevalence was 0.88 percent with 40 positives
•IgM positivity was associated with high DNA levels (median concn
105 IU/mL) indicating acute resolving infection
•IgG but not IgM positivity is indicative of chronic and persistent phase
of B19 infection
V. Shyamala 26

27. Characterization of the terminal regions of hepatitis C viral RNA:
Identification of conserved sequences in the 5' untranslated region
and poly(A) tails at the 3' end
J. H. Han, V. Shyamala, K. H. Richman, M. J. Brauer, B.
Irvine, M. S. Urdea, P. Tekamp-Olson, G. Kuo, Q.-L. Choo, and M.
Houghton
Chiron Corporation
Proc. Natl. Acad. Sci. USA 88, 1711-1715 (1991)
• The nucleotide sequence at the 5' and 3' termini of the hepatitis C
virus (HCV) genome has been determined, with the sequence in the 5'
untranslated region highly conserved among geographical isolates
•There are several features indicating relatedness of HCV to pestivirus
but not to other flavi viruses such as, (a) blocks of 5’ nucleotide
sequence and position of short open reading frames, (b) poly(A) tails
present on 3' subgenomic RNAs, (c) RNAs truncated at the 5’ and 3’
end.
•However, HCV also appears to be substantially different from pesti
virus with assignment to a separate viral genus.
V. Shyamala 27

28. Receptor recognition and specificity of interleukin-8 is determined by
residues that cluster near a surface-accessible hydrophobic pocket
M. E. Wernette-Hammond, V. Shyamala, M. A. Siani, C. A. Gallegos,,
P. H. Feucht, J. Abbott, G. Reza-Lapointe, M. Moghadam, H. Khoja, J.
Zakel, and P. Tekamp-Olson
Chiron Corporation
J. Biol. Chem. 271, 8228-8235 (1996)
•Chemokine IL8 (C-C) binds both R1 and R2 receptors and gro
gamma (CXC) binds only R2 receptor. Chimeric C-C and CXC ligands
were used to determine the specificity and affinity of binding to
recombinant R1 and R2 cell lines
•Substitution into C-C of CXC aa at the 1st beta sheet reduced binding
to both R1 and R2, and of 3rd beta sheet reduced binding to R1 but not
R2, with no effect of second beta sheet. Substitution into CXC of C-C
aa at the second beta sheet conferred high affinity binding to both R1
and R2, with no effect of 1st and 3rd beta sheets
•Individual aa substitutions were made and the results explained
through a homology model suggests that a hydrophobic pocket is
essential for both R1 and R2 binding, while surrounding residues play
an additional role for R1 binding.
V. Shyamala 28

29. Interleukin-8 receptors R1 and R2 activate mitogen-activated protein
kinases and induce c-fos, independent of Ras and Raf-1 in Chinese
hamster ovary cells
V. Shyamala, and H. Khoja
Chiron Corporation
Biochemistry 37, 15918-15924 (1998)
• Biological effects of interleukin-8 (IL-8) are realized by binding to the
two seven-transmembrane receptors IL-8 R1 and IL-8 R2.
• IL-8 R1 and R2 have been shown to interact with Galphai2 and
Galpha16, with activation of several mitogen-activated protein kinases
•In CHO cells stably expressing either IL-8 R1 or R2 receptors results
demonstrate that: (a) IL-8 activates ERK and ERK kinases (MEK)
through R1. Both IL-8 and GROalpha activate ERK and MEK through
R2, whereas MIP-1alpha, a beta chemokine, does not activate these
kinases through either of these receptors. (b) ERK activation is
inhibited by pertussis toxin and MEK1 inhibitor. (c) ERK activation is
independent of the upstream mediators Ras and Raf-1. (d) The
downstream effects of ERK activation result in increase of c-fos mRNA
through both R1 and R2 receptors.
V. Shyamala 29

30. High-throughput screening for ligand-induced c-fos mRNA
expression by branched DNA assay in chinese hamster ovary cells
V. Shyamala, H. Khoja, M. L. Anderson, J.-X. Wang, H. Cen, and W.
M. Kavanaugh
Chiron Corporation
Anal. Biochem. 266, 140-147 (1999)
• Cell based High-throughput screening requires use of standardized
cell lines for universal signal read-outs for use with a variety of targets.
• The screening assay for receptor agonists and antagonists is in
Chinese Hamster Ovary (CHO) cells overexpressing the relevant
receptors.
•A universal signal readout is of endogenous c-fos mRNA which
responds to a wide spectrum of stimuli.
•The signal readout was amplified with branched chain DNA (bDNA)
assay which is highly sensitive, quantifiable, amenable to high-
throughput analysis, and easy to execute.
•The combined benefit of the above three features was proven with
CHO cells overexpressing insulin receptor to compare conventional
signaling assays with the high-throughput c-fos branched DNA assay.
V. Shyamala 30

31. Tumor Necrosis factor alpha induced activation of c-jun N-terminal
kinase is mediated by TRAF2
C. Reinhard, B. Shamoon, V. Shyamala, and L. T. Williams
Chiron Corporation
EMBO J. 16,1080-1092 (1997)
• Tumor necrosis factor alpha (TNF alpha) a pro-inflammatory cytokine is
an endogenous mediator of septic shock, inflammation, anti-viral
responses and apoptotic cell death through 55 kDa (TNF-RI) and 75 kDa
(TNF-RII) receptors.
•TNF-RII specific signaling was examined by chimeric receptor with
extracellular domain mouse CD4 antigen and intracellular domain TNF-
RII, and activated it through anti-CD4 antibodies
•Results show that: (i) TNF-RII activates ERK and JNK; (ii)
Overexpression of TRAF2, a molecule that binds TNF-RII activates JNK ;
(iii) dominant-negative TRAF2 blocks JNK activation ; (iv) TRAF2 signals
activation of JNK and NF-kappaB through different pathways.
•TNF alpha-mediated JNK activation in fibroblasts is independent of the
cell death pathway.
V. Shyamala 31
.

32. Cloning of CCRL1, an orphan seven transmembrane receptor related
to chemokine receptors, expressed abundantly in the heart
H. Khoja, G. Wang, C. T. Ng, J.Tucker, T. Brown, V. Shyamala
Chiron Corporation
Gene 246, 229-238 (2000)B
Involved in stroke
• To identify novel chemokine receptor genes, cDNA expressed
sequence tags (EST) were analyzed for a significant homology with
mammalian chemokine receptors.
•One EST clones sequence was used to generate a full-length cDNA
encoding a putative seven transmembrane receptor, CCRL1-CC
chemokine receptor like 1, encoding a polypeptide of 350 amino acids
with 35% homology to the chemokine receptors CCR6 and CCR7.
Coupled transcription-translation of CCRL1 cDNA yielded a
glycosylated polypeptide of about 45kDa.
•Northern blot analysis indicates predominant expression of about 5.0,
2.0 and 1.3kb mRNA forms in human heart tissue. In-situ hybridization
confirmed the presence of CCRL1 mRNA in cardiac muscle cells.
•CCRL1 maps to chromosome 6 and has one intron in the 5'
untranslated region. V. Shyamala 32

33. Semirational design of a potent, artificial agonist of fibroblast growth
factor receptors
M.D. Ballinger, V. Shyamala, L. D. Forrest, M. Deuter-Reinhard, L. V.
Doyle, J. X.Wang, L. Panganiban-Lustan, J. R. Stratton, G. Apell, J. A.
Winter, M. V. Doyle, S. Rosenberg, W.M. Kavanaugh
Chiron Corporation
Nature Biotechnology 17, 1199 - 1204 (1999)B
Involved in stroke
• A 26 amino acid polypepetide with FGF receptor binding activity
unrelated to any known FGF was identified through phage display.
•A heparin binding, and dimerizaion enabling domain of c-jun leucine
zipper was tailored onto this peptide
•Such a synthetic polypeptide reproduced the intracellular kinase
cascade, and mitogenic and morphogenic properties of bFGF with
similar potency
•The synthetic peptide also reproduced corneal vascularization
properties of FGF
•Artificial FGFR peptide and non-peptide agonists may be useful
alternatives to FGF in the treatment of ischemic vascular disease.
V. Shyamala 33

34. Accomplishments (Selected)
Venkatakrishna Shyamala, PhD
•Consultant: Novartis Dx- Scientific Affairs;
Document writer - Molecular Diagnostics
•Innovative Biosensors, Inc.
•Senior VP, Research and Development
• Digene Corporation
•Director, Research and Development
• Chiron Corporation
•Associate Director, Research
• Univ of California, Berkeley
•Visiting Scientist
V. Shyamala 34

35. Amplification of bacterial genomic DNA by the polymerase chain
reaction and direct sequencing after asymmetric amplification:
application to the study of periplasmic permeases
V. Shyamala, and G. F.-L. Ames
Dept. of Biochemistry, UC Berkeley, CA
J. Bacteriol. 171, 1602-1608, (1989)
•The polymerase chain reaction (PCR) was used to amplify bacterial
genomic DNA and PCR amplicons of 4,400 base pairs obtained
•We discuss problems inherent in the direct sequencing of the
amplified product, and solved the problems by developing an
"asymmetric amplification" method in which one of the oligonucleotide
primers is used in limiting amounts, thus allowing the accumulation of
single-stranded copies of only one of the DNA strands.
•As an illustration of the use of PCR in bacteria, we have amplified,
sequenced, and subcloned several DNA fragments carrying mutations
in genes of the histidine permease operon. These mutations are part of
a preliminary approach to studying protein-protein interactions in
transport, and their nature is discussed.
V. Shyamala 35

36. Genome walking by single-specific-primer polymerase chain
reaction: SSP-PCR
V. Shyamala, and G. F. L. Ames
Division of Biochemistry and Molecular Biology, UC Berkeley, CA
Gene 84, 1-8, (1989)
•We have extended the use of the polymerase chain reaction (PCR) to
amplify double-stranded DNA when sequence information is available
only at one extremity sufficient to design a gene-specific primer.
• The unknown end is ligated to a vector and the gene-specific primer
is used in combination with a second generic vector primer.
•Restriction, ligation, amplification and sequencing of the products can
be achieved within three days.
•This method eliminates the laborious steps of shotgun cloning, colony
screening and culturing of cells.
•We demonstrate the usefulness of this technique for chromosome
walking in the absence of any restriction data.
V. Shyamala 36

37. Tandem chromosomal duplications: role of REP sequences in the
recombination event at the join-point
V. Shyamala, E. Schneider and G. F. L. Ames
Division of Biochemistry and Molecular Biology, UC Berkeley, CA
EMBO J. 9, 939 - 946 (1990)
•A family of prokaryotic repetitive sequences, called REP (repetitive
extragenic palindromic) is involved in the formation of chromosomal
rearrangements such as duplications.
•Here through SSP-PCR we have characterized the join-points of
seven RecA+ tandem duplications in Salmonella typhimurium, that
fuse the hisD gene to distant foreign promoters
• Such a recombination takes place even in a RecA-background. Thus,
REPs can recombine with each other by a RecA(-)-independent
mechanism
•Some RecA-duplications occurred outside of REP sequences by
recombination within a 7 bp homology.
•Possible roles for the known interaction between DNA gyrase and
REP in chromosomal rearrangements are discussed.
V. Shyamala 37
Blood

38. Genome walking by Single-Specific Primer Polymerase Chain Reaction
V. Shyamala, and G. F.-L. Ames
Division of Biochemistry and Molecular Biology, UC Berkeley, CA
Methods in Enzymology 217, Part H, 436 – 446 (1993) Ed. R. Wu B
ood
V. Shyamala 38

39. Use of exonuclease for rapid polymerase-chain-reaction-based
in vitro mutagenesis
V. Shyamala, and G. F.-L. Ames
Division of Biochemistry and Molecular Biology, UC Berkeley, CA
Gene 97, 1-6, (1991)
• In a previous publication we proposed Asymmetric Amplification as a
method to preferentially amplify one of the two PCR strands to
facilitate direct sequencing
•Here the extended application of Asymmetric amplification for in vitro
mutagenesis has been demonstrated
•We also demonstrate a second method for in vitro mutagenesis
following treatment of PCR fragments with lambda exonuclease. This
requires kinasing one of the primers.
•The entire procedure of kinasing the primer, amplification by PCR,
Exo lambda digestion and second step of PCR can be performed in
less than 6 h. to generate a number of mutations in the S. typhimurium
hisP gene of the histidine transport operon.
V. Shyamala 39

40. Antimalarial activity of optical isomers of quinacrine dihydrochloride
against chloroquine-sensitive and -resistant plasmodium falciparum
in vitro
R. V. Webster, J. C. Craig, V. Shyamala, G. C. Kirby, and D.
C. Warhurst
Dept. of Pharmacology, UCSF, Pacific Presbyterian Hospital
Biochem. Pharmacol. 42, S225-S227, (1991)
• Both enantiomers of quinacrine and the racemic form of the drug
showed equal activity in vitro against chloroquine-sensitive and -
resistant strains of Plasmodium falciparum, without detectable
stereoselectivity.
•This contrasts with observations on chloroquine, where a similar lack
of stereoselectivity in vitro is accompanied by a 10-fold loss of activity
against the resistant strain.
•The reported in vivo differences for the enantiomers of chloroquine
and the observations on the optically active metabolites of chloroquine
and quinacrine may be ascribed to a difference in the
pharmacokinetics of their enantiomers.
V. Shyamala 40