West Nile Virus: Pathogenesis and Diagnostic Tools Maria Rios, PhD - Senior Staff Fellow Laboratory of Molecular Virology DETTD/OBRR/CBER/FDA WNV epidemic in the US Since 1999 has reoccurred for 6 years Over 16,000 human cases and over 600 deaths Transmission by blood transfusion documented FDA & CDC action – blood screening assays implemented under IND within 8 months

Research Plans Panel development Studies on genetic evolution of WNV: Viral isolation and genetic characterization Viral Infectivity & Pathogenesis: Cellular tropism including partition of WNV in blood components - RBC; WBC; Platelets; Plasma Infectivity studies in animal model

WNV Panel Evaluation: Collaborative Studies Panel composed of 14 members including both NY99-FDA and FDA-Hu2002 strains 1000, 500, 100, 50, 10, 5 and 0 viral copies/mL (one concentration from each isolate) Distributed to seven independent laboratories Four laboratories performed quantitative assays Great variability in performance with low copy number panel members Qualitative assays performed better than quantitative assays Results

Studies on genetic evolution of WNV Viral mutation may affect assay performance, pathogenesis and impact in vaccine development Viral isolates were generated from 25 WNV- positive plasma samples identified by screening assays Virus propagation for up to 3 passages did not induce genetic change in the structural genes Viral genome of two isolates were fully characterized: FDA-Hu2002 (AY646354) and ARC (AY795965) The structural region of the other 23 isolates is fully sequenced

Future Plans on Genetic Evolution of WNV Continue studies in genetic evolution of WNV over time in the US We have a repository of over 300 WNV-positive plasma samples acquired in 2002, 2003 and 2004 Sequence studies – expand to samples obtained from patients Studied samples to date are biased because they were selected by blood screening assays Non-biased samples will allow to investigate whether there are isolates that could be missed by screening assays Microarray – use to expedite WNV genotype determination and classification

WNV Infectivity & Pathogenesis Cellular Tropism Cellular tropism of WNV infection following blood transfusion and the human blood cells that sustain viral replication are unknown Investigate the susceptibility of monocyte-derived macrophages (MDM) to WNV infection and replication in vitro system WNV tropism for other human cells PBMC, T-cells, B-cells and other cell lines Host factors (chemokines/cytokines) involved WNV pathogenesis

Summary MDM can be infected by WNV – supernatant testing demonstrated productive infection Viral replication persisted for up to 27 days - in one experiment up to 47 days Infected MDM showed no gross morphological changes (CPE negative) Positive supernatants have infectious particles The data suggest that human macrophages could play a role in initial viral replication following WNV transmission by transfusion

WNV Infectivity of non-Human Primates To define the minimal infectious dose in non-human primates and the infectivity of human plasma in presence and absence of antibodies WNV RNA (copies per mL) Days post infectious mosquito biteBusch, MP RNA IgM IgG ~7 days MP-NAT (50% LoD = ~80 copies/mL) ID-NAT (50% LoD = ~ 5 copies/mL) Stage-II ID NAT+ MP NAT- IgM- Stage-IV ID NAT+ MP NAT+ IgM+ Stage-V ID NAT + MP NAT- IgM+/IgG+ Stage-I ID NAT+/- MP NAT- IgM- Stage-III MP NAT+ IgM- IIIIIIIV V

Acknowledgments L. Kramer (NYSDH) R. Lanciotti (CDC) S. Stramer (ARC) S. Caglioti (BSL) K. Murthy (SPC) H. Alter (NIH) K. Chumakov (OVRR) V. Chizhikov D. Volokhov H. Nakhasi (DETTD) A. Grinev S. Daniel A. Dayton M. J. Zhang K. Srinivasan A. Machuca O. Wood S. Lee I. Hewlett