West Nile Virus: Pathogenesis and Diagnostic Tools

Maria Rios, PhD - Senior Staff FellowLaboratory 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 documentedFDA & 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 WNVStudies 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-10-02 (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

101

102

103

104

105

WN

V R

NA

(co

pies

per

mL

)

Days post infectious mosquito bite Busch, MP

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

RNA

IgM

IgG

~7 days MP-NAT(50% LoD = ~80 copies/mL)

ID-NAT(50% LoD = ~ 5 copies/mL)

Stage-IIID NAT+MP NAT-

IgM-

Stage-IVID NAT+MP NAT+

IgM+

Stage-VID NAT + MP NAT-

IgM+/IgG+

Stage-IID NAT+/-MP NAT-

IgM-

Stage-IIIMP NAT+

IgM-

I II III IV 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