West Nile virus: Research challenges and opportunities presented from this emerging flavivirus

1. West Nile virus: Research challenges and opportunities presented from this emerging flavivirus ADED rounds, 10:00-11:00 PST, March 16, 2004 E RNA M Harvey Artsob, PhD Director, Zoonotic Diseases and Special Pathogens National Microbiology Laboratory, Health Canada

2. West Nile virus

3. Genus – Flaviviruses • - Approximately 70 viruses, half of which • are associated with human disease • Some are mosquito-transmitted, • some tick-transmitted and some have • no known arthropod host • - Includes: • Yellow Fever • Dengue • Japanese Encephalitis • Tick-borne Encephalitis • West Nile Family : Flaviviridae Spherical enveloped viruses with a (+)-strand RNA genome of 10-11Kb Genus - Pestiviruses Bovine Viral Diarrhea Genus – Hepaciviruses Hepatitis C

4. Members of the Japanese encephalitis virus serocomplex Cacipacore virus Japanese encephalitis virus Koutango virus Murray Valley encephalitis virus Alfuy virus St. Louis encephalitis virus Rocio Stratford Usutu virus West Nile encephalitis virus Kunjin virus Yaounde virus

5. E RNA M Flavivirus Genome ns4a NC ns2a NC -3' NS5 NS3 C prM E NS1 5'- ns4b ns2b furin pr + M 7 non-structural genes/proteins 3 structural Genes/proteins From: Roehig, Fourth National Conference on West Nile Virus in the United States 2003.

6. Egypt 1951 Phylogenetic Relationships among West Nile Viruses France 1965 South Africa Israel 1952 Romania 1996 Kenya 1998 Senegal1993 Morocco 1996 Lineage 1: India, Australia, Europe Russia, Middle East, North Africa, Africa (Senegal, C. African Republic Ivory Coast) Lineage 2: Africa (Central and Southern including Madagascar) Italy 1998 Volgograd 1999 New York 1999 Israel 1998-A NY2000 3282 NY2000 3356 NY 1999 equine LIN-1 NY 1999 hum Conn 1999 MD 2000 NJ 2000 Israel 1999 H C.Afr.Rep 1989 Senegal 1979 Algeria 1968 Lanciotti et al. 2002. Virology 298,96-105 C.Afr.Rep 1967 Iv.Coast 1981 Kunjin 1960 Kunjin 1973 North American strain has not changed significantly to date Davis et al 2003 ( abstract-ASTMH meeting) tested over 30 isolates from 6 states and 5 provinces collected in 2001&2002 and found an average nucleotide divergence of only 0.18% Ebel et al 2003 ( abstract-ASTMH meeting) showed isolates from NY state from 2000-2002 remained relatively genetically homogeneous with a mean genetic distance of less than 0.002.. However strains collected in 2002 were more genetically diverse. Kunjin 1984b Kunjin 1991 Kunjin 1984a Kunjin 1966 Kunjin 1994 India 1955a India1980 India 1958 India 1955b Kenya Uganda Senegal 1990 Uganda 1937 C.Afr.Rep 1972a LIN-2 C.Afr.Rep 1983 Uganda 1959 C.Afr.Rep 1972b Madagascar1988 Madagascar 1986 Madagascar 1978 JE SA 14

7. West Nile virus studies proposed or under consideration Genotyping of WNV isolates/specimens from divergent sources (human, mosquito, bird) and different regions across Canada; comparisons with isolates from the United States, Europe, and Africa Analysis of proteomic changes in WNV infected cells Virulence studies/host infection with multiple WNV strains

8. West Nile human infections

9. West Nile Virus Activity in the Continental US Year Human Horses States 1999 62 25 4 2000 21 63 12 2001 66 733 27 4156 14901 44 9377 4636 45

10. West Nile Virus Activity in Canada Year Human Horses Provinces 1999 1(travel) 0 0 2000 0 0 0 2001 0 0 1 416 * 356 5 1335 445 7 *Several hundred additional suspect cases were identified

11. Human infection with WNV in 2003 vs 2002 Province B.C. Alberta Saskatchewan Manitoba Ontario Quebec New Brunswick Nova Scotia Yukon 2003 20* 272 792 (6 deaths) 141 (2 deaths) 89 (2 deaths) 17 1* 2* 1* 2002 0 2* 0 0 400+ (17 deaths) 17 (3 deaths) 0 0 0 *not locally acquired, travel history

13. Patterns of WN activity in North America, 2002 versus 2003Geographic patterns of human cases – north centralLargest concentration of human cases in 2002 seen within the geographically contiguous areas of the United States north central area (Illinois, Michigan, Indiana, etc) and Ontario.Few cases in that geographic region in 2003 Why? Climatic factors? Build up of immunity? Education/avoidance? Larviciding?

14. Patterns of WN activity in North America, 2002 versus 2003Geographic patterns of human cases - westLargest concentration of human cases in 2003 seen within the western part North America where Culex tarsalis is the most important mosquito vector species (Colorado, Nebraska, South Dakota, Prairies). There is a significantly greater number of cases than in 2002 with many “hot spots” of human cases within these states/provinces. Why? Can human serosurveys help shed some light on the question?

15. Is the WNV strain introduced into North America more virulent than most WNV strains? Humans Emerging clinical syndromes: movement disorders, Parkinsonism, - Flaccid paralysis, Rhabdomyolysis Short and long term sequelae Ratio of severe to mild and/or inapparent illness (< 1:100?) Infections associated with: transplantation, transfusion, breastfeeding, transplacental transmission

16. WNV Human Infection “Iceberg” Revisited 1 CNS disease case = ~100 total infections ~10% fatal (<0.1% of total infections) <?% CNS disease Febrile illness alone less frequent? ~?% “West Nile Fever” ~80% Asymptomatic

17. Factors related to the West Nile virus amplification cycle

18. VECTOR ENVIRONMENT HOST VIRUS From: Turell, Fourth National Conference on West Nile Virus in the United States 2003.

20. Mosquito field isolates (PCR-positive pools) • Isolates from more than 40 different mosquito species in North America including 10 from Canada

21. Potential vectors of West Nile virus based on laboratory vector competence studies Efficient Moderate Inefficient Ae. albopictus Ae. aegypti Ps. ferox Cx. salinarius Ae. vexans Cx. tarsalis Cx. nigripalpus Cq. perturbans Oc. atropalpus Cx. pipiens Oc. canadensis Oc. japonicus Cx. quinquefasciatus Oc. cantator Cx. restuans Oc. sollicitans Oc. triseriatus Oc. taeniorhynchus From: Turell, Fourth National Conference on West Nile Virus in the United States 2003.

22. Bionomics of potential mosquito vectors • Host preference • Population density • Biting behavior • Longevity • Feeding time • Seasonallity From: Turell, Fourth National Conference on West Nile Virus in the United States 2003.

23. Interpretation of mosquito data in relation to human risk - relevance of infection rate in different species with regard to risk for human disease - importance of bridging vectors - what are the important bridging vectors? - efficacy of control measures

25. Birds as reservoir hosts • There is currently evidence for infection of over 200 species of birds . • The reservoir potential of these birds varies greatly and is dependent upon: • the competency (days and level of viremia) and • relative number of infections (amount of birds in a given area, amount of infected mosquitoes feeding on these birds) of each bird species

26. Birds as reservoir hosts 1. Very competent species (viremia 3-5 days, peak viremia 8.5-11.0 logs per ml serum) e.g. blue jay, house sparrow, american crow 2. Moderately competent species (viremia 2.8-4.5 days, peak viremia 6.8-8.7 logs per ml serum) e.g. black billed magpie, American robbin 3. Weakly competent species (viremia 0.3-3.0 days, peak viremia 4.7-6.7 logs per ml serum) e.g. mallard, Canada goose, mourning dove 4. Non-competent species (viremia transient, < 4.6 logs per ml serum) e.g. chicken, rock dove From: Komar, Fourth National Conference on West Nile Virus in the United States 2003.

27. Dead bird surveillancein Canada • What worked? • Timely early warning on temporal & spatial distribution of WNV activity • Used to inform the public • Action point for intensified mosquito surveillance & control? • What needs to be addressed? • Continuous testing of corvids after widespread virus activity observed • Utility of dead bird sightings data or cluster analysis to predict human risk unknown

28. Alpaca Horses Big Brown Bat Little Brown Bat Cat Dog Grey Squirrel Llama Alligator Wolf Fox Squirrel Sheep Eastern Chipmunk Rocky Mountain Goat Striped Skunk Reindeer Domestic Rabbit Harbor Seal Non Avian animals shown to be infected in the United States, 1999-2002 From: Bunning, Fourth National Conference on West Nile Virus in the United States 2003.

29. Is the WNV strain introduced into North America more virulent than most WNV strains? Animals >200 spp of birds susceptible • Significant mortality in some e.g. American crow • Mammals: squirrels, dog, wolf, reindeer, mountain goat, etc • Reptiles: farmed alligators • Implications for wildlife populations, zoos and endangered species

30. Questions pertaining to natural cycle and ecology of West Nile virus • Will WNV spread to British Columbia? • Will WNV gain a foothold in Atlantic Canada? • What are the northern limits to the spread of WNV? (Presumed limitations to northern spread include a lack of Culex species to drive the cycles and degree day temperature limitations)

31. Most positive birds been found in the southernmost parts of Canadian provinces (Limitations around 55°N latitude?)

32. Questions pertaining to natural cycle and ecology of West Nile virus • What are the relative roles of overwintering versus virus introduction via migratory birds for risk for recurrence of West Nile in a given region of Canada?

33. Dissemination of WNV by migratory birds?

34. Questions pertaining to natural cycle and ecology of West Nile virus Overwintering mechanisms Mosquitoes – hibernating adult Culex - in mosquito larvae? Other arthropods? Vertebrate hosts?

35. ST LOUIS ENCEPHALITIS ACTIVITY 1975 & WEST NILE VIRUS ACTIVITY 2001, IN ONTARIO Why is WNV so much more successful than SLE in temperate areas of North America?

36. Questions pertaining to natural cycle and ecology of West Nile virus • Will West Nile virus become endemic in certain parts of Canada? If so, which parts? • Will immunity gradually build up in the bird and human populations in areas of continuous activity sufficient to limit WNV activity? Has this already started to happen in certain parts of North America?

37. Questions/Discussion Fight the bug