# Presentation: 711269

## Slide 1

## A Phylogenetic Approach to Following West Nile Virus

- John F. Anderson
- The Connecticut Agricultural
- Experiment Station
- New Haven, CT 06511

## ____________________________________________

- Possible Pathways of Introduction of WNV into the US

- Infected Human
- Human-transported vertebrate host
-  Legal
-  Illegal
- Human-transported mosquito vector
- Storm-transported vertebrate host (bird)
- Intentional introduction (terrorist event)

## Slide 4

## DISTRIBUTION OF WEST NILE VIRUS IN NORTH AMERICA

**DISTRIBUTION OF WEST NILE VIRUS IN NORTH AMERICA**

**1999**

**2000**

**2001**

## Genomic Structure of Flaviviruses

**C**

**prM**

**NS1**

**NS3**

**NS5**

**5’-**

**-3’**

**Genomic Structure of Flaviviruses**

**NC**

**NC**

**E**

**ns2a**

**ns2b**

**ns4a**

**ns4b**

**NC            noncoding (at ends)**
**C, M, E    structural proteins (3)**
**prM         precursor protein for M**
**NS            nonstructural proteins (7****)**

**Information from L.R. Peterson and J.T. Roehrig (2001)**

**921-nt**

## Primers Used to Amplify the 921-nucleotide Sequence of West Nile Virus from Positions 205-1125

- WN-233F (5’-GACTGAAGAGGGCAATGTTGAGC-3’)
- WN-1189R (5’GCAATAACTGCGGACYTCTGC-3’)

## Protocol Used to Develop a Phylogeny of West Nile Virus Isolates

## Protocol Used to Develop a Phylogeny of West Nile Virus Isolates

**A 921-nucleotide sequence using WNV primers 233F-1189R**

**Three different-sized sequences for each of 2 isolates, using primers 233F-540R, 451F-1189R, and 233F-1189R**

## Protocol Used to Develop a Phylogeny of West Nile Virus Isolates

## Slide 11

## Geographically Based West Nile Virus Mutation

***Geographically Based West Nile Virus Mutation***

## Significant Findings

- 30 of the 921 nucleotides showed variability
- 3 occurred in the nucleocapcid coding region
- 11 occurred in the premembrane coding region
- 12 occurred in the membrane coding region
- 4 occurred in envelope coding region
- 6 nucleotide positions showed identical changes in 2 or more isolates
- 34 isolates had sequences identical to WN-NY99
- 37 isolates had a single nucleotide change
- 8 isolates had two nucleotide changes
- 3 isolates had three nucleotide changes
- Amino acid changes occurred at 7 loci in 6 isolates

## Comparison of the 921-nt Sequence of WN-NY99 to WN Virus From Brain and Heart Tissues From Same Avian Host

-                   RT-PCR Sequences That Differ From WN-NY99
-     Host	              Brain		             Heart
- Crow	         456, C to T; 516, T to C     456, C to T; 516, T to C
- Crow           588, G to A; 1000, T to C   588, G to A; 1000, T to C
- Crow	         None                                   None
- Blue Jay      None			        None
- Crow           825, T to C                          825, T to C
- House
-   Sparrow    858, C to T                          858, C to T

## Comparison of the 921-nt Sequence of WN Virus in Different Passages in Vero Cell Culture

-                       RT-PCR Sequences That Differ From WN-NY99
-     Host               (Passage Number)	            (Passage Number)
- Crow             1032 T to C(1)                   1032 T to C(3)
- Crow             721 C to T; 905 G to A;     721 C to T; 905 G to A;
-                            940 T to C(1)                     940 T to C(3)
- Crow             673 A to G(1)                     673 A to G(3)
*Cx. pipiens*    933 C to T; 1047 C to T(0) 933 C to T; 1047 C to T (2)
- Crow             528 T to C; 543 A to G(0)  528T to C; 543 A to G(3)
*Cx. restuans*  None (0)                             None (2)
- Crow             726 A to G; 858 C to T(0)  726 A to G; 858 C to T(3)

## Slide 16

## Experiment Station StaffParticipating in This Study

- Charles Vossbrinck          Laura Mickowski
- Theodore Andreadis	     Jodie Ortega
- Andy Main		     Amanda Rahmann
- Melanie Baron		     John Shepard
- Terry Goodman		     Michael Thomas
- Bonnie Hamid		     Michael Vasil

## Participants in This Study FromOther Agencies

- CT DEPARTMENT OF PUBLIC HEALTH:
- 	William H. Beckwith, III
- 	Donald R. Mayo
- 	Skip Sirpenski
- 	Randall Nelson
- 	Lynn Wilcox
- USDA, ARS
- 	Daniel L. Kline
- UCONN:
- 	Richard A. French
- 	Meghan Tucker
- STAMFORD HEALTH DEPARTMENT:
- 	Anthony Iton
- STRATFORD HEALTH DEPARTMENT:
- 	Elaine O’Keefe