# Presentation: 012328

## Genetic variation in West Nile virus 

- Alan D. T. Barrett
- Department of Pathology,
- Sealy Center for Vaccine Development,
- Center for Biodefense and Emerging Infectious Diseases,
- University of Texas Medical Branch at Galveston

## ss (+) RNA genome 
Approximately 11 kb
5’-m7GpppAmp cap
Lacks 3’-polyA tail
Codes for
3 structural proteins
Capsid (C), membrane (prM/M), envelope (E)
7 non-structural proteins
NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5

- Approximately 11 kb
- 5’-m7GpppAmp cap
- Lacks 3’-polyA tail
- Codes for
- 3 structural proteins
- Capsid (C), membrane (prM/M), envelope (E)
- 7 non-structural proteins
- NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5

- Flavivirus Genome

##     

- 5’NCR	Structural proteins        Non-structural proteins       3’NCR

- RNA

- cap

- C   prM	  E      NS1 NS2A  NS2B     NS3 NS4A  2KNS4B   NS5

- Polyprotein

-   

- 

- 

- 

- 

- 

- C    prM	     E     NS1    NS2A   NS2B    NS3	     NS4A  2K    NS4B       NS5

**Co- and Post-translational Processing**

- pr

- M

- NS3’

- NS3”

- Signal peptidase site
- Unique site
- NS2B-NS3 protease site

- 

- NS3

- Protease, helicase, NTPase

- NS5

- Methyltransferase, RNA polymerase

##     

- 5’NCR	Structural proteins       Non-structural proteins      3’NCR

- RNA

- cap

- C   prM	  E      NS1 NS2A  NS2B     NS3 NS4A  2KNS4B   NS5

- Polyprotein

-   

- 

- 

- 

- 

- 

- C    prM	     E     NS1    NS2A   NS2B    NS3	     NS4A  2K    NS4B       NS5

**Post-translational Processing**

- pr

- M

- NS3’

- NS3”

- Signal peptidase site
- Unique site
- NS2B-NS3 protease site

- 

- NS3

- Protease, helicase, NTPase

- NS5

- Methyltransferase, RNA polymerase

## Lanciotti et al. 1999. Origin of the West Nile virus responsible for an outbreak of encephalitis in the northeastern U.S. [Science 286:2333-337.]

- ≥ 99.8% nucleotide homology with Israel 1998 over complete genome
- Lanciotti et al. 2002. Virology

## Phylogenetic studies of North American WN viruses 

- Anderson et al., PNAS 2001 and Ebel et al., EID 2001
- Partial nucleotide sequences (921 and 1,503 respectively) with 3 nucleotide mutations and 2 amino acid substitutions
- Lanciotti et al., Virology 2002
- Complete genomes of 1999 and 2000 isolates ≥99.8% identical by nucleotides and ≥99.9% amino acids to WN-NY99
- Huang et al., EID 2002
- Complete genome of human isolate from 2001: ≥ 99.7% nucleotide homology and ≥99.8% amino acid homology
**No sequence analyses of WNV isolates from 2002 had been reported and none were reported outside of the northeastern seaboard.**

## WN virus in Texas, 2002 (Beasley et al., 2003)

- First isolated by Bob Tesh (UTMB), Ray Parsons (Harris County) and Pushker Raj (TDH) in Houston, June 2002.
- Two “genetic variants”; one in Harris County and one on Bolivar Peninsula
- Harris County: 0.18% nucleotide divergence from New York 1999 strain 382-99.
- Bolivar Peninsula: 0.35% nucleotide divergence from New York 1999 strain 382-99.
- Harris County and Bolivar Peninsula isolates differ by 0.5%.

- 113, 114, 115, 116,118, 119, 123, 135

-     227

- 362

## Cladogram based on maximum parsimony analysis comparing a 2004-nt sequence of WN virus isolates collected during 2001-2003 to a homologous region of WN virus isolates collected in 1999, 2000, and 2001 from the northeastern U.S.

- 100

- 99

- 86

**Cladogram based on maximum parsimony analysis comparing a 2004-nt sequence of WN virus isolates collected during 2001-2003 to a homologous region of WN virus isolates collected in 1999, 2000, and 2001 from the northeastern U.S.**
**(Davis et al., 2003)**

**North America 2001-2003**

**Eastern U.S. 1999-2002**

**Southeast coastal Texas 2002**

## .=  1999-2000 (NE U.S.)

**Genetic distribution of WNV isolates, 1999-2003**

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- .=  1999-2000 (NE U.S.)

-  . =  2001-2003  (NA)

- .= 2002 (SE Texas)

- .

- .

- .

- .

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## SE coastal Texas strain(Granwehr et al., 2004)

| Residue | SE coastal Texas | Lineage I | Lineage II |
| --- | --- | --- | --- |
| E-76 | Ala | Thr | Thr |
| NS1-94 | Gly | Glu | Asn |
| NS2A-90 | Met | Met | Lys |
| NS2A-138 | Ile | Val | Val |
| NS4B-173 | Ile | Val | Val |
| NS5-526 | Ile | Thr | Thr |

## Overview of sequence studies

- Multiple introductions into TX during summer of 2002 and geographical clustering of variants (coastal vs. state-wide variants)
- Long-distance spread by birds with limited migration patterns (ie. circum-Gulf route)
- Localized spread by birds/mosquitoes over shorter-distance
- Identified nucleotide mutations at 31 positions (8 in prM, 23 in E) not previously observed in North American WN virus strains (no changes shared with NE United States strains)
- 7 amino acid substitutions (2 in prM, 5 in E)
- Revealed emergence of two distinct variants: 2001-2002 North America variant and coastal SE Texas variant
- Most divergent strains to date: individual isolates with 7 nucleotide mutations and 3 amino acid substitutions.
- Maximum nucleotide divergence from WN-NY99 was 0.35% (average of 0.18%)

## Evolution of WN virus in the United States

- Evidence suggests limited evolution between 1999 and 2002.
- Majority of nucleotide changes are transitions (U **** C).
- Strain isolated in 1999 and 2000 in NY, NJ, CT and MD differ _<_ 0.35% from prototype New York strain 382-99.
- SE coastal Texas isolates are the most divergent to date (0.55%).
- Distinct populations of virus evolving?
- No strong selection  genetic drift?

## Slide 14

## SN

**SN**

**NL**

## Cladogram based on maximum parsimony analysis comparing a 2004-nt sequence of WN virus isolates collected during 2001-2004 to a homologous region of WN virus isolates collected in 1999, 2000, and 2001 from the northeastern U.S.

- 99

- 86

**Cladogram based on maximum parsimony analysis comparing a 2004-nt sequence of WN virus isolates collected during 2001-2004 to a homologous region of WN virus isolates collected in 1999, 2000, and 2001 from the northeastern U.S.**

**North America 2001-2004**

**Eastern U.S. 1999-2002**

**Southeast coastal Texas 2002**

- 96

## Phylogram comparing a 2004-nt sequence of WN-NY99 with 35 WN virus isolates collected during 2001, 2002, and 2003.

## Eastern U.S. isolates, 1999-2002

- Southeast coastal Texas, 2002

- North America genotype

- 100

- 100

- 100

- Bayesian analysis of prM and E genes of 115 North American WNV isolates. The following criterion were used in  analysis:
- N gen= 150,000
- Print freq= 1000
- Sampling freq= 100
- N chains= 4
- burnin= 200

- 100

## Eastern U.S. isolates, 1999-2002

- Southeast coastal Texas, 2002

- North America genotype

- 100

- 100

- 100

- Bayesian analysis of prM and E genes of 115 North American WNV isolates. The following criterion were used in  analysis:
- N gen= 150,000
- Print freq= 1000
- Sampling freq= 100
- N chains= 4
- burnin= 200

- 100

## Maximum likelihood analysis of the complete genome of 48 WNV isolates. Bootstrap values represent 500 replicates performed with neighbor-joining analysis.

- Maximum likelihood analysis of the complete genome of 48 WNV isolates. Bootstrap values represent 500 replicates performed with neighbor-joining analysis.

- North America genotype

- Eastern U.S. isolates, 1999-2002

- Old World WNV isolates

- 100

- 92

- 100

- 94

- 86

## Maximum likelihood analysis of the complete genome of 48 WNV isolates. Bootstrap values represent 500 replicates performed with neighbor-joining analysis.

- Maximum likelihood analysis of the complete genome of 48 WNV isolates. Bootstrap values represent 500 replicates performed with neighbor-joining analysis.

- North America genotype

- Eastern U.S. isolates, 1999-2002

- Old World WNV isolates

- 100

- 92

- 100

- 94

- 86

## West Nile in the Americas

- United States
- Canada
- Mexico
- Dominican Republic
- El Salvador
- Jamaica

## Genomic sequence of TM-171 Mex03 isolate

- (Beasley et al., 2004)

- Isolate from dead raven at wildlife reserve in Villahermosa, Tabasco.
- RNA and, subsequently, Vero cell passaged virus sent to UTMB.

- 46 nucleotide differences (0.42%) from NY99; 4 amino acid differences:
- 	prM/M-141	Ile  Thr
- 	E-156 		Ser  Pro* (loss of glycosylation motif)
- 	NS4B-245	Ile  Val*
- 	NS5-898	Thr  Ile*

## Mixed sequence at the E glycosylation site of Mex03 isolate.

- Consensus sequences:
- NY99  AAC TAC TCC ACA   (NYST)
- MEX   AAC TAC CCC ACA   (NYPT)
- Cloned Mex03 PCR product and sequenced 5 clones:
-  three clones 	“CCC” – Pro
-  two clones 	“TCC” – Ser

- Forward

- Reverse

## Plaque purification of E glycosylation variants of Mex03.

-  Variants selected by two rounds of plaque purification in Vero cells.
-  Screened by Western blot (diff. in apparent mol. wt.)

-  Selection of variants confirmed by nucleotide sequencing.
-  gly+ variants grew faster (~24hrs earlier to harvest) and to ~10-fold higher titer, and had slightly larger plaque size.

## Mouse virulence of Mex03 glycosylation variants.

| Virus | E154-156 | i.p. LD50 (pfu) | A.S.T.  s.d. (days) | PD50(pfu) |
| --- | --- | --- | --- | --- |
| Mex03 | mixed | 0.5 | 8.2 1.8 | n.d. |
| Mex03-pp1 | NYP | >1000 | -- | 5.0 |
| Mex03-pp2 | NYP | 794 | 10.3  0.6 | 0.8 |
| Mex03-pp5 | NYS | 3.2 | 8.5  0.8 | 0.5 |
| Mex03-pp6 | NYS | 2.0 | 9.2  1.0 | 2.0 |

- n.d. – not determined

- All strains contained prM-141 IT; NS4B-245 IV; NS5-898 TI

## Neighbor-joining phylogenetic tree based on complete genome sequences of West Nile virus strains. The isolate from Tabasco, MX is designated TM171-03. 

- Neighbor-joining phylogenetic tree based on complete genome sequences of West Nile virus strains. The isolate from Tabasco, MX is designated TM171-03.

*Beasley et al., 2004. EID*

## Conclusions

- Evidence suggests limited, but continuing, divergence from isolates collected in 1999 and 2000
- Emergence of genetically distinct variants
- Dominant North America variant since at least 2002
- Coastal SE Texas variant in 2002 only  become extinct?
- Dominant variant has emerged throughout the majority of North America with >90% of isolates collected in 2002 and after belonging to the dominant clade
- Emergence of novel genotype corresponds with displacement or extinction of earlier genotypes
- Subclades are readily illustrated depending on year of isolation and collection location
- Higher degree of nucleotide identity within individual states and during the same transmission season
- Mexico 2003 isolate is most divergent from the Americas with 9 nt and 2 aa differences from New York 1999 isolates and has lost E protein glycosylation site.
- Genetic mutations could eventually lead to phenotypic changes in viral antigenicity or associated virulence (mouse model shows no attenuation in either variant)?

## Phenotypic variants – Texas 2003

- (Davis et al., 2004)

- Nucleotide sequencing and phenotypic comparisons of 29 WNV isolates collected by Harris Co. Mosquito Control Division in and around Houston, TX between 9 May – 8 Sept. 2003:
- 17 from dead birds
- 12 from mosquito pools (*Culex spp.*)
- Viruses isolated in Vero cells, amplified by one additional passage and plaque titrated on Vero cells.
- 6 strains (5 bird, 1 mosq.) with small plaque (SP) morphology

## Plaque morphology of WNV isolates

**WN-NY99 (382-99)**

**WNV 2002**

**WNV 2003 sp**

**In Vero cells, 72 hours post-infection.**
**Stained with crystal violet.**

**Plaque morphology of WNV isolates**

- Mean plaque size  1.5mm

- Mean plaque size < 1.0mm

## Phenotypic comparisons of LP/SP WNV strains

- Small plaque phenotype often a marker of attenuation of flaviviruses.
- Other phenotypic comparisons:
- temperature sensitivity at 39.5C
- mouse neuroinvasion
- 	- i.p. inoculation of 3-4 week old Swiss Webster mice

## Phenotypic comparisons of LP/SP WNV strains (cont.)

| Strain | Source | Plaque | TS index* | ip LD50 (pfu) | AST  sd | ic LD50 (pfu) |
| --- | --- | --- | --- | --- | --- | --- |
| 382-99 | Flamingo | L | -0.5 | 0.8 | 7.20.6 | 0.1 |
| TWN93 (02) | Bird | L | 0.3 | 0.5 | 8.01.0 | 0.1 |
| TWN301 (03) | Bird | L | -0.2 | 0.6 | 7.01.0 | 0.2 |
| TWN305 (03) | Bird | S | 0.3 | 51,000 | 9.04.0 | 0.1 |
| TWN274 (03) | Bird | S | -2.7 | 23,000 | 9.51.0 | 0.3 |
| TWN382 (03) | Mosq. | S | -1.8 | 645,000 | 8.33.0 | 0.1 |
| TWN269 (03) | Bird | S | -2.7 | 2,000 | 9.73.3 | nd |

- *TS index is log10 difference in virus titer at 39.5C compared with 37C; negative values indicate decreased titers.
- nd – not determined; L = large, S = small.

**Phenotypic comparisons of LP/SP WNV strains (cont.)**

## Viral growth curve in Vero cells of 2003 WNV isolates in comparison to isolates from 1999 and 2002

**sp and ts**

**sp**

## Slide 34

## Slide 35

## Conclusions

- First evidence of phenotypic variation in North American West Nile virus
- Attenuation of mouse neuroinvasiveness may not be indicative of attenuation in birds, horses, humans
- No indication that attenuated viruses persist in nature
- 		- No more SP/mouse attenuated isolates identified  another 	extinct lineage?
- Sequencing and reverse genetics studies can be used to identify molecular determinants of phenotypic variation

## Genetic and phenotypic variation in New York, 2002Ebel et al. 2004. AJTMH

- New genotype emerged in New York in 2002 (55% of all isolates in 2002 and 85% of all isolates in 2003)
- Genetically homogeneous to Texas isolates from 2002-2003 (prM and E)
- In vitro growth studies (C6/36 and Vero) showed no significant differences
- In vivo mosquito transmission studies (*Culex pipiens*)
- Significantly higher proportion of mosquitoes became infected (infectious virus in bodies) and developed disseminated infections (infectious virus in legs) following feeding with dominant genotype compared to WN-NY99
- Significantly higher proportion of mosquitoes were able to transmit (infectious virus in salivary secretions) at days 5 and 7 post-feed with dominant genotype
- Displacement of WN-NY99 genotype by dominant genotype may be due to differences in mosquito transmission efficiency (reduction of EIP with dominant genotype)

## Acknowledgements

- _Barrett lab_				_Other UTMB_
**David Beasley**				Bob Tesh + lab
- Mike Holbrook
**Todd Davis**				Steve Higgs + lab
- Amber Engel
- Li Li					Scott Weaver + lab
- Monica McArthur			Jose Estrada-Franco
**Melissa Whiteman**
- Jason Wicker
- Shuliu Zhang
**Bruno Granwehr**
- _Funding_
- CDC; State of Texas Advanced Research Program;Clayton Foundation; McLaughlin Foundation

## Collaborators

-   Harris County Mosquito Control Division
-   Texas Department of Health
-   CDC, Division of Vector-Borne Infectious Diseases
-   Illinois Natural History Survey
-   University of Alabama, Birmingham
-   Louisiana Dept. of Health
-   Florida Dept. of Health
-   Colorado State University
-   Nebraska Dept. of Health
-   University of California, Davis
-   New York State Dept. of Health
-   Health Canada
-   Mexico – Dept. de Salud Publico