# Presentation: 074249

## Update on flavivirus virulence studies

- Alan D.T. Barrett
- Department of Pathology,
- Center for Tropical Diseases,
- Sealy Center for Vaccine Development,
- University of Texas Medical Branch

## Important publications on West Nile

*Viral Immunology*, Volume 13, 2000.
*Emerging Infectious Diseases*, Volume 7, July-August, 2001.
*Annals of the New York Academy of Sciences*, Volume 951, December 2001.
*Current Topics in Microbiology and Immunology*, Volume 267, March 2002.

## Major Flavivirus Diseases

- Dengue
- Japanese encephalitis
- Tick-borne encephalitis
- West Nile
- Yellow fever

## West Nile virus

- Family: *Flaviviridae*
- Genus: *Flavivirus*
- Japanese encephalitis virus group
- 		*Cacipacore virus*
- 		*Koutango virus*
- 		***Japanese encephalitis virus***
- 		*Murray Valley encephalitis virus*						(Alfuy virus)
- 		***St. Louis encephalitis virus***
- 		*Usutu virus*
- 		***West Nile virus***
- 			(Kunjin virus)
- 		*Yaounde virus*

## Phylogeny of the Flavivirus genus (Gaunt et al., 2001)

- from Gaunt *et al.* (2001) *J. Gen. Virol.* **82**, 1867-76.

## West Nile Virus Transmission Cycle

- Mosquito vector

- Incidental infections

- Bird
- reservoir hosts

- Incidental  infections

## Pathogenesis

- Virus infects host via mosquito bite.
- Multiplication in tissues and lymph nodes near site of entry.
- Virus moves to blood via lymphatics; viremia detected early in infection.
- Infection of central nervous system takes place.

## How does West Nile virus invade the CNS?

*Four mechanisms to explain entry into brain*
- Neuronal route after infection of peripheral nerves.
- Virus enters brain via axonal transport through olfactory neurons.
- Virus crosses blood-brain barrier via replication in vascular endothelial cells in brain capillaries, transcytosis and release of virus into brain parenchyma.
- Diffusion of virus from vascular endothelial cells in situations where blood-brain barrier is leaky due to damage from related or unrelated trauma.

## Comparisons with St Louis encephalitis virus

** **observed a range of neuroinvasive phenotypes
- neuroinvasive, attenuated, non-invasive
- [Monath *et al.* 1980; AJTMH 29:948-962]
-  neuroinvasive phenotypes are linked to virus strain genotype
- [Trent *et al.*, 1981; Virology 114:319-332]
-  phenotypes are conserved in mouse and hamster models
- [Monath, Cropp & Harrison, 1983; Lab Invest 48:399-410]
-  similar presentation and progression of disease in animals
_**Neuroinvasion is via the olfactory nerve for SLE virus (and MVE virus?)**_

## Animal hosts

- Bird
- Horse
- Human
- Hamster
- Mouse

## Birds

- Primary vertebrate host of WN virus.
- Act as amplifying host; high viremias.
- Pathology: Meningoencephalitis and mycarditis
- Viral load in brain, kidney, and heart.

## Horses

- Polioencephalomyelitis type-disease with multifocal lesions.
- Humans
- Fatal cases have encephalitis or meningoencephalitis involving brainstem and spinal cord.

## Hamster model

- Xiao et al. EID 7, 714-721, 2001
- Used intraperitoneal route of inoculation.
- Histopathologic changes first in brain, followed by spinal cord.
- Direct virus infection responsible for neuronal damage.
- Focal distribution of viral antigen.
- Virus not found in olfactory bulbs  virus enters brain by crossing blood-brain barrier?

## Mouse

- Highly neurovirulent and neuroinvasive.
- Neuroinvasion not via olfactory route.
- Neuroinvasion different to SLE virus.

## WN virus strain virulence comparisons

- 19 strains of WN virus (inc. 2 Kunjin)
-  sequence 3’ non-coding region for phylogenetic analysis
-  i.p. LD50 in 3-4 wk female NIH Swiss mice
-  i.c. LD50 in 3-4 wk female NIH Swiss mice (selected strains)
-  i.p. inoculation in 3-4 wk female Golden Syrian hamsters (selected strains)
-  i.p. LD50 in 3-4, 7-8 and 15-16 wk female NIH Swiss mice (NY99 strain 385-99 [USA99b] only)
-  i.n. LD50 in 3-4 wk female NIH Swiss mice (selected strains)

## 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.]

## Slide 17

| Designation | Strain | Year | Group |
| --- | --- | --- | --- |
| CAR67 | ArB-310/67 | 1967 | I |
| NIG65 | IbAn7019 | 1965 | I |
| SEN79 | ArD-27875 | 1979 | I |
| USA99a | 31A | 1999 | I |
| USA99b | 385-99 | 1999 | I |
| IND68 | 68856 | 1968 | I |
| EGY50 | Egypt101 | 1950 | I |
| ETH | EthAn4766 | 1971 | I |
| AUS60 | MRM16 (Kunjin) | 1960 | KUNJIN |
| AUS91 | K6453 (Kunjin) | 1991 | KUNJIN |
| IND57 | IG-15578 | 1957 | INDIA |
| IND80 | 804994 | 1980 | INDIA |
| SEN90 | ArD-76104 | 1990 | II |
| CAR82 | ArB3573/82 | 1982 | II |
| SA58 | SAH-442 | 1958 | II |
| SA89 | SPU116-89 | 1989 | II |
| MAD88 | ArMg-979 | 1988 | II |
| CYP68 | Q3574-5 | 1968 | II |

## Lineage I

**Japanese encephalitis **

**CAR67**

** **

**NIG65**

**SEN79**

**USA99b**

**USA99a**

**IND68**

** **

**EGY50**

**ETH76**

**AUS60**

**AUS91**

** **

**IND80**

**IND57**

**SEN90**

**CA82**

**SA58**

**SA89**

**MAD88**

**CYP68**

**MAD78**

- 0.01 substitutions/site

**Lineage I**

**KUNJIN**

**INDIA**

**Lineage II**

## WN virus mouse neuroinvasion phenotypes

- (by i.p. inoculation)

**INVASIVE**
** **LD50 ranges from ~50 - <1 pfu  (majority <10 pfu)
**ATTENUATED**
** **scattered mortality over range of doses; LD50 not calculable
**NON-INVASIVE**
** **no morbidity/mortality at any dose; LD50  104 pfu

## WN VIRUS STRAINS HAVE SIMILAR MOUSE NEUROVIRULENCE CHARACTERISTICS

- (by i.c. inoculation)

- † for 1000 pfu dose of virus

## Intranasal inoculation of WN virus strains

| Virus | SMB passage | i.c. LD50(pfu) | i.n. LD50
(pfu) | i.p. LD50(pfu) |
| --- | --- | --- | --- | --- |
| USA99b | 0 | 0.1 | 200 | 0.5 |
| CYP68 | 2 | 0.3 | 1250 | >10,000 |
| SA58 | 4 | 0.3 | 200 | 3.2 |
| CAR67 | 10 | Not done | 5000 | 12.6 |
| EGY50 | 13 | 0.7 | 500 | 50 |

- SMB = suckling mouse brain

## Neuroinvasive phenotype of WN virus strains is conserved in a hamster model

**  ****Strain****	****# surviving ( out of 5)****	****  A.S.T. ******** s.d.**
**  ****USA99b****	****	****      0****	****	****	****     8.8 ******** 0.8**
**  ****SEN79****	****	****      0                                 9.2 ******** 0.4**
**   ****SA58****	****	****      0****	****	****	****     8.2 ******** 1.1**
**  ****IND80****	****	****      4****	****	****	****         12**
**  ****CYP68****	****	****      5****	****	****	****         n/a**
**  ****MAD78****	****	****      5****	****	****	****         n/a**

- Hamsters inoculated i.p. with 104 pfu of selected WN virus strains.

## Strains shown in white are neuroinvasive in mice

**Japanese encephalitis **

**CAR67**

** **

**NIG65**

**SEN79**

**USA99b**

**USA99a**

**IND68**

** **

**EGY50**

**ETH76**

**AUS60**

**AUS91**

** **

**IND80**

**IND57**

**SEN90**

**CA82**

**SA58**

**SA89**

**MAD88**

**CYP68**

**MAD78**

- 0.01 substitutions/site

**Lineage I**

**KUNJIN**

**INDIA**

**Lineage II**

**Strains shown in white are neuroinvasive in mice**

## Conclusions of mouse virulence studies

- WN virus strains differ in neuroinvasive phenotype in mouse and hamster models.
- Neuroinvasive phenotype is associated with particular subtypes within lineage I and II.
- Mouse virulence of neuroinvasive WN virus strains is high compared to other mosquito-borne flaviviruses
- closeness of i.p. and i.c. LD50 values
- lack of age-related resistance to infection in mice (USA99b)
- 4.  Lack of i.n. infectivity suggests the mechanism of neuroinvasion is probably via movement across the blood-brain barrier.

## 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 protein        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

## Attenuating Mutations

- Envelope protein.
- Deletions in the Capsid protein of tick-borne encephalitis virus.
- Deletions in the 3’ untranslated region of dengue-1,-2 and -4, West Nile and Langat viruses.
- Nonstructural proteins??

## E-protein

- Approximately 54 kDa
- Dimer positioned parallel to virus surface
- Three domains
- I-  Central domain
- II-  Dimerization domain
- III-  Immunogenic/Receptor binding domain
- 10.5 kDa
- Single disulfide bridge

## Variable residues in domain III of WN virus strains

| Residue | USA99b | Kunjin | SA58 |
| --- | --- | --- | --- |
| E310 | Lys | Arg | Lys |
| E312 | Leu | Leu | Ala |
| E332 | Thr | Thr | Lys |
| E338 | Val | Ile | Val |
| E365 | Ala | Ser | Ala |
| E369 | Ala | Ala | Ser |

- Side View

- Top View

## Neutralization escape variants

- virus
- +
- neutralizing
- monoclonal antibody

- Variability in virus populations allows the selection of escape variants.

- MAbR virus

## Membrane receptor preparation binding assays

**Another potential measure of variations in WN virus virulence??**
_**Previous MRP binding studies:**_
- Japanese encephalitis virus and mouse brain MRPs:
- 	- selected MRP binding escape variants with reduced 	virulence
- Yellow fever virus and monkey brain or liver MRPs:
- 	- observed differences in binding of neurotropic and 	   	viscerotropic strains
- 	- selected variants with attenuated mouse neurovirulence
- Langat virus and mouse or human brain MRPs:
- 	- selected variants with reduced mouse neurovirulence

## WN virus strain MRP binding characteristics

| Virus | i.p. LD50 (pfu) |  | Mouse brain MRP binding index* |  |
| --- | --- | --- | --- | --- |
| 
CAR67 | 12.6 |  | 1.2 |  |
| NIG65 | 3.2 |  | 1.2 |  |
| SEN79 | 0.2 |  | 2.1 |  |
| USA99a | 0.5 |  | 1.0 |  |
| USA99b | 0.5 |  | 1.0 |  |
| IND68 | 3.2 |  | >3.8 |  |
| EGY50 | 50 |  | 2.2 |  |
|  |  |  |  |  |
| AUS60 |  10,000 |  | 1.2 |  |
| AUS91

 |  10,000 |  | 0.2 |  |
| IND57 | n/a |  | 2.4 |  |
| IND80 | n/a |  | 1.3 |  |
|  |  |  |  |  |
| SEN90 | 50 |  | 1.2 |  |
| CAR82 | 0.8 |  | 1.0 |  |
| SA58 | 3.2 |  | 3.3 |  |
| SA89 | 5.0 |  | 1.2 |  |
| MAD88 | n/a |  | 2.7 |  |
| CYP68 | n/a |  | 3.5 |  |
| MAD78 |  10,000 |  | 0.9 |  |

**WN virus strain MRP binding characteristics**

- n/a – LD50 could not be calculated reliably* Binding index is log10 reduction in virus titer following incubation with MRP

## MRP binding assays and isolation of MRP variants

**MRP binding assays and isolation of MRP variants**

## Acknowledgements

**David Beasley**
- Li Li
- Mike Holbrook
- Jacqui Scherret
- Tom Solomon
- Miguel Suderman
- Shu-Yuan Xiao
- Hilda Guzman
- Steve Higgs
- Bob Tesh
- _Funding_
- CDC State of Texas Advanced Research Program