# Presentation: 711778

## Theoretical and practical aspects of flaviviral antigenic structure in diagnostic assays- Lessons from the past and promise for the future

## Diagnostic Assays for Arboviruses

**Mosquito pools, Tissues**

**Virus Detection**
**Assays**

**IgM ELISA**
**IgG ELISA**
**PRNT**
**CF**
**HI**
**IFA**
**Dipsticks**

**Virus isolation (cell culture, mice)**
**IFA**
**TaqMan RT-PCR**
**Ag-capture ELISA**
**RT-PCR / sequencing**
**Dipsticks**
**NASBA**

**Diagnostic Assays for Arboviruses**

**Serological**
**Assays for **
**Antibodies**

## Diagnostic Assays for Arboviruses

- Mosquito pools, Tissues

**Serological**
**Assays for **
**Antibodies**

**IgM ELISA**
**IgG ELISA**
**PRNT**
- CF
- HI
- IFA
- Dipsticks

- Virus isolation (cell culture, mice)
- IFA
- TaqMan RT-PCR
- Ag-capture ELISA
- RT-PCR / sequencing
- Dipsticks
- NASBA

**Diagnostic Assays for Arboviruses**

- Virus Detection
- Assays

## Approaches to Enhanced Testing 

- Increase specificity
- Increase sensitivity
- Reduce sample size
- Increase speed of testing
- Multiplexing

## Species in the Genus Flavivirus

**(family *****Flaviviridae*****)**

## Members of the Japanese encephalitis 

**virus serocomplex**

- Cacipacore virus

- Koutango virus

- Japanese encephalitis virus

- Murray Valley encephalitis virus

*Alfuy virus*

- St. Louis encephalitis virus

- Usutu virus

- West Nile encephalitis virus

*Kunjin virus*

- Yaounde virus

## Types of Antibody Reactivities(e.g., WNV)

_**Subtype**_ – reacts with some but not all WNVs
_**Type**_ – reacts with all WNVs
_**Subcomplex**_ – reacts with 2 or more members of the JEV complex
_**Complex**_ – reacts with all members of the JEV complex
_**Subgroup**_** **– reacts with 2 or more complexes
_**Group**_ – reacts with all flaviviruses

**High**

**Low**

**Specificity**

## Serum

**Serum**

**SLE**

**JE**

**WN**

**DEN2**

**YF**

**POW**

**1**

**4.96**

**7.75**

**16.74**

**2.45**

**1.82**

**1.56**

**2**

**4.8**

**13.77**

**16.68**

**4.13**

**2.14**

**1.75**

**3**

**5.45**

**9.67**

**16.08**

**4.09**

**1.61**

**1.44**

**4**

**4.76**

**10.07**

**17.19**

**3.32**

**1.62**

**1.3**

**Positive **

**Control**

**6.5**

**8.2**

**6.34**

**7.45**

**3.96**

**4.5**

*** 1:400 screening dilution of WNV patient serum**

## Complete Serological Analysis

**Patient**

**Days **

**P.I.**

**IgM **

**(WN)**

**IgG **

**(SLE)**

**WN**

**SLE **

**DEN2**

**JE**

**CSF**

**8**

**26.91**

**1.78**

**nd**

**nd**

**nd**

**nd**

**S1**

**9**

**9.1**

**4.16**

**160**

**20**

**<10**

**10**

**S2**

**34**

**6.7**

**4.62**

**1280**

**20**

**<10**

**20**

**Positive **

**Control**

**n.a.**

**9**

**6.5**

**>5120**

**2560**

**2560**

**320**

**ELISA**

**PRNT**

## What is the Best Way to Reduce Virus Cross-reactivity?

- Make antigens more specific
- Make antigens less cross-reactive

## Flavivirus Genome

**-3'**

**pr + M**

- furin

**NC**

**NC**

**ns2a**

**ns2b**

**ns4a**

**ns4b**

***C***

***prM***

***E***

***NS1***

***NS3***

***NS5***

**Flavivirus Genome**

**7 non-structural genes/proteins**

**3 structural**
**Genes/proteins**

## Virion Components

**=**

**NC**

**Capsid protein**
**12,000 dal**
**~120 a.a.**

**prM/M protein**
**20,000/8,000 dal**
**~165 a.a./75 a.a.**

**E-protein dimer**

**E-protein monomer**
**50-53,000 dal**
**~500 a.a.**

**ssRNA**
**~11,000 b.p.**

- 5'-

- -3'

- pr + M

- furin

**NC**

**NC**

**ns2a**

**ns2b**

**ns4a**

**ns4b**

***C***

***prM***

***E***

***NS1***

***NS3***

***NS5***

**Non-ionic**
**detergents**

**SDS**

**RNA extraction**

**SDS or   reduction**

## Homodimer of the Flavivirus E-protein

## General Antigenic Properties of Flaviviral Proteins - 1

- _E-glycoprotein_
- Most important flaviviral antigen
- Very immunogenic
- Most serological assays detect reactivity with this protein
- _Capsid_
- Elicits primarily group-reactive antibody
- _M-protein_
- Very small (75 a.a.)
- Embedded in the virion envelope membrane and not highly immunogenic

## General Antigenic Properties of Flaviviral Proteins - 2

- _NS1_
- Cell surface protein
- Secreted from cells and can be detected in serum samples during some flaviviral infections (*e.g*., dengue)
- Very immunogenic
- Can be used to differentiate infection from vaccination
- Has some intrinsic viral specificity
- _NS3 and NS5_
- Intracellular proteins, antigenic but not very immunogenic
- NS5 antibody is somewhat virus-specific
- NS3 antibody is cross-reactive

## Domains of the E-protein Monomer

**Domain III = B-domain**

**Domain I = C-domain**

**Domain II = A-domain**

**Domains of the E-protein Monomer**

## Serologic Cross-reactivities vs.Virus Neutralization

| Domain | Epitope | Specificity | PRNT |
| --- | --- | --- | --- |
| II | A1 | Group | + |
|  | A2 | Subcomplex | + |
|  | A3 | Type | - |
|  | A4 | Type | + |
|  | A5 | Subgroup | + |
| III | B1 | Type | + |
|  | B2 | Type | + |
|  | B3 | Subcomplex | - |
|  | B4 | Subcomplex | - |
| I | C1 | Subcomplex | - |
|  | C2 | Type | - |
|  | C3 | Type | - |
|  | C4 | Type | - |

**Serologic Cross-reactivities vs.Virus Neutralization**
**Activity of Dengue E-protein Epitopes**

## Specificity and Abundance of Neutralizing Epitopes

| Virus | Selecting Mab specificity | References |
| --- | --- | --- |
| DEN | 6 - Type
2 - Subcomplex | Lin 1994, Beasley 2001, Lok 2001 |
| JE | 5 –Type
1 - Subgroup | Hasegawa 1992, Cecelia 1991, Morita 2001 |
| LI | 3 - Subtype | Jiang 1993, Gao 1994 |
| MVE | 5 - Type | McMinn 1995 |
| TBE | 5 – Subtype
1 - Complex | Mandl 1989 |
| YF | 7 - Type | Lobigs 1987, Ryman 1997 |
| Totals | 31 – Subtype or Type
4 – Others |  |

**Specificity and Abundance of Neutralizing Epitopes**

## Location of Neutralization Domains on

**233**

**368**

**67-72, 112**

**124, 128**

**52,136,274-9**

**307-311,333,384-5 **

**155,158**

**171, 293**

**Location of Neutralization Domains on**
**the Flavivirus E-protein**

## Pitfalls of Modifying/Expressing the flavivirus E-protein

- Proper expression (conformation) of the E-protein requires co-expression of the prM protein when derived from acidic environments and requires glycosylation
- Domain I and Domain II epitopes are highly conformational; difficult to express on peptides or other small fragments
- Domain III has an essential S-S-bond for epitope expression
- Regions of sequence homology and uniqueness are interspersed throughout the protein.

## Summary

- Increase specificity?
- Increase sensitivity?
- Reduce sample size?
- Increase speed of testing?
- Multiplex?