# Presentation: 016098

## Update on West Nile Virus

**	****George J. Dawson, Ph.D.**
**	****Abbott Laboratories**

## West Nile Virus Update

**Abbott’s commitment to WNV **will continue as needed to aid in understanding evolution of markers associated with infection
**Abbott’s Research WNV IgM assay****:**** **
- Cutoff set based on human population studies, experimental infection studies in primates
- Repeatably reactive specimens were characterized with other serologic assays to confirm reactivity
- Supplemental tests from various sources have been utilized to aid in setting cutoff for Abbott’s test
**	**

## West Nile Virus Update

**Recent observations indicate:**** **
- Considerable overlap of WNV RNA detection and IgM detection in the early stage of infection
- Low level viremics may be negative by minipool NAT and reactive by WNV IgM
- Infectivity studies may be required to determine transmissibility of low level viremic specimens
- More sensitive IgM assays have greater overlap with WNV RNA detection than less sensitive assays
- As WNV IgM tests become more sensitive, there are fewer options for confirmatory tests with appropriate sensitivity

## West Nile Virus

**Specificity issues with WNV IgM assays for WNV**:
**	****1****.  Cross reactivity**
- 		A repeatably reactive test result may be induced
-  		by flaviviruses other than WNV due to relatively
- 		high degree of amino acid homology between 			proteins of WNV and other flaviviruses
**	****2.  Confirmatory testing**
- 		Low level reactives may be difficult to confirm 			Follow-up samples may be required

## Amino Acid Sequence Homology

| Region | Dengue* | YFV | TBEV | MV | JEV | SLE |
| --- | --- | --- | --- | --- | --- | --- |
| Core | 41.4 | 27.1 | ~21.9 | 65.9 | 61.8 | 60.3 |
| PreM/M | 37.6 | 38.6 | 30.2 | 77.2 | 74.2 | 65.9 |
| ENV | 50.1 | 45.2 | 44.1 | 79.3 | 78.8 | 73.3 |
| NS1 | 52.3 | 43.4 | 43.1 | 76.1 | 77.8 | 65.2 |
| NS2A | 28.5 | 27.9 | 21.3 | 64.8 | 65.6 | 49.8 |
| NS2B | 33.1 | 28.5 | 25.2 | 74.0 | 67.9 | 52.7 |
| NS3 | 63.6 | 52.1 | 46.7 | 80.5 | 81.7 | 72.9 |
| NS4A | 40.4 | 35.6 | 34.2 | 77.2 | 77.8 | 63.1 |
| NS4B | 40.7 | 32.0 | 27.1 | 64.6 | 65.5 | 56.9 |
| NS5 | 66.5 | 60.9 | 60.1 | 82.6 | 81.2 | 75.3b |

*** Average of Dengue type 1 through 4:  analyses performed at Abbott Laboratories**

**Amino Acid Sequence Homology**

**Comparison of percent identity of West Nile Virus vs. Dengue, Yellow Fever, Japanese Encephalitis and St. Louis Encephalitis Viruses**

## 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**

## Two-Step IgM Capture EIA

**Anti-IgM Coated Solid Phase**

**Serum Added**
***Diluent***

**Antigen Added**
***Diluent***
***Red***

**Conjugate Added**
***Diluent***
***Blue***

**Two-Step IgM Capture EIA**

**Timing:**            1** hr/37****o****C/Static                                  2 hr/37****o****C/Static**

## West Nile Virus

**West Nile Virus**

**Studies **
**Volunteer blood donors from 2002**
- Based on previous population studies the cut-off was provisionally set at S/N = 5.0
**Experimentally infected rhesus monkeys***
- Daily bleeds for 2 wks
- Natural history of WNV

- *In collaboration with Dr. Tesh and associates at UTMB Galveston, TX and Dr. Marion Ratterree and associates at the Tulane National Primate Research Center, Covington, LA

## Serologic Profile of WNV Infection in Experimentally Infected Monkeys

- *In collaboration with Dr. Tesh and associates at UTMB Galveston, TX and Dr. Marion Ratterree and associates at  the Tulane National Primate Research Center, Covington, LA

## Serologic Profile of Experimentally Infected Macaque #810

| Day # | Virus (log) | RT-PCT | IgM (S/N) | IgG S/N | HI (titer) | CF (titer) | PRNT (titer) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 0 | - | - |   1.0 | 1.0 | - | - | - |
| 1 | - | + |   1.0 | 1.0 | - | - | - |
| 2 | 2.0 | + |   1.0 | 1.1 | - | - | - |
| 3 | 1.0 | + |   1.0 | 1.1 | - | - | - |
| 4 | 1.7 | + |   1.0 | 1.2 | - | - | - |
| 5 | 1.7 | + |   1.0 | 1.1 | - | - | - |
| 6 | - | + |   1.3 | 1.0 | - | - | - |
| 7 | - | + |   1.6 | 1.0 | - | - | - |
| 8 | - | - |   2.1 | 1.0 | - | - | - |
| 9 | - | + |   3.3 | 1.0 | - | - | - |
| 10 | - | +/- |   8.3 | 1.1 | - | - | 1:10 |
| 11 | - | - |  18.3 | 1.1 | 1:20 | - | 1:20 |
| 12 | - | - |  45.2 | 1.3 | 1:160 | - | 1:20 |
| 13 | - | - | >65.6 | 1.5 | 1:640 | - | 1:40 |
| 14 | - | - | >65.6 | 2.0 | 1:640 | 16 | 1:20 |
| 21 | - | - | >65.6 | 7.1 | ND | ND | 1:80 |
| 28 | - | - | >65.6 | 8.2 | 1:1280 | 64 | ND |
| 45
63 | -
- | -
- | 38.7
21.6 | 9.0
15.9 | 1:640
1:1280 | 64
64 | ND
1:512 |

**Serologic Profile of Experimentally Infected Macaque #810**

- *In collaboration with Dr. Tesh and associates at UTMB Galveston, TX and Dr. Marion Ratterree and associates at the Tulane National Primate Research Center, Covington, LA

## Serologic Profile of WNV Infection in Experimentally Infected Monkeys

- *In collaboration with Dr. Tesh and associates at UTMB Galveston, TX and Dr. Marion Ratterree and associates at the Tulane National Primate Research Center, Covington, LA

## Serologic Profile of Experimentally Infected Macaque #J840

| Day # | Virus | RT-PCT | IgM (S/N) | IgG S/N | HI | CF | PRNT |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 0 | - | - |   1.0 | 1.0 | - | - | - |
| 1 | - | + |   0.8 | 0.9 | - | - | - |
| 2 | 1.7 | + |   1.1 | 1.0 | - | - | - |
| 3 | - | + |   0.8 | 1.0 | - | - | - |
| 4 | - | + |   1.0 | 1.0 | - | - | - |
| 5 | - | - |   0.9 | 0.9 | - | - | - |
| 6 | - | + |   1.1 | 1.0 | - | - | - |
| 7 | - | - |   1.4 | 1.0 | - | - | - |
| 8 | - | - |   1.5 | 0.9 | - | - | - |
| 9 | - | - |   2.3 | 1.1 | - | - | - |
| 10 | - | - |   3.3 | 1.3 | - | - | - |
| 11 | - | - |  4.4 | 1.4 | - | - | - |
| 12 | - | - |  8.4 | 1.6 | 1:20 |  8 | - |
| 13 | - | - | 15.2 | 2.2 | 1:40 | 32 | 1:10 |
| 14 | - | - | 27.6 | 2.6 | 1:80 | 32 | 1:20 |
| 21 | - | - | 27.9 | 9.6 | 1:320 | 64 | 1:80 |
| 28 | - | - | 9.3 | 9.2 | 1:160 | 64 | ND |
| 45
63 | -
- | -
- | 2.3
2.3 | 7.7
7.0 | 1:160
1:160 | 16
 8 | ND
1:1280 |

**Serologic Profile of Experimentally Infected Macaque #J840**

- *In collaboration with Dr. Tesh and associates at UTMB Galveston, TX and Dr. Marion Ratterree and associates at the Tulane National Primate Research Center, Covington, LA

## Summary of Serologic Profiles of Experimentally Infected Macaques

| Day # | Virus log PFU’s (pos/total) | RT-PCR (pos/total) | IgM (S/N) | IgG S/N | HI (titer) | CF (titer) | PRNT (titer) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 0 | - | - | 1.0 | 1.0 | - | - | - |
| 1 | - | 5/5 + | 0.9 | 1.0 | - | - | - |
| 2 | 1.7  (5/5+) | 5/5 + | 1.1 | 1.0 | - | - | - |
| 3 | 2.0  (3/5+) | 5/5 + | 1.0 | 1.0 | - | - | - |
| 4 | 1.0  (2/5+) | 5/5 + | 1.1 | 1.0 | - | - | - |
| 5 | 1.7  (2/5+) | 3/5+ | 1.1 | 1.0 | - | - | - |
| 6 | - | 3/5 + | 1.3 | 1.0 | - | - | - |
| 7 | - | 1/5+ | 1.5 | 1.0 | - | - | - |
| 8 | - | 0/5 + | 2.1 | 0.9 | - | - | - |
| 9 | - | 1/5 + | 3.3 | 1.0 | - | - | - |
| 10 | - | 1/5 +- | 11.9 | 1.0 | 1:20 | - | 1:10 |
| 11 | - | - | 18.3 | 1.1 | 1:40 | - | 1:20 |
| 12 | - | - | 30.9 | 1.3 | 1:80 | 8 | 1:20 |
| 13 | - | - | 40.2 | 1.5 | 1:160 | 16 | 1:40 |
| 14 | - | - | 50.8 | 2.1 | 1:320 | 16 | 1:40 |
| 21 | - | - | 46.1 | 7.1 | 1:320 | 32 | 1:80 |
| 28 | - | - | 27.0 | 8.2 | 1:160 | 64 | ND |
| 45 | - | - | 11.6 | 9.0 | 1:160 | 16 | 1:640 |
| 63 | - | - | 3.4 | 7.0 | 1:160 | 16 | 1:640 |

**Summary of Serologic Profiles of Experimentally Infected Macaques**
**	****	****	****	**Median Values

- *In collaboration with Dr. Tesh and associates at UTMB Galveston, TX and Dr. Marion Ratterree and associates at the Tulane National Primate Research Center, Covington, LA

## Experimentally Infected Macaques

**A total of 38 of the 41 IgM reactive specimens (S/N>5.0) were confirmed by one or more supplemental assay**
- 2 of 5 of the first bleed dates were confirmed by one or more supplemental assays
**A provisional cutoff was set an S/N greater than 5.0 and utilized for human studies**

## IgM Detection in a Selected Population*

**IgM Detection in a Selected Population***

- Volunteer blood donations from 2002, from centers considered at “high risk” for WNV
- Infection – Dr. L Tobler Blood Centers of the Pacific; Dr. Michael Busch, America’s Blood Centers

## Serologic Profile of Repeatably Reactive Human Specimens

| Number | IgM (S/N) | IgM (S/N)
retest | IgM 2 (S/N) | PRNT Titer | WNV IgG | WNV IgG
retest | Supplemental Testing |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | >155.5 | >111.1 | 48.8 | 640 | 14.5 | 20.1 | Pos |
| 2 | 142.4 | >111.1 | 45.0 | 1280 | 85.2 | 99.8 | Pos |
| 3 | 127.3 | >111.1 | 47.1 | 320 | 8.7 | 8.4 | Pos |
| 4 | 110.7 | 109.2 | 24.7 | 320 | 24.6 | 38.9 | Pos |
| 5 | 103.9 | 108.8 | 32.4 | 160 | 29.1 | 20.1 | Pos |
| 6 | 91.1 | 95.9 | 26.4 | 160 | 63.8 | 57.8 | Pos |
| 7 | 59.9 | 78.3 | 20.4 | 640 | 81.4 | 45.6 | Pos |
| 8 | 31.6 | 32.1 | 18.4 | Neg | 0.8 | ND | Neg* |
| 9 | 10.1 | 9.6 | 4.4 | Neg | 1.3 | ND | Neg* |
| 10 | 7.9 | 8.7 | 5.1 | Neg | 1.1 | ND | Neg* |
| 11 | 7.8 | 5.0 | 4.4 | Neg | 0.5 | ND | Neg* |
| 12 | 7.7 | 12.4 | 5.8 | Neg. | 0.5 | ND | Neg* |
| 13 | 5.5 | 10.4 | 4.4 | Neg | 0.6 | ND | Neg* |

**Serologic Profile of Repeatably Reactive Human Specimens**

- Additional supplemental testing is being performed

- Volunteer blood donations from 2002, from centers considered at “high risk” for WNV
- Infection – Dr. L Tobler Blood Centers of the Pacific; Dr. Michael Busch, America’s Blood Centers

## SUMMARY

**Assay Format Finalized**
- EIA on polystyrene beads
- Capture IgM completed in 3.5 hours at 37° C
**Preliminary evaluation of IgM testing indicates:**
- In experimentally infected primates, IgM was detected by day 10 post infection
- The provisional cutoff set at S/N 5.0 appears to provide adequate specificity
- Specificity improvements may be made in the future if needed
- Research studies will continue utilizing current provisional cutoff, while evaluating supplemental testing (confirmatory) algorithms