LABORATORY TESTING FOR ARBOVIRUSESLaura D. Kramer

Wadsworth CenterNew York State Department of Health

and State University of New York at Albany

Atlantic Monthly, 1997

o Arboviruses of medical importance•Alphaviruses•Flaviviruses•Bunyaviruses

o Surveillance protocols – clinical and field•Serologic assays•Viral assays

Why do we conduct surveillance? To detect and control the spread of infectious agents to prevent major outbreaks,

and to aid in investigative epidemiology

BF - Barmah Forest MAY - Mayaro VEE - Venezuelan Equine EncephalitisCE - California Encephalitis MVE - Murray Valley Encephalitis WEE - Western Equine EncephalitisCHIK- Chikungunya ONN – O’nyong-nyong WN - West NileCCHF - Congo-Crimean Hemorrhagic Fever ORO - Oropouche WSL - WesselsbronDEN - Dengue RVF - Rift Valley Fever YF - Yellow FeverEEE - Eastern Equine Encephalitis RR - Ross RiverJE - Japanese Encephalitis SLE - St. Louis EncephalitisKFD - Kyasanur Forest Disease SIN - SindbisLAC - LaCrosse Encephalitis TBE- Tick-Borne Encephalitis

DEN DENDEN

DEN

DEN DEN

DEN

DEN

DEN

DEN

DEN

DEN

DENDEN

DEN

DEN

DEN

DEN

DEN

DEN

JE

JE

JE

JEJE

JE

CHIKCHIK

CHIK

CCHF

CCHFCCHF

CCHF

MVE

RRBF

SINSIN

TBE

TBE

CE

WNWNWN

WN

WN

WNWN

WN

WN

WN

WSL

ONN

RVF

RVF

RVF

KFD

YF YF

YF YFYF ORO

ORO

OROMaYVEE

VEEVEE

EEELAC SLE

Global Emergence of Epidemic Arboviral Diseases

WN

WNWN

EEESLEWN

WNWN

DEN

DENDEN

DEN

DEN

DEN

POW

YF

JEDEN

DENRVF

WNV

Togaviridae: Alphaviruses

o Enveloped virion

o Single stranded positive sense RNA

o 3 disease patterns:• Arthropathy (Sindbis, Ross River, Chikungunya)• Systemic febrile illness (Semliki forest, VEE)• Encephalitis (EEE, WEE, VEE)

Paredes, et al. 1995

Geographic Distribution

WEEEEEVEE

Medically important Alphaviruses, USA

Chikungunya

CDC

Eastern Equine Encephalitis VirusNeuroinvasive Disease Cases Reported by Year, 1964-2010

4

70

36

16 ~265 cases5-6 cases/yr

Western equine encephalitisHuman cases 1964-1994

640 cases

Enzootic focus in FloridaOutbreak in Texas in 1970’sAerosol transmission possible

Venezuelan Equine Encephalitis Virus

o Viral factors• Enzootic & epizootic strains

o Environmental factors• Mosquito populations

o Herd immunity• Equine vaccine

Outbreaks are episodic

S Weaver and A Barrett, 2004

Potential to emerge

Chikungunya

A.M. Powers and C.H. Logue, 2007 J Gen Virol,

In Swahili, “chikungunya” : that which contorts or bends “up”

Disease:o High fever (103-104 F)o Rash o Severe incapacitating arthritis/arthralgia

• Generalized • Usually acute

oHemorrhagic manifestations have been reported

Vector: Ae aegypti and Ae albopictus

Chikungunya Virus Distribution

o Latin flavus: Yellow

o Grouped into a single family due to similarities in virion morphology, genome organization, and RNA replication strategy

o Enveloped virion containing single stranded positive sense RNA

o Disease: hemorrhagic, encephalitic, febrile

Flavivirus: Flaviviridae (~70 members)

Geographic Distribution

SLEVPOWVDENV

Medically important Flaviviruses, USA

Yellow feverTBE

WNV

WNVSLEV

Confirmed human cases in the U.S. during outbreak activity

1972

1973

1974

1975

1976

1977

1978

1979

0

2500

5000

7500

10000

year

case

s

1999

2000

2001

2002

2003

2004

2005

2006

0

2500

5000

7500

10000

year

case

s

Category No. Tested No. Positive No. States

Human* NA 29,121 47

Mosquito 23 million 65,135 48

Dead bird 170,961 62,694 48

Veterinary NA 25,293 47

Sentinel animals NA 9,708 30

1,162 human deathsLikely 1.5-2 million infections

WNV Surveillance Totals, U.S., 1999-2010

* 2010 human data included through August 31

FDA

Dengue

o > 2.5 billion people at risko 50-100 million cases of DFo 250,000 – 500,000 cases of DHFo Majority (90%) of DEN cases in

continental USA are traveler relatedo Hawaii 2001

•Local transmissiono Key West 2009 -

•Local transmission•27 cases 2009/22 cases 2010

Bunyaviridae

o Neg sense, segmented RNA genome (L, M, S)

o Envelopedo Structural: GI, G2, No Non-structural: L, NSs, NSm

Schmaljohn and Hooper 2001

*LACV (80-100 cases/yr)JCVCVSSHTVT

California Serogroup Viruses*Neuroinvasive Disease Cases Reported by Year 1964-2008

o Incubation period for RVF is relatively short (3-5 days in adult humans, 12 hours in young animals)

o Fever coincides with short viremiao Viremia 3-10 days in humanso Amplitude of viremia high (>108 PFU/ml) o Lifelong IgG and neutralization antibodies in

humanso Can be transmitted by aerosol, body fluids,

mechanically by vectors

Linthicum et al. 1999

Rift Valley Fever VirusDisease in Humans

II. Surveillance protocolsClinical and Field

Issues to consider in choice of assay

o Specimeno Sensitivityo Specificityo Speedo Staffingo Safetyo $$

Challenges to arbovirus detection

o >500 viruses (≈ 100 cause disease) o Worldwide travel and movement of goods

increasingo Cross-reactivity & antigenic sino Similar disease profiles among endemic

pathogens

Biosafety in Microbiological and Biomedical Laboratories (BMBL); 5th Ed

Biosafety issues

3

Cell culture assays : BSL-3

PRNT : BSL-3

PCR : Sample homogenization and centrifugation – BSC until lysis buffer added, then BSL-2

ELISA : BSC until serum is washed

Laboratory Techniques - Clinical

SPECIMEN TYPE ASSAYSSerum

IgM, IgG, IgA - ELISALuminex (MIA)

Confirmatory PRNT

TissueReal-time RT-PCRStandard RT-PCR

Virus isolation in cell cultureImmunofluorescent antibody

Human testSerum or CSF

Goat anti-Human IgM

MAC (IgM capture)-ELISA for clinical samples

HRP-conjugatedWNV Mab

WNVantigen

Flow Cell

QuantitativeMeasure of

ExtrinsicFluorescence

Bead Identification

Laser 1 Laser 2

Y

Y *

Microsphere immunoassay

Multiplex Assays

From Mandy et al, 2001

o Assay for multiplex WN, SLE, DEN 1-4, LAC, JE, POW, MVE, YF, RR, MAY, VEE, EEE, WEE, BF, CHIK (CDC).

WN Serological DataTypical Human WN Case

In primary flavivirus infections ;Martin et al 2002: IgM P/N to WN is 2-5X greater than SLE

CSF 8 26.91 ND NDNDNDND

Flavivirus Cross-reactivities of IgM from WN Patient 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

*1:400 screening dilution> 2 is positive CDC

Problem: Ab produced by many members of the flavivirus serogroup cross-react (eg. WNV, SLEV, DENV, YFV, JEV).

o Greater antibody specificityo TWO serum samples are required for accurate results:

• Acute: collected during acute infection (0-45 d)• Convalescent: collected after recovery (3-7 w)

o Performed with suspect and related arboviruses(patient location and travel history important in choosing challenge viruses)

Confirmatory PRNT

No neutralization

neutralization

Longevity of IgMHuman WN Virus-Reactive Serum

DaysP.I.

N Positive MAC ELISA Total(%)

Ave.P/N

(Range)PositiveNo. (%)

Equivocal

200 22 13 (60) 4 17 (77) 6.0(3.0-10.8)

300-400 21 9 (43) 2 11 (52) 4.0(3.1-6.5)

500 12 5 (42) 2 7 (58) 5.0(3.1-6.9)

WNVIgM

SLEIgM

WNVPRNT

SLEPRNT

JEPRNT

YFPRNT

CASE 1 7.1 5.8 1:2560 1:2560 1:5120 1:640

WNVIgM

DENIgM

WNVPRNT

SLEPRNT

DENPRNT

YFPRNT

CASE 2 33.2 2.4 1:2560 1:1280 1:640 1:640

WN Serological DataSecondary Flavivirus Infection

Original antigenic sin

IgM Cross-Reactivity of Human CHIK Alphavirus Cases

CHIK IgM Capture ELISA (P/N)CHIK RR ONN VEE MAY EEE12.3 1.5 15.3 0.89 1.9 1.2

10.6 1.2 13.9 1.1 1.9 1.3

14.5 1.7 17.3 1.1 3.1 0.89

20.4 0.92 20.7 1.5 7.1 1.8

26.6 4.9 27.4 2.2 1.7 1.5

21.2 1.5 24.7 2.2 1.2 1.6

15.3 1.5 8 1.6 1.9 2.2

31.3 1.5 24.6 1.7 2.9 1.8

22 1.6 15.9 1.8 1.4 1.4

18.8 0.59 13.1 1.2 1.5 1.1

34.3 3.7 22.6 1.6 2.8 9.1

Laboratory Techniques - field specimens

SPECIMEN TYPE ASSAYSSerum Indirect ELISA

Competitive ELISA Luminex (MIA)

Confirmatory PRNT

Dead vertebrate tissue Real-time RT-PCRStandard RT-PCR

Virus isolation in cell cultureAntigen capture assays (VecTest)

Immunofluorescent antibodyMosquito pools

Surveillance testing of field specimens

VertebrateTissue

LaboratoryMosquitoPools

RNA

RNA PURIFICATIONTecan or Qiagen RNeasy Kits

NUCLEIC ACID ANALYSES

HOMOGENIZATION(Mixer Mill)

Real-time RT-PCR(TaqMan)

CELL CULTURE ANALYSES

EXAMINE FOR CPE

Identify by RT-PCR, IFA and/ or sequence analysis

INOCULATE VERO CELLS

LIVE VIRUS

HOMOGENIZATION(Mixer Mill) IFA on frozen tissue sections

ANTIGENIC ANALYSESPROTEIN

Antigen capture assaysVecTest

o Standard RT-PCR• Agarose gels, nested

o Real time RT-PCR• Single\multiplex format• SYBR Green

o NASBA (nucleic acid sequence based amplification)• ECL, molecular beacons

o TMA (transcription-mediated amplification)

o LAMP (Loop-mediated isothermal amplification)

Nucleic Acid Amplification Assay Platform Options

Purification of RNA from vertebrate and mosquito homogenates

o Automated o Flexible Deck Space

(component based)o Flexibles Chemistries o 96-well format

Tecan Freedom Evo 150 Robotic Station

s s

Bind Antiviral MAb to well

Unknown virus sample

Antiviral MAb conjugate

Substrate addition

Antigen Capture Assay

Hunt et al.

o Mosquito homogenateo Avian tissueo Oral / cloacal swabs of avian

carcasses•Crows & blue jays•100% agreement with assay of

brain tissues

Rapid assay for viral antigen detection VecTest™ Dip Sticks

15 min

R Nasci 2002

Antigen-capture Assay

VecTest™ assay for arbovirus antigens

Control Zone: captures unbound Ab-gold complex, confirms migration of sample through test zone.

Test Zone: virus-specific antibodies immobilized on membrane, SLE (top) and WN (bottom) in this assay.

Reagent Zone: virus-specific antibodies conjugated to gold particle.

Specificity depends on antibodies

SLEWNV

Sensitivity Threshold in log10 PFU/mlWEEVEEEVSLEVWNV

4.75.33.43.7VecTest

0.1RT-PCR

0.6 – 1.0Plaque Assay

Results

Specificity: No evidence of cross reaction (false positives) in TaqMan and VecTest even at highest virus titer.

R Nasci

Antigen AssayFrozen section from kidney of American Crow

Second antibody = FITC labeled anti-mouse IgM

West Nile Virus E protein-specific Mab (H5.46 )

Rabies glycoprotein-specific Mab (3D7)

Evan’s Blue counterstain

Cell culture assays

Normal Vero monolayer Cytopathology

Cell cultureinoculation

Daily observation

for CPE

Identification of CPE-positive specimens

o IFA on infected cell scrapings, using panel of antibodies

o RT-PCR using panel of primers/probeso Sequence analysis

WNV E protein-specific Mab (H5.46 ) FITC labeled anti-mouse IgM

Evan’s Blue counterstain

Non-WNV Arbovirus IsolatesBunyavirus

Alphavirus

CaliforniaEncephalitis

Bunyamwera

Flavivirus

Rhabdovirus

YearPools

Tested*

EEEV

HJV

JCV

LACV

TVTV

SORV

CVV

POTV

WN

V

DTV**

FLAV

2006 3987 68 1 1 3 13 1 1 14 14 1

2007 2729 20 0 2 0 0 0 1 0 0 0

2008 3616 22 0 12 0 7 4 13 49 2 10 13

2009 4345 59 24 10 6 14 0 0 0 1 4 19

2010 4179 66 11 8 0 7 0 60 0 19 10

* Culex pipiens/restuans pools were not tested and are not included in this summary** Only tick specimens were tested (638 and 233 pools in 2008 and 2009, respectively)

Organism Vector Vertebrate host Geographic distribution

Syndrome Transfusiontransmission

described

Togaviridae

Chikungunya M Humans, primates Africa, Asia, Western Pacific

A No

O’nyong nyong M Humans, primates Africa A No

Venezuelan equine encephalitis

M Rodents Americas E No

Ross River M Marsupials Australia A No

Flaviviridae

Dengue 1–4 M Humans Worldwide in tropics HF Yes

Yellow fever M Humans, primates Africa, South America HF No

Japanese encephalitis M Birds, pigs Asia E No

St. Louis encephalitis M Birds Americas E No

West Nile encephalitis M Birds Asia, Africa, Americas, Europe

E Yes

Tick-borne encephalitis T Rodents Europe, Asia E Yes

Bunyaviridae

Rift Valley fever M Domesticungulates, rodents?

Africa HF, E No

Arboviral threats to the US

L. R. Petersen & M. P. Busch 2009M, mosquitoes; T, ticks; A, arthralgia; E, encephalitis; HF, haemorrhagic fever.

Global Aviation Network

Least frequent no. passengers / day Hufnagel et al, 2004 PNAS

ACKNOWLEDGEMENTSArbovirus Laboratoryo Elizabeth Kauffmano Mary Frankeo Susan Joneso Joe Maffeio Alan Dupuis

Funding Sourceso NYS Dept of Healtho CDC

CDCo Robert Lanciotti