Download DETECTION OF ST. LOUIS ENCEPHALITIS VIRUS IN

SEPTEMBER1992
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DETECTION OF ST. LOUIS ENCEPHALITIS VIRUS IN
MOSQUITOESBY USE OF THE POLYMERASE CHAIN REACTION
DANIEL K. HOWE,I MICHAEL H. VODKIN,IP ROBERT J. NOVAK,3 CARL J. MITCHELL4 AND
GERALD L, MCLAUGHLIN]
ABSTRACT. We recently developedan assay using the polymerase chain reaction (PCR) for the
specific detection of St. Louis encephalitis (SLE) virus RNA. This assaywas tested in a blind study on
7 samplesof pooled mosquitoes (50 mosquitoes/pool) which were also characterizedfor SLE virus by
plaque assayin Vero cell culture. One sample was positive for the SLE virus as determined by both the
PCR assay and a combination of the plaque assay and the indirect fluorescent antibody assay. The
remaining 6 sampleswere negative for the presenceof SLE virus as determined by both methods.These
data indicate that this PCR assay can be used to monitor for the presenceof SLE virus in pools of
homogenizedmosquitoes. This approach could provide early data on which to base diseasecontrol
decisions.
St. Louis encephalitis(SLE) virus is a major
mosquito-bornepathogen which causesperiodic
disease outbreaks throughout North, Central,
and South America (Monath 1990).Control of
SLE outbreakscurrently dependson monitoring
for SLE virus activity in the natural vertebrate
hosts and vectors (i.e., birds and mosquitoes)
and using mosquito abatement when the virus
is detected.The presenceof SLE virus has typically been detected by assaying for antibodies
to SLE virus in blood drawn from sentinel chickens or wild birds or by isolation and growth of
the virus from vertebrate hosts and mosquitoes
through intracranial inoculation of suckling
mice, infection of cell cultures, or enzyme immunoassay (Tsai et al. 1987, 1988). In various
ways, these techniques have had problems with
reliability or are laborious and time-consuming.
The polymerase chain reaction (PCR) is a
relatively new technique which has been usedto
specifically and sensitively detect a variety of
infectious pathogens. We recently developeda
PCR-based assay for the specific detection of
SLE virus RNA (Howe et al. 1992). This assay
was able to detect viral RNA spiked into a
backgroundof homogenizedmosquito abdomen.
Others have used the PCR for detecting the
RNA of SLE virus and additional flaviviruses
(Eldadahet al. 1991).The likely use for these
1Department of Veterinary Pathobiology,Schoolof
Veterinary Medicine, Purdue University, West Lafayette, IN 47907.
2Department of Veterinary Pathobiology, College
of Veterinary Medicine, University of lllinois, Urbana,
IL 61801.
3Illinois Natural History Survey, Center for Economic Entomology, Champaign, IL 61820.
n Medical Entomology-EcologyBranch, Division of
Vector-Borne Infectious Diseases,National Center for
Infectious Diseases,Centersfor DiseaseControl, Public Health Service, U.S. Department of Health and
Human Services,Fort Collins, CO 80522.
assaysis in the detection of virus RNA in mosquitoes since viremia levels in patients typically
disappearbefore clinical symptomsappear (Calisher and Monath 1988). In the current study,
we have utilized additional processingmethods
and tested our PCR-basedassayin a blind study
on pooled samples of mosquitoes collected in
August 1991 near Pine Bluff, Arkansas during
an SLE outbreak in that region (Savageet al.,
unpublished data).
Mosquitoes were collected by using either a
CDC light trap with dry ice or a CDC gravid
trap (Reiter 1987).Adult mosquitoeswere identified and pooled by speciesor taxonomic group
using dissectingmicroscopeson chill tables kept
at about 4'C. Mosquito pools (50 mosquitoesper
pool) were triturated in 2 ml of BA-1 diluent
(0.2M Tris, pH 8.0,0.15M NaCl, 1% BSA, 10
mg/l phenol red, 50 pg/ml gentamicinband one
p.g/mlFungizones)using cold mortars and pestles. Suspensionswere centrifugedat 14,000rpm
for 2 min. Supernatants were poured into 1dram screw-capvials and storedat -70"C. The
suspensionswere tested for virus by plaque assay in Vero cell culture as previously described
by Mitchell et al. (1987).
To assaythe samplesby the PCR, total RNA
was extracted from 100-pl aliquots of the mosquito suspensionsby using the RNaid PIus kit
(Bio 101, La Jolla, CA) according to the manufacturer's instructions. Briefly, the sampleswere
Iysed with guanidine isothiocyanate and extracted with acid-phenol. The RNA was bound
to a powdered-glassmatrix, washed and eluted
from the matrix in diethyl pyrocarbonate
(DEPC)-treatedwater. The RNA was eluted in
20 pl (l/5 the samplesoriginal volume, or 5x
the samples original concentration) of water,
and this 5x-concentratedRNA suspensionwas
then diluted 10-foldto 0.5Xconcentration.Twopl aliquots of the RNA suspensionswere assayed
by reversetranscription/PCR using the amplification primers SLE364.for (5'-GATCCATG-
SEPTEMBER 1992
Opnnatror.ler. eNp SctnNtrnlc NorEs
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