Susceptibility of Fox Squirrels (Sciurus niger )
to West Nile Virus by Oral Exposure
Sonthaya Tiawsirisup,1Bradley J. Blitvich,2Bradley J. Tucker,3Patrick G. Halbur,4
Lyric C. Bartholomay,3Wayne A. Rowley,3and Kenneth B. Platt2
Fox squirrels (Sciurus niger) (five of eight) were infected with West Nile virus (WNV) when challenged by the
oral route with 102.3or 103.4plaque forming units (PFU). The mean maximum serum WNV titer of infected fox
squirrels was 105.1PFU=mL and ranged from 104.6to 105.6PFU=mL. These levels of viremia are infectious for
several mosquito vectors of WNV. This virus was also isolated from swabs of the oral and rectal cavities, and
urine swabs between day 5 and 9 postexposure (p.e.) in amounts as high as 102.0, 102.8, and 102PFU, respectively.
WNV RNA was detected in salivary gland and=or kidney tissue of three squirrels between day 65 and 72 p.e. in
the presence of WNV neutralizing antibody, suggesting that long-term persistent infection occurs in fox squirrels.
These observations justify further studies to determine if nonarthropod transmission and long-term persistent
infection occur naturally in fox squirrels and contribute to trans-seasonal maintenance of WNV.
Key Words: Fox squirrels (Sciurus niger)—Oral exposure—West Nile virus.
of mosquitoes that subsequently have the potential to trans-
mit the virus to birds, humans, and other vertebrates (Root
et al. 2006, Padgett et al. 2007, Platt et al. 2008). Thus, fox
squirrels could have a role in the epidemiology of WNV, es-
pecially in peridomestic settings. Observations made by Root
et al. (2005) in areas of high WNV activity revealed that fox
53 fox squirrels representing six different locations were sero-
positive for WNV. This observation is consistent with the fact
that fox squirrels share common habitats with birds and are
study (Platt etal.2008)alsosuggestedthat foxsquirrels canbe
persistently infected with WNV, as the virus was recovered
on days 17 and 22 post exposure (p.e.) from urine and oral
swabs of two different squirrels with WNV-neutralizing
antibody. WNV RNA was also detected in kidney tissue col-
ox squirrels (Sciurus niger) can develop West Nile vi-
rus (WNV) serum titers sufficient to infect several species
lected from a third squirrel on day 29 p.e. Persistent infection
(PI) with WNV has been demonstrated in golden hamsters
(Mesocricetus auratus) and Rhesus macaques (Macaca mulatta).
Tesh et al. (2005) demonstrated WNV in hamster urine for up
to 247 days p.e. Pogodina et al. (1983) isolated WNV from
organs of rhesus monkeys, including the kidney for up to 161
days p.e. As such, it is conceivable that similar long-term PI
might occur in fox squirrels. If so, long-term PI of squirrels
periodic WNV shedding occurred in amounts sufficient to
infect WNV-antibody-free squirrels by close contact, includ-
ing the oral route. While fox squirrels are not considered
gregarious mammals, their normal behavior would facilitate
nonarthropod transmission of shed virus. For example, it is
not uncommon for fox squirrels to share the same tree-hole
cavity during winter months. There is also extensive interac-
tion among males during the breeding season which in
northern temperate latitudes generally occurs between the
1Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
2Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa.
3Department of Entomology, College of Agriculture and Life Sciences, Iowa State University, Ames, Iowa.
4Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames,
VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 10, Number 2, 2010
ª Mary Ann Liebert, Inc.
middle of December through June with peak activity in
December=January and June. Females generally produce two
litters of two to five individuals a year with the majority of
births occurring in early spring and mid-summer (Flyger and
Gates 1982). Thus, the purpose of this study was to establish
whether fox squirrels are susceptible to WNV infection per os.
conducted todeterminetheinfection rates, sheddingpatterns,
and viremia profiles after oral exposure to WNV. Fox squir-
rels were live-trapped in Story County, IA, in the spring of
2007 and housed in Biosafety Level 3 animal facilities at Iowa
State University (ISU). Squirrels were chemically restrained
with ketamine:acepromazine given intramuscularly, initially
at 0.44:0.44mg=kg. This dose was subsequently adjusted for
individual squirrels to provide restraint without abolishing
the pharyngeal reflex. Virus inoculum was WNV strain IA02-
crow (Erickson et al. 2006) in 0.5mL of CO2-independent
medium (Invitrogen, Carlsbad, CA) containing 1% fetal bo-
vine serum. The inoculum was delivered slowly to permit
swallowing. Five squirrels were challenged with 102.3plaque
forming units (PFU) of WNV, and the other five were chal-
because these amounts of WNV are frequently recovered
from swabs of the oral cavity, rectum, and urine swabs of
acutely infected squirrels (Root et al. 2006, Platt et al. 2008).
Blood and swabs of the oral cavity, rectum, and urine, when
possible, were collected as previously described (Platt et al.
2008) from all squirrels before inoculation and on alternate
days beginning on day 1 or 2 p.e., continuing through day 9
p.e., and intermittently thereafter including the day of death.
All samples were maintained at 48C after collection until
stored at ?708C. Swab and serum samples were tested for
WNV by virus isolation in African Green Monkey Kidney
(Vero) cells. Virus titers were measured by plaque assay, and
reduction neutralization test 90 (PRNT90) following standard
protocols (Platt et al. 2008).
Tissues representing brain, spinal cord, salivary glands,
lung, heart, liver, kidney, adrenal glands, spleen, pancreas,
small intestine, and large intestine were collected from all
eight squirrels at necropsy, processed for immunohistochem-
istry and histopathological examination using standardized
protocols at the Veterinary Diagnostic Laboratory (VDL) at
ISU, and evaluated blindly by a veterinary pathologist. Virus
isolation attempts were made on all tissues except intestines,
from WNV-infected squirrels with the exception of squirrel 4
that died on day 17 p.e. Fresh salivary gland and kidney tis-
sues from these squirrels were also assayed for WNV RNA,
because salivary gland and kidney tissue are likely sources of
potential virus shedding. These tissues were stored at ?708C
in RNAlater (Ambion, Austin, TX) until processed for total
RNA extraction using TRIzol LS Reagent (Invitrogen) fol-
lowing the manufacturer’s protocol. Real-time RT-PCR was
conducted by the Veterinary Diagnostic Laboratory on these
tissues using a standardized protocol based on the method
described by Lanciotti et al. (2000). All protocols were per-
formed in accordance with the ISU Institutional Animal Care
and Use Committee.
The response of fox squirrels to oral exposure to WNV is
summarizedin Table 1.Briefly, five squirrels developed WNV
viremias and three failed to become viremic. Two squirrels
died without a detectable viremia on day 4 p.e. during sam-
pling and were subsequently excluded from the study. The
mean maximum serum WNV titer for the five viremic squir-
WNV was recovered between day 5 and 9 p.e. from swabs of
the oral cavity, and rectum of four squirrels, and from urine
swabs of two of these four squirrels. The maximum amount of
WNV recovered from these swabs was 102.0, 102.8, and 102.3
the shedding pattern of fox squirrels infected with WNV by
needle or mosquito bite observed earlier (Platt et al. 2008),
except that the onset of shedding in orally infected squirrels
by real-time RT-PCR in tissues of the kidney (two squirrels)
Histological lesions or WNV antigen were not detected in any
squirrel except squirrel 4, which died on day 17 p.e. (Table 1).
WNV was not isolated from internal organs of any squirrel.
Fox squirrels are susceptible to oral infection by amounts of
WNV that are commonly recovered from the oral cavity,
rectum, and urine of squirrels with active infections. More
squirrels (Table 1) are sufficient to infect several competent
Table 1. Response of Fox Squirrels (Sciurus niger) to Per Os Exposure to WNV
Detection of WNV RNASerum WNV titer by day postexposure
on death dayKidney
aLog10PFU=mL delivered in 0.5mL of CO2-independent cell culture medium.
bHistological findings were mild multifocal gliosis and satellitosis in the brain and spinal cord, moderate multifocal lymphoplasmacytic
and histiocytic interstitial nephritis, and mild multifocal lymphoplasmacytic hepatitis.
PRNT90, plaque reduction neutralization titer90expressed as the reciprocal of the final serum dilution that reduced plaque counts by 90%;
–, not done.
208TIAWSIRISUP ET AL.
species of Culex and Aedes mosquitoes (Sardelis et al. 2001,
Turell et al. 2001, Goddard et al. 2002, Tiawsirisup et al. 2008).
It is important to note that the levels of WNV viremia that
occurred in these orally infected squirrels may have been
greater than observed because of sampling constraints. Al-
though the detection of WNV RNA in kidney and salivary
gland tissue at 65 to 72 days p.e. in the absence of infectious
virus is not proof of PI, it is suggestive and justifies further
studies to determine if long-term PI with periodic shedding
occurs in fox squirrels.
The research reported in this article was supported by the
Centers for Disease Control and Prevention Cooperative
Agreement Number 1 R01 C1000267-01 for Applied Research
in Emerging Infections: Investigations of West Nile virus.
This is a journal paper of the Iowa Agriculture and Home
Economic Experiment Station, Ames, IA (Project 5033), sup-
ported in part by the Hatch Act and State of Iowa funds. We
thank Flor Fabiosa for providing technical assistance.
The contents are the sole responsibility of the authors and
do not necessarily represent the official views of the Centers
for Disease Control and Prevention. No competing financial
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Address correspondence to:
Kenneth. B. Platt
Department of Veterinary Medicine and Preventive Medicine
College of Veterinary Medicine
Iowa State University
Ames, IA 50011
FOX SQUIRRELS’ ORAL EXPOSURE TO WEST NILE VIRUS209
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