Journal of Veterinary Diagnostic
The online version of this article can be found at:
2012 24: 333J VET Diagn Invest
Kate E. Pennick, Christy A. McKnight, Jon S. Patterson, Kenneth S. Latimer, Roger K. Maes, Annabel G. Wise and Matti
in formalin-fixed, paraffin-embedded brain tissue of horses
West Nile virus
equine encephalitis virus
Diagnostic sensitivity and specificity of in situ hybridization and immunohistochemistry for
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Journal of Veterinary Diagnostic Investigation
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Journal of Veterinary Diagnostic Investigation
24(2) 333 –338
© 2012 The Author(s)
Reprints and permission:
Eastern equine encephalitis virus (EEEV) and West Nile
virus (WNV) are arthropod-borne viruses (arboviruses) that
cause neurologic disease in horses, human beings, and other
vertebrates. Horses are considered dead-end hosts for both
viruses, but may present with clinical signs of disease.15
Neurologic signs due to EEEV are variable and may include
hyperexcitability or other sensory changes, anorexia, ataxia,
paresis, paralysis, and obtunded mentation.5,7,15 Brain lesions
in horses affected by EEEV are variably characterized by
suppurative or nonsuppurative encephalitis with neuronal
necrosis and neutrophilic neuronophagia (Fig. 1).9,12,13 Clini-
cal signs of WNV in horses may range from subclinical
infection to muscle fasciculations and motor abnormalities
that progress to complete paralysis.12 Lesion morphology
and distribution include a mild to moderate, nonsuppurative
encephalomyelitis with moderate to severe hemorrhage that
tends to involve the lower brain stem and ventral horns of the
thoracolumbar spinal cord (Fig. 2).2,3
Both viruses are endemic among horses in the United
States, with a most recent yearly mortality of 247 EEEV
cases and 125 WNV cases being reported by the U.S.
Division of Agriculture, Animal and Plant Health Inspection
Service.19,20 Transmission of EEEV and WNV is dependent
on mosquito vector viability (particularly Culex sp.) and the
presence of avian hosts.1,6 Vaccination has reduced the fre-
quency and spread of outbreaks for both diseases among the
horse population, but the potential for high morbidity and
mortality in susceptible horses and human beings generates
continued interest in these viruses.4,10
The objective of the current study was to evaluate the diag-
nostic sensitivity and specificity of automated in situ hybrid-
ization (ISH) and immunohistochemistry (IHC) for detecting
EEEV and WNV in formalin-fixed, paraffin-embedded
(FFPE) equine brain tissues using reverse transcription
polymerase chain reaction (RT-PCR) as a confirmatory test.
Because there are no pathognomonic clinical signs that distin-
guish EEEV infection from WNV infection or any other
encephalomyelitic disease, such as rabies, hepatic encepha-
lopathy, or equine protozoal myeloencephalitis, antemortem
diagnostics for EEEV and WNV are presumptive at best.8 An
accurate postmortem diagnostic test for identifying both
EEEV and WNV in routinely formalin-fixed material from
horses is critical to provide the correct diagnosis while mini-
mizing the zoonotic risk to laboratory personnel. While
RT-PCR is considered the gold standard for the confirmation
of viral infection in tissue or body fluid,19,20 IHC and ISH both
visualize the virus within the lesion of interest, which allows
the diagnostician to correlate the microscopic lesions directly
From the Diagnostic Center for Population and Animal Health,
College of Veterinary Medicine, Michigan State University, Lansing, MI,
USA (Pennick, McKnight, Patterson, Maes, Wise, Kiupel); and Covance
Laboratories Inc., Chantilly, VA (Latimer).
1Corresponding Author: Matti Kiupel, Diagnostic Center for
Population and Animal Health, College of Veterinary Medicine, Michigan
State University, 4125 Beaumont Road 152A, Lansing, MI 48910.
Diagnostic sensitivity and specificity of in
situ hybridization and immunohistochemistry
for Eastern equine encephalitis virus and
West Nile virus in formalin-fixed,
paraffin-embedded brain tissue of horses
Kate E. Pennick, Christy A. McKnight, Jon S. Patterson, Kenneth S. Latimer,
Roger K. Maes, Annabel G. Wise, Matti Kiupel1
Abstract. Immunohistochemistry (IHC) and in situ hybridization (ISH) can be used either to detect or to differentiate
between Eastern equine encephalitis virus (EEEV) and West Nile virus (WNV) within formalin-fixed, paraffin-embedded
(FFPE) brain tissue of horses. To compare the diagnostic sensitivity and specificity of ISH and IHC, FFPE brain tissue from
20 EEEV-positive horses and 16 WNV-positive horses were tested with both EEEV and WNV oligoprobes and EEEV- and
WNV-specific antibodies. Reverse transcription polymerase chain reaction (RT-PCR) for detection of EEEV and WNV was
used as the gold standard to confirm infection. All horses that tested positive for EEEV by RT-PCR also tested positive by IHC
and ISH, except for 1 case that was false-negative by ISH. In contrast, all horses that tested positive for WNV by RT-PCR
tested negative by IHC and only 2 horses tested positive by ISH. No false-positives were detected with either method for both
viruses. Both IHC and ISH are highly specific and sensitive diagnostic methods to detect EEEV in equine FFPE brain tissues,
although neither appear effective for the diagnosis of WNV in equine neurologic cases.
Key words: Eastern equine encephalitis virus; immunohistochemistry; in situ hybridization; West Nile virus.
334 Pennick et al.
Figure 1. Horse infected with Eastern equine encephalitis virus, brain. Nonsuppurative encephalitis with neuronal necrosis and
neutrophilic neuronophagia. Hematoxylin and eosin. Figure 2. Horse infected with West Nile virus, brain. Nonsuppurative encephalitis
with perivascular cuffing and glial nodules. Hematoxylin and eosin. Figure 3. Horse infected with Eastern equine encephalitis virus,
brain. Immunohistochemical labeling (red) of infected neurons and associated dendritic processes, along with fewer glial cells. Alkaline
phosphatase detection system using V Red chromogen with hematoxylin counterstain. Figure 4. American crow infected with West Nile
virus, heart. Immunohistochemical labeling (red) of infected cardiomyocytes and inflammatory cells. Alkaline phosphatase detection system
using V Red chromogen with hematoxylin counterstain. Figure 5. Horse infected with Eastern equine encephalitis virus (EEEV), brain.
Detection of EEEV nucleic acid by in situ hybridization in infected neurons and associated dendritic processes and few glial cells. Dig-
labeled oligoprobe with alkaline phosphatase red detection system and hematoxylin counterstain. Figure 6. Horse infected with West Nile
virus (WNV), brain. Detection of WNV nucleic acid by in situ hybridization in infected neurons and associated dendritic processes and few
glial cells. Dig-labeled oligoprobe with alkaline phosphatase red detection system and hematoxylin counterstain.
In situ hybridization for EEE in paraffin-embedded horse brain 335
with the cause of disease.2,5,9 There are few reported studies
that have evaluated IHC for detection of EEEV and WNV in
FFPE tissues and, to the authors’ knowledge, only 1 report of
ISH using DNA oligoprobes for detecting EEEV in FFPE
equine tissues.2,5,9,12,18 The current study was designed to
further evaluate the diagnostic utility for ISH and IHC in
detecting EEEV and WNV in FFPE brain tissues of clinically
Formalin-fixed brain tissues from 20 EEEV-positive
horses and 16 WNV-positive horses confirmed by RT-PCR
and virus isolation, either at the National Veterinary Services
Laboratory (NVSL; Ames, Iowa) or the Diagnostic Center
for Population and Animal Health (DCPAH; College of
Veterinary Medicine, Michigan State University, Lansing,
Michigan), were used in the present study. Tissues were rou-
tinely processed and paraffin embedded, with 5-μm sections
either cut onto positively charged slides or separately sub-
mitted for RT-PCR detection. For all testing methods, 1 or
more FFPE brain tissue sections were chosen for evaluation
based on tissue availability for each animal and included sec-
tions of cerebrum, cerebellum, and/or brainstem. Only tis-
sues with microscopic lesions were selected for the study,
and serial sections of these tissues from all horses were tested
by RT-PCR, IHC, and ISH for EEEV and WNV. The RT-PCR
for EEEV was performeda,b as previously described, using
EEEV primers that produce a 112–base pair (bp) amplicon
from the viral capsid gene.11 The WNV primers amplify a
155-bp region of the viral envelope glycoprotein gene.18
Controls consisted of positive and negative EEEV or WNV
FFPE equine brain tissue; all had been previously confirmed
by RT-PCR on both the fresh and formalin-fixed tissue, as
well as by virus isolation. The resulting RT-PCR findings
included detection of EEEV in all 20 of the EEEV-positive
cases (100%) and detection of WNV in all 16 of the WNV-
positive cases (100%). All 20 EEEV cases were negative for
WNV (100%) and all 16 WNV cases were negative for
For IHC, sections of equine brain tissue were evaluated
using an antibody detection system utilizing alkaline
phosphatasec,d as previously described.14,18 A mouse mono-
clonal anti-EEEV antibodye was used as the primary anti-
body for detection of EEEV (Fig. 3).14 A mouse monoclonal
anti-WNV antibody (clone 7H2), directed against an epitope
on domain III of the E protein of WNV,f was used as the
primary antibody for detection of WNV (Fig. 4).18 Positive
and negative control tissues (avian heart and brain tissue as
WNV positive controls, equine brain tissue as EEEV posi-
tive control, and uninfected equine brain as negative con-
trols) that had been previously confirmed by RT-PCR and
virus isolation were run concurrently with the different IHC
and ISH tests. Avian brain and heart tissue was used as a
positive control due to difficulties identifying a good immu-
nohistochemically WNV-positive equine brain sample. For
negative reagent controls, the primary antibodies were
replaced by homologous nonimmune sera.
Detection of hybridized viral RNA by ISH was performed
using an automated labeling system.g,h The EEEV probe con-
sisted of a 5’ fluorescein–labeled DNA sequencei compli-
mentary to the viral E1 glycoprotein (Fig. 5).9 The WNV
probe consisted of a 5’ fluorescein–labeled DNA sequencej
complimentary to the viral envelope protein (Fig. 6). Both
sequences were previously designed by the Infectious
Diseases Laboratory, Department of Pathology, College of
Veterinary Medicine, The University of Georgia (Athens,
The resulting IHC- and ISH-labeled slides were evaluated
microscopically by a single ACVP board-certified patholo-
gist (Kiupel). Cases were determined to be either positive or
negative, with weak labeling included as positive, based on
relative levels of specific cellular labeling when compared to
negative and positive controls.
All 20 horses that had tested positive for EEEV by RT-PCR
were also positive by IHC (Table 1). Immunohistochemistry
labeling detected antigen in areas of necrosis and inflamma-
tion within the brain tissues of all EEEV cases (Fig. 3).
Neurons and associated dendritic processes, along with
fewer glial cells and astrocytes, were also positive. Some
IHC cases demonstrated increased antigen reactivity, with
stronger labeling, when compared to the same inflammatory
regions in tissues that were EEEV positive using the ISH
method. None of the horses that tested positive for WNV and
negative for EEEV by RT-PCR were positive for EEEV by
IHC. Immunohistochemistry failed to detect WNV antigen
in any of the 16 horses that had been positive for WNV by
RT-PCR and did not detect WNV antigen in any of the
EEEV-positive brain tissue sections.
All but 1 of the 20 horses that tested positive for EEEV by
RT-PCR were also positive by ISH (Table 1). Positive label-
ing was detected within areas of inflammation in the brain,
specifically within the cytoplasm and dendritic processes of
necrotic neurons (Fig. 5). Occasional normal appearing neu-
rons were also positive, and associated axons, glial cells, and
astrocytes stained positive with a more prominent, well-
dispersed chromogen deposition. None of the horses that
were WNV-positive and EEEV-negative by RT-PCR tested
positive for EEEV by ISH. Two of the 16 horses that were
positive for WNV by RT-PCR tested positive for WNV using
ISH (Fig. 6). In the 2 positive cases, chromogen deposition
was observed in the cytoplasm of a few neurons within
inflammatory regions of the cerebrum. Several areas of
inflammation were present in brain sections from all cases,
but ISH reactivity was not observed within these areas in the
other 14 samples. In situ hybridization did not detect WNV
in any of the EEEV-positive brain tissue sections.
The goal of the current study was to evaluate the diagnos-
tic sensitivity and specificity of automated ISH and IHC for
detecting EEEV and WNV in FFPE equine brain tissues. For
EEEV, IHC showed 100% diagnostic sensitivity and speci-
ficity, and ISH showed 95% diagnostic sensitivity and 100%
diagnostic specificity when compared to the gold standard of
336 Pennick et al.
RT-PCR. A 2-tiered Fisher exact test confirmed that there
was no significant deviation between the ability for IHC and
ISH to detect both EEEV and WNV antigens using RT-PCR
as the gold standard for a positive result (P = 1.00 for EEEV,
P = 0.48 for WNV). None of the EEEV cases were negative
by IHC, and only 1 case tested negative for EEEV by ISH.
Since only 2 of the 16 WNV cases were positive by ISH, and
none of the cases were positive by IHC, these tests are mini-
mally effective as diagnostic tools for detecting WNV in
FFPE equine brain.
A DNA oligonucleotide probe, representing a conserved
region of EEEV, appears to be highly diagnostically sensi-
tive and specific for detecting EEEV in FFPE equine brain
tissues using ISH methods. However, 1 tissue sample previ-
ously confirmed to be EEEV-positive by RT-PCR also tested
positive by IHC, but did not demonstrate any reactivity using
the ISH method. The inability to detect viral nucleic acid by
ISH in this single sample may be due to variable lesion local-
ization in serial sections or RNA degradation.9 It is reason-
able to assume that the viral messenger RNA and encoded
protein concentrations vary in individual tissues.
In EEEV-positive tissues, there was a tendency for some
of the IHC-labeled tissues to exhibit stronger, more intense
labeling than their ISH counterparts. Neurons in an earlier
stage of EEEV infection likely contain virions that are repli-
cating at high enough rates to meet the threshold for detec-
tion required by the ISH probe targeting the envelope protein
that was used in this experiment. Infected neurons that are in
a later stage of virus replication, where more structural pro-
teins are being created in preparation for budding, would
also have more detectable E protein present. The probe for
the present study is complimentary to the genomic positive-
sense RNA, but should also be able to bind to the generated
messenger RNA. If the sensitivity of the IHC antibody for
detecting the envelope protein is high, then the concentration
of envelope proteins per virion in an infected cell versus the
amount of detectable RNA may explain the lack of reactivity
observed in the single EEEV case that was ISH-negative but
There is a strong possibility that prolonged fixation times
resulted in stronger cross-linking and degradation of viral
RNA within cells. Due to a scarcity of case material with
confirmed RT-PCR positivity, some of the cases used in the
current study were over 5 years old. This may explain why
the false-negative ISH test was positive by IHC, as RNA is
much more sensitive to degradation compared to protein.
The intensity of labeling for ISH, and perhaps IHC, may
have been affected by the extended storage time in paraffin.
Results of the current study indicate that both ISH and IHC
are insufficient for detecting WNV in FFPE equine brain tis-
sues. In situ hybridization had a diagnostic sensitivity of 12.5%
and a diagnostic specificity of 100% when detecting WNV in
FFPE tissue, while IHC failed to detect WNV in any of the
FFPE equine brain tissues. The results were not unexpected, as
WNV viral loads are reportedly low in equine neural tissues.2,15
Also, using neuronal inflammation and necrosis as morpho-
logical hallmarks to identify appropriate areas to test for viral
antigen or nucleic acid presence may be inappropriate consid-
ering the apoptotic effects of cluster of differentiation (CD)8
host immune response following WNV infection.16,17,21
However, observation of microscopic lesions in neuronal tis-
sues commonly results in request for additional diagnostic test-
ing of such tissues.
Previous studies that demonstrated detection of WNV
using IHC in brain sections of affected horses were designed
to study the pathogenesis of the virus and its cellular local-
ization and examined large numbers of tissue sections.2
However, IHC lacks reliable diagnostic sensitivity for detect-
ing WNV in FFPE equine brain tissue and therefore is not
useful as a diagnostic test for WNV on single tissues with
histopathologic lesions. For pathogenesis studies focused on
detection of WNV nucleic acid in conjunction with histo-
pathologic lesions, the ISH method could be used despite its
low diagnostic sensitivity.
No. of samples
EEEV detectionWNV detection
In situ hybridization
In situ hybridization
Table 1. Comparison of immunohistochemistry and in situ hybridization for detecting Eastern equine encephalitis virus (EEEV) and
West Nile virus (WNV) in formalin-fixed, paraffin-embedded equine brain tissue using reverse transcription polymerase chain reaction as
the gold standard.
In situ hybridization for EEE in paraffin-embedded horse brain 337
The authors would like to thank the Immunohistochemistry
Laboratory and Virology Laboratory at the Diagnostic Center
for Population and Animal Health, Michigan State University
for their expertise in both Immunohistochemistry and in situ
hybridization; the histopathology technicians at the Diagnostic
Center for Population and Animal Health, Michigan State
University for their technical assistance; the National Veterinary
Services Laboratory in Ames, Iowa, for their work with poly-
merase chain reaction; and Dr. Christopher R. Gregory and the
Infectious Disease Laboratory, Department of Pathology,
College of Veterinary Medicine at the University of Georgia for
their initial work developing the Eastern equine encephalitis
virus ISH test.
Sources and manufacturers
a. QuantiTect SYBR Green RT-PCR Detection Kit, Qiagen Inc.,
b. iCycler iQ™ Real-Time PCR Detection System, Bio-Rad
Laboratories, Hercules, CA.
c. Enhanced V Red (Alkaline Phosphatase Red) Detection
System, Ventana Medical Systems Inc., Tucson, AZ.
d. Benchmark Automated Staining System, Ventana Medical
Systems Inc., Tucson, AZ.
e. Mouse monoclonal anti-EEE antibody, concentration 1:500,
Centers for Disease Control and Prevention, Atlanta, GA.
f. Mouse monoclonal anti-WNV antibody (clone 7H2), concen-
tration 1:2,000, BioReliance, Rockville, MD.
g. Bond™ Automated Staining System, Vision BioSystems,
h. Preset Bond™ ISH Detection Program, Leica Biosystems
Richmond Inc., Richmond, IL.
i. EEE Probe 5’ fluorescein labeled DNA sequence ACG
Integrated DNA Technologies, Coralville, IA.
j. WNV Probe 5’ fluorescein labeled DNA sequence 5’-AA
DNA Technologies, Coralville, IA.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
The author(s) received no financial support for the research,
authorship, and/or publication of this article.
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