Vet Pathol 44:849862 (2007)

Equine Multinodular Pulmonary Fibrosis: A Newly Recognized

Herpesvirus-Associated Fibrotic Lung Disease
K. J. WILLIAMS, R. MAES, F. DEL PIERO, A. LIM, A. WISE, D. C. BOLIN, J. CASWELL, C. JACKSON,
N. E. ROBINSON, F. DERKSEN, M. A. SCOTT, B. D. UHAL, X. LI, S. A. YOUSSEF, AND S. R. BOLIN
College of Veterinary Medicine, Michigan State University, East Lansing, MI (KJW, RM, AL, AW,
NER, FD, MAS, SRB); School of Veterinary Medicine, New Bolton Center, University of
Pennsylvania, Kennett Square, PA (FDP); Livestock Disease Diagnostic Center, Lexington, KY (DCB);
Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada (JC); Equine Specialty
Hospital, Burton, OH (CJ); College of Natural Science, Michigan State University, East Lansing, MI
(BDU, XL); and Animal Health Laboratory, University of Guelph, Guelph, Ontario, Canada (SAY)
Abstract. Pulmonary fibrosis and interstitial lung disease are poorly understood in horses; the causes
of such conditions are rarely identified. Equine herpesvirus 5 (EHV-5) is a c-herpesvirus of horses that
has not been associated with disease in horses. Pathologic and virologic findings from 24 horses with
progressive nodular fibrotic lung disease associated with EHV-5 infection are described and compared
with 23 age-matched control animals. Gross lesions consisted of multiple nodules of fibrosis throughout
the lungs. Histologically, there was marked interstitial fibrosis, often with preservation of an alveolarlike architecture, lined by cuboidal epithelial cells. The airways contained primarily neutrophils and
macrophages. Rare macrophages contained large eosinophilic intranuclear viral inclusion bodies; similar
inclusion bodies were also found cytologically. The inclusions were identified as herpesviral-like particles
by transmission electron microscopy in a single horse. In situ hybridization was used to detect EHV-5
nucleic acids within occasional macrophage nuclei. With polymerase chain reaction (PCR), the
herpesviral DNA polymerase gene was detected in 19/24 (79.2%) of affected horses and 2/23 (8.7%) of
the control horses. Virus generaspecific PCR was used to detect EHV-5 in all of the affected horses and
none of the control horses. EHV-2 was detected in 8/24 (33.3%) of affected horses and 1/9 (11.1%) of the
control horses. This disease has not been reported before, and the authors propose that based upon the
characteristic gross and histologic findings, the disease be known as equine multinodular pulmonary
fibrosis. Further, we propose that this newly described disease develops in association with infection by
the equine c-herpesvirus, EHV-5.
Key words:

Equine herpesvirus 5; gamma-herpesvirus; horses; lung; pulmonary fibrosis.

Interstitial lung disease is a poorly understood

entity in adult horses (Equus caballus).35 As with all
veterinary species, a variety of toxic or infectious
agents are implicated in targeting the alveolus and
interstitium to induce interstitial pneumonia in
horses, but these agents are rarely identified at the
time of histologic diagnosis.35 Interstitial fibrosis
has been reported as a major component of
interstitial lung disease in individual horses, as well
as in a retrospective study of idiopathic interstitial
lung disease in 20 horses in Florida by Buergelt et
al.6,8,36 Additionally, Kleiboeker et al.13 reported on
a single donkey (Equus asinus) that had pulmonary
interstitial fibrosis among a group of donkeys with
asinine herpesvirus-associated interstitial pneumonia. Interstitial fibrosis is a prominent feature of
silicate pneumoconiosis in horses from the Monterey-Carmel peninsula of California,27 but there is

little evidence of other factors associated with

fibrotic lung disease in the horse.
Herpesviridae is a large family of DNA viruses
that is subdivided into three subfamilies (a, b, and c)
based upon genetic, biologic, and morphologic
characteristics.17 Eight herpesviruses have been
identified in equids: 5 in the a-herpesvirinae subfamily and 3 in the c-herpesvirinae subfamily. Of
those 8 viruses, 5 naturally infect the domestic horse
and 3 infect the donkey.22 In horses, the aherpesviruses equine herpesvirus 1 (EHV-1) and
EHV-3 clearly cause clinical disease. The role in
disease for the remaining a-herpesvirus, EHV-4, is
unclear, although it has been implicated in upper
respiratory infections.22 EHV-2 and EHV-5 are
closely related c-herpesviruses of horses.1,23,30
Whereas evidence of infection in horses with EHV2 and EHV-5 is reported as relatively common in

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850

Williams, Maes, Del Piero, Lim, Wise, Bolin, Caswell, Jackson, Robinson, Derksen,
Scott, Uhal, Li, Youssef, and Bolin

parts of Europe, Argentina, Australia, and New

Zealand, association between infection with these
viruses and disease is inconclusive at this
time.3,7,20,25,33
Herein we provide the first description of the
gross pathology, histopathology, and virology
findings of 24 horses with a newly recognized
fibrotic lung disease that is associated with infection with EHV-5. Whereas this association does
not imply causation, to our knowledge, this report
is the first to document an association between
a viral infection and lung fibrosis in any veterinary
species and the first to describe this disease. Based
on the characteristic gross and histologic findings
of this disease, the authors propose that the disease
be termed equine multinodular pulmonary fibrosis
(EMPF). Further, we propose that this newly
described disease develops in association with an
infection with the c-herpesvirus EHV-5.
Materials and Methods
Tissue Collection and Processing
All of the EMPF animals underwent euthanasia after
a period of severe respiratory disease that did not
respond to therapy (Table 1). When the clinical history
was available, the animals were described as being
mildly febrile (100.5102.8uF), with clinical signs referable to pulmonary disease; numerous discrete to coalescing nodular densities were visible within the lungs
when thoracic radiographs were taken. The horses
underwent necropsy examination, and a standard series
of tissues was collected in 10% neutral-buffered formalin
for histopathology. Frozen lung was collected from 11
EMPF-suspect horses at the time of necropsy (Table 2);
for the remaining 13 cases, histologic and virologic
analyses were performed on formalin-fixed, paraffinembedded lung (Table 2). Tissues from 23 age-matched
control horses (Table 3) were collected at the time of
necropsy for analysis (Table 4). The control group
consisted of 18 horses obtained from cases submitted
to Michigan State University, University of Pennsylvania, and University of Kentucky for pathologic examination. These animals were selected for inclusion in the
study based upon similar age to the affected horses and
having previously been diagnosed with pulmonary
fibrosis of unknown cause. An additional 5 control
horses were from a long-term study of recurrent airway
obstruction at Michigan State University. Routine
evaluation of the control lung tissues for viral agents
at the time of diagnostic workup did not identify any
significant pathogens; chronic bacterial pneumonia was
present in 2 of the control horses.
Tissues for histopathology were routinely processed
and embedded in paraffin for sectioning and placement
on glass slides. All tissue sections were stained with
hematoxylin and eosin. Masson trichrome staining was
performed to visualize the extracellular matrix within
the lung. Impression smears of the lung from horse

Vet Pathol 44:6, 2007

No. 4 of the affected group were prepared on glass slides

immediately after euthanasia for cytologic examination
and were treated with Wright-Giemsa stain.
Transmission Electron Microscopy
For examination of tissues for viral particles, fresh
lung was obtained from horse No. 4 and fixed in 4%
glutaraldehyde. The postfixation processing consisted of
rinsing in 0.1 mol/liter phosphate buffer and placement
in osmium tetroxide (Electron Microscopy Sciences,
Fort Washington, PA). The tissue was rinsed again in
the phosphate buffer, transferred into 2% uranyl
acetate, and rinsed in a graded series of ethanol. The
tissues were placed in propylene oxide (Electron
Microscopy Sciences) before being embedded in 2,4,6tris(dimethylaminomethyl)phenol and araldite 501
(Electron Microscopy Sciences). Sections were cut at
1-mm thickness using an LKB ultramicrotome
(Bromma, Sweden) and stained with toluidine blue.
Tissues of interest were sectioned at 60 nm, stained with
uranyl acetate and lead citrate (Electron Microscopy
Sciences), and examined on a Phillips 301 electron
microscope (Atlanta, GA).
Polymerase Chain Reaction (PCR) Assays

Extraction of DNA. DNA was extracted from

either fresh-frozen lung or formalin-fixed, paraffinembedded lung (Tables 2, 4). For fresh-frozen lung,
approximately 400500 mg of tissue with $1 clearly
visible fibrotic regions was macerated with a razor
blade, suspended in 4 ml of M199 cell culture medium,
and placed in a 15-ml conical tube containing 5 coppercoated ball bearings that were 4.5 mm in diameter. The
contents of the tube were then agitated in a vortex at
maximum power for 1 minute. After centrifugation at
1,700 3 g for 10 minutes, a 200-ml aliquot of supernatant was removed and placed in a 1.5-ml microcentrifuge tube. Twenty microliters of a 0.5% proteinase K
solution that contained 10 mM Tris buffer (pH 8.3),
50 mM KCl, and 0.5% v/v polyoxyethylene sorbitan
monolaurate was added to the tube, and the mixture was
incubated at 56uC overnight. For formalin-fixed paraffin embedded lung, three 20-mmthick sections of tissue
were placed in a 1.5-ml microcentrifuge tube. The
formalin-fixed tissue was digested by addition of 200
ml of ATL buffer, followed by the addition of 20 ml of
proteinase K solution (DNeasy tissue kit; QIAGEN
Inc., Valencia, CA). This mixture also was incubated at
56uC overnight. After digestion, the mixture from the
fresh-frozen or the formalin-fixed tissue was centrifuged
at 18,000 3 g for 3 minutes. For paraffin-embedded
tissues, the floated paraffin layer was removed with
a toothpick. Next 200 ml of AL buffer was added to the
microcentrifuge tubes, and the instructions for the
DNeasy Tissue Kit (Qiagen Inc.) were followed through
elution of the DNA in 50 ml of nuclease-free water.
PCR assays. Initially, all samples of DNA were
tested using a nested PCR that detects most members of
the Herpesviridae family of viruses by targeting con-

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Equine Herpesvirus-Associated Lung Fibrosis

Vet Pathol 44:6, 2007

Table 1. Characteristics of EMPF horses.*

Horse No.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

Age (years)

Gender

Breed

12
14
10
12
4
4
19
12
14
13
15
17
23
28
7
11
20
14
17
19
8
15
14
26

MC
MC
MC
MC
F
F
MC
F
F
MC
MC
F
F
F
F
F
MC
M
F
F
F
MC
F
M

Thoroughbred
Andalusian
Thoroughbred
Thoroughbred
Thoroughbred
Thoroughbred
Arabian
Quarter Horse
Warmblood
Oldenburg
Thoroughbred
Thoroughbred
Thoroughbred
Thoroughbred
Westphalian
Thoroughbred
Thoroughbred
Thoroughbred
Thoroughbred
Mixed breed
Belgian
Thoroughbred
Thoroughbred
Thoroughbred

* EMPF 5 equine multinodular pulmonary fibrosis; MC 5 male, castrated; F 5 female; M 5 male.

Table 2. Equine herpesvirus PCR data: EMPF horses.*

Horse No.

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24

Lung source

General HV

Gp EHV-5

Gp EHV-2

Packaging gene

FFPE
FFPE
Fresh
Fresh
FFPE
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
FFPE
FFPE
FFPE
FFPE
Fresh
FFPE
FFPE
FFPE
FFPE
Fresh
FFPE
FFPE

+
+
+
+
+
+
+
+
+
+
+
+
2
+
2
2
+
+
2
+
+
+
2
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

2
2
+
2
2
2
+
2
+
+
+
+
2
2
+
2
+
2
2
2
2
2
2
2

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

* PCR 5 polymerase chain reaction; EMPF 5 equine multinodular pulmonary fibrosis; FFPE 5 formalin-fixed paraffin
embedded; Gp 5 glycoprotein; EHV 5 equine herpesvirus.

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Williams, Maes, Del Piero, Lim, Wise, Bolin, Caswell, Jackson, Robinson, Derksen,
Scott, Uhal, Li, Youssef, and Bolin

852

Vet Pathol 44:6, 2007

Table 3. Characteristics of control horses.*

Horse No.

Age (years)

Gender

Breed

9
18
10.5
28
13
21
28
3
9
13
9.5
16
24
42
15
19
10
18
13
29
24
10
22

MC
MC
MC
MC
MC
F
F
MC
MC
MC
F
MC
F
M
MC
F
F
F
MC
F
MC
F
F

Quarter Horse
Quarter Horse
Quarter Horse
Mixed breed
Mixed breed
Quarter Horse
Quarter Horse
Standardbred
Thoroughbred
Warmblood
Mixed Breed
Tennessee Walker
Thoroughbred
Shetland Pony
Thoroughbred
Appaloosa
Miniature
Mixed pony
Oldenburg
Arabian
Mixed breed
Thoroughbred
Mixed breed

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

* MC 5 male, castrated; F 5 female; M 5 male.

Table 4.

Equine herpesvirus PCR data: control horses.*

Horse No.

Lung source

General HV

Gp EHV-5

Gp EHV-2

Packaging gene

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

FFPE
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
Fresh
FFPE
FFPE
FFPE
FFPE
FFPE
FFPE
FFPE
FFPE
FFPE
FFPE
Fresh
Fresh
Fresh
Fresh

2
+
2
2
2
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2

2
2
ND
2
ND
2
ND
2
2
ND
ND
ND
ND
ND
2
ND
2
ND
ND
ND
2
ND
2

ND
+
ND
2
ND
2
ND
2
2
ND
ND
ND
ND
ND
2
ND
2
ND
ND
ND
2
ND
2

2
+
2
+
2
+
2
2
2
2
2
2
2
2
+
2
+
2
2
2
2
2
+

* PCR 5 polymerase chain reaction; FFPE 5 formalin-fixed paraffin embedded; Gp 5 glycoprotein; EHV 5 equine
herpesvirus; ND 5 assay not done.

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Vet Pathol 44:6, 2007

853

Equine Herpesvirus-Associated Lung Fibrosis

served regions within the herpesviral DNAdirected

DNA polymerase gene.24,31 The primer sequences and
reaction conditions for this general herpesvirus PCR
assay and all other PCR assays used during this study
are given in Table 5. The 25-ml PCR mixture used to
detect the DNA polymerase gene consisted of PCR
buffer with 1.5 mM MgCl2, 200 mM each dNTP, 1.25
U of Taq polymerase (QIAGEN Inc.), 1.0 mM each
primer, 2.5 ml of DNA for the primary amplification (1
ml of the primary amplification product for the
secondary amplification), and molecular biologygrade
water to volume. The final amplification product was
approximately 210250 bp. Amplification products (10
ml) were analyzed in ethidium bromidestained 1.5%
agarose gels after electrophoresis in sodium borate
buffer (5 mM disodium borate decahydrate, pH adjusted to 8.5 with boric acid).4 Gel images were captured
with a Kodak EDAS 290 imaging system (Eastman
Kodak Co., Rochester, NY).
Samples of DNA were further tested using several
PCR assays specific for genera and subfamilies of
Herpesviridae. Those PCR assays targeted the viral
DNA packaging protein gene of equine c-herpesviruses,13 the viral glycoprotein H (gH) gene of EHV-2
or EHV-5,20 the viral glycoprotein B (gB) gene of EHV1,11 and the viral UL24 homolog gene of EHV-4 (inlaboratory development at Michigan State University).
The PCR mixture for the DNA packaging gene was the
same as above, and the product of the PCR was an
amplicon of approximately 297 bp. A hemi-nested set of
PCR primers was used to detect the gH gene of EHV-2,
and a single set of primers was used to detect the gH
gene of EHV-5.20 The PCR amplification products were
354 bp for EHV-2 and 344 bp for EHV-5. The PCR
mixtures for those assays contained 2X reaction buffer
with 1.5 mM MgCl2, 200 mM each dNTP, 0.625 U of
Taq polymerase (Promega, Madison, WI), 0.3 mM each
primer, 2.5 ml of DNA (1 ml of DNA for the secondary
amplification of EHV-2), and molecular biologygrade
water to a final volume of 25 ml.
Real-time PCR assays specific for the EHV-1 gB gene
or EHV-4 UL24 gene homolog were used to detect the
presence of these equine a-herpesviruses. The PCR assay
for EHV-1 used a TaqMan probe as a detector of PCR
amplification,11 whereas the PCR assay for EHV-4 used
SYBR Green followed by standard postamplification
melt-curve analysis. The reaction mixture for EHV-1 was
made from the Taq PCR master mix kit (QIAGEN Inc.)
with 500 nM each primer, 240 nM probe, 2.5 ml of 25 mM
MgCl2, 7 ml of DNA, and molecular biologygrade water
to a final volume of 25 ml. The reaction mixture for EHV4 was made from 25 ml of Quantitect SYBR Green PCR
mastermix (QIAGEN Inc.) with 500 nM each primer, 5 ml
of DNA, and molecular biologygrade water to a final
volume of 50 ml. The amplification product was 302 bp,
and the peak melting temperature was 84.5uC. The
iCycler iQ5 real-time detection system (Bio-Rad Laboratories Inc., Hercules, CA) was used for both of these
PCR assays. Positive controls consisted of DNA from
known isolates from EHV-1, EHV-2, EHV-4, and EHV-

5; negative controls consisted of the reaction mixture with

molecular-grade water and no DNA.
In Situ Hybridization
Complementary hybridization probes were designed
from the EHV-5 gH gene sequences derived in this
study, using the Clone Manager Professional Suite
version 7.11 software (Scientific & Educational Software, Cary, NC). Specificity of the probes was checked
against aligned sequences of the EHV-2 gH gene. The
probes were labeled on the 59 end with digoxigenin. The
probe sequences were synthesized by Integrated DNA
Technologies (Coralville, IA). A similarly labeled nonsense probe specific for the ISMAP02 gene of Mycobacterium avium subspecies paratuberculosis was used as
a control. In situ hybridization was performed on 6
EMPF horses and 5 controls essentially as described
previously.21,34 Briefly, deparaffinized slides were hybridized with the digoxigenin-labeled oligonucleotide
probes specific for the gH gene sequences. Then lung
sections were incubated with alkaline phosphatase
conjugated anti-digoxigenin antibodies, followed by
the NBT/BCIP detection system to amplify the chromogen signal. The digoxigenin-labeled probes used
were:
IS MAP02: 59-GCC ACC ACC GTC TTG ACC
GCC TCA AAG ATC -39
Glycoprotein H-1F: 59-TCT GAA AGC AAG CTG
GTT TCT AAC CGC ACC -39
Glycoprotein H-1R: 59-GGT GCT GTT AGA AAC
CAG CTT GCT TTC AGA -39
Virus Isolation
Virus isolation was performed on tissues from 7 of the
EMPF horses and 12 control horses, and previously
reported standard techniques were followed for EHV5.28 Bovine viral diarrhea virusfree rabbit kidney (RK13) cells were grown in Eagle minimum essential
medium supplemented with 10% fetal bovine serum,
0.29 mg/ml glutamine, 100 IU/ml penicillin, and 100 mg/
ml streptomycin. Semiconfluent monolayers were inoculated with a lung homogenate from 1 of the affected
horses, first clarified by low-speed centrifugation and
then passed through a filter with an average pore
diameter of 0.45 mm. The inoculum was adsorbed onto
the cultures in a small amount of medium for 1 hour at
room temperature. The cells were washed, re-fed, and
incubated at 37uC in an atmosphere of 5% CO2. The
inoculated cells were observed daily for the appearance
of cytopathic changes. Five passages of the inoculated
cell cultures were at an interval of 7 days between
passages. Confirmation of viral infection of the cells was
accomplished using the PCR methods outlined above.

Results
Summary of Cases

A total of 24 EMPF horses (Table 1) and 23 agematched control horses (Table 3) were included in
the study. The average age of the EMPF horses was

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Williams, Maes, Del Piero, Lim, Wise, Bolin, Caswell, Jackson, Robinson, Derksen,
Scott, Uhal, Li, Youssef, and Bolin

Vet Pathol 44:6, 2007

Table 5. Primers and reaction conditions for PCR assays.

PCR target/primer

pol gene*
DFA forward
ILK forward
KG1 forward
TGV forward
IYG reverse
Pkg gene{
EHVc forward
EHVc reverse

EHV-2 gH{
gHEF forward
gHEB reverse

gHEF forward
gHEB2 reverse
EHV-5 gH1
EHV-5/1 forward
EHV-5/2 reverse

EHV-1 gB||
EHV-1 forward
EHV-1 reverse
Probe#
UL24 gene"
EHV-4 forward
EHV-4 reverse

Primer sequences

Reaction conditions

First-round PCR
GAYTTYGCNAGYYTNTAYCC
TCCTGGACAAGCAGCARNYSGCN
MTNAA
GTCTTGCTCACCAGNTCNCANCC
YTT
Second-round PCR
TGTAACTCGGTGTAYGGNTTYCA
NGGNGT
CACAGAGTCCGTRTCNCCRTADAT
AACTCCTCSGACCAGACCA
SACCACCTTGTGCATGTTG

First-round PCR
ATGCTCTCTGACAAGAATCACTAC
TGTTGTAGACAATGGGAATCTG

Second-round PCR
ATGCTCTCTGACAAGAATCACTAC
CGAGACAAACATCTTTTTCTCT
TAACCTCCGCGACACGTTTTCA
TAGACATCACCGCAGAAACCACAA

GCTCTCAGGTTTTACGACATC
CTTTACCCAGGGCCCTTGAAA
TCAACGTGGACAATACCGCAGTGA
TTAT

1 cycle at 95uC for 15 minutes; 35 cycles at

95uC for 30 seconds, 46uC for 60 seconds,
and 72uC for 60 seconds; 1 cycle at 72uC
for 5 minutes
1 cycle at 95uC for 15 minutes; 40 cycles at
95uC for 30 seconds, 46uC for 60 seconds,
and 72uC for 60 seconds; 1 cycle at 72uC
for 5 minutes
1 cycle at 95uC for 15 minutes; 10 cycles at
95uC for 30 seconds, 67uC (21uC/cycle)
for 20 seconds, and 72uC for 60 seconds;
40 cycles at 95uC for 30 seconds, 57uC for
20 seconds, and 72uC for 60 seconds; 1
cycle at 72uC for 7 minutes
1 cycle at 94uC for 4 minutes; 5 cycles at
94uC for 30 seconds, 60uC (21uC/cycle)
for 30 seconds, and 72uC for 45 seconds;
30 cycles at 94uC for 30 seconds, 60uC for
30 seconds, and 72uC for 45 seconds; 1
cycle at 72uC for 5 minutes
1 cycle at 94uC for 4 minutes; 30 cycles at
94uC for 30 seconds, 55uC for 30 seconds,
and 72uC for 30 seconds; 1 cycle at 72uC
for 5 minutes; 1 cycle at 94uC for
4 minutes; 5 cycles at 94uC for 30 seconds,
60uC (21uC/cycle) for 30 seconds, and
72uC for 45 seconds; 30 cycles at 94uC for
30 seconds, 60uC for 30 seconds, and 72uC
for 45 seconds; 1 cycle at 72uC for
5 minutes
1 cycle at 95uC for 10 minutes; 45 cycles at
95uC for 15 seconds, 60uC for 60 seconds;
1 cycle at 95uC for 15 minutes; 40 cycles at
94uC for 30 seconds, 55uC for 30 seconds,
and 72uC for 30 seconds

CGGTTGATATCCCACAGCC
AACGAGGAATGCGGGTGTTG

PCR 5 polymerase chain reaction; EHV 5 equine herpesvirus.

* PCR primers that target the viral DNA polymerase gene and detect most herpesviruses.
{ PCR primers that detect the equine c-herpesvirus DNA packaging gene.
{ PCR primers that detect the EHV-2 glycoprotein H gene.
1 PCR primers that detect the EHV-5 glycoprotein H gene.
|| PCR primers that detect the EHV-1 glycoprotein B gene.
# TaqMan probe for the EHV-1 glycoprotein B gene.
" PCR primers that detect the EHV-4 UL24 gene homolog.

14.5 years; 11 were males and 13 were females.

There were 16 Thoroughbreds and 1 of each of the
following breeds: Andalusian, Arabian, Belgian,
mixed breed, Oldenburg, Quarter Horse, Standardbred, and Westphalian (Table 1). The average age

of the control horses was 17.5 years; 13 were males

and 10 were females (Table 3). There were 5
Quarter Horses, 5 mixed-breed horses, 4 Thoroughbreds, 3 ponies, and one of each of the
following breeds: Standardbred, Warmblood, Ten-

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Vet Pathol 44:6, 2007

Equine Herpesvirus-Associated Lung Fibrosis

nessee Walker, Appaloosa, Oldenburg, and Arabian (Table 3).

Gross Pathology

The most significant gross lesions were restricted

to the lungs and involved all lung regions. There were
2 distinct gross manifestations of EMPF, the more
common being numerous coalescing nodules of
fibrosis (Figs. 1, 2). The individual nodules ranged
from ,1 to 5 cm in diameter. The nodules were pale
tan-white and moderately firm; the borders were
discrete where they met unaffected lung. Little
unaffected lung is usually present when this form
of the disease is recognized. The less common gross
lesion consisted of multiple discrete nodules separated by grossly unaffected lung; these nodules may be
confused with a neoplastic process (Figs. 3, 4). These
masses of fibrosis were larger than the coalescent
form of the disease (up to 10 cm), pale tan-white, and
firm, and larger areas of grossly normal lung were
between the nodules. In each form of the disease, the
cut surface of the nodules was uniformly colored,
and the foci of fibrosis bulged slightly from the
surrounding tissue. Coincident with the lung lesions,
the bronchial lymph nodes in the affected horses
were markedly enlarged in 12/24 EMPF horses (not
shown). The gross lesions of EMPF were distinctive
and did not resemble the lesions of any of the control
horses. In the control horses, fibrosis was not the
primary gross manifestation of disease in most cases
and did not involve the extent of the lung field as seen
with EMPF (see histologic description below). In 5
of the control animals (Nos. 1, 10, 11, 12, and 16),
nearly diffuse fibrosis was prominent as part of
a granulomatous inflammatory process; unlike
EMPF, discrete granulomas with mineralization
were found in these 5 animals.
Histopathology and Cytology

Significant histologic findings in EMPF were

restricted to the lung and bronchial lymph nodes.
The lung lesions were largely confined to the
alveolar parenchyma and were similar regardless
of the gross pathology. Nodules were sharply
demarcated from adjacent less affected lung
(Fig. 5). The nodules consisted of marked interstitial expansion by well-organized mature collagen
(Figs. 68). The interstitium was infiltrated by
variable numbers of mixed inflammatory cells,
consisting primarily of lymphocytes, with smaller
numbers of macrophages, neutrophils, and occasional eosinophils. There was preservation of an
alveolar-like architecture, with the lumen of the
spaces lined by cuboidal cells (Figs. 68). Less
commonly, the fibrosis was less well organized,

855

with the collagen deposition arranged in broad

interlacing bundles that did not preserve the
alveolar-like architecture (Fig. 9). The lumen
contained moderate numbers of neutrophils and
macrophages, and rarely there were large macrophages with abundant eosinophilic cytoplasm and
a large oval eosinophilic intranuclear viral inclusion body (Fig. 10). The histology of the
bronchial lymph nodes consisted of lymphoid
hyperplasia, often with sinus histiocytosis and
occasional multinucleate giant cells (not shown);
viral inclusion bodies were not noted within cells
within the lymph nodes.
The imprints of fresh lung from horse No. 4 of
the affected group of horses were highly cellular;
intact cells were mostly neutrophils, macrophages,
and rafts of relatively uniform epithelial cells that
often appeared cuboidal and had moderately
basophilic cytoplasm. There were fewer mast cells
and rare small clusters of spindle cells. Macrophages were often vacuolated, and low numbers
were multinucleated. Low numbers of macrophages were enlarged (approximately 40 mm versus
20 mm for uninfected) and had large nuclei
containing a finely granular, centralized eosinophilic intranuclear inclusion (Fig. 11); nucleoli were
often located peripherally. Bacteria or other infectious agents were not found.
The histologic characteristics of the fibrosis of
EMPF (described above) were distinct from the
pattern of collagen deposition found in the control
animals. Fibrosis was a significant histologic
feature in 14/23 of the control horses. In the
control animals, 5 of the animals had lung fibrosis
as part of granulomatous pneumonia of unknown
cause (control horses Nos. 1, 10, 11, 12, and 16), 5
had chronic interstitial pneumonia with fibrosis of
unknown cause (control horses Nos. 13, 14, 15, 17,
and 18), 2 had fibrosis in the lung secondary to
chronic bacterial pneumonia (control horses Nos.
19 and 20), and fibrosis was a feature of a single
case of chronic hypertension (control horse No. 22)
and of metastatic adenocarcinoma to the lung
(control horse No. 21). The diagnoses associated
with the remaining control horses included lymphoma (control horse No. 9), chronic recurrent
airway obstruction (control horses Nos. 2, 4, 5, 6,
and 7), disseminated intravascular coagulation with
pulmonary thrombosis (control horse No. 3),
chronic Cushing disease (control horse No. 23),
and congestion and edema associated with acute
heart failure (control horse No. 8). All of the cases
of EMPF were histologically distinguishable from
control lungs with fibrosis based upon the nodular
pattern of collagen deposition, presence of luminal

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Fig. 1. EMPF lungs; horse No. 4. Gross pathology of EMPF, diffuse nodular form. The pulmonary
parenchyma fails to collapse, and rib impressions are visible on the pleural surface.
Fig. 2. EMPF lungs; horse No. 4. Gross pathology of EMPF, diffuse nodular form, lung cut section. The
parenchyma is nearly replaced by coalescent, variably sized, pale tan, moderately firm nodules of fibrosis.
Fig. 3. EMPF lungs; horse No. 5. Gross pathology of EMPF, discrete nodular form. Discrete nodules of
fibrosis can be seen scattered throughout the lung beneath the pleural surface (arrow).
Fig. 4. EMPF lungs; horse No. 5. Gross pathology of EMPF, discrete nodular form, lung cut section. The
discrete nodules of fibrosis are large and have sharp borders between adjacent nodules and grossly normal
lung (arrow).
Fig. 5. EMPF histopathology; horse No. 6. Histologically, there is a discrete border between the foci of
interstitial fibrosis and the relatively unaffected lung (arrow). HE. Bar 5 200 mm.

neutrophils and macrophages within the alveolarlike architecture, and finding intranuclear viral
inclusion bodies within the enlarged macrophages.
In Situ Hybridization

The digoxigenin label was localized only to the

affected areas of the lungs of the EMPF horses;
there was no specific labeling within the adjacent
unaffected lung or within the lungs of the control
horses. The label was found within the nuclei of
scattered macrophages within the alveolar-like
airspaces in the fibrotic lung (Fig. 12). There was no
specific labeling with the nonsense probe or when
the protocol excluded the virus-specific probe.

Transmission Electron Microscopy

Viral particles were identified only within the

nuclei of macrophages within the lumen of the
airspaces within the fibrotic lung. The chromatin of
the affected cells was at the periphery of the
nucleus, adjacent to the nuclear membrane
(Fig. 13). Some nuclei contained numerous fully
formed icosahedral capsids, with sizes (100 nm)
and hexagonal shapes consistent with herpesvirus
(Fig. 14).
PCR from Lung Tissue

All samples from control horses and EMPF

horses were screened in PCR assays with primers

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Equine Herpesvirus-Associated Lung Fibrosis

Vet Pathol 44:6, 2007

specific for Herpesviridae family (viral DNA polymerase gene), c-herpesvirus subfamily (viral DNA
packaging protein gene), or virus genera (EHV-1,
EHV-2, EHV-4, and EHV-5) (Table 5). PCR
analysis of fresh lung tissue or of tissue derived
from archived paraffin-embedded lung was used to
detect the presence of herpesviral DNA polymerase
gene in 19/24 (79%) affected horses, whereas 2/23
(8.7%) control horses were positive. Failure to
detect the herpesvirus DNA polymerase gene in the
affected horses was restricted to the formalin-fixed,
paraffin-embedded tissues; this is due to the
inefficiency of amplification of such large amplicons from DNA extracted from formalin-fixed,
paraffin-embedded tissues. When virus genera
specific PCR was performed, all of the affected
horses were positive for EHV-5 and 8/24 (33%)
affected horses were positive for EHV-2. None of
the horses in the control group that were positive
for the herpesvirus DNA polymerase gene or for
the c-herpesvirus DNA packaging protein gene
were positive for EHV-5. Tissue from 1 of the
horses in the control group was positive for EHV-2.
Nucleic acid sequencing (data not shown) of the
amplicons generated by the PCR assays confirmed
the specificity of the PCR products. Lung tissue
from all affected and control horses were negative
for EHV-1 and EHV-4 using the aforementioned
PCR assays. Six control horses were positive for
the c-herpesvirus packaging gene. Of these 6, 1
horse was positive for EHV-2; the other 5 were
negative for EHV-5 and EHV-2 using the aforementioned primer sets. Nucleic acid sequence
analysis of the putative c-herpesvirus detected in
those 5 horses is ongoing.
Virus Isolation

EHV-5 was isolated from 2/7 EMPF horses

(horses Nos. 2 and 6), and EHV-2 was isolated
from 1/7 (horse No. 8). Cytopathic changes were
first observed in the RK-13 cells on passage 3, and
they developed slowly. The cytopathic effect for
EHV-5 consisted of foci of infected cells, with some
evidence of ballooning (Fig. 15); these foci became
more numerous upon additional passages. The
EHV-5specific PCR was strongly positive for
DNA extracted from passage 5 cells. A virus stock
prepared with passage 5 material had a titer of 3.2
3 105 50% tissue culture infective dose per ml.
EHV-5 was not isolated from any of the control
horse lungs.
Discussion
Interstitial lung disease is an uncommon event
in adult horses, and interstitial fibrosis as a major

857

component of such lung disease is very poorly

understood.35 Interstitial lung fibrosis has been
reported as part of the granulomatous process in
an apparent silicate pneumoconiosis in horses
from the Monterey-Carmel peninsula of California,27 but there is little evidence of other factors
associated with fibrotic lung disease in the horse.
EHV-5 is a relatively recently described c-herpesvirus that, thus far, has not been associated with
clinical disease in equids.1,2,30 The current study
describes a newly recognized nodular fibrotic lung
disease of horses that is associated with pulmonary infection with EHV-5. The virus was
identified in all of the affected horses using virus
generaspecific PCR, was isolated from 2/7
affected horses, and was visualized using transmission electron microscopy. In addition, in situ
hybridization demonstrated viral genome within
the pulmonary lesions but not in adjacent unaffected lung or within the lungs of control horses.
Because of the characteristic gross and histologic
findings and the identification of c-herpesvirus
EHV-5 in affected animals, the authors propose
that this newly recognized disease be known as
EMPF; further, we propose that this disease is
associated with infection with EHV-5.
Idiopathic interstitial pneumonia with fibrosis,
sometimes referred to as idiopathic pulmonary
fibrosis, has been sporadically reported in individual horses, as well as in a retrospective study of 20
horses in Florida.6,8,35,36 In the report by Buergelt et
al.,6 6 of the 20 horses were older adults, and
interstitial fibrosis was described as a major feature
of the disease in this subset of animals. The authors
suggested that the fibrosis may be a sequela to
previous diffuse alveolar damage, as noted in the
series of foals from the same study.6 With the
description of EMPF in the current study and the
ability to identify the presence of the virus in
archived paraffin-embedded lung tissue, it will be
interesting to survey additional animals for this
newly recognized virus-associated pulmonary disease. Whereas this report is the first to thoroughly
describe the pathology of EMPF and its association with a viral infection, it seems likely that the
disease has been present for years; similar cases
have been reported by other pathologists for at
least 15 years (J. M. King, personal communication). The pattern of lung fibrosis, the paucity of
viral inclusion bodies, and an incomplete general
understanding of viral infections and tissue fibrosis
may have contributed to this disease going unreported for some time.
Recently Kleiboeker et al.13 reported on a series
of 17 donkeys (Equus asinus) with asinine herpes-

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Vet Pathol 44:6, 2007

Fig. 6. EMPF histopathology; horse No. 6. In most cases, there is preservation of an alveolar-like
architecture, which is lined by cuboidal epithelial cells. HE. Bar 5 100 mm.
Fig. 7. EMPF histopathology; horse No. 6. Higher magnification of interstitial fibrosis with inflammatory cells
within lumen of the airspace; the inflammatory cells are primarily neutrophils and macrophages. HE. Bar 5 50 mm.
Fig. 8. EMPF histopathology; horse No. 6. Histochemical staining of interstitial fibrosis. The fibrosis (blue) in

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Equine Herpesvirus-Associated Lung Fibrosis

859

Fig. 12. EMPF lung in situ hybridization; horse

No. 6. EHV-5 DNA within nuclei of scattered alveolar
macrophages (arrows). In situ hybridization with
digoxigenin-labeled DNA oligonucleotide probe for
the EHV-5 gH gene. Alkaline phosphatase/NBT/BCIP
chromogen, no counterstain. Bar 5 50 mm.

virus-associated interstitial pneumonia. The described gross and histopathologic lesions of the
asinine disease differ from EMPF. EMPF is
a distinctly nodular disease, with a relatively sharp
border of demarcation between the fibrotic and
unaffected lung, whereas asinine herpesvirus-associated interstitial pneumonia of donkeys is described as a coalescing inflammatory disease with
syncytial cell formation. The authors described
areas of interstitial fibrosis associated with the
disease but emphasized the inflammatory component rather than fibrosis as the primary finding
within the lungs of affected donkeys.13 Further,
viral inclusion bodies were not identified in the
donkeys, whereas low numbers of intranuclear viral
inclusions were regularly found in the affected
horses. Because of these differences, we consider
EMPF to be a distinct disease of the domestic horse
(Equus caballus).
The identification of this virus as EHV-5 is the
first published association of this virus with

Fig. 13. Lung ultrastructure EMPF; horse No. 4.

Electron micrograph of an alveolar macrophage with an
intranuclear inclusion; central viral capsids, and capsid
proteins; note marginated chromatin (arrow).

a disease. There is limited information on the

prevalence of EHV-5 infection in horses. Most
studies have identified the presence of the virus in
nasal swabs or peripheral blood leukocytes using
PCR, and there has been no clear association with
disease in the studied animals.3,20,23,33 There is no
information on the prevalence of EHV-5 infection
within the lower respiratory tract of horses. Earlier
studies found that EHV-5 was detected in peripheral blood leukocytes at a later age and much lower
frequency than EHV-2, suggesting that infection

r
most animals is well organized around the airspaces. Masson trichrome. Bar 5 50 mm.
Fig. 9. EMPF histopathology; horse No. 22. The fibrosis can be arranged in unorganized bands, without
appreciable preservation of alveolar-like airspaces. HE. Bar 5 50 mm.
Fig. 10. EMPF histopathology; horse No. 6. Large eosinophilic intranuclear viral inclusion body (arrow) in
a large macrophage within the inflammation in the airspaces of the nodular lesions. HE. Bar 5 20 mm.
Fig. 11. EMPF lung imprint cytology; horse No. 4. Vacuolated macrophages and neutrophils are intermixed.
One macrophage (left) is markedly enlarged and has a single large eosinophilic intranuclear inclusion that
marginates 2 nucleoli. Wright-Giemsa stain. Bar 5 10 mm.

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Vet Pathol 44:6, 2007

Fig. 15. Rabbit kidney cells (RK-13) infected with

EHV-5 (cytopathic effect). Cells surrounding a focus of
lysis (plaque) are swollen (ballooning degeneration;
arrow).

Fig. 14. Lung ultrastructure EMPF; horse No. 4.

Higher magnification electron micrograph of assembled
herpesviral capsids and surrounding capsid proteins;
note icosahedral shape of capsids (arrow).

occurs later in life in most horses and is of low

prevalence in equine populations.20 Recently
a study by Bell et al.3 detected EHV-5 in 64% of
nasal samples of young racehorses, and Wang et
al.3,33 detected EHV-5 in 48% of nasal swabs from
5- to 9-month-old Thoroughbred foals. This is an
interesting finding, given the age of the horses with
EMPF, and suggests that this disease, if related to
the viral infection, may have a prolonged lag period
between exposure to the virus and development of
the disease. Our studies failed to identify EHV-5 in
any of the control horses, but uniform infection in
the horses with EMPF was found, suggesting that
it may play a role in the development of the disease.
Detection of the c-herpesvirus packaging gene, but
neither of the two known c-herpesviruses of horses
(EHV-2 or EHV-5), in 5 of the control horses is an
interesting finding and suggests that there may be
additional unidentified c-herpesviruses in equine
lung tissue.
In situ hybridization identified viral genome
within the regions of fibrosis but not in the adjacent

unaffected lung or in the control horse lungs. Virus

was detected within luminal macrophages of the
EMPF lungs. This pattern of cellular infection is
unusual for a c-herpesvirus, for which the primary
target cells for infection are usually B and T
lymphocytes.2 Infection of alveolar epithelial cells
has been reported to occur following experimental
infection with the murine c-herpesvirus MHV-68,
and this virus has also been associated with
experimental fibrotic lung disease in mice.10,16,26
The appearance of the infected alveolar macrophages bearing the inclusion bodies is suggestive of
cytomegalovirus (CMV) infection. CMV belongs to
the b-herpesvirus subfamily of the Herpesvirinae
family.2 Inclusion bodies are not a common feature
of c-herpesviral infections, although there is a recent report of c-herpesvirus infection and intranuclear inclusion bodies within the epithelium of
genital carcinomas of California sea lions.14 Interestingly, EHV-5 was initially classified within the
b-herpesvirus subfamily based upon shared biologic characteristics and the structure of the
genome;5 it was only after analysis of nucleotide
and amino acid sequences that the virus was placed
within the c-herpesviruses.30 Our preliminary sequencing data from the amplicon from the DNA
polymerase gene (general herpesvirus primers)
indicates that the virus in question is a not a bherpesvirus but a c-herpesvirus. Work is underway
in our laboratory to further the molecular phylogenetic classification of this virus to determine its
relationship to the reported EHV-5.
This is the first report of an association between
a spontaneous, naturally occurring viral infection
and fibrotic lung disease in veterinary medicine.

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Equine Herpesvirus-Associated Lung Fibrosis

There is considerable interest and controversy as to

what role, if any, viral infections play in human
interstitial lung diseases and pulmonary fibrosis.15,32
In humans, a variety of viral infections have been
implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), including hepatitis C virus,
adenovirus, CMV, and Epstein-Barr virus.15 Of
these, there is the greatest amount of data concerning herpesviruses, especially Epstein-Barr, and
IPF.9,12,29 Tang et al.29 used PCR to identify
herpesviral DNA in 97% of human IPF cases
compared with only 36% of non-IPF cases. Of the
viruses that were investigated, human CMV, Epstein-Barr, and human herpesvirus 8 (HHV-8) were
most commonly detected within the IPF lung;29 both
Epstein-Barr and HHV-8 are c-herpesviruses,
whereas CMV is a b-herpesvirus. Similar to the
study by Tang et al.,29 we used PCR analysis to
survey both affected and control horses for 4
different equine herpesviruses (EHV-1, EHV-2,
EHV-4, and EHV-5); we detected EHV-5 only in
the EMPF cases, suggesting that EHV-5 is specifically associated with EMPF and is not an incidental
finding in diseased equine lungs. EHV-2 was found
as a co-infection in 8 of the EMPF horses; this is
likely not associated with development of EMPF, as
most affected horses did not carry this virus within
the lung. The actual prevalence of EHV-2 in the
control horses is likely higher than reported here, as
the nested PCR used to detect the virus is not as
effective with DNA extracted from formalin-fixed,
paraffin-embedded tissues.
How c-herpesviruses may drive pulmonary
fibrosis is not known at this time. In murine
models of pulmonary fibrosis, lung infection with
the murine c-herpesvirus MHV-68 can act synergistically with bleomycin to potentiate the degree of
fibrosis, although the virus alone did not result in
lung fibrosis in infected mice.16 In Th-2biased,
interferon-cR2/2 mice, MHV-68 infection can
result in the development of progressive lung
fibrosis, as well as increased expression of important markers of a profibrotic lung environment,
including increases in transforming growth factor
b, production of Th-2 cytokines, and myofibroblast
transformation.18 Interestingly, many c-herpesviruses encode viral homologs of a variety of
cytokines and chemokine receptors.19 These proteins may assist the virus in evading detection by
the immune system and push the immune response
away from a Th-1 response and toward a Th-2
response.19 This shift in cytokine and chemokine
profile may predispose the lung to a progressively
fibrotic phenotype. Work is ongoing to evaluate
the c-herpesvirus associated with EMPF for similar

cytokine and chemokine receptor homologs as

a beginning step toward understanding the pathogenesis of this disease.
Acknowledgements
We thank Dr. Elizabeth Howerth at the University of
Georgia for submitting a case for the study and Dr.
Perry Habecker at New Bolton Center for contributing
gross and histologic evaluation of several of the cases
from the University of Pennsylvania.

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Request reprints from Dr. Kurt J. Williams, G380 Veterinary Medical Center, Michigan State University, East
Lansing, MI 48824 (USA). E-mail: williamsk@dcpah.msu.edu.

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