http://jgv.sgmjournals.org/content/89/7/1699.full.pdf

Journal of General Virology (2008), 89, 1699–1708
DOI 10.1099/vir.0.2008/000554-0
Ovine herpesvirus 2 replicates initially in the lung of
experimentally infected sheep
Hong Li,1 Cristina W. Cunha,1 Christopher J. Davies,23
Katherine L. Gailbreath,1 Donald P. Knowles,1,2 J. Lindsay Oaks2
and Naomi S. Taus1
1
Correspondence
Animal Diseases Research Unit, United States Department of Agriculture-Agriculture Research
Service, Washington Sate University, Pullman, WA 99164, USA
Hong Li
[email protected]
Received 23 January 2008
Accepted 14 March 2008
2
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA
99164, USA
Ovine herpesvirus 2 (OvHV-2), a rhadinovirus in the subfamily Gammaherpesvirinae, is the
causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal
lymphoproliferative disease primarily of ruminants worldwide. Inability to propagate the virus in
vitro has made it difficult to study OvHV-2 replication. Aerosol inoculation of sheep with OvHV-2
from nasal secretions collected from naturally infected sheep during shedding episodes results in
infection of naive sheep, providing an excellent system to study OvHV-2 initial replication in
the natural host. In this study, we showed that OvHV-2 delivered through the nasal route by
nebulization resulted in infection in all lambs, but no infection was established in any lambs after
intravenous or intraperitoneal injection. In nebulized lambs, while it was not detected initially in any
other tissues, OvHV-2 DNA became detectable in the lung at 3 days post-infection (p.i.),
increased to about 900 copies per 50 ng DNA at 5 days p.i., reached peak levels (~7500 copies)
at 7 days p.i., and then declined to an average of 800 copies at 9 days p.i. Transcripts of OvHV-2
open reading frame 25 (coding for the capsid protein), an indicator of virus replication, were
only detected in lung tissues, and were positively correlated with OvHV-2 DNA levels in the lungs.
In addition, selected immune response genes were also highly expressed in the lung at 5 and
7 days p.i. The data indicate that lung is the primary replication site for OvHV-2 during initial
infection in sheep and suggest that viral replication is promptly controlled by a host defence
mechanism.
INTRODUCTION
Ovine herpesvirus 2 (OvHV-2) is a member of the
subfamily Gammaherpesvirinae, which includes a number
of important viruses that are associated with lymphoproliferative diseases and lymphoid tumours in humans and
animals, such as Epstein–Barr virus (EBV), Kaposi’s
sarcoma associated herpesvirus (KSHV) and murine
herpesvirus 68 (MHV68) (Roizman, 2000). Domestic and
wild sheep serve as reservoirs for the virus. Infection with
OvHV-2 in the reservoir species is usually subclinical.
However, infection often results in a fatal disease called
sheep-associated malignant catarrhal fever (SA-MCF)
when the virus is transmitted to other, less well-adapted
hosts, primarily ruminant species, such as cattle, bison and
3Present address: Department of Animal, Dairy and Veterinary Sciences,
Center for Integrated BioSystems, 4700 Old Main Hill, Utah State
University, Logan, UT 84322-4700, USA.
Primers used for real-time RT-PCR are presented in a table available
with the online version of this paper.
2008/000554
Printed in Great Britain
deer (Plowright, 1990; Crawford et al., 1999). SA-MCF has
been recognized as the cause of significant economic losses
in several species, including farmed deer in New Zealand
(Mackintosh, 1993), Bali cattle in Indonesia (Daniels et al.,
1988), farmed bison in North America (Li et al., 2006;
O’Toole et al., 2002; Schultheiss et al., 2000) and a wide
variety of ruminants in zoological collections (Heuschele &
Fletcher, 1984).
Increasing evidence has suggested that the pathobiology of
OvHV-2 may be different from that of alcelaphine
herpesvirus 1 (AlHV-1), a closely related MCF virus that
is carried by wildebeest, although both viruses cause similar
disease syndromes and lesions in clinically susceptible hosts
(Plowright, 1990). Earlier studies showed that viral
transmission and shedding patterns are different between
OvHV-2 and AlHV-1 in their natural hosts: sheep shed the
virus sporadically with a short-lived episode and new born
lambs are not the source of infection (Li et al., 2004), while
most newborn wildebeest calves are infected and shed virus
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1699
H. Li and others
continuously until 3–4 months of age and are the
primary source of transmission (Mushi & Wafula,
1983). Recent studies revealed that the transcripts of
the open reading frame (ORF) 25, a gene encoding a
capsid protein, were present in virtually all tissues of
cattle, bison (Cunha et al., 2007) and rabbits (Gailbreath
et al., 2008) with OvHV-2-induced MCF, but not present
in the tissues (spleen and lymph nodes) of rabbits with
AlHV-1-induced MCF (Dewals et al., 2008), suggesting
that virus lytic replication and latency are different in
clinically susceptible hosts. While AlHV-1 readily grows
in cell culture including several cell lines, OvHV-2 has
not been successfully propagated in vitro, suggesting that
their cell tropisms and/or in vitro culture requirements
are also different.
determined. This replication appeared to be localized,
which is consistent with the suggestion that this replication
is limited possibly to a single cycle of replication (Li et al.,
2004). This also suggests that virus shed from turbinate
cells may be incapable of reinfecting turbinate cells. If true,
OvHV-2 tropism may vary during the life cycle, with virus
shed from turbinate cells infecting other type(s) of cells in
the respiratory tract to establish infection in recipient sheep
via nasal transmission. To investigate this possibility, we
determined the tissue site(s) where OvHV-2 initial
replication takes place when virus from sheep nasal
secretions is transmitted by nebulization.
The lack of a cell culture system to propagate OvHV-2 in
vitro has constrained the ability to perform controlled
transmission studies and studies of pathogenesis. However,
much progress has been made in the past decade due to
advances in molecular technologies. OvHV-2 epidemiology
and transmission in its natural host, domestic sheep, were
studied using more advanced molecular tools (Li et al.,
1994, 2001; Baxter et al., 1993; Hussy et al., 2001).
Although still controversial, most data show that the
majority of lambs are not infected until after 2 months of
age under natural flock conditions (Li et al., 1998).
Placental transmission rarely occurs in sheep, and colostrum and milk from infected ewes have a very limited role
in viral transmission, even though they contain virusinfected cells (Li et al., 1998, 1999). Newborn lambs are
equally susceptible to infection as adults via aerosol
transmission (Taus et al., 2005), and thus the lack of
infection in the majority of lambs under 2 months of age is
probably due to the relatively low levels of virus shed by
adult sheep in the environment and inefficient infection
rates (Li et al., 2002, 2000). Experimental infection of sheep
has always been problematic, not only because of a lack of
infectious virus, but also due to the lack of OvHV-2-free
sheep. Recognition of the short window of time when
lambs are free of infection during early life under natural
flock conditions (Li et al., 1998) resolved the problem and
provided an opportunity to develop a programme for
production of OvHV-2-free sheep (Li et al., 1999). A study
of OvHV-2 shedding kinetics in sheep using quantitative
real-time PCR revealed that adolescent lambs between 6
and 9 months of age shed virus more frequently and
intensively than adults (Li et al., 2004). These studies have
made it possible to establish a source of infectious virus by
collection of nasal secretions from sheep during shedding
episodes. The virus collected by this method has been
successfully used for experimental infection in carrier
species (Taus et al., 2005), as well as in clinically susceptible
species (Taus et al., 2006; O’Toole et al., 2007). OvHV-2
predominantly replicates in cells in nasal turbinate when
naturally infected sheep experience intensive shedding
episodes (Cunha et al., 2007), although the specific type(s)
of cells that support lytic replication has not been
Animals and experimental groups. Twenty-two lambs approxi-
1700
METHODS
mately 3 months old were obtained from an MCF virus-free flock that
is maintained at Washington State University, Pullman, WA, USA in
accordance with an approved animal care and use protocol. All lambs
were seronegative for MCF viral antibody by competitive inhibition
ELISA (cELISA) and no OvHV-2 DNA was detected in their peripheral
blood leukocytes (PBL) using nested PCR. As shown in Table 1, the
lambs were divided into four groups: group 1, consisting of 13 lambs
(nos 1-IN+ to 13-IN+), was nebulized with 2 ml pooled nasal
secretions containing 107 OvHV-2 DNA copies; group 2 had five
animals (nos 14-IN2 to 18-IN2) nebulized with 2 ml nasal secretions
collected from OvHV-2 uninfected sheep as a control group; groups 3
and 4 had two lambs each, which were inoculated intravenously (group
3, nos 19-IV+ and 20-IV+) or intraperitoneally (group 4, nos 21-IP+
and 22-IP+) with the same dose of virus used in group 1. The
nebulization procedure was described previously (Li et al., 2004).
Sheep nasal secretion inoculum. Sheep nasal secretion inoculum
for this study was collected using the same protocol as described
previously (Taus et al., 2005). Briefly, nasal secretions were collected
daily for 3 months from 15 OvHV-2-positive sheep that were 6 months
of age at the beginning of the collection period. Nasal secretions were
collected from the sheep using multiple swabs within 5 to 6 h of the
initial sampling. High shedder sheep are defined as those sheep whose
initial samples contained ¢100 000 copies of OvHV-2 DNA per 2 mg
total DNA. Diluted secretions were clarified, aliquoted and stored in
liquid nitrogen, using 5 % chicken ovalbumin. After confirming the
presence of high viral DNA copy numbers, all preparations from
individual shedding sheep were combined to make a pooled inoculum,
and the inoculum was stored in liquid nitrogen. OvHV-2 DNA copies
from the pooled inoculum were quantified by real-time PCR. The
OvHV-2 infectivity of the pooled inoculum used in this study was
quantified in sheep using a protocol described previously (Taus et al.,
2005). The minimum infectious dose of this pooled inoculum for sheep
was about 103 OvHV-2 DNA copies, which was comparable to the
inoculum used in previous studies (Taus et al., 2005).
Sample collection and preparation. Lambs in group 1 nebulized
with sheep nasal secretions containing infectious OvHV-2 were
euthanized in pairs at 1, 3, 5, 7 and 9 days p.i., and the remaining
three lambs in the group were euthanized at 56 days p.i. The lambs
nebulized with nasal secretions from OvHV-2-negative sheep (group
2) were euthanized at 0 (nos 14-IN2 and 15-IN2) and 56 (nos 16IN2, 17-IN2 and 18-IN2) days p.i., respectively. Both lambs in each
group inoculated intravenously (19-IV+ and 20-IV+) or intraperitoneally (21-IP+ and 22-IP+) with virus were euthanized at 56 days
p.i. Blood and nasal secretion samples were collected daily from each
lamb for the first 9 days; only blood samples were collected weekly
until the termination of the experiment. Multiple tissues (n526) from
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Journal of General Virology 89
OvHV-2 replication in sheep
Table 1. Summary of the animal experimental groups
Group
Designated lamb ID*
1
2
3
4
+
1-IN
2-IN+
3-IN+
4-IN+
5-IN+
6-IN+
7-IN+
8-IN+
9-IN+
10-IN+
11-IN+
12-IN+
13-IN+
14-IN2
15-IN2
16-IN2
17-IN2
18-IN2
19-IV+
20-IV+
21-IP+
22-IP+
OvHV-2 inoculum
Route of inoculation
Days p.i. at termination
+D
+
+
+
+
+
+
+
+
+
+
+
+
2d
2
2
2
2
+
+
+
+
Intranasal
1
1
3
3
5
5
7
7
9
9
56
56
56
0
0
56
56
56
56
56
56
56
Intranasal
Intravenous
Intraperitoneal
*IN+, Intranasal inoculation with sheep nasal secretions containing infectious OvHV-2 by nebulization; IN2, intranasal inoculation with the nasal
secretions from OvHV-2-negative sheep; IV+, intravenous inoculation with sheep nasal secretions containing infectious OvHV-2; IP+,
intraperitoneal inoculation with sheep nasal secretions containing infectious OvHV-2.
D2 ml sheep nasal secretions containing 107 copies of OvHV-2 DNA.
d2 ml nasal secretions collected from OvHV-2-negative sheep.
various organs, especially those from the respiratory tract, were
collected during necropsy. The tissues of the most interest included
trigeminal ganglia, tonsil, pharynx, nasal mucosa, salivary gland,
turbinate from six locations (both left and right caudal, middle and
rostral), trachea from upper, middle and lower sections, and lung
from caudal, middle and cranial lobes. Additional tissues collected
were brain (periventricular and cortex), lymph nodes (retropharyngeal and mesenteric), spleen, liver, kidney, urinary bladder, large
intestine and small intestine. All collected tissues were snap frozen
and stored either at 280 uC or in liquid nitrogen for later use. The
tissues used for histopathology were fixed in 10 % neutral-buffered
formalin and then embedded into paraffin blocks.
Plasma from EDTA-blood samples was collected for the MCF viral
antibody assay and stored at 220 uC for later use. Total DNA for PCR
assays was extracted from PBL samples, nasal secretion samples, and
tissues using the FastDNA kit as described by the manufacturer
(QBiogene). Total RNA for ORF25 RT-PCR was extracted from
tissues using TRIzol Reagent as described by the manufacturer
(Invitrogen). After DNase treatment, the RNA was purified a second
time by TRIzol and stored at 280 uC until use. The total RNA used
for the cytokine real-time PCR was purified using TRIzol Plus RNA
Purification System (Invitrogen). The RNA was purified with TRIzol,
treated with DNase, then purified a second time with the PureLink
Micro-to-Midi Total RNA Purification System (Invitrogen). Purified
RNA was stored at 280 uC until use.
cELISA, nested PCR, real-time PCR, RT-PCR and real-time RTPCR. cELISA, which utilizes a monoclonal antibody against an
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epitope conserved among all MCF viruses examined to date (Li et al.,
1994), was used for the detection of MCF viral antibody. The protocol
for the cELISA was described previously (Li et al., 2001).
A nested PCR and a real-time PCR were used to detect and quantify,
respectively, OvHV-2 DNA. Both assays have been described
previously (Li et al., 1995; Traul et al., 2007) and used different
primer sets targeting the same region of OvHV-2 ORF75 (Baxter et
al., 1993; Hussy et al., 2001).
An RT-PCR assay using primers targeting OvHV-2 ORF25, which
codes for a major capsid protein, one of the OvHV-2 structural
proteins expressed in the late stage of replication, was used to identify
virus replication. RNA was reverse-transcribed and amplified in a
single step using the OneStep RT-PCR kit as described by the
manufacturer (Qiagen). The detailed procedure for the RT-PCR has
been described in a previous report (Cunha et al., 2007). Briefly,
RNA samples (100 ng) were added on ice to each 50 ml reaction
[16 Qiagen OneStep RT-PCR Buffer, 400 mM each dNTP, 16
Q-Solution, 0.4 mM specific ORF25 forward primer (59-ACTGCGGACGTGGCCTACTT-39), reverse primer (59-GTCCAGGAGGGCTCGGTGTG-39) and 2.0 ml Qiagen OneStep RT-PCR Enzyme Mix].
Negative RT control reactions, to confirm the absence of DNA
contamination, were performed by replacing the One Step RT-PCR
mix by a Hot Start Taq mix (Qiagen) in the reaction. The same RTPCR cycling conditions were used except that the transcription step
(50 uC for 30 min) was omitted. Amplification of cellular GAPDH
was used to ensure that RNA was present and of adequate quality to
be amplified.
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1701
H. Li and others
A real-time RT-PCR for analysis of the relative expression levels of
selected sheep immune response genes was developed. Total RNA
(1 mg) from each tissue sample was reverse-transcribed using
oligo(dT) primers and the Superscript III First-Strand Synthesis
System (Invitrogen) according to the manufacturer’s specifications.
The selected immune response genes, forward and reverse primers,
product lengths and GenBank accession numbers of the sequences
used to design the primers are listed in Supplementary Table S1
(available in JGV Online). All primer pairs were designed to target
areas with minimal secondary structure, to work at an annealing
temperature of 60 uC and, where feasible, to span two exons. Realtime PCR was performed by using SYBR GreenER Master Mix
(Invitrogen) in 25 ml reactions. Primers were used at a final
concentration of 200 nM. Reactions were performed in an iCycler
(Bio-Rad) with initial cycle (50 uC for 2 min, and 95 uC for 8 min
and 30 s), followed by 40 amplification cycles (95 uC for 15 s and
60 uC for 1 min). A melt curve was performed for each reaction to
confirm specificity of the amplicons; cycler parameters were 95 uC for
1 min, 55 uC for 1 min, followed by 80 cycles of increasing
temperature in 0.5 uC increments (10 s each) beginning at 55 uC.
All samples were run in duplicate. The data were analysed using the
22DDCT method (Livak & Schmittgen, 2001). Each value was
normalized to the values for the housekeeping genes, GAPDH and
b-actin, obtained from the same sample and the relative expression
levels (fold increase) were determined using the values from RNA
samples collected at 0 day p.i. as the calibrator.
RESULTS
MCF viral antibody in plasma
As shown in Fig. 1, MCF viral antibody was detected by
cELISA at 9 days p.i. and thereafter from the sheep that
were nebulized with the nasal secretions containing
infectious OvHV-2. MCF viral antibody was not detected
in any of the control sheep that were nebulized with the
nasal secretions collected from OvHV-2 uninfected sheep.
Both sheep inoculated intravenously with the virus
remained MCF viral antibody-negative until the termination of the experiment. However, one of two animals
inoculated intraperitoneally with the virus became transiently seropositive from 12 to 17 days p.i., while the other
remained seronegative (Fig. 1b).
OvHV-2 DNA in nasal secretions, PBL and tissues
OvHV-2 DNA was not detected by either real-time PCR or
nested PCR in any samples collected from the lambs that
were nebulized with nasal secretions from OvHV-2
uninfected sheep or from the animals inoculated either
intravenously or intraperitoneally with virus. In the group
of lambs nebulized with the nasal secretions containing
infectious OvHV-2, viral DNA was detected by nested PCR
in only a small percentage of tissues from 1 to 7 days p.i.,
ranging from 1.78 % (1 of 56) at 1 day p.i. to 17.87 % (10
of 56) at 7 days p.i., virtually all of which were lung tissue
(Table 2). However, the majority of samples (69.64 %, 39
of 56) had detectable OvHV-2 DNA at 9 days p.i. and all
(100 %, n581) were positive by nested PCR at the
termination of the experiment (56 days p.i.) (Table 2).
Using real-time PCR, OvHV-2 DNA was only detected in
1702
Fig. 1. MCF viral antibody in experimentally infected lambs. (a)
Lambs (nos 11-IN+, 12-IN+ and 13-IN+) were nebulized with
2 ml sheep nasal secretions containing 107 OvHV-2 DNA copies
and lambs (nos 16-IN”, 17-IN” and 18-IN”) were nebulized with
2 ml sheep nasal secretions collected from MCF virus-free sheep.
(b) Four lambs (two in each group) were intravenously (nos 19-IV+
and 20-IV+) or intraperitoneally (nos 21-IP+ and 22-IP+)
inoculated with 2 ml sheep nasal secretions containing 107
OvHV-2 DNA copies, respectively. MCF viral antibody was
measured by cELISA. The horizontal line represents the cut-off
value at 25 % inhibition.
the lung tissue from lambs at 3, 5, 7 and 9 days p.i., but not
at 1 day p.i. At 3 days p.i., only four of six lung samples
had detectable DNA copy number by real-time PCR, with a
mean of 13 copies per 50 ng tissue DNA, ranging from 10
to 53 copies. At 5 days p.i., all six lung samples had
detectable OvHV-2 DNA with a mean of 880 copies
(ranging from 8 to 1860 copies). The OvHV-2 DNA was
detected at the highest level, about 7500 copies (ranging
from 960 to 14 200 copies), at 7 days p.i. and then dropped
to a mean of 780 copies (ranging from 340 to 2540 copies)
at 9 days p.i. (Fig. 2). At the termination of the experiment
(56 days p.i.), 46 of 78 tissues, including lung, had
detectable OvHV-2 DNA by real-time PCR, ranging from
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Journal of General Virology 89
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Table 2. OvHV-2 DNA in tissues of experimentally infected lambs by nested PCR
Tissue
1 day p.i.
3 days p.i.
5 days p.i.
7 days p.i.
9 days p.i.
56 days p.i.
14-IN”D
15-IN”
1-IN+
2-IN+
3-IN+
4-IN+
5-IN+
6-IN+
7-IN+
8-IN+
9-IN+
10-IN+
16-IN+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
+
+
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
2
+
+
+
+
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
+
+
+
+
+
2
2
2
2
2
2
2
2
2
2
2
+
+
2
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
2
2
+
+
+
+
2
2
+
2
+
+
+
2
2
+
2
2
2
+
2
+
+
+
2
+
+
+
+
2
+
2
+
2
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0.60
1.78
7.14
14.20
*Lambs euthanized at 0 day p.i. were nebulized with the sheep nasal secretions collected from OvHV-2-negative sheep.
DLamb ID number.
NT, Not tested.
1703
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17.86
69.64
NT
17-IN+ 18-IN+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
NT
NT
100
OvHV-2 replication in sheep
Brain perivent.
Brain cortex
Trigeminal
Tonsil
Pharynx
Nasal mucosa
Turbinate L. caudal
Turbinate L. middle
Turbinate L. rostral
Turbinate R. caudal
Turbinate R. middle
Turbinate R. rostral
Trachea upper
Trachea middle
Trachea lower
Lung upper
Lung middle
Lung lower
Retro. LN
Mes. LN
Kidney
Spleen
Lg. intest.
Sm. intest.
Liver
Bladder
PBL
Nasal swab
Positive (%)
0 day p.i.*
H. Li and others
Fig. 2. OvHV-DNA and ORF25 transcripts in the lungs of
experimentally infected lambs. The lambs nebulized with 2 ml
sheep nasal secretions containing 107 OvHV-2 DNA copies were
euthanized at 1, 3, 5, 7 and 9 days p.i. and tissues were collected.
Two lambs euthanized at 0 day p.i. were nebulized with 2 ml sheep
nasal secretions collected from MCF virus-free sheep. The line
represents the mean OvHV-2 DNA levels in the lung samples
(n56) detected by real-time PCR. Error bars are 1 SD of error.
Bars represent the percentage of lung samples (n56) positive for
ORF25 transcripts by RT-PCR.
14 to 12 800 copies per 50 ng tissue DNA (mean51608
copies) (data not shown).
OvHV-2 ORF25 transcripts in lung and other
tissues
As shown in Figs 2 and 3, at 1 day p.i. OvHV-2 ORF25
transcripts were detected by RT-PCR in only one of six
lung samples (17 %) representing three different lobes of
lung from two lambs. Two lung samples (33 %) were
positive for the ORF25 transcripts from the lambs at 3 and
5 days p.i., respectively. The ORF25 transcripts were
detected in all six lung samples (100 %) at 7 days p.i.,
while only one sample (17 %) from one lamb at 9 days p.i.
was positive for the transcripts (Figs 2 and 3). ORF25
transcripts were not detected in any other tissues in the
respiratory tract (pharynx, nasal mucosa, turbinate,
trachea, tonsil or retropharyngeal lymph node) or in the
spleen or mesenteric lymph nodes from the infected lambs
at any time point (data not shown). GAPDH transcripts
were detected in all RNA samples prepared from the tissues
(data not shown). ORF25 transcripts were not detected in
control reactions without reverse transcriptase or without
template.
Transcripts of selected sheep immune response
genes in lung
Twenty-two sheep immune response genes, comprising 17
cytokines [granulocyte-macrophage colony-stimulating
factor (GM-CSF), interleukin (IL)-1b, IL-2, IL-4, IL-6,
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Fig. 3. Detection of ORF25 transcripts in the lungs of experimentally infected lambs. Lambs nos 1-IN+ to 10-IN+ were
nebulized with 2 ml sheep nasal secretions containing 107 OvHV2 DNA copies and lambs nos 14-IN” and 15-IN” were nebulized
with 2 ml sheep nasal secretions collected from MCF virus-free
sheep. Total RNA isolated from the tissues was analysed by RTPCR using specific primers for OvHV-2 ORF25. RT-PCR
products were electrophoresed through agarose gels containing
ethidium bromide and visualized using a UV transiluminator.
IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-23, alpha
interferon (IFN-a), IFN-c, transforming growth factor
(TGF)-b and tumour necrosis factor (TNF)-a], three
receptors (CD25, CD28 and CD152), one lytic protein
(granulysin) and one transcription factor (GATA-3), were
analysed for their expression levels in lung tissue from
lambs nebulized with OvHV-2 at different days p.i.
Eighteen of the immune response genes exhibited greater
than an eightfold (three cycle) increase in expression,
which is a conservative cut-off for a meaningful change in
expression, compared with the calibration value for the
lung samples from uninfected lambs euthanized at 0 day
p.i. (Fig. 4). Among these 18 genes, six (IL-8, IL-10, IL-13,
IL-17, TNF-a and CD28) had their highest expression
levels at 5 days p.i., while 11 (IL-1b, IL-4, IL-6, IL-15, IL18, IL-23, IFN-c, GM-CSF, CD152, granulysin and GATA3) had their highest expression levels at 7 days p.i. Only
IL-2 had its maximal expression at 1 day p.i., with a
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Journal of General Virology 89
OvHV-2 replication in sheep
Histopathology
Three sections of lung from each lamb from groups 1 and 2
were examined, including cranial, middle and caudal lobes.
All lambs had a few mild to moderate peribronchial and
peribronchiolar aggregates of mature lymphocytes and
macrophages. One infected lamb in group 1 had rare focal
areas of mild interstitial and alveolar inflammation with
minimal necrosis and oedema. One control lamb in group
2 had a similar focus of interstitial and alveolar
inflammation. Subtle differences between the two groups
would be difficult to detect due to the small sample size,
but based on the finding described here, there were no
significant differences between infected animals and
negative-control animals.
DISCUSSION
Fig. 4. Levels of various immune response gene transcripts
detected by real-time RT-PCR from lung samples of lambs
experimentally infected with OvHV-2. The cycle threshold (Ct)
values were normalized to the housekeeping genes, GAPDH and
b-actin, from the same lung samples and the average transcription
levels for two lambs per time point are reported as fold increases
relative to the lung samples from two uninfected lambs euthanized
at 0 day p.i. (a) Cytokine (IL-1b, IL-6, IL-8, IL-10, IL-12, IL-15, IL18, IL-23, IFN-a and TNF-a) genes expressed predominantly by
macrophages and dendritic cells. (b) Cytokine (IL-2, IL-4, IL-13,
IL-17, IFN-c, TGF-b and GM-CSF), receptor (CD25, CD28 and
CD152), lytic protein (granulysin) and transcription factor (GATA3) genes expressed predominantly by T lymphocytes. In each
graph the genes are arranged on the basis of their temporal pattern
of expression during an immune response. However, the genes in
each graph are not all expressed by the same antigen presenting
cells or T lymphocytes.
16.4-fold increase. Three additional genes (IL-12, TGF-b
and CD25) had greater than a fourfold increase in
expression. IFN-a was the only gene that did not exhibit
an increase in expression. By 9 days p.i., the expression of
all of the immune response genes had returned to
approximately baseline levels (ranging from 27- to 1.9fold increases).
http://vir.sgmjournals.org
It has been suggested that the respiratory tract is a primary
target for OvHV-2 during natural infection (Li et al., 2004).
In this study, OvHV-2 replication was detected only in the
lung tissue during initial infection by intranasal aerosolization, which mimics the natural nasal route of transmission.
Whether this method of nebulization may be favourable to
lung infection compared with a natural nasal route of
transmission was not determined. However, no replication
in any turbinate tissue was detected, suggesting that the
virus does not replicate in turbinate tissues during the
initial phase of infection. In contrast, OvHV-2 predominately replicates in the nasal turbinate of sheep during
intensive shedding episodes, and nasal secretions contain
infectious OvHV-2 virions (Cunha et al., 2007; Li et al.,
2004). It is not surprising that the OvHV-2 collected from
nasal secretions from shedding sheep may be incapable of
reinfecting cells in the turbinate; this is consistent with the
observation that OvHV-2 shedding from sheep is shortlived, usually lasting less than 24 h, implying a single cycle
of replication (Li et al., 2004). The data herein further
support that OvHV-2 in nasal secretions from productively
infected turbinate cells may not be capable of reinfecting
other turbinate cells, possibly due to a mechanism of cell
tropism switching similar to that reported for Epstein–Barr
virus (Borza & Hutt-Fletcher, 2002). The fact that OvHV-2
DNA, but not ORF25 transcripts, can be detected in the
majority of tissues, and PBL, by 9 days p.i. indicates that
the virus establishes latent infection in lymphocytes in the
lung and then the latently infected lymphocytes circulate in
the peripheral system.
It was unexpected that infection was not established when
sheep were inoculated with the cell-free virus from nasal
secretions intravenously or intraperitoneally, although a
transient antibody response was observed in one lamb
inoculated intraperitoneally. The transient response could
result from a low level of viral antigen stimulation without
viral replication or from a co-incident non-specific
antibody response detected by cELISA, which was documented in a previous study (Li et al., 2001). Failure to
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H. Li and others
induce infection by intraperitoneal inoculation of the cellfree virus is consistent with findings on experimental
infection of rabbits with OvHV-2 from sheep nasal
secretions (K. L. Gailbreath and others, unpublished
results). The reason for the failure is not clear. OvHV-2
could be immediately inactivated in the peripheral blood
and body fluids after injection. However, it is unlikely since
cell-free AlHV-1 from wildebeest nasal secretions can
establish infection in rabbit or cattle after intravenous
injection (Mushi & Wafula, 1983). A possible explanation
for the failure of inducing infection by intravenous and
intraperitoneal inoculation of the cell-free virus in sheep
could be that the susceptibility of lymphocytes to OvHV-2
requires replication in the lung to switch its cell tropism.
The data herein and from previous studies suggest that
OvHV-2 productive replication sites in sheep are different
for the entry and shedding events, and the virus probably
changes its cell tropism at three different stages during the
complete life cycle: turbinate (shedding), lung (entry) and
lymphocytes (latency). The likely scenario is that certain
type(s) of cells in the turbinate are susceptible and
permissive to the virus from some of the latently infected
lymphocytes that are switching to lytic infection in the
turbinate. The lymphocyte-originated virus may be capable
of infecting both lymphocytes (for latency) and specific
cells in the turbinate that permit it to replicate, producing a
sudden surge of infectious virus into the nasal secretions
for shedding, while maintaining latency in lymphocytes.
However, the turbinate-originated virus in nasal secretions
may not be capable of infecting lymphocytes or reinfecting
the cells in the turbinate. This is probably the reason why
the shedding is so transient, lasting less than 24 h (Li et al.,
2004). On the other hand, the turbinate-originated virus is
capable of infecting certain cells in the lung, and lungoriginated virus is capable of infecting lymphocytes.
Therefore, we propose that cell-free virus infection and
replication in the lung is required for OvHV-2 to establish
infection in sheep. If this is the case, the phenomenon of
cell tropism switching may explain why OvHV-2 has never
been successfully grown in vitro despite many attempts.
Determining whether lung-originated virus is capable of
reinfecting the cells in the lung is critical for the
development of an in vitro propagation system for
OvHV-2. The fact that the lambs nebulized with the virus
had ORF25 transcripts detected in a lung sample at 1 day
p.i. and all seroconverted at 9 days p.i. indicates that viral
replication occurred immediately following nebulization.
The rise in ORF25 transcript detection and viral DNA copy
numbers up to 7 days p.i. suggests there were several cycles
of viral replication in the lung. Since infectious OvHV-2
capable of infecting lung cells is available, identification of
its target, permissive cell type(s) is the first logical step to
establishing a cell culture system and is already in progress.
Regardless of how much difference there is between the
lung- and turbinate-originated virus, the virus generated
from lung cell cultures may have great potential for use not
only as virus stocks in animal experiments, but also for
1706
infecting lymphocytes in vitro, a system important for the
study of virus–lymphocyte interactions, specifically in the
latency stage.
The rapid drop of OvHV-2 DNA and ORF25 transcripts in
the lung at 9 days p.i. suggest that OvHV-2 replication may
be controlled by a host-defence mechanism. Preliminary
analysis of the expression levels of selected immune
response genes in the lungs of infected sheep showed that
expression levels of certain genes were inversely correlated
with the levels of viral DNA and transcripts in the lung,
strongly suggesting that this may be the case. Based on the
transcription profiles of the immune response genes, it
seems that innate, cell-mediated and humoral responses
were stimulated during early infection. Increased expression of IL-15, IL-6 and IL-8 at 1 day p.i. suggests that
pulmonary macrophages and/or dendritic cells were being
activated by an innate mechanism, probably involving a
Toll-like receptor (Fig. 4a). Likewise, increased expression
of IL-2, CD25 (the IL-2 receptor), CD28 (a lymphocyte
activation receptor) and IL-13 at 1 day p.i. suggests
activation of natural killer (NK) cells, NK T cells and/or
c/d T cells (Fig. 4b). At 5 days p.i., the strong macrophage
cytokine gene expression suggests activation of macrophages by T helper type 1 (Th1) lymphocytes with a
maximal response occurring at 7 days p.i. (Fig. 4a). The
cytokine expression profile also indicates that activated Th1
and T helper 2 (Th2) cells were present in the lungs of the
infected sheep from 5 to 7 days p.i. (Fig. 4b). Increased
expression of CD28 and IL-17 at 5 days p.i. is indicative of
an early Th1 response, while the effector phase of a Th1
response at 7 days p.i. is indicated by increased expression
of the genes encoding IFN-c, GM-CSF, granulysin and
CD152. Increased expression of IFN-c, GM-CSF and
granulysin could also be associated with a cytotoxic Tcell response. Increased expression of IL-13 at 5 days p.i.
suggests initiation of a Th2 response, while upregulation of
the GATA-3 and IL-4 genes at 7 days p.i. indicates a
definitive Th2 response also characterized by seroconversion (Fig. 1a). Presumably, the mature adaptive immune
response at 7 days p.i. was responsible for the rapid decline
in viral load that occurred between 7 and 9 days p.i., which
in turn led to resolution of the pulmonary immune/
inflammatory response by 9 days p.i.
Little has been reported about the host immune responses
in control of OvHV-2 infections in either carrier sheep or
clinically susceptible species, such as cattle and bison. Early
studies of the immune response to AlHV-1, a wildebeestassociated MCF virus, have reported only a humoral
response. Although these studies demonstrated that
neutralizing antibody responses developed in both experimentally infected cattle and rabbits with AlHV-1 (Rossiter
et al., 1977), the antibody response did not induce
protection against clinical disease (Plowright et al., 1975).
Similarly, studies of the humoral response to acute EBV
infection reveal that infectious mononucleosis (IM) develops in the presence of strong neutralizing antibody (Callan,
2004). Mice with deficient humoral immunity (lacking the
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Journal of General Virology 89
OvHV-2 replication in sheep
CD28 co-stimulatory receptor) control initial lytic infection in the lung normally and establishment of latency in
the spleen progresses normally (Lee et al., 2002). On the
other hand, a cell-mediated immune response has been
documented to play a critical role in the control of acute
lytic replication in EBV and MHV68. Control of acute lytic
replication is associated with strong, virus specific CD8+
T-cell responses during EBV-induced IM in humans
(Hoshino et al., 1999) and in mice after initial MHV68
infection (Stevenson et al., 1999). A recent study revealed
that MCF could be experimentally induced in sheep using a
much higher dose of the virus than in cattle or bison (Li
et al., 2005; Taus et al., 2005; O’Toole et al., 2007), suggesting
that host immune responses probably played an important
role in the control of initial replication of the virus, although
other factors such as inherent host susceptibility may also be
involved. Detailed characterization of how the immune
response controls OvHV-2 lytic replication in the sheep lung
will provide fundamental knowledge for the development of
vaccine strategies, which are desperately needed by producers, to protect clinically susceptible hosts, especially North
American bison, from SA-MCF.
ACKNOWLEDGEMENTS
This work was supported by USDA/ARS CWU 5348-32000-024-00D.
The authors thank Janice Keller, Lori Fuller, Shirley Elias and Dan
New for technical assistance, Emma Karel for animal care and
handling, and Danielle Nelson for helpful discussions. We also thank
Douglas Jasmer for critical review of the manuscript.
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