Isolation of a novel viral agent associated with porcine reproductive and neurological syndrome and reproduction of the disease

Article (PDF Available)inVeterinary Microbiology 131(1-2):35-46 · April 2008with18 Reads
DOI: 10.1016/j.vetmic.2008.02.026 · Source: PubMed
Abstract
Disease outbreaks characterized by reproductive failure and/or neurologic disorders, which are commonly referred as "Porcine Reproductive and Neurologic Syndrome (PRNS)", were observed in many swine farms in Iowa and other states. Although an infectious cause was suspected to account for the disease, no conclusive diagnosis had been reached with respect to conventional infectious agents. Extensive laboratory diagnostic investigation on suspect cases repeatedly resulted in the isolation of a cytopathic enveloped virus of 50-60nm in size from nervous and second lymphoid tissues and sera and, to reflect its unknown identity, named "Virus X". The presence of virus particle with morphological characteristics similar to Virus X in tissues from affected animals was also observed on thin-section positive-staining electron microscopy. Isolates of Virus X were not readily recognized by antibodies raised against any known viruses pathogenic to swine but by antisera collected from animals surviving clinical episode, indicating that Virus X is likely a previously unrecognized agent. Pregnant sows experimentally inoculated with Virus X (ISUYP604671) or homogenate (filtrate) of tissues from a clinically affected animal developed clinical signs and pathological changes similar to field observations including the loss of pregnancy. Furthermore, caesarian-derived, colostrum-deprived young pigs developed mild encephalomyelitis lesions in brains after experimental inoculation with the virus or the tissue homogenate although clinical neurologic signs were not observed. More importantly, Virus X was re-isolated from all inoculated animals while control pigs remained negative for the virus during the study. Collectively, Virus X is a novel viral agent responsible for PRNS and remains to be further characterized for taxonomical identity.

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Isolation of a novel viral agent associated with porcine
reproductive and neurological syndrome and
reproduction of the disease
Roman M. Pogranichniy
a,b
, Kent J. Schwartz
a
, Kyoung-Jin Yoon
a,
*
a
Department of Veterinary Diagnostic and Production Animal Medicine, Veterinary Diagnostic Laboratory,
College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
b
Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, W. Lafayette, IN, USA
Received 16 July 2007; received in revised form 14 February 2008; accepted 26 February 2008
Abstract
Disease outbreaks characterized by reproductive failure and/or neurologic disorders, which are commonly referred as
‘Porcine Reproductive and Neurologic Syndrome (PRNS)’’, were observed in many swine farms in Iowa and other states.
Although an infectious cause was suspected to account for the disease, no conclusive diagnosis had been reached with respect to
conventional infectious agents. Extensive laboratory diagnostic investigation on suspect cases repeatedly resulted in the
isolation of a cytopathic enveloped virus of 50–60 nm in size from nervous and second lymphoid tissues and sera and, to reflect
its unknown identity, named ‘‘Virus X’’. The presence of virus particle with morphological characteristics similar to Virus X in
tissues from affected animals was also observed on thin-section positive-staining electron microscopy. Isolates of Virus X were
not readily recognized by antibodies raised against any known viruses pathogenic to swine but by antisera collected from
animals surviving clinical episode, indicating that Virus X is likely a previously unrecognized agent. Pregnant sows
experimentally inoculated with Virus X (ISUYP604671) or homogenate (filtrate) of tissues from a clinically affected animal
developed clinical signs and pathological changes similar to field observations including the loss of pregnancy. Furthermore,
caesarian-derived, colostrum-deprived young pigs developed mild encephalomyelitis lesions in brains after experimental
inoculation with the virus or the tissue homogenate although clinical neurologic signs were not observed. More importantly,
Virus X was re-isolated from all inoculated animals while control pigs remained negative for the virus during the study.
Collectively, Virus X is a novel viral agent responsible for PRNS and remains to be further characterized for taxonomical
identity.
# 2008 Elsevier B.V. All rights reserved.
Keywords: Pig; Neurologic disorder; Reproductive failure; Novel pestivirus
1. Introduction
Wastage of pigs due to reproductive failure results
in significant economic losses to the swine industry.
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A
vailable online at www.sciencedirect.com
Veterinary Microbiology 131 (2008) 35–46
* Corresponding author. Tel.: +1 515 294 1084;
fax: +1 515 294 6619.
E-mail address: kyoon@iastate.edu (K.-J. Yoon).
0378-1135/$ see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2008.02.026
Author's personal copy
Death losses of sexually mature breeding animals and
market hogs add more economic burden to producers.
In many situations, both infectious and non-infectious
causes can account for reproductive problems and
mortality; infectious diseases are considerably more
important because of the rapid transmission between
animals and many times the lack of effective
preventive measures.
Since at least 1995, pork producers and swine
practitioners have reported disease outbreaks char-
acterized primarily by early infertility, hence com-
monly referred to as ‘sow infertility syndrome’ or
‘growth depression syndrome’ (Yoon and Zimmer-
man, 1998). Typically, producers have observed an
acute decline in farrowing rate due to increased early
or delayed returns followed by a prolonged period (up
to 2 years) of sub-optimal farrowing performance.
Loss of pregnancy most commonly occurs in the early
stages of gestation (i.e., 30–55 days), although
abortion and reproductive failure were reported on
sows at later gestations. Affected animals which were
confirmed pregnant at 30 days either aborted or
demonstrated irregular return to estrus. Besides
reproductive disorder, many of the affected animals
were presented with neurological signs, such as
posterior weakness/paralysis, ataxia, bar chewing,
and head pressing/banging (Yoon, 2003). The neuro-
logic symptoms in affected individuals progressed,
with death in 2 or 3 days as the usual outcome. Central
nervous signs were also observed in younger pigs such
as nursery and fattening pigs. To reflect various
clinical manifestations, the newly identified disease
has also been commonly referred to as ‘Porcine
Reproductive and Neurologic Syndrome (PRNS)’’.
Reproductive and neurologic problems were not,
however, always present simultaneously.
The incidence of the problem seems to continue
growing. The pattern of spread within and between
herds implies the involvement of an infectious agent.
To date, affected herds have been observed in both
PRRSV-infected and PRRSV-free swine herds in
many states including Iowa (Yoon, 2003). Although
many cases with similar clinical presentations have
been presented to the Iowa State University Veterinary
Diagnostic Laboratory (ISU-VDL), a conclusive
diagnosis could not be reached to date (Yoon and
Zimmerman, 1998). Laboratory testing including
serology, has not yet demonstrated the role of any
recognized reproductive or neurologic pathogens of
swine associated with the symptoms described above,
including foreign or exotic viral agents. No bacterial or
viral pathogens were consistently identified in index
cases. Furthermore, there is no evidence that the
condition is toxin-related, or associated with any
particular management practice or pig genotype.
Therefore, the disease was thought to be due to a
previously unrecognized agent. With that hypothesis,
the following extensive laboratory diagnostic investi-
gations were conducted to identify the causative agent.
2. Materials and methods
2.1. Case definition
Index animals were selected for diagnostic
investigations from herds showing the following
clinical presentations: Sows exhibiting an unusually
high early return or abortion after 30 but before 50
days of gestation; animals that were lethargic, restless,
and/or dyspneic particularly after exercise prior to
losing the pregnancy; some of the affected animals
were also febrile and pale. Besides reproductive
failure, some of the affected pregnant sows and
weaned pigs also showed neurological signs such as
posterior weakness, paresis, ataxia, lameness, head
pressing or banging and aggressive behavior often
leading to abrasions on the forehead, nose and front
legs as well as abortion. Neonates showed ‘shaking’
and many times appeared to be blind as those animals
could not walk in one direction. Central nervous signs
were also observed in grower-finishers. Some pigs
recovered but the majority of affected animals died
within several days of showing clinical signs. Some-
times affected animals died suddenly without any
overt proceeding clinical signs. To avoid any potential
confusion, animals from known PRRS-positive herds
were not included in the study.
2.2. Biological samples
Serum, whole blood and tissues (brain, secondary
lymphoid tissues, kidney, liver) were collected from
each animal and used for laboratory testing. Second-
ary lymphoid tissues included tonsil, regional lymph
nodes and spleen. When available, fetal thoracic fluid
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and tissues were collected from aborted or freshly
dead fetuses. Placenta and uterus were also collected
from sows.
Buffy coat cells were fractionated from EDTA
blood by gradient centrifugation and used for testing.
Sera and fetal thoracic fluids were tested as collected.
Tissues were tested after 20% (w/v) homogenate was
made in Earle’s balanced salt solution (Sigma
Chemical Co., St. Louis, MO, USA) and filter-
sterilized through 0.22-mm membrane filters (Fisher
Scientific Co, Houston, TX, USA).
2.3. Immunofluorescence microscopy on frozen
tissue sections
The FA test was performed on cryosections of
brain, tonsil, kidney and lymph nodes from clinically
affected animals submitted to ISU-VDL to detect viral
antigens. Sections were attached to prepared glass
slides and fixed by immersing in cold 100% cold
acetone. Fixed tissue sections were then stained with
many FA conjugates against different viruses. Fixed
tissues were also stained with commercially obtained
polyclonal antiserum for ruminant/caprine pestivirus
antibodies (440-BDV 9001) from the National
Veterinary Services Laboratory (Ames, IA, USA).
The bovine serum was diluted 1:80 in the 0.01 M
phosphate-buffered saline (PBS) at pH 7.4 for tissue
material from sows. Slides flooded with the antibodies
were then incubated at 37 8C for 1 h in a humid
condition and then rinsed with PBS three times. The
antigen–antibody reaction in tissues was visualized by
staining tissue sections with optimally diluted goat
anti-bovine IgG (H+L) conjugated with FITC (Kirke-
gaard and Perry Laboratories, Inc., Gaithersburg, MD,
USA). Swine sera collected from naturally or
experimentally infected pigs were used in the same
manner but FITC-labeled anti-porcine IgG (H+L) was
used instead of anti-bovine conjugate. Slides were
then observed under a fluorescence microscope.
2.4. Polymerase chain reaction assays
All clinical specimens were tested by polymerase
chain reaction (PCR) based tests for known swine viral
pathogens, such as porcine reproductive and respira-
tory syndrome virus (PRRSV) of European and North
American genotypes (type 1 and 2, respectively),
porcine circovirus (PCV) type 1 and 2, group 1–3
porcine enterovirus (PEV) (currently group 1 and 2
PEV are classified into porcine teschovirus, whereas
group 3 remains as enterovirus), influenza A virus,
porcine parvovirus (PPV), porcine reovirus type 1, 2
and 3, porcine cytomegalovirus (PCMV), pseudora-
bies virus (PRV), transmissible gastroenteritis virus
(TGEV), porcine respiratory coronavirus (PRCV),
porcine epidemic diarrhea virus (PEDV), Japanese
encephalitis B virus (JEV), porcine endogenous
retrovirus (PERV) type 1, porcine lymphotropic
herpesvirus type 1 (PLHV-1), swine hepatitis E virus
(sHEV), bovine viral diarrhea virus (BVDV), West
Nile virus (WNV), members of genus Pestivirus, and
members of alpha Togavirus. A multiplex PCR was
used for detecting PCV DNA as previously described
(Pogranichnyy et al., 2000). A PCR assays previously
described (Ellis et al., 1999) was employed for
detecting PPV DNA. Reverse transcription (RT)-PCR
assays were used for detecting RNA of PRRSV (Yoon
et al., 1999), group 1–3 PEV (Zell et al., 2000), CSFV
(Wirz et al., 1993; Hofmann et al., 1994), BVDV
(Ridpath and Bolin, 1998; Vilcek et al., 1994), JEV
and WNV (Scaramozzino et al., 2001), PERV
(Akiyoshi et al., 1998), and alpha-togaviruses (Powers
et al., 2001). Detection of SIV or TGEV/PRCV
genome in samples was attempted using multiplex RT-
PCR assays established in ISU-VDL (Harmon and
Yoon, 1999).
2.5. Virus isolation attempts
Virus isolation was attempted on all samples using
various cell lines and primary cells of porcine origin.
All cell lines and primary cells were confirmed by
virus isolation technique, polymerase chain reaction
(PCR) based assay and antigen-capturing ELISA
(Syracuse Bioanalytical, Inc., Ithaca, NY, USA) to be
free of bovine viral diarrhea virus (BVDV) prior to and
during use. All cell lines were also confirmed by a
commercial PCR test (American Tissue Culture
Collection, Manassas, VA, USA) to be free of
Mycoplasma spp.
Cells were prepared in 48-well plates and 25-cm
2
tissue culture flasks and grown in Minimum Essential
Medium (MEM, Mediatech, Inc., Herdon, VA, USA)
supplemented with 10% (v/v) BVDV-free fetal bovine
serum (Sigma Chemical Co., St. Louis, MO, USA) or
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5% (v/v) horse serum (Sigma Chemical Co.), 20 mM
L-glutamine (Gibco/BRL Life Science, Grand Island,
NY, USA), and an antibiotic-antimycotic mixture
(Sigma Chemical Co.). After the confluent monolayer
was formed, samples (0.25 ml/well and 1ml/flask)
were inoculated into each flask in duplicate or
triplicate. After 1-h incubation at 37 8C, cells were
rinsed with fresh growth medium and were incubated
further in a humid 37 8C incubator with 5% CO
2
supply. Alternatively, samples were inoculated in cell
suspension and left for 24 h before material was
removed. Inoculated cells were then observed daily
for cytopathic effect (CPE) until 7 days post
inoculation (PI). When CPE was evident in more
than 70% of cell monolayer, cell culture medium was
harvested and inoculated onto freshly prepared cells in
duplicate. For cases in which no CPE was evident by 7
days PI, cells were subjected to 2 cycles of freeze
thawing at 70 8C and 35 8C, respectively, and then
cell lysate was inoculated into freshly prepared cells in
the same manner as above.
At day 2 to 4 PI, regardless of the presence or
absence of CPE, one set of the inoculated cells was
fixed, after cell culture media were harvested, by
immerging them in cold 80% aqueous acetone and
subjected for an immunofluorescence microscopy
using fluorescent isothiocynate (FITC)-labeled anti-
bodies (USDA National Veterinary Services Labora-
tories, Ames, IA, USA; Rural Technologies, Inc.,
Brookings, SD, USA; DBA American Bioresearch,
Inc., Seymour, TN, USA) raised against known swine
viral pathogens, such as PRRSV type 1 and 2, PRV,
PCMV, PCV (type 1 and 2), PPV, encephalomyo-
carditis virus (EMCV), PEV, TGEV, hemagglutinating
encephalomyelitis virus (HEV), swine influenza virus
(SIV) of H1N1 and H3N2 subtypes, influenza A virus,
porcine reovirus, rabies virus and pestiviruses
(BVDV/BDV). In addition, the cells were reacted
with sera collected from animals affected but
surviving during the course of natural infection. For
pig and bovine sera, anti-porcine IgG (H+L) and anti-
bovine IgG (H+L) conjugated with FITC (Kirkegaard
and Perry Laboratories, Inc.) were used as secondary
antibody to visualize specific antigen–antibody com-
plex. At the same time another set of the inoculated
cells was subjected to freeze thawing and then cell
lysates were tested by PCR assays for known swine
viral pathogens described above under PCR.
Each cell culture fluid and corresponding unused
cell lysates that were negative for all viral agents
described above were combined for each sample and
inoculated again on each cell for further propagation
for use in other laboratory testing such as electron
microscopy (EM) or animal studies.
2.6. Electron microscopy
To assess morphological characteristic of the viral
isolate, cells inoculated with potential virus material
and cell culture fluid containing the virus as well as pig
tissues were examined by electron microscopic
techniques. Cells inoculated with virus material and
tissues were examined by thin-section positive-
staining EM. Inoculated cells were incubated at
37 8C in a humid 5% CO
2
atmosphere for up to 120 h
PI. At between 48 and 120 h PI, the cells were
harvested using a cell scraper and pelleted by low-
speed centrifugation. Each cell pellet was fixed in 2%
(w/v) glutaraldehyde and 2% (w/v) paraformaldehyde
in 0.05M phosphate-buffered saline (PBS, pH 7.2) for
48 h at 4 8C. Samples were rinsed once in PBS
followed by 2 washes in 0.1 M cacodylate buffer (pH
7.2) and then fixed in 1% osmium tetroxidate in 0.1 M
cacodylate buffer for 1 h at ambient temperature. The
samples then were dehydrated in a graded ethanol
series, cleared with ultra pure acetone, infiltrated and
embedded using a modified EPON epoxy resin
(Embed 812, Electron Microscopy Science, Fort
Washington, PA, USA). Resin blocks were polymer-
ized for 48 h at 70 8C. Thick and ultra-thin sections
were made using a Reichert Ultracut S ultra
microtome (Leeds Precision Instruments, Minneapo-
lis, MN, USA). Ultra-thin sections were collected onto
copper grids and counterstained with 5% uranyl
acetate in 100% methanol for 15 min followed by
Sato’s lead stain for 10 min. Images were captured
using a JEOL 1200EX scanning and transmission
electron microscope (Japan Electro Optic Labora-
tories, Akishima, Japan).
Viruses in cell culture fluid were examined by a
negative-staining EM as previously described
(Hsiung, 1982). Viral particles were pelleted by
centrifugation using SW41 rotor in an ultracentrifuge
(Optima LE 80K; Beckman, Fullerton, CA, USA) for
3 h at 160,000 g and resuspended in a small quantity
of PBS. Ten microliters of the virus suspension were
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applied to the carbon-coated grid. Excessive PBS was
dried out with an absorbent paper and stained with 4%
potassium phosphotungstate acid (PTA) for 5 min at
ambient temperature. Grids were then examined under
an electron microscope and digital images of virions
were captured using a JEOL 1200EX scanning and
transmission electron microscope (Japan Electro
Optic Laboratories).
2.7. Effect of DNA inhibitors on virus growth
Mitomycin C and 5-bromo-2-deoxyuridine
(BUDR) are known to inhibit the replication of
DNA viruses in cells. To determine if Virus X contains
a DNA genome, virus titration was performed using a
microtitration infectivity assay in the presence and
absence of these chemicals as previously described
(Benfield et al., 1992). In brief, confluent monolayer
of CL ISUVDL33 and CL ISUVDL99 cells were
prepared in 96-well plates and inoculated with virus
suspensions (100 ml/well) which were prepared by a
serial 10-fold dilution technique. After 1-hr of
absorption at 37 8C, the virus inoculum was removed
and cells replenished with MEM supplemented with
40 or 160 mg/ml of BUDR (Sigma Chemical Co.) or 2
or 20 mg/ml of mitomycin C (Sigma Chemical Co.).
Plates were incubated for an additional 48 h; virus titer
was determined by the presence of visible CPE and
then confirmed by IFA staining using rabbit antiserum
raised against virus isolate designated ISUYP604671
from a PRNS case. As controls for RNA and DNA
viruses, PRRSV and PRV, respectively were concur-
rently titrated under the same conditions described
above.
2.8. Animal inoculation studies
2.8.1. Inoculation of pregnant sows at 30 days of
gestation
Seven sows at 4th or 5th parity were purchased
from a high-health-status commercial vendor and
transferred to an animal holding facility with
farrowing crates and bred through artificial insemina-
tion. The facility was operated and managed at BSL2
compliance. Once pregnancy was confirmed by an
ultrasound technique, 5 sows were selected and used
for the study. Four of the 5 sows at 30 days of gestation
were inoculated intranasally, subcutaneously and
intramuscularly with one of the following biological
materials: (a) cell culture fluid containing virus isolate
designated ISUYP604671; (b) homogenate of tissues
collected from a clinically affected sow; (c) serum
collected from the clinically affected sow. All
inoculated animals were kept in the same room but
individually in farrowing crates for 30 days. The one
remaining sows served as sham-inoculated control and
was kept in a separate room. After inoculation, all
animals were monitored for changes in behavior and
any reproductive problems. In addition, the sows were
bled every seven days. At the termination of the study,
all sows were euthanized, and various tissues (brain,
tonsil, lung, spleen, lymph nodes, kidney, liver,
placenta, spinal cord, uterus, uterine fluid) and, if
present, fetuses were collected from each sow for
histopathology, virus isolation and/or serology.
2.8.2. Inoculation of CDCD pigs
Seven 7-week-old caesarian-derived-colostrum-
deprived (CDCD) pigs were used to evaluate the
clinical effect of Virus X on young swine. Animals
were divided to 3 groups. Group 1 (n = 3) was
inoculated with cell culture material containing Virus
X (ISUYP604671) via intranasal, intramuscular and
intravenous routes. Group 2 (n = 3) was inoculated via
the same routes with serum from an animal (Sow 800)
which was experimentally infected with the virus in
the previous animal study. Group 3 (n = 1) was
inoculated with virus-free cell culture fluid and served
as sham-inoculated negative control. All animals were
monitored for the first 2 weeks after inoculation for
changes in rectal temperature and behavior and for
clinical signs. One pig per group except group 3 was
euthanized and necropsied on days 7, 10 and 14 PI.
Tissues (brain, tonsil, lung, spleen, kidney, lymph
nodes) were collected from each pig for both virus
assays and histopathology. Animals were bled at days
0, 7, 10 and 14 PI to collect whole blood and serum. At
day 14 PI all remaining animals were euthanized and
necropsied.
2.9. Histopathology
Tissues were fixed by immersing in 10% neutral
buffered formalin immediately after collection. Fixed
tissues were processed, embedded in paraffin, and
sectioned according to the standard protocol established
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at ISU-VDL. Sections were then stained with hema-
toxylin and eosin for microscopic examination.
3. Results
3.1. Pathological evaluation of clinical cases
In many cases, external examination revealed
trauma to the face and head of clinically affected
pigs probably as a result of head-banging and head-
pressing behaviors. Traumatic lesions were also seen
in the extremities or shoulders possibly due to ataxia
or recumbency.
While affected animals showed distinct clinical
signs, gross lesions were not present except for the
presence of petichial hemorrhage in lymph nodes and
sometimes on the serosa of many organs. Micro-
scopically, some lesions limited to the brain were
detected as mild focal to locally extensive non-
suppurative perivascular cuffing, gliosis, and lympho-
plasmacytic encephalitis. Hepatitis and interstitial
pneumonia were other lesions frequently observed in
the affected animals.
3.2. Isolation and preliminary characterization of
virus isolates
A cytopathic agent that could pass through a
0.22 mm membrane filter was repeatedly and con-
sistently isolated from serum, tonsil, lymph nodes
and/or brain from clinically affected animals.
Because of its unknown identity, the agent was
tentatively named Virus X. Two initial isolates
were designated ISU-KJY96 and ISUYP604671,
respectively. Besides Virus X, several other viruses,
such as PEV, porcine reovirus and PRRSV, were also
inconsistently and infrequently isolated from suspect
animals.
Morphologically, Virus X was an enveloped virus
with the size of approximately 50–55 nm in diameter.
As shown in Fig. 1, the virion contains an icosahedral
core and acquires its envelope by budding through the
endoplasmic reticulum of infected cells. Overall,
Virus X resembles members of the family Arterivir-
idae, Flaviviridae or Togaviridae.
As summarized in Table 1, infection and replica-
tion of the virus ISUYP604671, like PRRSV, was not
negatively affected by the presence of BUDR and
mitomycin C in cell culture media, whereas PRV
replication was substantially inhibited by the treat-
ment.
Various cell lines and primary cells of porcine
origin were permissive to Virus X and supported
productive infection of the virus (Fig. 2). On
immunofluorescence tests, Virus X did not cross-
react with antibodies raised against PRRSV (type 1
and 2), PRV, SIV (both H1 and H3), TGEV, PPV, PCV
(type 1 and 2), porcine reovirus, EMCV, PEV, HEV,
PCMV and rabies virus. Interestingly, the virus
appeared to be recognized to a degree by the
polyclonal antiserum specific for BVDV as weak
positive fluorescence was observed when Virus X
infected cells were stained with the serum (Fig. 3).
However, PCR results for 5
0
UTR did not support that
Virus X was BVDV (both type 1 and 2) or CSFV. PCR
assays also demonstrated that Virus X was not PRRSV
(neither North American nor European genotypes),
PCV (neither type 1 nor 2), PEV (groups 1, 2 and 3),
influenza A virus, TGEV, PRCV, PEDV, PPV, sHEV,
WNV, JEV, PERV, PLHV-1 or alpha-togaviruses.
3.3. Outcom e of experimental inoculation
3.3.1. Sow study
Clinical, pathological and virological observations
on pregnant sows after experimental inoculation are
summarized in Table 2. During a 30-day observation
period, inoculated sows became viremic. Yet, all
animals remained normal in their behavior during the
study period. No abortion was observed in any of the
inoculated sows.
At the termination of the study, no lesions were
observed in the control sow. In contrast, gross lesions,
such as hemorrhages in inguinal lymph node and
decolorization of the uterus (green and brown), were
observed in the sows inoculated with materials
containing Virus X (ISUYP604671). Embryonic or
fetal death was also apparent in some of the inoculated
sows. One of the 4 inoculated sows did not have any
fetus at necropsy although no significant lesions were
observed. Microscopically, necrotic edema in the
lymph node, mineralization plaques in uterus and
necrotic debris in the lumen of the uterus were
observed, supporting the possibility of embryonic
death and/or fetal reabsorption.
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Virus X was recovered from tissues (spleen, liver,
jejunum, uterus, serum, tonsil) of the inoculated
sows, confirming the replication of Virus X in the
animals, particularly in secondary lymphoid
organs. In addition, the virus was also isolated
from fetal tissues such as lung, spleen, heart and
kidney, suggesting transplacental transmission of
the virus.
R.M. Pogranichniy et al. / Veterinary Microbiology 131 (2008) 35–46 41
Fig. 1. Electron photomicrographs of Virus X in CL ISUVDL13b cells (A) infected with isolate ISUYP604671, cell culture fluid (B) harvested
from the infected cells and nerve cell (C) of the brain from an experimentally infected pig.
Table 1
Effect of the presence of mitomycin C and 5-bromo-2 deoxyuridine (BUDR) in cell culture media on the replication of virus isolate (‘Virus X’)
from PRNS case, pseudorabies virus (PRV) and type 2 porcine reproductive and respiratory syndrome virus (PRRSV) in vitro as determined by
microtitration infectivity assay
Virus Type of nucleic acid Media only Media with BURD Media with
mitomycin C
40
b
160 2 20
PRRSV RNA 10
6a
10
6
10
6
10
6
10
6
Virus X Unknown 10
4
10
4
10
4
10
4
10
4
PRV DNA 10
8
10
6
10
3
10
6
10
3
a
Resulting virus titer (TCID
50
/ml) of the stock virus in the presence and absence of each chemical in the media.
b
Concentration (mg/ml) of each chemical in cell culture media.
Author's personal copy
3.3.2. CDCD pig study
All inoculated pigs became viremic during a 14-
day observation period but did not show any overt
clinical signs by the end of the study. The sham-
inoculated negative control pigs remained negative for
Virus X in the blood (Table 3).
At each necropsy, significant gross lesions were not
apparent although spleen and lymph nodes collected
from pigs at days 7 and 10 PI looked edematous.
Microscopically, nonsuppurative meningoencephalitis
was observed in 5 of the 6 inoculated pigs (Fig. 4).
Nonsuppurative interstitial pneumonia (Fig. 5) and
R.M. Pogranichniy et al. / Veterinary Microbiology 131 (2008) 35–4642
Fig. 2. Cytopathology in cell line CL ISUVDL44 infected with Virus X (A) in comparison to uninfected cells (B) at 200 magnification.
Fig. 3. Immunofluorescence photomicroscopy of CL ISUVDL13b cells infected (A, C) and not infected (B) with Virus X. The cells were fixed at
approximately 48 h post inoculation and stained with an anti-BVDV bovine polyclonal serum. Panel A and C are presented at 100
magnification, whereas panel C is presented at 200 magnification.
Author's personal copy
nonsuppurative periportal hepatitis were other lesions
commonly observed in the inoculated pigs. Neither
gross nor microscopic lesions were present in tissues/
organs of the control pig.
Virus X was recovered from tissues of all
inoculated pigs regardless of the presence or absence
of detectable lesions. Brain tissues from 4 of the 5 pigs
with meningitis or encephalitis were positive for Virus
X. The presence of virus particles with morphological
characteristics similar to Virus X in brain tissues was
confirmed by EM (Fig. 1). The virus was most
frequently recovered from tonsils. Spleens and lymph
nodes also harbored the virus.
4. Discussion
Porcine reproductive and neurologic syndrome
brought a challenge to the diagnostic community since
no definitive diagnosis as to the cause could be made.
Although death loss and reproductive failure in
breeding females is not an unusual event in many
farms for various reasons, clinical presentations of
reproductive problems along with neurologic dis-
orders were a unique feature of PRNS. Unfortunately,
diagnostic investigations focusing on all conventional
infectious agents that had been implicated in
reproductive and/or neurologic problems of pigs at
various ages has been unfruitful or yielded incon-
sistent results. With the hypothesis that a previously
unknown infectious agent could be responsible for the
disease, extensive laboratory testing resulted in
frequent isolation of a relatively small enveloped
cytopathic virus (‘Virus X’) from clinically affected
animals. Although one might say that Virus X is not
necessarily a causative pathogen, isolation of the virus
from nervous tissues collected from clinically ill
animals with CNS signs is a convincing evidence
R.M. Pogranichniy et al. / Veterinary Microbiology 131 (2008) 35–46 43
Table 2
Summary of reproductive record and pathological lesions of sows experimentally infected with Virus X isolate ISUYP604671 at early gestation
Sows (inoculum) Average # of litter
in previous gestations
No. of fetuses
recovered at
necropsy
Significant gross
lesions
a
Significant
microscopic lesions
a
Recovery of
Virus X from
sow/fetus
800 (serum) 11.4 3 Yes Yes Yes/Yes
21622 (virus isolate-P2) 13.3 0 No No Yes/NA
b
30021 (virus isolate-P3) 11.2 13 No No Yes/Yes
30172 (virus isolate-P1) 8.4 10 Yes Yes Yes/Yes
30120 (sham inoculum) 10.2 6 No No No/No
The virus inocula were prepared at various cell passages (P).
a
Significant gross lesions were hemorrhages in inguinal lymph node, decolorization of uterus (green and brown) and possible embryonic
death of fetuses. Microscopically, necrotic edema in the lymph node, mineralization plaques in uterus and necrotic debris in the lumen were
observed.
b
NA: not applicable.
Table 3
Summary of clinical, pathological and virological observations on 7-week-old caesarian-derived-colostrum-deprived pigs infected with Virus X
isolate (ISUYP604671), clinical specimen (serum) containing the virus or cell culture medium
Pig ID Inoculums DPI at necropsy
a
Clinical signs Lesions
b
Detection of Virus X
in serum/tissue
274 Serum 604671 7 No Yes Positive/positive
275 Isolate ISUYP604671 7 No Yes Negative/positive
302 Isolate ISUYP604671 10 No No Positive/positive
303 Serum 604671 14 No Yes Positive/positive
304 Serum 604671 10 No Yes Positive/positive
675 Isolate ISUYP604671 14 No Yes Positive/positive
273 Medium 14 No No Negative/negative
a
Day post inoculation.
b
Gross lesion: hemorrhagic, enlarged and swollen lymph node. Microscopic lesions: multifocal perivascular accumulations of mononuclear
cells in meninges, mild edema with occasional degenerating neurons, interstitial pneumonia.
Author's personal copy
suggesting that Virus X was likely responsible for the
disease. Observations from the animal inoculation
study with pregnant sows clearly indicate that Virus X
is capable of crossing the placental barrier and causing
the loss of pregnancy most likely due to embryonic or
fetal death as evident by recovery of the virus from
fetal tissues. Furthermore, development of meningitis
or encephalitis lesions in the brains of young CDCD
pigs after inoculation with Virus X also indicate that
the virus may be able to cause neurologic disorder in
infected animals, although the inoculated CDCD pigs
did not show clinical central nervous signs under
experimental conditions. Those lesions were due to
the attack by Virus X since the virus was recovered
from brain tissues. Since all these experimental
findings match well with field observations of PRNS,
Virus X is postulated to be the causative agent for
PRNS as Koch’s postulates were partially fulfilled. It
is proposed then that Virus X shall be named ‘PRNS
virus (PRNSV)’’.
Under a laboratory setting, PRNSV could not be
readily recognized by antibodies raised against
various viral agents that have been implicated in
diseases of swine except anti-BVDV polyclonal
antibody. The positive cross-reactivity of PRNSV
with anti-BVDV antibody is of particular interest with
respect to taxonomical identification of the virus. The
possibility that PRNSV could be CSFV was ruled out
at the beginning of the investigation since US swine
populations are known to be free of CSFV. Further-
more, diagnostic testing on some early PRNS suspect
cases at the Foreign Animal Disease Diagnostic
Laboratory of the National Veterinary Laboratory
Service ruled out the presence of CSFVor its infection
in those animals as well as the involvement of other
foreign animal disease agents (Data not shown). Pigs
are known to be susceptible to ruminant pestiviruses
such as BVDV and BDV besides porcine pestivirus
(i.e., CSFV). Although BVDV infection of pigs has
been demonstrated both in the field and under
experimental conditions, BVDV has not been con-
sidered to be a significant pathogen for pigs.
Considering that PRNSV is readily spread among
pigs and there is very minimum contact between pigs
R.M. Pogranichniy et al. / Veterinary Microbiology 131 (2008) 35–4644
Fig. 4. Photomicrographs of representative lesions in brains from CDCD pigs inoculated with Virus X. (A) Moderate multifocal perivascular
accumulations of mononuclear cells in meninges. (B) Occasional neurons appear in early stages of necrosis. (C) Mild edema with occasional
degenerating neurons. (D) Normal brain tissue.
Author's personal copy
and ruminants in the US, it would be hard to believe
that PRNSV is BVDV or BDV. This argument is
supported by negative PCR results (5
0
UTR) on
isolates of PRNSV for BVDV and CSFV. However,
based on its morphological similarity with pestiviruses
and cross-reactivity with anti-BVDV antibody, the
possibility that PRNSV is a pestivirus cannot be
completely ruled out until proven otherwise. At the
time of writing this manuscript, PRNSV is proposed to
be a novel swine pestivirus which remains to be further
characterized for its conclusive taxonomical identifi-
cation. As a pestivirus is suspected to be responsible
for PRNS, development of a specific diagnostic
reagent for PRNSV is prudent.
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