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Journal of Virology, April 2000, p. 3112-3121, Vol. 74, No. 7
Department of Molecular Genetics and
Biochemistry, School of Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261,1 and
Gluck Equine Research Center, Department of Veterinary
Science, University of Kentucky, Lexington, Kentucky
405462
Received 9 August 1999/Accepted 20 December 1999
Equine infectious anemia virus (EIAV) infection of horses
is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus
replication indefinitely. The dynamics of EIAV viremia and
its association with disease cycles have been well characterized, but
there has been to date no comprehensive quantitative analyses of the
specific tissue sites of EIAV infection and replication in
experimentally infected equids during acute disease episodes and during
asymptomatic infections in long-term inapparent carriers. To
characterize the in vivo site(s) of viral infection and replication, we
developed a quantitative competitive PCR assay capable of detecting 10 copies of viral DNA and a quantitative competitive reverse transcription-PCR assay with a sensitivity of about 30 copies of viral
singly spliced mRNA. Animals were experimentally infected with one of
two reference viruses: the animal-passaged field isolate designated
EIAVWyo and the virulent cell-adapted strain
designated EIAVPV. Tissues and blood cells were
isolated during the initial acute disease or from asymptomatic animals
and analyzed for viral DNA and RNA levels by the respective
quantitative assays. The results of these experiments demonstrated that
the appearance of clinical symptoms in experimentally infected equids
coincided with rapid widespread seeding of viral infection and
replication in a variety of tissues. During acute disease, the
predominant cellular site of viral infection and replication was the
spleen, which typically accounted for over 90% of the cellular viral
burden. In asymptomatic animals, viral DNA and RNA persisted in
virtually all tissues tested, but at extremely low levels, a finding
indicative of tight but incomplete immune control of EIAV
replication. During all disease states, peripheral blood mononuclear
cells (PBMC) were found to harbor less than 1% of the cellular viral
burden. These quantitative studies demonstrate that tissues, rather
than PBMC, constitute the predominant sites of virus replication during acute disease in infected equids and serve as resilient reservoirs of
virus infection, even in the presence of highly effective immune responses that maintain a stringent control of virus replication in
long-term inapparent carriers. Thus, these observations with EIAV, a predominantly macrophage-tropic lentivirus, highlight the role of tissues in sequestering lentiviral infections from host
immune surveillance.
Equine infectious anemia virus
(EIAV) is unique among lentiviruses in that the clinical
course of infection in equids results initially in a rapid and dynamic
series of clearly demarcated cycles of disease and associated viremia
that begin by 3 weeks postinfection and continue at irregular intervals
separated by weeks or months (reviewed in reference
25). Disease cycles last 3 to 5 days and are
characterized by fever, diarrhea, lethargy, edema, anemia, and
thrombocytopenia. This stage of disease, defined as chronic equine
infectious anemia (EIA), typically lasts about 8 to 12 months
postinfection, with the frequency and severity of clinical episodes
decreasing with time. In contrast to the progressive degenerative
disease associated with most lentiviral infections, horses infected
with EIAV typically make a transition during the first year
postinfection from chronic EIA to an inapparent infection in
which clinical symptoms are absent and viremia is usually undetectable
for the remainder of the animal's life span of up to about 20 years.
Thus, the EIAV systems provides a novel model in which to
examine the dynamics of lentivirus replication during clearly defined
cycles of disease and during long-term asymptomatic infections.
Several lines of evidence indicate that the control of EIAV
replication and disease in long-term inapparent carriers is
mediated by virus-specific host immune responses that evolve during the first year postinfection to achieve an enduring effective suppression of virus replication. For example, experimental infection of foals with
severe combined immunodeficiency results in a progressive infection
leading to death, demonstrating the necessity of the host immune system
in accomplishing the temporal control of virus replication associated
with infection of immunocompetent horses (29). In addition,
it has been shown that severe stress or treatment of long-term
inapparent carriers with immunosuppressive drugs can cause
recrudescence of viremia and disease, even after decades of clinical
quiescence (16, 48). Finally, it has been demonstrated that
transfer of whole blood from long-term inapparent carriers to naive
horses results in EIAV infection and disease in the recipient horses (12). Taken together, these observations demonstrate the lack of attenuation of the infecting virus during persistent infection and the importance of host immune responses in establishing and maintaining control of EIAV replication and disease. The
same experiments, however, also clearly demonstrate the persistence of
virulent EIAV infection in infected horses during
asymptomatic and nonviremic periods, either during chronic
EIA or in long-term inapparent infections. This persistence
of EIAV infection in the presence of enduring, robust, and
highly suppressive host immunity raises a number of interesting
questions about the reservoirs of EIAV infection that are
able to escape host immune surveillance.
The primary target of EIAV in vivo are cells of the
monocyte/macrophage lineage (25), although there has
recently been a report of limited infection of macrovascular
endothelial cells in the renal tissues of a long-term inapparent
carrier horse (22). Infection of blood monocytes with
EIAV results in a nonproductive infection, and
differentiation of virus-infected monocytes to macrophages is required
to activate virus replication (21, 43). This pattern of
infection has led to the model that virus-infected monocytes may serve
as "Trojan horses" that can widely disseminate EIAV
infection to tissues without detection by the host immune system,
suggesting that monocytes may sequester EIAV during
asymptomatic infection. The identification of EIAV infection
of macrovascular endothelial cells suggests that this cell type may
also serve as a reservoir for virus infection, at least in long-term
inapparent infections. The role, if any, of endothelial cell infection
in acute disease remains to be defined.
The primary sites of EIAV replication in persistently
infected horses has been examined by immunofluorescent techniques to detect viral antigens (23), by Southern blot analyses to
detect proviral DNA (35), and more recently by qualitative
PCR techniques to detect viral DNA or RNA (14, 27, 42). The
results of these studies uniformly demonstrate that the high levels of
viremia observed during acute EIA are associated with high
levels of virus replication in macrophage-rich tissues, including
liver, spleen, and kidney; peripheral blood mononuclear cells (PBMC)
and other tissues appeared to contain only minor levels of virus
infection, despite the high levels of virus in the blood. In contrast
to the abundance of viral antigens and DNA observed in tissues from EIAV-infected horses during acute disease, qualitative
analyses of EIAV infection sites by Southern blot
(35) and PCR (14, 42) in a limited number of
inapparent carrier horses indicate low levels of EIAV
infection associated with tissue macrophages and typically undetectable
levels of EIAV DNA in PBM. These general patterns of
EIAV infection were confirmed recently in an informative study by Oaks et al. (26) using a combination of PCR and in situ hybridization techniques to elucidate tissue macrophages as the
predominant site of EIAV infection during clinical and subclinical infections.
The previous studies of the sites of EIAV infection in
experimentally infected horses provide an important qualitative
description of virus infection in vivo, but there has been to date no
comprehensive quantitative analyses of EIAV DNA and RNA in
experimentally infected horses to ascertain the relative importance of
different tissues in virus production during acute disease or in
persistence during inapparent infections. In addition, the majority of
these published studies of EIAV infection during acute
disease are based on experimental infections with the highly virulent
EIAVWyo strain of virus that usually causes death
within several weeks postinfection, in marked contrast to the chronic
EIA observed in most EIAV infections in the field.
The highly virulent nature of the EIAVWyo raises
some concerns about the relevance of this experimental infection model to the more typical infections that result in chronic EIAV
that progresses to a long-term inapparent infection. Finally, the
characterization of EIAV replication in vivo has been limited
by the use of only one or two animals in many published studies.
Therefore, we sought in this current study to characterize viremia
levels and the tissue sites of EIAV infection and replication during symptomatic and asymptomatic infections by using newly developed
sensitive and quantitative PCR and reverse transcriptase PCR (RT-PCR)
techniques to measure viral DNA and RNA, respectively. For these
studies we employed the well-characterized EIAVPV
reference strain (25, 38), which has been used extensively
in our laboratory in experimental infections to reliably produce in
equids a clinical progression from chronic EIA to a long-term
inapparent infection. For comparison to previous studies, we also
examined virus infection and replication during acute disease in horses
inoculated with the highly virulent EIAVWyo. The
results of these studies provide for the first time a comprehensive
quantitative analysis of the in vivo sites of viral infection and
replication in various tissues from experimentally infected animals
during the initial acute disease and during long-term inapparent infection.
Virus strains and experimental animals.
The
highly virulent wild-type EIAVWyo strain, an
animal-passaged isolate, was produced as serum taken during acute
disease in an experimentally infected horse, as described earlier
(35). The titer of the EIAVWyo stock
was 106 horse infectious doses per ml. The cell-adapted
pathogenic EIAVPV strain was derived, and the
titers were determined as described elsewhere (49).
Infectious stocks of EIAVPV were grown, and
titers were determined in primary fetal equine kidney cell cultures. The titer of EIAVPV stock was 106.5
50% tissue culture infectious doses (TCID50) per ml.
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Tissue Sites of Persistent Infection and Active Replication of
Equine Infectious Anemia Virus during Acute Disease and Asymptomatic
Infection in Experimentally Infected Equids
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Clinical profiles and levels of EIAV in the
plasma of experimentally infected animals
Clinical samples.
Heparinized whole blood was collected by
jugular venipuncture from experimentally infected animals during
defined febrile episodes and from asymptomatic animals. Plasma and PBMC
were isolated by discontinuous density gradient centrifugation. Serum
samples were collected. Six animals were euthanized by administration of a lethal intravenous injection of Beuthanasia, at which time replicate tissues samples were collected and flash frozen immediately in liquid nitrogen. Tissue samples from the liver, spleen, medulla and
cortex of the kidney, peripheral lymph nodes including the sublingual
and mandibular nodes, and bone marrow were collected from all animals.
The cerebrum, medulla, and cerebellum from the brain were collected
from three animals (horse 94-07 and ponies 112 and 123). Tissue,
cellular, plasma, and serum samples were stored at 
80°C until
isolation of nucleic acids.
Nucleic acid purification from tissue samples.
Total
cellular DNA was purified by standard techniques (40).
Briefly, tissues were immersed in liquid nitrogen and immediately pulverized. Samples were then suspended in 1 ml of 0.1% sodium dodecyl
sulfate (SDS)-10 mM EDTA per 100 mg of tissue or per 106
PBMC, digested with 100 µg of proteinase K per ml at 37°C for a
minimum of 5 h, followed by phenol-chloroform extraction and ethanol precipitation. Pelleted DNA was resuspended in 10 mM
Tris-HCl-1 mM EDTA by gentle rocking at room temperature overnight.
The concentration of the DNA was determined spectrophotometrically, and
the quality of the DNA was assayed by gel electrophoresis. Samples were
stored at 20°C until quantification of the cellular genomic and
EIAV viral DNA content.
70°C until assayed for
viral and cellular RNAs.
Virion-associated genomic RNA was recovered from plasma samples by
ultracentrifugation to pellet virus and extracted as described earlier
(19). Briefly, a minimum of 1 ml of each plasma sample was
centrifuged at 100,000 × g for 45 min in a type 55.1 rotor (Beckman Instruments, Palo Alto, Calif.) at 4°C. The
supernatant was aspirated, and the pellet resuspended in 0.1% SDS. RNA
was extracted using 1 ml of TRIzol as described above. The RNA was precipitated in the presence of 20 µg of glycogen as carrier. RNA was
pelleted by centrifugation at 12,000 × g for 15 min at 4°C and resuspended in 10 to 20 µl of DEPC-treated water. All samples were stored at 70°C until quantification of the viral RNA.
Quantitative PCR analyses.
The viral DNA burden was
determined by using a competitive PCR (QC-PCR) assay to quantify viral
DNA (30). To optimize detection of variant quasispecies,
highly conserved gag-specific primers were chosen based on
alignment of all of the EIAV sequences in the database using
the Genetics Computer Group (Madison, Wis.) package analyses software
(10). A SmaI-XbaI fragment of the gag gene was cloned into a trpLE vector (3). The
competitor template containing a 55-bp deletion at the unique
ApaI site was generated by Bal31 exonuclease
digestion. The competitor template was quantified by spectrophotometry
and limiting-dilution analyses (data not shown). Amplification of
wild-type and competitor templates by using the sense gag11
(5'-ATGTATGCTTGCAGAGACATTG-3') and antisense gag34
(5'-GCTGACTCTTCTGTTGTATCG-3') primers yielded 310- and
255-bp products, respectively. The PCR reaction contained 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, 0.001% gelatin, 0.05 mM
concentrations of each deoxyribonucleoside triphosphate, 12.5 pmol of
each primer, 1 µCi of [
-32P]dATP (3,000 Ci/mmol;
NEN, Boston, Mass.), and 2.5 U of AmpliTaq DNA polymerase
(Perkin-Elmer, Norwalk, Conn.) in a final volume of 25 µl. The
thermal cycling conditions included an initial hot start at 99°C,
followed by 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min
for 30 cycles, with a final 10-min extension at 72°C. PCR products
were separated by electrophoresis on 6% acrylamide gels containing 8 M
urea. Gels were fixed in a 10% methanol-10% acetic acid solution and
dried. The radioactivity incorporated in each band was quantified by
using phosphorimaging technologies (Molecular Dynamics, Sunnyvale,
Calif.). Preliminary experiments were performed to determine the amount
of sample DNA that was within the linear range of the amplification
reaction. Addition of carrier DNA to maintain a constant mass had a
minimal effect on the amplification efficiency; therefore, carrier DNA
was not included in the reactions.
Quantitative RT-PCR analyses. To determine the levels of EIAV replication in various tissues, the amount of singly spliced viral mRNA was quantified by competitive RT-PCR (QC-RT-PCR [30]). A 910-bp cDNA fragment from singly spliced EIAV mRNA (33) was subcloned into a low-copy vector, pLG338 (6), modified to contain a T7 RNA promoter site. A 104-bp deletion was generated by ligation-mediated PCR techniques. Wild-type and competitor transcripts were generated by in vitro transcription using a commercially available T7 RNA polymerase kit (Ambion) according to the manufacturer's directions. Transcripts were subjected to a second RNase-free DNase (Promega) digestion, organic extraction, and ethanol precipitation. RNA was resuspended in DEPC-treated water and quantified by spectrophotometry. Reverse transcription of defined quantities of sample RNA and titered quantities of competitor transcripts was performed with Superscript II (BRL) according to the manufacturer's recommendations. cDNA was synthesized by using 12.5 pmol of an EIAV env-specific primer (5'-GTATTCCTCCAGTAGTTC-3'). Radiolabeled amplification of 2 µl of the RT reaction was performed by using the sense (5'-TGAACCTGGCTGATCGTAGGATCC-3') and antisense (5'-GTATTCCTCCAGTAGTTCCTGCTAAGC-3') primers. The concentration of the PCR reagents was as stated above for amplification of viral DNA. A 510-bp product and a 406-bp product were produced from the wild-type and competitor templates, respectively. PCR products were separated by polyacrylamide gel electrophoresis and quantified as described for viral DNA. Preliminary RT-PCR reactions were performed to determine the amount of sample RNA that fell within the linear range of the assay.
An equine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA clone was produced to provide a cellular marker gene. RT-PCR was performed using commercially available primers (Clontech) complementing highly homologous cross-species GAPDH-specific sequences. RT reactions were carried out as described above, except that 10 pmol of the antisense primer (5'-TCCACCACCCTGTTGCTGTA-3') and 1 µg of cellular RNA from an EIAVWyo-infected animal was used. Then, 2 µl of the RT product was amplified with 10 pmol of each of the sense (5'-ACCACAGTCCATGCCATCAC-3') and the above-described antisense primers and reagent concentrations analogous to the viral DNA reactions. The thermal cycling conditions were 94°C for 45 s, 45°C for 45 s, and 72°C for 2 min for 35 cycles, followed by a 10-min extension at 72°C. The resultant 452-bp product was purified by agarose gel electrophoresis and cloned into the pGEM T vector (Promega). The identity of an equine GAPDH cDNA was confirmed by gene sequencing. Tissue RNA samples were subjected to independent RT-PCR analyses for equine GAPDH transcripts to assess if the samples were amenable to RT-PCR analyses. The cDNAs were subjected to radiolabeled amplification and analyzed by denaturing acrylamide electrophoresis. The integrity of the tissue-derived RNAs was assayed by Northern blot hybridization with the equine GAPDH-specific probe (40). The viral burden in the plasma was determined by quantitative RT-PCR. A synthetic gag-specific RNA was employed as the external standard. The reaction conditions were as described elsewhere (19). Briefly, 1 to 4 µl of virion RNA was reverse transcribed with 10 pmol of gag34 primer as described above. Radiolabeled hot start PCR was performed as described for viral DNA analyses by using 10 pmol of each of the gag11 and gag34 primers. PCR products were separated by polyacrylamide gel electrophoresis, and the amount of radiolabel incorporated was quantified as described above. The copies of virion RNA per milliliter of plasma were calculated by using the equation of the line. At least two independent assays were performed for each sample.Measurement of infectious EIAV in plasma. To complement the measurement of viral RNA levels in plasma, we also determined the levels of infectious EIAV by using primary equine monocyte-derived macrophage (MDM) cultures. Monocytes were isolated by overnight adherence, cultured, and frozen as previously described (32). Thawed monocytes were plated at 105 cells per well in 48-well plates in minimal essential medium-alpha medium (Gibco BRL) supplemented with 50% heat-inactivated adult bovine serum (HyClone), 2 mM glutamine, 100 U of penicillin per ml, and 100 µg of streptomycin per ml. Two days after thawing, the MDM cells were infected for 2 h at 37°C in Mg2+- and Ca2+-free Hanks balanced salt solution (Gibco BRL). Six wells of MDM cells per dilution of plasma were infected. Tenfold serial dilutions of plasma samples, starting at a 1:10 dilution, were used. Serum-free medium was added after 0.5 h to permit readherence of cells. Cells were washed once with serum-free medium. Cultures were maintained in minimal essential medium-alpha supplemented with 10% heat inactivated horse serum (HyClone), penicillin, streptomycin, and glutamine. Supernatants were removed weekly for micro-RT assays (19), and the TCID50 was calculated 14 days postinfection by using the Kärber formula. At least two independent infections of MDM cells were performed for each plasma sample.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Clinical profiles of EIAV experimentally infected animals. Six animals were infected intravenously with either the EIAVWyo or EIAVPV strain (Table 1). All infected animals developed clinical disease symptoms, including febrile episodes, depression, and anorexia, within 1 month postinfection. Horses 298 and 94-07 infected with the highly virulent EIAVWyo strain became moribund and were euthanized 12 and 11 days postinfection, respectively. Four ponies were infected with the EIAVPV isolate. Ponies 112 and 123 were inoculated with EIAVPV and developed a fever associated with acute EIA at 9 and 13 days postinfection, respectively. The ponies were sacrificed at this time. Ponies 18 and 72 experienced multiple, irregularly spaced disease cycles typical of chronic EIA within 6 months of infection. Pony 18 resolved the chronic disease and remained clinically quiescent for more than 1 year. In contrast, pony 72 was clinically quiescent for 6 months and subsequently experienced two additional late disease cycles. These recrudescent febrile episodes were at 5 months and at 7 days prior to being euthanized, respectively. Ponies 18 and 72 were clinically normal (asymptomatic) at the time of sacrifice. Tissue samples and PBMC were isolated at the time of necropsy from all experimentally infected animals.
Quantification of EIAV in the plasma. As an initial measure of the systemic levels of virus replication associated with the various experimental infections, we assayed the levels of plasma viremia by using infectivity assays in cultured MDM cells. The results of these assays (Table 1) indicated similar plasma viremia levels ranging from 103.6 to 105.0 TCID50 per ml during acute disease caused by either the EIAVWyo (animals 298 and 94-07) or EIAVPV (animals 112 and 123) infections. In marked contrast to the levels of infectious EIAV in plasma during acute disease, no infectious virus was detected in repeated assays of plasma taken during the asymptomatic stage of EIA (animals 18 and 72). The levels of infectious virus associated with acute and asymptomatic EIA were in general agreement with previous studies from this laboratory and others using infectivity assays in FEK cells (11, 18, 31). However, these experiments represented the first direct comparison of plasma samples from EIAVWyo- and EIAVPV-infected animals made possible by the use of MDM cells.
As noted above, EIAV plasma viremia has to date been measured using virus infectivity assays in cell culture. With the development of a quantitative RT-PCR technique (19), we were able to also compare plasma viral RNA levels in EIAVWyo and EIAVPV infections in parallel (Table 1). The results of these assays indicated high levels of plasma viral RNA in the two EIAVWyo-infected horses with 3.1 × 108 and 3.6 × 107 copies per ml in animals 298 and 94-07, respectively. The EIAVPV acutely infected ponies were found to have 2.1 × 107 and 4.2 × 107 copies per ml for ponies 112 and 123, respectively. The amounts of viral RNA detected in these experimentally infected animals were within the range of RNA levels observed during acute EIA in our historical panel of experimental infections (32). Using both the measurements of infectious virus and plasma RNA levels, it can be calculated for the first time that one infectious unit of either the EIAVWyo or EIAVPV in blood corresponded to about 3,000 copies of viral RNA as measured by RT-PCR. In contrast to acute EIA, the asymptomatic animals had either undetectable or barely detectable levels of plasma viral RNA (Table 1). Interestingly, the level of suppression of EIAV replication was apparently similar in pony 18, which remained asymptomatic for over a year, and in pony 72, which had just recovered from a recrudescence of disease. One week prior to the euthanization of pony 72, the amount of viral RNA was determined to be about 4.2 × 107 copies of per ml of plasma. The precipitous drop of over 5 orders of magnitude in plasma RNA levels over the course of 1 week in an animal that had been infected for more than 1 year indicated the maintenance of rapid, potent host clearance mechanisms. Taken together, these quantitative viremia studies demonstrate that acute disease was associated with very high plasma virus levels as measured by plasma viremia in MDM cells and by quantitation of viral RNA. While the sample size was small, no association between disease severity and virus levels were observed, confirming previous observations (31). Additionally, the lack of detectable infectious virus and the low levels of viral RNA indicated that production of EIAV is tightly controlled in asymptomatic animals.Identification of the in vivo sites of EIAV
infection.
To determine the viral DNA burden in tissues and
thus identify the in vivo sites of viral infection, we developed a
sensitive and specific assay for quantifying viral DNA. To
ascertain the specificity of the QC-PCR, DNA from infected and
uninfected samples were amplified with viral
gag-specific primers. A 310-bp product was detected only
in samples from infected animals, indicating that the amplified DNA was
of viral origin (data not shown). Amplification of viral sequences was
additionally confirmed by restriction enzyme digestion, by Southern
blot hybridization using EIAV gag-specific probes,
and by sequencing (data not shown). To determine the sensitivity of the
quantitative reactions, competitive amplification experiments were performed using specific known quantities of cloned viral DNA. The calculated copies were in close agreement with the expected values (Fig. 1, R = 0.99), and the sensitivity of the assay was determined to be 10 copies of viral DNA.
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Determination of the tissue sites of viral replication.
To
determine sites of active viral replication during acute disease and of
continuous virus production in acutely infected and asymptomatic
animals, the amount of singly spliced EIAV mRNA was
quantified by directly radiolabeled QC-RT-PCR. The active sites of
lentivirus replication have been previously identified by
quantification of either genomic or singly spliced mRNA species (2, 24, 34, 39). Studies performed by our laboratory observed high levels of cell-free virus, up to 108
particles per ml of plasma, during disease episodes (Table 1 and
reference 32). Thus, levels of EIAV
replication were assayed based on quantification of a singly spliced
mRNA species to avoid any contribution of viral genomic RNA to these
measurements. Amplification of viral singly spliced mRNA sequences was
detected only in infected samples containing reverse transcriptase,
indicating that the reaction was specific for this viral RNA (Fig.
3). No amplified product was detected in
plasma RNA samples from febrile animals, indicating that virions do not
contain viral singly spliced mRNA (data not shown). Restriction enzyme
digestion and sequencing of the amplified products confirmed the
specificity of this assay (data not shown). To determine the
sensitivity of the QC-RT-PCR, experiments analogous to those described
for viral DNA were performed. There was excellent agreement between the
calculated copies and the determined copies of in vitro-transcribed
RNAs (Fig. 1, R = 100). The assay was sensitive (limit
of detection, 30 copies of viral singly spliced mRNA) and
reproducible (coefficient of variation, <30%).
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Determination of the overall EIAV nucleic acid load.
The above assays determine the concentration of viral DNA and RNA in
various tissues but do not reflect the total organ burden. To determine
the relative contribution of each organ to the total viral nucleic acid
burden, the amount of viral nucleic acids was calculated from
generalized measurements of equid organs and body weights, the number
of copies of viral nucleic acids, and the nucleic acid yields from each
specimen. The kidney, peripheral lymph nodes, bone marrow, liver,
spleen, and brain were estimated to account for approximately
0.25, 0.1, 3.5, 1, 0.4, and 0.1%, respectively, of the total body
weight in horses (15, 45) and were assumed to be similar in
ponies. The viral DNA loads in each organ revealed that the bone
marrow, liver, and spleen were the principal sites of infection in the
EIAVWyo and EIAVPV acutely
infected and asymptomatic animals, containing an average of
1011, 109, and 108 copies in each
organ, respectively (Table 4). Taken
together, the spleen, bone marrow, and liver accounted for 98% of the
viral DNA burden in all infected animals regardless of the disease
state. In contrast, the liver and spleen each had approximately
1012, 1011, and 109 copies of viral
singly spliced mRNA in the animals acutely infected with
EIAVWyo and EIAVPV and in
the asymptomatic animals, respectively (Table 4). Together, the spleen
and liver accounted for up to 98% of the viral singly spliced mRNA and
were the major producers of virus in all animals. In contrast to the
abundance of viral DNA and RNA localized to tissues, the PBMC contained
less than 0.1% of the total viral DNA or RNA during acute disease or
during asymptomatic infections. These data clearly demonstrate that
tissues constitute the predominant in vivo sites of EIAV
infection during symptomatic and asymptomatic infections.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The clearly demarcated disease cycles associated with EIAV infection provide a unique in vivo model for characterization of the tissue reservoirs of productive and latent lentiviral infection during clinical episodes and during long-term asymptomatic infections. The experiments described here present for the first time a rigorous quantitative analysis of the levels of both EIAV viral DNA as a measure of infection and singly spliced viral RNA as a measure of virus replication in various tissue compartments of experimentally infected equids. The results of these studies identified preferential sites of virus infection, replication, and latency associated with persistent EIAV infections.
The levels of EIAV DNA and singly spliced viral RNA measured in the experimentally infected equids differed markedly depending on the clinical status of the infected animals and the strain of virus used for the infection. In terms of plasma viremia, the current studies clearly demonstrate that acute EIAV infection is associated with very high levels of virus in the blood, as measured by infectivity in a recently developed quantitative equine macrophage assay (32). In marked contrast, infectious virus was undetectable in blood during asymptomatic EIAV infection. The current experiments for the first time extend the measurements of blood viral burden to include quantification of plasma viral RNA by RT-PCR. These data revealed plasma RNA levels of 107 to 108 copies of virion RNA per ml of plasma during acute disease and fewer than 100 copies per ml during asymptomatic stages of infection. Thus, the new combination of in vitro infectivity and PCR assays clearly illustrate the characteristic wave of viremia associated with EIAV disease episodes and the rigorous control of virus replication during clinical latency.
While these blood virus load measurements in general confirm previously reported viremia data derived from infectivity assays in equine fibroblastic cells, the new assays also provide new insights into the pattern of viremia during persistent EIAVPV infection. First, the viremia levels measured in equine macrophages are similar to viremia levels reported previously by infectivity assays in equine fibroblastic cells (11, 18, 31). This agreement indicates that the latter assays in fact provide a relevant measure of EIAVPV viremia, eliminating a lingering concern about the validity of using equine fibroblastic cells to measure EIAVPV infectivity. Second, the quantitative measurements of plasma viral RNA closely parallel viremia measurements in assessing viral load during acute disease and clinical latency. However, the viral RNA measurements demonstrate for the first time that the negligible levels of infectious EIAV associated with clinical latency is in fact due to an absence of viral particles in the blood and not to an inactivation of virus by neutralizing serum antibodies. This observation appears to indicate the efficacy of the host immune control of viral replication, presumably by the reduction of infected target cells. Finally, the viremia data summarized here indicates that the typically fatal outcome of EIAVWyo infection compared to the chronic EIA induced by EIAVPV is not reflected in substantial differences in plasma viremia levels, since the two infections produced similar plasma virus loads during the initial acute disease. As discussed below, however, the differences in disease severity may be more accurately associated with the tissue viral load rather than the levels of virus in blood.
During the initial stages of acute disease within 2 weeks postinfection, viral infection and replication were shown to be widely disseminated among all of the tissues examined in both the EIAVWyo- and EIAVPV-infected animals (Tables 2 to 4). In this regard, the results of the current studies are in agreement with previous analyses of EIAV replication in infected horses during acute disease using cultured cells to detect virus (17), fluorescent antibody (23), Southern blot (36), in situ hybridization (41), or PCR (13, 14, 26) procedures and with the pathological findings characteristic of EIAV infection (41). The results of these earlier qualitative analyses established an association between sites of virus infection and host tissues rich in macrophages. However, the quantitative assays employed here extend these studies to clearly identify the spleen as the predominant cellular site of EIAV infection and replication during acute disease. The concentration of proviral DNA and singly spliced viral RNA in the splenic tissues was typically 10 to 20 times greater than the concentration of EIAV DNA or RNA measured in any other tissue. In contrast, the asymptomatic animals had equivalent cellular loads of viral DNA and mRNA in the spleen and the liver (Tables 2 and 3). In all animals, estimation of total organ viral loads (Table 4) indicates further that the spleen and liver tissues contain up to 85% of the total viral DNA or mRNA that was detected in the tissues examined. It is assumed that the high levels of virus infection and replication in the splenic and hepatic tissues reflect the abundance of target macrophages in these organs. In addition, these quantitative analyses revealed that during the initial acute disease the EIAVWyo-infected animals contained 10- to 100-fold more viral DNA and singly spliced RNA in tissues compared to the levels measured in the EIAVPV-infected animals. This differential in tissue levels of viral DNA and singly spliced viral RNA is in contrast to the similar levels of plasma viral genomic RNA measured during acute disease in animals infected by the two viral strains. These observations indicate that the levels of tissue-associated viral DNA and singly spliced viral RNA, especially in the spleen and liver, constitute a more accurate correlate of disease severity than plasma viral RNA load.
Although the quantitative viral DNA and singly spliced viral RNA analyses in general identified macrophage-rich tissues as the predominant sites of viral infection during acute EIA, in agreement with pathological findings, these detailed analyses also revealed some unexpected aspects of EIAV infections. For example, a number of studies have reported that EIAV DNA and RNA is either very low or undetectable in PBMC isolated from horses infected with EIAV (14, 21, 42, 43). These observations support the widely accepted concept that infection of blood monocytes with EIAV results in latent infection and that cellular differentiation to macrophages is necessary for the production of virus, as proposed for other lentiviruses (8). However, in the current studies we observed substantial levels of viral DNA (2,000 to 16,000 copies per 100,000 CE) and viral singly spliced RNA (700 to 17,000 copies per µg of RNA) in PBMC in three of the four infected animals during acute disease. These findings suggest that blood monocytes can in fact serve as targets of EIAV infection and of virus production during waves of viremia associated with disease episodes, perhaps reflecting widespread activation of blood monocytes during cycles of EIAV disease.
A second unexpected observation is the relatively high level of viral DNA (Table 2) accompanied by a low level of viral singly spliced RNA (Table 3) in the bone marrow of the EIAVWyo acutely infected and the asymptomatic EIAVPV-infected animals. These observations extend previous findings from visna virus (9), simian immunodeficiency virus (4), and HIV-1 (7, 28) indicating that the less-differentiated cells of the bone marrow can serve as a reservoir of latently infected cells. Recent studies detected increased levels of inflammatory cytokines in the bone marrow during EIAV infection in vivo (47) and in bone marrow derived macrophages in vitro (46), which are hypothesized to suppress platelet production (5). Thus, cytokine dysregulation could result in thrombocytopenia, a clinical finding consistently associated with recrudescence of disease. Additionally, severe hemorrhaging is the principal contributing pathology in lethal EIAVWyo infection. Therefore, the organ-specific viral loads may directly or indirectly account for the pathological features manifest during disease cycles.
In striking contrast to the high levels of viral DNA and viral singly spliced RNA found in tissues from animals during acute disease, asymptomatic EIAV infections were characterized by extremely low levels of viral DNA (150 to 350 copies per 100,000 CE) and viral singly spliced RNA (50 to 750 copies per µg of RNA) concentrated predominantly in the liver and spleen. Other tissues examined contained fewer than 100 copies of DNA and usually <30 copies of singly spliced viral RNA. These observations demonstrate a rigorous control of EIAV replication that severely limits the number of EIAV-infected cells and the levels of virus production. However, it is important to note that despite the lifelong asymptomatic state usually maintained by EIAV-infected animals following 6 to 12 months of chronic EIA, the viral infection is never completely eliminated since tissue reservoirs of productively infected cells are sustained despite the high levels of immune control established by the host.
The ability of animals to routinely establish immunological control of EIAV infection and disease is considered the best example of natural immune control of a virulent lentivirus infection (25). The concept of immunologic control of EIAV replication in infected asymptomatic animals has been developed mostly based on measurements of viremia that indicate the absence of detectable virus in the blood of asymptomatic animals in marked contrast to the viremia levels of 105 to 106 TCID50 per ml of blood during clinical episodes (25). However, it has been shown that transfer of as little of 7 ml of whole blood from asymptomatic animals to naive equids produces infection of as least 80% of the recipient animals (12). Therefore, it has not been clear from these studies whether the lack of infectious virus in the blood actually reflected a lack of virus particles or inactivation of plasma virus by the high levels of EIAV-specific neutralizing antibodies present in asymptomatic animals (11). The RT-PCR assays described here demonstrate for the first time that the lack of infectious virus in the blood in asymptomatic animals is indeed reflective of a virtual absence of detectable viral genomic RNA in the plasma from these animals. Thus, the infectivity of blood from asymptomatic animals is most likely associated with the approximate 0.04% of PBMC containing EIAV proviral DNA.
The results described in the current study define the tissue sites of EIAV infection but do not specifically define the target cells within these tissues. In agreement with the well-established macrophage tropism of EIAV, the predominant sites of EIAV infection and replication during symptomatic and asymptomatic infections were determined to be tissues rich in macrophages, such as the spleen and liver. While a recent study (22) indicates that renal macrovascular endothelial cells may also serve as a reservoir of EIAV infection in a long-term inapparent carrier, the relative contributions of macrophages and endothelial cells in different tissues to viral persistence were not determined in this study. In addition, there has been no characterization of endothelial cell infection in experimentally infected horses during acute disease to assess the potential role of these cells in pathogenesis. The results of the current study seem to support the predominant importance of monocytes and macrophages in viral pathogenesis and persistence, since EIAV infection and replication correlated with the abundance of tissue macrophages in the respective organs.
These quantitative studies of EIAV infection and replication in vivo provide a detailed description of the patterns of virus replication associated with clinical and asymptomatic infections. While the data are specific for EIAV, the results have implications for broader issues related to the role of tissue reservoirs in lentiviral persistence, the potential for immune control or ultimate clearance of lentiviral infections, and for understanding the mechanisms of lentiviral pathogenesis. Based on the studies with EIAV, a predominantly macrophage-tropic lentivirus, it is clear that tissues can provide a widespread and resilient reservoir for lentiviral infection, even in the presence of potent host immune clearance mechanisms. Moreover, it appears that the extent of viral pathogenesis can be most closely associated with the levels of virus replication in tissues rather than plasma virus burden. Thus, it will be important to examine in more detail the potential role of tissue macrophages in the persistence of lentiviruses that are also lymphotropic and to define the mechanisms by which tissue macrophage infections lead to characteristic lentiviral pathologies such as fever, diarrhea, anemia, thrombocytopenia, and encephalitis.
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ACKNOWLEDGMENTS |
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We thank Drew Lichtenstein for stimulating discussions, Caroline Leroux for thoughtful review of the manuscript, and Gary Thomas and Brian Meade for excellent animal care.
This work was supported by National Institutes of Health grant R01 CA49296, by funds from the Lucille P. Markey Charitable Trust and the Kentucky Agricultural Experimental Station, and in part by a grant from the Pittsburgh Supercomputer Center through the NIH National Center for Research Resources (resource grant 2 P41 RR06009). S.M.H. was supported by NIH AIDS training grant 5T32 AI07487.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261. Phone: (412) 648-8869. Fax: (412) 383-8859. E-mail: rmont{at}pop.pitt.edu.
Present address: Ross Products Division, Abbott Laboratories,
Columbus, OH 43215.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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