tipyretics, but did not respond to treatment and was eutha- nized on ..... 135E:119-134. 29. Orrego, A., C. J. Issel, R. C. Montelaro, and W. V. Adams, Jr. 1982.
Vol. 64, No. 12
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0022-538X/90/125756-07$02.00/0 Copyright © 1990, American Society for Microbiology
Equine Infectious Anemia Virus Derived from Clone Persistently Infects Horses L.
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Molecular
WHETTER,1 D. ARCHAMBAULT,2 S. PERRY,' A. GAZIT,3 L. COGGINS,' A. YANIV,3 D. CLABOUGH,1 JOHN DAHLBERG,2 F. FULLER,'* AND S. TRONICK2
Department of Microbiology, Pathology & Parasitology, North Carolina State University, Box 8401, College of Veterinary Medicine, Raleigh, North Carolina 276061; Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 208922; and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel3 Received 11 May 1990/Accepted 20 August 1990
A full-length molecular clone of equine infectious anemia virus (EIAV) was isolated from a persistently infected canine fetal thymus cell line (Cf2Th). Upon transfection of equine dermis cells, the clone, designated CL22, yielded infectious EIAV particles (CL22-V) that replicated in vitro in both Cf2Th cells and an equine dermis cell strain. Horses infected with CL22-V developed an antibody response to viral proteins and possessed viral DNA in peripheral blood mononuclear cells, as determined by polymerase chain reaction assays. In addition, horses infected with CL22-V became persistently infected and were capable of transmitting the infection by transfer of whole blood to uninfected horses. However, CL22-V, like the parental canine cell-adapted virus, did not cause clinical signs in infected horses. Reverse transcriptase assays of CL22-V- and virulent EIAV-infected equine mononuclear cell cultures indicated that the lack of virulence of CL22-V was not due to an inability to infect and replicate in equine mononuclear cells in vitro.
properties of virus derived from this molecular clone with those of wild-type EIAV.
Equine infectious anemia virus (EIAV) is one of several animal lentiviruses that share homology with the human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) (4, 13, 14, 28, 34). EIAV possesses an Mg2'-dependent, RNAdirected DNA polymerase (1, 5) and an 8.2-kb RNA genome (6, 18, 37). In contrast to some other lentiviruses, EIAV can cause acute illness within 1 to 2 weeks after infection. The acute disease is characterized by a high-titer viremia, fever, thrombocytopenia, and edema due to vascular congestion. Few horses die from the acute illness, and all survivors become persistently infected with the virus. The persistent phase of EIA is typified by recurrent clinical episodes, with a novel antigenic variant of EIAV predominating during each one (3, 21, 31). Most of these episodes occur within 1 year of the initial infection, after which infected horses remain inapparent carriers of the virus (17). The infection can be transmitted by transfer of whole blood, washed leukocytes, cell-free serum, or plasma from asymptomatic infected carriers to other horses (7). Wild-type isolates of EIAV replicate in horses or equine leukocyte cultures (20, 22, 35), but establish a productive infection in equine fetal kidney cells, equine dermal fibroblasts, and canine fetal thymus cells only after multiple passages (adaptation). Adaptation to grow in the cultured cells leads to a loss of virulence (26, 29, 37), which can be restored in some cases by serial passage of the virus in horses (16, 29). To understand the molecular basis of EIAV virulence and to determine the extent to which mixed infections influence the pathogenesis of the disease, infectious molecular clones of the EIAV genome are required. Since previously characterized EIAV genomes have been defective, we have continued these efforts and have achieved the first successful isolation of an infectious molecular clone of this virus. In this paper we describe its isolation and compare the biological *
MATERIALS AND METHODS Cells and virus. The wild-type Wyoming virus strain 158 was obtained from L. 0. Mott (19) and propagated in horses. Serum was obtained from an infected horse during the peak febrile response, and the titer of this stock (hereafter referred to as WYO-WT) was determined to be approximately 106 infectious doses/ml (as determined by horse inoculation assay). The Malmquist strain of EIAV was derived from the wild-type Wyoming strain by adaptation of the wild-type virus in equine dermis fibroblast cells (26). Virus derived from the molecular clone (CL22-V; see below) was grown in equine dermis cells (ATCC CCL 57). The titer of the supernatant from these cultures was determined to be 106 50% tissue culture infective doses/ml in equine dermis cells. Equine dermis cells and canine fetal thymus cells, Cf2Th (ATCC CRL 1430), were cultured in Dulbecco modified Eagle medium with 10% fetal bovine serum.
Molecular cloning of the EIAV proviral genome. Cf2Th cells were infected with the Malmquist strain of EIAV (ATCC VR-778). Cf2Th cells were chosen as host cells for proviral cloning because these cells could be grown from single cells by limiting dilution, whereas equine dermis cells cannot. Replication of virus was detected by monitoring culture supernatants for EIAV gag antigen by radioimmunoassay (35). Two weeks after infection, high levels of virus antigen were detected and the cells were cloned by dilution in microtiter plates. Approximately 25% of the clones were positive for EIAV gag antigen. The DNA from Cf2Th cell clones producing infectious virus was isolated, digested with EcoRI (which does not cut within the EIAV genome), and analyzed by Southern blot hybridization. Restriction digestion patterns of the DNA from two cell clones were each found to contain a single band (both approximately 15 kb) that hybridized to a probe
Corresponding author. 5750
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derived from clone pEIAV-1369 (37). Both cell clones (designated 3E8 and 4H5) were recloned and screened for infectious virus production. A genomic library was prepared from one stable subclone (designated C5) that continued to produce infectious virus. An EMBL3 (Stratagene, La Jolla, Calif.) bacteriophage library of high-molecular-weight, EcoRI-cleaved genomic DNA was prepared by standard methods as described previously (37). Seven phage clones containing EIAV sequences were detected by hybridization, using the pEIAV1369 probe. Cf2Th and equine dermis cells were transfected with EIAV containing phage DNAs and tested for the release of infectious virus as described previously (37). The DNA from one phage, designated CL22, was used to transfect equine dermis cells, and the virus stock derived from this phage was designated CL22-V. In vivo infections. Horses were inoculated intravenously with either 1.0 ml of WYO-WT serum or 5.0 ml of CL22-Vinfected cell culture supernatant fluid. We were unable to use the same kind of in vitro infectivity assay on both WYO-WT and CL22-V. Therefore, we chose to equalize the inoculum by use of a reverse transcriptase assay which allows us to compare the two viruses by the same method. Rectal temperatures of infected horses were monitored twice a day, and blood and bone marrow samples were obtained at intervals pre- and postinfection. Whole blood was transferred from CL22-V-infected horses to recipients by removing 200 ml from donor horses (by venipuncture) into a blood collection bag (Fenwall Laboratories) and immediately transfusing the blood into the jugular vein of recipient horses through a straight type blood recipient large-capacity filter (Fenwall Laboratories). Western immunoblots. EIAV was harvested from the supernatant fluids of persistently infected equine dermis cell cultures by ultracentrifugation and concentrated by banding on sucrose gradients. EIAV proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (25) on 10% gels. The proteins were transferred to Immobilon-P membranes (Millipore Corp.) and reacted with pre- or posttest sera followed by phosphatase-labeled goat anti-horse immunoglobulin G (Kirkegaard & Perry Laboratories). Reactions were developed by using a model SK5200 alkaline phosphatase substrate kit II (Vector Laboratories Inc.). Agar gel immunodiffusion tests. Agar gel immunodiffusion tests were performed on horse sera as described by Coggins and Norcross (8) but with EIAV antigen as prepared by Malmquist et al. (26). The agar gel immunodiffusion test was used to detect antibody to the p26 core antigen in the sera of infected horses. Polymerase chain reaction assays. Mononuclear cells were obtained from whole venous blood by centrifugation through Sepracell (Sepracell Corp.). Bone marrow cells were obtained by sternal aspirate. DNA was isolated from these cells by the procedure described by Enrietto et al. (9). Briefly, cells were washed free of serum and lysed in a buffer (0.01 M Tris [pH 8.0], 0.1 M sodium chloride, 0.025 M EDTA) containing 0.5% sodium dodecyl sulfate and 100 jig of proteinase K per ml. Preparations were incubated overnight at 37°C and extracted with phenol-chloroform-isoamyl alcohol (25:24:1), and the DNA was ethanol precipitated. Polymerase chain reaction (PCR) amplification was performed on 1 ,ug of cellular DNA by using 100 pmol each of oligonucleotide primers U3 and GAG1. Primer U3 (5'-TGTG GGGTTTTTATGAGGGGTT-3') is complementary to the noncoding strand at nucleotides 1 to 22 of the 1369 EIAV
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proviral clone (18). Primer GAG1 (5'-AGGTGCTCTACATT GAC-3') is complementary to the coding strand at nucleotides 1662 to 1645, 260 bp upstream of the 3' end of the gag gene. Together, these primers amplify a 1.6-kb fragment of the EIAV provirus. After 40 cycles of amplification with Taq polymerase (Perkin Elmer-Cetus), DNA was resolved on a 1% agarose gel, transferred to nitrocellulose, and then hybridized with a 32P-labeled probe. The Southern hybridization probe used to detect the PCR product was prepared by nick translation of the same 1.6-kb region amplified with the U3 and GAG1 primers from an EIAV-containing plasmid. Southern blots were quantitated by analysis with an AMBIS
Radioanalytic Imaging System. In control experiments with known amounts of EIAV plasmid DNA as the template for amplification, we have determined that our threshold level of detection is about 6,000 copies of template per reaction. With 40 cycles of amplification there is an exponential relationship between the amount of template and the yield of amplified product up to approximately 6 x 106 molecules of template per reaction. The amount of product DNA obtained in the amplifications described here indicates that the amount of template DNA is in all cases fewer than 6 x 106 EIAV template molecules per reaction and therefore is in the exponential phase of the PCR as described by Wang and Mark (36). In vitro infections. Equine peripheral-blood mononuclear cells were separated from whole blood by centrifugation through Sepracell and washed twice in phosphate-buffered saline. Equine mononuclear cells were cultured in RPMI 1640 plus 50% fetal calf serum with 107 cells plated per well of a 24-well tissue culture plate. Cultures were incubated overnight at 37°C (5% C02), after which nonadherent cells were removed by washing with medium. Adherent cells were infected by substitution of their media with 0.5 ml of inoculum (WYO-WT serum, CL22-V supernatant, or medium) and incubation for 24 h, after which the inoculum was removed and replaced with fresh medium. Media on the cell cultures were changed every 2 days, and samples were collected for reverse transcriptase assay just prior to media change. Reverse transcriptase assays were performed as previously described by Carpenter and Chesebro (2). RESULTS Previous efforts to isolate infectious EIAV molecular clones have been unsuccessful (34, 37). In the present studies we isolated a clone of Cf2Th cells which contained a single copy of integrated proviral DNA and produced infectious virus. A stable, virus-producing, Cf2Th cell clone (CS) was obtained after multiple rounds of subcloning as described in Materials and Methods. A total of seven EMBL3 clones containing EIAV-specific sequences were isolated from the C5 genomic DNA library. Transfection of Cf2Th cells with one of the EMBL3 clones (CL22) resulted in detectable EIAV p149a9 in the cell supernatant fluid (as measured by radioimmunoassay) at 1 and 3 weeks posttransfection (Fig. 1). At 3 weeks posttransfection the supernatant fluids were used to infect equine dermis cells, and radioimmunoassays of supernatant fluids at 1 and 2 weeks postinfection indicated that EIAV was present. All seven of the EMBL3 clones produced infectious virions upon transfection of Cf2Th cells and passage of supernatant fluids to equine dermis cells (data not shown). Restriction enzyme maps of the DNA from each of these seven proviral clones were indistinguishable and identical to the map of the first reported full-length (but noninfectious) EIAV clone
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FIG. 3. Westem blot analysis of horses infected with WYO-WT or CL22-V and of transfusion recipients. Serum was analyzed for the of EIAV-specific antibody before or at various times postinfection. Lanes: A and V, serum from a horse chronically infected with EIAV; B to E, 0, 4, 10, and 18 days after infection of E403 with WYO-WT; F to I, 0, 3, 21, and 39 days after infection of E433 infected with CL22-V; J to M, 0, 4, 20, and 40 days after infection of E455 which received 200 ml of blood from E433 at 130 days postinfection; N to Q, 0, 3, 19, and 40 days after infection of E435 with CL22-V; R to U, 0, 3, 20, and 57 days after infection of E390 which received 200 ml of blood from E435 at 44 days postinfection. presence
days postinfection in a single experiment (Fig. 4F). In this analysis the PCR signal obtained from WYO-WT-infected horses was 12- to 45-fold greater than the signal obtained from CL22-V-infected horses. These data indicate either that fewer mononuclear cells became infected with CL22-V than with WYO-WT or that fewer copies of the viral genome were present per infected cell in CL22-V-infected mononuclear cells. Since some lentiviruses, notably visna virus, infect monocyte precursors in the bone marrow (11), we reasoned that EIAV might persist in the bone marrow of infected horses at times when virus was not detectable in the blood mononuclear cells. However, the PCR signal obtained from amplification of bone marrow cell DNA was, in each case, lower than or equal to that obtained from the blood mononuclear cells (Fig. 4A to C). Replication of WYO-WT and CL22-V in equine mononuclear cell cultures. Having established that CL22-V could infect equine mononuclear cells in vivo, we elected to test the hypothesis that the lack of virulence associated with CL22-V might be due to a decreased ability to replicate in monocyte-macrophages. Adherent equine mononuclear cell cultures were infected with WYO-WT or CL22-V as described above, and the cell culture supernatant fluid was tested for reverse transcriptase activity as a measure of virus production. CL22-V- and the WYO-WT-infected cultures had a comparable increase in reverse transcriptase activity by 7 to 8 days postinfection (Fig. 5). These findings suggest that the lack of virulence of CL22-V is not due to its inability to replicate in monocytes or macrophages.
DISCUSSION In order to study virulence factors and mechanisms of viral expression as they relate to pathogenesis and the development of immunity, we isolated an infectious molecular clone of EIAV and characterized its phenotype in vivo. Virus derived from this clone, CL22-V, was able to establish a persistent but asymptomatic infection in horses and, like the wild-type EIAV, infect blood mononuclear cells. We were able to demonstrate EIAV sequences in the DNA from peripheral blood mononuclear cells from two CL22-V-infected ponies at 10 days postinfection, which paralleled the wild-type infection. A persistent infection was established, and EIAV DNA was detected in the mononuclear cells for as long as 6 months postinfection in horse E433 and at least 40 days in horse E435. Furthermore, the virus was capable of being transferred from horse E433 to an uninfected horse by transfusion of blood at 4 months after infection, confirming that infectious EIAV was present. A similar transfer from a horse which had been infected for 40 days (E435) stimulated an immune response to EIAV proteins in the recipient (E390), confirming that EIAV proteins had been transmitted (Fig. 3, lane U), but did not result in detectable EIAV sequences in the mononuclear cell DNA, as measured by PCR. The absence of an EIAV-specific PCR signal may reflect a very low level of infection of blood mononuclear cells or may indicate that infected cells are not detected in the circulation in all infected animals. It has been reported that HIV-infected individuals may not all contain detectable HIV sequences in peripheral blood mononuclear
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FIG. 4. Southern blots of PCR assays of EIAV-specific sequences in blood and bone marrow cells from WYO-WT-infected, CL22-Vinfected and transfusion recipient horses. Each lane represents the DNA amplified in 10 ,ul of a 100-pl PCR mixture containing 1 ,ug of blood mononuclear (MN) or bone marrow (BM) template DNA. Exposure times were optimized for each blot (from 2 to 16 h, without intensifying screen). (A) WYO-WT-infected E403. (B) CL22-V-infected E433. (C) CL22-V-infected E435. (D) E455 transfusion recipient of E433. (E) E390 transfusion recipient of E435. (F) Mononuclear cell samples of CL22-V-infected horses (E433 and E435) and WYO-WT infected horses (E403 and E417) taken 10 days postinfection.
cell DNA when assayed by PCR (30, 33). Although the transfusion donor E435 has a strong PCR signal in blood mononuclear cells (Fig. 4C at 40 days postinfection), we cannot be certain that infectious EIAV was transferred to E390. We have observed that serum antibodies to EIAV declined to undetectable levels by 3 months postinoculation of E390 (data not shown). It is interesting that the intensity of the PCR signal obtained at 10 days postinfection was greater in each case with DNA from WYO-WT-infected horses than with DNA from CL22-V-infected horses (12 to 45 times greater [Fig. 4F], as determined by AMBIS scanner analysis). These results suggest that more viral replication occurred in the WYO-WT-infected horses than in the CL22-V-infected horses. This hypothesis is supported by the Western blot analysis, in which antibodies to EIAV were detected earlier in the WYO-WT-infected horse than in either of the CL22V-infected horses. The results from Fig. 4 indicated that there was a strong PCR signal in the bone marrow DNA samples from the WYO-WT-infected horse E403 and either a weak or no signal at 10 days postinfection from the CL22-V-infected horses E433 and E435. This could represent an important difference in the pathogenicity of these two virus strains. However, since blood contamination is a possibility when taking a bone marrow sample, we cannot be certain whether the WYO-WT PCR signal is from bone marrow or blood cell infection. We plan to address this question by in situ hybridization studies.
These data, combined with a report which indicated that virulent strains of EIAV replicate to a higher titer in horse mononuclear cell cultures than do avirulent strains (2), suggest that CL22-V would exhibit a decreased ability to replicate in equine mononuclear cell cultures when compared with WYO-WT. However, our data demonstrate that both viruses replicated to a similar titer, suggesting at least four possibilities: (i) the expression of EIAV in unstimulated mononuclear cell cultures does not approximate what occurs in vivo; (ii) there is not a direct association between virulence and mononuclear cell tropism; (iii) the ability to replicate to high titer in mononuclear cell cultures may be a strain characteristic; and (iv) reverse transcriptase activity in the supernatant fluid of equine mononuclear cells infected in vitro is not indicative of infectious virus production. In both visna virus and HIV infection, the pattern of virus replication in monocyte-macrophages is complex, with increased viral expression during cell maturation (10, 11, 15). Furthermore, various cytokines have been shown to increase HIV production from macrophage cultures (12, 23). We are currently investigating the role of cytokines and macrophage inducers in the growth of EIAV in equine macrophage cultures. Since CL22-V is clearly capable of infecting equine mononuclear cells, if there is a defect in its ability to replicate in these cells in vivo we are unable to determine what that defect could be with our unstimulated in vitro monocyte-macrophage cell cultures. An association between EIAV virulence and cell tropism has long been noted. Although virulent strains can be
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Day Post-Infection FIG. 5. WYO-WT and CL22-V infection and growth in equine peripheral blood mononuclear cell cultures. Equine peripheral blood mononuclear cells from a virus-negative horse were allowed to adhere overnight and were infected in culture with WYO-WT or CL22-V or mock infected at day 0. The growth medium was changed for all cultures at days 1, 3, and 5, and spent medium was collected and assayed (10 ,ul in duplicate) for Mg2+-dependent reverse transcriptase activity. The day 3 samples represent reverse transcriptase activity from days 1 to 3, day 5 represents activity from days 3 to 5, day 7 represents activity from days 5 to 7, and day 8 represents activity from days 5 to 8. A background reverse transcriptase value for medium alone was subtracted from the values. Symbols: *, WYO-WT culture 1; El, WYO-WT culture 2; O, CL22-V culture 1; ES, CL22-V culture 2.
maintained in equine leukocyte cultures, EIAV strains which have been adapted to grow in cultures derived from cells other than horse leukocytes lose their ability to cause clinical illness in horses. Avirulent non-leukocyte cultureadapted strains which revert to virulence when serially passaged in horses maintain their ability to replicate in the cell culture to which they were adapted (29; our unpublished observations). Thus, the properties of non-leukocyte culture adaptation and virulence are not mutually exclusive. It has not been demonstrated whether the ability of EIAV to replicate to high titer in mononuclear cell cultures is strain specific. HIV isolates from some patients grow more readily in macrophage cultures than do isolates from other patients, but this ability does not appear to correlate with the severity of clinical disease (12, 24). Additionally, it was found that serial passage in macrophage cultures greatly enhanced the ability of HIV (isolated from peripheral blood mononuclear cells) to replicate in macrophage cultures (12). We are attempting to culture EIAV from mononuclear cells taken from a WYO-WT-infected horse during viremia to determine whether this "monocyte-selected" virus is capable of replicating to a higher titer in mononuclear cell cultures than is virus from WYO-WT serum. While the monocyte-macrophage is assumed to be the sole target cell for EIAV replication (27, 32), it has recently been reported that megakaryocytes from HIV-1-infected patients express viral RNA (38). As in HIV infection, EIAV infection is characterized by a profound thrombocytopenia which often precedes other clinical signs. The role of bone marrow mononuclear cell precursors and/or megakaryocytes in EIAV infection is clearly of interest. Our studies indicate that EIAV DNA can be detected in the bone marrow of infected horses. However, since bone marrow samples can be easily contaminated with peripheral blood, we were not able to establish by PCR analysis whether bone marrow serves as a reservoir during persistent infection. In situ hybridization should provide a better tool with which to answer this question. EIAV presents a unique opportunity to study virulence factors in lentivirus diseases, since virulent EIAV, unlike
other lentiviruses, causes a prompt clinical illness which can be easily monitored by the presence of high fever. We have isolated a molecular clone of EIAV that upon transfection of permissive cells yields virus which can persistently infect horses but does not cause clinical disease. We plan to continue these studies by replacing portions of the molecular clone, CL22, with portions of the WYO-WT genome that we have cloned from PCR-amplified regions of the wild-type genome. We hope that this strategy will allow us to construct a molecular clone that will yield fully virulent EIAV and to further characterize factors associated with EIAV virulence.
ACKNOWLEDGMENTS This research was supported by Public Health Service grant R01-AI24904 from the National Institutes of Health. D. Archambault was supported by a fellowship from the Medical Research Council of Canada. We thank Michael Jones and Christine Kolmstetter for excellent technical assistance in these studies and Barbara Sherry for a critical reading of the manuscript. LITERATURE CITED 1. Archer, B. G., T. B. Crawford, T. C. McGuire, and M. E. Frazier. 1977. RNA-dependent DNA polymerase associated with equine infectious anemia virus. J. Virol. 22:16-22. 2. Carpenter, S., and B. Chesebro. 1989. Change in host cell tropism associated with in vitro replication of equine infectious anemia virus. J. Virol. 63:2492-2496. 3. Carpenter, S., L. H. Evans, M. Sevoian, and B. Chesebro. 1987. Role of the host immune response in selection of equine infectious anemia virus variants. J. Virol. 61:3783-3789. 4. Casey, J. M., K. Yangkil, P. R. Andersen, K. F. Watson, J. L. Fox, and S. G. Devere. 1985. Human T-cell lymphotropic virus type III: immunologic characterization and primary structure analysis of the major internal protein, p24. J. Virol. 55:417-423. 5. Charman, H. P., S. Bladen, R. V. Gilden, and L. Coggins. 1976. Equine infectious anemia virus: evidence favoring classification as a retrovirus. J. Virol. 19:1073-1079. 6. Cheevers, W. P., B. G. Archer, and T. B. Crawford. 1977. Characterization of RNA from equine infectious anemia virus. J. Virol. 24:489-497. 7. Coggins, L., and M. J. Kemen. 1976. Inapparent carriers of
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