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From www.bloodjournal.org by guest on April 28, 2017. For personal use only. Infectious Transmission of Human T-cell Lymphotropic Virus Type I1 in Rabbits By G.L. Cockerell, M.G. Weiser, J. Rovnak, B. Wicks-Beard, B. Roberts, A. Post, I.S.Y. Chen, and M.D. Lairmore To determine the susceptibility of rabbits t o experimental infection with human T-cell lymphotropic virus t y p e 4 (HTLV11). four separate groups of four weanling rabbits each were inoculated intravenously with lethally irradiated HTLV-IIinfected human cell lines Mo-T (HTLV-11,-infected T cells), WIL-NRA (an Epstein-Barr virus [EBVI-transformed B-lymphoblastoid cell line infected with HTLV-I,), 729pH6neo (an EBV-transformed lymphoblastoid cell line transfected with a or G12.1 (HTLV-ll-infected T molecular clone of HTLV-11,). cells from a Panamanian Guaymi Indian). Two additional groups of four rabbits each were similarly inoculated with control uninfected 729 or HUT 78 cells. Early and persistent seroconversion t o HTLV-II core antigen p24, as determined by Western immunoblot, occurred in all HTLV-ll-inoculated rabbits and was most intense in rabbits inoculated with G12.1 cells; seroreqctivity t o other HTLV-II gag or env antigens occurred later, with less intensity, or not in all inoculated rabbits. Peripheral blood mononuclear cells (PBMC) and other lymphoid cells from HTLV-ll-inoculated rabbits produced minimal p24 in vitro, as determined by enzyme immunosorbent capture assay. Virus was more readily detected by polymerase chain reaction amplification of HTLV-II pol sequences; this occurred most frequently in rabbits inoculated with Mo-T cells, and most frequently in PBMC as compared with other tissues tested (bone marrow, brain, and liver). No evidence of disease occurred in HTLV-IIinoculated rabbits observed for as long as 24 weeks. All control rabbits remained negative for evidence of HTLV-II infection, as determined by the same procedures. These results provide the first evidence of HTLV-II infection in a species other than humans, and demonstrate the usefulness of the rabbit as an animal model t o study the biologic response t o different isolates of this human retrovirus. 0 1991by The American Society of Hematology. T tions, 36 of 39 and 21 of 23 HTLV-infected individuals were confirmed by polymerase chain reaction (PCR) to be infected with HTLV-II.'4*'5In addition, HTLV-I1 infection has been documented in a population of Guaymi Indians in PanamaI6; this represents the first description of HTLV-I1 infection outside of traditional risk groups. The known pathogenicity of HTLV-I and the high seroprevalence of HTLV-I1 in certain risk groups justifies additional study of these human retroviruses. This should include the identification of appropriate auimal models to study the pathogenesis, prevention, and treatment of the infection and disease. Previous work has shown that rabbits can be infected with HTLV-I by the intravenous, intraperitoneal, and oral routes with HTLV-I-infected lymphoid cells of human or rabbit origin."-'' In addition, Miyamoto et a1 recently demonstrated the in vitro transformation of rabbit leukocytes cocultured with an HTLV-11-infected human T-cell line." However, to date, infection with HTLV-I1 in vivo has not been reported in any species other than humans. The purpose of the present study was to determine if rabbits were susceptible to infection with HTLV-I1 in vivo, and to determine if different HTLV-I1 isolates caused different biologic response patterns in this animal model similar to emerging findings with HTLV-I (Lairmore et al, submitted for publication, 1991). HE HUMAN T-CELL lymphotropic virus (HTLV) family, including both HTLV types I and 11, as well as simian T-cell leukemia virus (STLV) and bovine leukemia virus (BLV), comprise a group of oncovirinae that shares structural and functional characteristics and biologic effects distinct from all other mammalian and avian retroviruses.'4 HTLV-I and BLV are the causative agents of lymphoproliferative disorders referred to as adult T-cell leukemialymphoma (ATLL)' and as enzootic bovine lymphoma (EBL)6 in humans and cattle, respectively. The role of STLV in leukemogenesis in primates is less clear.' HTLV-I is also associated epidemiologically with a neurodegenerative disorder termed HTLV-I-associated myelopathy (HAM)'or tropical spastic paraparesis (TSP)9 in humans. In contrast, HTLV-I1 has not been linked to a distinct clinical syndrome in humans, and only occasional isolates of this virus have been made.'"." Limited systematic studies of the seroprevalence of HTLV in the United States have been performed, but such studies are complicated by the fact that enzyme immunoassays, the most widely used screening tests for HTLV antibodies, do not differentiate between infection with HTLV-I and HTLV-II.'2*'3However, recent evidence suggests that infection with HTLV-I1 may be more widespread than initially suspected, particularly in intravenous drug users (IVDU); in two separate studies of IVDU popula- MATERIALS AND METHODS From the Department of Pathology, Colorado State University, Fort Collins, CO; Retrovirus Disease Branch, Centers for Disease Control, Atlanta, GA; and the Department of Microbiology and Immunology and Department of Medicine, UCLA School of Medicine, Los Angeles, CA. Submitted February 11,1991; accepted May 17, 1991. Address reprint requests to G.L. Cockerell, D M , PhD, Depament of Pathology, Colorado State University, Fort Collins, CO 80523. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 sole4 to indicate this fact. 0 1991 by The American Society of Hematology. 0006-4971I91 l7806-0019$3.00/0 1532 Rabbits and HTLV-II-infected and control cell inocula. Weanling (8-week-old) specific-pathogen-free New Zealand White rabbits were obtained from a commercial rabbitry (Western Oregon Rabbit Co, Philomath, OR). Groups of rabbits were inoculated intravenously with 1.5 x lo7to 10 x lo7HTLV-11-infected cells or uninfected cells as shown in Table 1. HTLV-11-infected cells were 88% to 98% infected, as determined by fluorescent antibody assay using an HTLV-1/11 anti-p24 monoclonal antibody (MoAb)." In addition, soluble p24 antigen was quantified in 3-day culture supernatants (1 x 106 cellslml) of each cell line (except Mo-T) using an antigen capture enzyme immunosorbent assay (Coulter Immunology, Hialeah, FL). The range of p24 detected was 255 to 375 ng/mL. Control uninfected cells were negative by the same techniques. All cells were lethally irradiated (55 Gy) before inoculation and effectiveness of the irradiation was verified by Blood, Vol78, No 6 (September 15), 1991: pp 1532-1537 From www.bloodjournal.org by guest on April 28, 2017. For personal use only. HTLV-II INFECTION OF RABBITS 1533 Table 1. Inocula for induction of HTLV-I1 Infection in Rabbits Dose (~107) Cellular Inocula (no. of rabbits) WIL-NRA 1.5 (4) 729pH6neo 1.5 (4) G12.1 1.5 (4) Mo-T 10 (4) 729 1.5 (4) HUT78 1.5 (2) 10 (2) Origin of Cellular Inocula An EBV-transformedhuman B-lymphoblastoid cell line (WIL-2)infected with HTLVI, by cocultivationwith T cells from patient NRA with a T-cell variant of hairy cell leukemia" An EBV-transformed human lymphoblastoid cell line (729)transfected with a molecular clone of HTLV-II,," HTLV-ll-infected T cells from a Panamanian Guaymi Indian'6 HTLV-ll,,,-infected T-lymphoblastoidcell line from patient Mo with a T-cell variant of hairy cell leukemia'" Non-HTLV-infected EBV-transformed human B-lymphoblastoidcell line (used as control for WIL-NRA and 729pH6neo-infected cells) Non-HTLV-II-infected human T-lympho. blastoid cell line (used as control for G12.1 and Mo-T-infected cells) failure of the cells to grow in culture. Infectivity of the inocula was verified by transfer of infection after coculture with normal rabbit peripheral blood mononuclear cells (PBMC). Detection of anti-HTLV-IZ antibodies. Sera (1: 100 dilution) were tested by Western immunoblot assay (WIB) as previously de~cribed"~'~ using a commercially prepared HTLV-II,,-infected cell lysate (Hillcrest Biologicals, Cypress, CA) as target antigen. Detection of HTLV-II antigens. PBMC were collected from all rabbits at 4, 12, and 24 weeks postinoculation (PI), and single cell suspensions of spleen, bone marrow, and mesenteric lymph node were prepared from rabbits killed at each of these intervals. Cells were cultured at a concentration of 1 x 106/mLin RPMI 1640with 10% fetal bovine serum, 100 U/mL penicillin, and 100 pg/mL streptomycin, 2 mmol/L glutamine, 5 pg/mL phytohemagglutinin (PHA-M; Difco Labs, Detroit, MI), and 10% human interleukin-2 (IL-2;Advanced Biotechnologies Inc, Silver Spring, MD). Aliquots of culture supernatant were collected at 3, 7, and 14 days and tested for the presence of HTLV-I1 antigens by enzyme immunosorbent capture assay.I6 Briefly, supernatant was added to wells of microtiter plates precoated with a murine anti-HTLV-1/11 p24 MoAb (Coulter Immunology). Retention of p24 by the capture antibody was subsequently detected by the addition of biotinylated human anti-HTLV-1/11 antiserum, followed by a strepavidinhorseradish peroxidase complex and chromagen. Absorbance values were determined and compared with a standard curve prepared with known amounts of core protein p24 in the same assay. Detection of HTLV-II provirus. Serial samples of PBMC and other tissues collected at necropsy were assayed for HTLV-I1 by PCR as previously described" with minor modifications, using primers and probe specific for the HTLV-I1 pol region?' Briefly, each PCR amplification was performed using 1 pg genomic DNA (approximately 150,000 cells) in a 100 +L reaction volume. The amplification consisted of 34 repetitive three-step cycles with the following conditions: 25°C to 95T, and then 2 minutes each at 95"C, SYC, and 72°C per cycle in a thermal cycler (Perkin-Elmer Cetus, Norwalk, CT). The amplified products were electrophoresed on 1.8% agarose gels, transferred to nylon membranes, and hybridized with a "P-labeled oligonucleotide probe. RESULTS Serologic and clinicopathologic responses to HTL V-II. All HTLV-11-inoculated rabbits produced antibodies against HTLV-I1 within 2 to 4 weeks PI and remained persistently seropositive over the 12- to 24-week period of study. All control-inoculated rabbits remained seronegative (Table 2). The earliest and strongest HTLV-I1 seroreactivity occurred against p24 antigen in rabbits inoculated with each HTLV-I1 isolate. WIB-detectable seroreactivity to other HTLV-I1 gag (p21, pr53) and env (gp46) antigens occurred later, with less intensity, or not in all inoculated rabbits (Table 2 and Fig 1). Reactivity to gp61/68 was not evident by WIB. The time of onset, intensity, and pattern of seroreactivity varied with the HTLV-11-infected cell line used for inoculation. The earliest and most intense reactivity occurred in rabbits inoculated with G12.1 cells (Fig 1). No evidence of disease was observed in HTLV-IIinoculated rabbits studied through 24 weeks PI, as evidenced by differences in weight gains, hematologic profiles, or the gross or microscopic appearance of tissues between HTLV-11-inoculated and control-inoculated rabbits. HTLV-11 antigen production from cultured cells. There was no clear difference in HTLV p24 Antigen present in supernatants collected after 3,7, or 14 days of culture. Low levels of antigen were"detected only in rabbits inoculated with G12.1 cells; supernatants of PBMC from two of four rabbits tested at 4 weeks PI, and of spleen cells from one of two rabbits each tested at 4 weeks and 12 weeks PI contained 10 to 12 pg/mL HTLV p24 (data not shown). Levels of HTLV p24 antigen in other culture supernatants from other rabbits inoculated with other HTLV-11-infected cells or control cells were below cutoff levels. Detection of HTLV-IIprovirus in tissues. A summary of the detection of PCR-amplified HTLV-I1pol sequences in tissues of rabbits is shown in Table 3 and a representative Southern blot of the amplified products is shown in Fig 2. The PCR-amplified pol product was most frequently detected in PBMC from rabbits inoculated with Mo-T cells (three of four rabbits), and was distributed in multiple tissues of these rabbits (two of three bone marrows tested, one of four brains tested, and two of four livers tested). Interestingly, the most extensive PCR-amplifiedpol reactivity was derived from one rabbit (R841) among those inoculated with 729pH6neo cells containing a molecular clone of HTLV-II,, (Fig 2). DISCUSSION The results of this study show the infectious transmission of HTLV-I1 in rabbits and represent the first evidence of HTLV-I1 infection in vivo in a species other than humans. Further, the results suggest that rabbits infected with different isolates of HTLV-I1 express different biologic response patterns, which may be due at least in part to heterogeneity amongst the viral isolates. Finally, the demonstration of the in vivo infectivity of a molecular clone of HTLV-I1 provides a mechanism to map the viral genetic determinants responsible for different biologic responses in rabbits. From www.bloodjournal.org by guest on April 28, 2017. For personal use only. 1534 COCKERELL ET AL Table 2. Anti-HTLV-ll Humoral Immune Response (determined by WlB) in HTLV-ll-Inoculated Rabbits Cellular Inocula* WIL-NRA 729pH6neo G12.1 Mo-T 729 HUT 78 HTLV-II Antigen P21 P24 pr53 gP46 P21 P24 pr53 gp46 P21 P24 pr53 9P46 P21 P24 pr53 9P46 P21 P24 pr53 9P46 P21 ~ 2 4 pr53 gp46 Weeks PI 0 014t 014 014 014 014 014 014 014 014 014 014 014 014 014 014 014 012 012 012 012 013 01 1 013 013 1 014 014 014 114 2 014 414 014 314 011 011 Oil Oil 3 014 414 214 214 Oil 011 011 011 4 1I 4 414 214 214 1I 4 314 314 214 214 414 214 414 014 414 214 214 012 012 012 012 012 012 012 012 6 8 12 013 313 113 113 112 212 212 212 112 212 212 112 212 212 212 212 013 313 113 213 112 212 212 212 212 212 212 212 212 212 212 212 013 313 213 113 01 1 01 1 01 1 01 1 Oil 01 1 01 1 011 011 011 011 01 1 01 1 Oil 011 011 011 Oil 011 011 16 20 24 012 212 112 112 012 212 112 212 012 212 112 112 011 011 011 011 01 1 01 1 01 1 01 1 'Cellular inocula as per Table 1. tNumber of positive rabbitslnumber of rabbits tested. Open data points indicate not determined. In vivo infectivity was evidenced by an early (as early as 2 weeks PI) and persistent (as late as 24 weeks PI) antiHTLV antibody response and demonstration of viral antigens or amplifiable proviral sequences in tissues of inoculated rabbits. Similar to findings in HTLV-infected h ~ m a n s , ' the ~ . ~earliest and most intense serologic reactivity occurred against p24 antigen as determined by WIB. Seroreactivity to env (gp46) antigens was also detectable, despite the difficulties in using the WIB technique to detect envelope antibodies due to the expected loss of envelope viral components during antigen purification." The presence of antibodies to both p24 and gp46 in our rabbits met the criteria established by the US Public Health Service for serologic confirmation of HTLV infe~tion.'~ As compared with similar studies with HTLV-1'' (Lairmore et al, submitted for publication, 1991), the HTLV-I1 isolates tested appear to be less infectious and to replicate less efficiently in rabbits. Only low levels of HTLV-I1 antigen were detected in supernatants of short-term cultures of lymphoid cells, and only from 50% of rabbits inoculated with G12.1 cells. Since these studies were conducted, we have discovered that the sensitivity of the antigen detection technique can be increased by coculturing infected cells with normal mitogen-stimulated rabbit lymphoblasts. This modification of the technique should increase the value of data derived in future studies. Gene amplification by PCR provided a greater ability to detect the presence of virus in tissue. Of the tissues tested (including PBMC, bone marrow, brain, and liver), provirus was most readily detected in PBMC. These results parallel those of Miyamoto et al, in which HTLV-I1 were demonstrated by PCR, but not by indirect fluorescence, in in vitro HTLV-11-transformed rabbit leukocytes.*" No clinicopathologic evidence of disease was detected in HTLV-11-infected rabbits observed for as long as 24 weeks PI in this study, similar to results with HTLV-11-infected rabbits." The absence of disease in HTLV-infected rabbits parallels the situation that occurs in the vast majority of HTLV-infected humans.26It is possible that as increased numbers of HTLV-infected rabbits are studied for extended periods, disease may become apparent. Furthermore, it is possible that other viral isolates or cofactors are required for the development of disease in HTLV-infected rabbits, again paralleling the potential importance of these considerations in humans. The biologic response pattern of HTLV-11-infected rabbits varied depending on the viral isolate used for inoculation. This variation included the time of onset and pattern of antiviral antibody response and the frequency, distribution, and intensity of PCR amplifiable proviral sequences in tissues. The earliest seroconversion and the most intense seroreactivity occurred in rabbits infected with G12.1 cells, while provirus was most frequently detected in all tissues tested from rabbits infected with Mo-T cells. It is unlikely that these differences were due to differences in amount of virus administered, because all inocula contained approximately the same number of infected cells. However, the results may be explained by heterogeneity among the isolates, including properties such as replication competency, cell/tissue tropism, immunogenicity, or copy number From www.bloodjournal.org by guest on April 28, 2017. For personal use only. HTLV-II iNFECTlON OF RABBITS pr53 - SP46 - p24 p21 0 1 2 3 4 L ' 1535 812 6 0 4 812 0 4 812 0 4 812 4 0 812 0 2 3 61624 ULu - R35 R841 R843 R845 R839 R33 Mo-T 729pH6neo WILNRA G12.1 729 HUT 78 Fig 1. Representative WlBs illustratingreroreactivity of rabbits inoculatedwith different HTLV-ll-infected cells (Mo-T, 729pH6neo. WIL-NRA, and G12.1) or control uninfected cells (729 and Hut 78) as a function of weeks PI (shown at bottom of individual strips). Location of HTLV-II antigens is shown at the left margin. per infected cell. To provide further support for this supposition, additional studies will need to be performed using the same cell typc infected with different HTLV-I1 isolates. We have reported similar differences in the biological response pattern of rabbits infected with different isolates of HTLV-I, but we have becn unable to correlate these differences with disease expression in patients from which the isolates were derived (Lairmore et al. submitted for publication, 1991). To date, few structural differences have been described between HTLV-I isolates derived from patients with ATLL and those with HAM/TSP; however, this remains an incompletely studied and a potentially important mcchanism for explaining the variable disease outcome in HTLV-infected patients. Infectious molecular clones will be required for cxpcrimcnts to unambiguously map the genetic determinants responsible for spccific biologic activities. It is important Table 3. Detection of Provirus by PCR in HTLV-ll-lnoculated Rabbks PBMC (weeks PI1 Cellular Inocula. 4 12 WiL-NRA 729pH6neo G12.1 Mo-T 729 HUT70 014t 114 114 214 012 014 112 112 012 212 011 4 12 012 01 1 01 1 01 1 01 1 011 012 112 012 111 01 1 Liver (weeks PI) Brain lweeks PI) Bone Marrow (weeks PI) 24 24 111 4 12 012 012 012 011 01 1 011 012 112 012 011 'Cellular inocula as per Table 1 . tNumber of positive rabbitslnumberof rabbits tested. Open data points indicate not determined. 011 24 112 4 12 24 012 012 112 011 01 1 011 012 112 012 111 112 011 From www.bloodjournal.org by guest on April 28, 2017. For personal use only. 1536 COCKERELL ET AL A B C & - 143 bp IL PBMC u u Br Liv PBMC EM R34 Br R35 Liv EM Br PBMC Liv R36 I Br R841 I Mo-T EM 729pH6WO Liv ld3 id4 Mo-T HUT 78 Fig 2 Representatbe hybridizationsof PCR-ampltfied H N V - I I provirus in tissues from rabbit. using oligonucleotide primer pairs and probes specific for HTLV-IIpol sequences.Total genomic DNA was extracted from tissue samples and 1 pg of DNA (equivalentto approximately150,OOO cells) was used in each PCR amplification. (A) Tissues (PSMC; Br, brain; EM, bone marrow; Liv, liver) from three rabbits (R34, R35. and R36) inoculatedwith HTLV-ll-infected Mo-T cells, and assayed at either 12 weeks PI (R36).or 24 weeks PI (R34 and R35). (E) Tissues (seedesignations for A) from a rabbit (R841)inoculatedwith 729pH6neo cells containing a molecular clone of HTLV-II, and assayed at 12 weeks PI. (C) Positive control HTLV-II-infected Mo-T cells (10 and 10 ‘ dilutions of DNA extracts) and negative control Hut 78 cells. The location of the 143-bp sequence correspondingto the amplified HTLV-II pol gene is shown at the right margin. ’ and encouraging to notc, thcrcforc. that thc 729pH6nco ccll linc, which contains a molecular clonc dcrivcd from thc Mo-T isolatc of HTLV-11, was infectious for rabbits. Expcrimcnts similar to prcvious in vitro studies with dclction mutants or mutagcnizcd HTLV-I1,,,:7.:” can now bc pcrformcd in vivo to furthcr clucidatc thc rolc of spccific viral gcnc scgmcnts in viral rcplication and cellular transformation. ACKNOWLEDGMENT The authors express their appreciation to Dr Gary Toedter, Coulter Immunology, Hialeah, FL, for providing the HTLV-I/II antigen capture enzyme immunoahsorhent kits. REFERENCES I. Komuro A, Watanahe T, Miyoshi I, Hayami M, Tsujimoto H, IO. Kalyanaraman VS, Sarngadharan MG, Rohert-Guroff M, Seiki M, Yoshida M: Detection of simian retroviruses homologous Miyoshi I, Golde D, Gallo RC: A new suhtype of human T-cell to human T-cell leukemia virus type 1. Virology 138:373, 1984 leukemia virus (HTLV-11) associated with a T-cell variant of hairy cell leukemia. Science 218571. 1982 2. Oroszlan S, Sarngadharan MG. Copeland TD, Kalyanaraman VS. Gilden RV, Gallo RC: Primary structure analysisof the major 11. Rosenhlatt JD. Golde DW, Wachsman W. Giorgi W, Jacohs internal protein p24 of human type C T-cell leukemia virus. Proc A, Schmidt GM, Quan S, Gasson JC, Chen IS:A second HTLV-11 Natl Acad Sci USA 79:1291. 1982 isolate associated with atypical hairy cell leukemia. N Engl J Med 31532. 1986 3. Sagata N. Yasunaga T, Tsuzuku-Kawamura J, Ohishi K, Ogawa Y, lkawa Y: Complete nucleotide sequence of the genome 12. Lee TH, Coligan JE, McLane MF. 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Shimotohno K, Wachsman W, Takahashi Y, Golde DW, Miwa M, Sugimara T, Chen ISY: Nucleotide sequence of the 3’ region of an infectious human T-cell leukemia virus type I1 genome. Proc Natl Acad Sci USA 81:6657,1984 28. Chen ISY, Slamon DJ, Rosenblatt JD, Shah NP, Quan SG, Wachsman W The x gene is essential for HTLV replication. Science 229:54,1985 From www.bloodjournal.org by guest on April 28, 2017. For personal use only. 1991 78: 1532-1537 Infectious transmission of human T-cell lymphotropic virus type II in rabbits GL Cockerell, MG Weiser, J Rovnak, B Wicks-Beard, B Roberts, A Post, IS Chen and MD Lairmore Updated information and services can be found at: http://www.bloodjournal.org/content/78/6/1532.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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