Rabbit Cell Lines Research Articles

Oncolytic Virotherapy Synergism with Signaling Inhibitors: Rapamycin Increases Myxoma Virus Tropism for Human Tumor Cells

Myxoma virus is a rabbit-specific poxvirus pathogen that also exhibits a unique tropism for human tumor cells and is dramatically oncolytic for human cancer xenografts. Most tumor cell lines tested are permissive for myxoma infection in a fashion intimately tied to the activation state of Akt kinase. A host range factor of myxoma virus, M-T5, directly interacts with Akt and mediates myxoma virus tumor cell tropism. mTOR is a regulator of cell growth and metabolism downstream of Akt and is specifically inhibited by rapamycin. We report that treatment of nonpermissive human tumor cell lines, which normally restrict myxoma virus replication, with rapamycin dramatically increased virus tropism and spread in vitro. This increased myxoma replication is concomitant with global effects on mTOR signaling, specifically, an increase in Akt kinase. In contrast to the effects on human cancer cells, rapamycin does not increase myxoma virus replication in rabbit cell lines or permissive human tumor cell lines with constitutively active Akt. This indicates that rapamycin increases the oncolytic capacity of myxoma virus for human cancer cells by reconfiguring the internal cell signaling environment to one that is optimal for productive virus replication and suggests the possibility of a potentially therapeutic synergism between kinase signaling inhibitors and oncolytic poxviruses for cancer treatment.

Broad target cell selectivity of Kaposi's sarcoma-associated herpesvirus glycoprotein-mediated cell fusion and virion entry

The molecular mechanism of Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) entry is poorly understood. We tested a broad variety of cell types of diverse species and tissue origin for their ability to function as targets in a quantitative reporter gene assay for KSHV-glycoprotein-mediated cell fusion. Several human, non-human primate, and rabbit cell lines were efficient targets, whereas rodent and all human lymphoblastoid cell lines were weak targets. Parallel findings were obtained with a virion entry assay using a recombinant KSHV encoding a reporter gene. No correlation was observed between target cell activity and surface expression of α3β1 integrin, a proposed KSHV receptor. We hypothesize that target cell permissiveness in both the cell fusion and virion entry assays reflects the presence of a putative KSHV fusion–entry receptor.

Selection and characterization of canine, swine and rabbit cell lines resistant to bovine viral diarrhea virus

Contamination of cell cultures with adventitious viruses may pose serious risks for virology diagnosis, research and vaccine production. This article reports the selection and characterization of three cell lines resistant to bovine viral diarrhea virus (BVDV), a major contaminant of cell cultures. The resistant cells were obtained from canine (MDCK), swine (PK-15) and rabbit (RK-13) parental cell lines by expanding and cloning cells that survived lytic infection with BVDV. All three selected cell lines were resistant to three standard BVDV strains and to 10 field isolates as demonstrated by immunofluorescence for viral antigens and by co-cultivation with susceptible cells. Inoculation of selected cells with BVDV (10 TCID 50/cell) resulted in frequencies of infection of <10 −5 (MDCK-R, PK-15R) and 3.3 × 10 −4 (RK-13R). Comparing to the parental cell lines, these cells were >10,000-, >20,000- and 600-fold less susceptible to BVDV, respectively. Inoculation of resistant cells with BVDV in the presence of polyethylene–glycol increased the frequency of infection in the order of >437-, >346- and 87-fold, respectively, indicating that resistance is probably due to a block in viral entry. Nevertheless, each resistant cell line retained the susceptibility to three other viruses that replicate in the parental cells. Thus, these cells may be useful for many virology purposes, with a very low risk of BVDV contamination.

Species specificity of the Listeria monocytogenes InlB protein

InlA and InlB mediate L. monocytogenes entry into eukaryotic cells. InlA is required for the crossing of the intestinal and placental barriers. InlA uses E-cadherin as receptor in a species-specific manner. The human E-cadherin but not the mouse E-cadherin is a receptor for InlA. In human cells, InlB uses Met and gC1qR as receptors. By studying the role of InlB in vivo, we found that activation of Met by InlB is species-specific. In mice, InlB is important for liver and spleen colonization, but not for the crossing of the intestinal epithelium. Strikingly, the virulence of a DeltainlB deletion mutant is not attenuated in guinea pigs and rabbits. Guinea pig and rabbit cell lines do not respond to InlB, although expressing Met and gC1qR, but support InlB-mediated responses upon human Met gene transfection, indicating that InlB does not recognize or stimulate guinea pig and rabbit Met. In guinea pig cells, the effect of human Met gene transfection on InlB-dependent entry is increased upon cotransfection with human gc1qr gene, showing the additive roles of gC1qR and Met. These results unravel a second L. monocytogenes species specificity critical for understanding human listeriosis and emphasize the need for developing new animal models for studying InlA and InlB functions in the same animal model.

886. An Adenovirus Vector with a Chimeric Fiber Derived from Ovine Adenovirus for Novel Tropism Discovery

Gene therapy vectors based on human adenovirus (Ad) serotypes 2 and 5 continue to show increasing promise as gene therapy delivery vehicles, particularly in the context of cancer gene therapy. However, many clinically important tissues are refractory to Ad5 infection, including numerous cancer tissue types, due to reduced levels of the coxsackie and adenovirus receptor (CAR). Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Ovine adenovirus type 7, isolate 287 (OAdV7-287), has recently been described as the prototype of the new genus Atadenovirus, which contains several animal Ads. OAd287 encodes a fiber molecule containing 25 repeating units, and has a unique C-terminal knob domain of 121 amino acids. OAdV7-287 abortively infects several human, rodent and rabbit cell lines in vitro, and efficiently transduces human cells in vitro. Unlike Ad5, however, systemically administered OAd-based vectors do not predominately localize to the liver in rodents, but are distributed more evenly among the major organs. Based on these observations, we hypothesized that replacement of the Ad5 fiber with the non-human OAdV7-287 fiber would result in an Ad5 vector with a novel, CAR-independent, tropism in vitro and liver |[ldquo]|detargeting|[rdquo]| in vivo. We constructed an E1-deleted, luciferase transgene Ad5 vector encoding a chimeric fiber from ovine adenovirus 7-287 that includes the Ad5 tail region (Ad5/OAdV7), utilizing fiber-switching technology established in our laboratory. Viral particles were harvested from 911 cells, purified by standard cesium chloride gradient centrifugation and yielded titers in excess of 2.7|[times]|10e12 vp/mL. Characterization of Ad5/OAdV7 vector infectivity in CAR-deficient CHO Chinese hamster ovary cells, low-CAR MCF7 human breast cancer cells, T24 human bladder cancer cells and PC-3 human prostate cancer cells showed 10-to-30 fold greater luciferase reporter gene activities versus the isogenic AdLuc1 control vector. Furthermore, competitive inhibitory knob blocking assays in high-CAR cells using Ad5 recombinant knob protein demonstrated these increases to be independent of CAR receptor binding. Herein, we have demonstrated the construction, rescue, purification, and initial infectivity characterization of a novel CAR-independent, infectivity enhanced chimeric fiber Ad vector. These data strongly suggest that our genetic xeno-fiber paradigm alters Ad vector tropism, a key gene therapy vector design principle.

Toxicity and immunogenicity of purified Haemophilus ducreyi cytolethal distending toxin in a rabbit model

The cytolethal distending toxin of Haemophilus ducreyi (HdCDT) is a three-component toxin that induces the arrest of the mammalian cell cycle in the G2 phase. All of the individual gene products, CdtA, CdtB and CdtC, are required for toxic activity on cultured mammalian cells. The CdtB component alone exerts nuclease activity. The individual HdCDT components were purified by affinity chromatography or ion-exchange chromatography followed by gel-filtration. HdCDT was reconstituted and purified by the immobilization of a GST-CdtB fusion on a GSTrap column and the subsequent addition of cell sonicates from Escherichia coli recombinants that produced CdtA and CdtC. The purified HdCDT preparation contained all three CDT proteins, as detected by immuno-blotting, and had high cytotoxic activity (10 6 CPU/ml). Immunization of rabbits with the HdCDT complex and with the individual CdtA, CdtB and CdtC proteins elicited high titres of antibodies, as detected by ELISA. All of the immune sera had toxin-neutralizing activities. The pathological effects of the HdCDT complex were investigated in rabbits, since the proliferation of two rabbit cell lines, SIRC and RK-13, was inhibited by HdCDT. Intradermal injection of HdCDT (1, 10, 50 and 100 μg protein) into naive rabbits resulted in dose-dependent skin reactions (erythema) about 24 h after injection. Similar effects were not observed when the individual HdCDT proteins were injected. HdCDT injection into immune rabbits resulted in dose-dependent skin responses that were characterized by both erythema and oedema. Histological evaluation of the 24-h lesions in naive rabbits that were injected with HdCDT, revealed moderate levels of inflammatory cells, which were mainly granulocytes and macrophages, and dilatation of blood vessels. The skin reactions in HdCDT-injected immunized rabbits showed pronounced vascular changes and extensive infiltration of inflammatory cells, including eosinophils. All of the pathological changes healed after 3 days. In conclusion, purified HdCDT holotoxin is a complex of all three CDT proteins and all three components induce neutralizing antibodies when injected in rabbits. HdCDT causes dose-dependent pathologic skin reactions in both naive and immune rabbits, which is characterized by increased inflammatory responsiveness after each immunization.

Three soluble form messages of murine CD46 are produced through alternative mRNA splicing.

Murine CD46 (mCD46) is a type 1 membrane protein expressed predominantly in testicular germ cells, the distribution profile of which is in contrast to that of human CD46 showing a ubiquitous tissue distribution. We have identified an additional message of mCD46 that encodes a putative secretory form [Nomura et al. (1999) Immunogenetics 50, 245-254]. Here, we cloned three cDNAs encoding putative soluble CD46 from murine testis. These soluble form messages were yielded on insertion of unidentified nucleotide sequences, 77, 179, and 73 ntds, into the junctions between the SCR3 and SCR4 (variant 2), ST(c) and UK (variant 3), and SCR4 and ST(c) (variant 1) domains, respectively, the last one corresponding to the reported soluble form. The exons corresponding to these three inserts were identified in the murine CD46 genome, indicating that the alternative splicing of mRNA participates in the generation of these various CD46 messages. In normal mouse sera and cell lines, however, virtually no soluble CD46 was detected on immunoblotting. On Northern blotting analysis with specific probes, on the other hand, variant 1 was found to be predominantly expressed in the liver and heart. In addition, all variant messages were detected on PCR in all organs examined. When a rabbit cell line, RK13 cells, was transfected with cDNA of variant 1, protein synthesis was detected on immunoblotting. Although the mCD46 protein production was inefficient, this variant 1 exhibited factor I-cofactor activity as to inhibition of the complement cascade. Since the mCD46 protein was reported to be markedly up-regulated on infection of murine cells with mCMV, the soluble mCD46 proteins may act as a complement regulator that controls the systemic complement system under the conditions of a viral infection.

HTLV-1 cell lines differ in constitutively activated signaling pathways that can be altered by cytokine exposure.

Examination of signaling pathways used by HTLV-1-infected rabbit cell lines revealed differences between one, RH/K30, that mediates asymptomatic infection and another, RH/K34, that causes lethal experimental leukemia. Both lines are IL-2 independent; RH/K30 produces IL-4 while RH/K34 produces IL-10. Examination of the Jak/STAT (Janus kinase/signal transducer and activator of transcription) activation of the lines revealed constitutive phosphorylation of Jak1 in both STAT6 phosphorylation, not previously reported for HTLV-1 cells, was observed in RH/K30; STAT1 and STAT3 were phosphorylated in RH/K34. Treatment with cytokines altered the activation of the STAT proteins: IL-2 induced STAT5 phosphorylation in both lines. Supernatant from RH/K34 or IL-10 induced STAT3 phosphorylation in RH/K30 cells. Supernatant from RH/K30 or IL-4 induced STAT6 phosphorylation in RH/K34 cells, which could be reversed with a Jak kinase inhibitor--AG-490.

Recombinant human interleukin-6 enhances the immunoglobulin secretion of a rabbit-rabbit hybridoma.

The generation of stable rabbit-rabbit hybridomas is now possible by the recent development of a rabbit fusion partner. The ability to generate rabbit monoclonal antibodies (MAbs) can be advantageous because these rabbit immunoglobulins tend to exhibit higher affinity than murine MAbs. Furthermore, it has been observed that, in general, rabbits will elicit an immune response to antigens of limited immunogenicity in mice. Unfortunately, these rabbit-rabbit hybridomas secrete only 200 ng/mL to 5 microg/mL of immunoglobulin, which may limit larger scale production of rabbit antibodies. This study sought to determine if interleukin 6 (IL-6), which has been reported to have proliferative and secretory stimulating effects on some murine hybridomas, had any effect on a rabbit cell line that secretes a monoclonal IgG specific for estradiol. The results demonstrated that recombinant human IL-6 had a dose-dependent enhancing effect on the IgG secretion of the rabbit-rabbit hybridoma. The enhancing effect was consistent when the cells were continuously passed in the presence of IL-6. However, IL-6 did not affect the growth of the hybridoma. In contrast, no discernible effect was accomplished with recombinant mouse IL-6. Furthermore, no basal IL-6 activity was detected in the rabbit hybridoma extracellular medium. The IL-6 enhancement effect observed in this study may help to increase the immunoglobulin yield of rabbit hybridomas and to assist in the understanding of the mechanism(s) behind the lowered secretion level.

Cytotoxicity of potential ocular permeation enhancers evaluated on rabbit and human corneal epithelial cell lines

A series of prospective ocular permeation enhancers, benzalkonium chloride (BAC), cetylpyridinium chloride (CPC), ethylenediaminetetraacetic acid (EDTA), polyoxyethylene (20) stearyl ether (PSE) and polyethoxylated castor oil (PCO) were tested for cytotoxicity on cultures of rabbit (RCE) and human (HCE) corneal epithelial cells. The cells were treated for 5, 15 and 60 min with different concentrations of the test substances, in serum-free medium and in medium containing 15% foetal bovine serum (FBS). The cytotoxicity was evaluated by WST-1 test. The EC 50 values for HCE, after 15 min exposure and in the presence of FBS, indicate the following order of cytotoxicity: PSE≥BAC>CPC>EDTA>PCO. After 1 h exposure the order of decreasing cytotoxicity was PSE≥BAC>CPC>PCO>EDTA. In all cases the presence of FBS appeared to exert a protective effect against the cytotoxic effect.

A novel cellular protein, p60, interacting with both herpes simplex virus 1 regulatory proteins ICP22 and ICP0 is modified in a cell-type-specific manner and Is recruited to the nucleus after infection.

Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0). ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (gamma2) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells. We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22. This protein (apparent Mr of 60,000) has not been previously described and has no known motifs. Analyses of p60 revealed the following. (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R. Bruni and B. Roizman, J. Virol. 72:8525-8531, 1998) bound all forms of ICP22. The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms. (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22. (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm. In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent Mr but was not redistributed. Posttranslational processing required the presence of the genes encoding ICP22 and UL13 protein kinase. (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei. Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0. Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent Mr. The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the UL13 or US3 protein kinases. The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and UL13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding UL13 or ICP22.

Rabbit cells expressing human CD4 and human CCR5 are highly permissive for human immunodeficiency virus type 1 infection.

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.

Host Range and Cytopathogenicity of the Highly Attenuated MVA Strain of Vaccinia Virus: Propagation and Generation of Recombinant Viruses in a Nonhuman Mammalian Cell Line

Modified vaccinia virus Ankara (MVA), attenuated by over 500 passages in primary chick embyro fibroblasts (CEF), is presently being used as a safe expression vector. We compared the host ranges of MVA and the parental Ankara strain in CEF and 15 permanent cell lines. The cells could be grouped into three categories: permissive, semipermissive, and nonpermissive. For MVA, the permissive category consisted of primary CEF, a quail cell line derived from QT6, and the Syrian hamster cell line BHK-21. Only in BHK-21 cells did the virus yield approach that occurring in primary CEF. The semipermissive category included two African green monkey cell lines: BS-C-1 and CV-1. The nonpermissive category for MVA consisted of three human cell lines HeLa, 293, and SW 839; one rhesus monkey cell line FRhK-4; two Chinese hamster cell lines CHO and CHL; one pig cell line PK(15); and three rabbit cell lines RK13, RAB-9, and SIRC. The grouping for MVA with a restored K1L host range gene was similar except for the inclusion of RK13 cells among permissive lines. The grouping for the Ankara strain, however, was quite different with more permissive and semipermissive cell lines. Nevertheless, in cells that were permissive for MVA, the virus replicated to higher levels than Ankara, consistent with both positive and negative growth elements associated with the adaptation of MVA. The cell lines were also characterized according to their susceptibilty to MVA-induced cytopathic effects, expression of a late promoter regulated reporter gene by an MVA recombinant, and stage at which virion morphogenesis was blocked. Finally, the permissive BHK-21 cell line was shown to be competent for constructing and propagating recombinant MVA, providing an alternative to primary CEF.

Infectivity of chimeric human T-cell leukemia virus type I molecular clones assessed by naked DNA inoculation.

Two human T-cell leukemia virus type I (HTLV-I) molecular clones, K30p and K34p were derived from HTLV-I-infected rabbit cell lines. K30p and K34p differ by 18 bp with changes in the long terminal repeats (LTRs) as well as in the gag, pol, and rex but not tax or env gene products. Cells transfected with clone K30p were infectious in vitro and injection of the K30p transfectants or naked K30p DNA into rabbits leads to chronic infection. In contrast, K34p did not mediate infection in vitro or in vivo, although the cell line from which it was derived is fully infectious and K34p transfectants produce intact virus particles. To localize differences involved in the ability of the clones to cause infection, six chimeric HTLV-I clones were constructed by shuffling corresponding fragments containing the substitutions in the LTRs, the gag/pol region and the rex region between K30p and K34p. Cells transfected with any of the six chimeras produced virus, but higher levels of virus were produced by cells transfected with those constructs containing the K30p rex region. Virus production was transient except in cells transfected with K30p or with a chimera consisting of the entire protein coding region of K30p flanked by K34p LTRs; only the transfectants showing persistent virus production mediated in vitro infection. In vivo infection in rabbits following intramuscular DNA injection was mediated by K30p as well as by a chimera of K30p containing the K34p rex gene. Comparisons revealed that virus production was greater and appeared earlier in rabbits injected with K30p. These data suggest that several defects in the K34p clone preclude infectivity and furthermore, provide systems to explore functions of HTLV-I genes.

Variation in Cellular Glutathione Peroxidase Activity in Lens Epithelial Cells, Transgenics and Knockouts Does Not Significantly Change the Response to H 2O 2Stress

This investigation examines the contribution of glutathione peroxidase (GSHPx-1) in degrading H 2O 2in lens preparations. Rabbit (N/N1003A) and normal and GSHPx-1 transfected mouse (αTN4-1) lens epithelial cell lines and normal and GSHPx-1 transgenic and knockout mouse lenses were utilized. GSHPx-1 activity in the cell lines was increased from two-fold to about four-fold, in the lenses from transgenics more than four-fold and the lenses from knockouts had less than 3% of normal GSHPx-1 activity. The transgenic and knockout mice as well as their lenses appeared normal for up to 3 to 4 months, the longest period of observation. The preparations were subjected to oxidative stress by placing them either in a medium containing 120 or 300μ mH 2O 2or utilizing photochemical stress where the H 2O 2levels normally rise to about 100μ mover a few hours in the presence of a normal lens. With all preparations, it was found that either markedly increasing or eliminating GSHPx-1 activity had only a small effect on the system's ability to metabolize H 2O 2. 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase (GSSG Red) and 3-aminotriazole (3-AT), an inhibitor of catalase, also had little effect. However, the addition of both inhibitors caused a marked decrease in H 2O 2degradation. Examination of the distribution of GSHPx-1 in the lens indicated that the activity per milligram of protein was evenly distributed between the epithelium and the remainder of the lens in the normal lens and was about 1.7-fold greater in the epithelium of transgenic lenses than in the remainder of the lens. Surprisingly, the distribution of GSSG Red was quite different with eight- to ten-fold more activity in the epithelium. Catalase was also found to be concentrated in the epithelium. With H 2O 2exposure, a rapid loss of non-protein thiol (NP-thiol) was found in cell cultures and in the epithelia of cultured lenses. However, the remainder of the lens showed little change in NP-thiol. The variation of GSHPx-1 activity did not influence the NP-thiol changes which occurred more rapidly and to a greater extent in the presence of BCNU. The addition of BCNU also caused a decrease in total lens NP-thiol. Examination of thymidine incorporation and choline transport, indicators of nuclear and membrane function, also reflects the H 2O 2degradation data, showing little difference in the degree to which H 2O 2effects these parameters in lenses from normal and transgenic animals. Catalase activity is four- to six-fold greater than GSHPX-1 activity in the αTN4-1 cell lines, about three-fold lower in the rabbit cell line and, remarkably, about 18-fold lower than the peroxidase in the normal mouse lens. In spite of such observations, the consistent overall conclusion is that GSHPx-1 and catalase function together but when GSHPx-1 is knocked out or GSSG Red is inhibited, catalase is able to protect the system from H 2O 2stress. Indeed, the young mouse does not appear to require GSH Px-1 for normal function.

Immunization of a rabbit with beta 2-glycoprotein I induces charge-dependent crossreactive antibodies that bind anionic phospholipids and have similar reactivity as autoimmune anti-phospholipid antibodies.

A rabbit immunized with beta 2-glycoprotein I (beta 2-GPI) produced Abs that bind to negatively charged phospholipids and to beta 2-GPI. After affinity purification of the Abs to beta 2-GPI, the dual reactivity could still be detected. Adsorption studies with a phosphatidylserine affinity column depleted phospholipid-reactive Abs, but beta 2-GPI reactivity was retained. The same pattern of reactivity was found with culture supernatants from rabbit anti-beta 2-GPI splenocytes fused with an immortalized rabbit cell line. The reactivity to negatively charged phospholipids is likely to involve ionic interactions, as high ionic strength buffers eliminated binding to anionic phospholipids, but not to beta 2-GPI. Affinity-purified anti-phospholipid (aPL) Abs from four of seven autoimmune patients bound anionic phospholipids in the absence of beta 2-GPI. However, in high ionic strength buffer, this binding was abolished in three patients and significantly reduced in the fourth. In contrast, affinity-purified aPL Abs from seven autoimmune patients bound to beta 2-GPI-coated plates, and binding in high ionic strength buffer was reduced only moderately in three patients. Therefore, autoimmune-type aPL Abs display anti-beta 2-GPI reactivity and charge-dependent binding to anionic phospholipids similar to affinity-purified rabbit anti-beta 2-GPI Abs.

HTLV‐II Non‐integrated Malignant Lymphoma Induction in Japanese White Rabbits Following Intravenous Inoculation of HTLV‐II‐infected Simian Leukocyte Cell Line (Si‐IIA)

Lymphoma induction in rabbits by an unknown factor derived from an HTLV‐II‐producing simian (Cynomolgus) leukocyte cell line (Si‐IIA) is reported. Thirteen of 17 male Japanese white rabbits (76%) inoculated intravenously with Si‐IIA cells developed malignant lymphoma including Hodgkin‐like lymphoma between 62 and 167 days after inoculation. Historically, there was extensive diffuse or nodular infiltration of either large cell type or mixed type lymphoma cells in many organs, frequently involving the spleen, liver, lymph nodes and kidneys, and less frequently the thymus, bone marrow, lungs, heart, skin and gastrointestinal tract. Hodgkin‐like lymphoma was also observed in two rabbits. Chromosomal analysis of five cell lines established from tumor‐bearing rabbits revealed the male rabbit karyotype. The immunophenotype of these tumor cells was usually T‐cell (CD5+or, r RT1+, RT2+or‐, CD45+, CD4−, RABELA− and MHC class II‐DQ+) except for Hodgkin‐like lymphoma cells which expressed only CD45. However, integration of the HTLV‐II provirus genome could not be demonstrated in the tumor tissues or any of the rabbit cell lines by polymerase chain reaction or Southern blot analysis. Moreover, no lymphoma was induced by inoculation of HTLV‐IIC, MOT (other HTLV‐II‐producing human cell lines) or TALL‐1 (control). Two of four rabbits injected with cell‐free pellets from Si‐IIA cultures died of malignant lymphoma (15‐20 days). Five irradiated rabbit cell lines were inoculated but only one (Ra‐SLN) induced lymphoma in 1 of 3 rabbits at 27 days. Neither Herpesvirus saimiri nor Herpesvirus ateles (simian oncogenic viruses) was detected in Si‐IIA cells by immunofluorescence testing. These data suggest that the high rate of lymphoma induction in rabbits may be caused not by only HTLV‐II or well known simian oncogenic viruses, but rather by an unknown passenger agent derived from Si‐IIA or HTLV‐IIA, with which Si‐IIA was established.

Survey of cell lines in the American Type Culture Collection for bovine viral diarrhea virus

Cell lines originating from cattle, sheep, goat, deer, bison, swine, rabbit, hamster, cat, dog, monkey, human, and mosquito were obtained from the American Type Culture Collection and tested for contamination with bovine viral diarrhea virus (BVDV). Immunocytochemical procedures and polymerase chain reaction (PCR) amplification were used to detect viral antigen or viral RNA in 13 of 41 cell lines. The results of these procedures correlated exactly. Cell lines derived from cattle, sheep, goat, deer, bison, rabbit, and domestic cat were found contaminated with BVDV. Attempts were made to experimentally infect 14 swine, rabbit, hamster, cat, dog, monkey, and human cell lines that had been found free of virus. All swine cell lines, and most rabbit and cat cell lines, became infected with BVDV. Hamster, human, dog, and certain rabbit and cat cells were refractory to BVDV infection. Experimental infection of monkey cells produced variable results.

HIV-2 and SIV Infection of Nonprimate Cell Lines Expressing Human CD4: Restrictions to Replication at Distinct Stages

Mouse, rat, rabbit, cat, and mink cell lines expressing human CD4 restrict entry of HIV-1 strains at the cell surface. We now report that CD4+ cat and rabbit cell lines are permissive to cell fusion induced by several HIV-2 and SIV strains. CD4+ rat and mouse cells are only sensitive to HIV-2 and SIV fusion in the presence of active amphotericin B. HIV-2 and SIV replicate in CD4+ cat and CD4+ mink cells, but not in the CD4+ rabbit, rat, or mouse cells. These latter cell lines poorly express a transfected DNA molecular clone of HIV-2 DNA. CD4+ rat cells are sensitive to entry by HIV-2 (HTLV-I) pseudotypes and also to HIV-2 if amphotericin B is present. Early reverse transcription then occurs without subsequent viral gene expression, but the fusion-sensitive CD4+ rabbit cells are not permissive for HIV-2 reverse transcription. Our observations demonstrate (i) that the cat and mink cell lines express everything required except human CD4 for entry and replication by several HIV-2 and SIV strains and (ii) that distinct postpenetration blocks to viral replication are evident in rabbit and rodent cell lines.

Defective infection of rabbit peripheral blood monocyte cultures with human immunodeficiency virus type 1.

Human immunodeficiency virus type 1 (HIV-1) produces abortive infections of primary cultures of rabbit peripheral blood mononuclear cells (PMBCs). Mitogen activation of rabbit PBMCs or the addition of exogenous cytokines to the cultures does not change the level of release of the early HIV core protein, p24, into the culture medium. The amount of p24 increases steadily and reaches peak levels about 7 days after infection. HIV-1-specific DNA env sequences are also detected in infected PBMCs. However, reverse transcription of RNA of samples from activated infected cultures to cDNA, followed by amplification by the polymerase chain reaction, revealed transcription of gag, but not env, regions, suggesting that HIV-1 infection of rabbit PBMC does not lead to the replication and maturation of complete HIV-1 virions. In addition, neither CPE nor lytic infection was observed in HIV-1-infected rabbit cells and infectivity could not be transferred from rabbit cells infected with HIV for up to 2 weeks to MT-2 or H9 indicator cell lines. In addition, no CPE was seen in long-term cultures of the HTLV-I-transformed rabbit cell line PLT-1441, after inoculation with HIV. It is concluded that primary rabbit PBMCs may be infectable by HIV-1 but are not permissive for production of infectious virus. This conclusion is consistent with the apparent long-term latent infection seen after inoculation of rabbits with HIV-1 or HIV-1-infected cells.