Plasmonic Enhancement of Selective Photonic Virus Inactivation.

Femtosecond (fs) pulsed laser irradiation techniques have attracted interest as a photonic approach for the selective inactivation of virus contaminations in biological samples. Conventional pulsed laser approaches require, however, relatively long irradiation times to achieve a significant inactivation of virus. In this study, we investigate the enhancement of the photonic inactivation of Murine Leukemia Virus (MLV) via 805 nm femtosecond pulses through gold nanorods whose localized surface plasmon resonance overlaps with the excitation laser. We report a plasmonically enhanced virus inactivation, with greater than 3.7-log reduction measured by virus infectivity assays. Reliable virus inactivation was obtained for 10 s laser exposure with incident laser powers ≥0.3 W. Importantly, the fs-pulse induced inactivation was selective to the virus and did not induce any measurable damage to co-incubated antibodies. The loss in viral infection was associated with reduced viral fusion, linking the loss in infectivity with a perturbation of the viral envelope. Based on the observations that physical contact between nanorods and virus particles was not required for viral inactivation and that reactive oxygen species (ROS) did not participate in the detected viral inactivation, a model of virus inactivation based on plasmon enhanced shockwave generation is proposed.

Ultraviolet-light-inactivated Cas-Br-M murine leukemia virus induces a protective CD8+ cytotoxic T lymphocyte response in newborn mice.

Newborn NFS/N mice are susceptible to the neurological disease induced by infection with Cas-Br-M murine leukemia virus (Cas), and do not develop a protective cytotoxic T cell (CTL)-mediated response to Cas infection. Here we demonstrate that whole UV light-inactivated Cas (UV-Cas), inoculated in newborn NFS/N mice, induced a strong, Cas-specific CTL response detectable 2 weeks postinoculation and persisting in vivo for > or = 36 weeks. The magnitude of the UV-Cas-induced splenic CTL response, mediated by CD8+ T cells, inversely correlated with the level of proviral cas env sequences detectable in the spleen of the UV-Cas-inoculated mice, as revealed by PCR amplification of tissue DNA. The transfer of UV-Cas-primed splenocytes, with Cas-specific CTL activity, protected 100% of recipient newborn mice from the development of neurological disease induced by infection with live Cas, for more than 28 weeks, and reduced the level of viral replication in the recipients.

In vivo exposure to ultraviolet radiation enhances pathogenic effects of murine leukemia virus, LP-BM5, in murine acquired immunodeficiency syndrome.

LP-BM5 murine leukemia virus (MuLV) induces an immunodeficiency syndrome (MAIDS) in C57BL/6 mice which resembles immunological abnormalities observed in early stages of human AIDS. In our study, MAIDS virus-infected mice were exposed to low doses of ultraviolet radiation (UVR) before and after virus inoculation and compared with MAIDS-infected but not UVR-exposed mice. In all tested parameters (blood IgM levels; mitogenic responses to PHA, ConA, LPS and anti-mu; MLR; antigenic response to SRBC; enlargement and histopathologic changes of the spleen) we observed the same trend: changes due to MAIDS infection were more pronounced in the UVR-exposed group than in the unexposed group. Statistically significant differences between these two groups were seen for mitogenic responses at two different time points after virus inoculation. These results demonstrate that in vivo UVR exposure enhances the immunosuppressive effects of a retroviral infection. UVR exposure may affect the progression of AIDS in a similar manner.

Effect of cyclophosphamide, total body irradiation, and zidovudine on retrovirus proliferation and disease progression in murine AIDS.

Murine acquired immunodeficiency syndrome (MAIDS) develops when C57B1/6 mice are inoculated with LP-BM5 murine leukemia viruses. Disease progression in these animals is characterized by lymphadenopathy, polyclonal B-cell activation, severe immunodeficiency, and death. Mice with MAIDS have been used to examine the efficacy of antiretroviral therapies for possible use in AIDS patients. In the present work, MAIDS mice were employed to test the hypothesis that established retroviral infection might be cured by the combined use of a cytotoxic agent (cyclophosphamide) and total body irradiation--a regimen reported to have successfully cured HIV-1 infection in one AIDS patient. Results indicate that the ablation of retrovirus-infected lymphoid cells reduced but did not eliminate LP-BM5 infection. Moreover, this regimen was no more effective at controlling virus proliferation or preventing the polyclonal IgG activation characteristic of murine AIDS than was AZT alone.

Rejection of retrovirus-infected tumor cells in guinea pigs: effect on bystander tumor cells.

We studied the basis of rejection of retrovirus-infected tumor cells in guinea pigs by evaluating host response to injection of mixtures containing retrovirus-infected tumor cells and, antigenically and biologically distinct, uninfected tumor cells (line 10). After intradermal injection, line 10 grew progressively, metastasized to regional lymph nodes, and led to death of animals; line 107C3 4070A, a murine leukemia virus-infected fibrosarcoma cell line, grew for approximately 1 week and then regressed. Growth of the line 10 hepatoma was suppressed when the hepatoma cells were mixed with viable 107C3 4070A cells before injection into strain 2 guinea pigs. Viable virus-infected 107C3 cells were more effective than irradiated virus-infected cells in suppressing line 10 growth; mixture of line 10 with murine leukemia virus 4070A alone did not inhibit line 10 growth. Suppression of growth of line 10 cells by admixed murine leukemia virus 4070A-infected cells was less effective in animals with established viral immunity. Cyclophosphamide inhibited suppression of line 10 at sites of injection of virus-infected cells. Infection of line 10 with murine leukemia virus in vitro required cocultivation of line 10 cells with murine leukemia virus-infected fibrosarcoma cells; virus alone did not lead to acquisition of murine leukemia virus antigens by line 10 cells.

Absence of ecotropic or recombinant murine leukaemia virus in preleukaemic and leukaemic X-irradiated NZB mice.

NZB mice X-irradiated with a single dose of 630 R when they were 1-month old developed a high incidence of histologically defined lymphocytic leukaemia 8--25 weeks later. We have screened for murine leukaemia viruses (MuLV) in the lymphoid tissues of 8 of these leukaemic mice, and in 8 "preleukaemic", apparently healthy NZBs killed 1 month post irradiation. Xenotropic, but not ecotropic or recombinant MuLV, was detected by in vitro co-cultivation of bone marrow, spleen and thymus with selectively permissive cell lines, followed by the immunofluorescence test for MuLV gs antigen, and the XC test. Our results are not consistent, therefore, with the concept that the factor causing the leukaemias was an oncogenic virus activated by X-irradiation.

Abrogation of Fv-1b restriction with murine leukemia viruses inactivated by heat or by gamma irradiation.

Fv-1b restriction in BALB/3T3 cells is temporarily abrogated following infection with N-tropic murine leukemia virus. The mechanism of this phenomenon was investigated by comparing the inactivation rates for viral infectivity and for the ability of the same virus to abrogate Fv-1 restriction. Inactivation of the abrogating ability of N-tropic murine leukemia virus following graduated doses of gamma radiation proceeded at half the rate of that for viral infectivity. This result indicates that viral RNA must function in abrogating Fv-1b restriction but that only a portion of the viral genome is required. The inactivation kinetics of N-tropic murine leukemia virus were also determined following incubation of virus at 43 degrees C. Abrogating ability of N-tropic murine leukemia virus was found to be about six times as stable under these conditions as was viral infectivity. Interestingly, virion-associated reverse transcriptase activity was inactivated at the same rate as was viral infectivity, indicating that this enzyme may not need to function during abrogation. Virus heated at 43 degrees C was used to study the kinetics of the abrogation phenomenon itself. Abrogation was shown to be transient, requiring 6 to 9 h after virus infection to become maximally effective and beginning to disappear after about 18 h. The data reported here confirm the idea that abrogation of Fv-1 restriction can be separated experimentally from virus replication, and they raise the possibility that a separate biochemical pathway exists for incoming viral RNA in Fv-1 restrictive cells.

Differential susceptibility of spleen focus-forming virus and murine leukemia viruses to ansamycin antibiotics.

The streptovaricin complex (SvCx) and rifamycin SV derivatives display potent antiviral activity against the polycythemic strain of Friend leukemia virus (FV-P), as measured by a reduction in the number of spleen foci produced in mice. Such reductions may be explained by inactivation of functions of (i) the spleen focus-forming virus (SFFV), (ii) its "helper" murine leukemia virus (MuLV), or (iii) both viruses normally present in FV-P. We noted that preincubation of FV-P with fractionation products of SvCx, or derivatives of rifamycin SV, at low concentrations (3 to 5 mug/ml) reduces the number of spleen foci 80 to 97%, whereas titers of MuLV (from the same inoculum) remain unaffected (MuLV titers were measured by XC, S(+)L(-), and "helper activity" assays). Our findings indicate a remarkable biological selectivity of ansamycins, as well as nonansamycin components of SvCx, against the transforming and defective spleen focus-forming virus as compared to MuLV. Thus, the drugs might be useful in distinguishing other types of oncornaviruses.

Transient virus expression during murine leukemia induction by X-irradiation.

Most X-irradiation-induced thymomas in C57BL/6 mice are virus-free when assayed by immunofluorescence for the gs antigen (gsa) of murine leukemia virus (MuLV). Virus was induced transiently in bone marrow cells and later appeared in thymus cells. Six to 7 weeks post irradiation, thymocytes and bone marrow cells were MuLV gsa-negative and remained negative for the lifetime of most animals, whether or not they contracted overt leukemia. During the period when MuLV gsa-positive bone marrow cells were found, XC-positive syncytia-producing bone marrow cells were also found. Virus information was expressed, therefore, for a limited duration, long before any signs of leukemia in the animals were evident. MuLV gsa-positive thymocytes taken from mice 4 weeks after X-irradiation were cocultivated with a series of indicator cells. B-tropic virus, in addition to a xenotropic virus, was isolated from these cells. Ecotropic virus was not found in normal mouse thymocytes, in irradiated thymocytes a few days after termination of the X-irradiation sequence, or in most primary thymomas. All thymocytes produced only xenotropic virus in the cocultivation assays. Expression of the ecotropic virus was, therefore, transient, as assayed by immunofluorescence, XC syncytia formation, and virus isolation from MuLV gsa-positive thymus cells.

XC cell fusion by murine leukemia viruses: fusion from without.

Concentrated murine leukemia virus (MuLV) or MuLV producing cells induce XC cell fusion within an hour leading to syncytia formation. While MuLV inactivated by UV irradiation, beta-propiolactone or hydroxylamine treatment still caused cell fusion, Bromelin- or trypsin treated MuLV was no longer able to fuse XC cells. Though sonicated MuLV induced no XC cell fusion, it interfered with cell fusion as caused by untreated MuLV. XC cells infected by diluted MuLV of a titer lower than 1 X 10(5) PFU/ml formed no syncytia although they produced MuLV. The cell fusion mechanism is discussed.

Biological and serological characterization of radiation leukemia virus.

Radiation leukemia virus, isolated from radiation-induced lymphomas in C57BL/Ka mice and propagated in that strain, is thymotropic and leukemogenic in vivo but replicates poorly, if at all, in mouse and mink fibroblast cultures in vitro. Comparative studies indicate that this naturally occurring virus is distinct from the previously recognized classes of endogenous murine ecotropic and xenotropic C-type viruses which are capable of replication on fibroblasts (fibrotropic) but are neither thymotropic nor leukemogenic. These studies also demonstrate that a differentiation-specific restriction system governing the replication of the murine ecotropic C-type viruses operates in addition to the previously defined Fv-1 and SRV gene restriction systems.

Enhancement of infectivity and oncogenicity of a murine leukemia virus in adult mice by X-irradiation.

Infection of adult BALB/c mice with murine leukemia virus (MuLV) induces thymic lymphomas histologically indistinguishable from those caused by neonatal infection. X-irradiation permitted early and high levels of viral expression when given before or after MuLV administration and hastened the development of lymphomas. Expression of virus was assayed by using a radioimmune assay for murine p30, a virion core protein. Seventeen to 21 days after injection of MuLV into adult mice, there was 0.3 mug p30 per ml serum, approximately 5 times normal. Seventeen to 21 days after injection of MuLV into X-irradiated (600R) adult mice, there were 2.7 mug p30 per ml serum. The virus produced by infected adult mice was infectious and oncogenic when given to newborn mice.

Radiation-enhanced murine sarcoma virus genome rescue activity.

Although the doses of X-ray (312-2,500 R) used for irradiation of cells caused impairment of DNA synthesis and cell replication, co-cultivation of X-irradiated MuLV- carrier cells with un-irradiated nonproducer cells of MSV-induced tumour resulted in as much as 20-fold increase in MSV retrieval compared with the un-irradiated control. The enhancement was apparent also as a 3-fold increase in the number of cells producing MSV (infectious centers) in the co-cultivation plate. This suggested that the MSV genome rescue efficiency in terms of MSV per cell, as well as the number of cells producing MSV, increased markedly. By uridine-3H-labeling and focus assay experiments, evidence was presented which suggested that an increase in MSV/MuLV ratio in the culture fluid of co-cultivation plates was obtained when the MuLV-carrier cells were pre-irradiated. By contrast, X-irradiation of the nonproducer cells prior to co-cultivation caused only reductions of MSV genome rescue efficiency. However, use of X-irradiated MuLV-carrier cells for co-cultivation with X-irradiated nonproducer cells restored this efficiency to some extent. The dose-survival curve of the nonproducer cells was not much different from those of the MuLV-carrier cells after X-irradiation. It was suggested that the viability of nonproducer cells was required for replication of MuLV transferred from the carrier cells and for subsequent MSV genome rescue.

Evaluation of irradiation-plus-urethan-induced murine leukemia virus "release" using a new method for quantitation of oncornaviruses in tissues.

The quantity of C-type RNA tumor viruses in homogenate-sonicates of thymus-bone marrow tissues of C57BL/6J and RFM/Un mice 10 days after irradiation (X-rays or gamma-rays)-plus-urethan treatments is no greater than that in thymus-bone marrow homogenates from nontreated control mice. These results indicate that the leukemogenic activity, shown to be present in such thymus-bone marrow homogenates at this time after irradiation-plus-urethan treatment, is not due to change in quantity of C-type viruses as has been proposed. Virus quantity in tissues was evaluated by a new procedure that includes use of a microchamber with the sides situated on rotor radii so as to produce a uniform virus-containing sediment of tissue homogenate-sonicate that is evaluated by electron microscopic examination of this sections cut perpendicular to the membrane surface. Samples containing as little as 105 to 106 viruses can be relatively easily counted. Semipurified or purified viruses can also be counted after mixing with a tissue homogenate-bovine serum albumin diluent.