Semliki Forest Virus A7 Research Articles

MicroRNA-Attenuated Clone of Virulent Semliki Forest Virus Overcomes Antiviral Type I Interferon in Resistant Mouse CT-2A Glioma.

Glioblastoma is a terminal disease with no effective treatment currently available. Among the new therapy candidates are oncolytic viruses capable of selectively replicating in cancer cells, causing tumor lysis and inducing adaptive immune responses against the tumor. However, tumor antiviral responses, primarily mediated by type I interferon (IFN-I), remain a key problem that severely restricts viral replication and oncolysis. We show here that the Semliki Forest virus (SFV) strain SFV4, which causes lethal encephalitis in mice, is able to infect and replicate independent of the IFN-I defense in mouse glioblastoma cells and cell lines originating from primary human glioblastoma patient samples. The ability to tolerate IFN-I was retained in SFV4-miRT124 cells, a derivative cell line of strain SFV4 with a restricted capacity to replicate in neurons due to insertion of target sites for neuronal microRNA 124. The IFN-I tolerance was associated with the viral nsp3-nsp4 gene region and distinct from the genetic loci responsible for SFV neurovirulence. In contrast to the naturally attenuated strain SFV A7(74) and its derivatives, SFV4-miRT124 displayed increased oncolytic potency in CT-2A murine astrocytoma cells and in the human glioblastoma cell lines pretreated with IFN-I. Following a single intraperitoneal injection of SFV4-miRT124 into C57BL/6 mice bearing CT-2A orthotopic gliomas, the virus homed to the brain and was amplified in the tumor, resulting in significant tumor growth inhibition and improved survival. Although progress has been made in development of replicative oncolytic viruses, information regarding their overall therapeutic potency in a clinical setting is still lacking. This could be at least partially dependent on the IFN-I sensitivity of the viruses used. Here, we show that the conditionally replicating SFV4-miRT124 virus shares the IFN-I tolerance of the pathogenic wild-type SFV, thereby allowing efficient targeting of a glioma that is refractory to naturally attenuated therapy vector strains sensitive to IFN-I. This is the first evidence of orthotopic syngeneic mouse glioma eradication following peripheral alphavirus administration. Our findings indicate a clear benefit in harnessing the wild-type virus replicative potency in development of next-generation oncolytic alphaviruses.

PKR acts early in infection to suppress Semliki Forest virus production and strongly enhances the type I interferon response.

The double-stranded RNA-activated protein kinase (PKR) is a key regulator of protein translation, interferon (IFN) expression and cell survival. Upon infection of vertebrate cells in continuous culture, the alphavirus Semliki Forest virus (SFV) initiates apoptosis and IFN synthesis. To determine the effect of PKR on SFV infection, we studied the course of infection in wild-type (wt) mice, mice with a genetic deletion of PKR (PKR−/−) and mouse embryo fibroblasts (MEFs) derived from these mice. In MEFs, PKR delayed virus protein synthesis, production of infectious virus and caspase-3-activated cell death and reduced the yield of infectious virus by 90 %. Small interfering RNA suppression of PKR levels in NIH-3T3 cells also reduced virus production and apoptosis. In MEFs, PKR was not required for initiation of IFN-β gene transcription, but contributed strongly to the magnitude of this response. Levels of IFN-β transcripts in PKR−/− MEFs at 8 h were 80 % lower than those in wt MEFs and levels of functional IFN at 24 h were 95 % lower. Following infection of wt and PKR−/− mice, SFV4 and SFV A7(74) were avirulent. PKR increased levels of serum IFN and the rate of clearance of infectious virus from the brain. In summary, in response to SFV, PKR exerts an early antiviral effect that delays virus protein production and release of infectious virus and, whilst PKR is not required for induction of apoptosis or activation of the type I IFN response, it strongly augments the type I IFN response and contributes to clearance of infectious virus from the mouse brain.

The type I interferon system protects mice from Semliki Forest virus by preventing widespread virus dissemination in extraneural tissues, but does not mediate the restricted replication of avirulent virus in central nervous system neurons

Semliki Forest virus (SFV) infection of the mouse provides a powerful model to study the pathogenesis of virus encephalitis. SFV and other alphavirus-based vector systems are increasingly used in biotechnology and medicine. This study analysed the strong susceptibility of this virus to type I interferon (IFN) responses. Following intraperitoneal infection of adult mice, SFV strain A7(74) was efficiently (100 %) neuroinvasive. In contrast, SFV4 was poorly (21 %) neuroinvasive. Upon entry into the brain, both viruses activated type I IFN responses. As determined by quantitative RT-PCR, activation of the IFN-alpha gene was proportional to virus RNA load. An intact type I IFN system was required for protection against both strains of SFV. IFN strongly curtailed virus spread in many cell types and in many tissues. In mice with an intact type I IFN system, infected cells were rarely observed and tissue tropism was difficult to determine. In the absence of a functional type I IFN system, the tropism and the potential for rapid and widespread infection of this virus was revealed. Virus infection was readily observed in the myocardium, endocardium, exocrine pancreas, adipose tissue, smooth muscle cells and in the brain in meningeal cells, ependymal cells and oligodendrocytes. In the brains of mice with and without type I IFN responses, virus infection of neurons remained rare and focal, indicating that the previously described restricted replication of SFV A7(74) in neurons is not mediated by type I IFN responses.

Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models

Semliki Forest virus (SFV) is one of the latest candidates for a virotherapeutic agent against cancer, and recent studies have demonstrated its efficacy in tumor models. In the present study, we examined the antitumor efficacy of an avirulent SFV strain A7(74) and its derivative, a replication-competent SFV vector VA7-EGFP, in a partially immunodeficient mouse tumor model (subcutaneous A549 human lung adenocarcinoma in NMRI nu/nu mouse) and in an immunocompetent rat tumor model (intracranial BT4C glioma in BDIX rat). When subcutaneous mouse tumors were injected 3 times with VA7-EGFP, intratumorally treated animals showed almost complete inhibition of tumor growth, while systemically treated mice displayed only delayed tumor growth (intravenous injection) or no response at all (intraperitoneal injection). This was at least partially due to a strong type I interferon (IFN) response in the tumors. The animals did not display any signs of abnormal behavior or encephalitis, even though SFV-positive foci were detected in the brain after the initial blood viremia. Intracranial rat tumors were injected directly with SFV A7(74) virus and monitored with magnetic resonance imaging. Tumor growth was significantly reduced (p < 0.05) with one virus injection, but the tumor size continued to increase after a lag period and none of the treated animals survived. Three virus injections or T-cell suppression with dexamethasone did not significantly improve treatment efficacy. It appeared that the local virotherapy induced extensive production of neutralizing anti-SFV antibodies that most likely contributed to the insufficient treatment efficacy. In conclusion, we show here that SFV A7(74) is a potential oncolytic agent for cancer virotherapy, but major immunological hurdles may need to be overcome before the virus can be clinically tested.

Virus tropism, distribution, persistence and pathology in the corpus callosum of the Semliki Forest virus‐infected mouse brain: a novel system to study virus–oligodendrocyte interactions

The temporal course of cellular pathology in virus-infected oligodendrocytes in vivo is not well defined. Here we study these events in the mouse brain using a novel system in which large numbers of oligodendrocytes can be reproducibly infected. In the mouse, following extraneural inoculation, the A7(74) strain of the alphavirus Semliki Forest virus (SFV) is efficiently neuroinvasive and central nervous system (CNS) infection leads to predominantly perivascular lesions of immune-mediated demyelination. This study demonstrates that direct intracerebral inoculation with SFV A7(74) or the SFV1 vector results in dramatic, selective and widespread infection of the major white matter tract of the brain, the corpus callosum. Mature oligodendrocytes are the predominant cell type infected. Subsequent events are complex; early virus-induced necrotic death of infected cells is followed by apoptotic death of adjacent apparently uninfected cells. A strong inflammatory response and considerable myelin loss are evident from 10 days and virus-positive cells are not observed after this time. In contrast, in athymic nu/nu mice, in the absence of T-cell responses, no inflammatory infiltrates are observed and virus-infected cells persist for over 30 days with extensive vacuolation but less demyelination. The change from an early destructive to a potentially persistent infection of oligodendrocytes is likely to reflect activation of innate immune responses. Activation of peripheral innate defences by inoculation of poly I : C prior to CNS virus infection abrogates the widespread corpus callosum infection. This widespread infection of the corpus callosum provides a novel in vivo system in which to study virus-oligodendrocyte interactions.

41. Evaluation of Lung Cancer Virotherapy with Oncolytic Semliki Forest Virus Using Immunodeficient Rodent Tumor Model

Top of pageAbstract Virotherapy has been postulated as a promising approach for killing tumor cells. Unlike conventional gene therapy, it uses replication competent viruses that are able to spread through tumor tissue and replicate in cancer cells, eventually causing destruction of the tumor. Most of the virotherapy studies are focused on oncolytic adenoviruses, but in this study we wanted to evaluate the efficacy of replication competent alphavirus, Semliki forest virus (SFV) strain A7 (74). It is not considered a human pathogen and also, it causes only transient and benign CNS infection to adult mice. To test the efficacy of SFV A7 (74) virotherapy, we first conducted in vitro studies with different (A549, NCI-H661 and SW900) human non small cell lung cancer (NSCLC) cell lines representing all the three main types of NSCLC. The aim was to determine, how extensively different amounts (MOI 0.1, 1 and 10) of virus destroy the target cells. After in vitro studies we determined the efficacy of the virotherapy in vivo (in immunodeficient NMRI nu/nu mice) using a subcutaneous A549 tumor model. In this experiment we compared the efficacy of virotherapy when SFV A7 (74) virus was injected systemically (i.v. and i.p.) or locally (i.t.). Virus distribution analysis (serum, brain and tumor tissues) was carried out 16 h, 2 days, 4 days and one week after viral injections. SFV A7 (74) virotherapy appeared to be very efficient in vitro, inducing in all NSCLC cell lines almost 100% cell death 48 h after infection. In subcutaneous (lung adenocarcinoma A549) tumor model, the therapeutic efficacy was better when virus was injected locally. With this approach, the tumor growth rate was reduced significantly when compared to control group. When virus was administered systemically, the tumor growth was delayed, but the tumors reached the same size as the control tumors by the end of the experiment. The distribution study showed the neurotrophic nature of the Semilki forest virus: 7 days post-infection the virus was found in the brain, but not in serum or in tumors. However there were no differences in the behaviour or overall condition of SFV A7 (74) - treated mice and control mice. Even though the study was done with an immunocompromized mice model (lack of T cell response), our results suggest that the animals were able to elicit at least a partial immune response against the virus. This was seen particularly with the systemic delivery route: viruses did not find the tumors efficiently and were unable to initiate powerful intratumoral replication. Thus, to obtain high therapeutic efficacy with SFV virotherapy, the route of virus administration and the immunogenicity of the virus should be carefully evaluated.

MRNA expression of proinflammatory cytokines in mouse CNS correlates with replication rate of semliki forest virus but not with the strain of viral proteins.

Tissue expression in viral infection of immunological effector molecules may depend on virus structure or replication or both. We analyzed cytokine mRNA expression in the central nervous system (CNS) of Balb/c mice during viral infection with Semliki Forest virus (SFV) clones, which varied either in structure or virulence or both. Highly neurovirulent SFV4 effectively induced IFN-gamma, TNF-alpha, IL-6 and TGF-beta, but its avirulent derivative V4-opal with nsP3 arginine-476 to opal mutation, elicited only weak induction of these cytokines. Structurally different, avirulent rA774, obtained by cloning from avirulent SFV A7(74) strain, did not induce synthesis of proinflammatory Th1 or Th2 cytokines in murine CNS, but increased synthesis of TGF-beta transcripts. In contrast, structurally identical but moderately virulent rA774-arg virus with sense codon at opal position in nsP3, markedly stimulated synthesis of IFN-gamma, TNF-alpha, and IL-10 transcripts, without, however, reaching the levels elicited by lethal SFV4. The rA774-arg clone was more potent in attracting peripheral immune cells into the CNS than the completely avirulent strains. In conclusion, induction of proinflammatory cytokine mRNA in the CNS by SFV infection seemed to correlate with the rate of viral replication and was not significantly influenced by the virus envelope or nonstructural protein primary structure. The results also have relevance for development of CNS gene therapy vectors as SFV4 and A774 display differences in CNS infection characteristics.

A novel neurotropic expression vector based on the avirulent A7(74) strain of Semliki Forest virus.

Semliki Forest virus (SFV), an enveloped alphavirus of the family Togaviridae, infects a wide range of mammalian host cells. Most strains are neurotropic but differ in virulence. The authors took advantage of the nonpathogenic properties of SFV strain A7(74), cloned recently in their laboratory, and constructed a replication-proficient expression vector to target the central nervous system (CNS) for heterologous gene expression. The vector, termed VA7, was engineered to drive expression of foreign inserts through a second subgenomic promoter inserted in the viral 3' nontranslated region (NTR). Infectious virus was obtained by in vitro transcription and transfection into BHK cells, and was shown to direct synthesis of heterologous proteins in several mammalian cell lines. Although novel expression vehicle is not applicable for targeting specific cell populations within the CNS in its present form, in cultured rat hippocampal slices, VA7 encoding enhanced green fluorescent protein (EGFP) efficiently transduced pyramidal cells, interneurons, and glial cells. With prolonged time post infection, the number of EGFP-expressing neurons in hippocampal slices increased. Mice infected intraperitoneally with the recombinant virus remained completely asymptomatic but showed CNS expression of EGFP as evidenced by immunohistochemistry. SFV A7(74) is a nonintegrating virus, which gives rise to a randomly distributed, patchy infection of the adult CNS that is cleared within 10 days. With the advantage of noninvasive administration, the expression vector described in this work is thus applicable for short-term gene expression in the CNS.

Semliki Forest virus infection leads to increased expression of adhesion molecules on splenic T-cells and on brain vascular endothelium.

Semliki Forest virus A7 (SFV-A7) is a neurotropic alphavirus that leads to an asymptomatic encephalitis in adult immunocompetent mice. We studied the expression of leukocyte and endothelial cell adhesion molecules in the spleen and in the central nervous system (CNS) during SFV-A7 infection. Kinetics of the expression of LFA-1 alpha/CD11a, LFA-1 beta/CD18, Mac-1/CD11b, VLA-4/CD49d, ICAM-1/CD54 and L-selectin/CD62L was determined on splenic CD4+ and CD8+ T-cells and macrophages by flow cytometry. Time course of the expression of these antigens and VCAM-1/CD106 as well as viral antigens in the CNS was studied by immunoperoxidase staining. In the spleen, a sustained increase in LFA-1-expression and a temporary increase at day 7 in the expression of VLA-4, Mac-1 and ICAM-1 were detected on CD8+ T-cells. L-selection was down-regulated on CD4+ cells. Adhesion molecules on macrophages remained unchanged. In the CNS, expression of Mac-1+, VLA-4+ and LFA-1+ cells increased in parallel with the kinetics of the expression of their ligands ICAM-1 and VCAM-1 on brain vessels. Upregulation of adhesion of molecules peaked between days 5-8 and was most prominent in the cerebellar and brain stem white matter where viral antigens were most abundant. We conclude that the adhesion molecules profile of splenic T cells is altered during SFV-A7 infection which may influence their homing into the CNS. Macrophages are probably recruited non-specifically as a consequence of activation of the brain vascular endothelium in the inflamed areas of the brain.

Blood-brain barrier breakdown and increased intercellular adhesion molecule (ICAM-1/CD54) expression after Semliki Forest ( A7) virus infection facilitates the development of experimental allergic encephalomyelitis

This report describes two mechanisms by which virus infection can facilitate demyelinating autoimmune inflammation in the murine CNS. In the BALB/c mouse model of experimental allergic encephalomyelitis (EAE), peripheral infection with an avirulent strain (A7) of Semliki Forest virus (SFV) increased the morbidity to EAE by infecting endothelial cells and damaging the blood-brain barrier (BBB). An influx of hematogenous CD18 + (LFA-1 + and MAC-1 +) cells into the CNS compartment was followed by a local increase in intercellular adhesion molecule 1 (ICAM-1) expression on the vascular endothelium. Although SFV A7 infection without EAE induction caused multifocal cerebral vascular endothelial cell infection and BBB damage followed by cellular infiltration and transient increase of ICAM-1, inflammation and demyelination of CNS white matter with classical clinical signs of EAE was observed only in EAE-induced BALB/c mice, whereas the control mice remained neurologically healthy. The upregulation of ICAM-1 after virus infection was detected after the CD18 + (LFA-1 + and MAC-1 +) cells had infiltrated the CNS both after EAE induction and also in nonsensitized control mice. The observed increase in ICAM-1 expression was transient in nonsensitized SFV A7 infected mice just as in the cellular infiltrates in the CNS, but EAE induction resulted in prolongation in both the cellular infiltrates and upregulation of ICAM-1. Thus, SFV A7 infection causes BBB damage and prolongs increased ICAM-1 expression on brain endothelium. This results in increased and more rapid morbidity to EAE in mice which have been sensitized with neuroantigen. However, SFV A7-infected mice without neuroantigen sensitization remain neurologically healthy.

Role of immune responses in protection and pathogenesis during Semliki Forest virus encephalitis

The course of Semliki Forest virus (SFV) A7(74) infection in immunocompetent BALB/c, athymic nu/nu and severe combined immunodeficient (SCID) mice was compared. BALB/c mice remained healthy and exhibited transient viraemia and infectious virus in the brain from days 2 to 7. Antibodies were detectable by day 5. In comparison, SCID mice displayed a high incidence of paralysis and died: the average day of death was day 23. From infection until death, virus was present in blood and brain. No antibodies were detectable. Athymic mice were intermediate with a transient viraemia and a persistent (> 210 days) sub-clinical central nervous system (CNS) infection. These mice produced anti-viral IgM but not IgG. The pattern of infection in BALB/c or nu/nu mice could be recreated in infected SCID mice by transfer of immune serum from BALB/c or nu/nu mice with the important exception that although BALB/c immune serum could abolish infectivity titres in the CNS, scattered cells positive for viral RNA remained. Transfer of serum decreased mortality and delayed the onset of paralysis. Transfer to infected SCID mice of a non-neutralizing IgG anti-E2 monoclonal antibody did not affect the viraemia but could also reduce brain virus titres. Irrespective of specific immune responses, virus replication in CNS cells was restricted, was generally non-cytopathic and in the absence of specific immune responses could persist. From day 14 lesions of inflammatory, primary demyelination were observed throughout the CNS of BALB/c mice. In contrast, despite prolonged brain virus titres, no demyelinating lesions were observed in infected nu/nu or SCID mice. Lesions could be initiated in the latter by transfer of spleen cells but not antibody. In summary, the focal restricted infection in the CNS of adult mice infected with SFV A7(74) is independent of specific immune responses. IgM antibodies clear the viraemia. IgG antibodies including non-neutralizing antibodies reduce and clear infectious virus but cells positive for viral RNA remain. These may normally be cleared by T cell responses which are damaging and give rise to lesions of demyelination.

Immunocytochemical evidence for Semliki Forest virus antigen persistence in mouse brain

Semliki Forest virus (SFV) is neurotropic in mice. Mature virulent virus (strain L10) can be identified within the CNS by electron microscopy in adult mice. Inspite of high virus titres, avirulent SFV A7(74) cannot be visualised in the brain of adult mice. Immunocytochemical studies using monoclonal antibodies (MAbs) to A7(74) E 1 and E 2 proteins and viral envelope glycolipids, showed viral E 1 to be labelled in the cerebral capillaries, the E 2 and the putative envelope glycolipids were labelled in the cytoplasm of neurons, particularly in the hippocampal areas and glia in the cerebellum. By double labelling the presence of viral antigens in astrocytes and oligodendrocytes was demonstrated. Viral antigens were identified in the brain up to 183 days after infection. Paraffin sections from Bouin-fixed tissue were found to be the most suitable material for immunocytochemistry of SFV. The presence of life-long anti SFV antibody in the sera of animals after SFV infection, could be due to the persistence of viral antigens acting as constant stimuli to the immune system.