Preliminary investigation of the area under the L-lactate concentration-time curve (LACArea) in critically ill equine neonates.

BACKGROUND: A variety of measures of L-lactate concentration ([LAC]) in the blood of critically ill neonatal foals have shown utility as prognostic indicators. These measures, evaluating either the severity of hyperlactatemia or the duration of exposure to hyperlactatemia, perform fairly well and have correctly classified 75-80% of foals examined in several studies. The area under the L-lactate concentration versus time curve (LACArea) encompasses both severity and duration of hyperlactatemia and should improve correct classification of patient survival. HYPOTHESIS/OBJECTIVES: LACArea is larger in nonsurviving critically ill neonatal foals. ANIMALS: Forty-nine foals admitted for critical illness to 1 of 4 referral hospitals. METHODS: Whole blood was obtained at admission and 6, 12, 18, and 24 hours after admission for measurement of L-lactate using a handheld lactate meter. LACArea was calculated for: admission-6, 6-12, 12-18, 18-24 hours, and admission-24 hours using the trapezoidal method and summing the 6-hours interval areas to determine total 24 hours area. Differences between survivors and nonsurvivors were determined using robust regression and Kruskal-Wallis testing, P < .05. RESULTS: LACArea was significantly larger in nonsurviving foals (n = 9) than in surviving foals (n = 40) at all time periods examined. CONCLUSIONS AND CLINICAL IMPORTANCE: Differences in LACArea between surviving and nonsurviving critically ill neonatal foals are large and support further investigation of this method as an improved biomarker for survival in critically ill neonatal foals is indicated.

Semliki Forest virus-mediated gene therapy of the RG2 rat glioma.

AIMS: Glioblastoma multiforme is the most common and most malignant adult brain tumour. Despite numerous advances in cancer therapy there has been little change in the prognosis of glioblastoma multiforme, which remains invariably fatal. We examined the Semliki Forest virus virus-like particle (SFV VLP) expression system encoding interleukin-12 (IL-12) as a therapeutic intervention against the syngeneic RG2 rat glioma model. METHODS: Glioma-bearing rats were treated with IL-12-encoding SFV VLPs via an implanted cannula. Animals were treated with 5 × 107 (low-dose) or 5 × 108 (high-dose) VLPs per treatment and the effect on glioma growth and survival was assessed. RESULTS: Low-dose treatment produced a 70% reduction in tumour volume, associated with a significant extension (20.45%) in survival that was dependent upon IL-12 expression. High-dose treatment resulted in an 87% reduction in tumour volume, related to the oncolytic capacity of the SFV VLP system. VLP delivery to the central nervous system (CNS) demonstrated the potential of the vector system to induce lethal pathology that was unrelated to replication-competent virus or high-level IL-12 expression. Treatment-related death was pronounced in high dose-treated animals and appeared to be the result of inflammation, necrosis and oedema at the inoculation site. CONCLUSION: The efficacy of an IL-12 gene therapy approach for the treatment of the RG2 glioma model has been demonstrated in addition to the oncolytic capacity of the VLP vector system. Despite this, the broad tropism of the SFV-based expression vector may limit use as a CNS gene therapy vector unless this inherent limitation can be overcome.

Effect of intranasal administration of Semliki Forest virus recombinant particles expressing interferon-ß on the progression of experimental autoimmune encephalomyelitis.

The effect of intranasal (IN) administration of Semliki Forest virus (SFV) recombinant particles expressing interferon-ß [IFN-ß, a partially effective treatment for multiple sclerosis (MS)] on the progression of experimental autoimmune encephalomyelitis (EAE, a murine model for MS) was investigated. The murine IFN-ß gene was cloned from SFV-infected mouse brain by RT-PCR into an SFV-enhanced expression vector, pSFV10-E, from which IFN-ß-expressing recombinant particles (rSFV10-E-IFN-ß) were prepared. Expression studies using immunohistochemistry and viral inhibition assay in BHK and murine L929 cells confirmed increased expression of IFN-ß. High level expression in the central nervous system (CNS) following IN inoculation was confirmed by the excision of olfactory bulbs, brain and spinal cord, and the detection of IFN-ß levels in homogenised tissue by ELISA. rSFV10-E-IFN-ß particles were administered IN to C57/Bl6 mice that had been induced for EAE using the encephalogenic peptide myelin oligodendrocyte glycoprotein (MOG) 35-55. The progression of EAE was measured by clinical score, weight loss and pathology. As previously shown, treatment with empty rSFV10-E particles moderately exacerbated EAE, as did continuous treatment with rSFV10-E-IFN-ß particles. Inhibition of disease with rSFV10-E-IFN-ß particles was dependent on the number and timing of treatments. Fewer treatments, administered before the effector stage, led to an improvement in clinical and pathology score. In conclusion, the timing and frequency of IN administration of rSFV10-E-IFN-ß particles are critical to disease outcome, with treatment prior to the effector stage being most effective.

Inhibition of angiogenesis by a Semliki Forest virus vector expressing VEGFR-2 reduces tumour growth and metastasis in mice.

Inhibition of tumour angiogenesis has been shown to restrict primary tumour growth and metastatic spread. This study examines the active induction of immune responses against tumour endothelial cells following immunization with recombinant Semliki Forest virus (rSFV) particles encoding murine vascular endothelial growth factor receptor-2 (VEGFR-2). This approach was tested in two murine tumour models, CT26 colon carcinoma and 4T1 metastasizing mammary carcinoma. Tumour growth and metastatic spread were shown to be significantly inhibited in mice that were prophylactically vaccinated or therapeutically treated with rSFV particles coding for VEGFR-2. Microvessel density analysis showed that immunization with rSFV led to significant inhibition of tumour angiogenesis. Therapeutic efficacy was found to be associated with the induction of an antibody response against VEGFR-2. Co-immunization of mice with rSFV particles encoding VEGFR-2 and interleukin (IL)-12 completely abrogated both the antibody response and the antitumour effect. However, co-immunization of mice with VEGFR-2 and IL-4 encoding particles was shown both to induce higher titres of anti-VEGFR-2 antibodies and lead to enhanced survival following tumour challenge when compared to mice vaccinated with VEGFR-2 particles alone. These findings indicate that active immunization with rSFV particles coding for VEGFR-2 can break immunological tolerance and could potentially be used as part of a novel treatment for cancer.

Inhibition of murine K-BALB and CT26 tumour growth using a Semliki Forest virus vector with enhanced expression of IL-18.

The enhanced Semliki Forest virus vector (SFV10-E), an RNA-based suicide expression vector system, expresses foreign genes at levels up to 10x higher than the original SFV10 vector. This vector has been used previously to express interleukin-12 for a tumour treatment study in a BALB/c murine model. Interleukin-18, an IFN-gamma-inducing cytokine, plays a key role in the early induction of T helper1 (Th1) cell-mediated immune responses in addition to anti-angiogenic activity. In this study, the murine IL-18 gene along with an Ig-kappa leader sequence was cloned into the SFV10-E vector. The pSFV10-E-IL-18 construct was characterised in vitro for levels of expression and secretion, and the production of biologically active IL-18 was confirmed. An in vivo tumour treatment study using high titre rSFV10-E-IL-18 virus-like particles to treat subcutaneous K-BALB and CT26 tumours in BALB/c mice demonstrated therapeutic efficacy including the disappearance of tumour cells in a minority of treated animals. Tumour regression was associated with induction of avascular and suppurative necrosis.

Regression of mouse tumours and inhibition of metastases following administration of a Semliki Forest virus vector with enhanced expression of IL-12.

The Semliki Forest virus (SFV) vector is an RNA-based suicide expression vector that has been used experimentally for tumour therapy. Recently, a new enhanced vector pSFV10-E has been developed that expresses foreign genes at levels up to 10 times higher than the original vector. Interleukin-12 (IL-12), an immunomodulatory cytokine, plays a key role in the induction of T-helper1 responses. The two IL-12 gene subunits were cloned from mouse splenocytes and inserted into the pSFV10-E and pSFV10 (non-enhanced) vectors. Both constructs expressed and secreted biologically active murine IL-12. Administration of high titre rSFV10-E-IL12 particles intratumourally to treat implanted K-BALB tumours in BALB/c mice demonstrated complete tumour regression in comparison to control or rSFV10-IL12 treated groups. High titre rSFV10-E-IL12 particles were also effective in the CT26 tumour model. Histological and immunohistochemical studies revealed tumour necrosis in addition to aggressive influx of CD4+ and CD8+ T cells and other immune cells. Furthermore, inhibition of primary tumour growth and lung metastases of a metastatic (4T1) tumour model indicated the potential of high titres of rSFV10-E-IL12 particles as an efficient antitumour therapeutic agent.

Treatment of rapidly growing K-BALB and CT26 mouse tumours using Semliki Forest virus and its derived vector.

To assess the potential of immune stimulation in combination with apoptosis induction by Semliki Forest virus (SFV) and its derived vector for tumour treatment, we have utilized the poorly immunogenic and rapidly growing K-BALB and CT26 murine tumour models. Both cell lines underwent apoptosis and expressed viral antigen when infected with the SFV4 strain of SFV, or recombinant SFV (rSFV) virus-like particles (VLPs) encoding the p62-6k viral structural proteins. VLPs were used to immunize groups of BALB/c and BALB/c nu/nu mice prior to subcutaneous tumour induction and treatment. Direct intratumoral injection of VLPs or SFV4 resulted in an immediate and intense inflammatory reaction in immunized groups that was not observed in naive groups until day 5 of treatment, and was not observed in nu/nu groups. A significantly higher level of tumour growth inhibition was observed in immunocompetent groups than in athymic mice. For K-BALB tumours, SFV4 treated groups showed greater inhibition than that observed in VLP-treated groups, with immunization prior to treatment enhancing the overall antitumour effect and immune response. No significant difference was observed in CT26 tumours between VLP and SFV4-treated groups, but prior immunization considerably enhanced the antitumoural response. It is concluded that use of the inherent apoptosis-inducing capability of SFV or its vector, by perfusion in combination with immune stimulation, may have potential for the treatment of rapidly growing tumours.

Role of interferon-gamma and nitric oxide in the neuropathogenesis of avirulent Semliki Forest virus infection.

Semliki Forest virus (SFV) infection of mice provides a useful model for the analysis of viral neuropathogenesis. In this study, the roles of interferon (IFN)-gamma and nitric oxide (NO) in the pathogenesis of SFV infection were assessed using mice deficient in inducible nitric oxide synthase (iNOS-/-), an enzyme important in the production of NO, and mice deficient in IFN-gamma receptor (IFN-gammaR-/-). Gene-knockout and wildtype mice were infected intranasally with the avirulent A7 strain of SFV and neuropathological lesions were correlated with levels of IFN-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-10 in the olfactory bulbs and frontal cortex. Lesions in IFN-gammaR-/- mice were characterized by higher levels of neuronal necrosis than in wildtype mice. The higher levels of neuronal necrosis were associated with increased levels of SFV antigen in neurones and increased numbers of macrophages and B cells. Relative differences in the severity of demyelination between IFN-gammaR-/- and wildtype mice were not detected. Similar levels of neuronal necrosis and SFV antigen labelling occurred in iNOS-/- mice and wildtype mice and levels of demyelination and macrophage infiltration in the iNOS-/- mice were lower than those in the wildtype strain. A rapid, but transient increase in the concentration of IFN-gamma was demonstrated in the frontal cortex of all infected mice samples. IL-10 levels in the frontal cortex and olfactory bulbs of SFV-infected iNOS-/- mice exceeded those present in the wildtype mice. This study, taken with our previous reports, provides further evidence that type 1 T cell responses are important in the control of brain viral clearance and the prevention of neuronal necrosis, but not in the development of demyelination.

Avirulent Semliki Forest virus replication and pathology in the central nervous system is enhanced in IL-12-defective and reduced in IL-4-defective mice: a role for Th1 cells in the protective immunity.

Experimental infection of mice with avirulent Semliki Forest virus (SFV) has been used as a model of demyelinating disease in humans. A number of studies have shown that T cells may be important for mediating demyelination, but the role of T cells is still, unclear. Here, we show that neuronal necrosis, but not demyelination, was more severe in interleukin (IL)-12-defective mice compared with wild-type mice and this correlated with higher virus titers in the brain. In contrast, the severity of demyelination and neuronal depletion was reduced in IL-4-defective mice and this correlated with reduced brain virus titers and enhanced SFV-specific IFN-gamma production. The findings indicate that type 1 T cells play a role in the control of SFV replication but not directly in SFV-induced pathology in the CNS.

The pathogenicity of louping ill virus for mice and lambs.

Mice and lambs were infected with the LI/I, LI/31 or MA54 strain of louping ill virus (LIV) to provide information relevant to testing the efficacy and biosafety of a new generation of flavivirus vaccines based on a Semliki Forest virus (SFV) vector. Whereas clinical signs and neuropathological lesions were consistently severe in mice, the majority of lambs showed lesions of moderate severity and only lambs with severe lesions were clinically affected. For both species, dispersal of viral antigen occurred along neuronal cell processes, and neuronal degeneration and death were confirmed as central events after infection with LIV. In contrast to lambs, in which most lesions remained localized, mice showed widely dispersed lesions which were associated with less intense leucocytic infiltrates. Among the infiltrating cells, histiocytes predominated and apoptotic forms were prominent in severely affected animals. The intranasal route of infection provided an efficient avenue for entry of LIV into the brain and resulted in lesions which were more severe than those produced by subcutaneous or intraperitoneal inoculation.

Induction of apoptosis in BCL-2-expressing rat prostate cancer cells using the Semliki Forest virus vector.

The Semliki Forest virus (SFV) vector is a transient RNA expression vector that has an inherent p53-independent apoptosis-inducing property. It is administered as recombinant SFV particles (rSFV) that undergo 1 round of replication only and express a gene cloned into the multicloning site. In our study we have investigated the ability of the SFV vector to induce apoptosis and inhibit tumour growth in rat prostate cancer (AT3-Neo) cells expressing the Bcl-2 oncogene (AT3-Bcl-2 cells), which normally inhibits apoptosis. rSFV expressing the enhanced green fluorescent protein (EGFP) gene (rSFV-EGFP), or recombinant RNA transfected into cells by electroporation, induced delayed apoptosis in AT3-Bcl-2 cells. SFV-mediated expression of a cloned pro-apoptotic Bax gene by the vector, however, enhanced apoptosis induction both in AT3-Bcl-2 cells and standard BHK-21 cells. Such Bax-expressing particles could be produced only at low titers compared to EGFP-expressing particles under standard conditions for particle production, but lowering the incubation temperature for particle production to 33 degrees C partially alleviated this effect. Bax-expressing particles were shown to inhibit the growth of AT3-Neo and AT3-Bcl-2 tumours in nude mice, as did high titre EGFP-expressing particles. It is concluded that SFV recombinant particles have potential as anti-tumour agents to treat apoptosis-resistant tumours.

Progressive encephalomyelopathy and cerebellar degeneration in 10 captive-bred cheetahs.

Progressive ataxia, with head tremor, developed in 10 captive-born cheetah cubs under six months of age. The condition was usually preceded by coryza and an ocular discharge. Initially the ataxia and weakness affected the hindquarters, then the forelegs, and head tremor developed later. Significant pathological changes were confined to the central nervous system. There was widespread Wallerian degeneration in the funiculi of the spinal cord (except those in the dorsal columns), in the medulla and in the cerebellum. In the cerebellum there was degeneration of Purkinje cells and of the molecular and granular cell layers. There was chromatolysis in the Purkinje cells, the ventral horn cells of the spinal cord and in the neurons of the lateral vestibular nucleus. The olivary nucleus was necrotic. There were foci of inflammatory cells in the molecular layer of the cerebellum and in the medulla. The cause of the disease remains unknown.

A recombinant Semliki Forest virus particle vaccine encoding the prME and NS1 proteins of louping ill virus is effective in a sheep challenge model.

This study has examined the efficacy following intramuscular administration of a recombinant Semliki Forest virus (rSFV) vaccine, encoding the prME and NS1 proteins of louping ill virus (LIV), in sheep. Administration of rSFV-LIV vaccine resulted in transient detection at the injection site and draining lymph node only and no dissemination to distal sites. In addition, the recombinant vaccine offered complete protection against subcutaneous challenge with LIV, and partial protection following intranasal administration of LIV. Protected animals had no pathological changes normally associated with LIV infection, and had developed high antibody titres. In contrast, the two animals not protected exhibited classical clinical signs and neuropathological lesions of LIV infection. These findings indicate that rSFV-based vaccines have the potential to be developed as effective prototype vaccines for LIV.

Semliki Forest virus-based vaccines: persistence, distribution and pathological analysis in two animal systems.

This study has examined the persistence, distribution and pathological changes following intramuscular administration of Semliki Forest virus (SFV) vaccine vectors in mice and chickens. Administration of recombinant SFV RNA particles showed persistence at the injection site of mice up to 7 days, transient detection in secondary lymphoid organs and no dissemination to distal sites. In contrast, administration of a layered SFV DNA/RNA vector and a conventional standard naked DNA vector resulted in long-term persistence at the injection site, plasmid DNA being detected at 8 months post-inoculation in mice. Plasmid DNA was found distributed throughout the body, and tissues distal from the site of injection were positive up to 3 months. A similar pattern was observed in chickens. Mild pathological changes were observed at the injection site only, and plasmid DNA or recombinant RNA was not detected in mouse foetuses. These findings indicate that SFV-based vectors have the potential to be developed as safe vaccines.

Transient virus infection and multiple sclerosis.

Multiple sclerosis (MS) is a chronic, demyelinating disease of the CNS in which autoimmunity to myelin plays a role in pathogenesis. The epidemiology of MS indicates that it may be triggered by a virus infection before the age of adolescence, but attempts to associate a specific virus with MS have produced equivocal results. Many studies of the aetiology of MS have postulated that a persistent virus infection is involved, but transient virus infection may provide a plausible alternative mechanism that could explain many of the inconsistencies in MS research. The most studied animal model of MS is chronic relapsing experimental autoimmune encephalomyelitis (CREAE), which is induced in susceptible animals following injection of myelin components. While CREAE cannot provide information on the initiating factor for MS, it may mimic disease processes occurring after an initial trigger that may involve transient virus infection. The disease process may comprise separate triggering and relapse phases. The triggering phase may involve sensitisation to myelin antigens as a result of damage to oligodendrocytes or molecular mimicry. The relapse phase could be similar to CREAE, or alternatively relapses may be induced by further transient virus infections which may not involve infection of the CNS, but which may involve the recrudescence of anti-myelin autoimmunity. Although current vaccines have a high degree of biosafety, it is suggested that the measles-mumps-rubella vaccine in particular could be modified to obviate any possibility of triggering anti-myelin autoimmunity.

Inhibition of human lung carcinoma cell growth by apoptosis induction using Semliki Forest virus recombinant particles.

We have utilised cell cultures and growth of tumours in nude mice to assess further the potential of the Semliki Forest virus (SFV) vector as a cancer therapy agent. This vector is a transient RNA-based expression vector, and we have previously shown that SFV and its derived vector can induce p53-independent apoptosis by expression of the nonstructural region of the virus genome. Apoptosis induction is therefore an inherent property of the vector and is not dependent on heterologous gene expression. SFV recombinant suicide particles (rSFV) were shown to induce apoptosis in H358a cells, which are human non-small cell lung carcinoma cells deleted in p53. EGFP-expressing rSFV also inhibited the growth of developing H358a spheroids. Direct injection of rSFV into H358a tumours subcutaneously implanted as xenografts in nu/nu mice inhibited tumour growth, and in some cases caused complete regression. It is concluded that tumour growth suppression induced by rSFV was due to apoptosis induction and that the vector has an inherent cell death-promoting and antitumour activity. These results, as well as previous work by other authors on targeting and immune stimulation using alphavirus vectors, indicate that SFV recombinant particles in particular have considerable potential for further exploitation as a cancer therapy agent.

Recombinant Semliki Forest virus particles expressing louping ill virus antigens induce a better protective response than plasmid-based DNA vaccines or an inactivated whole particle vaccine.

Louping ill virus (LIV) infection of mice was used as a model to evaluate the protective efficacy of Semliki Forest virus (SFV)-based vaccines in comparison to a standard DNA vaccine and a commercial chemically inactivated vaccine. The recombinant SFV-based vaccines consisted of suicidal particles and a naked layered DNA/RNA construct. The nucleic acid vaccines expressed the spike precursor prME and the nonstructural protein 1 (NS1) antigens of LIV. Three LIV strains of graded virulence for mice were used for challenge. One of these was a naturally occurring antibody escape variant. All vaccines tested induced humoral immunity but gave varying levels of protection against lethal challenge. Only recombinant SFV particles administered twice gave full protection against neuronal degeneration and encephalitis induced by two of the three challenge strains, and partial protection against the highly virulent strain, whereas the other vaccines tested gave lower levels of partial protection.

Cell death mechanisms in the olfactory bulb of rats infected intranasally with Semliki forest virus.

Semliki Forest virus (SFV) infection of mice is used as a model to study pathogenic processes occurring in viral encephalitis. It has previously been shown that avirulent strains of SFV differ from virulent strains in showing restricted multiplication in neurones and in producing localized rather than widespread lesions in the central nervous system (CNS). Restricted neuronal damage is age-dependent and does not occur in neonatal animals. In this study, cell death mechanisms occurring in the CNS of adult rats infected intranasally (i.n.) with a virulent (SFV4) and an avirulent (A7) strain of SFV have been investigated. Although i.n. infection of rats was less efficient than that of mice, SFV4 reached a higher titre in the CNS of infected animals than A7. Neuronal destruction and leucocytic infiltration occurred throughout the forebrain of SFV4-infected rats. A7-infected rats remained clinically normal although degenerate neurons and inflammatory changes were present primarily in the olfactory system. Following infection with either A7-SFV or SFV4, TUNEL-positive nuclei were seen in areas of leucocytic infiltration and among the poorly differentiated cells of the rostral migratory stream. Migrating cells had condensed nuclear chromatin, compacted cytoplasm and intact cellular membranes, characteristic of apoptosis, and were sparsely immunolabelled for viral antigen. In SFV4-infected rats, large numbers of contiguous neurones in forebrain areas exhibited cytoplasmic eosinophilia and karyolysis and were surrounded by phagocytic cells. Such neurones contained dense intracytoplasmic deposits of viral antigen and showed weak cytoplasmic TUNEL staining; electron microscopy showed membrane disruption, organelle disintegration, irregular chromatin condensation and cytoplasmic aggregation of virus particles. Bcl-2 staining was similar in infected and control rats and was most intense in randomly distributed Purkinje cells in the cerebellum; neurons in the olfactory bulbs were unstained. These findings indicate that during SFV encephalitis, infiltrating leucocytes and neural precursor cells undergo apoptosis whilst productively infected neurons undergo necrosis.

Recombinant Semliki Forest virus particles encoding the prME or NS1 proteins of louping ill virus protect mice from lethal challenge.

Recombinant Semliki Forest virus (rSFV) vaccines encoding louping ill virus (LIV) genes prME and NS1 were examined. Cells transfected with rSFV-prME RNA showed correct processing of the precursor prME and the release into the medium of M and E proteins in particulate form, whilst rSFV-NS1-transfected cells secreted glycosylated, heat-labile NS1 dimers. Mice immunized with rSFV particles produced antibodies against prME and NS1 that were mainly of the IgG2a subtype, indicating that a T-helper 1 immune response was induced. Immunization with prME- or NS1-encoding particles induced T-cell proliferation. Mice vaccinated intraperitoneally (i.p.) with rSFV-prME and/or rSFV-NS1 were significantly protected from lethal i.p. challenge by two strains of LIV, the virulent LI/31 strain, from which the commercial LIV vaccine is derived, and the less-virulent LI/I antibody-escape variant. Intranasal (i.n.) vaccination was protective for rSFV-prME only against LI/31 challenge and not against challenge with LI/I. Immunization with rSFV-NS1 was protective against i.p. and i.n. challenge with both virus strains when given i.p., but was not protective when given i.n. For unvaccinated mice infected with LIV, all animals showing clinical signs had severe degenerative and inflammatory lesions in the central nervous system. None of the rSFV-vaccinated mice that survived challenge showed central nervous system pathology, with the exception of mild leptomeningitis in a minority of LI/31-infected mice. This suggests that protection following immunization with rSFV must occur at early stages of LIV infection.

The Semliki Forest virus vector induces p53-independent apoptosis.

Three deletion mutants of the structural protein region of the Semliki Forest virus (SFV) genome, including one which encompassed all the viral structural protein genes, induced apoptosis in BHK cells at 48 h after transfection, as shown by DNA laddering and TUNEL staining, as did the wild-type SFV4 RNA. A similar result was obtained for the SFV1 expression vector, which has a multicloning site inserted in place of the structural protein genes. However, in cells transfected with viral RNA containing a deletion of the nsP2 gene, neither viral RNA synthesis nor the induction of apoptosis occurred. Both SFV1 vector and wild-type SFV4 RNA induced apoptosis in human H358a lung carcinoma cells, which have a homozygous deletion of the p53 gene. It is concluded that the SFV vector encodes a function in the nonstructural coding region which induces p53-independent apoptosis and is dependent on viral RNA synthesis.