Brian J. Sheahan

Death mechanisms in cultured cells infected by Semliki Forest virus

We have investigated the induction of cell death in cultured cells by the virulent SFV4 and avirulent A7 strains of Semliki Forest virus (SFV). In BHK cells, death occurred by a typical apoptotic mechanism, as did the death of oligodendrocytes in glial cell cultures. For cerebellar neuron cultures, virus-induced death was due to necrosis. Although the SFV4 and A7 strains did not differ in the mechanism of induction of cell death, the virulent SFV4 strain did multiply to a higher titre in cultured neurons than the avirulent A7 strain. This is consistent with previous animal studies which indicate that the virulence of SFV strains is controlled by rapidity of multiplication in the CNS, leading to a lethal threshold of damage, rather than differential cell tropism or cell death mechanisms. The immune-mediated demyelination induced by avirulent strains may be triggered by apoptosis of oligodendrocytes, the consequences of which are obscured by death for virulent strains.

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.

The molecular pathogenesis of Semliki Forest virus: a model virus made useful?

IP: 54.70.40.11 On: Fri, 07 Dec 2018 19:28:21 Journal of General Virology (1999), 80, 2287–2297. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Therapeutic and prophylactic applications of alphavirus vectors.

Alphavirus vectors are high-level, transient expression vectors for therapeutic and prophylactic use. These positive-stranded RNA vectors, derived from Semliki Forest virus, Sindbis virus and Venezuelan equine encephalitis virus, multiply and are expressed in the cytoplasm of most vertebrate cells, including human cells. Part of the genome encoding the structural protein genes, which is amplified during a normal infection, is replaced by a transgene. Three types of vector have been developed: virus-like particles, layered DNA-RNA vectors and replication-competent vectors. Virus-like particles contain replicon RNA that is defective since it contains a cloned gene in place of the structural protein genes, and thus are able to undergo only one cycle of expression. They are produced by transfection of vector RNA, and helper RNAs encoding the structural proteins. Layered DNA-RNA vectors express the Semliki Forest virus replicon from a cDNA copy via a cytomegalovirus promoter. Replication-competent vectors contain a transgene in addition to the structural protein genes. Alphavirus vectors are used for three main applications: vaccine construction, therapy of central nervous system disease, and cancer therapy.

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.

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.

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.

Two mutations in the envelope glycoprotein E2 of semliki forest virus affecting the maturation and entry patterns of the virus alter pathogenicity for mice

The prototype strain of Semliki Forest virus (SFV) of known sequence and virus produced by the cDNA clone derived from it were lethal following intranasal (i.n.) infection of 40-day-old and intraperitoneal (i.p.) infection of pregnant BALB/c mice; this lethality was related to neuronal necrosis in the central nervous system (CNS). We conclude that the virulence of the prototype strain, and virus from the cDNA clone derived from it, is similar to that of L10 (the original SFV isolate). The effects of two mutations in the p62 envelope protein region of the clone were determined. Substitution of Glu for Lys at position 162 (mut64) extended the mean time of death following i.n. inoculation of 40-day-old mice. Pregnant mice infected with this virus survived but lethal infection of some fetuses did occur. Substitution of Leu for Arg at position 66 (mL), the cleavage site of the E2 and E3 proteins, results in the production of particles containing uncleaved p62. These particles were less virulent than the prototype strain when inoculated i.n. and induced immunity to virulent SFV challenge. The virus also induced the formation of multifocal glial nodules in the CNS of surviving mice. The differences in pathogenicity between the two mutants and the virulent parental virus are probably related to differences in the efficiency of virus multiplication in infected mice. The mut64 mutation attenuated the virus and allowed survival of pregnant mice infected i.p. so that the effects of fetal infection could be detected. The mL mutation allowed survival of i.n.-infected mice so that the later effects of virus multiplication in the CNS could be assessed. In the former case, this is probably a result of reduced virus release, whereas in the latter case it is due to inefficient entry of host cells. The results are consistent with our previous suggestion that lethality for virulent SFV infection results from a lethal threshold of damage to neurons in the CNS and that attenuating mutations may reduce neuronal damage below this threshold level.

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.

Semliki Forest Virus Infection of Mice: A Model for Genetic and Molecular Analysis of Viral Pathogenicity

Introduction. During the past two decades there has been a great increase in our knowledge of the mechanisms of virus multiplication in cultured cells. The cells utilized, however, have usually been undifferentiated standard cell lines, and little attention has been given to the interactions of viruses with differentiated cells, either in tissue culture or in the intact animal. Until recently, studies of viral pathogenicity have tended to be largely descriptive but a start has now been made in the analysis of the genetic and molecular control of virus pathogenicity for a number of model systems. Here we focus on one such system, Semliki Forest virus (SFV) infection of the laboratory mouse. This system has the initial advantages that the molecular biology of SFV and the closely related Sindbis virus has been extensively studied using standard tissue culture cell lines, that SFV is neurotropic, that strains of extremes of virulence for mice are available, and that inbred and immune-deficient mouse strains can be utilized.