Quantitative RT-PCR for titration of replication-defective recombinant Semliki Forest virus.

Virus titration may constitute a drawback in the development and use of replication-defective viral vectors like Semliki Forest virus (SFV). The standardization and validation of a reverse transcription quantitative PCR (qRT-PCR) method for SFV titration is presented here. The qRT-PCR target is located within the nsp1 gene of the non-structural polyprotein SFV region (SFV RNA), which allows the strategy to be used for several different recombinant SFV constructs. Titer determinations were carried out by performing virus titration and infection assays with SFVs containing an RNA coding region for the rabies virus glycoprotein (RVGP) or green fluorescent protein (GFP). Results showed that the standardized qRT-PCR is applicable for different SFV constructs, and showed good reproducibility. To evaluate the correlation between the amount of functional SFV RNA in a virus lot and its infectivity in BHK-21 cell cultures, a temperature mediated titer decrease was performed and successfully quantitated by qRT-PCR. When used for cell infection at the same multiplicity of infection (MOI), the temperature treated SFV-RVGP samples induced the same levels of RVGP expression. Similarly, when different SFV-GFP lots with different virus titers, as accessed by qRT-PCR, were used for cell infection at the same MOI, the cultures showed comparable amounts of fluorescent cells. The data demonstrate a good correlation between the amount of virus used for infection, as measured by its SFV RNA, and the protein synthesis in the cells. In conclusion, the qRT-PCR method developed here is accurate and enables the titration of replication-defective SFV vectors, an essential aid for viral vector development as well as for establishment of production bioprocesses.

Generation of recombinant alphaviral vectors.

The alphaviruses Semliki Forest virus and Sindbis virus have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in <2 d. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have showed reduced cytotoxicity and prolonged expression. Membrane proteins (which are generally difficult to express at high levels in recombinant systems) have generated high yields and facilitate applications in structural biology. Alphaviruses have also been applied in vaccine development and gene therapy. This protocol describes the production of recombinant alphaviral vectors. The SFV- and SIN-based expression systems apply two vectors for recombinant particle production. In addition to the RNA-based vectors described here, DNA vectors with cytomegalovirus or other RNA polymerase type II promoters can be used for direct plasmid DNA transfections. Cotransfection of SFV-based pSCA expression and pSCA helper vectors generates recombinant viral particles.

Purification and concentration of alphavirus.

The alphaviruses Semliki Forest virus and Sindbis virus have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. High-titer viral production is achieved in <2 d. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have showed reduced cytotoxicity and prolonged expression. Membrane proteins (which are generally difficult to express at high levels in recombinant systems) have generated high yields and facilitate applications in structural biology. Alphaviruses have also been applied in vaccine development and gene therapy. Generally, purification or concentration of alphaviruses is not necessary. However, for instance, the medium derived from baby hamster kidney cells is toxic to primary neurons in culture. Including a purification step substantially improves the survival of the transduced neurons. Viral concentration and purification may also be advantageous for in vivo studies in animal models and are mandatory for clinical applications. This protocol describes three methods for purification and concentration of alphavirus.

Determination of alphaviral titers.

The alphaviruses Semliki Forest virus and Sindbis virus have been used frequently as expression vectors in vitro and in vivo. Usually, these systems consist of replication-deficient vectors that require a helper vector for packaging of recombinant particles. Replication-proficient vectors have also been engineered. Alphaviral vectors can be used as nucleic-acid-based vectors (DNA and RNA) or infectious particles. The broad host range of alphaviruses facilitates studies in mammalian and nonmammalian cell lines, primary cells in culture, and in vivo. The strong preference for expression in neuronal cells has made alphaviruses particularly useful in neurobiological studies. Unfortunately, their strong cytotoxic effect on host cells, relatively short-term transient expression patterns, and the reasonably high cost of viral production remain drawbacks. However, novel mutant alphaviruses have showed reduced cytotoxicity and prolonged expression. Alphaviruses have also been applied in vaccine development and gene therapy. Before use in vitro or in vivo, it is essential to determine the titer of the generated alphaviral particles. Because defective alphaviruses do not produce plaques, their titers cannot be determined by conventional methods. However, viral titers can be determined readily in cases where the recombinant viruses express reporter genes such as green fluorescent protein or ß-galactosidase, as well as indirectly by immunofluorescence methods. The potency of viral stocks can also be evaluated by light microscopic analysis. Alphavirus-infected cells show a dramatic decrease in growth and can be easily distinguished from noninfected control cells through their rounded morphology.

Me Tri virus: a Semliki Forest virus strain from Vietnam?

Me Tri virus (MTV) is a member of the Semliki Forest virus (SFV) complex in the genus Alphavirus, first isolated from Culex tritaeniorhynchus mosquitoes in Vietnam in 1971 and described as a newly recognized alphavirus, based on antigenic characterization. However, based on a partial nucleotide sequence of the E1 envelope glycoprotein gene, it has recently been argued that MTV may represent a variant of SFV rather than a separate species. To enable definitive classification, we determined the complete genome sequence of MTV from original virus stock. Nucleotide homology, as well as phylogenetic analyses based on whole and partial genome sequences confirmed that MTV is an isolate of SFV. Notable differences to other reported SFV sequences included a 122 nt insertion at the 5' non-translated region (NTR), likely resulting from homologous recombination of part of the nsP2 gene, and differences in the sequence length of the 3' NTR. To our knowledge, this is the first and only documentation of SFV isolation outside Africa. Further research is needed to clarify whether SFV continues to circulate in Vietnam.

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.

Semliki forest virus infection of laboratory mice: a model to study the pathogenesis of viral encephalitis.

Semliki Forest virus (SFV) infection of the laboratory mouse provides an experimental system to study the pathogenesis of viral encephalitis. Following extraneural inoculation the virus is efficiently neuroinvasive and crosses the blood-brain barrier to initiate perivascular foci of infection in neurons and oligodendrocytes. The outcome of infection ranges from clinically unapparent mild encephalitis to fatal panencephalitis. SFV infections of the developing nervous system are always highly destructive and are generally fatal. In contrast, SFV infections of the mature nervous system can result in persistent infection with no apparent cell loss. This dramatic difference is attributable to developmental changes in the interactions between virus and CNS cells. Antibody responses clear the systemic infection and control the CNS infection. CD8+ T-cells are required to generate the lesions of inflammatory demyelination which can be a feature of the neuropathology. This article reviews the pathogenesis of SFV encephalitis, describing the neuropathology and the mechanisms which underlie it and which may be fundamental to many viral encephalitides.

Selective downregulation of Th1 response by Linomide reduces autoimmunity but increases susceptibility to viral infection in BALB/c and SJL mice.

Susceptibility to autoimmunity has been associated with polarization of Th1/Th2 balance in immune system towards the Th1-type of reactivity. We report here that orally administered quinoline-3-carboxamide (Linomide) selectively downregulates Th1 response in BALB/c and SJL mice, leading to reduction of autoimmunity in the BALB/c and SJL models of experimental allergic encephalomyelitis (EAE). This was shown by prevention of EAE in Th1 responding SJL mice and partial downregulation of EAE in Th2-prone BALB/c mice. In a BALB/c model of EAE, in which infection with Semliki Forest A7 virus (SFV-A7) is used for enhancement of autoimmunity, clinical signs of EAE were reduced while mortality due to viral infection in the CNS was enhanced. Selective downregulation of the Th1 response by Linomide also rendered initially resistant SJL mice susceptible to SFV-A7 CNS infection. This was shown by immunohistochemical detection of extensive deposits of viral antigen in numerous perivascular foci within the CNS and abolished virus antigen-specific lymphocyte reactivity in Linomide-treated SJL mice. In addition, analysis of spleen cell cytokine mRNA production profile revealed decreased number of IFN-gamma producing cells in both SJL and BALB/c mice, reduced number of IL-12p40 producing cells in SJL and increased number of 12p40 producing cells in BALB/c mice along with slightly increased IL-4 production in both strains of mice. These results indicate that oral treatment with Linomide induces selective downregulation of Th1 reactivity causing reduction of autoimmunity and increased susceptibility to SFV-A7 CNS infection. Selective downregulation of Th1 response is a desired effect in the treatment of autoimmune diseases but our results suggest that the benefits have to be balanced against the possible loss in immunoprotection against pathogens.

Sequence analysis of the avirulent, demyelinating A7 strain of Semliki Forest virus.

The nonstructural region of the genome of the avirulent A7 strain of Semliki Forest virus (SFV) has been sequenced, so that the complete nucleotide sequence is available. Compared to the virulent SFV4 strain (produced from the infectious clone pSP6-SFV4), A7 contains 226 nucleotide changes in the translated region, which result in 47 amino acid changes. The 5' nontranslated region has two nucleotide changes, and the 3' nontranslated region is longer in A7 than SFV4, and contains divergent and repeated sequences. Chimeras containing SFV4 and A7 sequences and an infectious clone of A7, pSP6-CA7, were constructed. The virulence of these was tested by intraperitoneal and intranasal infection of adult BALB/c mice. It was shown that determination of the avirulent phenotype of A7 was polygenic, and required the additive effect of sequences from both the structural and non-structural regions of the SFV genome.

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.

Long-term effects of Semliki Forest virus infection in the mouse central nervous system.

Semliki Forest virus (SFV) infection of mice is used as a model to study pathogenic processes occurring in viral encephalitis and demyelinating disease. In this study, the long-term effects of infection by the avirulent M9 mutant of SPV on the central nervous system (CNS) of BALB/c and SJL mice were determined. The presence of infectious virus, viral RNA and cytokine mRNA in the brains of individual mice and the presence of lesions in the spinal cords of the same mice up to 360 days post-infection (d.p.i.) were analysed in order to detect any correlation between these parameters of pathogenesis. Infectious virus could not be detected beyond 7 d.p.i. for either mouse strain. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the presence of the E2 and nsP1 regions of the virus genome and mRNA for interferon-gamma and tumour necrosis factor-alpha. Viral RNA could be detected up to 90 d.p.i. for both mouse strains. Cytokine mRNA could be detected up to 28 d.p.i. for BALB/c mice but up to 360 d.p.i. for SJL mice. Inflammatory lesions, which were associated with cytokine mRNA expression, were not detected in BALB/c mice beyond 28 d.p.i. but were detected in two SJL mice at 90 d.p.i. It is concluded that M9-SFV infection induces long-term prolonged expression of pro-inflammatory cytokines in the CNS of the majority of SJL (but not BALB/c) mice which is not associated with persistence of the virus genome. M9-SFV infection of SJL mice may be a relevant model for the pathogenesis of multiple sclerosis in man.

Efficient removal of viruses by a novel polyvinylidene fluoride membrane filter.

Virus removal by a novel filter (Ultipor VF DV50), comprising three layers of PVDF membrane, has been evaluated by infectivity studies using a range of viruses and conditions. The filter was able to remove at least 6 log of various viruses, i.e. Sindbis and Semliki Forest (40-70 nm), herpes simplex (120-200 nm) and vaccinia (200 x 350 nm), from cell-culture medium or phosphate buffered saline pH 6.8 containing 0.5% albumin. However, the removal of polio virus (25-30 nm) under these conditions was only limited, i.e. about 1 log. This filter is thus effective for removing viruses of about 50 nm or larger. Proteins as large as immunoglobulins (MW 160,000), were able to pass through the filter with recoveries of at least 85%. Due to its ability to remove viruses of medium to large size, this filter shows potential for increasing the safety of biological products where viruses such as hepatitis B, C, herpes and retroviruses are of concern.

Detection by a solid phase independent enzyme immunoassay of monoclonal anti-idiotypic antibodies against monoclonal antibodies neutralizing Semliki Forest virus and encephalomyocarditis virus.

Neutralization inhibition enzyme immunoassay (NI-EIA) was evaluated for its usefulness to detect monoclonal anti-idiotypic antibodies (ab2mAbs) against idiotypic monoclonal antibodies (ab1mAbs) neutralizing either Semliki Forest virus (SFV) or encephalomyocarditis virus (EMCV). Purified ab1mAbs were coupled to keyhole limpet hemocyanin (KLH) mixed with the adjuvant Quil A and then injected intracutaneously into homologous BALB/c mice. Successful fusions were performed 5, 6 and 7 days after intracutaneous booster immunizations of these mice. Ab2mAbs in hybridoma supernatant fluids were detected by their capacity to block virus neutralization by ab1mAbs in NI-EIA. Two stable ab2mAb producing hybridomas were obtained against SFV neutralizing mAb UM 1.13, and twelve against EMCV neutralizing mAb UM 21.1.

Polarized distribution of the viral glycoproteins of vesicular stomatitis, fowl plague and Semliki Forest viruses in hippocampal neurons in culture: a light and electron microscopy study.

We have shown previously using immunofluorescence microscopy that upon infection of polarized hippocampal cells in culture with vesicular stomatitis virus (VSV) and fowl plague virus (FPV) the VSV glycoprotein is delivered to the plasma membrane of the dendrites and of the cell body whereas the FPV hemagglutinin is transported to the axonal surface (Cell, 62 (1990) 63-72). In this work electron microscopy of infected rat hippocampal neurons showed that VSV progeny budded from the plasma membrane of the dendrites and the cell body. The location of the budding virions corresponded to the distribution of the VSV glycoprotein which was detected over the somatodendritic plasma membrane by immunoelectron microscopy. In contrast, no FPV formation was seen in the infected neurons although the FPV hemagglutinin was localized to the axonal surface by immunoelectron microscopy. In Semliki Forest virus (SFV) infected hippocampal cells we observed that the viral glycoproteins were exclusively present in the dendrites and cell body but not in axons.

An outbreak of human Semliki Forest virus infections in Central African Republic.

Semliki Forest (SF) virus was responsible for an outbreak of febrile illnesses in Bangui, Central African Republic (CAR), during October-December 1987. The virus was isolated at first from mosquitoes, mainly Aedes africanus, collected August-October in a gallery forest 100 km from Bangui. During October-December, 22 isolations of SF virus were made from serum samples collected from patients in Bangui presenting with fever, severe persistent headache, myalgia, arthralgia, and a convalescence marked by asthenia. During the same period, 8 SF virus isolates were obtained from mosquitoes collected in Bangui, mainly from Ae. aegypti. Europeans, particularly soldiers who had recently arrived from France, were affected. Antibodies to alphaviruses had been previously detected in a high proportion of resident human populations in CAR. We conclude that SF virus is a human pathogen.

The pathological effect on the central nervous system of mice following single and repeated infections of the demyelinating A7(74) strain of Semliki Forest virus.

The avirulent strain A7(74) of Semliki Forest virus was inoculated intraperitoneally into mice at weekly intervals for 7 weeks. Pathological, virological and serological studies were carried out twice weekly, after each infecting dose. Similar studies were performed on mice that had been given repeated inoculations at 2, 3 or 4 weekly intervals as were a group of control mice given a single dose of SFV. Results showed grossly enhanced central nervous system lesions, in particular the perivascular cuffing and demyelination, after the 2nd and 3rd weekly inoculation. With further injections there was no increase in severity of the lesions and by the eighth inoculation the pathological changes in the brain appeared to have recovered. The maximum and most persistent damage to the brain was seen after the 2nd and 3rd weekly inoculations. As the interval between two SFV inoculations was increased, the lesions in the central nervous system were reduced and protection increased. Virus in the blood was only detectable after the first inoculation and brain virus after the first and second inoculations. Peak IgG antibody levels were seen on day 46 after a single inoculation and day 35 in the multiple inoculations. It was concluded that repeated inoculations of SFV do not produce a relapsing demyelinating disease, but the 7th and 14th day inoculations do enhance the lesions which are seen to persist after the inoculation on the 21st day. In spite of the gross pathological changes inflicted, the brain damage appears to recover.

The effect of cycloleucine on SFV A7(74) infection in mice.

Cycloleucine (CL), a non-metabolizable amino acid analogue, was found to reduce thymus and spleen weights in Semliki Forest virus (SFV) strain A7(74) infected and control mice. The maximum effects were seen when three daily doses of CL were given to mice 24 h after an SFV A7(74) infection. In these mice thymus atrophy led to abolition of thymus dependent immune responses and changes in the pathological features of the viral infection--the most striking feature being prevention of demyelination. In addition virus titres in the brains of CL treated infected mice were increased and prolonged. These results show that demyelination following an SFV A7(74) infection is not a result of direct virus action, but of a T-cell mediated mechanism.