1 Institute of Virology, Medical University of Vienna, Kinderspitalgasse 15, A-1095 Vienna, Austria
2 Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
Correspondence
Christian W. Mandl
christian.mandl{at}univie.ac.at
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The most advanced replicon gene-expression systems are derived from members of the genus Alphavirus, family Togaviridae, i.e. Sindbis virus, Semliki Forest virus and Venezuelan equine encephalitis virus (VEEV) (Lundstrom, 2001; Perri et al., 2003
; Schlesinger, 2001
). Alphavirus expression systems take advantage of the efficient subgenomic promoter of these viruses, which normally drives the expression of the viral structural proteins. In alphavirus replicon vectors, the structural genes are replaced by the heterologous gene and this approach usually yields high expression levels of the foreign gene. Alphavirus vectors are used widely as vehicles for gene expression in cell culture and are being tested as experimental vaccines against various pathogens (Balasuriya et al., 2002
; Davis et al., 2000
; Gipson et al., 2003
; Hanke et al., 2003
; Hevey et al., 1998
; Vajdy et al., 2001
).
In contrast to alphaviruses, flaviviruses, i.e. members of the genus Flavivirus, family Flaviviridae, do not make subgenomic RNAs, but express all structural (C, prM/M and E) and non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) proteins as a single polyprotein that is co- and post-translationally cleaved into the individual proteins by the action of viral and cellular proteases (Lindenbach & Rice, 2001). Heterologous gene expression in flavivirus vectors can be achieved either by inserting the foreign gene in-frame with the viral proteins, flanked by appropriate proteolytic cleavage sites, or by adding to the genome a second cistron that is translationally controlled by an internal ribosomal entry site (IRES). Replicons derived from Kunjin virus, a mosquito-transmitted Australian flavivirus that is related closely to West Nile virus, have been developed during the past few years into a well-established and practical expression system (Varnavski & Khromykh, 1999
; Varnavski et al., 2000
). The recent advance of an efficient packaging system has further contributed to the value of Kunjin virus vectors as vehicles for in vivo applications (Harvey et al., 2004
). Heterologous genes have also been expressed from replicons of other flaviviruses, such as yellow fever virus (Molenkamp et al., 2003
), West Nile virus (Lo et al., 2003a
), dengue virus (Pang et al., 2001
) and tick-borne encephalitis virus (Hayasaka et al., 2004
). One study has highlighted the potential of such replicons not only for vaccination purposes, but also as screening systems for the development of antiviral substances (Lo et al., 2003b
). The infectious-vector approach, on the other hand, has only in a single case been used to express foreign genes from a flavivirus, i.e. Japanese encephalitis virus (Yun et al., 2003
).
In this study, we demonstrate expression of a reporter gene (enhanced green fluorescent protein, EGFP) by both infectious and replicon vectors derived from tick-borne encephalitis virus (TBEV), a tick-transmitted flavivirus that is endemic in many regions of Europe and Asia. Based on our studies with TBEV, we provide a direct comparison of the infectious-virus and replicon approaches with regard to the level and duration of heterologous gene expression. Moreover, we compare this flavivirus system with an established alphavirus replicon vector derived from VEEV (Balasuriya et al., 2000; Pushko et al., 1997
). Quantitative evaluation reveals that heterologous gene expression from the TBEV replicon reaches its maximum level later and remains at least 10-fold lower than that from the VEEV vector. In contrast to the VEEV vector, however, which destroys its host cell within a few days, expression by the TBEV vector is maintained at a significant level over a long time period.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmid pVR21-EGFP contains cDNA corresponding to a replicon derived from the alphavirus VEEV expressing EGFP under the control of the subgenomic promoter, as described originally by Pushko et al. (1997) with modifications described by Balasuriya et al. (2000)
. As is the case with the TBEV-specific plasmids, pVR21 contains bacterial plasmid sequences derived from pBR322 and a functional T7 promoter immediately upstream of the VEEV 5' non-coding region.
Plasmid pIRES2-EGFP was purchased from Clontech and contains the EGFP gene under the control of an encephalomyocarditis virus IRES sequence.
Manipulations of DNA and RNA.
The construction of EGFP-expressing TBEV vectors was performed in three steps. First, the variable region of the 3' non-coding region in plasmid pTNd/3' was replaced by an artificial multiple-cloning site (MCS), yielding plasmid pTNd/3'-MCS. This was achieved by taking advantage of unique cleavage sites for the restriction enzymes BssHII (at genome position 9880 in the NS5 gene) and AgeI (located at position 10796, exactly at the boundary between the variable and core regions of the 3' non-coding region). The BssHIIAgeI fragment was replaced by a PCR-generated fragment extending from the BssHII site to the stop codon terminating the long open reading frame (ORF; position numbers 1037510377), followed by artificial recognition sites for the enzymes SacII, AflII, NotI and AgeI. The primer sequences for this PCR were 5'-CCCTGGTGGTGCCGTGCCGA-3' (corresponding to TBEV genome positions 98369855) and 5'-TTACCGGTGCGGCCGCTTAAGCCGCGGTTAGATTATTGAGCTCTCCA-3' (nucleotides complementary to genome positions 1035810377 are shown in italics; the antisense stop codon is shown in bold; recognition sites for the restriction enzymes AgeI, NotI, AflII and SacII are underlined). In a second cloning step, an IRESEGFP cassette obtained by SacII and NotI digestion of plasmid pIRES2-EGFP (Clontech) was introduced into the MCS of plasmid pTNd/3'-MCS via the corresponding restriction sites, yielding plasmid pTNd/3'-EGFP. Finally, the BssHII (9880)NheI (11141) fragment from pTNd/3'-EGFP was recovered and used to replace the corresponding fragments of plasmids pTNd/c or pTNd/ME to yield the final plasmids pTNd/c-EGFP and pTNd/
ME-EGFP, respectively.
All plasmids were amplified in Escherichia coli HB101 and purified with commercially available systems (Qiagen). Sequence analysis of all PCR-derived fragments and surrounding recognition sites for restriction enzymes was performed in both orientations with an automated DNA sequencing system (Applied Biosystems) to confirm the presence of the desired sequences.
RNA was transcribed from 1 µg aliquots of plasmids pTNd/c-EGFP, pTNd/ME-EGFP and pVR-21-EGFP by T7 polymerase transcription, using commercially available reagents (Ambion) and conditions described in detail elsewhere (Mandl et al., 1997
). RNA was introduced into BHK-21 cells by electroporation with a Bio-Rad Gene Pulser, as described previously (Gehrke et al., 2003
; Mandl et al., 1997
).
Cell culture.
BHK-21 cells were grown in minimal essential medium supplemented with 5 % fetal calf serum (FCS). After RNA transfection, cells were seeded into six-well cluster plates and analysed for the expression of EGFP either by fluorescence microscopy or fluorescence-activated cell-sorting (FACS) analysis. To monitor the stability of EGFP expression from replicons, cells were grown to confluence and split at a ratio of 1 : 5 every 4 days. To analyse EGFP-expressing virus, supernatants were cleared from insoluble material by low-speed centrifugation and 1 ml aliquots were transferred to fresh cells every 4 days.
FACS analysis.
EGFP expression was measured with a FACScalibur flow cytometer (Becton Dickinson; 15 mW argon laser, 488 nm) with a 530/30 bandpass filter (FL-1) analysing 10 000 events per sample. For FACS analysis, BHK-21 cells were detached with trypsin from six-well cluster plates after rinsing them once with PBS and resuspended in 1 ml PBS supplemented with 5 % FCS. Measurement of forward scatter versus side scatter was performed routinely to exclude alterations of cell sizes and to confirm viability of the cells. Commercially available BD FACS EGFP calibration beads (BD Biosciences, Clontech) yielding standardized fluorescence intensities were used as internal controls along with each dataset. FL-1 geometric mean values were calculated by using CellQuestPro Software (Becton Dickinson). For quantitative comparison of expression levels, mean values were calculated for the 5 % of the cell population that exhibited the brightest EGFP fluorescence. Calculations including larger percentages of cells yielded lower absolute numbers, but did not change the proportions between the individual samples. For comparison of the VEEV replicon with the TBEV vectors, the instrument settings were adjusted to keep the bright fluorescence obtained from the VEEV vector within the measurable range of the flow cytometer. In all other experiments, untransfected cells were used as a reference for instrument settings. To determine the percentage of EGFP-expressing cells in a cell population, the cut-off value was set to exclude at least 99 % of the untransfected control cells.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Quantitative comparison of EGFP expression
To compare the efficiency of reporter-gene expression between the infectious and replicon TBEV vectors and the VEEV replicon vector, BHK-21 cells transfected with the corresponding RNAs were subjected to a standardized FACS analysis based on the quantitative determination of EGFP fluorescence for a constant number of cells. Fig. 3 shows results obtained at days 1, 2 and 3 post-transfection. Consistent with the visual intensity shown in Fig. 2
, the highest expression level was achieved with the VEEV replicon. In comparison, EGFP expression by the TBEV vectors was significantly lower and exhibited a delayed onset, with an approximately 10-fold increase of mean fluorescence values from days 1 to 2. On day 1, the difference between the VEEV replicon and the TBEV replicon was 175-fold, whereas on days 2 and 3, the differences in expression were not as large, amounting to only 24- and ninefold, respectively. Infectious TBEV produced between two- and eightfold less EGFP than the TBEV replicon at all times but, again, the biggest difference was observed on the first day. A comparison of the two TBEV vectors over an extended time period is described below. A quantitative evaluation of the VEEV expression level at later time points was not possible because of the rapid destruction of cells by CPE and the resulting disappearance of expressing cells from the total cell population, as analysed in more detail in the following section.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we constructed and evaluated infectious and non-infectious expression vectors from TBEV, a tick-borne flavivirus that differs from its mosquito-transmitted relatives in a number of molecular and biological properties (Lindenbach & Rice, 2001; Mandl et al., 1993
). We previously described the generation of subgenomic replicons of TBEV and demonstrated that a TBEV replicon lacking the proteins prM/M and E could be packaged into VLPs by trans-complementation using a cell line (CHO-ME cells) that expressed these two surface proteins constitutively (Gehrke et al., 2003
). By using a different strain of TBEV (belonging to the Far Eastern subtype of TBEV), Hayasaka et al. (2004)
recently also produced replicons and demonstrated expression of heterologous genes under the control of an IRES element inserted into the 3' non-coding region. Four days post-transfection, EGFP expression could be demonstrated in this system in <5 % of cells. Another replicon expressing a selectable marker protein (neomycin phosphotransferase) could be maintained under selection pressure in transfected cells for at least 50 days. In good agreement with our observations, the replicons derived from the Far Eastern subtype TBEV strain induced no apparent CPE in BHK-21 cells. Our study extends the evaluation of TBEV as a potential new expression vector by providing a quantitative comparison of the level and endurance of heterologous gene (EGFP) expression in the absence of a selectable marker in individual cells, achieved from infectious or non-infectious TBEV vectors, with expression of the same gene from an established non-infectious alphavirus vector.
The growing number of expression vectors calls for a direct comparison of their properties to enable a rational choice of the most appropriate vector for each particular application. Therefore, we performed a direct comparison between a well-established VEEV replicon and the newly developed TBEV replicon. From these results, it is clear that the alphavirus replicon used in this study is superior with respect to short-term expression, yielding a significantly higher expression level within the first 3 days (the difference decreased from 180-fold at day 1 to 10-fold at day 3). However, with respect to long-term expression, the TBEV-based system has the advantage that host cells carrying this replicon were able to survive and maintain expression for almost 4 weeks, and our data suggest that even longer expression times can be achieved even in the absence of a selection marker. Cells carrying the replicon underwent cell division at a rate similar to that of untransfected cells and distributed the replicon to their daughter cells. This indicates that the flavivirus causes little disturbance of fundamental host-cell functions. Expression levels were found to decline moderately at a steady rate, possibly reflecting inactivation of the IRESEGFP cassette by randomly occurring mutations. The alphavirus replicon would thus be the vector of choice for achieving a transiently high level of expression, whereas the TBEV vector might be considered for applications where long-term expression and prolonged survival of the host cells are required. Sustained expression, however, has also been achieved with modified, non-cytopathic alphavirus replicons (Agapov et al., 1998; Frolov et al., 1999
; Kong et al., 2002
; Perri et al., 2000
). Studies on flavivirus replicons derived from Kunjin virus have shown that insertion of an IRESheterologous gene cassette into the 3' non-coding region reduces replication efficiency of the replicon RNA (Khromykh & Westaway, 1997
). Moreover, expression of heterologous genes from the Kunjin viral promoter by replacing the genes encoding the structural proteins with the foreign gene yielded expression levels that were comparable to those obtained with two alphavirus systems, derived from Sindbis virus and Semliki Forest virus (Varnavski & Khromykh, 1999
; Varnavski et al., 2000
). Thus, it may also be possible to construct TBEV-derived vectors with higher expression efficiencies than those described in this study. Clearly, a larger number of comparative studies among the various vector systems will be necessary to provide a rational basis for choosing the optimal system for a particular application.
The infectious TBEV expressing EGFP was genetically stable for a few cell-culture passages. This finding demonstrated that substitution of the variable region of the 3' non-coding region by a foreign genetic element, which is more than three times larger and increases the total genome length by approximately 8 %, is still compatible with replication and packaging of the genome. Interestingly, the expression level achieved by infectious virus was somewhat lower than that observed with the replicon and increased more slowly to its maximum value. This suggests that the replicon RNA had possibly multiplied faster and to higher copy numbers within the cell than the full-length genome. In contrast to the replicon, expression by the infectious virus did not decline at a constant rate but, after a period of steady decline, expression vanished rapidly. We hypothesize that mutants carrying spontaneous deletions within the IRESEGFP cassette may have emerged and, due to somewhat better replication kinetics and/or packaging efficiency, outgrew the original vector virus. In contrast, similar mutations may also have occurred in the replicon but, as these could not spread to other cells, this had little effect on the overall EGFP-expression level in the total cell population. This leads to the conclusion that the stability of expression with the infectious TBEV vector depends on both the mutation rate and the impact that the insert has on the evolutionary fitness of the vector. In contrast, with the replicon vector, the stability of genes that do not provide a selective advantage or disadvantage for the host cell is determined mainly by the RNA-mutation rate of the viral polymerase. Recent work with yellow fever chimeric virus suggests that the flavivirus RNA polymerase exhibits a relatively high fidelity, compared with those of other RNA viruses (Pugachev et al., 2004); this may turn out to be a relevant advantage of flavivirus RNA-expression systems.
The TBEV expression system described in this study provides a tool for introducing heterologous genes into a wide variety of cells that support replication of this virus. In particular, the tropism of TBEV for ticks and tick-derived cell lines (Lawrie et al., 2004) can make this expression system a valuable tool for studying these arthropod vectors that, in addition to TBEV, also transmit other relevant human diseases, such as Lyme disease. Due to its inherent pathogenicity, the use of infectious TBEV vectors will certainly be restricted in terms of clinical applications, but will nevertheless be useful for in vitro and in vivo studies on assembly, tropism and pathogenesis of this flavivirus, as well as for the development of new antiviral agents.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anraku, I., Harvey, T. J., Linedale, R., Gardner, J., Harrich, D., Suhrbier, A. & Khromykh, A. A. (2002). Kunjin virus replicon vaccine vectors induce protective CD8+ T-cell immunity. J Virol 76, 37913799.
Balasuriya, U. B. R., Heidner, H. W., Hedges, J. F., Williams, J. C., Davis, N. L., Johnston, R. E. & MacLachlan, N. J. (2000). Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles. J Virol 74, 1062310630.
Balasuriya, U. B. R., Heidner, H. W., Davis, N. L. & 8 other authors (2002). Alphavirus replicon particles expressing the two major envelope proteins of equine arteritis virus induce high level protection against challenge with virulent virus in vaccinated horses. Vaccine 20, 16091617.[CrossRef][Medline]
Bredenbeek, P. J., Frolov, I., Rice, C. M. & Schlesinger, S. (1993). Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs. J Virol 67, 64396446.[Abstract]
Daemen, T., Riezebos-Brilman, A., Bungener, L., Regts, J., Dontje, B. & Wilschut, J. (2003). Eradication of established HPV16-transformed tumours after immunisation with recombinant Semliki Forest virus expressing a fusion protein of E6 and E7. Vaccine 21, 10821088.[CrossRef][Medline]
Davis, N. L., Caley, I. J., Brown, K. W. & 9 other authors (2000). Vaccination of macaques against pathogenic simian immunodeficiency virus with Venezuelan equine encephalitis virus replicon particles. J Virol 74, 371378.
Dufresne, A. T., Dobrikova, E. Y., Schmidt, S. & Gromeier, M. (2002). Genetically stable picornavirus expression vectors with recombinant internal ribosomal entry sites. J Virol 76, 89668972.
Frolov, I. & Schlesinger, S. (1994). Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells. J Virol 68, 17211727.[Abstract]
Frolov, I., Agapov, E., Hoffman, T. A., Jr, Prágai, B. M., Lippa, M., Schlesinger, S. & Rice, C. M. (1999). Selection of RNA replicons capable of persistent noncytopathic replication in mammalian cells. J Virol 73, 38543865.
Gehrke, R., Ecker, M., Aberle, S. W., Allison, S. L., Heinz, F. X. & Mandl, C. W. (2003). Incorporation of tick-borne encephalitis virus replicons into virus-like particles by a packaging cell line. J Virol 77, 89248933.
Gipson, C. L., Davis, N. L., Johnston, R. E. & de Silva, A. M. (2003). Evaluation of Venezuelan equine encephalitis (VEE) replicon-based outer surface protein A (OspA) vaccines in a tick challenge mouse model of Lyme disease. Vaccine 21, 38753884.[CrossRef][Medline]
Hall, R. A., Nisbet, D. J., Pham, K. B., Pyke, A. T., Smith, G. A. & Khromykh, A. A. (2003). DNA vaccine coding for the full-length infectious Kunjin virus RNA protects mice against the New York strain of West Nile virus. Proc Natl Acad Sci U S A 100, 1046010464.
Hanke, T., Barnfield, C., Wee, E. G.-T., Ågren, L., Samuel, R. V., Larke, N. & Liljeström, P. (2003). Construction and immunogenicity in a primeboost regimen of a Semliki Forest virus-vectored experimental HIV clade A vaccine. J Gen Virol 84, 361368.
Harvey, T. J., Anraku, I., Linedale, R., Harrich, D., Mackenzie, J., Suhrbier, A. & Khromykh, A. A. (2003). Kunjin virus replicon vectors for human immunodeficiency virus vaccine development. J Virol 77, 77967803.
Harvey, T. J., Liu, W. J., Wang, X. J. & 8 other authors (2004). Tetracycline-inducible packaging cell line for production of flavivirus replicon particles. J Virol 78, 531538.
Hayasaka, D., Yoshii, K., Ueki, T., Iwasaki, T. & Takashima, I. (2004). Sub-genomic replicons of Tick-borne encephalitis virus. Arch Virol 149, 12451256.[CrossRef][Medline]
Hevey, M., Negley, D., Pushko, P., Smith, J. & Schmaljohn, A. (1998). Marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates. Virology 251, 2837.[CrossRef][Medline]
Hewson, R. (2000). RNA viruses: emerging vectors for vaccination and gene therapy. Mol Med Today 6, 2835.[CrossRef][Medline]
Jia, Q., Liang, F., Ohka, S., Nomoto, A. & Hashikawa, T. (2002). Expression of brain-derived neurotrophic factor in the central nervous system of mice using a poliovirus-based vector. J Neurovirol 8, 1423.[Medline]
Khromykh, A. A. (2000). Replicon-based vectors of positive strand RNA viruses. Curr Opin Mol Ther 2, 555569.[Medline]
Khromykh, A. A. & Westaway, E. G. (1997). Subgenomic replicons of the flavivirus Kunjin: construction and applications. J Virol 71, 14971505.[Abstract]
Kong, W., Tian, C., Liu, B. & Yu, X.-F. (2002). Stable expression of primary human immunodeficiency virus type 1 structural gene products by use of a noncytopathic Sindbis virus vector. J Virol 76, 1143411439.
Lawrie, C. H., Uzcátegui, N. Y., Armesto, M., Bell-Sakyi, L. & Gould, E. A. (2004). Susceptibility of mosquito and tick cell lines to infection with various flaviviruses. Med Vet Entomol 18, 268274.[CrossRef][Medline]
Liljeström, P. & Garoff, H. (1991). A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 9, 13561361.[Medline]
Lindenbach, B. D. & Rice, C. M. (2001). Flaviviridae: the viruses and their replication. In Fields Virology, 4th edn, pp. 9911041. Edited by D. M. Knipe & P. M. Howley. Philadelphia, PA: Lippincott Williams & Wilkins.
Lo, M. K., Tilgner, M., Bernard, K. A. & Shi, P.-Y. (2003a). Functional analysis of mosquito-borne flavivirus conserved sequence elements within 3' untranslated region of West Nile virus by use of a reporting replicon that differentiates between viral translation and RNA replication. J Virol 77, 1000410014.
Lo, M. K., Tilgner, M. & Shi, P.-Y. (2003b). Potential high-throughput assay for screening inhibitors of West Nile virus replication. J Virol 77, 1290112906.
Lundstrom, K. (2001). Alphavirus vectors for gene therapy applications. Curr Gene Ther 1, 1929.[Medline]
Lundstrom, K. (2003). Alphavirus vectors for vaccine production and gene therapy. Expert Rev Vaccines 2, 445459.[CrossRef]
Mandl, C. W., Holzmann, H., Kunz, C. & Heinz, F. X. (1993). Complete genomic sequence of Powassan virus: evaluation of genetic elements in tick-borne versus mosquito-borne flaviviruses. Virology 194, 173184.[CrossRef][Medline]
Mandl, C. W., Ecker, M., Holzmann, H., Kunz, C. & Heinz, F. X. (1997). Infectious cDNA clones of tick-borne encephalitis virus European subtype prototypic strain Neudoerfl and high virulence strain Hypr. J Gen Virol 78, 10491057.[Abstract]
Mandl, C. W., Holzmann, H., Meixner, T., Rauscher, S., Stadler, P. F., Allison, S. L. & Heinz, F. X. (1998). Spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus. J Virol 72, 21322140.
Molenkamp, R., Kooi, E. A., Lucassen, M. A., Greve, S., Thijssen, J. C. P., Spaan, W. J. M. & Bredenbeek, P. J. (2003). Yellow fever virus replicons as an expression system for hepatitis C virus structural proteins. J Virol 77, 16441648.[CrossRef][Medline]
Pang, X., Zhang, M. & Dayton, A. I. (2001). Development of Dengue virus type 2 replicons capable of prolonged expression in host cells. BMC Microbiol 1, 18.[CrossRef][Medline]
Perri, S., Driver, D. A., Gardner, J. P., Sherrill, S., Belli, B. A., Dubensky, T. W., Jr & Polo, J. M. (2000). Replicon vectors derived from Sindbis virus and Semliki Forest virus that establish persistent replication in host cells. J Virol 74, 98029807.
Perri, S., Greer, C. E., Thudium, K. & 10 other authors (2003). An alphavirus replicon particle chimera derived from Venezuelan equine encephalitis and Sindbis viruses is a potent gene-based vaccine delivery vector. J Virol 77, 1039410403.
Proutski, V., Gaunt, M. W., Gould, E. A. & Holmes, E. C. (1997). Secondary structure of the 3'-untranslated region of yellow fever virus: implications for virulence, attenuation and vaccine development. J Gen Virol 78, 15431549.[Abstract]
Pugachev, K. V., Guirakhoo, F., Ocran, S. W. & 8 other authors (2004). High fidelity of yellow fever virus RNA polymerase. J Virol 78, 10321038.
Pushko, P., Parker, M., Ludwig, G. V., Davis, N. L., Johnston, R. E. & Smith, J. F. (1997). Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo. Virology 239, 389401.[CrossRef][Medline]
Rauscher, S., Flamm, C., Mandl, C. W., Heinz, F. X. & Stadler, P. F. (1997). Secondary structure of the 3'-noncoding region of flavivirus genomes: comparative analysis of base pairing probabilities. RNA 3, 779791.[Abstract]
Schlesinger, S. (2001). Alphavirus vectors: development and potential therapeutic applications. Expert Opin Biol Ther 1, 177191.[Medline]
Uhlirova, M., Foy, B. D., Beaty, B. J., Olson, K. E., Riddiford, L. M. & Jindra, M. (2003). Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis. Proc Natl Acad Sci U S A 100, 1560715612.
Vajdy, M., Gardner, J., Neidleman, J., Cuadra, L., Greer, C., Perri, S., O'Hagan, D. & Polo, J. M. (2001). Human immunodeficiency virus type 1 Gag-specific vaginal immunity and protection after local immunizations with Sindbis virus-based replicon particles. J Infect Dis 184, 16131616.[CrossRef][Medline]
Varnavski, A. N. & Khromykh, A. A. (1999). Noncytopathic flavivirus replicon RNA-based system for expression and delivery of heterologous genes. Virology 255, 366375.[CrossRef][Medline]
Varnavski, A. N., Young, P. R. & Khromykh, A. A. (2000). Stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic DNA-based Kunjin virus replicon vectors. J Virol 74, 43944403.
Wallner, G., Mandl, C. W., Kunz, C. & Heinz, F. X. (1995). The flavivirus 3'-noncoding region: extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virus. Virology 213, 169178.[CrossRef][Medline]
Westaway, E. G., Mackenzie, J. M. & Khromykh, A. A. (2003). Kunjin RNA replication and applications of Kunjin replicons. Adv Virus Res 59, 99140.[CrossRef][Medline]
Yun, S.-I., Kim, S.-Y., Rice, C. M. & Lee, Y.-M. (2003). Development and application of a reverse genetics system for Japanese encephalitis virus. J Virol 77, 64506465.
Received 7 October 2004;
accepted 21 December 2004.