yields, the quality is not always compatible with efficient production of high titer virus.
3. The length of gene inserts introduced into the expression
vector might affect the RNA yields, typically when inserts
exceed 4 kb. The RNA yields can be improved to some extent
by extension of the incubation time for in vitro transcription
reactions.
4. In comparison of the GFP and β-gal methods for the verification of virus titers, the fluorescence-based GFP approach is
easier to carry out as no fixing or staining of cells is required.
It also allows one to follow the duration of expression. The
drawback is the requirement of a fluorescence microscope.
5. Visualization of radioactively labeled proteins via SDS-PAGE
can efficiently be enhanced by application of film cassettes with
double-sided X-ray intensifying screens [44].
6. Due to the cytopathic effects caused by alphaviruses on host
cells, the time of host cell survival and transgene expression
levels might be reduced. To address these problems, several
mutant SFV [40] and SIN [41] vectors have been engineered.
Increased expression has also been achieved by applying alphavirus vectors with’ translation enhancement signals [42].
References
1. Strauss J, Strauss E (1994) The alphaviruses;
gene expression, replication and evolution.
Microbiol Rev 58:491–562
2. Liljestrom P, Garoff H (1991) A new generation of animal cell expression vectors based on
the Semliki Forest virus replicon. Bio/Technology 9:1356–1361
3. Xiong C, Levis R, Shen P, Schlesinger S, Rice
CM, Huang HV (1989) Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science 243:1188–1191
4. Davies NL, Brown KW, Johnston RE (1989)
In vitro synthesis of infectious Venezuelan
equine encephalitis virus RNA from a cDNA
clone: analysis of a viable deletion mutant. Virologie 171:189–204
5. Lundstrom K (2014) Alphavirus-based vaccines. Viruses 6:2392–2415
6. Schultz-Cherry S, Dybing JK, Davis NL,
Williamson C, Suarez DL, Johnston R et al
(2000) Influenza virus (A/HK/156/97) hemagglutinin expressed by an alphavirus replicon
system protects against lethal infection with
Hong Kong-origin H5N1 viruses. Virology
278:55–59
7. Fleeton MN, Chen M, Berglund P, Rhodes G,
Parker SE, Murphy M et al (2001) Selfreplicative RNA vaccines elicit protection
against influenza A virus, respiratory syncytial
virus, and a tickborne encephalitis virus. J
Infect Dis 183:1395–1398
8. Wang M, Jokinen J, Tretyakova I, Pushko P,
Luikashevich IS (2018) Alphavirus vectorbased replicon particles expressing multivalent
cross-protective Lassa virus glycoproteins. Vaccine 36:683–690
9. Wilson JA, Hart MK (2001) Protection from
Ebola
virus
mediated
by
cytotoxic
T-lymphocytes specific for the viral nucleoprotein. J Virol 75:2660–2664
10. Pushko P, Bray M, Ludwig GV, Parker M,
Schmaljohn A, Sanchez A et al (2000) Recombinant RNA replicons derived from attenuated
Venezuelan equine encephalitis virus protect
guinea pigs and mice from Ebola hemorrhagic
fever virus. Vaccine 19:142–153
11. Herbert AS, Kuehne AI, Barth JF, Ortiz RA,
Nichols DK, Zak SE et al (2013) Venezuelan
equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with
Ebolavirus. J Virol 87:4952–4964
12. Vogel AB, Lambert L, Kinnear E, Busse D,
Erbar S, Reuter KC et al (2018) Self-amplifying
Alphavirus-Based Antigen Preparation
79
3. The length of gene inserts introduced into the expression
vector might affect the RNA yields, typically when inserts
exceed 4 kb. The RNA yields can be improved to some extent
by extension of the incubation time for in vitro transcription
reactions.
4. In comparison of the GFP and β-gal methods for the verification of virus titers, the fluorescence-based GFP approach is
easier to carry out as no fixing or staining of cells is required.
It also allows one to follow the duration of expression. The
drawback is the requirement of a fluorescence microscope.
5. Visualization of radioactively labeled proteins via SDS-PAGE
can efficiently be enhanced by application of film cassettes with
double-sided X-ray intensifying screens [44].
6. Due to the cytopathic effects caused by alphaviruses on host
cells, the time of host cell survival and transgene expression
levels might be reduced. To address these problems, several
mutant SFV [40] and SIN [41] vectors have been engineered.
Increased expression has also been achieved by applying alphavirus vectors with’ translation enhancement signals [42].
References
1. Strauss J, Strauss E (1994) The alphaviruses;
gene expression, replication and evolution.
Microbiol Rev 58:491–562
2. Liljestrom P, Garoff H (1991) A new generation of animal cell expression vectors based on
the Semliki Forest virus replicon. Bio/Technology 9:1356–1361
3. Xiong C, Levis R, Shen P, Schlesinger S, Rice
CM, Huang HV (1989) Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science 243:1188–1191
4. Davies NL, Brown KW, Johnston RE (1989)
In vitro synthesis of infectious Venezuelan
equine encephalitis virus RNA from a cDNA
clone: analysis of a viable deletion mutant. Virologie 171:189–204
5. Lundstrom K (2014) Alphavirus-based vaccines. Viruses 6:2392–2415
6. Schultz-Cherry S, Dybing JK, Davis NL,
Williamson C, Suarez DL, Johnston R et al
(2000) Influenza virus (A/HK/156/97) hemagglutinin expressed by an alphavirus replicon
system protects against lethal infection with
Hong Kong-origin H5N1 viruses. Virology
278:55–59
7. Fleeton MN, Chen M, Berglund P, Rhodes G,
Parker SE, Murphy M et al (2001) Selfreplicative RNA vaccines elicit protection
against influenza A virus, respiratory syncytial
virus, and a tickborne encephalitis virus. J
Infect Dis 183:1395–1398
8. Wang M, Jokinen J, Tretyakova I, Pushko P,
Luikashevich IS (2018) Alphavirus vectorbased replicon particles expressing multivalent
cross-protective Lassa virus glycoproteins. Vaccine 36:683–690
9. Wilson JA, Hart MK (2001) Protection from
Ebola
virus
mediated
by
cytotoxic
T-lymphocytes specific for the viral nucleoprotein. J Virol 75:2660–2664
10. Pushko P, Bray M, Ludwig GV, Parker M,
Schmaljohn A, Sanchez A et al (2000) Recombinant RNA replicons derived from attenuated
Venezuelan equine encephalitis virus protect
guinea pigs and mice from Ebola hemorrhagic
fever virus. Vaccine 19:142–153
11. Herbert AS, Kuehne AI, Barth JF, Ortiz RA,
Nichols DK, Zak SE et al (2013) Venezuelan
equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with
Ebolavirus. J Virol 87:4952–4964
12. Vogel AB, Lambert L, Kinnear E, Busse D,
Erbar S, Reuter KC et al (2018) Self-amplifying
Alphavirus-Based Antigen Preparation
79