Download Rescue of Akabane virus (family Bunyaviridae) entirely from cloned

Journal of General Virology (2007), 88, 3385–3390
DOI 10.1099/vir.0.83173-0
Rescue of Akabane virus (family Bunyaviridae)
entirely from cloned cDNAs by using RNA
polymerase I
Yohsuke Ogawa,1,2 Keita Sugiura,1 Kentaro Kato,1 Yukinobu Tohya1
and Hiroomi Akashi1
Correspondence
1
Hiroomi Akashi
[email protected]
2
Received 21 May 2007
Accepted 28 July 2007
Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
National Institute of Animal Health, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-0856, Japan
Reverse-genetic systems are often used to study different aspects of the viral life cycle. To date,
three rescue systems have been developed for the family Bunyaviridae. These systems use T7
RNA polymerase, which is generally used in rescue systems for Mononegavirales. In the present
study, we describe a rescue system for Akabane virus (family Bunyaviridae) that uses cDNAs
and RNA polymerase I instead of T7 RNA polymerase. The utility of this system was demonstrated
by the generation of a mutant with a deletion of the non-structural protein (NSs) on the S RNA
segment. These results offer a new option for bunyavirus rescue.
INTRODUCTION
Reverse-genetic systems are often used to study different
aspects of the viral life cycle. In the family Bunyaviridae,
three rescue systems have been developed to date, for
Bunyamwera virus (BUNV), La Crosse virus and Rift
Valley fever virus (Bridgen & Elliott, 1996; Blakqori &
Weber, 2005; Ikegami et al., 2006). These rescue systems
use bacteriophage T7 RNA polymerase because this enzyme
localizes to the cytoplasm, which is the site of bunyavirus
replication. Similarly, negative-stranded RNA viruses
mainly replicate in the cytoplasm. Thus, most rescue
systems for Mononegavirales employ the T7 RNA polymerase-driven system. On the other hand, since influenza
virus replicates in the nucleus, the rescue system for
influenza virus uses cellular RNA polymerase I, which
transcribes rRNA that lacks both the 59 cap and 39 poly(A)
tail (Neumann et al., 1999). Although RNA polymerase I
localizes to the nucleus, transcripts generated by this
enzyme are exported from the nucleus to the cytoplasm
(Flick & Pettersson, 2001). Furthermore, these transcripts
are packaged into helper viruses and virus-like particles
(Flick & Pettersson, 2001; Blakqori et al., 2003; Overby
et al., 2006). Thus, these reports suggest that RNA
polymerase I may be useful in bunyavirus rescue systems.
Bunyaviruses are enveloped and are characterized by a
tripartite negative-strand RNA genome, the components of
which are designated S (small), M (medium) and L (large).
More than 300, mainly arthropod-borne, viruses are
contained in this family, which is subdivided into five
genera: Orthobunyavirus, Nairovirus, Phlebovirus, Hantavirus and Tospovirus (Schmaljohn & Hooper, 2001).
Genetic studies of orthobunyaviruses have shown that the
0008-3173 G 2007 SGM
S RNA segment codes for a nucleoprotein (N) and a nonstructural protein (NSs). N shares antigenic determinants
with some other species in the genus. NSs acts as an alpha/
beta interferon (IFN) antagonist and contributes to the
regulation of host protein synthesis (Weber et al., 2002).
The M RNA segment encodes two envelope glycoproteins
(Gn and Gc) and a non-structural protein (NSm). The
glycoproteins are responsible for viral neutralization and
attachment to cell receptors, while the function of NSm
remains unknown. Gc is a major neutralizing antigen. The
L RNA segment encodes an RNA-dependent RNA polymerase (L). With the exception of the genus Tospovirus,
viruses in each of the other genera infect mammalian
species and cause significant human and animal diseases.
Akabane virus (AKAV) is a member of the genus
Orthobunyavirus, family Bunyaviridae and was originally
isolated from mosquitoes, Aedes vexans and Culex
tritaneniorhynchus, in the central part of Japan in 1959
(Oya et al., 1961). The virus is the causative agent of
arthrogryposis–hydranencephaly syndrome in cattle, sheep
and goats, which is characterized by abortion, stillbirth,
premature birth and congenital deformities, and causes
considerable economic losses to the cattle industry (Inaba
& Matumoto, 1990). The virus is transmitted primarily by
biting midges of the genus Culicoides, and is widely
distributed throughout Australia, South-east Asia, East
Asia, the Middle East and Africa.
Recent studies have demonstrated that there are considerable antigenic and genetic variations among field isolates
of AKAV (Miyazato et al., 1989; Akashi & Inaba, 1997;
Akashi et al., 1997a, b; Ogawa et al., 2007a). However,
biological studies of AKAV as well as virus pathogenesis
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Y. Ogawa and others
studies have so far been limited. It is essential for research
into these areas to establish a reverse-genetic system. In the
present report, we describe a rescue system for AKAV that
uses RNA polymerase I instead of T7 RNA polymerase.
METHODS
Viruses and cells. The plaque-cloned OBE-1 strain of AKAV
(Akashi et al., 1997a) was used throughout this study. Hamster lung
cells (HmLu-1) and African green monkey kidney cells (Vero) were
cultivated in Dulbecco’s modified Eagle’s medium (DMEM) that was
supplemented with 5 % fetal bovine serum (FBS) at 37 uC. Baby
hamster kidney cells (BHK-21) were maintained in Eagle’s minimum
essential medium containing 5 % FBS. Medium without serum was
used for viral propagation.
Plasmid construction. The pcDNA/L and pcDNA/SDNSs plasmids
express the L and N of AKAV, respectively, under the control of the
CMV promoter (Ogawa et al., 2007b). The pcDNA/M plasmid, which
expresses the viral glycoproteins, was generated using a method
similar to that employed for pcDNA/L. Briefly, the full-length M RNA
segment was amplified by PCR using KOD Plus (Toyobo) and the
phosphorylated primers RF42-T-MF (59-aatCGTCTCgggggAGTAGTGAACTACCACAACAAAATG-39) and RF42-P-MR (59-aatCGTCTCtaggtAGTAGTGTTCTACCACAACAAATAATTAT-39) (the
restriction sites are underlined, the AKAV sequences are capitalized
and italicized). The PCR product was cloned into the mammalian
expression vector pcDNA3.1/myc-His version A (Invitrogen).
The pRF42/L, pRF42/M, pRF42/S and pRF42/SDNSs plasmids exhibit
RNA polymerase I-driven transcription of the respective genomic
RNAs. To generate pRF42/L and pRF42/M, pcDNA/L and pcDNA/M
were digested with Esp3I and cloned between the murine RNA
polymerase I promoter and the terminator of the pRF42 vector (Flick
& Pettersson, 2001). The S RNA segment was generated using the
primers RF42_T_SF (59-aatCGTCTCgggggAGTAGTGAACT-39) and
RF42_P_SR (59-aatCGTCTCtaggtAGTAGTGTTCT-39), and the amplified fragment was inserted into pRF42 using the Esp3I and BpiI sites,
to generate pRF42/S. In order to abrogate NSs expression in pRF42/S,
the ATG start codon (nt 59–61 of the S cRNA) was changed to ACG
by primer mutagenesis (Fig. 2a). This alteration does not affect the N
amino acid sequence. Briefly, two PCR products that contained
primer-encoded alterations in overlapping regions were generated
using the primer pairs RF42_T_ SF plus SdelNSs-R (59TCCGTTGTGGAACgTCGTTG-39) and RF42_P_ SR plus SdelNSs-F
(59-CAACGAcGTTCCACAACGGA-39). Both fragments served as
templates in a second round of PCR with the RF42_T_SF and
RF42_P_SR primers, to give the full-length N-coding region, which
was then cloned into the Esp3I and BpiI sites of pRF42, thereby
generating pRF42/SDNSs.
Rescue of recombinant AKAV. A subconfluent monolayer of
HmLu-1 cells was transfected with 0.5 mg pRF42/S or pRF42/SDNSs,
1.5 mg pRF42/M, 2 mg pRF42/L, 0.1 mg pcDNA/SDNSs and 0.4 mg
pcDNA/L, with or without 0.15 mg pcDNA/M, using TransIT-LT1
(2 ml mg21 DNA; Mirus) in 100 ml serum-free medium (OPTI-MEM;
Invitrogen). If no cytopathic effect (CPE) was observed, supernatants
were collected 5 days after transfection and aliquots were transferred
to fresh HmLu-1 cells, to monitor the development of CPE.
Growth kinetics. Subconfluent monolayers of HmLu-1, BHK-21 and
Vero cells were infected with the wild-type or recombinant virus at an
m.o.i. of 0.01 for 1 h at 37 uC. Uninfected virus was removed by
aspiration, and the infected cells were washed with PBS and overlaid
with serum-free medium. At different time points post-infection, the
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supernatants were harvested. The p.f.u. were determined in HmLu-1
cells. The experiments were performed in triplicate.
RT-PCR analysis. Viral RNA was extracted from the supernatant of
infected cell cultures using the QIAamp Viral RNA mini kit (Qiagen),
according to the manufacturer’s instructions. First-strand cDNA was
synthesized using the primer AKAcon (59-gggGCTCTAGAAGTAGTG-39). The S RNA segment was amplified by PCR using Ex Taq
(Takara Bio) with the primers SF (59-AGTAGTGAACTCCACTATTAACTACGC-39) and NSs_C_XhoI (59-ggggCTCGAGCTAAGTAGCCCGA-39). The amplicons were digested with HpyCH4IV.
The whole genomes of the rescued viruses were confirmed by direct
sequencing of the PCR products. The three segments of the
recombinant viruses were amplified by RT-PCR using the primer
set: RF42_T_SF plus RF42_P_SR for the S RNA segment, RF42_T_MF
plus RF42_P_MR for the M RNA segment, and RF42_T_LF
(59-aatCGTCTCgggggAGTAGTGTACCCCTAAATACAACATACA-39)
plus RF42_P_LR (59-aatCGTCTCtaggtAGTAGTGTGCCCCTAAATGCAATAATAT-39) for the L RNA segment. The sequencing reaction
was resolved on an ABI Prism 3100 automated sequencer (Applied
Biosystems).
Plaque morphology and size. To examine differences in plaque
morphology between the wild-type and recombinant viruses, HmLu1 or Vero cells were seeded into six-well plates. After virus adsorption
to HmLu-1 or Vero cells at 37 uC for 1 h, the inocula were removed
and the infected cells were washed with PBS and overlaid with DMEM
that contained 0.8 % agar and 2.5 % FBS. Three days later, the cells
were stained with neutral red. Fifty plaques were randomly selected
and sized using the NIH Image software. Statistical significance was
assessed by the Student’s t-test.
Experimental infections. Three groups of 12–14 one-day-old
BALB/c mice were inoculated intracerebrally with 0.02 ml wild-type
or recombinant viruses (TCID50, 105.25 0.1 ml21) and observed for
mortality over a period of 21 days. Mice that died within 1 day of
inoculation were not included in the results of these experiments. The
surviving mice were sacrificed 21 days after inoculation and tested for
the presence of antibodies against AKAV, to confirm that the animals
were infected with viruses. The animal experiments were conducted
according to the guidelines of the Animal Care Committee of the
University of Tokyo.
RESULTS
Rescue of AKAV from cDNAs using RNA
polymerase I
Although RNA polymerase I localizes to the cell nucleus,
transcripts generated by this enzyme are exported from the
nucleus to the cytoplasm, which is the site of bunyavirus
replication (Flick & Pettersson, 2001). Therefore, we
postulated that a rescue system for bunyaviruses could be
devised using RNA polymerase I. Essentially, we followed
the strategies described previously by Flick & Petterson
(2001) for constructing a bunyavirus minigenome system,
and that described previously by Neumann et al. (1999) for a
rescue system for influenza virus. To rescue AKAV from
cDNAs, plasmids that encode AKAV proteins (pcDNA/L,
pcDNA/SDNSs and pcDNA/M) and negative-sense viral
RNAs (pRF42/L, pRF42/M and pRF42/S) were constructed
(see Methods). Since the NSs of AKAV inhibits minireplicon
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Journal of General Virology 88
AKAV reverse genetics
activity, we used pcDNA/SDNSs, which expresses only N
(instead of pcDNA/S, which expresses both N and NSs;
Ogawa et al., 2007b). To recover infectious viruses,
HmLu-1 cells were transfected with pRF42/L, pRF42/M,
pRF42/S, pcDNA/L and pcDNA/SDNSs, with or without
pcDNA/M. At 4–5 days post-transfection, CPE was
observed regardless of the existence of the M polyprotein-expressing plasmid. This resulted in the recovery of
infectious AKAV (rAKAV) entirely from the cDNAs.
To characterize the rescue of AKAV, we measured the
development of virus titres (Fig. 1a–c). Although the titre
of rAKAV was slightly lower than that of the wild-type
virus at 12 h post-infection in HmLu-1 cells, the growth
kinetics of these viruses were similar (Fig. 1a). Similar
growth curves for these viruses were observed in BHK-21
(Fig. 1b) and Vero cells (Fig. 1c), although the virus titres
were generally lower in Vero cells. Furthermore, the
nucleotide sequence of the rAKAV genome was determined
by direct sequencing of PCR products. No mutations were
found in the rescued virus.
rAKAVDNSs entirely from cDNAs. Additionally, this
mutation was confirmed by direct sequencing of PCR
product of rAKAVDNSs. No other mutations were found
in the whole genome of rAKAVDNSs.
Plaque morphology and size
To characterize the recombinants, plaque assays were
performed. Although there was a great variation in the
plaque size of the wild-type virus on HmLu-1 cells, the
plaque size for rAKAV was strikingly smaller than that for
the wild-type virus (Fig. 1d). Similarly, the plaques for
rAKAVDNSs appeared to be significantly smaller than
those for rAKAV (P,0.01). Furthermore, the average sizes
of the plaques for the three viruses after five successive
passages were approximately similar to those before
passage (data not shown). In contrast, rAKAVDNSs formed
slightly smaller plaques than rAKAV on Vero cells but the
size difference was not significant (data not shown).
Pathogenesis
Rescue of AKAV with a deleted NSs gene
To test whether our rescue system could be used for the
introduction of functionally important mutations into the
AKAV genome, we generated a mutant virus with an
inactivated NSs gene. Since the S RNA segment encodes N
and NSs on overlapping reading frames, complete deletion
of the NSs gene without affecting the N gene is impossible.
Therefore, we applied a strategy that has been used for the
abrogation of BUNV NSs gene expression (Bridgen et al.,
2001) and changed the ATG start codon of NSs to ACG
(Fig. 2a). The resulting genomic AKAV S construct, pRF42/
SDNSs, expresses an unaltered N but no NSs.
For virus rescue, HmLu-1 cells were transfected with the
plasmids pcDNA/L, pcDNA/SDNSs, pRF42/L, pRF42/M and
the mutant construct pRF42/SDNSs. The supernatants were
collected 5 days post-transfection and aliquots were transferred onto fresh HmLu-1 cells. CPE was observed 4 days
post-infection. Analysis of the growth kinetics of the mutated
recombinant virus (rAKAVDNSs) in HmLu-1, BHK-21 and
Vero cells revealed slightly poorer multiplication compared
with the wild-type and rAKAV viruses (Fig. 1a–c).
To establish that rAKAVDNSs is derived from cDNA
plasmids, we distinguished the wild-type and rAKAVDNSs
viruses by HpyCH4IV restriction analysis of their S RNA
segments, as mutagenesis of the NSs start site created a
unique recognition site for this restriction enzyme (Fig. 2a).
RT-PCR amplification of the recovered viral RNA and
subsequent digestion of the PCR products with HpyCH4IV
showed that the recovered recombinant virus carried the
introduced HpyCH4IV site in the S RNA segment (Fig. 2b).
No PCR products were obtained from control reactions in
which the reverse-transcription step was omitted, indicating that no contaminating plasmid DNA was present in
the RNA preparations. This resulted in the recovery of
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Wild-type or recombinant viruses were inoculated intracerebrally into BALB/cAJcl suckling mice, and the inoculated
animals were observed over a period of 21 days. All of the
mice inoculated with wild-type or rAKAV viruses died, but
there was a clear difference in disease progression (Fig. 3).
The wild-type virus killed all the mice by 10 days postinoculation, whereas it took 14 days post-inoculation for the
rAKAV to kill all the mice. In contrast, the rAKAVDNSsinfected mice showed a survival rate .78 % during the
21 day observation period. These results suggest that
rAKAVDNSs is attenuated. All of the surviving mice carried
antibodies against each strain, with neutralizing antibody
titres greater than 1 : 2, which suggests that the animals were
infected with the recombinant virus (data not shown).
DISCUSSION
Current reverse-genetic systems for negative-strand RNA
viruses rely on the production of viral transcripts from
transfected plasmids by either bacteriophage T7 RNA
polymerase or cellular RNA polymerase I (reviewed by
Kawaoka, 2004). For T7 RNA polymerase-driven systems,
the viral transcripts are produced in the cytoplasm, whereas
RNA polymerase I is a cellular protein that synthesizes
unmodified RNA species with defined terminal sequences
in the nuclei of transfected cells. With regard to members
of the family Bunyaviridae, both RNA polymerases have
been utilized to develop minigenome systems for representatives of each of the four genera of the family
Bunyaviridae that infect animals (Dunn et al., 1995;
Lopez et al., 1995; Flick & Pettersson, 2001; Flick et al.,
2003a, b), although to date, only T7 RNA polymerasedriven systems have been established for the rescue of
infectious viruses from cDNA (Bridgen & Elliott, 1996;
Bridgen et al., 2001; Lowen et al., 2004; Blakqori & Weber,
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Y. Ogawa and others
Fig. 1. Growth kinetics and plaque size of
recombinant viruses. Monolayers of HmLu-1
(a), BHK-21 (b) and Vero (c) cells were
infected with each AKAV strain at an m.o.i. of
0.01. Supernatants were recovered at the time
points shown in each part, and the p.f.u. were
determined. The results are shown as
mean±SD (error bars) of three independent
experiments. (d) Plaque size of the recombinant viruses. The results are shown as
mean±SEM of the relative sizes (%) of 50
randomly selected plaques for each virus. The
average plaque size of wt virus was arbitrarily
set at 100. Asterisks indicate a significant
difference (P,0.01) compared with the mean
plaque size of the examined viruses.
2005; Ikegami et al., 2006). However, for influenza virus
and arenavirus, rescue systems that use both polymerases
have been established (Kawaoka, 2004; Flatz et al., 2006;
Sanchez & de la Torre, 2006).
As the 39 ends of the transcripts formed by T7 RNA
polymerase are correctly trimmed by the hepatitis delta
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virus ribozyme, the T7 RNA polymerase-driven system
appears to be necessary for both antigenomic and
supporting plasmids. However, in the case of the family
Bunyaviridae, small amounts of the antigenomic RNAs that
act as mRNA can be translated into viral proteins without
the supporting plasmids (Lowen et al., 2004; Blakqori &
Weber, 2005; Ikegami et al., 2006). Thus, in these systems,
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AKAV reverse genetics
Fig. 3. Pathogenicity of viruses in intracerebrally inoculated
suckling mice. Three groups of 12–14 1-day-old mice were
inoculated intracerebrally with each AKAV strain. The animals were
monitored for 21 days.
Fig. 2. Introduction of a genetic marker into the S RNA segment.
(a) Schematic representation of the NSs translational start site in
the AKAV S RNA segment. The NSs reading frame is expressed
from a +1-shifted reading frame within the N gene. The mutation
introduced to ablate NSs translation without affecting the N gene
is shown. (b) Genetic marker for the NSs-deleted rAKAV. The
mutation introduced to destroy the NSs reading frame without
affecting the N gene generates a new HpyCH4IV restriction site,
which is present only in rAKAVDNSs. After RNA was extracted
from the infected cells, PCR was performed with (+) and without
(”) the reverse-transcription step (RT), to amplify the 344 bp
fragment of the NSs ORF. HpyCH4IV digestion of the RT-PCR
product from rAKAVDNSs yields fragments of 60 and 284 bp.
it is not clear whether the M polyprotein-expressing plasmid
is necessary for bunyavirus rescue. Our results suggest that
the rescue of bunyavirus, as well as influenza and arenavirus,
is independent of the envelope-glycoprotein-expressing
plasmid. The genomic plasmids and minimum supporting
plasmids (N and L) that form the ribonucleoprotein
complex are necessary for this rescue system.
Although the growth kinetics of the wild-type and rAKAV
viruses were very similar in the three different cell lines used
in this study (Fig. 1a–c), their plaque sizes and the mortality
patterns of the virus-infected mice were slightly different
(Figs 1d and 3). Mutation frequencies of RNA viruses are
considered to be 1 mutation in 103–105 nt per replication,
while DNA viruses have rates of less than 1 in 108 (Leider et
al., 1988; Steinhauer et al., 1992). This difference is
attributed to a lack of proofreading ability in RNA
replication enzymes. Although plaque-cloning of the wildtype virus was performed, it seems likely that the wild-type
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virus still contains a quasispecies virus population. The
plaque size of the wild-type virus varied widely after five
successive plaque pickings (data not shown), and that of
rAKAV was within the range of the wild-type virus plaque
size. Furthermore, when the whole genomes of recombinant
viruses were compared to that of the original virus, no
mutations were found in rAKAV. We have also reported
previously that the genomes of the same AKAV strains with
different passage histories were slightly different (Ogawa et
al., 2007c). Thus, despite the slight differences in biological
characteristics between the wild-type and rAKAV viruses, we
conclude that rAKAV has a wild-type phenotype.
It has been reported that the NSs protein of BUNV acts as a
virulence factor by suppressing the production of IFN-a/b
(Weber et al., 2002). In the genus Orthobunyavirus, NSs
deletion mutants have been generated by a reverse-genetic
system (Bridgen et al., 2001; Blakqori & Weber, 2005). The
NSs deletion mutant of BUNV (BUNdelNSs) formed
smaller plaques in BHK cells than the wild-type virus.
When inoculated intracerebrally, BUNdelNSs killed BALB/
c mice more slowly and exhibited reduced cell-to-cell
spread, and the titres of virus in the brain were lower
compared with infection with the wild-type virus (Bridgen
et al., 2001). The attenuation of BUNdelNSs in infected
mice suggests that NSs acts as a virulence factor. Similarly,
rAKAVDNSs formed smaller plaques than the wild-type
virus (Fig. 1b). Furthermore, rAKAVDNSs-infected mice
had a survival rate .78 % (Fig. 3). Our results suggest that
rAKAVDNSs is also attenuated, and that the NSs of AKAV
plays the role of a virulence factor.
The present study describes for the first time, to our
knowledge, the establishment of an RNA polymerase Idriven system for the rescue of infectious viruses from the
cDNAs of the family Bunyaviridae. It is possible now to
choose between the T7 RNA polymerase-driven and RNA
polymerase I-driven systems. Since T7 RNA polymerase is a
foreign protein, this enzyme must be introduced by using
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Y. Ogawa and others
plasmids, stable cell lines or helper viruses, such as the
recombinant vaccinia virus, that express T7 RNA polymerase. In contrast, RNA polymerase I is a cellular protein
and all eukaryotes possess this enzyme, although it is
species-specific. Both T7 RNA polymerase- and RNA
polymerase I-driven systems of arenavirus efficiently
produced infectious viruses (Flatz et al., 2006; Sanchez &
de la Torre, 2006). In contrast, the efficiency of our RNA
polymerase I-driven system was still lower than that of the
T7 RNA polymerase-driven system for other bunyaviruses.
This might be due to the number of essential plasmids for
the arenavirus systems and bunyavirus T7 RNA polymerase-driven system. Although further development and
optimization of this system are still needed, it can facilitate
studies of the biology and pathogenesis of bunyaviruses.
This technology provides an important basis for investigating viral virulence determinants and molecular virology,
and for producing a live-attenuated vaccine candidate.
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ACKNOWLEDGEMENTS
Rift Valley fever virus can rescue viral ribonucleoproteins and transcribe synthetic genome-like RNA molecules. J Virol 69, 3972–3979.
This work was supported in part by a Grant-in-Aid from the Japan
Livestock Technology Association and the Ministry of Education,
Culture, Sports and Technology of Japan. We thank Ramon Flick of
the University of Texas Medical Branch and Ralf F. Pettersson of the
Karolinska Institute, Stockholm, Sweden, for providing pRF42, and
Hidetoshi Ikeda of the National Institute of Animal Health, Tsukuba,
Japan, for providing BALB/cAJcl mice.
Lowen, A. C., Noonan, C., McLees, A. & Elliott, R. M. (2004). Efficient
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