1 Laboratory of Public Health, Department of Environmental Veterinary Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
2 Department of Pathology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
3 Centre for Ecology and Hydrology, Oxford, UK
Correspondence
Takuya Iwasaki
tiwasaki{at}net.nagasaki-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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The flavivirus genome (single, positive-stranded RNA, approximately 11 kb in length) encodes three structural proteins [capsid (C) protein, precursor membrane (prM) protein, and envelope (E) protein] and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5), within a single long open reading frame (ORF). The 5' and 3' termini of the genome have predicted secondary structures (Gritsun et al., 1997; Proutski et al., 1997
; Rauscher et al., 1997
) and contain important elements for virus replication, translation and packaging of the genome (Chambers et al., 1990
; Lindenbach & Rice, 2001
).
A number of infectious cDNA clones of flaviviruses have been produced using different strategies to study the different aspects of flavivirus pathogenesis (Rice et al., 1989; Ruggli & Rice, 1999
; Sumiyoshi et al., 1992
; Kapoor et al., 1995
; Mandl et al., 1997
; Polo et al., 1997
; Gritsun & Gould, 1998
; Yamshchikov et al., 2001a
, b
; Bredenbeek et al., 2003
; Zhang et al., 2001
; Yun et al., 2003
; Puri et al., 2000
; Kinney et al., 1997
; Gualano et al., 1998
; Sriburi et al., 2001
; Lai et al., 1991
; Khromykh & Westaway, 1994
; Shi et al., 2002
; Campbell & Pletnev, 2000
; Pletnev, 2001
; Hurrelbrink et al., 1999
). There are two reports of infectious cDNA clones for TBEV, European subtype strains Neudoerfl and Hypr (Mandl et al., 1997
) and Siberian subtype strain Vasilchenko (Vs) (Gritsun & Gould, 1998
). The present study reports the construction and characterization of an infectious cDNA clone of the Far-Eastern subtype TBEV Oshima 5-10 strain, which was isolated in Japan in 1995 (Takashima et al., 1997
). Genetic and biological differences between the infectious clone and the parent Oshima 5-10 virus were revealed and the parent genotype was recreated from the infectious clone by reversion of all detected amino acid substitutions. Specific mutations within the E glycoprotein and the RNA-dependent RNA-polymerase (NS5) gene were shown to be responsible for modification of plaque phenotype, virus growth in cell culture and virulence for mice.
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METHODS |
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Reverse transcription and long-PCR.
Viral RNA extraction and long high-fidelity RT-PCR were described previously (Gritsun & Gould, 1995, 1998
). Briefly, viral RNA was extracted from 10 % MBS using Catrimox (Iowa Biotechnology Corp.). The RT reaction was carried out with Superscript II reverse transcriptase (Invitrogen) and 14-mer primer (AGCGGGTGTTTTTC). For the long PCR a mixture of thermostable Red Hot DNA polymerase (Advanced Biotechnologies) and Deep Vent DNA polymerase (NEB) was used for a hot start. The PCR programme included 30 cycles of 30 s at 94 °C and 10 min at 72 °C. Primers were designed on the basis of the nucleotide sequence of Oshima 5-10 strain (Hayasaka et al., 2001
; Goto et al., 2002
). The sense primer Oshi-5'-long included a NotI restriction enzyme site, SP6 promoter and 28 nucleotides of 5' untranslated region (UTR) (gtcgacGCGGCCGCatttaggtgacactatagAGATTTTCTTGCACGTGCATGCGTTTGC). The antisense primer Oshi-3'-long has a SpeI site and 35 nucleotides of 3'UTR (gcacgtACTAGTAGCGGGTGTTTTTCCGAGTCACTCATCACCTCCTT). PCR products were purified using a QIAquick PCR Purification Kit (Qiagen).
Cloning of full-length cDNA clone of TBEV Oshima strain.
Plasmid pGGVs209, based on pBR322 (Gritsun & Gould, 1998), was used for the cloning of full-length RT-PCR product amplified from Oshima 5-10 virus (Fig. 1
). Plasmid pGGVs209 was constructed from the infectious clone for TBEV Siberian strain, Vs, and contains the first 209 nucleotides of the Vs virus genome with the restriction site SpeI at position 80 and a NotI site upstream of the SP6 promoter. Plasmid pGGVs209 and purified long-PCR product were digested with NotI and SpeI enzymes, ligated with T4 ligase (NEB) and transformed into the low-copy Able-K strain of E. coli (Stratagene) by electroporation. Bacterial cells were incubated on TYM agar plates containing 100 µg ampicillin ml-1 at 37 °C. Small colonies were chosen and grown in liquid TYM at 28 °C overnight. Plasmid DNAs were extracted and those selected by size (about 15 kb) were verified by restriction analysis to confirm the insertion of virus cDNA.
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For electroporation, the transcription samples were treated with DNase; transcribed RNA after the precipitation with LiCl was dissolved in 10 µl of water. Trypsin-treated BHK cells were washed with cold PBS three times. About 10 µg of transcribed RNA was electroporated to 5x106 BHK cells in 500 µl of cold PBS in 0·4 cm cuvettes using a GenePulser apparatus (Bio-Rad), 1·3 kV, 25 µF and , pulsed two times. After incubation at room temperature for 10 min, cells were kept at 37 °C under 5 % CO2.
The identification of the virus in the mouse brain or in cell supernatant medium was carried out by indirect immunofluorescence microsopy using monoclonal antibodies to the E glycoprotein as described in Gritsun & Gould (1995). The full-length clone that produced infectious virus was nominated as O-IC (Oshima virus infectious clone). In the comparative experiments to be described below the virus stocks were derived from the supernatant medium of BHK cells.
Mutagenesis of the infectious clone.
A range of intermediate plasmids based on pGEM3 and pGEMT vectors (Promega) was constructed to produce reverse-mutations in the infectious clone (pGEM3-HS1, pGEM3-HS2, pGEMT-CprME and pGEMT-NS5, Fig. 1). Plasmid pGEM3-HS2 was constructed to introduce wild-type substitutions (NS5-T794, guanine10473 and adenine10922 in the 3'UTR) to produce a full-length plasmid and virus with genotype P40A378K674T794 (Fig. 1
).
To substitute P40 for S40, the parent virus genome fraction between nucleotides 2422295 was amplified by RT-PCR and cloned (with the introduction of two flanking SpeI restriction sites) to construct plasmid pGEMT-CprME. An AvrII restriction site (cohesive ends compatible with SpeI) was introduced into the plasmid P40A378K674T794 (D. Hayasaka and others, unpublished results). This intermediate plasmid and pGEMT-CprME were used to substitute the region 2422295 between two sets of restriction sites corresponding to AvrII or SpeI to produce a plasmid and virus with the genotype S40A378K674T794 (Fig. 1). Plasmid pGEM-3HS1 was constructed by subcloning the region between HindIII and SpeI (nucleotides 919711100) of the infectious clone O-IC. The S40A378K674T794 and plasmid pGEM3-HS1 were used to construct full-length plasmid and corresponding virus with genotype S40A378K674A794.
To recreate the infectious clone O-IC-pt, so that it was genetically identical to parent virus Oshima 5-10, the nucleotides 736511 100 of the NS5 gene were amplified from parent Oshima 5-10 virus by RT-PCR and cloned to construct a plasmid pGEMT-NS5 that was subsequently used to substitute the region between nucleotides 736511100 (AatII and SpeI sites) of the plasmid S40A378K674T794. All subcloned plasmids (Fig. 2) were fully sequenced to confirm their authenticity.
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Immunofluorescent antibody (IFA) focus assay and titration of viruses.
A fluorescent focus assay, developed previously (Takashima et al., 1997), was used to assess virus titres. Virus was adsorbed for 90 min at 37 °C on BHK cells. The inoculum was then replaced with EMEM and 5 % carboxymethyl cellulose. After incubation for 36 or 72 h, cells were fixed with methanol and incubated at 37 °C for 1 h with an appropriately diluted ascitic fluid taken from mice hyperimmunized with a tick-borne flavivirus. Following washing with PBS, fluorescein isothiocyanate-conjugated antibody against mouse IgG was added and incubated at 37 °C for 1 h. After washing with PBS, the numbers of fluorescent foci were estimated under a UV microscope. The virus titres were calculated and expressed as focus forming units per ml (f.f.u. ml-1).
Growth curve in cell cultures.
Subconfluent BHK cells were grown as 106 cells per well in 12-well plates and inoculated with each parent or engineered virus at an m.o.i of 0·01 f.f.u. Viruses were diluted with medium containing 4 % FCS. After incubation for 90 min, virus inoculum was removed and the cells were washed twice in PBS. Two ml of medium was added to each well and they were then incubated at 37 °C under 5 % CO2. The samples were harvested at 12, 24, 36 and 48 h post-inoculation and stored in aliquots at -80 °C prior to titration.
Virulence in mice.
Viruses were inoculated into 7-week-old male BALB/c mice (SLC) with body weights of about 20 g. Eight mice in each group received 50 f.f.u. of each virus intracerebrally or 1000 f.f.u. of each virus subcutaneously. Morbidity was defined as the appearance of more than 10 % weight loss or clinical signs ruffled fur and/or hunched back. Surviving mice were monitored for 28 days post-infection to obtain survival curves and mortality rates.
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RESULTS |
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To test for stability in bacteria, O-IC plasmids were transformed into E. coli strain HB101, grown for various incubation times, (8, 12, 16, 20 and 24 h) and plated on Luria Broth agar containing 100 µg ampicillin ml-1. The colony size on the plates was unaltered after five repeated passages and after each subculture, the infectious virus was successfully rescued (data not shown). Sequencing demonstrated that plasmid O-IC and the virus which was recovered from the infectious clone O-IC were identical.
Properties of the infectious clone
Formation of fluorescent foci.
Whilst carrying out the focus forming assay, we noticed that the parent Oshima 5-10 virus produced distinct fluorescent foci at 36 h post-infection (Fig. 3). However, O-IC produced only singly infected cells within the same time period and fluorescent foci, of the same size as those produced by the parent virus at 36 h, were visualized only after an incubation period of at least 72 h (Fig. 3
).
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A single amino acid substitution within the E protein reduces the size of fluorescent foci
Following the above analysis we carried out mutagenesis on the infectious clone to restore the original sequence of the parent virus and also to identify which mutation or mutations are responsible for reduction in size of fluorescent foci induced by the infectious clone. The strategy for the construction of mutant viruses is described in Methods and is illustrated in Fig. 1. The genotypes of recovered mutant viruses are presented in Fig. 2
. The viruses were compared in four different biological tests, viz. relative size of fluorescent foci, growth curve experiments, mouse neurovirulence and neuroinvasiveness.
The parent Oshima 5-10 virus and O-IC-pt virus which were genetically identical and contain serine in position 40 of the E protein (genotype S40V378R674T794), produced identical large fluorescent foci at 36 h post-infection (Fig. 3). Viruses S40A378K674T794 and S40A378K674A794, also with E-S40, but different from Oshima 5-10 virus and O-IC-pt virus in the NS5 gene and the 3'UTR, also formed large foci by 36 h post-infection. Therefore the three mutations in the NS5 protein, in conjunction with mutations in the 3'UTR, do not influence focus development. On the other hand, P40A378K674T794 virus with the single substitution E-S40
P in comparison with S40A378K674T794 virus, formed fluorescent foci only after incubation for at least 72 h. These results indicate that a single amino acid mutation within the E protein (E40) was responsible for the focus size reduction described in this report.
Virus growth curves in cell cultures
Several conclusions arise from the comparative growth characteristics of the mutated viruses (Fig. 5).
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Secondly, although newly synthesized virions began to appear for all viruses between 1214 h post-infection, the infectious yields at each time point remained higher for S40A378K674T794 virus than for P40A378K674T794 and since they differ by only one amino acid in position 40 of the E protein (E-S40 genotype against E-P40 respectively) these results imply that a single substitution in the E gene significantly reduces virus reproduction efficiency in cell cultures.
Thirdly, there was no significant difference in the growth curves produced by O-IC-pt (S40V378R674T794) and S40A378K674T794, despite the fact that they differ by two amino acids (NS5-V378A and NS5-R674
K).
Therefore these growth curve experiments demonstrate that only the mutation in the E protein exerts an effect on the phenotype of the virus in cell culture.
Virulence for mice
The results of neurovirulence tests for S40A378K674T794, P40A378K674T794, O-IC-pt and parent virus Oshima 5-10 are shown in Fig. 6. Following intracerebral inoculation, both parent virus Oshima 5-10 and genetically identical O-IC-pt virus produced 100 % mortality of mice within 12 days post-inoculation. In contrast, S40A378K674T794 virus, that is different from O-IC-pt virus by two amino acid substitutions in the NS5 gene (NS5-V378
A and NS5-R674
K) had lower neurovirulence, killing a maximum of 62·5 % of mice by day 18 post-inoculation. Virus P40A378K674T794, that is different from S40A378K674T794 virus by only one amino acid in the E protein, showed the lowest neurovirulence, killing about 25 % of mice. These experiments therefore demonstrate firstly, a biological effect of NS5-V378
A and NS5-R674
K mutations that was not detectable in cell culture, and secondly it shows the cumulative effect of E-S40
P, NS5-V378
A and NS5-R674
K on attenuation of the virus.
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DISCUSSION |
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To construct an infectious cDNA clone of Far-Eastern subtype TBEV, we used the Oshima 5-10 strain, which was isolated in 1995 in Japan (Takashima et al., 1997). Although only one case of human TBE has been confirmed in Japan, it is believed that TBEV emerged within the past few centuries and was distributed to a wide area of Hokkaido, the northern island of Japan (Hayasaka et al., 1999
; Takeda et al., 1999
). Oshima 5-10 virus shares more than 98 % amino acid identity with Sofjin virus, the prototype Far-Eastern TBEV (Goto et al., 2002
). In addition, Oshima 5-10 virus shows similar virulence in mice with the viruses recently isolated in Far-Eastern Russia (Chiba et al., 1999
; Hayasaka et al., 1999
, 2001
).
In this study we constructed an infectious clone using the strategy described for the Siberian strain of TBEV, Vs, (Gritsun & Gould, 1995, 1998
). Long high-fidelity PCR and one-step cloning procedures were employed to produce bacterial clones containing full-length molecules of TBEV. The bacterial cells were routinely propagated at 28 °C to reduce the mutation rate introduced by the bacteria. We checked 88 clones from bacterial colonies after transformation and ten contained full-length virus sequences. One clone (O-IC) that was infectious for mice and BHK cells was selected for further analysis. A separate non-infectious full-length clone that we sequenced had 15 nucleotide differences from the parent virus and a single nucleotide deletion at position 5120 in the NS3 protein region (data not shown). A similar proportion of 1/12 between infectious and non-infectious cDNA molecules was also detected after cloning the Siberian TBEV strain (Gritsun & Gould, 1998
). The fact that bacteria select in favour of non-infectious molecules has been explained by low level expression of regions of flavivirus polyprotein with transmembrane domains that could be toxic for bacteria; therefore clones that accidentally acquired lethal mutations might have a selective advantage (Yamshchikov et al., 2001a
). Nevertheless, although only one infectious clone was obtained, and it was stable in E. coli strain Able-K and also HB101 at both 28 °C and 37 °C. The original infectious clone O-IC had nine nucleotide substitutions compared with the parent virus Oshima 5-10 (see below), but other full-length infectious clones that were individually modified by reverse mutations to parent virus sequence (S40A378K674T794, P40A378K674T794, S40A378K674A794 and O-IC-pt) were also stable. Therefore, the infectious clone will be highly exploitable for genetic manipulations.
Four amino acid substitutions, one mapping within the E glycoprotein and the other three within the NS5 gene (RNA-dependent RNA polymerase), were revealed between parent Oshima 5-10 and the original infectious clone O-IC (Table 1). O-IC virus also differed from parent virus in the infectious focus test, producing infectious foci later than those of the parent virus Oshima 5-10 (Fig. 3
). We therefore carried out mutagenesis on the infectious clone to restore the original virus genotype and also to identify which mutations caused the delay in formation and reduction of focus size.
An infectious clone O-IC-pt was reconstructed from O-IC to restore the parent virus genotype. This was achieved using a cassette of intermediate plasmids that have also been used to engineer several viruses with different combinations of mutations (Fig. 1). The viruses were compared in focus assays, growth curves, mouse neuroinvasiveness and neurovirulence. The results of these tests demonstrated that substitution of conserved (among all flaviviruses) hydroxyl-containing amino acids serine or threonine in position 40 of the E protein for the aromatic hydrophobic amino acid proline of the infectious clone was solely responsible for the delay of TBEV focus formation, delayed growth curve characteristics and reduced neurovirulence (Figs 3, 5 and 6
). Examination of the crystal structure of the E protein (Rey et al., 1995
) shows that amino acid S40 is buried within the central domain I, i.e. it is not exposed on the surface of the protein. The effect of E-S40
P on virus reproduction might therefore be mediated by conformational changes that could affect virion adsorption, penetration or assembly. Proline is an aromatic amino acid, larger in size then threonine and serine, and it could induce important structural changes to the protein since proline residues are often located at the point of
-turns. Nevertheless one non-vectored flavivirus Apoi virus had an alanine in this position (Fig. 4
). Therefore it is possible to have a hydrophobic amino acid in this position, although it could change the properties of the protein. The E protein is the most studied protein of the flaviviruses because it is associated with the main biological characteristics of the virus, including virion adsorption, pH-dependent penetration, haemagglutination, induction of virus-neutralizing and protective antibodies, antibody-dependent enhancement and virulence properties (Barrett & Gould, 1986
; McMinn, 1997
; Heinz & Allison, 2000
; Heinz, 2003
). Many mutations have been identified that attenuate virus reproduction through different functional regions of the E protein (Lee & Lobigs, 2000
; Mandl et al., 2000
, 2001
; Hurrelbrink & McMinn, 2001
; Holzmann et al., 1997
; Allison et al., 2001
; Monath et al., 2002
; Cecilia & Gould, 1991
; Gritsun et al., 1995
, 2001
; Jiang et al., 1993
; Rey et al., 1995
). At this stage of the investigations it is not possible to define the function of the E protein (adsorption, penetration or virion assembly) that was affected by the E-S40
P mutation.
Two nucleotide substitutions were mapped in the 3'UTR. The mutation at position 10473 (position 97 after the stop codon in Oshima 5-10 virus sequence; Table 1) mapped in the hypervariable region of the flaviviruses (Gritsun et al., 1997
; Wallner et al., 1995
) that was shown not to influence flavivirus infectivity (Mandl et al., 1998
). The second mutation, guanine, in genome position 10922 (nucleotide 546 after the stop codon), mapped in a highly conserved region of the 3'UTR (Gritsun et al., 1997
; Proutski et al., 1997
; Rauscher et al., 1997
) where the nucleotide alignment revealed adenine for 25 tick-borne flaviviruses (data not presented). Nevertheless, folding of the 3'UTR of the infectious clone did not predict any change in RNA secondary structure following substitution of adenine for guanine (data not presented).
Similarly with the Siberian TBEV virus, Vs, mutations other than those in the E protein of the infectious clone O-IC, appeared to be responsible for the reduction of neurovirulence in mice, although they did not result in any biological consequences for plaque assays or growth cycle characteristics (Gritsun et al., 2001). Two viruses, O-IC-pt and S40A378K674T794 were genetically different by two amino acids within the NS5 protein (in positions 378 and 674, Fig. 2
and Table 1
) that affected only neurovirulence and neuroinvasive properties of the virus (Fig. 6
and Table 2
) with no effect on virus growth characteristics in BHK cell culture (Fig. 5
). These two substitutions within the NS5 protein were of a conserved nature (V378
A, R674
K), but both were located in functionally important domains of NS5 protein, one V378
A in the nuclear localization sequence (Forwood et al., 1999
) and the other R674
K in close proximity to the highly conserved GDD sequence (663665), which is an RNA-dependent RNA polymerase motif (Rice et al., 1985
; Khromykh et al., 1998
). Therefore the reduction of neurovirulence due to these two substitutions could be explained by the limitations imposed on protein tertiary structure in highly conserved domains of the NS5 protein. A similar observation for mutations within the NS5 protein and the 3'UTR was previously reported for the infectious clone of the Siberian TBEV Vs virus (Gritsun et al., 2001
).
Comparative analyses of three viruses in neurovirulence tests (Fig. 6), namely O-IC-pt (parent genotype), S40A378K674T794 (with substitutions V378
A and R674
K in comparison with parent genotype) and P40A378K674T794 (with substitution S378
P in comparison with S40A378K674T794 virus genotype) have also revealed the cumulative effect of different mutations on attenuating properties of TBEV, as has also been demonstrated for Siberian virus (Gritsun et al., 2001
).
Neuroinvasiveness was also compared by subcutaneous inoculation of mice (Table 2). All mice inoculated with parent Oshima 5-10 virus died within 812 days post-inoculation. Although mice inoculated with O-IC-pt virus survived until 28 days post-inoculation, some showed clinical signs such as paralysis or loss of body weight (Table 2
). Growth curve experiments also demonstrated smaller virus titres at 12 h post-infection for O-IC-pt virus in comparison with parent Oshima 5-10 virus (Fig. 5
). In view of these unexpected results, we sequenced the virus recovered from sick mice and confirmed that this O-IC-pt virus had the identical sequence to parent Oshima 5-10 virus. One possible explanation for this result may be that the parent Oshima 5-10 virus consists of a population of quasi-species. Since the parent Oshima 5-10 virus sequence had been determined by direct-sequencing from RT-PCR products (Hayasaka et al., 1999
; Goto et al., 2002
), the sequence would be a consensus. We therefore cloned parent Oshima 5-10 virus by plaque assay and noticed several different sizes of plaque (data not shown). We picked a large and a small plaque variant and determined the sequence of the E protein of each. A single but different amino acid substitution was detected in the E protein of each plaque variant. Both viruses showed less neuroinvasiveness than the parent virus (Goto et al., 2003
). Whilst we did not identify a plaque variant with the high neuroinvasiveness of the parent virus, these results combined with the concept that quasi-species may show a range of virulence characteristics, provide a rational explanation for the difference in neuroinvasiveness of parent Oshima 5-10 and O-IC-pt virus for mice. It is unlikely that other phenomena such as interferon or defective interfering particles can account for this difference in virulence because both virus stocks were prepared in the same way, that is in BHK cells. Moreover, the infectious clone was passaged only once in BHK cells which would be unlikely to generate a significant level of defective particles.
In summary, we have constructed an infectious clone based on the Far-Eastern subtype of TBEV. The original infectious clone contained four amino acid substitutions that were back-mutated to produce the infectious clone genetically identical to the parent virus. Site-directed mutagenesis on the infectious clone revealed that one amino acid, P40, in the E protein and two amino acids, A378 and K674, in the NS5 protein were responsible for the virus attenuation. We also demonstrated the cumulative effect of point mutations on attenuated characteristics of TBEV.
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ACKNOWLEDGEMENTS |
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Received 19 September 2003;
accepted 17 December 2003.