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Citation: Penghui Li, Chen Yao, Ting Wang, Tong Wu, Wenfu Yi, Yue Zheng, Yuanjiu Miao, Jianhong Sun, Zhongyuan Tan, Yan Liu, Xiaowei Zhang, Hanzhong Wang, Zhenhua Zheng. Recovery of a Far-Eastern Strain of Tick-Borne Encephalitis Virus with a Full-Length Infectious cDNA Clone [J].VIROLOGICA SINICA.  http://dx.doi.org/10.1007/s12250-021-00396-6

Recovery of a Far-Eastern Strain of Tick-Borne Encephalitis Virus with a Full-Length Infectious cDNA Clone

  • Corresponding author: Zhenhua Zheng, zhengzh@wh.iov.cn, ORCID: 0000-0002-4117-9364
  • Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s12250-021-00396-6.
  • Received Date: 17 July 2020
    Accepted Date: 15 March 2021
    Published Date: 30 June 2021
  • Tick-borne encephalitis virus (TBEV) is a pathogenic virus known to cause central nervous system (CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious cDNA clone of TBEV that was isolated in China (the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1 (NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect (CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.


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    Recovery of a Far-Eastern Strain of Tick-Borne Encephalitis Virus with a Full-Length Infectious cDNA Clone

      Corresponding author: Zhenhua Zheng, zhengzh@wh.iov.cn
    • 1. CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety MegaScience, Chinese Academy of Sciences, Wuhan 430071, China
    • 2. College of Life Sciences and Food Engineering, Hebei University of Engineering, Handan 056038, China

    Abstract: 

    Tick-borne encephalitis virus (TBEV) is a pathogenic virus known to cause central nervous system (CNS) diseases in humans, and has become an increasing public health threat nowadays. The rates of TBEV infection in the endemic countries are increasing. However, there is no effective antiviral against the disease. This underscores the urgent need for tools to study the emergence and pathogenesis of TBEV and to accelerate the development of vaccines and antivirals. In this study, we reported an infectious cDNA clone of TBEV that was isolated in China (the WH2012 strain). A beta-globin intron was inserted in the coding region of nonstructural protein 1 (NS1) gene to improve the stability of viral genome in bacteria. In mammalian cells, the inserted intron was excised and spliced precisely, which did not lead to the generation of inserted mutants. High titers of infectious progeny viruses were generated after the transfection of the infectious clone. The cDNA-derived TBEV replicated efficiently, and caused typical cytopathic effect (CPE) and plaques in BHK-21 cells. In addition, the CPE and growth curve of cDNA-derived virus were similar to that of its parental isolate in cells. Together, we have constructed the first infectious TBEV cDNA clone in China, and the clone can be used to investigate the genetic determinants of TBEV virulence and disease pathogenesis, and to develop countermeasures against the virus.

    • Tick-borne encephalitis virus (TBEV) is an enveloped virus that belongs to the Flavivirus genus (Kovalev and Mukhacheva 2017). TBEV is usually associated with central nervous system (CNS) diseases that are accompanied by high fever, vomiting and headache in humans (Zhang et al. 2016b; Sun et al. 2017). Annually, > 10, 000 cases were reported in Europe and Asia (Yoshii 2019). TBEV is usually subdivided into Far-Eastern (TBEV-FE), Siberian, and European subtypes (Zhang et al. 2016b). In general, the severity varies depending on the TBEV subtypes and strains, because each subtype contains low- and high-virulent strains. The mortality caused by TBEV was about 1%–2% in Europe (Kaiser 2008; Füzik et al. 2018). It was noted that TBEV-FE caused more severe encephalitis signs and higher mortality (5%–20%) than the other two subtypes (Suss 2008; Zhang et al. 2016b; Füzik et al. 2018), which, however, might be disturbed and exaggerated by the involvement of other virulence or disease severity factors apart from the subtype. Most of TBEV isolates in China belong to the FE subtype, which are chiefly transmitted to human beings by the bite of Ixodes persulcatus and maintained in the sylvatic cycle between the ticks and wild vertebrate animals (Lu et al. 2008; Yoshii et al. 2017; Dai et al. 2018; Boelke et al. 2019). Though commercial vaccines against TBEV have been available for decades, the rate of TBEV infection in the endemic countries is increasing on account of low vaccination coverage, climatic changes, agriculture changes, and socio-economic factors (Eyer et al. 2016; Lotrič-Furlan et al. 2017; Xing et al. 2017; Yang et al. 2019). To date, there is no antiviral treatment against TBEV infection, which has aggravated the current panic over the disease.

      The TBEV genome is a capped plus-strand RNA approximately 10.8 kb long, and contains an open reading frame (ORF) encoding a polyprotein, flanked by 5' and 3' untranslated regions (Zhang et al. 2016b; Litov et al. 2018; Selinger et al. 2019). The polyprotein is subsequently cleaved by host and viral proteases into capsid (C), membrane (M), envelope (E), and seven nonstructural proteins (NSs) including viral proteases and RNA-dependent RNA polymerase (RdRp) proteins (Zhang et al. 2016b; Füzik et al. 2018; Boelke et al. 2019; Eyer et al. 2019; Selinger et al. 2019). C, M and E protein form viral particles, among which E protein is the major surface protein and mediates attachment and entry of TBEV into host cells, while the NSs are necessary for virus replication, assembly, and innate immune suppression (Litov et al. 2018; Boelke et al. 2019; Yau et al. 2019).

      The availability of infectious cDNA clones generally determines the progress and trends in revealing the replication, dissemination, and pathogenesis of flaviviruses, as well as in the evaluation of antivirals and vaccines in vitro and in vivo for the treatment of viral infection in humans (Tsetsarkin et al. 2016; Li et al. 2018; Tamura et al. 2019; Wang et al. 2020). Infectious cDNA clones for several flaviviruses, including Japanese encephalitis virus, dengue virus, West Nile virus, TBEV, and Zika virus (ZIKV), have been generated (Shi et al. 2002; Zhu et al. 2007; Takano et al. 2011; Li et al. 2014, 2018; Ishikawa et al. 2015; Schwarz et al. 2016; Shan et al. 2016; Tsetsarkin et al. 2016). However, flavivirus cDNA were notoriously instable and difficult to propagate in Escherichia (E.) coli host, likely due to the bacterial toxicity of viral genome that harbored cryptic bacterial promoters (Pu et al. 2011; Aubry et al. 2015; Schwarz et al. 2016; Widman et al. 2017). To overcome these instability issues, several strategies have been devised to relieve the bacterial toxicity, including very-low-copy-number plasmids, multipiece systems, in vitro ligation approach, introns insertion, and alternative hosts such as yeast for plasmid propagation (Rice et al. 1989; Pu et al. 2011; Santos et al. 2013; Schwarz et al. 2016; Widman et al. 2017). In a previous study, we have isolated a Far-Eastern strain of TBEV designated WH2012 and revealed the first details regarding biological properties and pathogenicity of TBEV from China (Zhang et al. 2016b).

      We have developed stable infectious clones for ZIKVwt, codon pair-deoptimized ZIKVs, and the ZIKV-Nluc virus (Li et al. 2018; Wang et al. 2020). In view of the high genome-wide association between ZIKV and TBEV, the similar strategy was chosen to design a clone of our WH2012 strain of TBEV. In this study, we developed the first full-length cDNA clone of TBEV isolated in China by inserting a beta-globin intron at nt position 3099 to stabilize the TBEV genome in bacteria. This infectious clone showed high genetic stability in bacteria and produced infectious TBEVic (the rescued viruses from the full-length cDNA clone of TBEV) in mammalian cells.

    • Baby hamster kidney cells (BHK-21; ATCC CCL-10) were cultured at 37 ℃ with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Darmstadt, Germany) containing 10% fetal bovine serum (FBS) (Life Technology, Australia), 100 U/mL penicillin, and 100 lg/mL streptomycin and maintained. The WH2012 strain of TBEV (GenBank number: KJ755186.1) was isolated from ticks collected in northern China (Zhang et al. 2016b), and has been kept and passaged for several rounds in our lab (Fig. 1). The mouse polyclonal antibodies against TBEV NS5 protein were generated by immunization of BALB/C mice with purified TBEV NS5 protein. The TBEV NS5 polyclonal antibodies and a 4G2 mouse monoclonal antibody (MAb, ATCC) which showed cross-reactivity with flavivirus E protein were used as primary antibodies in the following immunological detection assays.

      Figure 1.  Characterization of the parental WH2012 virus. A Scheme of immunization of serial passages of the WH2012 virus in BHK-21 cells. B Apparent and similar CPE of TBEV Pn and TBEV Pn+5 on BHK-21 cells. C No plaque of the WH2012 strain on BHK-21 cells at 4 d.p.i. All the images were captured at a ×10 magnification. D Viral copies in the supernatants from serial passages were determined at 4 dpi by qRT-PCR. Data shown (D) are the mean ± SD analyzed by Student's t-test (two tailed) (*P < 0.05).

    • To generate the infectious cDNA of TBEV, the genomic RNA was extracted from the parental WH2012 strain with TRIzol reagent (TaKaRa, Dalian, China) and reverse transcribed with a PrimeScript RT reagent kit (TaKaRa, Dalian, China). PCR fragment 1 containing nt 1 to 1102 of the genome was overlapped with a cytomegalovirus (CMV) promoter and constructed into the low-copynumber plasmid pACYC177 at the KpnI and ClaI sites, yielding subclone A. PCR fragment 2 containing nt 1076 to 3098, the beta-globin intron from pACYC177-ZIKV-FL (Li et al. 2018), and PCR fragment 3 containing nt 3099 to 4343 were fused and cloned into pACYC177 at the ClaI and BlnI sites, yielding subclone B. Some hints and tips for the insertion site of the intron were from a previous study of ZIKV (Schwarz et al. 2016). PCR fragment 4 containing nt 4319 to 7946 was cloned into pACYC177 at the BlnI and XbaI sites, yielding subclone C. PCR fragment 5 containing nt 7906 to 10, 784, the hepatitis D virus ribozyme (HDVr) sequence-simian virus 40 (SV40) poly(A) (HDVr-SV40 poly(A) was directly amplified from pACYC177-ZIKV-FL) were fused and cloned into pACYC177 at the XbaI and XhoI sites, yielding subclone D. The four subclones were assembled step-by-step into the full-length infectious cDNA clone of TBEV (TBEV-FL), as shown in Fig. 2. All the primers used for cloning and qRT-PCR assay in the study were shown in Supplementary Table S1. A restriction map and complete sequencing were used to verify the clone of TBEV-FL.

      Figure 2.  Construction of the full-length infectious cDNA clone of TBEV. Four cDNA fragments (A to D) covering the complete TBEV genome were amplified from viral RNA of the parental WH2012 virus using RT-PCR and sequentially cloned into plasmid pACYC177 to form the full-length TBEV cDNA clone (TBEV-FL). The CMV promoter, synonymous substitutions, the positions of relevant restriction sites, and HDVr/SV40 poly(A) are shown.

    • Infectious viruses recovered from ICs have been described previously (Li et al. 2018; Wang et al. 2020). In brief, BHK-21 cells were seeded into 35 mm culture dishes and cultured at 37 ℃ with 5% for 24 h. BHK-21 cells at 85% confluence were transfected with ICs by lipofectamine 3000 (Life Technologies). The supernatant was harvested at 4 days post transfection (dpt), clarified by centrifugation, and stored at 80 ℃.

    • Plaque assay was performed as previously described (Li et al. 2018; Luo et al. 2020). In brief, BHK-21 cells at 85% confluence in 24-well plates were incubated with 100 μL viral samples of tenfold serial dilutions in DMEM. After 1.5 h of incubation, cells were overlaid with 1.25% methylcellulose-containing 2% FBS. After incubation for 4 days, cells were fixed for 0.5 h with 4% buffered formalin, and stained with 0.5% crystal violet solution. After rinsing with deionized water, plaque morphology and number were registered, and virus titrations were expressed as PFU/mL.

    • TCID50 assay was performed as previously described (Zhang et al. 2016a; Sun et al. 2020). Briefly, BHK-21 cells were seeded into 96-well plates, 5000 cells/well. Then, the cells were infected with 100 μL viral samples of tenfold serial dilutions in DMEM and incubated 37 ℃ for 4 days. The cytopathic effect (CPE) was thereby observed under the microscope, and the infectivity titer was determined by using the Reed-Muench calculation calculator software (Lindenbach 2009) and expressed as TCID50/mL.

    • BHK-21 cells at 85% confluence in 35 mm culture dishes were infected at an multiplicity of infection (MOI) of 0.1 in a volume of 500 μL. After incubation for 1.5 h at 37 ℃, the cells were washed once with 1 mL of phosphate-buffered saline (PBS), and then 1.5 mL DMEM with 2% FBS was added. A total of 200 μL of viral supernatants was sampled once every day, clarified, aliquoted, and stored until use. The infectious titers of the viral samples were quantitatively determined using a TCID50 assay.

    • Viral RNA copies from cell supernatants was quantified by using qRT-PCR, as described previously (Li et al. 2018). In brief, a universal pair of primers (TBEV qPCR-F and TBEV qPCR-R, Supplementary Table S1) was used to amplify the region spanning nt 2898 to 3013 in the NS1 gene, which is conserved in all virus strains. The TBEV NS1 gene was cloned into pGEM-T (Promega, WI, USA), and used as a standard. Each 20 μL of qRT-PCR reaction mix contains SYBR green master mix (Bio-Rad), cDNA, and a pair of primers. The assays were performed on the CFX96 touch real-time PCR detection system (Bio-Rad). Cycling conditions involved 95 ℃ for 3 min, followed by 39 cycles of 95 ℃ for 10 s, 55 ℃ for 10 s, and 65 ℃ for 30 s. The RNA copies were calculated based on the standard curve composed of tenfold serial dilutions of the standard DNA.

    • Western blot was performed as previously described (Tan et al. 2018; Sun et al. 2020). Briefly, cells were washed with PBS and lysed in RIPA buffer (Beyotime Institute of Biotechnology, China) in accordance with the manufacturer's instructions. After SDS-polyacrylamide gel electrophoresis, proteins were transferred to PVDF membranes (Millipore) in transfer buffer. After blocking with 5% bovine serum albumin (BSA) dissolved in TBST (TBS containing 0.1% Tween 20), membranes were incubated primary antibodies at 4 ℃ for 12 h. After washing with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at 37 ℃. After washing for five times, membranes were visualized with immobilon western chemiluminescent HRP substrate (Millipore) and analyzed by using Bio-Red Imaging System.

    • IFA was performed as previously described (Wang et al. 2020). Briefly, the cells infected with viruses or transfected with plasmids were washed with cold PBS and fixed with cold 4% buffered formalin for 30 min at room temperature (RT). After washing with PBS, the cells incubated with primary antibodies at 37 ℃ for 1.5 h. After washing, the cells were incubated with goat anti-mouse IgG conjugated to Texas-Red (Proteintech, Wuhan, China) at 37 ℃ for 1 h. After washing, the cell nuclei were stained with Hoechst 33258 at RT for 8 min. The images were photographed with a NIKON fluorescence microscope (Tokyo, Japan).

    • BHK-21 cells were pre-fixed with 2.5% glutaraldehyde at 4 ℃. After washing in PBS for three times, the cell samples were post-fixed with 1% osmium tetroxide. After washing, samples were dehydrated in acetone, and then embedded and sectioned. Ultrathin Sects. (100 nm) were examined by using a transmission electron microscope (Tecnai G2 20 TWIN).

    • Each experiment was reproducible and repeated in triplicate. All the data were analyzed by Student's t-test and analysis of variance (ANOVA). There were significant differences if probability (P) values were of less than 0.05, 0.01, and 0.001, which marked with *, **, and ***, respectively.

    • The WH2012 strain of TBEV has undergone several passages in our lab since it was first isolated. To determine the genetic homogeneity in the viral stock, RT-PCR products were amplified from the viral genome and cloned into the T vector. Multiple clones targeting at the same viral segment were sequenced. As shown in Table 1, there were thirteen non-synonymous substitutions that resulted in ten amino acid mutation in the parental WH2012 strain. In addition, there were also two nucleotide mutations and a deletion of two nucleotides in 5' UTR as well as twenty-three synonymous substitutions in the ORF region. As shown in Fig. 1, TBEV Pn replicated well and caused strong cytopathic effect (CPE) which could be used in endpoint dilution assay on BHK-21 cells (TCID50). However it did not produce plaques in BHK-21 cells (Fig. 1C). Even after five rounds of blind passages, TBEV Pn+5 still did not produce visible plaques in BHK-21 cells (Fig. 1A and 1C). In addition, qRT-PCR was also performed to analyze the viral copies of the serially passaged supernatants. Both TBEV Pn and TBEV Pn+5 reached viral loads of > 108 copies/mL with no significant differences (P > 0.05, Fig. 1D). Thus, there were a variety of variants in the present viral stock, the consensus sequence of the original WH2012 virus (each nucleotide only confirmed in more than 75% of the cloned T vectors was adopted as the consensus nucleotide) was used in the following construction of infectious TBEV cDNA clone.

      Base position WH2012 Sequencing resultsa Amino acid change Location
      50-52 - CA - 5'UTR
      71 C T - 5'UTR
      75 T C - 5'UTR
      3498-3499 TC CG S-R NS1347
      3597 G A G-R NS2A28
      5811 A G N-D NS3405
      6132 C G R-G NS3512
      6281 T C - NS3561
      6317 C T - NS3573
      6404 T C - NS3602
      7207 C T A-V NS4B100
      7408 C T P-L NS4B167
      7670 T C - NS52
      8561 C T - NS5299
      9468 C G R-G NS5602
      9471 A T T-S NS5603
      9475 A G E-G NS5604
      9503 C T - NS5613
      9506 T A - NS5614
      9612-9614 AAC GAT N-D NS5650
      9659 T C - NS5665
      9743 G A - NS5693
      9791 C T - NS5709
      9794 T C - NS5710
      9824 A G - NS5720
      9836 C T - NS5724
      9914 C T - NS5750
      9920 T C - NS5752
      9968 T C - NS5768
      10, 121 T C - NS5819
      10, 145 C T - NS5827
      10, 199 C T - NS5845
      10, 226 G A - NS5854
      10, 247 T C - NS5861
      10, 304 C T - NS5880
      10, 334 T C - NS5890
      aEach nucleotide substitution was observed and confirmed in more than 75% of the cloned T-vectors.

      Table 1.  Sequence differences in the parental WH2012 virus.

    • The full-length TBEV cDNA clone, TBEV-FL, was constructed based on the above consensus sequence of the WH2012 strain. As is shown in Fig. 2, we used a low-copy vector pACYC177 which works efficiently in the ZIKV infectious clone (Li et al. 2018). A cytomegalovirus (CMV) promoter was engineered right before the authentic 50 end of the viral sequence for in vivo transcription, and the connecting of a HDVr sequence and a SV40 poly(A) was placed downstream the 3' UTR to ensure that the 3' end of the viral RNA was generated correctly. Synonymous substitutions of C5859T, G8416A, and C9354T were introduced to delete the three redundant KpnI, XhoI, and KpnI sites. Synonymous substitutions of C1088A and G4464C were introduced to generate the single ClaI and BinI sites. During cloning of TBEV-FL, we found that fragment B, spanning the viral E-NS1-NS2A-NS2B genes, was unstable in E. coli, even with the use of pACYC177, leading to aberrant deletions/mutations in plasmids. To circumvent this issue, we inserted an intron in NS1 to disrupt the sequence of ORF, and postulated that the viral RNA would be spliced and restored in mammalian cells (Fig. 2). Overall, TBEV-FL assembled as depicted in Fig. 2 propagated stably E. coli, and sequence analysis of the plasmid revealed no nucleotide substitution.

    • BHK-21 cells were transfected with TBEV-FL to characterize its transcription in vivo and protein expression. Meanwhile, BHK-21 cells transfected with pACYC177 were used as the negative control, and BHK-21 cells infected with the wild-type TBEV (TBEVwt) at an MOI of 0.01 were used as the positive control. Three days later, total RNAs of the transfected cells were extracted and subjected to qRT-PCR analysis. As is shown in Fig. 3A, significant increasing copies of viral RNA were detected in the TBEV-FL-transfected cells. To further determine if splicing occurs in the transfected cells, total RNA was extracted from the supernatants at 4 d.p.t or infection with TBEVwt. A DNA fragment that contains the intron was amplified with a pair of primers surrounding the intron (Supplementary Table S1). As is shown in Fig. 3B, the similar single DNA fragments in size were amplified from the supernatants of both transfection with TBEV-FL and infection with TBEVwt. Sequence analysis of these RTPCRs showed that all the fragments from the supernatants of transfection with TBEV-FL were found in the absence of the inserted intron (Fig. 3C), which meant the introninterrupted fragments were recreated in BHK-21 cells.

      Figure 3.  Characterization of viral RNA replication and splicing in TBEV plasmid-transfected cells. A Viral copies in cells transfected with TBEV-FL were determined at 3 d.p.t. by qRT-PCR. B Agarose gel analysis of RT-PCR products from the supernatants of the TBEV-FL-transfected cells at 4 d.p.t. M, Marker; band 1, Water; band 2 and 3, Unspliced TBEV-FL; band 4, MOCK; band 5, Supernatants of transfection with TBEV-FL; band 6, Parental WH2012. C Sequence results of RT-PCR products from the supernatants of the cells transfected by TBEV-FL. The two reference sequences were parental WH2012 virus and unspliced TBEV-FL plasmid. Data shown (A) are the mean ± SD analysed by Student's t-test (two tailed) (***, P < 0.001).

    • To analyze the infectious TBEVic production, BHK-21 cells transfected with TBEV-FL were used to rescue infectious virion. Obvious CPEs were observed in the transfected cells since 2 d.p.t. (Fig. 4A). After harvesting the supernatant, the robust expression of NS5 was detected by Western blotting in cell samples (Fig. 4B), in another way, suggesting that the inserted intron was precisely excluded in mammalian cells, otherwise, the frameshift mutations would generate in the polyprotein following closely on the heels of the inserted intron. Though the parental WH2012 virus did not produced plaques (Fig. 1C), plaque assay was still performed in BHK-21 cells with the supernatant (TBEVic) harvested from transfected cells. Unexpectedly and interestingly, classic plaques with a size of 0.8–0.9 mm in diameter were observed at 4 d.p.i. (Fig. 4C, 4E) with a peak titer of 106.39±0.41 PFU/mL. Protein E and NS5 expression in BHK-21 cells infected with TBEVic could be also detected by IFA (Fig. 4D, 4F). To visualize the ultrastructural features of TBEVic directly, TEM assay was used to determine the infected cells. As is shown in Fig. 5, BHK-21 cells infected with TBEVic and TBEVwt presented with similar spherical particles ranging from 35 to 50 nm in diameter. Collectively, these data demonstrated that infectious TBEVic could be rescued by TBEV-FL cDNA clone.

      Figure 4.  Rescue of TBEVic from the infectious TBEV clone in cell culture. A The CPE on BHK-21 cells transfected with the TBEV-FL at 2 d.p.i. All the images were captured at a × 20 magnification. B Western bolt analysis of TBEV NS5 protein expression in BHK-21 cells transfected with the TBEV-FL. C and E The plaque morphology of the TBEVic virus in BHK-21 cells, visualized using 0.5% crystal violet solution following incubation for 4 days. D IFA of TBEV E protein expression in BHK-21 cells infected with either TBEVic or mock-infected at 2 d.p.i. (MOI = 0.02), Red represents TBEV E protein, and blue represents nuclei (stained with Hoechst 33, 258). F IFA of TBEV NS5 protein expression in BHK-21 cells infected with either TBEVic or mock-infected at 2 d.p.i. (MOI = 0.1). Red and blue represent TBEV NS5 protein and nuclei (stained with Hoechst 33258), respectively. D and F All the IFA images were captured at a × 10 magnification.

      Figure 5.  Transmission electron micrographs of BHK-21 cells infected with TBEVic and TBEVwt. Red arrows represented sphere particles in BHK-21 cells infected with the corresponding viruses. The right panels showed the enlarged views of the corresponding regions in the left panels. Scale bars are 2 lm and 500 nm for low magnification images and the high magnification images, respectively.

    • To compare the replicative fitness of the rescued and parental WH2012 virus, BHK-21 cells were infected by the two viruses at the same MOIs and the infectious titers were determined by a standard TCID50 assay and a qRT-PCR assay. As is shown in Fig. 6A, both TBEVic and the parental virus caused similar typical CPEs, such as cell rounding, necrosis, and detachment. Compared with the parental TBEVwt, TBEVic exhibited a slightly low replication kinetic in BHK-21 cells, however, there was no significant differences (P > 0.05) before 4 dpi (Fig. 6B). Both TBEVic and TBEVwt reached undifferentiated maximum viral titers (106.88±0.17 TCID50/mL and 107.13±0.12 TCID50/mL, P > 0.05) at 3 dpi (Fig. 6B). Only the endpoint titer of TBEVic was significant lower than that of the parental TBEVwt (P < 0.05, Fig. 6B). We next compared the viral RNA levels of TBEVwt and TBEVic at different time points after infection of BHK-21 cells. Results demonstrated that there were no significant differences between TBEVwt and TBEVic in RNA replication kinetics. Overall, infectious TBEVic reproduced from the infectious clone of TBEV-FL exhibited the similar replicative characteristic to its parental virus WH2012, enabled extensive analysis of the biological properties, potential virulent determinants, and immunogenicities of TBEV in vitro and in vivo.

      Figure 6.  Replication of TBEVic and the parental TBEVwt virus in cell culture. A Apparent and similar CPE of TBEVic and TBEVwt on BHK-21 cells. B and C The supernatants from BHK-21 cells transfected with the full-length TBEV-NS5DGDD (the conserved polymerase motif GDD in NS5 was mutated to GAA, G663G-D664A-D665A) cDNA clone were used as the replication-deficient negative control. B BHK-21 cells were infected with viruses at an MOI of 0.1. Virus titers were measured by a TCID50 assay on BHK- 21 cells. C BHK-21 cells were infected with viruses at an MOI of 0.0001. Viral RNA copies were measured by a qRT-PCR assay on BHK-21 cells. Data shown (B and C) are the mean ± SD analyzed by Student's t-test (two tailed) (*, P < 0.05).

    • The prevalence of TBEV in the Eurasian continent and the concomitant association with CNS diseases in humans highlighted the importance of studying the pathogenesis and replication, and developing countermeasures against it. In this study, we reported the construction of a stable infectious cDNA clone for the WH2012 strain of TBEV. The plasmid clone could be used to generate infectious TBEVic that exhibited comparable replicative properties to its parental version, which possessed a tractable platform in the fundamental and applied research of TBEV.

      Two main strategies have been reported to rescue TBEVs. One strategy was to obtain long PCR products by using long high-fidelity RT-PCR, and then to clone these products into the plasmid pBR322 or its derivate (pGGVS209) (Mandl et al. 1997; Gritsun and Gould 1998; Hayasaka et al. 2004; Takano et al. 2011). Another strategy was called infectious subgenomic amplicons (ISA), a bacterium-free approach (de Fabritus et al. 2016; Haviernik et al. 2020). The ISA was based on the infectious complete TBEV genome would be recombined and synthesized in susceptible cells that were transfected by several subgenomic overlapping DNA fragments covering the entire TBEV genome (de Fabritus et al. 2016; Haviernik et al. 2020). The method of ISA made it possible to rescue of infectious TBEV in days without the need for cDNA propagation in bacteria (Aubry et al. 2014; de Fabritus et al. 2016; Haviernik et al. 2020). In our first attempt, a low-copy vector pACYC177 which generates 15 copies per E. coli cell (Shan et al. 2016) was used as the backbone to construct the Full-Length TBEV cDNA Clone. However, the viral RNA of the WH2012 strain was genetically unstable in E. coli even with the use of pACYC177, and the sequencing results revealed aberrant deletions/mutations appeared in plasmids. We attributed this issue to the toxicity of cryptic promoters in the genome of the WH2012 strain for bacterial (Pu et al. 2011; Tsetsarkin et al. 2016). As the toxic sequence within viral genome could be disrupted by the insertion of an intron, along with that this strategy has been successful applied to the recovery of ZIKV in our previous study (Li et al. 2018), we inserted a beta-globin intron in the coding region of NS1 gene of the WH2012 isolate to improve the stability of viral genome in bacterial. We also launched viral RNA replication by the driving of CMV promoter in vivo, which, compared with conventional T7 or SP6 promoter that used for in vitro transcription, guaranteed the splicing of the intron from the viral genome (Tsetsarkin et al. 2016). Considering that intron retention is common for some kinds of viruses in nature (Rekosh and Hammarskjold 2018), we could not entirely exclude the possibility of intron retention in mature mRNAs of the transfected cells (Wong et al. 2016). In fact, not any retention of the beta-globin intron in NS1 was detected in our study. We believed that even if the inserted intron was not spliced and restored correctly in mammalian cells in rare cases, the resulting mRNAs would also be the "dead end" products (Rekosh and Hammarskjold 2018), and have little effect on the recovery of virus.

      Phylogenetic analysis showed that the WH2012 isolate is most closely associated with other Chinese TBEV isolates MDJ01, Senzhang, MDJ-02, Xinjiang-01, and MDJ-03 within the Far-Eastern subtype, may exhibit the conserved pathogenic properties among Chinese TBEV isolates (Zhang et al. 2016b). To date, all the TBEV strains isolated in China belonged to the Far-Eastern subtype (Zhang et al. 2016b; Sun et al. 2017; Yoshii et al. 2017; Dai et al. 2018), and only one Siberian subtype of TBEV was detected (not isolated) in the Xinjiang Uygur Autonomous Region (northwest China) (Liu et al. 2016). There were relatively abundant epidemiological characteristics of TBEV isolated from China, however, the relevant research regarding the virulence and other biological properties of was limited (Zhang et al. 2016b; Sun et al. 2017; Yoshii et al. 2017; Chen et al. 2019). In a previous study, we determined the potential CNS tropism of the WH2012 isolate, a Chinese TBEV strain, in both in vitro and in vivo models for the first time (Zhang et al. 2016b). The full-length TBEV cDNA clone of WH2012 strain described here could be important instrument in the investigation of these biological properties.

      Virus generated from the previously Sofjin-HO strain of TBEV reverse genetics systems exhibited no significant difference for growth kinetic with the parental viruses (Takano et al. 2011). Here, our rescued WH2012 virus also showed comparable growth curve to its parental isolate, although its endpoint titer was significantly lower in BHK-21 cells. The parental WH2012 virus replicated well and caused CPE in BHK-21 cells, however, even after n+5 rounds of blind passages, it still did not produce visible plaques in BHK-21 cells. A striking contrast was that the rescued WH2012 virus generated classic plaques with a size of 0.8–0.9 mm in diameter, which may possibly be interpreted by the presence of defective interfering particles in the original parent virus population. Because the clone-derived WH2012 virus harbored the actual consensus sequence of the original WH2012 virus, it could accurately represent most of the population of TBEVwt, and produced plaques, like other TBEV strains (Takano et al. 2011; Frey et al. 2012; Leonova et al. 2014; Luat le et al. 2014; Eyer et al. 2019). Based on quasispecies theory, the parental WH2012 virus population probably was not only a population of diverse mutants, but also a collection of interactive variants (Elena et al. 2006; Vignuzzi et al. 2006), which exhibited great genetic diversity, and contained few defective interfering particles. The interfering particles might have an effect on the host cells, and resulted in that no plaque formed in the infected cells, which warranted further investigations.

      In summary, we report the first, efficient, and stable infectious cDNA clone for TBEV isolated in China, which allow for generating allelic variants, chimeric viruses, reporter viruses, and functional subgenomes easily from different standpoints. Thus, the infectious clone of TBEV provided an essential platform for determining the mechanisms of epidemicity, transmission, virus-host interaction, replication, and pathogenesis of the virus, and furthering the new preclinical evaluation of vaccines, drugs, or therapeutic antibodies for TBEV.

    • This study was supported by grants from the National Key R&D Program of China (2018YFA0507201). We thank Hao Tang from biosafety level 3 (BSL-3) laboratory of the Wuhan Institute of Virology, Chinese Academy of Sciences, for support in BSL-3 laboratory operations. We would like to thank Pei Zhang and An-na Du from The Core Facility and Technical Support, Wuhan Institute of Virology, for their help with producing TEM micrographs.

    • ZHZ conceived and designed the study. PHL and CY developed the methodology and performed the experiment. PHL and ZHZ performed the formal analysis. T Wang, T Wu, WFY, YZ, YJM and JHS contributed to the investigation. YL and XWZ provided the resources. CY and PHL performed the data curation. PHL and ZHZ contributed to writing—original draft preparation. ZHZ and HZW helped perform writing—review and editing. ZHZ performed the supervision and conducted the project administration. All authors read and approved the final manuscript.

    • The authors declare that they have no conflict of interest.

    • This article does not contain any studies with human or animal subjects performed by any of the authors.

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