Initially we failed to generate minireplicon using the UTRs identical to that of SFTSV-WCH published in GenBank (data not shown). As UTR sequences were critical for constructing reverse genetic systems (Brennan et al. 2015), we then determined the UTRs sequences of adaptedSFTSV-WCH by 3' and 5' RACE. Sequencing of the RACE products revealed several nucleotide changes in all the L/M/S-UTRs of adapted-SFTSV-WCH (Fig. 1, shown in red bases) in comparison to the published SFTSV-WCH sequences (Fig. 1, shown in blue bases in the brackets). For the L segment, we found a change of A to G at position 6 in comparison to the SFTSV-WCH sequence in GenBank (JQ341188.1). For the M segment, two differences were detected at position 6 (A to G) and position 3353 (A to G) comparing to the GenBank sequence (JQ341189.1). For the S segment, we found an additional A at position 10 and one change at position 23 (A to G), compared to the GenBank sequence JQ341190.1 (Fig. 1). The terminal sequences of the 3'- and 5' UTR for SFTSV were complementary, which permits the formation of a 'panhandle' structure for each segment. The first 9 nt of both 3' and 5' termini were relatively conserved among all segments and comprised the conserved complementary region (Fig. 1, sequences in the red box). After these 9 nts, the sequence of the L, M, and S segments were segment specific but still exhibit terminal complementarity up to nucleotide positions 16, 18, and 23, respectively (Fig. 1, sequences in the blue box). In comparison to that of SFTSV-WCH, the adapted-SFTSV-WCH had a unpaired nucleotide at position 6 of the conservation complementary regions of L and M UTRs, and two nucleotides difference (position 10 and 23) at the variable complementary region of S UTR (Fig. 1).
Figure 1. Schematic of the 3'- and 5' UTRs of complementary viral RNA. The sequences of 5' UTR were present in capital letters, and the sequence of 3' UTR were present in lowercase letters. The corrected nucleotide of the adapted-SFTSV-WCH by RACE analysis was shown in red compared to these published SFTSV-WCH sequences (blue bases in the brackets). Potential to form Watson–Crick base pairs (dash in red) or noncanonical U-G pairings (dash in green) is indicated. The first 9 nt of both 3' and 5' termini are relatively conserved between all segments and comprise the conserved complementary region (red boxed sequences), except noncanonical U-G pairings. The variable complementary region of each segment is shown as blue boxed sequences.
Establishment of the minireplicon system is the first step to ensure that the cloned polymerase is functional and the replication signals are correct before attempting full virus rescue. Therefore, we firstly setup a minireplicon system for SFTSV. The plasmids expressing NP (pCAGGS-NP) and L proteins (pCAGGS-RdRp) were constructed as described in Material and Methods, and the sequences of the ORFs were confirmed to be consistent with those of SFTSV-WCH in the GenBank. The plasmids pT7-L-UTReGFP, pT7-M-UTR-eGFP, and pT7-delNSs: eGFP which contained a reporter gene eGFP in the negative sense between the corrected 3'UTR and 5'UTR of cRNA of the L, M and S segments were constructed as described in Materials and Methods (Fig. 2A).
Figure 2. Creation of the L/M/Sbased minigenome constructs. A Schematic diagram of the generation of L-, M-, and S-based reporter minigenomes. B Effect of increasing amounts of pCAGGS-RdRp (upper panel) or pCAGGS-NP (lower panel) on M-segment minigenome assay. BSR-T7 cells were co-transfected with pT7-M-UTR-eGFP (0.5 μg), and the indicated amounts of pCAGGS-RdRp and pCAGGSNP. C Generation of L/M/Sbased minigenomes. BSR-T7 cells were transfected with the minigenome plasmid of pT7-LUTR-eGFP, pT7-M-UTR-eGFP or pT7-delNSs: eGFP (UTReGFP); or co-transfected with the minigenome plasmid and pCAGGS-NP (NP + UTReGFP); the minigenome plasmid and pCAGGS-RdRp (RdRP + UTR-eGFP); or the minigenome plasmid with pCAGGS-NP and pCAGGSRdRp (RdRp + NP + UTReGFP). At 36 h post transfection, fluorescence of eGFP was observed. Scale bar: 400 μm.
To optimize the conditions for minigenome system, various ratios of the plasmids were used in order to maximize minigenome replication efficiency. M segmentsbased minigenome plasmid (500 ng pT7-M-UTR-eGFP), and 500 ng pCAGGS-NP with increasing amounts of pCAGGS-RdRp were co-transfected into BSR-T7 cells (Fig. 2B, upper panel). Increasing amounts of RdRp protein in the system generally led to an increase in minigenome activity, and superior effect was achieved with 1,000 ng pCAGGS-RdRp. Then 1,000 ng pCAGGS-RdRp and increasing amounts of pCAGGS-NP were transfected into BSR-T7 cells (Fig. 2B, lower panel), and the optimal effect was obtained with 500 ng pCAGGS-NP and 1,000 ng pCAGGS-RdRp. Therefore, the optimal amounts of the ribonucleoproteins were used for further experiments.
Then the L, M, S-based minigenomes were generated with this optimal amount of the ribonucleoproteins by cotransfection. At 36 h p.t., significant levels of eGFP were expressed from each of the minigenomes, and no green fluorescence was seen in the negative control groups (Fig. 2C). These results revealed that each minigenome RNA contained the correct cis-elements in UTRs, and pCAGGS-NP and pCAGGS-RdRp provided functional trans-factors required for transcription and replication.
In order to recover infectious SFTSV from cloned cDNA, the complete L, M, and S segments were cloned between a T7 promoter and a HDVR sequence in viral complementary orientation, resulting in pT7-L, pT7-M, and pT7-S, respectively as described in Material and Methods (Fig. 3A, left). Initial attempts for generating infectious virus by co-transfecting plasmids pT7-L, pT7-M, and pT7- S into BSR-T7 cells, as previously demonstrated successful for other bunyaviruses (Habjan et al. 2008), failed to rescue the SFTSV in our hands (data not shown). To improve the rescue efficiency of the system, we supplemented NP and RdRp via co-transfection with expression vectors pCAGGS-NP and pCAGGS-RdRp (Fig. 3A, left). A mixture of pCAGGS-NP, pCAGGS-RdRp, pT7-L, pT7-M, and pT7-S were co-transfected into monolayers of BSR-T7 cells, and the rescued virus at passages 0–3 (P0–P3) were collected as described in Materials and Methods. Infectivity of the P0–P3 viruses were detected in Vero cells by immune fluorescence assay using the anti-NP antibody. As showed in Fig. 3B, initially only a small amount of fluorescence signals were detected in P0 cells, but the amounts of infected cells increased with successive passaging, and the positive rate of viral infected cells had reached approximately 100% in the P3, indicating the infectious recombinant SFTSV virus has been successfully rescued. The rescued virus was named rSFTSV-WCH.
Figure 3. Rescue of infectious virus from cDNA. A General outline of the procedure to generate the rescued virus from cDNA. BSR-T7 cells were transfected pCAGGS-NP, pCAGGS-RdRp, pT7-S, pT7- M, and pT7-L. B Detection of the infectivity of the collected supernatants from passage 0 (P0) to P3 by immune fluorescence using the anti-NP antibody. The infectivity of the rescued virus was increasing with passaging from P0–P3, and the positive rate of fluorescent cells had reached 100% in P3. Scale bar: 400 μm.
To analyze the properties of rSFTSV-WCH, immunostaining assay was conducted using virus-containing supernatant collected from P8. The rescued virus showed foci with similar size and morphology to that of the adapted-SFTSV-WCH (Fig. 4A). Titration analyses revealed that repeated passaging of the rSFTSV-WCH substantially increased virus yield from 4.5 × 103 PFU/mL at P3 to 7.9 × 105 PFU/mL at P8, but the titer of P8 was still lower than that of the adapted-SFTSV-WCH which had a titer of 3 × 107 PFU/mL (Fig. 4B).
Figure 4. Characterization of rSFTSV-WCH in comparison to the adapted-SFTSV-WCH. A Comparison of immune-stained foci of rSFTSV-WCH and adapted-SFTSV-WCH. B The titration of rSFTSV-WCH collected at different passages. The titer of rSFTSVWCH increased from P3 to P8 but was lower than that of the adaptedSFTSV-WCH. C One-step growth curves of rSFTSV-WCH and adapted-SFTSV-WCH conducted in Vero cells at an MOI of 1. P values of < 0.01 indicated extremely statistical differences between two groups at any time point. D Time-course analysis of nucleoprotein (NP) expression in infected cells. Vero cells were infected with rSFTSV-WCH or adapted-SFTSV-WCH at an MOI of 1, and cellular samples were harvested at the indicated time points for Western blot analysis using anti-NP antibody.
One-step growth curves of rSFTSV-WCH and adaptedSFTSV-WCH were compared in Vero cells at an MOI of 1. As shown in Fig. 4C, the adapted-SFTSV-WCH grew more efficiently, achieving the maximal titers of 3 × 107 PFU/mL at 96 h p.i., while the rSFTSV-WCH grew to the highest titers of 5 × 105 PFU/mL at 120 h p.i.. And the titers between adapted-SFTSV-WCH and rSFTSV-WCH showed extremely significant difference (P < 0.01) at any time points of 12 h, 24 h, 48 h, 72 h, 96 h or 144 h p.i. (Fig. 4C).
Then, the time-course analysis of NP expression was performed by Western blotting. Cells were separately infected with rSFTSV-WCH and adapted-SFTSV-WCH at an MOI of 1, and cellular samples were harvested at 12, 18, 24 and 48 h p.i. As shown in Fig. 4D, NP could be detected at 12 h p.i. in cells infected with adapted-SFTSV-WCH, while it was not detected in rSFTSV-WCH infected cells until 18 h p.i. For both viruses, the intensity of the NP signal keep increasing until 48 h p.i. (Fig. 4D), but the expression level of NP was lower in the rSFTSV-WCH infected cells than that of the adapted-SFTSV-WCH. These results revealed that the rSFTSV-WCH had lower infectivity than the adapted-SFTSV-WCH.
During the construction of the reverse genetic system, all the inner sequences of the L, M, S segments were confirmed to be consistent to the SFTSV-WCH sequences in the GenBank, while the UTRs have been corrected according to the RACE results of the adapted-SFTSVWCH. To understand why rSFTSV-WCH had lower infectivity in comparison to that of the adapted-SFTSVWCH, both viruses were sequenced by RNA-seq and the results were summarized in Table 1. As expected, the UTR sequences of the rSFTSV-WCH were identical to that of adapted-SFTSV-WCH, and the internal L, M and S sequences of rSFTSV-WCH were identical to that of the SFTSV-WCH sequences in GenBank. The adaptedSFTSV-WCH, however, appeared to be a mixture that contained two different nucleotides at nine positions of the internal segments, including 3 positions in L, 5 positions in M, and 1 in S (Table 1). In each of these positions, one of the nucleotides was consistent with the published SFTSVWCH sequence in GenBank, while the other one appeared to be a new mutation. Apart from the mutation (G) at the 499 nt of M segment which has been appeared in other published SFTSV strains, the rest of the mutations have not been found in any SFTSV strains in the GenBank. Among the three mutations in the L segment, only the one at the 5967 nt position would result in amino acid change (G to E) in the encoding protein, which is not located in the functional motifs of RdRp (Amroun et al. 2017). Among the five mutations in the M segment, four (nts 117, 499, 2268 and 3028) resulted in amino acid changes in the encoding proteins. Two of the mutations are within domain I of Gn and one locates in the domain I of Gc (Table 1). Domain I of Gn forms the foundation bed with domain II, while domain I of Gc contributes to pH-induced conformational rearrangemen (Halldorsson et al. 2016; Wu et al. 2017). For inner sequence of S, there was one new mutation at the position of 556 resulting in amino acid changes (S to T) and this mutation was located between α10 and α11, relatively far from the RNA binding cavity (Jiao et al. 2013).
Segment Positiona Nucleotide (amino acid) Other strainsb Functional domainc SFTSV-WCH (GenBank) rSFTSVWCH Adapted-SFTSVWCH L 28 G (E) G (E) G/A (E/E) No - 2041 C (V) C (V) C-T (V/V) No - 5967 G (G) G (G) G/A (G/E) No Not in the functional motifs M 117 C (N) C (N) C/A (K/N) No Gn (domain I) 499 A (R) A (R) A/G (R/G) Yes Gn (domain I) 2268 C (S) C (S) C/A (S/R) No Gc (domain I) 2778 C (S) C (S) C/T (S/S) No - 3028 T (Y) T (Y) T/C (Y/H) No Gc (not in the functional domain) S 556 G (S) G (S) G/C (S/T) No Between α10 and α11 of NP aPosition in nucleotide sequence.
bWhether the mutations appeared in other reported SFTSV strains in GenBank.
cOnly the mutations which cause amino acid changes were indicated and whether they located in the functional domain of the respective protein or not; –indicated mutations did not cause amino acid changes.
Table 1. Comparison of the inner sequences of SFTSV-WCH, rSTFTSV-WCH and adapted-SFTSV-WCH.