Fig 1.
Growth of Colorado tick fever virus (CTFV) strains in various cell lines.
(A) Vero cells were infected with eight CTFV strains at a multiplicity of infection (MOI) of 0.01. The samples were collected 72 hours post-infection (hpi). (B) Plaque formations by the CTFV strains in Vero cells are shown. The cells were fixed and stained 7 days post-infection. The high-resolution picture was taken using a Cytation 5 Cell Imaging Multimode Reader and stitched with each picture is shown as a figure. The plaques are indicated with white arrow heads. (C) Various cell lines derived from rodents (yellow), monkeys (gray), humans (orange), and avian (blue) infected with the CTFV Florio strain at an MOI of 0.01. The samples were collected at 0, 24, and 48 hpi. (D) HeLa and L929 cells were infected with the CTFV Florio strain at an MOI of 0.01. The samples were collected at 0 hpi. The 0 hpi sample was defined as collection after infection for 1 h, followed by washing the cells and soaking them in complete medium. The viral titers of frozen–thawed samples were measured by performing a plaque assay. The limit of detection (LoD) is indicated as a broken line. The experiments were repeated at least twice to confirm the result. Three biological and two technical replicates were conducted for each independent repeat.
Fig 2.
Generation and characterization of the recombinant Colorado tick fever virus (CTFV).
(A) Schematic diagram of the reverse genetics system for CTFV established in this study. Each viral genome was cloned downstream of T7 RNA polymerase (T7P) and linked with the hepatitis D virus ribozyme (Rbz). (B) Nucleotide alignments of CTFV segment 7 (S7). The EcoRI digestion site is indicated by gray hatching. The synonymous mutation is indicated as a red, bold letter. (C) Schematic diagram of the amplicon obtained via RT-PCR, and images of the digested amplicons of S7 after electrophoresis. The black arrows indicate the primers. The electrophoresis was conducted using a 1% agarose gel. (D) Image of dsRNA genomes of CTFV strains after electrophoresis. The electrophoresis was conducted using a 6% polyacrylamide gel without SDS. (E) Growth kinetics of CTFV strains. Vero cells were infected with CTFV strains at a multiplicity of infection of 0.01, and samples were collected at each time point. The viral titers of frozen–thawed samples were measured by performing a plaque assay. The viral growth of rFlorio was compared with that of a parental CTFV Florio strain. The experiments were repeated at least twice to confirm the result. Three biological and two technical replicates were conducted for each independent repeat.
Table 1.
Reverse genetics system for CTFV using co-culture with various cell lines.
Fig 3.
Characterization of recombinant Colorado tick fever virus (CTFV) reassortment between the Florio strain and other CTFV strains.
(A) Schematic diagram of recombinant CTFV monoreassortants. The monoreassortants, in which segment 6 was reassorted between the Florio and R111081 strain (rFlorio/R111081-S6) or SS-18 (rFlorio/SS-18-S6), were generated using the reverse genetics system. (B) Image of dsRNA genomes of CTFV monoreassortants after electrophoresis. The electrophoresis was conducted using a 6% polyacrylamide gel without SDS. (C, D) Growth kinetics of CTFV monoreassortants. Vero cells were infected with CTFV strains at a multiplicity of infection (MOI) of 0.01. The samples were then collected at each time point. The viral titers of frozen–thawed samples were measured by performing a plaque assay. The viral growth of CTFV monoreassorted with R111081 (C) or SS-18 (D) strains was compared with that of a recombinant wild-type CTFV (rFlorio). (E) Growth of the CTFV monoreassortant in different cell lines. Vero and L929 cells were infected with CTFV strains at an MOI of 0.01, and samples were collected 48 hours post-infection. The viral titers of frozen–thawed samples were measured by performing a focus assay using an anti-VP5 antibody. (F) Predicted protein structural model of CTFV Florio strain VP6. The structure was developed using ColabFold v1.5.5. The P526 amino acid is highlighted by the red circle. Significant differences were determined by performing a two-way or one-way ANOVA. The experiments were repeated at least twice to confirm the result. Three biological and two technical replicates were conducted for each independent repeat. **0.005<p<0.01, ***p<0.005.
Fig 4.
Characterization of the recombinant Colorado tick fever virus (CTFV) harboring peptide tag-fused VP12.
(A) Schematic diagram of CTFV segment 12 encoding VP12. A recombinant CTFV harboring FLAG tag-fused VP12 (rFlorio/VP12-FLAG) was obtained by inserting a FLAG tag at the C-terminus of VP12. rFlorio/VP12-FLAG was generated using the reverse genetics system. The genome was amplified via RT-PCR using specific primers (black arrow). (B) Image of dsRNA genomes of rFlorio/VP12-FLAG after electrophoresis. The electrophoresis was conducted using a 6% polyacrylamide gel without SDS. Images of the amplicons of segment 12 after electrophoresis are shown below. (C) Growth kinetics of CTFV strains. Vero cells were infected with CTFV strains at a multiplicity of infection of 0.01. The samples were collected at each time point, and viral titers of frozen–thawed samples were measured by performing a plaque assay. The viral growth of rFlorio/VP12-FLAG was compared with that of a recombinant wild-type CTFV (rFlorio). (D) Western blotting of rFlorio/VP12-FLAG-infected cells. The FLAG tag-fused VP12 was detected using an anti-FLAG antibody. β-actin was detected as the loading control. The experiments were repeated at least twice to confirm the result. Three biological and two technical replicates were conducted for each independent repeat.
Fig 5.
Optimization of the reverse genetics system and characterization of the recombinant reporter Colorado tick fever viruses (CTFVs).
(A) Activity of T7 RNA polymerase in Expi293F-T7 cells. The cells were transfected with pT7-sNLuc plasmids encoding secreted NLuc gene. After incubation, the NLuc activity of the supernatant was measured. (B) Schematic diagram of the improved reverse genetics system for CTFV. Each viral genome was cloned downstream of T7 RNA polymerase (T7P) and linked with the hepatitis D virus ribozyme (Rbz). (C) Schematic diagram of the viral genomes of the recombinant reporter CTFVs. A recombinant CTFV harboring NLuc (rFlorio/VP12-2A-NLuc) or mStayGold (rFlorio/VP12-mSG) in VP12 were obtained by inserting the 2A peptide-linked NLuc or mStayGold genes at the C-terminus of VP12, respectively. These reporter CTFVs were generated using the improved reverse genetics system. (D) Growth kinetics of CTFV strains. Vero cells were infected with CTFV strains at a multiplicity of infection (MOI) of 0.01. The samples were collected at each time point, and viral titers of frozen–thawed samples were measured using a plaque assay. The viral growth of the reporter CTFVs (rFlorio/VP12-2A-NLuc, rFlorio/VP12-mSG) was compared with that of a recombinant wild-type CTFV (rFlorio). (E) NLuc activity of the cells infected with the recombinant CTFV. Vero cells were infected with CTFV strains at an MOI of 0.01. The samples were collected at each time point, and the NLuc activity of frozen–thawed samples was measured. (F) The localization of the mStayGold-fused VP12 in recombinant CTFV-infected Vero cells. The scale bar represents 10 μm. Significant differences were determined by performing a two-way or one-way ANOVA. The experiments were repeated at least twice to confirm the result. The experiments were repeated at least twice to confirm the result. Three biological and two technical replicates were conducted for each independent repeat. **0.005<p<0.01, ***p<0.005.