Fig 1.
Characteristics of passaged SFTS virus.
Confluent monolayers of HeLa cells were inoculated with the original or passaged Hp50-4 strain at a multiplicity of infection of 0.025 and cultured for 3 days. (a) Culture supernatants harvested at the indicated days were titrated in Vero cells. The means and standard deviations are shown (n = 3). Statistical comparisons were performed between the means for each day (Welch’s t-test). (b) Inoculated HeLa cells were fixed at 3 days post inoculation and stained with rabbit anti-NP serum. (c) Ifnar-/- mice were subcutaneously inoculated with the indicated 50% tissue culture infectious doses of the original or passaged Hp50-4 strain (five mice per group) and observed until 14 days post inoculation. Survival curves are shown.
Fig 2.
Determinants of Hp50-4 strain phenotypes (in vitro).
Recombinant viruses were produced using plasmids encoding SFTS viral genomes and used to infect HeLa cells. (a) Schematic segment combinations (L, M, and S) of the produced viruses are shown. Blue and orange squares are from the original and Hp50-4 strains, respectively. Note that the S segment sequence of the Hp50-4 strain was identical to that of the original strain; therefore, the same plasmid for the S segment of the original was used to produce the viruses. (b) Culture supernatants harvested at indicated days were titrated in Vero cells. The means and standard deviations are shown (n = 3). (c) Inoculated HeLa cells were fixed at 3 days post inoculation and stained with rabbit anti-NP serum.
Table 1.
Nucleotide and amino acid differences between the original and Hp50-4 strains.
Fig 3.
Determinants of Hp50-4 strain phenotypes (in vivo).
Ifnar-/- mice were subcutaneously inoculated with 102 50% tissue culture infectious doses of recombinant viruses (five mice per group) and observed until 14 days post inoculation. (a)(b) Survival curves are shown. Segment combinations of the recombinant viruses used in (a) are shown in Fig 2A. Schematic diagrams of the genome compositions and point mutations of recOri(U123A) and recLOriMHp(A123U) used in (b) are shown in (c).
Fig 4.
Glycosylation status of the GP of SFTS virus strains and C-type lectin usage upon infection.
(a) Nucleotide (position 115 to 123)/amino acid (position 33 to 35) sequences of the original (recOri) and Hp50-4 (recOri(U123A)) M segment/GP are shown. A nucleotide mutation observed in the Hp50-4 strain within the region and deduced amino acids are shown in orange. (b) Schematic diagram of the SFTS virus Gn and Gc with five potential N-linked glycosylation sites (black bars) are shown. (c) Western blotting analysis of lysates of Vero cells infected with recOri or recOri(U123A) recombinants were performed with (+) or without (-) glycosidase treatment and anti-Gn antibody. (d) Jurkat cells expressing a control molecule or one of the human C-type lectins (DC-SIGN, DC-SIGNR, and LSECtin) and Vero cells were inoculated with either recOri or recOri(U123A) strain at an MOI of 0.025. Ratios of viral antigen positivity in Jurkat cells to positivity in Vero cells are shown. Data shown are the means and standard deviations (n = 3). Statistical comparisons were performed between control recOri(U123A) and the others indicated (Dunnett’s test).
Fig 5.
iVLP infection in PMA-treated THP-1 cells.
(a) Human monocyte-derived THP-1 cells were cultured in the presence or absence of phorbol 12-myristate 13-acetate (PMA). The expressions of C-type lectins were analyzed by flow cytometry with antibody clones #120507 (DC-SIGN-specific), #120604 (DC-SIGNR-specific), DC28 (DC-SIGN/DC-SIGNR-dual specific), and SOTO-1 (LSECtin-specific). (b) PMA-treated (PMA+) and untreated (PMA-) THP-1 cells were inoculated with infectious viral particle (iVLP) carrying the original GP (iVLP-Ori) and reporter expression was analyzed by flow cytometry (FCM). (c) PMA-treated and untreated THP-1 cells were inoculated with iVLP carrying the GP from the original or recOri(123A) strains (iVLP-Ori or iVLP-Ori(U123A), respectively). Ratios of reporter positivity in treated cells to those in untreated cells are shown. Statistical comparisons were performed (Welch’s t-test). (d) PMA-treated THP-1 cells were pretreated or untreated with human IgG followed by the addition of the indicated concentration of Ab10. The cells were further inoculated with iVLP-Ori or iVLP-Ori(U123A). Reporter expression was analyzed by flow cytometry. All data shown are the means and standard deviations (n = 3).
Fig 6.
Viral growth and cytokine production in PMA-treated THP-1 cells.
Phorbol 12-myristate 13-acetate-treated THP-1 cells were inoculated with recOri or recOri(U123A) at a multiplicity of infection of 0.02 (left) or 2 (right) then cultured for 3 days. Culture supernatants were harvested daily and measured for viral titers (a), IL-6 (b), and TNFα (c). Data shown are the means and standard deviations (n = 3). (a, b, c) Statistical comparisons were performed between the means and p-values are shown (Welch’s t-test). ND, not detected. Detection limit for IL-6 (b) was 3.2 pg/mL and bars for ND were set as the detection limit value.
Fig 7.
Viral genome distribution and host response in the mouse model.
Ifnar-/- mice were subcutaneously inoculated with 102 50% tissue culture infectious doses of recOri or recOri(U123A) and blood sampling was performed upon euthanasia at the indicated days. Sera were used to measure the viral genome copy numbers, alanine aminotransferase, and cytokines. Whole blood was used to measure the numbers of white blood cells and lymphocytes. Blue and orange bars indicate recOri- and recOri(U123A)-infected mice, respectively. Black bars indicate mice injected with control media (6 days post injection). Three mice per group were used at each sampling point.
Fig 8.
C-type lectin usage and virulence of Heartland virus and recombinants.
Based on the genome sequence of the Heartland virus (HRTV) MO-4-NIID strain, two HRTV recombinants, HRTVrec and HRTVΔ1stNgly, were produced by reverse genetics. HRTVrec had no mutation but HRTVΔ1stNgly had an asparagine-to-glutamine substitution at position 35 of GP, resulting in the destruction of the N-linked glycosylation motif. (a) AG129 mice were subcutaneously inoculated with 102 focus forming units of HRTV MO4, HRTVrec, and HRTVΔ1stNgly (nine or ten mice per group) and observed for 14 days. Survival curves are shown. (b) Jurkat cells expressing control molecule or one of the human C-type lectins (DC-SIGN, DC-SIGNR, and LSECtin) and Vero cells were inoculated at a multiplicity of infection of 0.025. Ratios of viral antigen positivity in Jurkat cells to viral antigen positivity in Vero cells are shown. Data shown are the means and standard deviations (n = 3). Statistical comparisons were performed between control HRTVΔ1stNgly and the others indicated (Dunnett’s test).