Epidemiological and Clinical Features of Severe Fever with Thrombocytopenia Syndrome in Japan, 2013–2014 - Fig 2

<p>Serial concentrations of aspartate aminotransferase (A) and lactate dehydrogenase (B) in 15 fatal SFTS cases during hospitalization. The number in parenthesis of each patient indicates the days after disease onset when each patient died. A single result was available for the patient no.1. The normal ranges for each parameter are 10–35 IU/L for AST, and 120–220 IU/L for serum LDH.</p

Recovery of a recombinant virus expressing a fluorescence protein.

<p>(A) Schematic presentation of the S1 gene segment used for the recovery of rsMB/σC-ZsY (pT7-S1MB-σC-ZsY). The nucleotide sequence of 1001–1516 within the σC gene was replaced with the ZsYellow gene. (B) RT-PCR analysis of rsMB and rsMB/σC-ZsY. The S1 gene fragment was amplified by RT-PCR using viral dsRNA extracted from virions and primers specific for the MB S1 and ZsYellow sequences. The numbers show the S1 nucleotide position corresponding to the 5′ end of the S1-specific primers. (C) Growth kinetics of rsMB and rsMB/σC-ZsY in L929 cells. The cells were infected with the viruses at an MOI of 0.01 PFU/cell and incubated for various intervals. After freeze-thawing, the viral titer was determined by a plaque assay. (D) Expression of ZsYellow in cells infected with rsMB/σC-ZsY. Vero cells were infected with rsMB/σC-ZsY at an MOI of 0.05 PFU/cell and incubated for 24 h. Expression of ZsYellow in the syncytia of Vero cells was observed by confocal microscopy (yellow). Infected cells were fixed and stained using NBV-specific antiserum, followed by Alexa Fluor 633 Goat Anti-Mouse IgG second antibody (red). Cells were stained with DAPI to label nuclei (blue).</p

Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis

<div><p>Nelson Bay orthoreoviruses (NBVs) are members of the fusogenic orthoreoviruses and possess 10-segmented double-stranded RNA genomes. NBV was first isolated from a fruit bat in Australia more than 40 years ago, but it was not associated with any disease. However, several NBV strains have been recently identified as causative agents for respiratory tract infections in humans. Isolation of these pathogenic bat reoviruses from patients suggests that NBVs have evolved to propagate in humans in the form of zoonosis. To date, no strategy has been developed to rescue infectious viruses from cloned cDNA for any member of the fusogenic orthoreoviruses. In this study, we report the development of a plasmid-based reverse genetics system free of helper viruses and independent of any selection for NBV isolated from humans with acute respiratory infection. cDNAs corresponding to each of the 10 full-length RNA gene segments of NBV were cotransfected into culture cells expressing T7 RNA polymerase, and viable NBV was isolated using a plaque assay. The growth kinetics and cell-to-cell fusion activity of recombinant strains, rescued using the reverse genetics system, were indistinguishable from those of native strains. We used the reverse genetics system to generate viruses deficient in the cell attachment protein σC to define the biological function of this protein in the viral life cycle. Our results with σC-deficient viruses demonstrated that σC is dispensable for cell attachment in several cell lines, including murine fibroblast L929 cells but not in human lung epithelial A549 cells, and plays a critical role in viral pathogenesis. We also used the system to rescue a virus that expresses a yellow fluorescent protein. The reverse genetics system developed in this study can be applied to study the propagation and pathogenesis of pathogenic NBVs and in the generation of recombinant NBVs for future vaccines and therapeutics.</p></div

The C-terminal region of σC is required for cell attachment and infection in A549 cells.

<p>(A) Schematic presentation of σC protein structure as predicted by SWISS-MODEL (<a href="http://swissmodel.expasy.org/" target="_blank">http://swissmodel.expasy.org/</a>). NBV σC comprises tail, body, and head regions. (B) Expression and purification of 3 × FLAG-MB-σC, 3 × FLAG-Fd-σC-T, and 3 × FLAG-Fd-σC-BH proteins. 293T cells were transfected with p3×FLAG-MB-σC, p3×FLAG-Fd-σC-T, or p3×FLAG-Fd-σC-BH using 1 mg/ml polyethyleneimine solution. After purification of the recombinant proteins from the cell lysate, the proteins were analyzed by immunoblotting using anti-FLAG-M2 antibody. The molecular weights of the proteins are shown in kilodaltons (kDa). (C) The binding capacity of soluble truncated σC proteins to A549 cells. The cells were incubated with soluble σC proteins for 1 h, and the number of cells bound by the proteins was quantified by flow cytometry. The infectivity of the wild-type and rsMB/σC-Head-del viruses in A549 (D) and L929 cells (E). The viruses were used to infect A549 and L929 cells at MOIs of 10 and 30 PFU/cell, respectively. The infectivity of the viruses was analyzed by an indirect immunofluorescence assay using NBV-specific antiserum. Representative images are shown (upper). The infectivity rate was calculated as the ratio of the number of infected cells to the total cell population in the image (lower). Results are expressed as the mean infectivity of three fields of view. The error bars indicate standard deviations. Significant differences in comparison to rsMB were identified using Student’s <i>t</i>-test. NS: not significant; ***p < 0.0005.</p

NBV σC binds to the surface of A549 cells.

<p>Binding capacity of 3 × FLAG-MB-σC and 3 × FLAG-T3D-σ1 in culture cell lines. A549, L929, and CHO-K1 cells were incubated with the recombinant soluble proteins for 1 h, and the number of cells bound by the protein was quantified by flow cytometry.</p

Recovery of recombinant viruses incapable of expressing σC protein.

<p>(A) Schematic presentation of the plasmids encoding S1 segment used for the recovery of wild-type, rsMB/σC-del, and rsMB/σC-ACG viruses (pT7-S1MB, pT7-S1MB-σC-del, and pT7-S1MB-σC-ACG, respectively). The plasmid pT7-S1MB-σC-del lacks most of the nucleotide sequence in the ORF of σC. To generate the plasmid pT7-S1MB-σC-ACG, the start codon and other five AUG codons in the σC ORF were disrupted, and other five stop codons were inserted into the σC ORF. The black arrowheads indicate the ACG mutation sites, and the red arrowheads indicate the stop codon mutation sites. (B) The electropherotype of the dsRNA of rsMB, rsMB/σC-del, and rsMB/σC-ACG. The viral dsRNA was extracted from purified virions, electrophoresed, and visualized by ethidium bromide staining. Classes of gene segments based on their sizes are indicated. (C) Expression of σC and other NBV proteins in cells infected with the viruses. L929 cells were transfected with pCAG-MB-σC-FLAG or infected with rsMB, rsMB/σC-del, or rsMB/σC-ACG at an MOI of 0.1 PFU/cell and incubated for 48 h. The cell lysates were analyzed by immunoblotting using σC-specific polyclonal antiserum, NBV-specific antiserum, or antibody specific for actin. The molecular weights of the proteins are shown in kilodaltons (kDa).</p

Clinical characteristics of 48 case-patients with severe fever with thrombocytopenia syndrome at admission grouped according to outcome in Japan.

<p>Clinical characteristics of 48 case-patients with severe fever with thrombocytopenia syndrome at admission grouped according to outcome in Japan.</p

Generation of S1 monoreassortant viruses.

<p>(A) Electropherotype of dsRNA of rsMB/NB-S1 and rsMB/Mel-S1 compared with that of the wild-type viruses. The viral dsRNA was extracted from purified virion, electrophoresed, and visualized by ethidium bromide staining. Classes of gene segments based on their sizes are indicated. Growth kinetics of rsMB, rsMB/NB-S1, and rsMB/Mel-S1 in L929 cells. The cells were infected with the recombinant viruses at an MOI of 0.1 (B) or 0.01 (C) PFU/cell and were incubated for various intervals. After freeze-thawing, the viral titer in the cell lysate was determined by a plaque assay. Significant differences in comparison to rsMB were identified using Student’s <i>t</i>-test at 3 days post infection for an MOI of 0.1 PFU/cell and at 5 days post infection for an MOI of 0.01 PFU/cell. NS: not significant.</p

Infection by the wild-type virus is inhibited by treatment using σC-specific antiserum in A549 cells.

<p>Viruses were incubated with σC-specific antiserum for 1 h and used to infect A549 and L929 cells at MOIs of 30 and 10 PFU/cell, respectively. After incubation for 6–12 h, the infectivity of the viruses in A549 (A) and L929 cells (C) was analyzed by an indirect immunofluorescence assay using NBV-specific antiserum. The infectivity rate in A549 (B) and L929 cells (D) was calculated as the ratio of the number of infected cells to the total cell population in the image. Results are expressed as the mean infectivity of three fields of view. The error bars indicate standard deviations. Significant differences in comparison to the isotype control were identified using one-way ANOVA. NS: not significant; **p < 0.005.</p

Basic characteristics of 49 case-patients with severe fever with thrombocytopenia syndrome in Japan.

<p>Basic characteristics of 49 case-patients with severe fever with thrombocytopenia syndrome in Japan.</p