Additional file 1 of Can the use of azithromycin during labour reduce the incidence of infection among puerperae and newborns? A systematic review and meta-analysis of randomized controlled trials

Supplementary Material

SFTSV inhibits exogenous Type I IFN signaling pathway.

<p>(A)<b>SFTSV inhibits the phosphorylation of STAT1</b>. Hela cells were infected with SFTSV(MOI = 1.0) for2 hours and cultured for 36 hours before 100IU/ml IFNα was added for 15 minutes,and total protein were collected for WB and densitometry analysis for p-STAT1. (B)<b>SFTSV suppresses ISRE activity.</b> Hela cells were co-transfected with ISRE-luc plasmid 2μg per well and pRL-TK plasmid 2ng per well, then Hela cells were infected with SFTSV (MOI = 1.0) for 2 hours and cultured for 24hours, then we used 100IU/ml IFNα to stimulated the Hela cells for 24 hours, and samples were collected for dual-luciferase reporter assay. (C)<b>SFTSV down –regulates the expression of ISGs.</b> Hela cells were infected with 1.0MOI SFTSV for2 hours and culturedfor24hours, then treated with 100IU/ml IFNα for 12 hours, and total RNAs were isolated with Trizolreagent, The levels of ISG15, MxA, OAS3, GAPDH mRNA were measured by quantitative RT-PCR. Data were presented as mean± SD, n = 3. **p<0.01, ***p<0.001. Data are representative of at least 3 repeated experiments.</p

The list of primers for RT real-time qualitative PCR to detect the expression of IFNα, IFNß, TLR3, RIG-I and ISGs.

<p>The list of primers for RT real-time qualitative PCR to detect the expression of IFNα, IFNß, TLR3, RIG-I and ISGs.</p

SFTSV inhibits exogenous IFNα-induced Jak/STAT signaling pathway through NSs.

<p>(A)<b>NSs of SFTSV expresses in Hela cells successfully.</b> Hela cells were transfected with empty vector pcDNA3.1 plasmid or NSs plasmid for 48hours, and total RNA were prepared with Trizol reagent, cDNA were harvested with reverse transcriptase for real time quantitative PCR. Protein samples were harvested for WB analyzed. (B) <b>Expression of GFP protein in Hela cells.</b> GFP-expressing plasmid DNA was transfected into Hela cells for 48 hours and GFP expression was observed under immunofluorescence microscope (200×). (C)<b>NSs inhibits the phosphorylation of STAT1.</b>Hela cells were transfected with NSs plasmid or empty vector pcDNA3.1 for 36 hours before IFNα was added to stimulate the cells for 15minutes. Total proteins were collected with protein lysis buffer for WB and densitometry analysis of western blot datafor p-STAT1. (D)<b>NSs suppresses the ISRE activity</b>. Hela cells were co-transfected with pISRE-luc plasmid and pRL-TK plasmid together with NSs plasmid, GFP-expressing plasmid or empty vector pcDNA3.1 plasmid for 24hours, and IFNα (100IU/mL)were added to the cells for 24hours, then samples were collected for dual-luciferase reporter assay. (E) <b>NSs down-regulates the expression of several ISGs.</b> Hela cells were transfectd with NSs plasmid or empty vectorpcDNA3.1 plasmid for 36hours, then we added IFNα (100IU/ml) to the cells for 12hours,RNA were collected with Trizol reagent, cDNA were used for real time quantitative PCR. Data were presented as mean± SD, n = 3.*p<0.05, **p<0.01, ***p<0.001.Data were representative of at least 3 repeated experiments.</p

SFTSV replicates in Vero, Hela, Huh7.0, Huh7.5.1 cells efficiently.

<p>SFTSV infected Vero (A), Hela (B), Huh7.0 (C) and Huh7.5.1 (D) cells at MOI = 0.09. Cells and culture supernatant were collected at different time-points. Viral RNA levels was measured by Fluorescence Quantitative Polymerase Chain Reaction Diagnostic Kit as described in material and methods. Data were presented as means± SD, n = 3. Data were representative of at least 3 repeated experiments.</p

SFTSV activates host innate immunity in Hela cells. Endogenous IFNα/β were up-regulated by SFTSV in Hela cells but not in Huh7.0 0r Huh7.5.1 cells.

<p>Hela(A), Huh7.0(B) and Huh7.5.1(C) cells were infected with1.0 MOI SFTSV for 2 hours and cultured for 48 hours before RNA were collected with Trizol reagent following the manufacturer’s protocol. The levels of TLR3, RIG-I, IFNα, IFNβ, GAPDH mRNA and SFTSV mRNA were measured by quantitative RT-PCR. (D) Protein levels of IFNα/β were up-regulated in the culture medium of Hela cells stimulated by SFTSV. Hela and Vero cells were infected with SFTSV (MOI = 1.0) for 2 hours and cultured for 48 hours before the culture medium were collected, The protein levels of IFNα/β were assayed by the Elisa Kit. Data were presented as mean± SD (n = 3). *p<0.05,**p<0.01, ***p<0.001.Data are representative of at least 3 repeated experiments.</p

Figure 1. Phylogenetic analysis of the 14 clones derived from the Guilin recombinant compared with reference strains.

<p>GenBank accession numbers and clone numbers are showed on each tree, and the genotype is indicated after every accession number. Bootstrap values are showed at each nodes, and only bootstrap values of >70% are indicated. Phylogenetic trees comparing the 14 clones with 34 reference strains representing genotypes A–H, were constructed based on the S gene (A), pre-S region (B) and whole genomes (C). And the phylogenetic tree was constructed by comparing the Guilin recombinant with the Vietnam, Thailand and Long An recombinants as well as HBV C subgenotype based on the whole genomes (D).</p

Bootscan analysis demonstrating the complex recombination among genotypes A, C and G in the Guilin recombinant.

<p>The isolate from the female patient (A) and the isolate from the male patient (B) were subjected to bootscan analysis over the complete genome using the SIMPLOT program with a 500 bp window size, 10 bp step size and 100 bootstrap replicates, using gap-stripped alignments and neighbor-joining analysis, and were compared with three representative HBV genotypes: A (GenBank accession no. AB126580), C (GenBank accession no. AB050018) and G (GenBank accession no. AB064310). Woolly monkey was a known out-group (GenBank accession no. AF046996). Analysis was stared from nt 2700.</p

Nucleotide distances between the Guilin HBV recombinant and other reference genotype strains^*.

<p>The reference genotype strains used in the table are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084005#pone-0084005-g001" target="_blank">Figure 1C</a>.</p

The breakpoint positions and the arrangements of the genotype fragments of the Guilin, Vietnam, Long An and Thailand recombinants.

<p>The breakpoint positions and the arrangements of the genotype fragments of the Guilin, Vietnam, Long An and Thailand recombinants.</p