Table 1.
Complete open reading frame nucleotide and amino acid sequence identities of GRW/Aa with other DOBV and HTNV isolates.
Figure 1.
Maximum-likelihood phylogenetic trees of DOBV showing the phylogenetic placement of GRW/Aa.
The trees were constructed with TREE-PUZZLE software (Tamura-Nei evolutionary model) and are based on (A) complete S-segment ORF, (B) partial S-segment (559 nt, positions 377 to 938), (C) complete M- and (D) complete L-segment ORF sequences. Values above the branches represent PUZZLE support values, while values below the branches are bootstrap values of the corresponding maximum likelihood trees calculated with the MEGA5 software from 1000 bootstrap pseudoreplicates. Only values >70% (considered significant) are shown. Different DOBV clades are indicated by gray boxes. GRW/Aa positions in the tree are designated with an arrow. H169 patient-derived sequence is designated with a star. For accession numbers, see the materials and methods. DOBV, Dobrava-Belgrade virus; HTNV, Hantaan virus; SEOV, Seoul virus; THAIV, Thailand virus.
Figure 2.
Determination of DOBV GRW/Aa receptor usage by receptor blocking assays (A–C) and receptor binding experiment (D).
Vero E6 cells were treated with 40 µg/ml of indicated blocking antibodies for 1 hour. Then virus at multiplicity of infection 0.05 was added to the cells. After one hour cells were washed and new medium was added. One day later samples were collected. A) Viral S-segment RNA was measured by qPCR. B) Expression of viral nucleocapsid (N) protein was detected by Western blot. The density of bands on blots was quantified by ImageJ 1.41o programm (Wayne Rasband National Institutes of Health, USA). The percentages of antibody-mediated inhibition of viral infection were calculated in comparison to untreated but infected cells. Experiment was performed three times. Data are presented as the mean ± SD of the mean. C) Representative picture of the Western blot analyses summarized in part B. —, untreated cells; β1, cells pre-treated with β1 integrin specific monoclonal antibody (MAb); β3, cells pre-treated with β3 integrin specific MAb; DAF, cells pre-treated with DAF specific MAb. D) Binding of GRW/Aa to CHO cells stably expressing β3 integrins (CHO-β3cells) in comparison to control CHO cells. Virus binding was performed at 4°C for 1 hour. HTNV and PHV were used as controls. The amount of bound virus was measured through detection of viral RNA by specific qPCR. The binding affinity of virus particles to CHO-β3 cells is expressed as a ratio between virus genome equivalents detected on CHO-β3 cells and on the control CHO cells. Error bars represent standard deviations of the means from three experiments.
Figure 3.
Expression of mRNA and antiviral MxA protein in response to GRW/Aa infection.
A549 cells were infected at multiplicity of infection 1. Samples were taken at indicated time points post infection. A) Induction of IFN-β, IFN-λ1 and MxA mRNA (shown as fold of increase in comparison with uninfected cells) in response to GRW/Aa infection measured as virus genome copies by qPCR. Cells stimulated with poly I:C were transfected with 1.6 µg of poly I:C six hours prior infection. Experiment was performed three times. Data are presented as the mean ± SD of the mean. B) Expression of antiviral MxA protein detected by Western blot. NC (negative control), uninfected A549 cells. PC (positive control), A549 cells infected with HTNV for 4 days. Representative results of two independent experiments are shown.
Figure 4.
Influence of Vero E6-derived type III IFN on GRW/Aa MxA induction.
A) 100 µl of indicated Vero E6-derived virus stocks or medium (NC, negative control) were exposed to UV irradiation. Amount of IFN-λ1 present in virus stocks was measured by ELISA. Abbreviation “ucf” means that the virus stock was purified by the ultracentrifugation procedure. Data are presented as the mean between three independently prepared stocks ± SD from the mean B) GRW/Aa virus stock (∼50 µl) or recombinant proteins (rhIFN-λ1 or rhIFN-β) were preincubated with 1 µg of corresponding blocking antibody (anti-IFN-λ1 or anti-IFN-β). One hour after incubation the mixture was added on the top of A549 cells. Four days post infection MxA mRNA expression was measured by qPCR. NC (negative control), untreated A549 cells. Experiment was performed three times. Data are presented as the mean ± SD of the mean.