Antiviral effect of siRNA D3 in relation to the infectious dose of Langat.

<p>HeLa cells were infected with Langat virus at different infectious dose (MOI of 10, 1, 0.1 or 0.01) and one hour after infection, cells were treated with 200 nM siRNA sequence D3 or with nonsense siRNA. Virus titer was assessed by quantitative real-time RT-PCR 6 days after infection in the cell culture supernatant. The reduction of viral RNA in siRNA D3 treated cells is statistically significant compared to nonsense siRNA treated cells for all four MOI (p<0.05). The data are presented as mean ± SD of three independent experiments and significance calculated using an unpaired T-test.</p

Inhibitory effects of siRNA D3 on viral replication on HeLa cells over time.

<p>The replication profile for Langat virus was determined for the time period of 6 days. Indicated are the numbers of RNA copies (A) and the number of infectious particles (B) for cell cultures treated with nonsense siRNA or specific siRNA D3.</p

LGTV-specific siRNA sequences inhibit production of viral RNA in HeLa cells.

<p>Nineteen siRNA sequences (Q1–Q6 and D1–D13) targeting genes within the whole open reading frame of LGTV genome were analyzed for their antiviral potential on HeLa cells (A). After transfection with siRNA, cells were infected with Langat virus (MOI = 10) and six days later virus replication was assessed by quantitative real-time RT-PCR (B). Results are shown as a percentage of virus inhibition compared to the control cells transfected with the non-coding siRNA. Data are presented as mean ± SD of three independent experiments. Fourteen (Q2, Q3, Q4, Q5, Q6, D3, D4, D5, D6, D8, D9 D11 D12 and D13) out of 19 were significantly reduced (p<0.05) compared to a theoretical mean of 100% expressed by cells treated with nonsense siRNA; measured by One sample t test.</p

Modified WT amplification of non-clinical samples containing emerging virus.

<p>Purified viral RNA from a wide range of virus-positive cell culture supernatants (SN) or QCMD panel samples was amplified by WTA using the P-N6/SSIII RT-reaction. Virus-specific real-time PCR was performed before and after the amplification step, and fold increase was calculated using ΔC_t-values and dilution factors for each sample tested. (A) Fold increase of WT-amplified emerging viruses belonging to different virus genera. The two QCMD panel samples (WNV10-01 and WNV10-07) containing mixtures of different flaviviruses are highlighted. (B) The correlation between fold increase in WT amplification and viral sample content (C_t before WT amplification).</p

Microarray detection range on WT-amplified samples.

<p><b>NOTE</b>. Conc, Concentration; SN, supernatant; RVFV, Rift-Valley fever virus; WNV13-01, sample from QCMD EQA WNV panel 13-01; WNV, West Nile virus; JEV, Japanese encephalitis virus; TBEV, tick borne encephalitis virus; DENV13-01, sample from QCMD EQA DENV panel 13-01; DENV, Dengue virus; HCV, hepatitis C virus; ND, not detected; na, not analysed.</p>§<p>WNV13-10 contain additional viruses (DENV-1, DENV-2 and DENV-4).</p>#<p>WNV13-11 contain addition viruses (YFV, DENV-3).</p>95<p>results obtained using a 95 percentile threshold.</p>a<p>copies/ml (HCV; IU/ml).</p>b<p>ml (volume of sample for purification).</p>c<p>Number of copies into reaction.</p><p>*Difference in C_t-value in real-time PCR before and after WT amplification.</p><p>+Fold increase after WT amplification, calculated from ΔC_t combined with dilution factors for each sample.</p

Microarray results on clinical samples containing emerging viruses.

<p><b>NOTE</b>. Conc, Concentration; DENV; Dengue virus, WNV, West Nile virus; JCV, JC polyomavirus; BKV, BK polyomavirus; CCHFV, Crimean-Congo haemorrhagic fever virus; Neg. ctrl, Negative control; GBV-C, hepatitis GB virus C; JEV, Japanese encephalitis virus; CSF, Cerebrospinal fluid; HERV, human endogenous retrovirus; TTV, torque teno virus; ND, not determined. Bold represents correctly identified virus.</p><p>*Viruses are grouped based on nucleic acid content, according to the Baltimore Classification.</p>95<p>results obtained using a 95 percentile threshold.</p>a<p>Difference in C_t-value in real-time PCR before and after WT amplification.</p>b<p>Fold increase after WT amplification, calculated from ΔC_t combined with dilution factors for each sample.</p>c<p>Copies/ml (HCV; IU/ml).</p

Microarray results on non-clinical samples using two different data analysis methods.

<p><b>NOTE</b>. EEEV, Eastern equine encephalitis virus; WNV, West Nile virus; CCHFV, Crimean-Congo haemorrhagic fever virus; RVFV, Rift-Valley fever virus; TBEV, Tick borne encephalitis virus; OHFV, Omsk hemoratic fever virus; YFV, yellow fever virus; PV, poliovirus; HERV, human endogenous retrovirus; JEV, Japanese encephalitis virus; DENV, Dengue virus; DOBV, Dobrava-Belgrade virus; RV-A, rotavirus A; PRV-C, porcine rotavirus C; BEV, baboon endogenous virus; SRV-1, simian retrovirus 1; MuLV, murine leukemia virus; SV40, simian virus 40; MDEV, <i>mus dunni</i> endogeneous virus; MMTV, mouse mammary tumour virus; MRV, mammalian orthoreovirus; BVDV, bovine viral diarrhea virus; SV5, simian virus 5; pur. DNA, purified DNA; SN, cell culture supernatant; pur. RNA, purified RNA; WNV10-01, sample from QCMD EQA WNV panel 10-01; WNV10-07, sample from QCMD EQA WNV panel 10-07 Bold represents correctly identified virus.</p><p>∧Viruses with fragmented alignment plots.</p><p>*Viruses are grouped based on nucleic acid content, according to the Baltimore Classification.</p

CLiMax analysis detects Puumala virus in a non-clinical sample.

<p>The results of microarray analyses of WT-amplified viral DNA-samples, using CLiMax analysis. (A) Log-odds scores for a Puumala virus-positive sample. The lighter and darker-coloured portions of the bars represent the unconditional and conditional log-odds scores, respectively. The conditional log-odds scores shows the contribution from a target that cannot be explained by another, more likely target above it, while the unconditional score illustrates that some very similar targets share a number of probes. (B) Target sequence-probe alignment plots for segment L of the Puumala virus genome and for BVDV-1, showing probe intensity vs probe position in the viral genome. Plot symbol and color indicates positive (>99^th percentile), negative (<95^th percentile), or equivocal hybridisation signals; hollow symbols indicate probes found to hybridise non-specifically. The pattern seen for BVDV-1, in which positive probes are restricted to a few narrow genome regions, is a typical cross-hybridisation result.</p

Microarray analysis correctly identifies emerging viruses in clinical samples.

<p>The results of microarray analysis of WT-amplified virus-positive clinical samples, using the SSI analysis method. Graphs show the signal mean for the probe intensities for each detected virus. The bar across the graph demonstrates the signal threshold at the 99^th percentile of the random control intensities. (A) Microarray analysis of a Dengue-positive serum sample. (B) Microarray analysis of another Dengue-positive serum sample. (C) Microarray analysis of a WNV-positive urine sample. (D) Microarray analysis of another WNV-positive urine sample.</p

Improved WT amplification when using 5′-phosphorylated random hexamers in RT-reaction.

<p>Comparison of three different RT-reactions in the Whole Transcriptome Amplification (WTA) protocol. Purified viral RNA was amplified by WTA using VILO, T-Script or P-N6/SSIII RT-reaction. Virus-specific real-time PCR was performed before and after the amplification step, and fold increase was calculated using ΔC_t-values and dilution factors for each sample tested. (A) WTA-protocols tested with a 10-fold serial dilution of an HCV-positive serum sample with known concentration. (B) WTA-protocols tested with two different virus-positive cell culture supernatants, Puumala virus and Dobrava-Belgrade virus (DOBV), respectively. (C) WTA-protocols tested with five HCV-positive serum samples with estimated concentration (IU/ml). (D) WTA-protocols tested with five HCV-positive plasma samples with estimated concentration (IU/ml).</p