Flow cytometry analysis of DENV-2 infected human monocytes from one G6PD-deficient patient.

<p>Cells infected with New Guinea C strain (A) and 16681 strain (B) at an MOI of 0.1 and observed on day three post-infection. Black: Percent DENV-2 infected cells. White: Percent mock-infected cells (background)</p

Gender and age distributions of study populations including G6PD-deficient patients and healthy controls

*<p>IU/g Hemoglobin (IU/gHb)</p

Flow cytometry analysis of DENV-2 infected human monocytes from one healthy control.

<p>New Guinea C strain (A) and 16681 strain (B) of DENV-2 were infected at an MOI of 0.1 and their infection levels on day three post-infection were measured by flow cytometry. Black: Percent DENV-2 infected cells. White: Percent mock-infected cells (background)</p

Growth of DENV-2 in human monocytes from one healthy control.

<p>Monocytes were infected with DENV-2 New Guinea C (NGC) and 16681 strains at an MOI of 0.1 for five days. The virus titer in the cell culture supernatants were analyzed by plaque assay (—) and the intracellular replication of virus in the infected cells were detected by flow cytometry ( ---- ).</p

Detection of DENV in infected monocytes from one healthy control by using immunofluorescence staining.

<p>(A) Mock-infected cells. (B) DENV-2 infected monocytes. Monocytes were infected by DENV-2 New Guinea C strain at an MOI of 0.1 and then cultured for three days. Evans blue was added as a counter-stain to differentiate non-infected cells (shown by red color) from the dengue virus-positive infected cells (shown by green color).</p

Box plot for comparison of the <i>ex vivo</i> infection percentages of DENV-2 (New Guinea C and 16681 strain) in monocytes of PBMC obtained from 12 G6PD-deficient patients and 24 healthy controls.

<p>Box plot for comparison of the <i>ex vivo</i> infection percentages of DENV-2 (New Guinea C and 16681 strain) in monocytes of PBMC obtained from 12 G6PD-deficient patients and 24 healthy controls.</p

Selected HPAI H5N1 virus outbreaks and the minimum temperatures and dates at nearby WMO (World Meteorological Organization) stations.

<p>Selected HPAI H5N1 virus outbreaks and the minimum temperatures and dates at nearby WMO (World Meteorological Organization) stations.</p

Figure 1

<p>Variation of daily minimum temperatures at WMO stations near H5N1 virus outbreak areas. Each group and selected avian influenza H5N1 outbreaks events are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000191#pone-0000191-t001" target="_blank">Table 1</a>. Day 0 is the day with the lowest daily minimum temperature recorded; while the red dot represents the date of an outbreak of AIV was reported. For <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000191#pone-0000191-g001" target="_blank">Figure 1(a)</a> the day zero time point of group I was chosen as the time of the outbreak, not the time of the lowest temperature, since the former happened earlier.</p

Phenotypic and Genetic Characterization of Avian Influenza H5N2 Viruses with Intra- and Inter-Duck Variations in Taiwan

<div><p>Background</p><p>Human infections with avian influenza viruses (AIVs) have frequently raised global concerns of emerging, interspecies-transmissible viruses with pandemic potential. Waterfowl, the predominant reservoir of influenza viruses in nature, harbor precursors of different genetic lineages that have contributed to novel pandemic influenza viruses in the past.</p><p>Methods</p><p>Two duck influenza H5N2 viruses, DV518 and DV413, isolated through virological surveillance at a live-poultry market in Taiwan, showed phylogenetic relatedness but exhibited different replication capabilities in mammalian Madin-Darby Canine Kidney (MDCK) cells. This study characterizes the replication properties of the two duck H5N2 viruses and the determinants involved.</p><p>Results</p><p>The DV518 virus replicated more efficiently than DV413 in both MDCK and chicken DF1 cells. Interestingly, the infection of MDCK cells by DV518 formed heterogeneous plaques with great differences in size [large (L) and small (S)], and the two viral strains (p518-L and p518-S) obtained from plaque purification exhibited distinguishable replication kinetics in MDCK cells. Nonetheless, both plaque-purified DV518 strains still maintained their growth advantages over the plaque-purified p413 strain. Moreover, three amino acid substitutions in PA (P224S), PB2 (E72D), and M1 (A128T) were identified in intra-duck variations (p518-L vs p518-S), whereas other changes in HA (N170D), NA (I56T), and NP (Y289H) were present in inter-duck variations (DV518 vs DV413). Both p518-L and p518-S strains had the N170D substitution in HA, which might be related to their greater binding to MDCK cells. Additionally, polymerase activity assays on 293T cells demonstrated the role of vRNP in modulating the replication capability of the duck p518-L viruses in mammalian cells.</p><p>Conclusion</p><p>These results demonstrate that intra-host phenotypic variation occurs even within an individual duck. In view of recent human infections by low pathogenic AIVs, this study suggests possible determinants involved in the stepwise selection of virus variants from the duck influenza virus population which may facilitate inter-species transmission.</p></div

Evaluation of viral entry efficiency for p518-S and p518-L virus strains in 293T cells.

<p>The 293T cells were infected with p518-S and p518-L viruses at an MOI of 2. Cell lysates were collected at 0.5, 1.5 and 6 h p.i. and were examined by Western blot using anti-NP and anti-actin antibodies. Levels of protein and relative ratios of NP/Actin were analyzed by software Image J. <b>MOI</b>: multiplicity of infection</p