Polymerase activity of avian-human viral ribonucleoprotein (RNP) complexes.

<p>(A) A549 cells were transfected in duplicate with pPol1-NS-Renilla and pSV40-Luc reporter plasmids, together with plasmids expressing PB2, PB1, PA and NP from either WY03 (human symbol) or TH04 (chicken symbol) viruses. Cells were incubated at 33°C (hatched bars) or 37°C (solid bars) for 24 hours and cell lysates were analyzed to measure Renilla and firefly luciferase activities. The latter was used to normalize transfection efficiency. Values shown represent the activities of each RNP relative to that of WY03 measured at 37°C (100%). (B) Viral RNP activities derived from WY03 (human symbol) or VN04 (chicken symbol) viruses are shown as described in panel A.</p

Replication kinetics of avian-human reassortant viruses in differentiated human tracheobronchial epithelial (HTBE) cells.

<p>HTBE cells were infected in duplicate with parental TH04 and WY03 (A) or rH5N1 viruses (B, C, D) at an moi of 0.02; progeny viruses were collected and titrated on MDCK cells.</p

Replication of avian-human reassortant viruses in mice.

<p>Symbols and virus nomenclature are as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000072#ppat-1000072-g001" target="_blank">Figure 1</a>. The mouse infectious dose (MID₅₀) and lethal dose (LD₅₀) are expressed as the log₁₀ pfu required to give one MID₅₀ or one LD_50. Maximum mean weight loss was determined from five mice per group (percent weight loss relative to dpi 0) following intranasal infection with 10⁴ pfu. MST denotes the mean survival time in days following infection with 10⁴ pfu. Virus titer in lung, spleen, brain or nasal turbinate are geometric means of the log₁₀ pfu at 4 dpi of three mice infected with 10⁴ pfu. LD₅₀ values of rH5N1 in group A1 were significantly different from A2 and those from A1 and A2 were significantly different from TH04 WT (<i>P<</i>0.001) by analysis of variance. The — indicate that tissue titers were below limit of detection of the assay (0.7 log₁₀ pfu/ml). Viruses are listed in ascending LD₅₀ values. Viruses with identical LD₅₀ are listed by descending weight loss.</p

Characteristics of moderate to low yield avian-human reassortant viruses in cell culture.

<p>Symbols and virus nomenclatures are the same as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000072#ppat-1000072-g001" target="_blank">Figure 1</a>. Rescue efficiency represents virus titer (log₁₀ pfu/ml) from cell cultures at 72 hours after transfection; geometric mean from 3 independent experiments. Plaque formation by reassortant viruses with <100 pfu/ml rescue efficiency was not determined (ND).</p

Hemagglutinin clade annotation for avian influenza subtype A(H5N1) by LABEL.

*<p>Non-redundant H5N1, GISAID, Min. Len. 1200 bp, excluding previous sets, non-laboratory derived.</p>**<p>The cleavage site is not included.</p><p>Start and stop 9-mers with translated amino acids are given for each fragment relative to A/goose/Guangdong/1/96 (AF144305/EPI_ISL_1254); accuracy is the number of correct clade annotations over the total number of tested HA nucleotide sequences; BER is the balanced error rate.</p

Revised 2012 H9N2 nomenclature.

<p>Average pairwise distance (APD) values within and between closely related clades using a <i>p</i>-distance calculation with pairwise deletion. The C-value is the ratio of the between group APD to the within group APD.</p

Two-dimensional scatterplots of profile HMM negative log-likelihood scores for H5N1 hemagglutinins in clades <i>3</i> (green circles) and <i>4</i> (red triangles) along with those in a clade <i>3-like</i> group (blue stars).

<p>(A) Plot shows scores for the clade <i>3</i>-specific pHMM (Y-axis) versus scores for the clade <i>4</i>-specific pHMM (X-axis). (B) As in A, but with the X-axis containing scores for the <i>3-like</i> pHMM instead. Smaller (more negative) numbers are considered better fits for that clade or group.</p

Revised 2012 H9N2 nomenclature references.

*<p>Named for virus not in our dataset that clusters with clade VN222.</p

Hemagglutinin clade annotation for avian influenza subtype A(H9N2) by LABEL.

<p>Start and stop 9-mers with translated amino acids are given for each fragment relative to <i>A/chicken/Beijing/1/94</i> (AF156380/EPI_ISL_1270); accuracy is the number of correct lineage annotations over the total number of tested HA nucleotide sequences; BER is the balanced error rate.</p

Annotation tree for LABEL's H5N1 annotation module.

<p>Each internal node corresponds to an annotation level (classification step) within the hierarchical annotation process. Accordingly, HMM profiles and SVM classes used by the H5 module are represented by all non-root nodes (color circles). The “c-<i>X</i>” notation stands for “cluster <i>X</i>,” where <i>X</i> is some general group of clades. Exact correspondence with the H5N1 clade clustering <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086921#pone.0086921-WHOOIEFAO1" target="_blank">[5]</a> is not preserved in the annotation tree for the sake of algorithmic simplicity.</p