Characteristics of high yield avian-human reassortant viruses in cell culture.

<p>Human and chicken symbols denote the parental WY03 or TH04 source of each gene segment. The genotype of reassortants (r) is denoted by the genes derived from WY03 virus, designated by their segment number; 1:PB2. 2:PB1. 3:PA. 4:HA. 5:NP. 6:NA. 7:M. 8:NS. All others genes derived from TH04 do not bear a number. For example, r3/8 indicates the reassortant virus carries PA and NS genes from WY03 virus, and the remaining segments from TH04 virus. Rescue efficiency represents the virus titer (log₁₀ pfu/ml) from cell cultures at 72 hours after transfection; geometric mean from 3 independent experiments.</p

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 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

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

Maximum-likelihood (ML) tree of influenza A NA subtypes.

<p>A: N1; B: N2; C: N5; D: N8. The annotation for each lineage was labeled on the trees. Three lineages in N1 (1A, 1B and 1C), two lineages in N2 (2A and 2B), two lineages in N5 (5A and 5B), and two lineages in N8 (8A and 8B) were classified. The bootstrap values supporting the corresponding lineages are shown to the left of the major nodes. Scale bars indicate the numbers of nucleotide substitutions per site.</p

Two-dimensional scatterplots of profile HMM negative log-likelihood scores for H5N1 hemagglutinins in clades <i>1</i> (green circles) and <i>1.1</i> (red triangles) along with those in a <i>1.1-like</i> group (blue stars).

<p>(A) Plot shows scores for the clade <i>1.1</i>-specific pHMM (Y-axis) versus scores for the clade <i>1</i>-specific pHMM (X-axis). (B) As in A, but with the X-axis containing scores for the <i>1.1-like</i> pHMM instead. Smaller (more negative) numbers are considered better fits for that clade or group.</p

Annotation tree for LABEL's H9N2 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 H9 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. Clades are named according to historical names and for representative sequences.</p

Evolutionary History and Phylodynamics of Influenza A and B Neuraminidase (NA) Genes Inferred from Large-Scale Sequence Analyses

<div><p>Background</p><p>Influenza neuraminidase (NA) is an important surface glycoprotein and plays a vital role in viral replication and drug development. The NA is found in influenza A and B viruses, with nine subtypes classified in influenza A. The complete knowledge of influenza NA evolutionary history and phylodynamics, although critical for the prevention and control of influenza epidemics and pandemics, remains lacking.</p><p>Methodology/Principal findings</p><p>Evolutionary and phylogenetic analyses of influenza NA sequences using Maximum Likelihood and Bayesian MCMC methods demonstrated that the divergence of influenza viruses into types A and B occurred earlier than the divergence of influenza A NA subtypes. Twenty-three lineages were identified within influenza A, two lineages were classified within influenza B, and most lineages were specific to host, subtype or geographical location. Interestingly, evolutionary rates vary not only among lineages but also among branches within lineages. The estimated tMRCAs of influenza lineages suggest that the viruses of different lineages emerge several months or even years before their initial detection. The <i>d</i>_N<i>/d</i>_S ratios ranged from 0.062 to 0.313 for influenza A lineages, and 0.257 to 0.259 for influenza B lineages. Structural analyses revealed that all positively selected sites are at the surface of the NA protein, with a number of sites found to be important for host antibody and drug binding.</p><p>Conclusions/Significance</p><p>The divergence into influenza type A and B from a putative ancestral NA was followed by the divergence of type A into nine NA subtypes, of which 23 lineages subsequently diverged. This study provides a better understanding of influenza NA lineages and their evolutionary dynamics, which may facilitate early detection of newly emerging influenza viruses and thus improve influenza surveillance.</p></div

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