Combination index for IFN-α and IFN-λ1 against CCHF replication respectively in A549 and HuH-7 cells.

<p>*The Combination Index (CI) was calculated using the CompuSyn software (Chou, T.-C. and Martin, N. CompuSyn software for drug combinations and for general dose effect analysis, and user’s guide. ComboSyn, Inc. Paramus, NJ 2007. [<a href="http://www.combosyn.com" target="_blank">www.combosyn.com</a>]) which uses the method of Chou & Talalay. CI values <1, 1 and >1 indicate synergism, additive effect and antagonism, respectively.</p><p>Combination index for IFN-α and IFN-λ1 against CCHF replication respectively in A549 and HuH-7 cells.</p

Dose-dependent inhibition of CCHFV replication by recombinant IFN-α, IFN-λ1 and IFN-α+IFN-λ1.

<p>A549 <b>(a)</b> and HuH7 cells <b>(b)</b> were treated for 1 day with increasing amounts of either IFN type, alone or in combination, then infected with CCHFV at MOI 0.01; infectious virus yield was measured after overnight incubation. One out of three experiments is shown. Dotted lines: IFN-α (●) or IFN-λ1 (▲) used alone; continuous line: IFN-α and IFN-λ1 (■) used in combination.</p

A High Diversity of Eurasian Lineage Low Pathogenicity Avian Influenza A Viruses Circulate among Wild Birds Sampled in Egypt

<div><p>Surveillance for influenza A viruses in wild birds has increased substantially as part of efforts to control the global movement of highly pathogenic avian influenza A (H5N1) virus. Studies conducted in Egypt from 2003 to 2007 to monitor birds for H5N1 identified multiple subtypes of low pathogenicity avian influenza A viruses isolated primarily from migratory waterfowl collected in the Nile Delta. Phylogenetic analysis of 28 viral genomes was performed to estimate their nearest ancestors and identify possible reassortants. Migratory flyway patterns were included in the analysis to assess gene flow between overlapping flyways. Overall, the viruses were most closely related to Eurasian, African and/or Central Asian lineage low pathogenicity viruses and belonged to 15 different subtypes. A subset of the internal genes seemed to originate from specific flyways (Black Sea-Mediterranean, East African-West Asian). The remaining genes were derived from a mixture of viruses broadly distributed across as many as 4 different flyways suggesting the importance of the Nile Delta for virus dispersal. Molecular clock date estimates suggested that the time to the nearest common ancestor of all viruses analyzed ranged from 5 to 10 years, indicating frequent genetic exchange with viruses sampled elsewhere. The intersection of multiple migratory bird flyways and the resulting diversity of influenza virus gene lineages in the Nile Delta create conditions favoring reassortment, as evident from the gene constellations identified by this study. In conclusion, we present for the first time a comprehensive phylogenetic analysis of full genome sequences from low pathogenic avian influenza viruses circulating in Egypt, underscoring the significance of the region for viral reassortment and the potential emergence of novel avian influenza A viruses, as well as representing a highly diverse influenza A virus gene pool that merits continued monitoring.</p></div

Avian influenza viruses analyzed in this study with subtype, collection site (Governorate in Egypt), date of collection (DOC) and amino acid sequence of the cleavage site (*) of the HA0 protein.

<p>Avian influenza viruses analyzed in this study with subtype, collection site (Governorate in Egypt), date of collection (DOC) and amino acid sequence of the cleavage site (*) of the HA0 protein.</p

Phylogeny of HA gene sequences of H1 to H16 subtypes (A) and NA gene sequences of N1 to N9 subtypes (B).

<p>Viruses identified in this study from Egypt and Ukraine are in boldface. NJ trees were calculated with Mega4 and color coded for each subtype <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068522#pone.0068522-Tamura1" target="_blank">[25]</a>. For <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068522#pone-0068522-g001" target="_blank">Figure 1A</a> H2, H12, H15, H16 HA sequences and for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068522#pone-0068522-g001" target="_blank">Figure 1B</a> N5 NA gene sequences were included as reference only. Trees are rooted on an influenza B virus sequence (B/Yamagata/186/2005).</p

Number of influenza A virus PCR positive specimen and number of embryonated chicken egg isolates.

<p>Number of influenza A virus PCR positive specimen and number of embryonated chicken egg isolates.</p

Estimated calendar year (with decimals) of the most recent common ancestor (TMRCA) of phylogenetic groups 1 to 14 described in Figures 2 and 3.

<p>TMRCA of each internal gene segment group is shown with the 95% HPD in parenthesis.</p

Phylogenies and TMRCA of PB2 (A), PB1 (B), PA (C), NP (D), MP (E) and NS (F) genes inferred with BEAST [26].

<p>Posterior probabilities (>0.7) are shown at each node on the tree and a time scale in years is shown below each tree. Branches containing Egyptian viruses were collapsed around nodes and numbered according to the tree topology from top to bottom. All trees are rooted to the ancestral virus, A/equine/Prague/2/1956 H7N7, and the outlier branch was replaced by a black arrow except NS, which is midpoint rooted. The coloring of collapsed monophyletic groups (key shown in figure inset) corresponds to the predicted flyway (Red for East Asian-Australian flyway, purple for Black Sea-Mediterranean flyway, green for Central Asian flyway, blue for East African-West Asian flyway, orange for Nile Delta and yellow if the group contained 2 or more reference viruses collected from different flyways. Viruses that did not cluster with any other viruses are indicated with (#) behind the number. Abbreviations aq = aquatic, barhead = bar headed, ck = chicken, dk = duck, eq = equine, gar = garganey, gs = goose, magp = magpie, ml = mallard, ost = ostrich, qu = quail, pel = pelican, sho = shoveler, te = teal, tk = turkey, wi = wild, whisk = whiskered, wh-fr-gs = white fronted goose, EGY = Egypt, N3 = NAMRU3, UKR = Ukraine.</p

Phylogenetic groups of migratory flyways of viruses from this study.

<p>Viruses identified in this study and included in the TMRCA analysis are grouped according to their flyway in the dated phylogenetic tree (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068522#pone-0068522-g002" target="_blank">Figure 2</a>). Subtypes are also included. Color code is indicated in the figure legend and reflects the inferred flyway in which the Egyptian viruses grouped (red for East Asian-Australian flyway, purple for Black Sea-Mediterranean flyway, green for Central Asian flyway, blue for East African-West Asian flyway, orange for Nile Delta and yellow if the group contained 2 or more reference viruses collected from different flyways). Viruses from this study that did not cluster with any other viruses from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068522#pone-0068522-g002" target="_blank">Figure 2</a> are indicated with (#) behind the number and were assigned to flyways that was inferred from large trees (data not shown).</p