Figure 1.
Map indicating the locations of farms infected during the 2003 HPAI H7N7 epidemic.
Farms are represented by coloured dots, according to their location and inclusion in a cluster of infection. Black dots in the main map correspond to farm samples not analyzed in this study. Farm samples represented by coloured squares were used for the within-flock viral genetic analyses. In order to maintain the clarity of the figure, only the names of the farms mentioned in the main text are shown. All samples are described in details in Table S1.
Table 1.
Mean nucleotide substitution rates and estimation of TMRCA of the H7N7 epidemic.
Figure 2.
Phylogenetic trees of H7N7 viruses.
Time-scaled phylogenies (dates on the horizontal axis) inferred using Bayesian MCMC analysis from (A) HA gene; (B) NA gene; (C) PB2 gene. Nodes supported by ≥0.7 posterior probability are indicated by a grey dot. Posterior probability values from the time-scaled BMCMC method, the MrBayes BMCMC method, and the Maximum Likelihood method (1,000 ML bootstrap replications) are shown for nodes delimitating clusters of transmission (tsBMCMC/MrBMCMC/ML; noted Cluster I–IV). The three samples with discordant phylogenies are indicated by black square (F45), circle (F76), and triangle (F145). Nodes and branches are coloured according the geographical origin of the farm samples. Yellow, Gelderland area; Blue, Central area; Red, Limburg area; Green, Southwest area. Fully annotated trees are available online in supplementary figures S2A–C.
Figure 3.
Median-joining phylogenetic network of H7N7 viruses.
The median-joining network was constructed from the combined HA, NA and PB2 sequence data. This network includes all the most parsimonious trees linking the sequences. Each unique sequence genotype is represented by a coloured circle sized relative to its frequency in the dataset. Genotypes are coloured according to the location of the farm sample and its inclusion in a cluster of infection. Branches in black represent the shortest trees; Additional branching pathways are in grey. Each node is separated by a specific number of mutations represented by grey dots. Mutations corresponding to specific amino acid changes are indicated. For genotypes containing a deletion in the NA stalk region, the type of deletion is indicated between brackets beside the name of the isolate (see Table S1 for the description of deletion types). Names of farm samples involved in likely inter-farm transmission events are in red (see Table 2). (*) positively selected amino acids linked to adaptation to mammalian hosts. G1: group of samples including F38, F54, F64, F113, F162, F194, F199; G2: F134, F160, F166; G3: F122, F161, F164, F171, F182; G4: F2, F5, F12, F21, F43, F60, F91; G5: F39, F70, F92, F129; G6: F15, F29, F37; G7: F16, F19, F52; G8: F193, F217, F223, F231; G9: F36, F68, F167, F191(d1), F205(d5), F207(d2); G10: F203 (d3), F219(d3), F228(d3); G11: F197, F242, F232.
Table 2.
Summary of the most likely transmission events identified either from pair of farm samples exclusively sharing the same sequence genotype, or pair of farm samples having sequence genotypes unambiguously linked in the network analysis.
Table 3.
Values of Log-likelihood (lnL) and dN/dS for HA, NA and PB2 genes using different selection models in the CODEML analysis, and LRT tests comparing the two models.
Figure 4.
Recombination analysis on concatenated H7N7 virus sequences.
(A) Bootscan analysis on the full dataset; (B) Bootscan analysis on the dataset with the HA codon 143 removed. The Cluster IV virus group was used as query in the analysis, with an 800 bp window size and step size of 10 bp. A schematic diagram of the concatenated HA, NA and PB2 virus segments is shown on top.
Figure 5.
Schematic diagrams summarizing within-flock genetic diversity in 6 farm samples.
The sequence variants found by clonal sequencing of partial NA and HA genes in 6 different samples are represented by coloured circles sized relatively to their frequency. Total number of clones sequenced per sample (n) is indicated. The exact number of copies of each genetic variant is indicated when >1. Variants in black correspond to the sequence originally isolated in each farm. Each variant is separated by nucleotide substitutions represented by filled black dots (non-synonymous changes) or open dots (synonymous changes), and by deletions represented by squares. The exact position of the deletion in the NA gene is indicated. The red node represents a variant similar to the sequence obtained for the F192 sample (see main text). The white node represents a potential missing variant.
Table 4.
Summary of results obtained from clonal sequencing.