Effects of passage history and sampling bias on phylogenetic reconstruction of human influenza A evolution

In this paper we determine the extent to which host-mediated mutations and a known sampling bias affect evolutionary studies of human influenza A. Previous phylogenetic reconstruction of influenza A (H3N2) evolution using the hemagglutinin gene revealed an excess of nonsilent substitutions assigned to the terminal branches of the tree. We investigate two hypotheses to explain this observation. The first hypothesis is that the excess reflects mutations that were either not present or were at low frequency in the viral sample isolated from its human host, and that these mutations increased in frequency during passage of the virus in embryonated eggs. A set of 22 codons known to undergo such “host-mediated” mutations showed a significant excess of mutations assigned to branches attaching sequences from egg-cultured (as opposed to cell-cultured) isolates to the tree. Our second hypothesis is that the remaining excess results from sampling bias. Influenza surveillance is purposefully biased toward sequencing antigenically dissimilar strains in an effort to identify new variants that may signal the need to update the vaccine. This bias produces an excess of mutations assigned to terminal branches simply because an isolate with no close relatives is by definition attached to the tree by a relatively long branch. Simulations show that the magnitude of excess mutations we observed in the hemagglutinin tree is consistent with expectations based on our sampling protocol. Sampling bias does not affect inferences about evolution drawn from phylogenetic analyses. However, if possible, the excess caused by host-mediated mutations should be removed from studies of the evolution of influenza viruses as they replicate in their human hosts.

Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications.

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.