Phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy

Abstract

Human adenoviruses (HAdV) are responsible for a wide spectrum of diseases. The neutralization epsilon determinant (loops 1 and 2) and the hemagglutination gamma determinant are relevant for the taxonomy of HAdV. Precise type identification of HAdV prototypes is crucial for detection of infection chains and epidemiology. epsilon and gamma determinant sequences of all 51 HAdV were generated to propose molecular classification criteria. Phylogenetic analysis of epsilon determinant sequences demonstrated sufficient genetic divergence for molecular classification, with the exception of HAdV-15 and HAdV-29, which also cannot be differentiated by classical cross-neutralization. Precise sequence divergence criteria for typing (<2.5% from loop 2 prototype sequence and <2.4% from loop 1 sequence) were deduced from phylogenetic analysis. These criteria may also facilitate identification of new HAdV prototypes. Fiber knob (gamma determinant) phylogeny indicated a two-step model of species evolution and multiple intraspecies recombination events in the origin of HAdV prototypes. HAdV-29 was identified as a recombination variant of HAdV-15 (epsilon determinant) and a speculative, not-yet-isolated HAdV prototype (gamma determinant). Subanalysis of molecular evolution in hypervariable regions 1 to 6 of the epsilon determinant indicated different selective pressures in subclusters of species HAdV-D. Additionally, gamma determinant phylogenetic analysis demonstrated that HAdV-8 did not cluster with -19 and -37 in spite of their having the same tissue tropism. The phylogeny of HAdV-E4 suggested origination by interspecies recombination between HAdV-B (hexon) and HAdV-C (fiber), as in simian adenovirus 25, indicating additional zoonotic transfer. In conclusion, molecular classification by systematic sequence analysis of immunogenic determinants yields new insights into HAdV phylogeny and evolution.

FIG. 1.

Phylogenetic analysis of the nucleic acid sequences of ɛ determinant L1 (600 bp referring to HAdV-C1) (A) and L2 (283 bp referring to HAdV-C1) (B) hexon sequences (neighbor-joining tree, Kimura two-parameter matrix). The bootstrap values were generated with 1,000 pseudoreplicates. For nucleotide sequence accession numbers see Materials and Methods.

FIG. 2.

Phylogenetic analysis of the deduced amino acid sequences of L1 (A) and L2 (B) hexon sequences (neighbor-joining tree, Kimura two-parameter matrix). The bootstrap values were generated with 1,000 pseudoreplicates. Three HAdV-D subclusters which were used for calculation of similarity plots (Fig. 3) are depicted. For nucleotide sequence accession numbers see Materials and Methods.

FIG. 3.

Similarity plots of three clusters from species HAdV-D with a window of 10 amino acids. (A) Cluster 1; (B) cluster 2; (C) cluster 3. Members of the three clusters are shown in Fig. 2A. Hypervariable regions (HVR1 to HVR6) are marked in the similarity plots.

FIG. 4.

Distribution of pairwise nucleotide identity scores of L2. Frequency (y axis) represents the number of all nucleotide comparisons between different prototypes with an identical genetic diversity. Peak 1 corresponds to comparisons of heterologous strains (different serotype) of the same major phylogenetic cluster (same species). In contrast to this, peak 2 corresponds to comparison of heterologous strains (different serotype) of different phylogenetic clusters (different species). The asterisk represents the 99% identity between HAdV-D15 and HAdV-D29.

FIG. 5.

(A) Phylogenetic analysis of the deduced amino acid sequence from γ determinant (269-amino-acid fiber knob sequence referring to HAdV-C2), with a neighbor-joining method tree based on a Kimura two-parameter matrix. The bootstrap values were generated with 1,000 pseudoreplicates. For nucleotide sequence accession numbers see Materials and Methods. (B) Distribution of pairwise nucleotide identity scores of fiber knob (γ determinant). Frequency (y axis) represents the number of all nucleotide comparisons between different prototypes with an identical genetic diversity. Peak 1 corresponds to comparisons of heterologous strains (different serotype) of the same major phylogenetic cluster (same species). In contrast to this peak, peaks 2 and 3 correspond to comparison of heterologous strains (different serotype) of different phylogenetic clusters (different species). Peak 2 is formed only by interspecies identities between HAdV-C (including HAdV-E) and -D, as well as between HAdV-A and -F.

FIG. 6.

Phylogenetic analysis of generic hexon PCR sequences (nucleic acid sequences): amplicons of conventional PCR (4) (A) and real-time PCR (26) (B) with neighbor-joining method based on a Kimura two-parameter matrix. Both phylogenetic trees show that sequence divergence is sufficient for species identification as well as type identification of HAdV-A, HAdV-E, and HAdV-F viruses. For nucleotide sequence accession numbers see Materials and Methods.

FIG. 7.

Similarity plots of three clusters from species HAdV-B with a window of 10 amino acids. (A) Cluster formed by HAdV-B3 and -B7; (B) cluster formed by HAdV-B14, -B34, and -B35; (C) cluster formed by HAdV-B11, -B21, and -B35. Hypervariable regions (HVR1 to HVR6) are marked in the similarity plots.