Genetic diversity and evolution of human metapneumovirus fusion protein over twenty years

Virology Journal Genetic diversity and evolution of human metapneumovirus fusion protein over twenty years Chin-Fen Yang 3 Chiaoyin K Wang 3 Sharon J Tollefson 2 Rohith Piyaratna 2 Linda D Lintao 3 Marla Chu 3 Alexis Liem 3 Mary Mark 3 Richard R Spaete 3 James E Crowe Jr 0 1 2 John V Williams 0 1 2 0 Department of Microbiology and Immunology, Vanderbilt University School of Medicine , Nashville, TN , USA 1 Monroe Carell Jr Children's Hospital at Vanderbilt , Nashville, TN , USA 2 Department of Pediatrics, Vanderbilt University School of Medicine , Nashville, TN , USA 3 MedImmune Vaccines, Inc, Mountain View , CA , USA Background: Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in children. We examined the diversity and molecular evolution of HMPV using 85 full-length F (fusion) gene sequences collected over a 20-year period. Results: The F gene sequences fell into two major groups, each with two subgroups, which exhibited a mean of 96% identity by predicted amino acid sequences. Amino acid identity within and between subgroups was higher than nucleotide identity, suggesting structural or functional constraints on F protein diversity. There was minimal progressive drift over time, and the genetic lineages were stable over the 20-year period. Several canonical amino acid differences discriminated between major subgroups, and polymorphic variations tended to cluster in discrete regions. The estimated rate of mutation was 7.12 10-4 substitutions/site/year and the estimated time to most recent common HMPV ancestor was 97 years (95% likelihood range 66-194 years). Analysis suggested that HMPV diverged from avian metapneumovirus type C (AMPV-C) 269 years ago (95% likelihood range 106-382 years). Conclusion: HMPV F protein remains conserved over decades. HMPV appears to have diverged from AMPV-C fairly recently. - Background Human metapneumovirus (HMPV) is a recently described respiratory virus in the order Mononegavirales, family Paramyxoviridae, subfamily Pneumovirinae, genus Metapneumovirus [1]. HMPV is a leading cause of lower respiratory infection (LRI) in infants and children worldwide [2-13]. HMPV is also associated with severe disease in immunocompromised hosts or persons with underlying conditions [14-20]. Most reports of HMPV molecular epidemiology have included only a few seasons, and the genetic variability of HMPV over decades has not been determined. Candidate vaccines for HMPV are under development [21-25], and the fusion (F) protein is the major antigenic determinant of protection [22,24,26-28] Therefore, it is critical to understand the potential for immune escape through virus evolution over time, and the likelihood that immunity against a particular F protein included in a vaccine candidate will be broadly protective. The virus most closely related genetically to HMPV is avian metapneumovirus type C (AMPV-C) [1]. AMPV is an emerging pathogen of poultry that was identified in 1979. Subtypes AMPV-A and AMPV-B circulate in Europe and Africa, while AMPV-C was discovered in Minnesota and has been detected in the US and Korea [29,30]. Productive experimental infection of poultry with HMPV has not been successful, and serological studies have failed to detect evidence of human infection by AMPV [1]. Recent data suggest that F protein is responsible for this species restriction [31]. Thus, HMPV infection of humans may arise from a relatively recent trans-species transmission from AMPV-C. We analyzed full-length F gene sequences from 68 isolates of HMPV collected over a 20-year period from otherwise healthy children with respiratory disease and 17 published full-length F gene sequences from other regions of the world. Our data show that HMPV F is highly conserved geographically over several decades. Distinct amino acid changes were present between different genetic lineages, but these amino acids were conserved within lineages. Variations that were present clustered in discrete regions, suggesting antigenic sites possibly driven by selective immune pressure. However, HMPV F gene sequences did not display progressive drift over time, unlike influenza viruses. The mutation rate of HMPV was similar to that of other RNA viruses, and the time to most recent common ancestor suggested recent divergence from AMPV-C. Results Comparison of sequence identity between subgroups Full-length F gene sequences were obtained for 68 Tennessee strains of HMPV and assigned to one of the four proposed lineages (A1, A2, B1, or B2) based on phylogenetic analysis, discussed further below [32]. Of the 68 strains sequenced, 34 (50%) were of the B2 lineage, 18 (26%) A2, 7 (10%) B1 and 9 (13%) A1 lineage. Sequences obtained in this study were compared to 17 published full-length HMPV F gene sequences. The overall mean nucleotide identity between all 85 isolates was 89%, with a minimum identity of 83.7% (Table 1). The identity within major groups was higher, mean 96% (minimum 93.9%) between A1 and A2, and mean 97% (minimum 93.5%) between B1 and B2. The B2 lineage diverged more from the A lineages than the B1 lineage. B2 mean identity with A1 and A2 was 86.7% and 89.7%, respectively, while B1 identity with A1 and A2 was 91.3% and 94.7%, respectively. Mean nucleotide identity was >97% within all minor lineages, although the minimum identity for the B2 isolates was the lowest at 93.5%, showing more diversity within this lineage. Amino acid identity was more conserved than nucleotide identity between and within all groups, with overall minimum identity of 93.7% and mean identity 96.3%. Amino acid identity within major groups was 98.7% for A1 and A2, and 99.3% for B1 and B2. The minimum amino acid identity between all lineages was approximately 94%; the greater divergence of the B2 lineage at the nucleotide level was not represented in the amino acid sequence. Table 1: Comparison of nucleotide and amino acid identity of full-length human metapneumovirus F genes within or between subgroups. Number of sequences Minimum % nt identity Minimum % aa identity nt = nucleotide; aa = amino acid. Distinct and conserved amino acid changes between lineages There were a number of amino acid residues distinct to each group or subgroup (Table 2). The greatest number of divergent and subgroup-specific residues was identified in the F1 domain, between the two heptad repeat (HR) regions. At several positions all subgroups had either arginine or lysine but maintained a basic residue: 82, 348, 450, 479 and 518; only position 82 has been shown to be cleaved during infection [33,34]. Many subgroup-specific residues were similar biochemically between groups. Some variations, however, were unexpected, such as the presence of a proline at position 404 only in B subgroup viruses. Fourteen cysteine residues were conserved among all isolates except one Japanese sequence (JPS03.178) with a reported C292W variation [35]. Three potential Nglycosylation sites were conserved in (...truncated)