Molecular Evolution of Respiratory Syncytial Virus Fusion Gene, Canada, 2006–2010

To assess molecular evolution of the respiratory syncytial virus (RSV) fusion gene, we analyzed RSV-positive specimens from 123 children in Canada who did or did not receive RSV immunoprophylaxis (palivizumab) during 2006–2010. Resistance-conferring mutations within the palivizumab binding site occurred in 8.7% of palivizumab recipients and none of the nonrecipients.

H uman respiratory syncytial virus (RSV) is the most common cause of acute respiratory tract infections (RTIs) and a major cause of hospital admission and death among children <5 years of age worldwide (1). Risk for severe RSV-associated illness is highest among children born prematurely or with chronic medical disorders (2). Palivizumab immunoprophylaxis is the only available measure to prevent severe RSV disease.
The RSV fusion (RSV-F) surface glycoprotein mediates virus fusion to host cells. It is a major antigenic determinant that elicits neutralizing antibodies and cytotoxic T-lymphocyte immunity (3). Palivizumab (MedImmune, Gaithersburg, MD, USA) is a humanized mouse monoclonal antibody that inhibits RSV-F by binding to a defi ned epitope (residues 262-276) (4,5). Palivizumab immunoprophylaxis is recommended for the prevention of serious lower RTIs caused by RSV in children at high risk (6). RSV strains with mutations in key amino acid residues within the palivizumab binding site are resistant to this antibody (7)(8)(9); however, little is known about the prevalence of such mutations in clinical samples. Furthermore, despite its role in RSV pathogenesis, immunity, and prevention strategies, few data on RSV-F molecular evolution are available (10,11) because previous phylogenetic studies have focused on the RSV-G glycoprotein (12,13). Therefore, we monitored evolutionary changes in RSV-F, particularly potential resistance mutations in the palivizumab binding site, among strains from children who did and did not receive palivizumab.

The Study
This cohort study was approved by the Centre Hospitalier Universitaire de Québec Research Ethics Board. Participants were <3 years of age and either received medical attention at an outpatient pediatric clinic or were hospitalized at a pediatric center for acute RTI during 4 winter seasons (2006)(2007)(2008)(2009)(2010), in Québec City, Québec, Canada.
Clinical data were prospectively collected at study entry and after 1-month follow-up. For all patients, at the fi rst visit a nasopharyngeal aspirate was collected. The aspirate was frozen at −80°C until subsequent testing by a multiplex PCR/DNA hybridization assay that detects RSV genotype-A (RSV-A), RSV-B, and 22 other respiratory viruses (Infi niti RVP assay; Autogenomics, Carlsbad, CA, USA) (14). Along with the newly generated nucleotide sequences from the 123 children (23 palivizumab recipients; 100 nonrecipients), we analyzed 92 clinical RSV-F sequences provided by other investigators (10,11) or available from GenBank. Palivizumab exposure was unknown for these samples, and all originated outside Canada. We also included 10 unpublished RSV-F sequences from a 2004-2005 study of palivizumab recipients in Canada (15). Multiple-sequence alignment was performed with ClustalW in MEGA5 (www. megasoftware.net). A 1,524-bp region (positions 79-1602 in the prototype A2 and B1 RSV-F genes) was translated into amino acid sequences, and the palivizumab binding site (residues 262-276) was assessed for variations. We removed 55 redundant (identical) nucleotide sequences. The fi nal dataset comprised 170 unique sequences, including 89 that were newly generated in this study (GenBank accession nos. JF776691-JF776779).
Phylogenetic reconstructions were computed in MEGA5 ( Figure). Strains from Canada tended to group together. However, this segregation likely refl ects temporal evolution rather than geographic infl uence because all RSV-F sequences originating from other countries were collected >10 years ago. Previous RSV-G studies have demonstrated concurrent circulation of related lineages in distant areas (13). Overall, RSV-F was highly conserved (Table 1). RSV-A exhibited more genetic diversity than RSV-B. This difference may refl ect sampling bias because more RSV-A sequences originating from diverse locations and years were available; however, the greater genetic diversity of RSV-A compared with RSV-B has also been observed in a study conducted in South Korea (10).
Of the 23 RSV-F amino acid sequences from patients who received palivizumab ( Microneutralization assays were performed as described elsewhere, with minor modifi cations (9). RSV was incubated (for 2 h at 37°C) with serially diluted palivizumab, then cultured in Vero cells (for 5 d). RSV replication and 50% inhibitory concentrations (IC 50 ) were subsequently determined by F protein quantifi cation by using ELISA. The mean ± SD IC 50 of C0910-1006A, a N276S strain, was 0.33 ± 0.04 μg/mL, similar to that of RSV-A2 wild type (IC 50 0.46 ± 0.04 μg/mL) and therefore was considered susceptible. Position-272 variants did not grow in culture and were not tested.

Conclusions
We report the prevalence of resistance-conferring mutations in RSV-F among children receiving or not receiving palivizumab. Although infrequent (8.7% of infections in palivizumab recipients), residue 272 mutations were signifi cantly associated with palivizumab exposure and not observed at all in nonexposed patients.
We identifi ed 2 new clinical specimens with position 272 mutations (K272Q and K272M). We cannot exclude the possibility that additional specimens contained mixed viral populations with minor proportions of position-272 mutants not detectable by conventional sequencing methods. Changes at this position (from lysine to asparagine, glutamine, glutamic acid, methionine, or threonine) have produced palivizumab resistance in vitro (9) and in a cotton rat model (7). As previously reported, 1 (10.0%) of 10 sequences from our 2004-2005 study of palivizumab patients carried a 272 mutation (K272E) (15). The K272E substitution is the only substitution also demonstrated to confer resistance to motavizumab, an enhanced-potency monoclonal antibody developed by affi nity maturation of palivizumab (9). We could not perform neutralization assays on position-272 variants because they did not grow in culture. This fi nding suggests that such changes adversely affect viral replicative capacity (7,9).
Phylogenetic analysis demonstrated that mutations in the palivizumab binding site occur in diverse genetic backgrounds; all 3 strains with substitutions at residue 272 grouped to different clades (Figure). Furthermore, these mutant strains caused mild disease treatable in an outpatient clinic and severe illness requiring hospitalization.
From 3 Canadian communities we detected a lineage harboring an N276S mutation in 44.4% of RSV-A sequences  Although serious RSV RTIs during palivizumab prophylaxis remain uncommon, we observed an 8.7% prevalence of known resistance mutations among 23 medically attended patients receiving palivizumab. These fi ndings underscore the need for continued monitoring of RSV-F evolution. Figure. Phylogenetic analysis of 170 near-fulllength unique respiratory syncitial virus fusion (RSV-F) gene sequences (nt 79-1602). Panels A and B are detailed phylograms of the RSV-A and RSV-B taxa analyzed, respectively. One bovine RSV-F sequence was added to the dataset (GenBank accession no. AF295543.1) as the outgroup (not shown) and used for rooting the phylograms. Topology was inferred by using the neighbor-joining method, and evolutionary distances were computed by using the maximum-composite likelihood method in MEGA5 software (www.megasoftware.net). The topologic accuracy of the tree was evaluated by using 1,000 bootstrap replicates.