Molecular comparisons of full length metapneumovirus (MPV) genomes, including newly determined French AMPV-C and -D isolates, further supports possible subclassification within the MPV Genus

Abstract

Four avian metapneumovirus (AMPV) subgroups (A-D) have been reported previously based on genetic and antigenic differences. However, until now full length sequences of the only known isolates of European subgroup C and subgroup D viruses (duck and turkey origin, respectively) have been unavailable. These full length sequences were determined and compared with other full length AMPV and human metapneumoviruses (HMPV) sequences reported previously, using phylogenetics, comparisons of nucleic and amino acid sequences and study of codon usage bias. Results confirmed that subgroup C viruses were more closely related to HMPV than they were to the other AMPV subgroups in the study. This was consistent with previous findings using partial genome sequences. Closer relationships between AMPV-A, B and D were also evident throughout the majority of results. Three metapneumovirus "clusters" HMPV, AMPV-C and AMPV-A, B and D were further supported by codon bias and phylogenetics. The data presented here together with those of previous studies describing antigenic relationships also between AMPV-A, B and D and between AMPV-C and HMPV may call for a subclassification of metapneumoviruses similar to that used for avian paramyxoviruses, grouping AMPV-A, B and D as type I metapneumoviruses and AMPV-C and HMPV as type II.

Figure 1. Genetic relationships between previously published MPV genome sequences and the full length sequences of Fr-AMPV-C (•) and D (♦).

The tree was constructed as described in the text using the neighbor-joining method. Percentages at branch points represent the number of times the group to the right of that branch point occurred among 1000 trees generated by bootstrap from the original alignment.

Figure 2. Amino acid comparisons of the nucleocapsid ORF of AMPV-A, B, C and D and HMPV subgroups A and B.

Positions 44 and 137 were specific to Fr-AMPV-C. Grey shaded boxes represent four highly conserved regions across all MPVs. Boxes A, B and C are regions that have been reported to be conserved amongst pneumoviruses (Barr et al 1991). In metapneumoviruses domains B and C appear to be extended creating one single larger domain (B/C). *end of protein.

Figure 3. Amino acid comparisons of the phospoprotein ORF of AMPV-A, B, C and D and HMPV subgroups A and B.

A region resposible for the tetramerization of the P protein is shown together with a molecular recognition element (MoRE). Grey box represents a highly conserved region in MPVs in the MoRE. *end of protein.

Figure 4. Amino acid comparisons of the fusion ORF of AMPV-A, B, C and D and HMPV subgroups A and B. Previously identified biologicaly important domains are labled and underlined.

Diamonds indicate conserved cysteine residues. Open boxes highlight other domains discused in the relevent paragraph. Cleavage site (Clv) and integrin binding domain (Ibd). *end of protein.

Figure 5. Amino acid comparisons of the previously reported conserved domain III (Poch et al., 1990) in the L ORF of AMPV-A, B, C and D and HMPV subgroups A and B.

Four core motifs (A, B, C and D) described by Poch et al 1990 are underlined. A pentapeptide conserved in mononegavirales is highlighted in grey within motif C. Open boxes highlight regions in MPVs with 100% conservation. *end of protein.

Figure 6. Codon usage: the effective number of codons (Nc) used within the longest MPV gene (L). Sequences included as in Table 1.

The curve represents expected codon usage in relationship to the GC3 content, therefore at 50% GC content all synonymous codons should be used equally in the absence of other factors. Nc values range from 20 when only one of the possible synonymous codons is used for each amino acid, to 61 when all synonymous codons are used. The closer the Nc value is to 20, the stronger the codon usage bias is, and the higher the degree to which codons are used non-randomly. HMPVs (Nc = 41.9 to 43.2) AMPV-Cs (Nc = 47.1 to 47.5) and AMPV-A, B, D (Nc = 51.4 to 52.7).