Results
116 samples tested aMPV positive, all belonging to subtype B (n. 27 from France, n. 18 from Italy, n. 27 from Romania, n. 36 from Spain and n. 8 from the United Kingdom) and 116 partial G gene sequences were obtained. Sequence data have been submitted to the GenBank database under accession numbers MT432826-MT432923 and MT436220-MT436237 (Tables S3 to S7).
Phylogenetic analysis was carried out by reconstructing both a comprehensive tree (Figure 1) and five Country- specific trees (Figures S1 to S5).
Furthermore, a phylogenetic tree including selected European and extra-European aMPV-B sequences was generated (Figure 2).
Regardless of the Country of origin, aMPV strains detected from 2001 to 2019, herein referred to as “recent strains”, were distinguished from those clustering with vaccines and field strains detected prior to the 1990s, referred to as “older strains” (from which the vaccines were established by attenuation) and those forming independent clades.
As a rule, aMPV strains were referred to as “vaccine-derived strains” if the maximum nucleotide sequence identity with a reference vaccine strain was greater than or equal to 99 % and if they fell into the same phylogenetic cluster. The remaining strains were classified as “field strains”.
On the above basis, 70 out of 116 aMPV-B strains were labelled as field strains (60%); the remaining 46 strains (40%) were identified as vaccine-derived strains.
A certain degree of geographic clustering was observed among Romanian, Spanish, French and Italian field strains (Figure 1). Some Italian strains also clustered together with some recent Greek strains.
French aMPV field strains (Figure S1) were divided in two clusters. The largest cluster displayed a within-group mean p-distance of 0.004 and included chicken, turkey and guinea fowl strains detected from 2017 to 2019, while the smallest had a within-group mean p-distance of 0.000 and contains turkey strains detected in 2018. Vaccine-derived strains clustered respectively with vaccine strain PL21, 11/94 or VCO3 and were mostly detected in homologous-vaccinated turkeys or chickens, but also in unvaccinated guinea fowls.
Italian field strains were grouped in two main clusters (Figure S2): the larger one was composed of several sub-clusters including field strains detected from 2010 to 2019 in turkey, chicken, and guinea fowl flocks and showed a within-group mean p-distance of 0.011. The smaller one included field strains detected from 2001 to 2009 and in 2016 in turkeys and chickens, with a within-group mean p-distance of 0.004. Vaccine-derived strains clustering with aMPV-B vaccine strains VCO3 or 1062 were detected from vaccinated turkeys.
Romanian aMPV-B field strains (Figure S3), detected from turkeys or chickens, fell within a single phylogenetic group showing a within-group mean p-distance of 0.001. The vaccine-derived strains, all detected in vaccinated turkey flocks, clustered with vaccine strains PL21, 11/94 or VCO3.
In the Spanish tree (Figure S4) field strains fell within two main clusters: a larger one (within-group mean p-distance: 0.006) containing strains detected from 2014 to 2017; a smaller one (within-group mean p-distance: 0.003) containing viruses detected from 2014 to 2015. Spanish vaccine-derived strains clustered with vaccine strain 11/94 or PL21.
The British phylogenetic tree (Figure S5) exclusively displayed vaccine-derived strains, clustering with vaccine strain PL21, 11/94 or VCO3.
The tree reconstructed with representative aMPV-B sequences from all over the world (Figure 2) confirmed the Country-specific clustering trend seen for the European strains. Well-defined clusters were identified for field strains circulating in Brazil, Iran, Israel and Turkey. Vaccines, vaccine-derived and older strains clustered together and were showed in the figure compressed in two subtrees.