180-Nucleotide Duplication in the G Gene of Human metapneumovirus A2b Subgroup Strains Circulating in Yokohama City, Japan, since 2014

Abstract

Human metapneumovirus (HMPV), a member of the family Paramyxoviridae, was first isolated in 2001. Seroepidemiological studies have shown that HMPV has been a major etiological agent of acute respiratory infections in humans for more than 50 years. Molecular epidemiological, genetic, and antigenetic evolutionary studies of HMPV will strengthen our understanding of the epidemic behavior of the virus and provide valuable insight for the control of HMPV and the development of vaccines and antiviral drugs against HMPV infection. In this study, the nucleotide sequence of and genetic variations in the G gene were analyzed in HMPV strains prevalent in Yokohama City, in the Kanto area, Japan, between January 2013 and June 2016. As a part of the National Epidemiological Surveillance of Infectious Diseases, Japan, 1308 clinical specimens (throat swabs, nasal swabs, nasal secretions, and nasal aspirate fluids) collected at 24 hospitals or clinics in Yokohama City were screened for 15 major respiratory viruses with a multiplex reverse transcription-PCR assay. HMPV was detected in 91 specimens, accounting for 7.0% of the total specimens, and the nucleotide sequences of the G genes of 84 HMPV strains were determined. Among these 84 strains, 6, 43, 10, and 25 strains were classified into subgroups A2a, A2b, B1, and B2, respectively. Approximately half the HMPV A2b subgroup strains detected since 2014 had a 180-nucleotide duplication (180nt-dup) in the G gene and clustered on a phylogenic tree with four classical 180nt-dup-lacking HMPV A2b strains prevalent between 2014 and 2015. The 180nt-dup causes a 60-amino-acid duplication (60aa-dup) in the G protein, creating 23-25 additional potential acceptor sites for O-linked sugars. Our data suggest that 180nt-dup occurred between 2011 and 2013 and that HMPV A2b strains with 180nt-dup (A2b_180nt-dup HMPV) became major epidemic strains within 3 years. The detailed mechanism by which the A2b_180nt-dup HMPV strains gained an advantage that allowed their efficient spread in the community and the effects of 60aa-dup on HMPV virulence must be clarified.

Keywords: G gene; Human metapneumovirus; duplication; molecular epidemiology; surveillance.

FIGURE 1

Phylogenetic tree of 574 Human metapneumovirus (HMPV) strains, constructed with the maximum likelihood method. A phylogenetic tree was constructed from the G gene sequences of 84 HMPV strains and all other available HMPV G gene sequences. In total, 574 G gene sequences of HMPV strains were included. The HMPV A2b subgroup strains containing a 180-nt duplication (180nt-dup) in the G gene (A2b_180nt-dup HMPV) are shown in cyan boxes and the other HMPV strains detected in Yokohama City between January 2013 and June 2016 are shown in orange boxes.

FIGURE 2

Nucleotide sequence alignment of HMPV strains. An alignment of the plus-sense nucleotide sequences of the 180-nt duplicated regions and adjacent regions of the HMPV G genes of 15 A2b_180nt-dup HMPV strains, the hypothetical ancestral strain (Ancestor), and a representative classical A2b strain (P7015) is shown. The first 180-nt region (1st-180nt) and the second 180-nt region (2nd-180nt) are shown in green and pink boxes, respectively. The first nucleotide of the initiation codon of the G gene is deemed to be nucleotide position 1. The nucleotide sequence of the hypothetical ancestral strain was predicted by constructing a consensus sequence of the duplicated 180-nt sequences of all 15 A2b_180nt-dup HMPV strains. Dots indicate nucleotides identical to those of the hypothetical ancestral strain. Hyphens in yellow boxes indicate gaps in the sequence.

FIGURE 3

Phylogenetic tree constructed with 1st-180nt and 2nd-180nt in the duplicated region of 15 A2b_180nt-dup HMPV strains. The duplicated region contains the original 1st-180nt and the duplicated 2nd-180nt. A phylogenetic tree was constructed from the 1st- and 2nd-180nts of 15 A2b_180nt-dup HMPV strains, and the tree was rooted with HMPV A2a strain HMPV/Yokohama.JPN/P7011/2013. The labels at the branch nodes indicate their statistical significance, calculated with bootstrapping (50 replicates).

FIGURE 4

Deduced amino acid sequence of A2b_180nt-dup HMPV strain. A plus-sense nucleotide (nt) sequence alignment of a representative A2b_180nt-dup HMPV strain (P7450) and a classical A2b HMPV strain (P7015), together with their deduced amino acid (aa) sequences, are shown. The 1st-180nt and 2nd-180nt regions are shown in green and pink boxes, respectively. The first nucleotide of the initiation codon of the G gene is deemed to be nucleotide position 1. Dots indicate nucleotides identical to those in the hypothetical ancestral strain. Hyphens in yellow boxes indicate gaps in the sequence.

FIGURE 5

Amino acid sequence alignment of HMPV strains. Aligned amino acid sequences derived from the HMPV G genes of 15 A2b_180nt-dup HMPV strains, the hypothetical ancestral strain (Ancestor), and a representative classical A2b strain (P7015) are shown. The region between amino acid (aa) 1–30 is the cytoplasmic tail (CT) domain, and the region between aa 31–53 is the transmembrane (TM) domain. The remaining region is the extracellular ectodomain (EE). The 1st-180nt and 2nd-180nt regions are shown in green and pink boxes, respectively. The amino acid sequence of the hypothetical ancestral strain was predicted by constructing a consensus sequence of all 15 A2b_180nt-dup HMPV strains. Dots indicate amino acids identical to those of the hypothetical ancestral strain. Hyphens in yellow boxes indicate gaps in the sequence.