Human metapneumovirus (HMPV) was first discovered in 2001, but seroepidemiological studies indicate that HMPV has been a major etiological agent of acute respiratory infections (ARIs) in humans for more than six decades [1
]. HMPV circulates worldwide; about half of all children are infected by HMPV before 2 years of age, and most children are infected before 5 years of age [1
]. Unfortunately, individuals infected with HMPV usually do not develop lifelong immunity against this virus, and reinfection occurs frequently [1
]. Although HMPV generally causes mild-to-moderate ARIs in the healthy adult population, it causes severe ARIs in aged adults and in patients with underlying diseases, such as diabetes and cardiopulmonary disease [3
]. Fatal outbreaks of HMPV in a long-term care facility have been reported [6
HMPV is a non-segmented, negative-stranded RNA virus belonging to the family Pneumoviridae
. Its viral genome is approximately 13 kb in length and contains eight genes (N, P, M, F, M2, SH, G, and L). These genes encode a total of nine proteins, including three surface glycoproteins: F (fusion), SH (small hydrophobic), and G (glycol-) proteins. The F protein is an essential protein for viral adsorption and fusion to host cells, acting via RGD-bound integrins, such as αv
integrin, and glycosaminoglycans, such as heparan sulfate, and is a major target antigen for neutralizing antibodies [8
]. The SH protein may suppress the innate immune response of host cells via suppressing NF-κB activation [11
]. This protein also has properties consistent with those of a viroporin and can modulate viral fusogenic activity [12
]. The G protein of some HMPV lineages also binds to glycosaminoglycans on the host cell surface and contributes to HMPV infection [13
]. Additionally, the HMPV G protein associates with RIG-I, inhibits RIG-I-dependent gene transcription, and suppresses host innate immune responses [15
HMPV is classified into two antigenically distinct groups, A and B [16
]. The G protein is the most variable of the HMPV proteins, and mutations of the G protein accumulate predominantly in its extracellular domain [17
]. Each viral group is further divided into two subgroups—A1 and A2 in group A, and B1 and B2 in group B—based mainly on variations in the G gene [16
]. Furthermore, within the A2 subgroup, there are two phylogenetically distinct clades, A2a and A2b [19
]. We recently reported unique HMPV A2b strains with a 180- or 111-nucleotide duplication in the G gene (HMPV A2b180nt-dup
strains, respectively) [20
]. In addition, other studies have recently described novel HMPV clades A2b2 and A2c [22
]. The phylogenetic and evolutionary relationships between the HMPV A2b nt-dup strains, A2b2 strains, and A2c strains are still unclear. The present study conducted a genome-wide phylogenetic and evolutionary analysis of 161 HMPV strains to address this question.
Shortly after the initial discovery of HMPV, four subtypes (A1, A2, B1, and B2) were proposed [16
]. Subsequently, the HMPV subtype A2 was divided into two subtypes, A2a and A2b [19
]. A consensus has been reached among researchers regarding these five HMPV subtypes. However, individual researchers have used different methods and gene regions or lengths when proposing new subtypes, such as A2c, A2b1, and A2b2 [22
]. The “A2c” subtype was provisionally proposed based on analyses using only a short region (321 nucleotides in length) of the F gene and limited numbers of HMPV strains [22
]. The “A2b1” and “A2b2” subtypes were also proposed based on analyses using a short region (111 nucleotides in length) of the F gene [23
]. These provisional new subtypes of A2b strains were still classified as part of subtype A2b in some studies [32
]. Such variations in genotype nomenclature among researchers may cause confusion regarding HMPV molecular epidemiology. Here, we demonstrated that the HMPV A2b2 strains and A2c strains all belong to the same subtype, together with the HMPV A2b180nt-dup
strains, based on phylogenetic analyses conducted using partial regions of the F and G genes. Additionally, some of the HMPV A2b2 strains and A2c strains had identical nucleotide sequences in the analyzed region of the F gene. These data strongly suggest that the recently proposed HMPV subtypes A2c and A2b2 are separate descriptions indicating the same subtype. The genetic distance between the subtypes A2b1 and A2b2, which is twice as high as the highest intra-subtype p
distance, indicates that A2b1 and A2b2 are distinct subtypes.
Both HMPV novel subtypes A2b2 and A2c were first proposed in 2012 based on phylogenetic analyses using a partial sequence of the HMPV F gene [22
]. The phylogenetic trees of the two studies that proposed these HMPV subtypes differed from one another, especially in their subdivision of the subtype A2 strains. Nidaira et al. reported that strains belonging to subtype A2 were subdivided into three clusters: A2a, A2b, and A2c [22
]. In contrast, Regev et al. found that subtype A2 was first divided into subtypes A2a and A2b, and then subtype A2b was further divided into subtypes A2b1 and A2b2 [23
]. This difference in the constructed phylogenetic trees may reflect the differences in the viral strains and F gene regions used for the analyses. In the present study, we found that the HMPV A2 strains were first divided into A2a and A2b, and the A2b strains were then further divided into two distinct subtypes, A2b1 and A2b2. The presence of subtypes A2b1 and A2b2 within the subtype A2b was observed in the maximum likelihood tree constructed from the HMPV full-genome sequences as well as in all eight MCMC trees constructed based on the individual HMPV genes (Table 4
). These data support the A2b1 and A2b2 subtypes proposed by Regev et al. in their phylogenetic tree.
In the present study, estimates for the tMRCA of all the HMPV strains ranged from 1645 to 1858, whereas those for the tMRCA of individual HMPV subtypes (A1, A2, B1, and B2) were around the 1970s and 1980s. These data are consistent with findings from a previous study using all eight HMPV genes [38
]. The divergence time for the HMPV subtypes A2a and A2b was estimated as 1971–1985. In contrast, the estimated divergence time of the HMPV subtypes A2b1 and A2b2 was more recent (1989–1996). Compared with other subtypes, including A2a and A2b1, subtype A2b2 appeared to have the most recent tMRCA estimates (2002–2005). Our molecular evolutionary analyses indicate that subtypes A2b1 and A2b2 diverged from subtype A2b approximately a decade after subtype A2 became divided into subtypes A2a and A2b.
The recent subtype classification of HMPV A2 strains is not uniform among researchers. Here, we analyzed 161 full-genome sequences of HMPV strains, and the results show that the HMPV subtype A2b is divided into two subtypes that can appropriately be called A2b1 and A2b2. The HMPV A2b180nt-dup
strains, which are now the predominant HMPV strains, were found to belong to the HMPV subtype A2b2; therefore, HMPV strains belonging to the subtype A2b2 are likely to be continuously detected for several years, if not longer [32
]. Thus, a unified classification for the HMPV subtype A2 strains is important for future HMPV surveillance and epidemiological studies.
HMPV is currently divided into five agreed-upon subtypes: A1, A2a, A2b, B1, and B2. In 2012, the novel HMPV subtypes A2c, A2b1, and A2b2 were proposed based on phylogenetic analyses using a short region of the F gene and limited numbers of HMPV strains [22
]. Recently, individual researchers have used different methods and gene regions or lengths when describing new subtypes, such as A2c, A2b1, and A2b2 [31
]. However, a genome-wide phylogenetic and evolutionary analysis of recently proposed novel HMPV subtypes (A2c, A2b1, and A2b2) has not yet been performed, and detailed phylogenetic and evolutionary relationships between these subtypes are still unclear.
The present study conducted a genome-wide phylogenetic and evolutionary analysis of 161 HMPV strains and demonstrated that HMPV A2b strains were divided into two distinct subtypes, A2b1 and A2b2, as proposed by Regev et al. in 2012 [23
]. In addition, our data also demonstrated that the HMPV subtypes A2b2 and A2c are separate descriptions indicating the same subtype. These findings are essential for the unified classification of HMPV subtype A2 strains, which is important for future HMPV surveillance and epidemiological studies.