In this study, the genetic heterogeneity of HMPV was addressed by analysis of the nucleotide and predicted amino acid sequences of part of the F (n = 84), complete F (n = 4), and the complete G (n = 35) protein genes. Phylogenetic analysis of these sequences showed two main lineages (A and B) with each divided into two sublineages (1 and 2). As was described for HRSV and APV, the F protein was highly conserved, which is in agreement with F proteins of pneumoviruses having structural and functional constraints for amino acid mutations (34
). On the basis of the high percentage sequence identity for the complete F proteins of the prototype viruses of the four lineages, sequences for the complete F proteins of all 84 samples would probably demonstrate similar low variability. In contrast to the F protein, the nucleotide and predicted amino acid sequences of the complete G coding regions showed high sequence diversity (as low as 30%–37% aa identity). Besides the high amino acid sequence variation, we observed variation in length of the different G proteins. Where most of the length variation was due to nucleotide substitutions, two of the samples showed a change in reading frame due to deletion or addition of single nucleotides. Frame shift mutations and use of alternative reading frames have been described for HRSV (35
). As described for isolate NL/1/00, the G coding region of the 35 samples sequenced in the present work indicated long alternative ORFs. However, these secondary ORFs varied in length and position compared to the ones described (4
). Whether premature stop codons, the incidence of frame shift mutations, and possible use of alternative reading frames influence the antigenic properties of the viruses needs to be examined in more detail.
Phylogenetic analyses showed that the HMPV samples obtained from different years and from different countries were randomly distributed over all four sublineages. For HRSV it has also been reported that very similar viruses were isolated at different times and from geographically distant sites (36
). Different lineages within HRSV subgroup A and B have been found on the basis of the variation in the G protein. Within each subgroup, progressive accumulation of amino acid changes was noted, suggesting that the G protein of HRSV might be susceptible to immune pressure (36
). Analysis of the amino acid sequences of the HMPV samples described in this study did not indicate such accumulation over time. However the following observations indicate that the variation of the HMPV G protein might occur as a result of immunogenic pressure in a same manner as was postulated for the RSV G protein: 1) most of the amino acid sequence variation was found in the extracellular domain of the G protein, 2) the variation found at the amino acid sequence level was higher than that at the nucleotide sequence level, 3) the number and position of potential glycosylation sites were not conserved, and 4) deletions, additions and substitutions of single nucleotides resulted in premature stop codons and drastic changes of the carboxy terminal of the protein (18
). Until a larger number of more chronologically diverse HMPV samples have been examined, this issue remains inconclusive.
To address the antigenic relationship between members of the different HMPV lineages, we tested ferret sera raised against viruses from the four sublineages in virus neutralization assays. Serologic responses upon infections tend to broaden over time. On the basis of the relatively close genetic relationship between sublineages A1 and A2 or B1 and B2, we decided to collect serum samples at an early time point, to obtain large antigenic differences between the four sublineages. The low homologous virus neutralization titers in serum samples collected 21 days postinfection may explain the lower ratio between homologous and heterologous virus neutralization titers as compared to sera collected 28 days postinfection. The studies with serum samples collected at 21 days postinfection showed that viruses within one main lineage (e.g., A1 and A2 or B1 and B2) were antigenically closely related. The difference in homologous and heterologous virus neutralization titers between members of the two different lineages A and B titers (12- to 128-fold higher homologous titer than heterologous titer) indicate a difference in antigenicity between lineage A and B. Classic virology studies have used a definition of a homologous-to-heterologous virus neutralization titer ratio of >16 for defining serotypes. This same definition notes that if neutralization shows a certain degree of cross-reaction between two viruses in either or both directions (homologous-to-heterologous titer ratio of 8 or 16), distinctiveness of serotype is assumed if substantial differences in sequences are observed (29
). On the basis of our results, and based on the described definition, we propose defining the two main lineages of HMPV as serotypes A and B. The HMPV samples were obtained from different study populations, from different countries, and from patients with a wide spectrum of clinical signs. So far, we have no indication of an association between infection with either of the serotypes and a specific study group or with severity of disease. More epidemiologic studies are needed to address this issue.
The circulation of two serotypes of HMPV might have implications for the development of vaccines. Studies in cynomolgous macaques showed that reinfection is suppressed by high titers of virus neutralization antibodies against the homologous virus and far less by heterologous virus neutralization antibodies (data not shown). So far, one heterologous reinfection has been reported in humans (28
). However, children approximately >
5 years of age have higher virus neutralization antibody titers than those 1–2 years of age (25
), which suggests that reinfections may occur frequently, most likely with the viruses from the heterologous serotype. For RSV, the importance of difference in antigenicity between the two subgroups regarding protective immunity and vaccine development is still a subject of discussion. However, in animals and humans, the neutralizing capacity against homologous viruses is higher than that against heterologous viruses, and in animals high homologous virus neutralization titers protect against reinfection. In humans, reinfection often occurs with a strain from the heterologous group, and high homologous virus neutralization antibody titers protect against severe infection (13
). The two serotypes of HMPV might resemble the two subgroups of HRSV in immunogenic properties, although more extensive epidemiologic and immunologic studies have to prove this. The cross-reactive immunity provided by the F protein may be sufficient to overcome the effects of changes in the G protein. For HRSV the immune response against the F protein is cross-reactive between subgroup A and B, whereas the response against the G protein is subgroup (and sometimes even genotype) specific (14
). The prophylactic use of a virus neutralization monoclonal antibody preparation directed against the HRSV-F protein has been shown to decrease the severity of lower respiratory tract diseases caused by both subgroups of RSV (39
). In a similar way, the conserved F protein of HMPV could be a target for the development of monoclonal antibodies for treatment of HMPV-infected persons.
Our data support a technical description of two serotypes of HMPV in experimentally infected ferrets. The existence and relevance of these serotypes in other animal species, including humans, has yet to be determined.
Our results in combination with data published by others (26
) demonstrate that HMPV clusters in two globally distributed serotypes. However, the identification of two serotypes does not exclude the possible existence of more serotypes or sublineages. The described viruses were all identified by using primers against conserved regions in the genome of the four prototype viruses, but in order to allow identification of more diverse HMPV strains, virus isolation of original materials is a standard procedure in our laboratory.