The two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (F) protein is a major contributor to this antigenic relatedness

Abstract

The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) human metapneumovirus (HMPV) strains, which represent the two distinct HMPV genetic lineages and exhibit 5 and 63% amino acid divergence in the fusion (F) and attachment (G) proteins, respectively, were investigated in vitro and in rodents and nonhuman primates. Both strains replicated to high titers (> or =6.0 log(10)) in the upper respiratory tract of hamsters and to moderate titers (> or =3.6 log(10)) in the lower respiratory tract. The two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay with postinfection hamster sera, and infection with each strain provided a high level of resistance to reinfection with the homologous or heterologous strain. Hamsters immunized with a recombinant human parainfluenza virus type 1 expressing the fusion F protein of the CAN83 strain developed a serum antibody response that efficiently neutralized virus from both lineages and were protected from challenge with either HMPV strain. This result indicates that the HMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Both HMPV strains replicated to low titers in the upper and lower respiratory tracts of rhesus macaques but induced high levels of HMPV-neutralizing antibodies in serum effective against both lineages. The level of HMPV replication in chimpanzees was moderately higher, and infected animals developed mild colds. HMPV replicated the most efficiently in the respiratory tracts of African green monkeys, and the infected animals developed a high level of HMPV serum-neutralizing antibodies (1:500 to 1:1,000) effective against both lineages. Reciprocal cross-neutralization assays in which postinfection sera from all three primate species were used indicated that CAN75 and CAN83 are 64 to 99% related antigenically. HMPV-infected chimpanzees and African green monkeys were highly protected from challenge with the heterologous HMPV strain. Taken together, the results from hamsters and nonhuman primates support the conclusion that the two HMPV genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups as defined by reciprocal cross-neutralization in vitro.

FIG. 1.

Growth kinetics of HMPV strains CAN75 and CAN83 in the presence or absence of trypsin. LLC-MK2 monolayers were infected with the indicated HMPV strain at a multiplicity of infection of 0.01 and were incubated at 32°C with or without added trypsin. Aliquots of the medium supernatants were harvested at 24-h intervals until the monolayer became detached from the plate. The virus titer was quantified on LLC-MK2 monolayers at 32°C. The level of replication of a biologically derived HPIV2, or rHPIV1 or rHPIV3, was determined in separate experiments under similar conditions (HPIV2 and HPIV3 were grown in the absence of added trypsin, and HPIV1 was grown with added trypsin). The virus titer is expressed as log₁₀ TCID₅₀/ml.

FIG. 2.

Construction of an rHPIV1 vector expressing CAN83 F. (A) Diagram of the construction of rHPIV1-F₈₃. The HPIV1 genome was modified by the creation of an MluI restriction site (HPIV1 nt 113 to 118) 1 nt prior to the translational start codon of the N ORF (HPIV1 nt 119 to 121). The F ORF of HMPV strain CAN83 (1,620 nt in length and encoding a 539-amino-acid polypeptide; GenBank accession number AY297749) was engineered by PCR to be followed by the tetranucleotide TAAT (R6) and an HPIV1 gene junction consisting of a gene end signal, a CTT intergenic region, and a gene start signal. The length of the entire cassette was 1,656 nt and was designed, upon insertion into the MluI site, to conform to the rule of six and to maintain the HPIV1 gene start signal sequence phasing (23). Gene start and gene end signals are indicated. A portion of the predicted viral promoter that lies within the N gene is shown. (B) Indirect immunofluorescence of LLC-MK2 cells that were infected with rHPIV1-F₈₃, HMPV CAN83, or rHPIV1 or that were mock infected (uninfected), as indicated, incubated for 72 h, and analyzed by indirect immunofluorescence by using anti-HMPV (αHMPV) polyclonal hamster serum or a mixture of two mouse monoclonal antibodies to the HPIV1 HN protein (αHPIV1), as indicated, as the first antibody.

FIG. 3.

Seroprevalence of HMPV in captive chimpanzees. Sera obtained from 31 captive-bred chimpanzees were screened for the presence of neutralizing antibodies to HMPV strains CAN75 and CAN83. The serum neutralization titer (reciprocal log₂) to CAN75 or CAN83 is indicated for each sample screened. The values obtained are arranged in order of increasing neutralization titer, and the animals are numbered accordingly. The animals indicated with asterisks were chosen for an infection and challenge study (Table 5).

FIG. 4.

Level of replication of HMPV strains CAN75 and CAN83 in the upper and lower respiratory tract of African green monkeys. Four animals per group were infected IN and IT with 10^5.2 TCID₅₀ of the indicated virus, CAN75 (•) or CAN83 (▪), and NP swab (A) and TL (B) specimens were taken on the indicated days, and the titer of shed virus was quantified.