Consensus and variations in cell line specificity among human metapneumovirus strains

Abstract

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.

Fig 1. Phylogenetic tree of HMPV G gene sequence.

A phylogenetic tree was constructed using the G gene sequences of 41 A2b HMPV strains detected in Sendai and 97 strains obtained from the NCBI nucleotide sequence database. The tree was rooted with A2a HMPV strain HMPV/Yokohama.JPN/P7877/2015 and tested with bootstrapping (100 replicates). HMPV A2b_180nt-dup strains detected in Sendai, HMPV A2b_180nt-dup strains detected in Yokohama, HMPV A2b_180nt-dup strains detected in Spain, and HMPV A2b_111nt-dup strains detected in Yokohama are indicated with filled circles in pink, blue, yellow, and green, respectively. The cluster containing HMPV A2b_180nt-dup and A2b_111nt-dup strains is indicated by a bracket.

Fig 2. Phylogenetic tree of HMPV full genome sequence.

A phylogenetic tree was constructed using the full genome sequences of 9 HMPV strains isolated in Sendai and 140 strains obtained from the NCBI nucleotide sequence database. The tree was tested with bootstrapping (100 replicates). HMPV A2b_180nt-dup strains detected in Sendai are indicated with filled circles in pink.

Fig 3. Passage history and full-genome sequence data of hMPV/Sendai/0256/2015 and its recombinant MG0256-EGFP.

The hMPV/Sendai/0256/2015 was isolated using MNT-1 and VeroE6/TMPRSS2 cells. The recombinant MG0256-EGFP strain was generated based on the sequence of the hMPV/Sendai/0256/2015 strain. The full-genome virus sequence in the clinical specimen (the putative original virus sequence in the patient) and those of the isolate (hMPV/Sendai/0256/2015) and recombinant (MG0256-EGFP) after passages in various cell lines were determined. The numbers of passages in each cell line and mutations in each gene were indicated in parentheses.

Fig 4. Infectivity of various cell lines with clinical isolates of HMPV.

The infectivity of various cell lines (VeroE6, LLC-MK2, Vero, HeLa, MNT-1, HEK293, BEAS-2B, and A549) with thirteen clinical HMPV strains was analyzed. The numbers of HMPV infected cells in VeroE6 cells were set to 100%, and relative infectivity is presented as the average and standard errors from triplicated experiments. (A) Data of subtype A2b strains. (B) Data of subtype A1, A2a, B1, and B2 strains.

Fig 5. Infectivity of various cell lines with recombinant HMPV strains.

The infectivity of various cell lines (VeroE6, LLC-MK2, Vero, HeLa, MNT-1, HEK293, BEAS-2B, and A549) with six recombinant HMPV^GFP strains was analyzed. The numbers of HMPV infected cells in VeroE6 cells were set to 100%, and relative infectivity is presented as the average and standard errors from triplicated experiments.

Fig 6. Infectivity of A549/hSLAM and A549/hSLAM-NS1 cells with HMPV strains.

(A) A549/hSLAM and A549/hSLAM-NS1 cells were infected with VSV at an MOI of 1.0. At 6 hpi, total RNAs were extracted from VSV-infected or mock-infected cells. The levels of mRNA of IFN-β, MX1, and OASL were determined by qPCR, and the values were normalized to levels of the control gene HPRT1. Relative RNA copies are presented as the average and standard errors from triplicated experiments. Statistical significance was calculated using Student’s t test (*P < 0.05; **P < 0.01). (B) The infectivity of VeroE6 and 2 types of A549 (A549/hSLAM and A549/hSLAM-NS1) cells with 4 recombinant HMPV^GFP strains was analyzed. The numbers of HMPV-infected cells in VeroE6 cells were set to 100%, and relative infectivity is presented as the average and standard errors from triplicated experiments.