Date Published: April 23, 2019
Publisher: Public Library of Science
Author(s): Naganori Nao, Ko Sato, Junya Yamagishi, Maino Tahara, Yuichiro Nakatsu, Fumio Seki, Hiroshi Katoh, Aiko Ohnuma, Yuta Shirogane, Masahiro Hayashi, Tamio Suzuki, Hideaki Kikuta, Hidekazu Nishimura, Makoto Takeda, Oliver Schildgen.
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.
Human metapneumovirus (HMPV) is a major causative agent of acute respiratory infections especially in young children, older people, and patients with underlying conditions such as cardiopulmonary diseases and diabetes [1–3]. The virus is a member of the family Pneumoviridae and has a non-segmented negative sense RNA genome containing 8 genes in the order: 3′-N-P-M-F-M2-SH-G-L-5′. The genome encodes 9 viral proteins including three surface glycoproteins: F (fusion), SH (small hydrophobic), and G (glycol-) proteins. HMPV has been circulating worldwide for more than 6 decades , and about half of children are infected with HMPV before 2 years of age, and most children are infected before 5 years of age . HMPV is classified into two antigenically distinct groups, A and B. Based on nucleotide sequence variations, each group is further divided into two subgroups (A1 and A2 in group A, and B1 and B2 in group B) [5, 6]. Furthermore, in the A2 subgroup there are two phylogenetically distinct clades, A2a and A2b . In addition, currently unique A2b clade HMPV strains with a 180- or 111-nucleotide duplication (180nt-dup and 111nt-dup, respectively) in the G gene have been detected [8–10]. Although antigenic variations may contribute to repeat HMPV infections, antigenic changes are not required for HMPV to cause symptomatic reinfection, because HMPV infection usually does not result in lifelong protective immunity [4, 11].
The analysis of cell line specificity for clinical and recombinant HMPV strains demonstrated that VeroE6 cells generally showed the highest infectivity with HMPV strains. The infectivity of Vero-ATCC was also high, but it was 2- to 3-fold lower than that of VeroE6 cells. Vero and Vero-derived cell lines have a large deletion in the gene cluster of the interferon (IFN) genes . Thus, these cells do not produce type I IFNs and show high susceptibility to many kinds of viruses [53, 54]. Lack of IFN responses could be a major factor for VeroE6 cells having high infectivity with HMPV. However, suppression of the innate immune responses, including IFN production, by the influenza virus NS1 protein did not enhance the infectivity of A549 cells with HMPV at all. LLC-MK2 cells also demonstrated similarly high infectivity with clinical HMPV strains as did VeroE6 cells. Unlike VeroE6 cells, LLC-MK2 cells retain a functional IFN system and produce IFN in response to virus infections and exhibit an antiviral state [55–58]. Therefore, the quality or condition of the IFN system is not the main factor that determines the infectivity of each cell line with HMPV.