Effect of In Vitro Syncytium Formation on the Severity of Human Metapneumovirus Disease in a Murine Model

Abstract

Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infections (ARTI) in children, elderly individuals and immunocompromised patients. In vitro, different HMPV strains can induce variable cytopathic effects ranging from large multinucleated syncytia to focal cell rounding. In this study, we investigated the impact of different in vitro phenotypes of two HMPV strains on viral replication and disease severity in a BALB/c mouse model. We first generated two recombinant GFP-expressing HMPV viruses: C-85473, a syncytium-inducing strain (rC-85473) belonging to the A1 subtype and CAN98-75, a focal cell rounding-inducing strain (rCAN98-75) of the B2 subtype. We subsequently exchanged the F genes of both strains to create the chimeric viruses rC-85473_F and rCAN98-75_F. We demonstrated that the F protein was the sole protein responsible for the syncytium phenotype and that viruses carrying a syncytium-inducing F protein replicated to significantly higher titers in vitro. In vivo, however, the virulence and replicative capacity of the different HMPV strains did not appear to be solely dependent on the F gene but also on the viral background, with the strains containing the C-85473 background inducing more weight loss as well as increased lung viral titers, pro-inflammatory cytokines and inflammation than strains containing the CAN98-75 background. In conclusion, the F protein is the main determinant of syncytium formation and replication kinetics in vitro, although it is not the only factor implicated in HMPV disease severity in mice.

The role of human Metapneumovirus genetic diversity and nasopharyngeal viral load on symptom severity in adults

“…Interestingly, our findings corroborates with a previous report that demonstrated the differences in replication fitness between HMPV genotypes in vitro and in vivo [47]. The study by Aerts et al showed that HMPV genotype A replicates to a significantly higher titers than genotype B in LLC-MK2 cells and in the lungs of BALB/c mice on day 4 post-infection, but the viral titers of genotype A decreased more rapidly than genotype B after day 4 [47]. As observed in other viral genotypes/serotypes [48,49], the differences in replication capacity may contribute to the competitive, transmission and epidemiological fitness differences between HMPV genotypes [50], which in turn may dictate the spread and evolution of both genotypes in the human population.…”

“…However, the viral load of genotype A-infected patients who enrolled ≥6 days were observed to be significantly lower compared those who enrolled 3-5 days after symptom onset, due to the fact they enrolled much later during the course of infection, in which most viruses would have been cleared by immunity. Interestingly, our findings corroborates with a previous report that demonstrated the differences in replication fitness between HMPV genotypes in vitro and in vivo [47]. The study by Aerts et al showed that HMPV genotype A replicates to a significantly higher titers than genotype B in LLC-MK2 cells and in the lungs of BALB/c mice on day 4 post-infection, but the viral titers of genotype A decreased more rapidly than genotype B after day 4 [47].…”

Strain-dependent impact of G and SH deletions provide new insights for live-attenuated HMPV vaccine development

“…pSP72 plasmids (Promega) encoding the full-length genomic cDNA of HMPV B2/CAN98-75 or A1/C-85473 strains (GenBank accession numbers: AY145289.1 and KM408076.1, respectively) were constructed as previously described [29, 34]. To generate G- or SH-deleted viruses, plasmids encoding HMPV antigenomes were amplified with Phusion DNA polymerase (New England Biolabs) using primers (listed in Table 1) designed to match with the 3’ and 5’ regions of either G or SH genes, and taking care to conserve an intergenic sequence between flanking genes (as represented in Figure 1A ).…”

“…BSR-T7 cells were transfected with the HMPV antigenome constructions plus 4 supporting plasmids, encoding N, P, L, and M2-1 ORFs of CAN98-75 and LLC-MK2 cells were added for co-culture (2 to 3 days), as previously described [29]. Cells were harvested, sonicated, centrifuged and the supernatant was diluted to inoculate LLC-MK2 monolayers.…”

“…In that regard, many HMPV subtypes co-circulate each year with high genetic diversity among A and B subtypes, particularly in the case of the less conserved G and SH proteins, with approximately 37% and 59% amino acid sequence homologies between subtypes, respectively [9, 14]. In parallel, we and others previously demonstrated that HMPV viruses diverge in their in vitro and/or in vivo phenotypes in a strain-dependent manner, notably by considering the HMPV F and G proteins and their functions [18, 29-33].…”