title: Recovery of human metapneumovirus from cDNA: optimization of growth in vitro and expression of additional genes.
abstract: Human metapneumovirus (HMPV) is a recently recognized causative agent of respiratory tract disease in individuals of all ages and especially young infants. HMPV remains poorly characterized and has been reported to replicate inefficiently in vitro. Complete consensus sequences were recently determined for two isolates representing the two proposed HMPV genetic subgroups. We have developed a reverse genetic system to produce one of these isolates, CAN97-83, entirely from cDNA. We also recovered a version, rHMPV-GFP, in which the enhanced green fluorescent protein (GFP) was expressed from a transcription cassette inserted as the first gene, leaving the 41-nt leader region and first 16 nt of the N gene undisturbed. The ability to monitor GFP expression in living cells greatly facilitated the initial recovery of this slow-growing virus. In addition, the ability to express a foreign gene from an engineered transcription cassette confirmed the identification of the HMPV transcription signals and identified the F gene-end signal as being highly efficient for transcription termination. The ability to recover virus containing a foreign insert in this position indicated that the viral promoter is contained within the 3'-terminal 57 nt of the genome. Recombinant HMPV replicated in vitro as efficiently as biologically derived HMPV, whereas the kinetics and final yield of rHMPV-GFP were reduced several-fold. Conditions for trypsin treatment were investigated, providing for improved virus yields. Another version of HMPV, rHMPV+G1F23, was recovered that contained a second copy of the G gene and two extra copies of F in promoter-proximal positions in the order G1-F2-F3. Thus, this recombinant genome would encode 11 mRNAs rather than eight and would be 17.3 kb long, 30% longer than that of the natural virus. Nonetheless, the rHMPV+G1F23 virus replicated in vitro with an efficiency that was only modestly reduced compared to rHMPV and was essentially the same as rHMPV-GFP. Northern blot analysis showed that the increased number and promoter-proximal location of the added copies of the F and G genes resulted in a more than 6- and 14-fold increase in the expression of F and G mRNA, respectively, and sequence analysis confirmed the intactness of the added genes in recovered virus. Thus, it should be feasible to construct an HMPV vaccine virus containing extra copies of the G and F putative protective antigen genes to increase antigen expression or to provide representation of additional antigenic lineages or subgroups of HMPV.
authors.affiliation: Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-8007, USA.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Biacchesi
authors.forename: Mario H
authors.initials: MH
authors.lastname: Skiadopoulos
authors.forename: Kim C
authors.initials: KC
authors.lastname: Tran
authors.forename: Brian R
authors.initials: BR
authors.lastname: Murphy
authors.forename: Peter L
authors.initials: PL
authors.lastname: Collins
authors.forename: Ursula J
authors.initials: UJ
authors.lastname: Buchholz
mesh_terms: D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D029121:Metapneumovirus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016865:Reassortant Viruses; D011995:Recombination, Genetic; D013997:Time Factors; D014357:Trypsin
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D014357:Trypsin