http://repub.eur.nl/res/aut/4588/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/16401/ 2009-01-15T00:00:00Z The general composition of the metapneumovirus genome is similar to that of pneumoviruses but differs in the number of genes and their order in the genome (Figure 2) (237). Metapneumoviruses do not encode the nonstructural proteins NS1 and NS2, and the small hydrophobic (SH) and attachment (G) protein genes follow the fusion (F), M2.1 and M2.2 protein genes, whereas for pneumoviruses this is the other way around. In evolutionary terms, this difference is very striking since recombination for members of the subfamily Pneumovirinae has been observed in a laboratory setting only (218). The 3’ and 5’ ends of the genome consist of short extragenic regions, referred to as the leader and trailer regions, and approximately the terminal 12 nt of the leader and the terminal 12 nt of the trailer are complementary, most likely due to the fact that each contains the basic elements of the viral promoter (21, 57, 163). The 3’ end of the viral RNA (vRNA) directs both replication and transcription, while the 5’ end of the genome contains signals that direct the replication of the antigenome. Each gene is preceded by a gene start (GS) signal at which the synthesis of mRNA initiates. Subsequently, each gene terminates with a semiconserved gene end (GE) signal that directs polyadenylation and termination of the completed mRNA. The genomes of metapneumoviruses consist of non-segmented negative stranded RNA and are smaller (~13.000 kb) than those of pneumoviruses (~15.000 kb) (70). For members of the subfamily Paramyxovirinae, the nucleotide length of each genome is a multiplicity of six (“rule of six”), reflecting a requirement of the nucleocapsid for RNA binding (e.g., binding units of six nucleotides) (34). This rule does not apply for the subfamily Pneumovirinae (201), although the reasons underlying this discrepancy have not been determined. http://repub.eur.nl/res/pub/13460/ 2004-08-01T00:00:00Z Human metapneumovirus (hMPV) is a newly discovered pathogen associated with respiratory tract illness, primarily in young children, immunocompromised individuals, and the elderly. The genomic sequence of the prototype hMPV isolate NL/1/00 without the terminal leader and trailer sequences has been reported previously. Here we describe the leader and trailer sequences of two hMPV isolates, NL/1/00 and NL/1/99, representing the two main genetic lineages of hMPV. Minigenome constructs in which the green fluorescent protein or chloramphenicol acetyltransferase genes are flanked by the viral genomic ends derived from both hMPV lineages and transcribed using a T7 RNA polymerase promoter-terminator cassette were generated. Cotransfection of minigenome constructs with plasmids expressing the polymerase complex components L, P, N, and M2.1 in 293T or baby hamster kidney cells resulted in expression of the reporter genes. When the minigenome was replaced by a sense or antisense full-length cDNA copy of the NL/1/00 or NL/1/99 viral genomes, recombinant virus was recovered from transfected cells. Viral titers up to 10(7.2) and 10(5.7) 50% tissue culture infective dose/ml were achieved with the sense and antisense plasmids, respectively. The recombinant viruses replicated with kinetics similar to those of the parental viruses in Vero cells. This reverse genetics system provides an important new tool for applied and fundamental research. http://repub.eur.nl/res/pub/10320/ 2004-01-01T00:00:00Z The discovery of human metapneumovirus and its implications for respiratory tract disease have emphasized the need for a sensitive, specific, and rapid assay to detect this virus in a clinical setting. It recently became clear that human metapneumovirus can be grouped into at least four genetic lineages. Previously described assays for the detection of human metapneumovirus were developed by using limited sequence information and failed to detect viruses from all four genetic lineages with comparable sensitivities. Here we describe the development and evaluation of a real-time reverse transcriptase PCR assay that detects human metapneumovirus from the four known genetic lineages with equal specificities and sensitivities.