title: Detection of human metapneumovirus in respiratory secretions by reverse-transcriptase polymerase chain reaction, indirect immunofluorescence, and virus isolation in human bronchial epithelial cells.
abstract: Over two winters in Newcastle upon Tyne, respiratory secretions, negative by immunofluorescence staining for other respiratory viruses, were tested for the presence of human metapneumovirus (HMPV) by RT-PCR. In the second winter, specimens were also tested by immunofluorescence staining with an anti-HMPV polyclonal rabbit antiserum and immunofluorescence positive specimens were inoculated into a line of human bronchiolar cells, 16HBE140. Overall, 55 of 549 (10%) specimens tested were positive for HMPV by RT-PCR. Of 162 specimens tested by both RT/PCR and immunofluorescence staining, 23 were positive by both techniques. Of five specimens positive by RT-PCR alone, only one was confirmed with a second set of primers. Of three specimens positive by immunofluorescence alone, only one was confirmed by virus culture. All four previously recognized sub-genotypes of the virus were identified by both RT-PCR and immunofluorescence staining. Sub-genotype A1 was prevalent in the first winter and B1 prevalent in the second. HMPV replication and virus isolation rates were higher in 16HBE140 cells than in monkey kidney cells and did not require exogenous trypsin. Low passage isolates of both sub-genotypes A2 and B1 replicated slowly reaching peak titers only 12 days after inoculation. In summary, single round RT/PCR and immunofluorescence staining with a polyclonal rabbit antiserum proved of equal sensitivity in the diagnosis of HMPV infection in respiratory secretions both detecting 96% of confirmed positive specimens. 16HBE40 cells provided a significant improvement on monkey kidney cells for the isolation and propagation of the virus.
authors.affiliation: The School of Clinical Medical Sciences, The University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom.
authors.forename: R E
authors.initials: RE
authors.lastname: Ingram
authors.forename: F
authors.initials: F
authors.lastname: Fenwick
authors.forename: R
authors.initials: R
authors.lastname: McGuckin
authors.forename: A
authors.initials: A
authors.lastname: Tefari
authors.forename: C
authors.initials: C
authors.lastname: Taylor
authors.forename: G L
authors.initials: GL
authors.lastname: Toms
mesh_terms: D000818:Animals; D018893:Bronchoalveolar Lavage; D002460:Cell Line; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D005814:Genes, Viral; D006801:Humans; D029121:Metapneumovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009305:Nasopharynx / Q000378:metabolism / Q000821:virology; D018184:Paramyxoviridae Infections / Q000175:diagnosis* / Q000821:virology; D010802:Phylogeny; D020545:Respiratory Mucosa; D012137:Respiratory System / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013183:Sputum / Q000821:virology; D014132:Trachea / Q000378:metabolism / Q000821:virology; D006113:United Kingdom; D014764:Viral Proteins / Q000235:genetics; D014776:Virus Cultivation
chemical_list: D017931:DNA Primers; D014764:Viral Proteins