The assays were performed using the TaqMan ® PCR Core Kit. The final volume was 25 µl containing 500 nM of the forward primer (NL-N-forward (5'-CATATAAGCAT-GCTATATTAAAAGAGTCTC-3')), 250 nM of the reverse primer (NL-N-reverse (5'-CCTATTTCTGCAGCATATTTG-TAATCAG-3')) and 500 nM of the probe (NL-N-probe (5'-FAM-TGYAATGATGAGGGTGTCACTGCGGTTG-TAMRA-3', in which Y is either a C or a T residue). Nuclease-free water was used as negative control and a plasmid containing the N gene of hMPV (kindly provided by James Simon, VIRONOVATIVE, EUR Holding, Erasmus University Rotterdam) was used as a positive control in all PCR runs. Cycling parameters were as follows: 5 min at 95°C, 45 cycles of 30 s at 95°C and 1 min at 60°C. Amplification and detection of RNA from virus isolates or clinical specimens were performed using the GeneAmp ® 5700 Sequence Detection System (Applied Biosystems). The real-time PCR product was cloned with the QIAGEN ® PCR cloning kit (QIAGEN, Hilden, Germany) and this standard plasmid DNA was used for absolute quantification of hMPV viral load. Calculations were performed as previously described for absolute quantification of RSV viral load [23] .