Samples for genotyping were selected to represent all the different geographical regions of the country affected in a certain year. No other selection criteria such as age of the patients or sex were considered. DNA was extracted according to the manufacturer’s protocol from 200 µl of serum with the QIAamp DNA Blood Mini Kit (Qiagen). Viral DNA for sequencing was prepared by nested-PCR using previously described primers e1855f, e1863f, B19-R1 and B19-R2 for amplification of a 1,100-bp region spanning the NS1/VP1-unique region junction (NS1/VP1u)28,33. The PCR products were analysed in a 1.5% agarose gel stained with ethidium bromide. Products for sequencing were purified with the QIAquick PCR Purification Kit (Qiagen).

PCR products were sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies), using the nested PCR primers on a capillary sequencer (models 3130/3500 Avant, Applied Biosystems). Sequences were edited using SeqScape Software v3.0 (Applied Biosystems) and then aligned with references in ClustalW (integrated in MEGA version 5).

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