Early co-circulation of different clades of influenza A/H1N1v pandemic virus in Northern Italy

Introduction. The spatial diffusion over time of pandemic influenza A/H1N1 virus (A/H1N1v) was surveyed in Northern Italy (nearly 10 million inhabitants) from April to December 2009, and the molecular characteristics of circulating viruses were analyzed to identify the appearance of drift variants. About 45% of analyzed samples were laboratory-confirmed cases of A/H1N1v. Sporadic cases occurred until the middle of June 2009, then, case numbers began to increase delineating distinct epidemiological phases of viral circulation. Methods. RNA was extracted using RNeasy Mini kit (QIAGEN GmbH, Germany). Virological diagnosis of A/H1N1v infection was carried out by real-time RT-PCR assay. Sequence analysis of hemagglutinin (HA) gene was performed through a RT-PCR assay specific for a 995 bp fragment (nt. 64-1,058) in the HA1 domain. The nucleotide sequences were obtained by automated DNA sequencing. The HA1 sequences were aligned with other sequences collected from GenBank database by ClustalX software. The multiple sequence alignment was used to perform a basic phylogenetic analysis and a phylogenetic tree from HA sequences was constructed. Results. The HA gene sequences of A/H1N1v analyzed segregated into three genetically distinct clades and were characterized by the appearance of amino acid variations that were progressively fixed in the field viral population under scrutiny. Conclusions. These data suggest an early co-circulation of genetically distinct A/H1N1v variants and emphasize the importance of a close molecular surveillance to detect rapidly the spread of new viral variants and to define their epidemiological impact.