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Newly discovered viruses, such as human metapneumovirus (HMPV) and human bocavirus (HBoV) are important pathogens causing respiratory tract infection (RTI) in susceptible populations, particularly in children and the elderly.[1-4]
HMPV is a negative single stranded RNA virus belonging to the family of Paramyxoviridae, which was first isolated in 2001 from nasopharyngeal aspirates. It is responsible for about 5–15% of the worldwide respiratory tract infections, affecting both young children and adults, causing symptoms ranging from mild disease of the upper respiratory tract system to severe bronchiolitis and pneumonia. In infants, HMPV incidence is even higher, reaching 25%. Genetic analysis of HMPV isolates revealed two ‘major’ lineages (A and B) and four ‘minor’ lineages (A1, A2, B1 and B2), based on the sequence variability of the attachment (G) and fusion (F) surface glycoproteins. The existence of two further sublineages, A2a and A2b, has further been suggested.
HBoV, a single stranded DNA virus belonging to the family of Parvoviridae, was discovered in 2005 and has been detected in nasopharyngeal aspirates, sera and blood samples of patients with respiratory tract infection and in faecal specimens of patients with acute respiratory illness and/or gastroenteritis. HBoV affects, mostly, children younger than 2 years old, with its incidence ranging from 2·7 to 19% in respiratory and from 0·8 to 9·1% in faecal samples, obtained from patients with respiratory infection and gastroenteritis, respectively. Four species of HBoV have been recently discovered with HBoV1 being predominantly a respiratory pathogen, whereas HBoV2, HBoV3 and HBoV4 being found mainly in stool. Nucleotide sequence analysis of the two capsid proteins (VP1 and VP2), which show high variability compared with coding sequences of the two viral nonstructural proteins (NS1 and NP1), has further divided HBoV1 into genotypes St1 and St2. These two major HBoV genotypes correspond to the original St1 (Stockholm 1) and St2 (Stockholm 2) isolates.
Epidemiological and phylogenetic analysis of HMPV and HBoV has been carried out extensively in other countries but not in Greece, where only a few studies concerning newly recognized respiratory viruses have been published. Using molecular methods for identification, we recently reported the incidence of 13 respiratory pathogens, including HMPV and HBoV, in children presenting with influenza-like illness (ILI). In this study, we aimed to determine the genetic diversity of the HMPV and HBoV clinical strains identified within the Greek population by comparison with reference, as well as, wild strains described in other countries.
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This is the first study on the genetic diversity of HMPV and HBoV strains in children with respiratory tract infections in Greece, including paediatric patients who suffered from ILI as well as other respiratory infections.
Since its initial documentation in the Netherlands in 2001, HMPV has been reported to circulate worldwide.[22-24] We only found the B2 HMPV lineage circulating among the Greek population during the winter season of the three years examined, although cocirculation of all four viral lineages during the same period is not unusual.[25, 26] Reports from Croatia, Ireland and France have also indicated predominant circulation of lineage B2, during the winter periods of 2005–2006, 2006–2007 and the years 2006–2008, respectively.[27-29] Gaunt et al suggested a potential back-shift to lineage B2 in Scotland during the winter season of 2007-2008. The observation of only one circulating lineage among the study population was also mentioned in other studies. In Spain, Italy and China[33, 34] circulation of HMPV A2 lineage was reported. However, a number of studies have indicated that circulating predominant strains may vary on an annual basis and that their replacement may occur, on average, every 1–3 years.[35, 36]
As year-round surveillance was not performed and summer infections from HMPV and HBoV cannot be excluded, recirculation of the same HMPV lineage over three consecutive years within the Greek population could be the result of the maintenance of the virus throughout the year. Alternatively, HMPV lineage B2 could have been reintroduced each year in the Greek population with minor intralineage alterations causing infections to the naïve population.
Phylogenetic analysis of the HBoV full-length VP1 and VP2 proteins was performed on 26 HBoV-positive samples revealing limited sequence variations among them as well as in relation to available sequences of previously described HBoV strains. Genotype St2 was the only HBoV genotype, which circulated during the first winter period of 2005–2006, while both St1 and St2 cocirculated during the second and the third study period, with predominance of genotype St2. Interestingly, the majority of HBoV strains, detected in Italian children during winter periods of the years 2004–2008, also belonged to genotype St2 (62·5%). An even higher percentage (75·4%) of HBoV strains belonging to genotype St2 during the years 2002–2005 was reported from Germany.
Our study exposed the circulation of a third group of HBoV strains during the second surveillance period. Lin et al and Xu et al also reported the circulation of HBoV strains belonging to a third group different from St1 and St2, which resulted from recombination events between different circulating viral strains. Such genetic recombination events have been reported in parvoviruses[38, 39] and in different HBoV species.[40-42] Our data suggest that the strains HBoVs/Athens.GRC/35.07 and HBoVs/Athens.GRC/108.07 resulted from recombination events between a HBoVs/Athens.GRC/27.07-like and a HBoVs/Athens.GRC/24.08-like strains, with a breakpoint site in the VP1/VP2 region. To date, similar intraspecies recombination events involving HBoV1 have been reported before, with a breakpoint located in the NS1, VP1/VP2 and NS1 and VP1/VP2 regions. Taken together, these data suggest that intraspecies recombination could occur in HBoV1 at different positions of its genome. Further studies, including whole genome sequencing, are necessary to identify breakpoint patterns in these viruses, thus exploring their potential association with viral spread among populations.
Higher similarity observed between HBoV sequences to that observed between HMPV strains may be explained by the widespread assumption that mutation rates correspond to the fidelity of replication. DNA viruses, such as HBoV, mutate slower compared with RNA viruses, such as HMPV, because DNA polymerases possess proof-reading activity, which further reduces mutation rates during DNA replication. Moreover, the rate of generation and correction of genomic mutations may depend on a number of factors, including generation time, genomic architecture, replication speed, transmission and environmental effects.
Our study highlighted the heterogeneity of viral strains circulating within the Greek population by determination of the complete sequences of HMPV G gene and HBoV VP1/VP2 gene. The phylogenetic analysis performed aimed to monitor the genetic evolution of HMPV and HBoV during three consecutive years and revealed an individual predominant lineage for the former and a combination of genotypes for the latter.