the necessary ethics approval for this study was obtained from the royal children 's hospital ethics committee .
we collected nasopharyngeal aspirate ( npa ) specimens from january 2001 through december 2004 from patients with acute lower respiratory tract infection in queensland , australia .
patients were from 3 months to 93 years of age ( mean 8.2 years , median 1.37 years ) , and 78.1% of specimens were from children < 5 years of age .
nucleic acids were extracted from 0.2 ml of each npa specimen by using the high pure viral nucleic acid kit ( roche diagnostics , mannheim , germany ) , according to the manufacturer 's instructions .
extracts were analyzed for hmpv sequences by reverse transcriptase pcr ( 9 ) . for samples collected during 2001 and 2002 ,
other viral respiratory pathogens were detected by using a direct fluorescent antibody assay ( dfa ) in combination with a culture - augmented dfa method ( 10 ) . for samples collected in 2003 and 2004 ,
of 10,025 npa specimens tested , 707 were positive for hmpv , for an overall incidence of 7.1% during the 4 years .
the youngest hmpv - positive patient was 4 months old , and the oldest was 79 years . in children ( < 18 years of age ) the incidence of virus was 7.4% , and
the seasonal distribution of hmpv infection showed a distinctive pattern for each of the 4 years studied ( figure 1 ) . in 2001 , hmpv showed broad seasonal activity ; incidence was > 5% in 3 consecutive seasons ( autumn , winter , and spring ) and peaked at 10.6% in the spring ( september 
november ) . in 2002 and 2004 , most hmpv activity was in spring , ( incidence of 13.6% and 15.4% , respectively ) , with little evidence during autumn ( march  may ) . in 2003 , the peak incidence of 9.0% occurred in winter ( june  august ) and persisted into spring ( 5.4% ) .
in all years , virus was present well into summer ( december  february ) , with an incidence ranging from 2.5% to 5.2% . on examination of those samples collected in 2003 and 2004 , which were all previously analyzed for common respiratory viruses by pcr , hmpv was the most frequently detected respiratory virus in children during the spring of each year
. expressed as an annual average over the 4 years studied , the predominant viral pathogen was rsv ( 9.2% ) , followed by hmpv ( 7.1% ) , influenza a ( 3.5% ) , parainfluenza virus 3 ( 2.3% ) , and adenovirus ( 1.3% ) . in 6.8% of hmpv - positive cases ,
evidence of co - infection with another respiratory virus was seen ; 20 patients were concurrently infected with an adenovirus , 10 with influenza a virus , 8 with rsv , 9 with parainfluenza virus 3 , and 1 with parainfluenza virus 2 .
su , summer ( december  february ) ; au , autumn ( march  may ) ; w , winter ( june  august ) ; sp , spring ( september  november ) . amplification products generated directly from 640 hmpv - positive npa specimens were genotyped as previously described ( 11 ) ( genbank accession nos .
data showed that all 4 viral subtypes cocirculated during each of the 4 years studied ( table 1 , figure 2 ) .
however , a different subtype predominated during 3 of the 4 years : hmpv subtype a1 was dominant in 2001 , subtype a2 in 2002 and 2003 , and subtype b1 in 2004 ( table 1 ) .
phylogenetic analysis of the 182-nucleotide fragment of the phosphoprotein gene fragment of human metapneumovirus detected in respiratory samples collected in australia .
sequences of avian pneumovirus ( apv ) type c ( genbank accession nos . af176590 and af176591 ) were used as outgroups to root the tree .
nucleotide sequences were aligned by using bioedit version 7.0.0 and were subjected to neighbor - joining analysis with mega version 3.0 with 500 random bootstraps .
can and nc , canada ; jps , japan ; nl001 , the netherlands ; q , queensland , australia .
clinical records from 273 patients who were positive for hmpv were scrutinized , and data describing clinical features and length of hospital stay were recorded . of these patients , 203 ( 74.4% ) were admitted to hospital with a median length of admission of 3 days and a mean of 6.5 days .
the predominant clinical features were cough ( 63% ) , rhinorrhea ( 61% ) , respiratory crackles / crepitations ( 60% ) , and fever ( 57% ) ( table 2 ) .
ninety ( 33.9% ) of the 273 patients had a chest radiograph , and 77 ( 85.6% ) patients showed bilateral parahilar peribronchial infiltrates consistent with a lower respiratory tract infection .
disease severity of the 273 hmpv - positive patients was classified as mild ( 46.8% ) , moderate ( 42.5% ) , and severe ( 10.7% ) , based on the use of supplemental oxygen and fluids and length of hospital stay .
the results of recent , similar studies suggested that peak periods of infection with hmpv predominate during winter in the northern hemisphere .
however , this finding has not been extensively examined over an extended period with a large , continuous sample .
our study found that the peak period of hmpv infection in queensland , australia , occurs predominantly in spring ( august  october ) but that hmpv can be detected in every month .
this finding suggests that hmpv activity , like rsv activity , occurs in the community throughout the year , and peaks of infection are a result of seasonal environmental factors .
although rsv predominated in all years , hmpv was the second most frequently detected virus in each year studied .
the low rate of co - infection of hmpv with other respiratory viruses ( including rsv ) suggests that co - infection may not be common in our community .
when analyzing disease severity in this sample with a 2-sided test of proportions , we saw no significant difference between patients with a co - infection and those without . therefore , our data did not support the suggestion by others that co - infection of hmpv with rsv or other viral respiratory pathogens is a risk factor for severe disease ( 6 ) .
the shift in predominant hmpv genotype observed in this study was similar to those reported for rsv and influenza viruses ( 6 ) and can be attributed to changes in immunity of the population in response to antigenic differences between the predominant circulating strains ( 12,13 ) .
however , a relationship between genotype and disease severity , as previously established for rsv ( 14,15 ) , did not appear to apply for hmpv , but we plan to examine this relationship further .
the clinical features associated with hmpv infection in this study were not sufficiently distinctive to clinically differentiate it from other respiratory viral infections in children , particularly those attributed to rsv .
in addition , few patients ( 10.6% ) had severe disease , but most ( 76% ) were sufficiently ill to be admitted and treated in the hospital for > 3 days , which represents a substantial amount in healthcare costs .
finally , this comprehensive study , conducted for 48 months , is the first one aimed at establishing an accurate estimate of the incidence and seasonal distribution of hmpv infection and to determine the genetic variation of hmpv circulating in our population
. the clinical spectrum of infection in a substantial proportion of hmpv - positive patients has been described , and studies are continuing to fully elucidate the clinical effect of infection with this virus in our community .
 we examined 10,025 respiratory samples collected for 4 years ( 20012004 ) and found a 7.1% average annual incidence of human metapneumovirus . 
 the epidemic peak of infection was late winter to spring , and genotyping showed a change in predominant viral genotype in 3 of the 4 years . 