BACKGROUND
rapid detection and identification of infectious pathogens are necessary for proper treatment of infection and outbreak control measures.
many promising approaches are being developed enabling rapid identification of various infectious pathogens such as bacteria or viruses including multiplex -pcr amplification strategies or novel technologies such as high-performance electrospray ionization mass spectrometry  <cit> .
dna microarray technologies provide a mean to screen for thousands of different nucleic acid sequences simultaneously and have been used for the detection of many viruses such as hiv  <cit> , hepatitis b and d viruses  <cit> , human papillomaviruses  <cit> , rotaviruses  <cit> , vaccinia virus  <cit> , herpesviruses, enteroviruses, flaviviruses  <cit> , and measles virus genotypes  <cit> .
in addition to these microarrays, which have a spectrum of identification limited to a few viral species, major microarray platforms were developed for the detection of a much broader range of pathogens  <cit> .
in humans, influenza a viruses cause recurrent annual epidemics of moderate to severe respiratory diseases.
sixteen hemagglutinin  molecular types  and nine neuraminidase  molecular types  have been identified, although only a few of them occur in viruses causing human diseases.
identification and selection of sequences to be used on a microarray are particularly challenging, especially for rapidly mutating viruses.
antigenic drift is an ongoing process for influenza a viruses resulting in continuous evolution and annual epidemics.
moreover, the segmented nature of the influenza a virus genome can lead to reassortments during coinfection thus contributing to their vast genetic diversity and pandemic potential.
subtyping and molecular analysis of influenza virus strains were performed by different groups using low density microarrays  <cit> .
most of them were used to identify influenza a viruses h1n <dig>  h3n <dig>  h5n <dig> and influenza b viruses.
recently, combimatrix corporation  developed a semiconductor-based microarray and an integrated microfluidic array enabling the detection of  <dig> ha and  <dig> na  <cit> .
however, the cost per microarray is too high for routine experimental identification.
more recently, a rapid subtyping assay also identifying all the ha and na subtypes was developed using padlock probes  <cit> .
resequencing, using high-density oligonucleotide microarrays, is a technology that enables the rapid identification of genetic variants and the determination of their nucleotide sequence at single base-pair resolution.
this allows identification of pathogens with sequences similar but not identical to those represented on the microarray.
a resequencing microarray was designed in order to allow identification of genetically diverse human rna rhinoviruses and enteroviruses, with a minimum number of prototype 5'utr sequences  <cit> .
one respiratory pathogen resequencing microarray allowed the detection of geographically distant human h1n <dig>  h3n <dig> and avian h5n <dig> influenza virus strains covering the period between  <dig> and  <dig>  <cit> .
more recently, the same team developed a broad-range resequencing microarray for universal detection and identification of all the possible combinations of the  <dig> hemagglutinin and  <dig> neuraminidase types of avian influenza viruses in addition to three additional influenza a markers   <cit> .
their strategy was based on probe sequence selection using in silico modeling  <cit> .
a resequencing microarray called pathogenid v <dig>  has been developed by a consortium of laboratories of institut pasteur with affymetrix inc. it was used to explore various strategies of sequence selection for its design.
the part dedicated to influenza viruses, presented here, was based on consensus sequences specific for one gene.
within the global program, another strategy was explored for the part of the microarray specific for rhabdoviruses.
that strategy was based on the use of prototype sequences representative of each of the seven species described in the genus lyssavirus and chosen within a conserved region of the polymerase gene  <cit> .
consensus sequences generation was a posteriori used to analyse results.
our strategy presented in this study, uses consensus sequences generated from nucleotide sequence alignments to determine the sequences to be tiled.
these sequences characterised by a minimum size of  <dig> bp reduce the number of probes required for tiling yet still covering the wide diversity of influenza virus strains.
to validate our approach, influenza viruses from different host origins, isolated over a period of about  <dig> years, were chosen.
the dna microarray not only demonstrated its ability to identify the type and subtype of influenza viruses but also had a strong discriminatory power at the level of viral variants.
RESULTS
microarray design
the aim of the study was to detect influenza a viruses isolated over a long period of time, from a range of hosts  and with a minimum of sequences tiled on the array.
four genes including pb <dig>  m, ha and na were chosen in order to type and subtype influenza viruses.
different strategies for selecting the region to resequence were adopted depending on the particular gene.
characteristics of the sequences tiled on the microarray are detailed in table  <dig>  firstly, one partially conserved sequence of  <dig> bp covering the majority of influenza m segments, routinely employed in medical diagnostics, was used for genus identification, namely influenzavirus a, b and c. secondly, almost one hundred sequences of pb <dig> genes covering a large spectrum of influenza a viruses and hosts were selected from the genbank database.
five typical pb <dig> sequences representative of the diversity of pb <dig> sequences over a long period of time and for different host reservoirs were finally retained.
the five selected sequences are described in table  <dig>  the strategy adopted for the hemagglutinin  and the neuraminidase  genes involved the design of consensus sequences for the h <dig>  h <dig>  h <dig>  h <dig>  h <dig> and h <dig> genes and the n <dig>  n <dig> and n <dig> genes.
we have restricted the number of studied molecular subtypes of ha and na in order to make the confirmation of our hypothesis more straightforward and demonstrative without aiming to cover the whole set of serotypes.
the ha and na subtypes chosen to be tiled on the microarray are shared by all the human viruses and highly pathogenic avian viruses, and the has are further distributed into phylogenetic groups that gather all the  <dig> serotypes  <cit> .
for each major ha and na gene, sequences of influenza a virus were selected from different hosts including horses, pigs, humans and birds.
positions and sizes of the consensus ha and na sequences were determined and selected after blastn analysis of the sequence.
for each gene, a global alignment including all selected sequences was performed and clusters grouping a maximum number of sequences were established.
in each sequence cluster, a common region with the lowest genetic divergence was identified.
a consensus sequence was automatically obtained from this common region, characterised by a minimum size of  <dig> bases and a divergence between the consensus sequence and any given sequence belonging to the cluster of less than 15%.
twelve ha consensus sequences and  <dig> na consensus sequences were identified and their positions are detailed in table  <dig>  finally, the microarray based on the resequencing approach included  <dig> influenza viral sequences which corresponded to  <dig> bases.
for each relevant base of a given consensus or prototype sequence, the array contained eight  <dig> mer probes .
two of the eight probes represented perfect matches, while the others corresponded to possible mismatches at the central  position of the  <dig> mers.
all together,  <dig> probes were then tiled on the microarray, allowing subtyping of influenza viruses from different host species.
detection of type a influenza viruses
a total of  <dig> influenza virus samples were analysed by the resequencing microarray, including  <dig> swine,  <dig> human,  <dig> equine and  <dig> avian strains that originated from various locations worldwide and covered the period of time between  <dig> and  <dig>  all samples were viral isolates, propagated either in embryonated chicken eggs or in mdck cell cultures.
they were chosen from sufficiently distant points in time to cover a large diversity of influenza viruses, especially for human and swine viruses, which were subject to major antigenic drift over the years.
avian strains were derived from cloacal samples collected from ducks in baie de somme, marquenterre  in  <dig>  rna extracts were amplified by wta  and hybridised on the resequencing microarray .
results are summarised in table  <dig>  nucleotide sequences were determined from the fluorescent signals as previously described  <cit>  and compared with sequences in genbank by blastn analysis, to identify and subtype influenza virus strains.
results for the highest hit scores were taken as strain identification.
after hybridisation of wta amplified material on the dna microarray, all samples were positive for the presence of influenza a virus with a minimum call rate of 41% for the m gene and a call rate ranging from  <dig>  to  <dig> % for the pb <dig> gene, which is the percentage of bases determined by the resequencing algorithm.
subtypes of the different strains were identified with ha and na sequences, giving a call rate ranging from  <dig> % to 100%.
time ranges were obtained from blastn analysis.
the sequences showing the highest alignment score were considered and duplicated sequences representing different isolates of the same strain were eliminated.
the time range indicated the oldest isolate with high homology provided by the blastn algorithm and the most recent one.
the median was calculated based upon the sequences showing the highest alignment scores .
the call rate is the percentage of bases determined by the resequencing algorithm.
time ranges indicate the oldest isolate with high homology provided by the blastn algorithm and the most recent one.
the medians were calculated based upon the sequences showing the highest alignment scores.
for swine and human viruses, a minimum call rate of  <dig> % allowed identification of influenza virus with a correct temporality.
indeed, for the majority of strains the blastn result was close to that of the sample strain, as outlined by median analysis.
the median obtained for the na gene from the a/johannesburg/33/ <dig>  virus was relatively recent compared to the year of isolation of the strain, probably because the chosen consensus sequence for this gene was highly conserved and thus weakly discriminatory.
the time range obtained for the a/wyoming/3/ <dig>  virus was narrow and limited to the year  <dig>  probably due to the marked antigenic drift from the year  <dig> observed for the h <dig> gene in human influenza virus strains  <cit> .
the epidemiology of swine influenza has become increasingly complex over the last decades.
three major influenza a virus subtypes are currently circulating in swine worldwide, but the origins and the antigenic and genetic characteristics of these swine influenza viruses differ with continents or regions of the world.
a/swine/gent/7625/ <dig>  virus is the result of a multiple reassortment involving a swine influenza virus with avian-influenza-like internal segments and human h1n <dig> and h3n <dig> viruses isolated in  <dig>  <cit> .
blastn analysis for h1n <dig> swine virus reflected this multiple reassortment with blasted sequences originating from human, avian and swine hosts .
between  <dig> and  <dig>  swine h3n <dig> viruses appeared in italy.
they contained avian-like internal genes and ha and na genes from human h3n <dig> viruses  <cit> .
in blastn analysis, the vast majority of the sequences sorted by the algorithm for glycoprotein segments were from human or swine origins only.
all equine influenza virus strains were identified and subtyped with the dna microarray, with a minimum call rate of  <dig> %.
medians were often heterogeneous and far from the original strain.
this is probably due to  the paucity of equine influenza virus sequences available in the databases,  the limited genetic evolution of those viruses leading to equivalent result scores after blastn analysis,  the fact that ha and na sequences tiled on the microarray were shorter than pb <dig> sequences, due to their extreme genetic diversity in influenza a viruses.
for the h3n <dig> viruses, recent sequences corresponded to avian strains, outlining the fact that h3n <dig> equine viruses were reassortants between avian viruses and equine h7n <dig> viruses which have not been isolated in horses for more than  <dig> years.
all together, the results showed that the pathogenid v <dig>  dna microarray is able not only to provide correct full subtyping of various strains from different host origins but also to identify/detect, after blastn, an array of strains genetically related to them.
in all cases, the strains output by blastn belonged to the same time period thus giving a strong insight into the variant to which it is genetically, and very probably antigenically, related.
avian samples
cloacal samples were collected from ducks in baie de somme, marquenterre  in  <dig> and analysed by qrt-pcr specific for the m gene.
wta amplification of viral rna followed by hybridisation to the dna microarray confirmed the identification of influenza viruses.
two samples were identified as h1n <dig> viruses and one sample as h3n <dig> virus.
influenza virus strain  <dig> was identified as h3n <dig> virus with a call rate of  <dig> % for one of the h <dig> consensus sequences and  <dig> % for one of the n <dig> consensus sequences.
influenza virus strains  <dig> and  <dig> were both identified as h1n <dig> virus with call rates of  <dig> % and  <dig> % for the n <dig> gene and call rates of  <dig> % and  <dig> % for the h <dig> gene, respectively .
in order to confirm subtyping of the different viruses, primers were designed based upon resequencing sequences reconstructed by the microarray and used for rt-pcr .
amplified products were cloned and analysed by classical sequencing.
sequencing results confirmed the subtyping by the dna microarray of the  <dig> viral strains isolated from cloacal samples.
time ranges showed that the viral strains isolated in the environment are recent, except for the h <dig> gene of strain  <dig> .
the evolutionary rate of influenza viruses in the natural avian host is believed to be slower than in mammals, and this could explain the above result.
sequences were analysed with the blastn program and results with highest scores are represented in table  <dig>  the majority of the "classical" sequences were identical to those found after blastn analysis of sequences generated by the dna microarray.
the level of sequence information generated by the dna resequencing microarray is sufficient and equivalent to that obtained by classical sequencing.
sequences corresponding to highest scores are detailed.
asterisks indicate sequences that were the same as those given by the resequencing data.
CONCLUSIONS
the pathogenid v <dig>  microarray demonstrated its ability to type and subtype influenza viruses with a minimum number of tiled sequences and to give additional information close to the individual variant level.
identification of influenza viruses was obtained within the correct period of time in most cases.
however, this level of discrimination is limited by the number of sequences available in databases and their level of divergence as illustrated by results obtained with equine viruses.
the pathogenid v <dig>  microarray should also be able to detect a re-emerging influenza virus, that has already circulated in the population, as happened in  <dig> during the russian influenza outbreak  <cit> .
blastn analysis of the sequences generated by the dna microarray should allow the determination of the segment origin for pb <dig>  ha and na genes, even though the consensus sequence design was not based on the origin of the viral host.
the avian h3n <dig> virus isolated from the environment was identified and characterised as having an h <dig> consensus sequence different from that obtained for human h3n <dig> viruses.
recently, we have tested the novel human a pandemic virus of swine origin, derived from multiple reassortments, with the dna microarray and blastn analysis determined the segment origin for the pb <dig>  h <dig> and n <dig> genes, which were derived from avian, classical swine virus and avian-like swine virus, respectively  <cit> .
our data validated the strategy of using consensus sequences with all of the serotypes tested, including h <dig>  h <dig>  h <dig>  and h <dig>  <cit>   and n <dig>  n <dig> and n <dig> .
the simple design strategy used in this study, based on global alignements covering the whole gene, does not need the use of a specific algorithm as the one used for the tessarray rpm-flu resequencing microarray which was based on probe sequence selection  <cit> .
the consensus sequences which allowed to considerably limiting the number of probes tiled on the non-specialised pathogenid v <dig>  microarray would allow the determination of differential diagnosis with other respiratory viruses such as respiratory syncytial virus , sars-cov, and also potential co-infections, which is not possible with microarrays entirely dedicated to influenza viruses.
the use of wta amplification provides considerably better sensitivity and accuracy of detection compared to random rt-pcr  <cit>  which is compatible with the use of the pathogenid v <dig>  resequencing microarray for clinical samples.
furthermore, the use of random hexamers and multiple displacement amplification allowed detection of a new virus which can not be amplified with usual specific primers as shown by the study on the last novel h1n1pdm virus responsible for  <dig> influenza pandemic  <cit> .
however, the dna microarray failed to detect the n <dig> gene from h3n <dig> equine viruses.
here, it will be useful to tile other genes on the next generation microarray, including all ha and na subtypes or the pb <dig> gene, which has often been involved in reassortment in past pandemics.
in this way, one could detect as many reassortants as possible thereby impacting on public health measures.

 rapid detection and identification of infectious pathogens are necessary for proper treatment of infection and outbreak control measures. many promising approaches are being developed enabling rapid identification of various infectious pathogens such as bacteria or viruses including multiplex -pcr amplification strategies or novel technologies such as high-performance electrospray ionization mass spectrometry. dna microarray technologies provide a mean to screen for thousands of different nucleic acid sequences simultaneously and have been used for the detection of many viruses such as hiv, hepatitis b and d viruses, human papillomaviruses, rotaviruses, vaccinia virus, herpesviruses, enteroviruses, flaviviruses, and measles virus genotypes. in addition to these microarrays, which have a spectrum of identification limited to a few viral species, major microarray platforms were developed for the detection of a much broader range of pathogens. in humans, influenza a viruses cause recurrent annual epidemics of moderate to severe respiratory diseases. sixteen hemagglutinin molecular types and nine neuraminidase molecular types have been identified, although only a few of them occur in viruses causing human diseases. identification and selection of sequences to be used on a microarray are particularly challenging, especially for rapidly mutating viruses. antigenic drift is an ongoing process for influenza a viruses resulting in continuous evolution and annual epidemics. moreover, the segmented nature of the influenza a virus genome can lead to reassortments during coinfection thus contributing to their vast genetic diversity and pandemic potential. subtyping and molecular analysis of influenza virus strains were performed by different groups using low density microarrays. most of them were used to identify influenza a viruses h1n h3n h5n and influenza b viruses. recently, combimatrix corporation developed a semiconductor-based microarray and an integrated microfluidic array enabling the detection of ha and na. however, the cost per microarray is too high for routine experimental identification. more recently, a rapid subtyping assay also identifying all the ha and na subtypes was developed using padlock probes. resequencing, using high-density oligonucleotide microarrays, is a technology that enables the rapid identification of genetic variants and the determination of their nucleotide sequence at single base-pair resolution. this allows identification of pathogens with sequences similar but not identical to those represented on the microarray. a resequencing microarray was designed in order to allow identification of genetically diverse human rna rhinoviruses and enteroviruses, with a minimum number of prototype 5'utr sequences. one respiratory pathogen resequencing microarray allowed the detection of geographically distant human h1n h3n and avian h5n influenza virus strains covering the period between and. more recently, the same team developed a broad-range resequencing microarray for universal detection and identification of all the possible combinations of the hemagglutinin and neuraminidase types of avian influenza viruses in addition to three additional influenza a markers. their strategy was based on probe sequence selection using in silico modeling. a resequencing microarray called pathogenid v has been developed by a consortium of laboratories of institut pasteur with affymetrix inc. it was used to explore various strategies of sequence selection for its design. the part dedicated to influenza viruses, presented here, was based on consensus sequences specific for one gene. within the global program, another strategy was explored for the part of the microarray specific for rhabdoviruses. that strategy was based on the use of prototype sequences representative of each of the seven species described in the genus lyssavirus and chosen within a conserved region of the polymerase gene. consensus sequences generation was a posteriori used to analyse results. our strategy presented in this study, uses consensus sequences generated from nucleotide sequence alignments to determine the sequences to be tiled. these sequences characterised by a minimum size of bp reduce the number of probes required for tiling yet still covering the wide diversity of influenza virus strains. to validate our approach, influenza viruses from different host origins, isolated over a period of about years, were chosen. the dna microarray not only demonstrated its ability to identify the type and subtype of influenza viruses but also had a strong discriminatory power at the level of viral variants.  microarray design the aim of the study was to detect influenza a viruses isolated over a long period of time, from a range of hosts and with a minimum of sequences tiled on the array. four genes including pb m, ha and na were chosen in order to type and subtype influenza viruses. different strategies for selecting the region to resequence were adopted depending on the particular gene. characteristics of the sequences tiled on the microarray are detailed in table firstly, one partially conserved sequence of bp covering the majority of influenza m segments, routinely employed in medical diagnostics, was used for genus identification, namely influenzavirus a, b and c. secondly, almost one hundred sequences of pb genes covering a large spectrum of influenza a viruses and hosts were selected from the genbank database. five typical pb sequences representative of the diversity of pb sequences over a long period of time and for different host reservoirs were finally retained. the five selected sequences are described in table the strategy adopted for the hemagglutinin and the neuraminidase genes involved the design of consensus sequences for the h h h h h and h genes and the n n and n genes. we have restricted the number of studied molecular subtypes of ha and na in order to make the confirmation of our hypothesis more straightforward and demonstrative without aiming to cover the whole set of serotypes. the ha and na subtypes chosen to be tiled on the microarray are shared by all the human viruses and highly pathogenic avian viruses, and the has are further distributed into phylogenetic groups that gather all the serotypes. for each major ha and na gene, sequences of influenza a virus were selected from different hosts including horses, pigs, humans and birds. positions and sizes of the consensus ha and na sequences were determined and selected after blastn analysis of the sequence. for each gene, a global alignment including all selected sequences was performed and clusters grouping a maximum number of sequences were established. in each sequence cluster, a common region with the lowest genetic divergence was identified. a consensus sequence was automatically obtained from this common region, characterised by a minimum size of bases and a divergence between the consensus sequence and any given sequence belonging to the cluster of less than 15%. twelve ha consensus sequences and na consensus sequences were identified and their positions are detailed in table finally, the microarray based on the resequencing approach included influenza viral sequences which corresponded to bases. for each relevant base of a given consensus or prototype sequence, the array contained eight mer probes. two of the eight probes represented perfect matches, while the others corresponded to possible mismatches at the central position of the mers. all together, probes were then tiled on the microarray, allowing subtyping of influenza viruses from different host species. detection of type a influenza viruses a total of influenza virus samples were analysed by the resequencing microarray, including swine, human, equine and avian strains that originated from various locations worldwide and covered the period of time between and all samples were viral isolates, propagated either in embryonated chicken eggs or in mdck cell cultures. they were chosen from sufficiently distant points in time to cover a large diversity of influenza viruses, especially for human and swine viruses, which were subject to major antigenic drift over the years. avian strains were derived from cloacal samples collected from ducks in baie de somme, marquenterre in rna extracts were amplified by wta and hybridised on the resequencing microarray. results are summarised in table nucleotide sequences were determined from the fluorescent signals as previously described and compared with sequences in genbank by blastn analysis, to identify and subtype influenza virus strains. results for the highest hit scores were taken as strain identification. after hybridisation of wta amplified material on the dna microarray, all samples were positive for the presence of influenza a virus with a minimum call rate of 41% for the m gene and a call rate ranging from to % for the pb gene, which is the percentage of bases determined by the resequencing algorithm. subtypes of the different strains were identified with ha and na sequences, giving a call rate ranging from % to 100%. time ranges were obtained from blastn analysis. the sequences showing the highest alignment score were considered and duplicated sequences representing different isolates of the same strain were eliminated. the time range indicated the oldest isolate with high homology provided by the blastn algorithm and the most recent one. the median was calculated based upon the sequences showing the highest alignment scores. the call rate is the percentage of bases determined by the resequencing algorithm. time ranges indicate the oldest isolate with high homology provided by the blastn algorithm and the most recent one. the medians were calculated based upon the sequences showing the highest alignment scores. for swine and human viruses, a minimum call rate of % allowed identification of influenza virus with a correct temporality. indeed, for the majority of strains the blastn result was close to that of the sample strain, as outlined by median analysis. the median obtained for the na gene from the a/johannesburg/33/ virus was relatively recent compared to the year of isolation of the strain, probably because the chosen consensus sequence for this gene was highly conserved and thus weakly discriminatory. the time range obtained for the a/wyoming/3/ virus was narrow and limited to the year probably due to the marked antigenic drift from the year observed for the h gene in human influenza virus strains. the epidemiology of swine influenza has become increasingly complex over the last decades. three major influenza a virus subtypes are currently circulating in swine worldwide, but the origins and the antigenic and genetic characteristics of these swine influenza viruses differ with continents or regions of the world. a/swine/gent/7625/ virus is the result of a multiple reassortment involving a swine influenza virus with avian-influenza-like internal segments and human h1n and h3n viruses isolated in. blastn analysis for h1n swine virus reflected this multiple reassortment with blasted sequences originating from human, avian and swine hosts. between and swine h3n viruses appeared in italy. they contained avian-like internal genes and ha and na genes from human h3n viruses. in blastn analysis, the vast majority of the sequences sorted by the algorithm for glycoprotein segments were from human or swine origins only. all equine influenza virus strains were identified and subtyped with the dna microarray, with a minimum call rate of %. medians were often heterogeneous and far from the original strain. this is probably due to the paucity of equine influenza virus sequences available in the databases, the limited genetic evolution of those viruses leading to equivalent result scores after blastn analysis, the fact that ha and na sequences tiled on the microarray were shorter than pb sequences, due to their extreme genetic diversity in influenza a viruses. for the h3n viruses, recent sequences corresponded to avian strains, outlining the fact that h3n equine viruses were reassortants between avian viruses and equine h7n viruses which have not been isolated in horses for more than years. all together, the results showed that the pathogenid v dna microarray is able not only to provide correct full subtyping of various strains from different host origins but also to identify/detect, after blastn, an array of strains genetically related to them. in all cases, the strains output by blastn belonged to the same time period thus giving a strong insight into the variant to which it is genetically, and very probably antigenically, related. avian samples cloacal samples were collected from ducks in baie de somme, marquenterre in and analysed by qrt-pcr specific for the m gene. wta amplification of viral rna followed by hybridisation to the dna microarray confirmed the identification of influenza viruses. two samples were identified as h1n viruses and one sample as h3n virus. influenza virus strain was identified as h3n virus with a call rate of % for one of the h consensus sequences and % for one of the n consensus sequences. influenza virus strains and were both identified as h1n virus with call rates of % and % for the n gene and call rates of % and % for the h gene, respectively. in order to confirm subtyping of the different viruses, primers were designed based upon resequencing sequences reconstructed by the microarray and used for rt-pcr. amplified products were cloned and analysed by classical sequencing. sequencing results confirmed the subtyping by the dna microarray of the viral strains isolated from cloacal samples. time ranges showed that the viral strains isolated in the environment are recent, except for the h gene of strain. the evolutionary rate of influenza viruses in the natural avian host is believed to be slower than in mammals, and this could explain the above result. sequences were analysed with the blastn program and results with highest scores are represented in table the majority of the "classical" sequences were identical to those found after blastn analysis of sequences generated by the dna microarray. the level of sequence information generated by the dna resequencing microarray is sufficient and equivalent to that obtained by classical sequencing. sequences corresponding to highest scores are detailed. asterisks indicate sequences that were the same as those given by the resequencing data.  the pathogenid v microarray demonstrated its ability to type and subtype influenza viruses with a minimum number of tiled sequences and to give additional information close to the individual variant level. identification of influenza viruses was obtained within the correct period of time in most cases. however, this level of discrimination is limited by the number of sequences available in databases and their level of divergence as illustrated by results obtained with equine viruses. the pathogenid v microarray should also be able to detect a re-emerging influenza virus, that has already circulated in the population, as happened in during the russian influenza outbreak. blastn analysis of the sequences generated by the dna microarray should allow the determination of the segment origin for pb ha and na genes, even though the consensus sequence design was not based on the origin of the viral host. the avian h3n virus isolated from the environment was identified and characterised as having an h consensus sequence different from that obtained for human h3n viruses. recently, we have tested the novel human a pandemic virus of swine origin, derived from multiple reassortments, with the dna microarray and blastn analysis determined the segment origin for the pb h and n genes, which were derived from avian, classical swine virus and avian-like swine virus, respectively. our data validated the strategy of using consensus sequences with all of the serotypes tested, including h h h and h and n n and n. the simple design strategy used in this study, based on global alignements covering the whole gene, does not need the use of a specific algorithm as the one used for the tessarray rpm-flu resequencing microarray which was based on probe sequence selection. the consensus sequences which allowed to considerably limiting the number of probes tiled on the non-specialised pathogenid v microarray would allow the determination of differential diagnosis with other respiratory viruses such as respiratory syncytial virus, sars-cov, and also potential co-infections, which is not possible with microarrays entirely dedicated to influenza viruses. the use of wta amplification provides considerably better sensitivity and accuracy of detection compared to random rt-pcr which is compatible with the use of the pathogenid v resequencing microarray for clinical samples. furthermore, the use of random hexamers and multiple displacement amplification allowed detection of a new virus which can not be amplified with usual specific primers as shown by the study on the last novel h1n1pdm virus responsible for influenza pandemic. however, the dna microarray failed to detect the n gene from h3n equine viruses. here, it will be useful to tile other genes on the next generation microarray, including all ha and na subtypes or the pb gene, which has often been involved in reassortment in past pandemics. in this way, one could detect as many reassortants as possible thereby impacting on public health measures. 
the part dedicated to influenza viruses was based on consensus sequences specific for one gene generated from global alignments of a large number of influenza virus sequences available in databanks. influenza viruses were correctly identified, and in most cases with the accurate information of the time of their emergence. pathogenid v microarray demonstrated its ability to type and subtype influenza viruses, often to the level of viral variants, with a minimum number of tiled sequences. the versatility, rapidity and high discriminatory power of the pathogenid v microarray could prove critical to detect and identify viral genome reassortment events resulting in a novel virus with epidemic or pandemic potential and therefore assist health authorities to make efficient decisions about patient treatment and outbreak management. a resequencing microarray called pathogenid v has been developed and used to explore various strategies of sequence selection for its design. this validated the strategy of using consensus sequences, which do not exist in nature, for our microarray design. for each ha and na molecular type chosen to be tested, to consensus sequences were computed and tiled on the microarray. a total of influenza virus samples from different host origins and isolated over a period of about years were used in this study.
 a resequencing microarray called pathogenid v has been developed and used to explore various strategies of sequence selection for its design. the part dedicated to influenza viruses was based on consensus sequences specific for one gene generated from global alignments of a large number of influenza virus sequences available in databanks.  for each ha and na molecular type chosen to be tested, to consensus sequences were computed and tiled on the microarray. a total of influenza virus samples from different host origins and isolated over a period of about years were used in this study. influenza viruses were correctly identified, and in most cases with the accurate information of the time of their emergence.  pathogenid v microarray demonstrated its ability to type and subtype influenza viruses, often to the level of viral variants, with a minimum number of tiled sequences. this validated the strategy of using consensus sequences, which do not exist in nature, for our microarray design. the versatility, rapidity and high discriminatory power of the pathogenid v microarray could prove critical to detect and identify viral genome reassortment events resulting in a novel virus with epidemic or pandemic potential and therefore assist health authorities to make efficient decisions about patient treatment and outbreak management. 