For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents. There are sixteen recognized serological subtypes of type A influenza virus hemagglutinin (H1 through H16) and 9 type A neuraminidase subtypes (N1 through N9). Among the combinatorial diversity of 144 possible A/HN subtypes, relatively few subtypes have been identified as causes of human disease. Four pandemic outbreaks in the last century, one catastrophic, appear to have introduced the subsequently prevalent seasonal human influenza virus subtypes A/H1N1 , A/H2N2 , A/H3N2 , and A/H1N1 again . The current year 2009 has been marked by a late season pandemic-scale emergence of a novel A/H1N1 outbreak strain, raising immediate concerns for public health as well as for pork and poultry production industries worldwide.As with the few common subtypes of human type A influenza viruses, there are similarly few subtypes of type A influenza viruses that are associated with most influenza infections of swine, horses or dogs. In distinct contrast, wildfowl species are natural hosts and a global reservoir for the majority of possible influenza A/HN subtypes. Many of these variant strains appear to be associated with endemic infections, often asymptomatic in avian hosts Fortunately, infectious transmission of such avian influenza virus strains between humans continues to be limited. However, history suggests that further evolution of these or other type A influenza strains could emerge as a next pandemic strain. Similarly, variant type A influenza virus strains have emerged from time to time, imposing serious costs and burdens upon poultry and livestock production.Because the natural history and the molecular biology of influenza viruses reflect such viral genome diversity, there is a critical need for rapid, sensitive, specific, and informative assays to detect and characterize any subtype of influenza virus. Benchmark standard methods that employ propagation of virus in cell culture or in embryonating chicken eggs, with assays using panels of specific serological reagents, or reverse transcriptase polymerase chain reaction (RT-PCR)-based assays, using panels of short oligonucleotide primers and probes, are either slow and time consuming, or expensive. As prevailing strains of avian influenza continue to evolve and diverge, diagnostic assays that are based only on specific recognition of short signature sequences or peptide biomarker loci will increasingly fail, through false-positive and/or false-negative results. This will adversely impact critical decision-making.This report describes a re-sequencing pathogen microarray (RPM)-based assay for simultaneous detection, identification and characterization of any subtype of type A human or avian influenza virus, based on rapid, sensitive and specimen-specific determination of nucleotide sequences from viral hemagglutinin, neuraminidase, and other genes.All specimens described in this report that were originally obtained from human subjects were obtained with informed  consent of study participants allowing for further research use, following Institutional Review Board-approved research protocol NHRC.1999.0002, Triservice Population-Based Surveillance for Respiratory Pathogens Among High-Risk Military Personnel, CAPT Kevin Russell, MC, Principal Investigator. The NHRC IRB approved verbal consent for this protocol because of minimal risk to volunteers. Samples are collected for accredited diagnostic testing, and only used for further research in a de-identified manner with minimal demographic data , tested only for respiratory pathogens and analyzed in aggregate, and resulting data is not used for patient treatment or management. No part of this study was conducted as experimentation involving live vertebrate animals. Reference strains of avian influenza viruses were propagated in laboratory cultures for analysis of viral RNA.The Naval Health Research Center (NHRC) collects, analyzes, and archives throat-swab specimens from human subjects as part of respiratory infection surveillance at US basic military training facilities. Research use of donated specimens is permitted under the local Institutional Review Board's approved protocol-compliant informed consent (see Acknowledgments). Throat swabs are suspended in stabilizing transport media and archived frozen at −80°C. Viral culture and PCR-based testing protocols are used at the NHRC diagnostic laboratory to determine the presence or absence of influenza virus, and these assays are accredited by the College of American Pathologists as compliant with the Clinical Laboratory Improvement Amendments of 1988, and the Department of Defense Clinical Laboratory Improvement Program of 1994.Cloacal swabs and/or tracheal swabs from migratory birds (mostly waterfowl) and commercial poultry; tracheal and lung tissue samples from dead birds; human throat swabs and one human lung sample from a deceased patient were collected by Naval Medical Research Unit No. 3 . Avian influenza viruses in these samples were cultured using chicken eggs and/or Madin-Darby canine kidney cell cultures . Sample collection and viral culture techniques were as described The U.S. Department of Agriculture, Agriculture Research Service (USDA-ARS) Southeast Poultry Research Laboratory  selected specimens from its reference strain archive for blinded analysis by RPM-Flu. These samples comprised 20 representative avian influenza A/HN strains and also included 2 avian paramyxoviruses as controls. Total nucleic acid extractions and purifications  were performed following harvest from infected eggs at SEPRL. A 30 µl aliquot from the final preparation was frozen and forwarded for RPM-Flu analysis.in lieu of specimen total nucleic acid. Sources of control nucleic acid templates included type cultures of reference strain Eurasian-African A/H5N1 high pathogenicity influenza virus obtained from the Centers for Disease Control and Prevention . Reference control templates as mixtures of the A/H1N1, A/H3N2, and B strains of influenza virus were from live virus preparations , or from inactivated virus preparations . Synthetic DNA preparations were purchased from Blue Heron Biotechnology  as templates to represent other type A influenza hemagglutinin  and neuraminidase (N3 through N9) gene sequences, as well as other pathogen gene sequences that are used as detectors on the RPM-Flu array. In some cases, the NRL group outsourced  corroborative de novo gene sequence determinations from amplified products from hemagglutinin (HA), matrix (M), neuraminidase (NA), non-structural (NS1) and RNA-dependent RNA polymerase subunit (PB2) genes.The NRL group evaluated pathogen detection and identification capabilities of the RPM-Flu assay and kit components from TessArae, using control nucleic acid templates in assays The largest segment of the RPM-Flu microarray  is allocated to 40 detector tiles to represent the multiple subtypes of type A and type B HA and NA genes, selected type A M genes, and conserved NS and PB2 gene sequences from avian influenza virus A/H5N1.www.tessarae.com).Selected specimens were thawed to retrieve aliquots for extraction and purification of total nucleic acid for multiple assays, including RPM-Flu testing. Each RPM-Flu assay consumed about 30 µl (2%) of the originally archived specimen (about 1.5 ml). RPM-Flu analysis of specimen total nucleic acid followed the manufacturer's recommendations  of amplification products – 0.5 hourHybridization of labeled products to microarrays – 4 hour to 16 hour (overnight preferred)Washing and staining arrays – 1.5 hourScanning arrays for data acquisition – 0.15 hourThe abbreviated 4-hour hybridization time (5. above) reduces assay cycle time to single day, which may be helpful if same-day test results are a critical consideration. The overnight (∼16 hour) hybridization time is recommended and preferred to optimize both assay sensitivity and the quality and length(s) of assay-generated gene sequences from detected target pathogens.Analysis of RPM-Flu assay data now differs from earlier reported through the development of the prototype assay platform RPMv1 TessArray Sequence Analysis (TSEQ) software evaluates a “C3 Score” for each RPM-Flu detector tile, as a metric of detected DNA sequence quantity and quality. The C3 Score is the total number of GSEQ-identified nucleotides that appear in runs of three or more consecutive (non-N) base calls, expressed as percentage of the length (nucleotides) of each RPM-Flu detector tile sequence. This approach to definition of a detected sequence reduces background noise from spurious false-positive hybridizations of 25-base oligonucleotide probes that could lead to isolated and relatively uninformative single base calls.P<0.000003) above the mean of C3 Scores from thousands of detector tiles from assays performed in the absence of positive control templates.Relaxed and stringent target pathogen detection and reporting thresholds for the RPM-Flu assay have been statistically and empirically established for all of the resequencing detector tiles at C3 Score >10 or C3 Score ≥20, respectively. RPM-Flu assay results from hundreds of clinical and field specimens as well as laboratory reference strains and synthetic DNA templates (aggregate data not shown) have demonstrated that these detection thresholds often represent C3 Scores that are more than 6 standard deviations  , using a dedicated server-based implementation of the Basic Local Alignment Search Tool, BLAST, version 2.2.17 In order to qualify for reporting of target pathogen detection using the relaxed threshold (C3 Score >10), a BLAST alignment and sequence similarity search using the assay-generated sequence of basecalls must return one or more most similar sequence records from the search database (as TessArae's VRSD) that are concordant with the identity of the tentatively identified target pathogen suggested by the identity of the particular resequencing detector tile.Aliquots from two throat swab specimens collected during the 2008–2009 influenza were used for propagation of stocks by infection of MDCK cell cultures.A/California-NHRC/BRD10622N/2009(H1N1)A/California-NHRC/BRD10601N/2009(H3N2)7 TCID50/ml and 1.78×107 TCID50/ml, respectively.Infected culture lysates were then diluted for infections of replicate shell vial cultures to determine infectious titers . The resulting stocks of BRD10622N(H1N1) and BRD10601(H3N2) were determined to contain 3.16×100 (neat) to 10−3, and then three-fold dilutions were prepared from 1∶3,000 to 1∶2,187,000. The routine RPM-Flu protocol for total nucleic extraction and purification was performed on each of the triplicate diluted samples of the viral stocks for identity-blinded triplicate RPM-Flu assays.Serial ten-fold dilutions of viral stocks for RPM-Flu assays were prepared using fresh cell culture media as diluent, from 10The influenza virus subtype A/H1 gene detector tile ‘HA1(H1N1)’ on the RPM-Flu microarray is an array of 11,808 resequencing oligonucleotide probes (25-mers), representing the nucleotides 113 through 1,588 of the A/New Caledonia/20/99 (H1N1) strain hemagglutinin gene (GenBank Accession Number AY289929). This is the actual A/H1N1 strain of influenza virus representing the viral HA1 gene product in the 2004–2005 vaccine configuration.An aliquot of FluMist vaccine (2004–2005) was subjected to RPM-Flu assay, from which 1,351 specific base calls were determined across the HA1(H1N1) detector tile, resulting in C3 Score = 91.5. BLAST analysis returned AY289929 A/New Caledonia/20/99 (H1N1) as the most similar sequence record from alignment-based search across the entire GenBank, with the limit (minimum) expectation value of 0.0 .This example of the integrity of RPM-Flu assay-generated target pathogen gene sequencing for detection and identification is reinforced by results for the other specific hemagglutinin, neuraminidase, and matrix gene sequences from the same RPM-Flu assay of the same aliquot of FluMist vaccine. Similarly specific typing, subtyping and strain identification results to those shown in The results in Except for the vaccine sample, assays of each sample reported sequences from only one hemagglutinin detector tile and one neuraminidase detector tile, those corresponding precisely to the specimen's A/HN subtype or B type.The mean values of C3 Scores from the positive detector tiles in pos = 73.7±24.0 (N = 22)Mean C3neg = 3.2±2.3 (N = 261)Mean C3[A six-sigma (6σ) detection threshold for this data set is C3 = 17.0]pos = 13.7). The same assay of this specimen generated higher C3 Scores for sequences from the B-HA (C3 = 31) and B-M (C3 = 34) detector tiles that exceeded the stringent detection threshold. The type B-NA sequence from the assay met the relaxed detection threshold (C3 Score >10), in part because BLAST analysis of the sequence returned nine most similar type B influenza virus NA gene sequence records. This corroborated a tentative detection report (relaxed threshold) that might have been made on the basis of the NA gene resequencing result alone. The specimen was collected in 2006 at U S Army Fort Leonard Wood, MO, and each of these most similar sequence records represented a NA gene from various circulating type B influenza virus strains collected during the same influenza season.The lowest positive control result from this data set was for the NA gene of the NHRC-type B influenza virus-positive specimen , A/H3N2(human), A/H5N1(avian) and B(human) types and subtypes. The results in We have reviewed the lists of most similar sequence records returned by BLAST analysis of RPM-Flu assay-generated gene sequences from 12 different human  A/H1N1 strains and 31 different human  A/H3N2 strains.Among RPM-Flu assay results from the 12 A/H1N1 samples there were 478, 800, 201, 348 and 312 most similar sequence records returned from BLAST analysis of sequences generated from the HA1(H1N1), NA1(H1N1), M(H1N1), M(H3N2), and NS(H5N1) detector tiles, respectively. Without exception, each of these most similar sequence records represents an A/H1N1 strain, including the 660 sequence records found to match the RPM-Flu sequences generated from the RPM-Flu M(H3N2) and NS(H5N1) detector tiles. Among the 31 A/H3N2 samples there were 754, 189, 1267, and 1678 most similar sequence records returned from the HA3(H3N2), NA2(H3N3), M(H1N1), and M(H3N2) detector tiles, respectively, and every one of these sequence records represents an A/H3N2 strain.However, the NS (H5N1) detector tile generated 109 specimen-specific NS gene sequences from several of the A/H3N2 samples, and only 19 of the most NS gene sequence records were from A/H3N2 strains. The other NS gene sequence records that were found by BLAST to be most similar to the sequences generated from assays of A/H3N2 specimens represent subtypes A/H2N2 (12), A/H1N1 (73), A/H11N9 (2), A/H6N2 (2) and A/H3N6 (1). These results suggest that circulating strains of subtype A/H3N2 harbor diverse NS gene sequences, variations that presumably arise from frequent co-infections and genome segment reassortments in the field. On the other hand, the subtype A/H1N1 specimens led to RPM-Flu assay-generated NS gene sequences that are most similar to NS gene sequence records associated with subtype A/H1N1 strains.Although it appears that the RPM-Flu assay-generated M gene sequences may have some utility to differentiate human A/H1N1 and human A/H3N2 subtypes, the NS gene sequences do not appear to be so reliable for the purpose of inferring A/HN subtype. This consideration will appear again later in RESULTS with the subject of indirect, inferred A/HN subtyping of type A avian influenza viruses.The RPM-Flu assay was implemented in a blinded analysis of several hundred clinical specimens from the respiratory infectious disease archives at NHRC. Selected throat swab specimens had been collected over the period 2005–2007 from basic military trainees at several installations in the United States. The subjects were evaluated at the time of specimen collection and associated with one of three cohorts: apparently healthy and afebrile individuals; those diagnosed with febrile respiratory illness (FRI); and those diagnosed with pneumonia (confirmed by chest x-ray).A group of 298 specimens were analyzed using both the RPM-Flu assay and a validated benchmark RT-PCR assay for type A influenza virus described by Freed et al. Mycoplasma pneumoniae; results not shown).The benchmark RT-PCR type A influenza virus test used to assess clinical sensitivity and specificity of the RPM-Flu assay does not distinguish between the A/H1N1 and A/H3N2 subtypes. However the RPM-Flu assay is capable of simultaneous and mutually exclusive identification of a detected type A influenza virus as either seasonal subtype A/H1N1 or seasonal subtype A/H3N2. Results presented in In the course of increasing prevalence of oseltamivir-resistant strains of A/H1N1 and amantadine-resistant strains of A/H3N2, the NHRC has implemented a new RT-PCR panel to determine the A/H1 or A/H3 subtype for specimens already shown to be type A influenza virus-positive by the earlier RT-PCR panel . They have also validated the assay by demonstrating concordance of subtyping results from new RT-PCR panel with a cell culture/hemagglutinin immunofluorescence assay (HAI). Type B influenza virus-positive specimens (identified from the first tier RT-PCR typing panel) were negative controls for the second tier RT-PCR-based Type A subtyping panel.Twelve isolates from the 2008–2009 influenza season, six each testing positive for A/H1 or A/H3 by the RT-PCR panel , were selected for RPM-Flu analysis. All of the specimens tested definitively positive in the initial (and only necessary) RPM-Flu assay as either A/H1N1 or A/H3N2, matching the results of both the RT-PCR and HAI benchmark A/H1 and A/H3 assays. These results are summarized in The results for M gene sequences from both the M(H1N1) and the M(H3N2) detector tiles for both sets of specimens shown in Moraxella catarrhalis, Staphylococcus aureus, Streptococcus agalactiae, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas spp., Streptococcus mitis, and Streptococcus pneumoniae. There was no obvious correlation of the distributions of other co-infecting pathogens with the particular subtype of infecting influenza virus, or with particular specimen collection venue(s). There was no data on clinical presentation or outcomes to correlate with particular states of co-infection(s).All 30 of the influenza-positive specimens described above were also reported by the individual RPM-Flu assays to be positive for at least one and up to six other viral and/or bacterial respiratory pathogens, including adenovirus, rhinovirus, coronavirus, We considered whether or not there may be interference with the detection and identification of specific influenza viruses in specimens that test positive in RPM-Flu assays for other viral and bacterial respiratory pathogens. The background distribution of C3 Scores for detection and identification of influenza viruses was estimated from RPM-Flu assays of 14 different blank samples (water or transport media negative controls). The average C3 Score of the 39 type A and type B influenza virus detector tiles to be 1.7±1.1 .Another set of 25 specimens were found to be negative for influenza virus by both RPM-Flu and benchmark RT-PCR tests, while the same RPM-Flu assays reported the specimens to be positive with apparently high load (C3 Score>75) of at least one other viral or bacterial respiratory pathogen (results not shown).Chlamydophila pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Mycoplasma pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus (including MRSA strains), Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes.Simultaneous pathogen targets of the RPM-Flu assay that apparently do not interfere with detection and identification of influenza viruses include adenovirus , coronavirus , rhinovirus , metapneumovirus , parainfluenzavirus , respiratory syncytial virus , The 25 A/H3N2-positive clinical specimens from the ensemble of 298 specimens described in the preceding section were collected between January 2007 and March 2007, at five different military basic training venues . All but one of the RPM-Flu assay-generated HA3 gene sequences were most similar to BLAST-returned sequence records of A/H3N2 isolates that Nelson et al in vitro amantadine resistance At least five loci in the M2 trans-membrane peptide-encoding region of influenza virus M gene have been reported to confer some level of The M gene sequences detected by RPM-Flu assay of the 24 amantadine-resistant N-lineage or clade-a specimens all reveal -AAT- as asparagine (N) codon 31 of the M2 peptide, a genotype consistent with amantadine resistance. The matrix gene sequence of the outlier clade-b specimen (NHRC_393) reveals -AGT- as serine (S) codon 31 of the M2 peptide, a genotype consistent with an amantadine-sensitive phenotype.The five A/H1N1-positive clinical specimens from the ensemble of 298 specimens described in the preceding section were collected between January 2007 and March 2007, at two different military basic training venues . BLAST analysis of the five RPM-Flu-detected HA1 gene sequences returned most similar reference sequence records including those that Nelson et al. Neuraminidase gene sequences were detected and compared at this locus from RPM-Flu assays of specimens from the earlier 2006–2007 season and more recent 2007–2008 season specimens provided by NHRC for analysis. As shown in the Future iterations of the RPM-Flu microarray design could provide one or more alternative NA1 gene sequence detector subtiles. Such assay design iterations would enable future RPM-Flu assays to deliver more explicit first-assay characterization of the oseltamivir genotype and phenotype.7 TCID50/ml and 1.78×107 TCID50/ml, respectively.An isolate of subtype A/H1N1 and another of subtype A/H3N2 were selected by NHRC for single-passage propagation in MDCK cell cultures, followed by infectivity titrations in replicate shell vial cultures. The resulting neat stocks of BRD10622N(H1N1) and BRD10601(H3N2) were determined to contain 3.16×10These two titered stocks were used to determine the analytical sensitivity of the RPM-Flu, by blinded assays of samples extracted from triplicate three-fold serial dilutions in the range of 1∶1,000 to 1∶2,187,000. Background C3 Scores were used as thresholds for reporting detection of either A/H1N1 or A/H3N2 in these limit dilution assays . The background detector tiles represent 25 non-A/H1N1 and non-A/H3N2 detector tiles from each individual RPM-Flu assay, and a positive detection was reported if one of the target-specific detector tiles from the following lists had C3 score at least 3.29 standard deviations greater than the background mean C3 Score (p<0.001).50 for the A/H1N1 titered stock preparation and 6 TCID50 for the A/ H3N2 titered stock preparation.From these results RPM-Flu assay, the 95% positive endpoint dilution limits of detection (LoD) be 3 TCIDFrom the total of 41 independently extracted dilution specimens of the subtype-confirmed and titered A/H1N1 stock , 37 wereA collection of 23 reference strain specimens, representing diverse type A avian influenza virus subtypes, were propagated in embryonated eggs by SEPRL, then extracted for purification of total nucleic acid as input to RPM-Flu assays.The RPM-Flu assay of specimen USDA_13 was reported negative for detection of avian influenza virus, but the same assay also reported the specimen to be positive for avian metapneumovirus. This was revealed to be a correct result after unblinding of the specimen identification as a non-influenza control Type C avian metapneumovirus.Three avian influenza virus-negative RPM-Flu assay results were also concordant with subsequently unblinded specimen information. USDA_5 was RPM-Flu negative for avian influenza and later confirmed to have been from an unsuccessful inoculation of the egg culture.de novo gene sequencing and determined to be A/H11 strain. The RPM-detected neuraminidase sequence  unequivocally matched A/N9; the mean C3 Score of all other neuraminidase detector tiles in the assay was 3.1±1.6 (N = 10).USDA_8 was reported by RPM-Flu assay to be A/H11N9, and while the initial identification from SEPRL was subtype A/H9N2, the original specimen apparently represented a mixed infection of H11N9 and H9N2. The HA component of the specimen was subjected to USDA_16 was correctly negative by RPM-Flu assay for avian influenza, but unblinding revealed this specimen to be a second negative control, containing instead avian paramyxovirus type I (Newcastle disease virus). After receiving this information, another aliquot of the same nucleic acid preparation from specimen USDA_16 was tested using an alternative RPM-TEI assay Specimen USDA_23 was received later as a substitute for the failed inoculum specimen USDA_5. The RPM-Flu assay of this specimen resulted in detection of sequence for neuraminidase type N4, with most similar sequence record representing an A/H8N4 strain. However, no hemagglutinin gene sequences were detected that met even the relaxed detection threshold. The highest C3 Scores among all RPM-Flu hemagglutinin detector tiles were HA1(H1N1-1918), a very short detector tile, and FLUAHA7, but neither of these sub-threshold results triggered a positive detection report.Lin et al (2009) recently reported another study of avian influenza viruses using RPM-Flu assays for 24 AH5N1-positive field and clinical specimens collected by the NAMRU-3 group from infected birds and humans The RPM-Flu assay-generated HA5 gene sequences of all the A/H5N1-positive specimens from NAMRU-3 and also of the A/H5N2 reference strain specimen from SEPRL , USDA_17The hemagglutinin RPM-Flu assay-generated HA5 gene sequence for the A/H5N3 reference strain , USDA_14A/duck/Singapore/3/1997(H5N3) -PQRE----TR/GLF- (LP)A/Viet Nam/1203/2004 (H5N1) -PQRERRRKKR/GLF- (HP)These results demonstrate efficacy of RPM-Flu for unequivocal diagnostic detection and identification of human and avian influenza virus subtypes, based upon direct determination of specimen-specific sequences of the hemagglutinin and neuraminidase genes. The two negative control specimens, USDA_13 and USDA_16, demonstrated the capability of the RPM-Flu assay to identify alternative pathogens to avian influenza virus as components of a more complete differential diagnosis assay for cases of avian influenza-like illness.The results presented in BLAST analysis of the M gene sequences from the 19 avian influenza specimens returned 79 most similar sequence records, of which 40 (51%) were associated with different A/HN subgroups than determined from the corresponding specimens' HA and NA gene sequences. For five of the 19 specimens there were no most similar M gene sequences returned to match the specimen's actual A/HN subtype .BLAST analysis of the NS gene sequences from the 19 avian influenza specimens returned 63 most similar sequence records, of which 41 (65%) were associated with different A/HN subgroups than determined from the corresponding specimens' HA and NA gene sequences. For five of the 19 specimens there were no most similar NS gene sequences returned to match the specimen's actual A/HN subtype .BLAST analysis of the PB2 gene sequences from the 19 avian influenza specimens returned 58 most similar sequence records, of which 22 (38%) were associated with different A/HN subgroups than determined from the corresponding specimens' HA and NA gene sequences. Nine of the 19 specimens had no most similar PB2 gene sequences from the same A/HN subtype .These results suggest little predicate value of most type A influenza virus M, NS or PB2 sequences for determination of the A/HN subtype of the strain from which these sequences were identified.We referred earlier to the complementary set of field and clinical samples  of type A avian influenza viruses provided by NAMRU-3  and analyzed at NRL using RPM-Flu Eight samples from this previously referenced set of clinical and field specimen isolates of avian influenza virus represented subtypes A/H10N7 (4), A/H7N7 (2), A/H11 (1) or A/H13 (1). In marked contrast to results from RPM-Flu assay-generated HA and NA gene sequences, there was poor correspondence of the actual specimen A/HN subtypes with the subtypes of sequence records returned from BLAST analysis of the assay-generated M, and NS gene sequences. Of 30 most similar M gene sequence records only 5 were from A/HN subtypes matching the subtypes of the corresponding specimens . None ofexclusively represented subtype A/H5N1 avian influenza viruses, as 255 M gene, 146 NS gene and 310 PB2 gene sequence records.Twenty-one of the remaining specimens of the set reported by Lin et al human influenza virus sequence records that are found to be most similar to RPM-Flu assay-generated M, NS and PB2 gene sequences from the corresponding types and subtypes. It is interesting to consider that these results may reflect the highly biased over-representation of samples A/H5N1 sequences in the genome sequence database(s), reflecting much more of the natural diversity of non-HA and non-NA gene sequences than in more sparsely sampled  avian influenza virus subtypes.This result for A/H5N1strains is reminiscent of the results presented earlier for subtypes A/H1N1 and A/H3N2 and type B directly distinguishes each of them among the 9 different A/HN subtype categories, or from the background of 144 combinatorial variations of influenza virus A/HN subtypes.Rapid and cost-effective microarray-based resequencing (RPM) of influenza virus hemagglutinin and neuraminidase genes provides sensitive detection and specific identification for types and subtypes of previously known as well as emergent strains and variants. These capabilities of the RPM platform stand out very favorably on comparisons with the most recently adopted diagnostic methodologies that are based on real-time reverse-transcription, polymerase chain reaction methods (RT-PCR). The FDA-cleared CDC rRT-PCR Flu Panel (510(k) 080570) enables detection and discrimination of influenza viruses having HA subtype components A/H1, A/H3 and A/H5, and also type B influenza virus. However the test provides no assurance that a positive test result correctly identifies one of the inferred target subtypes A/H1N1, A/H3N2 or A/H5N1; there is no neuraminidase subtype component evaluated as part of the test. Results presented in this report demonstrate that the TessArray RPM-Flu detect and identifies these three targeted subtypes of interest, but also 50) and 5.8 TCID50, respectively. These strains have not been serially passaged at high multiplicity in either egg or MDCK cell cultures, so it is not likely that accumulations of defective viral particles would favorably bias determination of sensitivity with genome-based assays relative to infectious doses. Furthermore, these two strains were not selected a priori for favorable match of viral sequences with oligonucleotide primers used for target gene sequence amplification. The observed RPM-Flu LoD for each strain is of similar order of magnitude as comparable estimates of analytical sensitivity reported from RT-PCR based methods, The RPM-Flu assay leverages a multiplexed RT-PCR-like strategy to rapidly (∼1 hour) amplify trace quantities of specific target gene sequences so that they may be detected if actually present in a specimen. Results from this report demonstrated that the 95% endpoint limits of detection (LoD) for seasonal A/California-NHRC/BRD10622N/2009(H1N1) and seasonal A/California-NHRC/BRD10601N/2009(H3N2) influenza virus strains were 2.8 infectious doses (TCIDEach RPM assay of a single specimen is capable of generation from hundreds to thousands of nucleotides of multiple targeted pathogen gene sequence(s). Gene sequences generated directly from RPM-Flu assays may specify determinants of particular target pathogen phenotypes , or support forensic queries of identity of detected strains in different specimens, or support genome diversity-based epidemiological tracking in the course of an infectious disease outbreak.RPM-Flu assay-generated nucleotide sequences representing one or more target genes of each detected pathogen convey significantly more informative specificity than can be obtained from any singlet or multiples of RT-PCR tests. Some RT-PCR protocols are designed to glean additional detail regarding detected pathogen genotypes, through results from multiple  RT-PCR panels, but this approach imposes additional testing costs and time delays and yield far less specific information than actual target gene sequences provided from a single RPM assay.Some platforms claim to differentiate selected type A influenza virus subtypes based upon indirect detection or specification of non-HA and non-NA viral gene sequences The RPM-Flu assay panel simultaneously addresses 30 different categories of viruses and bacteria that are more or less likely to be encountered in specimens related to respiratory infections . Each asFor comparison, the recently FDA-cleared Luminex® Respiratory Viral Panel (RVP) Multiplex Nucleic Acid Detection Assay Respiratory Virus Panel (510(k) K063765) is a twelve-plex RT-PCR panel for detection and differentiation of a few strains and types of respiratory viruses including adenovirus, influenza A virus (as A/H1 or A/H3), respiratory syncytial virus, parainfluenza virus, metapneumovirus and rhinovirus . The RVP assay yields no specimen-specific viral gene sequences that could be used for more detailed identification and differentiation of detected virus strains and variants of detected virus(es).For human and avian type A influenza viruses in particular, the RPM-Flu provides direct detection and identification capability for any of the combinatorial 144 A/HN subtypes that may be present in a specimen, using an ensemble of 39 different influenza virus target gene resequencing detectors. The Luminex RVP multiplex RT-PCR panel uses three of its twelve primer-probe sets to detect type A influenza virus (M gene-specific primer pair) and distinguish A/H1 from A/H3 (HA1- and HA3-specific primer pairs). While this RT-PCR panel may infer influenza virus subtype, not even testing for the NA component, the assay result is only based on a pattern of signals from a small panel of RT-PCR primer-probes.FACT SHEET FOR HEALTHCARE PROVIDERS: INTERPRETING CDC HUMAN INFLUENZA VIRUS REAL-TIME RT-PCR DETECTION AND CHARACTERIZATION PANEL FOR RESPIRATORY SPECIMENS  AND VIRAL CULTURE TEST RESULTS (Authorized by FDA 02 May 2009),Some RT-PCR protocols require serial analysis with hierarchical panels of primer-probes in order to expand the practical multiplicity of pathogen targets or variants. This is the case with the benchmark PCR testing used in this report –a type A influenza virus-positive specimen may be identified in a first round RT-PCR panel, followed by testing with another panel to distinguish A/H1 from A/H3. Failure to establish subtype A/H1 or A/H3 in the follow-on test is a first tier screening results for possible identification of the 2009 Novel A/H1N1 influenza virus outbreak strain. According to the NPS, NS, TS, NPS/TS, NA) should be ordered to diagnose influenza A infections caused by influenza A or B and subtype determination of seasonal human influenza A virus  to be used as a first tier test for the in vitro qualitative detection of novel influenza A (H1N1) virus in patients suspected of having novel influenza A (H1N1) infection. If the test result is positive for influenza A and negative for seasonal H1 and H3 subtypes, the laboratory should test the specimen with the Swine Influenza Real-time RT-PCR Detection Panel (rRT-PCR Swine Flu Panel) for the presumptive detection of novel influenza A (H1N1) infection.”“The rRT-PCR Flu Panel  at different laboratory venues over a period likely longer than a single day can be either efficient or cost-effective. Far more diagnostic intelligence about the patient and specimen would be available in first-day same-day results from a single RPM-Flu test of the original specimen.Influenza infections do not necessarily preclude co-infection of the same individual by other viral and/or bacterial pathogens (including other strains of influenza virus). Such co-infections may confound a simple diagnosis of influenza, and may also compound morbidity and mortality for a co-infected patient. However it is not common practice that a positive result from a routine diagnostic influenza test would be followed by a call for one or more tests of possible secondary agents of infection. The single specimen single aliquot RPM-Flu assay simultaneously targets 30 different categories of viruses and bacteria, and these were each selected as a respiratory pathogens reported to cause “flu-like” symptoms at some stage of human infection.Implementation of a highly multiplexed differential diagnostic assay as RPM-Flu would be of great advantage in anticipation of highly likely future influenza outbreaks, epidemics and pandemics. For many decades, leaders in the area of influenza epidemic and pandemic risk assessment and outbreak management have reviewed the pathology of influenza with respect to rapid deterioration and patient deaths attributable to secondary bacterial infections and pneumonia twenty-five years from the first research reports of the polymerase chain reaction It has taken almost Table S1Analysis of 2009–2010 FluMist live virus trivalent vaccine as comparisons of Influenza type A and type B detector tile sequences to detection and identification of RPM-Flu assay-generated gene sequences. The strains configured in this vaccine are A/South Dakota/06/2007(H1N1), A/Uruguay/716/2007(H3N2) and B/Brisbane/60/2008. The vaccine matrix genes and other non-HA, non-NA genes are from master donor strains A/Ann Arbor/6/1960(H2N2) and B/Ann Arbor/1/66.(0.06 MB DOC)Click here for additional data file.Table S2Analysis of 2009–2010 Fluvirin inactivated virus trivalent vaccine as comparisons of Influenza type A and type B detector tile sequences to detection and identification of RPM-Flu assay-generated gene sequences. The strains configured in this vaccine are A/Brisbane/59/2007, IVR-148 (H1N1), A/Uruguay/716/2007(H3N2) and B/Brisbane/60/2008. The type A virus subtypes of the inactivated vaccines have matrix genes and other non-HA, non-NA genes derived from the master donor strain A/Puerto Rico/8/1934 (H1N1).(0.07 MB DOC)Click here for additional data file.Table S3Analysis of 2005–2006 inactivated virus trivalent vaccine as comparisons of Influenza type A and type B detector tile sequences to detection and identification of RPM-Flu assay-generated gene sequences. The strains configured in this vaccine are A/New Caledonia/20/99 (H1N1), A/New York/55/2004(H3N2) and B/Jiangsu/10/2003. The type A virus subtypes of the inactivated vaccines have matrix genes and other non-HA, non-NA genes derived from the master donor strain A/Puerto Rico/8/1934 (H1N1). RPM-Flu detector title prototype sequences C3 Score BLAST E-value SNPsa Most similar sequence records from BLAST/GenBank include: Hemagglutinin genes A/New Caldedonia/20/1999 (H1N1) 62.3 1e-180 4/934 A/New Caldedonia/20/1999 A/Canterbury/125/2005 (H3N2) 84.6 1e-180 11/1269 A/New York/55/2004 B/Malaysia/2506/2004 25.2 2e-89 23/226 B/Jilin/20/2003 b B/Shanghai/361/2002 55.8 1e-180 20/502 B/Jiangsu/10/2003 b Neuraminidase genes A/New Caldedonia/20/1999 (H1N1) 51.6 1e-180 5/619 A/New Caldedonia/20/1999 A/Canterbury/125/2005 (H3N2) 89.8 1e-180 2/1077 A/New York/55/2004 B/Malaysia/2506/2004 55.2 1e-180 21/662 B/Jiangsu/10/2003 Matrix genes A/Canterbury/100/2000 (H1N1) 54.1 1e-180 24/459 A/Puerto Rico/8/1934(H1N1) A/Canterbury/125/2005 (H3N2) 52.4 1e-180 29/445 A/Puerto Rico/8/1934(H1N1) B/Memphis/13/2003 91.6 1e-180 14/870 B/Jiangsu/10/2003 a SNPs are single base call discrepancies between detector tile sequence and assay generated sequence from labeled target DNA. The number of detected SNPs is shown relative to the number of bases called from the detector tile as contiguous runs of three or more base calls. b The B/Jilin/20/2003 and B/Jiangsu/10/2003 strains are equivalent B/Shanghai/361/2002-like strains as also used in 2004–2005 vaccine configurations see .(0.06 MB DOC)Click here for additional data file.Table S4Analysis of 2006–2007 inactivated virus trivalent vaccine as comparisons of Influenza type A and type B detector tile sequences to detection and identification of RPM-Flu assay-generated gene sequences. The strains configured in this vaccine are A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/2005(H3N2) and B/Malaysia/2506/2004. The type A virus subtypes of the inactivated vaccines have matrix genes and other non-HA, non-NA genes derived from the master donor strain A/Puerto Rico/8/1934 (H1N1).(0.06 MB DOC)Click here for additional data file.Table S5RPM-Flu assay detection and identification of avian influenza viruses in field and clinical specimens obtained from human and avian hosts.(0.11 MB DOC)Click here for additional data file.Table S6The A/HN subtypes associated with most similar sequence records for RPM-Flu assay-generated M gene sequences from 19 type A avian influenza viruses are not reliable indicators of the actual A/HN subtype. Forty of 79 (51%) most similar M gene sequence records are associated with different A/HN subtypes than independently determined from each specimen's specific HA and NA gene sequences (mismatches for 8 of 19 specimens).(0.07 MB DOC)Click here for additional data file.Table S7The A/HN subtypes associated with most similar sequence records for RPM-Flu assay-generated NS gene sequences from 19 type A avian influenza viruses are not reliable indicators of the actual A/HN subtype. Forty-one of 63 (65%) most similar NS gene sequence records are associated with different A/HN subtypes than independently determined from each specimen's specific HA and NA gene sequences (mismatches for 11 of 19 specimens).(0.07 MB DOC)Click here for additional data file.Table S8The A/HN subtypes associated with most similar sequence records for RPM-Flu assay-generated PB2 gene sequences from 19 type A avian influenza viruses are not reliable indicators of the actual A/HN subtype. Twenty-two of 58 (38%) most similar PB2 gene sequence records are associated with different A/HN subtypes than independently determined from each specimen's specific HA and NA gene sequences (mismatches for 12 of 19 specimens).(0.07 MB DOC)Click here for additional data file.Figure S1All combinatorial subtypes of type A influenza virus strains and 30 different viral and bacterial pathogens causing influenza-like illness as represented on the RPM-Flu 3.1 resequencing pathogen microarray.(0.49 MB TIF)Click here for additional data file.


The current pandemic (H1N1) 2009 virus remains transmissible among humans worldwide with cases of reverse zoonosis, providing opportunities to produce more pathogenic variants which could pose greater human health concerns. To investigate whether recent seasonal human or swine H1N1 vaccines could induce cross-reactive immune responses against infection with the pandemic (H1N1) 2009 virus, mice, ferrets or mini-pigs were administered with various regimens (once or twice) and antigen content  of a-Brsibane/59/07, a-CAN01/04 or RgCA/04/09xPR8 vaccine. Receipt of a-CAN01/04 (2-doses) but not a-Brisbane/59/07 induced detectable but modest (20–40 units) cross-reactive serum antibody against CA/04/09 by hemagglutinin inhibition (HI) assays in mice. Only double administration (7.5 µg HA) of both vaccine in ferrets could elicit cross-reactivity (30–60 HI titers). Similar antigen content of a-CAN01/04 in mini-pigs also caused a modest ∼30 HI titers (twice vaccinated). However, vaccine-induced antibody titers could not suppress active virus replication in the lungs (mice) or virus shedding (ferrets and pigs) of immunized hosts intranasally challenged with CA/04/09. Furthermore, neither ferrets nor swine could abrogate aerosol transmission of the virus into naïve contact animals. Altogether, these results suggest that neither recent human nor animal H1N1 vaccine could provide complete protectivity in all animal models. Thus, this study warrants the need for strain-specific vaccines that could yield the optimal protection desired for humans and/or animals. Influenza A virus is the cause of recurrent influenza epidemics and from time to time, global pandemics. In the past century, the world had experienced three devastating influenza pandemics which claimed hundreds of thousands to millions of lives globally: Spanish Flu , Asian Flu , and Hong Kong Flu  Detailed genomic sequence analysis of the pandemic (H1N1) 2009 virus reveals that it contains unique reassortment of genes that are of swine origin Vaccination is the primary measure to control influenza virus infections which come in two forms: inactivated or live-attenuated vaccine. Annually updated influenza virus vaccines typically contain three influenza viruses : one A (H3N2) virus, one A (H1N1) and one B virus as chosen by the WHO Global Influenza Surveillance Network In the present study, we made use of mice, mini-pigs, and ferret animal models to assess the immunogenicity, protective efficacy, and cross-reactivity of various regimens of vaccination with inactivated whole-virus vaccines intended for humans (a-Brisbane/59/07) or for swine (a-CAN01/04). Results were compared to data obtained with RgCA/04/09xPR8 immunization, a reverse genetics-generated vaccine. Immunogenicity and cross-reactivity of the vaccines were evaluated by hemeagglutination assays (HI) while cross-protection in vaccinated animals were determined by challenge with the A/California/04/2009 virus. We report here and provide evidence of the inability of recent human and animal influenza A/H1N1 vaccines to provide complete protection, including inhibition of virus replication and transmission, among vaccinated mammalian hosts.Serum specimens collected from children and adults that were immunized with recent seasonal influenza vaccines suggest that receipt of such vaccines is unlikely to elicit protective antibody immune response to the pandemic (H1N1) 2009 virus 5 50% tissue culture infective dose per milliliter (TCID50/ml) of the wild type CA/04/09 virus and monitored active virus replication in the lungs. A group of mock-vaccinated mice which only received phosphate-buffered saline (PBS) was included as control. Mice lungs were collected at 2, 4, 6, and 8 days post infection (dpi) (3 heads per day) for virus titration in 11-day-old embryonated chicken eggs. Experimentally challenged mice manifested clinical signs with influenza-like disease  which was more prominent in mock-vaccinated and single-shot groups. Mock-vaccinated mice produced high lung titers initially detected at 2 dpi and persisting up to 8 dpi . Compared to a-Brisbane/59/07, RgCA/04/09xPR8 demonstrated the most efficient suppression of active virus growth in mice lungs in all of the vaccine regimens. Essentially, the pandemic (H1N1) 2009 virus was cleared from the lungs as early as 6 dpi in two-shot regimens with antigen containing as low as 1.77 µg/dose. Virus clearance was delayed in single shot groups of the reverse genetics vaccine (at 8 dpi) (10 EID50 lower). However, viral titers are still considerably higher compared to groups that received the RgCA/04/09xPR8. These results suggest that a-Brisbane/59/07 could not induce cross-reactive antibodies, sufficient to provide protection against infection or abrogate replication of the heterologous pandemic (H1N1) 2009 virus in mice lungs.To investigate the protective efficacy of the vaccine, immunized mice were intranasally (i.n.) challenged with 30ul of 10t 8 dpi) . In contFerrets are broadly accepted as suitable animal models for influenza virus infection and transmission in humans 5 TCID50 two weeks after the last vaccination. Nasal wash specimens were collected from experimental animals on 2, 4, 5, 6, 7, and 8 dpi to examine the ability of the vaccines to hinder virus replication in the upper respiratory tract. To monitor transmission of the challenge virus from vaccinated to naïve animals through the air, two seronegative ferrets were added at 1 dpi in the same isolator constructed with perforated dividers preventing direct or indirect contact (5 centimeter distance) but could allow virus spread by aerosol transmission from infected ferrets. Elevated body temperatures were noted in a-Brisbane/59/07-immunized ferrets but not in RgCA/04/09xPR8 group from 1 to 4 dpi (39–40°C) (10 EID50 difference) indicating unhindered active replication in the upper respiratory tract. The higher nasal titers from seasonal human vaccine recipients were also accompanied by prolonged virus shedding up to 5 dpi from initial virus detection and successful virus transmission at 2 days of post contact (dpc)  2009 virus, ferrets vaccinated twice with 7.5 µg/doses HA of a-Brisbane/59/07 or RgCA/04/09xPR8 were subjected to virus challenge experiments. Two-dose vaccinated hosts received CA/04/09 virus challenge at titers 1039–40°C)  with somct (dpc) . Interes10 EID50 difference) were obtained compared to the mock-vaccinated group, a pattern similarly observed in seasonal human H1N1 vaccine recipients (The current pandemic (H1N1) 2009 virus had been traced to have originated from pigs cipients .10 EID50 at 2 dpi persisting up to 5 dpi (10 EID50).Among a-CAN01/04 vaccinated ferrets, receipt of two doses  elicited relatively high HI response (∼60 HI units) to the pandemic (H1N1) 2009 virus . In contto 5 dpi . This suPigs have demonstrated that they are susceptible to the pandemic (H1N1) 2009 virus under experimental settings 5 TCID50/ml two weeks after the last vaccination. Nasal swab specimens were collected from experimental animals on 2, 4, 5, 6, 7, and 8 dpi to examine the ability of the vaccine to impede virus replication in the upper respiratory tract. Except for loss of appetite, no other remarkable sign of disease or changes in body temperature was observed in all vaccinated swine (10 EID50) were obtained from a-CAN01/04-vacccinated animals that lasted up to 5 dpi compared to the RgCA/04/09xPR8 group (10 EID50). In contrast, RgCA/04/09xPR8-vaccine group was able to suppress aerosol transmission of the CA/04/09 virus, consistent to data obtained in ferrets (5 TCID50/ml of CAN01/04 as the test virus reversed the results. The a-CAN01/04 vaccine recipients blocked the transmission of the swine H1N1 virus (CAN01/04) which the RgCA/04/09xPR8 antigen failed to suppress  at 5 dpi. Gross lesions of a-CAN01/04-vaccinated mini-pigs already indicated apparent signs of regeneration. Both lungs are rather non-collapsed although there were diffused consolidation of cranial lobes and multifocal indications of bronchointerstitial pneumonia scattered through the accessory and caudal lobes . MinimalSince the causative pandemic (H1N1) 2009 influenza virus is considered a novel strain, it appears that currently available human influenza virus vaccines could not elicit cross-reactive antibodies to the current pandemic virus The Brisbane/59/07 virus is one of the recommended vaccine seed strain by the WHO against seasonal human H1N1 virus infections in the southern (2009 influenza season) and northern (2008–2009 and 2009–2010 seasons) hemispheres. On the other hand, the prototype swine H1N1 vaccine (a-CAN01/04) was prepared from a Korean swine virus isolated in 2004 10 EID50) were obtained in swine H1N1-vaccinated mice and ferrets than in seasonal human H1N1-vaccinated hosts. When the a-CAN01/04 was further tested in mini-pigs, it can only induce limited cross-reactive immunogenicity against the CA/04/09 virus  cells, administration of the reverse-genetics vaccine demonstrated efficient virus neutralizing activity which correlated comparably well with the vaccine-induced HI responses (data not shown). In contrast, neutralizing activities to the CA/04/09 virus was barely detected in either of the seasonal human or swine H1N1 vaccine recipients confirming poor cross-protection. However, this result is not so surprising due to identical antigenic match between the challenge and vaccine virus, but rather emphasizes the need for strain-specific vaccines. Sustained human-to-human transmission is a key requirement for pandemics and could result in the genesis of more pathogenic variants, as what happened with the 1918 pandemic virus (as reviewed in Reference 25). In response to live virus challenge, this important feature was considerably countered by the receipt of RgCA/04/09xPR8 in ferrets and pigs: virus growth and shedding in experimentally infected animals was limited which in turn reduced the chance of transmission of the test virus  . When imst virus  and 4. CReceipt of either trivalent inactivated vaccine (TIV) or live attenuated influenza vaccine (LAIV) is the currently acceptable immunization strategies for the prevention and control of influenza infection. Although both types of vaccine are effective, the use of LAIV is considered more effective for its potential to induce broader and more durable protection against influenza  In summary, we report that contemporary human seasonal and veterinary H1N1 vaccines are unlikely to induce immunologic responses that could inhibit growth or transmission of the current pandemic (H1N1) 2009 virus. Undeterred efficient dissemination of the pandemic (H1N1) 2009 virus among humans could allow opportunities to acquire adaptive mutations producing more pathogenic variants. Alternatively, establishment in a new host could also facilitate the production of progeny viruses with deleterious consequences of unknown magnitude. Despite the increasing number of countries reporting animal infections, most notably among swine herds All experiments involving animal subjects were conducted in strict accordance and adherence to relevant national and international guidelines regarding animal handling as mandated by the Animal Use and Care by Laboratory Animal Research Center (LARC) in Chungbuk National University, a member of the International Animal Care and Usage Committee (IACUC), and in Bioleaders Corp.50) were determined by infection in Madin-Darby canine kidney cells calculated by the method of Reed and Muench The human pandemic H1N1 virus, CA/04/09, was obtained from St. Jude Research Hospital, USA. CAN01/04 (H1N1) is a recent swine influenza virus strain isolated from a Korean swine farm in 2004 The reverse genetics RNA transcription vector, pHW2000, and eight plasmids containing the cDNAs of influenza A/Puerto Rico/8/34 (H1N1) (PR8) were kindly provided by Dr. Robert G. Webster. The RgCA/04/09xPR8 reassortant virus containing the HA and NA genes CA/04/09 in the background of PR8 was generated by plasmid-based reverse genetics as described previously The CAN01/04, Brisbane/59/07, and RgCA/04/09xPR8 viruses were propagated in the allantoic fluid of a 10-day old embryonated chicken egg and purified by ultracentrifugation through a 25% and 70% sucrose cushion, at 30,000 X g in 4°C for 3 hours, as described previously 5 TCID50 per milliliter of the CA/04/09 virus. Mini-pigs (n = 2 per group) and ferrets (n = 4 per group) were vaccinated intramuscularly with one or two doses each of inactivated vaccines that contain 7.5 µg/dose of HA with 2% of aluminum hydroxide adjuvant, administered 2 weeks apart. Intranasal instillation of 105 TCID50 CA/04/09 virus challenge in a 1.0 ml volume (divided between two plastic syringes for separate inoculation of each nostril) was done after 2 weeks of receiving their last immunization. For transmission studies in ferrets and mini-pigs, animals (n = 2) were co-housed in adjacent transmission cages fitted in the same isolator (at a distance of 5 centimeters apart) that prevented direct or indirect animal contact but allowed influenza virus spread through aerosol contact from experimentally infected animals. All viruses and animal experiments including serologic testing were handled in a BSL 3+ containment facility approved by the Korea Centers for Disease Control and Prevention.Four-week-old BALB/c mice were obtained from Samtaco , eight-week-old specific-pathogen-free (SPF) outbred miniature pigs were from PWG Genetics Korea, Ltd , and 15- to 16-week-old ferrets were purchased from Marshall Bio Resources . All animals were seronegative for influenza A viruses by serologic assay. Mice (12 heads per group) were vaccinated intramuscularly with 1 or 2 doses of inactivated vaccines containing 1.77 or 3.5 µg/dose of HA containing 2% of aluminum hydroxide adjuvant in 200 ul volume, administered 2 weeks apart. Two weeks after the last immunization, mice were i.n. challenged with 30 µl 10Sera from mice, mini-pigs, and ferrets were collected after 2 weeks of receiving their last vaccination, respectively, and stored at −82°C until use.10 50% egg infective dose per milliliter or gram of tissue collected (EID50/mL or EID50/g) 10 EID50/mL. Lungs of infected and contact pigs (one head per group) were harvested at 5 dpi for gross histopathological examination.Lung tissue samples of mice were collected at 2, 4, 6, and 8 dpi and homogenized in 1X phosphate-buffered saline (PBS) containing antibiotics. Tissue homogenates were clarified by centrifugation at 12,000 g and supernatants were transferred to new tubes. Nasal washes (ferrets) and swabs (mini-pigs) were collected in 1X PBS with antibiotics after days 2 to 8 of virus challenge. All samples were immediately serially diluted 10-fold and then inoculated into 11-day-old embryonated chicken eggs for virus titration as computed by the Reed and Muench method with results expressed as log50 of virus after incubation at 37°C for 72 h Hemagglutinin inhibition (HI) assays were done as described elsewhere


We have characterized and analyzed the efficiency and kinetics of this inducible E2AER system in the context of B cell development.E-proteins are transcription factors important for the development of a variety of cell types, including neural, muscle and lymphocytes of the immune system. E2A, the best characterized E-protein family member in mammals, has been shown to have stage specific roles in cell differentiation, lineage commitment, proliferation, and survival. However, due to the complexity of E2A function, it is often difficult to separate these roles using conventional genetic approaches. Here, we have developed a new genetic model for reversible control of E2A protein activity at physiological levels. This system was created by inserting a tamoxifen-responsive region of the estrogen receptor (ER) at the carboxyl end of the ER/ER mice demonstrated this predicted block in B cell development, and E2AER DNA binding activity was not detected in the absence of ligand. In vitro studies verified rapid induction of E2AER DNA binding activity upon tamoxifen treatment. While tamoxifen treatment of E2AER/ER mice showed inefficient rescue of B cell development in live animals, direct exposure of bone marrow cells to tamoxifen in an ex vivo culture was sufficient to rescue and support early B cell development from the pre-proB cell stage.B cell development has been shown previously to be blocked at an early stage in E2A deficient animals. Our E2AER system provides inducible and reversible regulation of E2A function at the protein level. Many previous studies have utilized over-expression systems to induce E2A function, which are complicated by the toxicity often resulting from high levels of E2A. The E2AER model instead restores E2A activity at an endogenous level and in addition, allows for tight regulation of the timing of induction. These features make our E2AER ex vivo culture system attractive to study both immediate and gradual downstream E2A-mediated events.The E2A Origina systems . E2A parE-proteins, including E2A, HEB, and E2-2 in mammals, function as dimers to bind DNA and regulate gene expression. E2A homodimers are the primary E-protein dimers functioning in B cells, whereas E2A-HEB heterodimers are the primary dimers functioning in T cells -6. Recentcfe2a, resulting in the production of E2AER fusion proteins. The use of tamoxifen inducible ER fusion proteins in mouse genetics has already been established as a valuable tool, especially with the vast use of the Cre recombinase-ER fusion protein for inducible gene knockout in mice [in vitro study of MyoD gene regulation [in vivo post transplantation of transduced cells into recipient animals [ER system now introduces an ER fusion with a bHLH transcription factor into the mouse genome for analysis of an endogenously expressed protein.Here we have developed a new genetic model to examine E2A function. We have established an inducible E2A mouse model by inserting a tamoxifen-responsive region of the estrogen receptor (ER) ligand binding domain at the carboxyl end of  in mice -15. In agulation  and in m animals . HoweverER/+ and E2AER/ER mice, along with E2AER protein function, are analyzed here in the context of B cell development. A block in B cell development at the pre-proB cell stage, prior to B lineage commitment, has been characterized by previous E2A-deficient mouse models [ER/ER pre-proB cells upon tamoxifen treatment would be a stringent test to verify inducible E2AER function.E2Ae models ,19. A reER system. E2AER protein activity was rapidly induced upon tamoxifen treatment and reversibly regulated by tamoxifen withdrawal. Even though we show effective induction of E2AER DNA binding activity, tamoxifen treatment of E2AER/ER mice did not efficiently restore B cells in vivo. However, tamoxifen treatment was able to rescue and support early B cell development from E2AER/ER pre-proB cells in an ex vivo culture system. Use of E2AER ex vivo culture systems may therefore be beneficial for the study of gene regulation in B cells and other E2A-regulated cell lineages.In this study, we provide the initial characterization of the E2AER/+ and E2AER/ER mice were generated by using a knock-in strategy for tamoxifen-inducible E2A function. The tamoxifen-responsive ligand binding domain of the mouse ER [tcfe2a to produce the E2AER allele . E2AER/ER animals also exhibit stunted growth and reduced survival  in the bone marrow through a series of developmental stages . The preER/ER mice revealed a block at the pre-proB cell stage, similar to that seen in the E2A-mutant E2AE47bm/E47bm mice J recombination, were used as negative controls. Analysis of Day 8 cultures demonstrated a clear V to DJ rearrangement product from tamoxifen treated E2AER/ER cells  ,19. We ne family , which rs Figure . V to DJER system provides an improved method for analyzing E2A function. Previous analysis using over-expression of E2AER fusion proteins by retroviral transduction has already demonstrated the value of inducible E2A activity [ER allele provides an attractive system for studying gene regulation and other E2A-regulated events in potentially any E2A-expressing cell type at a more physiological level.The E2Aactivity ,31-34. Hactivity ,35, and ex vivo culture system demonstrated a rescue from the pre-proB cell stage, but suggested delayed kinetics from E2AER/ER pre-proB cells compared to B cell development from control pre-proB cells. This result suggests that even though E2AER/ER and wild-type pre-proB cells are phenotypically similar, E2AER/ER pre-proB cells may not have equivalent developmental potential or may require additional cellular changes prior to entering the B cell lineage. It is possible that E2A-regulated events normally occurring earlier in development, as suggested in hematopoietic stem cell (HSC), lymphoid-primed multipotent progenitor (LMPP), and common lymphoid progenitor (CLP) stages [ER/ER B cells may not have expanded as rapidly as wild-type cells upon commitment to the B cell lineage. Our Western Blot analysis of E2A proteins in E2AER/+ Abelson cells  stages ,36,37, ws Figure  suggesteex vivo culture data clearly indicated a rescue of early B cell development to the proB cell stage. However, we have so far been unable to determine if tamoxifen treatment can rescue E2AER/ER B cells through the subsequent preB cell stage, when E2A is known to be critical for Ig kappa light chain (Igk) recombination [ex vivo culture system because this system primarily supports development at a stage prior to these events  or FACSVantage SE with DiVa option (BD Biosciences) and FlowJo software (Tree Star). FACSVantage SE with DiVa option was used for cell sorting.For sorting, bone marrow was harvested and pooled from 2-3 mice per genotype. Cells positive for lineage markers Mac-1, Gr-1, Ter-119, and CD3 were depleted with Dynal Dynabeads (Invitrogen) according to manufacturer's instructions. Dead cells stained with 7-aminoactinomycin D  were excluded. Pre-proB cells were further distinguished as B220Tamoxifen (Sigma) was prepared as a 1 mM stock (1000×) dissolved in cell culture grade dimethyl sulfoxide (DMSO) and stored at -20°C.ER/ER Abelson preB cell line was derived by Abelson Murine Leukemia Virus transformation of bone marrow cells from an E2AER/ER mouse. Briefly, whole bone marrow was plated on an S17 stromal layer in the presence of 1 uM tamoxifen and 10 ng/mL IL-7 in 5% FBS RMPI media. This culture was performed prior to transduction to ensure cells were proliferating and were at the optimal target stage for Abelson transformation. Once an expanding B cell population was observed, cells were infected with Abelson virus in the presence of 4 ug/mL polybrene. Abelson transformed cells were then removed from the stromal layer, and tamoxifen and IL-7 were withdrawn. The established E2AER/ER Abelson preB cell line was maintained in 10% FBS RPMI media  prior to experimental analysis. Additional Abelson lines were established as described previously [The E2Aeviously .Abelson preB cell lines were cultured without tamoxifen or with 1 uM tamoxifen for 24 hr prior to analysis. Cells were lysed in RIPA lysis buffer  with protease inhibitors. Whole cell lysates were resolved by SDS-PAGE and blotted with anti-E2A  and anti-ERK2  antibodies.ER/ER Abelson preB cells were cultured with or without 1 uM tamoxifen as indicated. For withdrawal analysis, tamoxifen-treated cells were washed and re-plated in the absence of tamoxifen for the indicated times. Nuclear extracts were incubated with a 32P-labeled μE5 oligonucleotide probe, with or without Yae anti-E2A monoclonal antibody , and resolved on a 5% polyacrylamide gel. Gels were dried and exposed to a phosphor screen for phosphorimager analysis (Amersham Biosciences). Oligos used for μE5 probe: 5'-TCGAAGAACACCTGCAGCAGCT-3' and 5'-TAGAGCTGCTGCAGGTGTTCTT-3'.E2AMice were treated with tamoxifen in the drinking water for 27 days. A 68 mg/mL tamoxifen in ethanol stock was used to bring the concentration in drinking water to approximately 26 ug/mL, resulting in 0.04% ethanol in water. A fresh bottle of tamoxifen water was given every 5 days.4 cells per well and cultured with 5% FBS RPMI hormone-free media containing 10 ng/mL IL-7. Hormone free media consisted of phenol-red free RPMI 1640 supplemented with 5% charcoal/dextran treated FBS (Hyclone), 100 units/mL penicillin, 100 ug/mL streptomycin, and 55 uM 2-mercaptoethanol. Treated wells contained 1 uM tamoxifen and untreated controls were given DMSO alone (0.1%). Cells received fresh media, cytokine, and tamoxifen or DMSO every other day. Cells were harvested at time points indicated, and samples were split in half for FACS analysis and RNA or DNA extraction.Sorted pre-proB cells were plated on an S17 stromal layer in 24-well plates at approximately 1.5 × 10ex vivo cultured as described above. DNA was extracted from Day 8 cultures and then analyzed for IgH V to DJ rearrangements using VH1 gene family and JH4 specific primers. A nested PCR strategy was used to amplify rearrangements involving VH1 family gene segments. The following primers were used for Round 1: VH1 ext 5'-AGRTYCAGCTGCARCAGTCT-3' [H4 YZB6 5'-TCCCTCAAATGAGCCTCCAAAGTCC-3' [H1 int 5'-GARGATRTCCTGYAAGGCTTC -3' [H4 YZB5 5'-CCTGAGGAGACGGTGACTGAGGTTCCTTG-3'[H1-DHJH4 PCR products were cloned into a pCR4 TOPO vector (Invitrogen) and sequenced . Rearrangement product sequences were analyzed by SoDA [Sorted pre-proB cells were AGTCT-3'  and JH4 AGTCC-3'  and for CTTC -3'  and JH4 TCCTTG-3'. CD14 prTCCTTG-3'. VH1-DHJ by SoDA .MEJ participated in the design of the study, carried out the studies, and drafted the manuscript. MK participated in the design of the study and assisted with cell sorting. YZ conceived of the study and participated in its design. All authors read and approved the final manuscript.Characterization of E2A gene-targeted mice. Phenotypes are descriptive of homozygous animals.Click here for fileUp-regulation of E2A protein levels during pre-proB to proB cell stage development. (A) E2AGFP allele. An E2A-GFP fusion protein is produced from this allele. Therefore, GFP expression can be used to monitor E2A protein levels. (B) E2AGFP/GFP and E2A+/+ control bone marrow was stained for B220, CD43, and CD19 surface expression. Cells are pre-gated on lymphocytes. GFP expression is shown for E2A+/+ B220+CD43+ (pre-proB + proB) control cells, and E2AGFP/GFP pre-proB (B220+CD43+CD19-) and proB (B220+CD43+CD19+) compartments.Click here for fileIn vivo BrdU labeling suggests similar expansion of E2AER/ER pre-proB cells compared to E2A+/+ pre-proB cells. Two mice from each genotype, E2AER/ER and E2A+/+ control, were IP injected with 1 mg BrdU and analyzed 4 hrs post injection. Bone marrow was stained for B220, CD43, and CD19 surface expression, then processed to analyze BrdU labeling with a FITC BrdU Flow Kit as per manufacturer's instructions (BD Pharmingen). All plots are pre-gated on lymphocytes. Graphs display percent BrdU+ cells within the pre-proB population (B220+CD43+CD19-) as labeled. An E2AER/ER mouse receiving no BrdU injection was used as a negative control (plot displays pre-proB cells). The proB population (B220+CD43+CD19+) from BrdU injected E2A+/+ #2 is shown as a positive control for BrdU incorporation.Click here for fileSequencing analysis of IgH V-DJ rearrangements. PCR products from the IgH V to DJ rearrangement analysis in Figure +/+ (WT) tamoxifen treated, and E2AER/ER (ER) DMSO treated and tamoxifen treated. The total numbers of colonies sequenced per sample are shown. Sequences encoding VH1 to DHJH4 rearrangement products were determined. Unique rearrangement products were defined as products using a unique set of V, D, and J gene segments or containing a unique number of nucleotide additions/deletions. The average length of unique rearrangement products from the internal (Round 2) VH1 and JH4 primers is also shown for each sample analyzed.Click here for filePhenotype of cultures utilized for IgH V-DJ rearrangement. Staining of Day 8 cultures for CD19 and IgM expression. Cells are pre-gated on 7AAD-B220+ lymphocytes. Relative percentages are displayed.Click here for file


Alpha 2 Macroglobulin family members have been studied extensively with respect to their roles in physiology and human disease including innate immunity and Alzheimer's disease, but little is known about a possible role in liver development loss-of-function in model systems.α2 macroglobulin-like (A2ML) gene and its specific expression in the liver during differentiation. Morpholino-based knock-down of A2ML did not block the initial formation of the liver primordium, but inhibited liver growth and differentiation.We report the isolation of the zebrafish This report on A2ML function in zebrafish development provides the first evidence for a specific role of an A2M family gene in liver formation during early embryogenesis in a vertebrate. Xenopus laevis, the A2M-related genes endodermin (EDD) and Panza are expressed in liver and in other endodermal derivatives alpha-2 macroglobulin-like (A2ML) gene, and show that it has a role in liver development in this organism. Morpholino-based knock-down of A2ML does not block the initial formation of the liver primordium, but growth and development of the liver is disrupted largely by reduced cell proliferation, without observable defects in pancreas and intestine development. Thus we present evidence for the role of an A2M family gene in liver formation during embryogenesis.The α2 macroglobulin (A2M) family includes conserved blood proteins with multiple functions, especially as protease inhibitors α2-macroglobulin like gene (A2ML) was listed under GenBank accession number BC125959 (IMAGE: 6996802), and is localized on chromosome 15 in the Sanger Center Zebrafish Genome Data base (Scaffold Zv7_scaffold1487 http://www.ensembl.org/Danio_rerio/). To extend this sequence we performed the RACE procedure, obtaining full-length A2ML cDNA (accession number EU689051). Zebrafish A2ML contains three domains that are conserved in the vertebrate A2M family  beneath the dorsal shield in the zebrafish gastrula, as verified by two color in situ hybridization with vent as a ventral marker A2ML was confirmed by RT-PCR transferrin a (tfa)A2ML in the liver starts around 3.5 days post fertilization (dpf)  , and conon (dpf) , while ton (dpf) . By 7 dpf) , similar to embryos injected with cont MO  tfa, and foxa3A2ML MO, while the pancreatic primordium is normal. Specificity of this MO was tested using an A2ML-GFP fusion construct, whose expression is effectively blocked by the morpholino (hhex and cp expression at 24 hpf and detected no changes in liver and pancreatic primodium development (data not shown). These results indicate that the development of the liver and pancreas is initiated normally in A2ML ATG MO-injected embryos, but that liver formation begins to be affected after 2 days of development.To study A2ML function in development, we used two anti-sense morpholinos (MO) to inhibit its expression. One MO is complementary to the 5′-UTR (UTR MO) while the other targets the translation start area (ATG MO). n =  33) . Becauserpholino . We alsoA2ML expression led to specific deficits in liver formation that became more pronounced with time, without causing widespread developmental defects. These results were confirmed with the aid of the transgenic line Tg(XlEef1a1:GFP) that allows visualization of intestinal organs A2ML mRNA tfa were greatly reduced after injection of A2ML UTR or ATG MO (cp (data not shown). The liver and pancreas form as protrusions from the gut primordium during early development This effect on liver formation was visualized with the aid of molecular markers at five to six dpf in We also monitored possible effects of A2ML MO injection on blood vessel formation in the liver by inspecting fliGFP transgenic animals, and found no substantial defect in blood vessel formation .A2ML MOs affect the formation of the gut and pancreas with the aid of specific markers for these tissues. Using the fabp2 as a marker A2ML MO-injected embryos islet1 (isl1)A2ML MO injected embryos s854/+, was obtained from Zebrafish International Resource Center (ZIRC. http://zebrafish.org/zirc/home/guide.php) and fli-gfp transgenic zebrafish were obtained from Dr. Brant Weinstein (NICHD/NIH).The A2ML sequence was completed by 5′- and 3′-RACE (BD Biosciences Clontech), using 5′-GCAGCGGTGAAACTCTTGCTCAGCATTC-3′ (5′-RACE) and 5′-CTTCACTGTCAAATACGATG GGCCAGAG-3′ (3′-RACE). Subcloning into pCS2+ used primers GAATTCGTCATCATGGCTCTGAA TGTTAGC5′- and CTCGAGTTAATT GATGTTCTTCAGTGCTTC3′-, introducing 5′ EcoRI and 3′ XhoI sites. Amino acid comparisons and phylogenic tree analysis were carried out with DNASIS MAX version 2.0 .The full-length Whole-mount in situ hybridization with alkaline phosphatase-based single or double color staining has been described A2ML ATG MO (5′-ACAACAGCTAACATTCAGAGCCATG-3′) and UTR MO (5′-CAGAGCCATGATGACGAGTGTCCAG -3′) were synthesized by Gene Tools. The injection doses were: cont MO, 10 ng; ATG MO, 7 ng; UTR MO, 2 ng; all were injected into one-cell stage embryos. For rescue, 30 pg of A2ML-eGFP RNA was used; the same level was used for specificity tests.ser10 at 1∶500 (Upstate), anti P-glycoprotein at 1:300 (Abcam), anti-bovine carboxypeptidase A at 1∶500 (Rockland Inc) Sections were cut after embedding in JB-4 plastic (Polysciences) at 7 µm, or in OCT medium for cryosectioning (Tissue-Tek) at 10 µm for immunohistochemistry (IHC). Embryos were fixed in 4% paraformaldehyde for 2 hours at 4°. For IHC, sections were soaked for 2 min in acetone, followed by 30 min in 10 mM sodium citrate (pH 6.0) in PBS (0.1% Triton X-100). Blocking was done with 2% goat serum in PBS-Tween 20 for 30 min at room temperature. Methylene blue staining has been described Figure S1Amino acid comparison of A2ML family. Amino acid comparison of α2ML family. A. Schematic representation of amino acid sequence comparison between species. The percentage identity to zebrafish α2 macroglobulin-like is given for N-terminal (N), middle (M), and C-terminal (C) regions, and for the entire proteins to the right. B. The bait regions (bold underline) are compared. C. The highly conserved thio ester region. A glycine and glutamine (asterisks) represent essential amino acids that form thio ester bonds. D. C-terminal highly conserved target binding domain (CHB) (bold underline). The accession number for A2M family are carp A2M-1 (AB026128); Xenopus laevis Panza (DQ080115); Xenopus laevis EDD (L63543); Human A2M (BC040071); Human A2ML1 (NM144670); Mouse A2M (BC072642).(5.97 MB TIF)Click here for additional data file.Figure S2Development of blood vessels, goblet cells, and exocrine pancreas. Embryo stages are 5 dpf for A–D, and 4dpf for E–F. A–B. Blood vessel formation of control MO (A) and A2ML MO (B) injected embryos as visualized in fli1-gfp Tg embryos. C–D. Confocal images of goblet cells in intestine were obtained using fluorescein-conjugated wheat germ agglutinin in control MO (C) and A2ML MO (D) injected embryos. E–F. Confocal images of immunoreactive carboxypeptidase A showing exocrine cells in the pancreas in cont MO (E) and A2ML MO (F) injected embryos. L, liver.(8.65 MB TIF)Click here for additional data file.