JCM Accepts, published online ahead of print on 6 June 2007 J. Clin. Microbiol. doi:10.1128/JCM.00737-07 Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1
Detection of respiratory viruses and subtype identification of influenza A
2
viruses by GreeneChipResp oligonucleotide microarray
3 Phenix-Lan Quan,1# Gustavo Palacios,1# Omar J. Jabado,1 Sean Conlan,1 David
5
L. Hirschberg,2 Francisco Pozo,3 Philippa J.M. Jack,4 Daniel Cisterna,5 Neil
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Renwick,1 Jeffrey Hui,1 Andrew Drysdale,1 Rachel Amos-Ritchie,4 Elsa
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Baumeister,5 Vilma Savy,5 Kelly M. Lager,6 Jürgen A. Richt,6 David B. Boyle,4
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Adolfo García-Sastre,7 Inmaculada Casas,3 Pilar Perez-Breña,3 Thomas Briese,1
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W. Ian Lipkin1*
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Jerome L. and Dawn Greene Infectious Disease Laboratory, Mailman School of
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1
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Public Health, Columbia University, New York, New York, USA ; Stanford School
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of Medicine, Palo Alto, California, USA2; Centro Nacional de Microbiologia,
14
Instituto de Salud Carlos III, Madrid, Spain3; CSIRO Livestock Industries,
15
Australian Animal Health Laboratory, Victoria, Australia4; Instituto Nacional de
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Enfermedades Infecciosas, ANLIS “Dr. Carlos G. Malbrán”, Buenos Aires,
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Argentina5; National Animal Disease Center, USDA, Ames, Iowa, USA6;
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Department of Microbiology and Emerging Pathogens Institute, Mount Sinai
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School of Medicine, New York, New York, USA7
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#
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Both authors contributed equally.
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* Corresponding Author:
25
W. Ian Lipkin
26
Jerome L. and Dawn Greene Infectious Disease Laboratory
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Mailman School of Public Health, Columbia University
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722 West 168th Street, Room 1801, New York, NY 10032
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Voice: (212) 342-9033; Fax: (212) 342-9044; Email:
[email protected]
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Running title: Respiratory virus detection by oligonucleotide microarray
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WORD COUNT: 3,089
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ABSTRACT WORD COUNT: 104
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ABSTRACT
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Acute respiratory infections are a significant cause of morbidity, mortality and
35
economic burden worldwide. Accurate, early differential diagnosis may alter
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individual clinical management as well as facilitate recognition of outbreaks that
37
have implications for public health. Here we report the establishment and
38
validation of a comprehensive and sensitive microarray system for detection and
39
speciation of respiratory viruses in clinical materials. Implementation of a set of
40
influenza enrichment primers facilitated subtyping of influenza A viruses through
41
differential recognition of hemagglutinins 1 through 16 and neuraminidases 1
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through 9. Twenty-one different respiratory virus species were accurately
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characterized including a recently identified novel genetic clade of rhinovirus.
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INTRODUCTION
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Acute respiratory infections (ARI) are a leading cause of childhood morbidity and
46
mortality worldwide, resulting in an estimated 1.9 million deaths in 2000 (8, 23,
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34). ARI account for 1-3% of deaths in children less than 5 years of age in
48
industrialized countries and 10-25% of deaths in developing countries (4). The
49
economic burden of ARI is profound. In the United States the annual economic
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impact of non-influenza related viral respiratory tract infections is estimated to be
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$40 billion (13); influenza alone is responsible for approximately $12 billion (26).
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Highly multiplexed, sensitive diagnostic methods are needed to address
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the challenges of ARI. Early recognition of a causative agent may enable specific
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interventions that reduce morbidity and mortality, personal and social burdens
55
associated with losses in productivity, and the potential resistance, toxicity and
56
expense associated with inappropriate therapy. Insights into the epidemiology of
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ARI may also be useful in directing vaccine and drug development and policy on
58
a larger scale. With the recent appreciation of the risk of pandemic influenza
59
there is an urgent need for establishing tools for diagnosis and surveillance (26).
60
Clinicians and public health practitioners must have the ability to discriminate
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between the worried well and individuals infected with pandemic influenza
62
strains, or other pathogens, in order to appropriately allocate limited resources
63
such as drugs and isolation facilities.
C A
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We and others have reported multiplex PCR assays whereby microflora in
65
clinical materials can be detected at the genus and species level (6, 7, 9, 12, 16,
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28, 29). Although these assays can facilitate rapid, sensitive differential diagnosis
67
of ARI, and recently enabled recognition of a novel genetic clade of rhinovirus
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(18), such assays are limited to 20-30 candidate pathogens and may be
69
confounded in the event that virus evolution results in mutations at primer binding
70
sites. DNA microarrays offer unprecedented opportunities for multiplexing;
71
however, they are not widely implemented in clinical microbiology laboratories
72
because of problems with sensitivity, throughput, validation and expense (11, 15,
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19, 20, 25, 30, 32, 33). Here we report design and validation of a comprehensive
74
microarray system, GreeneChipResp, that allows sensitive detection and
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speciation of a wide variety of respiratory viruses, including all influenza A
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hemagglutinin and neuraminidase subtypes: H1 through H16, and N1 through
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N9.
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METHODS
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Viruses
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The sources of viral reference strains used in this study are indicated in the
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legends of Table 3 and 4. With the exception of postmortem lung tissue (samples
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47 and 160) from two patients who died of SARS (severe acute respiratory
83
syndrome) at Mount Sinai Hospital, Toronto, all clinical samples were
84
nasopharyngeal aspirates collected by the Instituto de Salud Carlos III, Madrid,
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Spain. All nasopharyngeal aspirates were previously assayed for the presence of
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viral pathogens using multiplex reverse transcription nested-PCR assays (9, 10,
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21).
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C A
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Sample preparation
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RNA from virus isolates (culture supernatant) and clinical samples was isolated
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using TriReagent (Molecular Research Center, Cincinnati, OH). DNA was
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removed from RNA preparations by treatment with DNase I (DNA-free, Ambion,
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Austin, TX). Reverse transcription (RT) reactions were performed using the
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TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Foster City,
95
CA).
96
Two protocols were used to amplify template for GreeneChipResp
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hybridizations. In one protocol first strand synthesis was initiated with a random
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octamer linked to an specific artificial primer sequence, 5’ GTT TCC CAG TAG
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GTC TCN NNN NNN N 3’ (Sequence independent amplification, SIA Primer (5)).
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After RNAse H digestion, cDNA was amplified using a 1:9 mixture of the SIA
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primer and a primer targeting the specific primer sequence (5’ CGC CGT TTC
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CCA GTA GGT CTC 3’; the sequence CGCC at the 5’ end of the primer is
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included to enhance the annealing of the primer to the template and allow
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amplification at higher temperature to increase efficiency of the PCR reaction).
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Initial PCR amplification cycles were performed at a low annealing temperature
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(25oC); subsequent cycles used a stringent annealing temperature (55oC) to
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favor priming through the specific sequence. Products of this first PCR were then
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amplified in a second PCR using the specific primer sequence linked to a capture
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sequence for 3DNA dendrimers, that contain more than 300 fluorescent reporter
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molecules (Genisphere Inc., Hatfield, PA).
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When this approach failed with nasopharyngeal aspirates from infected
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individuals with influenza A virus (FLUAV), influenza B virus (FLUBV) or
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influenza C virus (FLUCV), we established a modified protocol wherein first
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strand synthesis was initiated with the SIA primer doped with a primer mixture
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containing the same specific sequence linked to FLUAV, FLUBV and FLUCV
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sequences representing the conserved termini of influenza virus genome
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segments (Influenza Enrichment [IE] primers, 5 pmol per primer).
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Design of Influenza Enrichment (IE) primers
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Conserved sequences (10-14 nt in length) at the 5’end or 3’end of the published
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FLUAV, FLUBV and FLUCV sequences were identified by computer-assisted
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analysis (MACAW version 32 software, 1995; NCBI). The minimum number of
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forward and reverse sequence sets to cover all available influenza sequences
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was identified (6 for FLUAV, 10 for FLUBV and 4 for FLUCV, Table S1 in
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supplemental material).
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Design of GreeneChipResp array probes
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The GreeneChipResp contains probes from the GreeneChipVr array (25) as well
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as additional probes for detecting and subtyping FLUAV viruses. Probes were
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selected for genera of viral families containing viruses known to cause respiratory
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illness or symptoms compatible with influenza such as fever and myalgia. Virus
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families represented on the GreeneChipResp include adenoviridae, arenaviridae,
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bunyaviridae,
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orthomyxoviridae, paramyxoviridae, parvoviridae, picornaviridae, polyomaviridae,
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poxviridae, reoviridae, rhabdoviridae. Probes were selected in conserved regions
135
of
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(http://www.sanger.ac.uk/Software/Pfam/) and the motif finding strategy MEME
137
(2). Coverage was deemed sufficient when all sequences within an alignment
138
were addressed by at least one probe with no more than 5 mismatches (25).
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FLUAV subtyping probes were designed using a database of HA and NA
140
segments constructed from a union of the LANL Influenza Sequence Database
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(http://www.flu.lanl.gov) and Genbank (http://www.ncbi.nlm.nih.gov/Genbank/). A
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two-step process was employed. In the first step, 60 nt sequences were
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generated for every sequence in the FLUAV database using a sliding window
144
strategy. In a second step, a set covering algorithm was applied to this sequence
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set to select the probes required to cover every sequence within a subtype with a
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minimum of 3 probes (14). The 60-mer oligonucleotide arrays were synthesized
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on 70-mm x 20-mm slides by using an inkjet deposition system (Agilent
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Technologies, Palo Alto, CA). Eight arrays can be printed on a single slide (8-
both
caliciviridae,
structural
coronaviridae,
non-structural
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herpesviridae,
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and
flaviviridae,
genes
using
Pfam
alignments
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plex format). The GreeneChipResp array comprises 14,795 viral probes, of which
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4,696 are FLUAV subtyping probes. In addition to the virus-specific probes
151
described above, the array also features 1000 Null probes for background
152
discrimination, landing light probes for accurate alignment during feature
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extraction, and internal positive control probes complementary to a green
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fluorescent protein transcript.
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D E
T P
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Microarray hybridization and processing
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Products of the second PCR were added to 30 µL of SDS-based hybridization
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buffer (Genisphere Inc., Hatfield, PA), heated for 10 min at 80ºC, and added to
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GreeneChipResp for hybridization for 16 hours at 65ºC. After 10 min washes at
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room temperature in 6X SSC, 0.005% Triton X-100 and 0.1X SSC, 0.005% Triton
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X-100, Cy3 3DNA dendrimers (Genisphere Inc, Hatflied, PA) were added at 65ºC
162
for 1 hour using the same hybridization conditions. Slides were washed as
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before, air dried and scanned (Agilent DNA Microarray scanner, Agilent
164
Technologies).
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E C
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GreeneLAMP analyses
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GreeneLAMP v1.0 was created to assess results of GreeneChip hybridizations
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(25). Briefly, BLASTN (1) was used to connect probe sequences on the
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GreeneChipResp to entries in a viral sequence database. Each sequence has a
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corresponding NCBI Taxonomy ID (TaxID), which is in turn mapped to a node in
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a phylogenetic tree constructed based on the ICTV taxonomy.
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Probe intensities were background corrected, log2-transformed and
173
converted to Z-scores (and the corresponding p-values). Positive events were
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selected as those with a fluorescent signal that was greater than two standard
175
deviations above the mean fluorescent signal. Candidate TaxIDs were ranked by
176
combining the p-values for individual probes (QFAST, (3)) for the positive probes
177
within
178
GreeneChipResp were grouped by subtype rather than TaxID. FLUAV subtyping
179
probes were then reanalyzed for FLUAV positive samples using the
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GreeneLAMP algorithm. The rank and positive probe distribution along each
181
gene was then used to determine the subtype.
that
TaxID.
For
FLUAV
subtyping,
probe
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sequences
on
the
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182 183
Quantitative Real-Time PCR
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For sensitivity assessments, real-time PCR assays were conducted to determine
185
the viral load in each sample. Reactions were performed in a 25 µL volume using
186
either a SYBR Green or a Taqman assay (Applied Biosystems, Foster City, CA).
187
The following cycling conditions were used: 50ºC for 2 min and 95ºC for 10 min,
188
followed by 45 cycles at 95ºC for 15 sec and 60ºC for 1 min. Real time PCR
189
assays were performed using previously published primers (22, 24, 27, 31, 35)
190
with the exception of the assay for human parainfluenza virus 2 where we
191
employed the primer set:
192
(forward), Taq-984R 5’ AGCATGAGAGCYTTTAATTTCTGGA 3’ (reverse), Taq-
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930T 5’ FAM- CATTGGCTCTTGCAGCATTYTCTGGG -TAMRA 3’ (probe),
194
labeled with the reporter FAM (6-carboxytetramethylrhodamine) (TIB Molbiol,
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Taq-908F: 5’ GGACTTGGAACAAGATGGCCT 3’
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Berlin, Germany). Thermal cycling was performed in an ABI 7300 real-time PCR
196
system (Applied Biosystems, Foster City, CA).
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RESULTS
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Validation of the GreeneChipResp array using reference strains and tissue
199
culture isolates
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To assess the capacity of the GreeneChipResp for detection and typing of
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influenza viruses, we tested 33 FLUAV and FLUBV reference strains of human
202
and animal origin (Table S2 in supplemental material). Human strains included
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ten FLUAV viruses (four H1N1, two H2N2, three H3N2, one H5N1) and two
204
FLUBV viruses. The avian strains represented chicken, duck, grey teal, gull,
205
mallard, tern, turkey, red- necked stint, rhea, mallard, shelduck, and comprised a
206
repertoire of subtypes including H3N8, H4N4, H5N2, H5N9, H6N2, H7N1, H7N3,
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H7N7, H8N4, H9N2, H10N7, H11N9, H12N9, H13N6, H14N5, H15N9 and
208
H16N3. We also tested H1N1 and H3N2 viruses isolated from swine. Avian,
209
swine and human influenza virus strains were accurately detected. All sixteen HA
210
(H1 through H16) and nine NA (N1 through N9) FLUAV subtypes tested were
211
correctly identified.
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Reference strains represent only a limited fraction of the genetic variability
213
of influenza viruses. Thus, we next tested a panel of 15 circulating human
214
influenza virus strains isolated worldwide since 1998. These included one H1N1
215
virus
216
A/Sydney/05/97(H3N2)
217
A/Korea/770/02(H3N2), A/Fujian/411/02(H3N2) or A/California/07/04(H3N2)-like
218
viruses); two H5N1 viruses; one H9N2 virus; and four FLUBV strains
219
(B/Yamanashi/166/98,
(A/Caledonia/20/999(H1N1)-like or
virus);
seven
H3N2
A/Panama/2007/99(H3N2)-like
B/Sichuan/379/99,
13
B/Hong
viruses viruses,
Kong/330/01
(four three
and
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197
220
B/Shangai/361/02-like strains) (Table S3 in supplemental material). All FLUAV
221
and FLUBV strains were accurately identified and correctly subtyped. Because swine are an important reservoir from which new reassortants
223
with the potential to infect humans may emerge, we assayed swine viruses
224
isolated in the United States between 1968 and 2004 (Table S3 in supplemental
225
material). All 11 isolates, comprising four H1N1, one H1N2, two H3N1 and four
226
H3N2 viruses, were accurately detected and subtyped.
D E
T P
227
The GreeneChipResp also accurately identified other human respiratory
228
viruses including human adenovirus C and E; human respiratory syncytial virus A
229
and B; human parainfluenza virus 1 and 3; human coronaviruses SARS, OC43
230
and 229E; and human enteroviruses A and B. The threshold for detection of
231
these viruses is indicated in Table 1.
232
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C A
In summary, a total of 69 viruses comprising 54 FLUAV and FLUBV
233
isolates from human, avian and swine hosts; and 15 human respiratory viruses
234
were tested, identified and subtyped.
235 236
Validation of the GreeneChipResp array using clinical respiratory
237
specimens
238
To further assess the utility of the GreeneChipResp array in diagnostics we
239
tested a panel of human respiratory specimens for which the viral burden was
240
known. Based on previous work with the panmicrobial array, GreeneChipPm, we
241
set a minimum threshold of 1,000 viral RNA copies for samples carried forward to
242
array analysis (25). By this criterion samples of human SARS coronavirus (n=2),
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human respiratory syncytial virus A (n=8), human respiratory syncytial virus B
244
(n=1), human enterovirus (n=3), human metapneumovirus (n=3), human
245
parainfluenza type 2 (n=1), FLUAV (n=18), FLUBV (n=11), and FLUCV (n=2)
246
were selected. All non-influenza viruses were amplified to 107 to 1010 copies after
247
random PCR and were detected in array hybridization as well as non-quantitated
248
specimens containing rhinovirus A (n=3), rhinovirus B (n=2) and a recently
249
identified novel rhinovirus clade (rhinovirus NY, n=1) (Table 2). In contrast,
250
influenza viruses amplified inefficiently and were not detected in array
251
experiments. The efficiency of random priming for amplification of specific targets
252
is influenced by the length of the target template and the presence of competing
253
nucleic acid template. Reasoning that lack of sensitivity might be due to
254
inefficiency of primer binding to cognate sequences in short genome segments in
255
the context of abundant host nucleic acid, we developed a strategy for
256
enrichment of influenza virus sequences. SIA primers were supplemented at the
257
RT step by primers designed to bind to the conserved terminal sequences of
258
FLUAV, FLUBV and FLUCV genome segments (influenza enrichment primers, IE
259
primers, Table S1). Using the modified influenza enrichment protocol, all 18
260
FLUAV, 11 FLUBV, and 2 FLUCV viruses were accurately detected on the
261
GreeneChipResp array (Table 3). In addition, all FLUAV specimens were
262
correctly subtyped as H3N2 (n=16) and H1N1 (n=2). The addition of IE primer
263
did not reduce the sensitivity for detection of other respiratory viruses (data not
264
shown).
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DISCUSSION
266
We have established a comprehensive, sensitive microarray that allows detection
267
of respiratory viruses and speciation of influenza virus. It has been validated with
268
isolates representing the 16 H and 9 N influenza virus A subtypes; influenza
269
viruses circulating in the human population since 1998; 30 nasopharyngeal
270
aspirates from individuals infected with FLUAV, FLUBV or FLUCV; 22
271
nasopharyngeal aspirates and 2 lung specimens from individuals infected with
272
other common respiratory viruses including human SARS coronavirus, human
273
enterovirus, human rhinovirus, human metapneumovirus, human respiratory
274
syncytial virus A and B, and human parainfluenzavirus 2. To our knowledge the
275
GreeneChipResp is the first platform with the capacity to identify all known
276
FLUAV subtypes.
277
D E
T P
E C
C A
Several DNA microarrays are reported for detection and characterization
278
of respiratory viruses (11, 15, 19, 20, 25, 30, 33). However, none other than the
279
GreeneChipResp addresses the full complement of viruses known to be
280
associated with respiratory disease. Furthermore, because other microarrays
281
designed to type and subtype influenza viruses have focused on the subset
282
comprising circulating strains currently implicated in human disease (H1N1,
283
H1N2, H3N2, H5N1 and FLUBV) they do not have the capacity to detect either
284
advent of a new strain representing one or more of the remaining thirteen HA and
285
seven NA FLUAV subtypes or H2N2, a subtype that circulated in the human
286
population during the interval 1957-1968 (17).
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Sensitivity is a critical parameter in the implementation of DNA array
288
technology. Thus, sample nucleic acids are typically processed for pathogen-
289
specific multiplex or random primed amplification prior to hybridization.
290
Pathogen-specific priming may be more sensitive but may not amplify targets in
291
instances of primer/template mismatch. Random primed amplification allows
292
detection of a wider variety of pathogen targets but may reduce sensitivity. The
293
system we describe circumvents the limitations of these earlier approaches:
294
random priming allows unbiased amplification of all templates in a sample; the
295
addition of agent-specific primers enriches for the presence of sequences that
296
are present in low copy number or that may fail to amplify efficiently due to
297
competition between the target of interest and other nucleic acids in the sample,
298
or, in viruses like influenza with segmented genomes, a template length too short
299
to allow robust priming with random primers.
300
D E
T P
E C
C A
The majority of respiratory arrays rely on reporter molecules that are
301
directly incorporated into primers or amplification products. In contrast, the
302
GreeneChip system uses an indirect dendrimer labeling method whereby signal
303
is enhanced by the presence of >300 fluorescent reporter molecules in each
304
probe-target hybridization. In concert, the use of primer pools designed for
305
influenza target enrichment and the application of dendrimer technology yield
306
sensitivity in the range of 1,000 RNA molecules with nasopharyngeal aspirates.
307
Despite these advantages the GreeneChipResp cannot be considered a
308
stand-alone diagnostic platform. Neither our array nor others used for microbial
309
surveillance is quantitative. In transcript profiling or genome copy microarrays,
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probes and amplification protocols have been optimized to allow signal intensity
311
to be used for estimating the relative concentration of a genetic target in a
312
sample. In contrast, signal intensity in microbial surveillance arrays reflects
313
differences in probe and target complementarity as well as the differences in the
314
efficiency of amplification of different targets; furthermore, there is no internal
315
standard for estimating viral burden based on relative signal intensity obtained
316
with microbial and control probes. A second limitation is the absence of probes
317
for non-viral pathogens. We recently reported a panmicrobial oligonucleotide
318
array (GreeneChipPm, (25)) that contains probes for vertebrate viruses, bacteria,
319
fungi and parasites. Although a comprehensive array could be fabricated that
320
includes the probes for broad detection, speciation and subtyping of all potential
321
respiratory pathogens, the cost of such an array might be a barrier to use. We
322
currently print eight GreeneChipResp arrays per 70-mm x 20-mm slide at a cost
323
of $75USD per array. Moving to the probe density required for inclusion of
324
respiratory bacteria and fungi would increase the cost to $300USD per array. A
325
third limitation is the absence of sequence information that may be important for
326
detailed phylogenetic analyses and monitoring vaccine efficacy or drug
327
resistance markers. This challenge has been elegantly addressed by Stenger
328
and colleagues through use of a tiling array (19, 33); however, the probe density
329
required for a tiling array for all respiratory pathogens cannot be achieved with
330
existing technology.
D E
T P E C
C A
331
We view DNA microarrays as one in a suite of tools to be used in
332
infectious disease diagnosis and surveillance. Our strategy is to begin with a
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highly multiplexed PCR method such as MassTag PCR (6) that can survey for
334
the presence of up to 30 different agents in six hours at a supply cost of $12USD
335
per assay. If this approach fails to yield candidate organisms we move to
336
microarray analyses wherein thousands of candidates are screened in 16 hours
337
at a cost of $100USD or $325USD per assay (total supply cost for the
338
GreeneChipResp
339
GreeneChip analyses are not fruitful we have used unbiased high throughput
340
sequencing systems to identify novel pathogens; however, the cost and effort
341
required to do so precludes such investment until after other strategies are
342
exhausted.
or
GreeneChipPm,
respectively).
In
D E
instances
where
T P
E C
343
Detection of a candidate pathogen is only one step to proving causation.
344
Judgment in assessing biological plausibility and relevance will become
345
increasingly important given the breadth and sensitivity of new surveillance
346
technologies, the myriad mechanisms for microbial pathogenesis and the fact
347
that infection may be asymptomatic.
C A
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333
348
Acknowledgments
349
The work reported here was supported by National Institutes of Health awards
350
AI062705,
351
U54AI57158 (Northeast Biodefense Center-Lipkin). We thank Ruben Donis,
352
Gerry Harnett, Anthony Mazzuli and David Williams for specimens used in assay
353
development and validation. We also thank Cassandra Kirk, Estela Fernandez
354
and Eric M. Leproust for technical support and advice.
U01AI070411,
HL083850-01,
AI51292,
AI056118,
C A
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D E
T P
E C
AI55466,
355
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Table 1. GreeneChipResp sensitivity for detection of non-influenza virus strains Virus a
Sensitivity
Mastadenovirus Mastadenovirus Pneumovirus Pneumovirus Pneumovirus Pneumovirus Respirovirus Respirovirus Coronavirus Coronavirus Coronavirus Enterovirus Enterovirus Enterovirus Enterovirus
10 4 10 4 10 4 10 4 10 4 10 4 10 4 10 3 10 3 10 3 10 3 10 3 10 3 10 3 10
T P E C C A
4
D E
* SARS, severe acute respiratory syndrome a American Type Culture Collection, Manassas, Virginia, USA b Department of Microbiology, Mount Sinai School of Medicine, New York, New York, USA c Jerome L. and Dawn Greene Infectious Disease Laboratory, Mailman School of Public Health, Columbia University, New York New York, USA
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Human adenovirus E (HAdV-4) a Human adenovirus C (HAdV-5) a Human respiratory syncytial virus A a Human respiratory syncytial virus B b Human respiratory syncytial virus A (A-2) b Human respiratory syncytial virus (CH18537) a Human parainfluenza virus 1 a Human parainfluenza virus 3 c Human SARS* coronavirus a Human coronavirus (OC43) a Human coronavirus (229E) a Human enterovirus A (HEV71) a Human enterovirus B (E25) a Human enterovirus B (E14) a Human enterovirus B (E30)
Genus
Table 2. Clinical samples containing common, non-influenza viruses analyzed by GreeneChipResp Virus
Origin
Human SARS coronavirus Human SARS coronavirus Human enterovirus Human enterovirus Human enterovirus Human metapneumovirus Human metapneumovirus Human metapneumovirus Human respiratory syncytial virus A Human respiratory syncytial virus A Human respiratory syncytial virus A Human respiratory syncytial virus A Human respiratory syncytial virus A Human respiratory syncytial virus A Human respiratory syncytial virus A Human respiratory syncytial virus B Human rhinovirus A Human rhinovirus A Human rhinovirus A Human rhinovirus B Human rhinovirus B *Human rhinovirus NY Human parainfluenza 2 Human parainfluenza 2
lung tissue lung tissue nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab nasal swab
Initial RNA # copy number 6 3.32x10 5 5.98x10 5 5.84x10 3 2.60x10 3 1.40x10 3 2.15x10 3 1.11x10 3 1.70x10 2 7.89x10 3 7.68x10 3 4.84x10 4 2.30x10 4 1.18x10 3 1.41x10 3 1.06x10 4 3.58x10 ND ND ND ND ND ND 5 1.42x10 4 2.53x10
Copy number $ after PCR 8 3.467x10 8 1.192x10 10 1.44x10 6 7.80x10 8 1.20x10 7 9.27x10 7 1.05x10 8 1.55x10 7 3.79x10 8 4.48x10 9 3.27x10 8 9.87x10 8 1.26x10 9 6.14x10 9 1.47x10 9 3.64 x10 ND ND ND ND ND ND 10 1.44x10 9 3.55x10
T P E C
D E
C A
ND: Not done *Novel genetic clade of rhinovirus recently identified in New York State # Viral RNA copy number measured by real time PCR $ Viral cDNA copy number measured by real time PCR after random amplification
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Sample ID 47 160 23 SO4475 SO4505 SO4705 SO4743 SO5197 SO4512 SO4606 SO4614 SO4632 SO4650 SO4695 SO4698 SO4713 SO4923 SO4928 SO4866 SO4898 SO4900 SO4897 SO4480 SO4504
Table 3. Clinical samples containing influenza virus analyzed and subtyped by GreeneChipResp Sample ID
Initial RNA # copy number
Copy number $ after PCR
Type
Subtype
H3N2 H3N2 H3N2 H3N2 H1N1 H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H3N2 H1N1 H3N2
FLUAV 4
9
6.95x10 3 9.25x10 3 4.89x10 4 5.55x10 5 2.88x10 5 1.71x10 6 1.45x10 5 1.09x10 5 4.37x10 5 1.15x10 7 3.58x10 3 8.83x10 4 1.25x10 4 4.58x10 4 1.69x10 4 6.34x10 4 2.21x10 3 3.67x10
2.48x10 9 1.10x10 9 2.79x10 9 8.41x10 10 1.07x10 9 8.60x10 10 7.96x10 9 2.76x10 10 2.61x10 9 2.05x10 10 1.79x10 8 6.69x10 10 1.02x10 10 2.11x10 9 1.23x10 10 2.11x10 10 4.64x10 10 6.50x10
FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV FLUAV
SO2667 SO2683 SO2693 SO2695 SO2696 SO2784 SO2833 SO2844 SO3800 SO3804 SO3822 FLUCV
6
7.94x10 4 3.45x10 3 1.47x10 5 6.34x10 4 5.94x10 5 2.06x10 4 2.08x10 3 3.67x10 4 4.17x10 3 8.90x10 4 5.32x10
3.21x10 8 9.67x10 8 3.11x10 8 5.43x10 8 7.90x10 9 2.31x10 8 5.20x10 8 5.56x10 9 8.10x10 8 6.57x10 9 9.65x10
9
FLUBV FLUBV FLUBV FLUBV FLUBV FLUBV FLUBV FLUBV FLUBV FLUBV FLUBV
NA NA NA NA NA NA NA NA NA NA NA
ND ND
ND ND
FLUCV FLUCV
NA NA
T P
E C C
A
SO3802 a SO4680
D E
ND, Not done NA, Not applicable a Sample 4680 contains FLUAV and FLUCV # Viral RNA copy number measured by real time PCR $ Viral cDNA copy number measured by real time PCR after random amplification in the presence of influenza enrichment primer
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SO2820 SO2951 SO3812 SO3813 SO3833 SO3917 SO3924 SO3937 SO3943 SO3945 SO4578 SO4616 SO4639 SO4649 SO4652 SO4669 SO5265 a SO4680 FLUBV