JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2009, p. 2779–2786
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 9
Rapid Semiautomated Subtyping of Influenza Virus Species during the
2009 Swine Origin Influenza A H1N1 Virus Epidemic
in Milwaukee, Wisconsin?
Michael E. Bose,1,2Eric T. Beck,1,2Nate Ledeboer,3,6Sue C. Kehl,1,3,4,5Lisa A. Jurgens,1,2
Teresa Patitucci,1,2Lorraine Witt,1,2Elizabeth LaGue,1,2Patrick Darga,1,2Jie He,1,2
Jiang Fan,1,2Swati Kumar,1,2,4,5and Kelly J. Henrickson1,2,4,5*
Midwest Respiratory Virus Program1and Departments of Pediatrics2and Pathology,3Medical College of Wisconsin,
Children’s Research Institute,4Children’s Hospital of Wisconsin,5and Dynacare Laboratories,6Milwaukee, Wisconsin
Received 19 May 2009/Returned for modification 7 July 2009/Accepted 23 July 2009
In the spring of 2009, a novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) emerged and
began causing a large outbreak of illness in Milwaukee, WI. Our group at the Midwest Respiratory Virus
Program laboratory developed a semiautomated real-time multiplex reverse transcription-PCR assay (Sea-
sonal), employing the NucliSENS easyMAG system (bioMe ´rieux, Durham, NC) and a Raider thermocycler
(HandyLab Inc., Ann Arbor, MI), that typed influenza A virus, influenza B virus, and respiratory syncytial
virus (RSV) and subtyped influenza A virus into the currently circulating H1 and H3 subtypes, as well as a
similar assay that identified H1 of S-OIV. The Seasonal and H1 S-OIV assays demonstrated analytical limits
of detection of <50 50% tissue culture infective doses/ml and 3 to 30 input copies, respectively. Testing of the
analytical specificities revealed no cross-reactivity with 41 and 26 different common organisms and demon-
strated outstanding reproducibility of results. Clinical testing showed 95% sensitivity for influenza A virus and
influenza B virus and 95 and 97% specificity compared to tissue culture. Comparisons of results from other
molecular tests showed levels of positive agreement with the Seasonal and H1 S-OIV assay results of 99 and
100% and levels of negative agreement of 98 and 100%. This study has demonstrated the use of a semiauto-
mated system for sensitive, specific, and rapid detection of influenza A virus, influenza B virus, and RSV and
subtyping of influenza A virus into human H1 and H3 and S-OIV strains. This assay/system performed well in
clinical testing of regular seasonal influenza virus subtypes and was outstanding during the 2009 Milwaukee
S-OIV infection outbreak. This recent outbreak of infection with a novel influenza A (H1N1) virus also
demonstrates the importance of quickly distributing information on new agents and of having rapid influenza
virus subtyping assays widely available for clinical and public health decisions.
In 2005 an adolescent in Wisconsin developed a brief respi-
ratory illness later determined to be caused by a novel influ-
enza A virus that was a mixture of swine, avian, and human
virus gene segments. This infection was thought to be zoonotic,
originating from pigs that the individual had helped butcher a
few days before. No other cases could be linked to this partic-
ular virus or individual. In the early spring of 2009, this sce-
nario appeared to occur again in Mexico. An outbreak of
respiratory illness caused by a novel influenza A (H1N1) virus
(swine origin influenza virus [S-OIV]) shown also to be a triple
reassortant began, with significant numbers of individuals be-
ing infected (2–4, 20, 21). Soon a widespread outbreak oc-
curred in Milwaukee, WI (13). Our group at the Midwest
Respiratory Virus Program laboratory was able to molecularly
confirm the first case of infection in Wisconsin on 29 April
2009 and worked closely with the state and city public health
officials to provide rapid influenza virus subtyping for a large
number of samples.
We have recently developed a number of rapid assays to type
and subtype influenza virus. During the 2 weeks prior to 27
April 2009, very little respiratory virus activity was detected in
our community or within the entire state of Wisconsin. Be-
cause of the concern over S-OIV, we began to perform con-
firmatory influenza virus typing and subtyping on influenza A
virus-positive specimens from the Children’s Hospital of Wis-
consin (CHW; E. T. Beck, L. A. Jurgens, S. C. Kehl, M. E.
Bose, T. Patitucci, E. LaGue, P. Darga, K. Wilkinson, L. M.
Witt, J. Fan, J. He, S. Kumar, and K. J. Henrickson, unpub-
lished data) and Dynacare Laboratories (DL) (6, 17) by using
multiplex real-time reverse transcription-PCR (rRT-PCR) as-
says for influenza A virus, influenza B virus, and respiratory
syncytial virus (RSV). The presence of these influenza virus
subtyping assays in our laboratory provided the necessary tools
for us to quickly respond to the emergence of a novel influenza
virus subtype within our community.
The Seasonal assay is a semiautomated multiplex rRT-PCR
assay that types influenza A virus, influenza B virus, and RSV
and subtypes influenza virus by targeting the H1 (human) and
H3 (human) hemagglutinin (HA) genes with a noncompetitive
RNA internal control (MS2 RNA phage). The FluPlex is a
large multiplex RT-PCR enzyme hybridization assay that types
influenza A virus and influenza B virus and identifies H1 (hu-
man), H2, H3, H5, H7, H9, N1 (human), N1 (animal), N2, and
N7 subtypes. We initially were able to use the Seasonal assay to
* Corresponding author. Mailing address: Pediatrics/Infectious Dis-
ease/CCC/Suite c450, Children’s Corporate Center, P.O. Box 1997,
Milwaukee, WI 53201-1997. Phone: (414) 337-7073. Fax: (414) 337-
7093. E-mail: Khenrick@mcw.edu.
?Published ahead of print on 29 July 2009.
identify influenza A virus samples that did not type as H1 or
H3. We then performed the FluPlex assay, which confirmed
the samples to be positive for influenza A virus and subtyped
them as negative for human HA and neuraminidase subtypes
and positive for animal (swine) N1 virus (7). Three days after
subtyping with the FluPlex began, we had developed an S-OIV-
specific semiautomated assay (the H1 S-OIV assay) with ex-
traction by the NucliSENS easyMAG system (bioMe ´rieux,
Durham, NC) and amplification by the Raider thermocycler
(HandyLab Inc., Ann Arbor, MI) and the same protocols and
formats used for our Seasonal assay. With these tools, we were
able to rapidly subtype the influenza A viruses sent to us from
two large clinical laboratories. This paper reports the use of the
semiautomated multiplex real-time typing and subtyping assays
during this outbreak.
MATERIALS AND METHODS
Primer and probe design. The Seasonal assay has primers and probes designed
to correspond to highly conserved regions of the influenza A virus matrix (M)
gene, the influenza B virus M gene, the RSV polymerase (L) gene, the HA genes
of the H1 and H3 subtypes of human influenza A virus, and the bacteriophage
MS2 (internal control) (Table 1). Influenza virus primers were designed using
the Influenza Primer Design Resource (http://www.ipdr.mcw.edu), and RSV
primers were designed by aligning the 16 RSV L gene sequences found in
GenBank (1). Primers and probes utilize proprietary superbases and a 5? minor
groove binder (Pleiades probes; Nanogen, Inc., Bothell, WA) (18). The H1
S-OIV multiplex rRT-PCR assay (H1 S-OIV assay) primer/probe set was de-
signed to detect the HA gene segment from the currently circulating S-OIV. In
silico coverage by the primer/probe sets was determined using an in-house
program. A sequence was considered to be hit by the primers if there were no
mutations within 5 bases from the 3? end, one or no mutation within 10 bases
from the 3? end, and two or fewer mutations in the whole region corresponding
to the oligonucleotide or to be hit by the probes if there were two or fewer
mutations in the whole region corresponding to the oligonucleotide. The number
of gaps was determined by looking at an alignment of the sequences for which
coverage was being determined and counting the number of sequences in the
alignment that did not have a full sequence in the target region for the primers
and probes. To calculate the percent coverage, the number of sequences hit was
divided by the total number of sequences with the number of gaps subtracted,
and the quotient was then multiplied by 100 [hits ? (total ? gaps) ? 100]. In
silico coverage rates for the primer/probe sets in the Seasonal assay were greater
than 95% for all of the targets in the assay. For the H1 S-OIV assay, the coverage
was 99.1% for all sequences available as of 11 July 2009 (Table 1). One of the
sequences not covered in silico was from one of our own isolates that we had
detected with the H1 S-OIV assay and then sequenced.
Sample preparation. A sample of 400 ?l was combined with 10 ?l of MS2
bacteriophage (5 ? 105PFU/ml) and 1 ml of lysis buffer and incubated at room
temperature for 10 min. After lysis, the samples were loaded onto the NucliSENS
easyMAG system (bioMe ´rieux, Durham, NC). Total nucleic acid extractions
proceeded according to the manufacturer’s protocol. Samples were eluted in 25
?l of elution buffer.
rRT-PCR and melt analysis. Following elution, 3.4 ?l of RNA was mixed with
4.6 ?l of supermix containing primers and probes, Platinum Tfi, and SuperScript
III (Invitrogen, Carlsbad, CA) to yield a one-step RT-PCR mixture with an 8-?l
TABLE 1. In silico coverage by the primers and probes used in the Seasonal assay and the H1 S-OIV assay
OrganismPrimer name Primer sequencea
Total no. of
Total for RSV
Influenza A virusINFA-L30
3,677 Total for influenza
Influenza B virusDIF430-L30
97.2Total for influenza
H1 (human) virusH1-L17
98.3Total for H1
Total for H3 virus
H1 virus (S-OIV) H1Sw_For652 ? 22
Total for H1 virus
a* indicates that the previous base is a proprietary superbase (Nanogen, Inc., Bothell, WA). MGB is a proprietary minor groove binder (Nanogen, Inc., Bothell, WA).
Fam is a proprietary fluorophore (Nanogen, Inc., Bothell, WA) similar to 6-Fam (Applied Biosystems Inc., Foster City, CA). AP-593 is a proprietary fluorophore
(Nanogen, Inc., Bothell, WA) similar to CalRed. EDQ is Eclipse dark quencher (Glen Research Corp., Sterling, VA). BHQ1 is Black Hole Quencher 1 (Biosearch
Technologies Inc., Novato, CA).
bOne of the sequences missed in silico was actually detected experimentally.
cThe MS2-AP593-6 probe is used in the S-OIV assay, and the MS2-AP593-7 probe is used in the Seasonal subtyping assay.
dNA, not applicable.
2780BOSE ET AL.J. CLIN. MICROBIOL.
final volume. The reaction mixture was manually loaded into a microfluidic
Raider cartridge and placed into the Raider high-speed thermocycler (Handy-
Lab Inc., Ann Arbor, MI). The Raider thermocycler utilizes a proprietary mi-
crofluidic cartridge that is approximately 1.5 mm thick and utilizes 4.2-?l reac-
tion wells allowing for rapid heating and cooling. Each cartridge can run up to 12
reactions. The cycling parameters used are as follows: 15 min at 50°C; 2 min at
95°C; 25 cycles of 1 s at 95°C, 15 s at 61°C, and 10 s at 76°C; 20 cycles of 1 s at
95°C, 15 s at 56°C, and 10 s at 76°C; a 60-s light-emitting diode warm-up; and a
subsequent melt analysis at 45 to 85°C with a melting rate of 0.3°C/s.
Analysis of results. Assay results were analyzed using an in-house-developed
Excel workbook (computational algorithm) that converts raw data into the final
output format. Data were analyzed and the tests were scored based on the
amplification and melting profiles of the sample. The melting profile was created
as the change in fluorescence versus temperature (Fig. 1) in two different chan-
nels. Influenza A virus, influenza B virus, and RSV were labeled with Fam (a
proprietary fluorophore created at Nanogen, Inc.). The H1 S-OIV probe was
labeled with 6-carboxyfluorescein (6-Fam; a dye similar to Fam) from Applied
Biosystems, Foster City, CA. In this article, Fam, 6-Fam, and FAM refer to the
fluorophore from Nanogen, Inc., the fluorophore from Applied Biosystems, and
the fluorescence channel on the Raider thermocycler that detects fluorescence of
the Fam and 6-Fam fluorophores. The H1 (human) virus and the H3 virus and
both internal control MS2 probes were labeled with AP-593 (a proprietary
fluorophore from Nanogen, Inc.). Samples were considered positive if the thresh-
old cycle (CT) value was ?40.0, the amplification curve shape was appropriate,
and the melting profiles yielded melting temperatures (Tm) within 2°C of those
expected for RSV (74°C), influenza B virus (66°C), influenza A virus (60°C), H1
(human) virus (60°C), H3 (human) virus (68°C), or H1 S-OIV (60°C). Sample
results were considered indeterminate if CTvalues were ?40.0, with an appro-
priate Tmand amplification curve shape. Samples were considered negative if the
internal control (MS2) Tmwas appropriate (73 ? 2°C in the AP-593 channel),
the AP-593 CTwas ?40.0 with the appropriate amplification curve shape, the
FAM CTwas ?40.0 with an incorrect or nonexistent Tmor an abnormal ampli-
fication curve, and no melt profiles in the AP-593 channel for H1 or H3 were
Analytical sensitivity (LODs). Serial 10-fold dilutions from 104to 10?2
TCID50/ml of different subtypes of influenza virus were prepared in M4 viral
transport medium (Remel, Lenexa, KS) and tested in the Seasonal and H1
S-OIV assays (Table 2). The limits of detection (LODs) for the Seasonal assay
were determined using a probit analysis based on 10 separate experiments to
calculate the LODs. Several of the S-OIV-positive samples were cultured and
quantitated using an in-house quantitative rRT-PCR assay that targets the matrix
gene of influenza A virus. Serial 10-fold dilutions of these virus isolates were
prepared in M4 and tested in the H1 S-OIV assay to determine the lowest
concentrations that could successfully be detected (Table 2). LODs for this assay
FIG. 1. Melting profile from the Seasonal subtyping assay. The melting curves for influenza A and influenza B viruses and RSV are visible on
the FAM channel, and the H1, H3, and MS2 melting curves are visible on the AP-593 channel. NTC, no-template (negative) control.
VOL. 47, 2009 MULTIPLEX rRT-PCR TYPING/SUBTYPING OF FLU VIRUS/S-OIV2781
were determined with only three replicates because time was of the essence
during the beginning of the pandemic.
Influenza A virus subtype specificity. Both assays were tested against influenza
A virus strains representing H1 to H15 and N1 to N9 at ?105TCID50/ml to
determine cross-reactivity with other influenza A virus subtypes.
Analytical specificity against other common respiratory organisms. M4 viral
transport medium was spiked with high concentrations (?104TCID50, PFU, or
CFU/ml) of common respiratory pathogens and commensal organisms and
tested in both of the assays (Table 3).
Reproducibility. Interrun reproducibility of the Seasonal assay results was
determined by calculating the standard deviations of the CTvalues and Tmof the
positive controls in seven runs with the same sets of samples representing each
of the targets in the assay. The positive controls consisted of quantitated virus
diluted in M4 at the following concentrations: 100and 10?3TCID50/ml (RSV-A)
and 103and 102TCID50/ml (influenza A [H1N1 and H3N2] virus and influenza
B virus) (see Table 5). Intrarun reproducibility was determined by calculating the
standard deviations of the CTvalues and Tmfor an influenza A H1N1 virus at 106
TCID50/ml and negative samples. The H1N1 virus samples and negative samples
were prepared in M4 and then divided into 400-?l aliquots. Five runs with six
H1N1 virus samples and six negative samples per run were performed. Intrarun
variability for each run was then assessed.
Clinical sensitivity and specificity. Three hundred and fifteen deidentified
nasopharyngeal swabs were collected at TriCore Laboratories (Albuquerque,
NM). Following collection, Dacron swabs were placed into M5 viral transport
medium (Remel, Lenexa, KS) and shipped to TriCore Laboratories. Upon ar-
rival, samples were inoculated into shell vials containing R-Mix cells. Positive
samples were analyzed at TriCore by a fluorescent-antibody assay for the pres-
ence of influenza A virus or influenza B virus. Influenza virus-positive samples
were then subtyped using type-specific serum. Samples were then frozen at
?80°C until testing with the Seasonal assay could be performed (as described
Subtyping of influenza A virus-positive samples. A total of 2,517 nasopharyn-
geal, nasal, and/or throat specimens submitted to the CHW or DL between 27
April and 11 May 2009 were tested for influenza A virus by using multiplex
rRT-PCR assays. Samples from the CHW were tested using a fully automated
multiplex RT-PCR on a Jaguar extractor/thermocycler (HandyLab, Inc., Ann
Arbor, MI), while samples from DL were tested using a semiautomated multi-
plex RT-PCR consisting of extraction on an easyMAG system and real-time
amplification on a smart cycler (Cepheid, Sunnyvale, CA) using Cepheid’s assay-
specific reagents. Both assays are capable of simultaneously detecting influenza
A virus, influenza B virus, and RSV (6, 17; Beck et al., unpublished). Three
hundred and five influenza A virus-positive specimens, 2 influenza B virus-
positive specimens, and 22 negative specimens were sent to the Midwest Respi-
ratory Virus Program lab for influenza virus subtyping. Raw specimens (from the
CHW) subjected to extraction as described above or total nucleic acid previously
extracted from 255 ?l of sample material on the easyMAG system with elution
in 55 ?l (from DL) was used in the assay. Influenza A virus-positive samples were
typed and subtyped with the Seasonal assay, the H1 S-OIV assay, and the FluPlex
(7). The FluPlex targets different genetic regions from those targeted by the
Seasonal or H1 S-OIV assay. The first 127 influenza A virus-positive and 22
negative clinical samples were tested by the Seasonal, FluPlex, and H1 S-OIV
TABLE 2. Analytical sensitivities of the Seasonal subtyping assay
and the H1 S-OIV assay
AssayVirus Analyte LODa
A/Hawaii/15/2001 (H1N1) Influenza A virus
A/Hawaii/15/2001 (H1N1) H1 virus
Influenza A virus 16
Influenza A virus 34
H3 virus 53
Influenza A virus8
Influenza B virus
Influenza B virus 12
aThe LODs for the Seasonal subtyping assay are expressed as the number of
TCID50per reaction, and those for the H1 S-OIV assay are expressed as the
number of copies per reaction.
TABLE 3. List of common respiratory organisms tested to
Organisms tested in both assays
Adenovirus type 3
Herpes simplex virus type 1
Human metapneumovirus (A1, A2, B1, and B2)
Human parainfluenza viruses 1 to 4
Human rhinovirus 1B
Organisms tested in the Seasonal assay only
Adenovirus types 1, 5, 7, 10, and 18
Coxsackievirus E9 and B5
Human rhinovirus 2, 14, and 16
Streptococcus groups B, C, F, and G
2782BOSE ET AL. J. CLIN. MICROBIOL.
assays. Thereafter, all influenza A virus-positive samples were subtyped with the
Seasonal and H1 S-OIV assays. All samples with discrepant results were tested
by the FluPlex. A segment of the H1 gene was sequenced using 13 random
clinical S-OIV-positive samples for subtype confirmation. For sequencing, 3 ?l of
nucleic acid was reverse transcribed in a 20-?l reaction mixture with murine
leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA). Ten
microliters of this reaction mixture was used for PCR with the following primers:
H1sw_For403 ? 21SQ (TGTAAAACGACGGCCAGTCCCAAGACAAGTTC
ATGGCCC) and H1sw_Rev906-21SQ (AGGAAACAGCTATGACCATAGCA
CCCTTGGGTGTTTGACA) (underlining indicates M13 primer binding se-
quences used during subsequent reactions with M13 primers). Following
amplification, PCR products were purified with the QiaQuick gel extraction kit
and sent to Retrogen, Inc. (San Diego, CA), along with the primers for sequence
analysis. In addition to being analyzed in our laboratory, 23 clinical samples
(collected early in the course of the outbreak) were sent to the Wisconsin State
Laboratory of Hygiene for confirmation of results by the CDC Laboratory
Response Network influenza virus typing and subtyping assays and the CDC
H1N1 S-OIV-specific assay.
Analytical sensitivity and subtype specificity. The analytical
sensitivities, or LODs, of the Seasonal assay and the H1 S-OIV
assay are shown in Table 2. The Seasonal assay was able to
detect ?50 TCID50/ml or fewer with all of the targets in the
assay. The H1 S-OIV assay had a sensitivity of 102to 103
copies/ml, or 3 to 30 copies/reaction. Testing of the two assays
with viruses representing 15 different HA types and 9 different
neuraminidase types showed no cross-reactivity with other sub-
types and no cross-reactivity between human H1 virus and H1
S-OIV (Table 4).
Analytical specificity with other common respiratory organ-
isms. Testing of the analytical specificities of both assays against
26 common respiratory organisms revealed no cross-reactivity
between the assay mixtures and the nonspecific targets. Addi-
tional testing of the Seasonal assay against 41 more respiratory
organisms also showed no cross-reactivity.
Reproducibility. The interrun variability of the Seasonal as-
say showed a standard deviation of the CTof less than 1 cycle
and of the Tm of less than 0.5°C for all of the targets except the
internal control, which had a standard deviation of 1.5 cycles
and 0.5°C (Table 5). The intrarun variability was similar with
an average standard deviation of 0.6 cycles for the influenza A
CT, 0.8 cycles for the MS2 Ct, 0.5°C for the influenza A Tm,
0.3°C for the H1 Tm, and 0.4°C for the MS2 Tm.
Clinical sensitivity and specificity. Of the 315 nasopharyn-
geal swabs tested, 20% (65) were positive for influenza A virus,
18% (57) were positive for influenza B virus, and 65% (205)
were negative for both viruses by tissue culture. Testing with
the Seasonal assay showed 95% sensitivity for both of these
viruses, with 95% specificity for influenza A virus and 97%
specificity for influenza B virus (Table 6). While these speci-
ficity numbers are excellent, it is probable that the real speci-
TABLE 4. Results from evaluation of the Seasonal subtyping assay
and the H1 S-OIV assay against other influenza virus subtypes to
A/New Caledonia/20/1999 (H1N1)
A H1 S-OIV
aThe S-OIV concentration is given in copies per milliliter; all other concen-
trations are in TCID50per milliliter.
bN indicates a negative result.
TABLE 5. Reproducibility of Seasonal assay CTand Tmresults in seven runs on the same day during a throughput study
FAM channelAP-593 channel
aAP-593 CTvalues for H1N1 and H3N2 viruses reflect simultaneous amplification of the HA subtype and the MS2 internal control amplicons.
bMeans and standard deviations for MS2 were calculated using negative, influenza B virus, and RSV samples in which the only target in the AP-593 channel
cNA, not applicable.
VOL. 47, 2009MULTIPLEX rRT-PCR TYPING/SUBTYPING OF FLU VIRUS/S-OIV2783
ficity is higher since RT-PCR is known to be more sensitive
than tissue culture. In addition, of the 60 samples called influ-
enza A virus by tissue culture and the Seasonal assay, all 60
gave a subtype result (50 H1 samples and 10 H3 samples).
Clinical testing of samples during the S-OIV infection out-
break. The results of the influenza virus typing and subtyping
can be seen in Table 7. The Seasonal and H1 S-OIV assay
results had outstanding agreement and correlation with each
other and with the results of the other molecular assays tested
(7; Beck et al., unpublished). In addition, 23 of 23 of the
clinical samples were confirmed to be positive for influenza A
H1N1 virus (19 for S-OIV and 4 for human virus) by Wisconsin
State Laboratory of Hygiene. Thirteen clinical isolates were
also confirmed by sequencing to be influenza A H1N1 S-OIV.
One influenza A virus sample (328) could not be subtyped in
any assay, nor could it be sequenced, while all other influenza
A virus isolates could be sequenced. This may indicate a sam-
ple with a low virus titer or an incomplete RNA genome.
We report the development and use of semiautomated rRT-
PCR assays to type and subtype influenza viruses and the ease
and efficiency with which this technology was adapted to detect
a novel influenza virus subtype very early in an outbreak. Ul-
timately, the ability to perform influenza virus subtyping on
large numbers of clinical samples each day led to improved
patient care and greatly facilitated timely and informed public
health decisions throughout the epidemic.
A goal of our laboratory has been to develop rapid, sensitive,
and specific semiautomated and automated multiplex assays
for the detection of common community-acquired respiratory
viruses (5, 9, 11, 14). Tissue culture had been the “gold stan-
dard” for respiratory virus detection until approximately the
late 1990s, when large multiplex RT-PCR assays first became
available clinically and commercially (5). RT-PCR was quickly
shown to be more sensitive than tissue culture and highly
specific for the detection of influenza virus and RSV (and most
other respiratory viruses) (5, 8–11, 14).
Just prior to the S-OIV infection outbreak, we had devel-
oped a rapid, sensitive, multiplex rRT-PCR assay (the Sea-
sonal assay) capable of detecting and differentiating influenza
A virus, influenza B virus, and RSV and identifying the H1 and
H3 subtypes of influenza A virus. We had also developed a
reflex (nonseasonal) assay for potential pandemic situations to
further subtype influenza A virus-positive samples as H5, H7,
or H9 if they were not subtyped by the Seasonal assay. These
avian subtypes had been identified as possible causes of the
next influenza pandemic, and many laboratories around the
world have been focusing their efforts on being able to detect
these subtypes, especially H5. The emergence of S-OIV and its
ability to efficiently spread from human to human has effec-
tively demonstrated that widely available influenza virus sub-
typing assays that include broader subtyping ability may be
critical in the next pandemic.
The LODs for the Seasonal assay are less then 102TCID50/ml
for RSV-A and RSV-B, influenza B virus, and H1N1 and
H3N2 viruses, and those for the H1 S-OIV assay are 103
copies/ml or less. These results compare well to those reported
for the FDA-approved ProFlu? (Prodesse Inc., Waukesha,
WI) and xTAG respiratory virus panel (Luminex Corp., Aus-
tin, TX) assays (12, 15, 16). Our assays, however, can be com-
pleted much faster than either of these two assays, with a time
from sample collection to result of just under 21⁄2 h, with the
ProFlu? assay taking 3.5 h and the xTAG respiratory virus
panel assay taking up to 8 h (12, 16, 17, 19). Throughput
studies with the Seasonal assay demonstrated that as many as
144 samples can be processed and tested in an 8-h shift by
using one easyMAG extractor and two Raider thermocyclers.
In addition to the impressive sensitivity, we demonstrated that
our assay has a high level of specificity, showing no cross-
reactivity with a panel of common respiratory organisms. We
also tested subtypes H1 to H15 of influenza A virus with both
the assays. All viruses were typed as influenza A virus, and only
H1 and H3 subtypes were positive in the Seasonal assay and
only H1 S-OIV was positive in the H1 S-OIV assay.
Testing of the Seasonal and H1 S-OIV assays demonstrated
outstanding clinical sensitivities, specificities, and agreement of
results with those of other molecular assays (7; Beck et al.,
unpublished). One advantage to real-time melt analysis for
influenza virus subtyping is that mutations in the probe region
can be readily seen by the shift of the melting curve. Two of the
288 S-OIV-positive clinical samples demonstrated a significant
melting-curve shift, suggesting mutations in the HA gene of
S-OIV. Upon HA gene sequence analysis for these two sam-
ples, different single mutations in the probe region were dis-
covered, explaining the observed shift in the Tm.
The easyMAG/Raider system developed for our Seasonal
assay demonstrated significant flexibility, allowing us to quickly
respond to the emergence of S-OIV infection in Milwaukee. We
were able to have a validated assay for the H1 gene of S-OIV up
and clinically available within 4 days of the first S-OIV se-
quence being available. The H1 S-OIV assay not only uses the
same internal control (MS2) but also runs on the same cycling
parameters and can be run at the same time in the same
microfluidic cartridges as the Seasonal assay. The only cur-
rently available FDA-approved influenza virus subtyping as-
says that are comparable in speed to our assay are the CDC
singleplex assays for H1, H3, and H5 and now their S-OIV
assay. These are only available through the Laboratory Re-
sponse Network and have not yet been distributed to other
laboratories in our area. The ability to rapidly test 329 samples
and provide specific influenza virus subtyping information in as
little as 3 h during the first 2 weeks of the S-OIV infection
outbreak allowed for timely and effective clinical and public
health decision making by health officials. On any one day, we
reported the presence of human H3N2 virus, H1N1 virus,
S-OIV, influenza B virus, and many other community-acquired
respiratory viruses. The fact that the influenza viruses have
TABLE 6. Performance characteristics of the Seasonal assay
compared to tissue culturea
Influenza A virus
Influenza B virus
a95% CI, 95% confidence interval; PPV, positive predictive value; NPV,
negative predictive value.
2784 BOSE ET AL.J. CLIN. MICROBIOL.
TABLE 7. Results of influenza virus subtyping during S-OIV infection outbreak in Milwaukee, WI, in 2009
(no. of samples)
Result for target (no. of samples) or no. of samples designated:
Agreement (95% CI)
Samples with resolved
Samples subjected to:
Influenza A/B virus-
Testing in Seasonal
Testing in H1
S-OIV assay (329)
Testing in FluPlex
aTesting was performed at the CHW (Beck et al., unpublished) and DL.
bMultiplex RT-PCR-enzyme hybridization influenza virus subtyping assay (4).
cSamples with discrepant results (no. 266, 301, 307, and 328): no. 301, influenza A virus (H1N1 S-OIV) positive in H1 S-OIV assay and FluPlex assay, with CTvalue just over the cutoff limit in the Seasonal assay; no.
328, influenza A (untyped) virus positive in FluPlex assay (only M gene detected); no. 266 and 307, subjected to repeated testing and found to be negative by the screening assay and negative by the Seasonal and FluPlex assays, with previous results all determined to be false positives.
dNA, not applicable.
eTwo influenza A H1N1 (human) virus samples (no. 39 and 107) were not subtyped by the Seasonal assay.
fOf the 110 H1 S-OIV assay-positive samples tested in the FluPlex, all 110 were positive for N1 S-OIV, showing a 100% correlation between the results of the two assays.
gPercent agreement is given for the following comparisons, from top to bottom: initial screening results versus Seasonal assay results for identification of influenza virus; Seasonal assay results versus H1 S-OIV results
for designation as negative for H1/H3 versus positive for S-OIV; and H1 S-OIV assay results versus FluPlex assay results. 95% CI, 95% confidence interval.
VOL. 47, 2009MULTIPLEX rRT-PCR TYPING/SUBTYPING OF FLU VIRUS/S-OIV2785
different antiviral susceptibilities makes rapid subtype report- Download full-text
ing critical to clinical management. Working closely with the
state and city public health officials, we provided rapid subtyp-
ing of S-OIV which helped them recognize the extent of the
Milwaukee outbreak earlier than would normally have been
possible. This recent outbreak of infection with a novel influ-
enza A (H1N1) virus demonstrates the importance of quickly
distributing information on new agents and of having rapid
influenza virus subtyping assays widely available.
We thank Jacob Metallo for his help with bioinformatics, Cecilia
Rebuffo-Scheer for help with assay development, and Jessica Trost and
Rose Chen for growing and quantitating the virus isolates used in this
This research was supported in part by grants UO1-AI77988, U01-
AI070428, and U01-AI066584 from the NIAID and by the Centers for
Disease Control and Prevention contract 200-2008-25466, which is a
cooperative agreement with Nanogen, Inc. (San Diego, CA) and Han-
dyLab, Inc. (Ann Arbor, MI).
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