Am. J. Trop. Med. Hyg., 85(5), 2011, pp. 961–963
Copyright © 2011 by The American Society of Tropical Medicine and Hygiene
Globally, influenza remains a leading cause of human mor-
bidity and mortality, largely as a result of the virus’s inher-
ent evasiveness from the immune response. 1 Coinfection
of viruses in birds or mammals, such as swine, increases the
chance for the emergence of new variants. 2, 3 Novel viruses
can emerge within a population, evade immunity, and result
in local epidemics or in some instances, pandemics. However,
recombination among subtypes remains rare. 4 In early 2009, a
novel influenza A/H1N1 virus (pH1N1) emerged in Mexico.
By October 2009, pH1N1 had become the predominant influ-
enza subtype infecting populations in most areas of the world. 5
Notwithstanding, in Southeast Asia, seasonal influenza viruses
as well as the avian influenza virus A/H5N1 continued to cir-
culate [World Health Organization (WHO) Pandemic (H1N1)
2009–Update 82; http://www.who.int/csr/don/2010_01_08/en/
index.html ). In the Southeast Asian nation of Cambodia, we
and others have shown that cases of influenza peak with the
monsoon between the months of July and December. 6, 7
In early October 2009, a 23-year-old man from central
Cambodia presented to the Ta Khmau health clinic with
influenza-like symptoms ( Table 1 ). Real-time reverse tran-
scriptase polymerase chain reaction (rRT-PCR) assays to
detect influenza A and B viruses were used to diagnose pH1N1
infection. 8 The patient received treatment to alleviate symp-
toms and recovered at his residence.
On October 14, 2009, three male (M) children, ages 13, 8,
and 4 years, who lived in the same home as the suspected index
case presented at the health clinic with fever (39°C), cough,
sore throat, headache, and symptoms of either nausea or vom-
iting ( Table 1 ). The students attended classes in a single-room
school. They reported neither recent extended contact with ani-
mals nor travel. A day after their illness, their teacher reported
febrile illness, and samples were obtained. Specimens from
one of three school children and the teacher indicated dual
infection with both seasonal A/H3N2 and pH1N1 viruses.
Virus isolation from collected clinical specimens was per-
formed in Madin-Darby canine kidney (MDCK) cells and
shell vials. Isolated viruses were analyzed on the Ibis T5000
by ESI-MS (Ibis Biosciences, Inc., Carlsbad, CA) analysis to
generate a specific mass measurement for each amplified PCR
product. Primer sequences and other PCR components were as
previously described. 9 The base composition signature for the
product was then compared with known sequences in a data-
base to generate an internally verified identification. Analyses
from six regions of the influenza genome confirmed pH1N1
infection in the 23-year-old man and dual A/H3N2 and pH1N1
infection in specimens from one of three children (8/M) and
the teacher (24/female [F]). Only A/H3N2 viruses were evi-
dent in samples from the 4/M and 13/M victims. Analysis did
not discern recombined signatures in gene segments within
the isolated viruses ( Table 2 ).
To fully characterize the gene segments of dual-infected
individuals, single-passaged viruses from two of the patients
and six of corresponding purified virus plaques were processed
for pyrosequencing using a specialized multisegment RT-PCR
procedure to amplify the genome of all subtypes of the influ-
enza A virus through degenerate primers. Sequencing, genome
assembly, and closure reactions were performed as previously
described. 10 Complete genomes (> 99% open reading frame
[ORF]) were obtained for all eight segments of each virus
isolate. A complete ORF region (100% genome length) was
obtained for all isolates. Sequences for the hemagglutinin
(HA) segment from the isolates were compared with known
sequences (data not shown). Relative to A/Perth/16/2009
(H3N2), the H3N2 vaccine component for 2010 and 2011, a
total of 3 aa substitutions were seen in the area sequenced,
I25V, P162T, and S214I, with an overall similarity of 99%. The
latter mutation corresponds to a previously identified anti-
body combining site. 11 More complete datasets for recent
swine strains allowed for a fuller comparison of pH1N1 HA
sequences. Comparison between the pH1N1 reference strain,
A/California/04/2009, and the full genome sequence of iso-
late material from 4/M revealed 4 aa changes from the vaccine
strain (P100S, S220T, I338V, and Y528H), with an overall sim-
ilarity of 99.3% over 566 aa. Phylogenetic analysis of the
HA genes of all analyzed plaques revealed a single genetic
sequence for both the A/H3N2 and pH1N1 strains. None of the
isolated plaques showed evidence of recombination between
Short Report : Dual Infection of Novel Influenza Viruses A/H1N1 and
A/H3N2 in a Cluster of Cambodian Patients
Christopher A. Myers , Matthew R. Kasper , Chadwick Y. Yasuda , Chin Savuth , David J. Spiro , Rebecca Halpin , Dennis J. Faix ,
Robert Coon , Shannon D. Putnam , Thomas F. Wierzba , and Patrick J. Blair *
Naval Health Research Center, San Diego, California; US Naval Medical Research Unit No. 2, Phnom Penh, Kingdom of Cambodia;
National Institute of Public Health, Ministry of Health, Phnom Penh, Kingdom of Cambodia; J. Craig Venter Institute, Rockville, Maryland
Abstract. During the early months of 2009, a novel influenza A/H1N1 virus (pH1N1) emerged in Mexico and quickly
spread across the globe. In October 2009, a 23-year-old male residing in central Cambodia was diagnosed with pH1N1.
Subsequently, a cluster of four influenza-like illness cases developed involving three children who resided in his home and
the children’s school teacher. Base composition analysis of internal genes using reverse transcriptase polymerase chain
reaction and electrospray ionization mass spectrometry revealed that specimens from two of the secondary victims were
coinfected with influenza A/H3N2 and pH1N1. Phylogenetic analysis of the hemagglutinin genes from these isolated
viruses showed that they were closely related to existing pH1N1 and A/H3N2 viruses circulating in the region. Genetic
recombination was not evident within plaque-purified viral isolates on full genome sequencing. This incident confirms
dual influenza virus infections and highlights the risk of zoonotic and seasonal influenza viruses to coinfect and possibly,
reassort where they cocirculate.
* Address correspondence to Patrick J. Blair, Department of Respi-
ratory Diseases, Naval Health Research Center, 140 Sylvester Road,
San Diego, CA 92106. E-mail: firstname.lastname@example.org
MYERS AND OTHERS
pH1N1 and A/H3N2, and all had full sequences for the eight
influenza segments from both strains.
Herein, we describe a cluster of influenza-like illness (ILI)
cases at a school in central Cambodia. Among the afflicted,
two were coinfected with A/H3N2 and pH1N1 influenza
viruses. The finding of coinfections has rarely been reported.
A recently study of over 2,000 clinical samples found no dual
infection. 12 However, coinfection of pH1N1 and A/H3N2 has
been reported in a 38-year-old woman from Singapore, 13 and
mixed infection was also evident in six individuals after an out-
break of influenza at a college campus near Beijing, China. 14 A
more recent New Zealand study collected and screened 1,044
clinical samples during the pandemic and found 11 coinfec-
tions with A/H1N1 seasonal viruses. 15
Transmission of pH1N1 at a time when seasonal influenza
viruses were circulating in Cambodia resulted in coinfection
and raised the possibility of reassortment. The generation of
novel influenza viruses through reassortment has occurred
when zoonotic viruses mix in birds, swine, and humans, and
gene segments are reshuffled. Pandemic strains often are the
result of emerging viruses from reservoirs to which humans
have little or no immunity. The A/H2N2 1957 and A/H3N2 1968
pandemics occurred after reassortment between human and
avian strains. 16, 17 The 1957 virus was generated when A/H1N1
1918 reassorted with avian viruses to pick up new PB1, HA,
and neuraminidase (NA) segments. Similarly, the novel virus
isolated from ILI cases in southern California in April 2009
contained genetic elements from four different sources, includ-
ing North American swine influenza viruses, North American
avian influenza viruses, human influenza viruses, and a Eurasian
swine influenza virus. 18 In our analysis , recombination was not
detected in viruses isolated from the Cambodian patients.
The clinical disease within the dual A/H3N2 and A/H1N1
Cambodian patients did not result in hospitalization nor did
these patients’ disease seem more severe than the disease
in the other patients with influenza. 19 Clinical findings were
broad, including upper respiratory and gastrointestinal symp-
toms. None of five patients in this outbreak had been vacci-
nated against either seasonal or pH1N1 influenza infections.
Indeed, in rural Cambodia, little seasonal influenza vaccination
is conducted, and use of therapeutics such as neuraminidase
inhibitors is rare 20 ; thus community-wide immunity is lacking.
Southeast Asia has proven to be a critical region for the
adaptation and emergence of variants of seasonal influenza
viruses 21 as well as an area of zoonotic virus transmission in
humans. Cases of A/H5N1 have largely been restricted to
the Near East and southeast Asia, with Cambodia suffering
15 confirmed human cases and 13 fatalities since 2005. The
endemicity of A/H5N1 in poultry in many areas of south-
east Asia provides increased opportunity for human expo-
sure and adaptation of a lethal virus suitable for sustained
human transmission. Our findings emphasize the importance
Demographics of Cambodian cases involved in influenza cluster
FSS08728 FSS08731 FSS08732FSS08733FSS09305
Clinical findings *
* Recorded on date of sample collection.
Base composition data from clinical samples
Patient Detection *
PB1NPM1 PA NS1NS2
A39 G32C24 T33
A41 G30C23 T34
A41 G30 C22 T35
A41 G20 C23 T34
A41 G30 C23 T34
A40 G31 C23 T34
A35 G21 C20 T25
A32 G24 C20 T25
A35 G21 C20 T25
A32 G24 C20 T25
A35 G21 C20 T25
A24 G28 C24 T29
A25 G29 C21 T30
A25 G27 C24 T29
A25 G29 C21 T30
A25 G29 C21 T30
A25 G27 C23 T30
A25 G29 C21 T30
A36 G25 C27 T24
A31 G25 C25 T31
A37 G24 C26 T25
A41 G22 C26 T23
A40 G23 C27 T22
A36 G36 C20 T28
A40 G30 C20 T29
A37 G34 C19 T29
A41 G29 C19 T30
A40 G30 C20 T29
A37 G34 C19 T29
A37 G25 C16 T27
A35 G28 C15 T27
A37 G25 C16 T27
A37 G25 C16 T27
A34 G29 C16 T26
A37 G25 C16 T27
ND = not determined.
* The listing of strain determinations made by the PlexID instrument based on the base compositions detected in the sample.
† The more recent version of the influenza surveillance plate that was used for sample 2 3/ M does not include an NS2 primer pair.
963 Download full-text
DUAL INFLUENZA INFECTIONS, CAMBODIA 2009
of national and international cooperation to survey for the
emergence of novel and/or reassorted influenza viruses.
Received February 16, 2011. Accepted for publication June 6, 2011.
Acknowledgments: This work would not be possible without the sub-
stantial daily efforts of the staff at the clinical sites in Kandal Province.
The authors thank the laboratories at Naval Health Research Center
and J. Craig Venter Institute and the US Naval Medical Research Unit
No. 2 and National Institute of Public Health, Kingdom of Cambodia,
for their contributions in diagnosing and characterizing resulting
viruses. This research has been conducted in compliance with all appli-
cable federal regulations governing the protection of human subjects
in research (Protocols NAMRU2.2005.0004 and NHRC.2010.0007).
Financial support: This work was funded in part by grants from the
US Department of Defense Armed Forces Health Surveillance
Center division of the Global Emerging Infections Surveillance and
Response System (AFHSC/GEIS) and the US Defense Advanced
Research Projects Agency, (DARPA) under work unit number
60941. A portion of this project was funded by the National Institute
of Allergy and Infectious Diseases, National Institute of Health,
Department of Health and Human Services under contract number
Disclaimer: The views expressed in this article are of the authors and
do not reflect the official policy or position of the Department of the
Navy, the Department of Defense, or the US Government.
Authors’ addresses: Christopher A. Myers, Dennis J. Faix, and Robert
Coon, Naval Health Research Center, San Diego, CA, E-mails: Chris
.Myers2@med.navy.mil , email@example.com , and robert.coon@
med.navy.mil . Matthew R. Kasper, Chadwick Y. Yasuda, Shannon D.
Putnam, and Thomas F. Wierzba, US Naval Medical Research Unit
No. 2, Phnom Penh, Kingdom of Cambodia, E-mails: Matthew.Kasper@
med.navy.mil , Chad@namru2.org.kh , firstname.lastname@example.org ,
and twierzba@IVI.int . Chin Savuth, National Institute of Public
Health, Ministry of Health, Phnom Penh, Kingdom of Cambodia,
E-mail: email@example.com . David J. Spiro and Rebecca Halpin,
J. Craig Venter Institute, Rockville, MD, E-mails: firstname.lastname@example.org
and email@example.com . Patrick J. Blair, Naval Health Research Center,
San Diego, CA and US Naval Medical Research Unit No. 2, Phnom
Penh, Kingdom of Cambodia, E-mail: firstname.lastname@example.org .
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