JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2010, p. 1438–1441 0095-1137/10/$12.00 doi:10.1128/JCM.01928-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 4
Outbreak of Febrile Respiratory Illness Associated with Adenovirus 11a Infection in a Singapore Military Training Camp䌤 Adriana E. Kajon,1* Laura M. Dickson,1 David Metzgar,2 Huo-Shu Houng,3 Vernon Lee,4 and Boon-Huan Tan5 Infectious Disease Program, Lovelace Respiratory Research Institute (LRRI), 2425 Ridgecrest Drive SE, Albuquerque, New Mexico 871081; Department of Respiratory Disease Research, Naval Health Research Center (NHRC), 140 Sylvester Rd., San Diego, California 92106-35212; Department of Virus Diseases, Walter Reed Army Institute of Research (WRAIR), 503 Robert Grant Avenue, Silver Spring, Maryland 209103; Biodefense Center, Headquarters for Medical Corps, Singapore Armed Forces, 701 Transit Road 04-01, Singapore 778910, Republic of Singapore4; and Detection and Diagnostics Laboratory, Defense Medical and Environmental Research Institute, DSO National Laboratories, 27 Medical Drive, Singapore 117510, Republic of Singapore5 Received 29 September 2009/Returned for modification 21 December 2009/Accepted 28 January 2010
Outbreak cases of acute respiratory disease (ARD) associated with subspecies B2 human adenovirus 11a (HAdV-11a) infection were detected during 2005 in a military basic training camp in Singapore. The Singapore HAdV-11a strain is highly similar to other Asian strains of HAdV-11, including strain QS-DLL, which is responsible for the recently described 2006 outbreak of ARD in China. among these patients. Recruits testing positive for HAdV were between 19 and 21 years old. The examination of clinical characteristics of the HAdV-positive cases of ARD showed that 23.3% of the recruits reported shortness of breath, 50% reported nasal congestion, 80% reported a headache, 80% reported body ache, and 13.3% reported signs of nausea or vomiting. The identified influenza A virus coinfection did not increase the severity of the respiratory symptoms. One patient presented with additional symptoms of conjunctivitis, but no eye swab samples were collected. Adenovirus isolation was accomplished for 27 of the 30 positive clinical specimens. All virus isolates were initially typed as species B HAdVs by PCR as described previously by Metzgar and colleagues (22). Isolates were further characterized by restriction enzyme analysis and sequencing of the hexon and fiber genes as described previously by Kajon and Erdman (13). Digestion with BamHI identified 26 of 27 isolates as belonging to subspecies B2 HAdVs and one isolate as being HAdV-3. Digestion of viral DNAs with BclI, BglII, BstEII, DraI, HindIII, PstI, SmaI, and XbaI determined that the 26 subspecies B2 isolates were identical and identified them as corresponding to genome type 11a (Fig. 2A) (17). The HAdV-3 isolate was identified as belonging to genome type 3a2 (Fig. 2B) (16). By using the primer sets described in Table 1, identical hexon and fiber sequences were obtained for three randomly selected HAdV-11a isolates (GenBank accession no. FJ607010 and FJ603103 for isolate SNG1218, accession no. FJ607011 and FJ603104 for isolate SNG1222, and accession no. FJ607012 and FJ603105 for isolate SNG1223). Alignment of the sequences of the hexon gene corresponding to hypervariable regions 1 to 7 using the Basic Local Alignment Search Tool (BLAST) optimized for highly similar sequences (megablast) against the NCBI GenBank database (http://www.ncbi .nlm.nih.gov) showed the three examined viruses to correspond to serotype 11 (19, 28). By using ClustalW implemented in Lasergene (DNASTAR, Inc., Madison, WI), the sequences
Due to unique risk factors that include crowding and increased physical and psychological stress, military recruits in training are highly susceptible to outbreaks of acute respiratory disease (ARD), which are most often caused by viruses (26, 29). Human adenovirus (HAdV) infections have been recognized for decades as being important causes of ARD among military trainees in North America, Europe, and Asia (5, 8, 10, 15, 25, 27, 30, 32). The HAdV serotypes most frequently associated with ARD in both military and civilian communities include subspecies B1 HAdV-3, HAdV-7, and HAdV-21 and species E HAdV-4. The association of subspecies B2 HAdV infection with ARD has historically been rarely reported and restricted mostly to closed community outbreaks, some of which were documented among military trainees (1, 2, 8, 24, 36). From 11 January 2005 to 14 October 2005, a total of 226 male participants aged 18 to 24 years were enrolled in a study designed to detect and characterize viral agents responsible for ARD in the Singapore military recruit population. Analysis of laboratory data collected during this period identified influenza virus as the primary causative viral agent of respiratory illness among Singapore recruits in training (51 influenza viruspositive isolates out of 226 tested cases [22.5%]). HAdV-associated ARD cases were detected sporadically between January and October 2005. Thirty symptomatic trainees (13.3%) tested positive for HAdV during this period by a PCR assay described previously by Echavarria and colleagues (6). The temporal distribution of confirmed HAdV-associated cases of ARD is shown in Fig. 1. Cases of adenovirus-associated ARD were detected between February and June 2005 and in October 2005. One case of coinfection with influenza A virus was detected
* Corresponding author. Mailing address: Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108. Phone: (505) 348-9159. Fax: (505) 348-8567. E-mail:
[email protected]. 䌤 Published ahead of print on 3 February 2010. 1438
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TABLE 1. Primers used for amplification and sequencing of hexon and fiber genes of Singapore HAdV-11 isolatesa Primer
Sequence (5⬘33⬘)
Hexon Forward hex1 ..................CGTCGACGCTGAGTTAC Reverse hex 6..................ACATCGGGATCATAACTGTCAAC HVR-7 forward...............GTCTTATGTACTATAACAGTACTGG HVR-7 reverse................GTGGTTGAATGGGTTGAC Fiber Forward............................AGCGGCATACTTTCTCCATAC Reverse ............................GGGAGGCAAAATAACTACTCG a
FIG. 1. Temporal distribution of HAdV-associated cases of ARD diagnosed among Singapore military recruits during 2005.
for both the hexon and the fiber genes of the Singapore HAdV-11a strain were found to be highly similar to those reported for Asian and Middle Eastern HAdV-11 strains circulating over the last 2 decades in association with respiratory disease (Table 2). In addition, the complete genomic sequence obtained at the Walter Reed Army Institute of Research for isolate SNG1222 (GenBank accession no. FJ597732) was
FIG. 2. (A) Restriction enzyme analysis of a representative HAdV11a isolate (SNG1222). (B) Restriction enzyme analysis of HAdV-3a2 isolate SNG1206. M, 1-kbp and 100-bp molecular markers.
Primers were used for amplification and sequencing.
found to be 99.9% identical to that reported previously for strain QS-DLL, isolated in China in 2006 during an outbreak of ARD (33) (GenBank accession no. FJ643676).The identified differences between these two genomes are listed in Table 2. The results of this study suggest that, in contrast to other geographic locations where HAdV-3, -4, -7, and -21 rank among the most prevalent serotypes, HAdV-11 may be a relatively more important causative agent of ARD in military training facilities in Singapore. The circulation of HAdV-11 in South East Asia has been documented since the early 1960s in association with conjunctivitis, pharyngoconjuctival fever, ARD, and hemorrhagic cystitis among immunocompromised individuals (7, 9, 14, 17, 24, 31, 34–36). The work of Wadell and colleagues demonstrated the existence of two main clusters of relative homology for HAdV-11 genome types: the cluster of prototype-like genomic variants with a tropism for the renal epithelium, and the a-like cluster, with a distinct tropism for the respiratory tract. In addition to their unique restriction site maps, the p-like and a-like genomes differ in the sequences of the fiber gene involving the receptor binding domains (17, 20, 21). As noted by others previously (33), the fiber of the 11a-like genomes is more closely related to the fiber of the prototype strain of HAdV-14, de Wit (99.5% identity), than to the fiber of the prototype strain of HAdV-11, Slobitski (94.4% identity), suggesting that this genomic variant of HAdV-11 is an intertypic recombinant 11-14. Except for strain BC34, isolated in Beijing, China, between 1965 and 1985 (17), none of the HAdV-11 strains with which the Singapore HAdV-11 isolates share high sequence similarities have been genotyped. However, the available sequence data indicate that all the Asian and Middle Eastern respiratory HAdV-11 strains used for comparison in this study represent closely related viruses likely belonging to genome type 11a, as described previously by Li and colleagues (17). Our data confirm the long-lasting prevalence of genome type HAdV-11a in South East Asia and, together with data from recent studies from China (33, 36), support the observation that HAdV-11a is an important respiratory pathogen in the region and the hypothesis that this HAdV-11 genomic variant is a recombinant between HAdV-11 and HAdV-14 ancestral strains. At present, the circulation of HAdV-11 has not been detected in association with respiratory illness outbreaks among U.S. military trainees, but HAdV-11 was confirmed to be the causative agent of a large outbreak of ARD in a job-training
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J. CLIN. MICROBIOL. TABLE 2. Comparative sequence analysis of Singapore HAdV-11a and other Asian strains of HAdV-11a
Strain or isolate
Origin
Hexon HVR1-7 QS-DDL China, 2006/ARD
Reference
36
GenBank % nt sequence accession no. identity
DQ874353
99.9
91-038T
Japan, 1991/throat Unpublished swab
AB162772
99.9a
R1332 RKI-2797/04
Kuwait, 2007 Turkey, 2004
Unpublished 4
EU755357 AY972815
100b 99.9c
20
L08232
99.9
Unpublished
AB162822
99.9
4
AY972816
99.8d
QS-DDL
China, 1965-1985/ ARD Japan, 1991/throat swab Turkey, 2004/ARD China, 2006/ARD
33
FJ643676
99.9
HAdV-11p Slobitsky HAdV-14p de Wit
United States, 1957 Netherlands, 1957/ARD
20
AY163756
94.4
AB065116
99.5
China, 2006/ARD
33
FJ643676
99.9
Fiber BC34 91-038T RKI-2797/04
Whole genome QS-DDL
Identified difference(s)
1 synonymous point mutation at nt position 1647 of the hexon gene nt deletions at positions 1583, 1590, and 1597; one nonsynonymous point mutation at position 1617 of the hexon gene None 1 nonsynonymous point mutation at nt position 1075 of the hexon gene 1 nonsynonymous point mutation at nt position 127 of the fiber gene 1 nonsynonymous point mutation at nt position 127 of the fiber gene 1 synonymous point mutation at nt position 138 of the fiber gene 1 nonsynonymous point mutation at nt position 127 of the fiber gene Multiple,g plus one nonsynonymous point mutation at nt position 127 of the fiber gene Nonsynonymous point mutations at nt positions 250, 346, and 412 of the fiber gene; 1 synonymous point mutation at nt position 774 Point mutations at positionse 134, 202, 1721, 1885, 2170, 2823, 8351, 9253, 11167, 11293, 13328, 18072, 19849, 22846, 24348, 25509, 26060, 27198, 28745, 28827, 29204, 29429, 30259, 30378, 30501, 30901, and 34617; insertion of sequence TTCCATATCCGTG downstream of the stop codon for E1A at nt position 1463; length of poly(A) tracts starting at positions 581, 3908, 29462c,f 33984; length of poly(A) signals for L1, L2
a
Sequence data available for the hexon gene of strain 91-038T comprise nucleotide (nt) 346 to nt 1635. Sequence data available for the hexon gene of strain R1332 comprise nt 991 to nt 1604. Sequence data available for the hexon gene of strain RKI-2797/04 comprise nt 107 to nt1541. d Sequence data available for the fiber of strain RKI-2797/04 comprise nt 95 to nt 675. e Nucleotide positions based on the genome of HAdV-11 strain QS-DLL (GenBank accession no. FJ643676). f c, complementary strand. g As described previously by Mei and Wadell (20) and Yang et al. (33) b c
facility in South Dakota in 1997, documenting the circulation of this serotype in association with respiratory disease in the United States (1). The detection of HAdV-11 in some collections of respiratory HAdVs gathered over the last 2 decades in South and North America, Asia, and the Middle East (3, 4, 11, 12, 22; A. Kajon, unpublished data) and the recently reported emergence of HAdV-14 in North America (2, 18, 23) suggest an increasing role for these subspecies B2 serotypes in the etiology of ARD in several areas of the globe. Continuous surveillance and molecular characterization of respiratory adenovirus isolates worldwide will contribute to the elucidation of the natural history and pathogenesis of species B2 HAdV respiratory infections. The protocol for this study was approved by the Joint Medical Committee for Research, Singapore Armed Forces, and participating institutions in the United States as IRB exempt. We acknowledge Shirley Seah Gek-kheng, Elizabeth Ai-Sim Lim, Jasper Chin-Wen Liaw (DSO), and Susan Core (LRRI) for technical assistance. Funding for this work was provided by the Global Emerging Infections Surveillance and Response System, a Division of the U.S. Armed
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