Molecular identification of adenovirus causing respiratory tract infection in pediatric patients at the University of Malaya Medical Center

  • Juraina Abd-Jamil1,

    Affiliated with

    • Boon-Teong Teoh1,

      Affiliated with

      • Eddy H Hassan1,

        Affiliated with

        • Nuruliza Roslan1 and

          Affiliated with

          • Sazaly AbuBakar1Email author

            Affiliated with

            BMC Pediatrics201010:46

            DOI: 10.1186/1471-2431-10-46

            Received: 17 November 2009

            Accepted: 2 July 2010

            Published: 2 July 2010

            Abstract

            Background

            There are at least 51 adenovirus serotypes (AdV) known to cause human infections. The prevalence of the different human AdV (HAdV) serotypes varies among different regions. Presently, there are no reports of the prevalent HAdV types found in Malaysia. The present study was undertaken to identify the HAdV types associated primarily with respiratory tract infections (RTI) of young children in Malaysia.

            Methods

            Archived HAdV isolates from pediatric patients with RTI seen at the University of Malaya Medical Center (UMMC), Kuala Lumpur, Malaysia from 1999 to 2005 were used. Virus isolates were inoculated into cell culture and DNA was extracted when cells showed significant cytopathic effects. AdV partial hexon gene was amplified and the sequences together with other known HAdV hexon gene sequences were used to build phylogenetic trees. Identification of HAdV types found among young children in Malaysia was inferred from the phylograms.

            Results

            At least 2,583 pediatric patients with RTI sought consultation and treatment at the UMMC from 1999 to 2005. Among these patients, 48 (< 2%) were positive for HAdV infections. Twenty-seven isolates were recovered and used for the present study. Nineteen of the 27 (~70%) isolates belonged to HAdV species C (HAdV-C) and six (~22%) were of HAdV species B (HAdV-B). Among the HAdV-C species, 14 (~74%) of them were identified as HAdV type 1 (HAdV-1) and HAdV type 2 (HAdV-2), and among the HAdV-B species, HAdV type 3 (HAdV-3) was the most common serotype identified. HAdV-C species also was isolated from throat and rectal swabs of children with hand, foot, and mouth disease (HFMD). Two isolates were identified as corresponding to HAdV-F species from a child with HFMD and a patient with intestinal obstruction.

            Conclusions

            HAdV-1 and HAdV-2 were the most common HAdV isolated from pediatric patients who sought treatment for RTI at the UMMC from 1999 to 2005. HAdV-B, mainly HAdV-3, was recovered from ~22% of the patients. These findings provide a benchmark for future studies on the prevalence and epidemiology of HAdV types in Malaysia and in the region.

            Background

            Respiratory tract infections (RTI) are common in adults and children worldwide. The disease varies in severity, presenting as uncomplicated, subacute, acute and chronic infection. RTIs can be life threatening depending on the causative agent and host condition. In children, a high incidence of RTI is caused either by: 1) heightened exposure of young children to RTI infectious agents from siblings, friends, and child care; 2) environmental factors; and 3) inherited disorders of the immune system [1]. In industrialized and developed countries, nearly 50% of pediatric consultations are RTI related [2, 3], and at least 1.9 million children died from acute RTI with 70% of them in Africa and Asia in 2000 [4].

            There are a number of infectious agents that cause RTI. Bacteria, such as Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, Mycoplasma pneumonia, and Chlamydia trachomatis are among the most common. However, 80%-90% RTI are caused by viruses, such as respiratory syncytial virus (RSV), influenza virus, parainfluenza virus, and adenovirus (AdV) [1, 5]. RSV is one of the most common agents of RTI in infants and young children in many countries. It is estimated to cause ~39% of all pneumonia cases and up to 6% of pneumonia-associated deaths [6]. Parainfluenza virus and influenza virus also are commonly isolated viruses from children with viral RTI.

            HAdV-associated RTI is reportedly low. It accounts for 4%-10% cases of pneumonia, 2%-10% cases of bronchiolitis, and 3%-9% cases of croup [7]. Severity of infection associated with HAdV varies with the different HAdV serotypes [79]. There are at least 51 immunologically distinct HAdV types classified into six species, designated A to F [10]. Viruses causing RTI are usually isolated in the laboratory from patients' nasal secretions and serotyped by immunological typing methods [1113]. In recent years, molecular identification of the virus has become more common, where distinction of the virus types can be made through specific genomic sequence amplification by polymerase chain reaction (PCR) and determination of the partial hexon gene sequence [14, 15]. Presently, there is no report of the prevalent HAdV types causing infections among Malaysians. This could be due to the low infection and fatality rates of the infection resulting in limited interest in typing the virus. The present study was undertaken to type HAdV of pediatric patients younger than 5 years seen at the University of Malaya Medical Center (UMMC), Kuala Lumpur, Malaysia.

            Methods

            Virus

            Twenty-seven archived HAdV isolates were recovered from the UMMC virology repository and used for the study. The isolates were derived mainly from the nasopharyngeal secretions (NPS) of children younger than 5 years diagnosed with RTI (Table 1). Virus isolation and propagation were performed using African green monkey kidney cells (Vero), human lung carcinoma cells (A549), and dog kidney cells (MDCK). The presence of HAdV was detected by immunofluorescence staining using specific antibodies (Cat. No. 5000; Light Diagnostics Inc., Salt Lake City, UT, USA) following manifestation of cytopathic effects. Virus inoculum was prepared and kept at -70°C until needed for genomic typing. Further maintenance and propagation of the HAdV isolates were performed in A549 cells.
            Table 1

            Human adenoviruses isolated at the University of Malaya Medical Centre from 1999 to 2005.

            Sample

            Year

            Isolation sitea

            Age

            Diagnosis

            Serotype

            AD01MY99

            1999

            NPS

            NAb

            Pneumonia

            B3

            AD02MY99

            1999

            NPS

            3 mth

            Chronic lung diseases

            B7

            AD04MY00

            2000

            RS

            7 mth

            HFMD

            F40

            AD05MY00

            2000

            TS

            1 yr

            HFMD

            C2

            AD06MY00

            2000

            TS

            8 mth

            HFMD

            C1

            AD07MY01

            2001

            RS

            6 mth

            Intestinal obstruction

            F41

            AD08MY00

            2000

            RS

            NA

            HFMD

            C5

            AD09MY01

            2001

            NPS

            I yr

            Acute bronchiolitis

            C1

            AD10MY01

            2001

            NPA

            1 yr

            Bronchiolitis

            C2

            AD11MY02

            2002

            NPS

            1 yr

            Bronchopnuemonia, hypochronic anemia

            C1

            AD12MY02

            2002

            NPS

            1 yr

            Bronchiolitis

            C1

            AD13MY03

            2003

            NPS

            10 mth

            Acute gastroenteritis

            C2

            AD14MY03

            2003

            NPS

            1 yr

            Viral fever

            C5

            AD15MY03

            2003

            NPS

            8 mth

            Acute bronchiolitis

            C2

            AD16MY03

            2003

            NPS

            NA

            Bronchopneumonia

            C2

            AD17MY04

            2004

            NPS

            2 yr

            Acute bronchiolitis

            C1

            AD18MY04

            2004

            NPS

            9 mth

            Pneumonia

            C5

            AD19MY04

            2004

            NPS

            5 mth

            Bronchiolitis

            B3

            AD20MY04

            2004

            NPS

            2 yr

            Pneumonia

            C2

            AD21MY04

            2004

            NPS

            3 yr

            Pneumonia

            C1

            AD22MY04

            2004

            NPS

            1 yr

            Bronchopneumonia

            C2

            AD23MY04

            2004

            NPS

            5 mth

            Bronchiolitis

            C6

            AD24MY04

            2004

            NPS

            3 yr

            Viral fever

            B3

            AD25MY04

            2004

            NPS

            8 mth

            Pulmonary collapse

            C5

            AD26MY04

            2004

            NPS

            1 yr

            Pneumonia

            B3

            AD27MY04

            2004

            NPS

            3 mth

            Acute bronchiolitis

            C2

            AD28MY05

            2005

            NPS

            2 yr

            Bronchopneumonia

            B3

            aNPS is nasopharyngeal secretion; RS is rectal swab; TS is throat swab

            bNot available

            PCR amplification and genome sequencing

            Viral genomic DNA was extracted using Tri Reagent® (Molecular Research Center Inc., Cincinnati, OH, USA) following the manufacturer's protocol. The partial hexon gene was amplified using the primer pair, AdTU7 (5'-GCCACCTTCTTCCCCATGGC-3') and AdTU4 (5'-GTAGCGTTGCCGGCCGAGAA-3') to amplify a 1,001 bp fragment of the partial hexon gene (position 20,733 - 21,734; Accession # NC_001405). A nested polymerase chain reaction (PCR) also was performed on the amplified fragment using the primers, AdU-S (5'-TTCCCCATGGCNCACAACAC-3') and AdU-A (5'-GCCTCGATGACGCCGCGGTG-3') which resulted in a 956 bp fragment. Amplification was performed for 36 cycles consisting of a denaturation step at 94°C for 1 min, an annealing step at 50°C for 1 min, and an extension step at 72°C for 2 min. The extension was continued at 72°C for 7 min. The amplified DNA fragment was separated in 1.5% agarose gel (Promega, Madison, WI, USA), and purified using QIA Quick gel extraction system (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. The DNA fragments were sequenced at Macrogen Inc. (Seoul, Korea).

            Sequence and phylogenetic analysis

            The partial hexon gene sequences were aligned and phylogenetic trees were drawn as previously described [16]. Briefly, the HAdV partial hexon gene sequences were analyzed using Sequencher 4.6 (Gene Codes Corporation, Ann Arbor, MI) and aligned against other available AdV sequences using ClustalX [17]. Phylogenetic trees were drawn using the maximum-likelihood method as implemented in PHYLIP 3.67 [18] and the maximum-parsimony method using MEGA4 [19]. Bootstrap values were obtained from a random sampling of 100 replicates. Reference HAdV sequences used to build the phylogenetic trees were obtained from the GenBank. Details on the reference sequences are shown in Table 2.
            Table 2

            Reference adenovirus (AdV) strains from the Genbank used for the typing of human AdV isolated at the University of Malaya Medical Centre.

            Accession Number

            Serotype

            Country/Strain

            HAdV_A_NC_001460

            A12

            ATCC

            HAdV_B_NC_004001

            B11

            Slobitski

            HAdV_C_NC_001405

            C2

            NAa

            HAdV_D_NC_002067

            D17

            NA

            HAdV_E_NC_003266

            E4

            Vaccine CL68578

            HAdV_F_NC_001454

            F40

            Dugan

            HAdV_F_L19443

            F40

            Dugan

            HAdV_3_AY878716

            B3

            Guangzhou, China

            HAdV_3_AF542108

            B3

            Korea

            HAdV_3_AF542129

            B3

            Korea

            HAdV_3_DQ086466

            B3

            NA

            HAdV_7_AF053085

            B7

            S-1058; Japan

            HAdV_7_AY769945

            B7

            95-81; Korea

            HAdV_7_AF515814

            B7

            China

            HAdV_7_AC_000018

            B7

            NA

            HAdV_7_AY769946

            B7

            Strain 99-95; Korea

            HAdV_7_AB243009

            B7

            Kyoto, Japan

            HAdV_11_AC_000015

            B11

            NA

            HAdV_11_AF532578

            B11

            Slobitski

            HAdV_11_AY163756

            B11

            Ad11p Slobitski

            HAdV_14_AY803294

            B14

            De Wit/ATCC VR1091

            HAdV_14_DQ149612

            B14

            NA

            HAdV_16_X74662

            B16

            ATCC CH.79

            HAdV_21_AB053166

            B21

            AV-1645

            HAdV_34_AY737797

            B34

            Compton/ATCC VR716

            HAdV_34_AB052911

            B34

            NA

            HAdV_35_AC_000019

            B35

            Old world monkey strain

            HAdV_35_AY128640

            B35

            Holden/ATCC VR718

            HAdV_35_AY271307

            B35

            35p

            HAdV_35_AB052912

            B35

            Japan

            HAdV_50_DQ149643

            B50

            NA

            HAdV_50_AY737798

            B50

            Wan/ATCC VR1502

            HAdV_1_AC_000017

            C1

            P1 C124G1

            HAdV_1_Y17244

            C1

            Ad71

            HAdV_2_AC_000007

            C2

            NA

            HAdV_2_AY224392

            C2

            Korea

            HAdV_2_AF542118

            C2

            Korea

            HAdV_2_AJ293905

            C2

            Germany

            HAdV_2_AJ293901

            C2

            United Kingdom

            HAdV_5_AF542130

            C5

            Korea

            HAdV_5_AF542128

            C5

            Korea

            HAdV_5_AF542124

            C5

            Korea

            HAdV_5_AC_000008

            C5

            NA

            HAdV_5_AF542109

            C5

            Korea

            HAdV_6_DQ149613

            C6

            NA

            HAdV_6_Y17245

            C6

            Ton66

            HAdV_40_X51782

            F40

            Dugan

            HAdV_41_X51783

            F41

            Tak

            HAdV_41_D13781

            F41

            Tak

            aNot Available

            The study was approved by the University Malaya Medical Centre Ethics Committee (Approval #794.6).

            Results

            A total of 2,583 pediatric patients with RTI were treated at the UMMC from 1999 to 2005. Of these patients, 48 (<2%) were positive for HAdV by either direct immunofluorescence staining of the patient's NPS, PCR amplification, or virus isolation. HAdV also was isolated from the throats of patients with HFMD. These HFMD patients usually did not present with RTI symptoms, but throat swabs were routinely collected in addition to rectal swabs.

            In the present study, the partial hexon gene sequences (nucleotide 20,734-21,737) of 27 HAdV isolates from UMMC were determined. The sequences were aligned and phylogenetic trees were drawn using the maximum-likelihood and maximum-parsimony methods. Only results from the maximum-likelihood method were presented, as trees drawn from both the methods were similar. The nucleotide sequence alignment clustered the UMMC isolates into HAdV-C species (n = 19), HAdV-B species (n = 6), and HAdV-F species (n = 2) with high bootstrap values. Within the HAdV-C species, eight isolates were HAdV-2 (42%); six isolates, HAdV-1 (32%); four isolates, HAdV-5 (21%);, and one isolate, HAdV-6 (5%; Figure 1a). When compared with other HAdV from the GenBank, most of the UMMC HAdV-5 and HAdV-2 isolates formed a separate subcluster of their own (Figure 1a). Within HAdV-B species, five isolates grouped into HAdV-3, and one was HAdV-7 (Figure 1b). HAdV-3 isolates from the study formed a separate subcluster together with an isolate from Guangzhou, China (Accession # AY878716) distinct from other known HAdV-3. This raised the possibility that the viruses may share a common ancestral lineage with the Malaysian isolate that was isolated in 1999. The HAdV-7 clustered together with isolates from the East Asian countries suggesting a possible widespread presence of the virus in the East Asian region. Four of the HAdV isolates sequenced in the study were from HFMD patients, and three of them clustered together with the HAdV-C species. One isolate clustered with known HAdV-40, whereas an isolate from the rectal swab of a patient diagnosed with intestinal obstruction clustered with known HAdV-41. No HAdV-B genotype previously associated with fatal cases of HFMD [20, 21] was detected in any of the HFMD patient samples. Isolates from HFMD patients were included in the study initially because no effort was made to discriminate the samples from those strictly with RTI. Patients with HFMD normally would not present with RTI symptoms.
            http://static-content.springer.com/image/art%3A10.1186%2F1471-2431-10-46/MediaObjects/12887_2009_Article_358_Fig1_HTML.jpg
            Figure 1

            Molecular typing of human adenoviruses (HAdV) isolated at the University of Malaya Medical Center (UMMC) from 1999 to 2005. HAdV-1 and HAdV-2 comprised most of the HAdV-C species isolated at the UMMC (a). HAdV-B species consisted of HAdV-3 and HAdV-7. HAdV-3 formed its own distinct cluster separate from the rest of the group (b). Two isolates from the study were grouped as HAdV-F serotype 40 and 41 (c), commonly implicated in gastroenteritis.

            Discussion

            Human adenovirus is most commonly associated with respiratory illnesses. However, depending on the infecting serotype, the virus also causes various other illnesses, including gastroenteritis, conjunctivitis, cystitis, and non- specific exanthemas [13, 22]. Symptoms of the respiratory illness associated with HAdV range from mild infection to severe pneumonia [8, 23]. Young children and immunocompromised patients are especially vulnerable to severe complications of HAdV infection [24, 25]. The findings that less that 2% of UMMC pediatric RTI is associated with HAdV respiratory infection is consistent with other reports that HAdV-associated respiratory infection is usually low in comparison to other viruses, such as RSV and parainfluenza virus. The infection also is generally milder and rarely leads to severe complications and deaths [8, 26]. The low number of HAdV isolation among pediatric patients seen at the UMMC also suggests that the virus is not associated with any major RTI outbreaks during the period from 1999 to 2005. This is perhaps among the reasons why there have not been many efforts to identify the HAdV species and types in children with RTI in many countries, including Malaysia. In addition, the low incidence of RTI caused by HAdV in the community hampered the effort to get enough representative isolates.

            In our study, HAdV partial hexon gene sequences were used to type the different HAdV isolates. This gene region contains the hypervariable region that confers HAdV serotype specificity. Using this molecular typing method, HAdV-C species, specifically type 1 and 2, were the most common HAdV isolated from the pediatric patients seen at UMMC from 1999 to 2005. In contrast, studies done in the United States of America, United Kingdom, Korea, and China, showed HAdV-B species as the most commonly isolated HAdV [2730]. The reasons for the marked differences are not known. It could be that HAdV-C is more common in the region in comparison to the more developed countries. However, the prevalence of HAdV-C species in the neighboring countries could not be compared because information from these countries are lacking.

            Overrepresentation of HAdV-C in UMMC pediatric patients could suggest a high prevalence of the virus in the community. There are reports that the virus could persist and cause asymptomatic latent infection in rabbits for as long as one year [31]. HAdV-C serotypes 1, 2, and 5 are the most common serotype of HAdV associated in latent infection of tonsils and adenoids of humans, which at times cause RTI in young children [32]. The prolonged presence of the virus in infected children increases its transmissibility, and this could contribute to the persistence of the virus of young children in Malaysia. The ubiquitous presence of the virus also could help explain isolation of the virus from patients with HFMD and nonspecific viral fever. On the other hand, the results also could reflect the higher tendency of children with HAdV-C species infection to seek medical attention, hence suggesting that the virus could cause more severe manifestations of RTI. Further studies, however, will be needed to verify this.

            Conclusions

            The present study is the first to report the prevalent and circulating HAdV types in Malaysia. It showed that HAdV-C species especially HAdV-1 and HAdV-2 were the most commonly isolated HAdV among pediatric patients seen at UMMC from 1999 to 2005, followed by HAdV-B species type 3. These viruses are common serotypes of HAdV causing acute RTI in pediatric patients. Because no such study has ever been reported in Malaysia, the present study provides a benchmark for future studies of HAdV infection in the country.

            Abbreviations

            HAdV: 

            human adenovirus

            HFMD: 

            hand, foot and mouth disease

            NPS: 

            nasopharyngeal secretion

            RTI: 

            respiratory tract infection

            UMMC: 

            University of Malaya Medical Center

            Declarations

            Acknowledgements

            We thank the Department of Medical Microbiology, University of Malaya for allowing the study to be undertaken using virus isolates from the UMMC Virology repository. None of the authors received specific funding for this study.

            Authors’ Affiliations

            (1)
            Tropical Infectious Diseases Research and Education Center (TIDREC), Department of Medical Microbiology, Faculty of Medicine, University of Malaya

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            33. Pre-publication history

              1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2431/​10/​46/​prepub

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            © Abd-Jamil et al. 2010

            This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.