Human adenoviruses (HAdVs) were initially characterized as respiratory tract pathogens in the early 1950s (Hllleman M R, et al., 1954; Rowe W P, et al., 1953). HAdV infections are responsible for a large spectrum of diseases in human, such as Acute Respiratory Disease (ARD), pharyngoconjunctivitis, acute hemorrhagic cystitis and infant gastroenteritis, with approximately 5-7% of respiratory illnesses in young children attributed to HAdVs (Carballal G, et al., 2001). Although most of respiratory infections caused by HAdVs are self-limiting, fatal infections also occur in children and adults (Carr M J, et al., 2011; Girouard G, et al., 2011; Rebelo-deAndrade H, et al., 2010).
To date, 68 genotypes of HAdVs have been identified (GenBank accession no. JN860678) (Dehghan S, et al., 2012) and classified into 7 species: species A to G (Jones M S, Ⅱ, et al., 2007). Human adenovirus genotypes 3, 4 and (HAdV-B3, -E4, and -B7) are the major HAdVs associated with ARD in children and adults and have caused many outbreaks of pneumonia and pharyngoconjunctivitis in China (Guo L, et al., 2012; Ou Z Y, et al., 2008; Tang L-y, et al., 2006; Tang L, et al., 2011; Zhang Q, et al., 2006). Therefore, it is of critical importance for clinic and disease prevention and control to identify and type HAdVs for the potential outbreaks.
Here, we developed a simple, rapid and practical detection and typing method for the surveillance of HAdVs in Guangzhou, Southern China. As the main ingredient of HAdV capsids, the hexon protein contains most of the type-specific epitopes that can stimulate the host to produce type-specific neutralizing antibodies. The differences amongst different types of HAdVs rely mainly on the hypervariable regions (HVRs) in the hexon gene. Based on the conserved region and HVRs, we designed one pair of universal primers and three pairs of type-specific primers, which can detect and type HAdV-B3, -E4 and -B7, predominant in China by traditional PCR. By using this method, the molecular epidemiology of HAdVs circulating in Guangzhou, Southern China during Oct 2010 to Dec 2011was performed.
The adenovirus strains of HAdV-B3, -E4, -B7 and -B14 were isolated, identified and preserved in our laboratory (Su X, et al., 2011; Zhang Q-W, et al., 2012; Zhang Q, et al., 2012; Zhang Q, et al., 2006). HAdV -F40 and -F41 were gifts that were isolated and identified by Professor Chen in Southern Medical University. Human rhinovirus and coxsackie virus type A16 were identified by Guangdong CDC. Human influenza A/H1N1, chlamydia and mycoplasma were identified by Guangzhou Women and Children's Hospital. Human respiratory syncytial virus strain Long was bought from the ATCC (Cat no. VR-26). One hundred throat swab specimens suspected to be associated with adenoviral infection were collected from the outpatient clinic and hospitalized children with bronchial pneumonia in Guangzhou Women and Children's Medical Center during Oct 2010 to Dec 2011, of which, 60 were male and 40 were female, and aged one month to eight years old. Taq Master Mix PCR kits and Viral DNA extraction kits were purchased from Omega Bio-Tek Inc. Corp (Norcross). DL1000 and DL2000 DNA Markers were the product of TAKARA Corp (Dalian).
Throat swabs were inoculated into A549 cells and cultured at 37 ℃ in an atmosphere containing 5% (v/v) carbon dioxide in Dulbecco's minimum essential medium supplemented with 100 IU penicillin per mL, 100 mg streptomycin per mL and 2% (v/v) fetal calf serum. The cells were observed for 1-2 weeks for CPE and identified by PCR or sequencing.
Viral nucleic acids from the throat swabs and adenovirus strains were extracted according to the E.Z.N.A. Viral DNA kit instructions (Omega Bio-tek, Inc.). Universal primers HexF and HexR were designed according to the conserved region of the hexon gene from all HAdV types, generating a 300 bp PCR product; Type-specific primers (Ad3F, Ad3R; Ad4F, Ad4R; Ad7F, Ad7R) amplifying the HVRs of the hexon gene were designed and optimized to correctly identify HAdV-B3, -E4 and -B7, respectively. The size of all the PCR products was about 300bp. All the primes used for detection and typing of HAdVs are shown in Table 1. The PCR reaction system was as follows: 2 × Taq Master Mix (10 μL), primer F (10 μmol/L, 0.5 μL), primer R (10 mmol/L, 0.5 μL), DNA template (2 μL) and sterile water (7 μL). PCR Reaction conditions for HAdV detection and typing is as follows: 94 ℃ for 1 min, 34 cycles of 94 ℃ for 30 s, 55 ℃ for 30 s and 72 ℃ for 20 s with a final extension of 72 ℃ for 7 min. Primers HVRF and HVRR were designed according to the conserved region of the hexon gene and used for PCR and the sequencing of all seven HVRs of 1685 bp (Table 1). PCR was performed as follows: 94 ℃ for 1 min, 34 cycles of 94 ℃ for 30 s, 55 ℃ for 30 s and 72 ℃ for 100 s with a final extension of 72 ℃ for 7 min.
Primer names Primer sequences Positions in
HexF (Allard A, et al., 1990) 5'-GCCCCAATGGGCATACATGCACATC -3' 18442-18466 DQ099432 HexR 5'-AGCACGCCGCGAATGTCAAAG-3' 18741-18721 DQ099432 Ad3F 5'-AAGACATTACCACTACTGAAGGAGAAGAA-3' 18933-18961 DQ099432 Ad3R 5'-CGCTAAAGCTCCTGCAACAGCAT-3' 19246-19224 DQ099432 Ad4F 5'-AGCAAAATGCATACCTTTGGGG-3' 18665-18686 AY594253 Ad4R 5'-ATAGTTAGGAGTGGTGGCGGCG-3' 18988-18967 AY594253 Ad7F (Xu W, et al., 2001) 5'-GGGAAAGACATTACTGCAGACA-3' 18890-18911 AY594256 Ad7R 5'-AAAAAGCGTCAGCAGCTTCT-3' 19190-19171 AY594256 HVRF 5'-CAGGATGCTTCGGAGTACCTGAG-3' 18473-18495 DQ099432 HVRR 5'-TTTCTGAAGTTCCACTCGTAGGTGTA-3' 20157-20132 DQ099432 Primer HexF was optimized based on the primer described elsewhere (Allard A, et al., 1990). Primer Ad7F was optimized based on the primer of Xu and Erdman (Xu W, et al., 2001).
Table 1. Primes used for the detection, typing and sequencing of HAdVs
One hundred throat swab specimens suspected of being associated with adenoviral infection, collected during Oct 2010 to Dec 2011, were detected for presence of adenovirus and typed using our described method. PCR-Positive specimens were further cultured for 1-2 weeks in A549 cells for CPE observation.
The HVRs of seven adenovirus isolates typed by type-specific primers were PCR amplified, purified and directly sequenced, including three of HAdV-B3, one of HAdV-E4, two of HAdV-B7 and one of HAdV-B14 selected randomly. The sequencing reaction was carried out by using an ABI Prism BigDye Terminator v3.1 Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase on an ABI 3730 DNA sequencer (Applied Biosystems). All of the sequences are sequenced at least three times.