The training base where the HAdV-B55 outbreak took place
On August 31, 2015, local public health authorities were informed about an outbreak of ARI among young students at a training base located in the Daxing District of Beijing. The training base consisted of two three-floor buildings for teaching and three three-floor dormitories with eight persons to a room. The training base recruits only male middle school graduates. Approximately 3,000 students majoring in Math, Chinese, and English were enrolled in a total of sixty classes. The training base employs 100 full time staff members.
Epidemiological investigation
On August 26, 2015, one student reported having symptoms of an ARI and had a body temperature of 38.4°C. For the purposes of our analysis, we defined ARI cases as individuals with a body temperature over 38.0°C and with at least one symptom of a respiratory tract infection, such as cough or sore throat. By August 30, a total of 12 ARI patients from the same class were reported by the local hospital. On August 31, the Daxing District Center for Disease Prevention and Control (CDC) began an epidemiological investigation, collecting demographic, clinical and laboratory data. Under the guidance of the CDC, the training base took precautionary measures, including quarantining the affected students, carrying out a routine cleaning and disinfection of living quarters, and morning body temperature checks. No further new cases were reported by September 08, 2015.
Patients and samples
Pharyngeal swab samples were obtained from each of the twelve students infected in this ARI outbreak. The specimens were collected in 3 mL vials containing transport medium and quickly transported on ice to the laboratory of the Daxing District CDC. The specimens were stored at -80°C until further use. Patient information and laboratory results are shown in Table 1.
Respiratory virus detection
Nucleic acid was extracted from 140 μL of each of the clinical samples using QIAamp Viral RNA mini Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Pharyngeal swab specimens were analyzed by real-time PCR multiplex assay using commercial kits (Uninovo Biological Technology, Zhenjiang, China) to screen for 16 common respiratory pathogens as described by Shi W et al.[23]. The 16 assayed pathogens are influenza viruses A (H3), pandemic influenza viruses A (H1N1), influenza viruses A and B (Flu A and B), parainfluenza viruses 1, 2, 3, and 4 (PIV1, 2, 3, and 4), human metapneumovirus (hMPV), humanbocavirus (HBoV), human coronavirus OC43/NL63, 229E/ HKU1, human respiratory syncytial virus (HRSV), human rhinovirus (HRhV) and HAdV.
HAdV isolation and typing
Hep-2 cells were inoculated with HAdV PCR-positive specimens and cultured in high-glucose Dulbecco’s Modified Eagles Medium (Gibco, NY, USA) containing 2% fetus bovine serum (Gibco, NY, USA), 100 U/mL penicillin (Gibco, NY, USA) and 100 mg/mL streptomycin (Gibco, NY, USA) at 37°C in a 5% CO2 incubator for one week following standard protocols [19]. The culture medium was harvested until cytopathic effects (CPE) were observed. Cultures with CPE were screened for specific HAdVs as described by Kim C et al. [24].
Molecular typing for HAdVs was also performed via conventional PCR using specific primers targeting the complete coding sequences of hexon, fiber and penton genes [25].Viral DNA was extracted from cultured medium with QIAamp RNA mini kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions [26]. Conventional PCR was conducted using high-fidelity DNA polymerase (Takara, Dalian, China) according to the manufacturer’s instructions. The hexon, fiber, and penton genes of HAdV were amplified as described previously [25]. For the hexon and penton genes, the PCR protocol was: 94°C for 5 min, followed by 35 cycles of 50 sec at 94°C, 50 sec at 55°C, 3 min at 72°C, and a final extension step of 72°C for 10 min. The PCR protocol for amplification of fiber gene fragments was identical, with the exception of the annealing temperature, which was 52°C instead of 55°C. The amplified PCR products were excised from agarose gels, purified using an Axyprep DNA gel extraction kit (Axygen, Hangzhou, China), and bi-directionally sequenced using the Sanger sequencing method by Invitrogen Biotechnology Co. Ltd (Invitrogen, Beijing, China) with an ABI 3730 DNA Analyzer (Applied Biosystems, Austin, TX, USA).
Whole-genome sequencing
To further analyze mutations in the genome sequences of the viruses isolated in this ARI outbreak, the whole genome of one isolate from the index case was sequenced using the Sanger method after PCR amplification of targeted 1-2 kb segments that covered the entire genome with overlapping sequences of about 200 bp. The 5′ and 3′ LTR of the genome were amplified and cloned into a plasmid T-vector and then sequenced. A set of 47 pairs of primers was designed in-house to amplify the whole genome according to the reference sequence (GenBank accession no. FJ643676) and then used for separate PCRs. All primer sequences are available upon request.
Whole-genome sequencing segments were amplified using high-fidelity polymerase (Takara, Dalian, China) using 1.0 mM of each primer. PCRs were carried out using a BioRad thermocycler (Applied Biosystems, Austin, TX, USA) with the following protocol: 94°C for 5 min, followed by 35 cycles of 50 sec at 94°C, 50 sec at appropriate annealing temperature for separate primers, 3 min at 72°C, and a final extension step of 72°C for 10 min.
The amplified segments were purified and bi-directionally sequenced. Gaps and ambiguous sequences were PCR-amplified using different primers and re-sequenced. DNA sequence fragments were assembled using the SeqMan program impleted in DNASTAR Lasergene 7.0 (DNASTAR, Inc. Madison, WI). ) into a single contig. The whole genomic sequence determined in this study was deposited in GenBank with accession numbers (GenBank accession number MK886831).
Sequence alignment and phylogenetic analysis
Nucleotide sequence homologies were identified using the Basic Local Alignment Search Tool (BLAST, https://blast.ncbi.nlm.nih.gov/). Multiple nucleotide sequence alignments were performed using the ClustalW program implemented in BioEdit. Comparisons of the whole genome sequence of the BJDX-01-2015 virus strain with those of other types of HAdVs were generated using CLC Genomics Workbench (Qiagen, Hilden, Germany).
Phylogenetic trees were constructed using the neighbor-joining method with the Kimura-2-parameters in the MEGA program (Version 5.05). One thousand bootstrap replications were used for distance estimation. Bootstrap values greater than 70% are shown for selected nodesFigures 2 (A) to 2 (D). Whole-genome sequences and hexon, fiber, and penton gene sequences from other HAdVs were downloaded from GenBank.