An Outbreak of Acute Respiratory Illness at a Training Base in Beijing, China, Due to Human Adenovirus Type B55

Background: An outbreak of acute respiratory illness (ARI) occurred at a training base in Beijing. A total of 12 students were diagnosed with symptoms of ARI from August 26 to August 30, 2015. The cause of the ARI outbreak was investigated. Methods: In partnership with the local center for disease control, we conducted an epidemiological investigation on the outbreak, collecting a total of twelve pharyngeal swab specimens as well as patient demographic for the affected patients. We used multiplex real-time PCR to screen for sixteen common respiratory viruses in these samples. To isolate the causative virus, we inoculated Hep-2 cells with the human adenovirus (HAdV)-positive samples and then carried out sequencing and phylogenetic analysis of the hexon, fiber and penton genes of the isolated adenoviruses. In addition, we analyzed the whole genome of one strain isolated from the index case to identify single-nucleotide substitutions. Results: We identified ten HAdV-positive students by multiplex real-time PCR. None of the students were co-infected with other viruses. We successfully isolated seven strains from the pharyngeal swab specimens. Genetic analysis showed that the coding sequences of the hexon, fiber, and penton genes obtained from those seven HAdV strains were identical, suggesting that they represented seven isolates of a single virus strain. One HAdV isolate obtained from the index case, BJDX-01-2015, was selected for whole genome analysis. From this isolate, we obtained a 34,774-nucleotide sequence. Phylogenetic analysis showed that the genome of BJDX-01-2015 clustered with HAdV-B55 and had 99.97% identity with human adenovirus 55 isolate HAdV-B/CHN/BJ01/2011/55 (GenBank accession no. JX491639).

Conclusions: We identified HAdV-B55 as the culprit of the ARI outbreak. This was the first reported outbreak in Beijing due to HAdV-B55 virus. Continuous surveillance of respiratory adenoviruses is urgently needed for understanding the epidemiological and evolutionary features of HAdV-B55 and could also find value in an epidemiological modeling approach.

Background
Human adenoviruses (HAdVs) cause a wide variety of clinical manifestations, including respiratory tract illnesses, gastroenteritis, kerato-conjunctivitis, acute hemorrhagic cystitis, nephritis, hepatitis, and encephalitis [1][2][3]. HAdVs are responsible for 2-5% of all respiratory illnesses and for 4-10% of pneumonias in children [4][5]. Most HAdV infections are mild, self-limiting, and indistinguishable from other viral infections. However, the diseases caused by HAdVs can be severe or even fatal and can result in substantial morbidity [6][7]. Outbreaks of HAdVassociated acute respiratory infection (ARI) usually occur in healthy children or in adults in enclosed or crowded settings [1,8].
HAdV was first reported as a viral pathogen in 1953 [6]. Since this initial identification, HAdVs have been classified into seven species (A to G), and the Human Adenovirus Working Group has identified 90 HAdV types as of July 2018 (http://hadvwg.gmu.edu/) [7][8][9][10][11][12]. Adenoviruses are non-enveloped icosahedral particles that contain genomes of linear double-stranded DNA with sizes ranging from 26 to 45 kb. The genomes of all adenoviruses have a similar genomic structure that is characterized by inverted terminal repeat sequences (LTR) with sizes ranging from 36 to over 200 bp [12][13].
Here we describe an outbreak of ARI caused by HAdV-B55 at a training base in the Daxing District of Beijing, China. To help identify the causative pathogen, we collected pharyngeal swab specimens from the affected students and carried out molecular detection and typing, phylogenic analysis, and whole-genome sequencing. This is the first reported outbreak of ARI in Beijing due to HAdV-B55.

Methods
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

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.

HAdV isolation and typing
Hep-2 cells were inoculated with HAdV PCR-positive specimens and cultured in highglucose 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% CO 2 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

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.

Clinical symptoms of the infected students
The clinical symptoms of the infected students are described in Table 1. All 12 infected individuals had a fever, while 9 (75%) of them had a sore throat and 7 (58.33%) had a headache. Other symptoms reported by the patients were cough, body ache, stuffy nose, and diarrhea. Laboratory diagnosis showed that all affected students had normal white blood counts. Most of the infected students were treated in outpatient clinics; only one patient, who had pneumonia, was hospitalized.

Virus isolation and HAdV typing
To isolate viruses, we inoculated Hep-2 cells with the HAdV-positive samples, then amplified the HAdV hexon gene using in-house primers. This approach isolated seven HAdV virus strains [24]. Using typing primers, which allow for the determination of viral type, we sequenced the hexon, fiber, and penton genes in the seven HAdV strains [25].
The hexon, fiber, and penton sequences from the seven HAdV isolates were 100% identical, suggesting that this outbreak was caused by a single viral strain. We next compared the hexon, fiber, and penton sequences the viral strain to other sequences in GenBank. A BLAST sequence analysis revealed that the hexon, fiber, and penton genes were 99.6%, 100%, and 100% identical to their counterparts in the HAdV-B55 reference strain isolate China/QS-DLL (Genbank accession number where we found seven nucleotide changes and six amino acid substitutions. We also found different numbers of poly "T" and ploy "A" tracts (Fig. 4B).

Discussion
This study describes an ARI outbreak with 12 students infected at a training base in the Daxing District of Beijing, China, in August, 2015. Based on epidemiological and laboratory investigation, we confirmed that the etiologic pathogen of this ARI outbreak was HAdV-B55.
In the 2006 HAdV-B55 outbreak in China, a total of 254 patients were infected, and one died [19]. Although the virus spread quickly in the 2015 outbreak reported here, only 12 individuals were infected, including one student who developed pneumonia and was hospitalized. This suggests that the HAdV-B55 strain that caused this outbreak is not as virulent as those reported previously [19,33], yet the strain should still be considered an urgent public health threat with need to take measures to contain or control it in order to prevent epidemics. (Genbank accession number JX491639) that was isolated from a severe pneumonia case in Beijing in 2011 [42,43]. However, the genome of BJDX-01-2015 is most similar to that of CHN-BJ01-2011, leading to milder ARIs. These findings imply that severe ARIs were not only caused by HAdV-B55 virus strains themselves, but are also correlated with the hosts, e.g. the age of individuals, their general state of health, and any underlying diseases or additional infections.
In this outbreak, we also found that both the index case and the hospitalized case HAdV-B14 strains. The role that poly "T"and poly "A" tracts play in the evolution of HAdVs also remains unclear and requires further research.
The increasing frequency of ARI outbreaks due to HAdV-B55 suggests that this reemergent virus poses a serious threat to public health. It is therefore urgent that the local CDC improve epidemiological and virological surveillance of HAdV-B55.

Conclusions
We identified HAdV-B55 as the culprit of a recent localized ARI outbreak. This incident was the first reported outbreak in Beijing that can be attributed to this re-

Ethics approval and consent to participate
This study was approved by the Ethics Committee of the Beijing Center for Disease Prevention and Control. Prior written consent form was obtained for all individuals who participated in this study.

Consent for publication
The parents of the patients had given their consent for publication of clinical details and written informed consent was obtained.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors have no conflicting interests.

Funding
This study was supported in part by the Research on Early Detection, Genetic Evolution and Risk Assessment for Novel Influenza Virus by Capital's Funds for Health Improvement and Research (2018-1-1012).

Authors' contributions
All authors made significant contributions to the data, analysis and draft of this

Consent for publication
The parents of the patients had given their consent for publication of clinical details and written informed consent was obtained.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors have no conflicting interests.

Funding
This study was supported in part by the Research on Early Detection, Genetic Evolution and Risk Assessment for Novel Influenza Virus by Capital's Funds for Health Improvement and Research (2018-1-1012).

Authors' contributions
All authors made significant contributions to the data, analysis and draft of this manuscript and approved the final version submitted. Guilan Lu wrote the manuscript. Guilan Lu, Xiaoming