Sporadic and Severe Lower Respiratory Illness-related Human Adenovirus Type 21 Infection in Southern China

Background: Human adenovirus type 21 (HAdV-21) is an important member of HAdV species B, but our understanding of this type is limited. Methods: We screened HAdV and 17 other common respiratory pathogens for 1,704 pediatric patients ( ≤ 14 years old) hospitalized with acute respiratory illness in Guangzhou, China in 2019. HAdV-21 infections were further conrmed by molecular typing from HAdV-positive patients, and their clinical manifestations, genomes, infectivity and pathogenicity in vitro were analyzed. Results: 151 of 1,704 cases (8.9%) were positive for HAdV, making it the third most frequently detected pathogen. Two sporadic HAdV-21 infections were identied in June and September. Both HAdV-21-positive patients presented with severe lower respiratory illness and had similar initial symptoms at onset of illness. The genome structure of HAdV-21 was found to be similar to that of other members of HAdV species B. The phylogenetic analysis showed that it was closely related to HAdV-B21 strain BB/201903 (MN686206) isolated in Bengbu, China in 2019, suggesting the possibility of the same source, and attention need to be paid to its prevention and control. In vitro, the infectivity and pathogenicity of HAdV-21 were lower than the main epidemic types 7 and 3. Plaques formed by HAdV-21, -7, and -3 were signicantly different in shape and size (p < 0.05), with plaques formed by HAdV-21 being the smallest and with poorly dened edges. There was no signicant difference between the plaques of the HAdV-21 isolates (p > 0.05). Conclusions: This study provides an important reference for the in-depth understanding of the epidemiology and pathogenicity of HAdV-21, and suggests the necessity of HAdV-21 research, prevention and control.


Introduction
Human adenoviruses (HAdVs) are non-enveloped, double-stranded DNA viruses of the family Adenoviridae. More than 100 genotypes of HAdVs have been identi ed, which are classi ed into seven species (A-G) (1,2). HAdVs are associated with a broad spectrum of clinical diseases, such as acute respiratory illness (ARI), conjunctivitis, gastrointestinal infections, and obesity (2)(3)(4)(5). Members of species B are known to cause human diseases, with HAdV types 3, 7, 14, and 55 being the most common causes of respiratory disease outbreaks (6).
HAdV-21 is a member of species B, and was rst isolated in 1956 from a 1-year-old child with trachoma and conjunctivitis in Saudi Arabia (7). HAdV-21 was later found to be associated with a variety of diseases, including ARI (8-12). Severe pneumonia, myocarditis, accid paralysis, and even fatal infections in both pediatric and adult patients have been reported (13)(14)(15)(16). The circulation of ARIassociated HAdV-21 has been reported among military recruits and civilians in several developed countries (8, [17][18][19], and caused nosocomial infections in lung transplant patients at a large tertiary care hospital (20). However, data on HAdV-21 are limited as HAdV-21 infections have rarely been reported in regions outside North America and Europe and are especially rare in China (21). To better understand the epidemiology of HAdV-21, infection data from different regions are of great importance.
In this study, we analyzed the distribution of respiratory pathogens in 1,704 pediatric patients with ARI in Guangzhou, China, in 2019. Two patients with HAdV-21 displaying severe lower respiratory illness (LRI) were identi ed. The clinical features of these infected patients were assessed, and the genomic characteristics and cyto-pathogenicity of HAdV-21 in vitro were analyzed.

Respiratory sample collection
Respiratory samples, including throat swabs, sputum, and bronchoalveolar lavage uid, from pediatric patients (≤ 14 years old) hospitalized with ARI were collected for routine screening of respiratory viruses Mycoplasma pneumoniae (MP) and Chlamydophila pneumoniae (CP) in accordance with established clinical protocols at the First A liated Hospital of Guangzhou Medical University between January and December 2019 (22). The samples were refrigerated at 2-8°C in viral transport medium, transported on ice to the State Key Laboratory of Respiratory Diseases, and analyzed immediately or stored at − 80°C before analysis, as previously described (23). The patients' clinical presentations were collected from the medical records.

HAdV-21 genome sequencing and annotation
HAdV-21-positive samples were cultured and harvested. Viral genomic DNA was extracted using a TaKaRa Mini BEST Viral RNA/DNA Extraction Kit Ver.5.0 (TaKaRa) according to the manufacturer's instructions. Next-generation sequencing was conducted with a Illumina NovaSeq 6000 sequencer following a protocol from Synbio-Technologies (paired-end, 2 × 150 bp). The complete genome of HAdV-21 was assembled using CLC Genomics Workbench 11.0. The complete genomes of the HAdV-21 isolates were annotated based on the annotation of HAdV-21 strain BB/201903 (accession no. MN686206). Complete genome sequences were logged in the GenBank database.

Phylogenetic analysis and HAdV sequences used
Phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis (MEGA) version 5.05 (25). Phylogenetic trees were constructed by the Neighbor-joining (NJ) method with 1,000 bootstrap replicates and default settings for all other parameters. HAdV sequences of the penton base, hexon, and ber genes, and the genomes for phylogenetic analyses retrieved from GenBank are summarized in Table   1.

Statistical analysis
Statistical analysis was performed using SPSS 19.0 (SPSS Inc., Chicago, IL). Differences between groups were calculated using the t-test and Mann-Whitney U test. A p-value < 0.05 (two-tailed) was considered statistically signi cant.

Genome features of HAdV-21 isolates
HAdV-21 isolates GZ06109 and GZ09107 were sequenced, annotated, and uploaded to the GenBank database with accession numbers MW091531 and MW151243, respectively. The identi ed genomes were 35,362 and 35,365 bp in length for GZ06109 and GZ09107, respectively, and had similar genomic organization and transcription maps (shown for strain GZ09107 in Fig. 2).

Phylogenetic features of the HAdV-21 isolates
Phylogenetic analysis of the genomes and three structural protein sequences showed that HAdV-B21 members are closely related to each other, forming a clade (Fig. 3). The percent identities of the genomes and three capsid protein genes of the two HAdV-21 isolates identi ed here were compared with the most closely related strain, HAdV-21-BB/201903 (98.82-100%), and the rst strain to be isolated, HAdV-21-AV-1645 (98.38-99.47%; Table 3). Compared with HAdV-21-BB/201903, GZ06109 contained one insertion mutation in the penton base (GCG, alanine) and two synonymous mutations in the non-coding region. No non-synonymous mutations were found in strain GZ09107 (Fig. 4).

Discussion
Among the more than 100 types and seven species (A-G) of HAdV, species B is of particular importance in ARI (http://hadvwg.gmu.edu/). Although HAdV-21 is an important member of HAdV species B, our understanding of this type is inadequate. From 2017 to 2019, HAdV strains were prevalent in China, causing outbreaks of severe pneumonia in children (http://www.chinacdc.cn/) (26). In this study, we analyzed the epidemiologic characteristics, genome features, and cyto-pathogenicity of HAdV-21 Of the 1,704 participants in this study, 8.9% of the patients were infected with HAdV, making it the third most prevalent pathogen detected (Fig. 1). This positive rate was higher than that reported for previous years in this region (5%, 213/4242) (22). The main HAdV types detected were type 7 (46.4%) and 3 (47.0%), which is largely consistent with previous reports (22,26,27). Two sporadic cases of HAdV-21, GZ06109 and GZ09107, were identi ed in June and September. The low prevalence of HAdV-21 may signify low immunity against this type in the general population, increasing its potential to cause an epidemic.
Both HAdV-21-positive patients presented with severe LRI (Table 2), which highlights the need for increased awareness of HAdV-21 infections. The HAdV-21 infections had similar initial symptoms at onset of illness, and multiple indexes exceeded the normal ranges, such as white cell count, PCT, CRP, AST, and D-Dimer. These factors may help physicians judge and screen for this pathogen (  (20), highlighting the importance of prevention and control of this type.
To improve our understanding of HAdV-21, we cultured the two HAdV-21 isolates, GZ06109 and GZ09107. Sequencing and annotation of their genomes revealed similar structures to other members of HAdV species B (28) (Fig. 2). By comparing and analyzing the genome sequences of HAdV-21 and other HAdV species (Table 1), we found that the HAdV-21 genome is comparatively stable and constitutes a clade (29) (Fig. 3). The two HAdV-21 strains in this study had highest genome identity (99.97%) with strain HAdV-21-BB/201903 (accession no. MN686206), which was isolated in Bengbu, China in 2019, and 98.86% similarity with the rst strain to be isolated, HAdV-21-AV-1645 (Table 3). In terms of the main structural protein genes, GZ09107 had 100% similarity to the Bengbu strain, with only two synonymous mutations (T antigen and 20k protein) and two non-coding region mutations (Fig. 4). Compared with the Bengbu strain, GZ06109 had 100% sequence identity for the ber and hexon genes (Table 3) and an alanine insertion in the penton base (Fig. 4). This suggests that HAdV-21 isolates prevalent in China have a high degree of kinship and are from the same source, although there is insu cient data to identify the potential source.
To analyze the pathogenicity of HAdV-21, plaque formation assays with the two HAdV-21 isolates in this study, reference HAdV-21-AV-1645, and severe pneumonia-related HAdV-3-Guangzhou01 and HAdV-7-CQ1198 were conducted (Fig. 5). Although the plaques formed by the three HAdV-21 strains showed similar characteristics (Fig. 5A) and were similar in size (p > 0.05; Fig. 5B), they were signi cantly smaller (p < 0.001) than plaques from HAdV-7 and HAdV-3, and had poorly de ned edges. It was also found that plaques formed by HAdV-7 were signi cantly larger than those of HAdV-3 (p < 0.05). These plaque features indicate that HAdV-21 < HAdV-3 < HAdV-7 with regard to virulence and infectivity. Compared with clinical research reports, the prevalence and pathogenicity of HAdV-7 and HAdV-3 are largely consistent with this result (30). There are too few reports on HAdV-21 to determine its overall pathogenic characteristics; thus, more research on this type is urgently needed.
The main limitation of this study is that selection bias may have occurred, because the sample comes from one hospital, and there is a lack of samples from outpatient clinics and healthy people. This may lead to deviations in the understanding of HAdV-21 infection, especially the epidemiological characteristics.

Conclusions
In this study, we investigated HAdV-21 infections in Guangzhou, China, and identi ed two patients infected with severe LRI-related HAdV-21 strains. Genomic analysis showed that the Chinese isolates of HAdV-21 showed a high degree of similarity, suggesting that attention should be paid to its prevention and control. Analyses of the clinical characteristics, genome structures, and cyto-pathogenicity of these strains revealed important information that will contribute to a deeper understanding of HAdV-21.

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

Competing interests
The authors declare that they have no competing interests.
Funding  Transcriptional map and genome organization of HAdV-21 strain GZ09107 The genome is indicated by the black horizontal line marked at 10,000 bp intervals. The transcription units are designated by gray arrows, while blue arrows designate coding regions. Arrows re ect the transcriptional orientation of the coding transcripts.

Figure 3
Phylogenetic analysis of HAdV-21 strains GZ06109 and GZ09107 The nucleotide sequences of the whole genome (A), hexon (B), penton base (C), and ber (D) genes of the HAdV strains were analyzed for their phylogenetic relationships using the Neighbor-Joining method with 1,000 bootstrap replicates implemented in the MEGA 5.0 software package. For reference, taxon names include the genome type, corresponding GenBank accession number, country of isolation, strain name, and year of isolation. The two HAdV-21 strains isolated in this study are marked with "▲"; "▼", strain isolated from Bangbu (BB/201903), China in 2019; "•", reference standard HAdV-21 isolated in Saudi Arabia in 1945.