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 identified 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 (Table 2), although definitive diagnosis will require laboratory screening as many respiratory viruses have similar manifestations. Because there are no specific treatments against adenoviruses, only symptomatic treatment can be used at present. Previous studies have shown that HAdV-21 can cause nosocomial infections in immune-compromised patients (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 first 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 fiber 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 insufficient 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 significantly smaller (p < 0.001) than plaques from HAdV-7 and HAdV-3, and had poorly defined edges. It was also found that plaques formed by HAdV-7 were significantly 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.