Human Adenovirus Subtype 21a Isolates From Children With Severe Lower Respiratory Illness in China

Figures

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  FIGURE 1. Detection of human adenovirus infection among 1,704 pediatric patients hospitalized with acute respiratory illness in Guangzhou, China in 2019. (A) Distribution of 18 respiratory pathogens in the children with ARD. (B) Monthly positive rate of HAdV infection in the hospitalized children.

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  FIGURE 2. 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 reflect the transcriptional orientation of the coding transcripts.

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  FIGURE 3. In silico restriction enzyme analysis (REA) of the HAdV-21 GZ06109 and GZ09107 sequences using the enzymes BamHI, BglI, BglII, KpnI, and SmaI. Comparison was made with the HAdV-21a NHRC-5, HAdV-21p AV-1645, and HAdV-21b OHT-006. Genome type designations are based on unique arrays of restriction profiles with a panel of 6 endonucleases.

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  FIGURE 4. Phylogenetic analysis of HAdV-21 strains GZ06109 and GZ09107. The nucleotide sequences of the whole genome of the HAdV strains were analyzed for their phylogenetic relationships using the Neighbor-Joining method with 1,000 bootstrap replicates implemented in the MEGA 11.0.8 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.

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  FIGURE 5. Plaque formation and size distribution of the HAdV-21 isolates and references HAdV-21-AV-1645, HAdV-3-Guangzhou01, and HAdV-7-CQ1198. (A) Plaque formation. (B) Size distribution. Plaque plates were incubated and stained with crystal violet for a total of 13 days in 6-well culture plates. ns, not significant; *, p < 0.05; ***, p < 0.001. (C) Growth curves of HAdV strains. Virus genome copies were quantified by Q-PCR after different hours post-infection of HAdVs in A549 cells.

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  FIGURE 6. Antigenic variability among HAdV-21 strains. (A) Multiple sequence alignment of several hexon proteins from subtypes HAdV-21a (GZ06109), 21b (OHT-006), and 21p (AV-1645). Only five hypervariable regions with substitution are shown. The number shows the position of amino acid in the hexon protein. *, conserved amino acid; ∙, either size or hydropathy is conserved; and:, both size and hydropathy are conserved. Gaps used to optimize alignments are indicated by dashes. (B) In vitro micro-neutralization of HAdV-21 with hyperimmune sera from mice. Antisera were collected from mice immunized with purified HAdV-21a (GZ06109) and 21p (AV-1645) (n = 4). Cross-neutralization tests against these two strains were performed. The anti-PBS sera were used as the negative control.