Re-emergent human adenovirus genome type 7d caused an acute respiratory disease outbreak in Southern China after a twenty-one year absence

Abstract

Human adenoviruses (HAdVs) are highly contagious pathogens causing acute respiratory disease (ARD), among other illnesses. Of the ARD genotypes, HAdV-7 presents with more severe morbidity and higher mortality than the others. We report the isolation and identification of a genome type HAdV-7d (DG01_2011) from a recent outbreak in Southern China. Genome sequencing, phylogenetic analysis, and restriction endonuclease analysis (REA) comparisons with past pathogens indicate HAdV-7d has re-emerged in Southern China after an absence of twenty-one years. Recombination analysis reveals this genome differs from the 1950s-era prototype and vaccine strains by a lateral gene transfer, substituting the coding region for the L1 52/55 kDa DNA packaging protein from HAdV-16. DG01_2011 descends from both a strain circulating in Southwestern China (2010) and a strain from Shaanxi causing a fatality and outbreak (Northwestern China; 2009). Due to the higher morbidity and mortality rates associated with HAdV-7, the surveillance, identification, and characterization of these strains in population-dense China by REA and/or whole genome sequencing are strongly indicated. With these accurate identifications of specific HAdV types and an epidemiological database of regional HAdV pathogens, along with the HAdV genome stability noted across time and space, the development, availability, and deployment of appropriate vaccines are needed.

Figure 1. Genomic organization and transcription map of human adenovirus 7d strain CHN/DG01/2011/7[P7H7F7]″ (DG01_2011).

The grey arrows indicate early, intermediate, and late transcription units; the red indicates coding regions. Arrows reflect the direction of the coding regions.

Figure 2. Identification of genome types and comparison of three recent HAdV-7d genomes in China.

The in-silico restriction endonuclease analysis (REA) pattern analysis of the HAdV-7d sequenced genomes from recently circulating strains DG01_2011, 0901HZ/ShX/2009, and CHN_CQ1198_2010 were generated using the Vector NTI 11.5 software (Invitrogen Corp.; San Diego). REA allows for comparisons with historical strains that were not sequenced but were characterized by REA patterns and descriptions. GenBank-available genome sequences of strains Gomen- the type 7 prototype (lane A; AY594255), DG01_2011 (lane B; KC440171), 0901HZ/ShX/2009 (lanes C; JF800905), and CHN_CQ1198_2010 (lane D; JX625134) were analyzed with BamHI, BclI, Bgll, BglII, BstEII, HindIII, HpaI, SmaI, EcoRI, SalI, XbaI, and XhoI, respectively, as described by Li, et al. (Li et al. J Med Virol, 1996, 49(3):170–177). M: molecular weight markers corresponding to a 1 kb DNA Ladder are provided for reference. All three recent outbreak genomes correspond to the genome type of HAdV-7d, and have identical REA profiles to each other, divergent from the HAdV-7 prototype (1952).

Figure 3. Phylogenetic analysis of HAdV-7d strain DG01_2011 isolated from Dongguan, Southern China (2011).

Nucleotide sequences of the archived HAdV-7 hexon genes (A) and whole genomes (B) are accessible from GenBank. These include five hexon gene sequences from Taiwan (TW; JX174426-JX174430); three from Hebei (JQ360620-JQ360622); one from Chongqing (JX625134); one from Shaanxi Province (JF800905); and one, this report, from Dongguan (DG) (KC440171). Additional HAdV-7 sequences from other global isolates were also analyzed. For reference, taxon names include the corresponding GenBank accession number, country of isolation, strain name, year of isolation (if available), and genome type (if available). Boot-strapped, neighbor-joining trees with 1,000 replicates were constructed using the MEGA 5.1.0 software (http://www.megasoftware.net) and by applying default parameters, with a maximum-composite-likelihood method. Bootstrap numbers shown at the nodes indicate the percentages of 1,000 replications producing the clade. A bootstrap value of 80 indicates robustness and confidence in the branching. The scale bar indicates units of nucleotide substitutions per site. Sequences from strains DG01_2011, 0901HZ/ShX/2009, and CQ1198_2010 are noted for reference (▴).

Figure 4. Genome recombination analysis.

The genomes of HAdV-B7d strain DG_2011 (Guangdong Province, China; 2011), CQ1198_2010 (Southwestern China; 2010, unpublished) and 0901HZ/ShX/2009 (Northwestern China; 2009) were analyzed for sequence recombination events along with HAdV species B genomes using the software Simplot (http://sray.med.som.jhmi.edu/SCRoftware/simplot/). For the recombination analysis, MAFFT software was used first to align the sequences using default parameters (http://mafft.cbrc.jp/alignment/server/). Default parameter settings for the Simplot software were used for analyzing the whole genomes, with the following input: window size (2000 nucleotides [nt]), step size (200 nt), replicates used (n 100), gap stripping (on), distance model (Kimura), and tree model (neighbor-joining). Genome nucleotide positions are noted along the x-axis, and the sequence similarities are indicated along the y-axis. For reference, select genome landmarks of the three major capsid protein genes are noted above each graph. Simplot analysis of the genome of strain DG_2011 (A) shows a lateral transfer of a small portion of the genome upstream of the penton base gene from HAdV-B16. This is also present in the genomes of HAdV-7d strains CQ1198_2010 and 0901HZ/ShX/2009 (B). Surprisingly, this recombination is not found in the genome from the prototype virus from which these strains descend, Gomen HAdV-B7 (Fort Ord, U.S.A.; 1955) (C). Colors: green, HAdV-B7p (AY594255); blue, HAdV-B3 (AY599834.1); magenta, HAdV-B16 (AY601636.1); light blue, HAdV-B21 (AY601633.1); dark blue, HAdV-B50 (AY737798.1); grey, HAdV-B11 (AY163756.1); brown, HAdV-B34 (AY737797.1); light grey, HAdV-B35 (AY271307.1); dark green HAdV-B14 (AY803294.1); and dark violet HAdV-B55 (FJ643676.1).