Molecular Typing and Rapid Identification of Human Adenoviruses Associated With Respiratory Diseases Using Universal PCR and Sequencing Primers for the Three Major Capsid Genes: Penton Base, Hexon, and Fiber

Abstract

Human adenoviruses (HAdVs) within species B, C, and E are responsible for highly contagious and potentially severe respiratory disease infections. The traditional method to type these pathogens was based on virus neutralization and hemagglutination assays, which are both time-consuming and difficult, particularly due to the nonavailability of reagents. Subsequent molecular typing based on the partial characterization of the hexon gene and/or the restriction enzyme analysis (REA) of the genomes is inadequate, particularly in identifying recombinants. Here, a rapid, simple, and cost-effective method for molecular typing HAdV respiratory pathogens is presented. This incorporates three pairs of universal PCR primers that target the variable regions of the three major capsid genes, i.e., hexon, penton base, and fiber genes, that span the genome. The protocol enables typing and characterization of genotypes within species B, C, and E, as well as of some genotypes within species D and F. To validate this method, we surveyed 100 children with HAdV-associated acute respiratory infections identified by direct immunofluorescence (Hong Kong; July through October, 2014). Throat swab specimens were collected and analyzed by PCR amplification and sequencing; these sequences were characterized by BLAST. HAdVs were detected in 98 out of 100 (98%) samples, distributing as follows: 74 HAdV-B3 (74%); 10 HAdV-E4 (10%); 7 HAdV-C2 (7%); 2 HAdV-C6 (2%); 1 HAdV-B7 (1%); 1 HAdV-C1 (1%); 2 co-infection (2%); and 1 novel recombinant (1%). This study is the first detailed molecular epidemiological survey of HAdVs in Hong Kong. The developed method allows for the rapid identification of HAdV respiratory pathogens, including recombinants, and bypasses the need for whole genome sequencing for real-time surveillance of circulating adenovirus strains in outbreaks and populations by clinical virologists, public health officials, and epidemiologists.

Keywords: Hong Kong; adenovirus; co-infection; epidemiology; molecular typing; recombination; universal primers.

Figure 1

(A) Schematic human adenoviruses (HAdV) genome, with the primer positions and the resultant PCR products noted for the three major capsid protein genes: penton base (left), hexon (middle), and fiber (right) genes. The relative locations of the capsid protein genes in the HAdV genome are also noted. These genes contain the genotyping and molecular characterization information for HAdVs. The purple arrows indicate the genes and their locations; the blue bars indicate the PCR products and their relative lengths; and other colored bars indicate important domains within each gene, including hypervariable regions (HVRs and RGD Loop), as well as the tail, shaft, and knob domains. (B) Alignment of HAdV universal primers for HAdV-A18, B3, -B7, B-11, -B14, -B16, -B21, -B34, -B35, -B50, -B55, -B66, -B68, -C1, -C2 -C5, -C6, -D9, -D19, -D37, -E4, -F41, and -G52. Divergence from the primer sequences is shown for the isolates tested. Each virus is identified with its genotype number as well as its species demarcation for reference. Dots represent identical bases, and base differences are noted. The bases are color-coded for visual comparisons.

Figure 2

One percent Agarose gel electrophoresis profiles of PCR products from HAdV-B3, -B7, -B11, -B14, -B21, -B55, -C5, -D19, -E4, and -F41 using universal primers for penton base, hexon, and fiber genes. (A) Penton base gene PCR amplification using primers Penton-F and Penton-R. (B) Hexon gene PCR amplification using primers HVR-F and HVR-R. (C) Fiber gene PCR amplification using primers Fiber-F and Fiber-R (Fiber-CR for HAdV-C5). Molecular weight sizing markers are in lane M. NTC, “no template” PCR control.

Figure 3

(A) HAdV genotype distribution in hospitalized pediatric patients with ARIs from July through October, 2014. Panels (B–D) display the phylogenetic relationships of the penton base, hexon, and fiber genes, respectively, of the 98 identified HAdV clinical isolates. Additional reference sequences were retrieved from GenBank to provide context and reference. The phylogenetic trees were generated based on the Tree-Bisection-Reconnection (TBR) model by MEGA 7.0 (www.megasoftware.net/) using the maximum parsimony method with 1,000 boot-strap replicates and default parameters. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The scale bar is in units of nucleotide substitutions per site.