Unique sequence features of the Human adenovirus 31 complete genomic sequence are conserved in clinical isolates

Abstract

Background: Human adenoviruses (HAdV) are causing a broad spectrum of diseases. One of the most severe forms of adenovirus infection is a disseminated disease resulting in significant morbidity and mortality. Several reports in recent years have identified HAdV-31 from species A (HAdV-A31) as a cause of disseminated disease in children following haematopoetic stem cell transplantation (hSCT) and liver transplantation. We sequenced and analyzed the complete genome of the HAdV-A31 prototype strain to uncover unique sequence motifs associated with its high virulence. Moreover, we sequenced coding regions known to be essential for tropism and virulence (early transcription units E1A, E3, E4, the fiber knob and the penton base) of HAdV-A31 clinical isolates from patients with disseminated disease.

Results: The genome size of HAdV-A31 is 33763 base pairs (bp) in length with a GC content of 46.36%. Nucleotide alignment to the closely related HAdV-A12 revealed an overall homology of 84.2%. The genome organization into early, intermediate and late regions is similar to HAdV-A12. Sequence analysis of the prototype strain showed unique sequence features such as an immunoglobulin-like domain in the species A specific gene product E3 CR1 beta and a potentially integrin binding RGD motif in the C-terminal region of the protein IX. These features were conserved in all analyzed clinical isolates. Overall, amino acid sequences of clinical isolates were highly conserved compared to the prototype (99.2 to 100%), but a synonymous/non synonymous ratio (S/N) of 2.36 in E3 CR1 beta suggested positive selection.

Conclusion: Unique sequence features of HAdV-A31 may enhance its ability to escape the host's immune surveillance and may facilitate a promiscuous tropism for various tissues. Moderate evolution of clinical isolates did not indicate the emergence of new HAdV-A31 subtypes in the recent years.

Figure 1

Map of the genome organization and transcription units of HAdV-A31. Early and late transcription units are represented in different colors, intermediate gene products in white. The block arrows represent the predicted protein, titled either by protein name or predicted molecular size. Orientation of the arrows indicates the direction of transcription.

Figure 2

Phylogenetic analysis of all available complete genomic HAdV sequences representing all human adenovirus species (A to G), including the newly generated HAdV-A31 sequence. The tree was generated with MEGA 3.1 using neighbor-joining method, bootstrap values (%) were generated with 1,000 pseudoreplicates. For nucleotide accession numbers see Methods section.

Figure 3

Global pairwise sequence alignment of the HAdV-A31 genome with representative types of each HAdV species. The x axis shows the genome position, the y axis shows the sequence conservation in percent. Arrows on top display the transcription units and the direction of their transcription.

Figure 4

Schematic view of the predicted E3 CR1 beta (A) and the E3 RID beta (B) proteins of HAdV-A31, -A12 and -F40 or -C5, respectively. (A): a V-Set domain (red box) was only predicted within the N-terminal region of the E3 CR1 beta protein of HAdV-A31. (B): N-terminal phosphorylation sites (small red boxes) were predicted both for the HAdV-A31 and -C5 RID beta proteins, but not for HAdV-A12. Domain predictions were carried out using web based Pfam, ProSite and BLASTp.

Figure 5

Multiple alignment of the protein IX amino acid sequences of HAdV-A31, -A12, -C5, -F40 and -G52. The RGD motif found in the protein IX of HAdV-A31 is highlighted.