Isolation and characterization of adenoviruses persistently shed from the gastrointestinal tract of non-human primates

Abstract

Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors.

Figure 1. Phylogenetic tree of adenoviruses that infect primates.

The tree was reconstructed from an alignment of the polymerase gene, using maximum likelihood under the HKY85 model of substitutions, as described in Materials and Methods. The names of simian isolates include the serotype nomenclature, the animal species of isolation (Hu: human, Ch: chimpanzee, Bo: bonobo, Go: gorilla, Cy: cynomolgus macaque, Rh: rhesus macaque), and the source of adenoviral isolation (ATCC: American Tissue Type Collection, JX: Jacksonville zoo, NI: New Iberia Research Center, MD: MD Anderson, SD: San Diego zoo, At: Atlanta zoo, LR: Little Rock zoo, BF: Buffalo zoo). Isolates that have a closely related hexon structure are referred to as variants of the same serotype (“.1” or “.2”). Names of novel sequences obtained in this study are shown in italics. Colors indicate the six species of viruses that infect higher primates (A, B, C, D, E, and F); grey is used to indicate viruses isolated from monkeys. The inset (upper left) shows the same tree with the inclusion of a tree-shrew isolate as an outgroup, after collapsing poorly supported bifurcations. Bootstrap values less than 80% or close to terminal leaves are suppressed.

Figure 2. Evidence for intra-species B recombination within the New Iberia chimpanzee colony.

A Simplot analysis of New Iberia species B adenoviral isolates aligned with a schematic annotated genomic structure of the SAdV-27.1 showing evidence of an incidence of recombination between viral isolates from the same compound. The analysis demonstrates homology on the left genomic end extending up to the DNA Binding Protein (DBP) open reading frame with various species B isolates from within one subspecies (represented by SAdV-32) from the New Iberia chimpanzee colony, followed by an 100K, 22K, 33K and partial E3 gene region of near complete identity to the structurally distinct New Iberia isolate SAdV-35.1 of a distinct subspecies within species B. To the right of this region of identity the homology transitions to the fiber region first to B isolate SAdV-33 and subsequently to SAdV-32 again. Further evidence is provided by SAdV-27.2, isolated from a gorilla in a different geographical location (Atlanta zoo). This genome is closely related to SAdV-27.1 and is almost identical in hexon but did not undergo the presumed recombination event between DBP and E3. The left and extreme right ends of the SADV-27.1 genome demonstrate close but not complete identity to SAdV-32 indicating a close relationship to the putative parental adenovirus of this event of homoplasy. The left junction of the recombination event is a clearly defined junction centrally in the DNA binding protein as indicated by the dotted blue line. The right end junction was less well-defined due to a region of lack of complete identity in the fiber-proximal part of the coding region of the E3 gene family. This region is indicated by the dotted blue lines in the sequence. The more distantly related species B isolate SAdV-35, species C isolate SAdV-31 and species E isolate SAdV-38 (all isolated from the New Iberia chimpanzee colony) are provided in the analysis as reference.