Sequence analysis of a novel Mastadenovirus isolated in a Pygmy Marmoset (Callithrix [Cebuella] pygmaea)

Abstract

Adenoviruses are present worldwide infecting a large variety of hosts each with their own specific target species. To date, there have been no reported incidents of the detection of an own Adenovirus infection within the Marmoset genera. The purpose of this study was firstly to identify and sequence the Adenovirus isolated from the Pygmy Marmoset specimens. Secondly to attempt to conclude if the now detected Mastadenovirus is as a result of a switch of the virus among host species, or is it an ancient representative of the new world monkeys AdV-s and thirdly to decipher the degree of the relatedness of the PMAdV with other Mastadenoviruses. From specimens taken from a Pygmy Marmoset virus isolation was performed. The viral DNA was then isolated and extracted from the organ samples and the cell culture supernatants. Primers were applied to the extracted viral DNA for the detection of the DNA virus, for the amplification of the polymerase gene which resulted in an amplicon of 1020 bps long, and then for the detection of the hexon gene. Once the virus was confirmed as a member of the Mastadenovirus, sequences from GenBank were downloaded and aligned, to aid with the design of further primers for the whole genome amplification. The “primer walking” sequencing strategy was performed along the whole virus genome using new specifically designed primers for sequencing, based on newly achieved genetic data of the PMAdV. This resulted in the generation of long amplicons between two relatively adjacent conserved genes. PMAdV amplified DNA-s were sequenced and the nucleotide sequences were compiled and compared using FASTA. Based on the results from this the four most closely related adenoviruses, CAdV-2 strain Toronto, CAdV-1 and BtAdV strain TJM and PPV-1 were identified. The common stretch of 181 bps of ITR and a conserved character of 94% similarity the E1a and E1b, IVa2 and polymerase (with a 75 % similarity to CAdV-2) genes led us to the grouping of this virus in the Mastadenoviruse genera. Based on this study the penton, hexon and protease proteins proved to be the most conserved genes among Mastadenoviruses. On the surrounding region of the fiber gene a BLAST hit noted the distant relatedness with BtAdV-s and CAdV-2 Toronto. In conclusion, the PMAdV proved to be an ancient Mastadenovirus with a distant relationship to BtAdV-s and CAdV-s. It also suggests the PMAdV might be an early split of Mastadenoviruses coevolving with the Pygmy Marmosets of the New World Monkeys.