A Novel Simian Adenovirus Associating with Human Adeno-virus Species G Isolated from Long-Tailed Macaque Feces

Abstract

Metagenomics has demonstrated its capability in outbreak investigations and pathogen surveillance and discovery. With high-throughput and effective bioinformatics, many disease-causing agents, as well as novel viruses of humans and animals, have been identified using metagenomic analysis. In this study, a VIDISCA metagenomics workflow was used to identify potential unknown viruses in 33 fecal samples from asymptomatic long-tailed macaques (Macaca fascicularis) in Ratchaburi Province, Thailand. Putatively novel astroviruses, enteroviruses, and adenoviruses were detected and confirmed by PCR analysis of long-tailed macaque fecal samples collected from areas in four provinces, Ratchaburi, Kanchanaburi, Lopburi, and Prachuap Khiri Khan, where humans and monkeys live in proximity (total n = 187). Astroviruses, enteroviruses, and adenoviruses were present in 3.2%, 7.5%, and 4.8% of macaque fecal samples, respectively. One adenovirus, named AdV-RBR-6-3, was successfully isolated in human cell culture. Whole-genome analysis suggested that it is a new member of the species Human adenovirus G, closely related to Rhesus adenovirus 53, with evidence of genetic recombination and variation in the hexon, fiber, and CR1 genes. Sero-surveillance showed neutralizing antibodies against AdV-RBR-6-3 in 2.9% and 11.2% of monkeys and humans, respectively, suggesting cross-species infection of monkeys and humans. Overall, we reported the use of metagenomics to screen for possible new viruses, as well as the isolation and molecular and serological characterization of the new adenovirus with cross-species transmission potential. The findings emphasize that zoonotic surveillance is important and should be continued, especially in areas where humans and animals interact, to predict and prevent the threat of emerging zoonotic pathogens.

Keywords: VIDISCA; long-tailed macaques; metagenomics; novel adenovirus; virus discovery.

Figure 1

Map of Thailand showing sites of long-tailed macaque fecal sample collection. Provinces where the samples were collected are indicated by black circles. Star (*) indicates Ratchaburi Province. Samples from this region were analyzed by VIDISCA NGS and by virus culture. Colored squares indicate types of viruses detected by PCR in each site. The color labeling for each virus is indicated in the figure.

Figure 2

Map of AdV-RBR-6-3 genome assembly. (A) The whole genome of AdV-RBR-6-3 is 34,144 bp coding for at least 11 groups of early (E) and late (L) proteins. (B) By de novo and reference sequence assembly of NGS contigs, whole genome of AdV-RBR-6-3 was initially retrieved with gaps. (C) Primers were designed for PCR and nucleotide sequencing to close the gap regions. Bars in (C) represent primer binding and PCR products. P1–P7 are primer names of which sequences are provided in Table S2. PV indicates site of VIDISCA PCR product in E3 region.

Figure 3

Phylogenetic analysis of (A) whole genome, (B) hexon, (C) fiber-1, and (D) CR1 genes of AdV-RBR-6-3 compared with reference sequences of Human adenovirus A to G (HAdV-A to -G), Simian adenovirus A (SAdV-A) and B (SAdV-B). Black triangle indicates AdV-RBR-6-3. Diamond indicates Human adenovirus 52, the only member of species HAdV-G isolated from human. Tree shrew adenovirus sequences were used as an outgroup for the whole genome and hexon trees. SAdV-A sequences were used as an outgroup for the fiber-1 and CR1 trees. According to variabilities in fiber and CR1 regions amongst mastadenoviruses, sequences with ≥30% identities were included in the trees. The trees were constructed using the maximum likelihood method with a bootstrap of 1000. Bootstrap values are shown at the node. The bar represents nucleotide substitutions per site.

Figure 4

Recombination analyses of AdV-RBR-6-3 complete genome. (A) Similarity plot and bootscan analyses to identify potential genetic recombination sites were performed with a sliding window size of 200 nucleotides and a step size of 20 nucleotides using SimPlot. The AdV-RBR-6-3 complete genome was used as a query against reference sequences of Human adenovirus G. (B) Recombination analysis using 3SEQ suggested recombination events among members of Human adenovirus G. Breakpoint intervals and genome regions are indicated for each recombinant. Color labels are indicated for adenovirus sequences. Rh AdV, Rhesus adenovirus; SAdV, Simian adenovirus.

Figure 5

Neutralizing antibody titers against AdV-RBR-6-3 in long-tailed macaque plasmas and human sera in Prachuap Khiri Khan Province. Antibody titer ≥10 was considered as positive.