RNA Deep-Sequencing Analyses for Detection and Characterization of Avian Orthoreovirus and Fowl Adenovirus Co-Infections in Layer Chickens

Avian orthoreovirus (ARV) and Fowl Adenovirus (FAdV) infections are pervasive in domestic poultry species, especially in chickens. Coinfections of the two viral pathogens could cause much severer symptoms on infected birds. In our recent research studies on application of Next-Generation Sequencing (NGS) techniques, we have identified two coinfection viruses of ARV (Reo/PA/Layer/27614/13 or Reo/PA27614) and FAdV (FAdV/PA/Layer/27614/13 or FAdV/PA27614) from one isolation from tendon tissue of 35-week-old commercial layer chickens. Among a total of 831,429 RNA-seq reads, 40,954 reads (4.92%) were confirmed to be ARV genome sequence, whereas an extremely small number of 566 reads (0.06%) were confirmed to be FAdV mRNA which transcribed by viral genome DNA. The de novo assembly of two types of viral reads generated 10 ARV contigs and 23 FAdV contigs, which according to 10 genome segments of ARV full genome and 14 mRNAs of partial FAdV transcriptome, respectively. Sequence comparison of nucleotide (nt) and amino acid (aa) sequences of Reo/PA27614 genome and FAdV/ PA27614 hexon gene revealed that the Reo/PA27614 field variant had 40.0-94.1% nt and 27.4-98.8% aa identities in comparison with ARV reference strains, and the FAdV/PA27614 variant had 73.6-98.2% nt and 83.1-98.8% aa identities in comparison with FAdV reference strains. Genome alignment and phylogenetic analysis revealed that the Reo/ PA27614 evolved distant from most ARV reference strains in three major outer capsid proteins, whereas the FAdV/PA27614 showed a close relationship with pathogenic reference strains of FAdV group C. Taken together, the NGS-based deep RNA sequencing techniques allowed us to identify the RNA virus and DNA virus co-infections at the same time and provided important epidemiological insights into ARV and FAdV co-infections in chickens.