Unbiased sequencing of RNA taken from six individuals suffering from beak deformities using the Illumina HiSeq platform yielded about 1.4×10 7 to 7.2×10 7 126-nt single-read read sequences per individual ( Table 1). Arizona Birds  Retrospective mapping of Sequenced Reads back to the genome of poliovirus identified the complete polyprotein code regions (7,588 Nt) of
the poliovirus in a single red-breasted
Nuthatch (929). It also exposed a small portion from the poliovirus polyprotein coding region that are of different sizes (789-3,166 in) within the remaining specimens that were tested (Fig. 2). Metagenomic sequencing in conjunction together with the bioinformatic pathogen identification method failed to identify any pathogen other than poliovirus in the samples tested.
Comparing the NCBI NRT and nonredundant protein databases revealed that the complete polyprotein sequence of Red-breasted Nuthatch 929 (validated through Sanger analysis) was closer to poecivirus than another known virus, with 96.5 percent identity between nts
and 98.2 percent amino acid identity with poecivirus that was isolated from the Alaskan Black-capped Chickadees (Zylberberg and colleagues. 2016). The genome of 7,588 nt discovered from the Red-breasted Nuthatch had 267 genetic mutations when compared
with that of the Black-capped
Chickadee poecivirus genome, as identified through Sanger sequencing. Of these of which there were 202 (75.7 percent) were confirmed by Illumina sequencing data , with an average 427 reads for each nt. 112 of them were not
synonyms. The negative controls we used and the high number of Illuminaand Sanger-confirmed variations discovered in this full-length polyprotein confirm that this sequence didn’t originate from contamination of pestiviruses that were previously amplified by this lab.
All nongenomic raw data that confirm the conclusions of this publication are available within the US Geological Survey data release (Zylberberg and others. 2020). Genomics data are available on NCBI (GenBank numbers. MN944596-MN944619).
DISCUSSION
The study examined whether poliovirus was found in people of a variety of species, who showed clinical signs that are consistent with AKD which is a condition characterized by severe beak defects. The simultaneous appearance of beak defects across species as well as the geographical proximity of clusters adds to the hypothesis that several species are affected by the same disease that shares the same etiology (Handel and
colleagues. 2010). Although we have previously established the strong link with poecivirus as well as AKD in the Black-capped Chickadee, our latest study has provided evidence that the same etiologic agent may be the cause of similar beak defects in other species of avian.
In line with the notion that the development of deformities in the beak in different species is directly related poecivirus was found being present in eight out of (100 percent) of the individuals tested with targeted PCR, followed by Sanger sequencing, but not uniformly across samples of different types. However, unblinded metagenomic analysis of the beak and cloacal tissues did not reveal a second potential pathogen. Although we can’t rule out
the possibility of additional
pathogens in different tissues, this finding is in line with our previous discovery of poecivirus, but there are no other pathogens were found in the beaks of the AKD-affected Black-capped chickadees.
We found the entire polyprotein coder region of virus from one specimen, the Red-breasted Nuthatch 929. We also retrieved more than 25 percent of the virus’s genome in Hairy Woodpecker 969 and American Crow 848 (2.5 kb and 3.2 Kb and 3.2 kb,
respectively). Despite our efforts to collect the entire polyprotein coding region that encode the virus in other samples, we were not able to achieve this. This is probably because of the inconsistent condition of the samples, which were collected randomly and subjected to various handling conditions prior process by the University of California. The analysis of
sequences revealed that the viruses
originated from Hairy Woodpecker 969, Red-breasted Nuthatch 929, and American Crow 848 (GenBank nos. MN944614-MN944618-MN944618, M MN944600-MN944602, and MN 944604 and MN 944604, respectively) are closely connected to the virus isolated from Black-capped Chickadees and displaying the 96, 94 in
addition to 94% identification as well as 94% identity in line with the reference genome for poecivirus (GenBank number. KU977108). Do Birds Have Ears For The Red-breasted Nuthatch and American Crow These data are a step forward from the results of earlier studies of more limited viral
sequences from these species (Zylberberg and colleagues. 2016). With the exception of the Red-breasted Nuthatch Next-generation sequencing of more well-preserved samples will be required for obtaining the complete viral genome of each host to prove that they are affected by poecivirus, not an unrelated but distinct type of virus.
