Abstract
BackgroundNewcastle disease viruses (NDV) are highly contagious and cause disease in both wild birds and poultry. A pigeon-adapted variant of genotype VI NDV, often termed pigeon paramyxovirus 1, is commonly isolated from columbids in the United States and worldwide. Complete genomic characterization of these genotype VI viruses circulating in wild columbids in the United States is limited, and due to the genetic variability of the virus, failure of rapid diagnostic detection has been reported. Therefore, in this study, formalin-fixed paraffin-embedded (FFPE) samples were subjected to next-generation sequencing (NGS) to identify and characterize these circulating viruses, providing valuable genetic information. NGS enables multiple samples to be deep-sequenced in parallel. When used on FFPE samples, this methodology allows for retrospective studies of infectious organisms.MethodsFFPE wild pigeon tissue samples (kidney, liver and spleen) from 10 mortality events in the U.S. between 2010 and 2016 were analyzed using NGS to detect and sequence NDV genomes from randomly amplified total RNA. Results were compared to the previously published immunohistochemistry (IHC) results conducted on the same samples. Additionally, phylogenetic analyses were conducted on the complete and partial fusion gene and complete genome coding sequences.ResultsTwenty-three out of 29 IHC-positive FFPE pigeon samples were identified as positive for NDV by NGS. Positive samples produced an average genome coverage of 99.6% and an average median depth of 199. A previously described sub-genotype (VIa) and a novel sub-genotype (VIn) of NDV were identified as the causative agent of 10 pigeon mortality events in the U.S. from 2010 to 2016. The distribution of these viruses from the North American lineages match the distribution of the Eurasian collared-doves and rock pigeons in the U.S.ConclusionsThis work reports the first successful evolutionary study using deep sequencing of complete NDV genomes from FFPE samples of wild bird origin. There are at least two distinct U.S. lineages of genotype VI NDV maintained in wild pigeons that are continuously evolving independently from each other and have no evident epidemiological connections to viruses circulating abroad. These findings support the hypothesis that columbids are serving as reservoirs of virulent NDV in the U.S.
Highlights
Newcastle disease viruses (NDV) are highly contagious and cause disease in both wild birds and poultry
There are at least two distinct U.S lineages of genotype VI NDV maintained in wild pigeons that are continuously evolving independently from each other and have no evident epidemiological connections to viruses circulating abroad
From 17 birds submitted for sequencing, nearly complete genomes were recovered from 12 Eurasian collared-doves (ECDO) and 2 Rock pigeons (ROPI) birds, which represents a total positive characterization rate of 82% (Table 1)
Summary
Newcastle disease viruses (NDV) are highly contagious and cause disease in both wild birds and poultry. Due to the rapid spread of the virus, mass mortalities of Eurasian collared-doves (Streptopelia decaocto, ECDO) and rock pigeons (Columba livia, ROPI) have occurred within the U.S [7, 8] Both species have the ability to interact with naïve species due to the lack of geographical separation, which justifies increased biosecurity within poultry production regions where these columbids abound. For these reasons, it is important to develop a robust and reliable diagnostic assay to provide detailed genomic characterization of NDV circulating in wild columbids in the United States
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