Whole genome sequencing of genotype VI Newcastle disease viruses from formalin-fixed paraffin-embedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the U.S

Authors: HE, YING - Guangxi University; Taylor, Tonya; DIMITROV, KIRIL - Consultant; BUTT, SALMAN - University Of Georgia; STANTON, JAMES - University Of Georgia; GORAICHUK, IRYNA - Consultant; FENTON, HEATHER - University Of Georgia; POULSON, REBECCA - University Of Georgia; ZHANG, JIAN - University Of Georgia; BROWN, CORRIE - University Of Georgia; IP, HON - National Wildlife Health Center; ISIDORO-AYZA, MARCOS - University Of Wisconsin; Afonso, Claudio

Submitted to: Virology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/18/2017
Publication Date: 1/12/2018
Citation: He, Y., Taylor, T.L., Dimitrov, K.M., Butt, S.L., Stanton, J.B., Goraichuk, I.V., Fenton, H., Poulson, R., Zhang, J., Brown, C.C., Ip, H.S., Isidoro-Ayza, M., Afonso, C.L. 2018. Whole genome sequencing of genotype VI Newcastle disease viruses from formalin-fixed paraffin-embedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the U.S. Virology Journal. 15:9. https://doi.org/10.1186/s12985-017-0914-2.
DOI: https://doi.org/10.1186/s12985-017-0914-2

Interpretive Summary: Newcastle disease is a world-wide problem of poultry caused by virulent isolates of Newcastle disease virus. This virus is commonly isolated from pigeons in the U.S., highlighting the importance of maintaining bio-security to keep the Newcastle disease free status. There is a need for the development of new detection tests for Newcastle disease viruses circulating in pigeons in the U.S. as these have the capacity to infect poultry. In this study, we have used a novel target-independent sequencing approach that is capable of detecting unknown avian pathogens on pigeon tissue samples from 10 different NDV mortality events in the U.S that have been fixed with formalin and stored in paraffin. The use of formalin fixed and paraffin embedded archive samples allowed investigations to be conducted outside of high containment laboratories, as well as, the possibility to study old historical samples. We demonstrated the applicability of this new approach by analyzing samples obtained from different mortality events in the U.S. and by obtaining complete genome sequences from these samples. These complete genome sequences provided data that was used in phylogenetic analyses to further study the circulation and evolution of endemic pigeon viruses in different U.S. regions. This study provides evidence that pigeons are serving as reservoirs of virulent NDV.

Technical Abstract: Background Newcastle 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. Methods FFPE 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. Results Twenty-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. Conclusions This 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.