Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples

Authors: BUTT, SALMAN - University Of Georgia; DIMITROV, KIRIL - Consultant; ZHANG, JIAN - University Of Georgia; WAJID, ABDUL - University Of Agriculture - Pakistan; BIBI, TASRA - University Of Veterinary And Animal Sciences; BASHARAT, ASMA - University Of Veterinary And Animal Sciences; BROWN, CORRIE - University Of Georgia; REHMANI, SHAFQAT - University Of Veterinary And Animal Sciences; STANTON, JAMES - University Of Georgia; Afonso, Claudio

Submitted to: Virus Genes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/7/2019
Publication Date: 5/14/2019
Citation: Butt, S.L., Dimitrov, K.M., Zhang, J., Wajid, A., Bibi, T., Basharat, A., Brown, C.C., Rehmani, S.F., Stanton, J.B., Afonso, C.L. 2019. Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples. Virus Genes. 55(4):505-512. https://doi.org/10.1007/s11262-019-01669-9.
DOI: https://doi.org/10.1007/s11262-019-01669-9

Interpretive Summary: Virulent strains of Newcastle disease virus cause significant disease in non-vaccinated and in poorly vaccinated poultry. The circulation of these viruses across the globe represent a constant threat to U.S. poultry industries. Due to the genetic variability of the virus, multiple cases where current diagnostic tools failed detection have been reported, highlighting the need of a more reliable approach to identify and to study the virus. Here we report the utilization of random sequencing on nucleic acids extracted from chicken tissue samples that have been fixed with formalin during disease outbreaks in 2015 in Pakistan. This approach allows identification of pathogens without any prior knowledge of the agent (it is targetindependent). This is the first successful case of complete genome sequencing of viruses obtained from formalin fixed paraffin embedded chicken tissue samples. This methodology is important because it facilitate studies of highly virulent viruses and select agents. Newcastle disease virus is classified as select agent and as such requires specific transportation, special permits and research under biosafety level 3. Formalin fixation inactivates the viruses in the samples and allows their safe and affordable international transport allowing the samples can be studied nearly anywhere under biosafety level 1. Our results demonstrate that virulent Newcastle disease viruses are still circulating in Pakistan.

Technical Abstract: Highly virulent Newcastle disease virus (NDV) is a threat to poultry production worldwide. The global risk of Newcastle disease (ND) requires safe and reliable approaches to elucidate its epidemiology. Next-generation sequencing (NGS) has emerged as a research tool for thorough genetic characterization of infectious organisms. While fresh tissue samples are preferred for diagnostics, their utility is limited by restrictions in transportation from outbreak sites to diagnostic laboratories within and across countries, and by limited preservation and storage at room temperature. Formalin-fixed paraffin-embedded (FFPE) tissues do not have such limitations; however, FFPE tissues are not typically used to differentiate between closely related viruses during disease outbreaks because of degradation and chemical modifications of nucleic acids imparted by formalin fixation. These changes result in only small genomic fragments that do not provide sufficient diagnostic and epidemiological information with commonly used methods. Here thirty-six FFPE tissues (spleen, lung, brain, and small intestine) obtained from 11 birds from 11 poultry flocks during a disease outbreaks in Pakistan were shipped to the U.S. Libraries were prepared from total RNA without using viral enrichment and sequenced on an Illumina MiSeq instrument. Raw data were analyzed using a custom bioinformatics pipeline that includes de novo assembly. Genomes of virulent NDV were detected in 10/11 birds: eight nearly complete (>95% coverage) and two partial genomes. Phylogeny of the NDV concatenated complete genome coding sequences provided better resolution of isolates as compared to phylogenies based on the more commonly used partial or complete fusion gene sequences. Two distinct lineages of sub-genotype VIIi NDV were identified to be simultaneously circulating in Pakistani poultry. Non-targeted NGS of total RNA from FFPE tissues coupled with de novo assembly provided a reliable, safe, and affordable method to conduct the epidemiological and evolutionary studies to facilitate management of ND in Pakistan.