Introduction Newcastledisease (ND) is one of the most important infectious diseases of poultry and isa major cause of economic losses to the poultry industry3. The virulent strains of Newcastledisease virus (NDV), also known as Avian paramyxovirus serotype-1 (APMV-1),recently named as Avian avulavirus-I (AAvV-I) cause ND. The global spread ofAPMVs, constantly evolving genetic variants and wide range of host avianspecies (chicken and wild migratory birds) are big challenges to the surveillanceand control of Newcastle Disease.

13. NDV belongs to the genus Avulavirus, family Paramyxoviridae, and orderMononegavirales which includes 9 accepted (APMV 1–9) 1 and 4 putative serotypes (APMV 10–13)of APMV and recently reported APMV-14  within the same genus 7, 21,27, 30. The nucleic acid of APMV-1 is single-stranded negativesense RNA 3, with approximate size of 15kb 11 and codes for at least six geneproducts: the nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F),hemagglutinin-neuraminidase (HN), and the RNA polymerase (L) 3 The genetic diversity in the viral genomesof APMV-1 has resulted into phylogenetically two distinct “classes”. 12. The nucleotide variation in codingsequence of Fusion gene is less among Class I viruses (~5.9%) compared to ClassII viruses (7.

8–28.9%) 13. Characterization of APMV-1 by sequence based methodshas been widely used. Genetic analysis of nucleotide sequences for the completecoding region of the fusion gene has recently been proposed as the basis to classifyAPMV-1 isolates into evolutionary related groups 12. Sometimes these groups are limited to only oneparticular region (XVI in North America) and some are highlymobile (V, VI, VII isolated in different continents) 13, 24.

The Fprotein is the major molecular determinant of pathogenicity and variation of nucleotide sequencearound the cleavage site of the Fusion gene protein has been explored for thecharacterization of NDV pathotypes. A polybasic amino acid sequence at thiscleavage site (113R-Q-R/K-R? F117) results in velogenic and mesogenic NDV. However, lentogenic NDV strains have a monobasiccleavage motif due to a leucine at position 117 (113K/R-Q-G/E-R ? L117)23. Conventionally, isolation of NDV inembryonated eggs and then further testing with multiple in vivo assay such as IntracerebralPathogenicity and Index Mean Death Time have been used for pathotypecharacterization 3, 9. Genotypic characterization of virulent genotypes has been performed withReverse Transcription-PCR coupled with Sanger sequencing and has been a goldstandard for DNA sequencing and genotypic characterization of NDV. 5. Currently, identification of NDV virusesby rapid molecular Polymerase Chain Reaction (PCR) based diagnostic assays,such as Matrix gene and Fusion gene assays for NDV is being practiced 19,22.

Earlier, PCR-based assays for identification ofAvirulent and virulent strains of NDV have been reported but these assayscannot differentiate among virulent genotypes of NDV, 28,29. Over past few years with growing application of NGStechnology, different sequencing platforms have been developed and available 4. Next generation sequencing has been widely applied forwhole genome sequencing14, transcriptomics and metagenomics 10,20 and for identification of novel viruses 6. However, high capitalinvestment bioinformatics training, specialized computing facilities andrelatively longer turnaround time ofdata are the limitations for rapid adaptability and availability of NGSplatforms to developing countries17. Rapid detection and identification is an importantcomponent of NDV diagnosis for implementation of quick control measures. The Oxford Nanopore SequencingTechnology (ONT) is a third-generation sequencing technology.

The importanttransformative advantages of ONT are sequencing of longer reads from viruses,bacteria (although despite of origin of sequence), the ability to performreal-time sequence analysis with short turnaround time once the desired targetsequence is detected during the real-time data analysis, the instrument can bestopped, data acquisition is fast, portable without the need of hardy laboratory requirements and over all shortturnaround time 16, 18. Relativelylow cost compared to other high throughput technologies, MinION sequencing willbe a very useful diagnostic tool for viral genomic studies especially in thedeveloping countries where high endemicity of disease and lack of resources areadditional challenges to monitoring and molecular studies of ND 25. so constantsurveillance is important to understand the spread of genotypes indifferent regions. Additionally, genotype matched vaccines provide betterprotection to the birds compared with heterologousvaccines 8. Generating genetic informationdirectly from clinical samples, and avoiding the need for culture, would betransformative step in disease diagnostics approaches15.

In this study we test a simple cost-efficient, sensitive,specific, rapid sequencing on MinION on 33 egg grown viruses 15 clinical swabsamples from chicken. The limit of detection, sensitivity, specificity andreproducibility of the assay compared to other current rapid diagnostic assays wastested. This assay demonstrates a single test to deliver the core informationfor simultaneous detection and characterization of NDV which providessub-genotype level resolution using portable MinION oxford NanoporeTechnologies (ONT)


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