Introduction and 15,198 nt 9 and codes for

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Newcastle disease (ND) is one of the most important
disease of poultry and is a major cause of economic losses to the poultry
industry. The virulent strains of Avian paramyxovirus serotype-1 (APMV-1) also
known as Newcastle disease virus (NDV), recently named as Avian avulavirus-I (AAvV-I)
cause ND. The global spread of APMVs, constantly evolving genetic variants and
wide range of host avian species (chicken and wild migratory birds) are big
challenges to surveillance and control of ND in developed and developing
countries 11. NDV belongs to the genus Avulavirus,
family Paramyxoviridae, and order Mononegavirales 1 There are 9 accepted
(APMV 1–9) 1 and 4 putative
serotypes (APMV 10–13) of APMV within the genus 6, 17, 21, 22. The
nucleic acid of APMV-1 is single-stranded negative sense RNA 2, has three genome
sizes 15,186 nucleotides (nt), 15,192 nt and 15,198 nt 9 and codes for at least
six gene products: the nucleoprotein (NP), phosphoprotein (P), matrix (M),
fusion (F), hemagglutinin-neuraminidase (HN), and the RNA polymerase (L) 2

analysis of nucleotide sequences for the complete coding region of the fusion
gene has recently been proposed as the basis for a unified nomenclature and
classification system for assigning APMV-1 isolates into evolutionary related
groups 10. A 10% mean nucleotide
difference of the fusion protein gene coding sequence is required to assign
phylogenetically distinct groups of viruses into genotypes. Based on genetic
diversity within genotypes, some genotypes are further divided into ‘sub-genotypes
10. The mean genetic
distance per site in the range of 3–10% and a bootstrap value N60 at a defining
node are used as cutoff values for assigning new sub-genotypes. The viruses of
Class I are 41.46.3% divergent from class II genotypes based on the complete
coding sequence of the fusion protein gene, with overall 44.2% distance between
these two major groups. Class I viruses are less diverse among themselves with
5.9% overall mean distance between all known isolates. Class II viruses are
more diverse with nucleotide distances between genotypes varying from 7.8–28.9%
at the fusion gene.

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identification and genetic characterization of circulating NDV genotypes is
also important as first, there
are genotypes which are limited to only one particular region (XVI in
North America) and some are highly mobile (V, VI, VII isolated in
different continents) 11 so it is important to know the spread of genotypes in the regions
where they are not expected to be. Second, there is scientific evidence
that genotype matched vaccines provide better protection to the birds
compared with heterologous vaccines 7. So, it is
important to know the genotypes/sub-genotypes for development of vaccine as
disease control measure.

characterization of viruses by molecular biological approaches has been widely
accepted across the world. Initially, Sanger sequencing has been a gold
standard for DNA sequencing and until recently used to confirm NGS results 4. Over past few years
with growing application of NGS technology, different sequencing platforms have
been developed and available 3. Next
generation sequencing has been widely applied for whole genome sequencing12, transcriptomics and metagenomics
of small RNA viruses 8, 16 and
for identification of novel viruses 5.
However, expensive piece of equipment, bioinformatics training, specialized
computing facilities, relatively longer
turnaround time of data and capital investment are still big hindrance of rapid
adaptability and availability of NGS platforms to developing countries13.

sequencing, PCR amplicon sequencing and target enrichment sequencing are three
most common approaches for virus sequencing from clinical samples14. Among these PCR-based
approach has advantage of cost and sensitivity over other approaches. Currently,
identification of viruses by rapid molecular diagnostic assays, such as Matrix
qRT-PCR and Fusion protein gene qRT-PCR assays for NDV is being practiced 15, 18. These
potentially culture-free, PCR-based tests rapidly identify Avirulent and
virulent strains of NDV, but cannot differentiate among different virulent
genotypes of NDV. However, these assays have limitations as genotypic
characterization of virulent genotypes is only being done with RT-PCR coupled
with Sanger sequencing, which is laborious, time consuming. Generating genetic
information directly from clinical samples, and avoiding the need for culture,
would be transformative step in disease diagnostics approaches. However, direct
samples contain highly variable amounts of virus nucleic acid, and mixed
population of infectious agents.

Oxford Nanopore Sequencing Technology (ONT) is a third-generation sequencing
technology. There important transformative advantages of ONT are sequencing of
longer reads from viruses, bacteria (although despite of origin of sequence), the
ability to perform real-time sequence analysis with short turnaround time once
the desired target sequence is detected during the real-time data analysis, the
instrument can be stopped, data acquisition is fast, portable without the need of hardy laboratory
requirements, short turnaround time and real-time sequence analysis. Relatively
low cost compared to other high throughput technologies, MinION sequencing will
be a very useful diagnostic tool for viral genomic studies especially in the developing
countries where high endemicity of disease and lack of resources are additional
challenges to monitoring and molecular studies of ND 19.

In this study we test a simple cost-efficient, sensitive,
specific, rapid sequencing on MinION on 33 egg grown viruses 15 clinical swab
samples from chicken.  We evaluate the
protocol in terms of RNA obtained, sub-genotypic resolution of the reads. These
data would enable a single test to deliver the core information for both detection
and characterization of NDV in < 12 hours using highly portable MinION oxford Nanopore Technologies (ONT). The limit of detection, sensitivity, specificity of the test was tested on egg grown viruses representative genotypes of APMV-1. We generated an amplicon mixture (6, 16, 33) of Avian paramyxoviruses (APMV,s). After sequencing target region spanning Matrix and Fusion gene of APMV-1, a consensus sequence was reconstructed using newly written alignment tool which is part of the data analysis pipeline which provides sub-genotype level resolution

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