Introduction

Newcastle
disease (ND) is one of the most important infectious diseases of poultry and is
a major cause of economic losses to the poultry industry3. The virulent strains of Newcastle
disease virus (NDV), also known as Avian paramyxovirus serotype-1 (APMV-1),
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 the surveillance
and control of Newcastle Disease. 13. NDV belongs to the genus Avulavirus, family Paramyxoviridae, and order
Mononegavirales 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 negative
sense RNA 3, with approximate size of 15kb 11 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) 3

The genetic diversity in the viral genomes
of APMV-1 has resulted into phylogenetically two distinct “classes”. 12. The nucleotide variation in coding
sequence of Fusion gene is less among Class I viruses (~5.9%) compared to Class
II viruses (7.8–28.9%) 13. Characterization of APMV-1 by sequence based methods
has been widely used. Genetic analysis of nucleotide sequences for the complete
coding region of the fusion gene has recently been proposed as the basis to classify
APMV-1 isolates into evolutionary related groups 12. Sometimes these groups are limited to only one
particular region (XVI in North America) and some are highly
mobile (V, VI, VII isolated in different continents) 13, 24.

The F
protein is the major molecular determinant of pathogenicity and variation of nucleotide sequence
around the cleavage site of the Fusion gene protein has been explored for the
characterization of NDV pathotypes. A polybasic amino acid sequence at this
cleavage site (113R-Q-R/K-R
? F117) results in velogenic and mesogenic NDV. However, lentogenic NDV strains have a monobasic
cleavage motif due to a leucine at position 117 (113K/R-Q-G/E-R ? L117)
23.

Conventionally, isolation of NDV in
embryonated eggs and then further testing with multiple in vivo assay such as Intracerebral
Pathogenicity and Index Mean Death Time have been used for pathotype
characterization 3, 9. 
Genotypic characterization of virulent genotypes has been performed with
Reverse Transcription-PCR coupled with Sanger sequencing and has been a gold
standard for DNA sequencing and genotypic characterization of NDV. 5. Currently, identification of NDV viruses
by 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 of
Avirulent and virulent strains of NDV have been reported but these assays
cannot differentiate among virulent genotypes of NDV, 28,
29. Over past few years with growing application of NGS
technology, different sequencing platforms have been developed and available 4. Next generation sequencing has been widely applied for
whole genome sequencing14, transcriptomics and metagenomics 10,
20 and for identification of novel viruses 6. However, high capital
investment bioinformatics training, specialized computing facilities and
relatively longer turnaround time of
data are the limitations for rapid adaptability and availability of NGS
platforms to developing countries17. Rapid detection and identification is an important
component of NDV diagnosis for implementation of quick control measures.

The Oxford Nanopore Sequencing
Technology (ONT) is a third-generation sequencing technology. The 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 and over all short
turnaround time 16, 18. 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 25. so constant
surveillance is important to understand the spread of genotypes in
different regions. Additionally, genotype matched vaccines provide better
protection to the birds compared with heterologous
vaccines 8. Generating genetic information
directly from clinical samples, and avoiding the need for culture, would be
transformative 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 swab
samples from chicken. The limit of detection, sensitivity, specificity and
reproducibility of the assay compared to other current rapid diagnostic assays was
tested. This assay demonstrates a single test to deliver the core information
for simultaneous detection and characterization of NDV which provides
sub-genotype level resolution using portable MinION oxford Nanopore
Technologies (ONT)

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