Keywords
Rabies; Rabies whole genome sequencing; Phylogenetic analysis; Genomic
surveillance; Nanopore sequencing
Introduction
Rabies is a fatal zoonotic disease caused by viruses of the Lyssavirus
genus (in the family Rhabdoviridae of the order Mononegavirales) that
kills tens of thousands of people world-wide annually, mostly in Asia
and Africa. An estimated 20,000 people die of rabies in India every
year, representing one third of the global burden and the highest for
any country. The prototype virus of the genus, Rabies lyssavirus (RABV)
is the most common causative agent, and dog-mediated transmission is
responsible for >99% of the human cases reported1.
To meet the global goal of “Zero human deaths due to dog-mediated
rabies by 2030” 2, recently the National Action Plan
for Dog Mediated Rabies Elimination (NAPRE) by 2030, has been developed
in India, to prioritize rabies as a zoonosis and provide a strategic
framework for control of rabies using an ‘One Health’ approach3. Systematic disease surveillance in humans and
animals is a vital component of effective rabies control and elimination
strategies. Laboratory confirmation and genomic characterization of the
virus can help identify circulating variants, the reservoir host species
and geographical distribution across various regions, which may help to
plan, optimize, monitor, and confirm rabies elimination, and track
outbreaks and transboundary incursions 4.
The RABV genome is about 12kb in size, comprising of a single-stranded,
non-segmented, negative sense RNA encoding five structural proteins; N
(nucleoprotein), P (phosphoprotein), M (matrix protein), G
(glycoprotein) and L (RNA-dependent RNA polymerase) 5.
RABV has been extensively studied by molecular epidemiological
tools-owing to its zoonotic spread, global public health impact and the
invariably fatal outcome of disease. Eight major phylogenetic lineages
of RABV circulating world-wide, including six canid-related lineages,
associated with particular host reservoirs and geographical range have
been reported 6.
Early molecular epidemiological studies of RABV are based on limited
regions of the genome, which could adequately identify viral variants
and their host reservoirs. However, whole genome sequencing has enhanced
our understanding of the taxonomy of lyssaviruses 7and is now considered a pre-requisite for confirmation of new species8. It has the potential to uncover genetic variations
in RABV strains which may influence host tropism and pathogenicity9. Compared to the Sanger sequencing method frequently
used for this purpose earlier, next generation sequencing (NGS) enables
full-genome sequencing of RABV with rapidity and high
cost-effectiveness, with a remarkable increase in available genetic
information, facilitating more accurate and precise molecular
epidemiological and phylogenetic analysis 10. In this
context, the recently available novel, portable real-time NGS sequencer,
MinION (Oxford Nanopore Technologies (ONT), Oxford, UK), enables direct
genome sequencing of micro-organisms (bacteria, viruses, fungi and
parasites) from biological samples, which has been successfully utilized
for viral discovery, diagnostics and genomic surveillance in outbreaks11-14.
Studies on the molecular epidemiology of RABV from India are limited and
mostly based on partial/complete single gene sequencing (such as N,G)15-24. All these studies have reported the circulation
of the Arctic/Arctic-like (AL) lineage predominantly across the country.
Co-circulation of the Indian subcontinental lineage, restricted to a few
southern states 20,24, and anecdotal evidence of
presence of the Cosmopolitan lineage 23 has also been
reported. The impact of non-RABV lyssaviruses is undefined in India due
to paucity of genomic data. Furthermore, while the vast majority of
rabies deaths are attributed to dogs, the role of wildlife and bats in
the transmission of fatal lyssaviral infections to humans, reported
globally 25 is significantly underreported in India.
This study was performed to assess the feasibility of direct sequencing
of clinical samples (archived human and canine brain tissues), for
detection and recovery of whole genome sequences of RABV, using the
single molecule nanopore sequencing technology (ONT, UK), and
characterization and phylogenetic analysis of the resulting full
genomes. Complete RABV genomes were recovered from all the 20 archived
human and canine brain tissue samples (from 2003 to 2019), from 4
southern states of India, sequenced in this study. The establishment and
validation of tiling primer-based amplicon sequencing using the
nanopore, directly from clinical samples will advance rabies diagnosis
and research in our setting, and improve our understanding about
evolution and spread of this neglected zoonotic pathogen in India.
Materials and Methods
Human and animal brain tissues
Post-mortem human brain tissues (n=10) were obtained from the Human
brain tissue repository (HBTR, Brain Bank), Department of
Neuropathology, NIMHANS, Bangalore, India. These brain tissues were
collected at autopsy (between 2003-2019) from patients who succumbed to
rabies, laboratory-tested for confirmation of rabies, frozen and stored
at -800C. The archived canine brains (n=10) used in
this study had been received for diagnostic confirmation (during
2017-2018) at the Neurovirology laboratory, NIMHANS, Bangalore as a part
of routine rabies surveillance from Goa state. Laboratory confirmation
of rabies on these samples was done by fluorescent antibody test (FAT)
for antigen detection using monoclonal antibodies to rabies
nucleoprotein (Light Diagnostics, USA) 26 and real
time PCR for detection of rabies viral RNA 27,28.Details of samples used for genomic sequencing are given in Table 1.
Table 1. Details of human and canine brain samples used in this study