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