Figure 2. Bayesian phylogenetic inference of rabies
whole-genome sequences: 2A: Phylogenetic inference and divergence
estimation of Indian rabies sequences with respect to other rabies
genomes reported from south Asian countries. 2B: Map showing different
south Indian states from which the samples were obtained. 2C: Divergence
estimation of regional Arctic-like (AL1a) sequences from south India.
Sequences from this study are colored based on the state from which the
corresponding samples were obtained. The colour scale refers to
posterior probability 0 to 1 of the nodes in the Bayesian tree.
4. Discussion
Despite being endemic for canine rabies and accounting for the highest
burden of human deaths due to rabies world-wide, there are limited
studies focusing on the molecular epidemiology of RABV from India. Most
of these reports are based on single gene sequencing15-24, with only a few reports of complete RABV
genomes 35-37. This report with 20 complete genomes,
recovered directly from archived human and canine brain samples,
provides an important starting point for analyzing the evolution and
spread of RABV in India. The tiling primer-based amplicon sequencing on
the MinION, used and validated as a method for detection and full length
sequencing of RABV from clinical samples will advance rabies diagnosis
and research in our setting. The successful retrieval of whole genomes
using this protocol from samples archived since 2003 in our laboratory
has immense potential to characterize more samples archived in
repositories to gain insights into the historical RABV diversity,
emergence of new variants, evolutionary mechanisms, spread and impact of
rabies control efforts in specific regions.
Full length genomes of lyssaviruses are increasingly available using
next generation sequencing (NGS) methods and provide more robust and
comprehensive data, which allow us to trace the evolution of the virus,
study outbreaks including the spatio-temporal pattern of viral
transmission, and explore the virus and host shifts in an efficient
manner 38,39. Whole-genome sequencing gives accurate
and precise information compared to partial gene sequences and is
important for validating diagnostic results, tracking virus
phylogenetics and monitor minor variants and mutations in genes that are
associated with virulence 40. Despite the
susceptibility of numerous mammalian species to RABV infection,
sustained transmission occurs only in a few species, suggesting barriers
to cross-species transmission 6,41. Extensive analysis
of RABV whole genome sequences world-wide can potentially reveal the
complex evolutionary dynamics and heterogeneity in evolutionary rates
among RABV in different hosts 6.
Interestingly, the genomes of 20 RABV sequences from the current study
clustered together and all the genetically related viruses were from the
four different states of South India. Absence of geography-associated
clustering and the wide dissemination of the same strain of rabies virus
within India suggests one pre-dominant enzootic transmission cycle in
dogs and a continuous viral gene flow across state borders with limited
regional evolution 4,42. Phylogenetic analysis also
revealed that all the 20 study sequences belonged to the Arctic-like 1a
lineage, as reported earlier 4,23. While dogs are the
principal reservoirs of the Arctic-like viruses, wild-life may have a
possible role in maintaining these viral populations in India24. Further genomic studies on the role of wild-life
in disease transmission is critical to rabies-control efforts in India.
Though the Arctic-like lineage is the major circulating lineage reported
across the country 15-24, co-circulation of the Indian
subcontinental lineage in some southern states has been reported in a
few studies 20, 24. Whole genome sequencing of a
rabies-infected wild dog from our laboratory recently also confirmed the
Indian subcontinent lineage 36. This emphasises the
importance of continuous genomic surveillance in a variety of hosts and
geographical areas. However, since the subcontinental lineage is not
reported to be circulating widely in human populations, the amplicon
sequencing used here was primarily designed for sequencing the
Arctic-like lineage; all the study sequences belonged to Arctic-like
lineage. We therefore recommend the use of Arctic lineage primers as a
primary method, and then gap filling using the Indian subcontinental
primers if required.
Our divergence estimates suggest that the sequence from this study
diverged from related strains about 60 years ago. This highlights the
need to sequence more circulating strains in India. The lack of complete
genomes from India is a particular problem because of the high burden of
disease. Adding more complete genomes will aid the construction of
phylogenetic history of rabies in the subcontinent, estimate viral
diversity and population dynamics and contribute to more focused
surveillance and rabies control measures.
We observed, the sequences within India have more recent ancestors
(TMRCA) while the difference between the Afghanistan and Indian
sequences is 200 years. Further studies on RABV in different parts of
India and neighbouring countries are important to understand the
dynamics of rabies in the region.
Genomic data has the potential to inform several stages of the complex
pathway leading to rabies elimination 43, such as to
characterize circulating variants and lineages, and identify reservoirs
to inform targeted vaccination 4,44,45, cross-species
transmission/spill over 46,47, identifying
trans-border incursions 4,48,49, monitoring threats to
bio-diversity conservation in canine-endemic regions36, and enhance surveillance during
end-game/post-elimination 4,50,51.
The nanopore sequencer (ONT, UK), a small, portable, low-cost sequencer
used in this study enables rapid detection and surveillance of RABV, and
other related lyssaviruses especially in field conditions and in low
throughput, peripheral laboratories. It is a promising tool for outbreak
investigations and point-of-care genomic analysis for various infectious
diseases in resource limited setting. Amplicon sequencing using primer
sets that span the RABV genome, employed in this study, is a convenient
and cost-effective method for obtaining whole genomes. However,
modification or re-designing of primers, as well as the protocol may be
required for optimal characterization of other lineages or non-RABV
lyssaviruses. A metagenomics approach, which is an unbiased method of
sequencing total nucleic acid content from clinical samples, is ideal
for detection and characterization of diverse or novel lyssaviruses.
However, this approach is relatively costly and may not yield complete
genomes, especially in samples with low viral titres43,52. A combination of amplicon sequencing and
metagenomics using the MinION is likely to emerge as a powerful tool for
molecular epidemiology of rabies in resource limited settings.
Genomic data is emerging as a powerful surveillance tool and can provide
unique insights into rabies spread and persistence that can direct
control efforts. A global initiative to achieve zero human deaths from
dog-mediated rabies by 2030 is now underway 2.
Integrated, economical and effective surveillance tools are critical to
achieve these goals in India.
Supplementary Materials: The following supporting information
can be downloaded at: www.mdpi.com/xxx/s1, Table S1: RABV Arctic Clade
Primers; Figure S1: Mapping of
Arctic primer set designed in the study with other RABV lineages
circulating in India; Figure S2: Base coverage of RABV genomes recovered
in the study
Author Contributions: Conceptualization, RSM.; methodology,
PKH., GY., PP., CP., and BRA.; software, PKH., CP. and BRA.; validation,
RSM., CP., and PKH.; formal analysis, PKH., PP., CP. and BRA.;
investigation, PKH., PP., CP., GY., AM., and MMSB; resources, RSM., GY.,
AM. and CP; data curation, PKH. and CP.; writing—original draft
preparation, PKH., CP. and RSM.; writing—review and editing, RSM;
supervision, RSM., AM. and MMSB.; project administration, RSM.
Funding: This research received no external funding.
Institutional
Review Board Statement: This study did not involve humans or animals.
Only post-mortem brain samples (from humans and dogs) were used in this
study. The human brain samples sourced from the human brain tissue
repository were collected post-mortem following written, informed
consent from next of kin of the deceased, and their use for research was
approved by the NIMHANS Institutional Ethics Committee. All canine brain
tissues used in this study were collected at necropsy from dogs that
died of suspected rabies, through the routine rabies surveillance
programme conducted by Mission Rabies, a non-governmental organization
and the Department of Animal Husbandry and Veterinary Services,
Government of Goa and sent to our rabies referral laboratory for
diagnostic confirmation. Therefore, approval from Institutional Animal
Ethics Committee was not required. The study protocol was approved by
the NIMHANS Institutional Ethics Committee (NIMH/DO/ETHICS SUB-COMMITTEE
MEETING/2017, dated June 19, 2017).
Informed Consent Statement: Not applicable
Data Availability Statement: The data that support the reported
results in this study are openly available in GenBank and
dx.doi.org/10.17504/protocols.io.3byl4jzb8lo5/v1.
Conflicts of Interest: The authors declare no conflict of
interest.
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