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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