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Crystal Gigante

Low-cost rabies virus sequencing in Goa, India using the Oxford Nanopore MinION

Note: Crystal presented disclaimers at the beginning of her talk; please view her presentation for more information.

Crystal Gigante (Centers for Disease Control and Prevention, USA) introduced how rabies is a global burden, present on all continents but Antarctica, which causes nearly 60,000 deaths each year. Post-bite vaccination and treatment with rabies immunoglobulin can prevent almost 100% of cases; however, once symptoms begin, there is no cure, and mortality rates are close to 100%. Crystal highlighted how India carries the greatest rabies burden of any country, both in terms of human deaths and disability-adjusted life years. It is estimated that 1/3 of global rabies deaths take place in India, and almost all of these cases are caused by a dog bite. Crystal noted that children are disproportionately affected. Controlling rabies in the canine population can reduce human rabies cases – Crystal highlighted how vaccinating only humans is insufficient, as the virus will continue to be maintained in dogs. Coordinated surveillance and canine vaccination campaigns have successfully controlled – even eliminated – rabies in several countries, and the WHO have set the goal of eliminating dog-mediated human rabies deaths by 2030. Crystal described how many regions with endemic canine rabies are enthusiastic to meet this goal but are still in the planning stages as they may not have the resources to monitor and track the virus. Furthermore, some populations may be difficult to access. Crystal therefore stressed the importance of cost and portability in rabies control.

Human and dog cases of rabies are underreported in many canine rabies-endemic countries; Crystal described that reliable surveillance data is necessary to estimate the prevalence of the virus and size of the canine population, procure funding, and plan control strategies. Rabies is caused by rabies virus; it can infect many mammalian host species, but has only established sustained transmission cycles in a few, including dogs. Sequencing can be used to genetically or antigenically distinguish the virus strains circulating in different reservoirs. It can also determine their geographical distribution: Crystal showed the variants circulating in non-bat reservoirs including fox and skunk in the USA. In combination with other surveillance data, sequencing data can be used to determine host reservoirs, characterise the geographical limits of circulation, identify importation/translocation events, inform outbreak and case investigations, guide control efforts, and confirm rabies-free status. Crystal showed an example phylogeny constructed using sequencing data for rabies virus isolates, displaying clades clustered by geographical location across the United States and Canada.

Crystal described how rabies is neglected in India, with no nation-wide canine vaccination program. Goa has been a test case for canine rabies elimination in India. Starting in 2014, the NGO Mission Rabies partnered with the government of Goa: through increased surveillance and lab capacity, education, and canine vaccination, a striking drop-off in human and canine rabies cases has been seen, with no human rabies cases in Goa reported in 2018 or 2019. As Goa moved towards rabies elimination, Mission Rabies and their collaborators became interested in sequencing the virus; however, Goa does not have local sequencing capacity, and it was not possible to ship samples out of the country. Crystal described how nanopore sequencing on the portable MinION allowed for in-country sequencing in a veterinary diagnostic lab that lacked molecular biology lab equipment. Rabies is caused by viruses in the Lyssavirus genus, most frequently Rabies lyssavirus. This is a negative-sense ssRNA virus, with a 12 kb genome encoding 5 proteins.

Crystal and her colleagues developed a wet lab protocol for rabies virus sequencing, prioritising low cost, maximal comparisons to public sequences, and ease of use. The method used a partial genome approach, employing RT-PCR to amplify complete nucleoprotein and glycoprotein genes. A single primer set was designed to work for any rabies sample. Samples were then barcoded and sequenced in multiplex on a MinION. This RT-PCR approach cost USD 6.57/sample. Sequencing 50 samples per run cost USD 14.90/sample, including library prep. Sequencing was performed overnight, and the data was basecalled and demultiplexed. BWA mem and CLC genomics workbench 11 were then employed for fast reference-based consensus generation on a laptop. The consensus sequences were polished with Nanopolish; an alignment-based manual correction method was then used. This produced a nucleoprotein gene consensus accuracy of 99.996% when using >50x read depth, compared with Sanger sequencing.

After developing this protocol, Crystal travelled to the Disease Investigation Unit, Directorate of Animal Health and Veterinary Services, Goa. As well as the MinION, she used portable solutions for the rest of the workflow, including the equipment for RT-PCR, centrifugation, and DNA quantification. From the 103 rabies-positive sequenced samples, the protocol enabled the production of 80 nucleoprotein sequences and 97 glycoprotein sequences, representing at least one sequence per sample. Phylogenetic analysis of these new isolates alongside previously available sequences, based on nucleoprotein gene sequence, revealed that all newly-sequenced isolates fell into the Arctic-like 1a rabies virus lineage. Most clustered into three groups: one mostly containing samples from northern Goa, one featuring samples collected throughout Goa, and one with samples from northern Goa and the neighbouring state Maharashtra. One isolate also displayed divergence from other isolates. Two samples fell out of these groups, and one had only 96% nucleotide identity to the others (versus >99% for the others). The latter sample was found to cluster with sequences from northern India, Nepal, and Pakistan. Further investigation revealed that this sample was collected from a sick dog that was transported from Rajasthan, northern India, to Goa. Crystal noted that clustering of samples did not correlate with host animal species, suggesting one predominant enzootic transmission cycle in dogs.

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