Rabies Prophylactic and Treatment Options: An In Vitro Study of siRNA- and Aptamer-Based Therapeutics.

If the goal of eliminating dog-mediated human rabies by 2030 is to be achieved, effective mass dog vaccination needs to be complemented by effective prophylaxis for individuals exposed to rabies. Aptamers and short-interfering RNAs (siRNAs) have been successful in therapeutics, but few studies have investigated their potential as rabies therapeutics. In this study, siRNAs and aptamers-using a novel selection method-were developed and tested against rabies virus (RABV) in a post-infection (p.i.) scenario. Multiple means of delivery were tested for siRNAs, including the use of Lipofectamine and conjugation with the developed aptamers. One siRNA (N53) resulted in an 80.13% reduction in viral RNA, while aptamer UPRET 2.03 demonstrated a 61.3% reduction when used alone at 2 h p.i. At 24 h p.i., chimera UPRET 2.03-N8 (aptamer-siRNA) resulted in a 36.5% inhibition of viral replication. To our knowledge, this is the first study using siRNAs or aptamers that (1) demonstrated significant inhibition of RABV using an aptamer, (2) tested Lipofectamine RNAi-Max as a means for delivery, and (3) produced significant RABV inhibition at 24 h p.i. This study serves as a proof-of-concept to potentially use aptamers and siRNAs as rabies immunoglobulin (RIG) replacements or therapeutic options for RABV and provides strong evidence towards their further investigation.

Lyssaviruses and the Fatal Encephalitic Disease Rabies.

Lyssaviruses cause the disease rabies, which is a fatal encephalitic disease resulting in approximately 59,000 human deaths annually. The prototype species, rabies lyssavirus, is the most prevalent of all lyssaviruses and poses the greatest public health threat. In Africa, six confirmed and one putative species of lyssavirus have been identified. Rabies lyssavirus remains endemic throughout mainland Africa, where the domestic dog is the primary reservoir - resulting in the highest per capita death rate from rabies globally. Rabies is typically transmitted through the injection of virus-laden saliva through a bite or scratch from an infected animal. Due to the inhibition of specific immune responses by multifunctional viral proteins, the virus usually replicates at low levels in the muscle tissue and subsequently enters the peripheral nervous system at the neuromuscular junction. Pathogenic rabies lyssavirus strains inhibit innate immune signaling and induce cellular apoptosis as the virus progresses to the central nervous system and brain using viral protein facilitated retrograde axonal transport. Rabies manifests in two different forms - the encephalitic and the paralytic form - with differing clinical manifestations and survival times. Disease symptoms are thought to be due mitochondrial dysfunction, rather than neuronal apoptosis. While much is known about rabies, there remain many gaps in knowledge about the neuropathology of the disease. It should be emphasized however, that rabies is vaccine preventable and dog-mediated human rabies has been eliminated in various countries. The global elimination of dog-mediated human rabies in the foreseeable future is therefore an entirely feasible goal.

The Role of Waste Management in Control of Rabies: A Neglected Issue.

Despite being vaccine preventable, the global burden of dog rabies remains significant, and historically it is the rural and marginalized communities in developing countries of Africa and Asia that are most threatened by the disease. In recent years, the developing world has been experiencing unprecedented increases in urbanization, with a correspondingly massive increase in municipal solid waste generation, among other things. Inefficient and inadequate waste collection and management, due to lack of resources and planning, led to significant increases in the volumes of waste on the streets and in open dumps, where it serves as food sources for free-roaming dogs. In this commentary, we discuss examples of poor waste management and the likely impact on rabies control efforts through the sustenance of free-roaming dogs in some dog rabies-endemic countries. We aim to stress the importance of implementing strategies that effectively address this particular issue as an important component of humane dog population management, as it relates to aspirations for the control and elimination of dog rabies per se.

Formation of the Asian Rabies Control Network (ARACON): A common approach towards a global good.

The drive towards the worldwide elimination of dog-mediated human rabies by 2030 is the first step towards the ultimate goal of dog rabies elimination - as dogs account for more than 99% of human rabies cases globally - and has gained considerable momentum since this resolution was taken at a global meeting in Geneva in December 2015. For dog rabies-endemic countries and regions, dedicated regional networks may offer unique opportunities to take advantage of this global momentum. Towards this goal, the Pan-African Rabies Control Network (PARACON) was created in 2015, and the past year has seen the formation of the Asian Rabies Control Network (ARACON). ARACON provides opportunities for member countries to share lessons learnt and challenges faced, while also introducing them to programmatic support tools such as the Stepwise Approach towards Rabies Elimination (SARE) assessment and the Rabies Epidemiological Bulletin (REB). During the inaugural ARACON meeting, member countries evaluated their progress and developed country-specific Practical Workplans based on their SARE outcomes. The results from the national-level SARE assessments were considered at the regional level and, after discussion among countries, consensual agreement was reached that the target date of regional freedom from dog-mediated human rabies by 2020 was not feasible, and a new regional target of 2030 was set. With this new regional target, ongoing support will continue to be provided to countries through regional structures such as ARACON. However, the responsibility remains with the countries to use the available tools and resources to progress towards the new regional goal of dog-mediated human rabies elimination by 2030.

Improved method for the generation and selection of homogeneous lumpy skin disease virus (SA-Neethling) recombinants.

Lumpy skin disease virus (LSDV) is being developed as a vector for recombinant vaccines against diseases of veterinary importance. A strategy for generating viral thymidine kinase (TK) gene-disrupted recombinants which are stable and homogeneous using the South African Neethling vaccine strain of LSDV as vector has been developed. To assist with the selection process, the Escherichia coli beta-galactosidase (lacZ) visual marker gene was incorporated into the constructs. However, the use of lacZ has certain limitations. An improved strategy was then devised substituting lacZ with the enhanced green fluorescent protein (EGFP) under control of the vaccinia virus (VV) P11K late promoter. The EGFP marker was found to enhance the selection process, and with the inclusion of additional sonication and filtration steps the number of passages required to select recombinants to homogeneity has been reduced. In support of the improved method for generation and selection of recombinants described, three different LSDV recombinants expressing the glycoprotein genes of bovine ephemeral fever virus, Rift Valley fever virus and rabies virus were prepared and characterised.

Epidemiology of Rabies in Lesotho: The Importance of Routine Surveillance and Virus Characterization.

Rabies is widespread throughout Africa and Asia, despite the fact that the control and elimination of this disease has been proven to be feasible. Lesotho, a small landlocked country surrounded by South Africa, has been known to be endemic for rabies since the 1980s but the epidemiology of the disease remains poorly understood due to limited sample submission, constrained diagnostic capabilities, and a lack of molecular epidemiological data. Considering the existing challenges experienced in Lesotho, we aimed to evaluate the direct, rapid immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis in Lesotho. Towards this aim, extensive training on the implementation and interpretation of the DRIT was hosted in Lesotho in April 2016 before both tests were applied to all samples subjected to routine rabies diagnosis at the Central Veterinary Laboratory (CVL). We found agreement between the DFA and DRIT assays in 90/96 samples (93.75%). The samples that produced inconsistent results (n = 6) were re-tested a further two times with both assays before being subjected to a real-time qPCR to confirm the diagnosis. Additionally, a statistically significant three-fold increase in the average number of samples submitted per month was observed after the DRIT implementation started, following continuous rabies awareness initiatives amongst the animal health professionals in the country over a 12-month period (p = 0.0279). Partial G-L intergenic regions of selected rabies-positive samples (n = 21) were amplified, sequenced, and subjected to phylogenetic analyses. Molecular epidemiological analyses, that included viruses from neighbouring provinces in South Africa, suggested that at least three independent rabies cycles within Lesotho were implicated in instances of cross-border transmission. This study has evaluated alternative methods for diagnosing and improving rabies surveillance in Lesotho, as well as providing new information that would be of importance in the planning of future disease intervention campaigns, not only in Lesotho, but also in neighbouring South Africa.