Rabies in a postpandemic world: resilient reservoirs, redoubtable riposte, recurrent roadblocks, and resolute recidivism.

Rabies is an ancient disease. Two centuries since Pasteur, fundamental progress occurred in virology, vaccinology, and diagnostics-and an understanding of pathobiology and epizootiology of rabies in testament to One Health-before common terminological coinage. Prevention, control, selective elimination, and even the unthinkable-occasional treatment-of this zoonosis dawned by the twenty-first century. However, in contrast to smallpox and rinderpest, eradication is a wishful misnomer applied to rabies, particularly post-COVID-19 pandemic. Reasons are minion. Polyhostality encompasses bats and mesocarnivores, but other mammals represent a diverse spectrum of potential hosts. While rabies virus is the classical member of the genus, other species of lyssaviruses also cause the disease. Some reservoirs remain cryptic. Although global, this viral encephalitis is untreatable and often ignored. As with other neglected diseases, laboratory-based surveillance falls short of the notifiable ideal, especially in lower- and middle-income countries. Calculation of actual burden defaults to a flux within broad health economic models. Competing priorities, lack of defined, long-term international donors, and shrinking local champions challenge human prophylaxis and mass dog vaccination toward targets of 2030 for even canine rabies impacts. For prevention, all licensed vaccines are delivered to the individual, whether parenteral or oral-essentially 'one and done'. Exploiting mammalian social behaviors, future 'spreadable vaccines' might increase the proportion of immunized hosts per unit effort. However, the release of replication-competent, genetically modified organisms selectively engineered to spread intentionally throughout a population raises significant biological, ethical, and regulatory issues in need of broader, transdisciplinary discourse. How this rather curious idea will evolve toward actual unconventional prevention, control, or elimination in the near term remains debatable. In the interim, more precise terminology and realistic expectations serve as the norm for diverse, collective constituents to maintain progress in the field.

The Effect of Mating Complexity on Gene Drive Dynamics.

AbstractGene drive technology promises to deliver on some of the global challenges humanity faces today in health care, agriculture, and conservation. However, there is a limited understanding of the consequences of releasing self-perpetuating transgenic organisms into wild populations under complex ecological conditions. In this study, we analyze the impact of three such complexities-mate choice, mating systems, and spatial mating network-on the population dynamics for two distinct classes of modification gene drive systems. All three factors had a high impact on the modeling outcome. First, we demonstrate that distortion-based gene drives appear to be more robust against mate choice than viability-based gene drives. Second, we find that gene drive spread is much faster for higher degrees of polygamy. Including a fitness cost, the drive is fastest for intermediate levels of polygamy. Finally, the spread of a gene drive is faster and more effective when the individuals have fewer connections in a spatial mating network. Our results highlight the need to include mating complexities when modeling the properties of gene drives, such as release thresholds, timescales, and population-level consequences. This inclusion will enable a more confident prediction of the dynamics of engineered gene drives and possibly even inform about the origin and evolution of natural gene drives.

Eroding norms over release of self-spreading viruses.

Risky research on lab-modified self-spreading viruses has yet to present credible paths to upsides.

A common gene drive language eases regulatory process and eco-evolutionary extensions.

BACKGROUND: Synthetic gene drive technologies aim to spread transgenic constructs into wild populations even when they impose organismal fitness disadvantages. The extraordinary diversity of plausible drive mechanisms and the range of selective parameters they may encounter makes it very difficult to convey their relative predicted properties, particularly where multiple approaches are combined. The sheer number of published manuscripts in this field, experimental and theoretical, the numerous techniques resulting in an explosion in the gene drive vocabulary hinder the regulators' point of view. We address this concern by defining a simplified parameter based language of synthetic drives. RESULTS: Employing the classical population dynamics approach, we show that different drive construct (replacement) mechanisms can be condensed and evaluated on an equal footing even where they incorporate multiple replacement drives approaches. Using a common language, it is then possible to compare various model properties, a task desired by regulators and policymakers. The generalization allows us to extend the study of the invasion dynamics of replacement drives analytically and, in a spatial setting, the resilience of the released drive constructs. The derived framework is available as a standalone tool. CONCLUSION: Besides comparing available drive constructs, our tool is also useful for educational purpose. Users can also explore the evolutionary dynamics of future hypothetical combination drive scenarios. Thus, our results appraise the properties and robustness of drives and provide an intuitive and objective way for risk assessment, informing policies, and enhancing public engagement with proposed and future gene drive approaches.

Natural copy number variation of tandemly repeated regulatory SNORD RNAs leads to individual phenotypic differences in mice.

Genic copy number differences can have phenotypic consequences, but so far this has not been studied in detail in natural populations. Here, we analysed the natural variation of two families of tandemly repeated regulatory small nucleolar RNAs (SNORD115 and SNORD116) in the house mouse (Mus musculus). They are encoded within the Prader-Willi Syndrome gene region, known to be involved in behavioural, metabolic, and osteogenic functions in mammals. We determined that the copy numbers of these SNORD RNAs show substantial natural variation, both in wild-derived mice as well as in an inbred mouse strain (C57BL/6J). We show that copy number differences are subject to change across generations, making them highly variable and resulting in individual differences. In transcriptome data from brain samples, we found SNORD copy-number correlated regulation of possible target genes, including Htr2c, a predicted target gene of SNORD115, as well as Ankrd11, a predicted target gene of SNORD116. Ankrd11 is a chromatin regulator, which has previously been implicated in regulating the development of the skull. Based on morphometric shape analysis of the skulls of individual mice of the inbred strain, we show that shape measures correlate with SNORD116 copy numbers in the respective individuals. Our results suggest that the variable dosage of regulatory RNAs can lead to phenotypic variation between individuals that would typically have been ascribed to environmentally induced variation, while it is actually encoded in individual differences of copy numbers of regulatory molecules.

Transcriptional effects of a positive feedback circuit in Drosophila melanogaster.

BACKGROUND: Synthetic systems that use positive feedback have been developed to control human disease vectors and crop pests. The tTAV system, which has been deployed in several insect species, relies on a positive feedback circuit that can be inhibited via dietary tetracycline. Although insects carrying tTAV fail to survive until adulthood in the absence of tetracycline, the exact reason for its lethality, as well as the transcriptomic effects of an active positive feedback circuit, remain unknown. RESULTS: We engineered the tTAV system in Drosophila melanogaster and investigated the effects of tTAV genome integration locus on the whole fly transcriptome during larval and adult life stages in four transgenic fly strains using gene expression microarrays. We found that while there were widespread effects on the transcriptome, the gene expression differences after removal of tetracycline were not consistent between integration sites. No specific region of the genome was affected, no common set of genes or pathways, nor did the integration site affect the transcripts in cis. CONCLUSION: Although the positive feedback tTAV system is effective at killing insect larvae regardless of where it is inserted in the genome, it does not exhibit a specific, consistent transcriptional signature. Instead, each insertion site is associated with broad, but different, transcriptional effects. Our results suggest that lethality may not be caused by a direct effect on transcription of a set of key genes or pathways. Instead, we propose that rather than a specific action of a tTAV protein, it is the stochastic transcriptional effects specific to each insertion site that contribute to the tTAV-induced mortality.

Automated Phenotyping Indicates Pupal Size in Drosophila Is a Highly Heritable Trait with an Apparent Polygenic Basis.

The intense focus on studying human height has done more than any other genetic analysis to advance our understanding of the heritability of highly complex phenotypes. Here, we describe in detail the properties of a previously unexplored trait in Drosophila melanogaster that shares many salient properties with human height. The total length of the pupal case varies between 2.8 and 3.9 mm among natural variants, and we report that it is among the most heritable traits reported in this species. We have developed a simple semiautomatic phenotyping system with which a single operator can reliably score >5000 individuals in a day. The precision of the automated system is 0.042 mm (± 0.030 SD). All phenotyped individuals are available to be mated in subsequent generations or uniquely archived for future molecular work. We report both broad sense and narrow sense heritability estimates for two biologically distinct data sets. Narrow sense heritability (h2) ranged from 0.44 to 0.50, and broad sense heritability (H2) ranged from 0.58 to 0.61. We present results for mapping the trait in 195 recombinant inbred lines, which suggests that there are no loci with >10% effect size in this panel. We propose that pupal size genetics in Drosophila could represent a model complex trait amenable to deep genetic dissection using the automated system described.

Engaging scientists: An online survey exploring the experience of innovative biotechnological approaches to controlling vector-borne diseases.

BACKGROUND: Pioneering technologies (e.g., nanotechnology, synthetic biology or climate engineering) are often associated with potential new risks and uncertainties that can become sources of controversy. The communication of information during their development and open exchanges between stakeholders is generally considered a key issue in their acceptance. While the attitudes of the public to novel technologies have been widely considered there has been relatively little investigation of the perceptions and awareness of scientists working on human or animal diseases transmitted by arthropods. METHODS: Consequently, we conducted a global survey on 1889 scientists working on aspects of vector-borne diseases, exploring, under the light of a variety of demographic and professional factors, their knowledge and awareness of an emerging biotechnology that has the potential to revolutionize the control of pest insect populations. RESULTS: Despite extensive media coverage of key developments (including releases of manipulated mosquitoes into human communities) this has in only one instance resulted in scientist awareness exceeding 50% on a national or regional scale. We document that awareness of pioneering releases significantly relied on private communication sources that were not equally accessible to scientists from countries with endemic vector-borne diseases (dengue and malaria). In addition, we provide quantitative analysis of the perceptions and knowledge of specific biotechnological approaches to controlling vector-borne disease, which are likely to impact the way in which scientists around the world engage in the debate about their value. CONCLUSIONS: Our results indicate that there is scope to strengthen already effective methods of communication, in addition to a strong demand by scientists (expressed by 79.9% of respondents) to develop new, creative modes of public engagement.

First steps towards underdominant genetic transformation of insect populations.

The idea of introducing genetic modifications into wild populations of insects to stop them from spreading diseases is more than 40 years old. Synthetic disease refractory genes have been successfully generated for mosquito vectors of dengue fever and human malaria. Equally important is the development of population transformation systems to drive and maintain disease refractory genes at high frequency in populations. We demonstrate an underdominant population transformation system in Drosophila melanogaster that has the property of being both spatially self-limiting and reversible to the original genetic state. Both population transformation and its reversal can be largely achieved within as few as 5 generations. The described genetic construct {Ud} is composed of two genes; (1) a UAS-RpL14.dsRNA targeting RNAi to a haploinsufficient gene RpL14 and (2) an RNAi insensitive RpL14 rescue. In this proof-of-principle system the UAS-RpL14.dsRNA knock-down gene is placed under the control of an Actin5c-GAL4 driver located on a different chromosome to the {Ud} insert. This configuration would not be effective in wild populations without incorporating the Actin5c-GAL4 driver as part of the {Ud} construct (or replacing the UAS promoter with an appropriate direct promoter). It is however anticipated that the approach that underlies this underdominant system could potentially be applied to a number of species.

Using underdominance to bi-stably transform local populations.

Underdominance refers to natural selection against individuals with a heterozygous genotype. Here, we analyze a single-locus underdominant system of two large local populations that exchange individuals at a certain migration rate. The system can be characterized by fixed points in the joint allele frequency space. We address the conditions under which underdominance can be applied to transform a local population that is receiving wildtype immigrants from another population. In a single population, underdominance has the benefit of complete removal of genetically modified alleles (reversibility) and coexistence is not stable. The two population system that exchanges migrants can result in internal stable states, where coexistence is maintained, but with additional release of wildtype individuals the system can be reversed to a fully wildtype state. This property is critically controlled by the migration rate. We approximate the critical minimum frequency required to result in a stable population transformation. We also concentrate on the destabilizing effects of fitness and migration rate asymmetry. Practical implications of our results are discussed in the context of utilizing underdominance to genetically modify wild populations. This is of importance especially for genetic pest management strategies, where locally stable and potentially reversible transformations of populations of disease vector species are of interest.

Evidence of susceptibility and resistance to cryptic X-linked meiotic drive in natural populations of Drosophila melanogaster.

There is mounting evidence consistent with a general role of positive selection acting on the Drosophila melanogaster X-chromosome. However, this positive selection need not necessarily arise from forces that are adaptive to the organism. Nonadaptive meiotic drive may exist on the X-chromosome and contribute to forces of selection. Females from a reference D. melanogaster line, containing the X-linked marker white, were crossed to males from 49 isofemale lines established from seven African and five non-African natural populations to detect naturally occurring meiotic drive. Several lines exhibited a departure from expected Mendelian transmission of X-chromosomes to the third generation (F2) offspring, particularly those from hybrid African male parents. F2 viability was not correlated with skewed chromosomal inheritance. However, a significant difference in viability between cosmopolitan and tropical African crosses was observed. Recombination analysis supports the presence of a male-acting meiotic drive element near the centromeric region of the X-chromosome and putative recessive autosomal drive suppression. There is also evidence of another female-acting drive element linked to white. The possible role meiotic drive may contribute in shaping levels of genetic variation in D. melanogaster, and additional ways to test this hypothesis are discussed.