Inclusion body disease (IBD) is an infectious fatal disease of snakes typified by behavioral abnormalities, wasting, and secondary infections. At a histopathological level, the disease is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues. To date, no virus or other pathogen has been definitively characterized or associated with the disease. Using a metagenomic approach to search for candidate etiologic agents in snakes with confirmed IBD, we identified and de novo assembled the complete genomic sequences of two viruses related to arenaviruses, and a third arenavirus-like sequence was discovered by screening an additional set of samples. A continuous boa constrictor cell line was established and used to propagate and isolate one of the viruses in culture. Viral nucleoprotein was localized and concentrated within large cytoplasmic inclusions in infected cells in culture and tissues from diseased snakes. In total, viral RNA was detected in 6/8 confirmed IBD cases and 0/18 controls. These viruses have a typical arenavirus genome organization but are highly divergent, belonging to a lineage separate from that of the Old and New World arenaviruses. Furthermore, these viruses encode envelope glycoproteins that are more similar to those of filoviruses than to those of other arenaviruses. These findings implicate these viruses as candidate etiologic agents of IBD. The presence of arenaviruses outside mammals reveals that these viruses infect an unexpectedly broad range of species and represent a new reservoir of potential human pathogens.

Summary1. Culicoides biting midges are vectors of internationally important arboviruses including bluetongue virus (BTV). The ecological constraints imposed by the small body size of these insects strongly influence the epidemiology of the diseases they can carry. Bluetongue virus recently emerged in northern Europe, and atmospheric dispersion models have subsequently been employed to simulate vector movement (and hence likely spread of BTV). The data underlying such models, however, have hitherto either been obtained from small-scale studies or from outside the northwestern Palaearctic. 2. The effects of seasonality and local meteorological conditions upon the daily presence and abundance of Culicoides vectors were examined using 2760 samples collected across a network of 12 different habitat types in England during 2008. Over 50 000 individuals were estimated to be in the samples with males constituting 62% of the total collection, allowing straightforward comparison between potential vector species in terms of their activity rates and seasonality. Culicoides abundance was linked to livestock density and land use. Farm-associated Culicoides species were recorded at all sites including species thought to be restricted to this ecosystem by larval habitat, suggesting a greater potential for dispersal over land than previously thought. 3. Synthesis and applications. The model developed has already been applied in a functional dispersion model to predict disease risk from wind-borne infected Culicoides incursion into the UK and elsewhere. The study has expounded the long-distance dispersal potential of Culicoides, essential for future prediction of the incursion and spread of Culicoides-borne pathogens. It has additionally contributed to the understanding of the ecology of highly dispersive insect vectors.

Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential.

ABSTRACT. On passing from clean air into a homogeneous cloud of sex pheromone in a wind tunnel flying male Adoxophyes orana (F.v.R.) (Lepidoptera: Tortricidae) turned more or less upwind and reduced the time and distance between their switchings of track from one side of the wind line to the other. These responses became adapted under the constant pheromone stimulation in the cloud, thereby arresting upwind progress; but the adapted moths would now ‘lock‐on’ to an added pheromone plume and advance upwind along it. Moths also locked‐on to the border of a pheromone cloud, not by turning back on losing the scent as previously supposed but by initiating the above programme of small‐amplitude, crosswind movements (reversing anemomenotaxis). The onset and cessation of the pheromone stimulus produced anemotactic responses that differed quantitatively within a continuum, not two distinct kinds of response as previously supposed. The behavioural mechanism whereby uniform permeation of an area with synthetic sex pheromone can prevent males from finding females is reconsidered.