Transcriptomics has the potential to discover new RNA virus genomes by sequencing total intracellular RNA pools. In this study, we have searched publicly available transcriptomes for sequences similar to viruses of the Nidovirales order. We report two potential nidovirus genomes, a highly divergent 35.9 kb likely complete genome from the California sea hare Aplysia californica, which we assign to a nidovirus named Aplysia abyssovirus 1 (AAbV), and a coronavirus-like 22.3 kb partial genome from the ornamented pygmy frog Microhyla fissipes, which we assign to a nidovirus named Microhyla alphaletovirus 1 (MLeV). AAbV was shown to encode a functional main proteinase, and a translational readthrough signal. Phylogenetic analysis suggested that AAbV represents a new family, proposed here as Abyssoviridae. MLeV represents a sister group to the other known coronaviruses. The importance of MLeV and AAbV for understanding nidovirus evolution, and the origin of terrestrial nidoviruses are discussed.

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Roadkill has gradually become a common factor that has contributed to the decline of amphibians, and traffic volume is an important parameter that can be used to determine the impacts of roads. However, few researchers have studied the effects of either daily or nightly traffic volume on amphibian roadkill in China. Hence, as an essential step for implementing mitigation measures, we conducted 77 road surveys along 10 km of road in the Wanglang National Nature Reserve (NNR) to determine the temporal and spatial distribution patterns of amphibian road mortality. In total, 298 dead individuals (Bufo andrewsi and Rana chensinensis) were observed on the road from April to October in 2017 and during June and August in 2015 and 2016. B. andrewsi had the highest number of records (85.2%) and was more vulnerable to road mortality than R. chensinensis. Amphibian fatalities mainly occurred during the breeding season in April, but there was an additional concentration of B. andrewsi roadkill in June and July. There was a significantly positive correlation between amphibian road mortality and mean night-time traffic volume. Roadkill hotspots were non-randomly distributed throughout the study area and were mainly concentrated in the road sections near the breeding pools. Therefore, to effectively mitigate the effects of road mortality in the Wanglang NNR, measures should be implemented both during hot moments and at hotspots. First, based on roadkill hot moments, during the breeding season (in April) and in June and July, the Wanglang NNR should establish temporary traffic restraints at night. Second, based on roadkill hotspots, culverts should be established in areas near breeding pools adjacent to roads, and barrier walls should be installed to guide amphibians into the culverts.