Peer Review #1 of "Discovery of novel astrovirus genotype species in small ruminants (v0.2)"

Abstract

Astroviruses are single-stranded, positive-sense RNA viruses, best known for causing diarrhea in humans and are also found in many other mammals; in those, the relevance in gastroenteritis remains unclear. Recently described neurotropic astroviruses showed associations with encephalitis in humans as well as in other mammals. In Switzerland, two different neurotropic astroviruses were identified in cattle, as well as one in a sheep. The high genetic similarity between the ovine and one of the bovine astroviruses strengthens the hypothesis of an interspecies transmission. In humans, astroviruses associated with encephalitis were found also in human stool samples, suggesting that in these patients the infection spreads from the gastrointestinal tract to the brain under certain conditions, such as immunosuppression. Whether a similar pathogenesis occurs in ruminants remains unknown. The aims of this study were 1) the investigation of the potential occurrence of neurotropic astroviruses in feces samples, 2) the discovery and analysis of so far unknown astroviruses in small ruminants and other ruminant species' fecal samples and 3) the examination of a potential interspecies transmission of astroviruses. To achieve the aims, RNA extraction out of 164 fecal samples from different ruminant species was performed and all samples were screened for known neurotropic astroviruses occurring in Switzerland, as well as for various astroviruses using RT-PCR. Positive tested samples were submitted to next generation sequencing (NGS). The generated sequences were compared to nucleotide-and amino acid databases, virus properties were identified, and phylogenetic analyses as well as recombination analysis were performed. The excretion of neurotropic astroviruses in small ruminants' feces could not be demonstrated, but this work suggests the first identification of astroviruses in goats as well as the discovery of multiple and highly diverse new genetic variants in small ruminants, which lead to a classification into novel genotype-species. Additionally, the prediction of multiple recombination events in four of five newly discovered full or almost full-length genome