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Abstract: Expanding international pet trade has emerged as one of the main introduction pathways of aquatic invasive species, with ornamental crayfish species commonly available on the EU and global markets. Besides most frequently studied crayfish pathogens, such as Aphanomyces astaci and white spot syndrome virus (WSSV), ornamental crayfish carry associated microbial communities, which may potentially lead to the emergence of known or even novel diseases following intentional or unintentional release of animals into the wild. This is especially problematic in the case of viruses, which represent an important, yet considerably understudied, group of crayfish pathogens. Here we analyzed viromes of hepatopancreas tissue of four crayfish species acquired in the international pet trade in Europe (Procambarus clarkii, Procambarus alleni, Cherax holthuisi, and Cherax quadricarinatus) using a high throughput sequencing based metagenomic approach. Seven different known viruses were identified, which were previously either directly associated with crayfish (WSSV, Cherax quadricarinatus reovirus, chequa iflavirus, athtab bunya-like virus) or with hosts from subphylum Crustacea or invertebrates associated with freshwater environment (Shahe ispoda virus 5, Dicistroviridae sp.). Additional sequences represented 8 potential novel and divergent RNA viruses, most similar to sequences belonging to members of Picornavirales, Elliovirales, Reovirales, Hepelivirales, Tolivirales and Ghabrivirales orders. We discuss our findings in relation to their phylogenetic relationships, geographical origins, and putative pathogenicity implications. The results highlight the need for further research into the risks related to disease emergence associated with the pet trade.
Keywords: virome, RNA viruses, ornamental crayfish, pet-trade, high throughput sequencing, ecology, molecular biology, pathology, virology, environmental risk assessment
Published in DiRROS: 23.05.2025; Views: 708; Downloads: 465
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Keywords: ToBRFV, ToMMV, tobamovirusi, epidemiologija, voda kot vir okužb, poročila
Published in DiRROS: 28.11.2024; Views: 931; Downloads: 737
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Abstract: CRISPR-Cas9 technology has emerged as a promising tool for genetic engineering of Streptomyces strains. However, in practice, numerous technical hurdles have yet to be overcome when developing robust editing procedures. Here, we developed an extension of the CRISPR-Cas toolbox, a simple and reliable cas9 monitoring tool with transcriptional fusion of cas9 nuclease to a beta glucuronidase (gusA) visual reporter gene. The Cas9-SD-GusA tool enables in situ identification of cells expressing Cas9 nuclease following the introduction of the plasmid carrying the CRISPR-Cas9 machinery. Remarkably, when the Cas9-SD-GusA system was applied under optimal conditions, 100% of the colonies displaying GusA activity carried the target genotype. In contrast, it was shown that the cas9 sequence had undergone major recombination events in the colonies that did not exhibit GusA activity, giving rise to “escaper colonies” carrying unedited genotype. Our approach allows a simple detection of “escaper” phenotype and serves as an efficient CRISPR-Cas9 optimisation tool.
Keywords: CRISPR-Cas9, gusA visual screening, "CRISPR escaper colonies", Streptomyces
Published in DiRROS: 17.07.2024; Views: 795; Downloads: 574
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