RNA viruses in aquatic environments remain poorly studied. Here, we analysed the RNA virome from approximately 10 l water from Yangshan Deep-Water Harbour near the Yangtze River estuary in China and identified more than 4,500 distinct RNA viruses, doubling the previously known set of viruses. Phylogenomic analysis identified several major lineages, roughly, at the taxonomic ranks of class, order and family. The 719-member-strong Yangshan virus assemblage is the sister clade to the expansive class Alsuviricetes and consists of viruses with simple genomes that typically encode only RNA-dependent RNA polymerase (RdRP), capping enzyme and capsid protein. Several clades within the Yangshan assemblage independently evolved domain permutation in the RdRP. Another previously unknown clade shares ancestry with Potyviridae, the largest known plant virus family. The ‘Aquatic picorna-like viruses/Marnaviridae’ clade was greatly expanded, with more than 800 added viruses. Several RdRP-linked protein domains not previously detected in any RNA viruses were identified, such as the small ubiquitin-like modifier (SUMO) domain, phospholipase A2 and PrsW-family protease domain. Multiple viruses utilize alternative genetic codes implying protist (especially ciliate) hosts. The results reveal a vast RNA virome that includes many previously unknown groups. However, phylogenetic analysis of the RdRPs supports the previously established five-branch structure of the RNA virus evolutionary tree, with no additional phyla.

Syndromic panels for infectious disease have been suggested to be of value in point-of-care diagnostics for developing countries and for biodefense. To test the performance of isothermal recombinase polymerase amplification (RPA) assays, we developed a panel of 10 RPAs for biothreat agents. The panel included RPAs for Francisella tularensis , Yersinia pestis , Bacillus anthracis , variola virus, and reverse transcriptase RPA (RT-RPA) assays for Rift Valley fever virus, Ebola virus, Sudan virus, and Marburg virus. Their analytical sensitivities ranged from 16 to 21 molecules detected (probit analysis) for the majority of RPA and RT-RPA assays. A magnetic bead-based total nucleic acid extraction method was combined with the RPAs and tested using inactivated whole organisms spiked into plasma. The RPA showed comparable sensitivities to real-time RCR assays in these extracts. The run times of the assays at 42°C ranged from 6 to 10 min, and they showed no cross-detection of any of the target genomes of the panel nor of the human genome. The RPAs therefore seem suitable for the implementation of syndromic panels onto microfluidic platforms.

Several real-time PCR approaches to develop field detection for Francisella tularensis , the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.

Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease caused by pirAB toxins encoded by a plasmid found in Vibrio parahaemolyticus. The pirAB toxins are the homologs of the Photorhabdus insect-related (Pir) toxins. Here, we report the complete sequences of the AHPND-causing plasmid isolated from V. owensii, as well as those of its 11 siblings (pVH family). In addition, we also included 13 related plasmids (pVH-r family) without the pirAB genes isolated from a variety of species within the Vibrio Harveyi clade. Furthermore, the pirAB-Tn903 composite transposon was identified in pVH, and both ends of the transposon appeared to have inserted simultaneously into the ancestor plasmid at different sites. The homologue counterparts of pirAB were also detected in a non-pVH plasmid in V. campbellii. Taken together, our results provide novel insights into the acquisition and evolution of pirAB as well as related plasmids in the Vibrio Harveyi clade.