Highly divergent CRESS DNA and picorna-like viruses associated with bleached thalli of the green seaweed <i>Ulva</i>

Marine macroalgae (seaweeds) are important primary producers and foundation species in coastal ecosystems around the world. Seaweeds currently contribute to an estimated 51% of the global mariculture production, with a long-term growth rate of 6% per year, and an estimated market value of more than US$11.3 billion. Viral infections could have a substantial impact on the ecology and aquaculture of seaweeds, but surprisingly little is known about virus diversity in macroalgal hosts. Using metagenomic sequencing, we characterized viral communities associated with healthy and bleached specimens of the commercially important green seaweed Ulva. We identified 20 putative new and divergent viruses, of which the majority belonged to the Circular Rep-Encoding Single-Stranded (CRESS) DNA viruses [single-stranded (ss)DNA genomes], Durnavirales [double-stranded (ds)RNA], and Picornavirales (ssRNA). Other newly identified RNA viruses were related to the Ghabrivirales, the Mitoviridae, and the Tombusviridae. Bleached Ulva samples contained particularly high viral read numbers. While reads matching assembled CRESS DNA viruses and picorna-like viruses were nearly absent from the healthy Ulva samples (confirmed by qPCR), they were very abundant in the bleached specimens. Therefore, bleaching in Ulva could be caused by one or a combination of the identified viruses but may also be the result of another causative agent or abiotic stress, with the viruses simply proliferating in already unhealthy seaweed tissue. This study highlights how little we know about the diversity and ecology of seaweed viruses, especially in relation to the health and diseases of the algal host, and emphasizes the need to better characterize the algal virosphere. IMPORTANCE Green seaweeds of the genus Ulva are considered a model system to study microbial interactions with the algal host. Remarkably little is known, however, about viral communities associated with green seaweeds, especially in relation to the health of the host. In this study, we characterized the viral communities associated with healthy and bleached Ulva. Our findings revealed the presence of 20 putative novel viruses associated with Ulva, encompassing both DNA and RNA viruses. The majority of these viruses were found to be especially abundant in bleached Ulva specimens. This is the first step toward understanding the role of viruses in the ecology and aquaculture of this green seaweed.</p

Onchocerciasis-associated epilepsy: an explorative case-control study with viral metagenomic analyses on Onchocerca volvulus [version 2; peer review: 2 approved]

Background A high prevalence of onchocerciasis-associated epilepsy (OAE) has been observed in onchocerciasis-endemic areas with high ongoing Onchocerca volvulus transmission. However, the pathogenesis of OAE remains to be elucidated. We hypothesise that the O. volvulus virome could be involved in inducing epilepsy. With this study, we aim to describe the O. volvulus virome and identify potential neurotropic viruses linked to OAE. Methods In Maridi County, an onchocerciasis endemic area in South Sudan with a high prevalence of OAE, we will conduct an exploratory case-control study enrolling 40 persons aged 12 years and above with palpable onchocerciasis nodules. Cases will be participants with OAE (n=20), who will be age- and village-matched with controls without epilepsy (n=20). For each study participant, two skin snips at the iliac crest will be obtained to collect O. volvulus microfilariae, and one nodulectomy will be performed to obtain adult worms. A viral metagenomic study will be conducted on microfilariae and adult worms, and the O. volvulus virome of persons with and without OAE will be compared. The number, size, and localisation of onchocerciasis nodules in persons with and without OAE will be described. Moreover, the pre- and post-nodulectomy frequency of seizures in persons with OAE will be compared. Ethics and dissemination The protocol has been approved by the Ethics Committee of the University of Antwerp and the Ministry of Health of South Sudan. Findings will be disseminated nationally and internationally via meetings and peer-reviewed publications. Registration ClinicalTrials.gov registration NCT05868551 (https://clinicaltrials.gov/study/NCT05868551). Protocol version 1.1, dated 09/05/2023

Adult <i>Culex</i> mosquitoes captured at the urban (A) and peri-urban (B) sites in Leuven, Belgium.

Adult Culex mosquitoes captured at the urban (A) and peri-urban (B) sites in Leuven, Belgium.</p

Prevalence of <i>Wolbachia</i> infection per species by detection of the <i>wsp</i> gene.

Prevalence of Wolbachia infection per species by detection of the wsp gene.</p

Infectious titer per saliva sample of <i>Culex pipiens</i> (<i>p</i>.) <i>pipiens</i> and <i>Culex modestus</i>.

Infectious virus titers were determined by plaque assay (PFU: plaque forming units). The bars show the median ± interquartile range; the grey dotted line represents the limit of detection (LOD) of the plaque assay.</p

Urban and peri-urban field collection sites in Leuven, Flemish Brabant, Belgium.

Map made using QGIS v3.18.3 [QGIS Development Team (2021). QGIS Geographic Information System. Open Source Geospatial Foundation Project. http://qgis.osgeo.org]. Open map data was obtained from OpenStreetMap contributors through the Wikimedia Commons unlabeled layer (accessed: 14 July 2022).</p

Viral genome copies in the bodies and head, wings, and legs of <i>Culex pipiens (p</i>.<i>) pipiens</i> (A-B) and <i>Culex modestus</i> (C-D).

The bars represent the median viral genome copies ± interquartile range; the grey dotted lines represent the limit of detection (LOD) of the qRT-PCR assays used. Statistical analysis was performed with the Mann-Whitney U test; the asterisk represents a p-value of <0.05; ns: non-significant.</p

Vector competence of <i>Culex pipiens (p</i>.<i>) pipiens</i> (A) and <i>Culex modestus</i> (B) for WNV and USUV.

The bars represent the rates of infection in the body (yellow), disseminated infection to the head, wings, and legs (orange), transmission potential in the saliva (red), and overall transmission efficiency (purple), determined by plaque assay. Grey labels above the bars indicate the number of positive mosquitoes over the total number of mosquitoes.</p

Detection of the <i>Wolbachia wPip</i> strain in <i>Wolbachia</i>-infected <i>Culex p</i>. <i>pipiens</i> by virus infection status in the mosquito midgut.

Detection of the Wolbachia wPip strain in Wolbachia-infected Culex p. pipiens by virus infection status in the mosquito midgut.</p