Biology subjects

Rosario, K.

Publications and source records attributed to Rosario, K..

6 recordsLinked to original sources

bioRxiv2025

Targeted metagenomic sequencing with spiked primers for enrichment of viruses in wastewater for pathogen surveillance

BackgroundWastewater surveillance offers an underutilized opportunity to identify high-risk viral pathogens that pose public health risks. Although metagenomic approaches have been increasingly adopted for human wastewater surveillance, little attention has been given to its application in rural agricultural settings. Untargeted metagenomic sequencing of wastewater poses considerable technical challenges for viral detection due to fragmented genomes and low viral abundance. While existing enrichment methods partially address these challenges, high costs and proprietary protocols limit adoption in resource-constrained settings. ObjectiveWe developed a fully open-source primer design algorithm (Open MSSPE Design) and evaluated Metagenomic Sequencing with Spiked Primer Enrichment (MSSPE)1, as a practical strategy for metagenomic surveillance of swine slurry and farm wastewater samples collected from rural agricultural settings. This non-invasive MSSPE strategy addresses the challenge of detecting extremely low-abundance viral targets amid samples dominated by a background of bacterial and eukaryotic nucleic acids. MethodologyWe generated fifteen primer sets targeting high-priority DNA and RNA viral pathogens affecting human and animal health in Southeast Asia using our open-source primer design algorithm. Twenty-five wastewater and swine slurry samples from smallholder farms in northern Thailand underwent parallel library preparation--untargeted mNGS and MSSPE--for direct comparison. Libraries were sequenced on Illumina platforms and analyzed using Chan Zuckerberg ID (CZ ID)2. Rarefaction analysis assessed performance at sequencing depths of 100,000-1.5 million reads per sample. ResultsWe detected multiple high-risk DNA and RNA viruses in wastewater samples from smallholder farm operations. MSSPE achieved substantial viral enrichment across nine pathogenic DNA and RNA viruses, with a median two-fold enrichment of reads per million, with variability across targets (IQR: 1.01-3.44x) and a nearly 10% median increase in breadth of genome coverage (IQR: 4.54-11.84%), while retaining sensitivity for untargeted pathogens. MSSPE also increased the odds of detecting targeted viruses (OR 1.35, CI 1.14-1.60), with the greatest advantage at shallow sequencing depths where MSSPE required fewer reads to identify targeted viral taxa relative to mNGS. ConclusionsMSSPE demonstrated the ability to enrich shallow-depth sequencing (<2M reads per sample) sufficiently to detect priority viruses without substantially increasing library preparation time or cost. This open-source workflow supports cost-effective metagenomic viral surveillance for resource-constrained settings, providing a non-invasive method for detecting low-abundance viral targets in high-background sample types at rural agricultural interfaces with elevated risk of zoonotic spillover.

genomics↗

bioRxiv2025

Diverse ssRNA viruses associated with Karenia brevis harmful algal blooms in Southwest Florida

Harmful algal blooms (HABs) caused by the dinoflagellate Karenia brevis frequently occur in the eastern Gulf of Mexico, where they negatively impact the environment, human health, and economy. Very little is known about viruses associated with K. brevis blooms, although viral infection of other HAB-forming phytoplankton species can play an important role in bloom dynamics. We used viral metagenomics to identify viruses in 11 pooled seawater samples collected from Southwest Florida, USA in 2021 during a severe, spatiotemporally dynamic K. brevis bloom. Assembled viral genomes were similar to published genomes from the order Picornavirales, family Marnaviridae, and genera Sogarnavirus, Bacillarnavirus, and Marnarnavirus. Viruses from these groups infect bloom-forming diatoms (Chaetoceros sp. and Rhizosolenia setigera) and the raphidophyte Heterosigma akashiwo. We also recovered unclassified Riboviria genomes related to a Symbiodinium positive-sense ssRNA virus sequenced from coral dinoflagellate symbionts. Reverse-transcriptase PCR assays were performed to monitor the occurrence of seven representative virus genomes in these 2021 samples and 43 seawater samples collected during a subsequent, typical bloom between November 2022 and May 2023. Over half of the samples contained multiple viruses, and at least one viral genome was detected in 44 of 54 samples, collected across seasons and years, highlighting the ubiquity of these viruses in this region. Alpha diversity was highest in the summer months and positively correlated with K. brevis cell counts. Multiple regression revealed month and the presence of unclassified Riboviria sequences most similar to dinoflagellate viruses as significant predictors of K. brevis cellular abundance. ImportanceHarmful algal blooms caused by the dinoflagellate Karenia brevis negatively impact the tourism, fisheries, and public health sectors. Anticipated impacts of climate change, nutrient pollution, and ocean acidification may sustain and/or exacerbate K. brevis blooms in the future, underscoring the need for proactive monitoring, communication, and mitigation strategies. This study represents a pioneering effort in monitoring viruses associated with K. brevis blooms. The findings lay the groundwork for studying the effects of environmental drivers on K. brevis blooms and their associated viruses, as well as for exploring the roles of viruses in bloom dynamics and potential applications of viruses as biocontrol agents for K. brevis blooms. Furthermore, the comparison of viral dynamics relative to local and regional bloom dynamics in this study helps inform future monitoring and modeling needs.

microbiology↗

bioRxiv2024

Simultaneous detection of pathogens and antimicrobial resistance genes with the open source, cloud-based, CZ ID pipeline

Antimicrobial resistant (AMR) pathogens represent urgent threats to human health, and their surveillance is of paramount importance. Metagenomic next generation sequencing (mNGS) has revolutionized such efforts, but remains challenging due to the lack of open-access bioinformatics tools capable of simultaneously analyzing both microbial and AMR gene sequences. To address this need, we developed the Chan Zuckerberg ID (CZ ID) AMR module, an open-access, cloud-based workflow designed to integrate detection of both microbes and AMR genes in mNGS and whole-genome sequencing (WGS) data. It leverages the Comprehensive Antibiotic Resistance Database and associated Resistance Gene Identifier software, and works synergistically with the CZ ID short-read mNGS module to enable broad detection of both microbes and AMR genes. We highlight diverse applications of the AMR module through analysis of both publicly available and newly generated mNGS and WGS data from four clinical cohort studies and an environmental surveillance project. Through genomic investigations of bacterial sepsis and pneumonia cases, hospital outbreaks, and wastewater surveillance data, we gain a deeper understanding of infectious agents and their resistomes, highlighting the value of integrating microbial identification and AMR profiling for both research and public health. We leverage additional functionalities of the CZ ID mNGS platform to couple resistome profiling with the assessment of phylogenetic relationships between nosocomial pathogens, and further demonstrate the potential to capture the longitudinal dynamics of pathogen and AMR genes in hospital acquired bacterial infections. In sum, the new AMR module advances the capabilities of the open-access CZ ID microbial bioinformatics platform by integrating pathogen detection and AMR profiling from mNGS and WGS data. Its development represents a critical step toward democratizing pathogen genomic analysis and supporting collaborative efforts to combat the growing threat of AMR.

microbiology↗

bioRxiv2023

Degenerate PCR primers for potexvirus detection in seagrasses

2.Turtlegrass virus X, which infects the seagrass Thalassia testudinum, is the only potexvirus known to infect marine flowering plants. We investigated potexvirus distribution in seagrasses using a degenerate reverse transcription polymerase chain reaction (RT-PCR) assay originally designed to capture potexvirus diversity in terrestrial plants. The assay, which implements Potex-5 and Potex-2RC primers, successfully amplified a 584 nt RNA-dependent RNA polymerase (RdRp) fragment from TVX-infected seagrasses. Following validation, we screened 74 opportunistically collected, apparently healthy seagrass samples for potexviruses using this RT-PCR assay. The survey examined the host species T. testudinum, Halodule wrightii, Halophila stipulacea, Syringodium filiforme, Ruppia maritima, and Zostera marina. Potexvirus PCR products were successfully generated only from T. testudinum samples and phylogenetic analysis of sequenced PCR products revealed five distinct TVX sequence variants. Although the RT-PCR assay revealed limited potexvirus diversity in seagrasses, the expanded geographic distribution of TVX shown here emphasizes the importance of future studies to investigate T. testudinum populations across its native range and understand how the observed fine-scale genetic diversity a?ects host-virus interactions. 3. Impact statementPotexviruses are widespread in terrestrial plants; however, the recent discovery of TVX in the seagrass Thalassia testudinum extends their host range to marine flowering plants. Here we use existing Potex-5 and Potex-2RC degenerate primers to explore potexvirus infections in several seagrass species. TVX sequence variants were detected in T. testudinum collected from the eastern Gulf of Mexico, uncovering previously unknown genetic diversity of this poorly understood virus. 4. Data summaryAll sequence data are available in NCBI GenBank under the accession numbers OR827692-OR827705, OR854648, OR863396, OR879052-OR879056, and PP430548-PP430571. The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2021

A world of viruses nested within parasites: Unraveling viral diversity within parasitic flatworms (Platyhelminthes)

Because parasites have an inextricable relationship with their host, they have the potential to serve as viral reservoirs or facilitate virus host-shifts. Yet, little is known about viruses infecting parasitic hosts except for blood-feeding arthropods that are well-known vectors of zoonotic viruses. Herein we uncover viruses of flatworms (Phylum Platyhelminthes, group Neodermata) that specialize in parasitizing vertebrates and their ancestral free-living relatives. We discovered 115 novel viral sequences, including 1 in Macrostomorpha, 5 in Polycladida, 44 in Tricladida, 1 in Monogenea, 15 in Cestoda and 49 in Trematoda, through data mining. The majority of newly identified viruses constitute novel families or genera. Phylogenetic analyses show that the virome of flatworms changed dramatically during the transition of Neodermatans to a parasitic lifestyle. Most Neodermatan viruses seem to co-diversify with their host, with the exception of rhabdoviruses which may switch host more often, based on phylogenetic relationships. Neodermatan rhabodviruses also have an ancestral position to vertebrate-associated viruses, including Lyssaviruses, suggesting that vertebrate rhabdoviruses emerged from a flatworm rhabdovirus in a parasitized host. This study reveals an extensive diversity of viruses in Platyhelminthes and highlights the need to evaluate the role of viral infection in flatworm-associated diseases.

microbiology↗

bioRxiv2019

Characterization of vertically and cross-species transmitted viruses in the cestode parasite Schistocephalus solidus

Parasitic flatworms (Neodermata) represent a public health and economic burden due to associated debilitating diseases and limited therapeutic treatments available. Despite their importance, there is scarce information regarding flatworm-associated microbes. We report the discovery of six RNA viruses in the cestode Schistocephalus solidus. None were closely related to classified viruses and they represent new taxa. Mining transcriptomic data revealed the broad distribution of these viruses in Alaskan and European S. solidus populations. We demonstrate through in vitro culture of S. solidus that five of these viruses are vertically transmitted. With experimental infections and field-sampling, we show that one of the viruses is transmitted to parasitized hosts. The impact of these viruses in parasite fitness and pathogenicity, and in host-parasite co-evolutionary dynamics remains to be determined. The detection of six novel viruses in this first characterization of viruses in Neodermatans likely represents a fraction of virus diversity in parasitic flatworms.

microbiology↗