Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species.

The incidence and global spread of dengue are reaching alarming levels. Thailand represents a critical disease epicenter and demands an understanding of the environmental and evolutionary pressures that sustain DENV transmission. Unlike most affected countries experiencing recurrent outbreaks of the same serotype or replacement of one serotype for another, Thailand is an ecological niche for all four serotypes. Favorable climate and mosquito vector availability maintain a landscape defined by stable, endemic circulation of genotypes, with minimal genetic variation attributed to sporadic, external introductions. This equilibrium is achieved through inter-serotype competition, characterized by reproductive fitness levels that maintain infections (Re>1) and elevated evolutionary rates ( ~ 10^-4), which steadily increase the genetic diversity of each serotype. This conclusion is reinforced by the identification of numerous positively selected mutations, skewed in the direction of non-structural proteins conferring replication and transmission advantages versus those present in structural proteins evading neutralizing antibodies. Precipitous drops in DENV cases following outbreaks of Chikungunya suggest that interactions with other arboviruses also impact DENV dynamics through vector competition, replication inhibition or partial cross-protection. Thailand is a major exporter of DENV cases and novel emergent lineages gaining fitness here are likely to spread internationally. Surveillance is therefore paramount to monitor diversification trends and take measures to avoid the establishment of similar sustained, local transmission in other countries.

: Between April and November 2023, 27 unexplained human deaths that presented with swelling of the arms, skin sores with black centers, difficulty in breathing, obstructed swallowing, headaches, and other body aches were reported in Kyotera District, Uganda by the Public Health Emergency Operations Center. Subsequently, the death of cattle on farms and the consumption of carcass meat by some residents were also reported. Field response teams collected clinical/epidemiological data and autopsy samples to determine the cause of deaths. Metagenomic next-generation sequencing (mNGS) and target enrichment sequencing conducted on postmortem samples confirmed Bacillus anthracis, the etiological agent of anthrax disease, as the cause of the deaths. Applying mNGS to autopsy specimens is useful as a retrospective tool for identifying high-consequence pathogens during suspected outbreaks of unknown etiology.

Probe-based capture represents a highly sensitive and cost-effective approach for overcoming host background and enriching viruses in metagenomic NGS (mNGS) libraries. Using clinical specimens collected globally from patients with fever or respiratory illness, we generated mNGS libraries by random priming and Nextera XT tagmentation, followed by target enrichment (teNGS) with Comprehensive Viral Research Panel (CVRP) probes. Capture pool sizes and total reads were optimized, and libraries were initially sequenced separately. Using only 3–4% of reads required for standard mNGS, teNGS achieved increased sensitivity, 100–10,000× increases in depth, and >50% genome coverage for pathogens with titers ≥ 1000 cp/mL. Application to >2000 clinical specimens from various matrices and to contrived samples containing viruses absent from the CVRP probe set enabled detection of diverse viral families and established a minimum 65% nucleotide identity for hybridization, respectively. To save time and resources, teNGS and mNGS libraries were then combined into one sequencing run: teamNGS. In addition to streamlining the workflow, teamNGS also improved genome recovery. Coupling methods maintain the sensitivity and coverage for viruses achieved by enrichment alone while also ensuring comprehensive recovery of non-viral microbes. teamNGS has the potential to improve patient management and lower the rates of unnecessary testing and antibiotic use.

: Undiagnosed acute febrile illness (AFI) remains elevated in the Peruvian Amazon. The contribution of Oropouche virus (OROV) to AFI in Loreto was quantified, and its recent circulation was contextualized using targeted molecular testing and phylogenomic analysis. Using a novel research-use-only OROV real-time quantitative polymerase chain reaction test across seven surveillance sites, 1,000 AFI samples were rescreened in a setting where undiagnosed presentations were common (70% lacked a confirmed etiology at enrollment). Oropouche virus was detected in 7.1% of cases, with nearly all consensus genomes recovered from specimens with a cycle threshold value ≤30, underscoring the importance of early-phase sampling for sequencing success. Positive results occurred at all seven locations, including the remote riverine community of San Lorenzo, indicating broad geographic distribution and supporting ongoing endemic transmission rather than a single localized outbreak. Exposure patterns were consistent with peridomestic, vector-mediated acquisition: animal contact was common (63.4%), whereas contact with symptomatic individuals (12.7%) and recent travel (35.7%) were infrequent, supporting local, not person-to-person or travel-related, transmission. Oropouche virus cases peaked in January to February 2024, temporally overlapping with the seasonal rise in AFIs, indicating synchronous circulation with other endemic pathogens. Phylogenetic arrangement of contemporary Peruvian strains within established South American diversity further supports ongoing local transmission consistent with regional patterns. Together, these findings indicate that OROV accounts for a meaningful portion of AFI in Loreto and that the routine inclusion of OROV testing, paired with genomic surveillance, can close diagnostic gaps, improve situational awareness, and guide targeted public health action.