Pathogens

Enteroviruses (EVs) are major pathogens transmitted via direct and indirect contact, with children being particularly susceptible. As EVs persist on surfaces, environmental hygiene is critical in communal environments. We investigated EVs presence on environmental surfaces in daycare centers from April to July 2024. Environmental samples (300) were collected from floors, toys, and desks. Viral RNA was extracted and analyzed using real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and ddPCR to detect pan-Enterovirus (pan-EVs) and Enterovirus D68 (EV-D68). EVs were detected in 45.3% of the samples. The detection rate refers to the combined results, including both ddPCR and real-time PCR. Specifically, pan-EVs were found in 88 samples (1.12–505 copies/20 μL) and EV-D68 in 104 samples (1.12–309 copies/20 μL). Floors (31%) were the most contaminated surfaces. Monthly analysis showed a gradual decrease in detection rates from 88.6% in April to 18.5% in July, appearing to align with the implementation of enhanced hygiene measures. However, this trend may also reflect multifaceted factors, including natural viral reduction, exclusion of symptomatic children, and increased hygiene awareness. Notably ddPCR (83.0%) exhibited nearly twice the detection rate of real-time RT-PCR (42.5%), identifying low-level viral persistence. These findings suggest that environmental surfaces serve as reservoirs for transmission, and integrating sensitive detection like ddPCR with proactive hygiene management may help mitigate EVs spread.

The family Flaviviridae has been expanded to include the highly divergent flavi-like viruses into three new families, Flaviviridae, Pestiviridae, and Hepaciviridae, in the order Amarillovirales. Classical flavivirids are small, enveloped viruses with positive-sense ssRNA genomes lacking a 3′ poly(A) tail and ~9.0–13.0 kb in length, with a single open reading frame (ORF) encoding structural proteins at the N-terminus and nonstructural proteins at the C-terminus. Members infect a wide range of mammals, birds, and insects, and many are host-specific and pathogenic. Although the RNA-directed RNA polymerase (RdRP) gene sequences of the flavi-like viruses group phylogenetically with those of classical flavivirids, flavi-like viruses often encode larger polyproteins and possess substantially longer genomes of up to ~40 kb, and some have a 3′ poly(A) tail. Their host range extends across the whole animal kingdom and angiosperm plants. This review describes the reported flavi-like viruses of aquatic animals, providing a meaningful update on all three new families in Amarillovirales that have been discovered using metagenomics in fish, crustaceans, mollusks, and echinoderms. These amarilloviruses include pathogenic viruses of aquatic animals, such as Cyclopterus lumpus virus (CLuV) detected in moribund lumpfish, and infectious precocity virus (IPV) found in iron prawn syndrome (IPS)-affected farmed giant freshwater prawns.

Scrub typhus, caused by Orientia tsutsugamushi, remains a neglected cause of acute febrile illness. Molecular testing of blood supports early diagnosis, yet once doxycycline is started, blood qPCR positivity can drop rapidly, complicating short-term follow-up and relapse surveillance. We compared detection across multiple clinical specimens and evaluated nasopharyngeal swabs (NPSs) as noninvasive supplementary specimens during treatment initiation. In a prospective single-center cohort from Hainan, China, we enrolled 20 patients with scrub typhus. Blood, NPS, urine, and stool were collected before doxycycline administration 24 h after the first dose and on day 5. qPCR was performed for the analysis of Orientia tsutsugamushi. qPCR-positive specimens were subjected to nested PCR targeting TSA56, and nested PCR-positive amplicons were Sanger sequenced for genotyping. Before treatment, O. tsutsugamushi DNA was detected in 15/20 blood samples (75.00%) and 5/20 NPS samples (25.00%), but 0/20 urine samples (0%) and 0/20 stool samples (0%). At 24 h after treatment, detection in blood was 0/20 (0%) while NPS samples were positive in 3/20 (15.00%). All specimens were negative by day 5 after treatment. Across sequenced NPS positives (n = 3), Karp 2/3 (66.77%) and Gilliam 1/3 (33.33%) predominated. In paired blood–NPS positives, inter-specimen homology was high (percentage nucleotide identity 100% for Karp and 100% for Gilliam). NPS is not sensitive enough for primary diagnosis; however, within the first 24 h after doxycycline it offers a practical, noninvasive supplementary specimen to support short-term follow-up and community-based sampling when venipuncture or transport are constrained. Larger, multi-center studies are warranted to refine sampling windows and diagnostic performance.