Volume 07 · Issue 02 · 2025

In 2022, a global outbreak of mpox was anticipated, with several cases reported in non-endemic countries in early May. Given the challenge of distinguishing the mpox virus (MPXV) from other pathogens based solely on symptoms, there is an urgent need for prompt and reliable MPXV detection methods. In this study, we developed assays using recombinase-aided amplification (RAA) to identify MPXV and evaluated their applicability with clinical samples. The assays were designed to detect the N4R gene of MPXV. All assays demonstrated detection limits of 1 copy/µL within the reaction system and exhibited no cross-reactivity with ectromelia or the TianTan strain of vaccinia virus, confirming their high specificity. Our established assay provides results in less than 50 min. Furthermore, we evaluated our assay using clinical samples from laboratory-confirmed mpox patients and demonstrated that the RAA-based assay is valuable for diagnosing MPXV infections in field and clinic settings, especially in areas with limited laboratory resources. Overall, three RAA-based nucleic acid assays for MPXV were established, providing a powerful tool for efficient, rapid, and specific detection of MPXV infection.

Rab proteins are involved in all facets of the vesicular transport process and play significant roles in different steps of the viral life cycle. Rab4b is a pivotal player in the endocytic recycling of proteins, whereas its roles in viral replication are still largely unknown. Our earlier work identified Rab4b as a host factor required to replicate the influenza A virus (IAV). Here, we further validated the impact of Rab4b on viral replication by silencing or overexpressing Rab4b. The results showed that silencing Rab4b significantly decreased IAV and influenza B virus (IBV) production. Overexpression of Rab4b enhanced IAV infection. We provided robust evidence to support the important role of Rab4b in facilitating IAV growth independent of the host innate immunity. Mechanism study revealed the involvement of Rab4b in the early steps of the IAV life cycle, including virus attachment, endocytosis of viral particles, virus-host membrane fusion, and nuclear import of viral nucleoprotein (NP). Furthermore, we found that Rab4b interacts with viral ribonucleoprotein (RNP) complexes, suggesting that Rab4b binds to RNP complex to facilitate viral replication. In summary, this work provided the first evidence to support the involvement of Rab4b in the IAV replication. Understanding the mechanisms underlying IAV and Rab4b interactions helps elucidate viral infection and pathogenesis and leads to the development of antiviral therapeutics.

Deoxyribonucleic acid (DNA) information storage has emerged as a promising solution to address the challenges of traditional silicon-based data storage systems. However, the biosafety implications of artificially synthesized DNA sequences in this technology remain understudied. This research evaluates the biosafety risks associated with five representative DNA storage encoding methods [Church, Goldman, DNA Fountain, Grass, and movable-type (MT) encoding] by analyzing their sequence similarities to natural biological DNA. Through Kraken2 taxonomic classification and Basic Local Alignment Search Tool for nucleotides (BLASTn) alignment analysis, we found that while most artificially designed DNA sequences showed significant differences from known biological sequences, specific encoding methods produced sequences similar to natural genomes. The MT encoding method showed the highest annotation rate (4.59 %) in Kraken2 analysis, while Goldman and Fountain methods demonstrated significant local sequence alignments in BLASTn analysis. Sequence length positively correlated with annotation rates, suggesting longer sequences pose potentially higher biosafety risks. Furthermore, aligned sequences often exhibited characteristics of tandem repeats, particularly in non-coding regions. These findings highlight the importance of incorporating biosafety considerations in DNA storage encoding method development and suggest that randomization strategies may help mitigate potential risks. Our study provides valuable insights into the safe advancement of DNA storage technology and emphasizes the need for comprehensive biosafety evaluation in synthetic biology applications.

Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening tick-borne disease characterized by cytokine dysregulation and immune-mediated hyperinflammation. This multicenter retrospective study analyzed the dynamics of 17 cytokines across acute and recovery phases using 287 serum samples collected between 2010 and 2023 from high-incidence regions of China, evaluating their associations with disease severity, sex, age, and antibody responses. The results demonstrated that elevations of interleukin (IL)-6, interferon (IFN)-α, IL-8, and IFN-γ-induced protein 10 (IP-10) during the acute phase were associated with hyperinflammation, while IL-10 balanced inflammatory control and may have contributed to viral persistence. During recovery, most cytokines declined; however, IL-8 and IP-10 remained elevated longer in some patients, reflecting heterogeneity in recovery trajectories. Severe cases exhibited significantly higher levels of IL-10, IFN-γ, IL-6, IFN-α, tumor necrosis factor (TNF)-α, IL-8, and IP-10, underscoring their potential as biomarkers for disease severity prediction. Sex-based differences revealed higher IFN-γ and IL-8 levels in females, potentially due to hormonal and genetic factors, while older patients exhibited elevated IL-10, IFN-γ, and IFN-α, reflecting immune dysregulation and age-related shifts in adaptive immunity. Correlation analysis revealed distinct immune response patterns, with IL-10 strongly correlating with IFN-γ and minimal antibody-cytokine associations observed during the acute phase. In contrast, in the recovery phase, immunoglobulin G (IgG) negatively correlated with IL-10, IFN-γ, and IP-10, and immunoglobulin M (IgM) positively correlated with IL-10, IFN-γ, IL-6, IFN-α, TNF-α, IL-8, and IP-10, reflecting dynamic immune regulation and the interplay between humoral and cellular immunity. These findings provide critical insights into the immunopathogenesis of SFTS, supporting the development of cytokine-targeted therapies and advanced diagnostic tools to improve clinical outcomes.

The metabolic activity of pathogens poses a substantial risk across diverse domains, including food safety, vaccine development, clinical treatment, and national biosecurity. Conventional subculturing methods typically require several days and fail to detect metabolic activity promptly, limiting their application in many areas. Consequently, there is an urgent need for a method capable of rapidly and accurately detecting this activity. This study builds upon an investigation of the effects of D₂O on Staphylococcus aureus (S. aureus), utilizing D₂O-probed single-cell Raman spectroscopy to detect the metabolic activity of S. aureus by the Carbon-Deuterium ratio (C-D_ratio). Then, it evaluates the performance of various machine learning models in classifying the metabolic states of the pathogen. Medium D₂O concentration below 50 % has no significant impact on the growth and reproduction of S. aureus or on the classification of metabolic states of S. aureus based on the fingerprint region by machine learning models. Additionally, as the metabolic activity of S. aureus decreases, both the C-D_ratio and the rate of viable cells also gradually decrease. The support vector machine model demonstrated an accuracy of 99.82 % in classifying viable and dead S. aureus, while the linear discriminant analysis model demonstrated an accuracy of 99.92 % in classifying S. aureus exhibiting distinct metabolic activities. Therefore, D₂O-probed single-cell Raman spectroscopy, combined with high-throughput technology, can rapidly, non-destructively, and accurately detect pathogen metabolic activity, offering valuable applications across multiple fields.

Given the overlapping endemic regions and clinical similarities between severe fever with thrombocytopenia syndrome (SFTS) and hemorrhagic fever with renal syndrome (HFRS), we developed a dual real-time fluorescence-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Recombinant plasmids and synthetic ribonucleic acid (RNA) were constructed to evaluate the specificity, sensitivity and reproducibility of the assay. Additionally, we assessed the specificity of the assay using samples from three distinct groups: individuals with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (n = 10), influenza A-positive individuals (n = 10), and healthy controls. Receiver operating characteristic (ROC) curves were used to assess diagnostic accuracy, while the Kappa coefficient and linear regression analysis were employed to evaluate clinical applicability. Our method exhibited specificity for both SFTSV and Hantaan virus detection, with detection limits of 333 and 1,022 copies/mL using plasmids, and 1,247 and 898 copies/mL using synthetic RNA, respectively. We evaluated 100 clinical samples from each of SFTS and HFRS. The Kappa coefficients for both diseases were 0.96. The areas under the ROC curves were 0.991 (P < 0.001) and 0.989 (P < 0.001), respectively. The linear regression equations were as follows: log (y) = 0.19 + 0.99 log (x) (R² = 0.95) for SFTS virus, and log (y) = 0.01 + 0.65 log (x) (R² = 0.92) for Hantaan virus. We established an in-house RT-qPCR method for the rapid quantification of both pathogens, making it an ideal tool for early clinical differentiation.

Brucellosis poses a significant health threat to the population, particularly to vulnerable populations, including infants. In this investigation, we retrospectively analyzed the infection source and potential transmission route in a three-month-old infant with febrile seizure. Bacteriology methods, epidemiological survey, Rose Bengal plate test (RBPT), and standard tube agglutination test (SAT) were used to diagnose the disease, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was applied to identify the strain. The study revealed that the infant’s parents had been diagnosed with brucellosis due to occupational exposure to infected sheep. The Brucella strain was isolated and identified from the infant’s blood sample, confirming brucellosis meningitis. Post-treatment serum analysis showed RBPT positivity and SAT titer of 1:200 (+ +). The infant had no direct contact with livestock, with breast milk as the only dietary source; however, the detailed transmission route remained undetermined. Maternal-fetal transmission or contamination through breastfeeding, parental hand contact, clothing exposure, or other passive contamination modes may be potential transmission routes. Notably, the parents had a history of brucellosis and given that the infant presented with a fever of unknown origin, screening for brucellosis should have been prioritized. Following diagnosis, the infant was treated with ceftriaxone sodium (2.0 g/day) and rifampicin (0.5 g/day) for four weeks, ultimately achieving full clinical recovery. This case highlights the importance of brucellosis screening in infants presenting with unexplained fever, especially in families whose members have previously been diagnosed with brucellosis in endemic regions.