Influenza, which causes respiratory tract infections and related complications, poses a major threat to global public health. However, the rapid and accurate detection of influenza viruses in controlling the flu pandemic remains challenging, as current diagnostic methods are static and unable to distinguish between viable and nonviable virus or directly monitor viral replication dynamics. Herein, we report viral luminogenic reporters (VLRs) with chemiluminescence/fluorescence dual-response for non-invasive imaging and urinalysis of the H1N1 virus protease. VLR comprises a bicyclic dioxetane chemiluminophore signaling scaffold caged by a N-acetylneuraminic acid, which further hooks a renal clearable moiety (2-hydroxypropyl)-β-cyclodextrin. VLR achieves a limit of detection of 2.62 CCID50/mL in H1N1 virus detection, which was 10.4-fold and 529.8-fold lower than that of ICA-qPCR and PMA-qPCR assays. After intratracheal administration into H1N1 virus-infected mice, VLR can efficiently accumulate in the lungs and specifically react with neuraminidase to restore its near-infrared chemiluminescent/fluorescent signals for real-time imaging. Leveraging the renal clearance (∼94% ID), VLR allows for remote detection of H1N1 virus infections and monitoring of antiviral therapeutic efficacy through in vitro urinalysis. Therefore, this study highlights a significant advance in addressing the critical gap in dynamic monitoring of virus replication activity and transforming virus-specific probes into urinary reporters.

The 2018-2020 Ebola virus disease (EVD) epidemic facilitated the emergence of viral mutations, enhancing the potential for host adaptation during sustained human transmission. Here, we identified the Ebola virus (EBOV) glycoprotein V75A (GP-V75A) substitution as a dominant variant during the epidemic. This substitution, located within the receptor-binding domain, emerged early in the outbreak and rapidly reached high prevalence. GP-V75A demonstrated enhanced infectivity in multiple cell lines and murine models. Mechanistically, GP-V75A increased viral GP binding affinity to the host receptor Niemann-Pick C1 (NPC1) and reduced the dependency on endosomal cysteine proteases for entry. Notably, GP-V75A also significantly reduced the efficacy of NPC1-targeting compounds and neutralizing antibodies. Epidemiological analysis indicated that the rise in GP-V75A prevalence coincided with the increase in case number during the outbreak. These findings provide crucial insights into the evolutionary adaptation of EBOV during large-scale outbreaks and underscore the importance of real-time genomic surveillance for improving epidemic preparedness.

BACKGROUND:Disruptor of telomeric silencing-1-like (DOT1L), a methyltransferase of H3K79, was observed to be amplified and overexpressed in certain malignancies. This work was aimed at investigating the differences in DOT1L expression and its regulatory mechanism in gastric cancer (GC) and healthy samples. METHODS:Immunohistochemistry was used to detect DOT1L levels in 101 cases of GC and marching adjacent normal tissues. DOT1L was inhibited by small interfering RNA (siRNA) and EPZ5676; a targeting drug. The ability of cells to proliferate were checked by cell counting kit-8 (CCK-8) and clone formation assays, with flow cytometry for observing the cell cycle. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot revealed the gene and protein profiles. Finally, the outcome of EPZ5676 administration was checked on a murine model. RESULTS:The expression of DOT1L is significantly increased in gastric malignant tumors that is related to the degree of differentiation, lymph node metastasis and TNM staging. DOT1L serves as an independent marker for the prognosis of overall survival (OS) with high levels implying worse prognosis. In addition, DOT1L regulates cyclin-dependent kinase (CDK) 4 (CDK4) and CDK6 through H3K79me2, which leads to a change in the cell cycle at G1, thereby affecting the proliferation of tumors in vitro and in vivo. CONCLUSIONS:This is a first clinical demonstration of the applicability of DOT1L overexpression in gastric tumors. The work is suggestive of altered proliferation of cells by DOT1L via regulating cyclins and H3K79 methylation. This indicates the role of DOT1L in the prognosis and possible medical intervention of GC.

Dengue virus (DENV) represents a significant public health threat, with its four serotypes estimated to account for approximately 96 million symptomatic infections annually. Currently, there are no antiviral agents available for the prevention or treatment of DENV infection. Here, we initially screened 12 diphyllin derivatives and identified C156-P1, a nitrogen-containing compound, as a potent agent against DENV infection. Further, C156-P1 exhibited inhibitory effects against the viruses of the Flaviviridae family, including four serotypes of DENV (DENV-1 to DENV-4) in multiple human and monkey cell lines, as well as Zika virus, Japanese encephalitis virus, yellow fever virus, and hepatitis C virus. In addition, C156-P1 also showed inhibition of the infections of herpes simplex virus type 1 and vesicular stomatitis virus, but not adenovirus and Sendai virus. Mechanistic studies demonstrated that C156-P1 inhibited DENV-2 after cell entry but before the endosomal membrane fusion step. C156-P1 inhibited vacuolar-type ATPase activity by perturbing the expression of ATP6V0A2 subunit, thereby suppressing endosomal acidification. Consequently, DENV was restricted in the late endosome, inhibiting virus fusion with endosomal membranes and resulting in infection inhibition. C156-P1 treatment also suppressed both IFN-I responses and endosomal TLR3 activation induced by DENV-2 infection. Furthermore, administration of C156-P1 in AG129 mice significantly reduced DENV-2 infection and effectively increased the survival rate of the mice. Taken together, our study demonstrates that the novel nitrogen-containing diphyllin derivative C156-P1 functions as a broad-spectrum antiviral agent by inhibiting endosomal acidification, thus representing a promising host-targeting antiviral candidate for future development.

Monkeypox virus (MPXV) poses a global health threat. Viral infection can alter host metabolic homeostasis, while its changes may influence disease severity and progression. The relationship between MPXV and metabolites has not been reported. we employed metabolomics to characterize the mpox specific metabolic features by comparing changes in urinary metabolites from patients with MPXV infection, HIV infection, and HIV-MPXV co-infection. The metabolic changes caused by MPXV were mainly concentrated in amino acid and hormone metabolism. Further analysis showed that 5'-dihydroadenosine and uric acid can serve as potential molecular markers for MPXV and HIV-MPXV co-infected patients, respectively. Confirmed by targeted metabolomics and ELISA methods, MPXV infection caused severe disorders in the metabolic pathways of steroid hormones such as testosterone and progesterone in humans. Our findings identify dysregulated metabolism as an underpinning of MPXV pathogenicity, and increasing the anti-inflammatory capacity of patients may play a role to some extent against mpox.

ABSTRACT Influenza A virus (IAV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and respiratory syncytial virus (RSV) are significant pathogens that induce respiratory symptoms in humans, significantly impacting healthcare systems, economic activities, and human life on a global scale. The clinical manifestations associated with SARS-CoV-2, IAV, and RSV exhibit similarities. Cases of clinical coinfection are prevalent, highlighting the necessity for rapid differential diagnosis to avoid treatment delays. Traditional reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods are often time-consuming and limited to central laboratories, necessitating the development of rapid and in situ screening techniques to accommodate broad and large-scale testing requirements. In light of the possibility of coinfection with multiple viral strains, a multiplex detection method known as triplex reverse transcription recombinase-aided amplification (RT-RAA) has been established to facilitate the simultaneous detection of these three pathogens. The triplex real-time RT-RAA assay operates at a constant temperature of 42°C and achieves detection within 30 minutes.The limits of detection (LOD) for IAV, SARS-CoV-2, and RSV were 138, 198, and 162 copies per reaction, respectively, comparable in order of magnitude to the 100 copies per reaction observed in the triplex RT-qPCR assay. Importantly, no cross-reactivity was observed with other significant viruses. In addition, compared with the triplex RT-qPCR assay, the triplex real-time RT-RAA assay demonstrated a high degree of concordance in the detection of IAV, SARS-CoV-2, and RSV in 58 clinical specimens. Overall, the triplex real-time RT-RAA assay holds considerable promise as a powerful tool for the rapid screening of respiratory diseases, owing to its advantages, including expedited detection, ease of operation, strong specificity, and high sensitivity.IMPORTANCEIt is well established that SARS-CoV-2, IAV, and RSV are significant pathogens that elicit respiratory symptoms in humans. In this study, we successfully developed a triplex real-time RT-RAA assay for the simultaneous rapid detection of SARS-CoV-2, IAV, and RSV. This assay is distinguished by its high specificity, sensitivity, and user-friendliness. It addresses the limitations associated with the necessity of specialized biological laboratories and the time-consuming, complex procedures that impede rapid diagnosis. This assay holds significant importance for the early diagnosis and epidemiological surveillance of respiratory tract infections, providing comprehensive and timely diagnostic information for clinical practice. Furthermore, this assay serves as a valuable tool for facilitating extensive and large-scale screening of SARS-CoV-2, IAV, and RSV.

Circular RNA (circRNA) is emerging as a highly promising technology in various biomedical applications, offering advantages over traditional linear RNA. The Twister-optimized RNA for the durable overexpression (Tornado) system has been widely investigated for generating circRNAs in mammalian cells; however, the use of the Tornado system for large RNA inserts, especially those containing the internal ribosome entry site (IRES) sequences, is hindered by low circularization efficiency and limited circRNA abundance. Therefore, developing novel strategies to enhance RNA circularization in cells is of critical importance. In this study, we present a modified Tornado system that significantly improves circRNA-based protein expression by incorporating an optimal distance between the IRES and the upstream CMV promoter. Furthermore, we elucidate the dual roles of HRV-B3 IRES in mammalian cells, demonstrating its negative regulatory effect on RNA abundance and its positive contribution to RNA circularization. Additionally, the integration of a truncated 5' long terminal repeat (LTR) from HIV-1 upstream of the HRV-B3 IRES, combined with the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), further enhances transcriptional efficiency in the Tornado system. This modified system holds great potential for advancing circRNA-based therapeutics and vaccines, and these findings provide valuable insights for refining the Tornado system and designing regulatory elements in synthetic biology applications.

Xanthomonas bacteria are responsible for disease outbreaks in several hundred plant species, causing significant economic losses. Xanthomonas phages have emerged as a promising biocontrol strategy in managing various important plant diseases caused by Xanthomonas bacteria. However, structural information for Xanthomonas phages has remained limited so far. Here, we present high-resolution cryo-electron microscopy (cryo-EM) structures of the Xanthomonas citri phage ΦXacJX1 from siphoviruses. These structures include atomic models for the head, head-to-tail connector and head-proximal portion of the tail. ΦXacJX1's head and head-to-tail connector components show significant protein sequence and structural homology with those of the model siphophage HK97. However, the in-situ structures of head-to-tail connector of phage HK97 remain unavailable. The presented structures of phage ΦXacJX1 enhance our understanding of Xanthomonas phages and the mature virion of phage HK97. They provide a valuable framework for future structural and functional studies on both Xanthomonas phages and phage HK97.

Ebola virus disease (EVD) caused by Zaire Ebolavirus (EBOV) infection is a major threat to public health in Africa and even worldwide, due to its extremely high mortality rate. However, there are still no effective antiviral therapies that can completely cure EVD. A comprehensive understanding of virus-host interactions would be beneficial for developing new antiviral agents. Here, we showed that CXCR4-induced macroautophagy/autophagy and was internalized to endosomes by interacting with glycoprotein (GP) on viral particles during EBOV infection; this promoted the EBOV attachment and entry, which was reduced by CXCR4 antagonist and neutralizing antibody. We also found that CXCR4 increased EBOV replication by downregulating cytotoxic GP to promote viral fitness instead of influencing the assembly of viral factory. Mechanistically, excessive EBOV GP could hijack CXCR4 sorting and transporting pathways by their interactions with HGS, one of the key components of the ESCRT machinery; subsequently GP could be carried back to the endoplasmic reticulum by CXCR4, where the E3 ubiquitin ligase RNF185 was recruited to polyubiquitinate GP in a K27- and K63-linked manner. Finally, polyubiquitinated GP was degraded in lysosomes via reticulophagy by interacting with RETREG1 (reticulophagy regulator 1), in an ATG3- and ATG5-dependent manner. Our findings revealed dual roles of CXCR4 in regulation of EBOV life cycle, either acting as an entry factor by interacting with GP on viral particles to facilitate viral entry or targeting excessive GP for reticulophagic degradation, providing new evidence that EBOV hijacked the host vesicular transportation system through efficient virus-host interactions to facilitate viral fitness.Abbreviations: Baf A1: bafilomycin A1; BDBV: Bundibugyo Ebolavirus; CHX: cycloheximide; CXCR4: C-X-C motif chemokine receptor 4; CLEC4M/DC-SIGNR: C type lectin domain family 4 member M; EBOV: Zaire Ebolavirus; EEA1: early endosome antigen 1; ER: endoplasmic reticulum; ERAD: ER-associated degradation; ESCRT: endosomal sorting complex required for transport; EVD: Ebolavirus disease; HAVCR1/TIM-1: hepatitis A virus cellular receptor 1; GP: glycoprotein; HGS: hepatocyte growth factor-regulated tyrosine kinase substrate; HIV: human immunodeficiency virus; IFL: internal fusion loop; ITCH/AIP4: itchy E3 ubiquitin protein ligase; LAMP: lysosomal associated membrane protein; LC-MS/MS: liquid chromatography mass spectrometry; PDIs: protein disulfide isomerases; RBD: receptor binding domain; RESTV: Reston Ebolavirus; RETREG1: reticulophagy regulator 1; RNF185: ring finger protein 185; SQSTM1/p62: sequestosome 1; SUDV: Sudan Ebolavirus; TAFV: Taï Forest Ebolavirus; TRIM21: tripartite motif containing 21; trVLPs: transcription- and replication-competent virus-like particles; Ub: ubiquitin.

e15146 Background: Vascular endothelial growth factor receptors (VEGFRs) are highly expressed in many solid tumors and plays a key role in angiogenesis, while the CSF1R/CSF-1 axis is implicated in tumor progression and poorer prognosis. TR64, a small molecule inhibitor of VEGFR, CSF1R, and c-KIT, has demonstrated promising antitumor activity in preclinical studies. This Phase I study evaluates its safety, tolerability, pharmacokinetics, and preliminary efficacy in patients with advanced malignant solid tumors. Methods: Patients received TR64 orally once daily in 28-day cycles from 25mg to 200mg dose groups. The regimen in 300mg and 400mg cohorts consisted of a 14-day continuous dosing period, followed by a 7-day break, and then a 21-day treatment phase until disease progression, unacceptable toxicity, or withdrawal occurred. Adverse events were assessed per NCI-CTCAE v5.0, and tumor response was evaluated using RECIST v1.1. Results: By January 13, 2025, 31 patients were enrolled, including those with small-cell lung cancer (SCLC, n = 10), breast cancer (n = 9), pancreatic cancer (n = 4), gastrointestinal stromal tumor (GIST, n = 2), colon cancer (n = 2), rectal cancer (n = 1), duodenal cancer (n = 1), sacral osteosarcoma (n = 1), and spindle cell sarcoma (n = 1). The median age was 57 (range 33–74) with 58% males, and most were heavily pretreated with a median of 3 previous lines of therapy. The most common TRAEs (all grades/Grade ≥3) included lymphocytopenia (48.4%/12.9%), leukopenia (35.5%/12.9%), neutropenia （35.5%/12.9%）, elevated aspartate transferase (32.3%,3.2%), anemia (29%/0%), elevated hydroxybutyrate dehydrogenase (25.8%,0%), proteinuria (25.8%,0%)，elevated lactate dehydrogenase (25.8%,0%), elevated lipase (22.6%,0%), hypokalemia (22.6%,12.9%). In the subset of 5 evaluable SCLC patients, the overall response rate was 40% and the disease control rate 60%. Notably, an SCLC patient with brain metastases who previously underwent three lines of systemic and whole brain radiation therapies achieved a partial response with a 53.1% reduction in target lesion diameter on a 300 mg dose, remaining on treatment for nearly one year. Additionally, a GIST patient on 200 mg with four lines prior therapies and a HER2-positive breast cancer patient on 100 mg previously treated with five lines of therapies both achieved stable disease, continuing treatment for over 17 and 21 months, respectively. Pharmacokinetic analysis showed that both AUC₀₋₂₄ h and C max increased proportionally with dose, and the median T max was 4 hours. Conclusions: TR64 exhibits a favorable safety profile and promising efficacy, particularly in SCLC. These findings warrant further investigation in the ongoing dose-expansion study (NCT05649345). Clinical trial information: NCT05649345 .

Ebola virus (EBOV) belongs to Filoviridae family possessing single-stranded negative-sense RNA genome, which is a serious threat to human health. Nowadays, no therapeutics have been proven to be successful in efficiently decreasing the mortality rate. RNA binding proteins (RBPs) are reported to participate in maintaining cell integrity and regulation of viral replication. However, little is known about whether and how RBPs participate in regulating the life cycle of EBOV. In our study, we found that RNA binding motif protein 4 (RBM4) inhibited the replication of EBOV in HEK293T and Huh-7 cells by suppressing viral mRNA production. Such inhibition resulted from the direct interaction between the RRM1 domain of RBM4 and the "CU" enrichment elements located in the PE1 and TSS of the 3'-leader region within the viral genome. Simultaneously, RBM4 could upregulate the expression of some cytokines involved in the host innate immune responses to synergistically exert its antiviral function. The findings therefore suggest that RBM4 might serve as a novel target of anti-EBOV strategy.

The rarity of Francisella novicida infection in humans is well-known, and the F. novicida cases occur in immunocompromised patients or those with underlying health problems. Herein, we report the case of a patient with long-term diabetes who died following F. novicida infection that caused multiple organ failure, although F. novicida was effectively eliminated using antimicrobial therapy. Microbiological confirmation of F. novicida infection relies on metagenomic next-generation sequencing (mNGS) and pdpD-2 gene-specific identification. This study highlights the importance of early pathogen diagnosis in severely infected patients, particularly in cases of F. novicida, and indicates that mNGS is a useful tool for early diagnosis.

The outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 highlight the need for countermeasures to prevent future coronavirus pandemics. Given the unpredictable nature of spillover events, preparing antibodies with broad coronavirus-neutralizing activity is an ideal proactive strategy. Here, we investigated whether SARS-CoV-2 infection and vaccination could provide cross-neutralizing antibodies (nAbs) against zoonotic sarbecoviruses. We evaluated the cross-neutralizing profiles of plasma and monoclonal antibodies constructed from B cells from coronavirus disease 2019 (COVID-19) convalescents and vaccine recipients; against sarbecoviruses originating from bats, civets, and pangolins; and against SARS-CoV-1 and SARS-CoV-2. We found that both SARS-CoV-2 infection and vaccination elicited broad cross-nAbs against multiple sarbecoviruses, and vaccination boosters significantly augmented the magnitude and breadth of nAbs to sarbecoviruses. Of the nAbs, several exhibited neutralization activity against multiple sarbecoviruses by targeting the spike receptor-binding domain (RBD) and competing with angiotensin-converting enzyme 2 (ACE2) binding. SCM12-61 demonstrated exceptional potency, with half-maximal inhibitory concentration (IC 50 ) values of 0.001–0.091 μg/mL, indicating its potential for combating new sarbecovirus outbreaks. Collectively, our findings suggest that both SARS-CoV-2 infection and current vaccination schemes elicit broad cross-neutralizing antibodies against diverse sarbecoviruses, enforcing prevention and therapeutic strategies for future sarbecovirus spillover events.

Objective To analyze the risk and prognostic factors for synchronous breast cancer(BC)and thyroid cancer(TC).Methods The Surveillance,Epidemiology,and End Results Program(SEER)2020 database was utilized to collect the information of patients with synchronous BC and TC(BC and TC group)and those with BC alone(BC group).Clinical data and survival were compared between two groups.Clinical data of patients with synchronous BC and TC(BC and TC group A)and those with BC alone(BC group B)admitted to a certain hospital were retrospectively analyzed.Clinical data and survival were also compared between two groups.Results ① Analysis of SEER database,482 patients in BC and TC group and 500 patients in BC group.Univariate analysis revealed that age at first diagnosis and progesterone receptor(PR)were the risk factors for synchronous BC and TC(both P＜0.05).Multivariate analysis found that age at first diagnosis(OR=1.800,95％CI:1.387-2.337,P＜0.001)and PR(OR=1.364,95％CI:1.023-1.818,P=0.034)were the independent risk factors for synchronous BC and TC.Excluding those with incomplete follow-up data,univariate analysis indicated that tumor diameter and PR were the prognostic factors for synchronous BC and TC(both P＜0.05);multivariate analysis revealed that tumor diameter was an independent prognostic factor for synchronous BC and TC(OR=4.328,95％CI:1.410-13.288,P=0.010).Univariate analysis found that age at first diagnosis and tumor diameter were the prognostic factors for BC alone(both P＜0.05);multivariate analysis identified that age at first diagnosis(OR=2.443,95％CI:1.014-5.889,P=0.047)and tumor diameter(OR=2.030,95％CI:1.039-3.969,P=0.038)were the independent prognostic factors for BC alone.(2)Analysis of inpatients,there were 40 patients each in BC and TC group A and BC group A.Univariate analysis indicated that menstrual status,PR,proliferation index Ki-67,and TT3 were the risk factors for synchronous BC and TC(all P＜0.05),multivariate analysis found that menstrual status(synchronous BC and TC versus BC alone,OR=0.175,95％CI:0.052-0.591,P=0.005),PR(OR=5.686,95％CI:1.677-19.282,P=0.005),Ki-67(OR=3.966,95％CI:1.133-13.875,P=0.031)were the independent risk factors for synchronous BC and TC.Eighty patients were subject to follow-up,6 patients died,27 survived,and 7 were lost to follow-up in BC and TC group A;2 patients died,29 survived,and 9 were lost to follow-up in BC group A.Cox regression analysis revealed no statistical significance in both groups.Conclusions Age at first diagnosis,menstrual status,PR,and Ki-67 are the risk factors for synchronous BC and TC.Tumor diameter is an independent prognostic factor for synchronous BC and TC.Age at first diagnosis and tumor diameter are the independent prognostic factors for BC alone.

Nipah virus (NiV), a highly pathogenic Henipavirus in humans, has been responsible for annual outbreaks in recent years. Experiments involving live NiV are highly restricted to biosafety level 4 (BSL-4) laboratories, which impedes NiV research. In this study, we developed transcription and replication-competent NiV-like particles (trVLP-NiV) lacking N, P, and L genes. This trVLP-NiV exhibited the ability to infect and continuously passage in cells ectopically expressing N, P, and L proteins while maintaining stable genetic characteristics. Moreover, the trVLP-NiV displayed a favourable safety profile in hamsters. Using the system, we found the NiV nucleoprotein residues interacting with viral RNA backbone affected viral replication in opposite patterns. This engineered system was sensitive to well-established antiviral drugs, innate host antiviral factors, and neutralizing antibodies. We then established a high-throughput screening platform utilizing the trVLP-NiV, leading to the identification of tunicamycin as a potential anti-NiV compound. Evidence showed that tunicamycin inhibited NiV replication by decreasing the infectivity of progeny virions. In conclusion, this trVLP-NiV system provided a convenient and versatile molecular tool for investigating NiV molecular biology and conducting antiviral drug screening under BSL-2 conditions. Its application will contribute to the development of medical countermeasures against NiV infections.

The convergence strategies of antigenic subunits and synthetic nanoparticle scaffold platform improve the vaccine production efficiency and enhance vaccine-induced immunogenicity. Selecting the appropriate nanoparticle scaffold is crucial to controlling target antigens immunologically. Lumazine synthase (LS) is an attractive candidate for a vaccine display system due to its thermostability, modification tolerance, and morphological plasticity. Here, the first development of a multivalent thermostable scaffold, LS-SUMO (SUMO, small ubiquitin-likemodifier), and a divalent nanovaccine covalently conjugated with Chikungunya virus E2 and Zika virus EDIII antigens, is reported. Compared with antigen monomers, LS-SUMO nanoparticle vaccines elicit a higher humoral response and neutralizing antibodies against both antigen targets in mouse sera. Mice immunized with LS-SUMO conjugates produce CD4+ T cell-mediated Th2-biased responses and promote humoral immunity. Importantly, LS-SUMO conjugates possess equivalent humoral immunogenicity after heat treatment. Taken together, LS-SUMO is a powerful biotargeting nanoplatform with high-yield production, thermal stability and opens a new avenue for multivalent presentation of various antigens.

ABSTRACT Monkeypox virus (MPXV) poses a global health threat. Droplet digital PCR (ddPCR) holds potential as an accurate diagnostic tool for clinical microbiology. However, there is limited literature on the applicability of ddPCR in clinical settings. In this study, the clinical features of patients with MPXV during the initial outbreak in China in June 2023 were reviewed, and an optimized ddPCR method with dilution and/or inhibitor removal was developed to enhance MPXV detection efficiency. Eighty-two MPXV samples were tested from nine different clinical specimen types, including feces, urine, pharyngeal swabs, anal swabs, saliva, herpes fluid, crust, and semen, and the viral load of each specimen was quantified. A comparative analysis was performed with qPCR to assess sensitivity and specificity and to investigate the characteristics of MPXV infection by analyzing viral loads in different clinical specimens. Consequently, common pharyngeal and gastrointestinal symptoms were observed in patients with MPXV. The optimized ddPCR method demonstrated relatively high sensitivity for MPXV quantification in the clinical materials, with a limit of detection of 0.1 copies/μL. This was particularly evident in low-concentration samples like whole blood, semen, and urine. The optimized ddPCR demonstrated greater detection accuracy compared with normal ddPCR and qPCR, with an area under the curve (AUC) of 0.939. Except for crust samples, viral loads in the specimens gradually decreased as the disease progressed. Virus levels in feces and anal swabs kept a high detection rate at each stage of post-symptom onset, and feces and anal swabs samples may be suitable for clinical diagnosis and continuous monitoring of MPXV. IMPORTANCE The ddPCR technique proved to be a sensitive and valuable tool for accurately quantifying MPXV viral loads in various clinical specimen types. The findings provided valuable insights into the necessary pre-treatment protocols for MPXV diagnosis in ddPCR detection and the potentially suitable sample types for collection. Therefore, such results can aid in comprehending the potential characteristics of MPXV infection and the usage of ddPCR in clinical settings.

The increase in emerging and reemerging infectious diseases has underscored the need for the prompt monitoring of intact infectious viruses and the quick assessment of their infectivity. However, molecular techniques cannot distinguish between intact infectious and noninfectious viruses. Here, two distinct methodologies have been developed for the expeditious and dependable quantification of intact infectious H1N1 virus, and several experiments have been conducted to substantiate their efficacy. One is an integrated cell absorption quantitative polymerase chain reaction (qPCR) method (ICA-qPCR), and the other is a combined propidium monoazide qPCR method (PMA-qPCR). The quantification limit is 100 cell culture infective dose 50 % (CCID50)/mL in ICA-qPCR following a 1.5-hour cell absorption or 126 CCID50/mL after a 15-minute incubation. For PMA-qPCR, the limit was 2,512 CCID50/mL. The number of genome copies quantified by the ICA-qPCR and PMA-qPCR methods was strongly correlated with the infectious titer determined by the CCID50 assay, thereby enabling the estimation of virus infectivity. The ICA-qPCR and PMA-qPCR methods are both suitable for the identification and quantification of intact infectious H1N1 virus in inactivated samples, wastewater, and biological materials. In conclusion, the ICA-qPCR and PMA-qPCR methods have distinct advantages and disadvantages, and can be used to quantify intact infectious viruses rapidly. These methodologies can facilitate the identification of the presence of intact infectious viruses in wastewater or on pathogen-related physical surfaces in high-level biosafety laboratories and medical facilities. Furthermore, these methodologies can also be utilized to detect other highly pathogenic pathogens.

Objectives Mpox continues to spread in China, and stakeholders' experiences may help inform prevention and control strategies. Study design Qualitative study. Methods A qualitative study across 14 Chinese cities recruited stakeholders from Centers for Disease Control and Prevention (CDCs), community-based organizations (CBOs), and hospitals involved in curbing mpox. Semi-structured interviews were conducted by telephone and analyzed using Colaizzi's phenomenological method. Results 15 CBOs workers, 14 CDCs staff, and 13 healthcare workers were recruited. Three theme categories were identified: “Efforts to curb mpox epidemic”, including CDCs' epidemic management and health education, hospitals' diagnosis, treatment, and care, CBOs’ counseling, publicity, and referrals. “Challenges to curb mpox epidemic”, including negative impacts of hospital-based quarantine, lack of specific antiviral drugs, gay identity disclosure concerns, psychological problems, contact tracing difficulties, and inadequate communication and collaboration. “Recommendations for curbing mpox epidemic”, including prioritizing supervised home-based quarantine, incorporating HIV-related indicators into hospital quarantine criteria, reducing the cost of hospital quarantine, accelerating the development of vaccines and drugs, enhancing patient privacy protection, psychological training for stakeholders, establishing a task force that comprises personnel who are experienced in contact tracing and strengthening communication and collaboration. Conclusions Effective control of mpox spread requires strengthening collaboration with CBOs and community healthcare centers (CHCs) and working out a flexible and contextualized mechanism. It also needs to reinforce patient privacy protection and integrate stigma reduction into strategies. Additionally, it is important to include HIV-related indicators in the quarantine evaluation and provide psychological training for stakeholders to help them manage their mental health and improve counseling skills.

Influenza A viruses continue to be a serious health risk to people and result in a large-scale socio-economic loss. Avian influenza viruses typically do not replicate efficiently in mammals, but through the accumulation of mutations or genetic reassortment, they can overcome interspecies barriers, adapt to new hosts, and spread among them. Zoonotic influenza A viruses sporadically infect humans and exhibit limited human-to-human transmission. However, further adaptation of these viruses to humans may result in airborne transmissible viruses with pandemic potential. Therefore, we are beginning to understand genetic changes and mechanisms that may influence interspecific adaptation, cross-species transmission, and the pandemic potential of influenza A viruses. We also discuss the genetic and phenotypic traits associated with the airborne transmission of influenza A viruses in order to provide theoretical guidance for the surveillance of new strains with pandemic potential and the prevention of pandemics.