Arboviruses such as dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV) are transmitted by Aedes mosquitoes and mainly circulate in tropical and subtropical regions. With global warming, their geographic range is expanding, increasing their threat to public health. Yunnan Province, China, bordering Southeast Asia, is a hotspot for viral importation due to intensive cross-border mobility. However, systematic surveillance for these arboviruses among acute febrile patients remains insufficient. We performed metagenomic sequencing on serum specimens from 990 acute febrile patients at the China-Myanmar border between 2017 and 2023. The pathogens were confirmed by PCR and viral isolation. Phylogenetic and spatiotemporal analyses were used to infer viral origins and transmission dynamics. In this study, a CHIKV outbreak was confirmed in 2019, with strains closely related to those from Myanmar and Thailand. Four DENV serotypes 1-4 were identified, with the predominant serotype varying annually. ZIKV was detected and closely related to strains from Myanmar. Co-infections were identified, including one case each of CHIKV with DENV-1, CHIKV with DENV-3, CHIKV with ZIKV, and DENV-1 with DENV-2. Bayesian spatiotemporal analysis of CHIKV reconstructed global transmission routes, indicating that the 2019 outbreak in China likely originated in India and spread sequentially through Bangladesh, Thailand, and Myanmar. In addition, we also detected enterovirus, hepatitis virus, Saffold virus, and rhinovirus. This study reveals a comprehensive spectrum of pathogens, including the co-circulation of DENV, CHIKV, and ZIKV, and underscores the potential risk of arbovirus importation into China, highlighting the need for strengthened border surveillance. IMPORTANCE:Arboviruses, including dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV), are expanding their range and threatening global public health. Yunnan, situated along the China-Southeast Asia border, is highly susceptible to viral introduction. By applying viral metagenomic sequencing to acute febrile patients, this study uncovered a comprehensive spectrum of pathogens and the co-circulation of DENV, CHIKV, and ZIKV. Phylogenetic analyses revealed that arboviruses were closely related to strains from Myanmar and Thailand, indicating possible frequent cross-border viral introductions. Meanwhile, we reconstructed the global transmission pathways of CHIKV through Bayesian spatiotemporal analysis, providing valuable insights for regional prevention and control of arboviruses. These findings demonstrate that Yunnan serves as a critical interface for viral importation and underscore the urgent need to strengthen border surveillance and early warning systems to mitigate the spread of arboviruses.

IntroductionViral calf diarrhea poses a significant challenge to the cattle industry worldwide due to its high morbidity and mortality rates, leading to substantial economic losses. The clinical symptoms associated with various diarrhea pathogens often overlap, complicating accurate diagnosis; thus, there is an urgent need for rapid and precise diagnostic methods to improve prevention and treatment efforts. In this study, we developed a one-step multiplex reverse-transcription quantitative real-time polymerase chain reaction (mRT-qPCR) that enables the simultaneous detection of three key viral pathogens responsible for calf diarrhea: bovine kobuvirus (BKoV), bovine astrovirus (BoAstV), and bovine torovirus (BToV). However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of viral calf diarrhea.MethodsSpecific primers and minor groove binder (MGB)-based probes were designed targeting the 3D region of BKoV, ORF1 region of BoAstV, and N region of BToV. The sensitivity, specificity, and reproducibility ability were evaluated for the mRT-qPCR. Further, 80 bovine fecal samples were subjected to the mRT-qPCR, and the results were verified using conventional reverse-transcription PCR (RT-PCR) or PCR methods and sequencing methods.ResultsThis novel method demonstrated high sensitivity and specificity,achieving a detection limit of 24 copies/mL for each pathogen. Furthermore, the assay exhibited excellent reproducibility, with coefficients of variation below 1.5%, a strong linear correlation (R2 > 0.996), and an amplification efficiency between 90% and 110%. Validation with 80 clinical samples from both diarrheic and non-diarrheic cattle across four farms in Shanghai showed a high degree of concordance with RT-PCR, with positive detection rates for BKoV, BoAstV, and BToV at 28.75%, 8.75%, and 3.75%, respectively, highlighting the predominance of BKoV and BoAstV. Notably, this study represents the first identification of BKoV, BoAstV, and BToV in the Shanghai region.DiscussionThe mRT-qPCR is a robust, rapid, and simple tool for identifying viral pathogens associated with calf diarrhea, facilitating the development of effective prevention and control measures that are vital for the future sustainability of the cattle industry.

IntroductionThe establishment of clinical breakpoints for antimicrobial drug is crucial for guiding appropriate therapeutic interventions. This study aims to identify the pharmacokinetic/pharmacodynamic (PK/PD) cut-offs for using sitafloxacin against target pathogens to support clinical breakpoint establishment for antimicrobial drug sensitivity testing.MethodsA population PK (PopPK) model was built (342 subjects) to calculate the dosing-regimen-dependent (50 mg q12 h, 100 mg q24 h and 100 mg q12 h) PK parameters of sitafloxacin-infected patients, which were combined with in vitro PD data and PK/PD target data. The probabilities of attainment (PTAs) and cumulative fraction of response (CFR) values for different sitafloxacin dosing regimens against Streptococcus pneumoniae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were calculated via Monte Carlo simulation.ResultsPopPK modelling revealed that the PK profile of sitafloxacin was consistent with a two-compartment model with first-order elimination. Creatinine clearance affected total clearance, bodyweight and age affected the central ventricular apparent volume of distribution, and food affected the sitafloxacin absorption rate. On the basis of the animal infection model target (fAUC24h/MIC = 11.56), the anti-Streptococcus pneumoniae sitafloxacin dosing regimen PTAs were >95% (MIC ≤ 0.06, ≤0.06, ≤0.125 mg/L; CFRs = 98.2∼99.3%). With a clinical study target of fAUC24h/MIC ≥ 30, the anti-Streptococcus pneumoniae dosing regimen PTAs were >95% (MIC ≤ 0.03, ≤0.03, ≤0.06 mg/L; CFRs = 89.2∼97.3%). For the other four strains, the dosing-regimen-dependent sitafloxacin PK/PD cut-offs were 0.06, 0.06 and 0.125 mg/L, respectively (CFRs = 56.3∼76.9%).DiscussionOur findings suggest that sitafloxacin PK/PD cut-offs of S ≤ 0.06 mg/L and R > 0.125 mg/L should be used against these five strains and that the sitafloxacin dosing regimens (50 mg q12 h, 100 mg q24 h and 100 mg q12 h) have the expected efficacy against Streptococcus pneumoniae-related infections, but the efficacy against Pseudomonas aeruginosa-associated infections needs to be verified in clinical practice.

In this study, we used baculovirus to express hemagglutinin (HA) and neuraminidase (NA) to prepare a novel genotype of H3N2 canine influenza virus particles (VLPs). The effectiveness of the H3N2 VLP vaccine was evaluated by detecting HI antibodies, the antiviral protection rate, antibody persistence and anatomical examination of the lungs.A challenge model has been established in a previous study for the study of canine influenza virus-like particle vaccines. A/Canine/Shanghai/0103/2019, with a challenge dose of 106 EID50, infects 10week-old healthy beagle dogs through nasal instillation and can cause severe clinical symptoms. Using a single dose of VLP vaccine for beagle dogs, the vaccine was tested at titers of 26 intranasally and 26 intramuscularly. One week after a single immunization, the HI titer promptly reached 28 among the immunized groups. The duration of antibody can persist for four months. We differentiated between CD4+ and CD8+ T cells in the peripheral blood. Four weeks after the single immunization, all beagles except those in the noninfected and nonimmunized groups were intranasally challenged with live H3N2 virus (1 x 106 EID50). All immunized beagles shed no virus at d 1-4 post-challenge. After the challenge, the placebo control beagles shed the virus on d 1 postchallenge (105.85 +/- 0.071 EID50). An anatomical examination of the lungs revealed that visible lesions were rarely detected in the lungs of the nasal immunization group, and the lungs were as healthy as those of the noninfected and nonimmunized groups were. The lung surfaces presented visible bleeding spots in the intramuscular immunization group and placebo-control group. Their effectiveness will provide a scientific basis for the promotion and use of these products.

Cell Division Cycle 42 protein (CDC42) is a pivotal member of the Rho family of small GTPases, integral to cell signaling and the regulation of cell polarity. CDC42 is extensively documented to participate in host immunity, particularly in processes such as phagocytosis and cell migration. Nevertheless, the precise mechanisms by which CDC42 affects host-bacteria interactions remain unclear in Echinoderms. In this study, we have isolated and characterized the CDC42 gene from Apostichopus japonicus, referred to as AjCDC42. The complete cDNA sequence of AjCDC42 extends 1282 base pairs, including a typical classical RHO domain, spanning amino acids from 6 to 179. Spatial expression analysis demonstrated that AjCDC42 is ubiquitously expressed across all examined tissues. Following infection with Vibrio splendidus and stimulation with LPS, the protein and mRNA levels of AjCDC42 were significantly upregulated in coelomocytes. Moreover, the knockdown of AjCDC42 significantly decreased V. splendidus-induced phagocytic activity in coelomocytes, leading to a reduced intracellular load of V. splendidus, as evidenced by flow cytometry and bacterial plate counting assays. Mechanistically, AjCDC42 was identified as an interacting partner of IQ Motif Containing GTPase Activating Protein 1 (AjIQGAP1) through the use of co-immunoprecipitation, immunofluorescence, and GST pull-down assays combined with mass spectrometry techniques. Additional functional analyses revealed that the knockdown of AjIQGAP1 similarly decreased the phagocytic activity and the intracellular load of V. splendidus in coelomocytes. Furthermore, we detected that decreased levels of AjCDC42 and AjIQGAP1 impaired cytoskeletal rearrangement and delayed lysosomal clearance of V. splendidus. In summary, our findings underscore the pivotal role of the AjCDC42-AjIQGAP1 axis in regulating coelomocytes phagocytosis in A. japonicus during V. splendidus infection.

As crucial biomarkers of food spoilage, portable and real-time monitoring of the biogenic amines (BAs) is essential to ensuring food safety. In light of this, a ratiometric fluorescent probe (Tb@HOF-BPTC) is developed, which exhibits distinct fluorescence response upon exposure to BAs. It demonstrates exceptional analytical performance with a low limit of detection ranging from 3.1 to 14.3 μM and remarkably rapid response times (<7.02 s). Notably, the differential responses of three BAs to the Tb@HOF-BPTC construct individualized "portrait" that enhance identification accuracy. Moreover, a machine learning-intelligent sensing platform is constructed by integrating back-propagation neural networks (BPNN) with smartphone-based RGB recognition. This platform utilizes a smartphone camera to capture fluorescence images, which are then processed by a BPNN model to classify the concentrations and the species of the BAs. Furthermore, for the purpose of overcoming the inconvenience of detecting BAs vapors in conventional fluorescence sensing, poly(vinyl alcohol) (PVA) hydrogel microneedles combined with Tb@HOF-BPTC are designed for real-time detection of BAs. Overall, our work shows the potential of real-time food freshness assessment by constructing an intelligent sensing platform based on PVA hydrogel microneedles.

Influenza, a highly pathogenic infectious disease, causes nearly half a million deaths annually worldwide. Thus, effective vaccine-based prevention and control are crucial. Although live attenuated influenza vaccines (LAIVs) can induce mucosal immunity, existing vaccines effectiveness remains relatively low, posing a significant threat to public health. Thus, we developed a novel mosaic H1N1 LAIV candidate by integrating mosaic antigen design with established LAIV technology. This vaccine incorporates most potential T-cell epitopes of hemagglutinin and neuraminidase antigens into an attenuated master donor strain, ensuring safety and broad immunity. We compared it with commercial monovalent attenuated and inactivated vaccines in mice. The mosaic H1N1 LAIV induced robust cross-reactive humoral and mucosal immune responses, enhanced antigen-specific cellular immunity, and established tissue-resident memory T and B cells in the respiratory tract. Challenge experiments confirmed its protective efficacy against homologous and heterologous strains. It provided complete protection against homologous strains with low epitope similarity and partial protection against the ancestral H3N2 virus. Our study highlights the mosaic H1N1 LAIV as an excellent universal vaccine candidate capable of inducing broad cross-reactive immune responses and providing robust protection against distinct influenza A viruses, demonstrating a promising strategy to address the limitations of current commercial vaccines.

The rising prevalence of extensively drug-resistant gram-negative infections necessitates safer polymyxin analogs, as conventional polymyxins are limited by nephrotoxicity and compositional heterogeneity. MRX-8, a next-generation polymyxin B1 derivative engineered to reduce toxicity while maintaining potent antibacterial activity (MIC90 <1 mg/L), demonstrates improved pharmacokinetic/pharmacodynamic (PK/PD) targets. To overcome challenges in preclinical blood sampling volume, we developed a LC-MS/MS method to quantify MRX-8 in dried blood spots (DBS). DBS samples (40 μL blood) collected on Whatman 903® cards were extracted with formic acid-water-acetonitrile (6:64:30, v/v/v) and analyzed using an AB Sciex QTRAP 5500/Shimadzu LC-30A system with MRM transitions at 617.5→155.1 (MRX-8) and 402.3→101.2 (polymyxin B1 internal standard). The method demonstrated linearity (0.0200-10.0 mg/L), precision (3.6-14.1 %), accuracy (87.4-107.8 %), recovery (90.1-102.2 %), and matrix effects (108.1-125.4 %). DBS stability was confirmed under room temperature (3 days), refrigerated (2-8 °C, 7 days), and frozen (-20 °C/-70 °C, 25 days) conditions. Adjusted DBS concentrations incorporating a plasma-blood distribution coefficient (0.59) correlated with plasma pharmacokinetics (R² = 0.976). Compared to conventional plasma sampling, this DBS approach reduces blood volumes and significantly minimizes animal burden. This validated microsampling method provides an ethical and efficient solution for preclinical PK evaluation of MRX-8, accelerating its development as a critical antibacterial therapy against drug-resistant pathogens.

Influenza A virus (IAV) has the characteristics of high infectivity and high pathogenicity, which makes IAV infection a serious public health threat. Identifying protein-protein interactions (PPIs) between IAV and human proteins is beneficial for understanding the mechanism of viral infection and designing antiviral drugs. In this article, we developed a sequence-based machine learning method for predicting PPI. First, we applied a new negative sample construction method to establish a high-quality IAV-human PPI dataset. Then we used conjoint triad (CT) and Moran autocorrelation (Moran) to encode biologically relevant features. The joint consideration utilizing the complementary information between contiguous and discontinuous amino acids provides a more comprehensive description of PPI information. After comparing different machine learning models, the eXtreme Gradient Boosting (XGBoost) model was determined as the final model for the prediction. The model achieved an accuracy of 96.89%, precision of 98.79%, recall of 94.85%, F1-score of 96.78%. Finally, we successfully identified 3,269 potential target proteins. Gene ontology (GO) and pathway analysis showed that these genes were highly associated with IAV infection. The analysis of the PPI network further revealed that the predicted proteins were classified as core proteins within the human protein interaction network. This study may encourage the identification of potential targets for the discovery of more effective anti-influenza drugs. The source codes and datasets are available at https://github.com/HVPPIlab/IVA-Human-PPI/.

Developing a sensitive analytical platform for monitoring tiopronin (MPG), its metabolite 2-mercaptopropionic acid (MPA), and the key liver biomarker glutathione (GSH) is crucial for liver health assessment. Here, an artificial intelligence-assisted bionic vision platform based on a dual-emission Tb3+-functionalized hydrogen-bonded organic framework (Tb@HOF-dobpdc) is constructed. The ratiometric fluorescent sensor exhibits a distinct ″turn-on″ response, enabling highly sensitive detection of MPG, MPA, and GSH with low limits of detection (0.20, 0.68, and 0.31 μM, respectively). For practical application, Tb@HOF-dobpdc can achieve rapid detection of target analytes in real serum and urine samples by combining with the hydrogel. To overcome the human eye's limitation in discerning subtle color changes, RGB channel processing is used to generate visually distinguishable pseudocolor signals. Furthermore, a backpropagation neural network (BPNN) is applied for accurately distinguishing analyte concentrations by identifying fluorescence images. Leveraging the strong correlation between these analytes and liver health, an AND logic gate diagnostic system is established, outputting ″healthy″ only when all biomarker levels are within safe ranges. This intelligent platform combines advanced material design and artificial intelligence, providing a powerful tool for point-of-care liver health diagnosis.

Background:Aerosolized polymyxin B delivery was a promising approach for the treatment of ventilator-associated pneumonia (VAP). However, there were little data on the concentrations of polymyxin B in epithelial lining fluid (ELF), which impedes the optimal use of aerosolized polymyxin B in clinics. Methods:We present four cases of patients diagnosed with VAP caused by Gram-negative bacteria, who enrolled in a prospective, therapeutic drug monitoring (TDM) study of polymyxin B. The patients were treated with aerosolized and intravenous administration of polymyxin B. Polymyxin B concentrations in both ELF and plasma were determined using validated LC-MS/MS methods. Results:All four patients achieved bacterial eradication, with three of them reaching clinical improvement or cure. Following aerosol administration (25 or 50 mg, q12h) and intravenous infusion (50-100 mg, q12h) of polymyxin B, it was observed that the concentrations of polymyxin B in ELF were significantly higher in ELF (20.6-97.6 mg/L) compared to those in plasma (1.19-5.16 mg/L) during the steady sate. The area under the concentration-time curve for 24 h (AUC24h,ELF) ranged from 283.6 to 1872.9 mg•h/L. Conclusions:This study presented polymyxin B concentrations in ELF following aerosolized delivery, supporting its clinical use from a PK/PD perspective. Following combined aerosol and intravenous administration, polymyxin B achieved notably higher concentrations in ELF than those observed in plasma.

Multiple subtypes of avian influenza virus (AIV), including H5N1, H5N6, and H5N8 viruses, are currently co-circulating in wild birds and poultry and causing sporadic human infections. Vaccine development is essential for pandemic preparedness. In this study, we constructed a candidate vaccine virus (CVV) using reverse genetics (RG) based on the sequence of the first human-infected H5N8 subtype AIV, A/Astrakhan/3212/2020 (H5N8). We evaluated the immunogenicity of the rH5N8/PR8 vaccine strain in combination with Alum, ISA51, and MF59 adjuvants, and we optimized immunization strategies including dosage, administration route, and immunization interval in BALB/c mice. Our results demonstrated that a 10 μg dose of inactivated rH5N8/PR8 with MF59 adjuvant, administered intramuscularly twice at 7-day intervals, induced the strongest immune response and effectively protected mice against challenge with wild-type H5N8 AIVs. Since pandemic influenza vaccines typically require tailored vaccination doses and routes specific to their characteristics, this study provides valuable insights for the development of similar vaccine strains with pandemic potential.

Aims/Background Mycoplasma pneumoniae pneumonia (MPP) is typically a benign and self-limiting disease. This study aimed to investigate the effect of early oral administration of doxycycline on macrolide resistance in children with MPP. Methods This study retrospectively analyzed the clinical data of 173 MPP children treated with macrolides at the Second Affiliated Hospital of Mudanjiang Medical University from March 2020 to March 2023. Nine cases that did not meet the inclusion criteria were excluded, leaving 164 children. They were divided into Group A (early oral administration of doxycycline + macrolide treatment) (n = 85) and Group B (macrolide treatment alone) (n = 79) based on whether early oral administration of doxycycline was given. Drug sensitivity results and adverse reactions after treatment were statistically analyzed. Based on the drug sensitivity results, the MPP children were classified as having either macrolide-resistant mycoplasma pneumoniae (MRMP) or macrolide-sensitive mycoplasma pneumoniae (MSMP) infections. A stratified analysis was performed to compare the disappearance time of fever, disappearance time of shortness of breath, disappearance time of rales, and symptom improvement time on chest X-ray examination, and to further explore the clinical efficacy of early oral administration of doxycycline in different groups of children. Results No significant differences were found in baseline data such as age, sex, and weight between the two groups (p > 0.05). A total of 112 out of 164 children developed macrolide resistance (68.29%), with 47 cases in Group A and 65 cases in Group B, indicating a significant difference between the two groups (p < 0.05). The two groups showed a significant difference in macrolide sensitivity levels after treatment (p < 0.05), with no significant difference in the incidence of adverse reactions (p > 0.05). After treatment, the time to the disappearance of febrile fever, time to disappearance time of fever, disappearance time of shortness of breath, disappearance time of rales, symptom improvement time on chest X-ray examination, and time to administration of macrolides after treatment were shorter in children with MRMP in group A than in children with MRMP in group B (p < 0.05). In contrast, MSMP children in both groups exhibited no significant differences in symptom disappearance time and duration of macrolides treatment (p > 0.05). Conclusion Early oral administration of doxycycline is a safe and effective treatment for MPP. It helps relieve symptoms in MRMP children, shortens the duration of macrolide use, and reduces the incidence of macrolide resistance.

Rhinovirus (RV), a prominent causative agent of both upper and lower respiratory diseases, ranks among the most prevalent human respiratory viruses. RV infections are associated with various illnesses, including colds, asthma exacerbations, croup and pneumonia, imposing significant and extended societal burdens. Characterized by a high mutation rate and genomic diversity, RV displays a diverse serological landscape, encompassing a total of 174 serotypes identified to date. Understanding RV genetic diversity is crucial for epidemiological surveillance and investigation of respiratory diseases. This study introduces a comprehensive and high-quality RV data resource, designated RVdb (http://rvdb.mgc.ac.cn), covering 26 909 currently identified RV strains, along with RV-related sequences, 3D protein structures and publications. Furthermore, this resource features a suite of web-based utilities optimized for easy browsing and searching, as well as automatic sequence annotation, multiple sequence alignment (MSA), phylogenetic tree construction, RVdb BLAST and a serotyping pipeline. Equipped with a user-friendly interface and integrated online bioinformatics tools, RVdb provides a convenient and powerful platform on which to analyse the genetic characteristics of RVs. Additionally, RVdb also supports the efforts of virologists and epidemiologists to monitor and trace both existing and emerging RV-related infectious conditions in a public health context.

Anaplastic lymphoma kinase (ALK) is a highly responsive therapeutic target for ALK-rearranged nonsmall cell lung cancer (NSCLC). However, patients with this cancer invariably relapse because of the development of ALK inhibitor resistance resulting from mutations within the ALK tyrosine kinase domain. Herein, we report the discovery of dEALK1, a small -molecule degrader of EML4-ALK fusion proteins, with capability of overcoming resistance to ALK inhibitor ceritinib. dEALK1 induces rapid and selective degradation of wild -type (WT) EML4-ALK and mutated EML4-ALKs acquiring resistance to ceritinib, leading to inhibition of cell proliferation and increase of apoptosis in NSCLC cells expressing WT EML4-ALK or ceritinib-resistant EML4-ALK mutants in vitro. Furthermore, dEALK1 also exerts a potent antitumor activity against EML4-ALK-positive xenograft tumors without or with harboring ceritinibresistant EML4-ALK mutations in vivo. Our study suggests that dEALK1-induced degradation of EML4-ALK fusion proteins is a promising therapeutic strategy for treatment of ALK-rearranged lung cancer with ceritinib resistance.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both alpha 2,3 and alpha 2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn. [GRAPHICS]

Urban stray cats are cats without owners that survive in the wild for extended periods of time. They are one of the most common stray animals in cities, and as such, monitoring the pathogens carried by urban stray cats is an important component of urban epidemiological surveillance. In order to understand the prevalence of respiratory diseases in urban stray cats in Shanghai and provide scientific evidence for the development of targeted prevention and control strategies for respiratory diseases in stray cats, we collected 374 ocular, nasal, and oropharyngeal swabs from urban stray cats in Shanghai from January 2022 to December 2022. After RNA extraction, we used real-time PCR to detect six respiratory pathogens, including influenza A virus, feline calicivirus, feline herpesvirus type 1, Mycoplasma, Chlamydia, and Bordetella bronchiseptica. The results showed that among the 374 samples, 146 tested positive, with a positivity rate of 39.04%. The highest positivity rate was observed for Mycoplasma felis at 18.72% (70/374), followed by Chlamydia felis at 11.76% (44/374), feline calicivirus at 3.74% (14/374), feline herpesvirus 1 at 3.48% (13/374), Bordetella bronchiseptica at 1.34% (5/374), and influenza A virus was not detected. The highest positivity rate for Mycoplasma felis was in Minhang District at 31.94% (23/72), while Chlamydia felis and Bordetella bronchiseptica had the highest positivity rates in Jiading District at 23.53% (8/34) and 5.88% (2/34), respectively. The highest positivity rates for feline calicivirus and feline herpesvirus 1 were both observed in Qingpu District, at 14.46% (12/83) and 9.64% (8/83), respectively. A total of 36 samples showed mixed infections with two or more pathogens, with Mycoplasma felis being involved in 32 of these mixed infections, with the highest number of mixed infections being with Chlamydia felis at 25 samples. Respiratory pathogen positivity was detected throughout the year, with peak detection rates in summer and winter. The positivity rates of cat respiratory pathogens in different seasons showed statistical differences (χ2 = 27.73, p < 0.01). There was no statistical difference in the positivity rates of respiratory pathogens between cats of different genders (χ2 = 0.92, p > 0.05). The positivity rates of respiratory pathogens in cats of different age groups showed statistical differences (χ2 = 44.41, p < 0.01). Mycoplasma felis and Chlamydia felis were the main pathogens causing respiratory infections in stray cats, with Mycoplasma felis showing a much higher positivity rate than other respiratory pathogens and often co-infecting with Chlamydia felis and feline calicivirus. The positivity rate of Mycoplasma felis was high in summer, autumn, and winter, with no statistical difference between seasons. These results indicate a serious overall prevalence of respiratory pathogens in urban stray cats in the Shanghai area, showing seasonal trends and mixed infections with other pathogens. These findings suggest the need for comprehensive prevention and control measures to address respiratory pathogen infections in urban stray cats in the Shanghai area.

A rapid and sensitive assay is essential for reliable surveillance and diagnosis of canine astrovirus (CaAstV). In this study, two real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays with high sensitivity, rapidity, and reliability were developed using fluorescence dye and FRET-based assimilating probes for real-time detection of CaAstV. These assays specifically amplified the ORF2 gene of CaAstV and did not amplify any sequences from canine enterovirus. The limit of detection (LOD) of both the probe-based and dye-based RT-LAMPs was 10 0 copies/μL. Fluorescence signals were generated within 30 min for the lowest concentration of a standard RNA sample, which was significantly faster than that achieved by real-time fluorescence quantitative PCR (qRT-PCR) assay. When clinical samples were tested, the positive and negative agreement of the dye-based RT-LAMP assay with qRT-PCR was 87.5% (14/16) and 93.55% (29/31), respectively. The positive and negative agreement of the probe-based RT-LAMP assay with qRT-PCR was 94.11% (16/17) and 96.55% (28/29), respectively. The RT-LAMP assays developed in this study showed strong potential for use as an on-site diagnostic assay for rapid, specific, and reliable detection of CaAstV in clinical samples.

BACKGROUND:The canine influenza virus (CIV) outbreak has garnered considerable attention as it poses a significant threat to dog health. During the H3N2 CIV evolution in beagles, the virus formed a new clade after 2019 and gradually became more adaptable to other mammals. Therefore, successfully elucidating the biological characteristics and constructing a canine influenza infection model is required for CIV characterization. METHODS:We performed genetic analyses to examine the biological characteristics and infection dynamics of CIV. RESULTS:The genotype of our H3N2 CIV strain (from 2019 in Shanghai) belonged to the 5.1 clade, which is now prevalent in China. Using MDCK cells, we investigated viral cytopathic effects. Virus size and morphology were observed using transmission electron microscopy. Beagles were also infected with 104, 105, and 106 50% egg-infectious doses (EID50). When compared with the other groups, the 106 EID50 group showed the most obvious clinical symptoms, the highest virus titers, and typical lung pathological changes. Our results suggested that the other two treatments caused mild clinical manifestations and pathological changes. Subsequently, CIV distribution in the 106 EID50 group was detected by hematoxylin and eosin (H&E) and immunofluorescence (IF) staining, which indicated that CIV primarily infected the lungs. CONCLUSIONS:The framework established in this study will guide further CIV prevention strategies.