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Natural killer (NK) cells are pivotal in immunotherapy due to their potent tumor-targeting capabilities. However, accessible in vitro 3D dynamic models for evaluating Tumor Infiltrating Natural Killer Cells (TINKs) remain scarce. This study addresses this gap by developing a novel pump-free microfluidic chip to investigate the interactions between NK-92 cells and prostate DU 145 tumor spheroids. The platform facilitates the separation of free NKs and TINKs for subtype characterization. The design integrates multiple planes with a multi-layer paper scaffold to accommodate tumor spheroids, allowing NK-92 cells to traverse Matrigel-coated barriers that mimic the extracellular matrix. The dual-channel pump-free device enables unidirectional circulation of NK-92 cells, allowing analysis of tumor spheroid movement and NK-92 cell interactions under flow conditions. Results demonstrate continuous fluid circulation in the dual-channel device by rocking the platform at tilt angles of 21° and 15°. Tumor spheroids show- enhanced migration under flow conditions compared to static culture. Although spheroids recruit slightly more NK-92 cells under flow conditions, CD56 and CD16 receptor expression on IL-2-activated free NK-92 cells and tumor-infiltrating NK-92 cells matches in vivo patterns in dynamic cultures. These findings suggest that tumor cells and fluid dynamics significantly influence NK cell subtypes. This pump-free microfluidic platform is a functional tool for simulating and studying immune cell-tumor interactions, providing valuable insights into NK cell dynamics with tumor spheroids in physiologically relevant environments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Emerging tick-borne orthonairovirus infections pose a growing global concern, with limited understanding of the viral ovarian tumor-like cysteine proteases (vOTUs) encoded by novel orthonairoviruses. These vOTUs, a group of deubiquinylases (DUBs), disrupt the innate immune response. Yezo virus (YEZV), a recently discovered pathogenic orthonairovirus, was first reported in Japan in 2021. In this study, we successfully isolated and identified YEZV and a new orthonairovirus, Jiànchuān tick virus (JCTV), from Ixodes persulcatus and Haemaphysalis montgomeryi ticks, respectively, in China. We found that the vOTU domains encoded by YEZV and JCTV exhibited both DUB and deISGylase activities, though with potentially less broad deISGylation compared to that of Crimean-Congo hemorrhagic fever virus (CCHFV) during natural infection. Phylogenetic analysis of global vOTUs, including 83 new sequences, revealed a high diversity of this domain. Interestingly, retrospective screening of tick-bite patients from 2012 to 2016 in northeastern China traced YEZV infections as far back as 2012, identifying four cases. Additionally, YEZV primarily infected I. persulcatus (31.4%) and Dermacentor nuttalli (10.5%) in northern China, while JCTV exhibited high infection rates in H. montgomeryi (81.3%) in southern China. In summary, our work emphasizes the active surveillance of orthonairovirus infections and the imperative need for the development of vOTU domain-targeted anti-virals, offering potential therapeutic solutions for a broad spectrum of orthonairoviruses.IMPORTANCEThe vOTUs, a group of DUBs, mimic the functions of host DUBs to enhance viral infectivity and may serve as potential drug targets. vOTUs from different orthonairoviruses exhibit distinct preferences toward ubiquitin (Ub) and ubiquitin-like protein interferon stimulated gene 15 (ISG15). In this study, we investigated the deubiquitinase and deISGylase functions of various orthonairoviral vOTUs using both an overexpression system and natural viral infections in vitro . Our findings illustrate that the vOTUs from YEZV and JCTV can cleave both Ub and ISG15 in an overexpression system, but these viruses exhibit potentially narrower deISGylation capacity than CCHFV during natural infection. This suggests that the diversity of vOTUs may have a potential relationship with the pathogenesis.

A half-dose influenza vaccine (7.5 μg hemagglutinin per strain) has been used for children under 3 years of age for a long time. However, several studies indicate that a full-dose influenza vaccine (15 μg hemagglutinin per strain) may bring more benefit to this population without increasing the risk of adverse reactions. We conducted a clinical study in children aged 6-35 months in China. Participants were randomized to receive two doses of full-dose quadrivalent influenza vaccine (F-QIV), half-dose quadrivalent vaccine (H-QIV), and two half-dose trivalent vaccines (H-TIV) in a 2:2:1:1 ratio. The safety and tolerability profile of the vaccine was evaluated for 6 months postvaccination. Hemagglutination inhibition (HI) antibody titers were measured for immunogenicity assessment. The primary objective was to assess whether the results of all vaccines met the criteria. A total of 1,980 participants were enrolled in the study. Both H-QIV and F-QIV were well tolerated after vaccination. Although the geometric mean increase (GMI), seroconversion rate (SCR), and seroprotection rate (SPR) for both H-QIV and F-QIV were achieved by the criteria, superior immunogenicity in terms of geometric mean titer (GMT) ratio was observed in F-QIV to H-QIV for A/H3N2 (GMT ratio (95% CI) of 1.37 (1.11 ~ 1.68)) and B/Yamagata (1.21 (1.05 ~ 1.39)). Antibody responses to the QIV were non-inferior to the response to the TIV for the matched strains. In conclusion, F-QIV and H-QIV were both safe and immunogenic for children. F-QIV induced a stronger immune response to influenza viruses and may provide more protection and benefit by promoting the use of F-QIV in children aged 6-35 months.

The lack of a robust system to reproducibly propagate HRV-C, a family of viruses refractory to cultivation in standard cell lines, has substantially hindered our understanding of this common respiratory pathogen. We sought to develop an organoid-based system to reproducibly propagate HRV-C, and characterize virus-host interaction using respiratory organoids. We demonstrate that airway organoids sustain serial virus passage with the aid of CYT387-mediated immunosuppression, whereas nasal organoids that more closely simulate the upper airway achieve this without any intervention. Nasal organoids are more susceptible to HRV-C than airway organoids. Intriguingly, upon HRV-C infection, we observe an innate immune response that is stronger in airway organoids than in nasal organoids, which is reproduced in a Poly(I:C) stimulation assay. Treatment with α-CDHR3 and antivirals significantly reduces HRV-C viral growth in airway and nasal organoids. Additionally, an organoid-based immunofluorescence assay is established to titrate HRV-C infectious particles. Collectively, we develop an organoid-based system to reproducibly propagate the poorly cultivable HRV-C, followed by a comprehensive characterization of HRV-C infection and innate immunity in physiologically active respiratory organoids. The organoid-based HRV-C infection model can be extended for developing antiviral strategies. More importantly, our study has opened an avenue for propagating and studying other uncultivable human and animal viruses., Competing Interests: Competing interests: J.Z., K.Y.Y., H.Clevers, C.L., and M.C.C. are listed as inventors on the patent of airway organoids (Patent No: ZL 2019 8 0037552.0), and nasal organoids (US 63/358,795). J.Z. is the founder of BiomOrgan Ltd. All other authors declare no competing interests., (© 2024. The Author(s).)

Hormographiella aspergillata is a rare hyaline mold causing invasive fungal infection in humans, until the frequent use of antifungal prophylaxis in immunocompromised hosts. Due to the high mortality of H. aspergillata infection, early recognition and treatment are crucial. Previous case reports suggested that serum (1,3)-beta-D-Glucan (BG) is one of the diagnostic aids for H. aspergillata infection. Here we report for the first time a case of pulmonary H. aspergillata infection with a negative serum BG but positive bronchoalveolar lavage fluid (BAL) BG. This may suggest that BAL BG is a useful and additional microbiological marker for prompt identification of this fatal invasive fungal infection (IFI). But it should be interpreted together with the clinical presentation, imaging, and other laboratory results., Competing Interests: Declarations. Ethics approval: The study was approved by the Ethics Committee of the University of Hong Kong-Shenzhen Hospital. Written informed consent was obtained from the patient’s husband. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Competing Interests: Declaration of interests The authors declare no conflict of interest.

Background: Liver involvement is a common complication of coronavirus disease 2019 (COVID-19), especially in hospitalized patients. However, the underlying mechanisms involved are not fully understood., Methods: Immunohistochemistry (IHC) staining of SARS-CoV-2 spike (S) and nucleocapsid (N) proteins was conducted on liver tissues from six patients with COVID-19. The 10x Genomics Visium CytAssist Spatial Gene Assay was designed to analyze liver transcriptomics. TCR CDR3 sequences were analyzed in DNA from liver tissues. Liver function indicators were retrospectively studied in 650 hospitalized patients with COVID-19., Findings: SARS-CoV-2 proteins were initially detected in the livers of naturally infected golden (Syrian) hamsters, prompting us to investigate the situation in clinical cases. Thus, we collected liver tissues from patients with abnormal liver biochemical values. Viral S and N proteins were detected in the livers of severe and deceased patients but not in those of moderate patients. We further demonstrated that hepatocytes and erythroid cells in hepatic sinusoids are major cells targeted by SARS-CoV-2. Immune cells, especially T cells, were enriched in surviving severe patients, characterized by enhanced CDR3α clonality and novel CDR3β recombination of the T-cell receptor. In contrast, hepatocyte apoptosis was triggered, and the transcription of albumin (ALB) was obviously impaired in the deceased patients. We then performed a retrospective study including patients with COVID-19. Serum aspartate aminotransferase (AST) and ALB levels at baseline significantly differed in the deceased cohort. However, AST regression did not decrease the risk of death. ALB recovery indicated clinical improvement, and declining or low serum ALB concentrations were associated with death., Interpretation: This study provides clinical evidence for liver infection with SARS-CoV-2, insight into the impact of SARS-CoV-2 on the liver, and a potential way to evaluate the risk of death via assessing serum ALB concentration fluctuations in patients with COVID-19., Funding: National Key R&D Program of China (2021YFC2300602), National Natural Science Foundation of China (92369110), National Natural Science Foundation of China (U23A20474), Shanghai Municipal Science and Technology Major Project (ZD2021CY001), Shanghai Jinshan District Medical and Health Technology Innovation Fund Project (2023-WS-31)., Competing Interests: Declaration of interests The authors declared no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Competing Interests: We declare no competing interests. The work conducted by the Pandemic Research Alliance is partly supported by the Emergency COVID-19 Project, Major Projects on Public Security, the National Key Research and Development Program (2021YFC0866100); the Emergency Collaborative Project of Guangzhou Laboratory (EKPG22-01); the National Key R&D Program of China (projects 2021YFC0866100 and 2023YFC3041600); the Tsinghua Spring Breeze (Chun Feng) Fund and Wanke Scientific Research Program (grant number 20221080056 to LZ); and donations from Richard Yu and Carol Yu, the Shaw Foundation Hong Kong, Michael Seak-Kan Tong, May Tam Mak Mei Yin, Lee Wan Keung Charity Foundation Limited, and Providence Foundation Limited (in memory of the late Lui Hac Minh). The Columbia unit acknowledges the Wu Family for support. SRL is supported by the National Health and Medical Research Council of Australia (APP 2026490), the Office of Health Protection (through the Australian Partnership for Infectious Disease Emergencies, APPRISE), the Medical Research Future Fund (APP2016144), mRNA VIC (GA-F3673757-3598), and the Cumming Global Centre for Pandemic Therapeutics. The Programme for Research in Epidemic Preparedness and Response is funded by the Singapore National Research Foundation. The funding sources had no role in the study design, data collection, analysis, interpretation, or writing of this report.

Sepsis (defined as sepsis 3.0) is a life-threatening organ dysfunction caused by a dysregulated host response to a variety of pathogenic microorganisms. Characterized by high morbidity and mortality, sepsis has become a global public health problem. However, there is a lack of appropriate diagnostic and therapeutic strategies for sepsis and current management rely on the limited treatment strategies. Recently, nanomedicines targeting and controlling the release of bio-active agents have shown excellent potency in sepsis management, with improved therapeutic efficacy and reduced adverse effects. In this review, we have summarized the advantages of nanomaterials. Also, the preparation and efficacy of the main categories of anti-sepsis nanomedicines applied in sepsis management are described in detail, including antibiotic-coated nanomaterials, antimicrobial peptides-coated nanomaterials, biomimetic nanomaterials, nanomaterials targeting macrophages and natural products loaded nanomaterials. These advances in nanomedicines establish the huge potential for nanomaterials-based sepsis management, especially in the improved pharmaceutical and pharmacological properties, enhanced therapeutic efficacy, controllable drug-targeting and reduced side effects. To further facilitate clinical translation of anti-sepsis nanomedicines, we propose that the issues involving safety, regulatory laws and cost-effectiveness should receive much more attention in the future., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Li, Wang, Li, Yan, Tan, Zeng, Ma, Zhu, Yin and Ma.)

Patients with type 2 diabetes (T2D) are more susceptible to severe respiratory viral infections, but the underlying mechanisms remain elusive. Here, we show that patients with T2D and coronavirus disease 2019 (COVID-19) infections, and influenza-infected T2D mice, exhibit defective T helper 1 (Th1) responses, which are an essential component of anti-viral immunity. This defect stems from intrinsic metabolic perturbations in CD4 + T cells driven by hyperglycemia. Mechanistically, hyperglycemia triggers mitochondrial dysfunction and excessive fatty acid synthesis, leading to elevated oxidative stress and aberrant lipid accumulation within CD4 + T cells. These abnormalities promote lipid peroxidation (LPO), which drives carbonylation of signal transducer and activator of transcription 4 (STAT4), a crucial Th1-lineage-determining factor. Carbonylated STAT4 undergoes rapid degradation, causing reduced T-bet induction and diminished Th1 differentiation. LPO scavenger ameliorates Th1 defects in patients with T2D who have poor glycemic control and restores viral control in T2D mice. Thus, this hyperglycemia-LPO-STAT4 axis underpins reduced Th1 activity in T2D hosts, with important implications for managing T2D-related viral complications., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Tsukamurella , a group of multi-drug resistant, Gram-positive, aerobic, and partially acid-fast bacteria, are emerging causes of bacterial conjunctivitis and keratitis. However, the pathogenesis of Tsukamurella keratitis is largely unknown. To address this, we used New Zealand White rabbits to develop the first eye infection model and conducted in vitro tests to study the pathogenesis mechanisms of Tsukamurella . There is increasing evidence that biofilms play a significant role in ocular infections, leading us to hypothesize that biofilm formation is crucial for effective Tsukamurella infection. In order to look for potential candidate genes which are important in biofilm formation and Tsukamurella keratitis. We performed genome sequencing of two ocular isolates, T. pulmonis -PW1004 and T. tyrosinosolvens -PW899, to identify potential virulence factors. Through in vitro and in vivo studies, we characterized their biological roles in mediating Tsukamurella keratitis. Our findings confirmed that Tsukamurella is an ocular pathogen by fulfilling Koch's postulates, and using genome sequence data, we identified tmytC, encoding a mycolyltransferase, as a crucial gene in biofilm formation and causing Tsukamurella keratitis in the rabbit model. This is the first report demonstrating the novel role of mycolyltransferase in causing ocular infections. Overall, our findings contribute to a better understanding of Tsukamurella pathogenesis and provide a potential target for treatment. Specific inhibitors targeting TmytC could serve as an effective treatment option for Tsukamurella infections.

Hepatitis E virus (HEV) variants infecting humans belong to two species: Paslahepevirus balayani (bHEV) and Rocahepevirus ratti (rat hepatitis E virus; rHEV). R. ratti is a ubiquitous rodent pathogen that has recently been recognized to cause hepatitis in humans. Transmission routes of rHEV from rats to humans are currently unknown. In this study, we examined rHEV exposure in cats and dogs to determine if they are potential reservoirs of this emerging human pathogen. Virus-like particle-based IgG enzymatic immunoassays (EIAs) capable of differentiating rHEV & bHEV antibody profiles and rHEV-specific real-time RT-PCR assays were used for this purpose. The EIAs could detect bHEV and rHEV patient-derived IgG spiked in dog and cat sera. Sera from 751 companion dogs and 130 companion cats in Hong Kong were tested with these IgG enzymatic immunoassays (EIAs). Overall, 13/751 (1.7%) dogs and 5/130 (3.8%) cats were sero-reactive to HEV. 9/751 (1.2%) dogs and 2/130 (1.5%) cats tested positive for rHEV IgG, which was further confirmed by rHEV immunoblots. Most rHEV-seropositive animals were from areas in or adjacent to districts reporting human rHEV infection. Neither 881 companion animals nor 652 stray animals carried rHEV RNA in serum or rectal swabs. Therefore, we could not confirm a role for cats and dogs in transmitting rHEV to humans. Further work is required to understand the reasons for low-level seropositivity in these animals.

Human coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 have recurrently emerged as significant pathogens, causing severe respiratory illnesses and presenting challenges to monoclonal antibody therapeutics due to their rapid evolution, particularly the diverse variants of SARS-CoV-2. In this study, we utilized "Knob-into-Hole" and "IgG-scFv" technologies to engineer bispecific antibodies (bsAbs) that target both the viral receptor and spike protein, enhancing their neutralization breadth and potency. Our bsAbs, combining anti-SARS-CoV-2 or anti-MERS-CoV antibodies with an anti-ACE2 antibody, demonstrated effective neutralization across a range of SARS-CoV-2 variants, SARS-CoV and MERS-CoV in both pseudovirus and authentic virus assays. Notably, the "IgG-scFv" bsAbs format exhibited superior binding and neutralization capabilities compared to the "Knob-into-Hole" configurations. The most effective of these, "IgG-scFv" H11B11_m336, displayed exceptional neutralization potency against a panel of 24 pseudotyped Beta-Coronaviruses, with IC 50 values ranging from 0.001-0.183 μg/mL. Overall, our findings underscore the potential of bsAbs as an effective strategy to meet the immediate challenges posed by existing and emerging pathogens, thereby enhancing global pandemic preparedness.

Background: Early in the coronavirus disease 2019 (COVID-19) pandemic, Sunda pangolins (Manis javanica) involved in the illegal wildlife trade in mainland China were identified as hosts of severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs). Although it is unconfirmed whether pangolins or other traded wildlife served as intermediate hosts for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the trafficking of pangolins presents a clear risk for transmission of viruses with zoonotic and epizootic potential regardless. We have investigated the origins of pangolin carcasses seized in Hong Kong and have evaluated their potential exposure to SARSr-CoVs, other coronaviruses, and paramyxoviruses, aiming to address a gap in our knowledge with regard to the role of wildlife trade in the maintenance and emergence of pathogens with zoonotic and epizootic potential., Results: Using a combination of virological and wildlife forensics tools, we investigated 89 Sunda pangolin carcasses seized by Hong Kong authorities during anti-smuggling operations in the territory conducted in 2013 (n = 1) and 2018 (n = 88). Swabs, organ tissues, blood, and other body fluids were collected during post-mortem examination. Two enzyme-linked immunosorbent assays (ELISAs), which employ a double-antigen sandwich format, were used to detect antibodies reactive against SARSr-CoVs. One individual was found to be seropositive with support from both methods, while five individuals exhibited a putatively seropositive result from one ELISA method. Polymerase chain reaction (PCR) screening for coronavirus and paramyxovirus ribonucleic acid (RNA) did not yield any positives. Based on genomic data, the seropositive individual was determined to have likely originated from Java, while the putatively seropositive individuals were determined to have originated from populations in Borneo, Java, and Singapore/Sumatra., Conclusions: While the role of pangolins in the evolution and ecology of SARS-CoV-2 is uncertain, our results suggest susceptibility and potential exposure of pangolins to SARSr-CoVs, occurring naturally or associated with the illegal trafficking of these animals. Complex dynamics between natural populations, traded individuals, and pathogen susceptibility complicate conclusions about the role of pangolins, as well as other host species, in the ecology of SARSr-CoVs and potentially zoonotic viruses with risk of future emergence., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

Humans experience many influenza infections over their lives, resulting in complex and varied immunological histories. Although experimental and quantitative analyses have improved our understanding of the immunological processes defining an individual's antibody repertoire, how these within-host processes are linked to population-level influenza epidemiology in humans remains unclear. Here, we used a multilevel mathematical model to jointly infer antibody dynamics and individual-level lifetime influenza A/H3N2 infection histories for 1,130 individuals in Guangzhou, China, using 67,683 haemagglutination inhibition (HI) assay measurements against 20 A/H3N2 strains from repeat serum samples collected between 2009 and 2015. These estimated infection histories allowed us to reconstruct historical seasonal influenza patterns in humans and to investigate how influenza incidence varies over time, space, and age in this population. We estimated median annual influenza infection rates to be approximately 19% from 1968 to 2015, but with substantial variation between years; 88% of individuals were estimated to have been infected at least once during the study period (2009 to 2015), and 20% were estimated to have 3 or more infections in that time. We inferred decreasing infection rates with increasing age, and found that annual attack rates were highly correlated across all locations, regardless of their distance, suggesting that age has a stronger impact than fine-scale spatial effects in determining an individual's antibody profile. Finally, we reconstructed each individual's expected antibody profile over their lifetime and inferred an age-stratified relationship between probability of infection and HI titre. Our analyses show how multi-strain serological panels provide rich information on long-term epidemiological trends, within-host processes, and immunity when analysed using appropriate inference methods, and adds to our understanding of the life course epidemiology of influenza A/H3N2., Competing Interests: DATC declares research funding from Merck, Sharp and Dohme and from Pfizer for research unrelated to this manuscript. All other authors have declared that no competing interests exist., (Copyright: © 2024 Hay et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Remdesivir (Veklury, Gilead Sciences, Foster City, CA, USA) and nirmatrelvir-ritonavir (Paxlovid, Pfizer, New York, NY, USA) were reported to improve the outcome of patients with mild-to-moderate COVID-19 symptoms. Preclinical data suggest that nirmatrelvir-ritonavir might be more effective than remdesivir alone or in combination with nirmatrelvir-ritonavir for people at high risk of severe COVID-19. We aimed to assess the safety and effectiveness of combining remdesivir and nirmatrelvir-ritonavir compared with using each drug alone for adults hospitalised with COVID-19., Methods: In this target trial emulation study, we used electronic health records of patients aged 18 years or older who received either combination treatment of nirmatrelvir-ritonavir and remdesivir or monotherapy of either drug between March 16 and Dec 31, 2022, within 5 days of hospitalisation for COVID-19 in Hong Kong. Inverse probability of treatment weighting was applied to balance baseline patient characteristics across the treatment groups. The primary outcome was all-cause mortality. Cox proportional hazards regression adjusting weighting was used to compare the risk of all-cause mortality, intensive care unit (ICU) admission, or ventilatory support for 90 days of follow-up between groups., Findings: Between March 16 and Dec 31, 2022, 18 196 participants were identified from electronic health records and assigned to receive remdesivir (n=4232), nirmatrelvir-ritonavir (n=13 656), or nirmatrelvir-ritonavir and remdesivir (n=308). By applying an inverse probability of treatment weighting, a weighted sample composed of 18 410 recipients of nirmatrelvir-ritonavir and remdesivir combination treatment, 18 178 recipients of remdesivir monotherapy, and 18 287 recipients of nirmatrelvir-ritonavir monotherapy was obtained. After a median follow-up of 84 days (IQR 45-90), risk of mortality was lower in patients who received nirmatrelvir-ritonavir monotherapy (hazard ratio [HR] 0·18 [95% CI 0·15 to 0·20]; absolute risk reduction [ARR] -16·33% [95% CI -16·98 to -15·68]) or remdesivir and nirmatrelvir-ritonavir combination therapy (HR 0·66 [95% CI 0·49 to 0·89]; ARR -6·52% [95% CI -7·29 to -5·74]) than in patients who received remdesivir monotherapy. Similar results were observed for ICU admission or ventilatory support (nirmatrelvir-ritonavir monotherapy: HR 0·09 [95% CI 0·07 to 0·11]; ARR -10·04% [95% CI -10·53 to -9·56]; combination therapy: HR 0·68 [95% CI 0·42 to 1·12]; ARR -3·24% [95% CI -3·84 to -2·64]). Compared with combination therapy, nirmatrelvir-ritonavir monotherapy was associated with lower risk of mortality (HR 0·27 [95% CI 0·20 to 0·37]; ARR -9·81% [95% CI -10·39 to -9·24]) and ICU admission or ventilatory support (HR 0·13 [95% CI 0·08 to 0·22]; ARR -6·80% [95% CI -7·22 to -6·39])., Interpretation: Our study highlighted the potential for reduced risk of mortality, ICU admission, or the need for ventilatory support in patients hospitalised with COVID-19 treated with nirmatrelvir-ritonavir as a monotherapy compared with treatment regimens based on nirmatrelvir-ritonavir and remdesivir combination therapy or remdesivir monotherapy. Further randomised controlled trials are needed to support the validity of the current results., Funding: The Health and Medical Research Fund Commissioned Research on COVID-19., Translation: For the Chinese translation of the abstract see Supplementary Materials section., Competing Interests: Declaration of interests EYFW has received research grants from the Food and Health Bureau of the Hong Kong Government, the Hong Kong Research Grants Council of the Hong Kong Government, Narcotics Division, Security Bureau of the Hong Kong Government, and National Natural Science Foundation of China, outside the submitted work. ICKW reports research funding from Amgen, Bristol Myers Squibb, Pfizer, Janssen, Bayer, GSK, Novartis, the Hong Kong Research Grants Council of the Hong Kong Government, the Hong Kong Health and Medical Research Fund, the UK National Institute for Health Research, the European Commission, and the Australia National Health and Medical Research Council; reports consulting fees from IQVIA and WHO; reports payment for expert testimony for Appeal Court of Hong Kong; is a non-executive director of Jacobson Medical in Hong Kong, Advance Data Analytics for Medical Science (in Hong Kong), Asia Medicine Regulatory Affairs Services, and OCUS Innovation (in Hong Kong, Ireland, and the UK); is a former director of Therakind in the UK, outside of the submitted work; and reports being a member of the Pharmacy and Poisons Board in Hong Kong, the Expert Committee on Clinical Events Assessment Following COVID-19 Immunisation, and the Advisory Panel on COVID-19 Vaccines of the Hong Kong Government. EWYC reports grants from the Hong Kong Research Grants Council of the Hong Kong Government, Research Fund Secretariat of the Food and Health Bureau, National Natural Science Foundation of China, Wellcome Trust, Bayer, Bristol Myers Squibb, Pfizer, Janssen, Amgen, Takeda, RGA Reinsurance Company, AstraZeneca, Narcotics Division of the Security Bureau of the Hong Kong Special Administrative Region, Innovation and Technology Commission of the Hong Kong Government, Novartis, and the Australia National Health and Medical Research Council, and honorarium from Hospital Authority, outside the submitted work. KYY is a shareholder of the Hong Kong Universal Biologicals Company and Hong Kong Universal Vaccine; reports and collaborating with Sinovac Biotech and the China National Pharmaceutical Group (Sinopharm); reports grants from Health@InnoHK, Innovation and Technology Commission of the Hong Kong Government, the Theme-Based Research Scheme (T11-709/21-N), Research Grants Council of the Hong Kong Government, Programme of Enhancing Laboratory Surveillance and Investigation of Emerging Infectious Diseases and Antimicrobial Resistance for the Hong Kong Department of Health, the China National Key Research and Development Program (projects 2021YFC0866100 and 2023YFC3041600), Sanming Project of Medicine in Shenzhen, China (SZSM201911014), the High Level-Hospital Program, Health Commission of Guangdong Province, China, and the Emergency Collaborative Project of Guangzhou Laboratory (EKPG22-01); and donations from the Shaw Foundation Hong Kong, Richard Yu and Carol Yu, Michael Seak-Kan Tong, May Tam Mak Mei Yin, Providence Foundation (in memory of the late Lui Hac Minh), Lee Wan Keung Charity Foundation, Hong Kong Sanatorium & Hospital, Hui Ming, Hui Hoy and Chow Sin Lan Charity Fund, The Chen Wai Wai Vivien Foundation, Chan Yin Chuen Memorial Charitable Foundation, Tse Kam Ming Laurence, Marina Man-Wai Lee, the Hong Kong Hainan Commercial Association South China Microbiology Research Fund, Pui-Sze Cheng, Perfect Shape Medical, Kai Chong Tong, Tse Kam Ming Laurence, Foo Oi Foundation, Betty Hing-Chu Lee, and Ping Cham So. IFNH is an advisory board member for Pfizer, MSD, GSK, Moderna, and Sinopharm; has received research grants from the Hong Kong Research Grants Council and the Hong Kong Health and Medical Research Fund; has received a travel grant from MSD and AstraZeneca to attend scientific meetings; reports a role as a co-convenor of the Expert Committee on Clinical Events Assessment Following COVID-19 Immunisation; is a member of the COVID-19 Expert Advisory Panel of the Hong Kong Government, the advisory panel on COVID-19 Vaccines of the Hong Kong Government, and the Scientific Committee on Vaccine Preventable Diseases of the Hong Kong Government; and is an Associated Editor for the journals Vaccine and Diagnostics. MHC, ART, and WMC declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

In this review, we delineate the unique set of characteristics associated with cryosphere environments (namely, ice and permafrost) which present both challenges and opportunities for studying ancient environmental microbiomes (AEMs). In a field currently reliant on several assumptions, we discuss the theoretical and empirical feasibility of recovering microbial nucleic acids (NAs) from ice and permafrost with varying degrees of antiquity. We also summarize contamination control best practices and highlight considerations for the latest approaches, including shotgun metagenomics, and downstream bioinformatic authentication approaches. We review the adoption of existing software and provide an overview of more recently published programs, with reference to their suitability for AEM studies. Finally, we summarize outstanding challenges and likely future directions for AEM research., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

SARS-CoV-2 JN.1 with an additional L455S mutation on spike when compared with its parental variant BA.2.86 has outcompeted all earlier variants to become the dominant circulating variant. Recent studies investigated the immune resistance of SARS-CoV-2 JN.1 but additional factors are speculated to contribute to its global dominance, which remain elusive until today. Here, we find that SARS-CoV-2 JN.1 has a higher infectivity than BA.2.86 in differentiated primary human nasal epithelial cells (hNECs). Mechanistically, we demonstrate that the gained infectivity of SARS-CoV-2 JN.1 over BA.2.86 associates with increased entry efficiency conferred by L455S and better spike cleavage in hNECs. Structurally, S455 altered the mode of binding of JN.1 spike protein to ACE2 when compared to BA.2.86 spike at ACE2 H34 , and modified the internal structure of JN.1 spike protein by increasing the number of hydrogen bonds with neighboring residues. These findings indicate that a single mutation (L455S) enhances virus entry in hNECs and increases immune evasiveness, which contribute to the robust transmissibility of SARS-CoV-2 JN.1. We further evaluate the in vitro and in vivo virological characteristics between SARS-CoV-2 BA.2.86/JN.1 and EG.5.1/HK.3, and identify key lineage-specific features of the two Omicron sublineages that contribute to our understanding on Omicron antigenicity, transmissibility, and pathogenicity., (© 2024. The Author(s).)

Yersinia pestis has a broad host range and has caused lethal bubonic and pneumonic plague in humans. With the emergence of multiple resistant strains and the potential for biothreat use, there is an urgent need for new therapeutic strategies that can protect populations from natural or deliberate infection. Targeting F1 has been proven to be the main strategy for developing vaccines and therapeutic antibodies, but data on anti-F1 antibodies, especially in humans, are scarce. To date, three human anti-F1 monoclonal antibodies (m252, αF1Ig2, and αF1Ig8) from naive populations have been reported. Here, we constructed an antibody library from vaccinees immunized with the plague subunit vaccine IIa by phage display. The genetic basis, epitopes, and biological functions of the obtained mAbs were assessed and evaluated in plague-challenged mice. Three human mAbs, namely, F3, F19, and F23, were identified. Their biolayer responses were 0.4, 0.6, and 0.6 nm, respectively. The dissociation constants (K D ) of the F1 antigen were 1 pM, 0.165 nM, and 1 pM, respectively. Although derived from distinct Ab lineages, that is, VH3-30-D3-10-JH4 (F3&F23) and VH3-43-D6-19-JH4 (F19), these mAbs share similar binding sites in F1 with some overlap with αF1Ig8 but are distinct from αF1Ig2. Each of them provided a significant protective effect for Balb/c mice against a 100 median lethal dose (MLD) challenge of a virulent Y. pestis strain when administered at a dose of 100 µg. No synergistic or antagonistic effects were observed among them. These mAbs are novel and excellent candidates for further drug development and use in clinical practice.IMPORTANCEIn this study, we identified three human monoclonal antibodies with a high affinity to F1 protein of Yersinia pestis . We discovered that they have relatively lower somatic hypermutations compared with antibodies, m252, αF1Ig2, and αF1Ig8, derived from the naive library reported previously. We also observed that these mAbs share similar binding sites in F1 with some overlapping with αF1Ig8 but distinct from that of αF1Ig2. Furthermore, each of them could provide complete protection for mice against a lethal dose of Yersinia pestis challenge. Our data provided new insights into the anti-F1 Ab repertories and their associated epitopes during vaccination in humans. The findings support the additional novel protective human anti-F1Abs for potential therapeutics against plaque., Competing Interests: The authors declare no conflict of interest.

Introduction: The frequent occurrence of mutations in the SARS-CoV-2 Spike (S) protein, with up to dozens of mutations, poses a severe threat to the current efficacy of authorized COVID-19 vaccines. Membrane (M) protein, which is the most abundant viral structural protein, exhibits a high level of amino acid sequence conservation. M protein ectodomain could be recognized by specific antibodies; however, the extent to which it is immunogenic and provides protection remains unclear., Methods: We designed and synthesized multiple peptides derived from coronavirus M protein ectodomains, and determined the secondary structure of specific peptides using circular dichroism (CD) spectroscopy. Enzyme-linked immunosorbent assay (ELISA) was utilized to detect IgG responses against the synthesized peptides in clinical samples. To evaluate the immunogenicity of peptide vaccines, BALB/c mice were intraperitoneally immunized with peptide-keyhole limpet hemocyanin (KLH) conjugates adjuvanted with incomplete Freund's adjuvant (IFA). The humoral and T-cell immune responses induced by peptide-KLH conjugates were assessed using ELISA and ELISpot assays, respectively. The efficacy of the S2M2-30-KLH vaccine against SARS-CoV-2 variants was evaluated in vivo using the K18-hACE2 transgenic mouse model. The inhibitory effect of mouse immune serum on SARS-CoV-2 virus replication in vitro was evaluated using microneutralization assays. The subcellular localization of the M protein was evaluated using an immunofluorescent staining method, and the Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) activity of the S2M2-30-specific monoclonal antibody (mAb) was measured using an ADCC reporter assay., Results: Seroconversion rates for ectodomain-specific IgG were observed to be high in both SARS-CoV-2 convalescent patients and individuals immunized with inactivated vaccines. To assess the protective efficacy of the M protein ectodomain-based vaccine, we initially identified a highly immunogenic peptide derived from this ectodomain, named S2M2-30. The mouse serum specific to S2M2-30 showed inhibitory effects on the replication of SARS-CoV-2 variants in vitro . Immunizations of K18-hACE2-transgenic mice with the S2M2-30-keyhole limpet hemocyanin (KLH) vaccine significantly reduced the lung viral load caused by B.1.1.7/Alpha (UK) infection. Further mechanism investigations reveal that serum neutralizing activity, specific T-cell response and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC) correlate with the specific immuno-protection conferred by S2M2-30., Discussion: The findings of this study suggest that the antibody responses against M protein ectodomain in the population most likely exert a beneficial effect on preventing various SARS-CoV-2 infections., Competing Interests: Author LD was employed by company Beijing Weimiao Biotechnology Co. Ltd. Authors LD, YT, SY, YC, CL, Z-WY, YH, and DL are inventors of the patent application related to this study Patent applicant: Hunan University; Application No.: CN202210366334.5. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Tang, Tang, Hu, Ye, Luo, Luo, Cao, Wang, Yue, Liu, Liu, Ge, Liu, Chen, Yuan and Deng.)

Background: Post-acute sequalae of COVID-19 defines a wide range of ongoing symptoms and conditions long after SARS-CoV-2 infection including respiratory diseases. The histopathological changes in the lung and underlying mechanism remain elusive., Methods: We investigated lung histopathological and transcriptional changes in SARS-CoV-2-infected male hamsters at 7, 14, 42, 84 and 120dpi, and compared with A (H1N1)pdm09 infection., Findings: We demonstrated viral residue, inflammatory and fibrotic changes in lung after SARS-CoV-2 but not H1N1 infection. The most prominent histopathological lesion was multifocal alveolar-bronchiolization observed in every SARS-CoV-2 infected hamster (31/31), from 42dpi to 120dpi. Proliferating (Ki67+) CK14+ basal cells accumulated in alveoli adjacent to bronchioles at 7dpi, where they proliferated and differentiated into SCGB1A+ club cell or Tubulin+ ciliated cells forming alveolar-bronchiolization foci. Molecularly, Notch pathway significantly upregulated with intensive Notch3 and Hes1 protein expression in alveolar-bronchiolization foci at 42 and 120dpi, suggesting Notch signaling involving the persistence of alveolar-bronchiolization. This is further demonstrated by spatial transcriptomic analysis. Intriguingly, significant upregulation of some cell-growth promoting pathways and genes such as Tubb4b, Stxbp4, Grb14 and Mlf1 were spatially overlapping with bronchiolization lesion., Interpretation: Incomplete resolution of SARS-CoV-2 infection in lung with viral residue, chronic inflammatory and fibrotic damage and alveolar-bronchiolization impaired respiratory function. Aberrant activation of CK14+ basal cells during tissue regeneration led to persistent alveolar-bronchiolization due to sustained Notch signaling. This study advances our understanding of respiratory PASC, sheds light on disease management and highlights the necessity for monitoring disease progression in people with respiratory PASC., Funding: Funding is listed in the Acknowledgements section., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The spread of emerging SARS-CoV-2 immune escape sublineages, especially JN.1 and KP.2, has resulted in new waves of COVID-19 globally. The evolving memory B cell responses elicited by the parental Omicron variants to subvariants with substantial antigenic drift remain incompletely investigated., Methods: Using the single B cell antibody cloning technology, we isolated single memory B cells, delineated the B cell receptor repertoire and conducted the pseudovirus-based assay for recovered neutralizing antibodies (NAb) screening. We analyzed the cryo-EM structures of top broadly NAbs (bnAbs) and evaluated their in vivo efficacy (golden Syrian hamster model)., Findings: By investigating the evolution of human B cell immunity, we discovered a new panel of bnAbs arising from vaccinees after Omicron BA.2/BA.5 breakthrough infections. Two lead bnAbs neutralized major Omicron subvariants including JN.1 and KP.2 with IC 50 values less than 10 ng/mL, representing ultrapotent receptor binding domain (RBD)-specific class I bnAbs. They belonged to the IGHV3-53/3-66 clonotypes instead of evolving from the pre-existing vaccine-induced IGHV1-58/IGKV3-20 bnAb ZCB11. Despite sequence diversity, they targeted previously unrecognized, highly conserved conformational epitopes in the receptor binding motif (RBM) for ultrapotent ACE2 blockade. The lead bnAb ZCP3B4 not only protected the lungs of hamsters intranasally challenged with BA.5.2, BQ.1.1 and XBB.1.5 but also prevented their contact transmission., Interpretation: Our findings demonstrated that class I bnAbs have evolved an ultrapotent mode of action protecting against highly transmissible and broad Omicron escape variants, and their epitopes are potential targets for novel bnAbs and vaccine development., Funding: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section., Competing Interests: Declaration of interests The other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Competing Interests: Declaration of interests The authors declare no competing interests.

Memory T cells play a key role in immune protection against cancer. Vaccine-induced tissue-resident memory T (TRM) cells in the lung have been shown to protect against lung metastasis. Identifying the source of lung TRM cells can help to improve strategies, preventing tumor metastasis. Here, we found that a prime-boost vaccination approach using intramuscular DNA vaccine priming, followed by intranasal live-attenuated influenza-vectored vaccine (LAIV) boosting induced higher frequencies of lung CD8+ TRM cells compared with other vaccination regimens. Vaccine-induced lung CD8+ TRM cells, but not circulating memory T cells, conferred significant protection against metastatic melanoma and mesothelioma. Central memory T (TCM) cells induced by the DNA vaccination were major precursors of lung TRM cells established after the intranasal LAIV boost. Single-cell RNA sequencing analysis indicated that transcriptional reprogramming of TCM cells for differentiation into TRM cells in the lungs started as early as day 2 post the LAIV boost. Intranasal LAIV altered the mucosal microenvironment to recruit TCM cells via CXCR3-dependent chemotaxis and induced CD8+ TRM-associated transcriptional programs. These results identified TCM cells as the source of vaccine-induced CD8+ TRM cells that protect against lung metastasis. Significance: Prime-boost vaccination shapes the mucosal microenvironment and reprograms central memory T cells to generate lung resident memory T cells that protect against lung metastasis, providing insights for the optimization of vaccine strategies., (©2024 The Authors; Published by the American Association for Cancer Research.)

Joint pain and osteoarthritis can occur as coronavirus disease 2019 (COVID-19) sequelae after infection. However, little is known about the damage to articular cartilage. Here we illustrate knee joint damage after wild-type, Delta and Omicron variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vivo. Rapid joint injury with cystic lesions at the osteochondral junction was observed in two patients with post-COVID osteoarthritis and recapitulated in a golden Syrian hamster model. SARS-CoV-2-activated endothelin-1 signalling increased vascular permeability and caused viral spike proteins leakage into the subchondral bone. Osteoclast activation, chondrocyte dropout and cyst formation were confirmed histologically. The US Food and Drug Administration-approved endothelin receptor antagonist, macitentan, mitigated cystic lesions and preserved chondrocyte number in the acute phase of viral infection in hamsters. Delayed macitentan treatment at post-acute infection phase alleviated chondrocyte senescence and restored subchondral bone loss. It is worth noting that it could also attenuate viral spike-induced joint pain. Our work suggests endothelin receptor blockade as a novel therapeutic strategy for post-COVID arthritis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

An extensive mosquito survey was carried out in Hong Kong from September to October 2022, employing a variety of collection methods. Specimens were identified using a combination of morphology and mitochondrial cytochrome C oxidase subunit 1 (COI) barcode sequences. Twenty-nine species, including three new records, i.e., Culex bicornutus (Theobald), Culex cinctellus Edwards, and Lutzia chiangmaiensis Somboon & Harbach, were collected. Phylogenetic analysis of COI sequences of Culex annulus Theobald and Culex vishnui Theobald collected in Hong Kong and elsewhere revealed that the sequences of the two nominal species are genetically very similar and are included in the same clade. Consequently, the synonymy of Cx. annulus with Cx. vishnui is reinstated. Lutzia halifaxii (Theobald) is removed from the list of species in Hong Kong and is replaced with Lutzia vorax Edwards, the identification of which is confirmed in the present study. The record of Culex spiculothorax Bram recorded in Hong Kong is replaced with the senior synonym Culex sasai Kano, Nitahara & Awaya. The occurrence of Anopheles fluviatilis James and Aedes aegypti (Linnaeus) is discussed. Finally, an updated checklist of the mosquitoes of Hong Kong, which now includes 76 species representing 14 genera, is provided, with notation of those species that vector pathogens of human diseases., (© The Author(s) 2024. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Gold standard detection of SARS-CoV-2 by reverse transcription quantitative PCR (RT-qPCR) can achieve ultrasensitive viral detection down to a few RNA copies per sample. Yet, the lengthy detection and labor-intensive protocol limit its effectiveness in community screening. In view of this, a structural switching electrochemical aptamer-based biosensor (E-AB) targeting the SARS-CoV-2 nucleocapsid (N) protein was developed. Four N protein-targeting aptamers were characterized on an electrochemical cell configuration using square wave voltammetry (SWV). The sensor was investigated in an artificial saliva matrix optimizing the aptamer anchoring orientation, SWV interrogation frequency, and target incubation time. Rapid detection of the N protein was achieved within 5 min at a low nanomolar limit of detection (LOD) with high specificity. Specific N protein detection was also achieved in simulated positive saliva samples, demonstrating its feasibility for saliva-based rapid diagnosis. Further research will incorporate novel signal amplification strategies to improve sensitivity for early diagnosis.

Human endogenous retroviruses (HERVs) are widely recognized as the result of exogenous retroviruses infecting the ancestral germline, stabilizing integration and vertical transmission during human genetic evolution. To date, endogenous retroviruses (ERVs) appear to have been selected for human physiological functions with the loss of retrotransposable capabilities. ERV elements were previously regarded as junk DNA for a long time. Since then, the aberrant activation and expression of ERVs have been observed in the development of many kinds of human diseases, and their role has been explored in a variety of human disorders such as cancer. The results show that specific ERV elements play respective crucial roles. Among them, long non-coding RNAs (lncRNAs) transcribed from specific long-terminal repeat regions of ERVs are often key factors. lncRNAs are over 200 nucleotides in size and typically bind to DNA, RNA, and proteins to perform biological functions. Dysregulated lncRNAs have been implicated in a variety of diseases. In particular, studies have shown that the aberrant expression of some ERV-derived lncRNAs has a tumor-suppressive or oncogenic effect, displaying significant functional bidirectionality. Therefore, theses lncRNAs have a promising future as novel biomarkers and therapeutic targets to explore the concise relationship between ERVs and cancers. In this review, we first summarize the role of ERV-derived lncRNAs in physiological regulation, mainly including immunomodulation, the maintenance of pluripotency, and erythropoiesis. In addition, pathological regulation examples of their aberrant activation and expression leading to carcinogenesis are highlighted, and specific mechanisms of occurrence are discussed.

Developing an effective Staphylococcus aureus ( S. aureus ) vaccine has been a challenging endeavor, as demonstrated by numerous failed clinical trials over the years. In this study, we formulated a vaccine containing a highly conserved moonlighting protein, the pyruvate dehydrogenase complex E2 subunit (PDHC), and showed that it induced strong protective immunity against epidemiologically relevant staphylococcal strains in various murine disease models. While antibody responses contributed to bacterial control, they were not essential for protective immunity in the bloodstream infection model. Conversely, vaccine-induced systemic immunity relied on γδ T cells. It has been suggested that prior S. aureus exposure may contribute to the reduction of vaccine efficacy. However, PDHC-induced protective immunity still facilitated bacterial clearance in mice previously exposed to S. aureus . Collectively, our findings indicate that PDHC is a promising serotype-independent vaccine candidate effective against both methicillin-sensitive and methicillin-resistant S. aureus isolates., Competing Interests: Competing interests statement:The authors declare no competing interest.

There are only eight approved small molecule antiviral drugs for treating COVID-19. Among them, four are nucleotide analogues (remdesivir, JT001, molnupiravir, and azvudine), while the other four are protease inhibitors (nirmatrelvir, ensitrelvir, leritrelvir, and simnotrelvir-ritonavir). Antiviral resistance, unfavourable drug‒drug interaction, and toxicity have been reported in previous studies. Thus there is a dearth of new treatment options for SARS-CoV-2. In this work, a three-tier cell-based screening was employed to identify novel compounds with anti-SARS-CoV-2 activity. One compound, designated 172 , demonstrated broad-spectrum antiviral activity against multiple human pathogenic coronaviruses and different SARS-CoV-2 variants of concern. Mechanistic studies validated by reverse genetics showed that compound 172 inhibits the 3-chymotrypsin-like protease (3CLpro) by binding to an allosteric site and reduces 3CLpro dimerization. A drug synergistic checkerboard assay demonstrated that compound 172 can achieve drug synergy with nirmatrelvir in vitro . In vivo studies confirmed the antiviral activity of compound 172 in both Golden Syrian Hamsters and K18 humanized ACE2 mice. Overall, this study identified an alternative druggable site on the SARS-CoV-2 3CLpro, proposed a potential combination therapy with nirmatrelvir to reduce the risk of antiviral resistance and shed light on the development of allosteric protease inhibitors for treating a range of coronavirus diseases., Competing Interests: The authors have no conflict to declare., (© 2024 The Authors.)

The mortality and hospitalization rate by COVID-19 dropped significantly currently, but its seasonal outbreaks make antiviral treatment still vital. The mortality and hospitazation rate by COVID-19 dropped significantly currently, but its seasonal ourbreaks make antiviral treatment still vital. In our study, syrian golden hamsters were treated with molnupiravir and interferons (IFNs) after SARS-CoV-2 infection. Their weight changes, pathological changes, virus replication and inflammation levels were evaluated. In the IFNs single treatment, only IFN-α group reduced viral load (p < 0.05) and virus titer in hamster lungs. The TNF-α expression decreased significantly in both IFNs treatment at 2dpi. Histological and immunofluorescence results showed lung damage in the IFNs groups were milder at 4dpi. In the molnupiravir/IFN-α combination treatment, weight loss and virus replication in lung were significantly decreased in the mono-molnupiravir group and combination group (p < 0.05), the expression of IL-6, TNF-α, IL-1β and MIP-1α also decreased significantly (p < 0.05), but the combination treatment was not more effective than the mono-molnupiravir treatment. Histological and immunofluorescence results showed the lung damage and inflammation in mono-molnupiravir and combination groups were milder. In summary, IFNs treatment had anti-inflammatory effect against SARS-CoV-2, only IFN-α showed a weak antiviral effect. Molnupiravir/IFN-α combination treatment was effective against SARS-CoV-2 but was not superior to mono-molnupiravir treatment. IFN-α could be considered for immunocompromised patients to stimulate and activate early immune responses., (© 2024 Wiley Periodicals LLC.)

Viruses often manipulate ubiquitination pathways to facilitate their replication and pathogenesis. CUL2 ZYG11B known as the substrate receptor of cullin-2 RING E3 ligase, is bound by SARS-CoV-2 ORF10 to increase its E3 ligase activity, leading to degradation of IFT46, a protein component of the intraflagellar transport (IFT) complex B. This results in dysfunctional cilia, which explains certain symptoms that are specific to COVID-19. However, the precise molecular mechanism of how ORF10 recognizes CUL2 ZYG11B remains unknown. Here, we determined the crystal structure of CUL2 ZYG11B complexed with the N-terminal extension (NTE) of SARS-CoV-2 ORF10 (2.9 Å). The structure reveals that the ORF10 N-terminal heptapeptide (NTH) mimics the Gly/N-degron to bind CUL2 ZYG11B . Mutagenesis studies identified key residues within ORF10 that are key players in its interaction with CUL2 ZYG11B both in ITC assay and in vivo cells. In addition, we prove that enhancement of CUL2 ZYG11B activity for IFT46 degradation by which ORF10-mediated correlates with the binding affinity between ORF10 and CUL2 ZYG11B . Finally, we used a Global Protein Stability system to show that the NTH of ORF10 mimics the Gly/N-degron motif, thereby binding competitively to CUL2 ZYG11B and inhibiting the degradation of target substrates bearing the Gly/N-degron motif. Overall, this study sheds light on how SARS-CoV-2 ORF10 exploits the ubiquitination machinery for proteasomal degradation, and offers valuable insights for optimizing PROTAC-based drug design based on NTH CUL2 ZYG11B interaction, while pinpointing a promising target for the development of treatments for COVID-19., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Authors.)

Emerging mycotoxins enniatins (ENNs) and beauvericin (BEA) pose potential health risks to humans through dietary exposure. However, research into their mechanisms of toxicity is limited, with a lack of comprehensive toxicological data. This study investigates from a hepatic lipid metabolism perspective, establishing a more precise and reliable 3D HepaRG hepatocyte spheroid model as an alternative for toxicity assessment. Utilizing physiological indices, histopathological analyses, lipidomics, and molecular docking techniques, it comprehensively elucidates the effects of ENNs and BEA on hepatic lipid homeostasis and their molecular toxicological mechanisms. Our findings indicate that ENNs and BEA impact cellular viability and biochemical functions, significantly altering lipid metabolism pathways, particularly those involving glycerophospholipids and sphingolipids. Molecular docking has demonstrated strong binding affinity of ENNs and BEA with key enzymes in lipid metabolism such as Peroxisome Proliferator-Activated Receptor α (PPARα) and Cytosolic Phospholipase A2 (cPLA2), revealing the mechanistic basis for their hepatotoxic effects and potential to impair liver function and human health. These insights enhance our understanding of the potential hepatotoxicity of such fungal toxins and lay a foundation for the assessment of their health risks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Coronaviruses remodel the intracellular host membranes during replication, forming double-membrane vesicles (DMVs) to accommodate viral RNA synthesis and modifications 1,2 . SARS-CoV-2 non-structural protein 3 (nsp3) and nsp4 are the minimal viral components required to induce DMV formation and to form a double-membrane-spanning pore, essential for the transport of newly synthesized viral RNAs 3-5 . The mechanism of DMV pore complex formation remains unknown. Here we describe the molecular architecture of the SARS-CoV-2 nsp3-nsp4 pore complex, as resolved by cryogenic electron tomography and subtomogram averaging in isolated DMVs. The structures uncover an unexpected stoichiometry and topology of the nsp3-nsp4 pore complex comprising 12 copies each of nsp3 and nsp4, organized in 4 concentric stacking hexamer rings, mimicking a miniature nuclear pore complex. The transmembrane domains are interdigitated to create a high local curvature at the double-membrane junction, coupling double-membrane reorganization with pore formation. The ectodomains form extensive contacts in a pseudo-12-fold symmetry, belting the pore complex from the intermembrane space. A central positively charged ring of arginine residues coordinates the putative RNA translocation, essential for virus replication. Our work establishes a framework for understanding DMV pore formation and RNA translocation, providing a structural basis for the development of new antiviral strategies to combat coronavirus infection., (© 2024. The Author(s).)

Human parainfluenza viruses (HPIVs) are the leading causes of acute respiratory tract infections (ARTIs), particularly in children. During the COVID-19 pandemic, non-pharmaceutical interventions (NPIs) significantly influenced the epidemiology of respiratory viruses. This study analyzed 19,339 respiratory specimens from pediatric patients with ARTIs to detect HPIVs using PCR or tNGS, focusing on the period from 2021 to 2023. HPIVs were identified in 1395 patients (7.21%, 1395/19,339), with annual detection rates of 6.86% (303/4419) in 2021, 6.38% (331/5188) in 2022, and 7.82% (761/9732) in 2023. Notably, both the total number of tests and HPIV-positive cases increased in 2023 compared to 2021 and 2022. Seasonal analysis revealed a shift in HPIV prevalence from winter and spring in 2021-2022 to spring and summer in 2023. Most HPIV-positive cases were in children aged 0-7 years, with fewer infections among those aged 7-18 years. Since June 2022, HPIV-3 has been the most prevalent serotype (59.55%, 524/880), whereas HPIV-2 had the lowest proportion (0.80%, 7/880). The proportions of HPIV-1 (24.89%, 219/880) and HPIV-4 (15.45%, 136/880) were similar. Additionally, the incidence of co-infections with other common respiratory pathogens has increased since 2021. This study highlights rising HPIV detection rates post-COVID-19 and underscores the need for continuous surveillance of HPIVs to inform public health strategies for future epidemic seasons.

Due to the increasing resistance of aerobic and facultative anaerobic Gram-negative rods, ceftazidime-avibactam and ceftolozane-tazobactam have been launched in the market in the last few years. In this study, we analyzed the susceptibility pattern of the major aerobic and facultative anaerobic Gram-negative rods in Hong Kong for ceftazidime-avibactam, ceftolozane-tazobactam, four other broad-spectrum antibiotics commonly used in Hong Kong and colistin. For 300 isolates collected from January to December 2021, non-ESBL-producing Enterobacterales, ESBL-producing Enterobacterales and Pseudomonas aeruginosa were highly susceptible to ceftazidime-avibactam (all 100%) and ceftolozane-tazobactam (98.7%, 99.7% and 94.3%). For 32 archived ESBL-producing Klebsiella pneumoniae isolates collected between January 2014 and March 2023, all were susceptible to ceftazidime-avibactam and ceftolozane-tazobactam. For 101 archived carbapenemase-producing Enterobacterales, their susceptibilities to ceftazidime-avibactam and ceftolozane-tazobactam varied depending on the type of carbapenemase produced. Both had high activities against OXA-producing strains (97.1% and 76.5%, respectively) but were 100% resistant for NDM-producing and NDM+OXA-producing strains. All KPC-producing strains were susceptible to ceftazidime-avibactam but resistant to ceftolozane-tazobactam. Ceftazidime-avibactam and ceftolozane-tazobactam are good alternatives for the management of infections caused by ESBL-producing Enterobacterales and selective strains of carbapenemase-producing Enterobacterales in Hong Kong.

We determined the dengue virus (DENV) serotypes and genotypes in archived serum samples that were collected during the 2014-2016 and 2021 dengue outbreaks in Dire Dawa City and the Somali region in Ethiopia. DENV serotype 1 (DENV-1) was predominant followed by DENV serotype 2 (DENV-2). Thirteen of the DENV-1 strains were assigned to Genotype-I, while the remaining two were found to be Genotype-III. All three DENV-2 strains were assigned the Cosmopolitan Genotype. The DENV strains responsible for the outbreaks are genetically closely related to the DENV strains that circulated in neighboring and Asian countries. The findings also showed continued local transmission of a monophyletic lineage and a co-circulation of DENV-1 and DENV-2 during the outbreaks. There is a need to strengthen DENV genomic surveillance capacity for the early detection of circulating serotypes, and prevent devastating consequences of future outbreaks due to the co-circulation of different serotypes.

Background: This study explored neutralizing IgG antibody levels against COVID-19 decline over time post-vaccination. We conducted this prospective cohort study to investigate the function of gut microbiota in the host immune response following three doses of BNT162b2., Methods: Subjects who received three doses of BNT162b2 were recruited from three centers in Hong Kong. Blood samples were obtained before the first dose and at the one-year timepoint for IgG ELISA to determine the level of neutralizing antibody (NAb). The primary outcome was a high immune response (NAb > 600 AU/mL). We performed shotgun DNA metagenomic sequencing on baseline fecal samples to identify bacterial species and metabolic pathways associated with high immune response using linear discriminant analysis effect size analysis., Results: A total of 125 subjects were recruited (median age: 52 years [IQR: 46.2-59.0]; male: 43 [34.4%]), and 20 were regarded as low responders at the one-year timepoint. Streptococcus parasanguinis (log 10 LDA score = 2.38, p = 0.003; relative abundance of 2.97 × 10 -5 vs. 0.03%, p = 0.001), Bacteroides stercoris (log 10 LDA score = 4.29, p = 0.024; relative abundance of 0.14% vs. 2.40%, p = 0.014) and Haemophilus parainfluenzae (log 10 LDA score = 2.15, p = 0.022; relative abundance of 0.01% vs. 0, p = 0.010) were enriched in low responders. Bifidobacterium pseudocatenulatum (log 10 LDA score = 2.99, p = 0.048; relative abundance of 0.09% vs. 0.36%, p = 0.049) and Clostridium leptum (log 10 LDA score = 2.38, p = 0.014; relative abundance of 1.2 × 10 -5 % vs. 0, p = 0.044) were enriched in high responders. S. parasanguinis was negatively correlated with the superpathway of pyrimidine ribonucleotides de novo biosynthesis (log 10 LDA score = 2.63), which contributes to inflammation and antibody production. H. parainfluenzae was positively correlated with pathways related to anti-inflammatory processes, including the superpathway of histidine, purine, and pyrimidine biosynthesis (log 10 LDA score = 2.14)., Conclusion: Among three-dose BNT162b2 recipients, S. parasanguinis , B. stercoris and H. parainfluenzae were associated with poorer immunogenicity at one year, while B. pseudocatenulatum and C. leptum was associated with a better response.

Background: COVID-19 can cause cardiac complications and the latter are associated with poor prognosis and increased mortality. SARS-CoV-2 variants differ in their infectivity and pathogenicity, but how they affect cardiomyocytes (CMs) is unclear., Methods: The effects of SARS-CoV-2 variants were investigated using human induced pluripotent stem cell-derived (hiPSC-) CMs in vitro and Golden Syrian hamsters in vivo., Results: Different variants exhibited distinct tropism, mechanism of viral entry and pathology in the heart. Omicron BA.2 most efficiently infected and injured CMs in vitro and in vivo, and induced expression changes consistent with increased cardiac dysfunction, compared to other variants tested. Bioinformatics and upstream regulator analyses identified transcription factors and network predicted to control the unique transcriptome of Omicron BA.2 infected CMs. Increased infectivity of Omicron BA.2 is attributed to its ability to infect via endocytosis, independently of TMPRSS2, which is absent in CMs., Conclusions: In this study, we reveal previously unknown differences in how different SARS-CoV-2 variants affect CMs. Omicron BA.2, which is generally thought to cause mild disease, can damage CMs in vitro and in vivo. Our study highlights the need for further investigations to define the pathogenesis of cardiac complications arising from different SARS-CoV-2 variants., (© 2024. The Author(s).)

Introduction: Emerging severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 causes a global health disaster and pandemic. Seeking effective anti-pan-CoVs drugs benefit critical illness patients of coronavirus disease 2019 (COVID-19) but also may play a role in emerging CoVs of the future., Objectives: This study tested the hypothesis that alisol B 23-acetate could be a viral entry inhibitor and would have proinflammatory inhibition for COVID-19 treatment., Methods: SARS-CoV-2 and its variants infected several cell lines were applied to evaluate the anti-CoVs activities of alisol B 23-aceate in vitro. The effects of alisol B 23-acetate on in vivo models were assessed by using SARS-CoV-2 and its variants challenged hamster and human angiotensin-converting enzyme 2 (ACE2) transgenic mice. The target of alisol B 23-acetate to ACE2 was analyzed using hydrogen/deuterium exchange (HDX) mass spectrometry (MS)., Results: Alisol B 23-acetate had inhibitory effects on different species of coronavirus. By using HDX-MS, we found that alisol B 23-acetate had inhibition potency toward ACE2. In vivo experiments showed that alisol B 23-acetate treatment remarkably decreased viral copy, reduced CD4 + T lymphocytes and CD11b + macrophages infiltration and ameliorated lung damages in the hamster model. In Omicron variant infected human ACE2 transgenic mice, alisol B 23-acetate effectively alleviated viral load in nasal turbinate and reduced proinflammatory cytokines interleukin 17 (IL17) and interferon γ (IFNγ) in peripheral blood. The prophylactic treatment of alisol B 23-acetate by intranasal administration significantly attenuated Omicron viral load in the hamster lung tissues. Moreover, alisol B 23-acetate treatment remarkably inhibited proinflammatory responses through mitigating the secretions of IFNγ and IL17 in the cultured human and mice lymphocytes in vitro., Conclusion: Alisol B 23-acetate could be a promising therapeutic agent for COVID-19 treatment and its underlying mechanisms might be attributed to viral entry inhibition and anti-inflammatory activities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

The unprecedentedly large size of the global SARS-CoV-2 phylogeny makes any computation on the tree difficult. Lineage identification (e.g. the PANGO nomenclature for SARS-CoV-2) and assignment are key to track the virus evolution. It requires annotating clade roots of lineages to unlabeled ancestral nodes in a phylogenetic tree. Then the lineage labels of descendant samples under these clade roots can be inferred to be the corresponding lineages. This is the ancestral lineage annotation problem, and matUtils (a package in pUShER) and PastML are commonly used methods. However, their computational tractability is a challenge and their accuracy needs further exploration in huge SARS-CoV-2 phylogenies. We have developed an efficient and accurate method, called "F1ALA", that utilizes the F1-score to evaluate the confidence with which a specific ancestral node can be annotated as the clade root of a lineage, given the lineage labels of a set of taxa in a rooted tree. Compared to these methods, F1ALA achieved roughly an order of magnitude faster yet with ∼12% of their memory usage when annotating 2277 PANGO lineages in a phylogeny of 5.26 million taxa. F1ALA allows real-time lineage tracking to be performed on a laptop computer. F1ALA outperformed matUtils (pUShER) with statistical significance, and had comparable accuracy to PastML in tests on empirical and simulated data. F1ALA enables a tree refinement by pruning taxa with inconsistent labels to their closest annotation nodes and re-inserting them back to the pruned tree to improve a SARS-CoV-2 phylogeny with both higher log-likelihood and lower parsimony score. Given the ultrafast speed and high accuracy, we anticipated that F1ALA will also be useful for large phylogenies of other viruses. Codes and benchmark datasets are publicly available at https://github.com/id-bioinfo/F1ALA., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)

The poor prognosis observed in elderly individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a serious clinical burden and the underlying mechanism is unclear, which necessities detailed investigation of disease characteristics and research for efficient countermeasures. To simulate lethal coronavirus disease 2019 (COVID-19) in senescent human patients, 80-week-old male hamsters are intranasally inoculated with different doses of SARS-CoV-2 Omicron BA.5 variant. Exposure to a low dose of the Omicron BA.5 variant results in early activation of the innate immune response, followed by rapid viral clearance and minimal lung damage. However, a high dose of BA.5 results in impaired interferon signaling, cytokine storm, uncontrolled viral replication, and severe lung injury. To decrease viral load and reverse the deterioration of COVID-19, a new bio-mimic decoy called CoVR-MV is used as a preventive or therapeutic agent. Administration of CoVR-MV as a preventive or therapeutic intervention in the early stages of infection can effectively suppress viral load, regulate the immune response, and rescue animals from death and critical illness. These findings underscore the risk associated with SARS-CoV-2 Omicron BA.5 exposure in senescent hamsters and highlight the importance of early intervention to prevent disease progression., Competing Interests: The authors declare no conflict of interests., (© 2024 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.)

Although vaccination with inactivated vaccines is a popular preventive method against pseudorabies virus (PRV) infection, inactivated vaccines have poor protection efficiency because of their weak immunogenicity. The development of an effective adjuvant is urgently needed to improve the efficacy of inactivated PRV vaccines. In this study, a promising nanocomposite adjuvant named as MIL@A-SW01-C was developed by combining polyacrylic acid-coated metal-organic framework MIL-53(Al) (MIL@A) and squalene (oil)-in-water emulsion (SW01) and then mixing it with a carbomer solution. One part of the MIL@A was loaded onto the oil/water interface of SW01 emulsion via hydrophobic interaction and coordination, while another part was dispersed in the continuous water phase using carbomer. MIL@A-SW01-C showed good biocompatibility, high PRV (antigen)-loading capability, and sustained antigen release. Furthermore, the MIL@A-SW01-C adjuvanted PRV vaccine induced high specific serum antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response compared with commercial adjuvants, such as alum and biphasic 201. In the mouse challenge experiment, two- and one-shot vaccinations resulted in survival rates of 73.3 % and 86.7 %, respectively. After one-shot vaccination, the host animal pigs were also challenged with wild PRV. A protection rate of 100 % was achieved, which was much higher than that observed with commercial adjuvants. This study not only establishes the superiority of MIL@A-SW01-C composite nanoadjuvant for inactivated PRV vaccine in mice and pigs but also presents an effective method for developing promising nanoadjuvants. STATEMENT OF SIGNIFICANCE: We have developed a nanocomposite of MIL-53(Al) and oil-in-water emulsion (MIL@A-SW01-C) as a promising adjuvant for the inactivated PRV vaccines. MIL@A-SW01-C has good biocompatibility, high PRV (antigen) loading capability, and prolonged antigen release. The developed nanoadjuvant induced much higher specific IgG antibody titers, increased splenocyte proliferation and cytokine secretion, and a more balanced Th1/Th2 immune response than commercial adjuvants alum and biphasic 201. In mouse challenge experiments, survival rates of 73.3 % and 86.7 % were achieved from two-shot and one-shot vaccinations, respectively. At the same time, a protection rate of 100 % was achieved with the host animal pigs challenged with wild PRV., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)