RNA viruses, exemplified by the COVID-19 pandemic, pose a significant threat to global health. Their rapid mutation and host adaptability highlight the need for advanced tools for efficient viral studies and timely countermeasure development. Imaging technologies, such as cryo-electron microscopy and super-resolution microscopy, have been pivotal in advancing our understanding of viral structures, infection mechanisms, and virus-host interactions. However, each technique has limitations in the field of view or resolution. Recent advancements have focused on developing integrated multiscale imaging to better understand RNA virus pathogenesis. In this review, we examine recent progress in RNA virus imaging across molecular, cellular, and tissue scales, including cryo-electron tomography and correlative multiscale imaging, which link structural mapping with functional insights.

Background Rodent-borne pathogens pose important global health risks, yet national-scale assessments linking host-pathogen ecology with disease dynamics remain limited, particularly in China. Methods We compiled global (>29,000 records) and China-specific (2235 records; 1950–2023) rodent-pathogen datasets to characterize host-pathogen networks, identify hyperreservoirs, and quantify prevalence heterogeneity using hierarchical meta-regression. We applied a two-layer framework integrating 1-km host suitability surfaces (stacking ensemble of boosted regression trees and random forest models) with province-level disease inference using generalized linear models for hemorrhagic fever with renal syndrome (HFRS), leptospirosis, and plague. Human exposure was estimated by overlaying suitability with gridded population data. Results Globally, 116 pathogens of concern were identified across 206 host species, including 30 spillover-risk viruses and 34 hyperreservoirs. Meta-regression identified sample source as the only consistently robust moderator of prevalence heterogeneity; other moderators (e.g., rodent family, region, and habitat type) showed inconsistent or non-robust associations. Host suitability was positively associated with HFRS incidence (incidence rate ratio = 1.36, P < 0.01) but negatively or not significantly associated with leptospirosis and plague. In China, the three diseases showed contrasting suitability patterns, with approximately 404 million people (29.3%) residing in areas with high suitability for at least one disease. Conclusions Our analysis elucidates disease-specific ecological drivers and identifies spatial priorities to inform targeted One Health surveillance and integrated interventions.

Merbecovirus is a subgenus of betacoronaviruses and exhibits high genetic diversity with a capacity for cross-species transmission. However, beyond Middle East respiratory syndrome coronavirus (MERS-CoV), our knowledge of the ecology and pathogenic potential of these viruses remains limited. Merbecoviruses were once thought to rely exclusively on dipeptidyl peptidase 4 for cell entry, but recent discoveries have revealed that several members can also engage with angiotensin-converting enzyme 2 or aminopeptidase N, expanding their receptor repertoire and potential host range. Here we summarize recent advances in understanding of the receptor usage of merbecoviruses and examine how these insights inform pandemic preparedness and risk assessment. We discuss the development of targeted diagnostics, broad-spectrum antivirals and vaccines, including pan-coronavirus strategies. Together, these advances provide a foundation for predictive surveillance and rational countermeasure design, enabling earlier detection and more effective containment of future merbecovirus spillover events before they escalate into epidemics.

Abstract Emerging and re-emerging zoonotic viruses pose enormous challenges to public health worldwide. As an important livestock animal, pigs play a vital role in the evolution and spread of many zoonotic viruses. Hence, with the development of globalization and large-scale intensive farming, close human-pig contact increases the threat of zoonotic virus transmission. In this review, to facilitate disease prevention and control efforts, we summarized the prevalence and transmission characteristics of zoonotic viruses associated with pigs, such as influenza virus, coronavirus, and pseudorabies virus. Additionally, we emphasized novel detection techniques including rapid diagnostic tests, biosensor-based detection technology, high-throughput sequencing, and systematic viral epitope scanning. These techniques are instrumental in enabling cost-effective and convenient rapid detection procedures for broader implementation across diverse regions for effective surveillance of viral epidemics. To enhance virus surveillance capabilities and improve strategies for disease prevention in pigs, the improvement of our understanding of viral transmission modes combined with advancements in diagnostic technology is necessary.

The natural host for avian influenza virus (AIV) is waterfowl. However, certain subtypes have breached species barriers, causing epizootics in many avian and mammalian species with occasional zoonotic infections in humans. The ongoing spread of highly pathogenic avian influenza (HPAI) A(H5N1) poses a significant and growing public health threat. Here, we discuss recent advances in viral detection and characterization technologies and their integration into the diagnostics and surveillance of AIV within a “One Health” framework.

Alphaviruses are mosquito-transmitted viruses that cause severe zoonotic diseases. Their envelope glycoproteins, E1 and E2, undergo cysteine acylation, a process critical for virus infection but previously undefined mechanistically. Using the Getah virus as a model, we found that E1 is acylated at Cys433 in the endoplasmic reticulum (ER), a modification beneficial for virus entry. E2 follows a unique stepwise acylation pattern: Cys385 is acylated in the ER, while Cys395, Cys415, and Cys416 undergo interdependent acylation in the Golgi. Palmitoylation of E2 Cys415/416 proved essential for budding. Acylation also facilitated cholesterol incorporation into virions independently of lipid rafts. A small interfering RNA (siRNA) screen identified distinct acyltransferases for E1 and compartment-specific enzymes for E2. Depletion of Golgi-localized Asp-His-His-Cys (DHHC)11, which modifies Cys415/416, significantly inhibited replication of multiple alphaviruses. This study establishes the spatiotemporal model of alphavirus glycoprotein acylation and identifies DHHC11 as a conserved target for pan-alphavirus therapeutics, with potential implications for reducing the public health burden of alphavirus infections.

ABSTRACT Japanese encephalitis is a viral disease caused by the Japanese encephalitis virus (JEV), primarily affecting rural areas of Asia and western Pacific region. China remains one of the main epicenters, experiencing a significant burden of human and animal cases despite vaccination efforts. The ecology of this arbovirus is complex, involving Culex mosquitoes as primary vectors, wading birds as natural reservoirs, and pigs as amplifying hosts. Given the virus’s epidemiological importance in China, combined with the country’s expanding pig farming industry and diverse climates, investigating the virus spread and its environmental drivers is needed to address its persistent burden. In this study, we conducted phylogeographic analyses by combining publicly available JEV envelope gene sequences from China and other regions. Our reconstructions revealed multiple introduction events leading to various circulating JEV clades in China, with one predominant clade. Additionally, our analyses showed a diffusion capacity of JEV exceeding previous estimates for co-circulating arboviruses. These differences could be attributed to pig trade or bird migration, calling for further investigations into the drivers of JEV spread. IMPORTANCE Japanese encephalitis virus (JEV) is the cause of Japanese encephalitis, a significant health concern in China. Despite being one of the most studied mosquito-borne viruses, no previous studies have combined genomic and geographic data to investigate the spatial epidemiology and dispersal capacity of the virus. In this study, we analyzed genomic, geographic, and environmental data to trace the dispersal history of JEV in China and explore the environmental factors influencing its distribution. Our findings show that JEV circulates predominantly in areas with higher temperatures, dense human and pig populations, and favorable conditions for Culex mosquitoes. Notably, our analyses showed a higher diffusion capacity of JEV compared to co-circulating viruses, possibly driven by factors like pig trade and bird migration. Our analysis calls for improved genomic surveillance and establishes a baseline for future studies on the effects of climate change, agricultural practices, and bird migration on JEV circulation.

Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide. Genome structure of influenza D virus (IDV) is identical to that of influenza C virus (ICV), and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology. To date, the prevalence of ICV and IDV in China is unclear, but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential. To efficiently monitor ICV and IDV, it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research. A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed. TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed. This method exhibited good specificity and sensitivity, and the detection limit reached 1 × 10 1 copies/μL of plasmid standards of each pathogen. Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method. One positive sample of IDV was detected, and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing. The duplex real-time PCR detection method represents a sensitive and specific tool to detect ICV and IDV. It provides technical support for virus research and clinical diagnosis of ICV and IDV. This information will benefit animal and human health.

Rodents represent the most abundant order of mammals, exhibiting remarkable diversity in morphology, habitats, behaviors, and hosted pathogens. Significant attention is currently focused on rodents as experimental animals for biomedical research. However, numerous aspects of rodents remain unexplored, such as their potential in unconventional biomedical models, molecular underpinnings of intriguing complex phenotypes, adaptations to environment or climate change, and host-pathogen interactions and arms race evolution. These challenges require a systematic framework to integrate the genomic variations among rodents with information on rodent-borne pathogens. To address this gap, we have established a comprehensive, freely accessible, and user-friendly atlas named Rodent Genome and Pathogen multi-Omics (RodentGPOmics), which provides comparative analysis of rodent genomes and information on zoonotic pathogen sequences in rodents. The RodentGPOmics Atlas provides: (i) basic information on 2706 rodent species; (ii) chromosome-level visualization of genomes, functional annotations, and genomic comparisons across 121 rodent species; (iii) epidemiological profiles based on 21 852 pathogen sequences reported in rodents and (iv) a few genomic tools for in-depth exploration of rodent multi-omics. This resource aims to advance the development of biomedical models for humans for promoting public health, as well as innovate the genetics, genomics, and molecular evolution in rodents, and offer valuable knowledge on rodent-borne emerging/re-emerging zoonotic infectious diseases. The resources are freely available and easy-to-use at http://RodentGPOmics.njau.edu.cn:8888/Rodent/index/homePage.

East, South, and Southeast Asia (together referred to as Southeastern Asia hereafter) have been recognized as critical areas fuelling the global circulation of seasonal influenza. However, the seasonal influenza migration network within Southeastern Asia remains unclear, including how pandemic-related disruptions altered this network. We leveraged genetic, epidemiological, and airline travel data between 2007-2023 to characterise the dispersal patterns of influenza A/H3N2 and B/Victoria viruses both out of and within Southeastern Asia, including during perturbations by the 2009 A/H1N1 and COVID-19 pandemics. During the COVID-19 pandemic, consistent autumn-winter movement waves from Southeastern Asia to temperate regions were interrupted for both subtype/lineages, however the A/H1N1 pandemic only disrupted A/H3N2 spread. We find a higher persistence of A/H3N2 than B/Victoria circulation in Southeastern Asia and identify distinct pandemic-related disruptions in A/H3N2 antigenic evolution between two pandemics, compared to interpandemic levels; similar patterns are observed in B/Victoria using genetic distance. The internal movement structure within Southeastern Asia markedly diverged during the COVID-19 pandemic season, and to a lesser extent, during the 2009 A/H1N1 pandemic season. Our findings provide insights into the heterogeneous impact of two distinct pandemic-related disruptions on influenza circulation, which can help anticipate the effects of future pandemics and potential mitigation strategies on influenza dynamics.

Frequent spillovers and recent geospatial expansion of avian influenza virus (AIV) pose significant economic and public health threats. Recent advances in vaccine technologies, bioinformatics, and artificial intelligence will provide newer approaches to target genetically diverse and rapidly evolving AIVs. Here, we review recent advances in, and perspectives on, developing universal vaccines needed for the effective control of AIVs.

Understanding wildlife-pathogen interactions is crucial for mitigating zoonotic risk. Through meta-transcriptomic sequencing we profiled the infectomes of 1,922 samples from 67 mammalian species across China, uncovering a remarkable diversity of viral, bacterial, fungal, and parasitic pathogens. Of the 195 pathogens identified, 62 were novel, including a bi-segmented coronavirus in diseased lesser pandas, which we propose represents a new genus - Zetacoronavirus. The orders Carnivora and Rodentia exhibited the highest pathogen diversity and were implicated in numerous host-jumping events. Comparative analysis of diseased versus healthy animals revealed a trend of higher pathogen loads in the former, with possible differences in tissue tropisms. In total, 48 zoonotic and 17 epizootic pathogens were identified, with frequent cross-species transmission, emphasizing the potential for emerging public health threats. This study highlights the urgent need for wildlife pathogen surveillance to inform proactive disease management strategies. ### Competing Interest Statement The authors have declared no competing interest.

Despite accumulating evidence that bat-derived coronaviruses often require intermediate hosts to facilitate transmission to humans1, the potential role of fur animals in zoonotic coronavirus spillovers has largely been overlooked2. Here we report the isolation and characterization of a previously undescribed mink respiratory coronavirus (MRCoV) from farmed minks with pneumonia. Notably, MRCoV uses angiotensin-converting enzyme 2 (ACE2) as an entry receptor and can infect mink, bat, monkey and human cells. Cryo-electron microscopy analyses revealed that the MRCoV receptor-binding domain (RBD) binds to the same interface on ACE2 receptors as the RBD of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) despite structural differences. We identify the key determinants on the RBD of MRCoV and ACE2 that confer efficient binding. HKU5-33S, a bat coronavirus closely related to MRCoV, uses ACE2 of the bat Pipistrellus abramus for cell entry and requires only two amino acid substitutions to adapt to mink ACE2. SARS-CoV-2 protease and polymerase inhibitors potently block MRCoV infection, thereby indicating a potential therapeutic strategy. Collectively, these findings enhance our understanding of coronavirus receptor dynamics and highlight their zoonotic potential. Given the risks posed by fur farms as reservoirs for emerging pathogens, our study underscores the need for enhanced surveillance to mitigate future coronavirus outbreaks.

Non-traditional farmed and wild mammals are often neglected in pathogen surveillance. Through metagenomic and metatranscriptomic sequencing of fecal and tissue samples from 973 asymptomatic mammals, we identified 128 viruses (30 novel), including a new coronavirus genus, 10,255 bacterial species (over 7,000 undescribed), 201 fungi, and 7 parasites. Farmed and wild mammals shared 13.3% of virus species, including canine coronavirus in Asiatic black bears and Getah virus in rabbits, while the 2.3.4.4b clade of H5N1 avian influenza virus was found in a wild leopard cat. We identified potential bacterial pathogen transmission between farmed and wild mammals and bacterial strains with high genetic similarity to those found in humans. We observed 157 clinically prioritized antibiotic resistance genes (ARGs) in mammalian microbiomes with greater than 99% identity to ARGs from human microbiomes, often co-occurring with mobile genetic elements. Overall, this work highlights cross-species risks at the human-animal interface.

Branch-specific substitution models are popular for detecting evolutionary change-points, such as shifts in selective pressure. However, applying such models typically requires prior knowledge of change-point locations on the phylogeny or faces scalability issues with large data sets. To address both limitations, we integrate branch-specific substitution models with shrinkage priors to automatically identify change-points without prior knowledge, while simultaneously estimating distinct substitution parameters for each branch. To enable tractable inference under this high-dimensional model, we develop an analytical gradient algorithm for the branch-specific substitution parameters where the computation time is linear in the number of parameters. We apply this gradient algorithm to infer selection pressure dynamics in the evolution of the BRCA1 gene in primates and mutational dynamics in viral sequences from the recent mpox epidemic. Our novel algorithm enhances inference efficiency, achieving up to a 90-fold speedup per iteration in maximum-likelihood optimization when compared to central difference numerical gradient method and up to a 360-fold improvement in computational performance within a Bayesian framework using Hamiltonian Monte Carlo sampler compared to conventional univariate random walk sampler.

Covering the surface of both host cells and enveloped viruses,glycans are in-evitable biomolecules for investigating virus-host interactions.However,due to the intricate role of glycans in the viral-host relationship,few glycan-related molecular mechanisms have been harnessed for antiviral therapies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV