Bacterial outer membrane vesicles (OMV) hold promise as vaccine platforms due to their natural adjuvant properties and nanoscale dimensions. However, their inherent inflammatory properties can induce excessive immune reactions, potentially harming the host, while their variability in size and low yield pose challenges for large-scale applications. To address these challenges, we developed synthetic bacterial vesicles (SyBV) through bacterial cell lysis followed by detergent treatment and sonication to eliminate nucleic acids, producing high-purity vesicles with minimal cytoplasmic residue. SyBV triggered comparable antigen-specific adaptive immunity but avoided strong inflammatory responses. We engineered SyBV to display the prefusion F protein (preF) of respiratory syncytial virus (RSV) with cytotoxic lysin A (ClyA). This approach effectively induced preF-specific antibodies and robust immune responses. Our findings suggest that SyBV could provide a safer, more efficient vaccine platform for preventing viral infections, overcoming the limitations associated with traditional OMV while preserving immunogenicity.

It is estimated that over 60% of known tailed phages are siphophages, which are characterized by a long, flexible, and non-contractile tail. Nevertheless, entire high-resolution structures of siphophages remain scarce. Using cryo-EM, we resolved the structures of T-series siphophage T1, encompassing its head, connector complex, tail tube, and tail tip, at near-atomic resolution. The density maps enabled us to build the atomic models for the majority of T1 proteins. The T1 head comprises 415 copies of the major capsid protein gp47, arranged into an icosahedron with a triangulation number of seven, decorated with 80 homologous trimers and 60 heterotrimers along the threefold and quasi-threefold axes of the icosahedron. The T1 connector complex is composed of two dodecamers (a portal and an adaptor) and two hexamers (a stopper and a tail terminator). The flexible tail tube comprises approximately 34 hexameric rings of tail tube. The extensive disulfide bond network along the successive tail rings may mediate the flexible bending. The distal tip of T1, which is cone-shaped and assembled by proteins gp33, gp34, gp36, gp37, and gp38, displays structural similarity to that of phage lambda. In conjunction with previous studies of lambda-like siphophages, our structure will facilitate further exploration of the structural and mechanistic aspects of lambda-like siphophages.

ABSTRACT Myophage Mu is a representative of contractile nanomachines with a simple tail baseplate. It has the capacity to infect a range of intestinal bacteria and has extensive applications in genetic engineering research. Nevertheless, a comprehensive understanding of the entire structure and contractile mechanisms of Mu remains elusive. Using cryo-electron microscopy (cryo-EM), we resolved the asymmetric structures of Mu in both its extended and contracted states, the latter of which lacked the tail baseplate, at near-atomic resolutions. We built the atomic models for the extended Mu, encompassing the head, the connector complex, the tail, and the simple baseplate. It is noteworthy that we identified the position and structure of the tail tube initiator protein gp43 (referred to as the DNA circularization protein). The protein gp43 plays a crucial role not only in the baseplate assembly and DNA circularization but also in stabilizing the wedge-hub connection and mediating tail contraction. Except for the baseplate structure, the structural comparison of Mu in its extended and contracted states revealed that only the tail sheath protein gp39 and the C-terminus of the tail terminator protein gp37 undergo notable conformational changes to accommodate the tail contraction, whereas the remaining protein components remained unchanged. Our structures exhibited conserved properties among the majority of myophages, thereby providing valuable insights into the contraction mechanisms across myophages and contractile injection systems (CISs). IMPORTANCE Despite extensive study, the asymmetric structures of phage Mu, a highly effective transposable myophage, remain unknown. In this study, we present the high-resolution structures of Mu in both its extended and contracted states. The comparison of the two structures allows for the illustration of detailed conformational changes of the head-to-tail complex during the process of tail contraction. The contraction mechanism of Mu is highly conserved and widely adapted to all contractile nanomachines that share common structural features with Mu.

Washington University polyomavirus (WUPyV), a member of the polyomavirus family capable of infecting the human respiratory tract, exhibits high prevalence in human populations. However, confirmation of its pathogenicity and aetiology has been hindered by challenging culture conditions. To investigate the infective endocytosis of WUPyV, a novel infection model was developed using human airway organoids (HAOs); viral replication dynamics, cell tropism, endocytosis mechanisms, and morphogenesis in human airway epithelium (HAE) were characterized. The results indicated that WUPyV replicates efficiently and infects multiple cell types within the respiratory epithelium. After JC polyomavirus, WUPyV is the second human polyomavirus known to enter cells via clathrin-mediated endocytosis, independent of caveolar/lipid raft-mediated endocytosis or macropinocytosis. Consistent with other polyomaviruses, WUPyV is transported to the nucleus for replication and assembly through a lipid-dependent pathway requiring tyrosine kinase activity and endosome/lysosome acidification. Assembled viral particles were observed in nuclear pores; they were ultimately released extracellularly through vesicles and schistocytes. High-titre WUPyV infection caused extensive structural damage to the HAE and its cilia. Our findings provide important insights into the endocytosis, morphogenesis, and ultrastructural damage induced by WUPyV, supporting the notion that WUPyV is an underestimated human pathogen.

Human norovirus (NoV) is one of the major pathogens causing acute gastroenteritis. The GII.20 NoV VP1 reveals a close evolutionary relationship with that of the widely prevalent GII.4 genotype based on sequence phylogenetic analysis. In this study, we characterized the antigenic properties and glycan binding profile of GII.20 VP1 protein. GII.20 VP1 protein was successfully produced using a baculovirus expression system, and self-assembled into virus-like particles (VLPs) with a diameter of approximately 38 nm. Immunological assays demonstrated that polyclonal sera generated against GII.20 VLPs exhibited strong binding activity at dilution up to 1:8,192,000, while maintaining detectable cross-reactivity with GII.4 VLP at 1:128,000. dilution. Reciprocal cross-reactivity was observed, with anti-GII.4 sera recognizing GII.20 VLP at 1:125,000 dilution. Blocking assays demonstrated mutual partial cross-blocking between the antisera, with half-maximal blocking dilutions of 36 (anti-GII.20 sera) and 297 (anti-GII.4). Blocking assays demonstrated mutual partial cross-blocking effects between the antisera, with half-maximal blocking dilution of 36 and 297 for anti-GII.20 and anti-GII.4 polyclonal sera, respectively. Saliva binding analysis revealed that GII.20 VLPs recognize A, B, and O blood type saliva. Sequence alignment showed conserved key amino acids in the putative glycan-binding region between GII.20 and GII.4 genotypes. Structural modeling of the GII.20 P domain revealed high similarity with the corresponding domains of both GII.17 and GII.4 NoVs. These findings establish that GII.20 VP1 shares functional characteristics with GII.4 in both glycan binding specificity and antigenic cross-reactivity, providing more insights into the molecular epidemiology of diverse NoV genotypes.

Shanxi Tick Virus 2(SXTV2), a Tamdy group member of Orthonairovirus genus, Nairoviridae family, was initially identified through Next Generation Sequencing, with its pathogenicity and risk profile remaining unclear. This study reports the first successful isolation of SXTV2 from Haemaphysalis longicornis ticks collected from Hunchun City, China-a tri-border region between China, Russia, and North Korea. The isolated SXTV2 strain replicated and produced cytopathic effects in both Vero (primate) and SW-13 (human) cell lines. Electron microscopy revealed that SXTV2 particles are enveloped, surface-spiked, pleomorphic, and approximately 100 nm in diameter. Experimental inoculation in neonatal mice led to significant weight loss, liver injury and 100% mortality. In conclusion, this study marks the first successful isolation of the SXTV2 strain and exploring the animal model for member of Tamdy group orthonairovirus. These findings suggest the need for enhanced surveillance of SXTV2 zoonotic exposure and disease epidemic risks.

The Rhabdoviridae family comprises a diverse range of negative-sense single-stranded ribonucleic acid (RNA) viruses, including significant human and mammalian viruses transmitted by various arthropod species. Herein, using Aedes albopictus (Ae. albopictus) samples collected in two urban parks during 2023 and 2024, through metagenomics sequencing, 16 sequences were identified as putative novel viruses, showing closest homology to insect-specific viruses, mycoviruses, or plant-associated viruses. Notably, two novel viruses, Aedes albopictus almendravirus GCCDC15 (Aealb-AlmV GCCDC15) and Aedes albopictus almendravirus GCCDC16 (Aealb-AlmV GCCDC16) were identified and successfully isolated. Both of these viruses belong to the genus Almendravirus within the Rhabdoviridae family. Phylogenetic analysis revealed that Aealb-AlmV GCCDC15 and GCCDC16 are distantly related to Coot Bay virus (the United States of America, 2013) and Menghai rhabdovirus (Yunnan Province, China, 2017). The genetic distances between these two viruses and their most similar viruses are marked by 59.85 % and 87.20 % of amino acid identity in the L protein, respectively, supporting their classification as two new species in the Rhabdoviridae family. Cytopathic effects and rod-like virions were observed in mosquito cells (C6/36) after inoculating with supernatants from the Ae. albopictus samples. To investigate the natural distribution and persistence of the novel almendraviruses, we conducted a specific reverse transcription-polymerase chain reaction (RT-PCR) screening of Ae. albopictus mosquitoes collected from two urban parks across different time points. The assays confirmed the presence of both Aealb-AlmV GCCDC15 and GCCDC16 in mosquito populations. Critically, these viruses were detected repeatedly over successive sampling periods and in mosquitoes from geographically distinct sites within the urban environment. In summary, our study delineates the virome characteristics of Aedes mosquitoes in the urban ecosystem and successfully isolated two novel rhabdoviruses. The recurrent detection provides clear evidence for the sustained circulation of Ae. albopictus-derived almendraviruses in urban parks, highlighting their ongoing transmission and establishment in these habitats.

In recent years, coxsackievirus A6 (CVA6) has surpassed enterovirus A71 to become the main pathogen causing severe Hand, Foot, and Mouth disease (HFMD) in China with a substantial disease burden. However, there is currently no commercial CVA6 vaccine. The D3a genotype of CVA6 is the predominant genotype in China. In this study, virus-like particles (VLPs) and mRNA vaccines based on the CVA6 sub-genotype D3a were successfully developed. The immunogenicity and protective effects of the VLP of CVA6 combined with Al(OH)3 and CpG adjuvant indicated that VLP-induced neutralizing antibodies against three CVA6 sub-genotype (D2, D3a, and D3b) strains in Institute of Cancer Research (ICR) mice, and the combination of the two adjuvants enhanced cellular immunity. Passive immunization with serum from mice immunized with VLPs protected suckling mice against CVA6 lethal challenge in both antiserum transfer and maternal immunization experiments. The immunogenicity and protective effects of the mRNA vaccine of CVA6 indicate that it induces robust T-cell immunity. T-cell immunity was found to cross-protect against coxsackievirus A10 infection in mice. This is the first trial of a CVA6 mRNA vaccine worldwide and the first comparison of the immunogenicity and protective effects of VLP and mRNA vaccines based on D3a CVA6. The study provides a theoretical basis for the development of enteroviruses vaccines and the formulation of immunization strategies.

Adenoviruses are double-stranded DNA viruses with broad relevance to human and animal health and considerable potential as therapeutic vectors. Despite extensive studies, the structural details of core and minor capsid proteins in adenoviruses remain poorly understood. In this study, the architecture of bovine adenovirus type 3 (BAdV-3), a member of the Mastadenovirus genus, was solved by cryo-electron microscopy. Our structure shows that BAdV-3 shares significant structural conservation with human adenoviruses. Atomic models were constructed for a previously uncharacterized region of the minor protein VI and for the core proteins V and Mu. The study revealed how core proteins bridge the genome and capsid, underscore the multifaceted roles of protein V in strengthening capsid stability and facilitating genome release.

Since the COVID-19 pandemic, there has been a documented rise in the incidence of neurological manifestations among individuals complicated with encephalitis or myelitis. The spectrum of neurological symptoms associated with HCoVs infections is expanding. However, the infection characteristics and pathogenesis of seasonal HCoVs to the central nervous system remain obscure. No pharmacological agents have demonstrated the capacity to specifically and efficaciously mitigate the neurological symptoms induced by HCoVs infections to date. We developed human cerebral organoids (HCOs) derived from human induced pluripotent stem cells and established a blood–brain barrier (BBB) HCOs co-culture model. We subjected these models to seasonal human coronavirus (HCoV) infections to investigate the viral characteristics within the central nervous system (CNS). Utilizing RNA sequencing, we conducted a preliminary exploration of the mechanisms underlying virus-induced inflammatory responses in the CNS. Furthermore, we assessed the efficacy of antiviral and anti-inflammatory drugs using the HCO model. Our results showed that among seasonal coronaviruses, HCoV-OC43 replicates efficiently within the organoids, primarily targeting neurons and astrocytes, and disrupts the barrier function of the BBB. RNA sequencing analysis revealed that HCoV-OC43 infection triggers an inflammatory response through the TNF and NF-κB signaling pathways, leading to cell death, impaired neuronal function, and disrupted interneuron signaling. Interestingly, Bardoxolone methyl (CDDO-Me) demonstrated antiviral effects comparable to remdesivir, reducing both inflammation and cell death. Conclusively, HCOs infected with HCoV-OC43 offer valuable insights into the pathogenesis of HCoVs in central nervous system (CNS), and might serve as a tool for developing novel therapeutic strategies for HCoVs infections, including COVID-19, especially on exploring treatment candidates.

Ectromelia virus (ECTV), a member of the Orthopoxvirus genus, serves as both a causative agent of mousepox and a pivotal surrogate model for studying highly pathogenic orthopoxviruses. Although genomic data on ECTV remains limited, we report the isolation and characterization of a novel strain, ECTV-C-Tan-GD01, obtained from rodents in Guangdong Province, China. Nanopore sequencing yielded a complete genome (199 annotated genes, including one gene truncated at the C-terminus) with inverted terminal repeats (ITRs) harboring a conserved hairpin structure. Notably, a frameshift-inducing "G" deletion in the EV159 gene resulted in the truncation of a semaphorin-like protein. In vitro assays demonstrated cell-associated viral replication kinetics, with maximum titers achieved earlier in Vero/HeLa cells (72 ​h) than in BHK-21/CEF cells (84 ​h). Murine challenge experiments revealed extreme virulence (LD50 ​< ​1 plaque-forming unit (PFU) via intranasal/footpad routes) and hepatosplenic tropism. Furthermore, ECTV-C-Tan-GD01 exhibited utility in evaluating orthopoxvirus countermeasures: a single dose of vaccinia virus Tiantan (VTT) or non-replicating vaccinia virus Tiantan (NTV) conferred cross-protection, while tecovirimat (ST-246), cidofovir (CDV), and brincidofovir (initially CMX001) significantly reduced viral loads and pathology. This study establishes ECTV-C-Tan-GD01 as a dual-purpose resource for probing orthopoxvirus evolution and advancing therapeutic development.

In the assembly pathway of tailed double-stranded DNA (dsDNA) bacteriophages and herpesviruses, a procapsid with a dodecameric portal for DNA delivery at a unique vertex is initially formed. Appropriate procapsid assembly requires the transient presence of multiple copies of a scaffolding protein (SP), which is absent in the mature virion. However, how the SP contributes to dodecameric portal formation, facilitates portal and coat protein incorporation, and is subsequently released remains unclear because of a lack of structural information. Here, we present the structure of the SP-portal complex within the procapsid of bacteriophage P22 at 3-9 Å resolutions. The AlphaFold2-predicted SP model fits well with the density map of the complex. The SP forms trimers and tetramers that interact to yield a dome-like complex on the portal. Two SP domains mediate multimerization. Each trimer interacts with two neighboring portal subunits. The SP has a loop-hook-like structure that aids in coat protein recruitment during viral assembly. The loops of those SP subunits on the portal are positioned in clefts between adjacent portal subunits. Conformational changes in the portal during phage maturation may trigger the disassembly and release of the SP complex. Our findings provide insights into SP-assisted procapsid assembly in bacteriophage P22 and suggest that this strategy is also implemented by other dsDNA viruses, including herpesviruses.

With the widespread application of HIV-1 nucleic acid testing (NAT) in China, particularly in the diagnosis of HIV-1 infection, ensuring the accuracy of NAT results through quality control has become critically important. However, existing HIV-1 NAT quality control materials (QCMs), such as clinical plasma samples and inactivated HIV-1 cell culture supernatants, have limitations in sustainability, biosafety risks, and costs. MS2-armed RNA (MS2) does not replicate the biological characteristics of natural viruses or the complexities of the extraction and detection processes associated with authentic viral particles. To address these limitations, this study developed a novel HIV-1 NAT QCM based on HIV-1 pseudovirus (PsV). HIV-1 PsV packaged using an improved four-plasmid lentiviral vector (LV) system could be generated with a high concentration of up to 10⁹ copies/mL. The HIV-1 PsV mimics the morphology of the real virus and is capable of only single-cycle infection, thereby ensuring biosafety. The HIV-1 PsV-based QCM demonstrated excellent homogeneity, absence of matrix effects, stability for 7 days at 4°C and -20°C, and the ability to withstand up to five freeze-thaw times. We further found that HIV-1 PsV outperformed inactivated HIV-1 and MS2 in terms of short-term stability and freeze-thaw stability, respectively. Additionally, the PsV-based QCM was successfully detected by 12 commercial HIV-1 NAT quantification kits in the Chinese market and demonstrated excellent performance in an external quality assessment (EQA) involving 60 laboratories. In summary, the novel HIV-1 PsV-based QCM can serve as a safe and sustainable alternative to existing HIV-1 NAT QCMs for EQA of HIV-1 NAT laboratories.IMPORTANCEThis study proposes a novel strategy to prepare HIV-1 nucleic acid testing (NAT) quality control material (QCM) using HIV-1 pseudovirus (PsV) packaged by an improved four-plasmid lentiviral vector (LV) system. The HIV-1 PsV-based QCM can simulate authentic virus particles and better monitor the entire HIV-1 NAT process, including nucleic acid extraction, amplification, and detection. The innovative HIV-1 NAT QCM possesses several desirable characteristics: biosafety, homogeneity, stability, and the ability to be prepared at high concentrations and on a large scale, significantly reducing production costs. Compared to commonly used QCMs such as inactivated HIV-1 and MS2, the HIV-1 PsV demonstrates superior stability and better meets the requirements for transportation, storage, and quality control applications of HIV-1 NAT laboratory. Particularly, the ability of HIV-1 PsV to accommodate the insertion of large nucleic acid sequences provides a solid technical foundation for developing more advanced quality control solutions in the future.

Emerging pathogenic tick-borne viruses (TBVs) have attracted a great deal of attention due to their significant impact on human and animal health. A novel orthonairovirus named Dadong virus (DDV) was isolated from Haemaphysalis concinna ticks in the Changbai Mountain region on the China-North Korea border. DDV can induce cytopathic effects in mammalian and human cell lines. Phylogenetic analysis showed that it belongs to the genus Orthonairovirus, family Nairoviridae, exhibiting 72.4%-81.3% nucleic acid identity to Tofla orthonairovirus, known to cause lethal infection in IFNAR KO mice. The first serological evidence of DDV circulating in cattle and mice was also obtained, with 4.0% (1/25) of cattle and 2.27% (1/44) of mice seropositive for DDV. Further investigations, including serological surveys using human samples, are required to assess the public health risk posed by DDV.

Due to the advantages of direct visualization and high resolution, transmission electron microscopy (TEM) technology has been widely used in the morphological identification of viruses. With the development of artificial intelligence (AI), there have been some studies on automated TEM virus identification using deep learning. However, to achieve effective virus identification results, a large number of high-quality labeled images are required for network training. In this work, we propose an automatic virus segmentation method based on few-shot learning. We use the Chikungunya virus, Parapoxvirus and Marburg virus, etc. to construct a pre-training virus dataset and train an attention U-Net-like network with an encoder module, relationship module, attention module and decoding module to realize severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) segmentation using few-shot learning. The experiment shows that the proposed few-shot learning methods yield 0.900 Dice and 0.828 Jaccard in 1-shot, 0.903 Dice and 0.832 Jaccard in 5-shot, which demonstrates the effectiveness of our method and outperforms other promising methods. Our fully automated method contributes to the development of medical virology by providing virologists with a low-cost and accurate approach to identify SARS-CoV-2 in TEM.

From January 2022 to November 2022, sporadic psittacosis occurred in Lishui city, China. The patients were presented with fever, cough, and pulmonary infiltration. Their clinical symptoms were not relieved after receiving cephalosporin, penicillin, beta-lactamase inhibitors, and quinolones. Metagenomic next-generation sequencing of bronchoalveolar lavage fluid samples from the patients revealed Chlamydia psittaci infection. Then, three C. psittaci strains were isolated from the patients. Their whole genome sequences (WGSs) were obtained, and a core genome multilocus sequence typing (cgMLST) method was developed to study the population structure of C. psittaci. Using the constructed cgMLST method, 72 WGSs were divided into four related groups and ten sub-clusters. The Lishui strains formed a unique population of C. psittaci, which might represent a new variant of C. psittaci. In vitro antimicrobial susceptibility testing suggested that the Lishui strains were sensitive to tetracycline, macrolides, quinolones, and no drug-resistance was observed.

Lytic podophages (VP1-VP5) play crucial roles in subtyping Vibrio cholerae O1 biotype El Tor. However, until now no structures of these phages have been available, which hindered our understanding of the molecular mechanisms of infection and DNA release. Here, we determined the cryoelectron microscopy (cryo-EM) structures of mature and DNA-ejected VP1 structures at near-atomic and subnanometer resolutions, respectively. The VP1 head is composed of 415 copies of the major capsid protein gp7 and 11 turret-shaped spikes. The VP1 tail consists of an adapter, a nozzle, a slender ring, and a tail needle, and is flanked by three extended fibers I and six trimeric fibers II. Conformational changes of fiber II in DNA-ejected VP1 may cause the release of the tail needle and core proteins, forming an elongated tail channel. Our structures provide insights into the molecular mechanisms of infection and DNA release for podophages with a tail needle.

Nanoparticles have gained attention as potential antiviral agents, but the effects of graphene oxide nanoparticles (GONPs) on influenza virus remain unclear. In this study, we evaluated the antiviral activity of GONPs against influenza virus strain A/Hunan-Lengshuitan/11197/2013(H9N2). Our results show that GONPs with a diameter of 4 nm exerted an antiviral effect, whereas those with a diameter of 400 nm had no effect. Treatment with 4-nm GONPs reduced viral titers by more than 99% and inhibited viral nucleoprotein expression in a dose-dependent manner. We also confirmed that 4-nm GONPs inhibited the infectivity of H9N2 in MDCK cells. A transmission electron microscopic analysis revealed morphological abnormalities in the GONP-treated virus, including the destruction of the envelope glycoprotein spikes and an irregular shape, suggesting that GONPs cause the destruction of the viral coat proteins. Our results highlight the potential utility of GONPs in the prevention and treatment of viral infections, especially those of emerging and re-emerging viruses.

T5 is a siphophage that has been extensively studied by structural and biochemical methods. However, the complete in situ structures of T5 before and after DNA ejection remain unknown. In this study, we used cryo-electron microscopy (cryo-EM) to determine the structures of mature T5 (a laboratory-adapted, fiberless T5 mutant) and urea-treated empty T5 (lacking the tip complex) at near-atomic resolutions. Atomic models of the head, connector complex, tail tube, and tail tip were built for mature T5, and atomic models of the connector complex, comprising the portal protein pb7, adaptor protein p144, and tail terminator protein p142, were built for urea-treated empty T5. Our findings revealed that the aforementioned proteins did not undergo global conformational changes before and after DNA ejection, indicating that these structural features were conserved among most myophages and siphophages. The present study elucidates the underlying mechanisms of siphophage infection and DNA ejection.