Feline herpesvirus type 1 (FHV-1) is a member of the Alphaherpesvirinae subfamily, which includes important pathogens affecting humans and animals, such as herpes simplex virus types 1 and 2, pseudorabies virus, and Marek's disease virus. While herpesviruses show promise as vaccine vectors for delivering foreign antigens, ensuring their safety is a significant concern that must be addressed alongside their efficacy. In this study, we report the development of a replication-defective FHV-1 ΔgL-based vaccine candidate, which is propagated exclusively in a specially modified F81 cell line. Vaccination of cats with the FHV-1 ΔgL-based candidate demonstrated a favorable safety profile and elicited protective immunity against FHV-1 challenge. More importantly, the FHV-1 ΔgL platform exhibits excellent efficacy in delivering the VP2 antigen of the feline panleukopenia virus (FPV), inducing high-titer neutralizing antibodies in cats. Furthermore, we evaluated a trivalent vaccine candidate, rQD1 ΔgL/gI/gE-VP2-VP1, in which an expression cassette for the feline calicivirus (FCV) VP1 was inserted to replace the gI/gE genes. Vaccination of cats with rQD1 ΔgL/gI/gE-VP2-VP1 produced a strong protective effect against challenges from FHV-1, FPV, and FCV. To support clinical manufacturing, we generated a stable cell line through site-specific integration of the gL gene into the Rosa26 safe-harbor locus of F81 cells. In conclusion, the FHV-1 ΔgL platform demonstrates safety, efficacy, and promising potential for future applications and serves as an example for constructing attenuated vaccines in alphaherpesviruses.IMPORTANCEHerpesviruses are highly effective at presenting foreign antigens and serve as excellent viral vectors, yet their safety warrants careful attention. We developed a replication-defective feline herpesvirus type 1 (FHV-1) ΔgL vector platform. Using this platform, a bivalent vaccine (rQD1 ΔgL-VP2) and a trivalent vaccine (rQD1 ΔgL/gI/gE-VP2-VP1) were constructed. Both vaccines demonstrated robust immunoprotection and favorable safety profiles in immunized cats. Furthermore, to facilitate the application of this platform, a stable cell line with site-specific integration, F81-gL-Rosa26, was generated by inserting the gL gene into the Rosa26 safe-harbor locus of F81 cells. The strategy of the FHV-1 ΔgL platform also provides a reference for other members of the herpesvirus family.

Classical molecules encoded by the major histocompatibility complex (MHC) are central to immune responses. Compared to typical mammals, the chicken MHC is small and simple, determining life or death from economically important pathogens like avian influenza virus and Marek's disease virus (MDV). Several genes within the tightly linked chicken MHC have been suggested to determine resistance and susceptibility to MDV, but it was a surprise to find that the dominantly expressed class II molecule from the resistant B2 haplotype employed a novel peptide-binding mode with a decamer core sequence compared to the susceptible B19 haplotype with a typical nonamer core. We examined the crystal structure of the dominantly expressed class II molecule from another resistant haplotype, B21, which is extremely frequent in commercial chicken flocks, to find that it bound the same MDV peptide with both nonamer and decamer cores, revealing an unexpected plasticity of binding that potentially increases the immune response to this devastating pathogen.IMPORTANCEThe meat and eggs of chickens are major food sources for both developed countries (where vast numbers are produced by the commercial poultry industry) and developing countries (where backyard and village chickens provide animal protein for the poorest members of society). Chickens are beset by many devastating viral diseases, and decades of genetics research has shown the particular haplotype of the major histocompatibility complex (MHC) can determine life or death for an individual chicken. One major economically important virus is the oncogenic herpesvirus that causes Marek's disease virus (MDV). Through structural studies, we find that the dominantly expressed class II molecule from an MHC haplotype that confers decisive resistance to MDV (and a number of other viruses) binds the same MDV peptide in two conformations, thus providing the basis for a wider immune response by T cells, which is of potential importance for both disease resistance and vaccine response.

Viral infections pose a significant threat to humanity, as exemplified by the COVID-19 pandemic. To mitigate the associated damage, understanding the biological characteristics of causative viruses is essential. In this study, we report a novel method for identifying host cell molecules (including virus receptors) required for viral attachment, referred to as host cell attachment factors (HCAFs). Our approach utilizes proximity labeling (PL) technology; pseudoviruses engineered to express a PL enzyme are applied to host cells, where they bind and initiate PL. Because HCAFs are expected to localize near viral attachment sites, candidate HCAFs were specifically tagged using the PL process. The analysis of influenza HCAFs enabled the identification of multiple candidate molecules. Subsequent viral attachment experiments using Chinese hamster ovary cells suggested that neuropilin-1 may serve as an HCAF for influenza viruses. Given its simplicity and rapid turnaround, this method is adaptable to a wide range of enveloped viruses, including pandemic viruses that require emergency analyses.IMPORTANCEIdentifying cellular receptors for viruses is not only crucial in conventional virology research but also plays a key role in addressing pandemic viruses. The significance of our work lies in establishing a standardized method for viral receptor identification, an area in which no widely accepted protocol has previously existed, by utilizing pseudovirus-mediated proximity labeling technology. Moreover, this approach provides a highly versatile platform that enables rapid and low-cost identification of viral receptors.

Swine orthopneumovirus (SOV), a recently identified porcine pneumovirus, has been detected in pig farms worldwide; however, its pathogenicity and molecular biology remain poorly understood. To facilitate the study of SOV replication and transcription, we developed a functional minigenome system based on consensus sequences from multiple strains of SOV and related pneumoviruses. Here, we constructed and optimized this system in BSRT7/5 cells, revealing that the RNA-dependent RNA polymerase (RdRp) activity depends on a conserved protein phosphatase 1 (PP1) binding site within the phosphoprotein P, as a single F131A substitution markedly reduced polymerase function. Additionally, we identified and characterized the M2-1 binding site on P, which is essential for viral transcription. These findings provide new insights into SOV polymerase complex requirements and establish a foundation for reverse genetics approaches to rescue infectious viruses, advancing our understanding of SOV biology and its potential role in porcine respiratory disease.IMPORTANCERecently, a newly identified porcine pneumovirus, swine orthopneumovirus (SOV), was detected in pig farms in different countries. Although detected mainly in sick animals, this virus has not been isolated yet and its pathogenicity remains to be determined. We started by setting up a minigenome system with a view to develop reverse genetics and rescue infectious virions. This minigenome system was used to study the functioning of the SOV RNA polymerase and compared it with RSV. Although some similarities exist between SOV and RSV, the RdRp of RSV cannot rescue the SOV minigenome. SOV seems to belong to another genus/genogroup of pneumoviruses, which includes PVM and the canine pneumovirus. Our functional minigenome paves the way for reverse genetics of SOV and determination of its pathogenicity in different host species.

Enterovirus uncoating, the events leading to the release of the viral genome into the cytoplasm, remains a poorly understood process. This review discusses two competing models: the "genome translocation model," where viral proteins form a pore for RNA passage into the cytosol, and the "particle escape model," where the entire virion crosses the endosomal membrane before uncoating. We discuss the combination of structural biology, in vitro reconstitution, and cellular assays required to definitively resolve this long-standing mystery.

Dengue viruses are mosquito-borne human pathogens whose global incidence in human infections is rising. Surveillance is hampered by the requirement for blood-based diagnostics. Interestingly, in several prior research studies, dengue virus nucleic acids and proteins could be detected in saliva, making saliva-based diagnostics a plausible alternative. However, the temporal relationships between exposure, viremia, salivary accumulation, and symptom onset remain poorly defined. To address this knowledge gap, nine participants were enrolled in clinical trial NCT04298138 to receive an inoculation with an attenuated dengue virus (DENV-3 strain CH53489). Matched blood and saliva specimens were collected over 10 days. Dengue virus genomes could be detected by RT-qPCR in both blood and saliva before the onset of symptoms in most individuals. The earliest time of detection in blood and saliva was 3.4 and 5.0 days post-infection, respectively. Using this sample set, we also measured host transcriptional responses to dengue infection in both biospecimens. Strikingly, far more human genes showed increased transcript abundance exclusively in saliva (14%) than in PBMCs (5%) or both compartments simultaneously (6%), revealing compartment-specific host responses. For 21 of the human transcripts that increased in response to dengue infection, we quantified their daily abundance across the course of DENV-3 infection in all study participants. Together, these findings demonstrate that sensitive dengue virus detection in saliva should be possible and establish that both blood and saliva capture early host responses to infection.IMPORTANCEDengue virus infections are detected using blood samples, either with nucleic acid amplification tests for the dengue virus genome (in the case of an active infection) or serology-based tests for antibodies recognizing dengue viruses (indicating a prior infection). The requirement for blood draws creates a barrier to dengue virus surveillance. Our data demonstrate that saliva can serve as an alternative biospecimen for detecting dengue virus genomes early in infection, with detection lagging blood by only 1.6 days-a modest and likely acceptable tradeoff given saliva's practical advantages. Unlike blood, saliva is non-invasive, self-collectible, requires no sharps, and is thermostable. We also identified host genes upregulated upon dengue infection, informing on the host response to infection. Our longitudinal study design enabled us to track the kinetics of transcript accumulation both by day post-infection and relative to symptom onset, providing high resolution into the host response to the virus.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT04298138.

HBV remains a major global health burden, with cccDNA serving as the transcriptional template for viral RNAs that sustain persistent infection. The low copy number of cccDNA in the nucleus and the lack of authentic in vitro models hinder its investigation and therapeutic development. Here, we established an M13 phage-derived single-stranded DNA approach to efficiently generate sequence-authentic HBV cccDNA in vitro. Upon transfection, the cccDNA molecules assembled into minichromosomes and were proven to be biologically active, producing viral RNAs more faithfully recapitulating infection-derived transcripts compared to recombinant cccDNA. The system also permits targeted mutagenesis for loss-of-function studies as well as site-specific labeling. These results provide insights into the nucleosome organization and transcriptional characteristics of the HBV cccDNA minichromosome, offering an authentic platform to investigate minichromosome functionality and host-virus interactions.

Zika virus and West Nile virus replication complexes, which are typically symmetrically distributed around the nucleus in the perinuclear region of an infected cell, cluster asymmetrically on one side of the nucleus in A549 and human neural progenitor cells as the infections progress. Analysis of the sequential cellular responses involved showed that microtubule acetylation increased and the unfolded protein response was activated; then, the levels of ubiquitinated protein and HDAC6 increased, followed by asymmetric concentration of the viral replication complexes, ubiquitinated proteins, and HDAC6 at the cellular microtubule-organizing center, where they became surrounded by a vimentin cage. These sequential steps are characteristic of aggresome formation, an additional protective cellular response that copes with the accumulation of toxic misfolded proteins. This response delayed activation of cell death pathways and extended virus production. Both cell type-specific and orthoflavivirus-specific characteristics determine whether a particular infection induces aggresome formation.

Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus, is the causative agent of bovine viral diarrhea-mucosal disease, one of the most significant infectious diseases affecting cattle worldwide. BVDV employs diverse mechanisms to evade host innate antiviral immune response, while the precise processes remain incompletely understood. Here, we reveal that BVDV infection drives glycolytic reprogramming through the ROS-HIF-1α axis, leading to the formation of an HK2/MAVS/VDAC1 complex. This complex impairs the interaction between RIG-I and MAVS, resulting in suppressed IFN production. Moreover, we show that lactate, produced via LDHA-mediated glycolysis, binds to MAVS, inhibiting its mitochondrial localization and subsequent association with RIG-I. Together, these mechanisms reveal how BVDV harnesses glycolytic remodeling to dampen RIG-I/MAVS signaling and facilitate viral replication. Our study not only uncovers a potential therapeutic target for combating pestivirus infection but also provides valuable insights into immune evasion strategies shared within the Flaviviridae family, particularly among pestiviruses.

Coronaviruses and influenza A viruses (IAV) can cause severe respiratory disease and have pandemic potential. Both viruses depend on priming of their glycoproteins by host cell proteases for the acquisition of infectivity, and the responsible enzymes represent potential targets for intervention. Initial studies suggested that these viruses may exploit redundant proteolytic systems. However, research conducted over the last two decades has pointed to a key role for a single enzyme in coronavirus and IAV priming, the transmembrane protease serine 2 (TMPRSS2). Interest in TMPRSS2 as a host dependency factor and therapeutic target intensified during the COVID-19 pandemic, prompting extensive investigation into its biology, substrate specificity, and pharmacological inhibition. Here, we review recent efforts to define the role of TMPRSS2 in coronavirus infection and to target this protease for antiviral intervention.