Arboviruses encompass a diverse spectrum of arthropod-transmitted RNA viruses that pose an extensive threat to human health and the economy. These obligate intracellular pathogens induce severe pathologies by strategically subverting the host's innate immune responses, often leading to a plethora of clinical manifestations in the host. The pathogen-associated molecular patterns (PAMPs) are recognized by host pattern recognition receptors (PRRs), which initiate innate immune responses by releasing interferons and cytokines. However, dysregulation can divert these responses and lead to exacerbated inflammation and immunopathology. The major families of arboviruses, particularly Flaviviridae, Togaviridae, Bunyaviridae, Reoviridae and Rhabdoviridae, exert well-documented immunomodulatory effects. In this context, modulation of host immune pathways serves as an effective approach not only to control viral infection but also to restore dysregulated immune responses. Integration of host-directed immunotherapeutic interventions with virus-targeted antiviral treatments may offer a potential strategy to manage immunopathogenesis while combating infection. This review comprehensively summarizes the potential of host immunomodulation as an alternative therapeutic strategy against vector-transmitted RNA viruses.

The poxviruses are large, epitheliotropic, double-stranded DNA viruses that are pathogens of invertebrates and the mammalian, avian and reptilian vertebrate taxa including the familiar poxviruses of humans and domestic animals such as the variola virus or smallpox virus and the related vaccinia virus used for the live smallpox vaccine, mpox virus (formerly monkeypox virus), cowpox virus of rodents, and orf virus of sheep and goats. Poxviruses have also previously been described from teleost fishes including the common carp Cyprinus carpio, ayu Plecoglossus altivelis, Atlantic salmon Salmo salar, Atlantic cod Gadus morhua, red or Japanese seabream Pagrus major, and black bullhead Ameiurus melas. This report documents a novel poxvirus infection in a captive population of the endangered Knysna seahorse Hippocampus capensis. Histopathological examination revealed an epidermal keratinocytic dermatopathy with hypertrophy and subsequent progressive degeneration and necrosis of the affected cells; karyomegaly with a prominent nucleolus was also a feature of the affected cells. Ultrastructural examination of the affected keratinocytes revealed large and complex, intracytoplasmic virions with elliptical to discoid profiles and an average size of 548.5 nm (SD = 100.58) by 205.65 nm (SD = 21.19) consistent with a typical poxvirus morphology. Furthermore, metagenomic analysis revealed distinct genomic features, whereas phylogenetic analysis of nine core poxvirus genes demonstrated that the seahorse poxvirus forms a unique branch within the teleost poxvirus clade supporting its classification as a novel poxvirus with a proposed species name of Syngnathid cutaneouspox virus. Finally, this study confirms the utility of next-generation sequencing as a methodology for obtaining phylogenetically useful genomic sequences from archival formalin-fixed paraffin-embedded tissues.

Colorectal cancer (CRC) is one of the most common malignancies globally and effective therapeutic strategies are essential. Oncolytic viruses present a promising therapeutic strategy in this regard. In a previous study, we synthesized two oncolytic herpes simplex virus type I (oHSV-1), oHSV-1.1, and oHSV-1.2. Both viruses were attenuated by deleting the virulence genes ICP34.5 and ICP47, while oHSV-1.2 was further armed with murine granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-12 (IL-12) for immune activation. Both viruses exhibit significant anti-tumor efficacy in a murine melanoma model. In the present study, we evaluated the oncolytic potential of oHSV-1.1 and oHSV-1.2, in an immunocompetent murine CRC model and in human tumor cell lines. oHSV-1.2 induced significant tumor inhibition and anti-tumor activity in vivo, as evidenced by tumor regression, enhanced neutrophil infiltration, activation of specific anti-tumor immune responses, and significant pathological damage in tumor tissue. Additionally, both oHSV-1.1 and oHSV-1.2 replicated and exerted cytotoxicity in various human tumor cells in vitro. This study provides valuable insights into the application of oHSV-1.2 for the treatment of CRC.

Metallo-β-lactamase-producing Pseudomonas aeruginosa (MBL-PA) poses a major therapeutic challenge. In search of alternative therapeutic approaches, bacteriophages against MBL-PA were isolated from environmental and clinical samples and thoroughly characterized. Samples from a sewage treatment plant (n = 50), rectal swabs (n = 50), and stool samples (n = 10) from patients were screened. Genomic features, microbiological properties, and growth-inhibition kinetics were determined for all isolated phages. The in vivo pharmacokinetics and therapeutic efficacy of intranasally administered phages, given alone or in combination with intraperitoneally administered amikacin, were evaluated in a murine lung infection model using a clinical MBL-PA isolate. The phages exhibited variable lytic activity against 23-70% of the isolates. The median (range) adsorption rate, time, latent phase, and burst size were 93 (87-95) %, 4 (2-12) min, 30 (20-40) min, and 73 (48-235) PFU/infected cell, respectively. All isolated phages strongly inhibited the growth of their host strains with a median (range) of 88 (56-128)% growth inhibition. In vivo, phage titers reached up to 10[7] PFU/lung in lung tissue and in bronchoalveolar lavage fluid, whereas no phages were detected in blood. Combination therapy with amikacin significantly reduced the bacterial burden by ≥ 2.8 log10 CFU/lung compared with monotherapies. Strong lytic activity and a relatively broad spectrum within the tested isolate collection were observed among the isolated phages against MBL-producing P. aeruginosa isolates. Selected phages exhibited high lytic activity even at the lowest multiplicity of infection tested, making them promising candidates for further investigation in efforts to control multidrug-resistant P. aeruginosa infections, particularly when combined with antibiotics.

Enteroviruses are positive-sense single-stranded RNA viruses that cause a wide spectrum of human diseases, ranging from mild febrile illness to severe neurological complications. Although most infections are self-limiting, certain strains cause significant morbidity, particularly in young children. The infection outcome largely depends on the balance between viral replication efficiency and host antiviral responses. Among host antiviral responses, type I interferon signaling plays a central role in restricting enterovirus replication by inducing interferon-stimulated genes. This review summarizes the current understanding of interferon-mediated restriction mechanisms targeting enteroviruses, with a particular focus on representative antiviral effectors, including IFITs, PKR, OAS/RNase L, ZAP, and shiftless (SHFL, also known as C19orf66). SHFL has become an important interferon-stimulated restriction factor with antiviral activity against several RNA viruses, including enteroviruses. In enteroviruses, current evidence supports the role of SHFL in targeting viral replication proteins (such as 3Dpol) and reducing viral RNA accumulation, while other mechanisms described in other RNA viruses, including their effects on viral translation, have been discussed as potential or context-dependent activities. In addition, enteroviruses have evolved mechanisms to counteract interferon signaling and restriction factor activity, highlighting the sustained interaction between virus replication and host intrinsic immunity. A comprehensive understanding of interferon-stimulated restriction factors and virus evasion strategies may provide new insights into the pathogenesis of enteroviruses and guide future host-directed antiviral research.

Zika virus (ZIKV) remains a significant global health concern owing to its association with congenital abnormalities and neurological disease. Although mammalian models have provided important insights into ZIKV biology, complementary systems are needed to enable rapid mechanistic studies and high-throughput genetic analyses. Drosophila melanogaster has emerged as a powerful translational in vivo platform because of its sophisticated genetic toolkit, conserved cellular pathways, and whole-organism experimental accessibility. This review highlights the utility of Drosophila for modeling ZIKV infection, emphasizing experimental approaches used to investigate viral pathogenesis and host responses. We discuss how fly-based studies have advanced understanding of neurodevelopmental defects, tissue tropism, metabolic reprogramming, and innate antiviral immunity while identifying conserved host factors that influence disease outcomes. Finally, we evaluate the strengths and limitations of the model and discuss future applications integrating Drosophila genetics with multi-omics technologies, vertebrate models, and antiviral drug discovery.

Porcine Circovirus type 2 (PCV2) is an important pathogen that threatens China's swine industry, and the continuous mutation and evolution of its genotypes pose serious challenges for disease prevention and control. Although southern China is a crucial production area for pig farming, systematic analyses of the genetic and evolutionary characteristics of the circulating PCV2 strains in this region are lacking. To elucidate the epidemiological status and biological characteristics of PCV2 variants in southern China, we analyzed 339 full-genome sequences of PCV2 isolates collected from the region between 2015 and 2024, along with 38 reference strains. Using bioinformatics software, we constructed a phylogenetic tree and performed nucleotide homology alignment, amino acid sequence alignment, selection pressure analysis, and antigenic epitope prediction to providing the first comprehensive overview of the genetic evolution of PCV2 in this region over the past decade. Our results demonstrate that the PCV2d genotype predominated at 56.5%, whereas PCV2b at 23.9% and PCV2a at 8.0% exhibited subdominant distributions, indicative of the co-circulation of multiple genotypes. The ORF2 gene is primarily under purifying selection; however, signals of genotype-specific positive selection are also present. Key amino acid variation sites are mainly located on the surface of the capsid protein and within antigenic epitope regions, suggesting that these sites may be subjected to host immune selection pressure. The predictions of B-cell epitopes indicate that the amino acid regions at positions 175-180 are highly conserved, providing targets for subunit vaccine design. Notably, we identified an independent lineage comprising two strains, which occupies a unique phylogenetic position and exhibits a specific amino acid mutation pattern, potentially representing an unclassified genetic cluster of PCV2. Furthermore, this study indicated that PCV2 in southern China exhibits characteristics of both genotype turnover and polymorphism. Persistent variation in the ORF2 gene drives viral evolution, and the discovery of the unclassified genetic cluster further enhances our understanding of the genetic diversity of PCV2, providing an important theoretical basis for formulating targeted prevention and control strategies as well as vaccine updates.

Ascoviruses are large double-stranded DNA viruses that mainly infect lepidopteran larvae, and may have potential for development as bioinsecticides. Compared with well-studied baculoviruses, interactions among ascovirus virion structural proteins remain unknown. The protein composition of virions of Heliothis virescens ascovirus 3h (HvAV-3h), an ascovirus isolate reported in China, was analyzed using mass spectrometry (MS) in our previous study; however, how the 29 common virion structural proteins interact with one another remained unknown. The primary aim of this study was to systematically identify protein-protein interactions among the 29 common virion structural proteins of HvAV-3h, and further explore the potential assembly pattern of the virion. These proteins were expressed using the Gal4 yeast two-hybrid (Y2H) system. A total of 841 (29 × 29) direct cross Y2H assays were performed, and 192 interactions were identified, including 126 non-self pairwise interactions and 14 self-associations, with the remaining interactions described in the text. The self-associations of 3H-43, 3H-53, and 3H-107 were further confirmed using pull-down assays. A theoretical interaction network of the 29 proteins was constructed using R software. Combined with previously obtained MS data, the possible arrangement patterns of these proteins in HvAV-3h virions produced in Mythimna separata, Spodoptera exigua, Spodoptera frugiperda, and Spodoptera litura larvae were predicted and compared. The results of this study provided a theoretical foundation for understanding ascovirus virion structure and differences in ascovirus pathogenicity among host larval species.

Primary infection by Epstein-Barr virus (EBV) often causes infectious mononucleosis (IM), which is frequently accompanied by acute hepatitis despite the absence of direct EBV infection of hepatocytes. Our previous studies using murine γ-herpesvirus 68 (MHV68), a murine model of EBV, demonstrated that intestinal bacteria contribute to hepatitis induced by MHV68 airway infection, although the underlying mechanism remains unclear. In the present study, we investigated the effect of MHV68 airway infection on intestinal barrier function. C57BL/6 mice were intratracheally inoculated with MHV68. Intestinal permeability was assessed by the FITC-dextran permeability assay. Total fecal IgA and mucin levels were assessed as intestinal barrier-associated parameters. Bacterial translocation to the liver was evaluated by recovering intrarectally administered GFP-expressing Escherichia coli from the liver. The effect of neomycin on bacterial translocation was also examined. Infected mice exhibited increased FITC-dextran permeability and reduced total fecal IgA and mucin levels, consistent with a leaky gut phenotype. Both GFP-expressing E. coli and endogenous bacteria were recovered from the liver, demonstrating bacterial translocation, albeit transiently, to the liver. Oral administration of neomycin reduced bacterial translocation and attenuated hepatic inflammation. To gain insight into a possible mechanism underlying intestinal barrier dysfunction, we also evaluated circulating cytokine responses during MHV68 infection. Collectively, we found intestinal barrier dysfunction upon MHV68 airway infection, possibly through systemic cytokine responses and resulting in transient intestinal bacterial translocation to the liver. This process likely contributes to MHV68-induced hepatitis and further supports that targeting intestinal bacteria represents a potential therapeutic strategy for IM-associated hepatitis. (250/250 words).

Pasteurella multocida serotype D is a major pathogen responsible for severe respiratory disease in swine, resulting in substantial economic losses worldwide. The increasing prevalence of antimicrobial resistance highlights the urgent need for alternative control strategies. In this study, we report the isolation and comprehensive characterization of a novel, strictly lytic phage, PGG01, that specifically infects P. multocida serotype D. Phage PGG01 is stable over a pH range of 5-9 and at temperatures up to 50 °C, reflecting conditions relevant to swine production environments. Host-range analysis shows its high specificity and potent lytic activity against serotype D strains. One-step growth curve analysis reveals a latent period of approximately 20 min followed by a distinct burst phase, indicating efficient replication dynamics. Whole-genome sequencing demonstrated that PGG01 possesses a 34.7-kb double-stranded DNA genome lacking genes associated with virulence, antibiotic resistance, or lysogeny, supporting its favorable safety profile. Phylogenetic and intergenomic analyses place PGG01 within a distinct evolutionary lineage of virulent P. multocida phages, clearly separated from their temperate counterparts. Collectively, these findings identify PGG01 as a promising and safe biocontrol candidate and provide a foundation for the development of phage-based strategies to control pasteurellosis.

Influenza B viruses (IBV) elicit weaker, less durable antibody responses than influenza A viruses (IAV) in ferrets. Using an ex vivo primary ferret nasal epithelial cell (FNEC) and peripheral blood mononuclear cell (PBMC) co-culture model, we demonstrate that this deficit is not caused by poor viral fitness. IAV and IBV replicate to equivalent apical titers (p = 0.190) and trigger equivalent basolateral interferon lambda (IFNλ) protein (p > 0.98) and induction of RIG-I, IFNλ3, and TSLP (p > 0.83) at 48 h post-infection (hpi). However, subsequent downstream signaling in recruited PBMC diverges. At 48 hpi, IBV induces uncoupled signaling featuring 1160.1-fold TYK2 induction (p = 0.032). By 72 hpi, IAV maintains pro-inflammatory output, whereas IBV shifts toward a regulatory phase marked by GZMA (3.24-fold; p = 0.001), STAT3 (7.59-fold, p = 0.003) and TGFβ (3.97-fold, p = 0.003). Spearman's analysis confirms the TYK2 vs. CCL5 axis is decoupled during IBV infection (rs = 0.5, p = 0.450) compared to robust coupling in IAV (rs = 0.89, p = 0.033). This signaling decoupling across the "epithelial-immune bridge" drives suboptimal IBV immunogenicity.

The function of many viral proteins encoded by the open reading frames of herpes simplex virus have been studied and characterized, while others remain obscure. UL45 encodes an 18 kDa (18K) non-glycosylated virion envelope protein that is dispensable for in vitro replication. Previous studies reported that it was a mediator of glycoprotein B cell-cell fusion in infected cells, and that gB driven fusion did not occur in the absence of 18K. Using a HSV-1 strain 17 bacterial artificial chromosome we generated a point mutation that removed the UL45 start site. An HA epitope tag was added to the C-terminus for detection in western blotting. Recombinant viruses containing either glycoprotein B (gB) or glycoprotein K (gK) mutations that confer a syncytial phenotype, and with or without UL45 expression, were generated to revisit the role of 18K in mediating cell fusion. We were unable to reproduce the previously reported phenotype and, using this new set of recombinant viruses, showed that UL45 expression was not required for either gB- or gK-driven syncytial plaque formation.

Discriminating virus particles from extracellular vesicles (EVs) is critical for studying their distinct roles in infection. However, it remains a longstanding challenge to discriminate viruses from EVs because both particle types can exhibit similarities in size and protein compositions. Here, we used flow virometry (FV) with a dual staining strategy that combines surface antibody labeling of host-derived antigens with intravirion staining of the viral capsid protein, HIV Gagp24. This approach revealed substantial heterogeneity in surface proteins present on EVs (Gagp24 negative particles) and viruses (Gagp24 positive particles) present within HIV-1 virus stocks. The dual staining approach was also able to successfully confirm immunodepletions of putative EVs and viruses that were targeted for their prominent surface antigens. Reduced Gagp24 concentration and decreased infectivity from the virus-depleted supernatants further validated the discernment of putative viruses from EVs using the dual staining approach. Furthermore, the persistence of particles within immunodepleted supernatants indicated the presence of EV and virus subsets bearing distinct repertoires of surface antigens. To our knowledge, this is the first demonstration of staining an antigen inside intact HIV-1 particles using FV. These FV-based dual staining approaches provide a powerful new tool to better differentiate HIV-1 particles from EVs, and could be useful in future studies aimed at characterizing the interplay of EVs and viruses in infection.

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is well documented in the clearance of various viral infections. Beyond direct virus neutralization, virus-specific antibodies confer protection through effector functions involving ADCC. NK cells have been identified as key contributors in chikungunya pathogenesis. This study evaluates NK cell-mediated ADCC involvement in chikungunya by assessing chikungunya virus (CHIKV)-specific ADCC responses in 56 acute, 56 prolonged, and 44 chronic chikungunya arthritis patients, 23 recovered individuals, and 50 healthy controls. A flow-cytometry-based antibody-dependent heterologous NK cell activation assay was employed, utilizing CD107a and IFN-γ as surrogate markers. Group comparisons were performed using the Mann-Whitney test, binary data were analyzed by Fisher's exact test, and correlations between ADCC response, CHIKV viral loads, total anti-CHIKV IgG, and IgG3 antibody levels were assessed by Spearman rank correlation. P values < 0.05 were considered significant. Persistence of ADCC response was observed in recovered individuals for durations ranging from 3 months to 18 years post-infection, while a progressive decline was noted in patients with chronic chikungunya arthritis. An inverse association between ADCC response and viral load was observed in a subset of acute patients. Recovered individuals demonstrated a positive correlation between ADCC response and plasma anti-CHIKV IgG and IgG3 antibody levels (IgG: r = 0.4503, IgG3: r = 0.4583, p-value < 0.01 for each). Collectively, these findings establish the role of NK cell-mediated ADCC response in chikungunya infection and provide a foundation for future antibody-based therapeutics for chikungunya, particularly utilizing IgG3 antibodies with functional ADCC capacity.

Among feline coronaviruses (FCoVs), feline enteric coronavirus (FECV) is a ubiquitous enteric pathogen in feline populations and responsible for asymptomatic to mild enteric disease. However, in a small number of cats, viral mutations within the host combined with impaired host immune responses may lead to feline infectious peritonitis (FIP), a fatal, systemic infection. Programmed death-1 (PD-1) is a major immune-checkpoint protein expressed on activated T cells, and its engagement by the ligands PD-L1 and PD-L2 on target cells leads to suppression of T-cell activity. Previously, we found that infection with FIPV, but not FECV, up-regulated the expression of PD-L1 in feline cell lines, which attenuated T-cell activities. We also demonstrated that feline interferons (IFNs), particularly IFN-γ, are potent inducers of PD-L1 in cells. In this study, we assess the expression of PD-L1 in association with FIPV in the tissues of four cats with naturally occurring FIP using histopathology, real-time RT-qPCR, and confocal microscopy. By confocal microscopy, we found that numerous cells co-localized with FCoV and PD-L1 in various tissues of all animals, with about 50% of FCoV-positive cells also stained with PD-L1. Interestingly, about 10% of PD-L1 positive cells were also positive for FCoV, which is likely due to severe inflammation and cytokines such as IFN-γ. These findings support a tissue-level association between PD-L1 expression and FCoV infection in some tissues from cats with FIP, while also indicating substantial variation among tissues and animals.