Rift Valley fever virus (RVFV) can cause severe disease in humans and livestock. Several vaccine candidates are immunogenic and protective in pre-clinical animal models. Reported measurements of humoral immunity following vaccination typically assess antigen-specific activity, such as virus binding or neutralization, but do not consider the role of the Fc region. To address this knowledge gap, multiplexed Fc array assays were developed to characterize the Fc profile of antigen-specific antibodies following RVFV vaccination. The RVFV nucleocapsid protein (N) and glycoproteins (Gn/Gc) were conjugated to magnetic microspheres, and monoclonal antibodies were used to optimize assay conditions. Sera from vaccinated mice were incubated with the conjugated microspheres to capture antigen-specific antibodies, and then various detection reagents were used to measure the isotype of the antibodies and their ability to bind Fc receptors (FcRs) or complement (C1q). Two studies involving mice vaccinated with live-attenuated RVFV ΔNSsΔNSm or ΔNSs were used to demonstrate the utility of these assays. In WT C57BL/6 mice, both ΔNSsΔNSm and ΔNSs induced a high magnitude of IgG2c antibodies, indicative of a Th1-biased response; these antibodies also bound the four murine FcRs and C1q. However, following ΔNSs infection of a panel of innate immune knockout (KO) mice, several strains of KO mice, especially TLR3 KO, exhibited a reduced magnitude of IgG2c antibodies, demonstrating the impact of innate immunity on the Th1-biased response. Overall, the multiplexed Fc array assays performed as expected and can be applied to other RVFV vaccine platforms in the future to help inform rational vaccine design.

Labial gland disease viruses (LGDVs) are recently identified dsDNA viruses infecting ants, but their diversity, genomic coherence, and taxonomic boundaries have remained unresolved. Here, we expanded LGDV members by mining public arthropod genome assemblies and sequencing field-collected ants. Novel LGDV genomes were recovered from Pheidole selathorax, Proformica mongolica, Messor orientalis, and geographically distinct Anoplolepis gracilipes populations. Comparative genomic analyses demonstrate that LGDVs retain the conserved core genes in the order Lefavirales, class Naldaviricetes, share additional conservative genes with Apis mellifera filamentous virus and related viruses (AmFV/AmFLV), and encode an LGDV-restricted conservative gene set. Amino acid identity, conservative site, and gene neighborhood analyses support the internal coherence of LGDVs while distinguishing them from AmFV/AmFLV, which retained only partial local gene neighborhood similarities and lacked detectable genome-wide macrosynteny with LGDVs. Phylogenies inferred by protein sequences and structures were broadly congruent, showing that predicted protein structures retained complementary lineage-discriminating signal across deeply divergent naldaviricetes, and recovering LGDVs as a monophyletic sister group to AmFV/AmFLV. Molecular dating further supported a deep separation between these lineages, indicating that LGDVs and AmFV/AmFLV represent distinct, long-term divergent groups rather than recent derivatives of one another. Collectively, these findings support LGDVs as an internally coherent, deeply divergent, and ant-associated virus family in the order Lefavirales, class Naldaviricetes.

Noroviruses are a leading cause of acute gastroenteritis and impose a substantial global disease burden, particularly in children younger than 5 years and in older adults. Although multiple vaccine candidates are in preclinical and clinical development, no targeted intervention has been approved. We previously reported that a pentavalent mRNA vaccine candidate encoding VP1 proteins from genotypes GI.1, GII.2, GII.3, GII.4, and GII.6 elicited robust humoral and cellular immune responses in mice and nonhuman primates. Because recent surveillance data indicate increasing prevalence of GI.3 and GII.17, we designed mRNAs targeting these genotypes and evaluated an expanded seven-valent formulation in preclinical studies. We first compared candidate GI.3 and GII.17 strains in mice and selected vaccine components based on immunogenicity and cross-reactivity. The resulting seven-valent mRNA vaccine elicited strong humoral responses in mice and African green monkeys, including serum IgG binding to norovirus virus-like particles (VLPs) and functional antibodies that blocked VLP binding to histo-blood group antigens (HBGAs). Importantly, incorporation of GI.3 and GII.17 broadened functional coverage, whereas multivalent formulation introduced only limited evidence of immune interference. These findings support the feasibility of surveillance-guided updating of multivalent norovirus mRNA vaccines and highlight the adaptability of the mRNA platform for addressing emerging variants.IMPORTANCENorovirus is a major cause of vomiting and diarrhea worldwide, but developing a broadly protective vaccine is difficult because many genetically distinct strains circulate, and their prevalence changes over time. This study demonstrates a flexible vaccine strategy in which mRNA components can be selected and added based on disease surveillance and experimental evidence. We expanded a five-component norovirus vaccine to include GI.3 and GII.17, two increasingly important genotypes. The resulting seven-component vaccine generated strong antibody responses in mice and African green monkeys and broadened the range of virus types targeted by functional antibodies. Although additional studies are needed to determine whether these responses protect humans, the findings show how the adaptability of mRNA technology could support periodic updates of multivalent norovirus vaccines as circulating strains change.

Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses have recently infected dairy cows in the United States, raising concerns about human spillover. The antiviral roles of bovine host defense factors, including myxovirus resistance proteins (bMx1 and bMx2), remain relatively understudied. We examined bMx1/bMx2 induction in differentiated bovine cells following HPAI H5N1 infection and assessed their antiviral activity using overexpression, knockdown, and mutagenesis approaches. Both bMx1 and bMx2 were more strongly induced in mammary cells than in tracheal cells. Overexpression of bMx1, but not bMx2, significantly inhibited seasonal influenza A viruses (IAVs), low-pathogenic, and highly pathogenic avian influenza viruses, including H5N1 2.3.4.4b, but not influenza B viruses. Conversely, knockdown of bMx1 resulted in enhanced IAV growth. Further, bMx1 inhibited early stages of viral replication, acting to reduce viral transcription and subsequent steps in the viral replication cycle. Known mutations that reduce human MxA (hMxA) activity also impaired bMx1 function, and mutations in the viral nucleoprotein (NP) associated with resistance to hMxA also reduced virus sensitivity to bMx1. Furthermore, phylogenetic analysis showed that a small minority (>0.03%) of avian H5N1 2.3.4.4b viruses already carry some of these NP mutations. These findings suggest that while current H5N1 viruses largely remain susceptible to bMx1, viral evolution or opportunistic reassortment could drive Mx resistance and increase pandemic risk.IMPORTANCEHighly pathogenic avian influenza viruses have spilled over into dairy cows and have been circulating on dairy farms in the United States since 2024. The innate antiviral immune responses of cows to influenza viruses have not been extensively studied. In this study, we focused on Mx proteins, a key part of the innate immune defense system toward viruses. We specifically looked at bovine Mx proteins and demonstrated antiviral activity toward different influenza viruses, including the highly pathogenic strains. We also showed that the mechanism of antiviral activity of bovine Mx1 was very similar to that of the human equivalent MxA protein. Further, we found that a small minority of these avian viruses have mutations associated with resistance to both bovine Mx1 and human MxA proteins. Overall, this work highlights the elevated pandemic risk for highly pathogenic influenza viruses through continual circulation in cows, a mammalian species in close proximity to humans.

Pseudorabies virus (PRV) is the etiological agent responsible for porcine pseudorabies, causing substantial financial losses in the global swine industry. Non-muscle myosin heavy chain IIA (NMHC IIA), as an actin-binding motor protein, has been recognized as a functional factor in herpesvirus entry. However, its precise mechanism in PRV invasion remains poorly defined. Our investigation demonstrated that targeted inhibition of NMHC IIA, using a specific chemical inhibitor, siRNA-mediated knockdown, C-terminus of NMHC IIA (sPRA, amino acids [aa] 1672-1981), or rabbit anti-sPRA serum, significantly decreases PRV infection in PK-15 cells in a dose-dependent manner. During the early stage of PRV infection, NMHC IIA underwent rapid and pronounced translocation from the cytoplasm to the cell surface, which is beneficial to the virus entry. The study identified that sPRA mediates PRV internalization by binding to viral glycoproteins gH and gL. Furthermore, we pinpointed the subdomain spanning aa 1831-1882 within sPRA as the critical domain for both mediating the interaction with gH/gL and facilitating viral internalization. These results advance our comprehension of the molecular pathways through which NMHC IIA supports PRV infection and highlight its potential as a therapeutic target for PRV intervention.

Human cytomegalovirus (hCMV) reactivations and disease frequently occur in hematopoietic stem cell transplant (HSCT) recipients as a consequence of the profound immunosuppression required to prevent graft rejection. Despite clear evidence of the role of hCMV-specific T cells in controlling hCMV replication, quantification of hCMV-specific T cells is not widely performed to inform the clinical management of hCMV reactivations after transplantation, largely due to the lack of a simple and reliable assay to assess hCMV-specific cellular immunity. Herein, we developed a simple and rapid whole blood IFN-γ/IL-2 release assay (CRA) that uses overlapping hCMV peptides to quantify the hCMV-specific T cell responses and validated it in the peripheral blood of 40 healthy donors and 20 HSCT patients post-transplant. We found that the CRA correlates well with classical IFN-γ ELISpot assays but demonstrates greater sensitivity in detecting hCMV-specific T cell responses in HSCT recipients. Notably, the CRA also captured dynamic changes in hCMV-specific T cell responses associated with hCMV reactivation. Collectively, these findings demonstrate that the CRA is a simple and rapid method for longitudinal monitoring of hCMV-specific T cell immunity in HSCT recipients, with potential to inform clinical management of hCMV reactivations.

Epstein-Barr virus (EBV) is a human gammaherpesvirus that establishes a lifelong, persistent infection in greater than 90% of people worldwide. EBV has lacked an animal model that depicts the viral lifecycle in B cells and epithelial cells, allows for the investigation of immune responses, and models persistence in a host. EBV establishes a robust infection in immunosuppressed rabbits, resulting in widespread infection of the spleen and the development of splenomegaly. Within the spleen, follicle formation was altered as a result of EBV infection. Similar magnitudes of infection were observed in immunosuppressed rabbits infected with the B95-8 and Akata viral strains. In immunocompetent rabbits, spleen morphology appeared similar to that of mock-infected rabbits. Rabbits infected with B95-8 and Akata viruses developed antibody responses to the EBNA family of proteins in a comparable manner to human infection. Akata infections were more consistent in both the immunosuppressed and immunocompetent studies, whereas B95-8 infections resulted in variable magnitudes of infection and immune responses. The persistence of EBV in rabbits and whether the full lifecycle of EBV in lymphocytes can be replicated remain to be further examined.

HIV-1 virus assembly is organized by the precursor Gag (PrGag) protein, which, from its N-terminus to its C-terminus, is composed of the matrix (MA), capsid (CA), spacer peptide 1 (SP1), nucleocapsid (NC), spacer peptide 2 (SP2), and p6 domains. Evidence indicates that the full-length, wild-type (WT) HIV-1 envelope (Env) protein is incorporated into virus particles by virtue of interactions of plasma membrane-associated immature lattices assembled by the PrGag MA domains, but that Env proteins with cytoplasmic tail deletions (ΔCT) or truncations do not require such an interaction for virion incorporation. Remarkably, during PrGag proteolytic processing and viral maturation, the free SP2 peptide binds to MA proteins and drives the formation of a distinct mature MA lattice. This lattice maturation is necessary for efficient function of WT but not ΔCT Env proteins. Based on these observations, we tested whether ectopic expression of SP2 variants might inhibit HIV-1 by inappropriately driving the formation of mature MA lattices. We have found that the expression of a chimeric protein composed of the green fluorescent protein (GFP) and SP2 in virus-producing cells causes the assembly of defective viruses in a MA-dependent fashion. Viruses produced in the presence of GFP-SP2 had aberrant morphologies and incorporated significantly less Env, regardless of whether the Env protein was WT or CT-truncated. Our results suggest that it might be possible to develop SP2 derivatives into antivirals.IMPORTANCEThe matrix domain of the HIV-1 structural protein forms a hexagonal lattice at the plasma membranes of infected cells, and this lattice helps direct the assembly of immature virus particles. The immature matrix lattice is also instrumental in facilitating the incorporation of viral envelope proteins into virus particles. Surprisingly, when the HIV-1 protease processes the structural protein into its mature components, one of the peptides produced, spacer peptide 2, drives the reorganization of the immature matrix lattice into a mature form. This maturation step appears essential for envelope proteins to acquire their full activities. We have found that expressing a spacer peptide 2 variant in virus-producing cells results in the assembly of defective viruses. The viruses have aberrant morphologies and contain significantly less envelope protein than wild-type viruses. Our results suggest that it might be possible to develop spacer peptide 2 derivatives into antivirals.

The innate immune system is rapidly activated following antigen exposure and plays a critical role in shaping the ensuing adaptive immune responses. In this study, we performed bulk RNA sequencing and proteomic profiling in a cohort of 25 rhesus macaques following Ad26.COV2.S immunization to characterize early innate correlates of vaccine immunogenicity and protection. Our results show that innate immune activation occurred as early as day 1 post-vaccination and demonstrated a systemic enrichment of antiviral interferon pathways, cytokine signaling, and activation of innate immune cell signatures. These early transcriptomic signatures correlated positively with humoral and cellular immune responses at 6 weeks following vaccination and correlated inversely with viral loads following SARS-CoV-2 challenge. Similar correlates of immunogenicity were observed in a cohort of 25 adult healthy participants vaccinated with Ad26.COV2.S. Taken together, these findings highlight the importance of early activation of the innate immune responses following Ad26.COV2.S vaccination in shaping the protective adaptive immune response.IMPORTANCEEarly innate immune responses may shape vaccine immunogenicity and protection. We evaluated responses to Ad26.COV2.S vaccination in rhesus macaques and humans and found that day 1 innate immune signatures correlated with subsequent adaptive responses. These findings suggest that early post-vaccination immune programming influences immunogenicity and protective efficacy months later.

In lytic infection, Epstein-Barr virus (EBV) proteins manipulate cellular processes in multiple ways to facilitate virion production and overcome host antiviral responses. Here, we screened a library of EBV lytic proteins for those that manipulate or localize to cellular processing bodies (PBs), known to play important roles in RNA surveillance and miRNA-mediated translational control. One EBV protein, BRRF1, was identified that partially localized to PBs, and affinity purification-mass spectrometry experiments showed that it interacted with the CCR4-NOT complex-a known component of PBs with multiple roles in post-transcriptional mRNA regulation. The CNOT9 subunit of CCR4-NOT was sufficient to bind BRRF1, and its downregulation diminished BRRF1 interaction with the CNOT1 scaffold protein, indicating its importance in mediating the CCR4-NOT interaction with BRRF1. A BRRF1 double-point mutant was generated that failed to bind CNOT9 or interact with CCR4-NOT and was used to assess the importance of the BRRF1-CCR4-NOT interaction. Luciferase reporter assays identified multiple roles for BRRF1 in post-transcriptional regulation, including polyadenylation-dependent destabilization effects and interference with miRNA function, the latter of which was dependent on BRRF1 binding to CCR4-NOT. In addition, mRNA sequencing experiments comparing cells with and without BRRF1 expression identified multiple transcripts affected by BRRF1, some of which were at least partly dependent on the BRRF1-CCR4-NOT interaction. These include Claudin 6, whose transcripts and protein were downregulated by BRRF1 expressed on its own, as well as in lytic infection. Thus, we have identified multiple roles for BRRF1 in post-transcriptional mRNA regulation, some of which involve interaction with CCR4-NOT.

Viral-vector-based tracers have proven invaluable for mapping neural circuits. Rabies virus (RV)-based retrograde trans-monosynaptic tracers have been widely used for depicting the input neural circuit of targeted neuronal populations. However, the cytotoxicity of the RV tracer limits its application. Here, we employed the synthetic attenuated virus engineering technology to recode the N gene of the RV SAD-B19 strain by codon pair deoptimization to generate attenuated RV tracers. The characteristics of the recoded RV tracers were tested both in vitro and in vivo. The full-length recoded N gene abolished viral rescue, whereas the partial recoded N gene supported successful viral recovery. Compared to its parent, the recoded RV trans-monosynaptic tracer exhibited lower cytotoxicity and labeled more input neurons. It can also robustly express proteins to visualize the neuronal morphology and manipulate the neural circuit activity. Collectively, our work generated a new retrograde trans-monosynaptic tool, which complements existing neural circuit tracers.

Subgroup J avian leukosis virus (ALV-J) is an oncogenic retrovirus posing significant threats to the global poultry industry. While host factors are known to regulate retroviral reverse transcription, the specific host proteins that support this critical step in ALV infection have remained completely unknown. In this study, we confirmed that overexpression or knockdown of eEF1A significantly enhanced or suppressed ALV-J proliferation, respectively. Co-immunoprecipitation and molecular docking demonstrated an association between eEF1A and the ALV-J reverse transcriptase (RT). Furthermore, three conserved residues in eEF1A domain I, including glutamate 124 (E124), lysine 129 (K129), and arginine 134 (R134), were identified as critical for the eEF1A-RT association. Alanine substitution at these EKR residues disrupted the eEF1A-RT association, markedly impairing viral reverse transcription and infectious virion production. From the viral perspective, a conserved tryptophan (W250) in the RT thumb domain acts as the key counterpart to the eEF1A EKR motif, and its mutation similarly abolishes the association and viral replication. Notably, this eEF1A-RT association and its functional significance are conserved across ALV subgroups A, B, K, and J. Together, our work elucidates that three residues E124, K129, and R134 within domain I of eEF1A mediate the association with ALV-J RT, promoting viral replication by enhancing reverse transcription efficiency. This finding provides the first mechanistic insight into host-dependent regulation of reverse transcription in ALV infection, broadens the understanding of how retroviruses co-opt host factors to facilitate this critical process, and provides a rational basis for genome editing of eEF1A to generate broad-spectrum ALV-resistant poultry lines.IMPORTANCEAvian leukosis viruses (ALVs) cause severe economic losses to the global poultry industry, and current disease-resistant strategies targeting viral entry receptors only work for specific subgroups. This study identifies that the host protein eEF1A associates with ALV reverse transcriptase via conserved key residues to promote viral reverse transcription and replication, and this regulatory mechanism is conserved across multiple ALV subgroups. These findings provide a novel target for developing broad-spectrum ALV-resistant poultry breeds through genome editing, with important implications for poultry disease control.

Orbiviruses comprise numerous species and are transmitted by insect vectors. They are non-enveloped viruses with a multilayered capsid and a segmented double-stranded RNA (dsRNA) genome. Bluetongue virus (BTV), the prototype species of the genus Orbivirus, is a significant pathogen of livestock and is widely distributed throughout the world, causing serious periodic outbreaks with substantial economic consequences. Consequently, BTV has been extensively studied as a model orbivirus, resulting in a comprehensive understanding of its molecular epidemiology, genetics, biochemistry, structure, and replication cycle. This includes detailed insights into virus entry into host cells, synthesis of progeny viral components, assembly of new virions, and subsequent cell-to-cell transmission. This review summarizes the current literature on each of these aspects and discusses how this knowledge has been applied to develop novel vaccines against BTV and related orbiviruses. In addition, the review highlights important gaps in our understanding of virus replication within insect vectors, particularly Culicoides biting midges (gnats), at the molecular level. Addressing these gaps will provide valuable insights into orbivirus evolution, transmission, and the emergence of new viral strains.

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic infectious agent and causes animal and human illness of the central nervous system. Japanese encephalitis virus inflicts severe harm, yet even with an approved vaccine, prevention and control remain daunting, and the molecular mechanism underlying the virus-host interplay remains incompletely understood. In this study, we found that the host peroxiredoxin 1 (PRDX1) protein inhibits JEV replication. PRDX1 targets nonstructural protein 1 (NS1) of JEV and mediates its degradation via the proteasome and autophagy pathways, thus inhibiting viral replication. By targeting TRAF6, an E3 ligase, the PRDX1 protein catalyzes the ubiquitination of NS1, leading to its recognition by NDP52, a cargo receptor, along with lysosomal degradation. We also found that the JEV NS3 protein can target and degrade PRDX1 to decrease the antiviral function of PRDX1, which is a tactic by which viruses confront the host. In conclusion, these findings showed that selective autophagy has dual functions in the host and JEV: it can degrade viral proteins to inhibit viral replication and host antiviral proteins to promote viral replication.IMPORTANCEJapanese encephalitis virus (JEV) is a flavivirus capable of inducing human encephalitis and dysgenesis in gestating sows. It has an extraordinary ability to hijack cellular systems and evade defenses, establishing infection and spreading it throughout the body to cause disease. During viral infection, the host initiates several antiviral responses and activates various host-restricted factors to resist viral invasion. In this study, we identified PRDX1 as a novel host restrictor that inhibits viral multiplication by binding to TRAF6 (an E3 ubiquitin ligase) and NDP52 (a cargo receptor) to degrade nonstructural protein 1 (NS1) of JEV. We also found that JEV infection can downregulate intracellular PRDX1 expression via nonstructural protein 3 (NS3), thus promoting viral replication. These results revealed that the virus-host interaction is a complex and dynamic process in which viruses restrict and compete with each other.

During the coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), antibody-based therapies such as COVID-19 convalescent plasma (CCP) and monoclonal antibodies (mAbs) emerged rapidly. However, many CCP clinical trials failed to show consistent benefits, and mAbs lost effectiveness as new SARS-CoV-2 variants emerged. These studies consistently indicated that antibody therapies are effective when given very early in disease and in patients with delayed or suppressed immune responses. Intravenous immunoglobulin (IVIG), which is pooled IgGs from thousands of donors, has been explored as an additional source of anti-SARS-CoV-2 antibodies to treat acutely ill patients because IVIG was shown to prevent disease and death in animal models against variants even without neutralizing activity. As antibodies can also protect via Fc-mediated activation of innate immune effector functions, in this study, we compared the qualitative and quantitative properties of CCP and IVIG to determine if IVIG might serve as a clinical therapeutic alternative to CCP. We measured SARS-CoV-2 spike (S)- and nucleocapsid (N)- specific antibody-binding titers across variants and evaluated their ability to induce antibody-dependent effector functions. We found that IVIG and CCP had comparable binding and Fc-effector activity against ancestral SARS-CoV-2. Remarkably, IVIG manufactured before the emergence of XBB.1.5, JN.1, KP.2, XEC, and XFG demonstrated strong binding and Fc-effector activity against these newer variants.IMPORTANCEDespite the availabilities of vaccines for SARS-CoV-2 for most individuals, there remain vulnerable populations for which vaccination is ineffective. As the emergence of SARS-CoV-2 variants has rendered many monoclonal antibody therapies ineffective, there remains a need to understand the role of polyclonal antibody therapies for SARS-CoV-2, such as COVID-19 convalescent plasma (CCP) and intravenous immunoglobulin (IVIG). While CCP is variable and donor-dependent, IVIG is pooled from thousands of donors with a range of vaccine- and natural infection-derived antibody responses and may represent a consistent source of polyclonal antibodies. We compared high neutralizing antibody titer CCP, previously shown to be associated with reduced time to hospital discharge, with IVIG produced post-2021. We find that IVIG offers expanded antibody binding to SARS-CoV-2 variants, including those that emerged after the IVIG was collected, and enhanced induction of Fc-mediated innate immune effector functions, highlighting the potential utility of IVIG as an adjunct treatment of SARS-CoV-2.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT04524507.