The highly conserved alphaherpesvirus inner tegument protein UL37 has diverse functions in viral replication and manipulation of the host innate immune response. Understanding species differences among alphaherpesviruses may help elucidate conserved functions of the inner tegument protein UL37. To compare functional differences of UL37 among alphaherpesviruses, we used bovine herpesvirus type 1 (BoHV-1), as a representative of genus Varicellovirus, to determine the role of UL37 in intracellular transport and innate immune regulation. The UL37 gene was modified by double-red recombination in conjunction with the viral genome cloned as a bacterial artificial chromosome (BAC). Insertion of premature double
stop codons within amino acids 34-35 resulted in a lack of UL37 expression as detected by western immunoblots using anti-BoHV-1 UL37-specific antibodies. Replication kinetics and plaque morphology analysis showed slightly delayed replication and decrease in plaque size for
BoHV-1 ΔUL37 mutant virus as compared to wild-type (WT). In MDBK cells, a slight delay in capsid transport to the nucleus was observed at 1 hpi in BoHV-1 ΔUL37 as compared to the WT, indicating the facilitative role of BoHV-1 UL37 in efficient intracellular transport. To analyze the role of BoHV-1 in inhibiting cGAS and type 1 interferon response, we used a FLAG-tagged BoHV-1 UL37 (FLAG-UL37) co-expressed with eGFP-tagged bovine cGAS (bocGAS) in 293 cells. Co-immunoprecipitation assay showed that FLAG-UL37 interacts with bocGAS in 293 cells. Under confocal microscopy, bocGAS appeared to be sequestered in perinuclear zones when co-expressed with FLAG- UL37. Analysis of 2’3’-cGAMP levels indicate that BoHV-1 UL37 targets cGAS function. Infection with BoHV-1 ΔUL37 resulted in significant increase in relative mRNA expression levels of cGAS, interferon alpha (IFNα) and interferon beta (IFNβ) in MDBK cells. This study shows that BoHV-1 UL37 is important in facilitating intracellular virion capsid transport and innate immune regulation in MDBK cells.

The known and unknown diverse virosphere demonstrated the importance of a rapid and adaptable vaccine development infrastructure in responding to any viral emergence and threat of a pandemic. Over the last century, public health authorities have raised concerns about the emergence or reemergence of arthropod-borne zoonotic agents – particularly of the genus Alphavirus, which significantly impact human and animal health. We demonstrate a rapid and responsive pipeline for B- and T-cell-specific multi-target vaccine development that leverages epitope identification through machine learning, protein modeling, and docking to render a collection of viral proteomes into a ranked list of peptide candidates scored for immunogenicity, allele coverage, solubility, and epitope stability in cleavage and processing. We evaluated epitope reactiveness by microarrays, and molecular dynamics simulations to assess the epitope's correct binding to the target host receptors. Finally, flow cytometry evaluated T-cell antigen-specific immunogenicity on mice and human peripheral blood mononuclear cells (PBMCs). Most epitopes elicited strong T-cell activation with secretion of IFN-ɣ, TNF-α, or IL-2 – varying by species and HLA allele and final candidates were selected by overall scores. Our data support the development of broadly protective pan-alphaviral vaccines and establishing efficient and tunable processes for vaccine development in a global setting.

The orthoflavivirus Bussuquara (BSQV) was discovered in Brazil in 1956, with sporadic virus isolations or antibody detections throughout the Americas in diverse mosquito and vertebrate species, including humans. Critical gaps in BSQV knowledge include its capacity for urban transmission and clinical pathogenesis outcomes. Comprehensive field and experimental studies are urgently needed to assess risk factors for emergence.

Consensus RNA sequences of all available BSQV strains were obtained through next-generation sequencing. Phylogenetic analysis and genome annotation were performed to assess orthoflavivirus evolutionary relationships and genome characteristics for each strain. BSQV-infected cells (mosquito, non-human primate) were imaged with transmission electron microscopy. Replication kinetics studies were performed with BSQV infection (MOI=0.01) across a panel of mosquito, mammal, rodent, avian, non-human primate, and human cell lines. Vector competence was assessed in peri-domestic and domestic Aedes and Culex mosquito colonies from Asia, Africa, and South and North America.

We described the initial morphologic, genomic, phylogenetic, and replication relationships of the four BSQV strains available for use. Morphologic (virion diameter, cytopathic effect) and genomic (size, organization, architecture) results were in line with canonical orthoflavivirus characteristics. One of four strains shared greater sequence homology to the related Naranjal virus. Kinetic studies demonstrated robust replications in most mosquito and all vertebrate cell lines. Moderate-to=extreme cytopathic effects were prominent in vertebrate cells, with minimal cytopathy observed in mosquito cells. Several mosquito colonies, particularly Culex, appear refractory to BSQV, while others demonstrated transmission potential with infectious virus present in saliva.

BSQV poses a threat to humans as a generalist arbovirus with a broad host range of susceptible vertebrate and mosquito vectors, with capacity for BSQV transmission across urban and rural transmission cycles.

Viruses are considered the best cellular biologists; with little genetic information they take control of the cell for self-perpetuation and spread worldwide. Learning how this occurs is extremely difficult because of the large number of cellular factors involved and the multifunctional nature of viral components. Alphaviruses (such as Chikungunya virus) have four non structural proteins which have been studied for decades accumulating loads of information on host interactors, enzymatic and structural data on single domains or partial complexes. However, the functional units of replication and host interactions are macromolecular complexes gathering viral RNA, cell membranes and viral and host proteins. I will present the architecture of in vitro reconstituted full replication complexes showing how the complexes can change protein nsPs composition and stoichiometry in order to form evolving membrane associated replication complexes within replication organelles. I will also present how nsP3, a major interactor with the host, forms tubular scaffolds structuring cytoplasmic Alphagranules which gather host and viral factors into a massif interaction hub. Overall, these data depict the mode of action of Chikungunya nsPs explaining with unprecedented detail how only four four viral proteins manage to deploy multiple functions at different times of the infection and reconfigures the cell cytoplasm landscape.

SARS-CoV-2 is an enveloped RNA virus with a genome of approximately 30,000 nucleotides.
From the ancestral form of the virus to the Omicron (Pango Lineage BA.1) variant, SARS-CoV-2
has accumulated a multitude of mutations and amino acid changes. Some of these have resulted in
changes in pathogenicity and in transmissibility. Since its appearance in late 2021, all circulating
genotypes have been sublineages of the Omicron variant, with the results of conserving, and even enriching, the
mutation profile of the virus.
Ancestral variants of SARS-Cov-2 have been observed to compromise genome integrity by causing
virus-induced DNA damage (VIDD) and by impeding DNA repair mechanisms. The effects
culminate in the activation of proinflammatory pathways and virus-induced senescence (VIS).
Three viral proteins (Orf6, NSP13, and N) play a significant role in these mechanisms. Specifically,
Orf6 and NSP13 promote the degradation of the DNA damage-response kinase, while the SARSCoV-2 N protein, by competing with 53BP1, leads to reduced DNA repair.
The objective of this study is investigate the impact of the Omicron mutations on VIDD, and to
better elucidate the role of the viral proteins in generating an inflammatory state by interfering with
the DNA damage repair mechanism.

Zika virus (ZIKV) is an arbovirus linked to severe complications and for which there is no approved vaccine yet. ZIKV is an enveloped virus, the virion is composed by three structural proteins: capsid (C), precursor-membrane (prM) and envelope (E), which is the glycoprotein responsible for mediating cell entry and, consequently, a major target of the antibody response. A relevant complication in the design of a ZIKV vaccine is its proximity to dengue virus and the risk to stimulate cross-reactive and poorly neutralizing antibodies, responsible for antibody-dependent enhancement of infection (ADE).

The often-incomplete maturation process of the E protein together with the metastability of its dimers are responsible for the dynamic behaviour of the virus, the so-called “viral breathing”, which is an essential feature for the virus biology, but it has also important consequences on its antigenicity.

We compared two ZIKV isolates belonging to the Asian lineage: the Brazilian PE243 and the Puerto Rican PRVABC59. Despite sharing high sequence similarity and identical in vitro growth kinetics, PE243 exhibited lower pathogenicity in mouse and poor dissemination capacity, both in mouse and in mosquito. Interestingly, PE243 was more susceptible to antibody-mediated neutralization when treated with polyclonal sera, whilst no differences were observed with monoclonal antibodies, suggesting a higher exposure of cryptic epitopes. Structural stability and dynamic properties of the two viruses were evaluated by molecular dynamics, taking into consideration several key structural descriptors. The comparative analysis indicated that PE243 exhibits a more destabilized and flexible profile.

These findings suggest that the mutations in PE243 may exert an influence on viral structure and dynamics, particularly affecting regions involved in membrane fusion. Tailoring down to the single mutations responsible for it may allow the design of more stable vaccines, able to elicit antibodies with stronger neutralization capacity and lower potential of stimulating Dengue ADE Dengue.

Chikungunya virus (CHIKV), an alphavirus endemic to tropical regions of Africa, Asia, and the Americas, causes a self-limiting febrile illness characterized by fever, headache, arthralgia, and rash. No specific antiviral treatment is currently available for CHIKV infection. AG129 mice, deficient in interferon α, β, and γ receptors, serve as a model to study viral pathogenesis and antiviral efficacy. This study evaluated infection outcomes in AG129 mice inoculated intraperitoneally with 10, 100, or 1000 plaque-forming units (PFU)/mL of CHIKV to identify the optimal viral dose for antiviral assays. Two mice per group were euthanized daily for seven days post-infection to collect serum, spleen, liver, kidney, and lower leg muscle for viral RNA quantification. Survival rates were 80% for mice infected with 10 PFU/mL and 0% for those infected with 100 or 1000 PFU/mL. Viral RNA was detected from day 2 post-infection in serum, spleen, kidney, and muscle in the 100 and 1000 PFU/mL groups, while RNA was detectable from days 3 to 6 in all tissues of the 10 PFU/mL group. These results support the use of 10 and 100 PFU/mL CHIKV to infect AG129 mice as models of mild and severe disease, respectively, for antiviral studies.

Viral infections continue to pose a significant challenge to global health, and the growing emergence of new pathogens underscores the need for innovative tools to study and combat viral threats. Many viruses reorganise the host cytoplasm into specialised membraneless compartments, known as viral factories, that serve as sites for genome replication and assembly. A growing body of evidence indicates that numerous RNA and DNA viruses form liquid–liquid phase separation (LLPS)– driven viral replication factories. These transient yet highly organised biomolecular condensate structures represent a central node in the virus–host interface, and their regulation could be used as a novel antiviral paradigm. Here, we introduce a novel conceptual and experimental framework based on the selective degradation of viral factories. By inducing the controlled disassembly of these replication hubs, we will reveal their contribution to viral replication and demonstrate the feasibility of targeting them as a universal antiviral strategy. This approach provides both a powerful tool to study virus–cell interactions in real time and a foundation for developing next-generation antivirals that function through precise spatial control of degradation within infected cells. Our findings highlight viral factories as an emerging paradigm for understanding and therapeutically exploiting the intracellular organisation of viral replication.

Rubella virus (RV) causes mild illness. However, RV infection during the critical stages of fetal organ development lead to congenital rubella syndrome. The host factors associated with the RV teratogenicity are unknown. In this work, the effect of infection with wild (w) and attenuated (att) variants of the laboratory strain C-77 of RV on gene expression in A549 lung epithelial cells was compared via RNA sequencing. Libraries for sequencing were prepared using the TruSeqRNA Library Preparation Kit v2 (Illumina, USA). Sequencing was performed on the NextSeq500 (Illumina) platform.

In cells infected with C-77att, 1,088 genes were down-regulated and 1,096 were up-regulated, compared with 825 down-regulated and 928 up-regulated genes in cells infected with C-77w. The most up-regulated genes in С-77att-infected cells were IFNL1-4, IFNB1, TLR3, CXCL10, CXCL11, IFIT2, RSAD2, IDO1, OASL, OAS2, TNFSF13B, CASP1, CCL4, CCL5, ISG15, ISG20, IFI27, TRIM22, XAF1, TNFSF10, NCF2, LGALS9, USP18, CD274, EGR1, BST2, KRT17, GBP1-5, UBA7, CX3CL1, HSPA1A, and HSPA1B. These genes are mainly responsible for the production of interferons and chemokines, post-interferon response, apoptosis signaling, and unfolded protein response. In contrast, the most differentially up-regulated genes in C-77w-infected cells were genes, namely, TXNIP, CA9, PTPRN2, TGFBI, PTPRN2, SMOC1, BMP6, NDR1, PTPRN2, CCN5, MAPT, NTRK3, NDRG1, ADGRG1, SDK2, SMOC1, and DKK1, involved in the processes of morphogenesis and embryogenesis, including neuronal, glial, heart, and visual system development, whereas infection with the attenuated variant did not affect the expression of these genes.

Thus, a comparative analysis of gene expression in RV-infected A549 cells revealed that the attenuated variant predominantly caused activation of genes responsible for the immune response, while the wild variant additionally activated the expression of genes involved in gliogenesis and morphogenesis, indicating molecular mechanisms that could potentially be involved in the teratogenic effect of RV.

Bovine respiratory disease (BRD) is a major burden to the cattle industry worldwide. It is a complex poly-microbial disease involving viruses, bacteria, and host stressors. In this study, we use publicly available metatranscriptome data from worldwide respiratory sample datasets to simultaneously investigate how the bovine nasal virome, microbiome, and host transcriptome are associated with disease. We detected fourteen BRD-associated virus species in the samples. Most of them, thirteen, were found in both healthy and diseased animals, and twelve of them were found in samples from at least two continents. Viral diversity and abundance differed significantly based on geographic location, and certain viral pairs co-occur non-randomly. Interestingly, some virus species were more abundant in healthy animals. When examining the bovine microbiome, differences in alpha diversity and beta diversity were observed when considering sampling location and disease state. Mannheimia spp. were detected more abundantly in animals with respiratory disease. Host gene expression analysis revealed that animals with respiratory disease have increased expression of immune effector genes and receptors involved in innate immunity and antiviral response, making host gene expression, and not a specific microbe, the best predictor of disease state in our samples. Our results underscore the complex nature of bovine respiratory disease and the fine balance between host mucosal immunity and respiratory microbial species.