Our research studies the interaction of the FeLV Env A5, previously identified through a RBD library screen, with its host receptor SLC35F2. The natural substrate for SLC35F2 has been identified to be the micronutrients queuine (q) and Queuosine, involved in modifying the tRNA of Asp, Tyr, Asn, and His at the wobble position. Our expanded studies verify and define the role of q/Q in the entry of the FeLV Env A5through competition studies of the drug YM155 with q/Q on SLC35F2 functions.

Using structural predictive methods, we have developed models of the SLC35F2 proteins with two conformations: open inwards towards the cytoplasmic face (EVSF->GGGG) and open outwards towards the extracellular space (AYL->GGG), if expressed on the cell surface. Based on these models, mutant SLC35F2 proteins were expressed in mammalian cells that are biased to each of these conformations. Differential functions have been identified for these two conformations, including the requirement for the virus to enter cells only when the SLC35F2 is open towards the extracellular space (AYL->GGG). Additional studies include subcellular localization to the mitochondria, transport of the drug YM155 and interactions with p53. Metabolomic studies on isolated mitochondria indicate that the principal component analysis of the WT SLC35F2 correlates with the mutant conformation bearing the EVSF-> GGGG changes.

Three Args within the 11 aa randomized RBD region were found to be essential for viral entry. Mutagenesis of the SLC35F2 surface exposed loops has identified acidic residues required for entry of the A5 Env virus. As viruses usurp cellular functions, it was found that amino acids in one of these acidic regions on the cell surface is required for YM155 transport in addition to residues located within the central pore.

Potent broadly neutralizing antibodies (bnAbs) can protect against immunodeficiency virus infection. However, it is presently unclear how to elicit bnAbs by vaccination. As an alternative, we evaluated adeno-associated virus (AAV) delivery of rhesus macaque antibodies to the SIV envelope glycoprotein for protection of macaques against mucosal SIV challenge. AAV vectors encoding a bnAb or an antibody that only mediates antibody-dependent cellular cytotoxicity (ADCC) against SIV-infected cells were administered individually or together to separate groups of eight rhesus macaques. Sustained antibody expression with minimal anti-drug antibody responses was achieved in most animals. After sixteen weeks, the animals underwent twelve weekly low-dose, intrarectal (IR) challenges with SIV_mac239. All animals that received vectors encoding a control antibody or the antibody that only mediates ADCC became infected after four challenges. However, fourteen of the sixteen animals that received the bnAb, either alone or in combination with the ADCC antibody, resisted all twelve challenges. A year later, serum concentrations of the vectored antibodies were unabated, so the protected animals and six naïve control animals were rechallenged by repeated, low-dose IR inoculation with SIV_mac239. While all of the control animals became infected after six challenges, the fourteen animals that received the bnAb remain uninfected after nine challenges. Thus, sustained expression of a potent bnAb can afford durable protection against pathogenic SIV challenge.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a lethal pathogen with pandemic potential. Clades A and B MERS-CoV strains have caused periodic outbreaks in the Middle East since 2012 when they spilled over from camels to humans. Meanwhile, Clade C MERS-CoV strains are found only in camels across Africa and do not appear to cause outbreaks in humans. Here, we sought to understand whether differences in viral entry pathways underlie the reduced spread from camels to humans. We characterized the replication and viral entry of a panel of six clade C strains from West and East Africa and found that both West and East clade C strains were attenuated for replication in human lung cell lines relative to clade A/B viruses. We report that clade C spike proteins are less well-cleaved at the S1/S2 boundary than clade A or B viral spikes during viral infection and that a majority of this clade C panel induce reduced syncytium formation. Additionally, we demonstrate that while West African clade C strains have a similar propensity to use the TMPRSS2-mediated pathway for viral entry, East African clade C strains are less able to utilize this protease for entry in both lung-derived cell lines and primary nasal epithelial cultures. We map the molecular determinants of this reduced TMPRSS2 usage to both the N-terminal domain (NTD) and subdomain 2 (SD2) of spike. We suggest that reduced usage of the TMPRSS2-mediated entry pathway may contribute to the reduced replication of East African clade C strains in humans by altering cellular and organ tropism; moreover this indicates geographically distinct selection pressures on spike among MERS-CoV strains circulating in camels. Deciphering the barriers to spillover of the camel reservoir of MERS-CoV in Africa will facilitate pandemic preparedness and improve our understanding of species-specific factors driving viral evolution.

Myxoma virus (MYXV), endemic in Spain since 1952, is the causative agent of myxomatosis, a highly prevalent disease in European rabbits. In mid-2018, a myxomatosis outbreak was detected in the Iberian hare, a species indigenous to the Iberian Peninsula. MYXV isolated from infected Iberian hare tissues was identified as a novel recombinant MYXV strain, denominated ha-MYXV. This virus showed significant differences compared to the MYXV genome, highlighting a novel insert (Ins-H1) of 2.8 kb (containing M157L, M158L, M159L and M160L genes) that interrupts the M009 ORF. As an emerging virus occupying a novel ecological niche, the genetic stability of ha-MYXV is of particular interest. Therefore, the aim of this study was to monitor the Ins-H1 region using 106 lagomorph specimens, covering the eight-year period since its emergence, collected from 28 provinces across Spain. In addition, genomic analysis of several recent isolates was conducted using Oxford Nanopore Technologies. No mutations were detected in M157L or M158L and, in general, the M160L gene was highly conserved. M159 showed the most variation in the isolates analysed, demonstrating hypervariable microsatellite regions that may play a role in the evolution of ha-MYXV. The gene suffered alterations in the form of triplet nucleotide insertions and deletions, always maintaining the predicted ORF. It is interesting to speculate that the extensions and reductions in gene length may play a role in the adaptation of this virus to its novel host and may even facilitate further attempts to jump to closely related species. The identification of microsatellite regions within the M159 ORF led us to investigate the presence of further regions in the genome of ha-MYXV. The information provided by this study defines genome regions that may be targeted in future molecular epidemiological studies regarding ha-MYXV.

Mitophagy selectively removes depolarised and damaged mitochondria, promoting cell viability. Some members of the Orthoflavivirus genus activate mitophagy to allow extended replication times in a surviving cell, while others inhibit mitophagy to favour viral spread. Viral genome replication is catalysed by non-structural protein 5 (NS5), the largest and most conserved protein in the orthoflaviviruses (OFVs). Here we used quantitative proteomics to identify host cell interactors of Zika virus (ZIKV), Usutu virus (USUV), and West Nile virus (WNV) NS5 proteins. A total of 141 cellular proteins were found of which 26 were shared in all three datasets. RNAi-mediated knockdown of those factors most significantly enriched revealed putative proviral and restriction factors. Knockdown of host myoferlin and LGALS3BP led to increases in the virus titres, suggesting that both positively promote viral replication. Silencing of mitophagy-related prohibitin 2 (PHB2) led to significant increases in ZIKV and USUV titres, and intracellular levels of genomic RNA, in agreement with an antiviral activity associated with PHB2. Supporting a connection between this restrictive activity and mitophagy, treatment with an inhibitor of this pathway (mdivi-1) resulted in larger virus titres and intracellular genomic RNA. During the course of an infection, PHB2 protein levels gradually decreased, suggesting that it is specifically targeted and degraded by virus-mediated activities. Ectopic expression of NS5 alone did not affect PHB2 intracellular levels in 293T cells, however a significant decrease was detected when NS2B/NS3 protease was expressed. Enhanced PHB2 degradation was observed when both NS2B/NS3 and NS5 proteins were co-expressed, suggesting that NS5 could be acting as a bridge between PHB2 and the viral protease. Overall, our results support that PHB2 is a restriction factor for orthoflaviviral replication which is targeted to degradation to promote viral genome replication.

Infection with Human Cytomegalovirus (HCMV) can result in a significant burden of disease in those that are immunocompromised or immunonaive. HCMV encodes a repertoire of glycoproteins that facilitate its extensive viral tropism, some of which remain to be characterized. Currently, there is no effective vaccine or cure for HCMV, therefore emphasizing the need to identify viral proteins of critical function. UL14 was selected as an ORF of interest due to its high scoring on an in silico prediction algorithm, as well as its conservation amongst human and primate CMVs. Our goal was to elucidate the function of this previously uncharacterized viral ORF.

We hypothesized that UL14 functions in the establishment of infection in epithelial cells, due to its predicted structural similarity to UL141. We first investigated the expression of the protein through the sequential generation of a 3X-Flag mutant, a Δ UL14 mutant, and a repair in the TB40/E background. UL14 is readily expressed and detected by means of Western blot in both fibroblast and epithelial cells. Furthermore, UL14 was demonstrated to be a glycosylated viral protein through Pngase and EndoH enzymatic digests. Additionally, we confirmed the presence of UL14 in fibroblast-derived virions via Western blotting. Growth curves revealed that in fibroblasts, UL14 is dispensable for infection; however, in epithelial cells, the deletion of UL14 results in attenuated viral growth. During the infection of epithelial cells, we observed that the deletion of UL14 significantly reduces the expression of IE1 when compared to wild-type infection, despite the same number of viral genomes being delivered to each cell. Additionally, when the Δ UL14 virus is propagated in UL14-expressing fibroblasts, the virus incorporates UL14 into the virion and rescues the epithelial IE1 phenotype. Taken together, our results suggest that UL14 functions in the early establishment of infection in an epithelial cell-specific manner.

After primary infection, herpes simplex virus type 1 (HSV-1) can reach the central nervous system and, in rare cases, cause herpes simplex encephalitis (HSE). The HSV-1 genome has also been detected in the brains of individuals without clinical symptoms, and markers of viral reactivation have been linked to a higher risk of developing Alzheimer’s disease (AD). Experimental studies suggest that recurrent HSV-1 reactivation activates neurotoxic pathways and promotes AD-like pathology, but the underlying mechanisms remain unresolved.

We aim to investigate whether HSV-1 infection or latency-associated transcript (LAT) expression promotes tunneling nanotube (TNT) formation and intercellular communication in neurons. We will also determine the relative contribution of viral latency versus productive infection to TNT formation, cell-to-cell spread, and protein aggregation. Finally, we will assess whether increased TNT formation facilitates the propagation of misfolded proteins to uninfected neurons, thereby contributing to AD pathology.

To address these questions, we have established two complementary experimental platforms: a reporter system that enables visualization and quantification of neuronal cell-cell communication, and a protocol to establish latency with controlled HSV-1 reactivation. Together, these tools will allow us to dissect the role of HSV-1 in TNT-mediated intercellular spread and its potential contribution to neurodegeneration.

Viral infections can trigger the activation of several cellular pathways, which can contribute to the restriction of virus replication but also induce stress-responses such as virus-induced senescence (VIS). Yet, the direct relationship between virus infection and senescence induction is far from being understood. Cellular senescence is a direct consequence of DNA damage response (DDR). While the interplay between DNA viruses and DDR has been studied, the impact of RNA viruses is less known.

Here, we show that Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection causes DNA damage and elicits an altered DDR in vitro, in vivo and in COVID-19 patients. SARS-CoV-2 causes a reduction in CHK1 and a consequent decrease in RRM2, causing dNTP shortage and impairment of the S-phase progression associated with DNA damage accumulation, cGAS activation and senescence. SARS-CoV-2 infection has been associated with neurological symptoms characteristic of long-lasting coronavirus disease (COVID). Activated glial cells are key players in response to central nervous system infection, yet are implicated in inflammation and neurodegeneration. Interestingly, glial cells showed signs of senescence and activation of the cGAS-STING pathway. Then, we investigated if glial cell activation could affect the function of neuronal networks. Primary rat cortical cultures seeded on multielectrode arrays (MEAs) were used to monitor electrical activity. Effective SARS-CoV-2 infection of the glia led to a major loss of synaptic connections, an increase in cGAS-associated pro-inflammatory response and an increment of DNA damage foci. Finally, we demonstrated that an antagonist of the cGAS-STING pathway was able to rescue the decrease in electrical activity post-infection.

Tick-borne encephalitis virus (TBEV) is a growing health concern and causes severe and long-lasting sequelae, including permanent neurological complications. Therefore, we studied neurons and glial cells during infection. Similar to COVID-19, TBEV induced senescence, activation of the pro-inflammatory cGAS-STING pathway, an increase in DNA damage foci and cellular senescence.

Rotaviruses (RV) are classified into nine species (A–D and F–J). Species A (RVA) is the best studied and primarily infects infants and young animals, while non-RVA species infect adults, various mammals, and birds. However, research on non-RVA species has been limited by the lack of suitable cell systems and molecular tools, leaving their replication mechanisms largely unexplored.

In RVA, replication occurs in cytoplasmic inclusions called viroplasms, composed mainly of NSP5, NSP2, and VP2. In uninfected cells, co-expression of NSP5 with either NSP2 or VP2 can generate viroplasm-like structures (VLSs) that mimic genuine viroplasms but do not produce viral progeny. These VLSs provide valuable models for investigating the molecular basis of viroplasm formation.

We investigated whether NSP5, NSP2, and VP2 from non-RVA species can form VLSs. NSP5 co-expressed with NSP2 produced globular VLSs in RVA, RVB, RVD, RVF, RVG, and RVI, but not in RVC, RVH, or RVJ. In contrast, VP2-induced VLSs formed across all species, with RVH and RVJ also recruiting their respective NSP2 proteins. All NSP5 proteins self-oligomerize, with their C-terminal “tail” regions required for interaction with NSP2 and VP2 and for VLS formation. Interestingly, VLSs formed more readily between closely related species.

Replacing the NSP5 tail in RVH and RVJ with that of RVA restored VLS formation, while mutations in conserved VP2 residues disrupted it. Moreover, swapping the VP2 N-terminus between RVB and RVG also supported VLS formation. These findings reveal conserved principles of viroplasm assembly that could guide strategies to prevent rotavirus infections across species A–J.

Bacteriophages, the most abundant biological entities on Earth, remain vastly understudied and are often referred to as microbial “dark matter” due to the challenges in isolating and characterizing them. This is especially true in complex matrices like anaerobic digestion sludge. The first part of the study presents a simple and cost-effective protocol for phage concentration from anaerobic digester samples, optimized for subsequent extraction of nucleic acids enabling virome analysis. The entire workflow requires less than one hour to process ten samples under optimal conditions (excluding further downstream processes), using as little as 1 mL of sludge per sample. Phages were concentrated using an ultrafiltration-based approach with protein concentrators (100K MWCO), eliminating the need for expensive instrumentation or chemical reagents. The second part compares the impact of cold storage conditions by refrigeration (4 °C) for one week and a single freeze–thaw cycle at −20 °C on viral community composition.

PCoA visualization and statistical analysis using PERMANOVA revealed no significant difference between ultrafiltered samples and unfiltered negative controls (p = 0.66), suggesting that ultrafiltration does not introduce bias in community profiling. On the other hand, refrigeration demonstrated minimal impact (p = 0.14), while a single freeze–thaw cycle significantly altered the community structure (p = 0.01).

Together, these findings demonstrate that the proposed protocol enables reliable phage recovery and emphasize the importance of storage conditions in preserving community integrity. The method’s scalability and minimal input requirements make it well-suited for broader application in environmental virology and wastewater research.