Viruses

Bacteriophage (phage) collections are essential resources for studying virus-host interactions in bacterial species. Here, we report six Escherichia coli-infecting phages that expand the Lund Collection of Bacteriophages. These phages were isolated in 2025 within the framework of the School of Molecular and Theoretical Biology for high-school students, from samples collected in Lake Taldykol, Astana, Kazakhstan, using E. coli strains MG1655{Delta}RM and EV36 as hosts. The isolated phages comprise Taldykol (LuPh6), a member of the genus Kagunavirus; Aidakhar (LuPh7) of the genus Phapecoctavirus; Samruk (LuPh8) of the genus Tequintavirus; the T-odd-like phage Baiterek (LuPh9) of the genus Vequintavirus; and two T-even-like phages Tulpar (LuPh10) and Shurale (LuPh11) that belong to the Tequatrovirus genus. This expanded phage collection enhances the toolkit for investigating phage-host interactions and their molecular mechanisms and highlights the use of phage isolation as a component of high school research education. ImportancePhage collections are a key resource for studying phage biology, phage-bacteria interactions and bacterial immune systems. Here, we extend the Lund Phage Collection through the isolation and characterisation of six E. coli-infecting phages, including three novel species (LuPh6, LuPh8 and LuPh11) as well as a member of the genus Phapecoctavirus that not represented in widely used collections such as BASEL (LuPh7). This study expands the resources available for probing phage-host interactions and demonstrates an example of integrating phage research into education of high school students.

Shrimp and other arthropods are capable of specific, adaptive immune responses to viruses based on viral copy DNA (vcDNA) fragments in the host genome called endogenous viral elements (EVE). These may produce negative sense RNA transcripts leading to an RNA interference (RNAi) defense response against cognate viruses. We first reported high-frequency-read sequences (HFRS) of white spot syndrome virus EVE (named WSSV-EVE 4,6,8) in a WSSV-free breeding stock of whiteleg shrimp (Penaeus vannamei). Here we describe screening for the same HFRS-EVE in a captured giant tiger shrimp (Penaeus monodon) breeding stock, also free of WSSV. WSSV-EVE 4,6,8 was detected in some of the P. monodon stock individuals with positive or negative RNA expression. Eight broodstock individuals were selected for mating in 4 crosses. The offspring from these crosses were grown sufficiently to allow tagging and pleopod sampling for DNA and RNA analysis prior to challenge with WSSV. This allowed for Mendelian analysis of EVE inheritance and for its expression or not in the offspring, together with analysis of their relationships to survival and WSSV infection level after challenge. The results revealed that EVE inheritance was Mendelian, but that their RNA expression or not was independently controlled. In Crosses 1 and 2, all the offspring died and none of them carried 2 or more of the expressed EVE in their parental shrimp. In contrast, 100% of 10 arbitrarily selected surviving shrimp from Cross 3 and 90% from Cross 4 carried and expressed 2 or more of the 3 expressed EVE transmitted from the parental shrimp. These results reveal a potential protocol for development of viral tolerant shrimp stocks.

Autophagy is a catabolic process used for the degradation of organelles and proteins. Macroautophagy involves the formation of autophagosomes and subsequent fusion with lysosomes to mediate cargo degradation. It also functions as a cellular defence mechanism, known as xenophagy, during infection. Previous studies show that different viruses manipulate the autophagy pathway of the host cell to assure successful replication and/or virion assembly. Vaccinia virus (VACV), the prototypic poxvirus, replicates exclusively in the cytoplasm of host cells. It is known that VACV infection causes LC3 lipidation and prevents autophagosome formation, yet the double membrane vesicles formed during autophagy do not serve as the source of the mature VACV membrane. To date the viral protein(s) causing increased LC3 lipidation have not been identified. Here we developed an image-based screening approach based on LC3 granularity to identify candidate VACV genes affecting its lipidation. We identify several candidate viral membrane proteins as effectors of LC3 lipidation, suggesting that the interplay between VACV and autophagy is more directed than previously thought.

The ubiquitin-conjugating enzyme UBE2J1 functions in the proteasomal degradation of proteins at the ER. Existing evidence suggests that it plays a role during viral infection, with elevated UBE2J1 levels generally associated with increased infection. This is particularly relevant for some RNA viruses; however, the regulation of UBE2J1 during infection has not been well studied. Here, we used a BHK21 cell model to demonstrate that UBE2J1 overexpression promotes the replication of Vesicular Stomatitis Virus (VSV), as indicated by a significant increase in viral titres. To better understand the underlying molecular processes, cells were co-transfected to express the VSV-G protein and wild-type UBE2J1 protein, and we observed a significant increase in the syncytial fusion area. This effect was not observed when catalytically inactive (C91S) or phospho-deficient (S184A) forms of the protein were used. Interestingly, overexpression of a truncated, non-ER localized form of UBE2J1 ({Delta}TM) led to the largest increase in the syncytial fusion area. This arose as a result of increased syncytia size, and may indicate an enhanced cellular role if soluble forms of UBE2J1 are not anchored to the ER. Additional studies using truncated, mutated and wild-type proteins confirmed that UBE2J1 increases VSV viral replication and is associated with an increase in the number of infection plaques. Considering the emerging evidence for UBE2J1 involvement in viral infection, our finding should help in understanding the role of this protein in viral pathogenesis and cellular processes linked to syncytialization.

Ebola virus is a single-stranded negative-sense RNA virus that can cause severe hemorrhagic fevers in humans. Ebola virus, along with other members of the filoviridae family, produce virions with a characteristic filamentous morphology. VP40 is the Ebola virus matrix protein, which is responsible for curving the host PM into filamentous buds. VP40 forms cytosolic homodimers via interactions in its N-terminal domain, while interactions in its C-terminal domain drive oligomerization into the 2D-crystalline matrix layer. While VP40 is expressed in the host cytosol and assembles on the inner leaflet of the plasma membrane (PM), trafficking between the cytosol and PM is not direct. Here, we characterize a series of VP40 mutants targeted to the molecular determinants of Ebola VP40 assembly and trafficking using confocal microscopy with genetically-encoded fluorescent tags. Using this approach, we characterize the subcellular distribution of these mutants, showing novel phenotypes for each. Mutants related to trafficking show aggregation dependent on membrane binding, suggesting a possible mode of VP40 trafficking.

Sudan virus (SUDV) is a member of the family Filoviridae, which comprises highly pathogenic viruses associated with unusually high case fatality rates. The development of medical countermeasures against filoviruses, including antivirals, vaccines, and therapeutic antibodies, requires preclinical evaluation in suitable animal models. C57BL/6J IFNAR-/- mice, which lack the type I interferon (IFN-/{beta}) receptor, have been reported to be susceptible to filovirus infections, although their impaired innate immune response may represent a potential limitation of the model. Here, we show that IFNAR-/- mice constitute a suitable model for SUDV infection. Following infection, animals developed a clear clinical disease characterized by significant weight loss and pronounced changes in behaviour and appearance. Mice reached the predefined clinical endpoint 3-5 days post infection. Post mortem analysis of terminal samples revealed high viral loads and viral genome copies in all tested organs as well as in serum, indicating widespread systemic dissemination. Importantly, infection was associated with a marked increase in several key chemokines and cytokines linked to systemic inflammation, consistent with the development of a cytokine storm-like response. Together, these findings demonstrate that SUDV infection in IFNAR-/- mice induces systemic viral dissemination and a pronounced inflammatory response, supporting the suitability of this model for investigating filovirus pathogenesis and infection-associated immune dysregulation.

This novel study detected persistent low level infections of Elephant Endotheliotropic Herpesviruses (EEHV), that can cause highly pathogenic Elephant Hemorrhagic Disease (EHD) in Loxodonta and Elephas, and co-infection of presumed less pathogenic Elephant Gammaherpesviruses (EGHV), in skin nodule biopsies, saliva and tissues collected from 43 wild L. africana (savannah elephant) in Botswana, Kenya, South Africa and Zimbabwe; in saliva from 25 wild L. cyclotis (forest elephant) in Gabon; and in saliva collected over seven years from 7 wild-born L.africana at Six Flags Safari Park, USA; and in saliva, blood and tissues from an additional 200 L. africana in USA zoos. DNA from these samples was extracted in our USA laboratories and amplified by conventional polymerase chain reaction using three-round nested primer sets designed specifically to screen for known EEHV and EGHV genes loci and to discover new species and subtypes. Sanger sequencing of purified DNA from nearly all samples yielded unambiguous positive genetic matches to previously known Loxodonta-associated EEHV2, EEHV3A, EEHV3B, EEHV6, EEHV7A, and EGHV1B, EGHV2, EGHV3B, EGHV4B, EGHV5B and discovered novel types EEHV3C-H and EEHV7B and the prototype EGHV1B. Many of the primer sets used could also have detected known Elephas-associated EEHV1A, EEHV1B, EEHV4, and EEHV5 if present in these samples, but they did not. Our extensive library of EEHV and EGHV sequences from wild and zoo Loxodonta, (as well as from 100 zoo Elephas maximus not discussed in this review), is a significant contribution to the elephant virology community, particularly for comparing subtypes types of EEHV found in pathogenic cases of EHD in zoos as well as determining and comparing species and subtypes of EEHV present in existing zoo herds, and in individual elephants being transported between zoos, and for importation of wild elephants into existing zoo herds.

Elephant endotheliotropic herpesviruses (EEHV) pose a significant threat to the conservation of Asian elephants (Elephas maximus) worldwide, with a high mortality rate in young elephants. However, several components of EEHV virology remain underexplored, particularly for EEHV1B. This study describes a fatal case of EEHV1B infection in a nine-year-old Asian elephant from an ex situ conservation herd, examining herd viral dynamics, tissue viral loads and comparative genomics. This elephant succumbed to haemorrhagic disease within three days of developing clinical signs, despite therapeutic intervention. Quantitative PCR (qPCR) was performed on serial trunk washes and whole-blood surveillance samples collected before and after the clinical event, as well as on post-mortem tissues preserved in different storage media (DNA/RNA Shield, RNALater, and viral transport medium). Metagenomic next-generation sequencing of infected tissues was performed to characterise the complete viral genome, analyse variation from other published EEHV genomes and assess for evidence of viral recombination between EEHV subspecies. The affected elephant demonstrated a marked viraemia at onset of clinical disease, with viral load peaking at 5.47 x 106 viral genome equivalents per mL of blood, one day after the onset of clinical signs. Samples stored in viral transport medium yielded the greatest viral and host DNA recovery by qPCR, although tissues stored at -80 {degrees}C without media were still suitable for molecular detection. Whole genome sequencing demonstrated 96.0% pairwise nucleotide identity between the assembled genome (EEHV1B_AUP_01_2023, GenBank accession: PX651398) and the previously reported EEHV1B sequence (KC462164), and a maximum of 90.9% identity to published EEHV1A genomes, with evidence of recombination between the viral subspecies at several genomic regions. Viral recombination between EEHV subspecies may have significant implications for the pathogenesis of EEHV disease, the reliability of molecular diagnostics and the efficacy of vaccinations and anti-viral therapy.

Rio Negro virus (RNV) is an enzootic alphavirus and a member of the Venezuelan equine encephalitis virus (VEEV) complex. Despite its wide circulation in South America, RNV remains a neglected pathogen with no established wild-type animal model to study its pathogenesis. In this study, we developed a lethal mouse model using 18-day-old wild-type C57BL/6 mice to characterize the systemic and neurological features of RNV infection. Following subcutaneous inoculation, RNV exhibited rapid systemic dissemination with a brief and low-titer viremic phase and high viral loads in lymphoid tissues, pancreas, brains, and lungs. Notably, infected mice developed progressive neurological signs, including ataxia and hindlimb paralysis, culminating in 100% lethality. Histopathological analysis revealed significant damage, highlighted by a striking collapse of the splenic architecture, inflammatory and remodeling changes in the lungs, and prominent inflammatory infiltrates with neurodegenerative changes in the brain. The splenic disruption was further evaluated by immunofluorescence analysis of the spleen, which showed a consistent loss of compartmentalization, characterized by an atypical infiltration of CD8+ T cells into B-cell follicles. The terminal stage of disease was characterized by extensive neuroinflammation and neurodegeneration. Histological examination of the brain revealed meningoencephalitis, robust astrogliosis, and widespread somatodendritic and terminal degeneration, particularly clustered around blood vessels. These findings were supported by cytokine analysis of brain homogenates, which showed a significant upregulation of IFN-{gamma}, IL-6, and MCP-1/CCL2 during symptomatic stages. Collectively, these findings establish a reproducible, non-genetically modified animal model that reveals the pathogenic potential of RNV in the context of immune immaturity characteristic of early life. By identifying these specific pathological and neuroinflammatory markers, our study provides a foundational experimental framework to investigate the mechanisms underlying RNV emergence and host-pathogen interactions within the VEEV complex. Author summaryMany viruses circulate silently in nature, hidden within animal populations, until environmental or social changes bring them into contact with humans. The Rio Negro virus is one such example. Despite being closely related to the better-known Venezuelan equine encephalitis virus and showing evidence of circulation in South America, RNV has remained largely overlooked by the scientific community and public health authorities. In this study, we established a new experimental model using infant mice that allowed us to observe how the virus spreads and causes damage. We found that the virus rapidly reaches the brain and other vital organs, causing severe inflammation in the brain, inflammatory changes in the lungs, and a breakdown of the immune systems organization in the spleen. By using a genetically unmodified model, we were able to observe the infection in a host with an intact but naturally immature immune system. This approach avoids the artificial conditions of genetic engineering while providing a more realistic window into how host age and developmental stages can influence the outcome of neglected viral infections. We believe our work is a first step toward understanding how this overlooked virus emerges and highlights the need to monitor and prepare for "silent" pathogens that could pose a risk to public health in an era of intensifying human activity and ecological pressure.

Defective interfering particles (DIPs) derived from the influenza A virus (IAV) are a promising antiviral agent due to their strong antiviral efficacy demonstrated in various animal models. OP7 is an unconventional IAV DIP with multiple point mutations in the viral RNA (vRNA) of genome segment 7, as opposed to the large internal genomic deletions typically found in conventional IAV DIPs. Further, OP7 showed an even higher interfering efficacy than conventional DIPs. However, the inhibitory effect of OP7 on standard virus (STV) replication has primarily been investigated in Madin-Darby Canine Kidney (MDCK) cells, which lack a functional myxovirus resistance (Mx)-mediated antiviral activity against IAV. In this study, we examined the antiviral activity and mechanism of antiviral action of OP7 in an interferon (IFN)-competent human lung carcinoma cell line (Calu-3) in vitro. We performed STV and OP7 co-infection experiments using a variety of infection conditions and measured the time-resolved dynamics in viral titer, vRNA, protein level, and host cell gene expression. We observed that OP7 co-infection results in enhanced type I IFN responses and markedly reduced infectious virus release, even at low doses. Additionally, we found that at a high STV multiplicity of infection (MOI), the replication interference of OP7, suppressing the replication of STV vRNA, appears to be the dominant mechanism of its antiviral action. At a low MOI, however, IFN induction seems to be more important. Furthermore, we examined the efficacious co-infection time window for potential prophylactic and therapeutic antiviral treatment. We observed an antiviral effect exerted by OP7 infection for up to seven days before STV infection and up to 24 hours after STV infection. Together, these findings demonstrate that OP7 is a potent antiviral DIP. Therefore, this work supports the further development of OP7 as a therapeutic and prophylactic antiviral agent.

Porcine reproductive and respiratory syndrome (PRRS) is a globally distributed disease caused by Betaarterivirus europensis (PRRSV-1) and Betaarterivirus americense (PRRSV-2). Its clinical presentation ranges from subclinical infection to severe disease, depending on viral evolution and the emergence of novel variants. The aim of this study was to characterize the genetic diversity and identify recombination events in the ORF7 (nucleocapsid, N) gene of ten PRRSV-2 strains circulating in pig farms in Lima, Peru. Bioinformatic analyses were performed using DNAMAN v10.0, MEGA 6, BepiPred-2.0, DnaSP v6, and RDP v4.101. Phylogenetic analysis revealed two well-defined lineages: eight strains clustered within lineage 1A (NADC34-like), and two within lineage 5A (VR2332-like), demonstrating the co-circulation of genetically distinct variants in the region. Comparative sequence analysis identified significant amino acid substitutions in eight strains (15, 16, 17, 20, 21, 22, 23, and 24), with strain 24 being the most divergent, accumulating multiple substitutions, including T81I, R109S, I115F, R116S, and A119K within the C-terminal region encompassing antigenic domains I-V. B-cell epitope prediction using BepiPred-2.0 identified six epitope patterns (A-F) comprising nine potential B-cell epitope regions (positions 5-19, 33-72, 33-73, 84-85, 87-98, 84-98, 87-97, 84, and 119). Patterns B, E, and F exhibited four to five predicted epitope sites and corresponded to strains 21, 22, 23, and 24. Recombination analysis using RDP v4.101 detected a statistically robust recombination event in strain 18_montana2020-R (lineage 5A), with strain 24_montana2020-WT (lineage 1A) identified as the putative major parent (100% similarity) and the vaccine-like VR2332 strain (lineage 5A) as the minor parent (99.3% similarity). Secondary evidence of the same recombination event was observed in strain 19_montana2020-R. Genetic diversity analysis of the ORF7 gene identified 50 polymorphic nucleotide sites and 52 mutations. Overall, these findings demonstrate substantial genetic variability in the ORF7 gene of PRRSV-2 circulating in Lima, Peru, characterized by lineage co-circulation and inter-lineage recombination. Continuous molecular surveillance is warranted to monitor viral evolution, assess potential antigenic implications, and support effective PRRS control strategies in the Peruvian swine industry.

The majority of emerging infectious diseases are zoonotic, having their origin in wildlife before spilling over into the human population. While small mammals are recognized as critical reservoirs for these viruses, their viral diversity remains largely uncharacterized across many African countries. We conducted molecular surveillance of synanthropic rodents and shrews in the Kibera informal settlement in Nairobi and the rural Taita Hills region of Kenya to detect and characterize potential zoonotic viruses. Tissue samples from 228 rodents and shrews were screened for six viral families using PCR assays. Rat hepatitis E virus (HEV) (Rocahepevirus ratti), a rodent-associated virus with potential for human spillover, was identified in Mus musculus and Rattus norvegicus from Kibera. NGS was conducted for the HEV positive samples, and we obtained two near-complete HEV genomes from Rattus norvegicus, which clustered within rodent-associated HEV genotypes in the phylogenetic analysis. The two sequences from the Rattus norvegicus cluster together, indicating a close genetic relationship. Paramyxoviruses belonging to the genera Jeilongvirus and Parahenipavirus were detected both from Taita and Kibera in nine different samples from Rattus norvegicus, Mus minutoides, Crocidura sp and Acomys ignitus. One paramyxovirus positive sample (Acomys ignitus) from Taita was selected for further sequencing with NGS, and a complete genome of a new jeilongvirus was assembled. Phylogenetic analysis of the detected viruses confirmed the close relation to previously known rodent-borne jeilongviruses but also revealed potentially novel jeilong- and parahenipavirus species. Our findings highlight the circulation of potentially zoonotic viruses in both urban and rural small mammals in Kenya. It emphasizes the necessity of continued genomic surveillance of zoonotic viruses to mitigate risks of their spillover into human populations. HighlightsO_LISurveillance reveals diverse rodent-borne viruses circulating in Kenya. C_LIO_LIRat-HEV was detected in Rattus norvegicus and Mus musculus from an urban low-income area. C_LIO_LIParamyxoviruses were detected across multiple rodent and shrew species, including novel Acomys ignitus jeilongvirus. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/719784v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@194e81eorg.highwire.dtl.DTLVardef@11342cdorg.highwire.dtl.DTLVardef@186ad97org.highwire.dtl.DTLVardef@eeb516_HPS_FORMAT_FIGEXP M_FIG C_FIG

The caliciviruses include important human and animal pathogens such as norovirus, sapovirus and feline calicivirus. Viral reverse genetics is performed to understand the fundamental biology of these viruses, as well as a potential route to generate live-attenuated vaccines. Calicivirus reverse genetics systems have typically relied on either on the production of in vitro-transcribed RNA or plasmid-based rescue either from a mammalian promoter, or through supplementing with helper enzymes through means of a helper virus. Here, we present a novel system integrating vaccinia capping enzymes D1R and D12L encoded on plasmids as part of a system for Murine Norovirus (MNV) reverse genetics. Addition of D1R, D12L and T7 RNA polymerase-expressing plasmids increases the viral titres of rescued MNV in both BSR-T7 cells and transgenic BSR-T7CD300LF cells, and viral polyprotein abundance. When the murine norovirus receptor is expressed in BSR-T7CD300LFcells, viral titres increased 100-1000-fold compared over standard BSR-T7 cells. This system offers a robust, high-throughput means of assessing viral mutants.

Bacteriophages play an important role in the pathogenicity of Staphylococcus aureus, an important human pathogen. Phages are involved in generalized and specialized transduction as well as a more specific process by which they mobilize elements known as phage-inducible chromosomal islands, of which S. aureus pathogenicity islands (SaPIs) are an important group. SaPIs are mobilized at high frequency through interactions with specific "helper" bacteriophages, such as 80, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We previously showed that portal protein expressed in E. coli forms tridecameric rings, while portals found in virions are always dodecamers. To understand the role of the portal in capsid assembly, DNA packaging and ejection, we have here examined this phenomenon further. We show that portals assembled at lower temperature form unclosed rings that may represent portal assembly intermediates. By analyzing portal protein deletion mutants, we demonstrate the involvement of the different functional domains in phage assembly and protein incorporation.

Thogotoviruses are a group of tick-borne, six-segmented, negative-sense single-stranded RNA viruses. These viruses encode an RNA-dependent RNA polymerase that recognizes promoter sequences located at the genomic termini to initiate RNA synthesis. The 5' and 3' ends of the genome bind to the polymerase and function as a promoter. Outside the catalytic center, they base-pair with each other to form a double-stranded RNA structure. This structure is referred to as the distal duplex and plays an important role in RNA synthesis. In this study, we investigated how the RNA sequence of the distal duplex influences polymerase activity using minigenome systems of two thogotoviruses, Oz virus (OZV) and Dhori virus (DHOV). Each virus exhibits distinct activities among its six segments. In OZV, one determinant of these differences is the base pair at positions 5'12 and 3'11 within the distal duplex, where promoter activity varies depending on whether the base pair is G:C or A:U. In contrast, the DHOV polymerase is not affected by this difference. These results indicate that, even within the genus Thogotovirus, viruses differ in whether they possess a mechanism that modulates promoter activity based on subtle sequence differences within the distal duplex. Furthermore, phylogenetic analysis and comparison of promoter sequences suggest that thogotoviruses can be divided into groups that do or do not regulate intersegment promoter activity via the base pair at positions 5'12 and 3'11. HighlightsO_LIMinigenome systems of Oz virus and Dhori virus reveal segment-specific differences in promoter activity C_LIO_LIThe distal duplex sequence modulates RNA synthesis in a virus-dependent manner C_LIO_LIThe base pair at positions 5'12/3'11 determines promoter activity in Oz virus but not in Dhori virus C_LIO_LIThogotoviruses can be divided into groups that do or do not regulate promoter activity via distal duplex sequence variation at positions 5'12/3'11 C_LI

In most paramyxoviruses, RNA editing in the P gene enables expression of the V protein. Human parainfluenza virus type 1 (HPIV-1) differs from most paramyxoviruses in that it lacks RNA editing and does not produce a functional V protein, although its genome retains sequences corresponding to the ancestral V reading frame. Here, we analyzed all HPIV-1 genome sequences available in the NCBI GenBank database to assess the evolutionary state of this V protein-specific region. Using Sendai virus (SeV) as a closely related reference with an identical P gene length, we defined a pseudo-V reading frame by virtually inserting a single nucleotide at the conserved RNA editing site. In this pseudo-V frame, HPIV-1 showed a marked excess of stop codons within the 253-amino-acid region corresponding to the post-editing sequence, far exceeding expectations under random codon usage. This pattern was not observed in other viral genes analyzed under the same definition, nor in SeV, nor was it reproduced by in silico evolutionary simulations under constraints preserving the primary open reading frame. These results are consistent with a virus-specific evolutionary trajectory following the loss of RNA editing, rather than with generic coding constraints acting on overlapping reading frames.

Alternative splicing is a key regulatory mechanism known to be altered upon viral infection. These alterations can arise both from direct viral interference with the splicing machinery and from cellular responses such as activation of innate immunity. Here, we investigated splicing changes shared between dengue virus (DENV) infection and interferon (IFN) treatment in cultured cells, the latter serving as a model for virus-independent innate immune activation. Among the common events, we identified an increased production of non-coding mRNA isoforms from the CLK1 gene, which gives rise to a kinase that phosphorylates splicing factors including SR proteins and spliceosomal components. Consistent with this finding, IFN treatment led to a reduction in CLK1 protein levels. Using stable cell lines with CRISPR/dCas9-mediated modulation of CLK1 expression, we found that silencing CLK1 enhanced the induction of immune response genes, while its over-expression attenuated it. Inhibition of CLK1 kinase activity with the pharmacological inhibitor TG003 further potentiated IFN-induced gene expression and reduced DENV replication. Altogether, these results identify CLK1 as a proviral negative regulator of IFN-stimulated gene expression and suggest that its inhibition could enhance antiviral defenses and become a target for therapeutic strategies.

Escherichia coli ST131 is a globally disseminated multidrug-resistant lineage frequently associated with recalcitrant urinary tract infections (UTIs) and bacteraemia. While bacteriophages offer a promising alternative treatment to antibiotics, their efficacy is often limited in physiologically relevant conditions in comparison to laboratory media. In this study, we have investigated the mechanisms by which the representative ST131 strain, EC958, evades elimination by a model phage, LUC4. We observed that in the urine environment, EC958 can transiently resist phage infection by a density dependent mechanism and by the production of protective polysaccharides. Based on this understanding, we developed a phage treatment strategy that can sterilise an EC958 culture in urine-based medium, even at high bacterial densities. The rational design of the successful phage therapy strategy utilises a tailored phage cocktail containing phage that encode depolymerase enzymes to degrade bacterial surface carbohydrates and the targeting of multiple receptors to prevent the emergence of fixed genetic resistant mutants. We found the addition of specific carbon sources renders the bacteria more susceptible to phage infection. By combining these findings with a simulated bladder wash to model voiding, we successfully achieved elimination of EC958 cultures in a urine environment. This study provides a framework for overcoming both fixed and reversible phage resistance, offering a translatable strategy for effectively treating urinary tract infections with phage. Author SummaryIn this study, we investigated how bacterial populations can overcome a phage infection. Phage are viruses that naturally kill bacteria and provide an alternative treatment to antibiotics. We focussed on a particularly aggressive and antibiotic resistant strain of E. coli, EC958, which belongs to a group of E. coli strains that are a leading cause of urinary tract infections and life-threatening bloodstream infections worldwide. We found that in a simulated bladder environment, these bacteria do not rely on genetic mutations to survive but they employ a range of hide and seek strategies. We showed that bacteria can coat themselves in a protective layer to block the phage and use social signalling to enter a dormant state when cell density is high. When they are in this sleep-like state the phage cannot successfully infect. To overcome these bacterial defences, we developed a treatment strategy combining effective phage with specific naturally occurring additives, that trick the bacteria into waking up and becoming vulnerable again to phage infection. By also simulating a clinical bladder wash to reduce bacterial numbers and therefore reduce social-signalling, we were able to eliminate the bacterial population. Our findings suggest that by understanding bacterial strategies we can design more effective and personalised phage therapies to treat bacterial infections.

Hepatitis D virus (HDV) is a satellite virus that utilizes hepatitis B virus (HBV) as a helper for infectious particle formation. HDV was originally identified as a novel antigen in liver biopsies of HBV patients, and later studies showed the "delta" antigen (DAg) to be the sole protein encoded by HDV. Until the discovery of HDV-like agents in birds and snakes in 2018, HDV was a unique example of animal satellite viruses. We identified Swiss snake colony virus 1 (SwSCV-1) in the brain of a Boa constrictor, and through comparison we found the genome organization of SwSCV-1 to resemble that of HDV. However, in addition to the DAg open reading frame (ORF), the genome of SwSCV-1 includes another >500 nt ORF, "ORF2". To study whether the putative ORF2-encoded protein plays a role in the SwSCV-1 life cycle, we established an infectious clone of the virus with a point mutation in the methionine initiation codon of ORF2. The mutation did not significantly affect initiation of replication, establishment of persistent infection, or infectious particle formation upon superinfection with a helper virus. Using additional methods, we gathered further evidence confirming that ORF2 is not actively translated in boa constrictor cells. We further showed that unlike HDV, SwSCV-1 expresses a single form of the DAg. Although the proteins encoded by SwSCV-1 and HDV only include one and two forms of the DAg, respectively, whether other kolmioviruses express additional forms of DAg or related proteins in some cell types or host species merits further research. IMPORTANCEApproximately 40 years after the discovery of hepatitis D virus (HDV), satellite viruses with similar genome organization were found in various animals, thereby giving rise to family Kolmioviridae. HDV encodes a single protein, the delta antigen (DAg), which comes in small and approximately 20 amino acids longer large form. The genome of some HDV species and many of the newly found kolmioviruses contains additional open reading frames (ORFs), potentially enabling protein expression. Here, we studied the viral proteins expressed during Swiss snake colony virus 1 (SwSCV-1) infection of boa constrictor cells. Our findings show that unlike HDV, SwSCV-1 encodes only a single form of DAg. In addition, our study suggests that, like in HDV, the additional ORF in SwSCV-1 genome does not give rise to a protein. Although we could not demonstrate expression of additional viral proteins during SwSCV-1 infection, it is important to study the proteome of other kolmioviruses.

Flat mites (Tenuipalpidae) are diverse phytophagous arthropods, among which Brevipalpus species are economically important pests capable of transmitting plant viruses. Brevipalpus-transmitted viruses (BTVs) cause localized infections in plants and are classified into two major groups based on cytopathology and genome organization: BTV-C (genera Cilevirus and Higrevirus, family Kitaviridae) and BTV-N (genus Dichorhavirus, family Rhabdoviridae). Despite their significance, the virome of tenuipalpid mite vectors remains poorly characterized. Using high-throughput sequencing (HTS), we analyzed virus populations associated with Brevipalpus and Dolichotetranychus mites collected from multiple plant hosts across two Hawaiian Islands. We identified a diverse assemblage of viral sequences affiliated with Kitaviridae, negeviruses, Picornavirales, Narnaviridae, Tombusviridae, Solemoviridae, Ourmiaviridae, Reoviridae, and Potyviridae. Near-complete genomes of citrus leprosis virus C2H and hibiscus green spot virus 2 (both BTV-C) were recovered, highlighting the utility of HTS-based viromics for surveillance of BTVs in mite vectors. In addition, multiple divergent virus-like contigs were identified based on viral hallmark genes and sequence divergence, including Brevipalpus-associated negevirus, Brevipalpus-associated bluner-like virus, and Dolichotetranychus-associated cile-like virus, all showing evolutionary affinities to BTV-C-related viruses. Phylogenetic analyses support evolutionary links between negeviruses and kitavirids, consistent with the hypothesis that Kitaviridae evolved from arthropod-associated ancestors. While some detected plant viruses may reflect ingestion rather than active replication in mites, this study establishes a robust framework for virome-based surveillance of tenuipalpid mites, advancing our understanding of plant virus evolution and supporting agricultural biosecurity and pest management efforts.