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.

Our recent study using in silico data mining identified novel culterviruses (family: Bornaviridae) in fish, including a variant of W[u]han sharpbelly bornavirus (WhSBV) in grass carp kidney and liver cell lines. Here, metagenomic sequencing of different fish cell lines revealed WhSBV in two cell lines from grass carp (Ctenopharyngodon idella; order: Cypriniformes). Using these cell lines, we investigated the ability of WhSBV to infect and establish persistent infection in other cell lines from bony fish (Cypriniformes, Chichliformes, Salmoniformes, Centrarchiformes and Spariformes), reptiles (Testudines and Squamata), birds (Galliformes) and mammals (Primates and Rodentia). WhSBV showed efficient replication and a time-dependent increase in viral RNA levels in cypriniform cells, whereas replication was limited, confined to single cells, and lacked a clear time-dependent increase in cells from other bony fish and reptiles. No replication was detected in avian and mammalian cells. In situ hybridisation and electron microscopy confirmed the presence of viral RNA and particles in infected cypriniform cells. Transcriptomic sequencing revealed minimal innate immune activation during early stages of infection and antiviral response only at later stages, suggesting that WhSBV establishes persistence by evading early immune recognition. In addition, we identified polycistronic viral mRNAs regulated by specific transcriptional start and termination sites and RNA splicing. Viral proteins were detected, confirming previous in silico predictions. These findings provide insights into the potential infectivity, persistence mechanisms and transcriptional strategies of WhSBV. This study validates previous findings from in silico data mining, further reinforcing its effectiveness as a powerful tool for discovering hidden viruses. IMPORTANCEUnderstanding the diversity and host range of viruses is crucial for assessing their ecological role, associated diseases and zoonotic potential. However, many newly discovered viruses are characterised using sequence data alone because isolates are often difficult to obtain. Using cell culture models, this study characterises W[u]han sharpbelly bornavirus (WhSBV), a member of the genus Cultervirus. Here we demonstrate its ability to establish persistent infection in cypriniform fish cell lines, while exhibiting restricted replication in certain non-cypriniform fish. The identification of polycistronic transcription, splicing events and immune evasion mechanisms advances our understanding of the molecular biology of WhSBV and culterviruses in general. By validating in silico predictions, this study highlights the power of computational approaches in uncovering viral diversity. As cypriniform fish include economically important species such as carp, understanding the dynamics of WhSBV host range and infection biology may be crucial for future aquaculture health management.

Orthohantaviruses, are emerging zoonotic pathogens causing life-threatening diseases in humans. The orthohantavirus genome consists of three RNA segment (vRNAs) of negative polarity, which are encapsidated by the viral nucleoprotein (N). To date, the precise subcellular behavior of vRNAs and N has not been fully elucidated. Here, we present a comprehensive analysis of orthohantavirus infections using Fluorescence in situ Hybridization (FISH) and multiple sequential FISH (Mu-Seq FISH), which enables simultaneous detection of viral RNAs, viral mRNAs, N, and cellular factors. Our experiments revealed distinct patterns of viral RNA clustering with varying degrees of N association. Moreover, we found a significant spatial correlation of virus vRNAs and N with cellular processing (P)-bodies, underlining their key role in orthohantavirus replication. Throughout the course of an infection, we observed an increasing dominance of N expression, while concomitantly P-body numbers grew significantly. We also found indications for a preferential 5-end degradation of viral mRNAs in P-bodies. Furthermore, we report that orthohantavirus infection is accompanied by a significant redistribution of cellular components: while filamentous actin and microtubules become enriched in the perinuclear region, P-bodies move to the cell periphery. Finally, co-localization analyses suggest a formation of viral factories containing N, vRNAs, and viral mRNAs, indicating an intricate orthohantavirus assembly hierarchy. AUTHOR SUMMARYIn this study we used advanced imaging techniques to observe the dynamics of viral components and key cellular structures during orthohantavirus infections. Our experiments show that orthohantaviruses cause significant changes within the cell, particularly involving P-bodies and components of the cytoskeleton, such as actin and microtubules. We also provided comprehensive spatiotemporal maps of orthohantaviral components, including visualization of the viral nucleoprotein, genomic RNA and mRNAs. Finally, we found indications for a 5end degradation of virus mRNA in P-bodies, thus adding to our understanding of intracellular host-pathogen crosstalk. In summary, our work highlights the intricate relationship between viruses and host cells, emphasizing the dynamic changes that occur during orthohantaviral infections.

The Omicron subvariant BA.1 of SARS-CoV-2 was first detected in November 2021 and quickly spread worldwide, displacing the Delta variant. In Mexico, this subvariant began spreading during the first week of December 2021 and became dominant in the next three weeks, causing the fourth COVID-19 epidemiological surge in the country. Unlike previous SARS-CoV-2 variants, BA.1 did not acquire local substitutions nor exhibited a geographically distinct circulation pattern in Mexico. However, a regional difference in the speed of the replacement of the Delta variant was observed, as some northern states showed persistence of Delta lineages well into February 2022. Mexican states were divided into four regions (North, Central North, Central South, and Southeast) based on the lineage circulation before the dominance of BA.1 to study possible causes for this difference. For each region, the time to fixation of BA.1, the diversity of Delta sublineages in the weeks preceding BA.1 entry, the population density, and the level of virus circulation during the inter-wave interval were determined. An association between a faster Omicron spread and lower Delta diversity, as well as fewer COVID-19 cases during the Delta-BA.1.x inter-wave period, was observed. For example, the North region exhibited the slowest spread but had the highest diversity of Delta sublineages and the greatest number of inter-wave cases relative to the maximum amount of the virus circulating in the region, whereas the Southeast region showed the opposite. Viral diversity and the relative abundance of the virus in a particular area around the time of the introduction of a new lineage seem to have influenced the spread dynamics. Nonetheless, if there is a significant difference in the fitness of the variants or the time allowed for the competition is sufficient, it seems the fitter virus will eventually become dominant, as observed in the eventual dominance of the BA.1.x variant in Mexico. Impact statementThe surveillance of lineage circulation of SARS-CoV-2 has helped identify variants that have a transmission advantage and are of concern to public health and to track the virus dispersion accurately. However, many factors contributing to differences in lineage spread dynamics beyond the acquisition of specific mutations remain poorly understood. In this work, a description of BA.1 entry and dispersion within Mexico is presented, and which factors potentially affected the spread rates of the Omicron variant BA.1 among geographical regions in the country are analyzed, underlining the importance of population density, the proportion of active cases, and viral lineage diversity and identity before the entry of BA.1. Data summaryThis work was carried out using data shared through the GISAID initiative. All sequences and metadate are available through GISAID with the accession EPI_SET_220927gw, accession numbers and metadata are also reported in the supplemental material of this article. Epidemiological data was obtained though the Secretaria de Salud website (https://www.gob.mx/salud/documentos/datos-abiertos-152127),

Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements (EVEs) have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such EVEs and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of EVEs. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for one generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific EVEs identified an element comprised of a 9,045 bp stretch of repeated, inverted and jumbled genome fragments of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) bounded by a repeated 591/590 bp host sequence. As only near complete linear ~4 kb IHHNV genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear EVE types. The existence of joined inverted IHHNV genome fragments also provides a means by which hairpin dsRNAs could be expressed and processed by the shrimp RNA interference (RNAi) machinery.

Bats are described as the natural reservoir host for a wide range of viruses. Although an increasing number of bat-associated, potentially human pathogenic viruses were discovered in the past, the full picture of the bat viromes is not explored yet. In this study, the virome composition from Miniopterus fuliginosus bats inhabiting the Wavul Galge cave, Sri Lanka, was analyzed. To assess different possible shedding routes, oral swabs, feces and urine were collected and analyzed individually by using metagenomic NGS. The data obtained was further evaluated by using phylogenetic reconstructions. Two different alphacoronavirus strains were detected in feces and urine samples. Furthermore, a paramyxovirus was detected in urine samples. Sequences related to Picornaviridae, Iflaviridae, unclassified Riboviria and Astroviridae were identified in feces samples, and further sequences related to Astroviridae in urine samples. No further viruses were detected in oral swab samples. The comparative virome analysis in this study revealed a diversity in the virome composition between the collected sample types which also represent different potential shedding routes for the detected viruses. At the same time, several viruses were detected for the first time in bats in Sri Lanka. The detection of two different coronaviruses in the samples indicates the potential general persistence of this virus species in M. fuliginosus bats. Based on phylogenetics, the identified viruses are closer related to bat-associated viruses with comparably low human pathogenic potential. In further studies, the seasonal variation of the virome will be analyzed to identify possible shedding patterns for particular viruses.

The influenza A H1N1pdm09 virus continues to be a significant pathogen, posing potential risks to both animal and human health due to its zoonotic potential. Italy, which has one of the largest swine populations in Europe, plays a crucial role in monitoring the evolution of influenza viruses in livestock. This study aims to address the existing knowledge gaps regarding the genetic diversity and transmission dynamics of H1N1pdm09 circulating in Italian swine populations. Utilizing whole genome sequencing and dynamic modeling, we conducted a comprehensive analysis of virus samples collected from swine farms across Italy. Our results reveal that multiple independent viral introductions have occurred into the country, with most cases resulting in self-limited infections and limited onward transmission. However, six distinct transmission clusters were identified, suggesting instances of sustained viral spread. These clusters were found across multiple regions of Italy, highlighting the broad geographic distribution of virus lineages. Our findings indicate that while many introductions led to localized containment, certain virus lineages were able to spread within specific regions of Italy. Through a detailed examination of selective pressures, we observed that most viral genes are under strong purifying selection in both swine and human hosts, as reflected by dN/dS ratios well below 1. The hemagglutinin (HA) gene exhibited a notably higher dN/dS ratio in swine ([~]0.28) compared to humans ([~]0.22), indicating slightly relaxed selection in swine. In contrast, other genes, such as neuraminidase (NA) and non-structural protein (NS), showed similarly strong purifying selection across both hosts. These results reflect a general trend of selective pressures affecting multiple viral components, rather than emphasizing specific genes. Our study emphasizes the importance of ongoing genomic surveillance in detecting viral circulation and mitigating risks to both animal and public health. Italys efforts contribute significantly to global influenza monitoring and highlight the importance of a One Health approach that integrates human, animal, and environmental health. These findings provide essential data to inform public health policies and enhance preparedness against future zoonotic influenza outbreaks.

Human rhinovirus (RV) is the most frequent cause of the common cold, as well as severe exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. Currently, there are no effective and accurate diagnostic tools or antiviral therapies. MicroRNAs (miRNAs) are small, non-coding sections of RNA involved in the regulation of gene expression and have been shown to be associated with different pathologies. However, the precise role of miRNAs in RV infection is not yet well established. This study aimed to analyse the impact of RV16 on miRNA expression across the viral life-cycle to identify a small panel with altered expression. We then aimed to specifically interrogate these results using our in-house developed modelling programme to identify specific genes regulated by these miRNAs during RV infection which can then be tested functionally. Our results first identified that three miRNAs, miR-155-5p, miR-140-3p, and miR-122-5p were potential biomarkers being differentially regulated at specific time points post infection. Our modelling program then linked these miRNAs to four genes (OLFML3, STAG2, SMARCA2, CD40LG) that play important roles in regulating the hosts antiviral responses and viral progression. Together, this work identifies a potential panel of biomarkers that could, along with our previous work, form clear diagnostic markers for RV16 infection and identifies specific targets that can be functionally interrogated helping to identify new cellular targets modified by RV16 infection that could inform future therapeutic design.

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.

Yellow fever virus (YFV) exhibits a sylvatic cycle of transmission involving wild mosquitoes and non-human primates (NHP). In Brazil, YFV is endemic in the Amazon region, from where waves of epidemic expansion towards other Brazilian states eventually occur. During such waves, the virus usually follows the route from North to the Central-West and Southeast Brazilian regions. Amidst these journeys, outbreaks of Yellow Fever (YF) in NHPs, with spillovers to humans have been observed. In the present work, we describe a surveillance effort encompassing the technology of smartphone applications and the coordinated action of several research institutions and health services that succeeded in the first confirmation of YFV in NHPs in the state of Minas Gerais (MG), Southeast region, in 2021, followed by genome sequencing in an interval of only ten days. Samples from two NHPs (one of the species Alouatta caraya in the municipality of Icarai de Minas and the other of the species Callithrix penicillata in the municipality of Ubai) were collected and the presence of YFV was confirmed by RT-qPCR. We generated three near-complete by Nanopore sequencer MinION. Phylogenetic analysis revealed that all viral genomes recovered are equal and related to lineage South America 1, clustering with a genome detected in the Amazon region (Para state) in 2017. These findings reveal the occurrence of a new wave of viral expansion in MG, six years after the beginning of the major outbreak in the state, between 2015-2018. No human cases were reported to date, showing the importance of coordinated work between local surveillance based on available technologies and support laboratories to ensure a quick response and implementation of contingency measures towards avoiding the occurrence of YF cases in humans.

The necrosis syndrome of freesia, first described in 1970 in Northern Europe, is still jeopardizing freesia cultivation all over the world. Although several viruses have been listed as possible causal agents, the etiology of the disease is still not clear and is possibly linked to a combination of different factors. In this study, a high-throughput sequencing virome analysis was performed on total RNA extracts derived from symptomatic freesia leaves; a novel virus putatively belonging to the recently ratified Konkoviridae family in the Bunyaviricetes class has been identified and characterized, for which we propose the name of freesia konkovirus 1 (FreKV-1). This family, officially listing only one genus and two species, has been expanded by exploring publicly available metatranscriptomic datasets through the Serratus Project Database and reconstructing new viral entities; the phylogenetic position of the Konkoviridae family has been investigated and new genera belonging to the family have been proposed. Moreover, a further previously unknown virus, putatively belonging to the Yueviridae family was partially characterized and its phylogenetic position was discussed. Overall, the analysis increased our knowledge of the number of viral agents infecting freesia and possibly involved in freesia necrosis syndrome.

The Zika virus (ZIKV), a pathogenic member of the orthoflavivirus family, is raising serious health concerns worldwide. Like Dengue (DENV) and Chikungunya (CHIKV) viruses, it is one of the arboviruses that poses an emerging threat to areas where its main vectors, Aedes mosquitoes, proliferate, well beyond tropical and subtropical regions. Although often asymptomatic or mild, ZIKV infection has been responsible for a worrying increase in serious congenital syndromes, including microcephaly. The ability of ZIKV to be transmitted sexually and its long persistence in body fluids suggests its incomplete clearance in particular tissues, linked to recurrent infection. Among its clinical presentations, ZIKV infection has been associated with ocular complications, including maculopathy, retinopathy, uveitis, and optic neuropathy, which can lead to lasting visual impairment. The blood-retinal barrier (BRB), primarily composed of retinal pigment epithelium (RPE) and endothelial cells, plays a crucial role in shielding the retina from pathogens. Its disruption has been linked to viral retinal infections. In vitro monitoring of infection on hTERT RPE-1 cells revealed an ability of ZIKV to persist for up to 30 days in nearly 10% of the cells. This prolonged infection was marked by low cytopathic effects but notable morphological changes throughout the cell layer, suggestive of an epithelial-mesenchymal transition (EMT). Long-lasting viral replication and production was associated with reduced expression of epithelial genes and increased expression of certain mesenchymal genes, suggesting that integrity of the RPE layer may be compromised. These results indicate that viral persistence and phenotypical transition observed in vitro in RPE cells could provide clues to understanding the late onset of ocular pathophysiological manifestations in Zika virus-related diseases.

Emerging and re-emerging infectious diseases have posed significant global health threats, with many attributed to zoonotic RNA viruses. These pathogens can, under some conditions, cross species barriers, facilitating transmission from animal hosts to humans. Bats, characterised by unique physiological and ecological features, and remarkable species diversity, are recognized to host numerous viruses with cross-species transmission potential. This study aimed to investigate the presence of RNA viruses from a broad diversity of Tanzanian bats while valorising archived biological samples. RNA was extracted from 125 samples (28 faeces and 97 oral swabs) of 17 bat species, followed by PCR amplification targeting five distinct viral genera (Filovirus, Coronavirus, Hantavirus, Paramyxovirus and Astrovirus). Overall, 1.6 % (3/125) of the samples from two bat species (Scotophilus dinganii and Miniopterus fraterculus) tested positive for astrovirus, with the coinfection of one bat with two AstV strains. No samples tested positive for Filovirus, Coronavirus, Hantavirus and Paramyxovirus. Phylogenetic analysis based on RNA-dependent RNA polymerase sequences revealed these sequences are respectively clustering with astroviruses detected in other bat species from the genus Scotophilus from East Asia and with astroviruses detected in Miniopterus bats from Africa and Asia. Altogether, these results are the first report of astroviruses in Tanzanian bats.

Virus growth depends on host performance in any given environment and has a profound impact spanning fields from climate change to human health. We investigated the intrinsic growth rate of two natural G4-like bacteriophages on four clones of their host (E.coli strain C) that differed in their thermal performance, with significant differences in their maximum growth rate and optimum temperature. The intrinsic population growth rate of the G4-like phage isolates was measured on the four host clones at three temperatures, reflective of a low, optimum and high temperature for one of the phage. The phages intrinsic growth rate was significantly affected by host-temperature and phage-temperature interactions, but not by the interaction between host and phage. Across temperatures, phage growth rate was consistently highest on the host clone with the highest maximum growth rate, but was not, contrary to our expectations, lowest on the host with the lowest growth rate. At any given temperature, phage growth rate followed a directional trend with host growth rate, but this trend was not as apparent when looking between temperatures. Our work demonstrates a significant impact of temperature and the host on phage growth rate, but much work remains to be done to disentangle how variation in host performance impacts virus performance across temperatures.

African swine fever virus (ASFV) and classical swine fever virus (CSFV) are important transboundary animal diseases (TADs) affecting swine. ASFV is a large DNA virus with a genome size of 170-190 kilobases (kB) belonging to the family Asfarviridae, genus Asfivirus. CSFV is a single-stranded RNA virus with genome size of approximately 12 kB belonging to the family Flaviviridae, genus Pestivirus. Outbreaks involving either one of these viruses result in similar disease syndromes and significant economic impacts from: (i) high morbidity and mortality events; (ii) control measures which include culling and quarantine; and (iii) export restrictions of swine and pork products. Current detection methods during an outbreak provide minimal genetic information on the circulating virus strains/genotypes that are important for tracing and vaccine considerations. The increasing availability and reduced cost of next-generation sequencing (NGS), allows for the establishment of vital NGS protocols for the rapid identification and complete genetic characterization of outbreak strains during an investigation. NGS data provides a better understanding of viral spread and evolution facilitating the development of novel and effective control measures. In this study, panels of primers spanning the genomes of ASFV and CSFV were independently developed to generate approximately 10kB and 6kB amplicons, respectively. The primer panels consisted of 19 primer pairs for ASFV and 2 primer pairs for CSFV providing whole genome amplification of each pathogen. These primer pools were further optimized for batch pooling and thermocycling conditions, resulting in a total of 5 primer pools/reactions used for ASFV and 2 primer pairs/reactions for CSFV. The ASFV primer panel was tested on viral DNA extracted from blood collected from pigs experimentally infected with ASFV genotype I and genotype II viruses. The CSFV primer panel was tested on 11 different strains of CSFV representing the 3 known CSFV genotypes, and 21 clinical samples collected from pigs experimentally infected with 2 different genotype 1 viruses. ASFV and CSFV amplicons from optimized PCR reactions were subsequently sequenced on the Oxford Nanopore MinION platform. The targeted protocols for these viruses resulted in an average coverage greater than 1000X for ASFV with 99% of the genome covered, and 10,000X-20,000X for CSFV with 97% to 99% of the genomes covered. The ASFV targeted whole genome sequencing protocol has been optimized for genotype II ASF viruses that have been responsible for the more recent outbreaks outside of Africa. The CSFV targeted whole genome sequencing protocol has universal applications for the detection of all CSFV genotypes. Protocols developed and evaluated here will be essential complementary tools for early pathogen detection and differentiation as well as genetic characterization of these high consequence swine viruses, globally and within the United States, should an outbreak occur.

BackgroundMerkel Cell Carcinoma (MCC) is an aggressive skin cancer that is three times deadlier than melanoma. In 2008, it was found that 80% of MCC cases are caused by the genomic integration of a novel polyomavirus, Merkel Cell Polyomavirus (MCPyV), and the expression of its small and truncated large tumor antigens (ST and LT-t, respectively). MCPyV belongs to a family of human polyomaviruses; however, it is the only one with a clear association to cancer. MethodsTo investigate the role and mechanisms of various polyomavirus tumor antigens in cellular transformation, Rat-2, 293A, and human foreskin fibroblasts were transduced with pLENTI MCPyV LT-t, MCPyV ST, TSPyV ST, HPyV7 ST, or empty pLENTI and assessed through multiple transformation assays, and subcellular fractionations. One-way ANOVA tests were used to assess statistical significance. ResultsSoft agar, proliferation, doubling time, glucose uptake, and serum dependence assays confirmed ST to be the dominant transforming protein of MCPyV. Furthermore, it was found that MCPyV ST is uniquely transforming, as the ST antigens of other non-oncogenic human polyomaviruses such as Trichodysplasia Spinulosa Polyomavirus (TSPyV) and Human Polyomavirus 7 (HPyV7) were not transforming when similarly assessed. Identification of structural dissimilarities between transforming and non-transforming tumor antigens revealed that the uniquely transforming domain(s) of MCPyV ST are likely located within the structurally dissimilar loops of the MCPyV ST unique region. Of all known MCPyV ST cellular interactors, 62% are exclusively or transiently nuclear, suggesting that MCPyV ST localizes to the nucleus despite the absence of a canonical nuclear localization signal. Indeed, subcellular fractionations confirmed that MCPyV ST could achieve nuclear localization through a currently unknown, regulated mechanism independent of its small size, as HPyV7 and TSPyV ST proteins were incapable of nuclear translocation. Although nuclear localization was found to be important for several transforming properties of MCPyV ST, some properties were also performed by a cytoplasmic sequestered MCPyV ST, suggesting that MCPyV ST may perform different transforming functions in individual subcellular compartments. ConclusionsTogether, these data further elucidate the unique differences between MCPyV ST and other polyomavirus ST proteins necessary to understand MCPyV as the only known human oncogenic polyomavirus.

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.

The hepatitis B virus is a partially double stranded DNA virus in the Hepadnaviridae family of viruses that infect the liver cells of vertebrates including humans. The virus replicates through the reverse transcription of an RNA intermediate by a viral poly-merase, akin to retroviruses. The viral polymerase has high replication capacity but low fidelity and no proofreading activity resulting in a high mutation rate. This contributes to the emergence of a cloud of mutants or quasispecies within host systems during infection. Several host and viral factors have been identified that contribute to mutations and mutation frequency in shaping viral evolution, however, because the dynamics of viral evolution cannot be understood from the fittest strain alone, the need exists to sequence and reconstruct intra-host diversity, recently made possible through next generation sequencing. Due to the extensive pipeline of bioinformatic analyses associated with next generation sequencing studies are needed to ascertain if quasispecies reconstruction methods and diversity measures accurately model known diversity. Here, next generation sequencing and various quasispecies reconstruction methods are used to model the natural evolution of viral populations across the full genome of hepatitis B virus strains from South Africa. This study illustrates that (i) different methods of quasispecies reconstruction reconstruct the same amount of diversity, (ii) intra-host diversity derived from full quasispecies analyses re-sembles diversity measures obtained from previous methods, (iii) inter-host diversity resembles the diversity between closely related quasispecies variants, (iv) diversity is increased in HIV-negative individuals, and (v) corroborate that seroconversion of HBV biomarkers increases mutation rates.

West Nile virus (WNV) is a zoonotic mosquito-borne Flavivirus, with bird populations reservoirs. Although often asymptomatic, infection in humans can cause febrile symptoms and, more rarely, severe neurological symptoms. Previous studies assessed environmental drivers of WNV infections, but most relied on notified West Nile Disease human cases, exposing them to (i) overlook areas with WNV circulation despite no reported case, and (ii) mixing mechanisms affecting hosts vs. vectors. Our objective was to generate a WNV Bird Risk Index (BRI), mapping the potential of WNV circulation in bird communities across Europe, in order to better understand the distribution of WNV infections. We first used a bird traits-based model to estimate WNV seroprevalence in European bird species. This allowed us to build a map of the WNV BRI across Europe. To validate this metric, we investigated its association with WNV human cases notified at the NUTS administrative region scale, using a Besag-York-Mollie 2 spatial model in a Bayesian framework. We first identified eco-ethological characteristics associated with higher WNV seroprevalence in wild birds. Second, we mapped the BRI that showed a strong spatial heterogeneity across Europe. At the NUTS level, the BRI was positively associated with the number of years with notified WNV human cases between 2016 and 2023. To conclude, we provide a map quantifying the suitability for WNV to circulate in the bird reservoir. This allows to target surveillance efforts in areas at risk for WNV zoonotic infections in the future.