Urban wildlife species have the potential to serve as links in disease transmission between wildlife, humans and domestic animals at the wildland-urban interface (WUI), contributing to both sustained cross-species transmission of pathogens and the emergence of diseases in susceptible populations. However, the relative roles of host and environmental factors in shaping the composition of pathogen communities in urban wildlife is understudied. In this study, we integrated DNA and RNA virome data with host genomic and GPS datasets to investigate factors shaping virome composition in bobcats (Lynx rufus ) at the WUI in the Tucson Mountains, Arizona, USA. Using a hybrid-capture approach for 31 scats and 17 buccal swabs, we identified multiple viruses that could affect carnivore health at the WUI, including canine parvovirus, feline astrovirus, Felis catus papillomaviruses 2 and 3 and Lyon-IARC polyomavirus. Models of virome composition and distribution of viral taxa indicated contributions of host genetic relatedness and factors relating to urbanisation (such as percentages of urban land cover, road and building densities and distances to roads). Genetic associations with virome compositions were particularly influenced by females. While females exhibit significant isolation by distance, partial Mantel tests revealed a significant correlation between beta diversity and host genetic distance in females only. To our knowledge, this study represents the first assessment of factors shaping virome composition in a wild felid. Our finding of known feline and canine pathogens in bobcats underscores the potential of the WUI to facilitate cross-species transmission between wild and domestic animals.

AbstractAlmost all cervical cancers are caused by human papillomaviruses (HPVs). In most cases, HPV DNA is integrated into the human genome. We found that tumor-specific, HPV-human DNA junctions are detectable in serum cell-free DNA of a fraction of cervical cancer patients at the time of initial treatment and/or at six months following treatment. Retrospective analysis revealed these junctions were more frequently detectable in women in whom the cancer later recurred. We also found that cervical cancers caused by HPV types outside of phylogenetic clade α9 had a higher recurrence frequency than those caused by α9 types in both our study and The Cancer Genome Atlas cervical cancer database, despite the higher prevalence of α9 types including HPV16 in cervical cancer. Thus, HPV-human DNA junction detection in serum cell-free DNA and HPV type determination in tumor tissue may help predict recurrence risk. Screening serum cell-free DNA for junctions may also offer an unambiguous, non-invasive means to monitor absence of recurrence following treatment.

Virus taxonomy, comprising classification and nomenclature, is regulated by the International Committee on Taxonomy of Viruses (ICTV). Taxon names are standardized to facilitate recognition and communication, with defined suffixes for each rank (e.g., the names of orders, families, and genera end in -virales, -viridae, and -virus, respectively). However, until recently, a standard format for species names was lacking. In 2021, following extensive discussion and community consultation, the ICTV decided to adopt a standardized binomial (Linnaean) format for virus species names, consisting of the genus name followed by a "freeform" species epithet. Previously assigned virus species names that were non-compliant with the binomial format have been fully updated. In contrast to taxon names regulated by the ICTV, the names of viruses, or "common" names, such as yellow fever virus or human immunodeficiency virus, are not under the remit of the ICTV and have not been changed.

Taxonomic classification of cellular organisms requires the publication of descriptions and proposed names of species and the deposition of specimens. Virus taxonomy is developed through a different system of annual submission of formal taxonomy proposals (TPs) that can be submitted by anyone but are typically prepared by a study group appointed by the International Committee on Taxonomy of Viruses (ICTV) and consisting of experts on a particular group of viruses. These are initially evaluated by an expert subcommittee and by the executive committee (EC) of the ICTV. EC-approved TPs are then submitted for evaluation and a ratification vote by the wider ICTV membership. Following ratification, the new taxonomy is annually updated in the Master Species List, associated databases and bioinformatic resources. The process is consistent, creates traceability in assignments and supports a fully evaluated, hierarchical classification and nomenclature of all taxonomic ranks from species to realms. The structure also facilitates large-scale and coordinated changes to virus taxonomy, such as the recent introduction of a binomial species nomenclature.TPs are available on the ICTV website after ratification, but they are not indexed in bibliographic databases and are not easily cited. Authors of TPs do not receive citation credit for adopted proposals, and their voluntary contributions are largely invisible in the published literature. For greater visibility of TPs and their authors, the ICTV will commence the annual publication of summaries of all TPs from each ICTV subcommittee. These summaries will provide a searchable compendium of all annual taxonomy changes and additions as well as direct links to the Master Species List and other ICTV bioinformatic resources. Their publication will provide due credit and citations for their authors, form the basis for disseminating taxonomy decisions and promote greater visibility and accessibility to taxonomy changes for the virology community.

Most human papillomavirus (HPV)-associated cancers harbor viral DNA integrated into the human genome as extrachromosomal circles, intrachromosomal segments, or both. Distinguishing intrachromosomal from identical-sequence extrachromosomal DNA (ecDNA) by sequencing alone is challenging, and the architecture of large-scale HPV-human DNA structures remains incompletely understood. To address this, we applied complementary genomic tools, spanning single-nucleotide to megabase resolution, to the HPV16-positive oropharyngeal cancer-cell line UM-SCC-47. These revealed that an initial integration event formed a 23 kb extrachromosomal heterocatemer circle comprising 7.5 kb of HPV16 DNA and 16 kb of the human TP63 gene. Subsequent genomic rearrangements generated heterocatemer tandem arrays extending to 0.6 megabases, plus additional large-scale rearrangements involving the HPV- TP63 structures, as revealed by long-read DNA sequencing and optical genome mapping. Fluorescent in situ Hybridization (FISH) showed that the heterocatemers were intrachromosomally localized at chromosome 3 at the TP63 locus in 100% of the cells. Long-read RNA sequencing further showed that these intrachromosomal templates produced spliced, polyadenylated transcripts. A subset of cells also harbored HPV16 ecDNA derived from the intrachromosomal HPV- TP63 DNAs. These findings define previously unrecognized higher-order architecture of integrated HPV DNA and highlight the power of FISH for distinguishing intrachromosomal from extrachromosomal DNA structures. GRAPHICAL ABSTRACT:

The cGAS/STING pathway is an important innate immune pathway that senses and responds to foreign DNA or damaged host DNA. cGAS recognizes DNA and generates the second messenger cGAMP which activates STING. STING then creates a platform for IRF3 to be phosphorylated before pIRF3 induces a type I interferon response resulting in transcription of anti-viral genes. We examined how HPV18 infection modulates cGAS/STING activation following DNA stimulation in primary human foreskin keratinocytes from three different donors. Following exogenous activation, HPV(+) cells produced higher levels of cGAMP compared to patient-matched HPV(-) cells yet phosphorylation of STING and IRF3 was reduced in the HPV(+) cells. The reduced STING and IRF3 activation corresponded with a selective dampening of type I interferon driven antiviral genes and pro-inflammatory cytokines in HPV(+) cells. Simultaneously, HPV(+) cells had a baseline increase in genes associated with epithelial proliferation and skin development, which remained elevated following cGAMP treatment. We demonstrate that both E6 and E7 are required and sufficient to drive increased cGAMP levels and attenuate STING activation. Our results characterize cGAS/STING pathway activation across time in HPV positive and negative cells and demonstrate that the HPV oncogenes cooperate to suppress early STING activation despite higher levels of cGAMP in these HPV(+) cells. These findings indicate that HPV modulates cGAS/STING signaling to blunt antiviral defenses without impacting other arms of the cGAS/STING pathway response. Importance In this study, we used primary human keratinocytes from multiple donors to track cGAS/STING activity over time following stimulation. We found that although HPV(+) cells produced more cGAMP, these cells showed reduced early activation of STING and IRF3. Downstream of cGAS/STING, many antiviral genes were suppressed by HPV however, epithelial proliferation-related genes had an HPV-mediated increase in baseline expression which remained higher than uninfected cells following cGAMP stimulation. We identified the HPV oncogenes E6 and E7 work together to enhance cGAMP levels and suppress parts of the downstream STING signaling response. These findings reveal that HPV attenuates cGAS/STING signaling and downstream events, selectively suppressing antiviral genes without inhibiting other parts of the pathway. Our results provide new insights into how HPV modulates innate immune responses. ### Competing Interest Statement The authors have declared no competing interest. National Institute of General Medical Sciences, https://ror.org/04q48ey07, R35GM152143 American Cancer Society, https://ror.org/02e463172, ACS RSG-22-054-01-IBCD National Institute of Allergy and Infectious Diseases, https://ror.org/043z4tv69, R01AI179713, R01AI165638, R21AI173916, R03AI187892 National Cancer Institute, https://ror.org/040gcmg81, P30CA023074

The sudden rise of the SARS-CoV-2 virus and the delay in development of effective therapeutics for mitigation made evident a need for ways to screen compounds that can block infection and prevent further pathogenesis and spread. However, identifying effective drugs that are efficacious against viral infection and replication with minimal toxicity for the patient can be difficult. Monoclonal antibodies were shown to be effective, but as the SARS-CoV-2 mutated, these antibodies became ineffective. Small-molecule antivirals were identified using pseudovirus constructs to recapitulate infection in nonhuman cells, such as Vero E6 cells. However, the impact was limited due to poor translation of these compounds in the clinical setting. This is partly due to the lack of similarity of screening platforms to the in vivo physiology of the patient and partly because drugs effective in vitro showed dose-limiting toxicities. In this study, we performed two high-throughput screens in human lung adenocarcinoma cells with authentic SARS-CoV-2 virus to identify both monoclonal antibodies that neutralize the virus and clinically useful kinase inhibitors to block the virus and prioritize minimal host toxicity. Using high-content imaging combined with single-cell and multidimensional analysis, we identified antibodies and kinase inhibitors that reduce viral infection without affecting the host. Our screening technique uncovered novel antibodies and overlooked kinase inhibitors (i.e., PIK3i, mTORi, and multiple RTKi) that could be effective against the SARS-CoV-2 virus. Further characterization of these molecules will streamline the repurposing of compounds for the treatment of future pandemics and uncover novel mechanisms viruses use to hijack and infect host cells.

The 56th meeting of the Executive Committee (EC) of the International Committee on Taxonomy of Viruses (ICTV) was held in Bari, Italy, in July/August, 2024, and 115 submitted taxonomy proposals were reviewed. A total of 112 were subsequently ratified by the ICTV membership. An additional 9 error correction proposals were also approved in August 2025. This article lists the taxonomy proposals that have now been incorporated into release 40 version v2 of the Master Species List ( https://ictv.global/msl ), the Virus Metadata Resource ( https://ictv.global/vmr ), and associated ICTV databases. In addition to the assignments of 1,563 new virus species, 243genera, 55 families, 11 orders, and 8 classes, there were substantial additions to higher taxonomic ranks. These include the creation of a new realm (Singelaviria), which is based on the recognition of a separate evolutionary origin for the hallmark capsid genes of members of the kingdom Helvetiavirae. These express capsid proteins forming a single jelly-roll fold that is structurally and evolutionarily distinct from those of members of the family Bamfordvirae, assigned to the realm Varidnaviria. Furthermore, the realm Varidnaviria underwent a major reorganization, including the addition of a new kingdom, Abadenavirae. Another notable change was the classification of the vertebrate-infecting single-stranded DNA anellovirids into a new phylum Commensaviricota (kingdom Shotokuvirae, realm Monodnaviria). Archaeal viruses infecting the hyperthermophilic Archaeoglobi were assigned to a new phylum Calorviricota, in the kingdom Trapavirae (realm Monodnaviria), whereas RNA viruses infecting hyperthermophilic bacteria were classified into a new phylum Artimaviricota (realm Riboviria). In recognition of his extensive and valuable contributions to virus taxonomic developments in Study Groups and over the period of his EC membership, Stuart Siddell was honoured as a new life member of the ICTV. The ICTV has created a new strategy for disseminating information on taxonomy advances through annual open-access publication of citeable taxonomy proposal summaries from each ICTV Subcommittee. A collective total of 354 co-authors of the seven summaries were drawn from members of each Subcommittee, the EC, and a very large number of contributors from the wider virology community.

This article reports changes to virus taxonomy and taxon nomenclature that were approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in April 2024. The entire ICTV membership was invited to vote on 203 taxonomic proposals that had been approved by the ICTV Executive Committee (EC) in July 2023 at the 55th EC meeting in Jena, Germany, or in the second EC vote in November 2023. All proposals were ratified by online vote. Taxonomic additions include one new phylum (Ambiviricota), one new class, nine new orders, three new suborders, 51 new families, 18 new subfamilies, 820 new genera, and 3547 new species (excluding taxa that have been abolished). Proposals to complete the process of species name replacement to the binomial (genus + species epithet) format were ratified. Currently, a total of 14,690 virus species have been established.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

ABSTRACT Science is humanity’s best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.

HPV infections are associated with a fraction of vulvar cancers. Through hybridization capture and DNA sequencing, HPV DNA was detected in five of thirteen vulvar cancers. HPV16 DNA was integrated into human DNA in three of the five. The insertions were in introns of human NCKAP1, C5orf67, and LRP1B. Integrations in NCKAP1 and C5orf67 were flanked by short direct repeats in the human DNA, consistent with HPV DNA insertions at sites of abortive, staggered, endonucleolytic incisions. The insertion in C5orf67 was present as a 36 kbp, human-HPV-hetero-catemeric DNA as either an extrachromosomal circle or a tandem repeat within the human genome. The human circularization/repeat junction was defined at single nucleotide resolution. The integrated viral DNA segments all retained an intact upstream regulatory region and the adjacent viral E6 and E7 oncogenes. RNA sequencing revealed that the only HPV genes consistently transcribed from the integrated viral DNAs were E7 and E6*I. The other two HPV DNA+ tumors had coinfections, but no evidence for integration. HPV-positive and HPV-negative vulvar cancers exhibited contrasting human, global gene expression patterns partially overlapping with previously observed differences between HPV-positive and HPV-negative cervical and oropharyngeal cancers. A substantial fraction of the differentially expressed genes involved immune system function. Thus, transcription and HPV DNA integration in vulvar cancers resemble those in other HPV-positive cancers. This study emphasizes the power of hybridization capture coupled with DNA and RNA sequencing to identify a broad spectrum of HPV types, determine human genome integration status of viral DNAs, and elucidate their structures.

Only persistent HPV infections lead to the development of cancer. Thus, understanding the virus-host interplay that influences the establishment of viral infection has important implications for HPV biology and human cancers. The ability of papillomaviruses to establish in cells requires the strict temporal regulation of viral gene expression in sync with cellular differentiation. This control primarily happens at the level of RNA splicing and polyadenylation. However, the details of how this spatio-temporal regulation is achieved still need to be fully understood. Until recently, it has been challenging to study the early events of the HPV lifecycle following infection. We used a single-cell genomics approach to identify cellular factors involved in viral infection and establishment. We identify protein arginine N-methyltransferase 1 (PRMT1) as an important factor in viral infection of primary human cervical cells. PRMT1 is the main cellular enzyme responsible for asymmetric dimethylation of cellular proteins. PRMT1 is an enzyme responsible for catalyzing the methylation of arginine residues on various proteins, which influences processes such as RNA processing, transcriptional regulation, and signal transduction. In this study, we show that HPV18 infection leads to increased PRMT1 levels across the viral lifecycle. PRMT1 is critical for the establishment of a persistent infection in primary cells. Mechanistically, PRMT1 inhibition leads to a highly dysregulated viral splicing pattern. Specifically, reduced PRMT1 activity leads to intron retention and a change in the E6 and E7 expression ratio. In the absence of PRMT1, viral transcripts are destabilized and subject to degradation via the nonsense-mediated decay (NMD) pathway. These findings highlight PRMT1 as a critical regulator of the HPV18 lifecycle, particularly in RNA processing, and position it as a potential therapeutic target for persistent HPV18 infections.

ObjectivePatients with pathogenic variants in the GATA Binding Protein 2 (GATA2), a hematopoietic transcription factor, are at risk for human papillomavirus-related (HPV) anogenital cancer at younger than expected ages. A female cohort with GATA2 haploinsufficiency was systematically assessed by two gynecologists to characterize the extent and severity of anogenital HPV disease, which was also compared with affected males.MethodsA 17-year retrospective review of medical records, including laboratory, histopathology and cytopathology records was performed for patients diagnosed with GATA2 haploinsufficiency followed at the National Institutes of Health. Student’s t-test and Mann-Whitney U test or Fisher’s exact test were used to compare differences in continuous or categorical variables, respectively. Spearman’s rho coefficient was employed for correlations.ResultsOf 68 patients with GATA2 haploinsufficiency, HPV disease was the initial manifestation in 27 (40%). HPV occurred at median 18.9 (15.2-26.2) years in females, and 25.6 (23.4-26.9) years in males. Fifty-two (76%), 27 females and 25 males, developed HPV-related squamous intraepithelial lesions (SIL) including two males with oral cancer. Twenty-one patients developed anogenital high-grade SIL (HSIL) or carcinoma (16 females versus 5 males, (59% versus 20%, respectively, p=0.005) at median 27 (18.6-59.3) years for females and 33 (16.5-40.1) years for males. Females were more likely than males to require >2 surgeries to treat recurrent HSIL (p=0.0009). Of 30 patients undergoing hematopoietic stem cell transplant (HSCT) to manage disease arising from GATA2 haploinsufficiency, 12 (nine females, three males) had persistent HSIL/HPV disease. Of these nine females, eight underwent peri-transplant surgical treatment of HSIL. Five of seven who survived post-HSCT received HPV vaccination and had no or minimal evidence of HPV disease 2 years post-HSCT. HPV disease persisted in two receiving immunosuppression. HPV disease/low SIL (LSIL) resolved in all three males.ConclusionFemales with GATA2 haploinsufficiency exhibit a heightened risk of recurrent, multifocal anogenital HSIL requiring frequent surveillance and multiple treatments. GATA2 haploinsufficiency must be considered in a female with extensive, multifocal genital HSIL unresponsive to multiple surgeries. This population may benefit from early intervention like HSCT accompanied by continued, enhanced surveillance and treatment by gynecologic oncologists and gynecologists in those with anogenital HPV disease.

Papillomaviruses (family Papillomaviridae) are non-enveloped, circular, double-stranded DNA viruses known to infect squamous and mucosal epithelial cells. In the family Papillomaviridae there are 53 genera and 133 viral species whose members infect a variety of mammalian, avian, reptilian, and fish species. Within the Antarctic context, papillomaviruses (PVs) have been identified in Adelie penguins (Pygoscelis adeliae, 2 PVs), Weddell seals (Leptonychotes weddellii, 7 PVs), and emerald notothen (Trematomus bernacchii, 1 PV) in McMurdo Sound and Ross Island in eastern Antarctica. Here we identified 13 diverse PVs from buccal swabs of Antarctic fur seals (Arctocephalus gazella, 2 PVs) and leopard seal (Hydrurga leptonyx, 3 PVs) in western Antarctica (Antarctic Peninsula), and vaginal and nasal swabs of Weddell seals (8 PVs) in McMurdo Sound. These PV genomes group into four genera representing 11 new papillomavirus types, of which five are from two Antarctic fur seals and a leopard seal and six from Weddell seals.

CD4 + T cell activation is driven by five-module receptor complexes. The T cell receptor (TCR) is the receptor module that binds composite surfaces of peptide antigens embedded within MHCII molecules (pMHCII). It associates with three signaling modules (CD3γε, CD3δε, and CD3ζζ) to form TCR-CD3 complexes. CD4 is the coreceptor module. It reciprocally associates with TCR-CD3-pMHCII assemblies on the outside of a CD4 + T cells and with the Src kinase, LCK, on the inside. Previously, we reported that the CD4 transmembrane GGXXG and cytoplasmic juxtamembrane (C/F)CV+C motifs found in eutherian (placental mammal) CD4 have constituent residues that evolved under purifying selection (Lee et al., 2022). Expressing mutants of these motifs together in T cell hybridomas increased CD4-LCK association but reduced CD3ζ, ZAP70, and PLCγ1 phosphorylation levels, as well as IL-2 production, in response to agonist pMHCII. Because these mutants preferentially localized CD4-LCK pairs to non-raft membrane fractions, one explanation for our results was that they impaired proximal signaling by sequestering LCK away from TCR-CD3. An alternative hypothesis is that the mutations directly impacted signaling because the motifs normally play an LCK-independent role in signaling. The goal of this study was to discriminate between these possibilities. Using T cell hybridomas, our results indicate that: intracellular CD4-LCK interactions are not necessary for pMHCII-specific signal initiation; the GGXXG and (C/F)CV+C motifs are key determinants of CD4-mediated pMHCII-specific signal amplification; the GGXXG and (C/F)CV+C motifs exert their functions independently of direct CD4-LCK association. These data provide a mechanistic explanation for why residues within these motifs are under purifying selection in jawed vertebrates. The results are also important to consider for biomimetic engineering of synthetic receptors.

Mitosis induces cellular rearrangements like spindle formation, Golgi fragmentation, and nuclear envelope breakdown. Similar to certain retroviruses, nuclear delivery during entry of human papillomavirus (HPV) genomes is facilitated by mitosis, during which minor capsid protein L2 tethers viral DNA to mitotic chromosomes. However, the mechanism of viral genome delivery and tethering to condensed chromosomes is barely understood. It is unclear, which cellular proteins facilitate this process or how this process is regulated. This work identifies crucial phosphorylations on HPV minor capsid protein L2 occurring at mitosis onset. L2’s chromosome binding region (CBR) is sequentially phosphorylated by the master mitotic kinases CDK1 and PLK1. L2 phosphorylation, thus, regulates timely delivery of HPV vDNA to mitotic chromatin during mitosis. In summary, our work demonstrates a crucial role of mitotic kinases for nuclear delivery of viral DNA and provides important insights into the molecular mechanism of pathogen import into the nucleus during mitosis.