Insect-specific viruses (ISVs) are integral components of mosquito viromes, influencing virus evolution, vector competence, and arbovirus ecology. Papua New Guinea (PNG) is a region of exceptional biodiversity, yet ISVs in local mosquito populations remain poorly characterized. Here, we present the first comprehensive metagenomic survey of ISVs in Culex mosquitoes collected across five provinces of PNG between 2019 and 2021. Using unbiased shotgun metagenomic sequencing, 13,871 female Culex mosquitoes representing five species were analyzed across 86 samples. Approximately 1.6 billion paired-end reads were generated, yielding ∼20 million viral read pairs. Overall, 231 viral species representing 38 viral families were detected. Of these, 120 were ISV species spanning 25 viral families, and ISVs accounted for 93.8% of species-assigned viral reads, with RNA viruses predominating. The most abundant families included Partitiviridae, Birnaviridae, Rhabdoviridae, and Iflaviridae. Approximately 40% of viral reads corresponded to viruses that are not currently assigned to recognized ICTV families. More than 150 complete viral genomes or genome segments were recovered, including divergent sequences that share <90% amino acid identity with known references. Phylogenetic analyses revealed several divergent viral lineages across major ISV groups. Within the Partitiviridae, previously unrecognized RNA-dependent RNA polymerase (RdRP)-capsid pairings enabled reconstruction of complete genome architectures for Broome partiti-like virus 1 and Sonnbo virus for the first time, together with additional divergent partitivirus lineages. Cambodia Culex birnavirus sequences from PNG, clustered within the known lineage but formed a distinct regional clade. Rhabdovirus analyses identified host-restricted lineages, including Culex tritaeniorhynchus rhabdovirus-like sequences confined to Culex annulirostris. Within the Iflaviridae, several viruses were detected for the first time in PNG. Hubei virga-like virus 2 was the most abundant ISV detected and formed a PNG-specific clade, suggesting local diversification. ISVs were detected in both blood-fed and unfed mosquitoes, indicating that they are stable constituents of the mosquito virome. Multiple viral species from the same family were detected within pooled samples, consistent with either co-infection of individual mosquitoes or heterogeneous infections among pooled individuals. Overall, this study reveals a diverse ISV landscape in PNG Culex mosquitoes, expanding the known geographic range and genetic diversity of multiple viral groups and providing a baseline for future investigations into ISV ecology and evolution in the Indo-Pacific region and mosquito-virus interactions globally.

Human herpesvirus infections span a wide variety of pathogenic outcomes, with individual representatives comprising a huge clinical relevance on human health. Translational research efforts are directed towards the improvement of broadly applicable preventive measures and detailed understanding of viral pathogenesis. Here, we studied rate-limiting steps in viral replication that show high mechanistic conservation among the Herpesviridae family members, in particular concerning the viral nucleocytoplasmic capsid egress. As characterized for several herpesviruses, viral nuclear egress is facilitated by the virus-induced local nuclear lamina rearrangements of infected cells. Hereby, the virus-mediated site-specific phosphorylation at lamin A/C Ser22, followed by cis/trans isomerization of the structural conformation, leads to the induction of lamina-depleted areas (LDAs). We have generated an LDA reporter system in primary human fibroblasts (HFFs), which conditionally express the autofluorescent lamin A/C-RFP fusion construct, for quantifying viral nuclear egress activity. This system was successfully applied for human and animal cytomegaloviruses as well as a selection of α-, β-, and γ-herpesviruses shown here. Our results illustrate that (i) the reporter system was effective in a quantitative way for all viruses analyzed; (ii) the process of LDA formation showed consistency among herpesviruses; (iii) the impairment of viral kinase activity, responsible for lamin A/C Ser22 phosphorylation, significantly reduced LDA formation; and (iv) inhibitory small molecules, directed either to the viral nuclear egress complex or the cellular isomerase activity, likewise provide an LDA-blocking effect leading to strong antiviral efficacy. Thus, the autofluorescent reporter cell model provides manifold options to study herpesviral productive replication, nuclear host interaction, and the pathogenesis-related impact of regulatory factors.

Respiratory syncytial virus (RSV) glycoprotein G is heavily glycosylated and of variable size following virus growth in transformed cell lines or primary cells. However, it is unknown if the cell culture medium can influence the patterns of glycosylation. Therefore, we tested whether the choice of culture medium can affect the glycosylation of RSV G protein, viral infectivity and antibody recognition. The RSV G ectodomain was produced in HEK293T cells cultured in three different media. Marked differences in molecular weight were observed that were abolished in glycosylation-deficient knockout cells. RSV produced in A549 cells cultured in the same media also differed in relative G protein signals in virus stocks and virus spread. Higher apparent G protein glycosylation was associated with lower binding signals of human F protein-specific monoclonal antibodies to RSV-infected cells. These data show that the cell culture medium can influence biological properties of RSV preparations with consequences for antibody recognition and viral spread.

Human herpesvirus infections cover a wide range of pathogenic outcomes and challenging situations in the medical field. All herpesviral α-, β-, and γ-subfamilies comprise representatives with a huge clinical impact on human health. The basic and translational research efforts are strongly directed towards the improved molecular understanding of infections and development of novel antiviral test systems and preventive measures. We utilized cell culture-based quantitative models for the assessment of antiviral prototype drugs against human and animal pathogenic herpesviruses. Our analysis focused on approved, developmental, and experimental compounds. All three investigated herpesviruses showed sensitivity against the selected antivirals. The single-drug antiviral treatments identified mean effective concentrations in the micromolar range, for abemaciclib (ABE) and the pharmacophore compound 020 (P-020), as well as submicromolar to nanomolar range, for brincidofovir (BCV) and cyclin-dependent kinase inhibitor of CDK7 (LDC4297). Time-of-addition experiments addressed questions of temporal potency of inhibition and effects on the expression of individual viral proteins. The results demonstrated that these drugs were mostly robust and did generally show moderate or no difference in antiviral activity when administered pre-, post-, or total duration-infection. By addressing the question of potential drug-drug synergies in combinatorial cotreatment settings, we identified effective conditions of combined antiherpesviral drug treatment (cAHT), such as ABE + LDC4297, as demonstrated for all three viruses. Thus, the study provides new information about antiherpesviral potency and characteristics of a selection of four antiviral prototype inhibitors. Especially, examples of broadness in synergistic combination treatment suggested options to improve antiherpesviral therapy strategies.

Accurate characterization of human papillomavirus (HPV) integration into the host genome is essential for understanding HPV-driven carcinogenesis. However, most previous integration studies relied on incomplete human reference genomes, potentially obscuring integration events occurring within highly repetitive genomic regions. Here, we reassessed HPV integration landscapes using the first complete telomere-to-telomere (T2T) human reference genome assembly (hs1). We reanalyzed datasets from three independent studies generated using complementary sequencing technologies, including short-read Illumina sequencing, PacBio continuous long reads (CLR), PacBio high-fidelity (HiFi) sequencing, and ultra-long Oxford Nanopore sequencing. Reanalysis using the T2T reference confirmed the majority of previously reported integration breakpoints while identifying numerous additional sites, increasing the total number of detected breakpoints from 446 to 799. Notably, newly detected integrations frequently localized to repeat-rich genomic regions, including centromeric and pericentromeric satellite sequences that were absent from earlier reference assemblies. Importantly, integration events within these regions were detectable not only in long-read datasets but also in short-read sequencing data when analyzed using the complete reference genome. Our findings demonstrate that incomplete reference assemblies have led to systematic underestimation of HPV integration events, particularly within structurally complex genomic regions. These results highlight the importance of adopting complete human genome references for viral integration analyses and suggest that reanalysis of existing datasets may reveal previously overlooked biologically and clinically relevant integration events.

Hantavirus is considered as a fatal zoonotic pathogen that can be commonly transmitted from rodents to humans. However, the Andes virus (ANDV), a type of New World hantavirus, can also be transmitted from human to human. It is the cause of the 2026 hantavirus outbreak on a cruise ship. Hantavirus can rapidly progress to a severe respiratory distress, known as hantavirus cardiopulmonary syndrome (HCPS). Since there is no specific treatment or approved vaccine for hantavirus infection, early diagnosis and critical care support are essential. In cases of HCPS, depending on the severity of the infection, it is important to administer antipyretics, vasopressors, fluid therapy, cardiopulmonary support, extracorporeal membrane oxygenation (ECMO), mechanical ventilation, and renal replacement therapy (RRT) in a timely manner. Currently, there is no approved vaccine in the United States or Europe. However, various vaccine technologies, including inactivated, DNA, mRNA, viral vector and protein-based vaccines, are in the preclinical and clinical phases. Preventive actions, including patient isolation and physical distancing, are crucial to avoiding widespread transmission.