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.

This paper systematically reviews paradigm shifts in diagnostic technologies for norovirus. Given the substantial burden of acute gastroenteritis caused by this virus and the current lack of licensed antiviral therapies, early and accurate diagnosis is of paramount importance. Diagnostic technologies have evolved from electron microscopy and immunological assays to molecular detection methods, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) currently recognized as the gold standard for molecular diagnostics. However, RT-qPCR is constrained by complex operational procedures, the need for specialized equipment, and limited suitability for rapid point-of-care applications. Emerging technologies, including isothermal amplification and clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies, have significantly improved diagnostic sensitivity, specificity, and turnaround time. Nevertheless, challenges related to standardization, quality control, and contamination prevention remain to be addressed before their widespread implementation. Furthermore, this review proposes a stratified, scenario-adaptive diagnostic framework in which rapid immunochromatographic assays are used for preliminary screening in community settings, highly sensitive PCR-based or multiplex molecular methods are employed for clinical diagnosis in healthcare facilities, and digital PCR (dPCR) is applied for precise quantification in food safety and environmental monitoring. Looking ahead,, advances in norovirus diagnostics will increasingly rely on interdisciplinary collaboration to accelerate the development of integrated and intelligent point-of-care testing (POCT) platforms. In particular, biosensors based on molecular recognition technologies and microfluidic platforms, which offer advantages such as, simple operation, rapid processing and high stability, are among the promising approaches for achieving instrument-free, on-site "sample-in, result-out" diagnostics.

Despite intensive vaccination efforts, Chicken Infectious Anemia Virus (CIAV) remains a formidable threat to the Egyptian poultry industry, primarily through the emergence of vaccine-escape variants. While most studies have focused on Northern Egypt, there is a critical knowledge gap regarding the viral landscape in the Southern provinces. This study provides the most extensive surveillance to date, spanning 10 diverse Egyptian governorates with a strategic focus on Upper Egypt (2024-2025). Out of 400 collected samples, TaqMan-based qPCR revealed a high prevalence of 30% (120/400). Five representative isolates were selected for whole-genome sequencing and molecular characterization. The isolates were classified as Genotype II by phylogenetic analysis, showing a notable genetic divergence (85-87% amino acid similarity) from traditional Genotype I vaccine strains. Notably, comprehensive recombination analysis identified isolate PX96998 as a mosaic variant, with breakpoints indicating intra-genotypic recombination between closely related genotype II lineages, highlighting complex evolutionary dynamics in the field. Furthermore, 3D homology modeling of the VP1 protein identified critical structural alterations in functional loop regions, potentially hindering the binding affinity of neutralizing antibodies and facilitating immune evasion. In an experimental setting, the genotype II isolate demonstrated hypervirulence in SPF chickens, causing severe development retardation and extensive atrophy of lymphoid organs (p < 0.0001). Pathognomonic pale, fatty bone marrow with a sharp drop in PCV% to 18 ± 0.58% indicated severe aplastic anemia. By the fifth week after infection, there was a complete humoral collapse with undetectable ELISA titers due to the huge systemic replication revealed by viral load measurement, which peaked in the thymus (9.50 ± 0.25 log10) and bone marrow (9.00 ± 0.22 log10). These results were supported by histopathology, which revealed intranuclear inclusion bodies and systemic lymphocyte depletion. These findings support the highly pathogenic genotype II CIAV's dominance and aggressive evolution in Egypt. The emergence of recombinant variants with altered structural epitopes underscores the urgent need to replace outdated vaccination plans with genotype-matched immunogens in order to lessen the severe consequences on Egypt's poultry industry.

Metagenomics or metaviromics is emerging as a powerful technology for pathogen surveillance and pandemic preparedness. Mosquitoes are important vectors for transmission of many emerging viruses responsible for numerous outbreaks. Monitoring mosquitoes becomes essential to investigate its virome which leads to understanding of disease dynamics and allow preventive actions. In this study, shotgun metagenomic methodology using Ion GeneStudio S5 System was optimized for exploration of viral diversity. A total of 1913 Aedes larvae were collected from Central India during post monsoon season of 2024. Aedes larvae reared to adulthood and processed for sequencing using Ion Torrent S5 platform. Computational analyses were performed using three bioinformatic pipelines: Chan Zuckerberg ID (CZ ID), Genome Detective Platform and the Galaxy Platform. A mock database of 11 known viruses was created as well as publicly available NCBI Sequence Read Archive (SRA) datasets were used to validate all three pipelines. In terms of detection accuracy, Genome Detective and CZ ID performed exceptionally well and therefore may be suitable for future mosquito virome surveillance studies. We found presence of viruses viz. Alphamesonivirus cavallynense and Phasivirus phasiense dominating in all samples. Dengue virus was detected in one sample by CZ ID, whereas Wenzhou sobemo-like virus, Hubei mosquito virus 2, Cell fusing agent virus found most commonly among samples. Other viruses found like Aedes anphevirus (AeAV; genus Glybovirus), Aedes totivirus, Verdadero virus and Chaq-like virus. Variation in the result among different pipelines are likely attributable to incorporation of different viral reference databases, classification algorithms, metrics and analysis parameters. To the best of our knowledge, this study represents the first metagenomic study of mosquitoes using Ion GeneStudio S5 platform in India. The findings provide a comparative evaluation of the metagenomic pipelines and elucidates detailed information of each pipeline and its working for future studies.