Search Results (723)

The repeated occurrence of SARS-CoV-2 variants, largely driven by virus–host interactions, was and will remain a public health concern. Spike protein mutations shaped viral infectivity, transmissibility, and immune escape. From February 2022 to April 2024, a local genomic surveillance program in Verona, Italy, was conducted on 1333 SARS-CoV-2-positive nasopharyngeal swabs via next generation full-length genome sequencing. Spike protein mutations were classified based on their prevalence over time. Mutations were grouped into five categories: fixed, emerging, fading, transient, and divergent. Notably, some divergent mutations displayed a “Lazarus effect,” disappearing and later reappearing in new lineages, indicating potential adaptive advantages in specific genomic contexts. This two-year surveillance study highlights the dynamic nature of spike protein mutations and their role in SARS-CoV-2 evolution. The findings underscore the need for ongoing mutation-focused genomic monitoring to detect early signals of variant emergence, especially among mutations previously considered disadvantageous. Such efforts are critical for driving public health responses and guiding future vaccine and therapeutic strategies. Full article

SARS-CoV-2 undergoes frequent mutations that drive viral evolution and genomic diversity, influencing transmissibility, immune escape, and disease severity. In this study, we performed whole-genome sequencing on SARS-CoV-2 isolates from patients in New York City and identified several globally rare mutations across multiple viral lineages. The isolates analyzed for rare mutations belonged to three lineages: B.1.1.7 (Alpha), B.1.526 (Iota), and B.1.623. We identified 16 rare mutations (global incidence <1000) in non-structural protein genes, including nsp2, nsp3, nsp4, nsp6, nsp8, nsp13, nsp14, ORF7a, and ORF8. Three of these mutations—located in nsp2, nsp13, and ORF8—have been reported in fewer than 100 individuals worldwide. We also detected five rare mutations in structural proteins (S, M, and N), including two—one in M and one in N—previously reported in fewer than 100 cases globally. We present clinical profiles of three patients, each infected with genetically distinct viral isolates from the three lineages studied. Furthermore, we illustrate a local transmission chain inferred from unique mutation patterns identified in the Omicron genome. These findings underscore the importance of whole-genome sequencing for detecting rare mutations, tracking community spread, and identifying emerging variants with clinical and public health significance. Full article

The rabies virus (RABV) phosphoprotein (P protein) has multiple functions, including acting as the essential non-catalytic cofactor of the viral polymerase (L protein) for genome replication and transcription; the principal viral antagonist of the interferon (IFN)-mediated innate immune response; and the chaperone for the viral nucleoprotein (N protein). Although P protein is known to undergo phosphorylation by cellular kinases, the location and functions of the phosphorylation sites remains poorly defined. Here, we report the identification by mass-spectrometry (MS) of residues of P protein that are modified by phosphorylation in mammalian cells, including several novel sites. Analysis of P protein with phospho-mimetic and phospho-inhibitory mutations of three novel residues/clusters that were commonly identified by MS (Ser48, Ser183/187, Ser217/219/220) indicate that phosphorylation at each of these sites does not have a major influence on nuclear trafficking or antagonistic functions toward IFN signalling pathways. However, phosphorylation of Ser48 in the N-terminus of P protein impaired function in transcription/replication and in the formation of replication structures that contain complexes of P and N proteins, suggestive of altered interactions of these proteins. The crystal structure of P protein containing the S48E phospho-mimetic mutation indicates that Ser48 phosphorylation facilitates the binding of residues 41–52 of P protein into the RNA-binding groove of non-RNA-bound N protein (N⁰), primarily through the formation of a salt bridge with Arg434 of N protein. These data indicate that Ser48 modification regulates the cycling of P-N⁰ chaperone complexes that deliver N protein to RNA to enable transcription/replication, such that enhanced interaction due to S48E phospho-mimetic mutation reduces N protein delivery to the RNA, inhibiting subsequent transcription/replication processes. These data are, to our knowledge, the first to implicate phosphorylation of RABV P protein in conserved replication functions of the P gene. Full article

Background: COVID-19 is associated with coagulopathy and increased mortality. The ABO blood group system has been implicated in modulating susceptibility to SARS-CoV-2 infection and disease severity, but its relationship with viral RNAemia, spike gene mutations, and thrombosis remains underexplored. Methods: We analyzed 446 hospitalized COVID-19 patients between 2021 and 2022. SARS-CoV-2 RNAemia was assessed via RT-qPCR on whole blood, and spike gene mutations were identified through whole-genome sequencing in RNAemia-positive samples. ABO blood groups were determined by agglutination testing, and thrombotic events were evaluated using coagulation markers. Statistical analyses included chi-square tests and Kruskal–Wallis tests, with significance set at p < 0.05. Results: RNAemia was detected in 26.9% of patients, with no significant association with ABO blood group (p = 0.175). Omicron was the predominant variant, especially in blood group A (62.5%). The N501Y mutation was the most prevalent in group O (53.2%), and K417N was most prevalent in group B (36.9%), though neither reached statistical significance. Thrombotic events were significantly more common in blood group A (OR = 2.08, 95% CI = 1.3–3.4, p = 0.002), particularly among RNAemia-positive patients. Conclusions: ABO blood group phenotypes, particularly group A, may influence thrombotic risk in the context of SARS-CoV-2 RNAemia. While no direct association was found between blood group and RNAemia or spike mutations, the observed trends suggest potential host–pathogen interactions. Integrating ABO typing and RNAemia screening may enhance risk stratification and guide targeted thromboprophylaxis in COVID-19 patients. Full article

Various SARS-CoV-2 remdesivir resistance-associated substitutions (RAS) have been reported, but a comprehensive comparison of their resistance levels is lacking. We identified novel RAS and performed head-to-head comparisons with known RAS in Vero E6 cells. A remdesivir escape polyclonal virus exhibited a 3.6-fold increase in remdesivir EC₅₀ and mutations throughout the genome, including substitutions in nsp12 (E796D) and nsp14 (A255S). However, in reverse-genetics infectious assays, viruses harboring both these substitutions exhibited only a slight decrease in remdesivir susceptibility (1.3-fold increase in EC₅₀). The nsp12-E796D substitution did not impair viral fitness (Vero E6 cells or Syrian hamsters) and was reported in a remdesivir-treated COVID-19 patient. In replication assays, a subgenomic replicon containing nsp12-E796D+nsp14-A255S led to a 16.1-fold increase in replication under remdesivir treatment. A comparison with known RAS showed that S759A, located in the active site of nsp12, conferred the highest remdesivir resistance (106.1-fold increase in replication). Nsp12-RAS V166A/L, V792I, E796D or C799F, all adjacent to the active site, caused intermediate resistance (2.0- to 11.5-fold), whereas N198S, D484Y, or E802D, located farther from the active site, showed no resistance (≤2.0-fold). In conclusion, our classification system, correlating replication under remdesivir treatment with RAS location in nsp12, shows that most nsp12-RAS cause moderate resistance. Full article

The Crimean–Congo hemorrhagic fever virus (CCHFV) is a single-stranded, segmented RNA virus belonging to the Nairoviridae family, and it is rapidly expanding across Africa, Asia, and southern Europe, probably favored by climate change and livestock trade. Its fatality rate in humans reaches up to 40%, and there is currently no specific treatment or vaccine available. Therefore, the development of therapies against CCHFV is essential, and their design requires understanding of the molecular evolution and genetic distribution of the virus. Motivated by these concerns, we present a comprehensive review of the molecular evolution, genetic characterization, and phylogeography of CCHFV, and we discuss their potential implications for therapeutic design. Specifically, we describe the virus’s capacity to increase its genetic diversity through numerous mutations, recombination events, and genomic reassortments, which affect fundamental viral functions such as RNA binding, host–virus interactions, viral entry, and polymerase activity. We also assess the presence of temporal heterogeneous rates of evolution and molecular adaptation among CCHFV coding regions, where purifying selection is generally predominant but diversifying selection is observed in molecular regions associated with host adaptation and transmission. We emphasize the importance of understanding the complex molecular evolution of CCHFV for the rational design of therapies and highlight the need for efforts in surveillance, evolutionary prediction, and therapeutic development. Full article

Scavenging domestic ducks significantly contribute to the transmission and maintenance of highly pathogenic H5N1 clade 2.3.4.4b avian influenza viruses in Bangladesh, a strain of growing global concern due to its broad host range, high pathogenicity, and spillover potential. This study investigates the molecular epidemiology and pathology of HPAI H5N1 viruses in unvaccinated scavenging ducks in Bangladesh, with the goal of assessing viral evolution and associated disease outcomes. Between June 2022 and March 2024, 40 scavenging duck flocks were investigated for HPAI outbreaks. Active HPAIV H5N1 infection was detected in 35% (14/40) of the flocks using RT-qPCR. Affected ducks exhibited clinical signs of incoordination, torticollis, and paralysis. Pathological examination revealed prominent meningoencephalitis, encephalopathy and encephalomalacia, along with widespread lesions in the trachea, lungs, liver, and spleen, indicative of systemic HPAIV infection. A phylogenetic analysis of full-genome sequences confirmed the continued circulation of clade 2.3.2.1a genotype G2 in these ducks. Notably, two samples of 2022 and 2023 harbored HPAIV H5N1 of clade 2.3.4.4b, showing genetic similarity to H5N1 strains circulating in Korea and Vietnam. A mutation analysis of the HA protein in clade 2.3.4.4b viruses revealed key substitutions, including T156A (loss of an N-linked glycosylation site), S141P (antigenic site A), and E193R/K (receptor-binding pocket), indicating potential antigenic drift and receptor-binding adaptation compared to clade 2.3.2.1a. The emergence of clade 2.3.4.4b with the first report of neurological and systemic lesions suggests ongoing viral evolution with increased pathogenic potential for ducks. These findings highlight the urgent need for enhanced surveillance and biosecurity to control HPAI spread in Bangladesh. Full article

Background: Parvovirus B19 (B19V) can cause severe relapsing episodes of pure red cell aplasia in immunocompromised individuals, which are commonly treated with intravenous immunoglobulins (IVIGs). Few data are available on B19V intra-host evolution and the role of humoral immune selection. Here, we report the dynamics of genomic mutations and subsequent protein changes during relapsing infection. Methods: Longitudinal plasma samples from immunocompromised patients with relapsing B19V infection in the period 2011–2019 were analyzed using whole-genome sequencing to evaluate intra-host evolution. The impact of mutations on the 3D viral protein structure was predicted by deep neural network modeling. Results: Of the three immunocompromised patients with relapsing infections for 3 to 9 months, one patient developed two consecutive nonsynonymous mutations in the VP1/2 region: T372S/T145S and Q422L/Q195L. The first mutation was detected in multiple B19V IgG-seropositive follow-up samples and resolved after IgG seroreversion. Computational prediction of the VP1 3D structure of this mutant showed a conformational change in the proximity of the antibody binding domain. No conformational changes were predicted for the other mutations detected. Discussion: Analysis of relapsing B19V infections showed mutational changes occurring over time. Resulting amino acid changes were predicted to lead to a conformational capsid protein change in an IgG-seropositive patient. The impact of humoral response and IVIG treatment on B19V infections should be further investigated to understand viral evolution and potential immune escape. Full article

The H1N1 swine influenza viruses CQ91 and CQ445, isolated from pigs in China, exhibited distinct virulence in mice despite sharing similar genomic constellations. CQ91 demonstrated higher pathogenicity (MLD₅₀: 5.4 log₁₀ EID₅₀) and replication efficiency in mice compared to CQ445 (MLD₅₀: 6.6 log₁₀ EID₅₀). Through reverse genetics, we found that the attenuation of CQ445 was due to a single substitution of glutamic acid (E) with lysine (K) at position 343 in the PB2 protein. Introducing the CQ445-PB2 (343K) into CQ91 significantly reduced viral replication and pathogenicity in mice, while replacing CQ445-PB2 with CQ91-PB2 (343E) restored virulence. In vitro studies showed that the K343E mutation impaired viral replication in MDCK and A549 cells and reduced polymerase activity in minigenome assays. Mechanistically, the amino acid at position 343 in the PB2 affects the transcription stage of the viral replication process. Structural modeling indicated that the charge reversal caused by E343K altered local electrostatic interactions without major conformational changes. Phylogenetic analysis revealed that PB2-343E is highly conserved (>99.9%) in human and swine H1/H3 influenza viruses, suggesting that PB2-343E confers an adaptive advantage. This study identifies PB2-343E as a critical determinant of influenza virus pathogenicity in mammals, highlighting its role in host adaptation. Full article

HPV-independent cervical cancers represent a small proportion of these types of cancers, predominantly glandular lesions. It should be noted that some cases may depend on diagnostic problems that lead to false negative cases. However, the most recent classifications distinguish cervical tumors into HPV-associated and HPV-independent cancers. HPV-negative cervical carcinomas (5–11% of all cases) mainly include rare adenocarcinomas (gastric, mesonephric, clear, serous, and endometrioid) and present distinct clinical and molecular features. These tumors usually affect older women and are diagnosed at more advanced stages than HPV-positive tumors, with an overall worse prognosis. This concerning and notably worse prognosis highlights the need for further research and understanding. Unlike HPV-positive carcinomas (which depend on the viral oncogenes E6/E7), HPV-independent tumors accumulate genomic mutations that activate oncogenes and inactivate suppressor genes. Therefore, a comprehensive overview of these aspects can be the key to a better understanding and developing personalized treatments. In the present review, the main mutated genes, the signaling pathways involved, the differences from HPV-positive tumors, the distinctive immunohistochemical markers, and the diagnostic and therapeutic implications are explored in depth. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the 2019 coronavirus disease pandemic. The virus primarily spreads through person-to-person contact via aerosols and droplets, contributing to high case numbers and related morbidities. SARS-CoV-2 targets the respiratory tract, causing acute respiratory distress syndrome, particularly in immunocompromised individuals such as those with cystic fibrosis (CF). CF is a life-threatening genetic disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, leading to impaired respiratory function and recurrent severe respiratory symptoms. Despite their potential vulnerability, CF patients have shown a lower incidence of severe COVID-19, suggesting protective factors against SARS-CoV-2. Differential expression of the ACE2 receptor, crucial for viral entry, and other host factors, such as TMPRSS2, may play a role in this resistance to SARS-CoV-2. Analyzing the genomics and transcriptomics profiles of CF patients could provide insights into potential resistance mechanisms. The potential resistance mechanisms include blood and extracellular ATP levels, a deleted/dysfunctional CFTR gene, ACE and ACE2 regulation and expression, ACE and ACE2 polymorphism effects, host proteins and SARS-CoV-2 interactions, and SMN1 and ACE/ACE2 interactions. This review discusses the underlying factors and potential resistance mechanisms contributing to CF patients’ responses to SARS-CoV-2 infection. The review provides an opportunity to further investigate future therapy and research through understanding the underlying potential resistance mechanisms exhibited by CF patients against SARS-CoV-2, including ACE and ACE2 polymorphisms. Full article

Persistent SARS-CoV-2 infections involve prolonged viral replication and immune system interactions, potentially driving viral evolution and immune escape. This study examines viral characteristics and host gene expression changes in persistent infections. The nasopharyngeal samples from four patients with persistent SARS-CoV-2 infections at Tottori University Hospital, Japan, were analyzed. Viral isolates were cultured, and infectivity was assessed using TCID₅₀ assays. To investigate host responses, RNA sequencing (RNA-seq) was performed to identify differentially expressed genes (DEGs), and Gene Ontology (GO) enrichment analysis mapped affected biological pathways. Viral genome sequencing detected mutations associated with prolonged infection. The results showed significant infectivity differences between early- and late-phase infection. Gene expression analysis revealed a strong early phase of pro-inflammatory response (IL6, TNF, IL1B, CXCL10) followed by immune suppression. GO enrichment analysis highlighted inflammation and cytokine-mediated immune pathways. Genomic sequencing identified mutations in ORF1ab and the spike (S) protein, potentially aiding immune escape. The findings underscore that SARS-CoV-2 adapts during persistent infections, altering infectivity and immune responses. These highlight the need for continued monitoring of prolonged infections to mitigate immune escape and viral evolution. Full article

Mpox, caused by the Mpox virus (MPXV), is a re-emerging zoonotic disease in the Poxviridae family. Since 2022, sub-Saharan Africa has experienced recurrent outbreaks, with the Democratic Republic of the Congo (DRC) accounting for 96% of the 567 confirmed cases reported in the African region by the World Health Organization as of June 2024. Despite MPXV’s endemic presence, its genomic diversity and evolutionary dynamics remain poorly characterized. We analyzed 270 MPXV genomes from 13 sub-Saharan African countries (2022–2024), representing the most geographically comprehensive regional dataset from the outbreak period. Phylogenetic analysis identified two geographically distinct clades: Clade I (East/Central Africa) and Clade II (West/Southern Africa). A marked disparity in APOBEC3-associated mutations was observed, with Clade IIb exhibiting significantly higher enrichment than Clade I, suggesting clade-specific host adaptation pressures. These mutations predominantly target genes involved in immune evasion and replication. Preliminary functional predictions indicated that selected missense mutations may impact on protein stability, underscoring the need for further experimental validation. Our findings provide the first pan–sub-Saharan analysis of MPXV clade divergence and reinforce the importance of sustained, regionally informed genomic surveillance to monitor viral evolution and guide outbreak response strategies across Africa. Full article

Tomato spotted wilt virus (TSWV), belonging to the genus Orthotospovirus, is a significant pathogen through its infection of economically vital crops including tomato, tobacco, pepper, and other species worldwide. Given its substantial influence on the agricultural industry, in-depth research on TSWV is of great necessity. Several studies have been conducted on the dinucleotide preference of TSWV previously; however, the information regarding codon usage bias (CUB) and the virus’s adaptive evolution remains inconclusive. Here, a thorough analysis of TSWV was performed by utilizing five protein-coding sequences in order to investigate the characteristics of CUB. It has been observed that the TSWV protein-coding sequences are AU-rich, and codons ending with A or U are also preferred in these sequences. A consistently stable and preserved genomic composition characterized by a lower codon usage preference was also observed. Principal Component Analysis (PCA), neutrality analysis, and ENC-plot indicated that, in comparison to mutational pressure, natural selection has a more dominant influence on the CUB of TSWV. The codon adaptation index (CAI) demonstrated a more significant correlation between TSWV and Nicotiana tabacum. Meanwhile, the relative codon deoptimization index (RCDI) indicated a stronger correlation between TSWV and Solanum lycopersicum. This study is the first to systematically characterize the CUB of TSWV based on its protein-coding sequences, providing critical insights into viral genetic diversity, evolution mechanisms, and host adaptation. The findings advance understanding of plant-virus coevolution and inform virus-resistant crop breeding and antiviral strategies. Full article

The Baculovirus Expression Vector System (BEVS) is an important protein and complex biologics production platform. The baculovirus GP64 protein is the major envelope glycoprotein that aids in virus entry and is required for cell-to-cell transmission in cell culture. Several studies have developed strategies around gp64 gene disruption in an attempt to minimize baculovirus co-production. Here, we investigate the result of transiently targeting the baculovirus gp64 gene with CRISPR-Cas9 during infection. Because not all genomes are effectively disrupted, we describe a variant calling methodology that allows the detection of the targeted mutations in gp64 even though these mutations are not the dominant sequences. Using a transfection-infection assay (T-I assay), the AcMNPV gp64 gene was targeted at six different locations to evaluate the effects of single and multiple targeting sites, and we demonstrated a reduction in the levels of baculovirus vectors while maintaining or enhancing foreign protein production when protein was driven by a p6.9 promoter. Viral genomes were subsequently isolated from the supernatant and cell pellet fractions, and our sequencing pipeline successfully detected indel mutations within gp64 for most of the single-guide RNA (sgRNA) targets. We also observed that 68.8% of variants found in the virus stock were conserved upon virus propagation in cell culture, thus indicating that they are not detrimental to viral fitness. This work provides a comprehensive assessment of CRISPR-Cas9 genome editing of baculovirus vectors, with potential applications in enhancing the efficiency of the BEVS. Full article