Alphacoronaviruses comprise human, livestock, and wildlife coronaviruses of importance, particularly to the livestock industry worldwide. Domestic livestock have the potential to serve as intermediate hosts of coronaviruses, given the contact they have with both humans and wildlife. With the aim of detection and subsequent complete genome sequencing of novel and known but previously undetected alphacoronaviruses, a screening RT-PCR assay was established. In vitro transcripts of two human alphacoronaviruses were generated to evaluate assay performance, and a collection of archived bat and livestock viral RNA samples was tested as part of assay validation. Confirmation of RT-PCR-positive outcomes was done by Sanger and high-throughput sequencing, before phylogenetic analysis. The assay can detect both human alphacoronaviruses with a detection limit of 46 copies/reaction of Alphacoronavirus amsterdamense and 504 copies/reaction of Alphacoronavirus chicagoense in vitro transcripts, as estimated by a probit regression analysis. Validation screening of domestic and peri-domestic livestock species, including cattle, sheep, goats, donkeys, pigs, rabbits, and grasscutters, did not yield any positive outcomes. Confirmed detections were made in bats (3.11%), comprising 1.84% alphacoronaviruses and 1.27% betacoronaviruses. Two complete genomes were obtained for an Alphacoronavirus chicagoense-related bat coronavirus and a putative member of the subgenus Hibecovirus. A near-complete genome was obtained for a Chaerephon spp. bat alphacoronavirus, which was found to be most closely related to a similar virus from Nigeria. This study presents a sensitive assay for coronavirus surveillance and provides additional complete genomes, thereby enhancing our understanding of coronavirus diversity.
Since 2016, mass mortality events characterized primarily by severe abdominal swelling and ascites have emerged in Chinese soft-shelled turtle (Pelodiscus sinensis) farms in China, leading to substantial economic losses. However, the cause of the disease has remained elusive, as no bacteria or parasites were detected in the diseased turtles. Tissue homogenate filtrate was used to infect Vero, GCO, TSK, TSL, and C6/36 cells, all of which produced cytopathic effects (CPE). Spherical, suspected viral particles with diameters approximately 30 nanometers (nm) were observed in the kidney tissue of diseased turtles using transmission electron microscopy (TEM). The complete viral genome was obtained through a combination of sequence-independent single-primer amplification (SISPA) and high-throughput sequencing, with rapid amplification of cDNA ends (RACE) employed to determine the terminal sequences. The genome is a positive-sense single-stranded RNA comprising 10,956 nucleotides (nt), exhibiting classic Orthoflavivirus characteristics and encoding a single open reading frame (ORF). Phylogenetic analysis indicated that the virus was most closely related to Trionyx sinensis flavivirus isolate YH2018, member of the Orthoflavivirus genus. Consequently, the virus has been named P. sinensis flavivirus isolate HZ2016 (PSFV-HZ2016). In an artificial challenge experiment, injection of tissue filtrate resulted in 100% mortality among healthy turtles within 16 days post-infection. Infected turtles displayed symptoms similar to those of the primary cases. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) revealed broad tissue tropism, with the highest viral load detected in the heart. Histopathological analysis confirmed multi-organ damage consistent with severe systemic infection. These findings identify PSFV-HZ2016 as a novel and highly virulent Orthoflavivirus responsible for ascites-associated mortality in P. sinensis, providing crucial insights for diagnosis, surveillance, and disease management in aquaculture.
Occult hepatitis B virus Infection (OBI) is a form of hepatitis B virus (HBV) persistence that is not accompanied by the presence of circulating HBsAg. It is generally defined as the presence of replication-competent viral DNA, mostly covalently closed circular DNA (cccDNA), in hepatocytes. Although viral load is low and serum markers are typically undetectable, OBI has clinical significance due to its association with transmission, reactivation under immunosuppression, HCV replicative levels, and the progression of liver fibrosis, cirrhosis, and hepatocellular carcinoma. The presence of cccDNA, a stable episomal minichromosome sustained through epigenetic regulation and immune evasion mechanisms, forms the basis for viral evasion of immune clearance and diagnostic detection. The molecular drivers of OBI involve complex interactions among three key factors: viral genetic mutations (particularly in the S region), epigenetic modifications (including cytosine methylation and histone modifications), and host immune responses that are often weakened or exhausted. This review integrates current knowledge regarding these molecular pathways and their contributions to OBI's persistence, reactivation, and associated diseases. A comprehensive understanding of these mechanisms is essential for improving diagnostic approaches, developing targeted therapies, and ultimately achieving the eradication of HBV in this resistant form of infection.
The identification of specific serological targets for Orthoflavivirus zikaense (ZIKV) remains challenging due to the high degree of structural similarity and antibody cross-reactivity among co-circulating arboviruses, particularly dengue virus (DENV). In this context, immunoinformatics-based approaches may contribute to the rational selection of candidate epitopes with potentially improved analytical specificity. In the present study, five conserved ZIKV peptides previously identified through an immunoinformatic pipeline were experimentally evaluated as candidate serological targets. Peptide reactivity was assessed by indirect ELISA using human serum samples positive for anti-ZIKV IgM and IgG antibodies. Distinct reactivity patterns were observed between antibody classes. IgM assays showed limited analytical discriminatory capacity, particularly due to reduced specificity for some peptides. In contrast, IgG-based analyses revealed more favorable analytical profiles. Among the peptides evaluated, a peptide identified in the capsid (PEP03) exhibited the most favorable analytical performance, with an area under the ROC curve (AUC) of 0.9183 (95% CI: 0.8301-1.000). An absence of detectable reactivity was observed among sera from DENV-positive patients, and minimal reactivity with chikungunya virus (CHIKV) positive samples under the experimental conditions employed. Other peptides tested in this study demonstrated variable analytical profiles, reinforcing the importance of rational epitope selection for serological applications. Overall, these findings provide preliminary analytical evidence that structurally derived ZIKV epitopes may exhibit distinct IgM and IgG recognition patterns and support the continued evaluation of capsid-derived epitopes as candidate targets for serological applications (IgG detection) in regions where multiple arboviruses co-circulate.
Canine parvovirus type 2 (CPV-2) remains one of the most significant viral pathogens causing infectious disease in domestic dogs worldwide, particularly causing severe hemorrhagic gastroenteritis in puppies. In recent years, canine circovirus (CanineCV) has emerged as an additional enteric virus of concern, although its role in co-infection with CPV-2 remains poorly characterized in Southeast Asia. This study determines the prevalence, co-infection rate, and molecular characteristics of CPV-2 and CanineCV co-infections in domestic dogs across three major regions of Vietnam. A total of 254 rectal swab samples were collected from 2023 to 2024 from dogs exhibiting clinical signs consistent with CPV-2 infection. All samples were tested for CPV-2 and CanineCV by real-time PCR, and CPV-2-positive samples were genotyped using a SimpleProbe® assay. CPV-2-only infection resulted in a prevalence of 77.17%, while CPV-2-CanineCV co-infection had a prevalence of 22.83%. Vaccinated canines have 11.36% co-infection, while those with unknown vaccination history has 28.79% and unvaccinated has 19.23%. Genotyping results demonstrated that CPV-2c was the dominant variant, accounting for 93.31% overall and 93.1% in co-infections at a 22.78% co-infection rate, whereas CPV-2a was infrequent, at 6.69% overall and 6.9% in co-infections at a 23.53% co-infection rate, and CPV-2b was not detected. The highest co-infection rate was recorded in the Central region of Vietnam at 50%, compared with 9.38% in the North and 4.55% in the South. Seasonal analysis showed a high co-infection rate during the fall with no significant difference. Age distribution revealed that younger puppies were most affected and vulnerable to co-infection, with 37.50% in < 2 months, 24.24% in 2-3 months, 23.20% in 3-6 months, and 18.18% in 6-12 months. These findings suggest that CanineCV co-circulates extensively with CPV-2, particularly in younger dogs. The results also highlight the continued dominance of CPV-2c in Vietnam and underscore the importance of including CanineCV in diagnostic, vaccines, and epidemiological surveillance of canine enteric diseases.
Adenoviruses are the pathogens of a widespread adenovirus disease that affect wide range of hosts. 100 K protein is an important scaffolding protein for hexon assembly of the adenovirus. In recent years, the 100 K in the adenovirus attract more attention. Therefore, the character, location, and function of the 100 K protein is summarized from the past ten years. The current 100 K research mainly focuses on hexon assembly. The role of 100 K protein in hexon assembly is illustrated in detail. At early and late stages of hexon assembly, 100 K protein has localized to the nucleus and the cytoplasm to exert different functions. Its role in human granzyme B (GrB) and viral mRNA translation are also very important but have received little attention. The suppression of GrB by 100 K promote viral mRNA translation through inhibiting cell apoptosis. 100 K protein promotes hexon assembly through inhibition of cellular mRNA translation and cell apoptosis. The 100 K protein in ELISA kit may be used in differentiation of infected chickens from FAdV vaccinated chickens, but that in the vaccine has little capacity to protect adenovirus infection. Meanwhile, the adenovirus vector with 100 K protein will hold promise for diverse gene therapy applications. This review will facilitate a better understanding of 100 K protein function during adenovirus infection.
Bacteriophages (phages), viruses that specifically infect and lyse their host bacteria, hold significant potential for application in the prevention of Vibrio infections and the treatment of vibriosis for aquaculture. In this study, a novel Vibrio harveyi phage, designated Vibrio Vibrio phage LRZ, was isolated from an abalone aquaculture farm in Lianjiang, Fujian Province. Its biological characteristics and complete genomic sequence were investigated. The results demonstrated that Vibrio phage LRZ exhibits robust tolerance to acidic/alkaline environments, as well as elevated temperatures. The phage exhibited a latent period of approximately 30 min and reached the plateau phase at approximately 190 min post-infection, with an estimated burst size of 134 plaque-forming units (PFU) per cell. Whole-genome sequencing using the Illumina platform revealed a genome length of 43,274 base pairs (bp) with a guanine-cytosine (GC) content of 39.45%. Annotation identified 51 open reading frames (ORFs), encoding proteins involved in phage structural assembly, host lysis, DNA replication, and metabolism. Bioinformatic predictions did not identify antibiotic resistance or virulence genes in the LRZ genome. BLAST comparative analysis indicated the highest similarity (75.47%) to Vibrio phage VPMS1, establishing Vibrio phage LRZ as a novel isolate of Vibrio phages. Phylogenetic analysis further confirmed its closest relationship to Vibrio phage VPMS1, placing them within the same genus in the class Caudoviricetes. Vibrio phage LRZ could serve as a valuable novel genomic resource for studying phage evolution and offers novel perspectives for the biocontrol of V. harveyi in aquaculture settings.
Deep sequencing and de novo assembly of small RNAs (sRNA analysis) provide a powerful approach to detect plant RNA and DNA viruses. Here, using sRNA analysis of Gentiana triflora (gentian) plants affected with kobu-sho disease, a serious disease of gentian, we found caulimovirus-like sequences, provisionally designated as gentian caulimovirus-like sequence (GCVS). Analysis of the complete nucleotide sequence indicated that GCVS is a novel virus with a genomic structure and organization typical of members of genus Caulimovirus (family Caulimoviridae). Based on Southern blot hybridization and rolling circle amplification analyses, GCVS seems to be integrated into the genomes of a wild gentian plant and three F₁ gentian cultivars and their respective parents and without any episomal viral forms in plants. Consistently, no episomal virus was detected in gentian plants with kobu-sho disease. To confirm GCVS integration in the gentian genome, we used a BLASTn analysis of the gentian draft genome (4,374 contigs; total: 3,657,985,820 nucleotides [nt]) using the GCVS sequence as a query and identified 1,929 GCVS-derived fragments (total: 3,116,680 nt) across 280 contigs. All fragments were shorter than the full-length GCVS genome (8,125 nt), and more than 90% were less than 4,000 nt long. Open reading frames in these fragments encoded proteins containing numerous amino acid substitutions and were frequently disrupted by premature stop codons. Collectively, these features indicate that GCVS represents a non-infectious endogenous pararetrovirus, defined as a pararetroviral sequence that has been integrated into the host genome and is no longer capable of autonomous replication or infection.
Noroviruses (NoVs) are a leading cause of acute gastroenteritis (AGE) worldwide, with genetic diversity driven by recombination and genotype replacement. This study investigated the prevalence and molecular characteristics of NoVs among Japanese children with AGE from July 2021 to June 2024. A total of 1,095 stool samples were collected from three prefectures (Kyoto, Osaka, and Shizuoka), and NoV RNA was detected in 456 cases (41.6%). Genogroup GII predominated (96.9%), while GI infections were rare. Genotyping identified eight genotypes, including two GI (GI.1, GI.6) and six GII (GII.2, GII.3, GII.4, GII.6, GII.7, GII.17). GII.4 remained the predominant genotype (71.7%) throughout the study period. Notably, GII.7 emerged in 2023-2024 and became the second most prevalent genotype. Analysis of the ORF1/ORF2 junction identified multiple RdRp-capsid combinations, with recombinant strains accounting for 92.5% of detections. The predominant recombinant lineages were GII.4[P16] and GII.4[P31], followed by GII.2[P16]. Phylogenetic analysis indicated close relatedness to strains circulating internationally. These findings demonstrate the sustained predominance of GII.4, the emergence of GII.7, and the high prevalence of recombinant strains among Japanese children, highlighting the importance of continuous molecular surveillance.
In this study, a novel double-stranded RNA (dsRNA) mycovirus, tentatively designated Fusarium solani partitivirus 4 (FsPV4), was isolated from the Fusarium solani strain GF7, a phytopathogenic fungus responsible for tobacco root rot. The genome of FsPV4 consists of two dsRNA segments, referred to as dsRNA1 (2312 bp in length) and dsRNA2 (2213 bp in length). The dsRNA 1 and dsRNA 2 were predicted to encode an RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. Sequence analysis revealed that the RdRp of FsPV4 showed significant sequence similarity to the RdRps of partitiviruses, with Aplosporella javeedii partitivirus 1 being the best match (identity: 60.76%). Phylogenetic analysis of the RdRp showed that FsPV4 clustered robustly within the genus Betapartitivirus of the family Partitiviridae. This represents the first report of a betapartitivirus infecting F. solani, providing a potential candidate for the biological control of F. solani-mediated plant diseases.
Spiraea plants showing symptoms of virus infection were investigated using high-throughput sequencing which resulted in the identification of two novel viruses: an emaravirus named Spiraea chlorotic leaf spot distortion virus (SCLSDV), and an alphacytorhabdovirus named Spiraea alphacytorhabdovirus 1 (SpCRV-1). The complete genome of SCLSDV comprises four RNA segments, each encoding a distinct protein: RNA-dependent RNA polymerase, glycoprotein precursor, nucleocapsid protein, and movement protein, respectively. SCLSDV shares 33.99-71.78% amino acid identity with pear chlorotic leaf spot-associated virus. Virus purification and electron microscopy confirmed pleomorphic, enveloped particles (80-100 nm) matching the morphology of members of the genus Emaravirus. The alphacytorhabdovirus shares 70% of nucleotide identity with Actinidia virus D and encodes eight putative proteins. Both novel viruses were detected in a mixed infection with Spiraea yellow leaf spot virus. Additionally, SCLSDV was consistently detected in a single infection in 50 out of 77 plant samples exhibiting yellow spot disease, characterized by chlorotic lesions, leaf distortion, and necrosis. These findings suggest that strong yellow spot symptoms and leaf distortion are likely associated with SCLSDV infection. Furthermore, eriophyid mites were collected from symptomatic plants and identified morphologically as Calepitrimerus sp. Given the known role of eriophyid mites in emaravirus transmission, their presence suggests it could be a potential vector for SCLSDV.
RNA viruses are highly abundant and diverse in marine ecosystems, yet the currently discovered viral species represent only a minute fraction of the total diversity, with viral communities in port ecosystems particularly understudied. To bridge this gap, in summer, approximately 100 L of surface water was collected from Yangshan Port. Viral particles were concentrated using tandem filtration and 50-kDa tangential-flow ultrafiltration, followed by metatranscriptomic sequencing of extracted RNA. This approach yielded 74 full-length RNA-dependent RNA polymerase (RdRp) sequences, which clustered into 72 distinct genus-level (or higher) taxonomic units-significantly expanding the known diversity of seaport RNA viruses. Phylogenetic and EFI-EST network analyses showed that 93% of these sequences belong to the two dominant phyla, Pisuviricota and Kitrinoviricota, forming a seasonally stable backbone consistent with a previously obtained winter dataset. Notably, five sequences formed a maximally supported (99% bootstrap) basal clade within Kitrinoviricota. They share only 8-24% amino-acid identity with representatives of the four ICTV-recognized classes and exhibit DALI Z-scores ≤ 20, precluding assignment to any currently established class. Three of these candidate novel-class viruses were already detectable in the winter metatranscriptome (bit-score ≥ 40, ≥ 98% amino-acid identity), indicating year-round persistence of this lineage in Yangshan Port. Their taxonomic novelty thus reflects a gap in the current ICTV framework rather than seasonal sporadicity. Two sequences represent true seasonal debutants absent from public databases. These sequences not only fill a notable gap in the summer virome but also lack a class-level taxonomic anchor within the ICTV hierarchy, jointly occupying a "season-new" and "taxon-new" niche that prioritizes them for downstream isolation and functional characterization.
Porcine epidemic diarrhea virus (PEDV) G2c subtype has emerged as an increasingly prevalent variant causing widespread epidemic, high mortality and severe economic losses in the Chinese swine industry. The lack of specific and efficient detection methods hinders its surveillance, early detection and control. In this study, a TaqMan-MGB probe-based duplex quantitative real-time reverse transcription PCR (RT-qPCR) assay was developed for simultaneous detection of pan-PEDV and differentiation of the G2c subtype. Universal primers/probe targeting the conserved N gene of PEDV and G2c-specific primers/probe targeting the unique mutation sites in the S gene were designed. The assay was validated for performance, including specificity, sensitivity, and repeatability, and further evaluated using artificial challenge models and clinical samples. The standard curves exhibited excellent linearity (R2 > 0.999) with amplification efficiencies of 99.1% (Pan-pedv) and 98.4% (G2c). The limits of detection (LOD) were 10 copies/µL for Pan-pedv and 100 copies/µL for G2c subtype. No cross-reactivity was observed with 11 common swine diarrhea-related pathogens, and the G2c-specific channel exclusively recognized the G2c subtype. Intra-assay and inter-assay coefficients of variation (CVs) were < 1.40% and < 0.62%, respectively, confirming the good repeatability. In the artificial challenge model, the assay detected dynamic changes in fecal viral load consistent with the singleplex RT-qPCR. In clinical sample testing (n = 18), 13 PEDV-positive samples were identified, with 6 G2c-positive cases validated by Sanger sequencing. Collectively, this duplex RT-qPCR assay is sensitive, specific, and reliable, providing a valuable tool for rapid diagnosis, epidemiological surveillance, and targeted control of PEDV G2c subtype outbreaks.
A highly divergent isolate of rice stripe virus (RSV, Tenuivirus oryzaclavatae) was identified by high-throughput sequencing (HTS) of volunteer durum wheat (Triticum turgidum subsp. durum (Desf.) Husn.) plants collected in summer 2023 in Loir-et-Cher (France). The complete sequence of all four genomic RNAs was obtained from a single plant, showing a genomic organization similar to that of known RSV isolates but with significant indel polymorphism in non-coding regions. Genomic RNAs showed between 81.6% (RNA4) and 88.3% (RNA3) nucleotide identity with RSV reference isolates, while encoded proteins showed between 86.3% (NSvc2) and 97.4% (L and NSvc4 (movement protein)) with those of the reference RSV isolate. The 97.4% identity in the L protein should be compared with the average pairwise identity of 99.0% +/- 0.1% among all RSV isolates present in GenBank, as well as with the ICTV species demarcation threshold of 5% divergence. This demonstrates that the French wheat variant is indeed an RSV isolate but a highly divergent one, which questions our current understanding of RSV geographic distribution, diversity and evolution history.
Enterovirus D68 (EV-D68) is a re-emerging respiratory pathogen that is increasingly associated with severe respiratory disease and occasional neurological complications, yet its circulation in Portugal remains poorly documented. In the present study, we investigated the presence of EV-D68 in respiratory samples collected in southern Portugal (Algarve region) from October 2024 to February 2025. Screening of 150 respiratory samples from patients (115 from children and 35 from adults), all previously positive for rhinovirus/enterovirus, revealed eight EV-D68-positive cases, corresponding to a detection rate of 5.44%, spanning a broad age range from infancy to the elderly. Clinical manifestations ranged from mild respiratory illness to severe conditions, including pneumonia and acute respiratory distress syndrome. Neurological manifestations, including complex febrile seizures and encephalomyelitis, were also observed in EV-D68-positive patients. Some cases involved coinfections, while others presented with EV-D68 as the sole detected respiratory pathogen. Genomic characterization from one isolate revealed a VP1 fragment belonging to clade B3, and a complete genome from another isolate clustered within clade A2. Phylogenetic analysis confirmed clustering with contemporary European isolates. These findings provide preliminary evidence of EV-D68 detection in southern Portugal and document the presence of clades A2 and B3 among the characterized samples. The results illustrate the heterogeneous clinical presentations observed in affected patients and highlight the value of continued molecular monitoring and genomic characterization of EV-D68.
Grapevine red blotch disease (GRBD) poses a serious threat to viticulture in the United States. Grapevine red blotch virus (GRBV), a member species of Grablovirus vitis (family Geminiviridae), the causal agent of GRBD, disrupts grapevine physiology and metabolism, thereby interfering with the natural processes of fruit ripening. To date, there is limited knowledge about how grape maturity stages influence the timing of changes in viral load and disease progression. This study elucidates the effect of fruit maturity stages on GRBV accumulation in Vitis vinifera L. cv. Merlot grapevines in a Central Coast vineyard in California. Petioles from six basal leaves at random from previously GRBV-infected vines were collected at pre-véraison, véraison, post-véraison, and harvest stages, across two years (2021 and 2022). The viral copy number was quantified using digital PCR. The study identified significant differences (p < 0.05) in GRBV copy number across different maturity stages in 2021, irrespective of the year of infection. The lowest viral load was observed during the pre-véraison stage, and the highest at harvest, indicating progressive viral accumulation as the grapevines development progressed. In 2022, however, no significant difference in viral load across maturity stages was detected, an outcome attributed to extreme heat spikes coinciding with sampling periods. Our findings highlight the interplay between GRBV accumulation, grape development, and environmental parameters.
Kiwifruit (Actinidia spp.) has long been appreciated for its desirable flavor and high nutritional value. In this study, leaf samples with chlorotic mottling symptoms were collected from kiwifruit plants in Hanzhong, Shaanxi Province of China. Using high-throughput sequencing, a novel totivirus tentatively designated Actinidia totivirus 1 (AcToV1) was identified. Its complete genome sequence was determined using RT-PCR and RACE techniques. The genome of AcToV1 is 5,060 nucleotides in length and contains two open reading frames (ORFs), encoding a coat protein (CP) of 793 amino acids and an RNA-dependent RNA polymerase (RdRp) of 841 amino acids. It also features a typical slippery heptanucleotide motif, a conserved element characteristic of the genus Totivirus. AcToV1 shares the highest genome-wide nucleotide sequence identity of 57.6% with Panax notoginseng virus A (PnVA; GenBank accession NO. KT388111). Its CP and RdRp also exhibit the highest amino acid sequence identities to PnVA, at 44.0% and 56.7%, respectively. Phylogenetic analysis further supported that AcToV1 clusters closely with PnVA and Hubei toti-like virus 2 (HTLV2). Therefore, AcToV1 be considered a new species of the genus Totivirus.
Here, we present the complete genome sequence of a highly pathogenic fowl adenovirus serotype 4 (FAdV-4) isolate (PK-1-SBL2021), collected in 2021, from an infected chicken in Pakistan. Analysis of the genome sequence revealed that PK-1-SBL2021 is closely related to highly pathogenic FAdV-4 isolates from neighboring countries (China and Iran). However, the genome is devoid of a large (1966 bp) deletion commonly found in highly pathogenic FAdV-4 isolates from neighboring countries (China and Iran). Other features of the genome, such as distinct amino acid substitutions in specific proteins encoded by the genome and repeat sequences, are similar to those found in highly pathogenic FAdV-4 isolates. To the best of our knowledge, PK-1-SBL2021 represents the first highly pathogenic FAdV-4 isolate identified in the region that lacks a 1966 bp deletion in its genome. Our sequence analysis reveals that the deletion is also lacking in the genomes of multiple pathogenic FAdV-4 isolates from Pakistan, suggesting that the genomic deletion is dispensable for increased virulence of FAdV-4.
The COVID-19 pandemic highlighted the potential of oral antiseptic agents to reduce viral transmission. Strategies that enhance and sustain antiviral activity in the oral cavity may further support control of respiratory viruses. In this study, we evaluated the virucidal activity of selected antiseptic agents against influenza A virus (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), individually and in combination, under controlled in vitro conditions. Cetylpyridinium chloride (CPC), benzethonium chloride (BZT), thymol, and tannic acid were assessed using a quantitative suspension assay in the presence of mucin and representative mucoadhesive polymers. Dynamic light scattering and zeta potential analyses were performed to characterize interactions between active agents and mucin or polymers. CPC, BZT, and thymol exhibited dose-dependent viral inactivation. CPC reduced IAV and SARS-CoV-2 titres by approximately 4 log₁₀ at > 200 µg/mL and 25 µg/mL, respectively, while thymol achieved comparable reductions at 1000 µg/mL and 750 µg/mL. Tannic acid showed minimal virucidal activity. Notably, CPC-thymol and BZT-thymol combinations achieved > 4 log₁₀ reductions at substantially lower concentrations than individual agents, indicating enhanced virucidal activity and improved cytocompatibility. Mucin markedly attenuated the virucidal activity of CPC-thymol combinations; however, selected polymers partially preserved antiviral efficacy under mucin-rich conditions. Physicochemical analyses suggest that electrostatic, hydrophobic, and hydrogen-bonding interactions contribute to these effects. These findings demonstrate the enhanced antiviral activity of quaternary ammonium compound-thymol combinations and highlight the influence of mucosal components and macromolecules interactions in modulating oral antiseptic activity.
Vaccinia virus (VACV) is a member of the Poxviridae family and served as the vaccine strain used to eradicate smallpox. As a large double-stranded DNA virus, VACV exhibits a broad host range and remains a model organism for studying viral evolution and gene expression. Codon usage bias (CUB) is a fundamental feature of genome evolution that reflects the interplay between mutational pressure and natural selection. In this study, we systematically analyzed codon usage patterns in 107 strains of Vaccinia virus (VACV) to uncover the driving forces behind its genomic evolution and host adaptation. Our findings revealed a strong A/T bias across all codon positions, particularly at the third codon position (AT3 = 66.59%), suggesting a mutational or selective preference for A/T-ending codons. RSCU analysis confirmed the predominance of A/T-ending codons, while dinucleotide abundance showed balanced usage without significant suppression, unlike host genomes such as Homo sapiens and Bos taurus. CAI and RCDI analyses demonstrated that VACV codon usage is better adapted to Homo sapiens, highlighting potential host-specific evolutionary pressures. The PR2 and neutrality plots further emphasized the dominant role of natural selection, with minor contributions from mutational bias. Correspondence analysis indicated that geographic origin had limited influence on codon usage variation, while correlation and ENC-GC3s analyses reinforced the interplay between mutational pressure and selection. Phylogenetic analysis revealed regional clustering of strains, particularly from Brazil and the USA, indicating localized viral evolution. Overall, our results suggest that natural selection is the primary force shaping VACV codon usage, facilitating its efficient replication and adaptation across host environments.