Pseudomonas aeruginosa (P. aeruginosa) is widely distributed in the environment. As an opportunistic pathogen, it commonly causes infections in immunocompromised individuals, including respiratory tract infections and burn wound infections. P. aeruginosa possesses multiple antibiotic resistance mechanisms, including efflux pumps, resistance genes, and population dynamics. Phage therapy is a potential approach for addressing drug-resistant P. aeruginosa infections; however, clinical experience and standardized guidelines for its application in severe pneumonia remain limited. A 14-month-old infant was hospitalized for pneumonia. Four days later, he developed acute pneumonia and was sent to the ICU for 38 days of antibiotic therapy; nonetheless, P. aeruginosa remained detectable in the patient’s respiratory secretions. During the clinical course, phage zjk6 was detected from a longitudinal P. aeruginosa isolate in the absence of phage therapy. This finding documents the coexistence of a naturally detected phage and MDR P. aeruginosa during prolonged pneumonia, but does not establish that the phage mediated bacterial clearance or clinical recovery. We performed whole-genome sequencing on P. aeruginosa isolates from patients to ascertain if they were infected by the same infection and assessed their antibiotic resistance using drug sensitivity testing. We isolated phages using the drip technique and double-layer plate method, examined their appearance by transmission electron microscopy, and assessed their biological properties through one-step growth curve analysis and lysis spectrum detection. Genome sequencing and comparative genomic analyses were performed to characterize phage zjk6 and representative bacterial isolates and to evaluate phage–host genomic relatedness. P. aeruginosa was isolated repeatedly during 49 days of treatment. Comparative genomic analysis of representative longitudinal isolates revealed multiple strain backgrounds, including distinct ST508 and ST266 lineages and a closely related ST836 lineage. Phage zjk6 was isolated from the fifth clinical isolate, which served as the propagation/reference host. This phage possesses an elongated tail and a limited lysis spectrum, which is capable of gradually lysing the fifth isolated P. aeruginosa strain. Genomic analysis showed that zjk6 formed plaques and displayed slow lytic behavior under the tested conditions, while also carrying lysogeny-associated regulatory modules, indicating temperate potential rather than a strictly lytic lifestyle. A naturally detected slow lytic Pseudomonas phage may coexist with MDR P. aeruginosa during prolonged infection. These findings support further study of phage–bacterium interactions in clinical infections, while the therapeutic significance of zjk6 requires additional validation.
The family Spiciviridae, belonging to the order Ghabrivirales, is currently composed of only one officially recognized genus, Spicivirus. However, metagenomic studies have revealed an increasing diversity of related viruses, suggesting that the current classification may underestimate their evolutionary complexity. In this context, the aim of the present study was to detect and characterize new viruses related to the Spiciviridae in the family Culicidae mosquitoes. A total of nineteen mosquito pools were sequenced and analyzed using metagenomic approaches and bioinformatics tools. Among these, two novel viruses were identified in four pools (S4, S11, S18, and S19), provisionally named Sabethes virus 1 (SV1) and Sabethes virus 2 (SV2). Phylogenetic inference revealed clades with strong statistical support and genetic divergences greater than 20
Norovirus is a major causative agent of pediatric acute gastroenteritis, whereas GII.8[P8] represents a rare, non-dominant genotype with limited publicly available whole-genome data. We characterized a GII.8[P8] norovirus strain, Harbin-Nov-076-2022, detected through routine surveillance of pediatric acute gastroenteritis in Harbin, China. The strain was obtained from a stool sample of a 15-month-old female patient and subjected to whole-genome sequencing using the DNBSEQ-T7 platform. Integrated analyses included phylogenetic reconstruction, amino acid entropy profiling, selection pressure assessment, homology modeling of the capsid P protein, and B-cell epitope prediction. The near-complete genome of Harbin-Nov-076-2022 is 7486 nt in length, with a GC content of 53.81
Newcastle disease virus (NDV) represents a major threat to the worldwide poultry industry, and the absence of approved antiviral therapeutics highlights the need for alternative intervention strategies. In this study, an integrated computational and experimental methodology to assess the interaction of selected plant-derived flavonoids against NDV. Molecular docking was conducted to evaluate the interactions of karanjin, mammeigin, and 3, 5-dihydroxy-4′,7-dimethoxyflavone (DHDM) with multiple NDV proteins, comprising hemagglutinin neuraminidase (HN), fusion protein (F), matrix protein (M), nucleoprotein (N), and phosphoprotein (P). Based on docking predictions, the NDV phosphoprotein was selected for experimental validation of biophysical interaction studies. Among the tested compounds, karanjin consistently exhibited the strongest binding affinities, with particularly favorable interactions observed for the phosphoprotein, a key regulator of viral transcription and replication. In silico pharmacokinetic analysis further supported the drug-likeness and oral absorption potential of karanjin. Based on docking predictions, the NDV phosphoprotein was selected for experimental validation. Recombinant phosphoprotein was expressed and purified, and flavonoid interactions were examined using UV–Vis absorption spectroscopy, fluorescence quenching, and isothermal titration calorimetry analysis. Karanjin displayed a clear and specific binding profile characterized by moderate-to-strong affinity and an enthalpy-driven interaction, whereas mammeigin and DHDM showed weak or nonspecific interactions. Spectroscopic analyses corroborated these findings, indicating stable complex formation without major structural perturbation. Collectively, these results identify karanjin as a promising flavonoid candidate targeting the NDV phosphoprotein.
The family Amalgaviridae comprises double-stranded RNA viruses with two partially overlapping open reading frames (ORFs), in which the RNA-dependent RNA polymerase is expressed through programmed ribosomal frameshifting. virus 1 (AnloV1), the founding member of the proposed genus “Anlovirus”, represents a poorly characterized viral lineage because only a limited number of genome sequences have been reported. Here, we systematically mined publicly available transcriptome datasets and identified 13 AnloV1-like genome sequences from four insect species and one mammalian fecal sample. Pairwise sequence comparisons indicated that these genomes represent nine putative viral species, substantially expanding the diversity of this lineage. Comparative genomic analyses showed that all newly identified viruses possess two overlapping ORFs and a highly conserved UUU_CNN motif at the ORF1/ORF2 overlap, further supporting this sequence as the consensus +1 programmed ribosomal frameshifting motif of the proposed genus “Anlovirus”. Phylogenetic analysis recovered a well-supported AnloV1-like clade distinct from the recognized genera of the family Amalgaviridae, providing additional evidence for the proposed genus “Anlovirus”. Transcriptome datasets from all four insect species also contained transcripts with high similarity to microsporidian proteins, suggesting that at least some AnloV1-like viruses may infect microsporidian parasites rather than the sampled animals. These findings substantially expand the genomic resources available for AnloV1-like viruses and provide an evolutionary framework that may facilitate the future establishment and classification of the proposed genus “Anlovirus”.
Wild red cowpea (Vigna angularis var. nipponensis) plants exhibiting systemic chlorotic mottles and mosaic patterns on trifoliate leaves were identified during a virus survey in South Korea. High-throughput sequencing of symptomatic leaf samples generated contigs related to members of the genus Soymovirus (family Caulimoviridae). The complete circular double-stranded DNA genome was subsequently determined and validated by PCR amplification and Sanger sequencing. The virus genome is 8,892-nt in length and was tentatively named Vigna yellow mottle soymovirus (VYMSV). Its genomic organization, including primer binding sites, gene arrangement, and open reading frame (ORF) sizes, is largely consistent with those of other soymoviruses, except that ORFI is split into three segments (Ia, Ib, and Ic). The genome contains 10 putative ORFs encoding a movement protein, a coat protein, a replicase polyprotein, a translational activator, and proteins of unknown functions. Pairwise nucleotide sequence comparisons of the reverse transcriptase and RNase H region showed 69.9
To investigate the positive rate and molecular epidemiological characteristics of human rhinovirus (HRV) in influenza-like illness (ILI) cases in Daishan, Zhejiang Province. Throat swab specimens from ILI cases were collected from a hospital in Daishan, Zhejiang Province, between January 2022 and December 2023. Real-time PCR was performed to identify 14 common respiratory pathogens. Nested PCR was performed to amplify the VP4/VP2 gene sequences of HRV-positive specimens. Phylogenetic analyses were conducted based on the VP4/VP2 regions of HRV. A total of 1,255 specimens were collected. The overall positive rate for respiratory pathogens was 54.34
Complete genome sequences were determined for three uncharacterized betabaculoviruses (Baculoviridae: Lefavirales) from noctuid moth species. These viruses included Eudryas unio granulovirus 1112 (EuunGV-1112), Eudryas unio granulovirus 1229 (EuunGV-1229), and Autographa gamma granulovirus Darmstadt (AugaGV-Darmstadt). Analysis of the features and open reading frame (ORF) contents of the genome sequences indicated that all three viruses were closely related to Helicoverpa armigera granulovirus (HearGV; Betabaculovirus helarmigerae) and Xestia c-nigrum granulovirus (XecnGV; Betabaculovirus xecnigri), two betabaculoviruses that kill larvae slowly and appear to suppress alphabaculovirus replication in coinfections. Phylogenetic inference with whole genome or core gene alignments grouped the EuunGV isolates in a clade with XecnGV. This clade was contained within a larger clade with AugaGV. Pairwise nucleotide distance estimation confirmed that AugaGV and the EuunGV isolates are viruses of Betabaculovirus xecnigri. Five ORFs were found to be unique to the EuunGV/AugaGV/HearGV/XecnGV group of betabaculoviruses, though what role the products of these ORFs play in the unusual features of their pathology remains to be determined. PCR analysis of cadavers from bioassays of AugaGV and EuunGV indicated that these viruses are able to infect and replicate in larvae of Helicoverpa zea and Spodoptera frugiperda, species which are susceptible to HearGV.
Rapidly evolving begomoviruses are a serious threat to cash crops worldwide. Perennial plants often harbor these viruses for a long time, making them accessible to crop plants. Perennial ornamental plants Duranta erecta variegata (Variegated Sky Flower) were found with typical begomoviral symptoms, including leaf curling, leaf deformation, and leaf shortening in the plant nurseries of Faisalabad, Pakistan, in 2021. Begomovirus and its associated satellite molecules of 2.8 and 1.4 kb, respectively, were amplified using rolling circle amplification (RCA). Sanger sequencing of cloned molecules revealed the presence of rose leaf curl virus (RoLCuV, Begomovirus rosae) accompanied by a novel recombinant DNA-B, tentatively named “rose leaf curl virus DNA-B,” along with hollyhock yellow vein alphasatellite (HYVA, Gosmusatellite alceae). Another plant sample showed the presence of agriculturally important papaya leaf crumple virus (PaLCrV, Begomovirus papayae). To our knowledge, this is the first report of RoLCuV, a novel DNA-B, PaLCrV, and HYVA infecting D. erecta variegata in Pakistan.
The dengue virus (DENV) belongs to the Flaviviridae family and is responsible for a febrile arbovirus disease of global relevance. In Brazil, there has been endemic circulation of the four viral serotypes since the last century, with DENV-3 being frequently associated with more severe clinical conditions and major epidemics. This study aimed to perform the motif characterization of DENV-3 strains circulating in Brazil and its epidemiology. We obtained cases information by serotype from DATASUS/TABNET from 2013 to 2025. Furthermore, 184 complete genomes of Brazilian samples available in the GenBank and GISAID databases were analyzed with the genome reference sequence of DENV-3 (NC_001475.2). The sequences were edited and analyzed with the MAFFT, BioEdit, Nextclade, and GeneDoc software to identify lineages and amino acid motifs and were subsequently submitted to phylogenetic analyses in the IQ-Tree and FigTree programs. The results indicated a historical predominance of serotype one, but with a considerable increase in DENV-2 and DENV-3 cases in 2024 compared to the previous year. By December 2025, DENV-3 cases had already surpassed those of DENV-1 for that year. Nine amino acid motifs were identified throughout the genome, especially sites of N-myristoylation, casein kinase II phosphorylation, protein kinase C phosphorylation, and N-glycosylation. The predominant lineages were 3III_C.2 and 3III_C.2.2 until 2022, when lineage 3III_B.3.2 was introduced in Brazil, being associated with the increase in cases. This genotype presented substitutions and the absence of protein motifs compared to previous lineages, suggesting that such alterations contributed to its replicative success.
Mitochondrial DNA (mtDNA) plays a pivotal role in cellular bioenergetics, immune regulation, and evolutionary adaptation, making it a uniquely informative genomic system for studying host–pathogen interactions. The cytochrome b (cyt b) gene, one of the most conserved mitochondrial protein-coding loci, is widely recognized for its utility in species identification, phylogenetics, and molecular evolution [1-3]. However, its potential role in shaping comparative host susceptibility to infectious diseases remains underexplored. In this study, we re-analyzed PCR–RFLP-derived mitochondrial cyt b restriction profiles targeting CCNNGG, GTAC, AGCT, TTAA, and CGCG motifs across seven vertebrate hosts: cattle, buffalo, pig, goat, rabbit, chicken, and quail. These motif architectures were compared with documented susceptibility patterns for Pasteurellosis, foot-and-mouth disease (FMD), Rinderpest, and Sarcoptes scabiei infestation [4, 6, 7]. The integrated motif matrix revealed distinct host-specific restriction profiles and four comparative genomic vulnerability clades. The findings support a model in which conserved mitochondrial motif architecture may reflect evolutionary genomic signatures relevant to pathogen–host range and susceptibility. The study is intended as a comparative genomic framework and does not claim direct causality between single restriction motifs and disease outcome.
It is crucial to conduct strict surveillance on the diversity of HIV-1 to aid the generation of evidence-based biomedical and behavioral prevention programs to HIV/AIDS. In the current study, the social demographic characteristics, HIV-1 viral diversity among migration populations between 2017 and 2021 were investigated comprehensively and were compared with those in the previous five-year, between 2012 and 2016. Epidemiological investigation indicated that the male-to-female ratio decreased from 10.34:1 to 5.12:1, indicating significantly more female were infected when compared to the previous five years (p = 0.004). Moreover, an inversion on both the gender and nationality was found when comparison between study participants from southeastern ports and northwestern ports (p < 0.05) during the ten-year study period. Molecular epidemiology study revealed that two subtypes C/BC and CRF01_AE dominated, with the emerging of novel unique recombinant forms, including CRF01_AE/C/BC, CRF03_AB/C/BC, A/D, CRF01_AE/C, CRF08_BC/B, CRF01_AE/B, and B/CRF01_AE/C between 2017 and 2021. In addition, a significant higher proportion of drug-resistant viruses was found among this population when compared with the previous five-year (p < 0.001). In conclusion, the altered social demographic characteristics, co-circulation of CRF01_AE, C/BC recombinants, multiple URFs, and increasing amount of multiple drug-resistant viruses, indicated a new trend of the HIV-1 molecular epidemic in this population, which called for enhanced public health responses and prioritization of resources.
The persistent incidence of Newcastle disease (ND) in vaccinated flocks highlights the limitations of conventional vaccination and necessitates improved strategies like in ovo vaccination. This study evaluated the protective efficacy of a live, thermostable Newcastle disease virus (NDV) strain D58 (genotype II) in two groups of chicks (each n = 12), either delivered in ovo on embryonic day 18, or by intraocular vaccination in 7-day-old chicks. Two more groups (each n = 12) vaccinated through the in ovo or intraocular routes with commercial F strain were included for comparison. An unvaccinated group (n = 12) was used as a control. All groups were challenged with the virulent NDV D162 strain (genotype XIII) at 35 days post-hatch to assess cross-protective efficacy. Serological responses, clinical protection, viral shedding, cell-mediated immunity, and histopathological changes were assessed. The in ovo D58 group exhibited early seroconversion with significantly higher antibody titers by day 7 post-hatch. This group demonstrated 100
Measles is a highly contagious infectious disease and remains a major cause of global morbidity and mortality. Molecular surveillance of measles virus (MV) is used for tracing transmission chains, by, for instance, distinguishing between repeated introductions and endemic circulation. WHO guidelines used in the global measles surveillance network recommend sequencing of a 450-nucleotide region at the C-terminal end of the N-gene (N450). Given the limited genetic variability of MV, whole-genome sequencing would increase the resolution and provide a more detailed molecular surveillance of circulating MV. In this study, we describe an amplicon-based nanopore sequencing protocol for generating near complete whole MV genome sequences (nWGS; 15,813 nucleotides). We applied this protocol to samples collected during an epidemic of measles in the Netherlands in 2013-2014, involving an estimated 30,000 cases, to quantify the sequence variation of nWGS during this epidemic. While this was considered an epidemic with very limited sequence variation based on analysis of the N450 region, different molecular clusters were identified by phylogenetic analysis of nWGS genomes. Notably, the number of single nucleotide variants (SNVs) between viruses detected during the early and late phase of the epidemic varied among molecular clusters. Four epidemiological clusters could be identified in the beginning of the epidemic, of which 2 were supported by molecular data. Analysis of nWGS genomes suggested that the epidemic started with either a single unnoticed introduction into the Netherlands and a few unnoticed generations or with at least 2 introductions at the same time, the latter option is supported by epidemiological data. These results can aid in interpreting MV sequence variation during transmission chains, outbreaks and epidemics in countries or regions approaching measles elimination.
The continuous emergence of novel coronaviruses, characterized by high mutation rates and frequent recombination events, poses severe threats to global “One Health.” Notably, the recent outbreak of the recombinant feline coronavirus (FCoV-23) and the persistence of SARS-CoV-2 variants underscore the urgent need to understand viral pathogenesis and cross-species transmission mechanisms. Reverse genetics technology serves as a critical platform for bridging genomic sequencing to functional virology, enabling targeted mutagenesis and the generation of recombinant viruses. However, the construction of reverse genetics systems for coronaviruses is often hampered by their exceptionally large genomes and the instability of viral cDNA sequences in bacterial hosts due to cytotoxicity. This review moves beyond a simple enumeration of methods to systematically compare current reverse genetics strategies—including in vitro ligation, bacterial artificial chromosome (BAC) systems, and transformation-associated recombination (TAR)—across different viral genera. Furthermore, we critically evaluate the application of these platforms in deciphering pathogenic mechanisms and developing next-generation vaccines, with a specific focus on overcoming technical bottlenecks and designing broad-spectrum countermeasures against emerging cross-species threats.
Aedes albopictus is a globally important mosquito species capable of transmitting a variety of viruses. In this study, a total of 440 Ae. albopictus individuals were collected from Fanchang, Anhui Province, and 22 tissue libraries were constructed for metagenomic sequencing. A total of 649,930,614 reads were obtained and assembled into 209,335 contigs, of which 18,339 showed similarity to known viral proteins, spanning 13 viral families including both DNA and RNA viruses. Because several DNA virus-related sequences were recovered from the dataset, we further focussed on CRESS-DNA virus-related sequences and members of the family Parvoviridae. Phylogenetic analysis showed that three CRESS-DNA virus-related sequences clustered within Smacoviridae and Genomoviridae, while two Parvoviridae genomes were assigned to Brevihamaparvovirus and Protoparvovirus. These findings provide a metagenomic overview of the Ae. albopictus-associated virome in Anhui Province and provide baseline information on mosquito-associated DNA virus-related sequences in this region.
Orthoflaviviruses constitute a diverse genus within the Flaviviridae family, comprising over 70 enveloped, single-stranded positive-sense RNA viruses that pose significant global health threats. Yellow fever virus (YFV) remains a significant public health concern in Nigeria, with suboptimal vaccination coverage and a persistent risk of transmission. Understanding the circulation of local orthoflaviviruses in mosquito vectors is crucial for disease surveillance and prevention strategies. Adult female mosquitoes were collected from the University of Ibadan’s dairy and teaching farms between June and August 2022 using stationary human-bait catches and miniature light traps. Mosquitoes were morphologically identified, pooled by species and collection site, and screened for orthoflaviviruses using hemi-nested reverse transcription PCR targeting the NS5 gene. Positive samples were sequenced and phylogenetic analysis was carried out. A total of 600 mosquitoes, representing six species from three genera, were collected, with Aedes aegypti predominating (60.67
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome is organized into two functional regions. The 5′ region comprises an open reading frame (ORF1a) that encodes the polyprotein pp1a and, through a programmed frameshifting event, enables the production of the extended polyprotein pp1ab. These polyproteins are processed by the viral proteases 3CLpro and PLpro into 16 nonstructural proteins (nsps). Nsps 1–11 participate in polyprotein processing, formation, and regulation of the replication–transcription complex, and modulation of host immune responses. Nsps 12–16 form the core of the viral RNA synthesis machinery and are primarily associated with RNA-dependent RNA polymerase activity, proofreading, capping, and RNA modification, while also functioning in coordination with additional nsps. The 3’ functional region of the genome encodes structural (S, E, M, and N) and accessory proteins (e.g., 3a, 6, 7a, 8, and 9b) that contribute to viral assembly, replication efficiency, and pathogenicity. Synthesis of pp1ab depends on a programmed -1 ribosomal frameshifting (-1 PRF) event mediated by cis-acting RNA elements that induce a one-nucleotide shift in the 5′ direction of the ribosome. This mechanism is critical for maintaining the stoichiometric balance of replication proteins. Here, we review recent current insights into the molecular mechanisms and structural dynamics of -1 PRF in SARS-CoV-2 and discuss its potential as a therapeutic target for antiviral intervention across clinically relevant coronaviruses.
