Search Results (67)

Porcine Rotavirus (PoRV), a predominant causative agent of neonatal diarrhea in piglets, shares substantial genetic homology with human rotavirus and represents a considerable threat to both public health and the global swine industry in the absence of specific antiviral interventions. The VP6 protein, an internal capsid component, is characterized by exceptional sequence conservation and robust immunogenicity, rendering it an ideal candidate for viral genotyping and vaccine development. In the present study, the recombinant plasmid pET28a(+)-VP6 was engineered to facilitate the high-yield expression and purification of the VP6 antigen. BALB/c mice were immunized to generate monoclonal antibodies (mAbs) through hybridoma technology, and the antigenic specificity of the resulting mAbs was stringently validated. Subsequently, a panel of truncated protein constructs was designed to precisely map linear B-cell epitopes, followed by comparative conservation analysis across diverse PoRV strains. Functional validation demonstrated that all three mAbs exhibited high-affinity binding to VP6, with a peak detection titer of 1:3,000,000 and exclusive specificity toward PoRVA. These antibodies effectively recognized representative genotypes such as G3 and X1, while exhibiting no cross-reactivity with unrelated viral pathogens; however, their reactivity against other PoRV serogroups (e.g., types B and C) remains to be further elucidated. Epitope mapping identified two novel linear B-cell epitopes, ¹²⁸YIKNWNLQNR¹³⁷ and ¹³⁸RQRTGFVFHK¹⁴⁷, both displaying strong sequence conservation among circulating PoRV strains. Collectively, these findings provide a rigorous experimental framework for the functional dissection of VP6 and reinforce its potential as a valuable diagnostic and immunoprophylactic target in PoRV control strategies. Full article

The HIV-1 capsid has emerged as a highly attractive drug target due to its highly conserved sequence and critical role in the viral life cycle. By disrupting interactions between capsid proteins and impairing the proper assembly or disassembly of the capsid, the inhibitors can effectively suppress HIV-1 replication and infection. Based on this mechanism, numerous small-molecule agents targeting the HIV-1 capsid protein have been developed to date. In this review, we report the latest advances in such inhibitors and delve into their molecular mechanisms of action. We find a focus on small molecules modulating capsid stability and their assembly/disassembly. Hopefully this study will further enhance the understanding of HIV-1 inhibition mechanisms, facilitating the future exploration of novel capsid inhibitors. Full article

This study investigated a suspected Feline calicivirus (FCV) outbreak at a veterinary facility in Zhengzhou, Henan Province, China. RT-PCR analysis confirmed the FCV presence, with subsequent CRFK cell culture propagation leading to the isolation and characterization of strain ZZ202306. Immunofluorescence and Western blot analyses validated the specificity of monoclonal antibodies targeting the FCV VP1 capsid protein. Transmission electron microscopy revealed non-enveloped virions of ~40 nm in diameter, exhibiting typical caliciviral architecture. Viral replication kinetics demonstrated exponential growth between 6 and 18 h post-inoculation, reaching a peak titer of 10^7.96 TCID₅₀/0.1 mL. Genomic sequencing coupled with phylogenetic reconstruction of the VP1 gene revealed a close genetic relation to domestic Chinese strains and international variants, while maintaining distinct evolutionary divergence from other calicivirus genera. Full article

Salmonella is one of the main foodborne pathogens. Irrational antibiotic management has led to an increase in the incidence of multidrug-resistant strains. Bacteriophages may be an alternative method of food biopreservation and contribute to reducing the number of food poisonings requiring pharmacotherapy. This study aimed to isolate a bacteriophage (phage) targeting indigenous multidrug-resistant (MDR) Salmonella strains, followed by their biological, morphological, and genomic characterization. In this study we isolated Salmonella phage KKP_3822, targeting MDR Salmonella Manchester strain KKP 1213. Salmonella phage KKP_3822 retained high activity in the temperature range from −20 °C to 40 °C and active acidity from pH 3 to 11. Temperatures of 70 °C and 80 °C and extreme pH values (2 and 12) significantly reduced the phage titer. Its activity decreased proportionally to the time of UV exposure. Genome analysis (linear dsDNA with a length of 114,843 bp) revealed the presence of 27 tRNA genes. Proteins encoded by the vB_Sen-IAFB3822 phage were divided into functional modules related to (i) phage structure/assembly, (ii) DNA replication/modification/regulation, (iii) phage lysis, and (iv) DNA packaging into the capsid. No genes associated with antibiotic resistance or integration into the host genome, markers of temperate bacteriophages, were annotated in the Salmonella phage KKP_3822 genome. Based on morphological features and whole-genome sequence analysis, the newly isolated Salmonella phage KKP_3822 shows the greatest similarity to representatives of tailed phages from the Caudoviricetes class, Demerecviridae family, and Epseptimavirus genus. Genome analysis confirmed the virulent nature of the Salmonella phage KKP_3822, making it a potential candidate for food biocontrol. Full article

Geminiviridae are ssDNA plant viruses whose control has both economical and agricultural importance. Their capsids assemble into two distinct architectural forms: (i) a T = 1 icosahedral and (ii) a unique twinned quasi-isometric capsid. Described here are the high-resolution structures of both forms of the maize streak virus using cryo-EM. A comparison of these two forms provides details of the coat protein (CP) and CP–CP and CP–genome interactions that govern the assembly of the architecture of the capsids. Comparative analysis of other representative members of Geminiviridae reveals structural conservation of 60–95% compared to a sequence similarity of 21–30%. This study provides a structural atlas of these plant pathogens and suggests possible antiviral-targetable regions of these capsids. Full article

Certain species D human adenoviruses (HAdV-D19, -D37, and -D64) are causative agents of epidemic keratoconjunctivitis. HAdV-D37 has previously been shown to bind CD46 (membrane cofactor protein) and sialic acid as adhesion receptors. HAdV-D64 is genetically highly similar to HAdV-D37, with an identical fiber protein sequence, but differs substantially in its penton base and hexon proteins, two other major capsid components, due to genetic recombination. Here, we demonstrate that, like HAdV-D37, HAdV-D64 virions bind directly to CD46 and that CD46 and sialic acid also function as receptors for HAdV-D64 on multiple cell types. Expression of CD46 on CD46-negative cells conferred susceptibility to HAdV-D64 entry. Specifically blocking HAdV-D64 binding to CD46 on the host cell surface strongly inhibits viral entry and gene delivery into multiple cell lines that represent target tissues. We show that CD46 is expressed on human conjunctival epithelial cells and directly binds to the HAdV-D64 virion. Our results suggest that HAdV-D64 may be used to deliver genes to target conjunctival cells and that interrupting HAdV-D64 entry through its interaction with CD46 may prevent or lessen adenovirus-associated ocular disease. Full article

Adeno-Associated Viruses (AAVs, genus Dependoparvovirus) are the leading gene therapy vector. Until recently, efforts to enhance their capacity for gene delivery had focused on their capsids. However, efforts are increasingly shifting towards improving the viral replication protein, Rep78. We discovered that Rep78 and its shorter isoform Rep52 contain a strictly C-terminal sequence motif, DDx3EQ, conserved in most dependoparvoviruses. The motif is highly negatively charged and devoid of prolines. Its wide conservation suggests that it is required for the life cycle of dependoparvoviruses. Despite its short length, the motif’s strictly C-terminal position has the potential to endow it with a high recognition specificity. A candidate target of the DDx3EQ motif might be the DNA-binding interface of the origin-binding domain of Rep78, which is highly positively charged. Published studies suggest that this motif is not required for recombinant AAV production, but that substitutions within it might improve production. Full article

African swine fever (ASF) is an acute, highly hemorrhagic viral disease in domestic pigs and wild boars. The disease is caused by African swine fever virus, a double stranded DNA virus of the Asfarviridae family. ASF can be classified into 25 different genotypes, based on a 478 bp fragment corresponding to the C-terminal sequence of the B646L gene, which is highly conserved among strains and encodes the major capsid protein p72. The C-terminal end of p72 has been used as a PCR target for quick diagnosis of ASF, and its characterization remains the first approach for epidemiological tracking and identification of the origin of ASF in outbreak investigations. Recently, a new classification of ASF, based on the complete sequence of p72, reduced the 25 genotypes into only six genotypes; therefore, it is necessary to have the capability to sequence the full-length B646L gene (p72) in a rapid manner for quick genotype characterization. Here, we evaluate the use of an amplicon approach targeting the whole B646L gene, coupled with nanopore sequencing in a multiplex format using Flongle flow cells, as an easy, low cost, and rapid method for the characterization and genotyping of ASF in real-time. Full article

Deformed wing virus (DWV) is known as one of the main viruses that affect honeybees’ health all around the world. The virus has two widespread genotypes, DWV-A and DWV-B (VDV-1), transmitted mainly by V. destructor mites. In this study, we collected honeycombs with covered broods from 73 apiaries in eight Lithuanian regions and initially investigated the prevalence of V. destructor mites. Mites were collected from May to the end of July in 2021 from 124 hives. The prevalence of V. destructor infestations in beehives reached 30% and 63% in investigated apiaries. The presence of DWV-A and DWV-B pathogens in mites and broods was examined by RT-qPCR targeting the CRPV-capsid region. The molecular characterization of the virus in mite samples was based on sequence analysis of the RNA-dependent RNA polymerase (RdRp) region. In addition, leader polypeptide (LP), structural protein (Vp3), Helicase, and RdRp genes were used for phylogenetic characterization of dual infection. The prevalences of DWV-B in mites and broods were 56.5% and 31.5%, respectively, while DWV-A was detected in 12.9% of mite samples and 24.7% of brood samples. Some of the examined mite samples harboured dual virus infections. Our findings showed that bee colonies from the same apiary were not always infected by the same viruses. Some bee colonies were virus-free, while others were highly infected. Phylogenetic analysis of 21 sequences demonstrated the presence of highly variable DWV-B and DWV-A genotypes in Lithuania and possible recombinant variants of the virus. This study represents the first molecular characterization of mite-borne pathogens hosted by honeybees (Apis mellifera) in Lithuania. Full article

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile. Full article

Adeno-associated virus (AAV) is characterized by non-pathogenicity, long-term infection, and broad tropism and is actively developed as a vector virus for gene therapy products. AAV is classified into more than 100 serotypes based on differences in the amino acid sequence of the capsid protein. Endocytosis involves the uptake of viral particles by AAV and accessory receptors during AAV infection. After entry into the cell, they are transported to the nucleus through the nuclear pore complex. AAVs mainly use proteoglycans as receptors to enter cells, but the types of sugar chains in proteoglycans that have binding ability are different. Therefore, it is necessary to properly evaluate the primary structure of receptor proteins, such as amino acid sequences and post-translational modifications, including glycosylation, and the higher-order structure of proteins, such as the folding of the entire capsid structure and the three-dimensional (3D) structure of functional domains, to ensure the efficacy and safety of biopharmaceuticals. To further enhance safety, it is necessary to further improve the efficiency of gene transfer into target cells, reduce the amount of vector administered, and prevent infection of non-target cells. Full article

The SARS-CoV-2 pandemic resulted in a scale-up of viral genomic surveillance globally. However, the wet lab constraints (economic, infrastructural, and personnel) of translating novel virus variant sequence information to meaningful immunological and structural insights that are valuable for the development of broadly acting countermeasures (especially for emerging and re-emerging viruses) remain a challenge in many resource-limited settings. Here, we describe a workflow that couples wastewater surveillance, high-throughput sequencing, phylogenetics, immuno-informatics, and virus capsid structure modeling for the genotype-to-serotype characterization of uncultivated picornavirus sequences identified in wastewater. Specifically, we analyzed canine picornaviruses (CanPVs), which are uncultivated and yet-to-be-assigned members of the family Picornaviridae that cause systemic infections in canines. We analyzed 118 archived (stored at −20 °C) wastewater (WW) samples representing a population of ~700,000 persons in southwest USA between October 2019 to March 2020 and October 2020 to March 2021. Samples were pooled into 12 two-liter volumes by month, partitioned (into filter-trapped solids [FTSs] and filtrates) using 450 nm membrane filters, and subsequently concentrated to 2 mL (1000×) using 10,000 Da MW cutoff centrifugal filters. The 24 concentrates were subjected to RNA extraction, CanPV complete capsid single-contig RT-PCR, Illumina sequencing, phylogenetics, immuno-informatics, and structure prediction. We detected CanPVs in 58.3% (14/24) of the samples generated 13,824,046 trimmed Illumina reads and 27 CanPV contigs. Phylogenetic and pairwise identity analyses showed eight CanPV genotypes (intragenotype divergence <14%) belonging to four clusters, with intracluster divergence of <20%. Similarity analysis, immuno-informatics, and virus protomer and capsid structure prediction suggested that the four clusters were likely distinct serological types, with predicted cluster-distinguishing B-cell epitopes clustered in the northern and southern rims of the canyon surrounding the 5-fold axis of symmetry. Our approach allows forgenotype-to-serotype characterization of uncultivated picornavirus sequences by coupling phylogenetics, immuno-informatics, and virus capsid structure prediction. This consequently bypasses a major wet lab-associated bottleneck, thereby allowing resource-limited settings to leapfrog from wastewater-sourced genomic data to valuable immunological insights necessary for the development of prophylaxis and other mitigation measures. Full article

Numerous human adenovirus (AdV) types are endowed with arginine–glycine–aspartic acid (RGD) sequences that enable them to recognize vitronectin-binding (αv) integrins. These RGD-binding cell receptors mediate AdV entry into host cells, a crucial early step in virus infection. Integrin interactions with adenoviruses not only initiate receptor-mediated endocytosis but also facilitate AdV capsid disassembly, a prerequisite for membrane penetration by AdV protein VI. This review discusses fundamental aspects of AdV–host interactions mediated by integrins. Recent efforts to re-engineer AdV vectors and non-viral nanoparticles to target αv integrins for bioimaging and the eradication of cancer cells will also be discussed. Full article

The three most important commercial bacterial insecticides are all derived from subspecies of Bacillus thuringiensis (Bt). Specifically, Bt subsp. kurstaki (Btk) and Bt subsp. aizawai (Bta) are used to control larval lepidopteran pests. The third, Bt subsp. israelensis (Bti), is primarily used to control mosquito and blackfly larvae. All three subspecies produce a parasporal body (PB) during sporulation. The PB is composed of insecticidal proteins that damage the midgut epithelium, initiating a complex process that results in the death of the insect. Among these three subspecies of Bt, Bti is unique as it produces the most complex PB consisting of three compartments. Each compartment is bound by a multilaminar fibrous matrix (MFM). Two compartments contain one protein each, Cry11Aa1 and Cyt1Aa1, while the third contains two, Cry4Aa1/Cry4Ba1. Each compartment is packaged independently before coalescing into the mature spherical PB held together by additional layers of the MFM. This distinctive packaging process is unparalleled among known bacterial organelles, although the underlying molecular biology is yet to be determined. Here, we present structural and molecular evidence that the MFM has a hexagonal pattern to which Bti proteins Bt152 and Bt075 bind. Bt152 binds to a defined spot on the MFM during the development of each compartment, yet its function remains unknown. Bt075 appears to be derived from a bacteriophage major capsid protein (MCP), and though its sequence has markedly diverged, it shares striking 3-D structural similarity to the Escherichia coli phage HK97 Head 1 capsid protein. Both proteins are encoded on Bti’s pBtoxis plasmid. Additionally, we have also identified a six-amino acid motif that appears to be part of a novel molecular process responsible for targeting the Cry and Cyt proteins to their cytoplasmic compartments. This paper describes several previously unknown features of the Bti organelle, representing a first step to understanding the biology of a unique process of sorting and packaging of proteins into PBs. The insights from this research suggest a potential for future applications in nanotechnology. Full article

A new calicivirus isolated from a walrus was reported in 2004. Since unknown marine mammalian zoonotic viruses could pose great risks to human health, this study aimed to develop therapeutic countermeasures to quell any potential outbreak of a pandemic caused by this virus. We first generated a 3D model of the walrus calicivirus capsid protein and identified compounds from marine natural products, especially phlorotannins, as potential walrus calicivirus inhibitors. A 3D model of the target protein was generated using homology modeling based on two publicly available template sequences. The sequence of the capsid protein exhibited 31.3% identity and 42.7% similarity with the reference templates. The accuracy and reliability of the predicted residues were validated via Ramachandran plotting. Molecular docking simulations were performed between the capsid protein 3D model and 17 phlorotannins. Among them, five phlorotannins demonstrated markedly stable docking profiles; in particular, 2,7-phloroglucinol-6,6-bieckol showed favorable structural integrity and stability during molecular dynamics simulations. The results indicate that the phlorotannins are promising walrus calicivirus inhibitors. Overall, the study findings showcase the rapid turnaround of in silico-based drug discovery approaches, providing useful insights for developing potential therapies against novel pathogenic viruses, especially when the 3D structures of the viruses remain experimentally unknown. Full article