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Background/Objectives: Hepatitis E virus (HEV) is an RNA virus recognized to be spread mainly by fecal-contaminated water. Its infection is known to be a serious threat to public health globally, mostly in developing countries, in which Africa is one of the regions sternly affected. An African-based vaccine is necessary to actively prevent HEV infection. Methods: This study developed an in silico epitope-based subunit vaccine, incorporating CTL, HTL, and BL epitopes with suitable linkers and adjuvants. Results: The in silico-designed vaccine construct proved immunogenic, non-allergenic, and non-toxic and displayed appropriate physicochemical properties with high solubility. The 3D structure was modeled and subjected to protein docking with Toll-like receptors 2, 3, 4, 6, 8, and 9, which showed a stable binding efficacy, and the dynamics simulation indicated steady interaction. Furthermore, the immune simulation predicted that the designed vaccine would instigate immune responses when administered to humans. Lastly, using a codon adaptation for the E. coli K12 bacterium produced optimum GC content and a high CAI value, which was followed by in silico integration into a pET28 b (+) cloning vector. Conclusions: Generally, these results propose that the design of an epitope-based subunit vaccine can function as an outstanding preventive vaccine candidate against HEV, although validation techniques via in vitro and in vivo approaches are required to justify this statement. [ABSTRACT FROM AUTHOR]

The zoonotic transmission of hepatitis E virus (HEV) genotypes 3 (HEV-3) and 4 (HEV-4), and rabbit HEV (HEV-3ra) has been documented. Vaccination against HEV infection depends on the capsid (open reading frame 2, ORF2) protein, which is highly immunogenic and elicits effective virus-neutralizing antibodies. Escherichia coli (E. coli) is utilized as an effective system for producing HEV-like particles (VLPs). However, research on the production of ORF2 proteins from these HEV genotypes in E. coli to form VLPs has been modest. In this study, we constructed 21 recombinant plasmids expressing various N-terminally and C-terminally truncated HEV ORF2 proteins for HEV-3, HEV-3ra, and HEV-4 in E. coli. We successfully obtained nine HEV-3, two HEV-3ra, and ten HEV-4 ORF2 proteins, which were primarily localized in inclusion bodies. These proteins were solubilized in 4 M urea, filtered, and subjected to gel filtration. Results revealed that six HEV-3, one HEV-3ra, and two HEV-4 truncated proteins could assemble into VLPs. The purified VLPs displayed molecular weights ranging from 27.1 to 63.4 kDa and demonstrated high purity (74.7–95.3%), as assessed by bioanalyzer, with yields of 13.9–89.6 mg per 100 mL of TB medium. Immunoelectron microscopy confirmed the origin of these VLPs from HEV ORF2. Antigenicity testing indicated that these VLPs possess characteristic HEV antigenicity. Evaluation of immunogenicity in Balb/cAJcl mice revealed robust anti-HEV IgG responses, highlighting the potential of these VLPs as immunogens. These findings suggest that the generated HEV VLPs of different genotypes could serve as valuable tools for HEV research and vaccine development. [ABSTRACT FROM AUTHOR]

The Semliki Forest virus capsid protein (C) is an RNA binding protein which exhibits both specific and unspecific affinities to single-strand nucleic acids. The putative use of the self-amplifying RNAs (saRNAs) of alphaviruses for biotechnological purpose is one of the main studied strategies concerning RNA-based therapies or immunization. In this work, a recombinant C protein from SFV was expressed and purified from bacteria and used to associate in vitro with a saRNA derived from SFV. Results showed that the purified form of C protein can associate with the saRNA even after high temperature treatment. The C protein was associated with a modified saRNA coding for the green fluorescent protein (GFP) and delivered to murine macrophage cells which expressed the GFP, showing that the saRNA was functional after being associated with the recombinant purified C protein. [ABSTRACT FROM AUTHOR]

The high genetic heterogeneity of small ruminant lentiviruses (SRLV) renders the genetic characterization of the circulating strains crucial for the epidemiological investigation and the designation of effective diagnostic tools. In Greece, research data regarding the genetic diversity of the circulating SRLV strains is scarce, hindering the implementation of efficient surveillance and control programs. The objective of the study was to genetically characterize SRLV strains isolated from intensive dairy sheep farms in Greece and evaluate the variability of the immunodominant regions of the capsid protein. For this reason, a total of 12 SRLV-infected animals from four intensive dairy sheep farms with purebred Chios and Lacaune ewes were used for the amplification and sequencing of an 800 bp gag-pol fragment. The phylogenetic analyses revealed a breed-related circulation of strains; Chios ewes were infected with strains belonging exclusively to a separate group of genotype A, whereas strains belonging to subtype B2 were isolated from Lacaune ewes. Immunodominant epitopes of capsid protein were quite conserved among the strains of the same genotype, except for the Major Homology Region which showed some unique mutations with potential effects on viral evolution. The present study contributes to the extension of the current knowledge regarding the genetic diversity of SRLV strains circulating in sheep in Greece. However, broader genetic characterization studies are warranted for the exploration of possible recombinant events and the more comprehensive classification of the circulating strains. [ABSTRACT FROM AUTHOR]

We engineered Saccharomyces cerevisiae to express structural proteins of foot-and-mouth disease virus (FMDV) and produce virus-like particles (VLPs). The gene, which encodes four structural capsid proteins (VP0 (VP4 and VP2), VP3, and VP1), followed by a translational "ribosomal skipping" sequence consisting of 2A and protease 3C, was codon-optimized and chemically synthesized. The cloned gene was used to transform S. cerevisiae 2805 strain. Western blot analysis revealed that the polyprotein consisting of VP0, VP3, and VP1 was processed into the discrete capsid proteins. Western blot analysis of 3C confirmed the presence of discrete 3C protein, suggesting that the 2A sequence functioned as a "ribosomal skipping" signal in the yeast for an internal re-initiation of 3C translation from a monocistronic transcript, thereby indicating polyprotein processing by the discrete 3C protease. Moreover, a band corresponding to only VP2, which was known to be non-enzymatically processed from VP0 to both VP4 and VP2 during viral assembly, further validated the assembly of processed capsid proteins into VLPs. Electron microscopy showed the presence of the characteristic icosahedral VLPs. Our results clearly demonstrate that S. cerevisiae processes the viral structural polyprotein using a viral 3C protease and the resulting viral capsid subunits are assembled into virion particles. Key points: • Ribosomal skipping by self-cleaving FMDV peptide in S. cerevisiae. • Proteolytic processing of a structural polyprotein from a monocistronic transcript. • Assembly of the processed viral capsid proteins into a virus-like particle. [ABSTRACT FROM AUTHOR]

We engineered Saccharomyces cerevisiae to express structural proteins of foot-and-mouth disease virus (FMDV) and produce virus-like particles (VLPs). The gene, which encodes four structural capsid proteins (VP0 (VP4 and VP2), VP3, and VP1), followed by a translational “ribosomal skipping” sequence consisting of 2A and protease 3C, was codon-optimized and chemically synthesized. The cloned gene was used to transform S. cerevisiae 2805 strain. Western blot analysis revealed that the polyprotein consisting of VP0, VP3, and VP1 was processed into the discrete capsid proteins. Western blot analysis of 3C confirmed the presence of discrete 3C protein, suggesting that the 2A sequence functioned as a “ribosomal skipping” signal in the yeast for an internal re-initiation of 3C translation from a monocistronic transcript, thereby indicating polyprotein processing by the discrete 3C protease. Moreover, a band corresponding to only VP2, which was known to be non-enzymatically processed from VP0 to both VP4 and VP2 during viral assembly, further validated the assembly of processed capsid proteins into VLPs. Electron microscopy showed the presence of the characteristic icosahedral VLPs. Our results clearly demonstrate that S. cerevisiae processes the viral structural polyprotein using a viral 3C protease and the resulting viral capsid subunits are assembled into virion particles. Key points: • Ribosomal skipping by self-cleaving FMDV peptide in S. cerevisiae. • Proteolytic processing of a structural polyprotein from a monocistronic transcript. • Assembly of the processed viral capsid proteins into a virus-like particle. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) vector has become the leading platform for gene delivery. Each serotype exhibits a different tissue tropism, immunogenicity, and in vivo transduction performance. Therefore, selecting the most suitable AAV serotype is critical for efficient gene delivery to target cells or tissues. Genome divergence among different serotypes is due mainly to the hypervariable regions of the AAV capsid proteins. However, the heterogeneity of capsid glycosylation is largely unexplored. In the present study, the N-glycosylation profiles of capsid proteins of AAV serotypes 1 to 9 have been systemically characterized and compared using a previously developed high-throughput and high-sensitivity N-glycan profiling platform. The results showed that all 9 investigated AAV serotypes were glycosylated, with comparable profiles. The most conspicuous feature was the high abundance mannosylated N-glycans, including FM3, M5, M6, M7, M8, and M9, that dominated the chromatograms within a range of 74 to 83%. Another feature was the relatively lower abundance of fucosylated and sialylated N-glycan structures, in the range of 23%–40% and 10%–17%, respectively. However, the exact N-glycan composition differed. These differences may be utilized to identify potential structural relationships between the 9 AAV serotypes. The current research lays the foundation for gaining better understanding of the importance of N-glycans on the AAV capsid surface that may play a significant role in tissue tropism, interaction with cell surface receptors, cellular uptake, and intracellular processing. [ABSTRACT FROM AUTHOR]

Successful assembly of bacteriophage MS2 virus-like particles (VLPs) from a secreted recombinant capsid protein in the culture medium of the Komagataella phaffii methylotrophic yeast has been shown. The yield of VLPs was 5 g/L and reached 30% of the total protein of the culture liquid supernatant. The VLPs were well-ordered icosahedral structures with a diameter of 26‒28 nm, which was confirmed by transmission electron microscopy. A simple method for the isolation and purification of the VLPs was proposed, which ensures the purity of the target protein structures of more than 90%. The results we obtained can be used for development of a technology for the large-scale production of recombinant vaccines based on bacteriophage MS2 VLPs. [ABSTRACT FROM AUTHOR]

Porcine circovirus 3 (PCV3) is a newly emerging virus associated with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive disorders, impacting global pig populations. Porcine circoviruses contain two major open reading frames (ORFs), and the ORF2 encodes the viral capsid protein (Cap). Cap is the most antigenic structural protein and an ideal candidate for the development of vaccines and diagnostic reagents. This study generated a monoclonal antibody (MAb) specific to PCV3 Cap, MAb CCC160, for diagnosis and pathogenesis studies of this novel virus. The MAb specifically recognized PCV3-infected swine lymph node tissue in an immunohistochemical analysis confirming its clinical diagnostic potential. In addition, a novel linear B-cell epitope recognized by MAb CCC160 was identified at the amino acid region 120–134 of Cap. Nuclear localization analysis of PCV3 Cap revealed a potential nuclear localization signal (NLS) in the middle region (aa 131–143) in addition to the dominant N-terminal NLS that is already known. A cell viability assay further demonstrated that the cytotoxicity of PCV3 Cap is correlated with its nuclear localization, indicating a crucial role of Cap in the pathogenic mechanism of PCV3. A full-length construct of PCV3 Cap was successfully expressed using a baculovirus expression system and purified recombinant proteins self-assembled into virus-like particles (VLPs). The protein constitution of the VLPs was confirmed by MAb CCC160 recognition, indicating the correct conformation and specificity of VLP and exhibiting the linear epitope aa 120–134 on the VLP surface. These results provide insights for developing diagnostic tools and potential VLP vaccines for PCV3, revealing its pathogenesis and antigenic properties. [ABSTRACT FROM AUTHOR]

Hepatitis E virus (HEV) is the notable causative agent of acute and chronic hepatic, renal, pancreatic, neurological, and hematopoietic blood cell infections with high risk in immunocompromised patients. Hepatic failure is mostly documented among adults, pregnant women, and patients with preexisting liver disease. HEV is a positive sense RNA virus of 7.2 kb genome size with typically three open reading frames (ORFs) which play essential roles in viral replication, genome assembly, and transcription. The mutational substitution in the viral RNA genome makes more it difficult to understand the actual relationship in the host–virus association. ORFs of HEV encode different structural and non-structural proteins and one of them is the capsid protein which is coded by ORF2. The capsid protein mediates the encapsulation of the viral genome as well as being involved in virion assembly. In the current study, the ligand-based docking approach was employed to inhibit the active amino acids of the viral capsid protein. Depending upon S-score, ADMET profiling, and drug scanning, the top ten tetrapeptides were selected as potential drug candidates with no toxicity counter to HEV receptor protein. The S-score or docking score is a mathematical function which predicts the binding affinities of docked complexes. The binding affinity of the predicted drug–target complexes helps in the selectivity of the desired compound as a potential drug. The best two selected peptides (i.e., TDGH with S-score of −8.5 and EGDE with S-score of −8.0) interacted with the active site amino acids of the capsid protein (i.e., Arg399, Gln420, and Asp444). The molecular dynamics simulations of RMSD trajectories of TDGH–capsid protein and EDGE–capsid protein have revealed that both docked complexes were structurally stable. The study revealed that these tetrapeptides would serve as strong potential inhibitors and a starting point for the development of new drug molecules against the HEV capsid protein. In future, in vivo studies are needed to explore selected peptides as potential drug candidates. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses (SRLV) infect sheep and goats resulting in significant economic losses. This study evaluated for the first time the predicted conformational structure of the SRLV-capsid-protein 25 (SRLV-p25) and analyzed the antigenicity of recombinant protein (SRLV-rp25) in mice by coupling to an immunostimulatory complexes based on glycyrrhizinic acid liposomes (GAL) and tested plasma from goats and sheep naturally infected. Analysis in silico and conformational structure of SRLV-p25 (genotype B-FESC-752) showed similar characteristics to other lentiviral capsids. The efficient expression of SRLV-rp25 was confirmed by Western blot. The humoral immune responses in mice showed an increased level of antibodies from day 21 to 35 of the SRLV-r p25-GAL and SRLV-rp25-ISCOM® groups and the cellular immune response showed no significant difference in IL-10 levels (P > .05), however, a significant difference (P < .001) was observed when comparing SRLV-rp25-GAL with SRLV-rp25 groups. Immunoreactivity toward SRLV- r p25 revealed 61% of positive samples from naturally infected goats and sheep. [ABSTRACT FROM AUTHOR]

Genome packaging is a critical step during the viral maturation process. Viruses employ a distinct approach to package their genetic materials inside capsid: ranging from very simple strategy of nucleation of capsid proteins onto genome to complex segro-packasome machinery. The majority of small plant viruses, which falls under type I passive system, package their genome into stable virions in the cytoplasmic compartment, where chances of co-packaging of host RNA is very high, indicates viruses evolved the mechanism of selective and stringent packaging of their genomes. Recent discoveries of the unique ATPase fold in the capsid proteins of smaller plant viruses and their direct or indirect role during genome packaging have changed the perception about genome packaging in type I system. We feel that viruses of type I system have acquired unique and independent innovations for genome packaging over the course of evolution. The molecular interactions, intriguingly, cross-talk between capsid proteins and conserved signal sequence situated at the end of genome, plays an important role while viral genome packaging and translocation. Strategy utilized by smaller plant viruses for enhancement of selective genome packaging does not depend simply on nucleation of capsid proteins over genome but interestingly, configuration of viral genome, replicase, tRNA, viral encoded movement proteins are the other important key players that regulate genome packaging. The main aim of this review is to discuss and revisit the mechanism of genome packaging among viruses of agronomic importance. This study will be also useful for understanding the origin and evolution of viral genome packaging apparatuses and their roles in eukaryogenesis. [ABSTRACT FROM AUTHOR]

BACKGROUND & OBJECTIVE: Dengue virus (DENV) genomic study is crucial for understanding how it spreads and presents and becomes dangerous. Three structural and seven non-structural proteins are encoded by the positive-stranded RNA DENV. Capsid protein (C) is one of the structural proteins which helps in the encapsidation of viral RNA. The objective of the study is to identify the DENV serotypes and the most common geographic specific mutations in the C protein circulating in the Punjab province of Pakistan. METHODOLOGY: It was a cross-sectional study in which about 120 DENV isolates were selected by the procedure of temporal sampling done in the peak season of dengue fever in 2022 from the major tertiary care hospitals for whole genomes sequence analysis. Among these only 23 whole genome sequences were performed after virus isolation, quantification, and cDNA synthesis. RESULTS: All the samples were found to be of genotypes 1 and 2, in which 44 non-synonymous mutations were detected in the C protein. The most common mutations were; N90S (n=11) and G70S (n=11) followed by V26G (n=5) and R10Q (n=4), in which G70S is novel. CONCLUSION: Mutations in the current study are of particular interest to the design of DENV vaccine projects in the future. Genomic epidemiology during each outbreak of DENV in different locations is critical for better public health and for designing new policies for future outbreaks. [ABSTRACT FROM AUTHOR]

T Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122GYVAGFL128 and 227SLYNDSL233. Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122GYVAGFL128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227SLYNDSL233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227SLYNDSL233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122GYVAGFL128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures. [ABSTRACT FROM AUTHOR]

Nuclear entrance and stability of porcine circovirus type 2 (PCV2), the smallest virus in mammals, are crucial for its infection and replication. However, the mechanisms are not fully understood. Here, we found that the PCV2 virion maintains self-stability via the host importin 5 (IPO5) during infection. Coimmunoprecipitation combined with mass spectrometry and glutathione S-transferase pulldown assays showed that the capsid protein (Cap) of PCV2 binds directly to IPO5. Fine identification demonstrated that the N-terminal residue arginine24 of Cap is the most critical to efficient binding to the proline709 residue of IPO5. Detection of replication ability further showed that IPO5 supports PCV2 replication by promoting the nuclear import of incoming PCV2 virions. Knockdown of IPO5 delayed the nuclear transport of incoming PCV2 virions and significantly decreased the intracellular levels of overexpressed PCV2 Cap, which was reversed by treatment with a proteasome inhibitor or by rescuing IPO5 expression. Cycloheximide treatment showed that IPO5 increases the stability of the PCV2 Cap protein. Taken together, our findings demonstrated that during infection, IPO5 facilitates PCV2 replication by directly binding to the nuclear localization signal of Cap to block proteasome degradation. IMPORTANCE Circovirus is the smallest virus to cause immune suppression in pigs. The capsid protein (Cap) is the only viral structural protein that is closely related to viral infection. The nuclear entry and stability of Cap are necessary for PCV2 replication. However, the molecular mechanism maintaining the stability of Cap during nuclear trafficking of PCV2 is unknown. Here, we report that IPO5 aggregates within the nuclear periphery and combines with incoming PCV2 capsids to promote their nuclear entry. Concurrently, IPO5 inhibits the degradation of newly synthesized Cap protein, which facilitates the synthesis of virus proteins and virus replication. These findings highlight a mechanism whereby IPO5 plays a dual role in PCV2 infection, which not only enriches our understanding of the virus replication cycle but also lays the foundation for the subsequent development of antiviral drugs. [ABSTRACT FROM AUTHOR]

Nervous necrosis virus (NNV) capsid protein plays an important role in producing viral particles without any genetic elements. Thus, NNV is a promising candidate for vaccine development and is widely used for constructing vaccines, including DNA, recombinant proteins, and virus-like particles (VLPs). Our study aimed to investigate the potential of NNV capsid protein (NNV) and NNV capsid protein fused to enhanced green fluorescent protein (NNV-EGFP) through VLP formation and whether their application can induce specific antibody responses against certain antigens. We focused on producing DNA and recombinant protein vaccines consisting of the genes for NNV, EGFP, and NNV-EGFP. The approach using NNV-EGFP allowed NNV to act as a carrier or inducer while EGFP was incorporated as part of the capsid protein, thereby enhancing the immune response. In vitro studies demonstrated that all DNA vaccines expressed in HINAE cells resulted in varying protein expression levels, with particularly low levels observed for pNNV and pNNV-EGFP. Consequently, structural proteins derived from HINAE cells could not be observed using transmission electron microscopy (TEM). In contrast, recombinant proteins of NNV and NNV-EGFP were expressed through the Escherichia coli expression system. TEM revealed that rNNV was assembled into VLPs with an approximate size of 30 nm, whereas rNNV-EGFP presented particles ranging from 10 nm to 50 nm in size. For the vaccination test, DNA vaccination marginally induced specific antibody responses in Japanese flounder compared to unvaccinated fish. Meanwhile, NNV and NNV-EGFP recombinant vaccines enhanced a greater anti-NNV antibody response than the others, whereas antibody responses against EGFP were also marginal. These results indicate that NNV capsid protein-based antigens, presenting as particles, play an important role in eliciting a specific anti-NNV antibody response and have the potential to improve fish immune responses. • Using NNV capsid protein as a carrier to deliver EGFP and enhance immune responses. • The E. coli -derived rNNV and rNNV-EGFP emerged as virus-like structures. • Antigen-presenting particles elicited the anti-NNV antibody response. [ABSTRACT FROM AUTHOR]

Porcine circovirus type 2 (PCV2) is capable of causing porcine circovirus-associated disease (PCVAD) and is one of the major threats to the global pig industry. The nucleocapsid protein Cap encoded by the PCV2 ORF2 gene is an ideal antigen for the development of PCV2 subunit vaccines, and its N-terminal nuclear localization sequence (NLS) structural domain is essential for the formation of self-assembling VLPs. In the present study, we systematically expressed and characterized full-length PCV2 Cap proteins fused to dominant T and B cell antigenic epitopes and porcine-derived CD154 molecules using baculovirus and found that the Cap proteins fusing epitopes were still capable of forming virus-like particles (VLPs). Both piglet and mice experiments showed that the Cap proteins fusing epitopes or paired with the molecular adjuvant CD154 were able to induce higher levels of humoral and cellular responses, particularly the secretion of PCV2-specific IFN-γ and IL-4. In addition, vaccination significantly reduced clinical signs and the viral load of PCV2 in the blood and tissues of challenged piglets. The results of the study provide new ideas for the development of a more efficient, safe and broad-spectrum next-generation PCV2 subunit vaccine. [ABSTRACT FROM AUTHOR]

In the context of the carboxyl-terminus (C-terminus) of the capsid protein of porcine circovirus type 2a (PCV2a) and PCV2a vaccines, this study aimed to explore its unrevealing cryptic epitope and its relation to PCV2-infected herd immunity. To discover the C-terminus of the capsid protein of PCV2a, monoclonal antibodies (mAbs) were generated in this work. Two mAbs bound the two minimal linear epitopes (229PPLKP233 and 228DPPLNP233 (or 229PPLNP233)), which were located at the C-terminus of the capsid proteins of PCV2a and PCV2b, respectively. One mAb bound to the minimal linear epitope (220QFREFNLK227, peptide P82), but it neither bound the virus-like particle (VLP) of PCV2a nor produced positive staining in PCV2a-infected cells by immunofluorescence assay. Further, the residues 220–227 were not accessible on the surface of the VLP on the three-dimensional model, but the residues 228–231 extend toward the VLP exterior. Immunoassays were conducted in this study to screen anti-viral peptide-specific IgGs, which could differentiate vaccinated pigs from non-vaccinated ones. The data show two 220QFREFNLKDPPLKP233-containing peptides had a significantly higher binding reactivity with sera from PCV2-infected pigs in the control group than with sera from the VLP-vaccine group, particularly seen in sera from swine aged 15 weeks to 24 weeks. However, the peptide P82 had not this phenomenon in that test. This study confirmed that C-terminal epitopes play an important role in PCV2-induced decoy of swine humoral immunity. [ABSTRACT FROM AUTHOR]

Background: The existing detection methods for porcine circovirus type 2 (PCV2) specific antibodies in serum cannot determine the infection status, thus it is necessary to establish a method for detecting PCV2 antigen. The capsid protein (CAP) of PCV2, as a major structural protein that plays a significant role in viral replication and in inducing host's immune response, is an ideal target antigen to monitor PCV2 infection. Therefore, a gold immunochromatographic assay (GICA) for rapid detection of PCV2 antigen based on the polyclonal antibodies (PAbs) against PCV2-CAP will be developed. Results: The truncated CAP protein (dCAP) was used to immunize rabbits to generate anti-serum. After preliminary purification by caprylic acid/ammonium sulfate precipitation (CAAS), specific PAbs were purified by affinity chromatography column coupled with dCAP and its titer was about two-fold higher than preliminary purified PAbs. Colloidal gold-PAbs conjugate was synthesized under the optimum conditions. The specific anti-dCAP PAbs and goat anti-rabbit antibody (GAR) were then sprayed onto nitrocellulose (NC) membrane as a test line (TL) and a control line (CL), respectively. The visual limit detection (vLOD) of the GICA strips was 5 ng/mL. Specificity assay indicated that the GICA strips had specifically detected PCV2 and was not reactive for porcine epidemic diarrhea virus (PEDV), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV) or classic swine fever virus (CSFV). A total of 36 porcine serum samples were detected by this GICA and commercial enzyme-linked immunosorbent assay (ELISA) Kit, 9 positive samples were found by the developed strip with the rate of 25.0% comparing with 11 positive samples detected by the commercially ELISA Kit which positive rate was 30.5%, and the receiver operating characteristic (ROC) curve revealed that the relative sensitivity and specificity of this GICA strip were 72.7 and 96.0%, respectively, with an area of 87.2%. Conclusions: This study established an efficient detection method with high sensitivity and specificity for the clinical diagnosis of PCV2 antigen, that will facilitate a rapid and convenient way to evaluate the infection status of vaccinated pigs. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are highly conserved and dynamic intracellular organelles. Their functions are not limited to serving as neutral lipid reservoirs; they also participate in non-energy storage functions, such as cell lipid metabolism, protection from cell stresses, maintaining protein homeostasis, and regulating nuclear function. During a Zika virus (ZIKV) infection, the viruses hijack the LDs to provide energy and lipid sources for viral replication. The co-localization of ZIKV capsid (C) protein with LDs supports its role as a virus replication platform and a key compartment for promoting the generation of progeny virus particles. However, in view of the multiple functions of LDs, their role in ZIKV infection needs further elucidation. Here, we review the basic mechanism of LD biogenesis and biological functions and discuss how ZIKV infection utilizes these effects of LDs to facilitate virus replication, along with the future application strategy of developing new antiviral drugs based on the interaction of ZIKV with LDs. [ABSTRACT FROM AUTHOR]

Porcine circovirus type 4 (PCV4) is a novel virus associated with porcine dermatitis and nephropathy syndrome (PDNS)-like signs identified firstly in China in 2019. However, the details of the molecular epidemiology of PCV4 are unclear at this time. A total of forty-two related sequences were selected from the GenBank database to explore the spread of PCV4 and its rule in genetic evolution. Of the selected strains, 41 were from south China in 2019 to 2021 and the other was a foreign representative strain. Phylogenetic tree construction, nucleotide and amino acid (aa) sequence alignment, gene recombination and antigen structure prediction were performed on the collected sequences using bioinformatics softwares. The 42 PCV4 strains were divided into two subgenotypes: PCV4a (35/42) and PCV4b (7/42), according to the constructed genetic evolution tree. PCV4a is the main epidemic strain, and it can be further divided into two different gene clusters: PCV4a-1 (22/35) and PCV4a-2 (13/35). The pairwise comparison analysis showed that the complete genome sequence similarity of the 42 PCV4 strains ranged between 97.9% and 100%, and the aa sequences of the Cap proteins of 42 PCV4 strains had three major heterogenic or hypervariable regions—27–28, 96 and 212—all located near the antigenic epitope of the Cap protein. The results of this study can provide some basis for further studying the spread and epidemic growth of PCV4, and the prevention and control of PCV4 infection in China. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) establishes persistent infections in multiple human tissues, a phenomenon that likely plays a role in its ability to cause congenital birth defects and neurological disease. Multiple nonstructural proteins encoded by ZIKV, in particular NS5, are known to suppress the interferon (IFN) response by attacking different steps in this critical antiviral pathway. Less well known are the potential roles of structural proteins in affecting the host immune response during ZIKV infection. Capsid proteins of flaviviruses are of particular interest because a pool of these viral proteins is targeted to the nuclei during infection and, as such, they have the potential to affect host cell gene expression. In this study, RNA-seq analyses revealed that capsid proteins from six different flaviviruses suppress expression of type I IFN and IFN-stimulated genes. Subsequent interactome and in vitro ubiquitination assays showed that ZIKV capsid protein binds to and prevents activating ubiquitination of RIG-I CARD domains by TRIM25, a host factor that is important for the induction arm of the IFN response. The other flavivirus capsid proteins also interacted with TRIM25, suggesting that these viral proteins may attenuate antiviral signaling pathways at very early stages of infection, potentially even before nonstructural proteins are produced. [ABSTRACT FROM AUTHOR]

Post-transcriptional gene silencing (PTGS) is an evolutionarily conserved plant defense mechanism against viruses. This paper aimed to evaluate a dsDNA construct (77 bp) as a template for in vitro production of virus-derived artificial small hairpin RNAs (shRNAs) and test for their potential to trigger the RNAi mechanism in Nicotiana benthamiana plants against CMV after their foliar infiltration. This approach allowed for the production of significant amounts of shRNAs (60-mers) quickly and easily. The gene silencing was confirmed using polymerase chain reaction (PCR), immunological-based assays, and real-time PCR (qPCR). The highest levels of gene silencing were recorded for mRNAs coding for replication protein (ORF1a), the viral suppressor of RNA silencing (ORF2b), and the capsid protein (ORF3b), with 98, 94, and 70% of total transcript silencing, respectively. This protocol provides an alternative to producing significant shRNAs that can effectively trigger the RNAi mechanism against CMV. [ABSTRACT FROM AUTHOR]

Biological and molecular properties of five Prune dwarf virus cherry isolates were studied. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) proved PDV infection in the respective source trees and absence of six other viruses infecting the cherry. Two of infected source trees were symptomless, and the other three trees showed deformed and elongated leaves with chlorotic lines and spots. The reaction of greenhouse-grown seedlings of 22 herbaceous test plant species was investigated. The woody indicators Prunus tomentosa and Prunus cerasifera were chip-budded. Cucumis sativus cv. Amelia, C. sativus cv. Levina, C. sativus cv. Tessa and Prunus tomentosa appeared as the most suitable test plants for bioassay of studied PDV isolates. The nucleotide sequences corresponding to the full-length coat protein (CP) of 657 bp were obtained. The identity among studied isolates in CP was 95.5–100% and 97.4–100% at nucleotide and amino acid levels, respectively. The movement protein (MP) sequences were 753 nucleotides long, translating 251 amino acids and shared 94.1–98.4% nucleotide and 96.8–99.2% amino acid identity. Constructed trees with the nucleotide sequences of both MP and CP genes of studied isolates and referent PDV cherry variants showed the formation of two phylogroups in the two genomic regions. All studied isolates separated into two clusters in group I. No correlation between the phylogenetic grouping and the biological properties, as well the geographical origin, was observed. [ABSTRACT FROM AUTHOR]

The assembly of HIV-1 particles is a concerted and dynamic process that takes place on the plasma membrane of infected cells. An abundance of recent discoveries has advanced our understanding of the complex sequence of events leading to HIV-1 particle assembly, budding, and release. Structural studies have illuminated key features of assembly and maturation, including the dramatic structural transition that occurs between the immature Gag lattice and the formation of the mature viral capsid core. The critical role of inositol hexakisphosphate (IP6) in the assembly of both the immature and mature Gag lattice has been elucidated. The structural basis for selective packaging of genomic RNA into virions has been revealed. This review will provide an overview of the HIV-1 assembly process, with a focus on recent advances in the field, and will point out areas where questions remain that can benefit from future investigation. [ABSTRACT FROM AUTHOR]

Potato leafroll virus (PLRV) uses powerful molecular machines to package its genome into a viral capsid employing ATP as fuel. Although, recent bioinformatics and structural studies have revealed detailed mechanism of DNA packaging, little is known about the mechanochemistry of genome packaging in small plant viruses such as PLRV. We have identified a novel P-loop-containing ATPase domain with two Walker A-like motifs, two arginine fingers, and two sensor motifs distributed throughout the polypeptide chain of PLRV capsid protein (CP). The composition and arrangement of the ATP binding and hydrolysis domain of PLRV CP is unique and rarely reported. The discovery of the system sheds new light on the mechanism of viral genome packaging, regulation of viral assembly process, and evolution of plant viruses. Here, we used the RNAi approach to suppress CP gene expression, which in turn prevented PLRV genome packaging and assembly in Solanum tuberosum cv. Khufri Ashoka. Potato plants agroinfiltrated with siRNA constructs against the CP with ATPase domain exhibited no rolling symptoms upon PLRV infection, indicating that the silencing of CP gene expression is an efficient method for generating PLRV-resistant potato plants. In addition, molecular docking study reveals that the PLRV CP protein has ATP-binding pocket at the interface of each monomer. This further confirms that knockdown of the CP harboring ATP-binding domain could hamper the process of viral genome packaging and assembly. Moreover, our findings provide a robust approach to generate PLRV-resistant potato plants, which can be further extended to other species. Finally, we propose a new mechanism of genome packaging and assembly in plant viruses. [ABSTRACT FROM AUTHOR]

• HEV ORF2 p551 protein forms virus-like particles upon expression in E. coli. • Common marmosets are susceptible to infection with HEV genotypes 1 and 3. • Immunization with p551 protein protects common marmosets against HEV challenge. • Antibody levels peak after two doses of p551 and remain stable for over 2.5 years. Hepatitis E virus (HEV) is a major causative agent of acute hepatitis worldwide, prompting continuous HEV vaccine efforts. Vaccine development is hampered by the lack of convenient animal models susceptible to infection with different HEV genotypes. We produced recombinant open reading frame 2 protein (pORF2; p551) of HEV genotype (GT) 3 and assessed its immunogenicity and protectivity against HEV challenge in common marmosets (Callithrix jacchus , CM). p551 with consensus sequence corresponding to amino acid residues 110–660 of HEV GT3 pORF2 was expressed in E. coli and purified by affinity chromatography. CMs were immunized intramuscularly with 20 μg of p551 VLPs with alum adjuvant (n = 4) or adjuvant alone (n = 2) at weeks 0, 3, 7 and 19. At week 27, p551-immunized and control animals were challenged with HEV GT1 or GT3 and thereafter longitudinally screened for markers of liver function, anti-HEV IgG and HEV RNA in feces and sera. Purified p551 formed VLPs with particle size of 27.71 ± 2.42 nm. Two immunizations with p551 induced anti-HEV IgG mean titer of 1:1810. Immunized CMs challenged with homologous and heterologous HEV genotype did not develop HEV infection during the follow-up. Control CMs infected with both HEV GT1 and GT3 demonstrated signs of HEV infection with virus shedding and elevation of the levels of liver enzymes. High levels of anti-HEV IgG persisted in vaccinated CMs and control CMs that resolved HEV infection, for up to two years post challenge. CMs are shown to be a convenient laboratory animal model susceptible to infection with HEV GT1 and GT3. Immunization with HEV GT3 ORF2/p551 triggers potent anti-HEV antibody response protecting CMs from homologous and heterologous HEV challenge. This advances p551 in VLPs as a prototype vaccine against HEV. [ABSTRACT FROM AUTHOR]

Interactions between the N-terminal (assembly) domain (NTD), the linker region of the hepatitis B virus (HBV) capsid protein, and the large (L) envelope protein are required for virion formation, which occurs via budding of cytoplasmic mature nucleocapsids (NCs) containing the relaxed circular (RC) DNA genome into an intracellular membrane compartment containing viral envelope proteins. L-capsid interactions also negatively regulate covalently closed circular (CCC) DNA formation, which occurs after RC DNA release from mature NCs and nuclear import. We have now found that L could increase RC DNA in cytoplasmic mature NCs that are destabilized due to mutations in the NTD or the linker, even in those that apparently fail to support secretion of complete virions extracellularly. Other mutations in the capsid linker could block the effects of L on both cytoplasmic NC DNA and nuclear CCC DNA. Furthermore, the maturity of RC DNA in cytoplasmic NCs that was enhanced by L or found in secreted virions was modulated by the capsid linker sequence. The level and maturity of the cytoplasmic RC DNA were further influenced by the efficiency of extracellular virion secretion dependent on viral genotype-specific envelope proteins. These results suggest that interactions between the capsid and envelope proteins regulate one or more steps during virion secretion beyond initial capsid envelopment and highlight the critical role of the capsid linker in regulating capsid-envelope interaction, including the timing of envelopment during NC maturation. [ABSTRACT FROM AUTHOR]

Different types of cancer including cervical (>90%), anal (~88%), vaginal (~40%), and penile (~40%) cancers are associated with human papillomaviruse (HPV) infections. Three prophylactic vaccines (Cervarix, Gardasil, and Gardasil‐9) were approved to provide immuno‐protection against certain types of HPVs. Currently, next‐generation HPV vaccines such as L1/L2‐based vaccines are being developed to provide broad‐type HPV protection. In this study, we introduced a comprehensive framework for design of L1/L2 polyepitope‐based HPV vaccine candidate. This framework started with protein sequence retrieval and followed by conservancy analysis between high‐risk HPVs, MHC‐I and MHC‐II epitope mapping, and B‐cell and T‐cell epitope mapping. Subsequently, we performed Tap transport and proteasomal cleavage, population coverage, antigenicity, allergenicity and cross‐reactivity. After that, peptide‐MHCI/II flexible docking and comprehensive conservancy analysis against all HPV types were carried out. The next steps were prediction of interferon‐gamma and interleukin‐10 inducing epitopes, epitope selection and construct design, tertiary structure prediction, refinement and validation, discontinuous B‐cell epitope prediction, vaccine‐TLR4 molecular docking, and codon optimization. Our data showed that two designed vaccine constructs harboring 8 L1 peptides or 7 L2 peptides, individually were highly conserved between all well‐known HPV types. In addition, the combination of in silico/in vivo approaches indicated the potential ability of L1 and L2 polyepitope constructs for development of next generation prophylactic/therapeutic HPV vaccine. [ABSTRACT FROM AUTHOR]

While the capsid of viruses in the Alphachrysovirus genus is built of subunits of a single coat protein, the capsid of viruses grouped in the Betachrysovirus genus may consist of subunits of two different proteins. For four of these betachrysoviruses, the detected molecular weights of the putative coat proteins differ from the sizes deduced from the nucleic acid sequence. The origin of these modifications remained unclear and it was hypothesized that the coat proteins undergo unspecific degradation. In our study, we show that these modifications are based on processing steps performed by unknown factors present in extracts of several eukaryotic organisms. Furthermore, we show that the C-terminal domain of P3 is fully degraded after capsid processing and particle assembly. • Coat proteins from FgV-ch9 are processed by eukaryotic factors. • The C-terminus of P3 of FgV-ch9 is fully degraded during processing. • Eukaryotic factors are processing P3 of the related betachrysovirus FodV1. [ABSTRACT FROM AUTHOR]

Porcine astrovirus (PAstV) is prevalent in pigs worldwide and could cause clinical symptoms such as diarrhea and encephalitis. The capsid protein (Cap) of PAstV plays a determinant role for virus immunological characteristics. In this study, the major antigenic regions of PAstV1 Cap were expressed through prokaryotic expression systems and immunized to BALB/c mice. Finally, two anti-Cap monoclonal antibodies (named mAb F4-4 and D3F10) were screened by indirect immune-fluorescence assay (IFA). A series of truncated GST-fused or artificially synthesized peptides were used to detect their reactivity with the mAbs and PAstV positive serum. Two novel B cell epitopes (120-GNNTFG-125, 485-RISDPTWFSA-494) were identified by using these two mAbs. Moreover, sequence alignment result showed that epitope 120-GNNTFG-125 was highly conserved in type 1 PAstV capsid protein. Cross-reactivity analysis further confirmed the genotype-specificity of mAb F4-4. The results of this study demonstrated to be the first description of monoclonal antibody preparation and B-cell epitope mapping on PAstV capsid protein, which may provide new information on the biological significance of PAstV capsid protein and lay a foundation for the development of PAstV immunological tests and genotype diagnostic methods. • Four monoclonal antibodies (mAbs) against PAstV1 were successfully developed. • Two mAbs could specifically react with the N terminal conserved and C terminal hypervariable region of PAstV Cap. • The B cell epitopes on PAstV Cap were further identified as 120GNNTFG125 and 485RISDPIWFSA494. • The epitope120GNNTFG125 was highly conserved and mAb F4-4 was PAtsV1 type-specific. [ABSTRACT FROM AUTHOR]

Capsid protein (CP) of Chikungunya virus (CHIKV) is a multifunctional protein with a conserved hydrophobic pocket that plays a crucial role in the capsid assembly and virus budding process. This study demonstrates antiviral activity of thymoquinone (TQ), a natural compound targeting the hydrophobic pocket of CP. The binding of TQ to the hydrophobic pocket of CHIKV CP was analysed by structure-based molecular docking, isothermal titration calorimetry and fluorescence spectroscopy. The binding constant K D obtained for TQ was 27 μM. Additionally, cell-based antiviral studies showed that TQ diminished CHIKV replication with an EC50 value 4.478 μM. Reduction in viral RNA copy number and viral replication as assessed by the qRT-PCR and immunofluorescence assay, confirmed the antiviral potential of TQ. Our study reveals that TQ is an effective antiviral targeting the hydrophobic pocket of CHIKV CP and may serve as the basis for development of a broad-spectrum therapy against alphaviral diseases. • Thymoquinone, a natural compound binds to the hydrophobic pocket of CHIKV CP. • Thymoquinone binding to purified CP was validated by isothermal titration calorimetry and fluorescence spectroscopy. • Cell-based antiviral assay revealed that thymoquinone diminished CHIKV replication with EC50 value of 4.478 μM. • Thymoquinone use as a broad spectrum antiviral against alphavirus has been proposed. [ABSTRACT FROM AUTHOR]

[Objective] Prokaryotic expression of African swine fever virus (ASFV) minor capsid penton apex protein (H240R) and study its immunogenicity. [Method] Using bioinformatics software to predict and analyze the physical and chemical properties, secondary and tertiary structure of H240R protein. After the codon optimization of the target gene sequence, the whole gene is synthesized, and the recombinant vector pET28a/H is constructed by gene recombination technology, and the target protein is expressed through the prokaryotic system, and the optimal induction temperature and induction time are screened. The target protein (inclusion body) is purified by washing with 2M urea washing buffer and affinity chromatography. The purified target protein was renatured by gradient dialysis, and the renatured target protein was identified by Western and Dot-ELISA. Refolding protein and adjuvant ISA201 were emulsified to immunize mice to evaluate the immunogenicity of H240R protein and the law of antibody growth and decline induced in mice. [Results] Bioinformatics analysis showed that the molecular weight of H240R protein was about 27 kDa, the isoelectric point was 9.40, there was no transmembrane domain, and the antigen index was high. The E. coli engineering strain BL21(DE3)/pET28a/H was successfully constructed, and the H240R protein was expressed in the form of inclusion bodies under different temperature induction conditions. After washing and affinity chromatography purification, the inclusion body protein has a purity of over 85%. Approximately 65% of purified protein can be renatured by gradient dialysis. Western showed that His antibody could bind to the fusion protein to develop color, indicating the correct expression of the target protein. Dot-ELISA showed positive serum and renatured target protein reaction, indicating that the renatured H240R protein has the correct conformation. After immunizing mice with refolding protein, they can stimulate the mice to produce antibodies, and reach a plateau 10 days after the second immunization, which can be stable for about 30 days. [Conclusion] ASFV capsid penton apex protein H240R is expressed in the form of inclusion bodies in the prokaryotic system. The purified and refolded target protein has good immunogenicity and can react with positive serum. These can provide references and ideas for further research on the structure and function of H240R protein and the development of ASFV subunit vaccines. [ABSTRACT FROM AUTHOR]

Pepper crops in Israel are infected by poleroviruses, Pepper vein yellows virus 2 (PeVYV-2) and Pepper whitefly-borne vein yellows virus (PeWBVYV). Herein we characterize the transmission of PeWBVYV and the aphid-transmitted PeVYV-2, and show that PeWBVYV is specifically transmitted by MEAM1 species of the whitefly Bemisia tabaci, with a minimum latency period of 120 h, and not by the Mediterranean (MED). PeWBVYV and PeVYV-2 were detected in the hemolymph of MED and MEAM1, respectively, however, amounts of PeWBVYV in the hemolymph of MED or PeVYV-2 in MEAM1 were much lower than PeWBVYV in hemolymph of MEAM1. Moreover, we show that PeWBVYV does not interact with the GroEL protein of the symbiont Hamiltonella and thus does not account for the non-transmissibility by MED. An insect glycoprotein, C1QBP, interacting in vitro with the capsid proteins of both PeWBVYV and PeVYV-2 is reported which suggests a putative functional role in polerovirus transmission. • Poleroviruses are strictly transmitted by aphids, yet a newly discovered poleroviruses has been associated with whiteflies. • The new polerovirus is transmitted by MEAM1 but not MED species of the whitefly Bemisia tabaci. • Transmission by MEAM1 but not MED is not associated with symbiotic bacteria. • The hemolymph plays a critical role in the inability of MED to transmit the new polerovirus. • The insect C1QBP protein interacts with the capsid proteins of both the aphid and whitefly transmitted poleroviruses. [ABSTRACT FROM AUTHOR]

Alphaviruses are positive sense, RNA viruses commonly transmitted by an arthropod vector to a mammalian or avian host. In recent years, a number of the Alphavirus members have reemerged as public health concerns. Transmission from mosquito vector to vertebrate hosts requires an understanding of the interaction between the virus and both vertebrate and insect hosts to develop rational intervention strategies. The current study uncovers a novel role for capsid protein during Chikungunya virus replication whereby the interaction with viral RNA in the E1 coding region regulates protein synthesis processes early in infection. Studies done in both the mammalian and mosquito cells indicate that interactions between viral RNA and capsid protein have functional consequences that are host species specific. Our data support a vertebrate-specific role for capsid:vRNA interaction in temporally regulating viral translation in a manner dependent on the PI3K-AKT-mTOR pathway. • Disruption of capsid-vRNA interactions affect chikungunya virus replication in a host specific manner. • Capsid-vRNA interactions regulate viral translation in mammalian cells. • Disruption of these interactions leads to increased sensitivity to inhibition of the PI3K-AKT-mTOR pathway. [ABSTRACT FROM AUTHOR]

This study is an attempt to find a suitable therapy using antimicrobial peptides (AMPs) by identifying peptide-protein interaction of AMPs and nucleocapsid protein of SARS and SARS-CoV- 2. The AMPs were shortlisted from the APD3 database (Antimicrobial peptide database) based on various physicochemical parameters. The binding efficacy of AMPs was measured using the lowest energy score of the docked complexes with 10 selected AMPs. For SARS-CoV, AP00180 showed the best pose with a binding affinity value of − 6.4 kcal/mol. Prominent hydrogen bonding interactions were observed between Lys85 (nucleocapsid receptor) and Arg13 (antimicrobial peptide ligand) having the least intermolecular distance of 1.759 Å. For SARS-CoV-2, AP00549 was docked with a binding affinity value of − 3.4 kcal/mol and Arg119 and Glu14 of receptor nucleocapsid protein and ligand AMP having the least intermolecular distance of 2.104 The dynamic simulation was performed at 50 ns to check the stability of the final docked complexes, one with each protein. The two best AMPs were AP00180 (Human Defensin-5) for SARS and AP00549 (Plectasin) for SARS-CoV-2. From positive results of dynamic simulation and previously known knowledge that some AMPs interact with the nucleocapsid of coronaviruses, these AMPs might be used as a potential therapeutic agent for the treatment regime of SARS-CoV-2 and SARS infection. [ABSTRACT FROM AUTHOR]

The capsid protein purity of adeno-associated virus (AAV) is considered a critical quality attribute of AAV-based gene therapy products. However, the analytical methods currently available to monitor the viral capsid proteins, which are present in extremely low concentrations, have limited sensitivity and robustness, thus limiting their general applicability. As a result, there is an urgent need to develop robust separation methods with highly sensitive detection. In this article, we describe the first denaturation and fluorescence labeling procedure for AAV capsid proteins using the pyrylium dye Chromeo™ P503, enabling the establishment of the first capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) method combined with laser-induced fluorescence (LIF) detection for AAV. Upon optimization using a quality-by-design approach, the newly developed method features a simple and robust one-step sample preparation workflow resulting in consistently labeled and denatured viral protein samples, which can subsequently be separated and quantified by CE-LIF. The method has been validated to be accurate and precise with a linear range of 50–150% of the nominal concentration of 2.0 × 1011 vector genomes per mL (vg/mL). The detection limit and quantitation limit were established to be 8.0 × 107 vg/mL (∼0.8 ng/mL) and 4.2 × 108 vg/mL (∼4 ng/mL), respectively, representing the highest sensitivity achieved for AAV capsid protein quantitation reported to date and a linear dynamic range of 8.0 × 107–3.0 × 1011 vg/mL. A comparison of the CE-SDS LIF method with existing methods, such as CE-SDS ultraviolet and sodium dodecyl sulfate–polyacrylamide gel electrophoresis with SYPRO Ruby stain, indicated that the new method has superior resolution and a significant increase in signal intensity. Capsid protein purity analysis of multiple AAV serotypes, including AAV5, scAAVrh10, AAV2, and AAV6, has been demonstrated for the first time using the same method, indicating the newly developed AAV labeling procedure and CE-LIF analysis could serve as a Quality Control-friendly platform and best-in-class analytical method for the control of AAV capsid protein purity. [ABSTRACT FROM AUTHOR]

Sapovirus (SaV) is a nonenveloped RNA virus that causes acute gastroenteritis in humans. Although SaV is a clinically important pathogen in children, an effective vaccine is currently unavailable. The capsid protein VP1 of SaVs forms the outer shell of the virion and is highly diverse, as often seen in the virion-surface proteins of RNA viruses, creating an obstacle for vaccine development. We here report a unique phenomenon pertaining to the variation of SaV VP1. Phylogenetic and information entropy analyses using full-length VP1 sequences from a public database consistently showed that the amino acid sequences of the VP1 protein have been highly conserved over more than 40 years in the major epidemic genotype GI.1 but not in GI.2. Structural modeling showed that even the VP1 P2 subdomain, which is arranged on the outermost shell of the virion and presumably exposed to anti-SaV antibodies, remained highly homogeneous in GI.1 but not in GI.2. These results suggest strong evolutionary constraints against amino acid changes in the P2 subdomain of the SaV GI.1 capsid and illustrate a hitherto unappreciated mechanism, i.e., preservation of the VP1 P2 subdomain, involved in SaV survival. Our findings could have important implications for the development of an anti-SaV vaccine. • We examined variations in sapovirus virion surface protein VP1 by multiple methods. • We found an unusual amino acid preservation of variable VP1 in genotype GI.1. • Our results disclose evolutionary constraints on sapovirus for the first time. • Novel mechanism(s) underlying sapovirus survival were also suggested. • Our results could have critical implications for the development of anti-SaV vaccines. [ABSTRACT FROM AUTHOR]

Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1–4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses. • Capsid protein (CP) of GETV undergoes phase separation in vitro and in cells. • Two regions of the GETV genomic RNA (gRNA) form co-condensates with CP. • The lysine-rich Link region of CP is essential for its phase separation. • K-R replacement in Link region increases CP-gRNA binding and improves viral functionality. [ABSTRACT FROM AUTHOR]

[Display omitted] • Recent literature review on electroanalytical diagnostics of viral diseases. • Review mainly focuses on genetic material (DNA/RNA) and capsid proteins detection. • Critical discussions including challenges in electroanalytical biosensing assays. • Advantages of various biorecognition elements were described in viral detection. Viral pandemic diseases have disruptive global consequences leading to millions of deaths and a severe impact on the global economy. Inadequate preventative protocols have led to an overwhelming demand for intensive care leading to uncontrollable burdens and even breakdown of healthcare sectors across many countries. The rapid detection of viral disease helps in the understanding of the relevant intricacies, helping to tackle infection with improved guidelines. Portable biosensor devices offer promising solutions by facilitating on-site detection of viral pathogens. This review summarizes the latest innovative strategies reported using electroanalytical methods for the screening of viral antigens. The structural components of viruses and their categories are presented followed by the various recognition elements and transduction techniques involved in biosensors. Core sections focus on biosensors reported for viral genomic detection(DNA and RNA) and antigenic capsid protein. Strategies for addressing the challenges of electroanalytical biosensing of viral components are also presented. The advantages, and disadvantages of biorecognition elements and nanozymes for the detection of viral disease are highlighted. Such technical insights will help researchers working in chemistry, and biochemistry as well as clinicians working in medical diagnostics. [ABSTRACT FROM AUTHOR]

Getah virus (GETV) is an emerging mosquito-borne alphavirus that can infect horses, pigs and other animals. Given the public health threat posed by GETV, research on its pathogenesis, diagnosis and prevention is urgently needed. In the current study, prokaryotic expression systems were used to express the capsid protein of GETV. This protein was then used to immunize BALB/c mice in order to generate monoclonal antibodies (mAbs). Subsequently, hybridoma cells secreting a mAb (2B11-4) against the capsid protein were obtained using the hybridoma technique. A B cell linear epitope, 18-PAYRPWR-24, located at the capsid protein's N-terminal region was identified using western blotting analysis with the produced mAb, 2B11-4. Sequence alignment indicated that this epitope was highly conserved in group III (GIII) strains of GETV, but varied among the other genotypes. Western blotting showed that mAb 2B11-4 could discriminate Group III GETVs from other genotypes. This study describes the preparation of a mAb against the GETV capsid protein and the identification of the specific localization of B-cell epitopes, and will contribute towards a better understanding of the biological importance of the GETV capsid protein. It will also pave the way for developing immunological detection methods and genotype diagnosis for GETVs. • Monoclonal antibody against GETV capsid protein was produced. • The precise linear B-cell epitope 18-PAYRPWR-24 on GETV capsid protein was identified. • The epitope 18-PAYRPWR-24 is prevalent among GIII strains. [ABSTRACT FROM AUTHOR]

Background: Viruses cause significant economic losses to shrimp aquaculture worldwide. In severe cases, they can lead to 100% mortality within a matter of days, hence the aquaculture industry requires antiviral strategies to minimize economic impacts. Currently, a double-stranded RNA (dsRNA)-based platform has been proven effective at a laboratory scale. The bottleneck for its industrialization is the lack of low-cost, efficient and practical delivery approaches. In an effort to bridge the gap between laboratory and farm applications, virus-like particles (VLP) have been used as nanocarriers of dsRNA. However, the implementation of this approach still suffers from high costs and a lengthy procedure, co-expression of subunits of VLP or capsid proteins (CPs) and dsRNA can be the solution for the problem. CP and dsRNA are traditionally expressed in two different E. coli hosts: protease-deficient and RNase III-deficient strains. To condense the manufacturing of dsRNA-containing VLP, this study constructed a novel E. coli strain that is able to co-express viral capsid proteins and dsRNA in the same E. coli cell. Results: A novel bacterial strain DualX-B15(DE3) was engineered to be both protease- and RNase III-deficiency via P1 phage transduction. The results revealed that it could simultaneously express recombinant proteins and dsRNA. Conclusion: Co-expression of viral capsid proteins and dsRNA in the same cell has been shown to be feasible. Not only could this platform serve as a basis for future cost-effective and streamlined production of shrimp antiviral therapeutics, it may be applicable for other applications that requires co-expression of recombinant proteins and dsRNA. [ABSTRACT FROM AUTHOR]

• PCV2b- and PCV2d-based virus-like particles (VLPs) were successfully generated. • This VLPs induced highly neutralizing antibodies in pigs naturally infected with PCV2d. • Interestingly, PCV2d viremia level was significantly reduced in the Bac-2dCP group. • The Bac-2dCP protein is a potential vaccine candidate for prevent against PCV2d. The pathogenic porcine circovirus type 2 (PCV2) causes significant economic losses in pig production. Emergence of the PCV2d genotype has been linked with PCV2-associated disease (PCVAD) outbreaks. However, no study has been conducted efficacy of an experimental PCV2d-based subunit vaccine in pigs. Therefore, PCV2b- and PCV2d-based capsid (CP) proteins were generated using a baculovirus (Bac) expression system, and we evaluated the protective immune responses in a commercial pig farm where predominant PCV2d is circulating. Eighteen 3-week-old pigs with maternal antibodies were randomly divided into four groups, and were immunized with purified Bac-2dCP, mixed 1:1 ratio with purified Bac-2bCP and Bac-2dCP (Bac-mCP), a commercial PCV2a-based subunit vaccine (VAC) or phosphate-buffered saline (PBS) as controls. The Bac-2dCP and Bac-mCP groups had significantly higher PCV2b- or PCV2d- specific IgG and neutralizing antibody without interference by maternal antibody compared to control group in pigs naturally infected with PCV2d. Interestingly, not only serum IL-4 level was significantly increased in the Bac-2dCP group, but also PCV2d viremia level was significantly reduced than the control group. The recombinant Bac-2dCP subunit vaccine is a good candidate for the effective reduction against PCV2d infection. [ABSTRACT FROM AUTHOR]

A liquid chromatography–tandem mass spectrometry method was developed to determine nine types of capsid proteins simultaneously in nine‐valent human papillomavirus vaccines. Signature peptides were optimized in terms of specificity, repeatability, determination accuracy and sensitivity. As a result, three signature peptides per capsid protein were obtained. The linear calibration curves were achieved in the range of 11.6–373.6 nmol/L (R2 > 0.998). Compared to our previous liquid chromatography–tandem mass spectrometry method, the current method was more sensitive (3.18‐fold) and it can be used for quality evaluation of nine‐valent human papillomavirus vaccines, unlike the previous method, which could only be used for bivalent human papillomavirus vaccines. Then, they were utilized to determine nine types of capsid proteins in nine‐valent human papillomavirus vaccines from four different manufactures. Intraday and interday precision values for the determination of capsid proteins in nine‐valent human papillomavirus vaccines were less than 6.8 and 9.1%, respectively. Recovery rates of all capsid proteins investigated were in the range of 80–120%. In addition, the current assay was used for determination of free capsid protein in nine‐valent human papilloma virus vaccines, and the results were used to evaluate the adsorption rate of the adjuvant. [ABSTRACT FROM AUTHOR]

Circoviruses represent a rapidly expanding group of viruses that infect both vertebrate and invertebrate hosts. Members are responsible for diseases of veterinary and economic importance, including postweaning multisystemic wasting syndrome in pigs, and beak and feather disease (BFD) in birds. These viruses are associated with lymphoid depletion and immunosuppressive conditions in infected animals leading to systemic illness. Circoviruses are small nonenveloped DNA viruses containing a single-stranded circular genome, encoding two major proteins: the capsid-associated protein (Cap), comprising the entirety of the viral capsid, and the replication-associated protein (Rep). Cap is the only protein component of the virion and plays crucial roles throughout the virus replication cycle, including viral attachment, cell entry, genome uncoating, and packaging of newly formed viral particles. Rep mediates recognition of replication origin motifs in the viral genome sequence and is responsible for endonuclease activity enabling nicking of the circular DNA and initiation of rolling-circle replication (RCR). Porcine circovirus 2 (PCV2) was the first circovirus capsid structure to be solved at atomic resolution using X-ray crystallography. The structure revealed an assembly comprising 60 monomeric subunits to form virus-like particles. Each Cap monomer harbors a canonical viral jelly roll domain composed of two, four-stranded antiparallel β-sheets. Crystal structures of two distinct macromolecular assemblies from BFD virus Cap were also resolved at high resolution. In these structures, the exposure of the N-terminal arginine-rich motif, responsible for DNA binding and nuclear localization is reversed. Additional structural investigations have also elucidated a PCV2 type-specific neutralizing epitope, and interaction between the PCV2 capsid and polymers such as heparin. In this review, we provide a snapshot of the structural and functional aspects of circovirus proteins. [ABSTRACT FROM AUTHOR]

Biological and molecular properties of six Prunus necrotic ringspot virus (PNRSV) isolates originating from sweet and sour cherry trees were studied. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) showed PNRSV infection in leaf samples from the respective source trees. While one of the infected source trees was symptomless, the other five showed symptoms, ranging from chlorotic to necrotic spots on the leaves. The reaction of greenhouse-grown seedlings of 22 herbaceous test plant species was investigated. The woody indicators Prunus serrulata cv. Kwanzan, wild cherry Prunus avium, P. cerasus, P. cerasifera and P. tomentosa were chip-budded. Chenopodium quinoa, Cucumis sativus cv. Amelia, C. sativus cv. Levina and P. tomentosa appeared as the most suitable test plants for the bioassay of the studied PNRSV isolates. The nucleotide sequences corresponding to the full-length coat (CP) and movement (MP) proteins were obtained. The nucleotide sequence identity among the studied isolates both in CP and MP genes was from 94% to 100%. The CP and MP amino acid sequence identities were from 95% to 100% and from 94% to 100%, respectively. Phylogenetic analyses performed with MP and CP nucleotide sequences showed that three isolates belonged to PV32-I and the other three to PV96-II phylogroups. Comparative amino acid analyses of MP and CP genes, together with the symptoms on naturally infected trees showed a higher affiliation of the studied isolates with the mild type than with the rugose types of isolates. The molecular variability correlated with the symptomatology on the naturally infected trees. [ABSTRACT FROM AUTHOR]

Despite their diversity, most double-stranded-RNA (dsRNA) viruses share a specialized T 1 capsid built from dimers of a single protein that provides a platform for genome transcription and replication. This ubiquitous capsid remains structurally undisturbed throughout the viral cycle, isolating the genome to avoid triggering host defense mechanisms. Human picobirnavirus (hPBV) is a dsRNA virus frequently associated with gastroenteritis, although its pathogenicity is yet undefined. Here, we report the cryoelectron microscopy (cryo-EM) structure of hPBV at 2.6-Å resolution. The capsid protein (CP) is arranged in a single-shelled, ~380-Å-diameter T 1 capsid with a rough outer surface similar to that of dsRNA mycoviruses. The hPBV capsid is built of 60 quasisymmetric CP dimers (A and B) stabilized by domain swapping, and only the CP-A N-terminal basic region interacts with the packaged nucleic acids. hPBV CP has an helical domain with a fold similar to that of fungal partitivirus CP, with many domain insertions in its C-terminal half. In contrast to dsRNA mycoviruses, hPBV has an extracellular life cycle phase like complex reoviruses, which indicates that its own CP probably participates in cell entry. Using an in vitro reversible assembly/disassembly system of hPBV, we isolated tetramers as possible assembly intermediates. We used atomic force microscopy to characterize the biophysical properties of hPBV capsids with different cargos (host nucleic acids or proteins) and found that the CP N-terminal segment not only is involved in nucleic acid interaction/packaging but also modulates the mechanical behavior of the capsid in conjunction with the cargo. [ABSTRACT FROM AUTHOR]

Porcine circovirus type 3 (PCV3) is a novel circovirus identified in sows with PDNS-like clinical signs and reproductive failure. The capsid protein (CAP) of PCV3 is expected to be an effective vaccine candidate. Here, we expressed the original capsid protein, truncated capsid protein without anterior highly repetitive arginine (ΔCAP) and their codon-optimized counterparts in E. coli. These results showed that lots of repeated arginine could severely lower the expression of PCV3 capsid protein in E. coli. At the same time, the recombined truncated PCV3 capsid protein forms typic virions. The efficient expression of capsid protein is expected to serve the development of PCV3 vaccines and other studies of PCV3 capsid protein. [ABSTRACT FROM AUTHOR]

Background: Porcine circovirus type 2 (PCV2) is recognized as virulent porcine pathogen and has been linked to porcine circovirus diseases (PCVD). However, there remain many unknowns regarding the spread and epidemic growth of PCV2. Methods: To assess the genetic diversity of PCV2 in the southern China, a total of 92 sequences of PCV2 strains from this region were retrieved from GenBank and were subjected to amino acid variation and phylogenetic analyses together with 28 representative sequences, based on the sequence of the ORF2 gene, from different swine‐producing countries. Results: All 92 PCV2 strains shared between 93.7% and 100% sequence similarity and could be divided into four genotypes (PCV2a, PCV2b, PCV2d and PCV2h), of which PCV2d had surpassed PCV2b and became the most prevalent PCV2 genotype in this region. Alignment of the deduced amino acid sequences of the capsid protein revealed that the obtained PCV2 strains possess two major heterogenic regions/hypervariable regions (positions 52–68 and 185–191), which were within or close to the epitopic regions in the capsid (Cap) protein. Meanwhile, the 92 PCV2 sequences also show evidence of at least five unique recombination events. Conclusion: The data in this study indicate that the PCV2 strains in the southern China are undergoing constant genetic variation and that the predominant strain and its antigenic epitopes in this area have been gradually changing in recent years. [ABSTRACT FROM AUTHOR]

• The structure and function of flaviviral capsid are very flexible. • The capsid gene contains conserved RNA secondary structures. • Both steps of assembly and dissociation of nucleocapsid complexes are obscure. • Capsid mutant viruses are highly attenuated and immunogenic. • ΔC-replicon and single-round infectious particles are promising vaccine approaches. The arthropod-borne flaviviruses cause a series of diseases in humans and pose a significant threat to global public health. In this review, we aimed to summarize the structure of the capsid protein (CP), its relevant multiple functions in the viral life cycle and innovative vaccines targeting CP. The flaviviral CP is the smallest structural protein and forms a homodimer by antiparallel α-helixes. Its primary function is to package the genomic RNA; however, both steps of assembly and dissociation of nucleocapsid complexes (NCs) have been obscure until now; in fact, flaviviral budding is NC-free, demonstrated by the subviral particles that generally exist in flavivirus infection. In infected cells, CPs associate with lipid droplets, which possibly store CPs prior to packaging. However, the function of nuclear localization of CPs remains unknown. Moreover, introducing deletions into CPs can be used to rationally design safe and effective live-attenuated vaccines or noninfectious replicon vaccines and single-round infectious particles, the latter two representing promising approaches for innovative flaviviral vaccine development. [ABSTRACT FROM AUTHOR]