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The available prophylactic vaccines for human papillomavirus (HPV) in the market are only effective against specific types of HPV, rendering them ineffective for other types of HPV infections. The objective of this research is to investigate the stability of the recombinant protein constructed, namely chimeric L1/L2 protein from HPV type 52, with improved cross-neutralization ability. The 3D model, predicted using Alphafold, Robetta, I-Tasser, and refined with Galaxy Refinement, is validated using Ramachandran plot analysis. The stability is verified through molecular dynamics simulations, considering parameters such as RMSD, RMSF, Rg, and SASA, where stable conditions are observed. The chimeric L1/L2 protein from HPV type 52 is purified using affinity chromatography, and the His-tag is cleaved using SUMO protease to obtain pure chimeric protein with the size of ~ 55 kDa. Western blot analysis confirms binding to anti-L1 HPV type 52 polyclonal antibody. The obtained vaccine candidate can be utilized as an effective prophylactic vaccine against HPV.

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing "empty" capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The utilization of biomaterials for the separation of rare earth elements (REEs) has attracted considerable interest due to their inherent advantages, including diverse molecular structures for selective binding and the use of eco-friendly materials for sustainable systems. We present a pioneering methodology for developing a safe virus to selectively bind REEs and facilitate their release through pH modulation. We engineered the major coat protein of M13 bacteriophage (phage) to incorporate a lanthanide-binding peptide. The engineered lanthanide-binding phage (LBPh), presenting ∼3300 copies of the peptide, serves as an effective biological template for REE separation. Our findings demonstrate the LBPh's preferential binding for heavy REEs over light REEs. Moreover, the LBPh exhibits remarkable robustness with excellent recyclability and stability across multiple cycles of separations. This study underscores the potential of genetically integrating virus templates with selective binding motifs for REE separation, offering a promising avenue for environmentally friendly and energy-efficient separation processes.

The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Norovirus-associated diseases are the most common foodborne illnesses worldwide. Polymerase chain reaction-based methods are the primary diagnostics for clinical samples; however, the high mutation rate of norovirus makes viral amplification and genotyping challenging. Technological advances in mass spectrometry (MS) make it a promising tool for identifying disease markers. Besides, the superior sensitivity of MS and proteomic approaches may enable the detection of all variants. Thus, this study aimed to establish an MS-based system for identifying and typing norovirus. We constructed three plasmids containing the major capsid protein VP1 of the norovirus GII.4 2006b, 2006a, and 2009a strains to produce virus-like particles for use as standards. Digested peptide signals were collected using a nano-flow ultra-performance liquid chromatography mass spectrometry (nano-UPLC/MS E ) system, and analyzed by ProteinLynx Global SERVER and TREE-PUZZLE software. Results revealed that the LC/MS E system had an excellent coverage rate: the system detected more than 94% of amino acids of 3.61 femtomole norovirus VP1 structural protein. In the likelihood-mapping analysis, the proportions of unresolved quartets were 2.9% and 4.9% in the VP1 and S domains, respectively, which is superior to the 15.1% unresolved quartets in current PCR-based methodology. In summary, the use of LC/MS E may efficiently monitor genotypes, and sensitively detect structural and functional mutations of noroviruses.

A unique region of human parvovirus B19 virus‑VP1 (B19V‑VP1u) has been linked to a variety of cardiac disorders. However, the precise role of B19V‑VP1u in inducing cardiac injury remains unknown. The present study investigated the effects of B19V‑VP1u and different regions of B19V‑VP1u, including B19V‑VP1uA (residues 1‑60), B19V‑VP1uB (residues 61‑129), B19V‑VP1uC (residues 130‑195) and B19V‑VP1uD (residues 196‑227), on inducing cardiac injury in naïve mice by zymography, immunoblotting, H&E staining and cytokine immunoassay. A significantly higher MMP‑9/MMP‑2 ratio and increased levels of inflammatory cytokines, including IL‑6 and IL‑1β, were detected in the left ventricles of the mice injected with B19V‑non‑structural protein 1 (B19V‑NS1) and B19V‑VP1u, accompanied by increased expression levels of phosphorylated (p‑)ERK and p‑P38. Significantly upregulated expression levels of atrial natriuretic peptide (ANP), heart‑type fatty acid‑binding protein (H‑FABP) and creatine kinase isoenzyme‑MB (CK‑MB), which are well‑known cardiac injury markers, as well as increased infiltration of lymphocytes, were detected in the left ventricles of the mice injected with B19V‑VP1, B19V‑NS1 and B19V‑VP1u. Moreover, a significantly higher MMP‑9/MMP‑2 ratio and increased levels of IL‑6 and IL‑1β were observed in the left ventricles of the mice injected with B19V‑VP1u, B19V‑VP1u‑A, B19V‑VP1u‑B and B19V‑VP1u‑C, accompanied by upregulated p‑ERK and p‑P38 expression. Notably, significantly lower levels of IL‑6 and IL‑1β were observed in the left ventricles of the mice injected with B19V‑VP1uD. Furthermore, significantly increased ANP, H‑FABP and CK‑MB expression levels were detected in the left ventricles of the mice injected with B19V‑VP1u, B19V‑VP1u‑A and B19V‑VP1u‑B, along with enhanced infiltration of lymphocytes. Significantly higher serum IL‑1β, IL‑6, TNF‑α and IFN‑γ levels were also detected in the mice injected with B19V‑VP1u, B19V‑VP1u‑A and B19V‑VP1u‑B. To the best of our knowledge, the findings of the present study were the first to demonstrate that the N‑terminal region (residues 1‑129) of B19V‑VP1u induces an increase in the levels of cardiac injury markers, thus providing evidence for understanding the possible functional regions within B19V‑VP1u.

Enteritis caused by CPV-2 antigenic variants (CPV-2a, 2b, and 2c) is frequently reported in dogs worldwide leading to significant morbidity and mortality. Here, we describe about a simple, single-step, ARMS-PCR strategy targeting the mutant 426 amino acid of VP2 to differentiate CPV-2 antigenic types. A total of 150 fecal samples were subjected to ARMS-PCR of which 18 were typed as CPV-2a, 79 were typed as CPV-2b, and 6 were typed as CPV-2c. The ARMS-PCR results were validated by randomly sequencing partial VP2 gene of 14 samples. Phylogenetic analysis of partial VP2 gene sequencing of each of the CPV-2 variants revealed that CPV-2a and CPV-2b isolates formed a separate clade of Indian lineage, while CPV-2c shared common evolutionary origin with Asian lineage. The developed technique is first of its kind, one-step, rapid, sequencing independent method for typing of CPV-2 antigenic variants., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Despite the global roll-out of rotavirus vaccines (RotaTeq/Rotarix / ROTAVAC/Rotasiil), mortality and morbidity due to group A rotavirus (RVA) remains high in sub-Saharan Africa, causing 104,000 deaths and 600,000 hospitalizations yearly. In Cameroon, Rotarix™ was introduced in March 2014, but, routine laboratory diagnosis of rotavirus infection is not yet a common practice, and vaccine effectiveness studies to determine the impact of vaccine introduction have not been done. Thus, studies examining RVA prevalence post vaccine introduction are needed. The study aim was to determine RVA prevalence in severe diarrhoea cases in Littoral region, Cameroon and investigate the role of other diarrheagenic pathogens in RVA-positive cases., Methods: We carried out a study among hospitalized children < 5 years of age, presenting with acute gastroenteritis in selected hospitals of the Littoral region of Cameroon, from May 2015 to April 2016. Diarrheic stool samples and socio-demographic data including immunization and breastfeeding status were collected from these participating children. Samples were screened by ELISA (ProSpecT™ Rotavirus) for detection of RVA antigen and by gel-based RT-PCR for detection of the VP6 gene. Co-infection was assessed by multiplexed molecular detection of diarrheal pathogens using the Luminex xTAG GPP assay., Results: The ELISA assay detected RVA antigen in 54.6% (71/130) of specimens, with 45, positive by VP6 RT-PCR and 54, positive using Luminex xTAG GPP. Luminex GPP was able to detect all 45 VP6 RT-PCR positive samples. Co-infections were found in 63.0% (34/54) of Luminex positive RVA infections, with Shigella (35.3%; 12/34) and ETEC (29.4%; 10/34) detected frequently. Of the 71 ELISA positive RVA cases, 57.8% (41/71) were fully vaccinated, receiving two doses of Rotarix., Conclusion: This study provides insight on RVA prevalence in Cameroon, which could be useful for post-vaccine epidemiological studies, highlights higher than expected RVA prevalence in vaccinated children hospitalized for diarrhoea and provides the trend of RVA co-infection with other enteric pathogens. RVA genotyping is needed to determine circulating rotavirus genotypes in Cameroon, including those causing disease in vaccinated children.

Rotavirus is the important etiological agents of infectious diarrhea among children under 5 years old. Rotaviruses are divided into 10 serogroups (A-J) and each group is based on genetic properties of major structural protein VP6. We designed a novel VP6 sequence optimization to increase the expression level of this protein. Numerous factors such as codon adaptation index, codon pair bias, and guanine-cytosine content were adapted based on Escherichiacoli codon usage. In addition, the ribosome binding site (RBS) of pET-15b was redesigned by the RBS calculator and the secondary structure of VP6 messenger RNA was optimized in the whole length of the coding sequence. Various factors including isopropyl beta- d-thiogalactoside (IPTG) concentration, temperature, and induction time were analyzed for the optimization of the best expression in E. coli by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The recombinant VP6 (rVP6) protein was purified by the Ni-sepharose and then the hyperimmune sera were generated against rVP6 in rabbits. Among three different temperatures, IPTG concentrations, and postinductions, the level of rVP6 was higher at 37°C, 1 mM of IPTG, and 8 h, respectively. Also, the high expression level of rVP6 was obtained in the insoluble aggregate form (43.8 g/L). After purification, the yield of rVP6 was 10.83 g/L. The rVP6 specific antiserum was confirmed by both immunofluorescent and western blotting. The versatile sequence optimization was the reason to produce a high level of rVP6 compared to other reports and can potentially apply to produce cheaper commercial kits to diagnose serological tests and new rotavirus vaccines., (© 2020 Wiley Periodicals LLC.)

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

Enzymatic nanoreactors were obtained by galactose-1-phosphate uridylyl-transferase (GALT) encapsulation into plant virus capsids by a molecular self-assembly strategy. The aim of this work was to produce virus-like nanoparticles containing GALT for an enzyme-replacement therapy for classic galactosemia. The encapsulation efficiency and the catalytic constants of bio-nanoreactors were determined by using different GALT and virus coat protein ratios. The substrate affinity of nanoreactors was slightly lower than that of the free enzyme; the activity rate was 16 % of the GALT free enzyme. The enzymatic nanoreactors without functionalization were internalized into different cell lines including fibroblast and kidney cells, but especially into hepatocytes. The enzymatic nanoreactors are an innovative enzyme preparation with potential use for the treatment of classic galactosemia., (© 2021 Wiley-VCH GmbH.)

Expression of viral capsomeres in bacterial systems and subsequent in vitro assembly into virus-like particles is a possible pathway for affordable future vaccines. However, purification is challenging as viral capsomeres show poor binding to chromatography media. In this study, the behavior of capsomeres in unfractionated bacterial lysate was compared with that for purified capsomeres, with or without added microbial DNA, to better understand reasons for poor bioprocess behavior. We show that aggregates or complexes form through the interaction between viral capsomeres and DNA, especially in bacterial lysates rich in contaminating DNA. The formation of these complexes prevents the target protein capsomeres from accessing the pores of chromatography media. We find that protein-DNA interactions can be modulated by controlling the ionic strength of the buffer and that at elevated ionic strengths the protein-DNA complexes dissociate. Capsomeres thus released show enhanced bind-elute behavior on salt-tolerant chromatography media. DNA could therefore be efficiently removed. We believe this is the first report of the use of an optimized salt concentration that dissociates capsomere-DNA complexes yet enables binding to salt-tolerant media. Post purification, assembly experiments indicate that DNA-protein interactions can play a negative role during in vitro assembly, as DNA-protein complexes could not be assembled into virus-like particles, but formed worm-like structures. This study reveals that the control over DNA-protein interaction is a critical consideration during downstream process development for viral vaccines., (© 2021 Wiley Periodicals LLC.)

Modular virus-like particles and capsomeres are potential vaccine candidates that can induce strong immune responses. There are many described protocols for the purification of microbially-produced viral protein in the literature, however, they suffer from inherent limitations in efficiency, scalability and overall process costs. In this study, we investigated alternative purification pathways to identify and optimise a suitable purification pathway to overcome some of the current challenges. Among the methods, the optimised purification strategy consists of an anion exchange step in flow through mode followed by a multi modal cation exchange step in bind and elute mode. This approach allows an integrated process without any buffer adjustment between the purification steps. The major contaminants like host cell proteins, DNA and aggregates can be efficiently removed by the optimised strategy, without the need for a size exclusion polishing chromatography step, which otherwise could complicate the process scalability and increase overall cost. High throughput process technology studies were conducted to optimise binding and elution conditions for multi modal cation exchanger, Capto™ MMC and strong anion exchanger Capto™ Q. A dynamic binding capacity of 14 mg ml -1 was achieved for Capto™ MMC resin. Samples derived from each purification process were thoroughly characterized by RP-HPLC, SEC-HPLC, SDS-PAGE and LC-ESI-MS/MS Mass Spectrometry analytical methods. Modular polyomavirus major capsid protein could be purified within hours using the optimised process achieving purities above 87% and above 96% with inclusion of an initial precipitation step. Purified capsid protein could be easily assembled in-vitro into well-defined virus-like particles by lowering pH with addition of calcium chloride to the eluate. High throughout studies allowed the screening of a vast design space within weeks, rather than months, and unveiled complicated binding behaviour for Capto TM MMC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Symmetrical protein complexes are ubiquitous in biology. Many have been re-engineered for chemical and medical applications. Viral capsids and their assembly are frequent platforms for these investigations. A means to create asymmetric capsids may expand applications. Here, starting with homodimeric Hepatitis B Virus capsid protein, we develop a heterodimer, design a hierarchical assembly pathway, and produce asymmetric capsids. In the heterodimer, the two halves have different growth potentials and assemble into hexamers. These preformed hexamers can nucleate co-assembly with other dimers, leading to Janus-like capsids with a small discrete hexamer patch. We can remove the patch specifically and observe asymmetric holey capsids by cryo-EM reconstruction. The resulting hole in the surface can be refilled with fluorescently labeled dimers to regenerate an intact capsid. In this study, we show how an asymmetric subunit can be used to generate an asymmetric particle, creating the potential for a capsid with different surface chemistries.

Background: Human noroviruses (HuNoVs) are a major cause of nonbacterial gastroenteritis in all age groups worldwide. HuNoVs can be detected in vitro using molecular assays such as RT-PCR and RT-qPCR. However, these molecular-based techniques require special equipment, unique reagents, experienced personnel, and extended time to obtain results. Besides, the diversity of viral genotypes is high. Therefore, methods that are rapid, broad-range and effective in the detection of HuNoVs are desiderated for screening the feces or vomit of infected people during outbreaks., Results: In this study, a colloidal-gold-based immunochromatographic assay (ICA) was developed for effective detection of HuNoVs in clinical samples. Monoclonal antibodies (MAbs) against the shell (S) domain in the major capsid protein of HuNoVs were used in the ICA. The limitations of detection for HuNoVs in clinical samples were 1.2 × 10 6 genomic copies per gram of stool sample (gc/g) and 4.4 × 10 5 gc/g for genogroup I and II (GI and GII) HuNoVs, respectively. A total of 122 clinical samples were tested for HuNoVs by ICA and compared against RT-qPCR. The relative sensitivity, specificity and agreement of ICA was 84.2% (95% CI: 83.6-84.8%), 100.0% (95% CI: 98.5-100.0%) and 87.7% (95% CI: 85.6-89.8%), respectively. No cross-reaction with other common enteric viruses or bacteria was observed. The ICA detected a broad range of genotypes, including GI.1, GI.3, GI.4, GI.6, GI.14, GII.2, GII.3, GII.4, GII.6, GII.13, and GII.17 HuNoVs., Conclusions: This study demonstrates that ICA targeting the S domain of VP1 is a promising candidate for effectively identifying the different genotypes of HuNoVs in clinical samples with high sensitivity and specificity.

Introduction: Rotavirus A is one of the leading causes of acute gastroenteritis in children in the first years of life. Rotavirus infection is currently classified as a preventable infection. The most abundant rotavirion protein is VP6., Material and Methods: Phylogenetic analysis and calculation of phylodynamic characteristics were carried out for 262 nucleotide sequences of the VP6 gene of rotavirus species A, isolated in Russia, using the BEAST v.1.10.4 software package. The derivation and analysis of amino acid sequences was performed using the MEGAX program., Results: This study provides phylodynamic characteristics of the rotaviruses in Russia based on the sequences coding VP6 protein. Bayesian analysis showed the circulation of rotaviruses of three sublineages of genotype I1 and three sublineages of genotype I2 in Russia. The level of accumulation of mutations was established, which turned out to be similar for genotypes I1 and I2 and amounted to 7.732E-4 and 1.008E-3 nucleotides/site/year, respectively. The effective population sizes based on nucleotide sequences of the VP6 I1 and I2 genotypes are relatively stable while after the 2000s there is a tendency of its decreasing. Comparative analysis of the amino acid sequences in the region of the intracellular neutralization sites A (231-260 aa) and B (265-292 aa) made it possible to reveal a mutation in position V252I in a proportion of Russian strains of genotype I1 some strains of genotypes I1 and I2 had mutation I281V. These substitutions were not associated with any sublineages to which the strains belong. The analysis of three T-cell epitopes revealed four amino acid differences (in aa positions 305, 315, 342, 348) that were associated with the first or second genogroup., Conclusion: Based on the phylodynamic characteristics and amino acid composition of antigenic determinants, it was concluded that the VP6 protein is highly stable and could potentially be a good model for development of a rotavirus vaccine.

Hepatitis E virus (HEV) is a nonenveloped virus causing an emerging zoonotic disease posing a severe threat to the public health in the world, especially to pregnant women. In this study, a truncated form (aa 368-606) of the open reading frame 2 of the capsid protein ( t ORF2-HEV), a major structural protein of HEV, was expressed in Escherichia coli . This work characterizes for the first time, the fused Glutathione-S-Transferase-tagged t ORF2 (GST- t ORF2) and t ORF2-HEV forms in E. coli . The fusion protein was purified by affinity chromatography with a purity higher than 90% and to yield about 27% after thrombin digestion. The purified GST- t ORF2 protein was then characterized by western blot, using anti-GST antibodies, and CD spectroscopy. The GST- t ORF2 and t ORF2-HEV proteins were shown to be efficient to develop an ELISA test to detect anti-HEV IgG in mice sera immunized with a recombinant full length ORF2 protein. Sera showed a significant increase of the absorbance signal at 450 nm, in plate wells coated with a quantity of 0.5, 1 and 2 µg of proteins. ELISA plates coated with the purified GST- t ORF2 and t ORF2-HEV showed similar response when compared to the HEV ELISA where total insect cell lysate, infected with the recombinant baculovirus expressing full ORF2, was used as positive control.

The capsid of human papillomavirus (HPV) consists of two capsid proteins - the major capsid protein L1 and the minor capsid protein L2. Assembled virus-like particles, which only consist of L1 proteins, are successfully applied as prophylactic vaccines against HPV infections. The capsid subunits are L1-pentamers, which are also reported to protect efficiently against HPV infections in animals. The recombinant production of L1 has been previously shown in E. coli, yeast, insect cells, plants and mammalian cell culture. Principally, in E. coli-based expression system L1 shows high expression yields but the protein is largely insoluble. In order to overcome this problem reported strategies address fusion proteins and overexpression of bacterial chaperones. However, an insufficient cleavage of the fusion proteins and removal of co-purified chaperones can hamper subsequent down streaming. We report a significant improvement in the production of soluble L1-pentamers by combining (I) a fusion of a N-terminal SUMO-tag to L1, (II) the heterologous co-expression of the chaperon system GroEL/ES and (III) low expression temperature. The fusion construct was purified in a 2-step protein purification including efficient removal of GroEL/ES and complete removal of the N-terminal SUMO-tag. The expression strategy was transferred to process-controlled high-cell-density fermentation with defined media according to the guidelines of good manufacturing practice. The produced L1 protein is highly pure (>95%), free of DNA (260:280 = 0.5) and pentameric. The production strategy yielded 5.73 mg of purified L1-pentamers per gram dry biomass. The optimized strategy is a suitable alternative for high yield L1-pentamer production and purification as a cheaper process for vaccine production., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A promising candidate for tumor targeted toxins is the chicken anemia-derived protein apoptin that induces tumor-specific apoptosis. It was aimed to design a novel apoptin-based targeted toxin by genetic fusion of apoptin with the tumor-directed ligand epidermal growth factor (EGF) using Escherichia coli as expression host. However, apoptin is highly hydrophobic and tends to form insoluble aggregates. Therefore, three different apoptin-EGF variants were generated. The fusion protein hexa-histidine (His)-apoptin-EGF (HAE) was expressed in E. coli and purified under denaturing conditions due to inclusion bodies. The protein solubility was improved by maltose-binding protein (MBP) or glutathione S-transferase. The protein MBP-apoptin-EGF His (MAEH) was found favorable as a targeted toxin regarding final yield (4-6 mg/L) and stability. MBP was enzymatically removed using clotting factor Xa, which resulted in low yield and poor separation. MAEH was tested on target and non-target cell lines. The targeted tumor cell line A431 showed significant toxicity with an IC 50 of 69.55 nM upon incubation with MAEH while fibroblasts and target receptor-free cells remained unaffected. Here we designed a novel EGF receptor targeting drug with high yield, purity and stability., (Copyright © 2020 Elsevier Inc. All rights reserved.)

All Neisseria gonorrhoeae strains contain multiple copies of integrated filamentous phage genomes with undefined structures. In this study, we sought to characterize the capsid proteins of filamentous N. gonorrhoeae bacteriophage NgoΦ6 and phagemids propagated in different bacteria. The data demonstrate that purified phage contain phage-encoded structural proteins and bacterial host proteins; host proteins consistently copurified with the phage particles. The bacterial host proteins associated with the phage filament (as identified by mass spectrometry) tended to be one of the predominant outer membrane components of the host strain, plus minor additional host proteins. We were able to copurify a functional ß-lactamase, a phagemid-encoded protein, with phage filaments. We used protein modeling and immunological analysis to identify the major phage encoded structural proteins. The antigenic properties of these proteins depended on the bacterium where the phages were propagated. Polyclonal antibodies against N. gonorrhoeae phage NgoΦ6 recognized phage-encoded proteins if the phage was propagated in N. gonorrhoeae or H. influenzae cells but not if it was propagated in Salmonella or E. coli. We show that the phage filaments isolated from gonococci and Haemophilus are glycosylated, and this may explain the antigenic diversity seen. Taken en toto, the data demonstrate that while the neisserial filamentous phage are similar to other Inovirus with respect to overall genomic organization, their ability to closely associate with host proteins suggests that they have unique surface properties and are secreted by a here-to-fore unknown secretory pathway., Competing Interests: The authors have declared that no competing interests exist.

Purpose: We assessed the frequency of triple-positive serology (viral capsid antigen [VCA]-immunoglobulin G [IgG], VCA-immunoglobulin M, Epstein-Barr nuclear antigen-IgG) for Epstein-Barr virus (EBV) in a small number of patients with angioimmunoblastic T-cell lymphoma (AITL) at disease onset., Methods: Nine patients with newly diagnosed AITL were retrospectively enrolled in the present study. For all of them, EBV serology data were available., Results: Of 9 patients, 7 (77.7%) had a triple-positive serology (VCA-IgG, VCA-IgM, EBNA-IgG ) for EBV. These patients were characterized by bone marrow involvement, high incidence of thrombocytopenia, and poor prognosis according to Revised International Prognostic Index and Prognostic Index for Angioimmunoblastic T-cell Lymphoma scores., Conclusion: Assessment of both viremia and serology for EBV could be useful in patients with clinical and laboratory data suggesting lymphoma diagnosis; furthermore, although our data need to be validated in a larger cohort of patients, triple positivity for EBV serology might help to direct the diagnosis toward AITL.

The applications of viral protein cages have expanded rapidly into the fields of bionanotechnology and materials science. However, the low-cost production of viral capsid proteins (CPs) on a large scale is always a challenge. Herein, we develop a highly efficient expression system by constructing recombinant Pichia pastoris cells as a "factory" for the secretion of soluble cowpea chlorotic mottle virus (CCMV) CPs. Under optimal induction conditions (0.9 mg/mL of methanol concentration at 30 °C for 96 h), a high yield of approximately 95 mg/L of CCMV CPs was harvested from the fermentation supernatant with CPs purity >90%, which has significantly simplified the rest of the purification process. The resultant CPs are employed to encapsulate Ruthenium (Ru) nanoparticles (NPs) via in-vitro self-assembly to prepare hybrid nanocatalyst, i.e. Ru@virus-like particles (VLPs). The catalytic activity over Ru@VLPs was evaluated by reducing 4-nitrophenol (4-NP) to 4-aminophenol (4-AP). The results indicate that, with the protection of protein cages, Ru NPs were highly stabilized during the catalytic reaction. This results in enhanced catalytic activity (reaction rate constant k = 0.14 min -1 ) in comparison with unsupported citrate-stabilized Ru NPs (Ru-CA) (k = 0.08 min -1 ). Additionally, comparatively lower activation energy over Ru@VLPs (approximately 32 kJ/mol) than that over Ru-CA (approximately 39 kJ/mol) could be attributed to the synergistic effect between Ru NPs and some functional groups such as amino groups (-NH 2 ) on CPs that weakened the activation barrier of 4-NP reduction. Therefore, enhanced activity and decreased activation energy over Ru@VLPs demonstrated the superiority of Ru@VLPs to unsupported Ru-CA., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Selective therapy has always been the main challenge in cancer treatments. Various non-replicative oncolytic viral systems have revealed the safety and efficacy of using viruses and these products. The aim of this paper is to examine the impact of recombinant apoptin on the proliferation of lung cancer and breast cancer cell lines., Methods: The present study consisted of two steps of expression of recombinant apoptin and its anti-proliferative effects on normal and cancer cells. In the first step, following bioinformatics and optimizing apoptin gene sequencing and synthesis, it was expressed using vector PET28a and E. coli BL21 (DE3). The expressed recombinant apoptin was confirmed by analytical SDSPAGE and then purified using Ni affinity chromatography. In the second step, the antiproliferative effects of recombinant apoptin on lung cancer, breast cancer and primary cell lines were determined using MTT assay., Results: According to the results of SDS-PAGE gel assay, recombinant apoptin was visible in the 14 kDa band. Also, the MTT assay results indicated that the antiproliferative effects of recombinant apoptin in cancer cell lines was different compared with the primary cell line, and followed a dose-dependent manner in both cell lines. The highest cytotoxicity (lowest cell viability) groups were 0.2 mg/mL in lung cancer (0.32 ± 0.015) ( P <0.001), and in breast cancer (0.33 ± 0.031) ( P <0.001) and 0.032 mg/mL in primary cells (0.17 ± 0.004) ( P <0.01), as compared to the control groups., Conclusion: Our results confirmed that recombinant apoptin can induce antiproliferative effects in lung cancer and breast cancer cell lines, but not in normal monkey kidney cell line Vero; thus, it can be introduced as a promising novel specific antitumor agent after further evaluation in clinical trials., (© 2020 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.)

The present study was aimed at comparing different E. coli strains in expressing the capsid protein of Porcine Circovirus 2 (PCV2). Full length capsid protein could be expressed only in Rosetta-gami 2 (DE3) pLysS strain using pET32b (+) vector. This confirmed that only those strains which possess tRNAs for rare codons can express the full length capsid protein. Purification of full length capsid protein could not be achieved even after several attempts using native and denaturing conditions. Subsequently, an attempt was made for expression of N-terminal truncated capsid protein using the same expression system. Truncated capsid protein was successfully expressed, purified and characterized by western blotting. The truncated capsid protein was also shown to be efficacious in testing serum samples using an optimized indirect ELISA, wherein a diagnostic sensitivity of 88.89% and specificity of 90.82% was obtained as compared to commercially available GreenSpring® porcine circovirus (PCV2) ELISA test kit. Thus, the expressed truncated capsid protein appears to be a promising diagnostic agent for PCV2. The comparative analysis suggests that cluster of arginine residues at N-terminal of capsid protein not only affects its expression in some E. coli strains but also its purification by Ni-NTA chromatography, when expressed as a histidine tagged fusion protein., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Porcine circovirus type 3 (PCV3) is an emerging swine pathogen associated with acute porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, reproductive failure, and multisystemic inflammation. Current evidence shows that PCV3 is spread worldwide, and its high incidence may pose a threat to the global pig industry. Capsid (Cap) protein is the sole structural protein which plays an important role in inducing protective immunity against PCV3 infection. In this study, monoclonal antibodies (mAbs) against Cap protein of PCV3 were produced by the hybridoma technique. Subsequently, 12 serial overlapping peptides (P1 to P12) spanning the entire region of Cap were synthesized to determine the B cell epitope regions using the mAbs. Results from dot-blot and peptide ELISA identified that P3, P9, and P10 were the major B cell antigenic regions. Fine mapping by shorter N- and C-terminal truncated peptides confirmed that the motifs 57 NKPWH 61 , 140 KHSRYFT 146 , and 161 QSLFFF 166 were linear B cell epitopes, which were highly conserved among different PCV3 strains. Interestingly, we found that the motif 140 KHSRYFT 146 was highly conserved in all reported types of PCVs (i.e., PCV1, PCV2, PCV3, and PCV4), except for the substitution (Y → K → R) of the first residue. This is the first research to identify B cell epitopes of PCV3 Cap, and these findings may lead to a better understanding of the antibody-antigen interaction and provide some guidance for PCV3 vaccine design.Key points• The recombinant Cap protein of PCV3 was expressed and purified in soluble form. • PCV3 Cap-specific mAbs prepared in this study had no cross-reactivity with PCV1/PCV2 Cap. • This is the first report of three conserved linear B cell epitopes on PCV3 Cap. • The minimal residues of the epitopes were 57-61 aa, 140-146 aa, and 161-166 aa.

Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella) production and results in high mortality in China. To obtain a genetically engineered oral vaccine against GCRV, the cholera toxin B subunit (CTB) of Vibrio cholerae was fused to VP7 (CTB-VP7) and transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified rCTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. The monomeric nature of rCTB-VP7 through multistage purification showed a binding affinity for GM1, a receptor for biologically active CTB. rCTB-VP7 is not vulnerable to disassembly by SDS but is vulnerable to disassembly by 2-mercaptoethanol. rCTB-VP7 is stable and highly active at room temperature. The binding affinity experiment between rCTB-VP7 and GM1 also confirms the effects of acid and alkalinity in solution on the structure of rCTB-VP7. rCTB-VP7 bound to GM1 with different affinities under different temperatures and pH values. Prokaryotic expression of rCTB-VP7 was characterized by high expression and easy purification and had a strong binding force with GM1 at 37 °C and pH 7.4. Our results suggest that rCTB-VP7 has the potential as an oral vaccine for protection against GCRV in aquaculture., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The HML2 (HERV-K) group constitutes the most recently acquired family of human endogenous retroviruses, with many proviruses less than one million years old. Many maintain intact open reading frames and provirus expression together with HML2 particle formation are observed in early stage human embryo development and are associated with pluripotency as well as inflammatory disease, cancers and HIV-1 infection. Here, we reconstruct the core structural protein (CA) of an HML2 retrovirus, assemble particles in vitro and employ single particle cryogenic electron microscopy (cryo-EM) to determine structures of four classes of CA Fullerene shell assemblies. These icosahedral and capsular assemblies reveal at high-resolution the molecular interactions that allow CA to form both pentamers and hexamers and show how invariant pentamers and structurally plastic hexamers associate to form the unique polyhedral structures found in retroviral cores.

To explore virus-like particles formation of dengue virus serotype type 2 (DENV-2) structural proteins of, C, prM, E were expressed in silkworm larvae using recombinant Bombyx mori nucleopolyhedroviruses (BmNPV). Each recombinant BmNPV bacmid coding the 2C-prM-E polypeptide and E protein fused with the signal peptide of bombyxin from B. mori was injected into silkworm larvae. The expressed proteins were collected from hemolymph and fat body, and purified using affinity chromatography. E protein was observed at 55 kDa. The DENV virus-like particles (DENV-LPs) with a diameter approximately 35 nm was observed using transmission electron microscopy (TEM) and immunogold-labelling TEM analysis. The binding of each partially purified proteins to heparin, one of receptors for DENV was confirmed. DENV-LPs were secreted in silkworm larval hemolymph even still low amount, but the E protein and heparin binding function were confirmed.

For many viruses, capsids (biological nanoparticles) assemble to protect genetic material and dissociate to release their cargo. To understand these contradictory properties, we analyzed capsid assembly for hepatitis B virus; an endemic pathogen with an icosahedral, 120-homodimer capsid. We used solution X-ray scattering to examine trapped and equilibrated assembly reactions. To fit experimental results, we generated a library of distinct intermediates, selected by umbrella sampling of Monte Carlo simulations. The number of possible capsid intermediates is immense, ∼10 30 , yet assembly reactions are rapid and completed with high fidelity. If the huge number of possible intermediates were actually present, maximum entropy analysis shows that assembly reactions would be blocked by an entropic barrier, resulting in incomplete nanoparticles. When an energetic term was applied to select the stable species that dominated the reaction mixture, we found only a few hundred intermediates, mapping out a narrow path through the immense reaction landscape. This is a solution to a viral application of the Levinthal paradox. With the correct energetic term, the match between predicted intermediates and scattering data was striking. The grand canonical free energy landscape for assembly, calibrated by our experimental results, supports a detailed analysis of this complex reaction. There is a narrow range of energies that supports on-path assembly. If association energy is too weak or too strong, progressively more intermediates will be entropically blocked, spilling into paths leading to dissociation or trapped incomplete nanoparticles, respectively. These results are relevant to many viruses and provide a basis for simplifying assembly models and identifying new targets for antiviral intervention. They provide a basis for understanding and designing biological and abiological self-assembly reactions.

Background & Objectives: West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and in areas where multiple flaviviruses are endemic. Diagnosis of WNV infection is primarily based on serodiagnosis, followed by virus isolation and identification. The aim of this study was to develop and evaluate a highly sensitive and specific immunoglobulin M (IgM) ELISA using the recombinant CprM protein (rWNV-CprM) for rapid, early and accurate diagnosis of WNV., Methods: The gene coding for the CprM protein of WNV was cloned and expressed in pET 28a vector followed by purification. An indirect IgM microplate ELISA using purified rWNV-CprM protein was optimized having no cross-reactivity with healthy human serum and serum samples obtained from patients with dengue and Japanese encephalitis viruses infection., Results: The comparative evaluation of this rWNV-CprM protein-specific IgM ELISA with plaque reduction neutralization test using 105 blood samples collected from patients suspected to have acute WNV infection revealed 98 per cent concordance with sensitivity and specificity of 100 and 97 per cent, respectively., Interpretation & Conclusions: The recombinant CprM protein-based WNV-specific ELISA reported in this study may be useful for rapid screening of large numbers of blood samples in endemic areas during outbreaks., Competing Interests: None

Objective To prepare a polyclonal antibody against S3 of Nelson Bay virus (NBV). Methods The E.coli BL21 (DE3) competent cells were transfected with the constructed Pris His MB-S3 recombinant plasmid. Protein expression was induced by IPTG. The target protein was purified by Ni-NTA column affinity chromatography to obtain a large amount of fusion recombinant protein, which was tested and used as the antigen for producing the S3 polyclonal antibody in rats. Results The relative molecular mass (M r ) of Pris His MB-S3 protein was around 39 000, in the form of inclusion bodies. NBV S3 polyclonal antibody was successfully produced from the immunized rats. The titer of the antibody was up to 1:64 000 determined by indirect ELISA. The indirect immunofluorescence assay verified that the S3 protein was successfully expressed in cells and distributed in a granular form in the cytoplasm. Conclusion The highly reactive and specific S3 protein polyclonal antibody is successfully prepared.

Chicken anemia virus (CAV) infection has been reported in various poultry industries worldwide. Since CAV infection is becoming increasingly prevalent, especially in local chickens of China, rapid CAV detection has become essential. The conventional diagnostic methods are time consuming and need special expertise. Therefore, in this study, we developed a specific and sensitive loop-mediated isothermal amplification (LAMP) assay for CAV detection by using multiple sequence alignment of VP2. This assay was performed at 61°C for 1 h, and there was no non-specific reaction to common avian disease viruses. The detection limit was 65 copies of viral DNA; thus, this assay showed similar sensitivity to quantitative polymerase chain reaction (qPCR) but it was more sensitive than conventional PCR. Moreover, this assay was performed using clinical samples. The LAMP assay results were 83.6% correlated to the PCR results of the clinical samples, indicating that this method is an effective tool for the rapid detection of CAV., (© 2018 Poultry Science Association Inc.)

By the age of 5 years, virtually all children have been infected by group A rotavirus (RVA), which is responsible for around half million mortality annually prior to vaccination. Relatively high rate of the morbidity and mortality highlights the necessity of applying preventive procedures particularly in developing countries. Two live attenuated RVA vaccines (Rotarix and RotaTeq) are licensed and now being used in many countries worldwide. Although these vaccines are shown to reduce the mortality up to 50%, several key questions yet remained to answer. Indeed, the licensed RV vaccines were found to be less effective in countries of sub-Saharan Africa and Southeast Asia. Therefore, developing next generation RVA vaccines is warranted. VP6 is highly abundant and conserved protein that forms the middle layer of RV particles and was shown to be both antigenic and immunogenic. Although it does not induce neutralizing antibodies, different VP6 preparations were found to induce homologous and cross-reactive immune responses with partial protection from RVA replication. Although the molecular mechanisms are not fully elucidated, VP6-based RVA vaccine candidates are worthy of further consideration. This review aims to focus on different aspects of VP6 protein and its potentiality for an alternative RV vaccine against RV disease., (© 2019 John Wiley & Sons, Ltd.)

Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1-252 and 253-371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity., Competing Interests: The authors have declared that no competing interests exist.

Adeno-associated virus (AAV) vectors currently represent the most attractive platform for viral gene therapy and are also valuable research tools to study gene function or establish disease models. Consequently, many academic labs, core facilities, and biotech/pharma companies meanwhile produce AAVs for research and early clinical development. Whereas fast, universal protocols for vector purification (downstream processing) are available, AAV production using adherent HEK-293 cells still requires time-consuming passaging and extensive culture expansion before transfection. Moreover, most scalable culture platforms require special equipment or extensive method development. To tackle these limitations in upstream processing, this study evaluated frozen high-density cell stocks as a ready-to-seed source of producer cells, and further investigated the multilayered CELLdisc culture system for upscaling. The results demonstrate equal AAV productivity using frozen cell stock-derived cultures compared to conventionally cultured cells, as well as scalability using CELLdiscs. Thus, by directly seeding freshly thawed cells into CELLdiscs, AAV production can be easily upscaled and efficiently standardized to low-passage, high-viability cells in a timely flexible manner, potentially dismissing time-consuming routine cell culture work. In conjunction with a further optimized iodixanol protocol, this process enabled supply to a large-animal study with two high-yield AAV2 capsid variant batches (0.6-1.2 × 10 15 vector genomes) in as little as 4 weeks.

Cryogenic transmission electron microscopy (cryo-EM) is widely used to determine high-resolution structures of symmetric virus capsids. The method holds promise for extending studies beyond purified capsids and their symmetric protein shells. The non-symmetric genome component has been addressed in dsRNA cypoviruses and ssRNA bacteriophages Qβ and MS2. The structure of human herpes simplex virus type 1 capsids has been determined within intact virions to resolve capsid-tegument interactions. Electron tomography under cryogenic conditions (cryo-ET), has allowed resolving an early membrane fusion intermediate of Rift Valley fever virus. Antibody-affinity based sample grids allow capturing of virions directly from cell cultures or even clinical samples. These and other emerging methods will support studies to address viral entry, assembly and neutralization processes at increasingly high resolutions and native conditions., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

JC polyomavirus (JCPyV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system in immunocompromised patients. Archetype JCPyV circulates in the human population. There have been several reports of archetype JCPyV replication in cultured cells, in which propagation was not enough to produce high titers of archetype JCPyV. In this study, we carried out cultivation of the transfected cells with archetype JCPyV DNA MY for more than 2 months to establish COS-7 cells (designated COS-JC cells) persistently producing archetype JCPyV. Moreover, JCPyV derived from COS-JC cells was characterized by analyzing the viral propagation, size of the viral genome, amount of viral DNA, production of viral protein, and structure of the non-coding control region (NCCR). Southern blotting using a digoxigenin-labeled JCPyV probe showed two different sizes of the JCPyV genome in COS-JC cells. For molecular cloning, four of five clones showed a decrease in the size of complete JCPyV genome. Especially, clone No. 10 was generated the large deletion within the Large T antigen. On the other hand, the archetype structure of the NCCR was maintained in COS-JC cells, although a few point mutations occurred. Quantitative PCR analysis of viral DNA in COS-JC cells indicated that a high copy number of archetype JCPyV DNA was replicated in COS-JC cells. These findings suggest that COS-JC cells could efficiently propagate archetype JCPyV MY and offer a useful tool to study persistent infection of archetype JCPyV in a kidney-derived system., (© 2018 The Societies and John Wiley & Sons Australia, Ltd.)

Trichodysplasia spinulosa-associated polyomavirus (TSPyV) has been linked to a rare and recently characterized skin disease occurring in immunocompromised patients. In analogy with other polyomaviruses, the major capsid protein VP1 of TSPyV can self-assemble into virus-like particles (VLPs). VLPs are increasingly applied for the vaccination and diagnostics. Mostly, non-scalable and labor intensive ultracentrifugation-based techniques are used for the purification of VLPs. In this work, we developed a purification procedure for TSPyV VP1 VLPs based on two chromatographic steps, ion-exchange monolith and core bead chromatography. Prior to chromatography, ammonium sulfate precipitation was used for the initial purification of TSPyV VP1 VLPs from yeast lysate. The VLPs were further purified using CIMmultus QA ion-exchange monolith in bind-elute mode. Most of TSPyV VP1 VLPs bound to the monolith and were subsequently eluted by a linear NaCl gradient. After ion-exchange monolith chromatography, the purity of TSPyV VP1 protein was about 75%. The final purification step of TSPyV VP1 VLPs was core bead chromatography using Capto Core 700 resin in flow-through mode. After core bead chromatography, 42% of TSPyV VP1 protein was recovered with a purity of 93%. The assembly of purified TSPyV VP1 protein into VLPs approximately 45-50 nm in diameter was confirmed by electron microscopy analysis. The purification procedure for TSPyV VP1 VLPs described here could be a scalable alternative to the conventional ultracentrifugation-based purification methods., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm-baculovirus expression vector system (silkworm-BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.

Aqueous two-phase system (ATPS) has great potential in industrial applications of bio-separations and bio-reactions. However, its large-scale application is limited by recovery difficulty of phase systems. In this paper, a recyclable ATPS was prepared by two pH-responsive copolymers (P ADB4.99 and P MDM7.08 ) and applied for purification of porcine circovirus type 2 Cap protein fermentation broth (PCV2 Cap protein). Phase separation mechanism was studied by using low-field nuclear magnetic resonance (LF-NMR). The results showed that relatively desirable ATPS were formed successfully when two copolymer concentrations were 4%-6% (w/w) with pH 7.5-8.6. The main parameters, such as copolymer concentration, temperature, pH, type and concentration of salts, were investigated. The results demonstrated that the best ATPS consisted of 4% (w/w) P ADB4.99 and 6% (w/w) P MDM7.08 , the optimal partition coefficient (K) and extraction recovery (ER) of PCV2 cap protein were 0.25 and 94.2% in the presence of 50 mM Li 2 SO 4 , 3.53 and 93.4% in the presence of 40 mM KCl, respectively, at 30 °C, pH 8.1. The maximum purity of PCV2 Cap protein was 88.4%. In addition, the two phase-forming copolymers could be recycled by adjusting solution pH to their isoelectric points (pI), with recoveries of over 97.5%., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A previous study showed that a single amino acid difference in the cucumber mosaic virus (CMV) capsid protein (CP) elicits unusual symptoms. The wild-type strain (CMV-R) induces green mosaic symptoms and malformation while the mutant strain (CMV-R3E79R) causes chlorotic lesions on inoculated leaves and strong stunting with necrosis on systemic leaves. Virion preparations of CMV-R and CMV-R3E79R were partially purified from Nicotiana clevelandii A. Gray and analysed by two-dimensional gel electrophoresis. Their separated protein patterns showed remarkable differences at the 50-75 kDa range, both in numbers and intensity of spots, with more protein spots for the mutant CMV. Mass spectrometry analysis demonstrated that the virion preparations contained host proteins identified as ATP synthase alpha and beta subunits as well as small and large Rubisco subunits, respectively. Virus overlay protein binding assay (VOPBA), immunogold electron microscopy and modified ELISA experiments were used to prove the direct interaction between the virus particle and the N. clevelandii ATP synthase F1 motor complex. Protein-protein docking study revealed that the electrostatic change in the mutant CMV can introduce stronger interactions with ATP synthase F1 complex. Based on our findings we suggest that the mutation present in the CP can have a direct effect on the long-distance movement and systemic symptoms. In molecular view the mutant CMV virion can lethally block the rotation of the ATP synthase F1 motor complex which may lead to cell apoptosis, and finally to plant death., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ranaviruses. Here we report the development and partial validation of a TaqMan real-time qPCR assay. The primers and TaqMan probe targeted a conserved region of the major capsid protein (MCP) gene. A series of experiments using a 10-fold dilution series of Frog virus 3 (FV3) MCP plasmid DNA revealed linearity over a range of 7 orders of magnitude (107-101), a mean correlation coefficient (R2) of >0.99, and a mean efficiency of 96%. The coefficient of variation of intra- and inter-assay variability ranged from <0.1-3.5% and from 1.1-2.3%, respectively. The analytical sensitivity was determined to be 10 plasmid copies of FV3 DNA. The qPCR assay detected a panel of 33 different ranaviral isolates originating from fish, amphibian, and reptile hosts from all continents excluding Africa and Antarctica, thereby representing the global diversity of ranaviruses. The assay did not amplify highly divergent ranaviruses, members of other iridovirus genera, or members of the alloherpesvirus genus Cyprinivirus. DNA from fish tissue homogenates previously determined to be positive or negative for the ranavirus Epizootic hematopoietic necrosis virus by virus isolation demonstrated a diagnostic sensitivity of 95% and a diagnostic specificity of 100%. The reported qPCR assay provides an improved expedient diagnostic tool and can be used to elucidate important aspects of ranaviral pathogenesis and epidemiology in clinically and sublinically affected fish, amphibians, and reptiles.

In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20-30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.

Hepatitis E virus (HEV) is associated with acute hepatitis disease. Numerous truncated HEV capsid proteins have been successfully expressed using different expression systems. Among these, p495, a protein truncated at its N- and C-termini by 111 and 54 amino acids (aa), respectively (HEV ORF2 aa 112-606) can self-assemble into T = 1 virus-like particles (VLPs) when expressed by insect cells. A shorter p239 (aa 368-606) protein is a particulate antigen that we have previously used in our commercialized HEV vaccine, Hecolin. Here, we sought to express p495 in its soluble form (named Ep495) in E. coli and in baculovirus-infected Tn5 insect cells (named BTp495) as a back-to-back control. Characterization of p495 particles derived from these two expression systems showed similarities in particle size, morphology, and sedimentation coefficient. Antigenicity assays using a panel of anti-HEV monoclonal antibodies also showed similar strong reactivities for Ep495 and BTp495, as well as similar binding profiles that were congruent with p239. Mouse immunization results showed that Ep495 particles had comparable immunogenicity with that of BTp495 VLPs, as well as p239. Overall, our findings suggest that p495 particles produced in E. coli are ideal for the development of next-generation prophylactic vaccines against hepatitis E., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Nervous necrosis virus (NNV), also known as betanodavirus, has been recently implicated in mass mortalities of cultured marine fish. An effective vaccine is urgently needed to protect fish against this virus. However, parenteral immunization methods are very stressful. Individual immunization for thousands of fish is very labor intensive and expensive. Therefore, we expressed NNV coat protein in tobacco chloroplasts and used it as an oral vaccine to induce immunities in fish followed by challenges with NNV. Our results revealed that mice (IgG and IgA) and fish (IgM) immunized with the oral vaccine developed significantly higher antibody titers against the NNV coat protein. Fish were partially protected against viral challenge. Taken together, our results demonstrated that a plant-based vaccine could effectively induce immune response and protect groupers against NNV. The present method could be used to develop oral fish vaccine in the future.

The controlled self-assembly of protein cages is vital for the use of these nanocompartments in biomedical and nanotechnological applications. Recently, we showed that by combining different structural peptide elements, it is possible to assemble viral capsid proteins in distinct well-defined morphologies. In this chapter, a triblock copolypeptide is discussed, consisting of a metal ion-coordinating hexahistidine tag, a stimulus-responsive elastin-like polypeptide and a pH-responsive self-assembling viral capsid protein. This protein is able to form two different types of capsids, depending on the assembly pathway that is followed. Here, we focus on the metal ion-induced assembly process and describe the relevant experimental procedures to induce and utilize this assembly behavior.

Background & Aims: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system., Methods: We performed studies with gt3 HEV cell culture-produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay., Results: We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture-produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients., Conclusions: We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

In cellular systems, compartmentalization plays an important role in the protection and regulation of enzymes. Controlled encapsulation of enzymes in nanocompartments is crucial in understanding biocatalytic processes in the cellular environment. We have recently described an enzymatic method to covalently attach enzymes, equipped with a small recognition peptide, to the interior of viral capsids. Viral capsids are especially interesting in this respect, as they form very well-defined nanoparticles with a uniform size and shape. Here, we describe the relevant experimental procedures to encapsulate a model enzyme into the interior of a viral capsid, purify the resulting viral capsids, and measure the catalytic activity of the encapsulated enzymes.