KIF5B-mediated internalization of FMDV promotes virus infection.

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease, that is caused by the FMD virus (FMDV). Although the cell receptor for FMDV has been identified, the specific mechanism of FMDV internalization after infection remains unknown. In this study, we found that kinesin family member 5B (KIF5B) plays a vital role during FMDV internalization. Moreover, we confirmed the interaction between KIF5B and FMDV structural protein VP1 by co-immunoprecipitation (Co-IP) and co-localization in FMDV-infected cells. In particular, the stalk [amino acids (aa) 413-678] domain of KIF5B was indispensable for KIF5B-VP1 interaction. Moreover, overexpression of KIF5B dramatically enhanced FMDV replication; consistently, knockdown or knockout of KIF5B suppressed FMDV replication. Furthermore, we also demonstrated that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating. KIF5B also promotes the transmission of viral particles to early and late endosomes during the early stages of infection. In conclusion, our results demonstrate that KIF5B promotes the internalization of FMDV via regulating clathrin uncoating and intracellular transport. This study may provide a new therapeutic target for developing FMDV antiviral drugs.

A novel VP1-based enzyme-linked immunosorbent assay revealed widespread Enterovirus G infections in Guangxi, China.

Enterovirus G (EV-G) has recently been shown to affect weight gain and cause neurological symptoms in piglets. However, the serological investigation of EV-G is limited. In this study, we developed a novel serological detection method based on the structural protein, VP1 of EV-G. The intra-assay and inter-assay coefficient variations were 3.2-8.9% and 2.6-8.0%, respectively. There was no cross-reaction of the VP1-based enzyme-linked immunosorbent assay (ELISA) with antisera against the other known porcine viruses. In addition, a comparison was made with other methods including the developed indirect ELISAs based on VP2 and VP3 proteins and western blot (WB) analysis, which demonstrated the reliability of the novel method. Using the VP1-based ELISA, we carried out the first seroepidemiological survey of EV-G in China by testing 1041 serum samples collected from different pig farms in Guangxi from 2019 to 2021. Our results showed that 68.78% of the serum samples and 100% of the pig farms were positive for EV-G, with a relatively high incidence of seropositivity in pigs of different ages. This was specifically evident in fattening pigs and sows, which may suggest that the piglets have experienced an infection with EV-G during their growth process. Our data provide the first serological evidence of EV-G infections in pigs from China and reveal the widespread presence of EV-G infections in Guangxi, China.

The analysis of the genotype of Sapovirus outbreaks in Zhejiang Province.

BACKGROUND: Sapovirus (SaV) infection is increasing globally. Concurrently, several SaV-outbreaks were observed in children of Zhejiang province, China, in recent years, In this study, the genotypes of Sapovirus from seven outbreaks in the Zhejiang province were analysed. METHODS: A total of 105 faecal samples were collected from children aged between 4 and 17 years from the Zhejiang Provincial Center for Disease Control and Prevention between October 2021 and February 2023. Genotypes were processed using reverse transcription polymerase chain reaction and Sanger sequencing, while next-generation sequencing was used to generate a complete viral genome. Deduced amino acid sequences were analysed to detect VP1 gene mutations. RESULTS: In total, 60 SaV-positive patients were detected at a 57.14% (60/105) positivity rate. Positive rates in the seven outbreaks were: 22.22% (2/9), 15.00% (3/20), 93.10% (27/29), 84.21% (16/19), 28.57% (2/7), 53.33% (8/15) and 33.33% (2/6), respectively. Four genotypes were identified in the seven outbreaks, of which, GI.1 accounted for 14.29% (1/7), GI.2 accounted for 14.29% (1/7), GI.6 and GII.5 accounted for 14.29% (1/7), and GI.6 accounted for 57.14% (4/7). All patients were children and outbreaks predominantly occurred in primary schools and during cold seasons. Additionally, the complete sequence from the GI.6 outbreak strain showed high homology (identity: 99.99%) with few common substitutions (Y300S, N302S and L8M) in VP1 protein. CONCLUSIONS: SaV genotype diversity was observed in the seven outbreaks, with GI.6 being the main SaV genotype in Zhejiang province. It demonstrated high homology and may provide a platform for SaV prevention and control measures.

Immune escape of bovine parvovirus by VP1 inhibiting IFN-β production through the RIG-I-like receptor pathway / Escape inmunológico del parvovirus bovino mediante la inhibición de VP1 de la producción de IFN-β a través de la vía del receptor tipo RIG-I

Objective: The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-β) production and to reveal further molecular mechanism of BPV immune escape. Method: The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293 T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-β mRNA were detected using qPCR. Results: The expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 104. Expression levels of IFN-β mRNA of the BPV VP1 group in HEK 293 T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-β expression levels in HEK 293 T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. Conclusion: pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-β. Overexpression of pCMV-Myc-BPV-VP decreased IFN-β mRNA expression in HEK 293 T cells and inhibited IFN-β production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.(AU)

Reverse Vaccinology and Immunoinformatic Approach for Designing a Bivalent Vaccine Candidate Against Hepatitis A and Hepatitis B Viruses.

Hepatitis A and B are two crucial viral infections that still dramatically affect public health worldwide. Hepatitis A Virus (HAV) is the main cause of acute hepatitis, whereas Hepatitis B Virus (HBV) leads to the chronic form of the disease, possibly cirrhosis or liver failure. Therefore, vaccination has always been considered the most effective preventive method against pathogens. At this moment, we aimed at the immunoinformatic analysis of HAV-Viral Protein 1 (VP1) as the major capsid protein to come up with the most conserved immunogenic truncated protein to be fused by HBV surface antigen (HBs Ag) to achieve a bivalent vaccine against HAV and HBV using an AAY linker. Various computational approaches were employed to predict highly conserved regions and the most immunogenic B-cell and T-cell epitopes of HAV-VP1 capsid protein in both humans and BALB/c. Moreover, the predicted fusion protein was analyzed regarding primary and secondary structures and also homology validation. Afterward, the three-dimensional structure of vaccine constructs docked with various toll-like receptors (TLR) 2, 4 and 7. According to the bioinformatics tools, the region of 99-259 amino acids of VP1 was selected with high immunogenicity and conserved epitopes. T-cell epitope prediction showed that this region contains 32 antigenic peptides for Human leukocyte antigen (HLA) class I and 20 antigenic peptides in terms of HLA class II which are almost fully conserved in the Iranian population. The vaccine design includes 5 linear and 4 conformational B-cell lymphocyte (BCL) epitopes to induce humoral immune responses. The designed VP1-AAY-HBsAg fusion protein has the potency to be constructed and expressed to achieve a bivalent vaccine candidate, especially in the Iranian population. These findings led us to claim that the designed vaccine candidate provides potential pathways for creating an exploratory vaccine against Hepatitis A and Hepatitis B Viruses with high confidence for the identified strains.

Designing a Multi-Epitope Subunit Vaccine against VP1 Major Coat Protein of JC Polyomavirus.

The JC polyomavirus virus (JCPyV) affects more than 80% of the human population in their early life stage. It mainly affects immunocompromised individuals where virus replication in oligodendrocytes and astrocytes may lead to fatal progressive multifocal encephalopathy (PML). Virus protein 1 (VP1) is one of the major structural proteins of the viral capsid, responsible for keeping the virus alive in the gastrointestinal and urinary tracts. VP1 is often targeted for antiviral drug and vaccine development. Similarly, this study implied immune-informatics and molecular modeling methods to design a multi-epitope subunit vaccine targeting JCPyV. The VP1 protein epitopic sequences, which are highly conserved, were used to build the vaccine. This designed vaccine includes two adjuvants, five HTL epitopes, five CTL epitopes, and two BCL epitopes to stimulate cellular, humoral, and innate immune responses against the JCPyV. Furthermore, molecular dynamics simulation (100 ns) studies were used to examine the interaction and stability of the vaccine protein with TLR4. Trajectory analysis showed that the vaccine and TLR4 receptor form a stable complex. Overall, this study may contribute to the path of vaccine development against JCPyV.

Inhibitory effects and mechanisms of proanthocyanidins against enterovirus 71 infection.

Proanthocyanidins (PC), a natural flavonoid compound, was reported to possess a variety of pharmacological activities such as anti-tumor and anti-viral effects. In this study, the anti-Enterovirus 71 (EV71) activities and mechanisms of PC were investigated both in vitro and in vivo. The results showed that PC possessed anti-EV71 activities in different cell lines with low toxicity. PC can block both the adsorption and entry processes of EV71 via directly binding to virus VP1 protein. PC may competitively interfere with the binding of VP1 to its receptor SCARB2. PC can also regulate three different MAPK signaling pathways to reduce EV71 infection and attenuate virus induced inflammatory responses. Importantly, intramuscular therapy of EV71-infected mice with PC markedly improved their survival and attenuated the severe clinical symptoms. Therefore, the natural compound PC has potential to be developed into a novel anti-EV71 agent targeting viral VP1 protein and MAPK pathways.

Immune escape of bovine parvovirus by VP1 inhibiting IFN-ß production through the RIG-I-like receptor pathway.

OBJECTIVE: The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-ß) production and to reveal further molecular mechanism of BPV immune escape. METHOD: The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293 T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-ß mRNA were detected using qPCR. RESULTS: The expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 104. Expression levels of IFN-ß mRNA of the BPV VP1 group in HEK 293 T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-ß expression levels in HEK 293 T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. CONCLUSION: pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-ß. Overexpression of pCMV-Myc-BPV-VP decreased IFN-ß mRNA expression in HEK 293 T cells and inhibited IFN-ß production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.

Evaluating the Immunogenicity of recombinant VP1 protein from the foot-and-mouth disease virus encapsulated in nanoliposome in guinea pig animal model.

Foot-and-mouth disease (FMD) is considered as contagious in livestock, which is caused by the Picornavridae virus family known as FMD virus (FMDV). In the present study, the VP1 gene from FMDV (O strain) was expressed and purified. In addition, nanoliposomes were utilized as an adjuvant. After formulating the nanoliposomes with DMPC, DMPG, and cholesterol, the recombinant VP1 protein was encapsulated in the nanoliposomes. Further, the intended nanoliposomes in the sizes of 400 and 200 nm, nanoliposome-inactivated FMDV, Freund's adjuvant-inactivated FMDV, and Freund's adjuvant-recombinant VP1 mixtures, and PBS buffer (negative control) were injected to six groups of five guinea pigs. Furthermore, the guinea pig serums were analyzed through using ELISA and serum neutralization tests after four boosting vaccinations. Based on the results, the immunogenicity of the 200 nm-nanoliposomes encapsulating recombinant VP1 protein was more than that of 400 nm-ones so that 200 nm-nanoliposomes could trigger the immune response against FMDV in guinea pigs.

Preparation of monoclonal antibody and identification of two novel B cell epitopes to VP1 protein of porcine sapelovirus.

Porcine sapelovirus (PSV) is an important emerging swine pathogen that causes diarrhoea, respiratory distress, severe reproductive system and neurological disorders in pigs, posing huge threat to swine industry. However, there are no effective serological diagnostic products and the epitope characterization of PSV VP1 protein is still largely unknown. In current study, we successfully expressed recombinant His-VP1 protein by prokaryotic expression system and the recombinant VP1 protein had good immunogenicity. BALB/C mice were then selected and immunized with purified recombinant VP1 protein, and two monoclonal antibodies (Mabs) 9F10 and 15E4 against VP1 were successfully prepared by hybrioma technology. The isotype of these two Mabs were identified and showed that Mab 9F10 with the heavy chain subtype was IgG1 and the light chain subtype was kappa. Mab 15E4 was identified as IgG2 for the heavy chain subtype and Kappa for the light chain subtype. The antigen epitopes of prepared two VP1 Mabs were clearly identified. The minimal unit of B cell specific epitope recognized by Mab 15E4 was 203YDGDG207 and conserved in different strain genotypes of PSV, indicating this epitope may be a good target for serological detection of PSV. However, the epitope recognized by Mab 9F10 was 8QAIVNRT14 and varied greatly among different PSV strains. Structural modeling analysis showed that the identified two novel B cell epitopes were located on the surface of VP1. Our study provides useful tool for the establishment the serological detection methods of PSV and may support the study of VP1 protein function.

Preparation and Characterization of Nanoliposome Containing Isolated VP1 Protein of Foot and Mouth Disease Virus as a Model of Vaccine.

Foot-and-mouth disease (FMD) is an acute and highly contagious disease in livestock, such as cattle, sheep, and pigs, leading to a lot of economic losses. The current FMD vaccines formulated by inactivated whole-virus and adjuvant successfully reduce disease outbreaks in many regions of the world. Immunological studies on FMD viruses revealed that the dominant epitope in arising neutral antibody response is amino acid residues constructing the G-H loop, constituting a surface loop of the structural protein, termed VP1. Liposomes as one of the most well-known vehicles are considered an important carrier in vaccine development, and their function is used to encapsulate purified VP1 protein based on their size, charge, and lipid content. Accordingly, the VP1 protein was isolated from the FMD virus. This study aimed to compare four methods of VP1 protein encapsulation in the liposome and the extruding effect, as follows: 1) VP1 protein was dissolved in dimethyl sulfoxide and added to the lipid film hydrated by ethanol, 2) the lipid film was hydrated by VP1 protein with 7M urea, 3) the lipid film was hydrated by VP1 protein and freeze-thawed, and 4) the lipid film was hydrated by VP1 protein. The highest encapsulation efficiency was 91% in the second method which purified protein-containing urea. The VP1 protein in the prepared liposome (1, 2-dimyristoyl-sn-glycero-3-phosphocholine: 1, 2-dimyristoyl-sn-glycero-3-phosphocholine: cholesterol) released more than 90% of protein content after 240 h.

Porcine circovirus-like virus P1 activates pancreatic secretion pathway by interacting with CHRM3 protein.

The porcine circovirus-like virus P1, a member of the circovirus family, causes post-weaning multisystemic wasting syndrome (PMWS) in weaned piglets with progressive wasting as the main clinical symptom. The pancreatic secretion pathway induces pancreatic acinar cells to secrete various digestive enzymes and as such is an important signaling pathway for the digestive system and somatic growth. This study examined the effects and mechanism of P1 virus infection on the pancreatic secretion pathway. The experiment was conducted by transfecting double-copy plasmid P1 into PK-15 and 3D4 cells and by infecting cells with the P1 virus. Samples were collected at various times after transfection or infection. The pathway's transcription and translation levels of CHRM3, Gq, PLC-ß2, PRKCA, Rab3D, RhoA, Rac1, and amyA proteins were detected by real-time PCR and Western blots; these analyses confirmed that the P1 virus infection could upregulate the expression level of key pancreatic secretion signaling molecules. Then, we confirmed that the VP1 protein of the P1 virus could interact with the pathway initiation protein CHRM3 using Co-IP, pull-downs, and confocal fluorescence microscopy. Finally, we demonstrated that the VP1 protein activates the pancreatic secretory pathway through the CHRM3 protein. In conclusion, this study demonstrated that the P1 virus can interact with the CHRM3 receptor protein to activate the pancreatic secretion pathway and promote the secretion of various digestive enzymes downstream of the pathway, thereby providing a basis for P1 virus pathogenesis.

Lewis fucose is a key moiety for the recognition of histo-blood group antigens by GI.9 norovirus, as revealed by structural analysis.

Noroviruses have been identified as major causative agents of acute nonbacterial gastroenteritis in humans. Histo-blood group antigens (HBGAs) are thought to play a major role among the host cellular factors influencing norovirus infection. Genogroup I, genotype 9 (GI.9) is the most recently identified genotype within genogroup I, whose representative strain is the Vancouver 730 norovirus. However, the molecular interactions between host antigens and the GI.9 capsid protein have not been investigated in detail. In this study, we demonstrate that the GI.9 norovirus preferentially binds Lewis antigens over blood group A, B, and H antigens, as revealed by an HBGA binding assay using virus-like particles. We determined the crystal structures of the protruding domain of the GI.9 capsid protein in the presence or absence of Lewis antigens. Our analysis demonstrated that Lewis fucose (α1-3/4 fucose) represents a key moiety for the GI.9 protein-HBGA interaction, thus suggesting that Lewis antigens might play a critical role during norovirus infection. In addition to previously reported findings, our observations may support the future design of antiviral agents and vaccines against noroviruses.

Construction of Recombinant Lactococcus lactis Strain Expressing VP1 Fusion Protein of Duck Hepatitis A Virus Type 1 and Evaluation of Its Immune Effect.

With the continuous development of duck farming and the increasing breeding density, the incidence of duck hepatitis A virus type 1 (DHAV-1) has been on the rise, seriously endangering the development of duck farming. To reduce the use of antibiotics in duck breeding, susceptibility risks and mortality, and avoid virulence recovery and immune failure risk, this study aims to develop a new type of mucosal immune probiotics and make full use of molecular biology techniques, on the level of genetic engineering, to modify Lactococcus lactis (L. lactis). In this study, a secretory recombinant L. lactis named MG1363-VP1 with an enhanced Green Fluorescent Protein (eGFP) and translation enhancer T7g10L was constructed, which could express the VP1-eGFP fusion protein of DHAV-1. The animal experiment in ducklings was performed to detect the immune response and protection effect of oral microecologics by recombinant L. lactis. The results showed that oral L. lactis MG1363-VP1 significantly induced the body's humoral immune system and mucosal immune system to produce specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) for DHAV-1 in ducklings, and cytokines including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon gamma (IFN-γ). The mortality rate was monitored simultaneously by the natural infestation in the process of production and breeding; notably, the ducklings vaccinated with L. lactis MG1363-VP1 were effectively protected against the nature infection of DHAV-1. The recombinant L. lactis MG1363-VP1 constructed in this study provides a new means of preventing and controlling DHAV-1 infection in the future.

Mass Spectrometry-Based System for Identifying and Typing Norovirus Major Capsid Protein VP1.

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/MSE) system, and analyzed by ProteinLynx Global SERVER and TREE-PUZZLE software. Results revealed that the LC/MSE 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/MSE may efficiently monitor genotypes, and sensitively detect structural and functional mutations of noroviruses.

Establishment of a two-step purification scheme for tag-free recombinant Taiwan native norovirus P and VP1 proteins.

The protruding (P) domain of the major capsid protein VP1 of norovirus (NoV) is the crucial element for immune recognition and host receptor binding. The heterologous P protein expressed by Pichia pastoris self-assembles into P particles. However, tag-free NoV protein purification schemes have rarely been reported due to the low isoelectric point of NoV proteins, which leads to highly competitive binding between the target protein and yeast host cell proteins at alkaline pH. In this study, a two-step purification scheme based on surface histidines and the charge on the NoV GII.4 strain P protein was developed. Using HisTrap and ion exchange chromatography, the P protein was directly purified, with a recovery of 28.1% and purity of 82.1%. Similarly, the NoV capsid protein VP1 was also purified using HisTrap and gel filtration chromatography based on native surface histidines and self-assembly ability, with 20% recovery and over 90% purity. Dynamic light scattering and transmission electron microscopy analyses of the purified NoV P revealed that most of these small P particles were triangle-, square- and ring-shaped, with a diameter of approximately 14 nm, and that the purified NoV VP1 self-assembles into particles with a diameter of approximately 47 nm. Both the purified NoV P and VP1 particles retained human histo-blood group antigen-binding ability, as evidenced by a saliva-binding assay.

The immune response to a recombinant Lactococcus lactis oral vaccine against foot-and-mouth disease virus in mice.

OBJECTIVE: Development of an effective mucosal vaccine to induce specific immune responses against Foot-and-mouth disease virus (FMDV). RESULTS: For this purpose, the FMDV VP1 gene (SPVP1) was optimized and synthesized based on the codon bias of Lactococcus lactis (L. lactis), and then incorporated in the plasmid pNZ8148. L. lactis NZ9000 containing the pNZ8148-SPVP1 recombinant plasmid was used as an oral delivery vehicle to induce anti-FMDV mucosal and systemic immune responses in mice. After confirmation that the SPVP1 protein was expressed successfully in the recombinant L. latic, the mice were orally challenged with NZ9000-pNZ8148, NZ9000-pNZ8148-SPVP1, phosphate-buffered saline as a mock infection group, or with inactivated vaccine as a positive group. Mice immunized with NZ9000-pNZ8148-SPVP1 produced high levels of mucosal secretory IgA (sIgA), antigen-specific serum IgG, IgA, and neutralizing antibodies, and developed stronger cell-mediated immune reactions and significant T spleen lymphocyte proliferation. Furthermore, the recombinant group generated much higher levels of IFN-γ, IL-2, IL-4, IL-5, and IL-10 than the other groups. CONCLUSIONS: Potent immune responses were successfully elicited in mice with FMDV VP1 delivered through L. lactis.

Effective production of recombinant Δ60VP1 chicken anemia virus protein in Escherichia coli and its application to a serodiagnostic indirect ELISA.

Chicken anemia virus (CAV) causes severe anemia and immunosuppression in chickens. VP1 is the main capsid protein, and is suitable for diagnostic kit development, however, it has 24 arginine residues in the first forty N-terminal amino acids of the protein causing toxicity to bacteria leading to reduced prokaryotic expression. In this study, a 60 amino acid N-terminally truncated VP1 (Δ60VP1) which removes the toxic region was expressed in Escherichia coli and the resultant insoluble recombinant protein was purified by Ni-NTA affinity chromatography with anionic denaturing detergents. The high amounts of purified Δ60VP1 produced (150 mg/L) retained appropriate antigenicity and the antigen was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of CAV. One hundred fifty-two chicken serum samples (n = 152) were evaluated using the newly developed Δ60VP1 indirect ELISA (cutoff value = 7.58 % S/P). The sensitivity and specificity of the Δ60VP1 indirect ELISA were 87.50 % and 95.31 %, respectively, while the agreement between the Δ60VP1 indirect ELISA and the commercial IDEXX CAV ELISA was 90.79 % (kappa = 0.814). In this study, we have developed an alternative VP1 production platform in E. coli by truncating the N-terminal 60 amino acids (Δ60VP1) and using anionic denaturing detergents during the purification to successfully solubilize the insoluble Δ60VP1. The antigen was purified with high yield and good immunoreactivity, and an indirect ELISA was developed. The assay could potentially be applied to large-scale CAV serosurveillance.

Soluble FMDV VP1 proteins fused with calreticulin expressed in Escherichia coli under the assist of trigger factor16 (Tf16) formed into high immunogenic polymers.

Foot and mouth disease virus (FMDV) is a highly contagious pathogen propagating among cloven-hoofed animals. As a major immunogenic protein, VP1 plays a pivotal role in the induction of neutralizing antibodies, which therefore is an ideal target for developing subunit vaccines. In current study, four prokaryotic expression clones (rV4C, rC4V, rV5F and rF5V) were constructed by fusing truncated calreticulin (CRT) (120-250 aa or 120-308 aa) at the N/C terminal of vp1 gene, and co-expressed with chaperone trigger factor 16 (Tf16) in E.coli, respectively. The soluble recombinant CRT-fused VP1 proteins could form into homogeneous reactive polymers with average hydrodynamic diameters around 100 nm according to the dynamic light scattering (DLS) data. Immunization of guinea pigs with 10 µg purified CRT-fused VP1 proteins induced high levels of antibodies against naked-VP1 through indirect ELISA. Sandwich ELISA showed that only rC4V could elicit the same level of antibody against FMD virus as commercial inactivated vaccine after booster. The lymphocyte cytokines secretion of immunized rC4V was higher than the other CRT-fused VP1 proteins in guinea pigs. These results showed that the soluble CRT-fused VP1 proteins, especially rC4V, expressed with Tf16 in E. coli might have potential to be used as subunit vaccine candidate against FMDV.

Effect of myricetinon enterovirus 71 infection in vitro / 中国药理学通报

Aim To investigate the antiviral activity and mechanism of myricetin against enterovirus 71 (E V 7 1) infection. Methods The cytopathic effect (CPE) and plaque assay were used to observe the antiviral effect of myricetin against EV71 in Vero cell. The cells were treated with myricetin at different concentrations combined with crystal violet staining to detectthe cytotoxicity of myricetin. The effect of myricetin on VP1 protein expression was detected by Western blot. The effect of myricetin on VP1 gene expressionwas evaluated byRT-PCR. Results Myricetin pretreatment at 2. 5-20 fimol L-1' significantly inhibitedcell death induced by EV71 infection in a dose-dependent manner with the IC50 value of 5. 6 jxmol • L-1. Compared to virus control group, myricetin could significantly reduce the viral titer at the concentration of 2. 5 ~ 20 u,mol • L-1. The results of Western blot and RT-PCR showed that myricetin could markedlyreduce the gene and protein expression levels of viral capsid protein VP1. Conclusion Myricetin has significant antiEV71 activity in vitro.