Evaluation of BVDV E2 proteins based on recombinant baculovirus expression system production as diagnostic antigens and immunogens.

Bovine viral diarrhea virus (BVDV) is a significant immunosuppressive pathogen that has a major impact on the global cattle industry. Research efforts are currently focused on the envelope glycoprotein E2 of BVDV to improve immune responses. However, the full-length E2 protein is not ideal as an immune antigen and diagnostic tool, leading to the exploration of alternative strategies. In this study, we optimized the E2 gene using IDEB and ExpOptimizer software, then expressed the E2 gene using both baculovirus and E. coli expression systems. Subsequently, we assessed the immunogenicity of the purified E2 protein in mice and its application in indirect ELISA assays. Our findings showed that the Bac-E2 protein produced by the baculovirus system induced higher levels of antibody production and splenic lymphocyte proliferation in mice compared to the E. coli system. Moreover, the indirect ELISA assay developed using Bac-E2 protein exhibited superior specificity, sensitivity, and accuracy in comparison to the E. coli-expressed E2 ELISA. Overall, our study demonstrates that the optimized E2 protein generated through a baculovirus expression system elicits robust humoral and cellular immune responses in mice, making it a promising candidate for vaccine development. Furthermore, the optimized E2 protein ELISA assay shows enhanced sensitivity and accuracy, suggesting its potential as a valuable diagnostic antigen.

Heterologous immunization targeting the CST1 antigen confers better protection than ROP18 in mice.

Aim: To evaluate the protective efficacy induced by heterologous immunization with recombinant baculoviruses or virus-like particles targeting the CST1 and ROP18 antigens of Toxoplasma gondii.Materials & methods: Recombinant baculovirus and virus-like particle vaccines expressing T. gondii CST1 or ROP18 antigens were developed to evaluate protective immunity in mice upon challenge infection with 450 Toxoplasma gondii (ME49).Results: Immunization with CST1 or ROP18 vaccines induced similar levels of T. gondii-specific IgG and IgA responses. Compared with ROP 18, CST1 vaccine showed better antibody-secreting cell response, germinal center B cell activation, and significantly reduced brain cyst burden and body weight loss.Conclusion: Our findings suggest that CST1 heterologous immunization elicited better protection than ROP18, providing important insight into improving the toxoplasmosis vaccine design strategy.

[Box: see text].

Efficient Simulation of Viral Transduction and Propagation for Biomanufacturing.

The design of biomanufacturing platforms based on viral transduction and/or propagation poses significant challenges at the intersection between synthetic biology and process engineering. This paper introduces vitraPro, a software toolkit composed of a multiscale model and an efficient numeric technique that can be leveraged for determining genetic and process designs that optimize transduction-based biomanufacturing platforms and viral amplification processes. Viral infection and propagation for up to two viruses simultaneously can be simulated through the model, considering viruses in either the lytic or lysogenic stage, during batch, perfusion, or continuous operation. The model estimates the distribution of the viral genome(s) copy number in the cell population, which is an indicator of transduction efficiency and viral genome stability. The infection age distribution of the infected cells is also calculated, indicating how many cells are in an infection stage compatible with recombinant product expression or viral amplification. The model can also consider the presence of defective interfering particles in the system, which can severely compromise the productivity of biomanufacturing processes. Model benchmarking and validation are demonstrated for case studies of the baculovirus expression vector system and influenza A propagation in suspension cultures.

Raman laser intensity and sample clarification on biochemical monitoring over Zika-VLP upstream stages.

In the current biopharmaceutical scenario, constant bioprocess monitoring is crucial for the quality and integrity of final products. Thus, process analytical techniques, such as those based on Raman spectroscopy, have been used as multiparameter tracking methods in pharma bioprocesses, which can be combined with chemometric tools, like Partial Least Squares (PLS) and Artificial Neural Networks (ANN). In some cases, applying spectra pre-processing techniques before modeling can improve the accuracy of chemometric model fittings to observed values. One of the biological applications of these techniques could have as a target the virus-like particles (VLP), a vaccine production platform for viral diseases. A disease that has drawn attention in recent years is Zika, with large-scale production sometimes challenging without an appropriate monitoring approach. This work aimed to define global models for Zika VLP upstream production monitoring with Raman considering different laser intensities (200 mW and 495 mW), sample clarification (with or without cells), spectra pre-processing approaches, and PLS and ANN modeling techniques. Six experiments were performed in a benchtop bioreactor to collect the Raman spectral and biochemical datasets for modeling calibration. The best models generated presented a mean absolute error and mean relative error respectively of 3.46 × 105 cell/mL and 35 % for viable cell density (Xv); 4.1 % and 5 % for cell viability (CV); 0.245 g/L and 3 % for glucose (Glc); 0.006 g/L and 18 % for lactate (Lac); 0.115 g/L and 26 % for glutamine (Gln); 0.132 g/L and 18 % for glutamate (Glu); 0.0029 g/L and 3 % for ammonium (NH4+); and 0.0103 g/L and 2 % for potassium (K+). Sample without conditioning (with cells) improved the models' adequacy, except for Glutamine. ANN better predicted CV, Gln, Glu, and K+, while Xv, Glc, Lac, and NH4+ presented no statistical difference between the chemometric tools. For most of the assessed experimental parameters, there was no statistical need for spectra pre-filtering, for which the models based on the raw spectra were selected as the best ones. Laser intensity impacts quality model predictions in some parameters, Xv, Gln, and K+ had a better performance with 200 mW of intensity (for PLS, ANN, and ANN, respectively), for CV the 495 mW laser intensity was better (for PLS), and for the other biochemical variables, the use of 200 or 495 mW did not impact model fitting adequacy.

Safety pharmacology of human endogenous retrovirus-enveloped baculoviral DNA vaccines against SARS-CoV-2 in Sprague-Dawley rats and beagle dogs.

The coronavirus disease 2019 (COVID-19) emerged as a major global health crisis, posing significant health, economic, and social challenges. Vaccine development has been a crucial response to the severe-acute-respiratory-syndrome-related coronavirus-2 pandemic owing to the critical role of immunization in controlling infectious diseases, leading to the expedited development of several effective vaccines. Although mRNA platform-based COVID-19 vaccines authorized under emergency-use authorization have been administered globally, concerns regarding the vaccines have increased owing to the occurrence of various side effects. The present study aimed to evaluate the safety of a non-replicating recombinant baculovirus expressing the human endogenous retrovirus envelope gene (AcHERV) vaccine encoding SARS-CoV-2 antigens. Owing to the limited number of existing safety pharmacology studies on AcHERV as a viral vector vaccine, we conducted neurobehavior (Modified Irwin's Test), body temperature, and respiratory function studies in rats and cardiovascular system studies in male beagle dogs, which were administered the AcHERV-COVID-19 vaccine using telemetry. The safety assessment revealed no significant toxicological alterations. However, in rats, both sexes administered with the AcHERV-COVID-19 vaccine exhibited a temporary increase in body temperature, which normalized or showed signs of recovery. In conclusion, AcHERV-COVID-19 demonstrates a sufficient safety profile that supports its potential evaluation in future clinical trials.

Efficient and accurate BmNPV bacmid editing system by two-step golden gate assembly.

The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130 kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2 %. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.

The mechanical properties of chimeric silk are improved by expressing the full-length Trichonephila clavipes major ampullate spidroin gene in the silkworm Bombyx mori via recombinant AcMNPV.

Spider silk is a type of natural protein fiber with excellent toughness and tensile strength. The mechanical properties of chimeric silk have been improved by integrating the spider silk protein gene into the silkworm (Bombyx mori) genome, but this strategy requires a long time to produce genetically modified silkworms. In this study, to rapidly produce chimeric silkworms/spider silk with improved toughness and tensile strength, recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV), AcMNPV-FHP-MaSp-G, harboring a full-length Trichonephila clavipes major ampullate spidroin G (MaSp-G) gene driven by the silkworm fibroin heavy chain (Fib-H) promoter, was constructed, in which the signal peptide sequence of the MaSp-G gene was replaced by the signal peptide sequence of the Fib-H gene. Western blot and LC-MS/MS results showed that MaSp-G was successfully expressed in the posterior silk gland of silkworm larvae infected with AcMNPV-FHP-MaSp-G and secreted into the cocoon. Mechanical property tests revealed that the average maximum breaking stress and the average maximum elastic strain of chimeric silkworms/spider silk were 497.867 MPa and 14.824%, respectively, which were 36.53% and 23.55% greater than those of silk produced by normal silkworms. Fourier transform infrared (FTIR) spectroscopy revealed that the proportions of ß-sheets, α-helices, and ß-turns in the chimeric silk increased by 18.22%, 16.92%, and 18.72%, respectively. These results indicate that the mechanical properties of the chimeric silk produced by silkworms infected with AcMNPV-FHP-MaSp-G were significantly improved, which provides a new method for rapid production of chimeric silk in a genetically modified/genome-edited silkworm-independent manner.

Assessment of adeno-associated virus purity by capillary electrophoresis-based western.

A rigorous analytical assessment of recombinant adeno-associated virus (rAAV)-based drug products is critical for their successful development as clinical candidates. It is especially important to ascertain high purity while simultaneously ensuring low levels of impurities in the final drug product. One approach to evaluate the purity of rAAV drug products is to determine the relative stoichiometry of the three viral proteins (VPs) that comprise an rAAV capsid, and the levels of impurities in the final drug product. Here we present two capillary electrophoresis-western (CE-western) assays for quantifying (1) the relative stoichiometry of VP using the anti-AAV B1 antibody, and (2) residual levels of a baculovirus protein impurity, GP64, using the anti-GP64 antibody. In each assay, various purified samples from diverse AAV serotypes were analyzed to determine their VP ratio or GP64 levels. The ratio of VP3/VP1 in rAAV samples was correlated with biological activity, and the clearance of GP64 from the manufacturing process was demonstrated. The results obtained from both assays were further supported by liquid chromatography-mass spectrometry analyses. Overall, we report that CE-western is a high-throughput platform that utilizes low sample volumes for a rapid, sensitive, and robust assessment of the identity, composition, and purity of rAAV drug products.

Insect cell production of chimeric virus-like particles based on human immunodeficiency virus GAG proteins and yellow fever virus envelope protein.

The yellow fever virus (YFV) is a single stranded RNA virus belonging to the genus Orthoflavivirus that is capable of zoonotic transmissions that infect nonhuman and human primates. It is endemic in Brazil with recurrent epidemics of the disease, and it is transmitted through mosquitoes. The detection and immunization against YFV and other flaviviruses are fundamental for the management of the impacts of the disease in human environments. In an ongoing effort to develop new approaches for diagnostics and immunizations, we expressed VLPs displaying the yellow fever virus envelope protein (YFE) using recombinant baculovirus in insect cells. By co-expressing HIV-1 Pr55Gag protein (GAG) together with YFE we were able to generate chimeric VLPs containing a GAG core together with an envelope containing the YFE protein. The YFE and the chimeric GAG-YFE VLPs have potential as vaccine candidates and as reagents for serological assays in the detection of these viruses in human sera.

Viral and cellular determinants of polarized trafficking of viral envelope proteins from insect-specific and insect-vectored viruses in insect midgut and salivary gland cells.

Systemic viral infection of insects typically begins with the primary infection of midgut epithelial cells (enterocytes) and subsequent transit of the progeny virus in an apical-to-basal orientation into the hemocoel. For insect-vectored viruses, an oppositely oriented process (basal-to-apical transit) occurs upon secondary infection of salivary glands and is necessary for virus transmission to non-insect hosts. To examine this inversely oriented virus transit in these polarized tissues, we assessed the intracellular trafficking of two model viral envelope proteins (baculovirus GP64 and vesicular stomatitis virus G) in the midgut and salivary gland cells of the model insect, Drosophila melanogaster. Using fly lines that inducibly express either GP64 or VSV G, we found that each protein, expressed alone, was trafficked basally in midgut enterocytes. In salivary gland cells, VSV G was trafficked apically in most but not all cells, whereas GP64 was consistently trafficked basally. We demonstrated that a YxxØ motif present in both proteins was critical for basal trafficking in midgut enterocytes but dispensable for trafficking in salivary gland cells. Using RNAi, we found that clathrin adaptor protein complexes AP-1 and AP-3, as well as seven Rab GTPases, were involved in polarized VSV G trafficking in midgut enterocytes. Our results indicate that these viral envelope proteins encode the requisite information and require no other viral factors for appropriately polarized trafficking. In addition, they exploit tissue-specific differences in protein trafficking pathways to facilitate virus egress in the appropriate orientation for establishing systemic infections and vectoring infection to other hosts. IMPORTANCE: Viruses that use insects as hosts must navigate specific routes through different insect tissues to complete their life cycles. The routes may differ substantially depending on the life cycle of the virus. Both insect pathogenic viruses and insect-vectored viruses must navigate through the polarized cells of the midgut epithelium to establish a systemic infection. In addition, insect-vectored viruses must also navigate through the polarized salivary gland epithelium for transmission. Thus, insect-vectored viruses appear to traffic in opposite directions in these two tissues. In this study, we asked whether two viral envelope proteins (VSV G and baculovirus GP64) alone encode the signals necessary for the polarized trafficking associated with their respective life cycles. Using Drosophila as a model to examine tissue-specific polarized trafficking of these viral envelope proteins, we identified one of the virus-encoded signals and several host proteins associated with regulating the polarized trafficking in the midgut epithelium.

New Cydia pomonella granulovirus strain with high entomopathogenic activity.

We report a genome of CpGV from the bioresource collection of the Federal Research Center of Biological Plant Protection "State Collection of Entomoacariphages and Microorganisms." Its sequence is 123,862 bp. The genome under study demonstrates a degree of similarity of more than 99% with reference NC_002816 from the NCBI RefSeq database.

Expression of F1L, a vaccinia virus H3L transmembrane protein analogue of orf virus, and its successful purification as a diagnostic antigen.

Orf or contagious ecthyma is a highly contagious, zoonotic, and economically important global viral disease of small ruminants and is endemic in India. Vaccination of susceptible goats/sheep along with suitable recombinant protein-based serological assay will be useful in the control of the infection. In this study, the full-length and truncated versions of F1L encoding gene (ORF 059) of orf virus were cloned into pFasBac HT A vector, transformed in DH10Bac cells, and expressed in insect cells. The full-length and truncated recombinant F1L proteins were expressed as a 6 × histidine-tagged fusion protein for ease of purification by Ni-NTA affinity chromatography under denaturing conditions. A protein with ~ 40 kDa and ~ 35 kDa for full-length and truncated F1L protein, respectively, were expressed and confirmed by SDS-PAGE and western blot. The protein reactivity evaluated by western blot analysis and indirect ELISA using ORFV hyperimmune serum was also found to be reactive. The results of the present study showed that the purified recombinant F1L protein can be used as a diagnostic antigen in sero-surveillance of ORFV infection in small ruminants. To the best of authors' knowledge, this is the first report on the expression of ORFV F1L in insect cells using a baculovirus vector and its successful purification to use as the potential diagnostic antigen in ELISA.

Baculovirus-mediated endostatin and angiostatin activation of autophagy through the AMPK/AKT/mTOR pathway inhibits angiogenesis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly vascularized carcinoma, and targeting its neovascularization represents an effective therapeutic approach. Our previous study demonstrated that the baculovirus-mediated endostatin and angiostatin fusion protein (BDS-hEA) effectively inhibits the angiogenesis of vascular endothelial cells and the growth of HCC tumors. However, the mechanism underlying its anti-angiogenic effect remains unclear. Increasing evidence suggests that autophagy has a significant impact on the function of vascular endothelial cells and response to cancer therapy. Hence, the objective of this research was to investigate the correlation between BDS-hEA-induced angiogenesis inhibition and autophagy, along with potential regulatory mechanisms. Our results demonstrated that BDS-hEA induced autophagy in EA.hy926 cells, as evidenced by the increasing number of autophagosomes and reactive oxygen species, accompanied by an upregulation of Beclin-1, LC3-II/LC3-I, and p62 protein expression. Suppression of autophagy using 3-methyladenine attenuated the functions of BDS-hEA-induced EA.hy926 cells, including the viability, proliferation, invasion, migration, and angiogenesis. Moreover, BDS-hEA induced autophagy by downregulating the expression of CD31, VEGF, and VEGFR2, as well as phosphorylated protein kinase B (p-AKT) and phosphorylated mammalian target of rapamycin (p-mTOR), while concurrently upregulating phosphorylated AMP-activated protein kinase (p-AMPK). The in vivo results further indicated that inhibition of autophagy by chloroquine significantly impeded the ability of BDS-hEA to suppress HCC tumor growth in mice. Mechanistically, BDS-hEA prominently facilitated autophagic apoptosis in tumor tissues and decreased the levels of ki67, CD31, VEGF, MMP-9, p-AKT, and p-mTOR while simultaneously enhancing the p-AMPK expression. In conclusion, our findings suggest that BDS-hEA induces autophagy as a cytotoxic response by modulating the AMPK/AKT/mTOR signaling pathway, thereby exerting anti-angiogenic effects against HCC.

The uncleaved signal peptide of Bombyx mori nucleopolyhedrovirus GP64 preferentially activates BmSpz7 expression, contributing to protein secretion.

Bombyx mori nucleopolyhedrovirus (BmNPV) significantly impacts silkworm sericulture, causing substantial economic losses. The GP64 protein, a primary envelope protein of BmNPV budded virus (BV), retains its signal peptide (SP) in the mature form, crucial for its translocation to the plasma membrane (PM) and viral infectivity. This study investigates the role of the uncleaved SP of GP64 in activating the expression of BmSpz7, a novel Spätzle family member identified through RNA-seq analysis. We cloned and characterized BmSpz7, demonstrating its upregulated expression in BmN cells and silkworm larvae infected with BmNPV containing GP64 with an uncleaved SP. Additionally, transient expression of GP64's SP significantly enhanced BmSpz7 expression and protein secretion. These findings suggest that the uncleaved SP of GP64 plays a pivotal role in activating BmSpz7, providing new insights into the molecular interactions between BmNPV and its host, and revealing potential targets for antiviral strategies in sericulture.

Development of a Recombinant Protein-Based Immunoassay for Detection of Antibodies Against Karolinska Institute and Washington University Polyomaviruses.

To develop polyomavirus VP1 recombinant protein-based immunoassay, the expression of two polyomavirus (Karolinska Institute Polyomavirus; KIPyV, and Washington University Polyomavirus; WUPyV) VP1s in insect cells was investigated using an improved baculovirus system (BacMagic). The reliability of the purified VP1 to serve as antigens in serological tests was confirmed by the establishment of an enzyme-linked immunosorbent assay (ELISA). Two panels of serum samples were used, with Panel I comprising 60 sera (20 KIPyV-positive, 20 WUPyV-positive, and 20 negative) and Panel II consisting of 134 sera with unknown status. The seroprevalence of KIPyV and WUPyV in the study population was determined to be 62% and 50%, respectively. Antibody-negative sera exhibited low reactivities in both ELISAs, whereas antibody-positive sera displayed high reactivity with median optical density values of 1.37 and 1.47 in the KIPyV and WUPyV ELISAs, respectively. The differences in seroreactivities between antibody positive and negative for each virus were statistically significant (p < 0.0001; with 95% confidence interval). The study suggests that seroconversion for KIPyV and WUPyV occurs in childhood, with KIPyV seropositivity reaching 70% and WUPyV seropositivity reaching 60% after the age of 5 years. Adult seroprevalence for polyomaviruses was high, with more than 64% and 51% of the adult population being seropositive for KIPyV and WUPyV, respectively. The constant prevalence of KIPyV and WUPyV antibody in the age groups suggested that this antibody persists for life. The fact that antibody titers were generally stable over time revealed a persistent infection of polyomaviruses in the human population. The insect cell-derived recombinant VP1-based ELISA has been demonstrated to be valuable as a serological assay, offering a valid, reliable, fast, nonlaborious, and economical procedure.

Baculovirus expression and purification of virion core and envelope proteins of goatpox virus to evaluate their diagnostic potential.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of ~30 kDa, ~31 kDa, and ~32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89% and 94% for goats and 98% and 91%, for sheep, respectively. No cross-reactivity was observed with other related viruses. The recombinant-A33R-based indirect ELISA developed in the present study shows that it has potential for the detection of antibodies in GTPV and SPPV infected/vaccinated animals.

Effectively Evaluating a Novel Consensus Subunit Vaccine Candidate to Prevent the H9N2 Avian Influenza Virus.

The enormous effects of avian influenza on poultry production and the possible health risks to humans have drawn much attention to this disease. The H9N2 subtype of avian influenza virus is widely prevalent among poultry, posing a direct threat to humans through infection or by contributing internal genes to various zoonotic strains of avian influenza. Despite the widespread use of H9N2 subtype vaccines, outbreaks of the virus persist due to the rapid antigenic drift and shifts in the influenza virus. As a result, it is critical to develop a broader spectrum of H9N2 subtype avian influenza vaccines and evaluate their effectiveness. In this study, a recombinant baculovirus expressing the broad-spectrum HA protein was obtained via bioinformatics analysis and a baculovirus expression system (BES). This recombinant hemagglutinin (HA) protein displayed cross-reactivity to positive sera against several subbranch H9 subtype AIVs. An adjuvant and purified HA protein were then used to create an rHA vaccine candidate. Evaluation of the vaccine demonstrated that subcutaneous immunization of the neck with the rHA vaccine candidate stimulated a robust immune response, providing complete clinical protection against various H9N2 virus challenges. Additionally, virus shedding was more effectively inhibited by rHA than by the commercial vaccine. Thus, our findings illustrate the efficacy of the rHA vaccine candidate in shielding chickens against the H9N2 virus challenge, underscoring its potential as an alternative to conventional vaccines.

Development of Fusion-Based Assay as a Drug Screening Platform for Nipah Virus Utilizing Baculovirus Expression Vector System.

Nipah virus (NiV) is known to be a highly pathogenic zoonotic virus, which is included in the World Health Organization Research & Development Blueprint list of priority diseases with up to 70% mortality rate. Due to its high pathogenicity and outbreak potency, a therapeutic countermeasure against NiV is urgently needed. As NiV needs to be handled within a Biological Safety Level (BSL) 4 facility, we had developed a safe drug screening platform utilizing a baculovirus expression vector system (BEVS) based on a NiV-induced syncytium formation that could be handled within a BSL-1 facility. To reconstruct the NiV-induced syncytium formation in BEVS, two baculoviruses were generated to express recombinant proteins that are responsible for inducing the syncytium formation, including one baculovirus exhibiting co-expressed NiV fusion protein (NiV-F) and NiV attachment glycoprotein (NiV-G) and another exhibiting human EphrinB2 protein. Interestingly, syncytium formation was observed in infected insect cells when the medium was modified to have a lower pH level and supplemented with cholesterol. Fusion inhibitory properties of several compounds, such as phytochemicals and a polysulfonated naphthylamine compound, were evaluated using this platform. Among these compounds, suramin showed the highest fusion inhibitory activity against NiV-induced syncytium in the baculovirus expression system. Moreover, our in silico results provide a molecular-level glimpse of suramin's interaction with NiV-G's central hole and EphrinB2's G-H loop, which could be the possible reason for its fusion inhibitory activity.

A σC-protein-based indirect enzyme-linked immunosorbent assay for clinical detection of antiavian reovirus antibodies.

Avian reovirus (ARV) is the causative agent of avian viral arthritis and causes significant economic losses to the global poultry industry. For clinical diagnosis, detecting ARV-specific antibodies is crucial. We successfully expressed the ARV-σC protein in insect cells using the baculovirus expression vector system, achieving an expression level of approximately 200 mg/L. We developed an indirect enzyme-linked immunosorbent assay (iELISA) using the ARV-σC protein as a coating antigen to detect antibodies against it. The inter-batch and intrabatch coefficients of iELISA variation were less than 10%. Its sensitivity (1:12,800 diluted in serum) was 4 times higher than that of the indirect immunofluorescence assay (IFA; 1:3200 diluted in serum), and it showed no cross-reactivity with antibodies against other common avian viruses (such as Infectious bursal disease virus, Newcastle disease virus). The practicality of the iELISA was further evaluated using clinical samples. 300 clinical sera from chickens vaccinated with the ARV attenuated vaccine and 20 SPF sera were tested using both the iELISA and the IFA, demonstrating a 100% conformity rate. In conclusion, these results suggest that the iELISA developed in this study is a rapid, sensitive, and specific method that could serve as an effective diagnostic tool for monitoring and controlling avian viral arthritis.

Characterisation of extracellular vesicles in baculovirus infection of Spodoptera frugiperda cells.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is an enveloped DNA virus of the Baculoviridae family. This baculovirus is widely exploited for the biological control of insect pest species and as an expression platform to produce recombinant proteins in insect cells. Extracellular vesicles (EVs) are secreted by all cells and are involved in key roles in many biological processes through their cargo consisting of proteins, RNA or DNA. In viral infections, EVs have been found to transfer both viral and cellular cargo that can elicit either a pro- or antiviral response in recipient cells. Here, small EVs (sEVs) released by Spodoptera frugiperda (Sf) insect cells were characterised for the first time. Using S. frugiperda (SfC1B5) cells stably expressing the baculovirus gp64, the viral envelope protein GP64 was shown to be incorporated into sEVs. Sf9 cells were also transfected with a bacmid AcMNPV genome lacking p6.9 (AcΔP6.9) to prevent budded virus production. The protein content of sEVs from both mock- and AcΔP6.9-transfected cells were analysed by mass spectrometry. In addition to GP64, viral proteins Ac-F, ME-53 and viral ubiquitin were identified, as well as many host proteins including TSG101-which may be useful as a protein marker for sEVs.