Expression of recombinant Florida clade 2 hemagglutinin in baculovirus expression system: A step for subunit vaccine development against H3N8 equine influenza virus.

Background: Equine influenza (EI) is a transmissible viral respiratory sickness of the Equidae family. Two viruses, H7N7 and H3N8 caused EI; however, H7N7 has not been detected for decades. H3N8 has circulated and bifurcated into Eurasian and American lineages. The latter subsequently diversified into Kentucky, South America, and Florida sub-lineages. Florida clade 1 (FC1) and Florida clade 2 (FC2) strains are the only circulating EI viruses (EIVs) in the meantime. Immunization is considered the major means for the prevention and control of EI infection. Using disparate technologies and platforms, several vaccines have been developed and commercialized. According to the recommendations of the World Organization for Animal Health (WOAH), all commercial vaccines shall comprise representatives of both FC1 and FC2 strains. Unfortunately, most of the commercially available vaccines were not updated to incorporate a representative of FC2 strains. Aim: The purpose of this research was to develop a new EI vaccine candidate that incorporates the hemagglutinin (HA) antigen from the currently circulating FC2. Methods: In this study, we report the expression of the full-length recombinant HA gene of FC2 in the baculovirus expression system. Results: The HA recombinant protein has been proven to maintain its biological characteristics by hemadsorption (HAD) and hemagglutination tests. Moreover, using a reference-specific serum, the specificity of the HA has been confirmed through the implementation of immunoperoxidase and western immunoblotting assays. Conclusion: In conclusion, we report the expression of specific biologically active recombinant HA of FC2, which would act as a foundation for the generation of an updated EI subunit or virus vector vaccine candidates.

Regulatory Mechanisms, Protein Expression and Biological Activity of Photolyase Gene from Spodoptera littoralis Granulovirus Genome.

One of the most important factor that affects the efficient using of baculoviruses as a biopesticide is their sensitivity to UV irradiation. In this study, a photolyase gene (phr) of 1.4 kbp DNA fragment was cloned and characterized from Spodoptera littoralis granulovirus, an Egyptian isolate (SpliGV-EG1). A sequence of 466 amino acid were deduced when the gene was completely sequenced with a predicted molecular mass of ~ 55 kDa. Transcriptional regulation analyses revealed that phr transcripts were detected early at 6-h post-infection (hpi) and remained detectable until 72 hpi, suggesting their transcriptional regulation from a putative early promoter motif. An approximately ~ 55 kDa protein fragment was expressed from phr-induced bacterial culture and detected by SDS-PAGE and western blotting. In addition, direct exposure to UV irradiation resulted in a twofold decrease in SpliGV-EG1 occlusion bodies activation compared with Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) occlusion bodies which decreased with about 129-fold after exposure to UV irradiation based on median lethal concentration value (LC50). The obtained results suggested that the presence of photolyase gene possibly alters the inactivation of SpliGV-EG1-occluded bodies by UV irradiation. These results support the role and application of the photolyase protein to improve the damaged DNA repair mechanism as well as resistance of SpliGV to UV light inactivation.

A gold nanoparticles-based lateral flow assay utilizing baculovirus expressed recombinant nucleocapsid and receptor binding domain proteins for serodetection of IgG and IgM against SARS-CoV-2.

Serological assays for SARS-CoV-2 are being utilized at an exponential rate for surveillance programs. This enterprise was designed to develop and validate a qualitative immunochromatographic test, via the Lateral Flow Assay (LFA), for detection of immunoglobulins M and G (IgM and IgG) against both nucleocapsid (N) and the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Both targeted proteins were cloned and expressed in baculovirus expression system utilizing insect cells Sf9. The recombinant RBD and N proteins were purified and conjugated with gold nanoparticles (AuNPs) to set up the coating antigens pad. Both anti-human IgG and IgM were dispensed on nitrocellulose membrane to capture human antibodies in serum samples. A home-made dispensing system was developed to draw identical test and control lines. The validity of the developed LFA was verified by testing serum samples from 103 convalescent COVID-19 patients who were PCR positive for SARS-CoV-2 along with 28 control serum samples. The developed strips showed distinctive bands for IgM and IgG of both proteins (RBD and N) in positive samples. The sensitivity of RBD-based LFA was 70.9% and 39.8% for IgG and IgM, respectively, with a specificity of 100% for both. The N-based LFA exhibited a sensitivity of 73.8% and 35.9% for IgG and IgM, respectively, while its specificity was 75% and 100% for IgG and IgM, respectively. Our developed LFA could afford a tool for surveillance programs in low-resource countries. Moreover, it might be functional for rapid and inexpensive monitoring of the anti-SARS-CoV-2 antibodies in the sera of vaccinated individuals.

Nano-insecticides against the black cutworm Agrotis ipsilon (Lepidoptera: Noctuidae): Toxicity, development, enzyme activity, and DNA mutagenicity.

Frequent applications of synthetic insecticides might cause environmental pollution due to the high residue. In addition, increasing insecticide resistance in many insect pests requires novel pest control methods. Nanotechnology could be a promising field of modern agriculture, and is receiving considerable attention in the development of novel nano-agrochemicals, such as nanoinsectticides and nanofertilizers. This study assessed the effects of the lethal and sublethal concentrations of chlorantraniliprole, thiocyclam, and their nano-forms on the development, reproductive activity, oxidative stress enzyme activity, and DNA changes in the black cutworm, Agrotis ipsilon, at the molecular level. The results revealed that A. ipsilon larvae were more susceptible to the nano-forms than the regular forms of both nano chlorine and sulfur within the chlorantraniliprole and thiocyclam insecticides, respectively, with higher toxicities than the regular forms (ca. 3.86, and ca.2.06-fold, respectively). Significant differences in biological parameters, including developmental time and reproductive activity (fecundity and hatchability percent) were also observed. Correspondingly, increases in oxidative stress enzyme activities were observed, as were mutagenic effects on the genomic DNA of A. ipsilon after application of the LC50 of the nano-forms of both insecticides compared to the control. These promising results could represent a crucial step toward developing efficient nanoinsecticides for sustainable control of A. ipsilon.

Removal of 3C protease from the 3ABC improves expression, solubility, and purification of the recombinant 3AB of foot-and-mouth disease virus.

During an ongoing outbreak of Foot-and-Mouth Disease Virus (FMDV), it is crucial to distinguish naturally infected from vaccinated seropositive animals. This would support clinical assessment and punctual vigilance. Assays based on 3ABC non-structural protein as an antigen are reliable for this intention. However, the insolubility and degradation of recombinant 3ABC during expression and purification are serious challenges. In this study, alternatively to expressing the recombinant 3ABC (r3ABC), we expressed the 3AB coding sequence (~672 bp) as a recombinant protein (r3AB) with a molecular mass of ~26 KDa. Analytical data from three-dimensional structure, hydrophilicity, and antigenic properties for 3ABC and 3AB exhibited the 3C protein as a hydrophobic, while 3AB as a hydrophilic and highly antigenic protein. The expressed r3AB was recovered as a completely soluble matter after merely native purification, unlike the full expressed r3ABC. Immunoreactivity of r3AB to anti-FMDV antibody in infected sera with different FMDV serotypes was confirmed by the western blot and indirect ELISA. Besides, the authentic antigenicity of purified r3AB was demonstrated through its ability to induce specific seroconversion in mice. Summarily, the removal of 3C: has influenced neither 3D structure nor antigenic properties of the purified r3AB, overcame insolubility and degradation of the r3ABC, and generated a potential superior antigen (r3AB) for herd screening of animals to any FMDV serotype.

Expression of 1B capsid protein of Foot-and-mouth disease virus (FMDV) using baculovirus expression system and its validation in detecting SAT 2- specific antisera.

Foot-and-mouth disease virus (FMDV) is one of the most devastating animal viruses that affect livestock worldwide. The 1B capsid of FMDV has been widely used to detect and confirm the infection. In the present study, the sequence coding for 1B subunit of FMDV capsid was expressed in insect cells using the baculovirus expression system under the polyhedrin (polh) promoter. The expression of 1B capsid protein was validated in the culture filtrate of insect cells using SDS-PAGE and western blotting. The culture filtrate containing recombinant 1B capsid (r1B) was used as a coated antigen in an indirect enzyme-linked immunosorbent assay (ELISA). The antigenicity and specificity of r1B against SAT 2 serotype-specific antibodies were assessed. Our results revealed that a protein concentration as low as 25 ng could detect SAT 2-specific antibodies in ELISA. The results highlight the application of insect cells developed r1B protein in the detection of FMDV. Further studies are required to determine the ability of r1B to detect other FMDV serotypes.

Characterization, cloning, expression and bioassay of vip3 gene isolated from an Egyptian Bacillus thuringiensis against whiteflies.

Throughout the vegetative life of Bacillus thuringiensis, vegetative insecticidal proteins (Vip) are produced and secreted. In the present study, the vip3 gene isolated from Bacillus thuringiensis, an Egyptian isolate, was successfully amplified (2.4 kbp) and expressed using bacterial expression system. The molecular mass of the expressed protein was verified using SDS-PAGE and western blot analysis. Whiteflies were also screened for susceptibility to the expressed Vip3 protein (LC50). In addition, ST50 was determined to assess the kill speed of the expressed Vip3 protein against whiteflies compared to the whole vegetative proteins. The results showed that the potency of whole B. thuringiensis vegetative proteins against whiteflies was slightly higher than the expressed Vip3 protein with 4.7-fold based on LC50 value. However, the ST50 parameter showed no significant difference between both the B. thuringiensis vegetative proteins and the expressed Vip3 alone. The results showed that the vip3 gene was successfully expressed in an active form which showed high susceptibility to whiteflies based on the virulence parameters LC50 and ST50. To our knowledge, this study showed for the first time the high toxicity of the expressed Vip3 proteins of B. thuringiensis toward whiteflies as a hopeful and promising bio-control agent.

Expression of tomato yellow leaf curl virus coat protein using baculovirus expression system and evaluation of its utility as a viral antigen.

DNA encoding the coat protein (CP) of an Egyptian isolate of tomato yellow leaf curl virus (TYLCV) was inserted into the genome of Autographa californica nucleopolyhedrovirus (AcNPV) under the control of polyhedrin promoter. The generated recombinant baculovirus construct harboring the coat protein gene was characterized using PCR analysis. The recombinant coat protein expressed in infected insect cells was used as a coating antigen in an indirect Enzyme-linked immunosorbent assay (ELISA) and dot blot to test its utility for the detection of antibody generated against TYLCV virus particles. The results of ELISA and dot blot showed that the TYLCV-antibodies reacted positively with extracts of infected cells using the recombinant virus as a coating antigen with strong signals as well as the TYLCV infected tomato and beat plant extracts as positive samples. Scanning electron microscope examination showed that the expressed TYLCV coat protein was self-assembled into virus-like particles (VLPs) similar in size and morphology to TYLCV virus particles. These results concluded that, the expressed coat protein of TYLCV using baculovirus vector system is a reliable candidate for generation of anti-CP antibody for inexpensive detection of TYLCV-infected plants using indirect CP-ELISA or dot blot with high specificity.

Genome-wide In Silico Analysis, Characterization and Identification of Microsatellites in Spodoptera littoralis Multiple nucleopolyhedrovirus (SpliMNPV).

In this study, we undertook a survey to analyze the distribution and frequency of microsatellites or Simple Sequence Repeats (SSRs) in Spodoptera littoralis multiple nucleopolyhedrovirus (SpliMNPV) genome (isolate AN-1956). Out of the 55 microsatellite motifs, identified in the SpliMNPV-AN1956 genome using in silico analysis (inclusive of mono-, di-, tri- and hexa-nucleotide repeats), 39 were found to be distributed within coding regions (cSSRs), whereas 16 were observed to lie within intergenic or noncoding regions. Among the 39 motifs located in coding regions, 21 were located in annotated functional genes whilst 18 were identified in unknown functional genes (hypothetical proteins). Among the identified motifs, trinucleotide (80%) repeats were found to be the most abundant followed by dinucleotide (13%), mononucleotide (5%) and hexanucleotide (2%) repeats. The 39 motifs located within coding regions were further validated in vitro by using PCR analysis, while the 21 motifs located within known functional genes (15 genes) were characterized using nucleotide sequencing. A comparison of the sequence analysis data of the 21 sequenced cSSRs with the published sequences is presented. Finally, the developed SSR markers of the 39 motifs were further mapped/localized onto the SpliMNPV-AN1956 genome. In conclusion, the SSR markers specific to SpliMNPV, developed in this study, could be a useful tool for the identification of isolates and analysis of genetic diversity and viral evolutionary status.

Possible functional co-operation of palindromes hr3 and hr4 in the genome of Cydia pomonella granulovirus affects viral replication capacity.

After previous studies had shown that natural transposon insertion between the two homologous regions hr3 and hr4 of the genome of the Mexican (M) strain of Cydia pomonella granulovirus (CpGV-M) resulted in a loss of viral competitiveness, the function of these homologous regions was investigated. A CpGV-based bacmid (CpBAC) was constructed and mutants with deleted hr3 and hr4 palindromes (CpBAChr3/hr4KO) and a construct (CpBAChr3-kan-hr4) with physically separated hr3 and hr4 repeats were generated to investigate their involvement in in vivo replication. Based on median lethal concentration (LC50) and median survival time (ST50) of the mutant viruses vCpBAChr3/hr4KO and vCpBAChr3-kan-hr4 it was found that the infectivity of both mutants for codling moth Cydia pomonella L. (Lep.: Tortricidae) larvae was not influenced compared with the parental virus vCpBAC. Co-infection experiments with vCpBAChr3-kan-hr4 and vCpBAC using different virus ratios revealed that vCpBAChr3-kan-hr4 was efficiently out-competed by vCpBAC during in vivo replication. These findings suggested that the separation of hr3 and hr4 resulted in a replication disadvantage of the mutant similar to the observation made in previous co-infection experiments using the transposon-carrying mutant CpGV-MCp5 and WT CpGV-M. It was concluded that the palindromes hr3 and hr4 may play a non-essential but co-functional role in the replication of CpGV-M.