Detection and molecular characterization of leptospira spp. from environmental samples in Kelantan

Leptospirosis is an important worldwide zoonotic disease caused by pathogenic leptospires. In Malaysia, Leptospira spp. have been detected in humans, livestock, environmental samples and rodents. Saprophytic species was usually associated with the environment. However, a novel pathogenic species, Leptospira kmetyi has been isolated from the soil in Malaysia. Therefore, the aim of this study is to isolate Leptospira spp. from the soil and water in selected environment, to detect the pathogenic isolates and to determine their genetic relationship. This is a cross-sectional descriptive study. Soil and water samples were collected from well known markets and recreational areas in Kelantan. All samples were filtered and inoculated into modified Ellinghausen and McCullough medium supplemented with additional 5-fluorouracil. The cultures were incubated at 30°C for 30 days and examined under dark field microscope. Microscopic Agglutination Test (MAT) was performed to determine the serovar of the positive cultures. Positive cultures were then subjected to PCR using G1/G2, B64-I/B64-II and Sapro1/Sapro2 primers. The presence of virulence gene lipL32 was also determined. Partial sequences of 16S rRNA gene of the isolates were obtained for molecular identification of the isolates. Phylogenetic analysis was carried out to determine the genetic relatedness among isolates. A total of 144 samples comprised of water (market, n=36; recreational area, n=36) and soil (market, n=36; recreational area, n=36) were collected. Based on darkfield microscopic observations, 10% water and 36% soil cultures were positive for Leptospira spp. All isolates were negative for the hyperimmune sera tested in MAT. A total of 18 out of 33 cultures gave positive PCR assay results using G1/G2 and B64-I/B64-II primers. LipL32 gene was not detected in all of the isolates. 16S rRNA sequencing results showed that 31 out of 33 isolates were identified as Leptospira spp. There were one pathogenic species, Leptospira alstonii and eight intermediate species, L. wolffii (n=7), and L. licerasiae (n=1). Twenty two isolates were identified as nonpathogenic species, L. meyeri. The remaining two isolates were identified as species from other closely related genus, Leptonema illini. Based on phylogenetic analysis, the leptopsiral isolates were clearly separated to form three major clades namely pathogenic, intermediate and nonpathogenic clades. In conclusion, only one pathogenic leptospires, L. alstonii was isolated from environment in selected areas in Malaysia. The remaining isolates were intermediate and saprophytic groups. All isolates were found not to contain one of the highly conserve putative leptospiral virulence gene LipL32

Optimization of Gene Design, PCR Assembly and Site-Directed Mutagenesis for the Synthesis of Beauveria Bassiana Protease Gene

Synthetic gene construction is one of the components of synthetic biology. It can be used for various purposes such as to optimize gene expression. In this study, we proposed six predetermined criteria for designing oligos for the synthesis of the Beauveria bassiana protease gene. These criteria were set up to optimize the cost and to accommodate the oligos assembly. A total of 44 overlapping oligos were designed and synthesized 0.5 µM of oligos mixture was used in assembly PCR together with high fidelity DNA polymerase to produce 1.1 kbp fragment. The gene was visualized by agarose gel electrophoresis before subcloned into pCR™2.1-TOPO. The sequence of the gene was verified by DNA sequencing. Site-directed mutagenesis was performed to repair errors resulted from the gene synthesis. A sharp and distinguished band of the expected size of the protease gene was observed in agarose gel electrophoresis. Errors in the sequence which was detected by DNA sequencing were successfully repaired using our simplified site-directed mutagenesis protocol. The result indicated long DNA sequences (>1 kbp) can be synthesized with less error by using our method. Additionally, this method was easy to perform because it would require minimum optimization to synthesize other genes by following our guidelines

Current Status of Genetically Modified Baculovirus Insecticide for Pest Control

Baculovirus is an insect specific virus which is harmless to human. This feature has made it suitable to be applied as biopesticide. It has been used to control the insect pest particularly in agriculture sector for half a century and several success stories have been shared. Nevertheless, this insecticide still cannot compete with the synthetic pesticides owing to its slow killing speed and deficiency of compatible hosts. Genetically engineered baculovirus has improved pathogenicity against insect by incorporating foreign genes. These foreign genes encode neurotoxin, hormones, enzymes, and antisense DNA. Expression of these genes can enhance the insecticidal activities of the recombinant baculovirus. Nonetheless, the genetically modified baculovirus still has not been commercialised until today. This might be associated with the concern about the release of the genetically modified organism (GMO) into the environment as the environmental impact of the genetically modified virus is not well understood. Furthermore, it has been found to have effect on certain parasitoid. In conclusion, genetic modifications of the baculovirus have successfully improved its insecticidal activities but insufficient knowledge about its safety has limited its use in the field.</jats:p

Application of Baculovirus Expression Vector system (BEV) for COVID-19 diagnostics and therapeutics: a review

Abstract Background The baculovirus expression vector system has been developed for expressing a wide range of proteins, including enzymes, glycoproteins, recombinant viruses, and vaccines. The availability of the SARS-CoV-2 genome sequence has enabled the synthesis of SARS-CoV2 proteins in a baculovirus-insect cell platform for various applications. Main body of the abstract The most cloned SARS-CoV-2 protein is the spike protein, which plays a critical role in SARS-CoV-2 infection. It is available in its whole length or as subunits like S1 or the receptor-binding domain (RBD). Non-structural proteins (Nsps), another recombinant SARS-CoV-2 protein generated by the baculovirus expression vector system (BEV), are used in the identification of new medications or the repurposing of existing therapies for the treatment of COVID-19. Non-SARS-CoV-2 proteins generated by BEV for SARS-CoV-2 diagnosis or treatment include moloney murine leukemia virus reverse transcriptase (MMLVRT), angiotensin converting enzyme 2 (ACE2), therapeutic proteins, and recombinant antibodies. The recombinant proteins were modified to boost the yield or to stabilize the protein. Conclusion This review covers the wide application of the recombinant protein produced using the baculovirus expression technology for COVID-19 research. A lot of improvements have been made to produce functional proteins with high yields. However, there is still room for improvement and there are parts of this field of research that have not been investigated yet. </jats:sec

Application of Baculovirus Expression Vector system (BEV) for COVID-19 diagnostics and therapeutics: a review

Abstract Background The baculovirus expression vector system has been developed for expressing a wide range of proteins, including enzymes, glycoproteins, recombinant viruses, and vaccines. The availability of the SARS-CoV-2 genome sequence has enabled the synthesis of SARS-CoV2 proteins in a baculovirus-insect cell platform for various applications. Main body of the abstract The most cloned SARS-CoV-2 protein is the spike protein, which plays a critical role in SARS-CoV-2 infection. It is available in its whole length or as subunits like S1 or the receptor-binding domain (RBD). Non-structural proteins (Nsps), another recombinant SARS-CoV-2 protein generated by the baculovirus expression vector system (BEV), are used in the identification of new medications or the repurposing of existing therapies for the treatment of COVID-19. Non-SARS-CoV-2 proteins generated by BEV for SARS-CoV-2 diagnosis or treatment include moloney murine leukemia virus reverse transcriptase (MMLVRT), angiotensin converting enzyme 2 (ACE2), therapeutic proteins, and recombinant antibodies. The recombinant proteins were modified to boost the yield or to stabilize the protein. Conclusion This review covers the wide application of the recombinant protein produced using the baculovirus expression technology for COVID-19 research. A lot of improvements have been made to produce functional proteins with high yields. However, there is still room for improvement and there are parts of this field of research that have not been investigated yet

Molecular Characterization of Leptospira spp. in Environmental Samples from North-Eastern Malaysia Revealed a Pathogenic Strain, Leptospira alstonii

The presence of pathogenic Leptospira spp. in the environment poses threats to human health. The aim of this study was to detect and characterize Leptospira spp. from environmental samples. A total of 144 samples comprised of 72 soil and 72 water samples were collected from markets and recreational areas in a north-eastern state in Malaysia. Samples were cultured on Ellinghausen and McCullough modified by Johnson and Harris media. Leptospires were positive in 22.9% (n=33) of the isolates. Based on partial sequences of 16S rRNA, a pathogenic leptospire, Leptospira alstonii (n=1/33), was identified in 3% of the isolates followed by intermediate leptospire (L. wolffii, n=1/33, and L. licerasiae, n=7/33) and nonpathogenic leptospire, L. meyeri (n=22/33) in 24.2% and 66.7%, respectively. This study demonstrates the presence of a clinically significant pathogenic L. alstonii in the environments which could pose health risks to the occupants and visitors

Additional file 1 of Application of Baculovirus Expression Vector system (BEV) for COVID-19 diagnostics and therapeutics: a review

Additional file 1: Table S1. The specific queries for PubMed, ScienceDirect, Scopus and Google Scholar databases