Molecular characterisation of rice tungro bacilliform virus isolated from Bario, Sarawak

Rice tungro disease is one of the most damaging and destructive diseases of rice in South and Southeast Asia. The disease is caused by the co-infection of two viruses, the Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). The symptoms and severity of the disease depend on these two viral agents, if rice is coinfected by both viruses, it will show the typical severe symptoms of yellow-orange leaf discoloration, plant stunting and reduced in yield. On the other hand, if rice is infected only with RTBV, it shows milder symptoms and in contrast, rice plants will show no symptoms if they are infected only with RTSV. The disease had been detected in Malaysia since the 1930s. However, the first incursion of the disease was only reported in Sarawak in 2012. Since the disease was not seen in the Sarawak until recently, very little information on local virus isolate is available. This study was conducted to obtain and record the nucleotide sequence of partial coat protein gene of two primary isolates of RTBV collected from Bario, Sarawak in 2012 and 2013. Methodology and results: Based on the phylogenetic analysis, the isolates cluster with the Southeast Asia group with sequence identity at nucleotide and amino acid level of 91.1 to 95.1% and 98.6 to 99.5% respectively. Conclusion, significance and impact of study: This study provide the first genetic information on RTBV isolates from Sarawak. This data is important for future reference of the virus variants and diversity for epidemiological and diagnosis purposes

Interaction of antigenic recombinant coat proteins against the affinity purified antibody of rice tungro viruses: A preliminary study

Rice tungro disease (RTD) is a major destructive rice viral disease. To date, the common practice of detecting RTD is still based on symptoms visualisation. This is because methods like polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) which are conducted in the lab are limited for many farmers. As paddy planting is more dynamic in the rural region, sending samples is inconvenient as facilities providing the services are located far from the farms. Therefore, an easy and rapid method that can be utilized at point-of-need is advantageous to help manage RTD. This study reports on the use of antigenic recombinant coat proteins of the tungro viruses; Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) to develop a rapid serological for the detection of RTD. The aim is to produce affinity purified antibody against the coat proteins (CPs) of the tungro viruses. In our previous study, we have successfully cloned the RTBV CP and RTSV CP3. Here we report the result of the reactivity of the IgG purified polyclonal antibodies of the RTBV CP and RTSV CP3 tested in Western Blot and indirect ELISA. The binding affinity of the antibody to the antigen was evaluated in these two assays. The result showed similar pattern in reactivity between the two IgG antibodies of the RTSV CP3 and RTBV CP sera to the corresponding recombinant coat proteins (rCPs) in the immunoblots. The indirect ELISA result shows that the RTSV CP3 protein reacted strongly to both IgG antibodies, however it was noticed that the binding affinity started to decrease at higher concentration of the antigenic protein. This differs with the reactivity of the RTBV CP protein as binding of the IgG antibodies for the protein gradually increased with protein concentration. In conclusion, the IgG purified polyclonal antibodies against the rCPs of RTSV and RTBV show potential binding ability to be used in a rapid serological assay. However, the sensitive immunoassay can be further developed by optimizing the conditions of the rCPs and the affinity purified antibodies. Keywords: Coat protein, rice tungro viruse

Partial characterization of bacteriocin-like compound (BLIS) produced by Burkholderia stagnalis strain K23/3 against Burkholderia pseudomallei

Aims: Burkholderia pseudomallei, the human pathogen that causes melioidosis, is intrinsically resistant towards a wide range of antibiotics and there have been reports of acquired resistance towards antibiotics used for melioidosis treatments. Antimicrobial peptides (AMP) such as bacteriocins are gaining the interests of researchers as alternative for treating infections caused by multidrug resistant bacteria. In this study, we aimed to identify Burkholderia spp. isolated from soil in Sarawak that possess the potential in inhibiting the growth of B. pseudomallei and to further characterize the antagonistic compound produced. Methodology and results: A total of 50 Burkholderia spp. isolates of environmental origin and two isolates of Ralstonia solanacearum were screened against five clinical isolates of B. pseudomallei using spot-on-lawn assay and flip streak method. Burkholderia stagnalis isolate K23/3 showed clear zones of inhibition (ZOI) in both preliminary tests. Cell-free supernatant (CFS) was obtained from B. stagnalis K23/3 broth culture and was tested via agar well diffusion assay (AWDA). The antagonistic compound secreted at the early log phase of the bacterial growth was shown to be stable in a wide range of temperatures and pH. Treatment with different enzymes revealed that it was sensitive towards proteinase K, suggesting that it is proteinaceous. The bacteriocin-like-substance (BLIS) was subjected to ammonium sulfate precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE gel was overlaid with indicator B. pseudomallei isolates where the active protein was shown to be less than 7.1 kDa. Conclusion, significance and impact of study: Burkholderia stagnalis isolate K23/3 was able to secrete bacteriocinlike-substance (BLIS) that has the potential in biocontrol of B. pseudomallei in the environment or as potential treatment for melioidosis

Expression and the antigenicity of recombinant coat proteins of tungro viruses expressed in Escherichia coli

Rice tungro disease (RTD) is a recurring disease affecting rice farming especially in the South and Southeast Asia. The disease is commonly diagnosed by visual observation of the symptoms on diseased plants in paddy fields and by polymerase chain reaction (PCR). However, visual observation is unreliable and PCR can be costly. High-throughput as well as relatively cheap detection methods are important for RTD management for screening large number of samples. Due to this, detection by serological assays such as immunoblotting assays and enzyme-linked immunosorbent assay are preferred. However, these serological assays are limited by lack of continuous supply of antibodies as reagents due to the difficulty in preparing sufficient purified virions as antigens. This study aimed to generate and evaluate the reactivity of the recombinant coat proteins of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) as alternative antigens to generate antibodies. The genes encoding the coat proteins of both viruses, RTBV (CP), and RTSV (CP1, CP2 and CP3) were cloned and expressed as recombinant fusion proteins in Escherichia coli. All of the recombinant fusion proteins, with the exception of the recombinant fusion protein of the CP2 of RTSV, were reactive against our in-house anti-tungro rabbit serum. In conclusion, our study showed the potential use of the recombinant fusion coat proteins of the tungro viruses as alternative antigens for production of antibodies for diagnostic purposes

Generation of DNA aptamers against envelope 2 (E2) protein of Chikungunya virus by in vitro systematic evolution of ligands for exponential enrichment (SELEX) for diagnostic application

Abstract Introduction: Chikungunya virus (CHIKV) causes febrile illnesses in human and these cases have rapidly expanded across the globe in recent years. The current antibody-based tests for CHIKV such as ELISA have a variety of limitations associated with the molecules such as batch-to-batch variation, high cost and less stable. Aptamers are single-stranded DNA or RNA that have high affinity and specificity against a wide variety of target molecules. Compared to antibodies, aptamers are cheaper, produced in vitro, no batch-to-batch variations and thus serve as a good molecular recognition element for the development of diagnostic tests for CHIKV. Methods: Cloning, expression and purification of the recombinant CHIKV E2 was carried out and its identity was verified with western blot analysis. The purified protein was subjected to 9 SELEX cycles, the resulting nucleic acid pools were cloned and sent for sequencing. The secondary structure of the aptamer was predicted using Mfold web server and the performance of the aptamer was determined by enzyme-linked aptamer assay (ELAA). Result: The 24kDa recombinant E2 proteins were successfully cloned and purified. The protein was reactive against anti-CHIKV positive sera and anti-CHIKV polyclonal antibody with no cross reactivity with anti-dengue positive pool sera. Sequencing result revealed there were 6 potential candidates of DNA aptamers. DNA aptamer candidate with the highest frequency (61.9%) showed two loops in their predicted secondary structures. ELAA analysis revealed a binding affinity (Kd) of 177.5 nM and limit of detection was 3.3 nM. Conclusion: DNA aptamers were successfully generated and it has great potential as a feasible tool in CHIKV detection. Keywords: Chikungunya virus, E2 protein, DNA aptamer, SELE

A novel silver nanoparticles-based sensing probe for the detection of Japanese Encephalitis virus antigen

A novel silver nanoparticles (Ag NPs)-based optical sensing probe has been developed for the detection of Japanese Encephalitis virus (JEV). Ag NPs were initially deposited onto amine functionalized glass slides. Subsequently, JEV antibodies were self-assembled onto surfaces of Ag NPs to form optical sensing probes. The detection of JEV antigen was observed via changes in light absorbance by Ag NPs upon occurrence of JEV antigen-antibody bindings. A highly sensitive and rapid optical sensing probe for JEV antigen with a detection limit of 12.8 ng/mL (for S/N ratio = 3) and an analysis assay time of 1 h had been demonstrated

Carbon Nanoparticles Based Electrochemical Biosensor Strip for Detection of Japanese Encephalitis Virus

We reported a disposable and sensitive electrochemical biosensor strip based on carbon nanoparticles modified screen-printed carbon electrode (SPCE) for rapid and sensitive detection of Japanese Encephalitis Virus (JEV). Amino group functionalized carbon nanoparticles were prepared from preformed chitosan nanoparticles. Japanese Encephalitis Virus (JEV) antibody was immobilized onto the surfaces of carbon nanoparticles through amide bonds formation between amino groups of carbon nanoparticles and carboxylic groups of JEV antibody. The analytical performance of SPCE electrochemical biosensor strip was characterized using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). SPCE electrochemical biosensor strip exhibited a linear detection range of 1–20 ngmL−1 with a low detection limit of 0.36 ngmL−1 (at S/N = 3) for JEV, detection sensitivity was 0.024 ngmL−1 for JEV, and analysis results were obtainable within 10 minutes. The potential clinical application of this SPCE electrochemical biosensor strip was demonstrated by the detection of JEV in human serum