Foot and mouth disease is an infectious and highly contagious viral disease of domestic and wild ... more Foot and mouth disease is an infectious and highly contagious viral disease of domestic and wild cloven-hoofed animals. Vaccination is a major means to control FMD in most endemic areas. Current FMD vaccines are typically produced by inactivation of cell culture virus with binary ethylenimine and formulated with oil adjuvant. This vaccine has a subset of limitations such as requirement for frequent booster doses, poor cellular response, improper inactivation of virus. The focus has been shifted for production of an alternative, more desirable vaccine formulations like live attenuated vaccines, live vectored vaccines, DNA vaccines, subunit vaccines, virus like particles etc., which are achieved through rDNA technology.
al.; licensee Canadian Journal of Biotechnology. This is an open access article distributed as pe... more al.; licensee Canadian Journal of Biotechnology. This is an open access article distributed as per the terms of Creative Commons Attribution-NonCommercial 4.0 International (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract FMD is an economically devastating livestock disease of global importance. The disease is caused by FMD virus of genus Aphthovirus of family picornaviridae. It affects both domestic and wild cloven-hoofed animals. Many alternate vaccine strategies such as peptide vaccines, DNA vaccines, viral-vectored vaccine, VLPs are attempted to date. The limited success of alternate strategies was attributed to either poor immune response or instability of antigen during storage. The FMDV capsid is reported to be thermo-and acid-labile. In this study, we report engineering and evaluation of FMDV capsid for enhanced thermostability. In this study, five mutations (TS1, TS2, TS3, TS4 and TS5) with a single amino acid change were introduced in VP2 region, by PCR-mediated site-directed mutagenesis and generated recombinant bacmids by transposition method, transfected in insect cells to produce recombinant baculovirus expressing FMDV empty capsids. The expressed VLPs were analyzed for antigenic thermostability, by storing the samples at-20 o C, 4 o C, 24 o C and 37 o C and subjecting to s-ELISA at 15 days interval. Purified VLPs were also investigated based on T m from DSF stability assay after preheating at 37 o C for 1hr, 45 o C and 56 o C for 30 min and varying pH conditions like 6, 7 and 8. The expressed protein purified by 15 to 60% sucrose gradient, on analysis by TEM showed the intact empty capsid particles of ~25nm. The s-ELISA of stored samples revealed the consistency in reactivity of TS1, TS2 and TS3 samples stored at-20 o C, 4 o C, 24 o C and 37 o C for more than 75 days, while the drop in antigen reactivity was noticed for wild-type after 30 days and for TS5 after 45 days when stored at the different temperatures. TS4 showed stability when stored at-20 o C, 4 o C for more than 75 days but the sudden decrease in antigen reactivity was seen after 45 days at 24 o C and after 30 days at 37 o C. In DSF stability assay, TS3 exhibited a constant high T m after heating at 37 o C for 1hr, 45 o C for 30min and under pH conditions 6, 7 and 8 in comparison to other mutants and wild-type. However, the complete dissociation of capsid was seen in all mutants and wild-type when heated at 56 o C for 30 min. TS3 capsids exhibited relatively more stability compared to all other mutant capsids. Further studies evaluating TS3 mutant capsids in eliciting a protective immune response are needed to investigate its potential as a vaccine candidate.. Mutation of VP2 capsid protein of Foot-and-mouth disease virus and its expression in the insect cell system for increased thermostability [Abstract]. In:
Foot and mouth disease (FMD) is a globally important livestock disease causing an economic havoc.... more Foot and mouth disease (FMD) is a globally important livestock disease causing an economic havoc. Much of the research was done and ongoing for control. In this article we described about the current scenario of FMD control in India, the conventional vaccines available and modern alternative strategies being in research for control of FMD. The world has taken its step forward in control of FMD with an effective vaccination strategies.
The present study describes a sophisticated technique to amplify gene fragment directly from the ... more The present study describes a sophisticated technique to amplify gene fragment directly from the baculovirus without isolating genome. The study is carried out at FMDRL, IVRI, Bengaluru during January to March 2017. The study was performed with a recombinant baculovirus which was propagated in Trichoplusia ni (Tn5) insect cell lines. Here we PCR amplified the gene fragment directly from the recombinant baculovirus without the need for isolation of genomic DNA and sequenced it. Five out of five PCR fragments sequenced shows no changes in the nucleotide sequence which indicates that there was 100% efficiency of the direct PCR approach for amplifying gene fragment from recombinant baculovirus is high. The use of direct PCR approach for amplifying gene fragment from recombinant baculovirus helps to cut short the time and expenses for amplification of gene fragment from baculovirus for sequencing.
In molecular biology, we most often handle with bacmid when working with baculovirus expression s... more In molecular biology, we most often handle with bacmid when working with baculovirus expression system. The cloning involves a two-step process and the recombinant bacmids are screened by blue-white lac operon system, white colonies are thought to be positive for transposition. But in our study with PCR based approach, we found that not all the white colonies are recombinants. Hence, it is necessary to be cautious while screening for recombinant bacmids and may be it is good too screen by two-way approach, by blue-white lac operon system followed by PCR approach using one bacmid specific and one gene specific primers.
The genomic DNA extraction and PCR amplification of gene fragment from whole blood sample is a cu... more The genomic DNA extraction and PCR amplification of gene fragment from whole blood sample is a cumbersome process. Here in this study we described a direct PCR amplification of gene fragment from the whole blood sample of sheep bypassing the genomic DNA extraction steps. The detailed technique is explained and the results are suggestive of high accuracy in gene amplification. This can be used to cut short the time and is economic for gene amplification from whole blood sample with the same available reagents as for the conventional method. Introduction The world is advancing with implementation of the rapid and cost effective techniques but still there is a need to develop a proper strategy for rapid PCR amplification of genomic DNA. Friedrich Miescher for the first time isolated DNA in 1869 [1]. The blood is the main source for many gene related studies in mammals [2]. Many advancements have been done by using different preservatives for blood preservation and effective DNA isolation [3-6]. Colony PCR was the one technique that is used for direct PCR amplification of gene fragment from bacterial cells [7]. In this study we explored the advantage of this technique for direct PCR amplification without isolating a genomic DNA.
Foot and mouth disease (FMD) is a globally important livestock disease causing an economic havoc.... more Foot and mouth disease (FMD) is a globally important livestock disease causing an economic havoc. Much of the research was done and ongoing for control. In this article we described about the current scenario of FMD control in India, the conventional vaccines available and modern alternative strategies being in research for control of FMD. The world has taken its step forward in control of FMD with an effective vaccination strategies.
Foot and mouth disease is an infectious and highly contagious viral disease of domestic and wild ... more Foot and mouth disease is an infectious and highly contagious viral disease of domestic and wild cloven-hoofed animals. Vaccination is a major means to control FMD in most endemic areas. Current FMD vaccines are typically produced by inactivation of cell culture virus with binary ethylenimine and formulated with oil adjuvant. This vaccine has a subset of limitations such as requirement for frequent booster doses, poor cellular response, improper inactivation of virus. The focus has been shifted for production of an alternative, more desirable vaccine formulations like live attenuated vaccines, live vectored vaccines, DNA vaccines, subunit vaccines, virus like particles etc., which are achieved through rDNA technology.
al.; licensee Canadian Journal of Biotechnology. This is an open access article distributed as pe... more al.; licensee Canadian Journal of Biotechnology. This is an open access article distributed as per the terms of Creative Commons Attribution-NonCommercial 4.0 International (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract FMD is an economically devastating livestock disease of global importance. The disease is caused by FMD virus of genus Aphthovirus of family picornaviridae. It affects both domestic and wild cloven-hoofed animals. Many alternate vaccine strategies such as peptide vaccines, DNA vaccines, viral-vectored vaccine, VLPs are attempted to date. The limited success of alternate strategies was attributed to either poor immune response or instability of antigen during storage. The FMDV capsid is reported to be thermo-and acid-labile. In this study, we report engineering and evaluation of FMDV capsid for enhanced thermostability. In this study, five mutations (TS1, TS2, TS3, TS4 and TS5) with a single amino acid change were introduced in VP2 region, by PCR-mediated site-directed mutagenesis and generated recombinant bacmids by transposition method, transfected in insect cells to produce recombinant baculovirus expressing FMDV empty capsids. The expressed VLPs were analyzed for antigenic thermostability, by storing the samples at-20 o C, 4 o C, 24 o C and 37 o C and subjecting to s-ELISA at 15 days interval. Purified VLPs were also investigated based on T m from DSF stability assay after preheating at 37 o C for 1hr, 45 o C and 56 o C for 30 min and varying pH conditions like 6, 7 and 8. The expressed protein purified by 15 to 60% sucrose gradient, on analysis by TEM showed the intact empty capsid particles of ~25nm. The s-ELISA of stored samples revealed the consistency in reactivity of TS1, TS2 and TS3 samples stored at-20 o C, 4 o C, 24 o C and 37 o C for more than 75 days, while the drop in antigen reactivity was noticed for wild-type after 30 days and for TS5 after 45 days when stored at the different temperatures. TS4 showed stability when stored at-20 o C, 4 o C for more than 75 days but the sudden decrease in antigen reactivity was seen after 45 days at 24 o C and after 30 days at 37 o C. In DSF stability assay, TS3 exhibited a constant high T m after heating at 37 o C for 1hr, 45 o C for 30min and under pH conditions 6, 7 and 8 in comparison to other mutants and wild-type. However, the complete dissociation of capsid was seen in all mutants and wild-type when heated at 56 o C for 30 min. TS3 capsids exhibited relatively more stability compared to all other mutant capsids. Further studies evaluating TS3 mutant capsids in eliciting a protective immune response are needed to investigate its potential as a vaccine candidate.. Mutation of VP2 capsid protein of Foot-and-mouth disease virus and its expression in the insect cell system for increased thermostability [Abstract]. In:
Foot and mouth disease (FMD) is a globally important livestock disease causing an economic havoc.... more Foot and mouth disease (FMD) is a globally important livestock disease causing an economic havoc. Much of the research was done and ongoing for control. In this article we described about the current scenario of FMD control in India, the conventional vaccines available and modern alternative strategies being in research for control of FMD. The world has taken its step forward in control of FMD with an effective vaccination strategies.
The present study describes a sophisticated technique to amplify gene fragment directly from the ... more The present study describes a sophisticated technique to amplify gene fragment directly from the baculovirus without isolating genome. The study is carried out at FMDRL, IVRI, Bengaluru during January to March 2017. The study was performed with a recombinant baculovirus which was propagated in Trichoplusia ni (Tn5) insect cell lines. Here we PCR amplified the gene fragment directly from the recombinant baculovirus without the need for isolation of genomic DNA and sequenced it. Five out of five PCR fragments sequenced shows no changes in the nucleotide sequence which indicates that there was 100% efficiency of the direct PCR approach for amplifying gene fragment from recombinant baculovirus is high. The use of direct PCR approach for amplifying gene fragment from recombinant baculovirus helps to cut short the time and expenses for amplification of gene fragment from baculovirus for sequencing.
In molecular biology, we most often handle with bacmid when working with baculovirus expression s... more In molecular biology, we most often handle with bacmid when working with baculovirus expression system. The cloning involves a two-step process and the recombinant bacmids are screened by blue-white lac operon system, white colonies are thought to be positive for transposition. But in our study with PCR based approach, we found that not all the white colonies are recombinants. Hence, it is necessary to be cautious while screening for recombinant bacmids and may be it is good too screen by two-way approach, by blue-white lac operon system followed by PCR approach using one bacmid specific and one gene specific primers.
The genomic DNA extraction and PCR amplification of gene fragment from whole blood sample is a cu... more The genomic DNA extraction and PCR amplification of gene fragment from whole blood sample is a cumbersome process. Here in this study we described a direct PCR amplification of gene fragment from the whole blood sample of sheep bypassing the genomic DNA extraction steps. The detailed technique is explained and the results are suggestive of high accuracy in gene amplification. This can be used to cut short the time and is economic for gene amplification from whole blood sample with the same available reagents as for the conventional method. Introduction The world is advancing with implementation of the rapid and cost effective techniques but still there is a need to develop a proper strategy for rapid PCR amplification of genomic DNA. Friedrich Miescher for the first time isolated DNA in 1869 [1]. The blood is the main source for many gene related studies in mammals [2]. Many advancements have been done by using different preservatives for blood preservation and effective DNA isolation [3-6]. Colony PCR was the one technique that is used for direct PCR amplification of gene fragment from bacterial cells [7]. In this study we explored the advantage of this technique for direct PCR amplification without isolating a genomic DNA.
Foot and mouth disease (FMD) is a globally important livestock disease causing an economic havoc.... more Foot and mouth disease (FMD) is a globally important livestock disease causing an economic havoc. Much of the research was done and ongoing for control. In this article we described about the current scenario of FMD control in India, the conventional vaccines available and modern alternative strategies being in research for control of FMD. The world has taken its step forward in control of FMD with an effective vaccination strategies.
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