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Open Access
Evaluation of Safety and Efficacy of Commercially Available Live Attenuated Infectious Bursal Disease Vaccines
(SKUAST Kashmir, 2022) Jan Mohd Muneeb; Nadeem Shabir
Background and Aim: Infectious bursal disease (IBD) or Gumboro disease is an economically important viral disease of poultry caused by non-enveloped dsRNA virus from Birnaviridae family. This disease mostly affects young chickens and causes considerable economic losses in terms of high morbidity and mortality losses. Along with strict biosecurity measures, live-attenuated and/or inactivated IBD vaccines are globally used to prevent the disease. However, previous studies have indicated that live-attenuated IBD vaccines are genetically diverse and might possess a different degree of attenuation, because of which these vaccines are often reported to either possess reduced safety and/or offer compromised cross-protection. The safety and cross-protective efficiency of these vaccines remain a cause of concern, thus warranting the need for evaluation of these aspects in commercially-available live-attenuated IBD vaccines in use in specific geographical regions. Keeping this in view, the current research study was aimed to evaluate and compare the safety and cross-protection features of three commercially available live-attenuated IBD vaccines (intermediate and intermediate-plus strains) being currently used in Jammu and Kashmir. Materials and Methods: An experimental study was carried out at the instructional poultry farm, division of livestock production and management (LPM) at the Faculty of Veterinary Sciences of SKUAST-K university, in which a total of 90 one-day old chicks were housed in the farm and allocated to four different groups, each containing 21 birds and housed separately. Each group was further subdivided into three replicas, with each replica containing seven birds. At fourteen days of age, birds were vaccinated with three different commercially available live-attenuated IBD vaccines (A, B and C) via nasal route with 100μl (50μl per nares) of 103EID50. Vaccine A belonged to intermediate type of IBD vaccine while as Vaccine B and Vaccine C belonged to intermediate-plus type of IBD vaccines. Serum from six birds per group was collected at 3-, 7-, 14- and 21-days post vaccination (dpv) for evaluation of neutralizing antibody titers raised by different vaccines against three different isolates of IBD viruses viz. FVSKG1, FVSKG2 and FVSKG4 using serum virus neutralization assay (VNA). Six birds per group at 3- and 7-days post vaccination and nine birds per group at 21-days post vaccination were euthanized to collect bursa and spleen for evaluation of bursal-body weight ratio, bursal-body weight index, histopathological analysis, residual viral load in bursa and cytokines mRNA expression. In addition, clinical scoring, feed conversion ratio and average weekly weight gain were also evaluated to check the safety profile of the live-attenuated IBD vaccines used in the current study. Results: Severe bursal atrophy at 7-days post vaccination (dpv) was observed in birds of Vaccine-C group which consequently resulted in significantly (p≤0.05) lower bursal-body weight ratio (BBR) and bursal-body weight index (BBI) in birds of Vaccine-C group when compared to those caused in Vaccine-B group. Further, the histopathological lesion scores in bursa and spleen were significantly (p≤0.05) higher in Vaccine-C group at 3-,7- and 21-dpv than those found in Vaccine-A group. The residual viral load (TCID50/mL equivalent) evaluated in bursa of birds was found to be significantly (p≤0.05) higher in Vaccine-C group than that found in all other study groups at 3 and 21dpv. In addition, clinical scores measured at different time points were found to be relatively higher in birds of Vaccine-C group than those found in Vaccine-A and Vaccine-B groups. Vaccine B raised significantly (p≤0.05) higher neutralizing antibody (Ab) titers against FVSKG1 at 3dpv, when compared to that raised by Vaccine C, and at 14dpv when compared to that raised by Vaccine A. However, no significant differences were observed in neutralizing Ab titers raised by different vaccines against FVSKG2 measured at different time points of the study. Vaccine A raised significantly (p≤0.05) higher neutralizing Ab titers against FVSKG4 at 3 and 7dpv than those raised by Vaccine C. Further, Vaccine A raised significantly (p≤0.05) higher homologous neutralizing Ab titers when compared to those raised by Vaccine C at 3 and 7dpv. However, Vaccine C raised significantly (p≤0.05) higher homologous neutralizing Ab titers than those raised by Vaccine B at 21dpv. A significantly (p≤0.05) higher mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β and IL-12) in bursa of birds of Vaccine-C group was observed at 3dpv, however, bursal mRNA expression of IL-6 was significantly (p≤0.05) higher in birds of Vaccine-A. mRNA expression of type- I interferons (IFN-α and IFN-β) was significantly higher in bursa of birds vaccinated with Vaccine A and C at 3dpv, while the IL-10 bursal mRNA expression was significantly (p≤0.05) higher in all vaccinated groups at 3dpv. At 3dpv, no significant difference was found in the mRNA expression of pro-inflammatory cytokines in spleens, however, IL-12 mRNA expression was significantly (p≤0.05) higher in birds of Vaccine-C group when compared with all other study groups. Similarly, no significant (p>0.05) difference was observed between the study groups towards the mRNA expression of Type-I interferons (IFN-α and IFN-β) and IL-10. However, mRNA expression of IL-2 was significantly (p≤0.05) higher in the spleen of birds of Vaccine-B group when compared to that expressed in birds of Vaccine-A groups. IL-6 mRNA expression was significantly (p≤0.05) higher in spleens of Vaccine-A group when compared to that expressed in Vaccine-B group. Vaccine A elicited a significantly (p≤0.05) higher mRNA expression of TNF-α in spleen than that expressed in all other study groups at 21dpv. Further, the mRNA expression of IL-1β was significantly (p≤0.05) lower in spleen of birds in all vaccinated groups at 21dpv. The mRNA expression of Type-I interferons (IFN-α and IFN-β) and Th1 cytokine (IL-2) was expressed at significantly (p≤0.05) higher levels in the spleens of Vaccine-A group in comparison to that expressed in Vaccine C group. Conclusion: Vaccine-A induced relatively higher homologous neutralizing antibodies however, all vaccines were found to induce high titers of heterologous neutralizing antibodies which could likely counter a breakthrough IBD infection. Vaccine-A group showed lowest HLS in bursa as well as in spleen among all the vaccinated groups. While as Vaccine-B group had higher BBR and higher BBI at 7 dpv. Vaccine-C group showed the highest residual viral load in bursa at 3 and 21 dpv. A significantly (p≤0.05) higher expression of IFN-α and IFN-β was observed in bursa at 3dpv or in spleen at 21dpv either in Vaccine- A or Vaccine-C group. A significantly (p≤0.05) higher expression of type-I interferons was observed in bursa at 3 dpv or in spleen at 21 dpv either in vaccine A and or Vaccine-C group. IL-2 was significantly (p≤0.05) expressed in bursa and spleen of vaccine-A group at 21 dpv, while as its expression in spleen at 3dpv was significantly higher in vaccine B group.
Open Access
Lethal mutagenesis for enhancing genetic stability of infectious bursal disease virus
(SKUAST Kashmir, 2022) Towseef Akram; Shah, Riaz Ahmad
Background: Infectious Bursal Disease Virus (IBDV) is a double-stranded (ds) RNA virus belonging to genus, Avibirnavirus and family, Birnaviridae, causing infectious bursal disease. It is an immunosuppressive viral disease of poultry leading to secondary infections as well, therefore has a huge economic impact on the poultry industries globally. Despite the wide use of live attenuated vaccines to prevent this disease, lack of safety of these vaccines has always been a concern. The major safety concern of these vaccines is their possible reversion to virulence, which leads to widespread immunosuppression, disease transmission or evolution of new viral serotypes. The reversion to virulence in live-attenuated vaccines used against RNA viruses is thought to be because of lack of proof-reading activity of their RNAdependent RNA polymerases and consequently, the attenuation markers of a vaccine can revert back to their virulent/wild-type form after vaccination. Therefore, it is imperative to overcome these shortcomings to reduce the economic losses caused due to safety issues of existing live attenuated infectious bursal disease vaccines. Previously, mutagen-driven lethal mutagenesis approaches have been used in single stranded RNA viruses to develop mutants with enhanced fidelity or resistance against mutations. In the present study, mutagen-driven lethal mutagenesis approach was employed to generate IBDV mutants with higher fidelity or higher stability against mutagens or mutations and further explored the putative genetic stability markers. Results: Four mutagens viz. ribavirin, 5- fluorouracil, 5-azacytidine, and amiloride were used in the current study. The cytotoxicity and anti-IBDV assay of each mutagen was performed in primary chicken embryo fibroblasts (CEFs). Among all the mutagens tested in the current study, ribavirin was found to be least cytotoxicity on CEFs and had a significant antiviral effect on replication of three isolates of IBDV viz. FVSK-G1, FVSK-G2 and FVSK-G3 in CEFs. Consequently, these IBDV isolates were serially passaged in sub-cytotoxic concentrations of ribavirin in order to facilitate emergence of ribavirin-resistant mutants. During serial passages, two ribavirinresistant mutants, viz. RFVSK-G1 and RFVSK-G3 emerged from FVSK-G1 at passage-7 in presence of 0.05mM ribavirin and from FVSK-G3 at passage-14 in presence of 0.1 mM ribavirin, respectively. In RNA-dependent RNA polymerases region of RFVSK-G1, two unique amino acid changes, Threonine to Lysine and Alanine to Valine at position 50 and 890, respectively were found. In segment A of RFVSK-G1, an amino acid change of Glycine to Glutamic acid at position 320 was found. Further the mechanism of action of ribavirin on inhibition of FVSK-GI replication was evaluated. The mutational rate in FVSK-G1 was found to increase by four times during serial passages of the virus in 0.05 mM ribavirin. The possible anti-IBDV mechanism of ribavirin through Guanosine pool depletion via inosine monophosphate dehydrogenase (IMPDH) inhibition was also investigated in the current study. The results revealed that with the addition of 40 mM Guanosine, the replication of FVSK-G1 in 40 μM ribavirin or 1 μM mycophenolic acid reached to around 104TCID/mL in CEFs. Further, the in silico docking studies revealed binding affinity of -8.1 and -5.9 Kcal/mol of ribavirin and mycophenolic acid with IMPDH, respectively. A significant increase in the expression of cytokines viz. IFN-α, TNF- α, IL-2, IL-12 and IL-10 was observed in CEFs with the addition of 0.05mM ribavirin. Conclusion: In conclusion, ribavirin was the least cytotoxic on chicken embryo fibroblasts among the tested mutagens and efficiently inhibited the replication of IBDV. Ribavirin resistant mutants emerged at 0.05 mM and 0.1 mM concentration of ribavirin. In those resistant mutants, three putative genetic stability markers, viz. T13K, A857V, and G320E were identified. Mechanism of action of ribavirin on the inhibition of replication of IBDV showed a cumulative effect of mutagenic and nonmutagenic effect. After further validation, these genomic stability markers can be incorporated into vaccines through a reverse genetics platform to achieve the goal of safer vaccines.
Open Access
Effect of Lotus (Nelumbo nucifera) leaf extract on growth and immune response in Rainbow trout (Oncorhynchus mykiss): A Nutrigenomics Approach
(SKUAST Kashmir, 2022) Hakim, Mudasir Maqsood; Ganai, Nazir Ahmad
The present study was conducted to assess growth performance, haemato-immunological response, immune gene expression and survival of rainbow trout(Oncorhynchus mykiss) fingerlings fed with crude Nelumbo nucifera leaf extract (NNLE). Under in vitro conditions, the Indian lotus leaf extract or NNLE showed strong anti-oxidant activity and significantly inhibited the fish bacterial pathogens viz Aeromonas hydrophila, Streptococcus aureus, Pseudomonasfluorescens and E. coli. Whereas, in in-vivo experiment, a purified diet comprising 0 percent NNLE (control), 0.25 percent NNLE (T1), 0.75 percent NNLE (T2), 1.25 percent NNLE (T3), and 1.75 percent NNLE (T4) was fed to each fish group (n=150). A total of 750 fingerlings (20g  0.5) were randomly distributed in triplicates among five separate treatment groups with 50 fish in each replicate. The fish were fed at a rate of 5% of their body weight twice a day. The results revealed that concentration of oxidative enzymes (SOD & CAT) lowered significantly (p<0.05) with increasing levels of NNLE in the diet. NNLE fed groups showed high TLC, respiratory burst activity, lysozyme activity and phagocytic activity compared to control. Serum protein level increased significantly (p<0.05) in T3 and T4 groups. A similar trend was observed during the post-challenge period wherein the weight gain percent, SGR, FCR, and PER were not significantly affected by NNLE. Also, no discernible effect on the ALP & ACP enzymes was observed during the study period. In T4 (1.75% NNLE) group, significant (p<0.05) mRNA expression of defensin and hepcidin genes were observed throughout the experimental period highlighting their role in innate immune defense mechanism. During the pre-challenge phase, proinflammatory cytokines (IL-1 & TNF-α) were significantly (p<0.05) down-regulated in spleen and head-kidney, whereas, during post-challenge phase, IL-1 and TNF-, showed a significant (p<0.05) up-regulation in T4 (1.75%) group, in spleen, head-kidney and liver. Regulatory cytokines (IL-10, TGF-β and IL-12p40) mRNA levels remained up-regulated during pre-challenge period to balance the anti-inflammatory cytokines. Interestingly, T3 and T4, showed the highest survival rate after challenge with pathogenic bacteria Aeromonas hydrophila. Based on the current findings, it can be concluded that supplementation of Indian lotus leaf extract or NNLE up to a dietary inclusion level of 1.75% significantly (p<0.05) enhances survival, immune response and overall biomass of rainbow trout. This study underlines the importance of plant based bioactive substances as a sustainable and effective alternative to synthetic antimicrobials in enhancing the overall fish welfare.
Open Access
Lethal mutagenesis for enhancing genetic stability of infectious bursal disease virus
(SKUAST Kashmir, 2022) Towseef Akram; Shah, Riaz Ahmad
Background: Infectious Bursal Disease Virus (IBDV) is a double-stranded (ds) RNA virus belonging to genus, Avibirnavirus and family, Birnaviridae, causing infectious bursal disease. It is an immunosuppressive viral disease of poultry leading to secondary infections as well, therefore has a huge economic impact on the poultry industries globally. Despite the wide use of live attenuated vaccines to prevent this disease, lack of safety of these vaccines has always been a concern. The major safety concern of these vaccines is their possible reversion to virulence, which leads to widespread immunosuppression, disease transmission or evolution of new viral serotypes. The reversion to virulence in live-attenuated vaccines used against RNA viruses is thought to be because of lack of proof-reading activity of their RNA- dependent RNA polymerases and consequently, the attenuation markers of a vaccine can revert back to their virulent/wild-type form after vaccination. Therefore, it is imperative to overcome these shortcomings to reduce the economic losses caused due to safety issues of existing live attenuated infectious bursal disease vaccines. Previously, mutagen-driven lethal mutagenesis approaches have been used in single stranded RNA viruses to develop mutants with enhanced fidelity or resistance against mutations. In the present study, mutagen-driven lethal mutagenesis approach was employed to generate IBDV mutants with higher fidelity or higher stability against mutagens or mutations and further explored the putative genetic stability markers. Results: Four mutagens viz. ribavirin, 5- fluorouracil, 5-azacytidine, and amiloride were used in the current study. The cytotoxicity and anti-IBDV assay of each mutagen was performed in primary chicken embryo fibroblasts (CEFs). Among all the mutagens tested in the current study, ribavirin was found to be least cytotoxicity on CEFs and had a significant antiviral effect on replication of three isolates of IBDV viz. FVSK-G1, FVSK-G2 and FVSK-G3 in CEFs. Consequently, these IBDV isolates were serially passaged in sub-cytotoxic concentrations of ribavirin in order to facilitate emergence of ribavirin-resistant mutants. During serial passages, two ribavirin- resistant mutants, viz. RFVSK-G1 and RFVSK-G3 emerged from FVSK-G1 at passage-7 in presence of 0.05mM ribavirin and from FVSK-G3 at passage-14 in presence of 0.1 mM ribavirin, respectively. In RNA-dependent RNA polymerases region of RFVSK-G1, two unique amino acid changes, Threonine to Lysine and Alanine to Valine at position 50 and 890, respectively were found. In segment A of RFVSK-G1, an amino acid change of Glycine to Glutamic acid at position 320 was found. Further the mechanism of action of ribavirin on inhibition of FVSK-GI replication was evaluated. The mutational rate in FVSK-G1 was found to increase by four times during serial passages of the virus in 0.05 mM ribavirin. The possible anti-IBDV mechanism of ribavirin through Guanosine pool depletion via inosine monophosphate dehydrogenase (IMPDH) inhibition was also investigated in the current study. The results revealed that with the addition of 40 mM Guanosine, the replication of FVSK-G1 in 40 μM ribavirin or 1 μM mycophenolic acid reached to around 104TCID/mL in CEFs. Further, the in silico docking studies revealed binding affinity of -8.1 and -5.9 Kcal/mol of ribavirin and mycophenolic acid with IMPDH, respectively. A significant increase in the expression of cytokines viz. IFN-α, TNF- α, IL-2, IL-12 and IL-10 was observed in CEFs with the addition of 0.05mM ribavirin. Conclusion: In conclusion, ribavirin was the least cytotoxic on chicken embryo fibroblasts among the tested mutagens and efficiently inhibited the replication of IBDV. Ribavirin resistant mutants emerged at 0.05 mM and 0.1 mM concentration of ribavirin. In those resistant mutants, three putative genetic stability markers, viz. T13K, A857V, and G320E were identified. Mechanism of action of ribavirin on the inhibition of replication of IBDV showed a cumulative effect of mutagenic and non- mutagenic effect. After further validation, these genomic stability markers can be incorporated into vaccines through a reverse genetics platform to achieve the goal of safer vaccines.
Open Access
Effect of Lotus (Nelumbo nucifera) leaf extract on growth and immune response in Rainbow trout (Oncorhynchus mykiss): A Nutrigenomics Approach
(SKUAST Kashmir, 2022) Hakim, Mudasir Maqsood; Ganai, Nazir Ahmad
The present study was conducted to assess growth performance, haemato-immunological response, immune gene expression and survival of rainbow trout(Oncorhynchus mykiss) fingerlings fed with crude Nelumbo nucifera leaf extract (NNLE). Under in vitro conditions, the Indian lotus leaf extract or NNLE showed strong anti-oxidant activity and significantly inhibited the fish bacterial pathogens viz Aeromonas hydrophila, Streptococcus aureus, Pseudomonasfluorescens and E. coli.Whereas, inin-vivo experiment,a purified diet comprising 0 percent NNLE (control), 0.25 percent NNLE (T1), 0.75 percent NNLE (T2), 1.25 percent NNLE (T3), and 1.75 percent NNLE (T4) was fed to each fish group (n=150). A total of 750 fingerlings (20g  0.5) were randomly distributed in triplicates among five separate treatment groups with 50 fish in each replicate. The fish were fed at a rate of 5% of their body weight twice a day. The results revealed thatconcentration of oxidative enzymes (SOD & CAT) lowered significantly (p<0.05) with increasing levels of NNLE in the diet. NNLE fed groups showed high TLC, respiratory burst activity, lysozyme activity and phagocytic activity compared to control. Serum protein level increased significantly (p<0.05) in T3 and T4 groups. A similar trend was observed during the post-challenge period wherein the weight gain percent, SGR, FCR, and PER were not significantly affected by NNLE. Also, no discernible effect on the ALP & ACP enzymes was observed during the study period. In T4 (1.75% NNLE) group,significant (p<0.05) mRNA expression of defensin and hepcidin genes were observed throughout the experimental period highlighting their role in innate immune defense mechanism. During the pre-challenge phase, proinflammatory cytokines (IL-1 & TNF-α) were significantly (p<0.05) down-regulated in spleen and head-kidney, whereas,during post-challenge phase, IL-1 and TNF-, showed a significant (p<0.05) up-regulation in T4 (1.75%) group, in spleen, head-kidney and liver. Regulatory cytokines (IL-10, TGF-β and IL-12p40) mRNA levels remained up-regulated during pre-challenge period to balance the anti-inflammatory cytokines. Interestingly, T3 and T4, showed the highest survival rate after challengewith pathogenic bacteria Aeromonas hydrophila. Based on the current findings, it can be concluded that supplementation of Indian lotus leaf extract or NNLE up to a dietary inclusion level of 1.75% significantly (p<0.05) enhances survival, immune response and overall biomass of rainbow trout. This study underlines the importance of plant based bioactive substances as a sustainable and effective alternative to synthetic antimicrobials in enhancing the overall fish welfare.
Open Access
Recombinant Lactic acid bacteria-based vaccine against poultry salmonellosis ---A novel approach
(SKUAST Kashmir, 2020) Khan, Shabir Ahmad; Andrabi, Syed Mudasir
This thesis describes a work of the M.V.Sc program aimed for developing oral mucosal vaccine based on recombinant Lactic acid bacteria expressing the heterologous(FimH) antigen. Lactic acid bacteria are considered an attractive candidate for antigen delivery because of their “Generally Regarded As Safe” (GRAS) status and probiotic effects on the host. The FimH antigen of salmonella is highly immunogenic (kisiela et al., 2009) and its uptake through the glycoprotein receptor of M-cell initiates a mucosal immune response also (Hase et al., 2009). The plasmid pTRK892, a kind of gift from Addgene, plasmid depository, USA was selected for being E. coli-Lactobacillus shuttle vector. Gibson assembly reaction mixture was used to construct our own designed plasmid (pTRK722) by joining pTRK892 plasmid backbone (plasmid pTRK892 digested by removing promoter pgm as well as reporting gene β-glucuronidase) with the FimH gene (salmonella), anchor motiff CWA2 (Lactobacillus plantarum), promotor Ppgm (phosphoglycerate mutase) and signal peptide SP1, LP0373 (Lactobacillus plantarum) DNA sequence. E. coli MC1061 cell strain having the presence of a functional recA gene (recA+) found suitable strain for replication of pTRK892 derived plasmids. These constructs (pTRK722 and pTRK722Histag) were first established in E. coli MC1061 and then transformed into L.plantarum ATCC 8014 and L.rhamnosus GG. Lactobacillus plantarum ATCC8014 was engineered for expression, secretion and subsequent anchoring of FimH on its external surface by LPxTG motif cell wall anchor. The Signal peptide (LP-0370) was also incorporated between promotor Pgm (Ppgm1) and FimH region for secretion of heterologous protein in Lactobacillus plantarum. The expression of FimH antigen in Lactobacillus plantarum was evaluated by Western blotting which demonstrated that FimH protein is expressing from Lactobacillus plantarum under pgm promoter. Chicken Intestinal epithelial cell cultures were used as an in vitro model to investigate the ability of recombinant Lactobacillus plantarum (FimH protein expression) for promoting internalization. The recombinant L. plantarum showed increased internalization compared to the control which is considered one of the important parameter for better and efficient delivery of antigen to the Peyer’s patches of the gastrointestinal tract and shall elicit the immune response to properly safeguard the vaccinated animal. However, for checking its efficacy under in vivo conditions the field experiments are needed.
Open Access
Protein extraction from low quality animal fibers for protein film preparation.
(SKUAST Kashmir, 2018) Nazir, Saba; Bhat, Hina F.
Municipal waste includes keratinized tissues such as animal hair, nail, horn, feather etc that consist largely of Keratin and Keratin associated proteins. There is a need to make good use of low quality animal fibers such as Goat hair, Human hair, Sericultural waste in order to increase their economic value. The current study is thus aimed at exploring some novel applications for these low quality animal fibers so as to aid in their value addition. We extracted proteins from low quality animal fibers (Goat hair, Human hair and Sericultural waste) using three different methods/buffers (shindai, Conventional and modified). According to our study Conventional method/buffer of protein extraction was found to be more effective as the yield and quality of the protein was better as compared to shindai and modified method. Due to unique biological and chemical properties of proteins extracted from these natural fibers, they were utilized for preparing flexible protein films or biodegradable films. Pre-cast and Post–cast methods have been used for film preparation. In our study the native proteins films were too fragile to be used for any medical application, but addition of stabilizers (Glycerol and Gelatin) to extracted proteins increased its flexibility and strength. We studied the basic characterization like thickness, surface morphology and tensile strength of prepared protein films from goat hair and human hair. The obtained results suggested that the addition of Glycerol or Gelatin (crosslinking agents) influenced the surface morphology and improved the physical properties of films like tensile strength. In our study structural analysis of fibers has been done using Scanning electron microscopy after proper cleaning of the fibers in order to differentiate their surface morphology before and after delipidization. Since we standardized a method for the maximum extraction of proteins from low quality fibers, which were further used for protein film preparation that may have biomedical role. Thus our work was directed towards the value addition of low quality animal fibers and make full use of keratins and keratin associated proteins obtained from otherwise discarded low quality fibers.
Open Access
Effect of DNA modifying agents on in vitro development of interspecies cloned Cattle-Buffalo embryos
(SKUAST Kashmir, 2019) Beig, Haris Rasool; Shah, Riaz Ahmad
Somatic cell nuclear transfer is one of the most advanced and least exploited technology among assisted reproductive technologies. It has immense potential in the conservation of endangered species as well as revival of extinct species. However, on account of limited oocyte availability from such species, a modified approach of interspecies somatic cell nuclear transfer (iSCNT) has been devised. In addition, iSCNT also provides a window of study on nuclear reprogramming and embryonic stem cell production for clinical and research purposes. Somatic cell nuclear transfer embryos as a whole and iSCNT embryos, in particular, have low efficiency to develop into transferable quality embryos which has been attributed to abnormal reprogramming of the donor somatic cell mediated by DNA methylation and histone acetylation. Current investigation was carried out to determine the efficiency of iSCNT cloned Cattle-Buffalo embryo production using Hand Made Cloning (HMC) technique and to compare in vitro development of iSCNT cloned Cattle-Buffalo embryos following donor cell treatment with epigenetic re-modelling drugs or chromatin modifying agent like DNA demethylation agents and histone deacetylase inhibitors (HDAC) inhibitors i.e. 5-aza- 20-deoxycytidine (5-aza-dC) and Scriptaid. A total of 1591 abattoir derived buffalo ovaries in 16 replicates were used for the experiments. 3347 COC’s were harvested upon aspiration of surface follicles (size ranging from 2mm - 8mm). A total of 2378 (71.05%) COC’s were of usable quality, therefore, selected for IVM. COC’s with not less than2 layers of surrounding cumulus were selected for in vitro maturation (IVM). Three types of IVM media were used, IVM media supplemented with with 1) pFSH + β estradiol, 2) pFSH +β estradiol+Follicular fluid and 3) follicular fluid only. The maturation percentage as determined by cumulus expansion and polar body extrusion was found to be higher when IVM medium supplemented with pFSh + 17 βestradiol + Follicular fluid was used as compared with supplementation of Pfsh+17 βestradiol or follicular fluid only (94.07% ±3.65% v 86.25% ±4.86% and 79.42%±1.37% respectively) but higher maturation percentage observed in IVM medium supplemented with pFSh + 17 βestradiol + Follicular fluid was statistically non-significant (P>0.05).The percentage of iSCNT cloned Cattle-Buffalo embryos without any treatment of donor cells which cleaved on day 2 was 26.25%±0.95%. 2-4 cell stage, 8 cell stage and 16-32 cell stage embryo percentages obtained on Day 7 were 42.86%±10.13%, 33.33%±7.01% and 23.81%±8.96%, respectively. The percentage of cleaved embryos as compared to control, was found to be significantly higher in all treatment groups (26.25%±0.95% vs 35.29%±0.74%, 41.79%±3.23%, 35.53%±3.77%). Highest cleavage rates were found in Sriptaid treated group but was non-significant in comparison with 5-aza-dC and combination treatment group. Other developmental stages were comparable and did not show any significant variation between the various treatment groups or from the control group. The embryos did not progress beyond 32-cell stage in any treatment group.
Open Access
Identification and in vitro validation of potential inhibitors of RNA-Dependent RNA Polymerase of Infectious Bursal Disease Virus
(SKUAST Kashmir, 2018) Khan, Mahak; Nadeem, Shabir
Infectious Bursal Disease (IBD) is an acute, host-specific, highly pathogenic, immunosuppressive viral disease of poultry that is reported to cause huge economic impact on the poultry industry worldwide. Currently, live attenuated vaccines are used against this virus. However, there is a safety concern involved with the use of these vaccines which primarily includes reversion of this live vaccine to wild-type virus that can lead to immune-suppression in vaccinated birds, emergence of new serotypes and disease transmission. Thus, alternate strategies to combat the disease need to be explored. Previous studies using computer-aided drug designing (CADD) have explored potential natural and synthetic compounds that can block the active sites of viral proteins involved in viral synthesis or its replication. In the current study computer–aided drug designing was employed to explore inhibitor compounds against RNA-dependent RNA polymerase of IBD virus from two compound libraries, one consisting of 66,609 natural compounds and the other consisting of 120 bioactive compounds of western Himalayanregion. Four conserved active sites of RNA-dependent RNA polymerase of infectious bursal disease virus viz. ADN-403, ASP-402, ASP-416 (involved in polymerization) and SER-166 (involved in self-guanylation) were used for the in silico drug designing. This in silico part of the drug designing was conducted in three steps viz., a) Virtual screening of each compound for free binding energy against each site using ArgusLab v 4.0.1. b) Top compounds with least free binding energy were evaluated for toxicity and mutagenecity using PreADMET software. c) Those compounds with least levels of toxicity were subjected to more stringent docking using AutoDock 4.0 software. Further, the best docked top two lead compounds with best docked conformation and least binding energies were selected for in vitro assays. Cytotoxicity assay of these compounds was performed in Chicken Embryo Fibroblasts (CEFs) using MTT assay following which the multi-step growth curve analysis of infectious bursal disease virus culture (0.1 Multiplicity of Infection) treated with two compounds at four different sub-cytotoxic concentrations was evaluated at 0, 24, 48, 72, 96 and 144 Hours Post-Compound Treatment. After virtual screening and stringent molecular docking of compounds from two libraries used in the present study, Azulene and Alantolactone were found to be the top two common lead compounds for all the selected target sites. The concentrations of Azulene and Alantolactone were found to be significantly cytotoxic in chicken embryo fibroblasts at ≥300 µM and ≥30 µM, respectively. Following multi-step growth curve analysis, it was found that Azulene significantly reduced the overall replication of infectious bursal disease virus at 200 µM, 250 µM as compared to the infectious bursal disease virus control. Thus, it was concluded that Azulene can significantly inhibit the replication of infectious bursal disease virus in vitro at concentrations more than 200 µM.

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