Arvind A. Sonwane

A sindbis virus replicon-based DNA vaccine encoding the rabies virus glycoprotein elicits immune responses and complete protection in mice from lethal challenge

A sindbis virus replicon-based DNA vaccine encoding rabies virus glycoprotein (G) was developed by subcloning rabies G gene into a sindbis virus replicon-based vaccine vector (pAlpha). The self-amplification of RNA transcripts and translation efficiency of rabies G was analyzed in pAlpha-Rab-G-transfected mammalian cells using RT-PCR, SDS-PAGE and Western blot analysis. The transfected cells also showed induction of apoptosis which is an important event in the enhancement of immune responses. Further, immune responses induced with replicon-based rabies DNA vaccine (pAlpha-Rab-G) was compared with conventional rabies DNA vaccine and commercial cell culture vaccine (Rabipur) in intramuscularly injected mice. The mice immunized with replicon-based rabies DNA vaccine induced humoral and cell mediated immune responses better than conventional rabies DNA vaccine however, comparable to Rabipur vaccine. On challenge with rabies virus CVS strain, replicon-based rabies DNA vaccine conferred complete protection similar to Rabipur. These results demonstrate that replicon-based rabies DNA vaccine is effective in inducing both humoral and cellular immune responses and can be considered as effective vaccine against rabies.

Intracerebral delivery of small interfering RNAs (siRNAs) using adenoviral vector protects mice against lethal peripheral rabies challenge.

Abstract To investigate the potential of RNA interference (RNAi) as antiviral agent against rabies, two small interfering RNAs (siRNAs) targeting rabies virus (RABV) nucleoprotein (N) and polymerase (L) genes were designed and evaluated. Both siRNAs knockdown or silenced the target RABV genes as evaluated in a plasmid based transient expression model. For efficient delivery, adenoviruses expressing the siRNAs were constructed and antiviral potential of the delivered siRNAs was investigated in BHK-21 cells. When cells treated with adenoviruses expressing siRNAs were challenged with RABV, there was 88.35±2.4% and 41.52±9.3% reduction in RABV multiplication in infected cells with siRNAs targeting RABV-N and L genes, respectively. Relative quantification of RABV transcripts using real-time PCR revealed knockdown of both RABV-N and L gene transcripts, however, significant reduction was observed only with adenovirus expressing siRNA against RABV-N. When mice treated intracerebrally with adenoviruses expressing siRNAs were challenged peripherally with lethal RABV by the intramuscular route in masseter muscle, there was 66.6% and 33.3% protection with adenoviruses expressing siRNAs against RABV-N and L genes, respectively. These results demonstrated that adenovirus expressing siRNA against RABV-N efficiently inhibited the RABV multiplication both, in vitro and in vivo and conferred significant protection against lethal RABV challenge. This supported the hypothesis that RNAi, based on siRNA targeting RABV-N gene can prevent RABV infection and holds the potential of RNAi as an approach to prevent rabies infection.

Inhibition of rabies virus multiplication by siRNA delivered through adenoviral vector in vitro in BHK-21 cells and in vivo in mice

To evaluate antiviral potential of adenoviral vector-delivered small interfering RNA (siRNA) against rabies, recombinant, replication-defective adenoviral vectors (rAdV) encoding siRNAs targeting rabies virus (RV) polymerase (L) and nucleoprotein (N) genes were developed. The siRNAs were delivered as small hairpin RNAs (shRNAs) through these vectors. Treatment of BHK-21 cells with rAdV expressing siRNA targeting L gene (rAdV-L) and N gene (rAdV-N) (100 MOI) and their subsequent infection with RV (0.001 MOI, RV PV-11), reduced RV fluorescent foci by 48.2% (mean±SEM; 48.17±0.6540, N=6) and 41.8% (mean±SEM; 41.83±0.3073, N=6), respectively, with respect to that of BHK-21 cells treated with rAdV expressing negative control siRNA (rAdV-Neg) indicating inhibition of multiplication of RV in BHK-21 cells in response to adenoviral vector mediated siRNA delivery. Also, the similar treatment of BHK-21 cells with rAdV-L and rAdV-N and similar subsequent infection of them with RV resulted in reduction in RV mRNA transcript levels for their respective targets (RV L gene for rAdV-L and N gene for rAdV-N). mRNA transcript level for RV L gene was reduced by 17.88-fold (mean±SEM; 17.88±0.06638, N=6) in cells treated with rAdV-L and that for RV N gene was reduced by 5.7-fold (mean±SEM; 5.7±0.04472, N=6), in cells treated with rAdV-N, in comparison with that in cells treated with rAdV-Neg, as analyzed by using real-time PCR. These in vitro studies showed that between these two, adenoviral vector mediated delivery of siRNA targeting RV L gene was comparatively more effective in inhibiting RV multiplication in BHK-21 cells than that of siRNA targeting RV N gene (p<0.0001). Localized treatment (intramuscular injection in masseter muscle) of mice with 10(7) plaque forming units of either rAdV-L or rAdV-N and subsequent lethal RV infection (15-20LD(50) of CVS-11) at the same site, through the same route, although resulted in 50% protection (3 out of 6 mice survived) against lethal rabies, the survival patterns for groups of mice treated with either rAdV-L or rAdV-N and that treated with rAdV-Neg did not differ significantly (p=0.5234). These results indicated that adenoviral vector mediated siRNA delivery, in vitro in BHK-21 cells inhibited RV multiplication in vitro in BHK-21 cells; siRNA targeting RV L gene used in this study was comparatively more efficient in doing this than that targeting RV N gene used in this study; in vivo in mice inhibited RV multiplication in mice and imparted partial protection against lethal rabies and so it may have a potential to be used as an alternative antiviral approach against rabies, although further study is required to establish its efficacy for this purpose.

Induction of immune responses and protection in mice against rabies using a self-replicating RNA vaccine encoding rabies virus glycoprotein

A self-replicating RNA vaccine encoding rabies virus glycoprotein gene was developed utilizing sindbis virus RNA replicon. The in vitro transcribed RNA (Sin-Rab-G RNA) was transfected in mammalian cells and analysed for self-replication and expression of rabies glycoprotein. To generate immune responses against rabies, mice were immunized with 10microg of Sin-Rab-G RNA and immune responses developed were compared with mice immunized with rabies DNA vaccine and commercial cell culture vaccine (Rabipur). The self-replicating rabies RNA vaccine generated cellular and humoral IgG responses similar to rabies DNA vaccine. On challenge with rabies virus CVS strain, rabies RNA vaccine conferred protection similar to rabies DNA vaccine. These results demonstrated that replicon-based self-replicating rabies RNA vaccine with 10microg dose was effective in inducing immune responses and protection similar to rabies DNA vaccine.

Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis)

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.

Molecular Characterization of Lapinized Classical Swine Fever Vaccine Strain by Full-Length Genome Sequencing and Analysis

The complete genome of a lapinized classical swine fever virus (CSFV) vaccine strain was amplified into nine overlapping fragments by RT-PCR, and nucleotide sequences were determined. Complete genome sequence alignment and phylogenetic analysis indicated 92.6–98.6% identities at the nucleotide level with other reported CSFV strains and could be grouped into subgroup 1.1 along with other attenuated strains of CSFV. The 5′-UTR demonstrated >97.0% nucleotide similarity with most of vaccine CSFV strains from China. Further, its 3′-UTR sequence indicated a length similar to all the CSFV strains from China with >98.0% nucleotide similarity, although high length heterogeneity of 3′-UTR was reported among different CSFV strains. There was 12 nt (TTTTCTTTTTTT) insertion in 3′-UTR similar to other reported attenuated vaccine strains. However, secondary structure of 3′-UTR indicated that Indian CSFV strain requires further passage to obtain a 3′-UTR structure similar to most of the attenuated strains.

Single nucleotide polymorphisms in toll-like receptor genes and case-control association studies with bovine tuberculosis

Aim: Toll-like receptor 2 (TLR2) and TLR4 genes play critical roles in host recognition of Mycobacterium bovis infection and initiation of innate and adaptive immune response. The present study was aimed at exploring the association of seven single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 genes with susceptibility/resistance against bovine tuberculosis (bTB) infection in cattle. Materials and Methods: A case-control resource population of 35 positive and 45 negative animals was developed after screening with single intradermal tuberculin test for bTB. Resource population was screened for SNPs in TLR2 and TLR4 genes using polymerase chain reaction-restriction fragment length polymorphism. The PROC LOGISTIC procedure of SAS 9.3 was used to find an association of allelic and genotypic frequencies with bTB. Results: In TLR2 gene, two of SNPs under study (rs55617172 and rs68268253) revealed polymorphism while in the case of TLR4 gene all four SNPs under investigation (rs8193041, rs207836014, rs8193060, and rs8193069) were found to be polymorphic in case-control population. SNP locus rs55617172 in TLR2 gene was found significantly (p<0.01) associated with susceptibility/resistance to TB in cattle. Conclusion: These findings indicate the presence of SNPs in TLR2 and TLR4 genes in our resource population. Upon validation in independent, large resource population and following biological characterization, SNP rs55617172 can be incorporated in marker panel for selection of animals with greater resistance to bTB.

Differential expression of ten candidate genes regulating prostaglandin action in reproductive tissues of buffalo during estrous cycle and pregnancy

Prostaglandins (PGs) are the key mediators of several female reproductive functions, including luteolysis, ovulation, fertilization, implantation, pregnancy, and parturition. The present study was conducted in buffalo endometrial and luteal tissues between nonpregnant and two stages of pregnancy (29-38 days of pregnancy, 48-56 days of pregnancy) tissue samples. The genes involved from synthesis upto receptor level effect of PGs (PGF2α and PGE2) were studied for their relative mRNA expression. We have collected the endometrial and luteal tissues from slaughtered animals and confirmed the stages by external examination and crown vertebral rump length measurement of the foetus. The mRNA expression of COX-2 and PGFS genes revealed high significant rise in the transcript at pregnancy stage I as compared to the late luteal phase of nonpregnant. However, EP2 and EP3 genes were highly upregulated in pregnancy stage II. The expression of PLA2G4A and PGT genes showed difference in their transcripts in pregnancy, however, the difference was nonsignificant as compared to the nonpregnant stage. The findings emerged from this study also suggested the strict regulation at COX-2 mRNA level than at synthase enzymes level. Among the four subtypes of EP gene, we have observed highly significant expression difference in EP2 followed by EP3 after implantation.

Red flour beetle (Tribolium castaneum): From population genetics to functional genomics

Tribolium castaneum is a small and low maintenance beetle that has emerged as a most suitable insect model for studying developmental biology and functional genetic analysis. Diverse population genetic studies have been conducted using Tribolium as the principal model to establish basic facts and principles of inbreeding experiments and response to the selection and other quantitative genetics fundamentals. The advanced molecular genetic studies presently focused on the use of Tribolium as a typical invertebrate model for higher diploid eukaryotes. After a whole genome sequencing of Tribolium, many areas of functional genomics were unraveled, which enabled the use of it in many technical approaches of genomics. The present text reviews the use of Tribolium in techniques such as RNAi, transgenic studies, immune priming, immunohistochemistry, in situ hybridization, gene sequencing for characterization of microRNAs, and gene editing using engineered endonuclease. In contrast to Drosophila, the T. castaneum holds a robust systemic RNAi response, which makes it an excellent model for comparative functional genetic studies.

An allogenic therapeutic strategy for canine spinal cord injury using mesenchymal stem cells: BHAT et al.

This study was conducted to characterize canine bone marrow‐derived mesenchymal stem cells (BMSCs); in vivo tracking in mice, and therapeutic evaluation in canine clinical paraplegia cases. Canine BMSCs were isolated, cultured, and characterized in vitro as per International Society for Cellular Therapy criteria, and successfully differentiated to chondrogenic, osteogenic, and adipogenic lineages. To demonstrate the homing property, the pGL4.51 vector that contained luciferase reporter gene was used to transfect BMSCs. Successfully transfected cells were injected around the skin wound in mice and in vivo imaging was done at 6, 12 and 24 hr post MSCs delivery. In vivo imaging revealed that transfected BMSCs migrated and concentrated predominantly toward the center of the wound. BMSCs were further evaluated for allogenic therapeutic potential in 44 clinical cases of spinal cord injuries (SCI) and compared with conventional therapy (control). Therapeutic potential as evaluated by different body reflexes and recovery score depicted significantly better results in stem cell‐treated group compared to control group. In conclusion, allogenic canine BMSCs can serve as potent therapeutic candidate in cell‐based therapies, especially for diseases like SCI, where the conventional medication is not so promising.