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Abstract: 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 107 plaque forming units of either rAdV-L or rAdV-N and subsequent lethal RV infection (15–20LD50 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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 (PGF 2α and PGE 2 ) 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 enzyme's level. Among the four subtypes of EP gene, we have observed highly significant expression difference in EP2 followed by EP3 after implantation. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]