V.K. Chaturvedi

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.

Effect of Zinc Supplementation from Different Sources on Growth, Nutrient Digestibility, Blood Metabolic Profile, and Immune Response of Male Guinea Pigs

Forty weaned male guinea pigs of 208.20±6.62 g mean body weight were divided into 4 groups of 10 animals in a randomized block design. All of the guinea pigs were fed a basal diet [25% ground maize hay, 30% ground maize grain, 22% ground chickpea (Cicer arietinum L.), 9.5% deoiled rice bran, 6% soybean meal, 6% fish meal, 1.45% mineral supplement (without Zn) and 0.05% ascorbic acid] and available green fodder. Group I served as the control (no Zn supplementation), whereas 20 ppm Zn was added in the diet in groups II, III, and IV either as zinc sulfate (ZnSO4), zinc amino acid complex (ZAAC), and ZnSO4+ ZAAC in equal parts, respectively. Experimental feeding lasted for 70 d, including a 3-d digestibility trial. Blood was collected through cardiac puncture from four animals in each group at d 0 and subsequently at the end of experimental feeding. After 40 d of experimental feeding, four animals from each group were injected with 0.4 mL of Brucella abortus cotton strain-19 vaccine to assess the humoral immune response of the animals. After 10 wk of study, four animals from each group were sacrificed to study the concentration of Zn, Cu, Co, Fe, and Mn in the liver, pancreas and spleen. Results revealed no significant difference in the feed intake, body weight gain, and digestibility of the nutrients, except for crude protein (CP) digestibility, which was significantly (p<0.05) lower in group IV. Although concentrations of serum glucose, Ca, and P and the albumin:globulin (A:G) ratio were similar in the different groups, the total protein, albumin, and serum alkaline phosphatase activity were higher in all of the Zn-supplemented groups on d 70. The serum Zn levels at the end of experimental feeding were significantly higher in groups II and III, whereas serum Mn levels were found to be significantly (p<0.05) higher in groups III and IV. The organ weights (as percentage of body weights) did not show any differences among the treatment groups. Although the Mn concentration was significantly (p<0.05) higher in the pancreas, the Cu concentration was significantly (p<0.05) reduced in the spleen in all of the Zn-supplemented groups. The humoral immune response (antibody titer values) on d 14 of vaccination was significantly (p<0.05) higher in all of the Zn-supplemented groups. It was concluded that the 20-ppm level of Zn in the diet might be adequate for growth and nutrient utilization in guinea pigs, but supplementation of 20-ppm zinc significantly improved the immune response and impact was more prominent with the ZAAC (organic source) compared to ZnSO4 (inorganic source).

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.

Poly(lactide-co-glycolide) microspheres: A potent oral delivery system to elicit systemic immune response against inactivated rabies virus

Rabies is an endemic, fatal zoonotic disease in the developing countries. Oral vaccination strategies are suitable for rabies control in developing countries. Studies were performed to investigate the suitability of poly(lactide-co-glycolide) (PLG) microspheres as an oral delivery system for beta-propiolactone inactivated concentrated rabies virus (CRV). Immune responses induced by encapsulated (PLG+CRV) and un-encapsulated inactivated rabies virus after oral and intraperitoneal route administrations were compared. The anti-rabies virus IgG antibody titer, virus neutralizing antibody (VNA) titers obtained by mouse neutralization test (MNT) and IgG2a and IgG1 titers of mice group immunized orally with PLG+CRV showed significantly (p<0.001) higher response than the group immunized orally with un-encapsulated CRV. There was no significant difference (p>0.05) between groups inoculated by intraperitoneal route. The stimulation index (SI) obtained by lymphoproliferation assay of PLG+CRV oral group also showed significantly (p<0.001) higher response than the group immunized orally with un-encapsulated CRV, suggesting that oral immunization activates Th1-mediated cellular immunity. Immunized mice of all experimental groups were challenged intracerebrally with a lethal dose of virulent rabies virus Challenge Virus Standard (CVS). The survival rates of mice immunized orally with PLG+CRV and CRV alone were 75% and 50%, respectively, whereas intraperitoneally immunized groups showed 100% protection. The overall results of humoral, cellular immune response and survival rates of mice immunized orally with PLG+CRV were significantly (p<0.001) higher than those of mice immunized orally with CRV alone. These data suggest that the PLG encapsulated inactivated rabies virus can be used for oral immunization against rabies.

Development of a simple and rapid method for the specific identification of organism causing anthrax by slide latex agglutination

A specific latex agglutination test (LAT) based on anti‐PA (protective antigen) antibodies having detection limit of 5 × 104 formalin treated Bacillus anthracis cells or 110 ng of PA was optimized in this study. The optimized LAT could detect anthrax toxin in whole blood as well as in serum from the animal models of anthrax infection. The protocol is a simple and promising method for the specific detection of bacteria causing anthrax under routine laboratory, as well as in field, conditions without any special equipments or expertise.

Construction of infectious cDNA clone derived from a classical swine fever virus field isolate in BAC vector using in vitro overlap extension PCR and recombination.

To develop reverse genetics system of RNA viruses, cloning of full-length viral genome is required which is often challenging due to many steps involved. In this study, we report cloning of full-length cDNA from an Indian field isolate (CSFV/IVRI/VB-131) of classical swine fever virus (CSFV) using in vitro overlap extension PCR and recombination which drastically reduced the number of cloning steps. The genome of CSFV was amplified in six overlapping cDNA fragments, linked by overlap extension PCR and cloned in a bacterial artificial chromosome (BAC) vector using in vitro recombination method to generate full-length cDNA clone. The full-length CSFV cDNA clone was found stable in E. coli Stellar and DH10B cells. The full-length RNA was transcribed in vitro using T7 RNA polymerase and transfected in PK15 cells using Neon-tip electroporator to rescue infectious CSFV. The progeny CSFV was propagated in PK15 cells and found indistinguishable from the parent virus. The expression of CSFV proteins were detected in cytoplasm of PK15 cells infected with progeny CSFV at 72 h post-infection. We concluded that the in vitro overlap extension PCR and recombination method is useful to construct stable full-length cDNA clone of RNA virus in BAC vector.

Development of a simple method for the rapid identification of organisms causing anthrax by coagglutination test

A protective antigen (PA) based coagglutination test was optimized in the present study for the specific and sensitive identification of bacteria causing anthrax in a cost effective and less risky manner. The test showed 100% specificity and sensitivity up to 9 × 10(3) formalinized vegetative cells or 11 ng of PA. The optimized test also detected anthrax toxin directly from the serum as well as blood of anthrax infected animals indicating the potential application for direct diagnosis of anthrax under field conditions.

Prokaryotic Expression of Ovis aries Conglutinin Encoding Neck and Carbohydrate Recognition Domain and its Functional Characterization

Conglutinin, a soluble pattern recognition receptor of innate immune system in bovines is known for its potential defensive activity against microorganisms either by direct agglutination in the presence of calcium or by acting as opsonin. In the present study, sheep (Ovis aries) conglutinin encoding neck and carbohydrate recognition domain (rSCGN) was expressed in the E coli BL21 expression host. The recombinant conglutinin revealed molecular weight of 27 kDa in SDS PAGE and also in western blotting using antibuffalo conglutinin polyclonal serum. The protein was characterized further for its functional activity in various assays. In ELISA based sugar and LPS binding assay, the rSCGN revealed its high binding activity toward N-acetyl glucosamine and E. coli LPS in the presence and the absence of calcium ions, respectively. Hemagglutination of chicken red blood cells caused by Newcastle disease virus was not inhibited in the presence of rSCGN as it lacked complete collagenous region present in the native protein. In virus neutralization test, the recombinant protein was found to reduce multiplication of bovine herpes virus-1 propagated in MDBK cells. This prokaryotically expressed 27 kDa recombinant sheep conglutinin can serve as antigen in future studies to develop sandwich ELISA for assessing the level of native conglutinin in sheep serum.

Complete Genome Sequence of an Indian Field Isolate of Classical Swine Fever Virus Belonging to Subgenotype 1.1

ABSTRACT We report the complete genome sequence of an Indian field isolate of classical swine fever virus (CSFV) belonging to predominant subgenotype 1.1 prevalent in India. This report will help in understanding the molecular diversity of CSFV strains circulating worldwide and to select and develop a suitable vaccine candidate for classical swine fever (CSF) control in India.

Effect on immune response against Pasteurella multocida capsular type a:1 and a:4 on supplementation with protein purified derivatives of Mycobacterium bovis in chicken

The adjuvant potential of tuberculo-protein of Mycobacterium bovis and Montanide ISA-206 adjuvants in different combination with Pasteurella multocida serotype A:1 and A:4 whole cell antigens have been evaluated in chicken model. Four different groups of chicken were immunized with different combination of adjuvants and humoral immune response was assessed by indirect ELISA. The immune response study revealed that chicken immunized with whole cell antigen in combination with Protein precipitated derivative (PPD) as well as montanide ISA-206 elicited a robust humoral immune response. To assess the protective ability two groups of chicken immunized with P.multocida A:1 were challenged with 1 MLD of Pasteurella multocida A:1; whereas two other groups of chicken immunized with P.multocida A:4 bacterin were challenged with 100 MLD of P.multocida A:4. Challenge studies indicated that both the groups of chicken immunized with P. multocida A:1 whole cell antigen adjuvanted with both Montanide ISA-206 and Mycobacterium bovis PPD as well as only Montanide ISA-206 conferred 100 % protection against P multocida A:1. However two other groups immunized with P.multocida A:4 bacterins could not sustain 100 MLD dose of direct challenge and there were 100% death of all the birds in both the immunized group, but the group immunized with both Montanide and PPD showed delayed death. * Corresponding author