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1.
J Adv Res ; 5(6): 695-704, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25685538

ABSTRACT

The Toll-like receptor 2 (TLR2) gene of old world camels (Camelus dromedarius and Camelus bactrianus) was cloned and sequenced. The TLR2 gene of the dromedary camel had the highest nucleotide and amino acid identity with pig, i.e., 66.8% and 59.6%, respectively. Similarly, the TLR2 gene of the Bactrian camel also had the highest nucleotide and amino acid identity with pig, i.e., 85.7% and 81.4%, respectively. Dromedary and Bactrian camels shared 77.9% nucleotide and 73.6% amino acid identity with each other. Interestingly, the amidation motif is present in camel (Dromedary and Bactrian) TLR2 only, and the TIR domain is absent in Dromedary camel TLR2. This is the first report of the TLR2 gene sequence of Dromedary and Bactrian camels.

2.
Mol Biotechnol ; 56(2): 91-101, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23877894

ABSTRACT

The antiviral potential of small interfering RNAs (siRNAs) targeting rabies virus (RV) polymerase (L) and nucleoprotein (N) genes delivered through lentiviral vector was investigated. For in vitro evaluation, siRNAs expressing BHK-21 cell lines (BHK-L and BHK-N) were developed using transduction with Lenti-L and Lenti-N lentiviruses encoding siRNAs against RV-L and N genes, respectively. When these cell lines were challenged in vitro with RV Pasteur virus-11 (PV-11) strain, there was reduction in number of RV-specific foci and target gene transcripts indicating inhibitory effect on RV multiplication. For in vivo evaluation, mice were treated intracerebrally with lentiviruses and challenged with 20 LD50 of RV challenge virus standard-11 (CVS-11) strain by intramuscular route in masseter muscle. Five out of eight mice treated with Lenti-N survived indicating 62.5 % protection. The control and Lenti-L-treated mice died within 7-10 days indicating lethal nature of challenge virus and no protection. These results demonstrated that siRNA targeting RV-N could not only inhibit RV multiplication, but also conferred protection in mice against lethal RV challenge. These findings have implication on therapeutic use of siRNA targeting RV-N against RV infection.


Subject(s)
Antiviral Agents/pharmacology , DNA-Directed RNA Polymerases/metabolism , Lentivirus/genetics , Nucleocapsid Proteins/metabolism , RNA, Small Interfering/pharmacology , Rabies virus/drug effects , Viral Proteins/metabolism , Animals , Cell Line , Cricetinae , DNA-Directed RNA Polymerases/genetics , Genetic Vectors/administration & dosage , HEK293 Cells , Humans , Mice , Nucleocapsid Proteins/genetics , Rabies/therapy , Rabies virus/genetics , Rabies virus/metabolism , Viral Load , Viral Proteins/genetics , Virus Replication/drug effects
3.
Res Vet Sci ; 93(2): 1089-97, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22349592

ABSTRACT

A replicon-based DNA vaccine encoding VP2 gene of canine parvovirus (CPV) was developed by cloning CPV-VP2 gene into a replicon-based DNA vaccine vector (pAlpha). The characteristics of a replicon-based DNA vaccine like, self-amplification of transcripts and induction of apoptosis were analyzed in transfected mammalian cells. When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. The virus neutralization antibody and lymphocyte proliferative responses were higher than conventional CPV DNA vaccine and commercial CPV vaccine. These results indicated that DNA-launched replicon-based CPV DNA vaccine was effective in inducing both CPV-specific humoral and cellular immune responses and can be considered as effective alternative to conventional CPV DNA vaccine and commercial CPV vaccine.


Subject(s)
Capsid Proteins/metabolism , DNA, Viral/immunology , Parvovirus/metabolism , Vaccines, DNA/immunology , Viral Vaccines/immunology , Animals , Antibodies, Viral/blood , Apoptosis , Capsid Proteins/genetics , Dogs , Gene Expression Regulation, Viral , Injections, Intradermal , Parvovirus/genetics , Plasmids , RNA, Messenger/genetics , RNA, Messenger/metabolism
4.
Virus Res ; 163(1): 11-8, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21864591

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.


Subject(s)
Adenoviridae/genetics , Antiviral Agents/metabolism , Biological Products/metabolism , Brain/virology , RNA, Small Interfering/metabolism , Rabies virus/drug effects , Rabies/prevention & control , Animals , Antiviral Agents/administration & dosage , Biological Products/administration & dosage , Biological Therapy/methods , Brain/immunology , Cell Line , Cricetinae , Disease Models, Animal , Drug Carriers , Genetic Vectors , Mice , RNA, Small Interfering/genetics , Rabies/mortality , Rabies virus/genetics , Survival Analysis , Transduction, Genetic
5.
Res Vet Sci ; 93(1): 498-503, 2012 Aug.
Article in English | MEDLINE | ID: mdl-21767857

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 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.


Subject(s)
Antiviral Agents/therapeutic use , RNA, Small Interfering/therapeutic use , Rabies virus/drug effects , Rabies/drug therapy , Adenoviridae/genetics , Animals , Cell Line , Cricetinae , Female , Genetic Vectors/genetics , Humans , In Vitro Techniques , Male , Mice , RNA, Small Interfering/genetics , Rabies virus/genetics , Rabies virus/physiology , Virus Replication/drug effects
6.
Anim Biotechnol ; 22(2): 111-7, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21500112

ABSTRACT

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.


Subject(s)
Classical Swine Fever Virus , Classical Swine Fever/virology , Vaccines, Attenuated/genetics , Viral Vaccines/genetics , 3' Untranslated Regions/genetics , 5' Untranslated Regions/genetics , Animals , Base Sequence , China , Classical Swine Fever/genetics , Classical Swine Fever Virus/classification , Classical Swine Fever Virus/genetics , Genome, Viral , India , Nucleic Acid Conformation , Nucleotides/genetics , Phylogeny , RNA, Viral/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , Swine , Viral Vaccines/classification
7.
Vet Microbiol ; 136(1-2): 36-44, 2009 Apr 14.
Article in English | MEDLINE | ID: mdl-19081687

ABSTRACT

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.


Subject(s)
Glycoproteins/immunology , Rabies Vaccines/pharmacology , Rabies virus/immunology , Rabies/veterinary , Viral Envelope Proteins/immunology , Animals , Antibodies, Viral/blood , Cricetinae , Cytokines/immunology , Flow Cytometry , Immunity, Cellular/immunology , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Immunophenotyping , Mice , Rabies/immunology , Rabies/prevention & control , Rabies/virology , Rabies Vaccines/immunology , Rabies virus/genetics , Survival Analysis , Vaccination/methods , Vaccination/veterinary
8.
Vaccine ; 26(51): 6592-601, 2008 Dec 02.
Article in English | MEDLINE | ID: mdl-18848857

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.


Subject(s)
Antigens, Viral/immunology , Glycoproteins/immunology , Rabies Vaccines/immunology , Rabies virus/immunology , Rabies/prevention & control , Viral Envelope Proteins/immunology , Animals , Antibodies, Viral/blood , Apoptosis , Cell Proliferation , Cells, Cultured , Cricetinae , Cytokines/immunology , Immunization, Secondary , Immunoglobulin G/blood , Mice , Rabies Vaccines/genetics , Replicon , Sindbis Virus/immunology , Vaccines, DNA/immunology
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