Rabies Virus Glycoprotein Gene Research Articles

Genetic analyses of rabies virus glycoprotein and nucleoprotein gene sequences reveal the emergence of multiple lineages in animals in Arkhangai province, a central region of Mongolia.

Genetic characterizations of rabies viruses circulating in carnivore and non-carnivore animals were investigated for the first time in Arkhangai province, a central region of Mongolia. Also, glycoprotein gene of the rabies virus was sequenced for the first time in Mongolia. The nucleotide sequences of the glycoprotein and nucleoprotein genes were analysed, revealing the presence of multiple lineages in this area. Of particular concern are the lineages identified in carnivores, which might emerge to spread throughout Mongolia, further facilitating transboundary transmission to neighbouring countries, including China and Russia.

Molecular genetic characterization of rabies virus glycoprotein gene sequences from rabid dogs in Bangkok and neighboring provinces in Thailand, 2013-2014.

Because of its association with dogs, rabies virus (RABV) is still endemic in Thailand, where it is a serious public health problem. The genetic characterization of RABV in Thailand is limited. Therefore, in this study, we investigated the molecular epidemiology and genetic diversity of RABV in the endemic area. Viral RNA from 48 brain specimens from rabid dogs, collected in Bangkok and seven neighboring provinces in 2013-2014, was extracted and sequenced. The complete rabies glycoprotein (G) gene sequences (1575 nt) were aligned, and a phylogenetic analysis was performed using the maximum-likelihood method. All of the Thai rabies virus isolates belonged to lyssavirus genotype 1 and clustered in the same lineage as isolates from South East Asia (SEA) and China. The Thai rabies virus isolates formed two distinct clades, THA-1 and THA-2. Clade THA-1 was the predominant clade and could be divided into two subclades, THA-1A and THA-1B. Clade THA-2 was closely associated with human Thai isolates collected in a previous study. The overall mean rate of evolution based on the G gene was approximately 1.56 × 10(-4) substitutions/site/year. The genetic identities among the isolates from Thailand and other SEA countries were >88.4 % at the nucleotide sequence level and 95 % at the amino acid sequence level. The deduced amino acid sequences of the G proteins of the RABV isolates were compared. A single amino acid change (N194T) in subclade THA-1A distinguished the Thai RABV isolates from other RABV isolates. Our results suggest that these Thai dog RABV isolates share a common ancestor with the RABV isolates circulating in the endemic regions of SEA countries and China. Furthermore, there were strong genetic relationship to RABV from Cambodia, Vietnam and Laos. These data extend our understanding of the relatedness and genetic variation of RABV in Thailand.

Enhancement of immunogenicity and efficacy of a plasmid DNA rabies vaccine by nanoformulation with a fourth-generation amine-terminated poly(ether imine) dendrimer

PurposeDelayed onset of, and low magnitude of, protective immune responses are major drawbacks limiting the practical utility of plasmid vaccination against rabies. In this study we evaluated whether nanoformulation with the novel poly(ether imine) (PETIM) dendrimer can enhance the immunogenicity and efficacy of a plasmid-based rabies vaccine.Materials and methodsA plasmid vaccine construct (pIRES-Rgp) was prepared by cloning the full-length rabies virus glycoprotein gene into pIRES vector. Drawing upon the results of our previous study, a dendriplex (dendrimer-DNA complex) of pIRES-Rgp was made with PETIM dendrimer (10:1 w/w, PETIM:pIRES-Rgp). In vitro transfection was done on baby hamster kidney (BHK)-21 cells to evaluate expression of glycoprotein gene from pIRES-Rgp and PETIM-pIRES-Rgp. Subsequently, groups of Swiss albino mice were immunized intramuscularly with pIRES-Rgp or PETIM-pIRES-Rgp. A commercially available cell culture rabies vaccine was included for comparison. Rabies virus neutralizing antibody (RVNA) titers in the immune sera were evaluated on days 14, 28, and 90 by rapid fluorescent focus inhibition test. Finally, an intracerebral challenge study using a challenge virus standard strain of rabies virus was done to evaluate the protective efficacy of the formulations.ResultsProtective levels of RVNA titer (≥0.5 IU/mL) were observed by day 14 in animals immunized with pIRES-Rgp and its dendriplex. Notably, PETIM-pIRES-Rgp produced 4.5-fold higher RVNA titers compared to pIRES-Rgp at this time point. All mice immunized with the PETIM-pIRES-Rgp survived the intracerebral rabies virus challenge, compared with 60% in the group which received pIRES-Rgp.ConclusionOur results suggest that nanoformulation with PETIM dendrimer can produce an earlier onset of a high-titered protective antibody response to a plasmid-based rabies vaccine. PETIM dendriplexing appears to be an efficacious nonviral delivery strategy to enhance genetic vaccination.

The feasibility of rabies virus-vectored immunocontraception in a mouse model

Immunocontraceptive vaccines may be an alternative to surgical sterilization. Dual rabies vaccination and dog population management is a helpful tool for rabies prevention. A synthetic gonadotropin-releasing hormone (GnRH) peptide coupled to a carrier protein or T cell epitope is efficacious in inducing immunocontraception in a variety of mammals. However, virus-vectored GnRH recombinant vaccines have advantages over the conjugation method. In a previous in vitro study, we were able to insert a GnRH-coding sequence into the rabies virus (RABV) glycoprotein (G) gene, and the recombinant viruses grew to high titers in cells. Here, we further focused on the RABV G in accepting various copy numbers of GnRH. We demonstrated although RABV G protein with up to 4 copies of GnRH was well expressed, the recombinant virus was recovered only when 2 copies of GnRH (20 amino acids) were incorporated into the G, indicating a possible insertion limit in making a full infectious clone. The investigation provides insight into the utility of RABV G as a carrier for small peptides and its suitability for vaccine studies. Following our previous study, we selected ERAg3p/2GnRH and tested the construct in mice. The vaccine induced ⩾80% infertility after three doses without any adjuvant, in live (8 of 10 mice infertility) or inactivated (13 of 14 mice infertility) formulations; while the pregnancy rate was 100% (10 of 10 mice) in the controls. This initial success of immunocontraception in mice is promising, and we are now optimizing the vaccine formulation by using adjuvants and exploring novel delivery methods to minimize the dosage.

Development of Genetically Recombinant Rabies Vaccine

Purpose: Pakistan is one of the few countries where Rabies is endemic and a great threat to humans as well as live stocks. A large number of individuals died of rabies exposure due to either the limited access to rabies vaccines or the side-effects of sheep brain -originated Sample vaccine. Cell culture-derived rabies vaccines are mostly unaffordable to the needed population under rabies threat. Development of a safer and more affordable vaccine is necessary in Pakistan. Here, we developed two novel recombinant rabies virus vaccines using a local Pakistan rabies virus glycoprotein gene, and tested the efficacy of vaccines in mice. Methods: The glycoprotein gene (RVG) of vector ERAg3p and ERAg3m was substituted, respectively, with a modified Pakistani RVG. The resulting recombinant vectors were applied for reverse genetics to recover two vaccine viruses, PK-SG and PK-DG. The efficacy of PK-SG and PK-DG were tested in mice by intramuscular injection and oral delivery. Results: All mice survived the challenge after intramuscular vaccination using PK-SG, or PK-DG. In the oral vaccination groups, 80% mice with PK-SG and 90% mice with PK-DG survived the challenge. Meanwhile, 80% of the unvaccinated control mice succumbed after challenge. The mean rabies virus neutralizing antibody titers was ≥0.5 IU/ml in all vaccinated groups. Conclusion: Our results demonstrated the efficacy of PK-SG and PK-DG in rabies vaccination in mice. The two recombinant virus strains may be good vaccine candidates for the target animals and humans in Pakistan. Detailed investigations are necessary in the future.

Pyrosequencing of the rabies virus glycoprotein gene to demonstrate absence of vaccine-associated rabies cases following oral vaccination

Replication competent vaccines have been used successfully for the control of terrestrial rabies, mainly in wildlife; however, these vaccine strains occasionally may induce rabies. In this study, a pyrosequencing protocol for the rapid identification of vaccine-associated rabies viruses was applied to the 2008-2011 Italian epidemic. There was no evidence of vaccine-associated rabies cases following oral vaccination of foxes with the SAG2 and SADB19 vaccine strains.

Cloning and Expression of Rabies Virus Glycoprotein Gene into Eukaryotic System

Cloning and Expression of Rabies Virus Glycoprotein Gene into Eukaryotic System

Coexpression of double or triple copies of the rabies virus glycoprotein gene using a ‘self-cleaving’ 2A peptide-based replication-defective human adenovirus serotype 5 vector

Rabies virus glycoprotein (RVG) is a major structural protein and antigen of rabies virus that induces a highly immunogenic response. In the present study, we have used 2A self-cleaving sequence of the foot-and-mouth disease virus (FMDV) to express double or triple copies of the RVG from a single open reading frame derived from human adenovirus 5 (AdHu5). The recombinant adenoviruses produce similar virus titers, indicating that the insertion of double or triple copies of the RVG gene linked with the FMDV 2A sequence does not affect virus replication. The RVG was efficiently expressed by constructs containing the 2A sequence and retained its antigenic property. The 2A self-cleaving peptide mediated efficient generation of individual glycoprotein in transient expression assay and did not lead to an altered surface distribution of RVG. Flow cytometry demonstrated that the expression levels of RVG were improved in recombinant Ads carrying multiple RVG gene copies. We conclude that ribosome skipping induced by the FMDV 2A sequence is an effective strategy to express multiple glycoprotein genes of rabies virus in adenoviruses and 2A-containing recombinant Ads may represent an attractive alternative to other coexpression strategies for multiple gene expression.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1 L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 °C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained ∼1.5–3 × 10 7 cells/mL after 3–4 days of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 μg/10 7 cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO 4 induction attaining specific productivities of 1.5–2 μg/10 7 cells at days 4–5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 μg/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5–3 mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function.

Oral immunization of raccoons and skunks with a canine adenovirus recombinant rabies vaccine

Oral vaccination is an important part of wildlife rabies control programs. Currently, the vaccinia-rabies glycoprotein recombinant virus is the only oral rabies vaccine licensed in the United States, and it is not effective in skunks. In the current study, captive raccoons and skunks were used to evaluate a vaccine developed by incorporating the rabies virus glycoprotein gene into a canine adenovirus serotype 2 vector (CAV2-RVG). Seven of 7 raccoons orally vaccinated with CAV2-RVG developed virus neutralizing antibodies and survived lethal challenge. Five of 5 and 6 of 6 skunks in 2 experimental groups receiving 10-fold different dilutions of CAV2-RVG developed neutralizing antibodies and survived challenge. The results of this preliminary study suggest that CAV2-RVG stimulates protective immunity against rabies in raccoons and skunks.

Safety studies on an adenovirus recombinant vaccine for rabies (AdRG1.3-ONRAB ®) in target and non-target species

A replication-competent human adenovirus vector in which the rabies virus glycoprotein gene was inserted (AdRG1.3-ONRAB ®) was given by direct instillation into the oral cavity to representatives of three wildlife vector species of concern in Ontario (red fox, raccoon and striped skunk) and to a variety of non-target wildlife species, domestic and laboratory species. Despite use of a relatively high dose of vaccine, no untoward clinical signs were observed. Subsequent to vaccine exposure, detection of vaccine virus in lung, spleen, intestine, liver, kidney and brain of each animal was attempted using an ONRAB ®-specific assay combining PCR with Southern blotting (PCR–SB). Of the 1280 tissue samples obtained from vaccinates or contact animals, 18 (1.4%) were found to be PCR–SB positive. Virus isolation attempts were performed utilizing cell culture for all PCR–SB positive tissues and a selection of PCR–SB negative tissues. Histological examination performed on all PCR–SB positive tissues failed to identify lesions attributed to the vaccine. A quantitative real-time PCR was used to determine the excretion of the vaccine in feces and in the oral cavity with 0.8% of oral swabs and 6.8% of fecal specimens found to be positive. The low rates of recovery of vaccine virus from tissues, feces and the oral cavity suggest that the likelihood of ONRAB ® causing a negative impact on wildlife species is unlikely.

Rabies virus glycoprotein expression in Drosophila S2 cells. I: Design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression

The cDNA encoding the rabies virus glycoprotein (RVGP) gene was cloned in expression plasmids under the control of the inductive metallothionein promoter. They were designed in order to bear or not a secretion signal (i) and a cDNA coding for the selection hygromycin. These vectors were transfected into S2 cells, cell populations selected and subpopulations were then obtained by reselection with hygromycin. Cell cultures were examined for kinetics of cell growth, detection of RVGP mRNA and expression of RVGP. All cell populations were shown to express the RVGP mRNA upon induction. S2MtRVGPHy cell population, transfected with one vector that contains RGPV gene and selection gene, was shown to express higher amounts of RVGP as evaluated by flow cytometry (∼52%) and ELISA (0.64 μg/10 7 cells at day 7). Subpopulation selection allowed a higher RVGP expression, specially for the S2MtRVGPHy + (5.5 μg/10 7 cells at day 7). NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MtRVGPHy + (8.4 μg/10 7 cells at day 7). SF900II medium leading to a higher S2MtRVGPHy +cell growth allowed a higher final RVGP synthesis in this cell culture. RVGP synthesis may be optimized by the expression/selection vectors design, cell subpopulations selection, chromatin exposure and culture medium employed.

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 10 μg 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 10 μg dose was effective in inducing immune responses and protection similar to rabies DNA vaccine.

Immune modulating effect by a phosphoprotein-deleted rabies virus vaccine vector expressing two copies of the rabies virus glycoprotein gene

The type of immune response induced by a vaccine is a critical factor that determines its effectiveness in preventing infection or disease. Inactivated and live rabies virus (RV) vaccine strains elicit an IgG1-biased and IgG1/IgG2a-balanced antibody response, respectively. However, IgG2a antibodies are potent inducers of anti-viral effector functions, and therefore, a viral vaccine vector that can elicit an IgG2a-biased antibody response may be more effective against RV infection. Here we describe the humoral immune response of a live replication-deficient phosphoprotein (P)-deleted RV vector (SPBN-ΔP), or a recombinant P-deleted virus that expresses two copies of the RV glycoprotein (G) gene (SPBN-ΔP-RVG), and compare it to a UV-inactivated RV. Mice inoculated with UV-inactivated RV induced predominantly an IgG1-specific antibody response, while live recombinant SPBN-ΔP exhibited a mixed IgG1/IgG2a antibody response, which is consistent with the isotype profiles from the replication-competent parental viruses. Survivorship in mice after pathogenic RV challenge indicates a 10-fold higher efficiency of live SPBN-ΔP compared to UV-inactivated SPBN-ΔP. In addition, SPBN-ΔP-RVG induced a more rapid and robust IgG2a response that protected mice more effectively than SPBN-ΔP. Of note, 10 3 ffu of SPBN-ΔP-RVG-induced anti-RV antibodies that were 100% protective in mice against pathogenic RV challenge. The increased immune response was directed not only against RV G but also against the ribonucleoprotein (RNP), indicating that the expression of two RV G genes from SPBN-ΔP-RVG enhances the immune response to other RV antigens as well. In addition, Rag2 mice inoculated intramuscularly with 10 5 ffu/mouse of SPBN-ΔP showed no clinical signs of rabies, and no viral RNA was detected in the spinal cord or brain of inoculated mice. Therefore, the safety of the P-deleted vectors along with the onset and magnitude of the IgG2a-induced immune response by SPBN-ΔP-RVG indicate that this vector holds great promise as either a therapeutic or preventative vaccine against RV or other infectious diseases.

Glycoprotein gene relocation in rabies virus

Unlike vesicular stomatitis virus, rabies virus glycoprotein gene has not been successfully relocated closer to promoter–proximal regions by reverse genetics. Here we describe an efficient system for the Evelyn-Rokitnicki-Abelseth (ERA) rabies virus with the glycoprotein gene switched with the matrix protein gene, creating a reshuffled virus ERAgm (gene order N-P-G-M-L). With the aid of an autogene plasmid, the T7 RNA polymerase containing a nuclear location signal from the SV40 large T antigen facilitated virus recovery. The rearranged ERAgm rabies virus replicated as well as the parental ERA (gene order N-P-M-G-L) virus, reaching 109ffu/ml in infected BSR cells. The altered glycoprotein gene position in viral genome presented an alternative way to study the pathogenicity of rabies virus. This also provides a potential novel method for rabies vaccine development.

Generation and evaluation of a recombinant modified vaccinia virus Ankara vaccine for rabies

Modified vaccinia virus Ankara (MVA) has become a vaccine vector of choice for recombinant vaccine development. A MVA-based rabies vaccine would be advantageous for use as a vaccine for dogs (and wildlife), particularly if it proves innocuous and efficacious by the oral route. Here, the generation and immunological testing of a recombinant MVA expressing a rabies virus glycoprotein gene is described. In a murine model, higher dosages of recombinant MVA were needed to induce equivocal immune responses as with Vaccinia Copenhagen or Vaccinia Western Reserve recombinants, when administered by a parenteral route. The MVA recombinant was not immunogenic or efficacious when administered per os in naïve mice. The ability of the recombinant MVA to induce anamnestic responses in dogs and raccoons was also investigated. Recombinant MVA boosted humoral immune responses in these animals when administered peripherally, but not when administered orally.

Genetic transformation of cantaloupe melon (Cucumis melo L.) with the rabies virus glycoprotein gene (PRGSpRgp) and immunisation studies in mice

SummaryVaccination is the only means to control rabies, a deadly neuroviral disease with no cure. The use of genetically-altered plants to produce vaccines is gaining importance as it is cost-effective and guarantees freedom from human or animal pathogens. This study reports on Agrobacterium-mediated transformation of Cucumis melo with a rabies virus (strain ERA) glycoprotein gene (PRGSpRgp). The presence of the rabies glycoprotein gene in the DNA of transformed melon leaves was confirmed by PCR amplification. Synthesis of the rabies glycoprotein was detected in transformed cantaloupe fruit by SDS-PAGE and western immunoblotting. Partially purified, recombinant rabies glycoprotein from transformed cantaloupe melon fruit was injected intramuscularly and intraperitoneally into mice, and resulted in the induction of anti-PRGSpRgp antibodies.When these mice were challenged with rabies virus strain ERA they survived infection due to the accumulation of sufficient quantities of anti-rabies glycoprotein antibodies (0.6 – 1.6 IU ml–1). These results demonstrate the accumulation of rabies glycoprotein in edible transgenic cantaloupe melon fruit, which offers the promise of biomedical exploitation.

Rabies Vaccine Baits, Pennsylvania

To the Editor: Oral rabies vaccine (ORV) programs control rabies in terrestrial reservoir species by distributing vaccine in baits (1). The current US-licensed ORV consists of a rabies virus glycoprotein gene inserted into the thymidine kinase gene of an attenuated strain of the Copenhagen vaccinia virus (V-RG) (2). Safety experience includes extensive animal studies (2,3) in which significant adverse effects were seen only with parenteral (but not mucosal) exposure of nude mice to V-RG (4). Usage monitoring (4,5) found only 1 human adverse complication to date (6).

A simple immuno-capture ELISA to estimate rabies viral glycoprotein antigen in vaccine manufacture

Rabies is an endemic, fatal zoonotic disease in the developing countries. Prevention and post-exposure therapy require safe and efficacious vaccines. The vaccine potency depends on the amount of immunogenic rabies viral glycoprotein antigen in the vaccine preparation. In order to estimate the rabies viral glycoprotein antigen, a specific monoclonal antibody was developed and used in an immuno-capture ELISA (IC-ELISA). The monoclonal antibody binds a conformational epitope on the natively folded rabies viral glycoprotein as indicated by specific, membrane fluorescence on unfixed, rabies virus infected murine neuroblastoma (MNA) cells and glycoprotein gene encoding plasmid transfected COS cells. In addition, the monoclonal antibody competes with and blocks a glycoprotein antigen site III binding monoclonal antibody (mAb-D1, Institut Pasteur, Paris, France). The monoclonal antibody was used in an IC-ELISA using an in-house standard to quantify the rabies viral glycoprotein antigen in 12 vaccine preparations with potency values ranging from 4 to 18 IU. The results indicated a good correlation with the NIH mouse potency assay ( r = 0.83). The immuno-capture ELISA described in this study can be used to quantify the immunogenic rabies viral glycoprotein antigen in the inactivated rabies viral antigen preparation in a simple and rapid format, which enables better vaccine formulation.

Genetic analysis of dog rabies viruses circulating in Bangkok

The genetic diversity of the rabies virus glycoprotein (G) gene isolated from individual rabid dogs (inter-hosts) and within a single infected dog (intra-host) has been analyzed in an effort to better understand selective pressures and population shifts among rabies viruses circulating in Bangkok. Comparison of individual master sequences among inter-hosts revealed that the dog virus isolates circulating in Bangkok were phylogenetically closely related. The ectodomain of the glycoprotein was highly conserved among the virus isolates. Furthermore, the genetic diversity of the G gene within an intra-host was assessed by comparing the cloned sequences in the virus population. The comparisons revealed that rabies virus circulating in an intra-host consisted of closely related heterogenous populations with minor substitutions at nucleotide (0.19%) and amino acid levels.