CVS Strain Of Rabies Virus Research Articles

Вплив температурних режимів зберігання та складу захисних середовищ на збереженість вірусу сказу штаму CVS

The impact of protective media supplemented with sucrose, glycerol, and maltose on the rabies virus CVS strain preservation during storage at 37, 5, –20, –80, and –196ºC has been studied. The temperature regimen of 37ºC was found as not suitable for virus storage, but temperature of 5ºC could be used for a short-term storage in corresponding protective medium in case of production need. It was shown that storage at –80 and –196ºC in DMEM-based preservation media containing 0.5% fetal bovine serum, supplemented with either 5% sucrose, or 5% glycerol, or 5% maltose, or sucrose and glycerol mixture provided high levels of the rabies virus CVS strain infectious activity within 24 months (observation period). At –20ºC, in a growth medium with sucrose and mixture of sucrose and glycerol, after 6 months 79–82% of the initial virus activity were preserved, and after 12 months that was 69–72%. Upon reaching –80ºC and during subsequent storage of virus samples at this temperature in the studied preservation media, the secondary crystallization of concentrated dissolved substances was prolonged. Probl Cryobiol Cryomed 2020; 30(2): 148–157

Хранение в течение года стандартного фиксированного штамма вируса бешенства CVS в защитных средах с сахарозой, глицерином и мальтозой при разных температурах

Хранение в течение года стандартного фиксированного штамма вируса бешенства CVS в защитных средах с сахарозой, глицерином и мальтозой при разных температурах

Development of an immunoenzyme test system for diagnosis if rabies

Rabies remains one of the most serious and pressing problems for veterinary science and medicine. According to the data of Rosselkhoznadzor Information and analytical center, the Russian Federation has been an area of natural focal endemicity for many years [13]. An important stage in the control and eradication of rabies in its laboratory diagnosis by detection of virus antigen in the pathological material. The aim of the study was to design an enzyme-linked immunosorbent assay (ELISA) test kit intended for the laboratory diagnosis of rabies in agricultural, domestic, and wild carnivores. Obtaining specific components for the kit, the GNKI “Sheep” master seed strain of rabies virus, the standard CVS strain of rabies virus, and white mice of the BALB line were used.In order to obtain high-activity of specific immunoglobulins (Igs ), the processes of antirabies serum production, immunoglobulin isolation, and purification have been improved as well as the conditions for immunoglobulin conjugation with peroxidase. It was found that the binding ratio should not to be lower than 0.45–0.6.A method of inactivation of rabies virus antigen has been developed, which eliminates the risk of being infected for laboratory workers and shows high specific activity in serological tests. The optimal concentrations of the reactants (immunoglobulin, antigen, conjugated) were determined; basic conditions for conducting the reaction were identified (pH of the carbonate/bicarbonate coating buffer, the optimum temperature, and duration of incubation) that ensure the required level of accuracy and sensitivity of the method.As a result of the conducted studies, a "Kit of preparations for the laboratory diagnosis of animal rabies in animals by ELISA method" was developed. The correlation between results obtained by the immunofluorescence (IF) method and the enzyme-linked immunoadsorbent assay (ELISA) methods was determined; the compliance coefficient constituted 98.8%, the sensitivity threshold of ELISA method being 3.3 Ig LD50 / ml. The sensitivity, specificity, and reproducibility of the developed test system were determined through laboratory and production tests.Â

An effective and simplified DO-stat control strategy for production of rabies glycoprotein in Pichia pastoris

The glycoprotein (G-protein) of rabies virus is responsible for viral attachment to the host cell surface and induces virus neutralization antibodies. In the present study, the G-protein gene of rabies virus CVS strain was cloned, sequenced and expressed in the yeast, Pichia pastoris, as a secreted protein, using a simplified DO-stat control feeding strategy. This strategy involves the addition of methanol when the dissolved oxygen (DO) level rises above the setpoint avoiding methanol accumulation and oxygen limitation. The G-protein expression was evaluated by SDS-PAGE, ELISA, and western blot assays. Like native G-protein, the recombinant G-protein was found reactive when it was challenged against specific antibodies. The data indicate that the recombinant G-protein can be easily expressed and isolated, and may be useful as a safe source in the production of diagnostic kits and subunit vaccines to prevent rabies.

Rabies virus matrix protein induces apoptosis by targeting mitochondria

Apoptosis, as an innate antiviral defense, not only functions to limit viral replication by eliminating infected cells, but also contribute to viral dissemination, particularly at the late stages of infection. A highly neurotropic CVS strain of rabies virus induces apoptosis both in vitro and in vivo. However, the detailed mechanism of CVS-mediated neuronal apoptosis is not entirely clear. Here, we show that CVS induces apoptosis through mitochondrial pathway by dissipating mitochondrial membrane potential, release of cytochrome c and AIF. CVS blocks Bax activation at the early stages of infection; while M protein partially targets mitochondria and induces mitochondrial apoptosis at the late stages of infection. The α-helix structure spanning 67–79 amino acids of M protein is essential for mitochondrial targeting and induction of apoptosis. These results suggest that CVS functions on mitochondria to regulate apoptosis at different stages of infection, so as to for viral replication and dissemination.

Rabies virus-like particles comprised of G and M proteins protect BALB/c mice from lethal dose challenging

Virus-like particles (VLPs) were super-molecular aggregates without any replicative genetic information, so they have been suggested to be a promising vaccine approach with several commercial practices. In this study, we tried to constructed rabies VLPs with recombinant baculovirus contained G and M proteins from CVS strain of rabies virus. Expression products can be easily purified by ultrafiltration and gradient centrifugation, and then identified with ELISA, West-blotting and TEM, respectively. A continuous high serum neutralizing antibody titers extended weeks had been measured with RFFIT, after mice primary immunized with rabies VLPs and boost 7 days later. Furthermore, Similar to same titers inactivated rabies vaccine purified from Vero cell, nearly 100% protection from lethal CVS strains and another wild type strains can be observed 7 days after boost. Taken together, these finding indicates that rabies VLPs deserved as a more effective, flexible, costly and safer vaccine candidates.

The Role of Nuclear Factor- B in Oxidative Stress Associated with Rabies Virus Infection (P03.263)

Objective: To investigate the role of NF-κB in rabies virus-induced oxidative stress in neurons. Background Recent studies in an experimental mouse model of rabies have shown that there are major structural changes in the brain involving neuronal processes that are associated with severe clinical disease. Cultured adult dorsal root ganglion (DRG) neurons are a good in vitro model for studying mechanisms involved in rabies virus – induced degeneration of neurites, because, unlike other neuronal cell types, these neurons are permissive to rabies virus infection. DRG neurons infected with the CVS strain of rabies virus show axonal swellings and immunostaining for 4-hydroxy-2-nonenal (4-HNE), indicating evidence of lipid peroxidation associated with oxidative stress, and also reduced axonal growth vs. mock-infected DRG neurons. Design/Methods: The expression and localization of NF-κB were evaluated by Western immunoblotting and immunofluorescence, respectively, in mock- versus CVS-infected rat DRG neurons. The effects of ciliary neurotrophic factor (CNTF), a potent activator of NF-κB, and SN50, a peptide inhibitor of NF-κB, were also evaluated. Results: CNTF had no effect on mock-infected rat DRG neurons, and was neuroprotective for CVS-neurons in reducing the number of 4-HNE-labeled puncta. SN50 and CVS infection had an additive effect in producing axonal swellings, suggesting that NF-κB is neuroprotective. CVS infection induced the expression of protein for the p50 subunit of NF-κB. The fluorescent signal for p50 was quantitatively evaluated in the nucleus and cytoplasm of mock- and CVS-infected rat DRG neurons. At 24 hrs p.i. there was a significant increase in the nucleus:cytoplasm ratio, indicating increased transcriptional activity of NF-kB, perhaps as a response to stress, whereas at both 48 and 72 hrs p.i. there was significantly reduced nuclear localization of NF-kB. Conclusions: CVS infection may induce oxidative stress by preventing nuclear activation of NF-κB. A rabies virus protein may directly inhibit NF-kB activity. Supported by: Canadian Institutes of Health Research. Disclosure: Dr. Jackson has nothing to disclose. Dr. Kammouni has nothing to disclose. Dr. Hasan has nothing to disclose. Dr. Fernyhough has nothing to disclose.

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.

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.

Protection of Mice from Rabies by Intranasal Immunization with Inactivated Rabies Virus

The mucosal immunization method is a needle-free alternative way of vaccination. This study evaluated the efficacy of mucosal immunization for rabies. Mice were intranasally administered five times with inactivated and concentrated rabies virus antigen (CRV) supplemented with or without cholera toxin (CT). The anti-rabies virus antibody titer of mice intranasally immunized with CRV plus CT (CRV/CT) was comparable to that of mice intraperitoneally immunized twice with the same amount of CRV. Virus neutralizing (VNA) titers of mice immunized intranasally with CRV/CT were slightly lower than those of intraperitoneally immunized mice. Both anti-rabies virus ELISA antibody and VNA titers of mice immunized with CRV without CT were significantly lower than those of mice immunized with CRV/CT. In mice intranasally immunized with CRV/CT, and intraperitoneally immunized mice, high levels of IgG(2a) antibody were detected, suggesting the activation of Th1-driven cellular immunity by the two ways of immunization. All immunized mice were challenged intracerebrally with a lethal dose of virulent rabies virus CVS strain. The survival rates of mice immunized with CRV/CT and CRV without CT were 67% and 17%, respectively, while the rate of intraperitoneally immunized mice was 100%. Antigen-specific whole IgG and IgG(2a), and VNA titers of survived mice were significantly higher than those of dead mice at the challenge day. These data suggest the possibility of intranasal immunization with inactivated antigen as a rabies vaccination strategy and the importance of a mucosal adjuvant such as CT.

Rabies Virus Infection of Primary Neuronal Cultures and Adult Mice: Failure To Demonstrate Evidence of Excitotoxicity

Cultures derived from the cerebral cortices and hippocampi of 17-day-old mouse fetuses infected with the CVS strain of rabies virus showed loss of trypan blue exclusion, morphological apoptotic features, and activated caspase 3 expression, indicating apoptosis. The NMDA (N-methyl-D-aspartate acid) antagonists ketamine (125 microM) and MK-801 (60 microM) were found to have no significant neuroprotective effect on CVS-infected neurons, while the caspase inhibitor Ac-Asp-Glu-Val aspartic acid aldehyde (25 microM) exerted a marked neuroprotective effect. Glutamate-stimulated increases in levels of intracellular calcium were reduced in CVS-infected hippocampal neurons. Ketamine (120 mg/kg of body weight/day intraperitoneally) given to CVS-infected adult mice produced no beneficial effects. We have found no supportive evidence that excitotoxicity plays an important role in rabies virus infection.

A novel rapid fluorescent focus inhibition test for rabies virus using a recombinant rabies virus visualizing a green fluorescent protein

Virus-neutralizing antibodies (VNAs) against rabies virus play a major role in protection from rabies. The rapid fluorescent focus inhibition test (RFFIT) has been internationally recognized as a standard in vitro test for measuring the VNA. CVS strain of rabies virus is used as challenge virus and the infected cells are indirectly detected by staining with fluorescein isothiocyanate (FITC)-conjugated rabies antibody which is expensive and high-quality products are often in short supply. In this study, a recombinant rabies virus strain carrying a green fluorescent protein (GFP) gene, rHEP-GFP, was used as a challenge virus in the virus neutralization assay. Expression of the GFP could be readily detected in the infected cells under a fluorescent microscope. This novel RFFIT modification RFFIT-GFP is a neutralization test, and it is based on the sound principle of the standard RFFIT using 96-well plates. VNA titers in 25 human, 18 canine and 15 horse sera have been compared between the RFFIT and RFFIT-GFP methods. The results obtained by the both methods showed good agreement between both methods in all sera investigated (coefficient of correlation, r = 0.98). It allowed direct detection of virus by expression of GFP and might be applicable for other viruses. The novel method is convenient, economical and a reliable tool not requiring expensive FITC-conjugated antibody for routine rabies VNA assays.

In vitro inactivation of the rabies virus by ascorbic acid

Objective: The current recommended inactivating agent for the rabies virus, beta propiolactone (BPL) is very expensive and potentially carcinogenic. There is a need to evaluate alternative chemicals, which will inactivate the virus without affecting its antigenicity. In this study the effect of ascorbic acid on the infectivity of the rabies virus has been investigated. Method: Vero cell grown fixed rabies virus CVS strain was treated with 0.1 mg/ml, 0.5 mg/ml and 1 mg/ml final concentrations of ascorbic acid and 5 μg/ml of copper sulfate and kept at 4 °C along with untreated virus material. Each aliquot was titrated after various intervals for viral infectivity using both mice inoculation and titration in vero cells. The antigenicity of the virus material was determined by antibody induction in mice and modified NIH tests in parallel with virus material inactivated with a 1:4000 concentration of BPL. Results: An optimal concentration of 0.5 mg/ml of ascorbic acid and 5 μg/ml of copper sulfate completely inactivated the virus after 72 hours. The inactivated virus retained good antigenicity and potency value, which was comparable with using BPL. Conclusion: These findings suggest that ascorbic acid can be used as an inactivating agent for fixed rabies virus grown in cell culture particularly for the preparation of diagnostic reagents. Further studies are required to evaluate its effect on the cell associated virus, probable therapeutic potential and feasibility of replacing BPL in production of inactivated rabies vaccine.

Absence of the p55 Kd TNF-alpha receptor promotes survival in rabies virus acute encephalitis.

We investigated the role played by inflammation in acute encephalitis following infection with a neurotropic virus by comparing the disease caused by the CVS strain of rabies virus in C57BL/6 and mice deficient for the p55 Kd TNF-alpha receptor (p55TNFR-/-). Morbidity (weight loss and paralysis) and mortality of infected mice were associated with viral propagation, cytokine (IL-6, IL-10, TNF-alpha and IFN-gamma) production, induction of apoptosis and infiltration of inflammatory cells. Mortality occurred later in p55TNFR-/- (than in C57BL/6 mice. In contrast, morbidity and the number of cells undergoing apoptosis were similar in C57BL/6 and p55TNFR-/- mice.) This suggests that morbidity and mortality are independently regulated and that the death of the animal was not due to CNS apoptosis. Delayed mortality correlated with: a reduction in viral load on day 9 p.i., an increase in IFN-gamma and IL-10 concentrations and a reduction in inflammatory cell infiltration in the CNS. Thus, these data indicate that CVS infection elicits an inflammatory response within the CNS and suggest that cytokines signaling via the p55 Kd TNF-alpha receptor is deleterious for the survival of the host. These results strongly suggest that, the modulation of TNF-alpha and upregulation of IFN-gamma would be a powerful anti-virus strategy in cases of viral encephalitis.

Polygenic control of antibody production and correlation with vaccine induced resistance to rabies virus in high and low antibody responder mice.

The amplification of "high" (H) and "low" (L) multispecific antibody responses achieved respectively by H and L lines of selection GP represents a valuable tool in the genetic study of host-infection interactions. These lines were obtained by bidirectional selective breeding for high (HGP) or low (LGP) antibody production to natural complex antigens. HGP and LGP parental lines and reciprocal F1 hybrids, as well as their F2 segregants and backcrosses were submitted to immunization and challenge with rabies virus CVS strain. Acquired resistance was 1000-fold higher in HGP than LGP mice, with a dominance effect to low antibody production observed in F1 hybrids. An association between high antibody response and acquired resistance (P < 0.001) in F2 segregant mice was noticed. The genetic study was performed in these several populations, with a single dose of 104.5-fold LD50 CVS. We could demonstrate 3 independent loci regulating the anti-rabies antibody production, that are distinct, at least in part, from the 10 genes controlling the antigen selection response (sheep erythrocytes) of selection GP.

Spread of the CVS strain of rabies virus and of the avirulent mutant AvO1 along the olfactory pathways of the mouse after intranasal inoculation

After intranasal instillation in the mouse, rabies virus (CVS strain) selectively infected olfactory receptor cells. In the main olfactory bulb (MOB), infection was observed in periglomerular, tufted, and mitral cells and in interneurons located in the internal plexiform layer. Beyond the MOB, CVS spread into the brain along the olfactory pathways. This infection is specific to chains of functionally related neurons but at the death of the animal some nuclei remain uninfected. CVS also penetrated the trigeminal system. The avirulent mutant AvOl, carrying a mutation in position 333 of the glycoprotein, infected the olfactory epithelium and the trigeminal nerve as efficiently as CVS. During the second cycle of infection, the mutant was able to infect efficiently periglomerular cells in the MOB and neurons of the horizontal limb of the diagonal band, which indicates that maturation of infective particles is not affected in primarily infected neuronal cells. On the other hand, other neuronal cells permissive for CVS, such as mitral cells or the anterior olfactory nucleus, are completely free of infection with the mutant, indicating that restriction is related to the ability of AvO1 to penetrate several categories of neurons. From these observations, we concluded that CVS should be able to bind several different receptors to penetrate neurons, while the mutant would be unable to recognize some of them.

Invasion of the peripheral nervous systems of adult mice by the CVS strain of rabies virus and its avirulent derivative AvO1.

The penetration of the CVS strain of rabies virus and its avirulent derivative AvO1 into peripheral neurons was investigated after intramuscular inoculation into the forelimbs of adult mice. It was found that CVS directly penetrates both the sensitive and motor routes with equal efficiency, without prior multiplication in muscle cells. Infected neurons became detectable 18 h after infection. The second cycle of infection occurred within 2 days, and at day 3 there was a massive invasion of the spinal cord and sensory ganglia. In sensory ganglia, where it was possible to identify cell outlines, it was evident that the infection did not proceed directly from cell body to cell body. The avirulent strain AvO1 penetrated motor and sensory neurons with the same efficiency as CVS. Restriction of viral propagation was observed from the second and third cycles onwards. No further development of the infection could be seen after day 3, and by that time the lysis of primarily infected neurons seemed to occur.

Antigenic site II of the rabies virus glycoprotein: structure and role in viral virulence

Twelve monoclonal antibodies neutralizing the CVS strain of rabies virus were used to characterize antigenic site II of the viral glycoprotein. Nineteen antigenic mutants resistant to neutralization by some of these antibodies were selected; some continued to normally or partially bind the antibody, whereas others did not. Mutations conferring resistance to neutralization by site II-specific monoclonal antibodies were localized into two clusters, the first between amino acids 34 and 42 (seven groups of mutants) and the second at amino acids 198 and 200 (three groups of mutants). Two intermediate mutations were identified at positions 147 and 184. Four mutations resulted in reduced pathogenicity after intramuscular inoculation of the virus in adult mice. One of the mutants, M23, was 300 times and the others were 10 to 30 times less pathogenic than CVS. In three cases the attenuated phenotype was related to an important modification of antigenic site II, whereas the other known antigenic sites were unchanged.

Characterization of a double avirulent mutant of rabies virus and its potency as a vaccine, live or inactivated

A strain bearing two mutations which abolish the virulence for adult animals after intracerebral or intramuscular inoculation has been constructed from the CVS strain of rabies virus. This apathogenic phenotype is stable after three successive passages of the double mutant in suckling mice brain. Trials of vaccination performed on mice in parallel with the double mutant and CVS both inactivated with β-propiolactone indicate that the mutant is at least as efficient as CVS. This strain has also been found to be efficient as a live virus vaccine.

Infection of the lens by AvO 1, an avirulent mutant of the CVS strain of rabies virus

Infection of the lens by AvO 1, an avirulent mutant of the CVS strain of rabies virus