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Journal of Virological Methods Aug 2019Rabies is an infectious viral disease, characterized as a neglected zoonosis, responsible for nearly 60,000 deaths annually. The virus is transmitted mainly by dogs in...
Rabies is an infectious viral disease, characterized as a neglected zoonosis, responsible for nearly 60,000 deaths annually. The virus is transmitted mainly by dogs in Africa and Asia, and wildlife in Europe and the Americas, to all mammals' species, causing severe encephalitis almost always fatal after the onset of neurological symptoms. Human rabies can be prevented through extensive vaccination of dogs and pre/post-prophylaxis treatments in humans with inactivated rabies vaccines. The vaccine manufacture involves a series of quality control assays using laboratory animals, which are mandatory to exclude the presence of viable residual virus. The quality controls must be carried out in various steps during the vaccine production, which demands the use of a large number of animals. In this study, we standardized a real-time quantitative RT-PCR duplex assay to be used during intermediate stages of the vaccine production. This assay was done for the quantification of vaccine strain rabies virus, targeting rabies nucleoprotein, and β-actin mRNA of BHK-21 cells as an internal endogenous control. The results showed specific amplification, with the analytical sensitivity ranged from 10 to 10 TCID/mL with high repeatability rate for the quantification of rabies virus in inactivated vaccine samples. Global organizations are engaged to develop new approaches to determine viable residual virus, and this assay can be applied in combination with traditional in vitro methods for the release of intermediate batches of vaccines during the production process, keeping the in vivo tests only for final release.
Topics: Fluorescent Antibody Technique, Direct; Quality Control; Rabies Vaccines; Rabies virus; Real-Time Polymerase Chain Reaction; Technology, Pharmaceutical; Vaccines, Inactivated
PubMed: 31047970
DOI: 10.1016/j.jviromet.2019.04.025 -
Acta Virologica 2019Rabies virus is an enveloped negative-stranded RNA virus belonging to the family Rhabdoviridae. It can be successfully controlled by vaccination however, there are still...
Rabies virus is an enveloped negative-stranded RNA virus belonging to the family Rhabdoviridae. It can be successfully controlled by vaccination however, there are still tens of thousands of deaths each year caused by rabies virus due to its mutations and complexity. A better understanding of the interaction between the rabies virus and the host might help solve this problem. Therefore, in this study, we used two-dimensional electrophoresis to investigate the protein expression of rabies virus-infected mice. This can help us to understand the impact of rabies virus on host protein expression during infection. For our experiment, two-dimensional electrophoresis was used to analyze the differential proteomics of the brain of 10- and 20-day-old suckling mice infected with attenuated rabies virus strain SRV9. The results showed that the expression levels of 10 protein spots had been up- or down-regulated at least 2-fold. Using MALDI-TOF-MS, we identified 8 differentially expressed proteins. We have identified proteins, namely hnRNP L, DPYSL3, NECAPs, and transaldolase that might be closely related to the susceptibility of SRV9 in suckling mice. Keywords: rabies virus; attenuated strain; suckling mouse; two-dimensional electrophoresis; proteomics.
Topics: Animals; Animals, Suckling; Brain; Mass Spectrometry; Mice; Proteomics; Rabies; Rabies virus
PubMed: 31802685
DOI: 10.4149/av_2019_406 -
Antiviral Research Apr 2021ABMA and its analogue DABMA are two molecules of the adamantane family known to perturbate the endosomal pathway and to inhibit cell infection of several RNA and DNA...
ABMA and its analogue DABMA are two molecules of the adamantane family known to perturbate the endosomal pathway and to inhibit cell infection of several RNA and DNA viruses. Their activity against Rabies Virus (RABV) infection has already been demonstrated in vitro. (Wu et al., 2017, 2019). Here, we describe in more details their mechanism of action by comparison to Arbidol (umifenovir) and Ribavirin, two broad spectrum antivirals against emerging viruses such as Lassa, Ebola, influenza and Hantaan viruses. ABMA and DABMA, delivered 2 h pre-infection, inhibit RABV infection in vitro with an EC of 7.8 μM and 14 μM, respectively. They act at post-entry, by causing RABV accumulation within the endosomal compartment and DABMA specifically diminishes the expression of the GTPase Rab7a controlling the fusion of early endosomes to late endosomes or lysosomes. This may suggest that ABMA and DABMA act at different stages of the late endosomal pathway as supported by their different profile of synergy/antagonism with the fusion inhibitor Arbidol. This difference is further confirmed by the RABV mutants induced by successive passages under increasing selective pressure showing a particular involvement of the viral G protein in the DABMA inhibition while ABMA inhibition induces less mutations dispersed in the M, G and L viral proteins. These results suggest new therapeutic perspectives against rabies.
Topics: Adamantane; Animals; Antiviral Agents; Benzylamines; Cell Line; Drug Resistance, Viral; Drug Synergism; Endosomes; Indoles; Mutation; Rabies virus; Ribavirin; Viral Proteins; Virus Internalization
PubMed: 33444703
DOI: 10.1016/j.antiviral.2021.105016 -
Journal of Clinical Microbiology Feb 1980The inactivation dynamics of rabies virus (PV strain) by binary ethylenimine, and the immunogenic properites and the stability of the vaccines prepared using this agent,...
The inactivation dynamics of rabies virus (PV strain) by binary ethylenimine, and the immunogenic properites and the stability of the vaccines prepared using this agent, were studied. Binary ethylenimine at a final concentration of 0.01 M was prepared wtih 2-bromoethylamine hydrobromide in alkaline solutions, either separately from or in suspensions of rabies virus propagated in BHK cells. The infectivity of virus suspensions containing more than 108 plaque-forming units per 0.1 ml was inactivated in 2 h when the inactivating agent was prepared before its addition to the suspensions, and in3 h when prepared directly in the suspensions. Liquid vaccines prepared in this manner and stored at different temperatures maintained potency for 1 month at 37 degrees C and for 6 months at 4 degrees C and 22 to 25 degrees C. Lyophilized vaccine maintained its potency for 6 months at the three temperatures. The inactivated vaccine mixed with aluminum or oil adjuvant at high dilutions protected guinea pigs against challenge. This safer procedure for rabies virus inactivation offers promise for the production of effective vaccines for the immunization of dogs and cattle.
Topics: Adjuvants, Immunologic; Animals; Antigens, Viral; Antiviral Agents; Aziridines; Azirines; Guinea Pigs; Rabies; Rabies Vaccines; Rabies virus; Vaccination; Viral Plaque Assay
PubMed: 7358836
DOI: 10.1128/jcm.11.2.120-122.1980 -
Biologicals : Journal of the... Apr 2021Rabies is a zoonotic disease that can be prevented by vaccination. The confirmation of rabies virus inactivation is a critical step during the vaccine quality test;...
Rabies is a zoonotic disease that can be prevented by vaccination. The confirmation of rabies virus inactivation is a critical step during the vaccine quality test; however, the current protocol conducted in Japan requires a large number of mice. The development and introduction of animal-free alternative assays are essential from the perspective of the 3Rs (reduction, refinement, and replacement) of animal testing. Here, we propose a novel inactivation assay for confirming the complete inactivation of the viable rabies virus using cultured Neuro-2a cells and an enzyme-linked immunosorbent assay (ELISA). The detection ability of ELISA was similar to that of a direct immunofluorescence assay, with the detection limit of ELISA being as low as 0.014 focus forming units/test. These results suggest that the assay could be used as a viral inactivation test. In comparison with a traditional in vivo assay, this assay has a higher detection ability, an objective interpretation, and would shorten the test duration from 25 days to 8 days.
Topics: Animal Testing Alternatives; Animals; Antibodies, Viral; Enzyme-Linked Immunosorbent Assay; Mice; Rabies; Rabies Vaccines; Rabies virus; Vaccines, Inactivated
PubMed: 33518433
DOI: 10.1016/j.biologicals.2021.01.002 -
Antiviral Research Dec 2018Rabies is a fatal and viral zoonosis that causes acute, progressive encephalitis and remains an important concern in public health. In the last few years, there has been...
Rabies is a fatal and viral zoonosis that causes acute, progressive encephalitis and remains an important concern in public health. In the last few years, there has been a change in the epidemiological profile of rabies after implementing canine rabies control in the Americas, which has led to a significant increase in both human and pet cases of rabies associated with insectivorous bats. Thus, it is important to understand the pathogenesis caused by Rabies virus (RABV) isolates from insectivorous bats. Viral growth kinetics, cell-to-cell spread and virus uptake in vitro were analyzed for RABV isolates from Eptesicus furiralis and Myotis nigricans. For pathogenesis evaluation, mice were inoculated with RABV isolates from Eptesicus furiralis and Myotis nigricans, and clinical signs were observed for 40 days. We observed that the insectivorous bat strains showed a higher replication rate, faster cell-to-cell spread and delayed virus uptake in N2a cells. Furthermore, after the first sign of a clinical infection, mice infected with Myotis nigricans and Eptesicus furiralis isolates succumbed rapidly (6 ± 9 days) compared with RABV strains associated with other reservoirs. Our results show that the insectivorous bat RABV strains are less pathogenic for mice than strains associated with other reservoirs. In addition, this study also indicates that the differences in the biological characteristics of the RABV strains are important to their pathogenicity. An enhanced understanding of rabies pathogenesis may be important for the development of novel therapies for humans and in the implementation of rabies control strategies.
Topics: Animals; Chiroptera; Disease Models, Animal; Mice; Rabies; Rabies virus; Survival Analysis; Time Factors; Virulence; Virus Internalization; Virus Release; Virus Replication
PubMed: 30393124
DOI: 10.1016/j.antiviral.2018.10.023 -
International Journal of Infectious... Jan 2004The current recommended inactivating agent for the rabies virus, beta propiolactone (BPL) is very expensive and potentially carcinogenic. There is a need to evaluate...
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 1mg/ml final concentrations of ascorbic acid and 5 microg/ml of copper sulfate and kept at 4 degrees 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 microg/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.
Topics: Animals; Antibodies, Viral; Ascorbic Acid; Biological Assay; Chlorocebus aethiops; Copper Sulfate; Female; Mice; Rabies; Rabies Vaccines; Rabies virus; Vaccines, Inactivated; Vero Cells; Virus Activation; Virus Replication
PubMed: 14690777
DOI: 10.1016/j.ijid.2003.09.002 -
Current Opinion in Virology Oct 2014Despite its ability to infect all mammals, Rabies virus persists in numerous species-specific cycles that rarely sustain transmission in alternative species. The... (Review)
Review
Despite its ability to infect all mammals, Rabies virus persists in numerous species-specific cycles that rarely sustain transmission in alternative species. The determinants of these species-associations and the adaptive significance of genetic divergence between host-associated viruses are poorly understood. One explanation is that epidemiological separation between reservoirs causes neutral genetic differentiation. Indeed, recent studies attributed host shifts to ecological factors and selection of 'preadapted' viral variants from the existing viral community. However, phenotypic differences between isolates and broad scale comparative and molecular evolutionary analyses indicate multiple barriers that Rabies virus must overcome through adaptation. This review assesses various lines of evidence and proposes a synthetic hypothesis for the respective roles of ecology and evolution in Rabies virus host shifts.
Topics: Adaptation, Biological; Animals; Biological Evolution; Host Specificity; Host-Pathogen Interactions; Mammals; Rabies; Rabies virus
PubMed: 25064563
DOI: 10.1016/j.coviro.2014.07.004 -
Archives of Virology Nov 2016Sub-passaging of QS-05, a street rabies virus (RABV) isolate, in non-neuronal cells resulted in a virus with higher pathogenicity, QS-BHK-P7. Four full-length cDNA...
Sub-passaging of QS-05, a street rabies virus (RABV) isolate, in non-neuronal cells resulted in a virus with higher pathogenicity, QS-BHK-P7. Four full-length cDNA plasmids were constructed and the corresponding recombinant viruses were recovered: rQS-05, rQS-BHK-P7 and rQS05-2475G/rQS-BHK-P7-2475A (made by switching of intergenic P-M between these two backbones). rQS-BHK-P7-2475 A virus had eight instead of seven adenosines in its poly(A) sequence. Interestingly, mutant viruses with 6 or 8 adenosines infected more neuroblastoma cells than their parental ones. Mice that were infected intracerebrally and intramuscularly with rQS05-2475G and rQS-BHK-P7 exhibited highest mortality. However, mice infected with rQS-BHK-P7-2475AA had the shortest survival time. This study demonstrates that modifications in the non-coding region may play a role in determining the virulence of RABV.
Topics: Animals; Cell Line; Female; Injections, Intramuscular; Mice, Inbred ICR; Neurons; RNA, Messenger; RNA, Untranslated; RNA, Viral; Rabies; Rabies virus; Survival Analysis; Time Factors; Virulence; Virus Cultivation
PubMed: 27558122
DOI: 10.1007/s00705-016-2990-9 -
Viruses Aug 2020The rabies virus strain Komatsugawa (Koma), which was isolated from a dog in Tokyo in the 1940s before eradication of rabies in Japan in 1957, is known as the only...
The rabies virus strain Komatsugawa (Koma), which was isolated from a dog in Tokyo in the 1940s before eradication of rabies in Japan in 1957, is known as the only existent Japanese field strain (street strain). Although this strain potentially provides a useful model to study rabies pathogenesis, little is known about its genetic and phenotypic properties. Notably, this strain underwent serial passages in rodents after isolation, indicating the possibility that it may have lost biological characteristics as a street strain. In this study, to evaluate the utility of the Koma strain for studying rabies pathogenesis, we examined the genetic properties and in vitro and in vivo phenotypes. Genome-wide genetic analyses showed that, consistent with previous findings from partial sequence analyses, the Koma strain is closely related to a Russian street strain within the Arctic-related phylogenetic clade. Phenotypic examinations in vitro revealed that the Koma strain and the representative street strains are less neurotropic than the laboratory strains. Examination by using a mouse model demonstrated that the Koma strain and the street strains are more neuroinvasive than the laboratory strains. These findings indicate that the Koma strain retains phenotypes similar to those of street strains, and is therefore useful for studying rabies pathogenesis.
Topics: Animals; Dog Diseases; Dogs; Genome, Viral; Male; Mice; Phenotype; Phylogeny; Rabies; Rabies virus; Tokyo; Viral Proteins
PubMed: 32825306
DOI: 10.3390/v12090914