Rabies Virus Strains Research Articles

Deactivation of the antiviral state by rabies virus through targeting and accumulation of persistently phosphorylated STAT1.

Antagonism of the interferon (IFN)-mediated antiviral state is critical to infection by rabies virus (RABV) and other viruses, and involves interference in the IFN induction and signaling pathways in infected cells, as well as deactivation of the antiviral state in cells previously activated by IFN. The latter is required for viral spread in the host, but the precise mechanisms involved and roles in RABV pathogenesis are poorly defined. Here, we examined the capacity of attenuated and pathogenic strains of RABV that differ only in the IFN-antagonist P protein to overcome an established antiviral state. Importantly, P protein selectively targets IFN-activated phosphorylated STAT1 (pY-STAT1), providing a molecular tool to elucidate specific roles of pY-STAT1. We find that the extended antiviral state is dependent on a low level of pY-STAT1 that appears to persist at a steady state through ongoing phosphorylation/dephosphorylation cycles, following an initial IFN-induced peak. P protein of pathogenic RABV binds and progressively accumulates pY-STAT1 in inactive cytoplasmic complexes, enabling recovery of efficient viral replication over time. Thus, P protein-pY-STAT1 interaction contributes to ‘disarming’ of the antiviral state. P protein of the attenuated RABV is defective in this respect, such that replication remains suppressed over extended periods in cells pre-activated by IFN. These data provide new insights into the nature of the antiviral state, indicating key roles for residual pY-STAT1 signaling. They also elucidate mechanisms of viral deactivation of antiviral responses, including specialized functions of P protein in selective targeting and accumulation of pY-STAT1.

Preparation and the assessed efficacy of oral baits for the vaccination of free-roaming dogs against rabies.

Background and Aim:Rabies is considered a highly fatal zoonotic disease and many deaths in humans have been associated with dog bites. This study was designed to prepare an oral anti-rabies vaccine in the form of baits to eliminate the disease in free-roaming dogs and subsequently protect humans from dog bites.Materials and Methods:The Evelyn Rokintniki Abelseth (ERA) rabies virus strain was propagated in baby hamster kidney cell cultures and adjusted to the recommended dose for application. Four forms of oral baits were employed with the rabies vaccine, which was evaluated for safety, acceptability, and potency in different dog groups. Enzyme-Linked Immunosorbent Assay (ELISA) and the serum neutralization test (SNT) were used to determine the protective rabies antibody titer in the sera of vaccinated dogs.Results:According to the results, a dose of 3 mL of the ERA strain, containing a viral titer of 107.6 TCID50/mL, induced a mean antibody titer of 25.6 by SNT, and the PI% was 75.7 by Block ELISA, providing a protective level of the rabies antibody in 100% of vaccinated dogs. All used baits were found to be safe, inducing no abnormal general post-vaccination signs (the signs are limited to mild fever, mild loss of appetite, and mild-to-moderate loss of energy for 24-36 h after vaccination).Conclusion:It was found that most of the accepted and highly potent bait types consisted of a mixture of wheat flour, vegetable oil, sodium alginate, corn starch, meat meal, cellulose gum, and water. This dog meal was covered with bran and edible wax to seal the bait cavity after inserting the vaccine sachet. This bait was able to induce a protective level of rabies antibodies in 100% of vaccinated dogs after receiving one bait/dog. Hence, such a bait could be recommended for use in the protection of free-roaming dogs and the elimination of the disease.

Rabies as a Potential Cause of Mass Mortality Events in North American Bat Species.

Rabies-association bat mortality events are rarely reported, suggesting that rabies is not a significant cause of bat mass mortality. Three bat die-offs in National Park Service units were attributed to rabies, highlighting the value of including rabies, and rabies virus strain spillover events, as a differential in mass mortality events.

Glu333 in rabies virus glycoprotein is involved in virus attenuation through astrocyte infection and interferon responses

SummaryThe amino acid residue at position 333 of the rabies virus (RABV) glycoprotein (G333) is a major determinant of RABV pathogenicity. Virulent RABV strains possess Arg333, whereas the attenuated strain HEP-Flury (HEP) possesses Glu333. To investigate the potential attenuation mechanism dependent on a single amino acid at G333, comparative analysis was performed between HEP and HEP333R mutant with Arg333. We examined their respective tropism for astrocytes and the subsequent immune responses in astrocytes. Virus replication and subsequent interferon (IFN) responses in astrocytes infected with HEP were increased compared with HEP333R both in vitro and in vivo. Furthermore, involvement of IFN in the avirulency of HEP was demonstrated in IFN-receptor knockout mice. These results indicate that Glu333 contributes to RABV attenuation by determining the ability of the virus to infect astrocytes and stimulate subsequent IFN responses.

Complete Genome Sequences of Five Rabies Virus Strains Obtained from Domestic Carnivores in Liberia.

ABSTRACTAs in other African countries, canine rabies is endemic in Liberia. However, data concerning the genetic diversity of rabies virus isolates circulating in this country remain limited. We report here the complete genome sequences of five rabies viruses obtained from domestic animals. All of them belonged to subgroup H within the Africa 2 clade.

Capacity Building Efforts for Rabies Diagnosis in Resource-Limited Countries in Sub-Saharan Africa: A Case Report of the Central Veterinary Laboratory in Benin (Parakou).

Rabies has been listed as a priority zoonotic disease in many African countries and the countdown to reach the goal of eliminating dog-mediated human rabies deaths by 2030 means that disease control measures need to be applied fast. In this context, an essential pillar of any national plan to control rabies is the implementation of reliable diagnostic techniques to ensure the success of field surveillance systems. Although many African countries have received international support for the control of rabies–some countries, like Benin, have not received a similar level of support. Indeed, until 2018, Benin was not able to diagnose rabies and rabies diagnosis in animals as well as humans relied solely on observed clinical symptoms. Although the Central Veterinary Laboratory (CVL) of Parakou had the equipment to implement two recommended tests, the lack of specific reagents and skills prevented the implementation of a rabies diagnostic service. Here we present the joint efforts of the national authorities in Benin, intergovernmental agencies, and non-governmental organizations to assess the strengths and weaknesses of the government's rabies control efforts. We have applied the Stepwise Approach toward Rabies Elimination (SARE) analysis, implemented rabies diagnostic capacities at the CVL of Parakou, characterized strains of rabies virus circulating in Benin, and finally integrated an inter-laboratory comparison program.

Nearly Complete Genome Sequences of Eight Rabies Virus Strains Obtained from Domestic Carnivores in the Democratic Republic of the Congo.

ABSTRACTIn this report, we describe eight nearly complete genome sequences of rabies virus strains collected in the Democratic Republic of the Congo from domestic carnivores in 2017 and 2018. All of them clustered into a specific phylogroup among the Africa 1b lineage in the Cosmopolitan clade.

АНАЛИЗ ВОЗДЕЙСТВИЯ ПРИРОДНЫХ ФАКТОРОВ НА ЦИРКУЛЯЦИЮ ВИРУСА БЕШЕНСТВА СРЕДИ РЕЗЕРВУАРНЫХ ХОЗЯЕВ

Wildlife is a reservoir for many pathogens, including the rabies virus. While carnivores (Carnivora) and bats (Chiroptera) are the main orders of mammals acting as natural reservoirs of rabies virus (RABV), the role of most other orders of mammals remains less well known. As a result of the evolution of the pathogen, climatic, landscape changes, anthropogenic impact on wildlife, the characteristics of the main hosts of the rabies virus change, new reservoirs of the virus appear. It has been established that environmental factors affect the circulation of the rabies virus among reservoir hosts in various zones of the global nosoareal of the disease. RABV strains circulate in most terrestrial reservoirs of lissavirus. In North and South America, all variants of the causative agent of the disease are currently strains of rabies virus (RABV). Here, bats are an autonomous source of rabies in humans and pets. There is no RABV in bats on other continents. RABV strains circulate in most terrestrial reservoirs of lissavirus, with the exception of Africa, where IKOV and MOKV have been detected in addition to RABV. Western and part of Central Europe, North America are considered to be free of terrestrial RABV among dogs and foxes as a result of actively implemented national vaccination programs for these animal species, the impact of climatic conditions has affected the survival tactics of the rabies virus. As an adaptation to real conditions, both a slowdown in virus replication and an increase in the number of viral generations due to the growth of the host population can be observed. The adaptation of the virus to the ecology of susceptible animals is manifested in the peculiarities of the pathogenesis of the disease, in the spread of the virus to new geographical territories, in the activation of RABV in previously prosperous territories and in the expansion of the potential range of reservoirs. Currently, RABV is being activated in previously safe territories and the expansion of the modern nosoareal of rabies of wild animals is taking place. New animal species are increasingly being included in the epizootic and epidemic processes of rabies. The problem of unrecognized reservoir species present in the mammalian animal population may have negative consequences for the prediction, prevention and control of rabies.

Establishment of a reverse genetics system for rabies virus strain Komatsugawa.

The rabies virus strain Komatsugawa isolated from a dog in Tokyo in the 1940s retains biological properties as a field strain, providing an effective model for studying rabies pathogenesis. To facilitate molecular studies on the pathogenesis, this study aimed to establish a reverse genetics system for the Komatsugawa strain. By transfecting the full-length genome plasmid of this strain, infectious virus with artificially introduced genetic markers in its genome was rescued. The recombinant strain had biological properties similar to those of the original strain. These findings indicate that a reverse genetics system for the Komatsugawa strain has successfully been established.

Recombinant Rabies Virus With a Multiple Cloning Site: A Platform Capable of Immunological Alterations

Background: Rabies is fatal encephalitis, i.e., preventable by appropriate vaccination. Reverse genetics has proved promising for manipulating the rabies virus immunological characteristics. The insertion or deletion of a gene from the rabies genome could render specific functions to the rabies virus. Materials and Methods: A multiple cloning site including 111 nucleotides long harboring 10 single-cut restriction sites have been designed. The designed fragment was cloned between the G and L genes of the rabies virus genome. The recombinant rabies virus was rescued, and its infectivity was confirmed in the BHK-21 cell line. The recombinant virus propagation was compared with the initial rabies virus strain. Statistical analysis was performed using GraphPad Prism. Results: The cloning and localization of the multiple cloning site were verified by nucleotide sequencing. The recombinant virus properly propagated and rescued in the BHK-21 cell line. Comparing the recombinant virus with the initial rabies virus has shown that both viruses had similar functionality and propagation rate. Conclusion: The recombinant virus obtained in the present study could facilitate further cloning experiments. Examples include constructing a marker virus, carrying green fluorescent protein to be used either in rabies immunity assays or tracking the virus infection in relevant tissues.

Molecular Characterization of Canine Rabies Virus Strains Circulating in Mali

Rabies are one of the major zoonoses in Mali. The control of this disease is based on the use of effective diagnostic techniques, the management of human cases after exposure and medical prophylaxis of animals. Diagnosis is usually made by detection of viral antigen by direct immunofluorescence (IF). Due to the increasing variability of the genus Lyssavirus observed, it was found necessary to use a complementary technique, namely reverse transcription in polymerase chain reaction (RT-PCR). The objective of the present study was to assess the diagnostic performance of IFD and RT-PCR for the diagnosis of animal rabies in Mali with a view to characterizing the different strains identified at the molecular level (gene of rabies virus nucleoprotein N). Thus, the analysis of forty (40) brain samples from dogs suspected of having rabies, revealed 36 positive cases for IFD, i.e. 90% and 33 for RT-PCR, i.e. 82.5.% and a concordance rate of 82.5%. Analysis of the segment of the gene of nucleoprotein (N) showed that the rabies viruses circulating in Mali belong to the Africa 2 lineage (Africa 2). Statistical analyzes showed the existence of a significant difference (P=0.001<0.005) between the performance of the two techniques. The present study shows the usefulness of the complementarity of the two tests as an alternative for confirming the diagnosis of rabies.

Comprehensive Analysis of Protein Acetylation and Glucose Metabolism in Mouse Brains Infected with Rabies Virus.

Rabies, caused by rabies virus (RABV), is a widespread zoonosis that is nearly 100% fatal. Alteration of the metabolic environment affects viral replication and the immune response during viral infection. In this study, glucose uptake was increased in mouse brains at the late stage of infection with different RABV strains (lab-attenuated CVS strain and wild-type DRV strain). To illustrate the mechanism underlying glucose metabolism alteration, comprehensive analysis of lysine acetylation and target analysis of energy metabolites in mouse brains infected with CVS and DRV strains were performed. A total of 156 acetylated sites and 115 acetylated proteins were identified as significantly different during RABV infection. Compared to CVS- and mock-infected mice, the lysine acetylation levels of glycolysis and tricarboxylic acid (TCA) cycle enzymes were decreased, and enzyme activity was upregulated in DRV-infected mouse brains. Metabolomic analysis revealed high levels of oxaloacetate (OAA) in RABV-infected mouse brains. Specifically, the OAA level in CVS-infected mouse brains was higher than that in DRV-infected mouse brains, which contributed to the enhancement of the metabolic rate at the substrate level. Finally, we confirmed that OAA could reduce excessive neuroinflammation in CVS-infected mouse brains by inhibiting JNK and P38 phosphorylation. Taken together, this study provides fresh insight into the different strategies the host adapts to regulate glucose metabolism for energy requirements after different RABV strain infections and suggests that OAA treatment is a strategy to prevent neural damage during RABV infection. IMPORTANCE Both viral replication and the host immune response are highly energy dependent. It is important to understand how the rabies virus affects energy metabolism in the brain. Glucose is the direct energy source for cell metabolism. Previous studies have revealed that there is some association between acetylation and metabolic processes. In this study, comprehensive protein acetylation and glucose metabolism analysis were conducted to compare glucose metabolism in mouse brains infected with different RABV strains. Our study demonstrates that the regulation of enzyme activity by acetylation and OAA accumulation at the substrate level are two strategies for the host to respond to energy requirements after RABV infection. Our study also indicates the role OAA could play in neuronal protection by suppressing excessive neuroinflammation.

Comparative effectiveness of liquid and freeze-dried vaccines for oral vaccination of dogs against rabies

Currently, vaccination is the main measure to combat rabies in domestic and wild animals. Inactivated and live vaccines are used for this purpose. Oral vaccines for wild carnivores contain live attenuated rabies viruses in liquid or freeze-dried form, which are packaged inside edible baits. Since there are no consistent data showing advantages of liquid and freeze-dried oral products for vaccine-induced immunity against rabies in animals, we compared effectiveness of these rabies vaccines produced from rabies virus strain VRC-RZ2. Immunogenicity was tested in mongrel dogs aged 3 months and older that are seronegative for rabies virus antigens. The animals were randomly divided into three groups: two experimental and one control group. Group One was fed a block-type bait containing a blister with liquid virus-containing suspension, Group Two was given a block-type bait containing a gelatin capsule with freeze-dried virus suspension. On Day 21 post vaccination, blood samples were taken from all the animals and the obtained sera were examined in virus neutralization test to measure virus neutralizing antibodies titers. The level of the immune response against rabies in the vaccinated dogs was assessed by intracerebral infection of animals with virulent rabies virus strain CVS. The carried out research demonstrated that both groups of the vaccinated dogs had approximately the same titers of virus neutralizing antibodies that ranged from 3.25 to 4.33 log 2 . The virus neutralizing antibodies observed in the immunized dogs ensured good protection from virulent CVS strain. All animals of the control group died after infection demonstrating clinical signs of paralytic rabies. The results obtained show that both forms of the oral rabies vaccines are effective.

BIOTECHNOLOGICAL RESEARCH IN THE CREATION AND PRODUCTION OF ANTIRABIC VACCINES

Rabies is a neurological disease of a viral nature, leading to death. Rabies virus is an RNA virus that invades the central nervous system, leading to neuronal dysfunction. Timely vaccination can prevent the diseases development. Aim. The article is devoted to immunobiotechnological research aimed at creating antirabic vaccines. Results. The history of the antirabic vaccines creation from the first inactivated vaccines obtained from nervous tissue to the cultivation of the virus on animal cell cultures is considered. The article presents commercially available anti-rabies vaccines: their composition, the used rabies virus strains, cell cultures, the methods of inactivation and purification. The technology of producing an anti-rabies vaccine based on a Pitman Moore virus strain and a chicken fibroblast cell culture is presented. The advantages of different vaccine types are considered: live attenuated, peptide, liposomal, RNA vaccines, vaccines based on viral vectors, transgenic plants and reverse genetics methods. Conclusions. The development of biotechnology, immunology and virology makes it possible to improve constantly vaccine preparations, including those against rabies, increasing their effectiveness and safety.

Differential pathogenesis of intracerebral and intramuscular inoculation of street rabies virus and CVS-11 strains in a mouse model.

Objective(s):The mechanisms of rabies evasion and immunological interactions with the host defense have not been completely elucidated. Here, we evaluated the dynamic changes in the number of astrocytes, microglial and neuronal cells in the brain following intramuscular (IM) and intracerebral (IC) inoculations of street rabies virus (SRV).Materials and Methods:The SRV isolated from a jackal and CVS-11 were used to establish infection in NMRI-female mice. The number of astrocytes (by expression of GFAP), microglial (by Iba1), and neuronal cells (by MAP-2) in the brain following IM and IC inoculations of SRV were evaluated by immunohistochemistry and H & E staining 7 to 30 days post-infection.Results:Increased numbers of astrocytes and microglial cells in dead mice infected by SRV via both IC and IM routes were recorded. The number of neuronal cells in surviving mice was decreased only in IC-infected mice, while in the dead group, this number was decreased by both routes.The risk of death in SRV-infected mice was approximately 3 times higher than in the CVS-11 group. In IC-inoculated mice, viral dilution was the only influential factor in mortality, while the type of strain demonstrated a significant impact on the mortality rate in IM inoculations.Conclusion:Our results suggested that microglial cells and their inflammatory cytokines may not contribute to the neuroprotection and recovery in surviving mice following intracerebral inoculation of SRV. An unexpected decrease in MAP2 expression via intramuscular inoculation indicates the imbalance in the integrity and stability of neuronal cytoskeleton which aggravates rabies infection.

Analyses of cell death mechanisms related to amino acid substitution at position 95 in the rabies virus matrix protein.

We previously reported that the avirulent fixed rabies virus strain Ni-CE induces a clear cytopathic effect in mouse neuroblastoma cells, whereas its virulent progenitor, the Nishigahara strain, does not. Infection with Nishigahara and Ni-CE mutants containing a single amino acid substitution in the matrix protein (M) demonstrated that the amino acid at position 95 of M (M95) is a cytopathic determinant. The characteristics of cell death induced by Ni-CE infection resemble those of apoptosis (rounded and shrunken cells, DNA fragmentation), but the intracellular signalling pathway for this process has not been fully investigated. In this study, we aimed to elucidate the mechanism by which M95 affects cell death induced by human neuroblastoma cell infection with the Nishigahara, Ni-CE and M95-mutated strains. We demonstrated that the Ni-CE strain induced DNA fragmentation, cell membrane disruption, exposure of phosphatidylserine (PS), activation of caspase-3/7 and anti-poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, an early apoptosis indicator, whereas the Nishigahara strain did not induce DNA fragmentation, caspase-3/7 activation, cell membrane disruption, or PARP-1 cleavage, but did induce PS exposure. We also demonstrated that these characteristics were associated with M95 using M95-mutated strains. However, we found that Ni-CE induced cell death despite the presence of a caspase inhibitor, Z-VAD-FMK. In conclusion, our data suggest that M95 mutation-related cell death is caused by both the caspase-dependent and -independent pathways.

Rabies virus infection is associated with alterations in the expression of parvalbumin and secretagogin in mice brain.

Infection with the deadly rabies virus (RABV) leads to alteration of cellular gene expression. The RABV, similar to other neurodegenerative diseases may be implicated in neuronal death due to an imbalance in Ca2+ homeostasis. Parvalbumin (PV) and Secretagogin (Scgn), two members of the Calcium-Binding Proteins (CBPs) are useful neuronal markers responsible for calcium regulation and buffering with possible protective roles against infections. This study investigated whether infection with rabies virus causes variance in expression levels of PV and Scgn using the Challenge virus standard (CVS) and Nigerian Street Rabies virus (SRV) strains. Forty-eight, 4-week-old BALB/c mice strains were divided into two test groups and challenged with Rabies virus (RABV) infection and one control group. The presence of RABV antigen was verified by direct fluorescent antibody test (DFAT) and real-time quantitative PCR (qRT-PCR) was used to assess PV and Scgn gene expression. Infection with both virus strains resulted in significant (p < 0.05) increases in expression during early infection. Mid-infection phase caused reduced expression for both genes. However, as infection progressed to the terminal phase, a lower increase in expression was measured. Gene expression and viral load correlation indicated no positive relationship. Neurons with these CBPs may have a greater capacity to buffer calcium and be more resistant to degenerative changes caused by RABV. This implies that, when PV and Scgn expression levels are kept adequately high, the integrity of neurons may be maintained and degeneration caused by RABV infection may be prevented or stopped, hence, these are possible constituents of effective rabies therapy.

Evaluation of Multiplicity of Infection (MOI) and Harvesting Time on the Production of CVS-11 Strain of Rabies Virus in BSR Cell Line

Evaluation of Multiplicity of Infection (MOI) and Harvesting Time on the Production of CVS-11 Strain of Rabies Virus in BSR Cell Line

A recombinase polymerase amplification assay for rapid detection of rabies virus

Rabies is a generally fatal encephalitis caused by a negative-sense single-stranded RNA lyssavirus transmitted to humans mainly from dog bite. Despite the recommendation by WHO and OIE to use the direct immunofluorescence test as standard method, molecular diagnostic assays like reverse transcription quantitative polymerase chain reaction (RT-qPCR) are increasing as a confirmatory method. However, both technologies are inaccessible in resource-limited settings. Moreover, the available point-of-need molecular assay is of poor detection limit for African strains. Herein, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay as potential point-of-need diagnostic tool for rapid detection of various strains of rabies virus including locally isolated African strains. The sensitivity and specificity of the method was evaluated using a molecular RNA standard and different Rabies-related viruses belonging to the Rhabdoviridea family, respectively. The RABV-RPA performances were evaluated on isolates representative of the existing diversity and viral dilutions spiked in non-neural clinical specimen. The results were compared with RT-qPCR as a gold standard. The RABV-RPA detected down to 4 RNA molecules per reaction in 95% of the cases in less than 10 min. The RABV-RPA assay is highly specific as various RABV isolates were identified, but no amplification was observed for other member of the Rhabdoviridea family. The sample background did not affect the performance of the RABV-RPA as down to 11 RNA molecules were identified, which is similar to the RT-qPCR results. Our developed assay is suitable for use in low-resource settings as a promising alternative tool for ante-mortem rabies diagnosis in humans for facilitating timely control decisions.

Development of Monoclonal Antibodies for Detection of Conserved and Variable Epitopes of Large Protein of Rabies Virus.

Rabies virus (RABV) causes fatal neurological encephalitis and results in approximately 6000 human death cases worldwide every year. The large (L) protein of RABV, possessing conserved domains, is considered as the target for detection. In this study, three monoclonal antibodies (mAbs), designated as 3F3, 3A6 and L-C, against L protein were generated by using the recombinant truncated L protein (aa 1431–1754) and the epitopes were also identified using a series of overlapping truncated polypeptides for testing the reactivity of mAbs with different RABV strains. The 1479EIFSIP1484, 1659RALSK1663 and 1724VFNSL1728 were identified as the minimal linear epitopes recognized by mAbs 3F3, 3A6 and L-C, respectively. Amino acid alignment showed epitope 1724VFNSL1728 recognized by mAb L-C is completely conserved among RABV strains, indicating that mAb L-C could be used to detect all of the RABV strains. Epitope 1479EIFSIP1484 is highly conserved among RABV strains except for a P1484S substitution in a China I sub-lineage strain of Asian lineage, which eliminated the reactivity of the epitope with mAb 3F3. However, the epitope 1659RALSK1663 was only completely conserved in the Africa-2 and Indian lineages, and a single A1660T substitution, mainly appeared in strains of the China I belonging to Asian lineage and a Cosmopolitan lineage strain, still retained the reactivity of the epitope with mAb 3A6. While both A1660T and K1663R substitutions in a China I lineage strain, single K1663R/Q substitution in some China II strains of Asian lineage and some Arctic-like lineage strains and R1659Q mutation in a strain of Africa-3 lineage eliminated the reactivity of the epitope with mAb 3A6, suggesting mAb 3A6 could be used for differentiation of variable epitopes of some strains in different lineages. Thus, variability and conservation of the three epitopes of L protein showed the reactive difference of mAbs among RABV strains of different lineages. These results may facilitate future studies in development of detection methods for RABV infection, the structure and function of RABV L protein.