Street Virus Research Articles

Characterization of a human-mouse chimeric monoclonal antibody targeting rabies virus glycoprotein.

At present, the horse or human rabies immunoglobulin (RIG) used for postexposure prevention of human rabies (PEP) has high cost and limited availability. It is strongly encouraged to replace RIG with equivalent or more effective and safer products. Mouse and human monoclonal antibodies have been shown to protect rodents from lethal rabies virus (RABV) attacks. In this study, we reported a human-mouse chimeric monoclonal antibody, 12-2A12, which showed a strong neutralization potency and a wide breadth against multiple street viruses of RABV in vitro. The antibody binded the viral glycoprotein (G) with nanomolar affinity. The complex structure of 12-2A12 bound to RABV G revealed that the antibody recognizes an epitope that partially overlaps with the recognition region for the nicotinic acetylcholine receptor (nAChR). The antibody therefore would interfere with the nAChR/G interaction to block the viral receptor binding. In addition, comparison of our complex structure with the G structure in the acidic state reveals a clear steric clash, highlighting that the antibody would further prevent the conformational changes of the viral glycoprotein that are essential for membrane fusion. In light of these functional and structural data, we believe that 12-2A12 might be developed to be included in an antibody cocktail for potential use in human rabies PEP.

The role of assaying recombinant Beclin1 by in vitro and in vivo tests

BackgroundRabies is an infectious viral disease without any cure. Autophagy is considered as a method for reducing the damage to the cell and its organelles. In some virus-infected cells, autophagy plays an antiviral effect, while it acts in an opposite manner in other cells. Beclin1 (Atg6/Vps30) is considered as a main regulator of autophagy. The present study aims to build recombinant beclin1 for stimulating autophagy and evaluating its effects in the mice infected with street rabies virus. MethodsFirst, pIRES2-EGFP-beclin1 vector was made by PCR. Then, rabies street virus (SRABV), pIRES2-EGFP-beclin1 and pIRES2-EGFP were injected into the brain of mice. In addition, the mice were sacrificed and the samples were taken for PCR, FFA, real-time PCR, and Tunnel assay. ResultsThe accuracy of pIRES2-EGFP-beclin 1 vector was confirmed by colony PCR and digestion by restriction enzymes. Then, its in vitro expression was tested by gene sequencing, SDS-PAGE, and Western blot. Further, the function of pIRES2-EGFP-beclin 1 vector in in vivo and in vitro was investigated by transfecting the vector into the cells of CHO and mouse brain. The result of beclin1sequence blasting with existing beclin1 sequences in GenBank database revealed high levels of homology without any mutation. ConclusionThe made recombinant vector plays a correct and appropriate role in in vivo and in vitro and can be used by researchers for research purposes.

Analysis Strategies for Rabies Prevention and Control in China

Objective To analyze the current epidemiological characteristics of rabies in China, grasp the law of epidemic changes, and provide scientific basis for rabies prevention and control strategies in China. Methods The data of human rabies prevention and control in China were systematically collected, combined with the genetic sequences of rabies virus street virus epidemic strains in various provinces of China, and statistical and molecular biological methods were used for statistics and analysis. Results The number of rabies cases and deaths in China has decreased year by year. At present, the main method of human rabies transmission in China is dog injury. Post-exposure treatment showed an increase in the rate of self-treatment, a decline in the overall immunity rate of the vaccine, and an increase in the rate of passive immunity. Human rabies vaccines are currently available to meet vaccination needs. In addition, the immune rates of dogs and cats did not change significantly. The rabies street virus strains in China are mainly divided into 6 species including China Ⅰ-China Ⅵ. Conclusion In terms of infrastructure, it is still necessary to increase the number of primary clinics, improve the professional capabilities of medical staff, and strengthen publicity and education on rabies prevention and treatment. In the areas with high incidence of cases, vaccines similar to street strain should be selected for immunization of human and animal.

The Serine/Threonine Kinase AP2-Associated Kinase 1 Plays an Important Role in Rabies Virus Entry.

Rabies virus (RABV) invades the central nervous system and nearly always causes fatal disease in humans. RABV enters cells via clathrin-mediated endocytosis upon receptor binding. The detailed mechanism of this process and how it is regulated are not fully understood. Here, we carried out a high-through-put RNAi analysis and identified AP2-associated kinase 1 (AAK1), a serine/threonine kinase, as an important cellular component in regulating the entry of RABV. AAK1 knock-down greatly inhibits RABV infection of cells, and AAK1-induced phosphorylation of threonine 156 of the μ subunit of adaptor protein 2 (AP2M1) is found to be required for RABV entry. Inhibition of AAK1 kinase activity by sunitinib blocked AP2M1 phosphorylation, significantly inhibiting RABV infection and preventing RABV from entering early endosomes. In vivo studies revealed that sunitinib prolongs the survival of mice challenged with RABV street virus. Our findings indicate that AAK1 is a potential drug target for postexposure prophylaxis against rabies.

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Lyssavirus phosphoproteins increase mitochondrial complex I activity and levels of reactive oxygen species.

We have previously demonstrated that serine residues at positions 162 and 166 of the rabies virus (RABV) phosphoprotein (P) are critical for oxidative stress induced by CVS in cultured cells. We have now evaluated the P of two street RABV variants and Mokola (MOK) virus. The P of these viruses, like CVS, induces an increase in complex I activities and reactive oxygen species levels in transfected cells. Although the sequence homology of P is only 45% with MOK (higher for street viruses) and CVS, serine residues are conserved at positions 162 and 166, suggesting their potential importance in oxidative stress.

Comparison of immune responses to attenuated rabies virus and street virus in mouse brain.

Rabies is a lethal neurological disease caused by the neurotropic rabies virus (RABV). To investigate the innate immune response in the brain during rabies infection, key gene transcripts indicative of innate immunity in a mouse model system were measured using real-time RT-PCR. Mice were infected via the intracerebral or intramuscular route with either attenuated rabies virus (SRV9) or pathogenic rabies virus (BD06). Infection with SRV9 resulted in the early detection of viral replication and the rapid induction of innate immune response gene expression in the brain. BD06 infection elicited innate immune response gene expression during only the late stage of infection. We measured Na-fluorescein uptake to assess blood-brain barrier (BBB) permeability, which was enhanced during the early stage of SRV9 infection and significantly enhanced during the late stage of BD06 infection. Furthermore, early SRV9 replication increased the maturation and differentiation of dendritic cells (DCs) and B cells in the inguinal lymph nodes and initiated the generation of virus-neutralizing antibodies (VNAs), which cooperate with the innate immune response to eliminate virus from the CNS. However, BD06 infection did not stimulate VNA production; thus, the virus was able to evade the host immune response and cause encephalitis. The rabies virus phosphoprotein has been reported to counteract IFN activation. In an in vitro study of the relationship between IFN antagonism and RABV pathogenicity, we demonstrated that SRV9 more strongly antagonized IFN activity than did BD06. Therefore, there is no positive relationship between the IFN antagonist activity of the virus and its pathogenicity.

RAPID CONFIRMATION OF RABIES ANTIGEN IN FORMALIN-FIXED PARAFIN-EMBEDDED TISSUE BY IMMUNOPATHOLOGIC DIAGNOSIS

Skunks experimentally infected with street virus rabies intramuscularly were used to demonstrate rabies viral antigen in paraffin-embedded tissue by immunopathology diagnosis approach, the avidin biotin complex (ABC) method. The use of ABC method provides a simple and sensitive method to localize rabies virus antigen in formalin- fixed tissues. The results of the present study also demonstrated that a procedures using formalin-fixed parafin-embeded tissue and stained immunopathologicalH by the ABC method was an excellent method for both preservation of morfological details of ceils/tissues and demonstration of rabies virus antigen.

Contribution of the interaction between the rabies virus P protein and I-kappa B kinase ϵ to the inhibition of type I IFN induction signalling.

The P protein of rabies virus (RABV) is known to interfere with the phosphorylation of the host IFN regulatory factor 3 (IRF-3) and to consequently inhibit type I IFN induction. Previous studies, however, have only tested P proteins from laboratory-adapted fixed virus strains, and to the best of our knowledge there is no report about the effect of P proteins from street RABV strains or other lyssaviruses on the IRF-3-mediated type I IFN induction system. In this study, we evaluated the inhibitory effect of P proteins from several RABV strains, including fixed and street virus strains and other lyssaviruses (Lagos bat, Mokola and Duvenhage viruses), on IRF-3 signalling. All P proteins tested inhibited retinoic acid-inducible gene-1 (RIG-I)- and TANK binding kinase 1 (TBK1)-mediated IRF-3-dependent IFN-β promoter activities. On the other hand, the P proteins from the RABV street strains 1088 and HCM-9, but not from fixed strains Nishigahara (Ni) and CVS-11 and other lyssaviruses tested, significantly inhibited I-kappa B kinase ϵ (IKKϵ)-inducible IRF-3-dependent IFN-β promoter activity. Importantly, we revealed that the P proteins from the 1088 and HCM-9 strains, but not from the remaining viruses, interacted with IKKϵ. By using expression plasmids encoding chimeric P proteins from the 1088 strain and Ni strain, we found that the C-terminal region of the P protein is important for the interaction with IKKϵ. These findings suggest that the P protein of RABV street strains may contribute to efficient evasion of host innate immunity.

Antibody Response of Dogs to Ethiorab Rabies Vaccine

Rabies is 100% fatal, but it is preventable. More than 95% of human rabies cases occur in improperly treated individuals. This is partly due to the fact that modern post-exposure rabies prophylaxis is expensive and therefore not readily available in many endemic regions. Nervous tissue vaccine has been in use for more than 100 yrs. These vaccines have now been superseded in purity, potency, immunogenicity and safety. The efficacy and immunogenicity of inactivated tissue culture vaccine, produced in Ethiopia was evaluated. Twelve experimental dogs from local breed were duly conditioned during a quarantine period and assigned to two groups randomly. Animals in group I (cases) were vaccinated subcutaneously with 1 ml of our experimental vaccine. Dogs in group II served as non-vaccinated controls. The immune response of each dog was monitored for 120 days. On the day 120 after final sampling, all dogs were challenged in the masseter muscle with a rabies street virus of canine origin. To evaluate the titer of the rabies virus neutralizing antibodies (VNA), sera were analyzed by Fluorescent Antibody Virus Neutralization (FAVN) Test. Geometric Mean Titers (GMT) to rabies virus was determined at days 7, 15, 21, 30, 60, 90 and 120. Geometric mean titers were equal to 1.59, 1.73, 2.19, 3.58, 3.17, 3.35 and 3.56 IU/ml respectively. All dogs showed VNA titers higher than the 0.5 IU/ml mandated WHO recommended threshold. 83.3% vaccinated dogs, survived the challenge virus. In contrast, all dogs in the control (non-vaccinated group), developed rabies and died. This study indicated cell culture-based anti-rabies vaccine manufactured in Ethiopia is efficacious and immunogenic.

Genetic Analysis of Glycoprotein Gene of Indonesian Rabies Virus

The amino acid sequences of the Glycoprotein gene (G gene) of field rabies virus SN01-23 from Indonesiawas determined. This isolate showed homology of 93% in the ectodomain of the Glycoprotein gene to that of theRC-HL strain, which is used for production of animal vaccine in Japan. The high identity in the ectodomainbetween this field isolate and strain RC-HL suggest that the rabies animal vaccine used in Japan will be effectivefor rabies street viruses in Indonesia. Result of phylogenetic analysis using the nucleotide sequences of the Ggenes of rabies street viruses showed that SN01-23 from Indonesia is more closely related to a rabies virus fromChina than to viruses from Thailand and Malaysia. This genetic data and historical background suggest thatrabies viruses in China had been transferred to Indonesia through dogs brought by humans migrating from Chinato Indonesia.Keywords : Rabies virus, Glycoprotein gene, Ectodomain, Phylogenetic analysis

Ability of Indian street rabies virus isolates to induce apoptosis by in-vitro

An attempt had been made in this study to know the ability of Indian street rabies virus isolates to induce apoptosis in murine neuroblastoma cells. Thirty Indian street virus isolates were tested by fluorescent antibody technique (FAT) among which 20 samples was positive. These positive samples were used to infect murine neuroblastoma (MNA) cells. Four street rabies virus isolates and challenge virus strain (CVS) were used in this study to show their ability to induce apoptosis. Total RNA was isolated and cDNA was synthesized by using gene specific primers pertaining to Caspase-1, BAD (Proapoptotic gene) and Glycoprotein gene of rabies virus. Challenge Virus Standard has been used as control in the amplification as well as in the demonstration of apoptosis. The expression of Caspase-1, BAD and glycoprotein genes in the murine neuroblastoma cells in comparision with CVS was measured by using densitometer. Street virus isolates expressed higher levels of Caspase-1 and BAD in comparison with CVS. Further TUNEL staining method showed higher apoptotic index when compared to CVS. This study is an initial attempt to exhibit the ability of Indian rabies street virus isolates to induce apoptosis in murine neuroblastoma cells (in vitro).

Glycoprotein from street rabies virus BD06 induces early and robust immune responses when expressed from a non-replicative adenovirus recombinant.

The rabies virus (RABV) glycoprotein (G) is responsible for inducing neutralizing antibodies against rabies virus. Development of recombinant vaccines using the G genes from attenuated strains rather than street viruses is a regular practice. In contrast to this scenario, we generated three human adenovirus type 5 recombinants using the G genes from the vaccine strains SRV9 and Flury-LEP, and the street RABV strain BD06 (nrAd5-SRV9-G, nrAd5-Flury-LEP-G, and nrAd5-BD06-G). These recombinants were non-replicative, but could grow up to ~10(8) TCID50/ml in helper HEK293AD cells. Expression of the G protein was verified by immunostaining, quantitative PCR and cytometry. Animal experiments revealed that immunization with nrAd5-BD06-G can induce a higher seroconversion rate, a higher neutralizing antibody level, and a longer survival time after rabies virus challenge in mice when compared with the other two recombinants. Moreover, the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly higher in mice immunized with nrAd5-BD06-G, which might also contribute to the increased protection. These results show that the use of street RABV G for non-replicative systems may be an alternative for developing effective recombinant rabies vaccines.

Genetically modified rabies virus ERA strain is safe and induces long-lasting protective immune response in dogs after oral vaccination

Oral immunization in free-roaming dogs is one of the most practical approaches to prevent rabies for developing countries. The safe, efficient and long-lasting protective oral rabies vaccine for dogs is highly sought. In this study, rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain wild-type (rERA) and a genetically modified type (rERAG333E) containing a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) were generated by reverse genetic. The recombinant virus rERAG333E retained growth properties of similar to the parent strain rERA in BHK-21 cell culture. The G333E mutation showed genetic stability during passage into neuroblastoma cells and in the brains of suckling mice and was significantly reduced the virulence of rERA in mice. rERAG333E was immunogenic in dogs by intramuscular inoculation. Mice orally vaccinated with rERAG333E induced strong and one year longer virus neutralizing antibodies (VNA) to RABV, and were completely protected from challenge with lethal street virus at 12months after immunization. Dogs received oral vaccination with rERAG333E induced strong protective RABV VNA response, which lasted for over 3years, and moderate saliva RABV-specific IgA. Moreover, sizeable booster responses to RABV VNA were induced by a second oral dose 1year after the first dose. These results demonstrated that the genetically modified ERA vaccine strain has the potential to serve as a safe and efficient oral live vaccine against rabies in dogs.

Association between RABV G Proteins Transported from the Perinuclear Space to the Cell Surface Membrane and N-Glycosylation of the Sequon Asn(204).

In this study, G proteins of the rabies virus (RABV) Kyoto strain were detected in the cytoplasm but not distributed at the cell membrane of mouse neuroblastoma (MNA) cells. G proteins of CVS-26 were detected in both the cell membrane and perinuclear space of MNA cells. We found that N-glycosylation of street RABV G protein by the insertion of the sequon Asn(204) induced the transfer of RABV G proteins to the cell surface membrane. Fixed RABV budding from the plasma membrane has been found to depend not only on G protein but also on other structural proteins such as M protein. However, the differing N-glycosylation of G protein could be associated with the distinct budding and antigenic features of RABV in street and fixed viruses. Our study of the association of N-glycan of G protein at Asn(204) with the transport of RABV G protein to the cell surface membrane contributes to the understanding of the evolution of fixed virus from street virus, which in turn would help for determine the mechanism underlying RABV budding and enhanced host immune responses.

Efficiency of live attenuated and inactivated rabies viruses in prophylactic and post exposure vaccination against the street virus strain.

Rabies remains an enigmatic and widely discussed global infectious disease and causes an increasing number of deaths. The currently used highly effective prophylactic and post exposure (p.e.) vaccination depends solely upon inexpensive, effective and safe vaccines to counteract the spread of the disease. In this study, the potential of an attenuated Chinese rabies vaccine (SRV9) strain in prophylactic and p.e. vaccination against the street strain of rabies virus (RV) was evaluated in mice. Prophylactic vaccination consisting of one intramuscular (i.m.) dose of SRV9 protected 100% of mice from intracerebral (i.c.) challenge with alethal dose of the street virus. The latter was detected in the brain of mice at day 6 post challenge by RT-PCR. Post exposure vaccination was performed at days 1, 2, 3, 4, 5 and 6 post infection (p.i.) with either SRV9 or inactivated rabies vaccine. The survival rates after i.m. inoculation of SRV9 at the indicated days were 70%, 50%, 30%, 20%, 10%, and 0%, respectively; the corresponding survival rates for the inactivated rabies vaccine were 30%, 20%, 10%, 0%, 0%, and 0%, respectively. However, 100%, 90%, 70%, 50%, 20%, 10%, and 10% of mice survived after i.c. inoculation of SRV9 at the indicated days. The increased permeability of the blood-brain barrier and the infiltration of CD19+ Bcells into the central nervous system after i.c. inoculation of SRV9 are regarded as prerequisites for the clearance of the street virus. The obtained data suggest that SRV9 is apromising candidate for prophylactic and p.e. vaccination against rabies infection and that it exhibits apotential for the control of rabies in China.

Genetic characterisation of the rabies virus vaccine strains used for oral immunization of foxes in Poland to estimate the effectiveness of vaccination.

The main reservoir of rabies virus in Poland has been the red fox. To control rabies in wildlife, oral immunization of foxes was introduced in 1993. The vaccine is effective when it confers immunity against the virus circulating in the environment. To assess the above issue, a study of the molecular characteristics of 570-bp fragments of the N and G genes of vaccine strains SAD B19 and SAD Bern against street virus strains was performed. The results confirmed the similarity of the vaccine strains and rabies virus strains circulating in the environment and also demonstrate the genetic stability of vaccine strains that have been distributed in Poland for 20 years.

Comparative Analysis of the Pathogenic Mechanisms of Street Rabies Virus Strains with Different Virulence Levels

ObjectiveTo characterize two strains of street rabies virus (RABV) isolated from the brain tissue of cattle from Inner Mongolia. Differences in the histopathological and ultrastructural changes in the brain tissue of infected mice were determined to reveal variation in the pathogenesis of infection between street rabies virus strains. MethodsTen-day-old mice were intracranially inoculated with one of three virus strains and brain tissue harvested when the mice were moribund. Various histopathological and ultrastructural markers of disease were then compared between the groups. ResultsInfection with the street virus strain CNM1101C resulted in severe neuronal dendrites damage, but only mild cell apoptosis, T lymphocyte infiltration and microglial activation. Infection with the other street virus strain, CNM1103C, was characterized by cell apoptosis, T lymphocyte infiltration and microglial activation as well as dendrites damage. However, in comparison, infection with the attenuated virus strain CTN caused severe T lymphocyte infiltration, microglial activation and cell apoptosis, but left the neuronal dendrites intact. ConclusionThe two street rabies virus strains isolated from cattle from Inner Mongolia had different levels of virulence and caused distinct pathological changes in infected mice. Therefore, we concluded that different pathogenic mechanisms exist between different RABV strains.

Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB®, an adenovirus-rabies recombinant vaccine

Twenty-seven red foxes (Vulpes vulpes) were each offered a bait containing ONRAB®, a recombinant oral rabies vaccine that uses a human adenovirus vector to express the immunogenic rabies virus glycoprotein; 10 controls received no vaccine baits. Serum samples collected from all foxes before treatment, and each week post-treatment for 16 weeks, were tested for the presence of rabies virus neutralizing antibody (RVNA). In the bait group, a fox was considered a responder to vaccination if serum samples from 3 or more consecutive weeks had RVNA ≥0.5IU/ml. Using this criterion, 79% of adult foxes (11/14) and 46% of juveniles (6/13) responded to vaccination with ONRAB®. Serum RVNA of adults first tested positive (≥0.5IU/ml) between weeks 1 and 3, about 4 weeks earlier than in juveniles. Adults also responded with higher levels of RVNA and these levels were maintained longer. Serum samples from juveniles tested positive for 1–4 consecutive weeks; in adults the range was 2–15 weeks, with almost half of adults maintaining titres above 0.5IU/ml for 9 or more consecutive weeks. Based on the kinetics of the antibody response to ONRAB®, the best time to sample sera of wild adult foxes for evidence of vaccination is 7–11 weeks following bait distribution. Thirty-four foxes (25 ONRAB®, 9 controls) were challenged with vulpine street virus 547 days post-vaccination. All controls developed rabies whereas eight of 13 adult vaccinates (62%) and four of 12 juvenile vaccinates (33%) survived. All foxes classed as non-responders to vaccination developed rabies. Of foxes considered responders to vaccination, 80% of adults (8/10) and 67% of juveniles (4/6) survived challenge. The duration of immunity conferred to foxes would appear adequate for bi-annual and annual bait distribution schedules as vaccinates were challenged 1.5 years post-vaccination.

Feasibility of reducing rabies immunoglobulin dosage for passive immunization against rabies: Results of In vitro and In vivo studies

Passive immunization is a crucial parameter for prevention of human rabies. Presently as World Health Organization (WHO) strongly advocates local infiltration of rabies immunoglobulin in and around the bite wound, we feel that there is no basis for calculating the dose of immunoglobulin based on body weight. Keeping this in view we conducted both in vitro and in vivo studies to know whether the dose of immunoglobulin can be reduced and still obtain complete neutralization of the virus. In vitro neutralization studies were conducted using CVS strain of virus and BHK 21 cells. In vivo experiments were conducted in 4 weeks old Swiss albino mice by initial challenge with CVS followed by infiltration with increasing dilutions of either human rabies immunoglobulin (HRIG) and equine rabies immunoglobulin (ERIG). In vitro studies showed that a dose of 100 FFD 50 of CVS was neutralized by increasing dilution of both HRIG and ERIG and 100% neutralization was observed with HRIG and ERIG in as low quantities as 0.025 IU. In mice studies there was 100% survival of mice infiltrated with 0.025 IU of both HRIG and ERIG compared with 100% mortality in mice infiltrated with normal saline. These results suggest that it is possible to reduce the dose of rabies immunoglobulins by at least 16 times the presently advocated dose. These findings needs to be further evaluated using larger animal models and street viruses prevalent in nature but cannot serve as recommendations for use of RIG for passive immunization in humans