Challenge Virus Standard Strain Research Articles

Internalization of rabies virus glycoprotein differs between pathogenic and attenuated virus strains.

The zoonotic rabies virus (RABV) is a non-segmented negative-sense RNA virus classified within the family Rhabdoviridae, and is the most common aetiological agent responsible for fatal rabies disease. The RABV glycoprotein (G) forms trimeric spikes that protrude from RABV virions and mediate virus attachment, entry and spread, and is a major determinant of RABV pathogenesis. A range of RABV strains exist that are highly pathogenic in part due to their ability to evade host immune detection. However, some strains are disease-attenuated and can be cleared by host defences. A detailed molecular understanding of how strain variation relates to pathogenesis is currently lacking. Here, we reveal key differences in the trafficking profiles of RABV-G proteins from the challenge virus standard strain (CVS-11) and a highly attenuated vaccine strain SAD-B19 (SAD). We show that CVS-G traffics to the cell surface and undergoes rapid internalization through both clathrin- and cholesterol-dependent endocytic pathways. In contrast, SAD-G remains resident at the plasma membrane and internalizes at a significantly slower rate. Through engineering hybrids of CVS-G and SAD-G, we show that the cytoplasmic tail of CVS-G is the key determinant of these different internalization profiles. Alanine scanning further revealed that mutation of Y497 in CVS-G (H497 in SAD-G) could reduce the rate of internalization to SAD-G levels. Together, these data reveal new phenotypic differences between CVS-G and SAD-G proteins that may contribute to altered in vivo pathogenicity.

Use of rapid fluorescent focus inhibition test (RFFIT) for in vitro evaluation of anti-rabies activity.

Even in the twenty-first century, rabies remains one of the most dreaded diseases in many parts of the world. An effective chemotherapeutic still remains elusive. The present study was aimed at in vitro evaluation of crude extracts of Allamanda cathartica and Cynodon dactylon for their potential anti-rabies activity based on the principle of immunofluorescence. The extracts were tested for cytotoxicity and screened for the presence of phytochemicals. While A. cathartica extracts were found to be non-toxic, the CC50 of C. dactylon (water and methanol) cold extracts were found to be 8.17 and 9.20mg/mL respectively on BHK-21 cell line. Rapid Fluorescent Focus Inhibition Test (RFFIT) was used to evaluate anti-rabies activities of these plants against the rabies challenge virus standard strain. We observed 50% inhibition of 10 TCID50 CVS at 5mg/mL (IC50) whereas florescence (no inhibition) was observed with A. cathartica extracts. The present study highlights the use of modified RFFIT as a method of choice for testing anti-rabies activity over assays based on evaluation of cytopathic effect.

Alum adjuvanted rabies DNA vaccine confers 80% protection against lethal 50 LD50 rabies challenge virus standard strain.

Rabies is a serious concern world-wide. Despite availability of rabies vaccines for long; their efficacy, safety, availability and cost effectiveness has been a tremendous issue. This calls for improvement of rabies vaccination strategies. DNA vaccination has immense potential in this regard. The DNA vaccine pgp.LAMP-1 conferred 60% protection to BALB/c mice against 20 LD50 rabies challenge virus standard (CVS) strain challenge. Upon supplementation with Emulsigen-D, the vaccine formulation conferred complete protection against lethal challenge. To assess the feasibility of this vaccine formulation for human use, it was tested along with other FDA approved adjuvants, namely, Alum, Immuvac, Montanide ISA720 VG. Enhanced immune response correlated with high IgG antibody titer, Th2 biased response with a high level of rabies virus neutralizing antibodies (RVNAs) and IgG1/IgG2a ratio >1, observed upon alum supplementation of the rabies DNA vaccine. The total IgG antibody titer was 2IU/ml and total RVNA titer was observed to be 4IU/ml which is eight times higher than the minimum protective titer recommended by WHO. Furthermore, it conferred 80% protection against challenge with 50 LD50 of the rabies CVS strain, conducted in compliance with the potency test for rabies recommended by the National Institutes of Health (NIH), USA. Previously, we have established pre-clinical safety of this vaccine as per the guidelines of Schedule Y, FDA as well as The European Agency for evaluation of Medicinal Products. The vaccine showed no observable toxicity at the site of injection as well as at systemic level in Wistar rats when administered with 10X recommended dose. Therefore, supplementation of rabies DNA vaccine, pgp.LAMP-1 with alum would lead to development of a non-toxic, efficacious, stable and affordable vaccine that can be used to combat high numbers of fatal rabies infections tormenting developing countries.

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

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

Utility of human embryonic kidney cell line HEK-293 for rapid isolation of fixed and street rabies viruses: comparison with Neuro-2a and BHK-21 cell lines

A confirmatory rabies diagnosis can be achieved by rapid virus isolation in cell culture using brain tissue from the suspect animal. Several cell lines have been used for this purpose and the murine neuroblastoma cell line Neuro-2a has been found to be the most sensitive. The human embryonic kidney cell line HEK-293 is known to express several neuronal proteins and is believed to be of neuronal origin. We hypothesized that this cell line could be susceptible to rabies virus, which is highly neurotropic. First we tested the sensitivity of HEK-293 cells to the laboratory strain, challenge virus standard (CVS). We then tested 120 brain samples from different animals and humans suspected to have died of rabies by fluorescent antibody test (FAT). Both FAT-positive and FAT-negative brains were tested for virus isolation using Neuro-2a, BHK-21, and HEK-293 cell lines and also by mouse inoculation. There was 100% correlation between FAT, virus isolation in Neuro-2a and HEK-293 cells, and mouse inoculation. However, the rate of virus isolation in the BHK-21 cell line was only 28% when compared to the other cell lines. The sensitivity of HEK-293 to CVS strain of virus was similar to that of Neuro-2a. We conclude that the HEK-293 cell line is as sensitive as the Neuro-2a cell line for the rapid isolation of rabies virus and may serve as an alternative cell line for rabies diagnosis and future research.

Intracerebral vaccination suppresses the spread of rabies virus in the mouse brain

To investigate the efficacy of intracerebral (IC) immunization in preventing viral spread in the brain, we immunized mice with inactivated rabies virus via the subcutaneous (SC) or IC route, followed by administration of a lethal dose of rabies virus (challenge virus standard strain), directly into the brains of immunized mice. Progressive paralytic neurological signs were observed in control and 75% of SC immunized mice, whereas only 20% of IC immunized mice exhibited symptoms. Neutralizing antibody titers in blood plasma were significantly elevated in SC and IC immunized mice, with the highest levels seen in IC immunized mice. Analysis of whole brain lysates revealed a strong induction of immunoglobulin in the brains of IC immunized mice that had virus neutralizing activity. Histopathological examination of brain tissue revealed mild encephalitis and disseminated viral antigen in control and SC immunized mice, but rare in IC immunized mice. These results suggest that IC immunization induces a preventive humoral immune response against intracerebrally inoculated rabies virus. Induction of neutralizing antibody in cerebrospinal fluid represents a putative therapeutic measure for the treatment of rabid animals and humans.

English

Rabies remains an important public health problem in the world due to uncontrolled enzootic rabies, lack of safe efficient vaccines and poor information on the risk of contracting rabies post animal exposure. The lethality and mutagenic potential of challenge virus standard (CVS) was evaluated in mice. Mice were intracerebrally infected (MIC) with low, medium and high viral LD50 (MICLD50). Mice were subjected to immunomodulation using interferon (IFN
-2a) pre- infection. The infected groups pretreated with IFN-
2a showed a higher survival rate than the infected group. Statistically significant increase in structural and numerical chromosomal aberrations and a decreased mitotic activity of mice bone marrow cells was observed post infection. Pretreatment of the infected groups with IFN
-2a showed a marked and significant reduction of these cytogenetic changes. The increased survival rate and reduced cytogenetic changes suggest that protection induced by interferon against rabies virus activity could be, at least partially, attributable to blockage of the replication of CVS strain of rabies virus. It could be concluded that interferon can be used as an immune enhancer to the application of vaccine administration.

Proteomics analysis of BHK‐21 cells infected with a fixed strain of rabies virus

Rabies is a neurotropic virus that causes a life threatening acute viral encephalitis. The complex relationship of rabies virus (RV) with the host leads to its replication and spreading toward the neural network, where viral pathogenic effects appeared as neuronal dysfunction. In order to better understand the molecular basis of this relationship, a proteomics study on baby hamster kidney cells infected with challenge virus standard strain of RV was performed. This cell line is an in vitro model for rabies infection and is commonly used for viral seed preparation. The direct effect of the virus on cellular protein machinery was investigated by 2-DE proteome mapping of infected versus control cells followed by LC-MS/MS identification. This analysis revealed significant changes in expression of 14 proteins, seven of these proteins were viral and the remaining were host proteins with different known functions: cytoskeletal (capping protein, vimentin), anti-oxidative stress (superoxide dismutase), regulatory (Stathmin), and protein synthesis (P0). Despite of limited changes appeared upon rabies infection, they present a set of interesting biochemical pathways for further investigation on viral-host interaction.

Neuronal apoptosis in immunodeficient mice infected with the challenge virus standard strain of rabies virus by intracerebral inoculation

The challenge virus standard-11 strain (CVS) of fixed rabies virus produces neuronal apoptosis in widespread areas of the brain of mice after intracerebral inoculation. The role of the adaptive immune response in producing neuronal apoptosis in this model was evaluated by comparing the infections in adult C57BL/6J mice with nude mice (T cell deficient) and Rag1 mice (T and B cell deficient). Both strains of immunodeficient mice showed very similar clinical disease and neuropathological findings, including marked neuronal apoptosis. The adaptive immune response is unlikely of fundamental importance in producing neuronal apoptosis in the brains of mice in this model.

Lyssavirus P Gene Characterisation Provides Insights into the Phylogeny of the Genus and Identifies Structural Similarities and Diversity within the Encoded Phosphoprotein

A comprehensive phylogenetic analysis of the Lyssavirus genus, employing P gene sequences from 128 isolates recovered globally, is presented. While confirming prior suggestions of the genetic distinctness of the Australian bat lyssaviruses, these data also revealed a clear division within the rabies virus clade (Genotype 1) between globally distributed viruses circulating predominantly in canid species (subgroup 1-1), and American strains harbored by both chiropteran and terrestrial hosts (subgroup 1-2). Nucleotide substitution patterns within the P locus suggested differential selection operating along the length of the open reading frame (ORF) between rabies viruses of these two subgroups. Comparison of the deduced primary sequences of the encoded phosphoproteins of all isolates provided insights into the product's structural organization. Two conserved (CD1,2) and two variable (VD1,2) domains were evident; high variation in the VD2 region was reflected by differences in hydropathic profiles. Only two of five serine residues reported to function as phosphate acceptors in the P protein of the rabies challenge virus standard (CVS) strain were absolutely conserved; similarly, out of four internal methionines reported to direct internal translation initiation of the CVS strain to produce N-terminally truncated P proteins, only Met20 was universally retained. In contrast, two sequence motifs, one believed to confer binding to the cytoplasmic dynein light chain LC8, and a lysine-rich sequence probably contributing to N protein binding, were conserved throughout the genus. Most rabies viruses of the carnivora (1-1) contain a potential C ORF in alternate frame to that of P, a feature limited or absent in most other isolates of the genus, an observation interpreted with consideration to the predicted course of lyssavirus evolution.

Rabies virus ocular disease: T-cell-dependent protection is under the control of signaling by the p55 tumor necrosis factor alpha receptor, p55TNFR.

Following brain infection, the Challenge Virus Standard strain of rabies virus infects the retina. Rabies virus ocular infection induces the infiltration of neutrophils and predominantly T cells into the eye. The role of tumor necrosis factor alpha (TNF-alpha)-lymphotoxin signaling in the control of rabies virus ocular infection and inflammatory cell infiltration was assessed using mice lacking the p55 TNF-alpha receptor (p55TNFR(-/-) mice). The incidence of ocular disease and the intensity of retinal infection were greater in p55TNFR(-/-) mice than in C57BL/6 mice: the aggravation correlated with less neutrophil and T-cell infiltration. This indicates that cellular infiltration is under the control of the p55 TNF-alpha receptor and suggests that inflammatory cells may protect the eye against rabies virus ocular infection. The role of T cells following rabies virus ocular disease was assessed by comparison of rabies virus infection in nude mice with their normal counterparts. Indeed, the incidence and severity of the rabies virus ocular disease were higher in athymic nude mice than in BALB/c mice, indicating that T lymphocytes are protective during rabies virus ocular infection. Moreover, few T cells and neutrophils underwent apoptosis in rabies virus-infected retina. Altogether, these data suggest that T lymphocytes and neutrophils are able to enter the eye, escape the immune privilege status, and limit rabies virus ocular disease. In conclusion, rabies virus-mediated eye disease provides a new model for studying mechanisms regulating immune privilege during viral infection.

Experimental rabies virus infection of p75 neurotrophin receptor-deficient mice.

The low-affinity neurotrophin (NT) receptor, p75(NTR), has complex biologic functions. A recent report provided evidence that the p75(NTR) is a rabies virus receptor in cultured BSR cells. We studied the experimental infection of 6-day-old p75(NTR)-deficient mice with the challenge virus standard strain of fixed rabies virus inoculated intracerebrally. The mice developed a fatal encephalitis. There were morphologic changes of apoptotic cell death involving neurons in widespread areas of the brain, which were associated with in situ evidence of oligonucleosomal DNA fragmentation. The findings were very similar to those that we previously reported in wild-type ICR mice of the same age. If the p75(NTR) is an important receptor of rabies virus in animal hosts, then a greater effect on the clinical and pathologic features of rabies virus-infected p75(NTR)-deficient mice would have been expected.

Gene gun particle-mediated vaccination with plasmid DNA confers protective immunity against rabies virus infection

Accell ® gene gun particle-mediated immunization with DNA encoding the glycoprotein gene of the challenge virus standard strain of rabies virus was evaluated for its ability to elicit protective levels of serum anti-rabies virus neutralizing antibody. Strong primary and booster neutralizing antibody responses were detected in mice following immunization with 2 μg of DNA coated on 2.6-μm gold beads. Protective levels of antibody persisted for over 300 days. Mice challenged intraplantarly 315 days post-primary immunization (225 days post-booster vaccination) survived lethal rabies virus challenge. Our data demonstrate a potentially significant role for gene gun-based delivery of DNA in the field of rabies virus vaccination.

Rabies virus infects mouse and human lymphocytes and induces apoptosis.

Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response.

The CVS strain of rabies virus as transneuronal tracer in the olfactory system of mice

The sequential distribution of transneuronally infected neurons was studied in the olfactory pathway of mice after unilateral inoculation of the challenge virus standard (CVS) strain in the nasal cavity. A first cycle of viral multiplication was observed in a subpopulation of receptor cells scattered in the main olfactory epithelium and in the septal organ. No viral spread from cell body to cell body was reported even in later stages of infection. The second round of viral replication which took place in the ipsilateral main olfactory bulb at 2 and 2.5 days post-inoculation (p.i.), involved second order neurons and periglomerular cells, known to be directly connected with the axon terminals of receptor cells. Also reported as a result of a second cycle of viral replication, was surprisingly the spread of CVS at 2 and 2.5 days p.i. in bulbar interneurons located in the internal plexiform layer and in the superficial granule cell layer, as well as that of 2 ipsilateral cerebral nuclei, the anterior olfactory nucleus and the horizontal limb of the diagonal band. From day 3, a rapid spread of CVS was suggested by detection of virus in all ipsilateral direct terminal regions of the second order neurons and in most tertiary olfactory projections. The locus coeruleus, a noradrenergic nucleus which sends direct afferents to the olfactory bulb, never appeared immunoreactive. In spite of a certaine inability of CVS to infect some neuron types, the virus appears relevant to provide new information regarding the complex network of olfactory-related neurons into the CNS.

Basis of neurovirulence of avirulent rabies virus variant Av01 with stereotaxic brain inoculation in mice

Av01 is a variant of the challenge virus standard strain of fixed rabies virus that was selected with a neutralizing anti-glycoprotein monoclonal antibody, and has a single amino acid change in the glycoprotein. It is avirulent after both intracerebral and peripheral routes of inoculation in adult mice. In this study, Av01 was found to be neurovirulent with stereotaxic brain inoculation in either the striatum or cerebellum of adult mice. Mice that had been inoculated simultaneously with Av01 by the intracerebral and intrastriatal routes recovered. More infectious virus was present in the brains of mice inoculated intrastriatally than intracerebrally, and more neurons contained rabies virus antigen. However, the topographical distribution of infected neurons was similar with both routes. Serum neutralizing antibodies against rabies virus were produced later and in smaller quantities after intrastriatal inoculation. Av01 is probably neurovirulent after stereotaxic brain inoculation because this route produces both a direct site of viral entry into the central nervous system and a low level of immune stimulation.

Protection of mice with vaccinia virus recombinants that express the rabies nucleoprotein

The role of rabies virus nucleoprotein (N) in protection against rabies was examined with recombinant vaccinia viruses expressing the N of the Challenge Virus Standard strain. Two chimeric plasmids were constructed with the open reading frame of the N gene placed downstream of the vaccinia P 7.5 promoter (early/late class) or the vaccinia P 11 promoter (late class), with each expression cassette flanked by vaccinia thymidine kinase (TK) sequences to enable marker rescue by TK insertional inactivation. Two recombinants were isolated that expressed the rabies N in infected cells as determined by radioimmunoprecipitation and immunofluoresence microscopy with an anti-N monoclonal antibody. Two groups of 25 ICR mice inoculated intradermally with the recombinants and challenged with 75 MFPLD 50 of street rabies virus showed high survival ratios ( 22 25 and 21 25 ). Intramuscular inoculation, however, was not protective against 25 MFPLD 50. The intradermally vaccinated mice developed non-neutralizing antibodies against rabies N.

Characterization of a New Temperature-sensitive and Avirulent Mutant of the Rabies Virus

A temperature-sensitive (ts) mutant, tsG1, has been isolated from the CVS (Challenge Virus Standard) strain of rabies virus. The ts mutation affects the glycoprotein (G protein); it consists of an amino acid substitution (leucine to phenylalanine) at position 132. tsG1 exhibits a slightly reduced pathogenicity when administered via the intracerebral route and complete avirulence after intramuscular inoculation, associated with a very high protective power for adult mice. The ts mutation does not seem to block the transport of the G protein to the plasma membrane at the non-permissive temperature (39.6 degrees C). It abolishes the c.p.e. of the virus in cell cultures.

Temperature-sensitive mutants of rabies virus: Behaviour following inoculation into mouse and fox

Twenty-nine temperature sensitive (ts) mutants of the rabies challenge virus standard strain (CVS) were administered intramuscularly and intracerebrally to adult mice. All led to either rapid death or protection, with the response being dose-dependent. In mice, nine ts mutants (tsO 6, 31, 52, 55, 96, 103, 106, 116 and 117) exhibited a high level of protection for the lowest level of pathogenicity. This could not be correlated with (1) the type of mutagen which induced it, (2), inhibition of multiplication at the host temperature, (3) a weak ability to multiply in nervous cells in vitro, or (4) a specific viral function affected by the mutation. Since the tsO 55 showed the highest level of protection while at the same time remaining apathogenic in mice, it was chosen for testing in foxes. No difference in reaction was observed either intramuscularly or orally between this strain and the original CVS strain. The reasons for this lack of correlation in the response of mice and foxes are discussed. Vingt-neuf mutants thermosensibles (ts) du virus rabique, isolés à partir de la souche «challenge virus standard (CVS), ont été inoculés à des souris adultes par les voies intramusculaire et cérébrale. Tous les mutants provoquent une mort rapide ou induisent une protection; ces deux résultats étant fonction de la dose utilisée. Neuf mutants ts (tsO 6, 31, 52, 55, 96, 103, 106 et 117) induisent un haut niveau de protection pour un faible pouvoir pathogène. Ces propriétés n'ont pu être corrélées ni au type du mutagène qui les a induites, ni à une inhibition de la multiplication due à la température interne de l'hôte ni à un faible taux de multiplication dans des cellules nerveuses in vitro, ni à une fonction virale spécifique affectée lors de la mutation. Comme le mutant tsO 55 produit un haut niveau de protection en l'absence totale de pouvoir pathogène, ce mutant a été choisi pour être testé chez le renard ainsi que la souche CVS dont il est issu. Aucune différence de réaction n'a été observée entre les deux souches ni par voie musculaire ni par voie orale. Les raisons de ce manque de corrélation de la réponse du mutant ts chez la souris et le renard sont discutées.