Rabies Virions Research Articles

Characterization of a human antibody fragment Fab and its calcium phosphate nanoparticles that inhibit rabies virus infection with vaccine.

Recombinant antibody phage display technology has been used to mimic many aspects of the processes that govern the generation and selection of high-affinity natural human antibodies in the human immune system, especially for infectious disease prophylaxis. An anti-rabies virus immunized phage-display Fab library was constructed from peripheral blood lymphocytes from vaccinated volunteers. The immunized antibody library, with a diversity of 6.7×108, was used to select and produce antibodies that bound to rabies virus glycoprotein. After five rounds of immobilized fixed rabies virion panning, four unique DNA sequences were found in the higher binding clones, and only one, Fab094, showed neutralization activity. Fab094 components were analyzed by ELISA, immunoprecipitation and immunofluorescent staining. ELISA and immunofluorescence showed that Fab094 bound specifically to rabies virions. Immunoprecipitation and mass spectrometry showed that Fab094 reacted with rabies virus glycoprotein. To improve the penetration power of Fab094 antibodies, we developed Fab094 calcium phosphate nanoparticles (Fab094-CPNPs) and tested their efficacy. The rapid fluorescent focus inhibition test indicated that the neutralizing antibody titers of Fab094 and Fab094-CPNPs were reached at 200.17 IU/Kg and 246.12 IU/Kg, respectively. These findings were confirmed in vivo in a Kunming mouse challenge model. Our results demonstrate that human Fab094 and Fab094-CPNPs are efficacious candidate drugs to replace rabies immunoglobulin in post-exposure prophylaxis (PEP).

Isothermal amplification of rabies virus gene.

A sensitive and specific in situ amplification technique is needed to elucidate the dynamics of rabies virus in the body during the long incubation period after infection. To overcome the disadvantage of using the traditional reverse transcription (RT)-PCR in in situ studies, an isothermal nucleic acid sequence-based amplification (NASBA) technique was developed for detection of the rabies virus gene. The NASBA technique involves the use of 4 enzymatic activities to produce multiple RNA copies of the target sequence by means of double-strand cDNA intermediates under an isothermal condition without thermocycling. The amplified cDNA intermediates from the genomic RNA in the rabies virion and the total RNA in the infected cells in NASBA reaction were analyzed by Southern hybridization assays. The specific amplified products of the rabies viral gene with the expected length were detected after 8 hr of incubation in NASBA using both of the RNAs as templates. The NASBA system used in this study was less sensitive than the general RT-PCR technique. This may have been because we employed Southern hybridization for the amplified cDNA intermediates, not many RNA copies, to evaluate the NASBA results. In conclusion, we successfully amplified the rabies viral gene in the NASBA reaction under an isothermal condition. The unique character of this technique would make it particularly valuable for in situ studies not only on rabies virus but also on other RNA viruses.

The rabies virion-associated 100-kDa polypeptide (VAP100) is a host-derived minor component of the viral envelope.

We investigated a minor polypeptide component of 100-kDa detected in the rabies virion (referred to as VAP100) by using a monoclonal antibody (mAb), #16743, which was shown to recognize the SDS-denatured VAP100 antigen by immunoblot analyses. Although the VAP100 antigen was hardly detectable in the cell by usual immunoblot methods with this mAb, we could detect the antigen by a luminescent immunoblot method as well as by immunoprecipitation from the metabolically radiolabeled cell lysates and virions. Fluorescent antibody (FA) staining with mAb #16743 detected the uniformly distributed antigen on the formalin-fixed normal BHK-21 cells, while slight accumulation of the antigen was also seen in the Golgi area when the cells were permeabilized by treatment with Triton X-100 after fixation. Rabies virus infection induced alteration of the behavior of VAP100 to show a spotted distribution pattern in virus-infected cells. Double FA staining with mAb #16743 and rabbit antibody against the rabies virus envelope antigen demonstrated colocalized distribution of the viral envelope antigens and VAP100 in the cell. From these results, we think that VAP100 is a membrane-associated component of the cell, and its colocalized distribution with the viral envelope antigens in the cell implicates an intimate association of the VAP100 with viral envelope protein(s) and a reflection of possible involvement in the efficient incorporation of VAP100 into the virion.

The 21-kDa polypeptide (VAP21) in the rabies virion is a CD99-related host cell protein.

In our monoclonal antibody (MAb) stocks prepared against the BHK-21 cell antigens, two (#11875 and 28276) recognized a 21-kDa polypeptide (referred to as VAP21) which is efficiently incorporated into the rabies virion. By using these MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids from our BHK-21 cell cDNA library. Based on the following evidence, the cDNA was assumed to encode a full-length sequence of VAP21 antigen: i) expression of the cDNA in animal cells resulted in the production of a polypeptide recognized by the two MAbs, and its electrophoretic mobility was the same as that of authentic VAP21 antigen; and ii) immunization with the products from the cDNA-transformed E. coli cells raised specific antibodies in rabbits that recognized a 21-kDa polypeptide in the virion. From the deduced amino acid sequence, it is suggested that the VAP21 antigen has a molecular structure of type-I transmembrane protein containing characteristic proline-rich and glycine-rich regions in its ectodomain. Homology searches resulted in finding homologous sequences (totally about 40% homology) in the human MIC2 gene product (CD99; 32-kDa) of T lymphocytes. These results suggest that the VAP21 antigen in the rabies virion is a cellular CD99-related transmembrane protein.

Immunological studies of a 21 kDa cellular component efficiently incorporated into rabies virion grown in a BHK-21 cell culture.

To investigate cellular components incorporated into the rabies virion, monoclonal antibodies (MAbs) were screened based on their reactivity with additional virion components. Two of the MAbs we prepared recognized a virion-associated 21 kDa polypeptide (referred to as VAP21) from a BHK-21 cell. Since the MAbs precipitated the rabies virion and trypsin digestion eliminated the VAP21 antigen from the virion but alkaline treatment (pH 11) did not, VAP21 seems to be anchored into the viral envelope and exposed on the virion surface. Although quantitative immunoblot analyses indicated an apparently increased concentration of VAP21 in the virion, the ratio of the content of VAP21 to that of viral glycoprotein (G) was several times decreased as compared to the ratio of those in the cell. These data suggest that sorting of VAP21 occurs during the viral budding process on the cell but that it might be inefficient, probably due to a more intimate association of VAP21 with the viral envelope proteins. This assumption seems to be consistent with the results of immunofluorescence studies; that is, VAP21 displayed colocalized distribution with viral envelope antigens in the cell. From these results, it is suggested that VAP21 closely associates with the viral envelope proteins in the cell, and this association might cause passive but relatively efficient incorporation of VAP21 into the virion.

Ambisense gene expression from recombinant rabies virus: random packaging of positive- and negative-strand ribonucleoprotein complexes into rabies virions.

We have recovered from cDNA a rabies virus (RV) containing identical, transcriptionally active promoters at its genome (negative-strand) and antigenome RNA and directing efficient expression of a chloramphenicol acetyltransferase (CAT) reporter gene from the antigenome. Transcription of the antigenome CAT gene was terminated by a modified RV gene junction able to mediate transcription stop and polyadenylation but not reinitiation of downstream transcripts. While in standard RV-infected cells genome and antigenome RNAs were present in a 49:1 ratio, the ambisense virus directed synthesis of equal amounts of genome and antigenome RNA (1:1). Total replicative synthesis was reduced by a factor of less than 2, revealing an unexpectedly high level of replication activity of the transcriptionally active promoter in the absence of the parental antigenome promoter. Successful packaging of ambisense ribonucleoprotein complexes (RNPs) into virions demonstrated that the parental 5' end of the RV genome RNA does not contain putative signals required for incorporation into virions. As determined both for standard RV and ambisense RV, virus particles contained genome and antigenome RNPs in the same ratios as those present in infected cells (49:1 and 1:1, respectively), indicating indiscriminate incorporation of RNPs independent of signals in the RNA. Ambisense expression of multiple foreign genes from RV vectors may circumvent problems with transcriptional attenuation of rhabdovirus housekeeping genes.

Rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes.

SummaryRecent studies have reported the detection of rabies viral antigens and virions in astrocytes and microglia of rabies-infected animals. As a first step toward understanding whether these glial cells may be involved in rabies virus replication, persistence, and/or pathogenesis, we explored their potential to be infected in vitro. Primary cultures of murine, feline, and human microglia and astrocytes were infected with several different rabies viruses: two unpassaged street virus isolates, a cell culture-adapted strain, and a mouse brain-passaged strain. Infection, as determined by immunofluorescence, was detected in 15 of the 16 (94%) virus-glial cell combinations. Replication of infectious virus, determined by infectivity assay, was detected in 7 of the 8 (88%) virus-cell combinations. These results show that astrocytes and microglia can be infected by rabies viruses, suggesting that they may have a potential role in disease, perhaps contributing to viral spread, persistence and/or neuronal dysfunction.

Cellular actin-binding ezrin-radixin-moesin (ERM) family proteins are incorporated into the rabies virion and closely associated with viral envelope proteins in the cell

Cellular ezrin-radixin-moesin (ERM) family proteins, members of the actin-binding proteins of the band 4.1 superfamily, were detected in the virions of enveloped viruses, such as rabies, vesicular stomatitis, Newcastle disease, and influenza viruses. To elucidate the mechanism of ERM protein incorporation, we investigated possible association of ERM proteins with viral components in rabies virus-infected BHK-21 cells. Double immunofluorescence studies demonstrated that the ERM proteins are concentrated in the microvilli, where the colocalized viral G protein was also seen. Viral G protein expressed in the G cDNA-transfected COS-7 cells also displayed similar distributions to those seen in the virus-infected cells. Both the ERM and viral envelope proteins were coprecipitated by anti-viral G antibody from lysates of the virus-infected cells, while the anti-ERM antibody coprecipitated viral G and ERM proteins. These observations suggest that the ERM proteins are closely associated with viral envelope proteins in the cell, which would be involved in the selective incorporation of cellular actin into the virion.

Infectious rabies viruses from cloned cDNA.

The generation of infectious rabies virus (RV), a non-segmented negative-stranded RNA virus of the Rhabdoviridae family, entirely from cloned cDNA is described. Simultaneous intracellular expression of genetically marked full-length RV antigenome-like T7 RNA polymerase transcripts and RV N, P and L proteins from transfected plasmids resulted in formation of transcriptionally active nucleocapsids and subsequent assembly and budding of infectious rabies virions. In addition to authentic RV, two novel infectious RVs characterized by predicted transcription patterns were recovered from modified cDNA. Deletion of the entire non-translated pseudogene region, which is conserved in all naturally occurring RVs, did not impair propagation of the resulting virus in cell culture. This indicates that non-essential genetic material might be present in the genomes of non-segmented RNA viruses. The introduction of a functional extra cistron border into the genome of another virus resulted in the transcription of an additional polyadenylated mRNA containing pseudogene sequences. The possibility of manipulating the RV genome by recombinant DNA techniques using the described procedure--potentially applicable also for other negative-stranded viruses--greatly facilitates the investigation of RV genetics, virus-host interactions and rabies pathogenesis and provides a tool for the design of new generations of live vaccines.

Both the N- and the C-Terminal Domains of the Nominal Phosphoprotein of Rabies Virus Are Involved in Binding to the Nucleoprotein

The interaction of the nominal phosphoprotein (NS) and the nucleoprotein (N) of rabies virus (Evelyn Rokitnicki Abelseth strain) was investigated by expressing these proteins in insect cells and in an in vitro coupled transcription-translation system. The N and NS proteins individually expressed in insect cells interacted with each other in vitro and formed complexes at a N:NS ratio similar to those found in rabies virions. In the in vitro transcription-translation system, both the N and NS proteins when systhesized simultaneously formed N-NS complexes which could be immunoprecipitated with either anti-N or anti-NS antibodies. NS mutant proteins with C terminal deletion of up to 166 amino acids were still able to form complexes with N protein when synthesized simultaneously. However, when the NS mutant proteins and the N protein were synthesized individually and then mixed together, only the intact NS protein and NS mutant protein with 24 amino acids deleted from the C-terminus bound to the N protein, whereas deletion of 47 or more amino acids from the C-terminus of tile NS protein resulted in total loss of binding to the N protein. NS mutants with N-terminal deletions of up to 68 amino acids bound to the N protein when synthesized either simultaneously or individually. These results indicate that both the N- and C-terminal domains of the NS protein of rabies virus are involved in the binding to rabies virus N protein but not in a mutually dependent manner. The interaction of the N-terminal domain of the NS protein with N protein occurs only during simultaneous synthesis of both proteins, whereas the C-terminal region of the NS protein can bind to the N protein when these two proteins are synthesized either simultaneously or separately. The two binding sites of the NS protein to N protein might have important functions in regulating virus transcription and replication as well as in virus assembly.

Rescue of synthetic genomic RNA analogs of rabies virus by plasmid-encoded proteins

Proteins entirely expressed from cDNA were used to rescue synthetic RNA genome analogs into infectious defective particles of rabies virus (RV). Synthetic negative-stranded RNAs containing 3'- and 5'-terminal RV sequences and transcriptional signal sequences were transcribed from plasmids transfected into cells expressing T7 RNA polymerase from recombinant vaccinia virus. After simultaneous expression of RV N, P, and L proteins from plasmids containing a T7 RNA polymerase promoter, the synthetic genomes were encapsidated, replicated, and transcribed by the RV polymerase proteins. Insertion of the bacterial chloramphenicol acetyltransferase gene or beta-galactosidase (lacZ) gene between the 3' and 5' termini containing transcriptional signal sequences resulted in transcription of mRNAs and expression of chloramphenicol acetyltransferase and beta-galactosidase, respectively. Upon simultaneous expression of N, P, M, G, and L proteins, virions carrying the foreign genes were assembled and released into the supernatant. The possibility of rescuing cDNA into rabies virions by proteins also expressed entirely from cDNA opens the possibility of studying the functions of each RV protein and analyzing cis-acting signals of the RV genome.

Identification of heat shock protein 70 in the rabies virion

We investigated a 73-kDa polypeptide (p73), a minor component of the rabies virion (HEP-Flury and ERA strains), accounting for as much as 1 % of total virion proteins. Two-dimensional gel electrophoresis and immunoblotting with the antiserum against the heat shock protein 70 (hsp70) demonstrated that p73 was identical to a constitutive type of cellular hsp70. The antiserum also detected p73/hsp70 in the purified virions of other negative-stranded RNA viruses, such as vesicular stomatitis virus (New Jersey serotype), Newcastle disease virus (Miyadera strain), and influenza A virus (PR8 strain), among which, however, the contents were variable.

Putative antigenic domains in glycoprotein G of rabies virus: Is the RGK sequence involved in virus adsorption to cellular receptors?

Computer analysis of rabies virus glycoprotein G provides a means for identification of functional domains in the viral glycoprotein. The computer analysis suggested 22 putative antigenic domains, of which three are RG-containing amino acid sequences that might be involved in the binding of rabies virions to cellular receptors. Synthetic RG peptides may be able to interfere with rabies virus adsorption to cellular receptors.

Immunogenic and protective properties of rabies virus glycoprotein expressed by baculovirus vectors

The gene encoding the glycoprotein of rabies virus (G protein, CVS strain) has been cloned and inserted into the baculovirus transfer vector pAcYM1 derived from the nuclear polyhedrosis virus of Autographa californica (AcNPV). The gene was placed under the control of the AcNPV polyhedrin promoter and expressed to high levels by the derived recombinant virus using a Spodoptera frugiperda cell line. It has been established that the antigenic characteristics of the protein were conserved by comparison with those of the native glycoprotein of rabies virions. The immunogenicity of the expressed product was also demonstrated. Intraperitoneal or intramuscular injection of G antigen conferred protection to mice and was associated with the induction of high titers of neutralizing antibodies. The availability of large quantities of antigenically and immunogenically reactive rabies G protein may make feasible crystallographic studies and the safe preparation of a low cost subunit vaccine for the disease.

The Anterograde Transport of Rabies Virus in Rat Sensory Dorsal Root Ganglia Neurons

We have previously described the capacity of neurites extending from cultured rat sensory dorsal root ganglia (DRG) neurons to transport rabies virus through axoplasm in the retrograde direction. Here we report the infection of cultured neurons derived from the DRG and the subsequent anterograde transport of rabies virus from the infected cell somas through the extending neurites to its release into the culture supernatant. Viral transport was monitored by titration of the virus yield in the external compartment. Both early and late transport mechanisms of rabies virions were identified. The first one occurred a few hours post-infection and was undetectable 6 h later, before the initiation of viral replication. The velocity of this first wave of infective virions was in the range of 100 to 400 mm/day. The early viral transport was probably the result of a direct translocation of infective virions from the somatic site of entry to the neuritic extensions and subsequent release into the culture medium without replication in the cellular perikaryon. The second virus transport peak was detected 48 h post-infection. In this case, the virions detected in the neuritic compartment were presumably the progeny of the inoculated virus which had replicated in the perikaryon before the viral transport occurs. Using a four-compartment culture device we were able to demonstrate, simultaneously, retrograde and anterograde transport of the virus. The presence of antirabies serum in contact with the exposed neurites did not inhibit either the retrograde or the anterograde transport mechanisms. The viral release from the neuritic extensions after the fast anterograde transport was evaluated to be in the range of 150 to 300 infectious virions per bundle of neurites per day.

Vaccination challenge studies with variants of street rabies virus isolated in Nigeria

In a preliminary study it was observed that adult ICR mice immunized with serial dilutions of an inactivated experimental human rabies vaccine from the Pitman-Moore (PM) vaccine virus were well protected against challenge with homologous virulent PM virus and challenge virus standard (CVS). However only one of five variant representatives in five of seven groups of 41 isolates of street rabies virus from Nigeria characterized by hybridoma monoclonal antibodies specific for the nucleocapsid and glycoprotein antigens of rabies virion was protected for by the vaccine. Guinea pigs immunized with a live attenuated low egg passage (LEP, Flury strain) vaccine currently used in canine vaccination in Nigeria protected against challenge with all five variants. The LEP vaccine protected against the variants and CVS quite well even when 1:125 dilution of the vaccine was used.

Cloning of rabies virus matrix protein mRNA and determination of its amino acid sequence

A cDNA clone of mRNA for rabies virus matrix (M) protein has been identified. The clone hybridizes to an mRNA species from rabies virus-infected cells, whose size correlates to the size of the M protein in rabies virions, and selects an mRNA that translates into a polypeptide corresponding in size to M protein. The nucleotide sequence of the cloned cDNA was determined and from this a complete amino acid sequence for M protein was deduced. The deduced sequence of 202 amino acids bears no detectable sequence homology with vesicular stomatitis virus M protein although these proteins may share functional homology.

Amplification of rabies virus-induced stimulation of human T-cell lines and clones by antigen-specific antibodies.

The effect of antigen-specific antibodies on the response of human T-cell lines and clones to rabies virus was studied. Plasmas from rabies-immune vaccine recipients, but not those from nonimmune individuals, enhanced the proliferative response of rabies-reactive T cells to whole inactivated virus or to the purified glycoprotein and nucleocapsid from the rabies virion. Rabies-immune plasma also increased the antigen-induced production of gamma interferon by the rabies-specific T-cell lines. Experiments performed on T-cell clones specific for either rabies glycoprotein or nucleocapsid showed that immune plasma as well as antiglycoprotein and antinucleoprotein murine monoclonal antibodies possessed the capacity to increase significantly the antigen-induced proliferative responses of these clones. The overall results indicate that this in vitro effect of antigen-specific antibodies on the response of regulatory T lymphocytes to rabies virus could be an important factor in the development of effective immune responses in vivo to rabies virus.

Phosphorylation of the N and M1 proteins of rabies virus.

Phosphorylation of rabies virus proteins was followed in vivo and in vitro. The N and M1 proteins were both found to be phosphorylated. The M1 protein was present in the virion in two phosphorylated states, but only the hypophosphorylated form of M1 was found in infected cells. The hypothesis that some of the M1 molecules become hyperphosphorylated during the maturation process by a membrane-bound kinase was examined. The phosphorylation of the viral proteins by the kinase present in purified rabies virions was studied using an in vitro transcriptase assay: under the conditions of the assay, additional phosphate groups were rapidly attached to the N protein. The M1 protein was similarly hyperphosphorylated although more slowly. Whether the hyperphosphorylation of the N protein is responsible for the poor efficiency of the in vitro transcriptase reaction is not clear. No detectable change in the phosphorylation of cellular proteins was observed in the course of rabies virus infection.

A sensitive bioassay system for detecting defective interfering particles of rabies virus

Rabies virus has been known to produce CPE after several days incubation. By increasing the incubation temperature to 39–41° after an initial period at 34–36°, we found a rapid CPE accompanied by a cessation of viral replication. When this procedure was utilized with a mixed infection of infectious and DI rabies virions, the surviving cells infected with DI particles stood out against a background of lysed cells which had been infected with infectious virions alone. With this finding, an interference focus-forming assay of rabies virus DI particles was devised. The critical m.o.i. of helper infectious virus was determined to be 0.2 PFU/cell. When a sample had many infectious virions which interfered with this narrow limit of the optimal m.o.i., that problem was overcome by UV irradiation, which inactivated infectious virions at a much higher rate than DI particles. Numbers of interference focus-forming units (IFU) were in linear proportion to the input dose of DI particles. Under optimal conditions, this temperature increase procedure was sensitive enough to measure as few as 20 IFU/ml, almost equal in sensitivity to the plaque assay.