Validation of the inactivant binary ethylenimine for inactivating rabies virus for veterinary rabies vaccine production.

The rabies vaccine is produced by inactivation of rabies virus propagated on BHK21 cells. In the rabies inactivation process, BEI is added at a final concentration of 1.6 mM to the viral harvest at 37 degrees C, followed by a second dose of BEI at 24 h post-inactivation. Inactivation was confirmed by the mice innocuity test and tissue culture amplification test as per B.P (Vet) 2004. Validation of test procedure is essential as per cGMP requirement. The dose of BEI was validated by using lower and higher concentrations of BEI in inactivation process. The study indicated that BEI at a lower concentration (0.4 mM) was able to inactivate the rabies virus within 30 h and the routine concentration (1.6 mM) of BEI is effective in inactivating rabies virus within 18 h. The amplification test used for confirming the inactivation of the live virus was validated by spiking the sample with different dilutions of pretitrated live rabies virus. The test revealed that the amplification method is sensitive to detect live rabies virus if present in the inactivated sample. The validation of BEI as an inactivant and the amplification test are discussed.     

Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein.

Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.     

Consideration of inactivated rabies vaccines as oral immunogens of wild carnivores.

An experimental beta-propiolactone (BPL)-inactivated rabies virus vaccine was evaluated for the oral immunization of captive raccoons (Procyon lotor) and red foxes (Vulpes vulpes). None of 10 red foxes administered a single 1.0 ml dose of BPL-inactivated rabies virus vaccine (PM strain; 100 or 500 micrograms protein) per os developed detectable anti-rabies virus-neutralizing antibodies (VNA) at any time over 8 wk of observation. Foxes were excluded from further study. In two different groups of five to six raccoons, each administered a single 1.0 ml dose of BPL-inactivated rabies virus vaccine (ERA strain) per os, at concentrations of 100 or 400 micrograms protein, only a single animal in each group demonstrated evidence of seroconversion within 4 wk. In contrast, instillation of a single dose (500 micrograms protein) of BPL-inactivated rabies virus vaccine (ERA strain), directly into the small intestine via fiberoptic endoscope, or ERA vaccine (800 micrograms protein) instillation to the buccal cavity by needle-less syringe, resulted in the production of rabies-specific VNA and protection against lethal rabies infection in three of six, and in four of six raccoons, respectively; all seven control raccoons succumbed to street virus challenge. These preliminary challenge studies, while somewhat encouraging, demonstrate that considerable quantities of purified viral antigen are required for even minimal oral efficacy against lethal rabies infection. At the present time, therefore, potent, self-replicating, attenuated, or recombinant viruses offer the most versatile, economic, efficacious, and safe solutions to terrestrial rabies control of free-ranging carnivores.     

Failure of interferon alfa and tribavirin in rabies encephalitis.

OBJECTIVE--To test the effect of interferon alfa and tribavirin (ribavirin) in patients with rabies encephalitis. DESIGN--An open trial of chemotherapy and intensive care in patients with early rabies. SETTING--The intensive care unit of a Bangkok hospital. PATIENTS--Four conscious men with clinical rabies encephalitis. INTERVENTIONS--Rapid virological diagnosis of rabies. Treatment with intravenous and intraventricular injections of high doses of lymphoblastoid interferon alfa in three patients and tribavirin in one patient. Intensive care was given throughout. MAIN OUTCOME MEASURES--Rabies infection confirmed by antigen detection and virus isolation. Rabies neutralising antibody and specific IgM sought in serum and cerebrospinal fluid. Interferon concentrations monitored before and during treatment in three patients. RESULTS--Interferon alfa treatment produced high concentrations in serum and cerebrospinal fluid. All four patients died after 5 1/2 to 12 1/2 days of treatment with no evidence of virostatic or clinically beneficial effects from either treatment. CONCLUSION--Interferon alfa treatment is not effective in rabies encephalitis. The use of tribavirin warrants further study, possibly combined with new therapeutic methods.     

Protection of mice from rabies by intranasal immunization with inactivated rabies virus.

The mucosal immunization method is a needle-free alternative way of vaccination. This study evaluated the efficacy of mucosal immunization for rabies. Mice were intranasally administered five times with inactivated and concentrated rabies virus antigen (CRV) supplemented with or without cholera toxin (CT). The anti-rabies virus antibody titer of mice intranasally immunized with CRV plus CT (CRV/CT) was comparable to that of mice intraperitoneally immunized twice with the same amount of CRV. Virus neutralizing (VNA) titers of mice immunized intranasally with CRV/CT were slightly lower than those of intraperitoneally immunized mice. Both anti-rabies virus ELISA antibody and VNA titers of mice immunized with CRV without CT were significantly lower than those of mice immunized with CRV/CT. In mice intranasally immunized with CRV/CT, and intraperitoneally immunized mice, high levels of IgG(2a) antibody were detected, suggesting the activation of Th1-driven cellular immunity by the two ways of immunization. All immunized mice were challenged intracerebrally with a lethal dose of virulent rabies virus CVS strain. The survival rates of mice immunized with CRV/CT and CRV without CT were 67% and 17%, respectively, while the rate of intraperitoneally immunized mice was 100%. Antigen-specific whole IgG and IgG(2a), and VNA titers of survived mice were significantly higher than those of dead mice at the challenge day. These data suggest the possibility of intranasal immunization with inactivated antigen as a rabies vaccination strategy and the importance of a mucosal adjuvant such as CT.     

Characterization of saturable binding sites for rabies virus.

A specific, saturable receptor for rabies virus was analyzed on cultured cells of neural or non-neural origin. Viral attachment kinetics were enhanced by DEAE-dextran, an effect which in turn enhanced the apparent infectivity of the virus inoculum. Under optimized conditions, the attachment of metabolically labeled ERA strain rabies virus obeyed the laws of mass action, whereby the amount of virus bound to cells varied proportionally with the concentration of cells or virus. Attachment was sensitive to changes of temperature and pH, did not require divalent cations such as Mg2+ or Ca2+, and occurred despite prior treatment of cells with proteolytic or sialic acid-specific enzymes. Saturation of the cell surface with rabies virus could be accomplished with 3 X 10(3) to 15 X 10(3) attached virions per cell. Competition for the rabies receptor occurred with rabies nonpathogenic variant virus, RV194 -2, and vesicular stomatitis virus. Reovirus type 3, another neurotropic virus, failed to inhibit rabies virus binding, and West Nile virus only slightly inhibited rabies virus binding, suggesting independent cellular receptors were recognized by these viruses. Isolated rabies virus glycoprotein failed to compete in an equivalent manner. However, solubilization of BHK-21 cells with octylglucoside yielded a chloroform-methanol-soluble extract which blocked rabies virus attachment. The binding inhibition activity of this extract was resistant to proteases but could be destroyed by phospholipases and neuraminidase, suggesting a phospholipid or glycolipid component at the receptor site. These data provide evidence for a rhabdovirus-common mechanism for cellular attachment to cells in culture.     

Progress towards rabies control.

Although safe and efficacious tissue-culture-derived rabies vaccines are available in developed countries, much of the world still depends on vaccines derived from neural tissue which were introduced half a century ago. Considerable advances have been made in our understanding of the molecular biology of rabies virus, and genetically engineered recombinant viruses (vaccinia-rabies virus glycoprotein) have been developed. These may facilitate the control of rabies in some species by oral vaccination campaigns.     

Experimental inoculation of raccoons (Procyon lotor) with rabies virus of skunk origin.

To determine raccoon (Procyon lotor) susceptibility and serum neutralizing antibody response to a skunk salivary gland rabies virus, raccoons were inoculated with a rabies virus isolated from a naturally-infected striped skunk (Mephitis mephitis). Raccoons were divided into four groups of three animals each. A dilution of the rabies virus suspension, 10(2.4), 10(3.4), or 10(4.8), mouse intracerebral lethal dose50 (MICLD50), was administered into the masseter muscles of each animal. Three negative control animals received only diluent. Saliva and sera were collected on post-inoculation days 35, 63 and 92 for virus isolation and determination of serum neutralizing antibody titer. All animals survived the 92 day observation period and none exhibited the behavioral changes classically associated with clinical rabies virus infections. Rabies virus was not detected in the saliva of any raccoon and two of the three animals receiving the highest inoculum developed serum neutralizing antibodies (SNA). On day 92, a challenge suspension of New York City/Georgia (NYC/GA) strain rabies virus in fox salivary glands (10(3.2) MICLD50) was administered to all 12 raccoons. All animals succumbed to rabies virus except the two animals that had earlier developed SNA. The results of this study provided evidence about the susceptibility of raccoons to a skunk rabies virus and demonstrated that exposed raccoons could survive for at least 92 days following exposure. Furthermore, animals developing SNA under such circumstances were capable of withstanding challenge with rabies virus that was fatal for seronegative raccoons.     

Vaccination of small Asian mongoose (Herpestes javanicus) against rabies

Oral vaccination of free-ranging wildlife is a promising technique in rabies control. The small Asian mongoose (Herpestes javanicus) is an important reservoir of rabies on several Caribbean islands, but no vaccines have been evaluated for this species. Captive mongooses were used to test the safety and efficacy of the commercially licensed vaccinia-rabies glycoprotein (V-RG) recombinant vaccine and a newly developed genetically engineered oral rabies virus vaccine (SPBNGA-S). In one study using V-RG, no vaccinated animals developed detectable rabies virus-neutralizing antibodies, and all but one died after experimental challenge with rabies virus. In contrast, all animals given SPBNGA-S demonstrated seroconversion within 7 to 14 days after vaccination and survived rabies virus challenge. On the basis of these preliminary results indicating the greater efficacy of SPBNGA-S vs. V-RG vaccine, additional investigations will be necessary to determine the optimal dose and duration of vaccination, as well as incorporation of the SPBNGA-S vaccine into edible bait.     

Presence of rabies neutralizing antibodies in wild carnivores following an outbreak of bovine rabies.

In an outbreak of bovine rabies in Argentina, a study was made of vampire bats (Desmodus rotundus) and wild carnivores. Rabies antibody rates of high prevalence were found in the bats, foxes (Dusicyon gymnocercus) and skunks (Conepatus chinga). The outbreak was part of an extensive continuing epizootic of vampire transmitted bovine rabies which may have also involved other vectors in the area of this study. Consumption of dead and dying bats by the carnivores is the suggested means of passage of rabies virus from vampire bats to foxes and skunks. Given optimum conditions it is conceivable that some outbreaks in carnivores may begin in this way.     

Appraisal of rabies vaccine potency by determination of in vitro, specific interleukin-2 production

The potency of different rabies vaccines was measured via cell mediated immunity (CMI) assessed by the production of interleukin-2 (IL-2) by CD4+CD8- lymphocytes. IL-2 production by splenocytes from mice immunized with various vaccines was measured following in vitro stimulation with antigens from different rabies and rabies-related strains. IL-2 production was specific, reproducible and correlated with the vaccine protective activity as determined by the pre-exposure NIH test. Our results suggest that measurement of IL-2 production could be used for the appraisal of rabies vaccine potency.     

Wildlife rabies in Zambia.

Wildlife species made up 26 (2.0%) of 1,304 positive rabies cases received between 1969 and 1976. The jackal (Canis adustus) was the predominate wildlife species involved (69%) and played a role in the epidemiology of bovine rabies in remote farm areas. Rabies appears to be absent from the intact wildlife communities in Zambia, especially the National Parks; this is considered in the light of the epidemiology of the disease in wildlife.     

Protective action of rifampicin in experimental rabies infection of albino mice

The paper presents the results of the experimental study of the action of rifampicin on the process of rabies infection in albino mice contaminated with 1-10 LD50 of the fixed rabies virus. Exposure to rifampicin in doses of 250 and 500 micrograms/mouse (35-70 mg/kg) resulted in survival of 66.7 and 83.4 per cent of the animals respectively while in the controls it did not exceed 16.6 and 25.0 per cent. The average life-span of the albino mice treated with the antibiotic increased 1.6-2.4-fold in comparison with the controls. The chemotherapeutic index of rifampicin representing the ratio of the maximum tolerance dose to the minimum dose providing the protective action was equal to 20. The protective action was observed either after administration of the antibiotic according to the treatment-and-prophylaxis scheme or after administration of its 2- or 3-fold dose once a day immediately after the contamination.     

Human rabies immunoglobulin assayed by the rapid fluorescent focus inhibition test suppresses active rabies immunization

The rabies antibody content of each of ten lots of human rabies immunoglobulin was titrated by both the mouse neutralization test and the rapid fluorescent focus inhibition test. The two tests did not give comparable results, the antibody titres obtained by the mouse neutralization test being 1·4–9·6 times higher than those obtained by the rapid fluorescent focus inhibition test. This titre difference was associated with a consistently lower antibody response in human volunteers who had received post-exposure rabies vaccine treatment which included the administration of RIG assayed by the RFFIT.     

Overexpression of cytochrome C by a recombinant rabies virus attenuates pathogenicity and enhances antiviral immunity

The pathogenicity of individual rabies virus strains appears to correlate inversely with the extent of apoptotic cell death they induce and with the expression of rabies virus glycoprotein, a major inducer of an antiviral immune response. To determine whether the induction of apoptosis by rabies virus contributes to a decreased pathogenicity by stimulating antiviral immunity, we have analyzed these parameters in tissue cultures and in mice infected with a recombinant rabies virus construct that expresses the proapoptotic protein cytochrome c. The extent of apoptosis was strongly increased in primary neuron cultures infected with the recombinant virus carrying the active cytochrome c gene [SPBN-Cyto c(+)], compared with cells infected with the recombinant virus containing the inactive cytochrome c gene [SPBN-Cyto c(-)]. Mortality in mice infected intranasally with SPBN-Cyto c(+) was substantially lower than in SPBN-Cyto c(-)-infected mice. Furthermore, virus-neutralizing antibody (VNA) titers were significantly higher in mice immunized with SPBN-Cyto c(+) at the same dose. The VNA titers induced by these recombinant viruses paralleled their protective activities against a lethal rabies virus challenge infection, with SPBN-Cyto c(+) revealing an effective dose 20 times lower than that of SPBN-Cyto c(-). The strong increase in immunogenicity, coupled with the marked reduction in pathogenicity, identifies the SPBN-Cyto c(+) construct as a candidate for a live rabies virus vaccine.     

Evaluation of rabies virus neutralizing antibody titres induced by intramuscular inoculation of rabies DNA vaccine in mice and Bonnet monkeys (Macaca radiata)

A rabies DNA vaccine consisting of plasmid DNA expressing the rabies virus surface glycoprotein was injected (im) twice at two week interval to outbred swiss mice or Bonnet monkeys (Macaca radiata) and the levels of rabies virus neutralizing antibody (VNA) titres were examined over a one year period. In mice, the VNA titre was maintained above the minimum protective level (0.5 I.U./ml) up to 10 months after primary immunization, while in monkeys, the titre dropped below the protective level by 6 months. An anamnestic B cell response was seen in both mice and monkeys following the administration of a booster dose, 10 and 6 months after the primary immunization, respectively. These results indicate that im injection of rabies DNA vaccine induces VNA in nonhuman primates and mice unlike intradermal (id) immunization, which was shown to induce VNA only in mice but not in monkeys. This is the first report on the induction of VNA in nonhuman primates by im inoculation of rabies DNA vaccine.     

Epidemiology of raccoon rabies in Virginia, 1984 to 1989.

Geographical and temporal trends in reports of rabid raccoons (Procyon lotor) in Virginia were summarized for 1984 to 1989; 3,256 raccoons were submitted for rabies testing, of which 1,053 (32.3%) had rabies. Both the absolute number of rabid raccoons and the percent of rabid raccoons (number rabid divided by number submitted) were examined for seasonal and yearly trends. Geographically, the epidemic moved eastward and southward in the state. The seasonal trend showed bimodal peaks in late winter and early fall and a seasonal low in summer. The percent of rabies positive raccoons peaked 1 mo earlier than the absolute number of rabies positive raccoons. The peak in the number of rabies positive raccoons occurred in 1987, while the percent of rabies positive raccoons peaked in 1986. These trends were used to recommend timing and placement of oral vaccine as one strategy to control raccoon rabies in wildlife.     

Immobilization of specific antibody on SAM functionalized gold electrode for rabies virus detection by electrochemical impedance spectroscopy

The rabies constitutes one of the most dangerous viruses causing many death cases every year. Each year approximately 55,000 people die of rabies, with high percentage of children. High percentages (99%) of the registered cases were in Asia and Africa. In order to fight this dangerous disease, many techniques are usually used for diagnostic but are usually complex, heavy, expensive, difficult to implement, requiring high-qualified personnel, and this is a necessity of developing new detection process. In this work, we describe the development of an immunological sensor based on functionalized gold electrode allowing rabies antigen detection. The biosensor is based on the immobilization of specific rabies antibodies onto functionalized gold microelectrode. The affinity interaction of the immobilized antibody with the specific antigen can be measured with low limit detection and with a good reproducibility with impedance spectroscopy. The non-specific interaction has been tested using the Newcastle antigen.     

Current rabies epizootiology

Situation in rabies in the Russian Federation (RF) remains to be tense and is characterized by important specific features. Central Russia and the Moscow region have essential differences in the epizootic situation, the epizootological structure of rabies and other indices as compared with the Russian Federation. In the course of the last 25 years the ecological stereotype of rabies has undergone considerable transformations, becoming natural focal infection with the circulation of the infective agent among wild carnivores, which is now particularly obvious in the Moscow region. In 1998 a sharp rise in rabies morbidity occurred in Central Russia: peak values exceeded average annual values 2 times for the RF, 4 times for Central Russia and more than 10 times for the Moscow region. The situation in rabies in the Moscow region permits to use it as a model in the study of today rabies.     

Induction and biological properties of defective interfering particles of rabies virus.

A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.