Comparisons of nucleotide and deduced amino acid sequences of the glycoprotein genes of a Chinese street strain (CGX89-1) and a Chinese vaccine strain (3aG) of rabies virus.

We analyzed the glycoprotein gene sequences of a Chinese street rabies virus strain (CGX89-1) and a Chinese human rabies vaccine strain (3aG). The complete glycoprotein gene sequence of each strain has 1575 nucleotides and encodes a polypeptide of 524 amino acids. The overall nucleotide homology of these glycoprotein genes is 84.5%, and the deduced amino acid homology is 89.5%. Twenty-one percent of the base changes result in amino acid substitutions. Comparison of the homologies of the glycoprotein genes showed that the most conserved region is the ectodomain, whereas the most variable regions are the transmembrane and cytoplasmic domains. The overall nucleotide homologies of the 3aG glycoprotein and the CGX89-1 glycoprotein compared with the Pasteur virus glycoprotein are 91.2% and 84.1% respectively. The glycoprotein gene sequences presented here, the first from isolates of Chinese origin, provide insights into the biologically significant regions of this rabies gene.

Molecular characterization of carrier rabies isolates.

We compared the genomes of nine dog rabies virus isolates using two molecular methods. The viruses used in the comparison included three Ethiopian rabies strains from carrier dogs, a street strain from a rabid dog from the same geographic area, two saliva isolates made from an experimentally infected carrier dog, the virus isolated from the tonsil of this carrier dog at necropsy, and two laboratory strains. We produced overlapping polymerase chain reaction (PCR) segments spanning 97% of the genome. Restriction analysis of these PCR products with AvaII, Bcll, and BamHI detected 39 variable sites representing 668 nucleotides (nt) or 5.5% of the genome. We also compared the DNA and the deduced peptide sequences of a 200-nt segment of the 3' end of the rabies nucleoprotein gene. Previous work with these Ethiopian carrier viruses and the endemic street strain had failed to show any differences among them. Both restriction mapping and sequence analysis of 200 nt of the nucleoprotein gene allowed us to individually identify these isolates. Phylogenetic analyses of these data sets showed only the two saliva isolates of the experimentally infected carrier dog to be identical. Each of the viruses in this study, including the one isolated from the tonsil of the experimentally infected carrier dog, could be distinguished by these techniques.

Intermittent excretion of rabies virus in the saliva of a dog two and six months after it had recovered from experimental rabies.

A dog inoculated with a rabies virus isolate from the saliva of an apparently healthy Ethiopian dog was followed for more than 9 months. Saliva and blood specimens were collected three times weekly and cerebrospinal fluid weekly. Saliva samples collected on days 42 and 169 after the dog's recovery produced fatal rabies infections in mice inoculated intracerebrally.

Use of anti-glycoprotein monoclonal antibodies to characterize rabies virus in formalin-fixed tissues.

Seventy anti-rabies virus monoclonal antibodies (Mabs) were tested for reactivity with rabies and rabies-related viruses in formalin-fixed (FF) tissues. Forty-three of the Mabs were directed against the glycoprotein and 27 were directed against the nucleocapsid as determined by enzyme immunoassays and neutralization tests. Twenty of the anti-glycoprotein Mabs and one of the anti-nucleocapsid Mabs reacted with the rabies challenge virus strain (CVS) in FF tissue. These 21 Mabs were screened against other lyssaviruses in FF tissues: five rabies virus strains (coyote, skunk, raccoon, red bat, and silver-haired bat), and four rabies-related viruses (Australian bat lyssavirus, Duvenhage virus, Lagos bat virus, and Mokola virus). One of the anti-glycoprotein Mabs was reactive with all the virus strains screened. Another of the anti-glycoprotein Mabs reacted with all of the rabies virus strains tested, but not with any of the rabies-related virus strains tested. The remaining Mabs had reactivity patterns that could be useful for characterizing lyssaviruses in FF tissues.

Procedures for reproducible detection of rabies virus antigen mRNA and genome in situ in formalin-fixed tissues.

Procedures allowing the reproducible in situ detection of rabies virus antigen and RNAs (both genome and message) in formalin-fixed tissue are described. These procedures can be used on sequential tissue sections and thereby permit comparison of results from tests detecting both antigen and RNA in the same tissue. This antigen-detecting procedure has also been used to identify both the phylogenetically distant rabies viruses from silver-haired bat and vampire bat and the rabies-related viruses Mokola, Duvenhage, and Lagos bat. One of the critical steps in these procedures is the digestion (and the resulting exposure of the target molecules) with proteinase K. These methods may be useful for the identification of other viruses of public health importance. Because in many situations only formalin-fixed tissue is available for postmortem diagnosis, the technical ability to identify a virus antigen and nucleic acid in such tissues greatly extends potential diagnostic capabilities.

Use of the avidin-biotin peroxidase system to detect rabies antigen in formalin-fixed paraffin-embedded tissues.

We stained rabies-infected nervous and salivary-gland tissues fixed in formalin or acetone and embedded in paraffin with the avidin-biotin peroxidase system. With this system, rabies-virus antigen was detected in neurons, glandular acinar cells, and vascular endothelial cells more effectively than by immunofluorescence, especially when tissues were enzyme-digested with pronase before immunoperoxidase staining. The avidin-biotin peroxidase system should be useful for routine diagnosis, retrospective studies of rabies, and identification of specific cells involved in the spread of virus in rabies-infected hosts.

A comparative study of the fluorescent antibody test for rabies diagnosis in fresh and formalin-fixed brain tissue specimens.

Many diagnostic methods have been used to detect rabies virus antigen. The preferred method for routine diagnosis of rabies in fresh or frozen brain tissues is the fluorescent antibody test (FAT). In this study, the FAT was used to evaluate the rabies status of fresh/frozen brain specimens from more than 800 rabies-suspected cases, in more than 14 different species of animals. A comparable brain specimen from each case was fixed in 10% buffered formalin and examined by the FAT. The evaluation of rabies status between fresh and formalin-fixed tissues was in agreement in more than 99.8% of the cases. When fresh tissue is not available for testing, these results validate the use of this procedure for routine diagnosis of rabies in formalin-fixed brain tissues.

Recovery from clinical rabies of 2 dogs inoculated with a rabies virus strain from Ethiopia.

Two dogs, inoculated with a strain of rabies virus from Ethiopia, showed typical signs of rabies 8 days after inoculation. After 3 or 4 days with a deterioration in the physical condition, both animals began to recover, as shown by increased muscular movements, reaction to stimuli, awareness of surroundings, and attempts to rise. Both animals recovered completely, although 1 then died of Pseudomonas bacterial pneumonia. An increase in serum-neutralizing antibody and in CSF or brain-neutralizing antibody was noted in both animals. Such concentrations have been noted only in animals or persons that recovered from rabies.

Peripheral distribution of virus in dogs inoculated with two strains of rabies virus.

Forty-seven Beagles were inoculated IM with an Ethiopian strain or a Mexican strain of rabies virus to study the pathogenesis of street rabies virus in dogs. Thirty-nine dogs died of rabies, with incubation periods lasting 9 to 69 days. Of the dogs that died, 82% had shown typical signs of rabies, but 18% died without any noticeable signs of illness. Eight dogs that remained healthy during an observation period lasting more than 2 years did not produce detectable amounts of rabies virus-neutralizing antibodies; however, when challenge exposed with a large dose of the homologous rabies virus inoculum, these 8 dogs responded with high antibody titers, but challenge-exposed control dogs died of rabies. Infective virus was isolated from the saliva and cerebrospinal fluid of dogs before any signs of rabies were noticed; rabies virus-neutralizing antibodies were not detected in the serum and cerebrospinal fluid before illness. In this study, viral antigen was not detected in the skin biopsy specimens taken before signs of rabies were noticed. At necropsy of the 39 dogs, rabies virus was detected in most tissues examined. Viral antigen was detected in the skin tissues of 14 (36.8%) of the 38 dogs examined. The presence of viral antigen in the skin seemed to correlate with the presence of virus in the salivary glands, but virus in the salivary glands did not indicate the presence of virus in the skin. Eleven (44%) of the 25 dogs which had virus in the salivary glands did not have any detectable amount of viral antigen in the skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral vaccination of skunks with raccoon poxvirus recombinants expressing the rabies glycoprotein or the nucleoprotein.

Twenty nine skunks (Mephitis mephitis) were vaccinated orally with raccoon poxvirus (RCN) recombinants: 10 with a recombinant expressing the rabies virus glycoprotein (RCNRG), 10 with RCNRG mixed with a recombinant expressing the rabies virus nucleoprotein (RCNRN) and nine with RCN alone. Rabies virus neutralizing antibodies were detected in six of the 20 skunks; five skunks (three given RCNRG, two given a mixture of recombinants) survived a rabies challenge that was lethal for nine skunks vaccinated with RCN alone.

Canine rabies.

Dog rabies is still epizootic in most countries of Africa, Asia and South America and in these countries dogs are responsible for most human deaths from the disease. The incubation period in dogs may vary from one week to several months and may be influenced by the site of infection and the virus dose and strain. Diagnosis by clinical signs alone is inadequate since many rabid dogs develop dumb rabies which can easily be overlooked and others die without showing signs of rabies. Rabies virus may be excreted in the saliva before clinical signs appear and may lead to infection of an unsuspecting and untreated bite victim. Dogs may recover from clinical rabies and may then intermittently excrete virus in the saliva. Prevention of human rabies depends on the control of canine rabies which can only be achieved by mass-immunization and control of stray dog populations.

Immunogenicity and efficacy of Fermi-type nerve tissue rabies vaccine in mice and in humans undergoing post-exposure prophylaxis for rabies in Ethiopia.

Rabies is an acute viral encephalitis that is invariably fatal following the manifestations of clinical signs. To subvert the course of the disease, rabies post-exposure prophylaxis (PEP) is widely utilized. The immunogenicity and efficacy of Fermi-type rabies vaccine produced in Ethiopia was determined in mice subjected to intracranial challenge with rabies virus, and in humans undergoing rabies PEP in Ethiopia. Mice were randomly assigned into 5 groups. Group 1 received 0.25 ml each of phenolized saline intraperitoneally for 14 consecutive days. Mice in groups 2-5 received 0.25 ml of rabies vaccine for human PEP for the same period of time. Blood samples were drawn from the retro-orbital vein of all mice on designated days for the determination of rabies virus neutralizing antibody (VNA) using the mouse serum neutralization test. Mice were subsequently challenged intracranially with rabies virus at a concentration of 64 MICLD50 90 days post initial vaccination. Rabies neutralizing antibody titers in the sera of immunized mice ranged from 4.6 to 25 IU/ml. Booster vaccine doses did not seem to induce significant increases in the immune response of vaccinated mice, all of whom withstood intracranial challenge with rabies virus. Rabies VNA was further determined in 12 patients vaccinated in accordance with the prescribed dosage of Fermi-type vaccine for human rabies PEP. Most had > 0.5 IU/ml of rabies VNA by day 14, and none detectable at day 1. In contrast to mice, booster doses of vaccine may contribute to slightly higher rabies VNA titers in humans but our small sample size, on top of significant defaulter rates in the study participants, limits our interpretation of the effects of booster vaccine doses. The results of this study are the first documentation of the efficacy and immunogenicity of the Ethiopian Fermi type nerve tissue vaccine in both humans and mice.

Possible human-to-human transmission of rabies in Ethiopia.

This report describes two unusual human rabies patients, a 41 year old woman and a 5 year old boy. The only known source of exposure for both patients was to family members who died of rabies. The clinical histories of these two patients suggest the possibility of naturally occurring human-to-human transmission of rabies.

Temporal trends in the incidence of HIV infection in antenatal clinic attendees in Addis Ababa, Ethiopia, 1995-2003.

BACKGROUND: The HIV incidence data are relevant in depicting the current dynamics and trend of the epidemic. Using a new laboratory method for HIV-1 incidence, we aimed at estimating a 10-year trend in HIV-1 incidence in Addis Ababa, Ethiopia. METHODS: We determined the temporal trends in HIV incidence based on a total of 7744 serum specimens from pregnant women who attended antenatal clinics in Addis Ababa between 1995 and 2003. HIV incidence was determined by IgG-capture HIV-1 BED incidence enzyme immunoassay following a validation using a well-characterized panel of serial serum specimens from subtype C-infected seroconverters. FINDINGS: Of the 1350 HIV+ specimens tested as part of the annual sentinel survey between 1995 and 2003, a total of 1332 (98.7%) were tested by BED HIV-1 incidence assay. The incidence rate of HIV-1 infection declined significantly from 7.7% (95% CI, 3.9-11.5%) in 1995 to 2.0% (95% CI, 0.7-3.3%) in 2003. Although there was a trend, amongst the age group of 15-29 years, in age-specific decline in incidence, it was not statistically significant. No change in HIV incidence rate was observed for the group aged above 30 years. INTERPRETATION: A corresponding decline in the incidence of HIV infection was observed with the decline in the prevalence of HIV infection between 1995 and 2003 in Addis Ababa City. Whether the declines were because of changes in sexual behaviours or other reasons needs to be explored. The BED HIV-1 incidence assay provides a valuable tool in obtaining information on recent HIV-1 infection.

Rabies in Ethiopia.

Rabies is one of the most severe infectious diseases in Ethiopia, with many cases of the disease diagnosed in various parts of the country. The dog is the species most responsible for human exposure, with over 98% of the human cases and vaccinations due to the bite of rabid or suspected rabid dogs. Most of the treatments are due to stray dogs that bite, escape and are not available for observation. Most of the people who die of rabies are under 40 years of age, and among adults, the majority of these are males, suggesting that the close contact the young men have with dogs causes them to have a higher exposure rate and more deaths from rabies.

Pathogenesis of rabies virus infection in dogs.

Most dogs experimentally infected with street rabies virus showed clinical signs of rabies before death, but up to 18% of the dogs died without showing detectable signs of illness. In dogs showing signs, rabies was not invariably fatal. Up to 20% of dogs recovered without any supportive treatment. Some dogs inoculated with American (southern Texas) or Ethiopian canine street virus excreted virus in their saliva up to 14 days before signs appeared. There was no relation between the time of excretion of virus in the saliva and the titer of virus in the salivary glands at death. One dog that recovered from rabies intermittently excreted rabies virus in its saliva for a long time. The carrier state in rabies may play a significant role in the perpetuation and survival of the virus and may become a source for rabies outbreaks whenever a new generation of rabies susceptibles reaches critical density.

Adjuvant activity of non-toxic Quillaja saponaria Molina components for use in ISCOM matrix.

It is well established that ISCOMs function efficiently as an antigen-presenting system and protective immunity has been evoked against a variety of infectious agents. The built-in saponin adjuvant from Quillaja saponaria Molina is responsible for the strong immunoenhancing activity displayed by the ISCOM. However, to allow the use of ISCOMs in human vaccines it is necessary to determine the immunological properties and toxicity of chemically defined Quillaja components. Thus, the present study was carried out in a mouse model to determine the adjuvant activity and toxicity of "free", isolated Quillaja components, as well as formulated into particles, i.e. ISCOM matrix. The purified Quillaja components and the ISCOM matrix formulations were examined for their adjuvant activity in a model system consisting of purified influenza virus antigen and Quillaja saponins. It was demonstrated that a Quillaja component, designated QH-C, either as a "free" component or in an ISCOM matrix, has a strong adjuvant activity, but little or no toxicity in the doses tested. In addition, QH-C in the form of ISCOM matrix does not induce any local reactions at the site of injection. Thus, ISCOMs containing the QH-C component, devoid of toxicity, but with strong adjuvant activity, can prove to be useful in adjuvant formulations for human use.

An evaluation of immunofluorescence and PCR methods for detection of rabies in archival Carnoy-fixed, paraffin-embedded brain tissue.

Direct immunofluorescence and PCR detection methods were compared for sensitivity in evaluating the rabies status of archival specimens of Carnoy-fixed, paraffin-embedded brain tissue. The material consisted of 23 samples obtained during a rabies outbreak in Finland in 1988, and one sample isolated from a bat researcher who died of rabies in Finland in 1985. These results were compared with the original diagnoses performed on the fresh tissues. The immunofluorescence assay detected 100% (12/12) of the rabies-positive archival cases. A PCR assay designed to detect a 139-bp target near the 5' end of the rabies nucleoprotein gene also detected 100% (12/12) of the samples identified as positive in the fresh tissue specimens. A PCR assay designed to detect a 304-bp target spanning the 139-bp target of the first assay detected only 67% (8/12) of the original cases. No false positives were recorded. Both immunofluorescence detection of antigen and PCR detection of a short region of the nucleoprotein gene are useful in determining the rabies status of fixed, paraffin embedded (archival) material.