Treatment of vulvar lichen sclerosus with topical corticosteroids in children: a study of 72 children.

Treatment of vulvar lichen sclerosus (VLS) in children by topical corticosteroids gives control of symptoms and some resolution of physical signs, but large studies are limited. We report the largest study of 72 prepubertal girls with VLS, 62 of whom were prospectively treated with daily application of an ultrapotent topical corticosteroid (UPTC), clobetasol propionate 0.05% ointment, for 3 months, with a follow-up period of 4-8 years [the remaining 10 patients responded to mild to moderate potency topical corticosteroids (MPTCs)]. The results were compared with a retrospective study of 31 prepubertal girls with VLS treated with MPTCs. MPTCs led to symptom clearance in 32.2% of patients, whereas UPTC led to symptom clearance in 72.6% of patients. Improvement in clinical signs following UPTC occurred in 90.3% of children at 3 months, with total resolution of clinical signs occurring in 29.2% at the 4-year follow-up or at puberty. No serious adverse effects occurred with UPTC treatment. In children with VLS, UPTCs relieve symptoms, resolve signs and possibly prevent scarring. UPTCs should therefore be the treatment of choice for VLS in children.

Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada.

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.

Phylogeographic patterns exhibited by Ontario rabies virus variants.

A previous study on N gene variation of rabies viruses circulating in Ontario red foxes identified four viral variants. This study confirms the geographical localization of these variants and extends the analysis to the less conserved G gene of these viruses. A greater number of regionally localized variants was revealed and their phylogenetic relationships have been examined. Ongoing surveillance on recent disease outbreaks revealed that variants do not always persist in specific areas. The distribution of these variants did however appear to be influenced by topographical features of the study area likely to affect host animal movements and contacts. The majority of G gene base changes were synonymous and limited glycoprotein sequence variation predominantly to the C-terminal transmembrane and endo-domains. These data are most readily explained by random appearance of genetic viral variants followed by their spread throughout sub-populations of the fox host according to the easiest routes of transmission.

The long incubation period in rabies: delayed progression of infection in muscle at the site of exposure.

The striped skunk (Mephitis mephitis) is a host of rabies in large areas of Canada and the United States. In each of two experiments, equal numbers of skunks in two groups were inoculated intramuscularly with low doses of a field strain of rabies virus (street rabies virus). In each experiment, skunks in one group surviving to 2 months were killed at this time and selected tissues were used for examination by the polymerase chain reaction (PCR) method or by immunohistochemistry for rabies antigen. Results of detailed examinations using PCR technology (experiment 1) indicated that muscle at the inoculation site contained viral RNA at 2 months postinoculation, when other relevant tissues on the route of viral migration and early entrance into the central nervous system were negative. The cellular location of virus/antigen, as determined immunohistochemically in experiment 2, was striated muscle fibers and fibrocytes. Our results indicate a major role of muscle (tissue) infection at the inoculation site in the long incubation period of rabies in skunks. These and related findings will be useful in rabies control and, if applicable to other species, will be relevant in postexposure treatment.

Duration of immunity in foxes vaccinated orally with ERA vaccine in a bait.

Red foxes (Vulpes vulpes) vaccinated orally with the ERA strain of rabies vaccine in a bait were challenged after 83 mo. Ten of 11 foxes that had seroconverted following vaccination resisted challenge with a virulent rabies virus which produced clinical signs of rabies in 6 of 6 unvaccinated foxes. Five of 11 vaccinated animals retained titers of rabies virus neutralizing antibody throughout the period. Although 6 of 11 had no detectable antibody at the time of challenge, 5 of these 6 resisted challenge and had an anamnestic response, as indicated by elevated titers of antibody when measured at day 77 postchallenge. These results show that foxes can be immunized successfully with a single oral dose of ERA vaccine, probably with protection against a lethal rabies challenge, for at least 7 y.

Cluster of rabies cases of probable bat origin among red foxes in Prince Edward Island, Canada.

Between 15 November and 13 December 1993, three cases of rabies of probable bat origin were confirmed in red foxes (Vulpes vulpes) from the same area of Prince Edward Island, Canada, previously thought to be free of rabies in terrestrial mammals. Such clusters have rarely been described in North America.

Early events in rabies virus infection of the central nervous system in skunks (Mephitis mephitis).

Twenty-four striped skunks were inoculated intramuscularly (long digital extensor muscle of right pelvic limb) with street rabies virus. Groups of two clinically normal skunks were killed at various times after inoculation; skunks that developed rabies were killed in early stages of the clinical signs. Four clinically normal skunks (numbered 1-4) had slight infection in lumbar spinal ganglia, spinal cord and brain. These four skunks were used for detailed immunohistochemical (rabies antigen) studies that included examination of sections from every segment of the spinal cord, most of the spinal ganglia from the 2nd cervical to the 2nd coccygeal (sections at 25-microns intervals of lumbar, sacral and coccygeal ganglia) and brain (sections at 50-micron intervals). In skunks 1-4, there was increasing distribution of antigen-containing neurons that was not correlated with the time elapsed since inoculation. In three skunks (nos. 1, 2 and 3), antigen-containing neurons were predominantly in caudal regions of the spinal cord, caudal right lumbar and sacral spinal ganglia and certain nuclei/regions of the brain (medial reticular formation, right interpositus and lateral vestibular nuclei, left red nucleus, left motor cortex, and left reticular nucleus of the thalamus). Skunk 4 had more extensive infection than skunks 1-3, but the previous pattern was still evident. The results are consistent with viral entrance into the lumbar spinal cord, initial replication mainly at the L2 and L3 levels, local spread in the cord by propriospinal neurons and early transit to the brain via long ascending and descending fiber tracts (bypassing the grey matter of the rostral spinal cord). These mechanisms could provide for early and rapid dissemination in the brain before a significant immune response develops and could induce behavioral changes before the animal is incapacitated by extensive spinal cord infection. Based on the distribution of antigen-containing neurons, the tracts considered most likely to serve as viral transitways from spinal cord to brain include: rubrospinal, corticospinal, spinothalamic, spino-olivary, vestibulospinal and/or spinovestibular, reticulospinal and/or spinoreticular, cerebellospinal and/or spinocerebellar, and dorsal column pathways.

A molecular epidemiological study of rabies virus in central Ontario and western Quebec.

Rabies persists in Ontario wildlife in two predominant species: the red fox (Vulpes vulpes) and the striped skunk (Mephitis mephitis). A protocol applying reverse transcription/polymerase chain reaction (RT/PCR) and restriction endonuclease analysis (REA) to the rabies virus nucleoprotein gene was previously reported by Nadin-Davis et al. (Journal of General Virology 74, 829-837, 1993) to be useful for discrimination of rabies virus variants in Ontario. Four main types, which showed no host species specificity but which did exhibit different geographical distributions, were identified. Between 1989 and 1992 an area north and west of the city of North Bay experienced unusual and substantial rabies activity. In this report we describe the use of these molecular techniques to investigate the epidemiology of this recent rabies outbreak in central Ontario. It is shown that two of the four previously identified variants had invaded this region from the south and east, but in addition viruses very closely related to arctic isolates of rabies virus were found. The nucleoprotein and glycoprotein genes of this arctic type were sequenced and compared to those of its more southerly neighbours.

Identification of regional variants of the rabies virus within the Canadian province of Ontario.

Although rabies outbreaks in most parts of the world tend to be host species-specific the rabies currently enzootic in the Canadian province of Ontario is hosted by two wildlife species, the red fox and the striped skunk. Previous studies employing monoclonal antibody panels failed to identify any host-specific differences in Ontario rabies virus street isolates, but certain observations suggested the existence of more than one viral strain in terrestrial mammals of this region. The extent of variation of the rabies virus circulating within this region has been re-examined using molecular biology techniques. The N gene of several independent isolates was amplified using PCR and the resulting products were compared by restriction enzyme analysis and, in some cases, by DNA sequencing. This analysis confirmed that there was indeed no host-specific variation in the portion of the viral genome under study but there were, however, very clear and consistent differences in the virus from distinct geographical regions.

Oral rabies vaccination of skunks and foxes with a recombinant human adenovirus vaccine.

A new recombinant rabies vaccine (human adenovirus 5 containing the rabies glycoprotein gene) was given to striped skunks (Mephitis mephitis) and red foxes (Vulpes vulpes). Groups of skunks received the vaccine in baits, by direct instillation into the mouth, or intramuscularly. Foxes were given vaccine by direct instillation into the oral cavity (DIOC). Selected groups of vaccinated skunks and foxes were challenged with street rabies virus. There were high rates of seroconversion (generally with high antibody titers) in both foxes and skunks, with survival of all challenged vaccinated animals (all challenge controls developed rabies). In skunks, vaccine given DIOC was effective over a broad range of doses (10(8.7), 10(7.6) and 10(6.4) median tissue culture infective doses). There was no evidence of pathogenic effects. The results indicate that this adenovirus recombinant has considerable potential as a wildlife oral rabies vaccine.

Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies.

Ineffectiveness and comparative pathogenicity of attenuated rabies virus vaccines for the striped skunk (Mephitis mephitis).

Three attenuated rabies virus vaccines (SAD-B19, ERA/BHK-21, AZA 2) were compared for efficacy and safety in the striped skunk (Mephitis mephitis) by the oral and intranasal routes. The SAD-B19 and ERA/BHK-21 vaccines were given orally; all three vaccines were given intranasally. Oral administration of SAD-B19 and ERA/BHK-21 vaccines induced neither seroconversion nor significant protection against rabies challenge. One skunk which consumed a SAD-B19 vaccine-laden bait succumbed to vaccine-induced rabies. Intranasal instillation of the three vaccines resulted in the deaths of two of six (AZA 2), three of six (ERA/BHK-21) and six of six (SAD-B19) skunks.

Mutants of rabies viruses in skunks: immune response and pathogenicity.

In studies to develop an oral rabies vaccine for wildlife, the immune response to and pathogenicity of two types of mutants of rabies viruses were examined. Forty-five small plaque mutants were selected from cultures of ERA rabies virus treated with 8-azaguanine or 5-fluorouracil and tested for pathogenicity in mice. Two of these mutants AZA 1 and AZA 2 (low pathogenicity in mice) were given to skunks by oral (bait), intestinal (endoscope) and intramuscular routes. Additionally, challenge virus standard (CVS) rabies virus and mutants of this and ERA rabies virus (CVS 3766 and 3713, and ERA 3629) that were resistant to neutralization by specific antiglycoprotein monoclonal antibodies (and apathogenic in mice) were tested by various routes in skunks. Skunks given AZA 1 and AZA 2 were challenged at three months postinoculation with street rabies virus. After oral administration, there were very low rates of seroconversion with AZA 1 and AZA 2 and on challenge only 2/7 given AZA 1 and 1/8 given AZA 2 survived. None of the skunks given the other mutants orally seroconverted. AZA 2 produced a high rate of seroconversion (8/8) by the intestinal route and all challenged skunks in this group survived (7/7). All skunks vaccinated intramuscularly with AZA 1 (4/4) or AZA 2 (4/4) developed high levels of rabies neutralizing antibodies and survived challenge. The mutant CVS 3766, while apathogenic when given intracerebrally to adult mice, was consistently pathogenic by this route (and intranasally) in skunks. These results demonstrate that skunks are highly resistant to oral immunization by live rabies virus vaccines and that pathogenicity (by intracerebral route) of the mutant CVS 3766 is markedly different in mice and skunks.

Persistent infections of a field strain of rabies virus in murine neuroblastoma (NA-C1300) cell cultures.

Rabies virus from the brain of a striped skunk (Mephitis mephitis) from Ontario was inoculated into murine neuroblastoma (NA-C1300) cell cultures. These cultures were incubated and the cells were subcultured every three to four days. The presence of viral antigen in the cell cultures was monitored by direct immunofluorescent staining and in the culture fluids by titration in either baby hamster kidney (BHK/C13) or NA cells or in experimental mice. The virus-infected NA cultures evolved from an initial high viral concentration in supernatant fluid through a period of decreasing titers of infectious virus in the supernatant fluids to a final phase where no infectious virus has been found following cell culture and animal inoculation methods attempted although the persistently infected cells remained 95-100% viral nucleocapsid antigen-positive. Possible mechanisms involved in the perpetuation of this infection are discussed. This is the first report of a persistent infection of cell cultures by a field strain of rabies virus.

Skunk rabies.

In North America, the number of cases of rabies diagnosed in skunks generally exceeds that in either raccoons or foxes. Enzootic skunk rabies occurs mainly in four geographic regions: (1) southern Ontario and Quebec and upper New York State; (2) the north central United States and the Canadian provinces of Manitoba, Saskatchewan, and Alberta; (3) California; and (4) south central United States (Texas and several adjacent states). Rabies in these areas (in skunks and, to a large extent, in other terrestrial mammals) is caused mainly by three street virus variants, as determined by monoclonal antibody testing (one variant for areas 2 and 3 and separate variants for each of areas 1 and 4). Experimental studies suggest that the species specificity (e.g., raccoon vs. skunk) of enzootic rabies is due, at least partly, to differences in the pathogenicity of variants of rabies virus.

Growth characteristics in cell culture and pathogenicity in mice of two terrestrial rabies strains indigenous to Canada.

Two strains of street rabies virus from striped skunks (Mephitis mephitis) were used to infect either a murine neuroblastoma (NA 1300) or a baby hamster kidney (BHK-21/C13) cell culture and the cell infection rates were noted during 4 days postinfection. These cultures were then passaged for four consecutive passages, and the viruses obtained in the supernatant fluids of passage 4 were then treated as original isolates and used to infect both neuroblastoma and baby hamster kidney cells. The mortality period in Swiss white mice caused by the various virus suspensions was noted. The virus strain from the brain of skunks from Saskatchewan infected neuroblastoma and baby hamster kidney cells equally well, produced similar virus titres in supernatant fluids after four subcultures in both cell types, and appeared to produce similar mortality periods in mice from either the original brain tissue or from cell culture supernatant fluids. On the other hand, the virus from the brains of skunks from Ontario readily infected neuroblastoma but poorly infected baby hamster kidney cell cultures. Passage of this strain through four subcultures in both cell types produced virus titres in the supernatant fluids of equal magnitude. However, reisolation of the virus from the supernatant fluid of passage 4 in neuroblastoma cell cultures showed a similar pattern to that from the original brain, while the virus from baby hamster kidney cell passage supernatant fluid was considerably altered. Although the mortality period in mice was similar with virus from the brain and neuroblastoma cell cultures, this period was shortened when mice were inoculated with baby hamster kidney culture supernatant virus.(ABSTRACT TRUNCATED AT 250 WORDS)