A need for standardized rabies-virus diagnostic procedures: effect of cover-glass mountant on the reliability of antigen detection by the fluorescent antibody test.

The direct fluorescent antibody test is a sensitive and specific procedure used in the routine diagnosis of rabies. However, given the critical role of the rabies diagnostic laboratory in patient management and public health decision-making, the use of a standardized national rabies diagnostic procedure is highly recommended. Seemingly small variations in test procedures may have dramatic effects on sensitivity. For example, two independent reports of diminished staining performance of two lots of a commercial anti-rabies conjugate were investigated in this study. The diminished staining occurred only with a single rabies-virus variant, associated with big brown bats, Eptesicus fuscus, in the southwestern United States. Similarly diluted and prepared diagnostic reagents provided bright staining on all other variants of rabies-virus tested. Subsequent evaluation disclosed that the phenomenon was associated with the relative concentrations of glycerol used in the mounting media by the reporting laboratories. These findings, related to the proper selection of an optimal cover-glass mountant for use in the immunofluorescence procedure, demonstrate the potential for erroneous results with severe implications for patient health, when uncontrolled variations in protocol occur. This paper underscores the necessity for all rabies diagnostic laboratories to follow one standard protocol. Such a protocol has been placed on the websites maintained by the Centers for Disease Control and Prevention: .

Emerging epidemiology of bat-associated cryptic cases of rabies in humans in the United States.

In the United States, during the past half-century, the number of humans to die of rabies dramatically decreased to an average of 1-2 per year. Although the number of deaths is low, most deaths occur because individuals are unaware that they had been exposed to and infected with rabies virus, and, therefore, they do not seek effective postexposure treatment. Molecular epidemiological studies have linked most of these cryptic rabies exposures to rabies virus variants associated with insectivorous bats. In particular, virus variants associated with 2 relatively reclusive species, the silver-haired bat (Lasionycteris noctivagans) and the eastern pipistrelle (Pipistrellus subflavus), are the unexpected culprits of most cryptic cases of rabies in humans.

Bat lyssaviruses (Aravan and Khujand) from Central Asia: phylogenetic relationships according to N, P and G gene sequences.

Bat lyssaviruses Aravan and Khujand were isolated in southern Kyrgyzstan in 1991 and in northern Tajikistan in 2001, respectively. Preliminary studies with anti-nucleocapsid monoclonal antibodies suggested that the viruses were distinct from other lyssavirus serotypes. These data were supported by sequencing of the N gene of Aravan virus. In the present study, we sequenced the entire N, P and G genes of both Aravan and Khujand viruses and compared them with respective sequences of other lyssaviruses available from GenBank. The results suggested that each virus should be considered as a newly recognized genotype according to the current approaches for genotype definition (amount of nucleotide identity of the N gene and bootstrap support of joining to certain phylogenetic groups). Use of different phylogenetic methods and comparison of different parts of the genomes generally suggested that Khujand virus was mainly related to genotype 6, while Aravan virus, on the one hand, was related to Khujand virus, and, on the other hand, demonstrated moderate similarity to genotypes 4, 5 and 6. The potential significance of these new lyssaviruses for veterinary and public health should not be underestimated.

Rabies among infrequently reported mammalian carnivores in the United States, 1960-2000.

Most cases of rabies reported annually in the United States occur among three groups of carnivores--raccoons (Procyon lotor), skunks (Mephitis, Spilogale, and Putorius), foxes (Vulpes, Urocyon, and Alopex)--and among bats (numerous species). However, between 1960 and 2000, a total of 2,851 cases of rabies in 17 other carnivore taxa were reported to the Centers for Disease Control and Prevention, Atlanta, Georgia (USA), from 49 states, the District of Columbia, and Puerto Rico. Three species of these other carnivores (mongooses [Herpestes javanicus], coyotes [Canis latrans], and bobcats [Lynx rufus]) accounted for 92% (2,624/2,851) of the cases reported among other canivorous mammals (OCMs). Most OCMs demonstrated temporal or spatial variation in numbers of reported cases. Tests of specimens from OCMs infected in the United States identified variants of the rabies virus that corresponded with variants associated with the major terrestrial reservoirs within their respective regions of origin. Variants of the rabies virus in samples from mongooses in Puerto Rico could not be distinguished from those in samples from dogs in Puerto Rico by virus typing methods.

Molecular epidemiology of terrestrial rabies in the former Soviet Union.

Fifty-five rabies virus isolates originating from different regions of the former Soviet Union (FSU) were compared with isolates originating from Eurasia, Africa, and North America according to complete or partial nucleoprotein (N) gene sequences. The FSU isolates formed five distinct groups. Group A represented viruses originating from the Arctic, which were similar to viruses from Alaska and Canada. Group B consisted of "Arctic-like" viruses, originating from the south of East Siberia and the Far East. Group C consisted of viruses circulating in the steppe and forest-steppe territories from the European part of Russia to Tuva and in Kazakhstan. These three phylogenetic groups were clearly different from the European cluster. Viruses of group D circulate near the western border of Russia. Their phylogenetic position is intermediate between group C and the European cluster. Group E consisted of viruses originating from the northwestern part of Russia and comprised a "northeastern Europe" group described earlier from the Baltic region. According to surveillance data, a specific host can be defined clearly only for group A (arctic fox; Alopex lagopus) and for the Far Eastern part of the group B distribution area (raccoon dog; Nyctereutes procyonoides). For other territories and rabies virus variants, the red fox (Vulpes vulpes) is the main virus reservoir. However, the steppe fox (Vulpes corsac), wolf (Canis lupus), and raccoon dog are also involved in virus circulation, depending on host population density. These molecular data, joined with surveillance information, demonstrate that the current fox rabies epizootic in the territory of the FSU developed independently of central and western Europe. No evidence of positive selection was found in the N genes of the isolates. In the glycoprotein gene, evidence of positive selection was strongly suggested in codons 156, 160, and 183. At these sites, no link between amino acid substitutions and phylogenetic placement or specific host species was detected.

Rabies in Sri Lanka: splendid isolation.

Rabies virus exists in dogs on Sri Lanka as a single, minimally divergent lineage only distantly related to other rabies virus lineages in Asia. Stable, geographically isolated virus populations are susceptible to local extinction. A fully implemented rabies-control campaign could make Sri Lanka the first Asian country in >30 years to become free of rabies virus.

Bat rabies, Texas, 1996-2000.

Bats submitted to the Texas Department of Health (1996-2000) were speciated and tested for rabies virus antigen by direct immunofluorescence microscopy. Antigenic analysis of rabies virus-positive specimens was performed with monoclonal antibodies against the nucleoprotein of the virus; atypical or unexpected results were confirmed by genetic analysis of nucleoprotein sequence.

Emerging pattern of rabies deaths and increased viral infectivity.

Most human rabies deaths in the United States can be attributed to unrecognized exposures to rabies viruses associated with bats, particularly those associated with two infrequently encountered bat species (Lasionycteris noctivagans and Pipistrellus subflavus). These human rabies cases tend to cluster in the southeastern and northwestern United States. In these regions, most rabies deaths associated with bats in nonhuman terrestrial mammals are also associated with virus variants specific to these two bat species rather than more common bat species; outside of these regions, more common bat rabies viruses contribute to most transmissions. The preponderance of rabies deaths connected with the two uncommon L. noctivagans and P. subflavus bat rabies viruses is best explained by their evolution of increased viral infectivity.

Serologic evidence of Lyssavirus infections among bats, the Philippines.

Active surveillance for lyssaviruses was conducted among populations of bats in the Philippines. The presence of past or current Lyssavirus infection was determined by use of direct fluorescent antibody assays on bat brains and virus neutralization assays on bat sera. Although no bats were found to have active infection with a Lyssavirus, 22 had evidence of neutralizing antibody against the Australian bat lyssavirus (ABLV). Seropositivity was statistically associated with one species of bat, Miniopterus schreibersi. Results from the virus neutralization assays are consistent with the presence in the Philippines of a naturally occurring Lyssavirus related to ABLV.