on may 11 , 2009 , an african civet ( civettictis civetta ) displaying clinical signs consistent with rabies was killed by rangers in ikoma ward within serengeti national park ( figure 1 ) .
rangers were contacted because the civet had bitten a child on the right leg in an unprovoked attack .
the wound was washed with soap and water , and the child received postexposure rabies vaccination but no rabies immunoglobulin .
brain samples from the civet were tested multiple times ( as part of a training course ) at the central veterinary laboratory in tanzania .
results of the fluorescent antibody test and a direct rapid immunohistochemistry test were positive for lyssavirus - specific antigen .
when testing was complete , the samples were sent to the animal health and veterinary laboratories agency ( ahvla , weybridge , uk ) for additional confirmation of results and molecular analysis .
blue dot indicates location of ikoma lyssavirus  infected african civet within ikoma ward in northwest tanzania .
top left , map of africa indicating study area in tanzania ( gray box ) .
rna was extracted by using trizol reagent ( invitrogen , paisley , uk ) , and a pan - lyssavirus reverse transcription pcr yielded a specific 606-bp amplicon ( 9 ) .
bayesian reconstructions were used for phylogenetic analysis of the nucleoprotein gene region ( 405 bp ) and included representatives of all species from the lyssavirus genus ; results showed that the sequence was unique and most closely related to wcbv ( figure 2 ) .
a canine rabv biotype from tanzania and a mongoose rabv biotype from southern africa were included in the dataset .
nucleotide comparisons indicated similar divergence from all lyssavirus species ( minimum identity 62.2% australian bat lyssavirus , maximum identity 68.6% wcbv ) , including 12 canine rabv sequences from domestic and wild animals in the serengeti ecosystem ( 64.1%65.1% identity ) .
the posterior probabilities indicated that the ikov and wcbv grouping was strongly supported , despite low sequence identity .
further phylogenetic analysis of representatives from other rhabdoviruses demonstrated that ikov is a member of the lyssavirus genus ( 41.6%50.9% identity to representative rhabdovirus sequences that are available for this region of the genome ) ( data not shown ) .
phylogenetic relationships between all currently identified lyssaviruses compared with ikoma lyssavirus ( ikov ; shown in boldface italics ) , as determined on the basis of partial nucleoprotein gene sequences ( 405 bp ) .
relationships are presented as an unrooted phylogram based on bayesian markov chain monte carlo ( mcmc ) analysis .
the mcmc analysis was performed in mrbayes ( version 3.1.2 ; http://mrbayes.sourceforge.net/ ) , by using the general - time reversible model with a proportion of invariable sites and a gamma - shaped distribution of rates across sites , treating values for model parameters as unknown variables with uniform priors to be estimated in each analysis .
four simultaneous mcmc chains ( 3 heated and 1 cold , according to the default settings ) were run for 10 generations ; trees were sampled every 100 generations .
the first 25,000 trees were discarded as the burn - in phase ; the remaining trees were used to estimate consensus phylograms and bayesian posterior probabilities .
posterior probability values represent the degree of support for each node on the tree : only values > 0.90 are shown ; values > 0.95 are shown in boldface .
scale bar indicates branch length , expressed as the expected number of substitutions per site .
arav ,  aravan virus ; ablv ,  australian bat lyssavirus ; bblv ,  bokeloh bat lyssavirus ; duvv ,  duvenhage virus ; eblv-1 and eblv-2 ,  european bat lyssavirus type 1 and 2 ; irkv ,  irkut virus ; khuv ,  khujand virus ; lbv ,  lagos bat virus ( lineages a , b , c and d ) ; mokv ,  mokola virus ; rabv ,  rabies virus ; shibv ,  shimoni bat virus ; wcbv ,  west caucasian bat virus .
we describe evidence , based on genomic sequences obtained from the brain sample of an african civet with clinical signs consistent with rabies , for the existence of a novel lyssavirus designated ikov ( ikoma lyssavirus ) . the sample was frozen and thawed several times before being sent to ahvla , which had a detrimental effect on the sample quality and resulted in viral rna degradation and loss in viral viability .
the results of confirmatory fats performed at ahvla were inconclusive , and attempts to isolate virus by using the rabies tissue culture inoculation test and the mouse inoculation test were unsuccessful . despite the lack of isolated virus , the pan - lyssavirus sensitivity and specificity of the fluorescent antibody test ( the test prescribed by the world organisation for animal health as the standard for rabies testing ) and direct rapid immunohistochemistry test support the assertion that a novel lyssavirus exists in the region . in addition , despite the poor quality of the sample , molecular techniques identified a lyssavirus - specific amplicon that was confirmed to be unique by phylogenetic analysis and to be highly divergent from known circulating rabv strains .
a real - time pcr also detected this unique lyssavirus sequence , confirming that both molecular tests are pan - lyssavirus specific and are sufficient for the detection of highly divergent novel lyssaviruses ( 11 ) .
the child who was bitten by the african civet received appropriate wound care and postexposure rabies vaccination . at the time of this report
we can not , however , draw any conclusions as to whether the african civet was shedding virus when it bit the child or whether postexposure vaccinations are effective against ikov .
this case of rabies in an african civet in the center of serengeti national park was highly unexpected . since 2000
, the park had been free of rabies and no cases had been detected within a 30-km radius .
however , subsequent molecular characterization demonstrated that this case of rabies had not been caused by a rabv from a canine source .
thus , a breach had not occurred ; instead , a novel lyssavirus with an unknown reservoir had caused the infection .
previously published data on lyssavirus infection in african civets ( n = 6 ) was restricted to the rabv mongoose lineage ( 12 ) .
although african civets can be infected with rabv and ikov , infrequent detection of lyssaviruses in this species suggests that they are more likely to be incidental hosts .
the nocturnal , opportunistic foraging behaviors of african civets imply that contact with bats is possible , particularly at roosts where interactions with a grounded rabid bat are more likely to occur . in the absence of virus
surveillance for rabies in bats and other mammals in tanzania and typing of all lyssavirus - positive samples is necessary to determine the distribution and prevalence of ikov .
bats in neighboring kenya could be informative , given the strong posterior probability values on the grouping of ikov and wcbv in the bayesian analysis ( figure 2 ) ( 13 ) .
additional genomic and evolutionary analysis is underway to support ikov as a new lyssavirus species , potentially grouping with wcbv in phylogroup iii ( 14 ) , and to determine the antigenic diversity of the glycoprotein ( 15 ) .
given that ikov is highly distinct from rabv ( more genetically distinct than wcbv from rabv ) and that a human has been bitten by an infected animal , the effectiveness of current rabies vaccines needs to be further investigated .
 evidence in support of a novel lyssavirus was obtained from brain samples of an african civet in tanzania . 
 results of phylogenetic analysis of nucleoprotein gene sequences from representative lyssavirus species and this novel lyssavirus provided strong empirical evidence that this is a new lyssavirus species , designated ikoma lyssavirus . 
The Study
Conclusions