Among more than 160 species of rhabdoviruses identified to date, fewer than 10 have been isolated from humans 
. In addition, while human infection by rhabdoviruses has previously been associated with encephalitis, vesicular stomatitis, or “flu-like” illness, the discovery of BASV is the first time that a member of the Rhabdovirus
family has been associated with hemorrhagic fever in humans with a fulminant disease course and high fatality rate. To our knowledge, this is also the first successful demonstration of de novo
assembly of a novel, highly divergent viral genome in the absence of a reference sequence and directly from a primary clinical sample by unbiased deep sequencing.
Several lines of evidence implicate BASV in the hemorrhagic fever outbreak among the 3 patients in Mangala. First, this virus was the only credible viral pathogen detected in the blood of the lone survivor during his acute hemorrhagic illness by exhaustive deep sequencing of over 140 million reads. Analysis of the Illumina deep sequencing reads for the presence of other viral pathogens yielded only endogenous flora or confirmed laboratory contaminants (Table S1
and Fig. S2
). Some enteric pathogens, such as E. coli O157:H7, Campylobacter, Shigella, and Salmonella, are diagnosed through fecal laboratory testing and not blood, and have been associated with hemorrhagic diarrhea 
. However, these outbreaks are typically foodborne and associated with larger clusters and much greater numbers of clinical cases than reported here 
. Furthermore, enteric diarrheal cases rarely present with systemic symptoms such as fever or generalized mucosal hemorrhage, with bleeding most often limited to the gastrointestinal tract, and overall mortality rates are generally low 
. Thus, the clinical syndrome observed in 3 patients with hemorrhagic fever in the DRC, a region endemic for viral hemorrhagic fevers, is much more consistent with infection by a VHF disease agent. BASV is a plausible hemorrhagic fever candidate because it is a novel, highly divergent infectious virus, thus of unknown pathogenicity, and was detected at a titer of >1 million copies/mL in blood from an acutely ill individual. In addition, there is ample precedent for hemorrhagic disease from rhabdoviruses, as members of the genus Novirhabdovirus
cause severe hemorrhagic septicemia in fresh and saltwater fish worldwide 
(). The detection of BASV seropositivity in an asymptomatic close contact () is not surprising given that up to 80% of patients infected with Lassa virus do not exhibit any hemorrhagic fever symptoms 
Prior to the BASV outbreak, no hemorrhagic disease cases had been reported in Boma Bungu Health Zone. BASV was also not detected in 43 serum samples from unknown, filovirus-negative cases or outbreaks of hemorrhagic fever from 2008–2010 spanning 9 of the 11 provinces in the DRC (). In addition, a serosurvey of 50 random blood donors from Kasai-Oriental province in central DRC was negative for prior exposure to BASV (). Taken together, these data suggest that the virus may have emerged recently and locally from Boma Bungu in Bas-Congo, DRC.
We were unable to isolate BASV despite culturing the RNA-positive serum in a number of cell cultures and inoculation into suckling mice. One explanation for these negative findings may be that the virus inoculation titers of <50 µL were insufficient, although this is surprising given the concentration of >1 million copies per mL of BASV in blood from the lone survivor. A more likely explanation is viral inactivation resulting from the lack of adequate cold chain facilities in remote Boma Bungu. Viral RNA can often still be detected by RT-PCR in sera that is culture-negative 
. In support of this premise, we have observed that the BASV-G/VSVΔG-GFP pseudotyped virus efficiently infects and replicates in a variety of insect and mammalian (including human) cell lines (Steffen, et al.
, manuscript in preparation). In the absence of a positive culture, a “reverse genetics” approach to produce recombinant BASV particles, if successful, would greatly facilitate further study of the virus, as established previously for other rhabdoviruses such as VSV 
Based on our findings, some speculations on the origin of and routes of transmission for BASV can be made. All 3 patients became ill with acute hemorrhagic fever over a 3-week period within the same 2500-m2
area of Mangala village, suggesting that all 3 cases were infected with the same pathogen. Waterborne or airborne transmission would be expected to result in more numerous cases than the 3 reported. There were no reports of animal die-offs that would suggest potential exposures to infected wild animals or livestock. Taken together, these observations suggest that an unknown arthropod vector could be a plausible source of infection by BASV. This hypothesis is consistent with the phylogenetic and structural relationship of BASV to rhabdoviruses in the Tibrogargan group and Ephemerovirus
genus, which are transmitted to cattle and buffalo by Culicoides
biting midges 
. In addition, the recent discovery of Moussa virus (MOUV), isolated from Culex
mosquitoes in Cote d'Ivoire, Africa 
, implies the presence of hitherto unknown arthropod vectors for rhabdoviruses on the continent. Nevertheless, at present, we cannot exclude the possibility of other zoonotic sources for the virus or even nosocomial bloodborne transmission (as Patients 1 and 2 have not clearly been established to be BASV cases by serology or direct detection), and the natural reservoir and precise mode of transmission for BASV remain unknown. A community-based serosurvey in Boma Bungu and an investigation to track down potential arthropod or mammalian (e.g. rodents and bats) sources for BASV are currently underway.
Although we cannot exclude the possibility of independent arthropod-borne transmission events, our epidemiologic and serologic data do suggest the potential for limited human-to-human transmission of BASV. Patient 3, a nurse, had directly taken care of Patients 1 and 2 at the health center, and another nurse (Contact 5), who had taken care of Patient 3 (but not Patients 1 or 2) had serologic evidence of asymptomatic BASV infection. We present a hypothetical model for BASV transmission during the hemorrhagic fever outbreak in which the initial infection of two children in Mangala (Patients 1 and 2) was followed by successive human-to-human transmission events involving two healthcare workers (Patient 3 and Contact 5) (). This pattern of transmission from the community to health care workers is also commonly seen in association with outbreaks of Ebola and Crimean-Congo hemorrhagic fever 
Proposed model for BASV transmission during the hemorrhagic fever outbreak in Mangala.
While rhabdoviruses are distributed worldwide, some authors have suggested that the Rhabdoviridae
family probably originated from tropical regions of the Old or New World 
. The discovery of BASV in Central Africa suggests that additional rhabdoviruses of clinical and public health importance likely await identification, especially in these poorly investigated geographic regions. Active epidemiological investigation and disease surveillance will be needed to fully ascertain the clinical and public health significance of BASV infection in humans, as well as to prepare for potentially larger human outbreaks from this newly discovered pathogen.