Serological and virological evidence documents the exposure of various East African bat species to several arboviruses including Rift Valley fever, dengue, and yellow fever virus [1
], however, little is known about the potential role of bats as arbovirus reservoirs or potential amplifying hosts. Orbiviruses (Reoviridae:Orbivirus
) are 10-segmented, dsRNA, vector-borne viruses that cluster phylogenetically by arthropod vector group [3
]. Recent research has also reported a novel orbivirus that may represent the first recognized insect-specific virus in the genus Orbivirus
]. While mostly recognized as veterinary pathogens (i.e., bluetongue virus, African horse sickness), several orbiviruses have been associated with neurologic disease in humans [5
Prior to this study, four orbiviruses had been isolated from wild bats. Japanaut virus (JAPV) was isolated from the blood of a southern blossom bat (Syconycteris crassa
) and a pool of mixed culicine mosquitoes in the Sepik District, New Guinea in 1965 [9
]. Heramatsu orbivirus was isolated from the blood of an eastern long-fingered bat (Myotis macrodactylus
) trapped in a mine in Heramatsu, Kagoshima, Japan in 1965 [11
]. The genome of Heramatsu orbivirus (isolate KY-663) has been partially sequenced [12
]. Eight isolates of Ife virus (IFEV) were isolated from the blood and organs of straw-colored fruit bats (Eidolon helvum
) in Nigeria, Cameroon, and the Central African Republic in 1971 and 1974 [13
]. Gambian pouched rats (Cricetomys gambianus
), African grass rats (Arvicanthis niloticus
), and domestic ruminants in Nigeria were also found to be seropositive for IFEV [14
]. Fomede virus (FOMV) was isolated from the brain, liver, and spleen of a dwarf slit-faced bat (Nycteris nana
) in Kindia, Guinea in 1978, and has been repeatedly isolated from Nycteridae bats in Guinea [17
]. Additionally, serologic evidence exists for exposure of Bolivian bats (genera Myotis
) to Matucare virus, an orbivirus isolated from Ornithodoros
ticks in 1963 [20
]. Australian fruit bats were found to be seropositive to Elsey virus, a serotype of the mosquito-borne Peruvian horse sickness virus [21
In 2013, a novel orbivirus, tentatively named Bukakata orbivirus (BUKV), was isolated from an Egyptian fruit bat (Rousettus aegyptiacus leachii
(A. Smith, 1829)) (ERB) captured in Kasokero Cave, Uganda (Figure 1
). This is the fifth orbivirus isolated from bats; however, aside from partial sequencing of Heramatsu, no bat-associated orbivirus has been genetically characterized. The specific aims of this project were to 1) determine the genome sequence of the four bat-associated orbiviruses without published existing genetic information (JAPV, IFEV, FOMV, & BUKV) and conduct phylogenetic analyses to ascertain their potential arthropod associations, as orbiviruses cluster phylogenetically based on their arthropod vector, 2) determine the replication kinetics of BUKV in multiple vertebrate cell types, and 3) determine the prevalence of BUKV RNA in additional archived field samples from Uganda.
Bats are known to host a number of emerging zoonotic viruses highly pathogenic to humans in the absence of overt pathology within the bat host. Limited information exists, however, about the ability of bat species to harbor and transmit medically important arboviruses. Despite extensive serologic evidence suggestive of exposure of multiple bat species to arboviruses from different families (Flaviviridae, Togaviridae, and Bunyavirales), few studies have resulted in the isolation of arboviruses from wild-caught bats. Molecular and in vivo characterization of novel viruses isolated from wild-caught bats, in addition to enhanced surveillance efforts targeting suspected hosts helps clarify the role of bats as reservoirs for emerging arboviruses.
This study provides complete coding sequences of three previously uncharacterized orbiviruses isolated from bats (JAPV, IFEV, and FOMV) and one novel bat-associated orbivirus, BUKV, isolated from an ERB. Phylogenetic analyses place BUKV and FOMV in the tick-borne orbivirus clade, and IFEV and JAPV cluster with the Culicoides/sandfly-borne orbiviruses. This study also provides documentation for in vitro propagation of a bat-associated orbivirus in a number of different vertebrate cell lines, one of which was derived from the ERB. The screening of additional archived bat and tick samples resulted in negative findings, but reflects a critical step in the investigation process into the host-vector relationships supported by phylogenetic analyses.
Of the three segments analyzed, the topology of the phylogenetic analysis consistently placed BUKV and FOMV within the tick-borne orbivirus subclade along with Chobar Gorge virus (CGV). Past studies indicated that FOMV is a serotype of the Chobar Gorge virus
species based on results of complement fixation, and it is considered as such by the International Committee on the Taxonomy of Viruses [3
]. This is consistent with past isolations of FOMV in field-caught Ixodid ticks [17
]. CGV has been isolated from Ornithidoros
spp. ticks in Nepal, and antibodies have been detected in humans and domestic ruminant species in the same region [49
]. Additionally, the clustering of BUKV, FOMV, and CGV within the same subclade of tick-borne orbiviruses and high degree of nucleotide and amino acid similarity regardless of protein analyzed suggests they are three different serotypes of the same species Attoui et al. suggested that the amino acid identity for T2 of <91% should be the criteria for designating a species within the genus Orbivirus [50
]. According to that criterion BUKV and FOMV viruses are on the border for consideration as new species. BUKV possesses 95.27% amino acid similarity to FOMV and 91.56% similarity to CGV, and Fomede possesses 90.87% amino acid similarity to CGV. Bukakata and Fomede viruses may be ecologically unique from Chobar Gorge in having been isolated from bats, however it is not known whether or not Chobar Gorge virus is also found in bats. Further characterization into the evolutionary relationship of these bat-associated and potentially tick-borne orbiviruses should involve exploration into in vitro growth kinetics in invertebrate cell lines in addition to the potential for serologic cross-reactivity and in vitro reassortment potential.
JAPV and IFEV cluster with the Culicoides
/sandfly-borne orbivirus clade and have not yet been approved as species of orbiviruses, but novel genetic sequence obtained during this study indicates that their listing should be revised. Neither JAPV nor IFEV possess the requisite >76% nucleotide identity to any other orbivirus in their conserved T2 gene, indicating they are each their own individual species [3
] (Figure 5
). In the analysis of all three segments, IFEV is very distantly related to all other orbiviruses and may represent its own species due to the low level of nucleotide (maximum 55.7%) and amino acid similarity (maximum 59.3%) to any other orbivirus when analyzing the gene encoding sub-core shell T2 protein (Figure 5
). Interestingly, the BLASTX results for IFEV virus segments reveal that it is most closely related to Heramatsu orbivirus. Heramatsu virus was obtained from a Japanese eastern long-fingered bat in 1965 and was partially sequenced in 2013 [11
]. However, due to lack of complete genome information and access to an archive isolate, this virus was not included in our phylogenetic analyses.
Due to their segmented genome, orbiviruses are known to undergo reassortment during co-infection [51
]. Comparing the placement of JAPV within the VP1 and T2 phylogenies to its placement in the T13 phylogeny suggests that it may have undergone reassortment; however, definitive conclusions surrounding its potential as a reassortant virus are difficult to make due to low bootstrap values and posterior probabilities (Figure 2
, Figure 3
and Figure 4
, Figures S2–S4
). Interestingly, our phylogenetic analyses indicate JAPV clusters with the Culicoides
/sandfly-borne orbiviruses, though it was isolated from a pool of Culicine mosquitoes in New Guinea [10
]. Further investigation is required to better characterize potential vector-host associations for JAPV and its potential as a reassortant orbivirus.
BUKV grew to high titers in all three vertebrate cell lines in which multi-step growth curves were conducted. Two of these cell lines, Vero cells and BHK-21 [C-13] cells, are deficient for the interferon pathway, while the R06E pathway has an intact interferon response [53
]. The Type I interferon response is the first line of antiviral defense in the mammalian immune system [54
]. Interestingly, viral titers in interferon-competent R06E cells were comparable to interferon-deficient BHK-21 [C13] and Vero cells (Figure 6
). The immune system of some bat species is highly unique in its constitutive expression of IFN-α [55
]. A recent study by Pavlovich and colleagues indicate that unlike Pteropus alecto
, transcriptomic analysis of the ERB does not provide evidence of constitutive interferon expression [56
]. Analysis of interferon expression over the course of infection in bat cells and other interferon-competent vertebrate lines would be an informative way to analyze the presence of this constitutive expression in existing bat cell lines.
None of the 171 bat samples or 513 tick pools tested positive for BUKV viral nucleic acid. However, GAPDH mRNA was tested for each bat sample and samples for which amplification of the GAPDH was not obtained were not included in the denominator of the total tested samples. While all samples were negative for BUKV RNA, some of the bat samples had very high CT values or were nearing the cycle threshold and as such, were considered to be suspect and subjected to a nested PCR protocol. The six suspect samples tested using this nested PCR were also confirmed negative. Samples types screened (spleen and/or liver) are consistent with the organs from which the virus was originally isolated. Testing additional bat species for viral RNA could yield additional information on the circulation of this virus.
While this study provides valuable information regarding potential vector-host associations among the orbiviruses, limitations restrict certain conclusions. Each orbivirus segment contains 1–2 genes, with untranslated regions on either end of the ORF. Due to decreased coverage at the ends of the reads, variable coverage was achieved throughout the length of each segment and only the complete coding genome of IFEV, JAPV, and FOMV were obtained. The coding complete sequence of segments 1–2 and 4–10 were obtained for BUKV but due to low coverage at the 5′ end of segment 3, the start codon was not obtained (Table 2
). Individual orbivirus species possess conserved 5′ UTR and 3′ UTR terminal sequences and as such, higher coverage in the untranslated regions would have provided additional information surrounding level of relatedness between these and previously sequenced orbiviruses [3
]. Field surveillance efforts were opportunistic and retrospective, and only ERB RNA was tested. The testing of additional bat species from nearby geographic areas sharing similar ecological habitats would provide additional information surrounding vertebrate host range. The tick pools tested were also opportunistic and retrospective, and originated in Python Cave, a cave with analogous ecological characteristics to Kasokero Cave, where BUKV was isolated, yet 213 km away (Table 1
, Figure 1