A large number of arthropod-borne viruses are endemic to East Africa. As a part of the process of undertaking a systematic characterization of the mosquito fauna of Uganda, we examined mosquitoes collected from 2008 through early 2012 for known and novel viruses. In all, 8,288 mosquito pools containing 157,554 mosquitoes were tested. Twenty-nine isolations of 11 different viruses were made from mosquitoes of nine distinct species and from pools identified only to genus Culex. Identified viruses were from family Togaviridae, alphaviruses Sindbis and Babanki viruses; family Rhabdoviridae, hapaviruses Mossuril and Kamese viruses; family Flaviviridae, flaviviruses West Nile and Usutu viruses; family Phenuiviridae, phlebovirus Arumowot virus; and family Peribunyaviridae, orthobunyaviruses Witwatersrand, Pongola, and Germiston viruses. In addition, a novel orthobunyavirus, provisionally named Mburo virus, was isolated from Coquillettidia metallica (Theobald). This is the first report of Babanki, Arumowot, and Mossuril virus isolation from Uganda.

The International Committee on Taxonomy of Viruses (ICTV) has implemented numerous changes to the taxonomic classification of bunyaviruses over the years. Whereas most changes have been justified and necessary because of the need to accommodate newly discovered and unclassified viruses, other changes are a cause of concern, especially the decision to demote scores of formerly recognized species to essentially strains of newly designated species. This practice was first described in the seventh taxonomy report of the ICTV and has continued in all subsequent reports. In some instances, viruses that share less than 75% nucleotide sequence identity across their genomes, produce vastly different clinical presentations, possess distinct vector and host associations, have different biosafety recommendations, and occur in nonoverlapping geographic regions are classified as strains of the same species. Complicating the matter is the fact that virus strains have been completely eliminated from ICTV reports; thus, critically important information on virus identities and their associated biological and epidemiological features cannot be readily related to the ICTV classification. Here, we summarize the current status of bunyavirus taxonomy and discuss the adverse consequences associated with the reclassification and resulting omission of numerous viruses of public health importance from ICTV reports. As members of the American Committee on Arthropod-borne Viruses, we encourage the ICTV Bunyavirus Study Group to reconsider their stance on bunyavirus taxonomy, to revise the criteria currently used for species demarcation, and to list additional strains of public and veterinary importance.

While serological and virological evidence documents the exposure of bats to medically-important arboviruses, their role as reservoirs or amplifying hosts is less well-characterized. We describe a novel orbivirus (Reoviridae:Orbivirus) isolated from an Egyptian fruit bat (Rousettus aegyptiacus leachii) trapped in 2013 in Uganda and named Bukakata orbivirus. This is the fifth orbivirus isolated from a bat, however genetic information had previously only been available for one bat-associated orbivirus. We performed whole-genome sequencing on Bukakata orbivirus and three other bat-associated orbiviruses (Fomede, Ife, and Japanaut) to assess their phylogenetic relationship within the genus Orbivirus and develop hypotheses regarding potential arthropod vectors. Replication kinetics were assessed for Bukakata orbivirus in three different vertebrate cell lines. Lastly, qRT-PCR and nested PCR were used to determine the prevalence of Bukakata orbivirus RNA in archived samples from three populations of Egyptian fruit bats and one population of cave-associated soft ticks in Uganda. Complete coding sequences were obtained for all ten segments of Fomede, Ife, and Japanaut orbiviruses and for nine of the ten segments for Bukakata orbivirus. Phylogenetic analysis placed Bukakata and Fomede in the tick-borne orbivirus clade and Ife and Japanaut within the Culicoides/phlebotomine sandfly orbivirus clade. Further, Bukakata and Fomede appear to be serotypes of the Chobar Gorge virus species. Bukakata orbivirus replicated to high titers (10⁶–10⁷ PFU/mL) in Vero, BHK-21 [C-13], and R06E (Egyptian fruit bat) cells. Preliminary screening of archived bat and tick samples do not support Bukakata orbivirus presence in these collections, however additional testing is warranted given the phylogenetic associations observed. This study provided complete coding sequence for several bat-associated orbiviruses and in vitro characterization of a bat-associated orbivirus. Our results indicate that bats may play an important role in the epidemiology of viruses in the genus Orbivirus and further investigation is warranted into vector-host associations and ongoing surveillance efforts.

: After confirmation of two human cases of Rift Valley fever (RVF) in March 2016 in the Kabale district of Uganda, an entomological investigation was conducted with a focus on mosquito species composition and abundance of known and potential mosquito vector species, and virus testing to identify species most likely involved in Rift Valley fever virus transmission. This information could be used to forecast risk and facilitate improvement of prevention and response tools for use in preventing or controlling future outbreaks. From these collections, two virus isolates were obtained, one each from a pool of Aedes tricholabis and Ae. gibbinsi. Next-generation sequencing identified both isolates as Wesselsbron virus, family Flaviviridae, a neglected arbovirus of economic importance. These are the first reported Wesselsbron virus isolates from Uganda since 1966.

The reservoir for zoonotic o’nyong-nyong virus (ONNV) has remained unknown since this virus was first recognized in Uganda in 1959. Building on existing evidence for mosquito blood-feeding on various frugivorous bat species in Uganda, and seroprevalence for arboviruses among bats in Uganda, we sought to assess if serum samples collected from bats in Uganda demonstrated evidence of exposure to ONNV or the closely related zoonotic chikungunya virus (CHIKV). In total, 652 serum samples collected from six bat species were tested by plaque reduction neutralization test (PRNT) for neutralizing antibodies against ONNV and CHIKV. Forty out of 303 (13.2%) Egyptian rousettes from Maramagambo Forest and 1/13 (8%) little free-tailed bats from Banga Nakiwogo, Entebbe contained neutralizing antibodies against ONNV. In addition, 2/303 (0.7%) of these Egyptian rousettes contained neutralizing antibodies to CHIKV, and 8/303 (2.6%) contained neutralizing antibodies that were nonspecifically reactive to alphaviruses. These data support the interepidemic circulation of ONNV and CHIKV in Uganda, although Egyptian rousette bats are unlikely to serve as reservoirs for these viruses given the inconsistent occurrence of antibody-positive bats.

Usutu virus (USUV; Flavivirus ), a close phylogenetic and ecological relative of West Nile virus, is a zoonotic virus that can cause neuroinvasive disease in humans. USUV is maintained in an enzootic cycle between Culex mosquitoes and birds. Since the first isolation in 1959 in South Africa, USUV has spread throughout Africa and Europe. Reported human cases have increased over the last few decades, primarily in Europe, with symptoms ranging from mild febrile illness to severe neurological effects. In this study, we investigated whether USUV has become more pathogenic during emergence in Europe. Interferon α/β receptor knockout ( Ifnar1 ^-/- ) mice were inoculated with recent USUV isolates from Africa and Europe, as well as the historic 1959 South African strain. The three tested African strains and one European strain from Spain caused 100% mortality in inoculated mice, with similar survival times and histopathology in tissues. Unexpectedly, a European strain from the Netherlands caused only 12% mortality and significantly less histopathology in tissues from mice compared to mice inoculated with the other strains. Viremia was highest in mice inoculated with the recent African strains and lowest in mice inoculated with the Netherlands strain. Based on phylogenetics, the USUV isolates from Spain and the Netherlands were derived from separate introductions into Europe, suggesting that disease outcomes may differ for USUV strains circulating in Europe. These results also suggest that while more human USUV disease cases have been reported in Europe recently, circulating African USUV strains are still a potential major health concern. Author Summary: Usutu virus (USUV) is an emerging mosquito-borne virus that causes severe neuroinvasive disease in humans. USUV was first detected in Africa in 1959, and cases of human disease have increased in recent years. Most USUV disease cases are now reported in Europe, where the virus currently circulating. One possibility for the increase in case numbers is that USUV strains have become more pathogenic over time during its spread from Africa into Europe. We compared the pathogenesis of five USUV isolates from Africa and Europe in a mouse model. Three isolates from Africa and one isolate from Europe caused 100% mortality in mice. Surprisingly, one isolate from Netherlands caused only 12% motality. Significantly less histopathology was observed in tissues from mice inoculated with the Netherlands strain compared to mice inoculated with the other four strains. Our results suggest that, even though more human USUV disease cases have been reported in Europe recently, African USUV strains are also highly pathogenic.