Exposure of Egyptian Rousette Bats (Rousettus aegyptiacus) and a Little Free-Tailed Bat (Chaerephon pumilus) to Alphaviruses in Uganda.

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.

Discovery and Characterization of Bukakata orbivirus (Reoviridae:Orbivirus), a Novel Virus from a Ugandan Bat.

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.

Neutralizing antibodies against flaviviruses, Babanki virus, and Rift Valley fever virus in Ugandan bats.

Introduction: A number of arboviruses have previously been isolated from naturally-infected East African bats, however the role of bats in arbovirus maintenance is poorly understood. The aim of this study was to investigate the exposure history of Ugandan bats to a panel of arboviruses. Materials and methods: Insectivorous and fruit bats were captured from multiple locations throughout Uganda during 2009 and 2011-2013. All serum samples were tested for neutralizing antibodies against West Nile virus (WNV), yellow fever virus (YFV), dengue 2 virus (DENV-2), Zika virus (ZIKV), Babanki virus (BBKV), and Rift Valley fever virus (RVFV) by plaque reduction neutralization test (PRNT). Sera from up to 626 bats were screened for antibodies against each virus. Results and Discussion: Key findings include the presence of neutralizing antibodies against RVFV in 5/52 (9.6%) of little epauletted fruit bats (Epomophorus labiatus) captured from Kawuku and 3/54 (5.6%) Egyptian rousette bats from Kasokero cave. Antibodies reactive to flaviviruses were widespread across bat taxa and sampling locations. Conclusion: The data presented demonstrate the widespread exposure of bats in Uganda to arboviruses, and highlight particular virus-bat associations that warrant further investigation.

Arboviruses Isolated From Mosquitoes Collected in Uganda, 2008-2012.

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.

Detection of Entebbe Bat Virus After 54 Years.

Entebbe bat virus (ENTV; Flaviviridae: Flavivirus), closely related to yellow fever virus, was first isolated from a little free-tailed bat (Chaerephon pumilus) in Uganda in 1957, but was not detected after that initial isolation. In 2011, we isolated ENTV from a little free-tailed bat captured from the attic of a house near where it had originally been found. Infectious virus was recovered from the spleen and lung, and the viral RNA was sequenced and compared with that of the original isolate. Across the polypeptide sequence, there were 76 amino acid substitutions, resulting in 97.8% identity at the amino acid level between the 1957 and 2011 isolates. Further study of this virus would provide valuable insights into the ecological and genetic factors governing the evolution and transmission of bat- and mosquito-borne flaviviruses.

Epidemiology of meningitis in an HIV-infected Ugandan cohort.

There is limited understanding of the epidemiology of meningitis among human immunodeficiency virus (HIV)-infected populations in sub-Saharan Africa. We conducted a prospective cohort study of HIV-infected adults with suspected meningitis in Uganda, to comprehensively evaluate the etiologies of meningitis. Intensive cerebrospiral fluid (CSF) testing was performed to evaluate for bacterial, viral, fungal, and mycobacterial etiologies, including neurosyphilis,16s ribosomal DNA (rDNA) polymerase chain reaction (PCR) for bacteria, Plex-ID broad viral assay, quantitative-PCR for HSV-1/2, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Toxoplasma gondii; reverse transcription-PCR (RT-PCR) for Enteroviruses and arboviruses, and Xpert MTB/RIF assay. Cryptococcal meningitis accounted for 60% (188 of 314) of all causes of meningitis. Of 117 samples sent for viral PCR, 36% were EBV positive. Among cryptococcal antigen negative patients, the yield of Xpert MTB/RIF assay was 22% (8 of 36). After exclusion of cryptococcosis and bacterial meningitis, 61% (43 of 71) with an abnormal CSF profile had no definitive diagnosis. Exploration of new TB diagnostics and diagnostic algorithms for evaluation of meningitis in resource-limited settings remains needed, and implementation of cryptococcal diagnostics is critical.

Sunguru virus: a novel virus in the family Rhabdoviridae isolated from a chicken in north-western Uganda.

Sunguru virus (SUNV), a novel virus belonging to the highly diverse Rhabdoviridae family, was isolated from a domestic chicken in the district of Arua, Uganda, in 2011. This is the first documented isolation of a rhabdovirus from a chicken. SUNV is related to, but distinct from, Boteke virus and other members of the unclassified Sandjimba group. The genome is 11056 nt in length and contains the five core rhabdovirus genes plus an additional C gene (within the ORF of a phosphoprotein gene) and a small hydrophobic protein (between the matrix and glycoprotein genes). Inoculation of vertebrate cells with SUNV resulted in significant viral growth, with a peak titre of 7.8 log10 p.f.u. ml(-1) observed in baby hamster kidney (BHK) cells. Little to no growth was observed in invertebrate cells and in live mosquitoes, with Anopheles gambiae mosquitoes having a 47.4% infection rate in the body but no dissemination of the virus to the salivary glands; this suggests that this novel virus is not arthropod borne as some other members of the family Rhabdoviridae.

Ticks and tick-borne viruses from livestock hosts in arid and semiarid regions of the eastern and northeastern parts of Kenya.

Biodiversity and relative abundance of ticks and associated arboviruses in Garissa (northeastern) and Isiolo (eastern) provinces of Kenya were evaluated. Ticks were collected from livestock, identified to species, pooled, and processed for virus isolation. In Garissa, Rhipicephalus pulchellus Gerstacker (57.8%) and Hyalomma truncatum Koch (27.8%) were the most abundant species sampled, whereas R. pulchellus (80.4%) and Amblyomma gemma Donitz (9.6%) were the most abundant in Isiolo. Forty-four virus isolates, comprising Dugbe virus (DUGV; n = 22) and Kupe virus (n = 10; Bunyaviridae: Nirovirus), Dhori virus (DHOV; n = 10; Orthomyxoviridae: Thogotovirus),and Ngari virus (NRIV; n = 2; Bunyaviridae: Orthobunyavirus), were recovered mostly from R. pulchellus sampled in Isiolo. DUGV was mostly recovered from R. pulchellus from sheep and cattle, and DHOV from R. pulchellus from sheep. All Kupe virus isolates were from Isiolo ticks, including R. pulchellus from all the livestock, A. gemma and Amblyomma variegatum F. from cattle, and H. truncatum from goat. NRIV was obtained from R. pulchellus and A. gemma sampled from cattle in Isiolo and Garissa, respectively, while all DHOV and most DUGV (n = 12) were from R. pulchellus sampled from cattle in Garissa. DUGV was also recovered from H. truncatum and Amblyomma hebraeum Koch from cattle and from Rhipicephalus annulatus Say from camel. This surveillance study has demonstrated the circulation of select tick-borne viruses in parts of eastern and northeastern provinces of Kenya, some of which are of public health importance. The isolation of NRIV from ticks is particularly significant because it is usually known to be a mosquito-borne virus affecting humans.

Deletion of the NSm virulence gene of Rift Valley fever virus inhibits virus replication in and dissemination from the midgut of Aedes aegypti mosquitoes.

BACKGROUND: Previously, we investigated the role of the Rift Valley fever virus (RVFV) virulence genes NSs and NSm in mosquitoes and demonstrated that deletion of NSm significantly reduced the infection, dissemination, and transmission rates of RVFV in Aedes aegypti mosquitoes. The specific aim of this study was to further characterize midgut infection and escape barriers of RVFV in Ae. aegypti infected with reverse genetics-generated wild type RVFV (rRVF-wt) or RVFV lacking the NSm virulence gene (rRVF-ΔNSm) by examining sagittal sections of infected mosquitoes for viral antigen at various time points post-infection. METHODOLOGY AND PRINCIPAL FINDINGS: Ae. aegypti mosquitoes were fed an infectious blood meal containing either rRVF-wt or rRVF-ΔNSm. On days 0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 post-infection, mosquitoes from each experimental group were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and examined for RVFV antigen by immunofluorescence assay. Remaining mosquitoes at day 14 were assayed for infection, dissemination, and transmission. Disseminated infections were observed in mosquitoes as early as three days post infection for both virus strains. However, infection rates for rRVF-ΔNSm were statistically significantly less than for rRVF-wt. Posterior midgut infections in mosquitoes infected with rRVF-wt were extensive, whereas midgut infections of mosquitoes infected with rRVF-ΔNSm were confined to one or a few small foci. CONCLUSIONS/SIGNIFICANCE: Deletion of NSm resulted in the reduced ability of RVFV to enter, replicate, and disseminate from the midgut epithelial cells. NSm appears to have a functional role in the vector competence of mosquitoes for RVFV at the level of the midgut barrier.

Isolation and molecular characterization of Fikirini rhabdovirus, a novel virus from a Kenyan bat.

Zoonotic and vector-borne pathogens have comprised a significant component of emerging human infections in recent decades, and bats are increasingly recognized as reservoirs for many of these disease agents. To identify novel pathogens associated with bats, we screened tissues of bats collected in Kenya. Virus isolates were identified by next generation sequencing of viral nucleic acid preparations from the infected cell culture supernatant and characterized. Here we report the identification of Fikirini rhabdovirus, a novel rhabdovirus isolated from a bat, Hipposideros vittatus, captured along the Kenyan coast.

Search strategy has influenced the discovery rate of human viruses.

A widely held concern is that the pace of infectious disease emergence has been increasing. We have analyzed the rate of discovery of pathogenic viruses, the preeminent source of newly discovered causes of human disease, from 1897 through 2010. The rate was highest during 1950-1969, after which it moderated. This general picture masks two distinct trends: for arthropod-borne viruses, which comprised 39% of pathogenic viruses, the discovery rate peaked at three per year during 1960-1969, but subsequently fell nearly to zero by 1980; however, the rate of discovery of nonarboviruses remained stable at about two per year from 1950 through 2010. The period of highest arbovirus discovery coincided with a comprehensive program supported by The Rockefeller Foundation of isolating viruses from humans, animals, and arthropod vectors at field stations in Latin America, Africa, and India. The productivity of this strategy illustrates the importance of location, approach, long-term commitment, and sponsorship in the discovery of emerging pathogens.

Identification of host blood from engorged mosquitoes collected in western Uganda using cytochrome oxidase I gene sequences.

Emerging infectious disease events are frequently caused by arthropod-borne viruses (arboviruses) that are maintained in a zoonotic cycle between arthropod vectors and vertebrate wildlife species, with spillover to humans in areas where human and wildlife populations interface. The greater Congo basin region, including Uganda, has historically been a hot spot for emergence of known and novel arboviruses. Surveillance of arthropod vectors is a critical activity in monitoring and predicting outbreaks of arboviral disease, and identification of blood meals in engorged arthropods collected during surveillance efforts provides insight into the ecology of arboviruses and their vectors. As part of an ongoing arbovirus surveillance project we analyzed blood meals from engorged mosquitoes collected at five sites in western Uganda November 2008-June 2010. We extracted DNA from the dissected and triturated abdomens of engorged mosquito specimens. Mitochondrial cytochrome c oxidase I gene sequence was amplified by PCR and sequenced to identify the source of the mosquito host blood. Blood meals were analyzed from 533 engorged mosquito specimens; 440 of these blood meals were successfully identified from 33 mosquito species. Species identifications were made for 285 of the 440 identified specimens with the remainder identified to genus, family, or order. When combined with published arbovirus isolation and serologic survey data, our results suggest possible vector-reservoir relationships for several arboviruses, including Rift Valley fever virus and West Nile virus.

Infection and transmission of Rift Valley fever viruses lacking the NSs and/or NSm genes in mosquitoes: potential role for NSm in mosquito infection.

BACKGROUND: Rift Valley fever virus is an arthropod-borne human and animal pathogen responsible for large outbreaks of acute and febrile illness throughout Africa and the Arabian Peninsula. Reverse genetics technology has been used to develop deletion mutants of the virus that lack the NSs and/or NSm virulence genes and have been shown to be stable, immunogenic and protective against Rift Valley fever virus infection in animals. We assessed the potential for these deletion mutant viruses to infect and be transmitted by Aedes mosquitoes, which are the principal vectors for maintenance of the virus in nature and emergence of virus initiating disease outbreaks, and by Culex mosquitoes which are important amplification vectors. METHODOLOGY AND PRINCIPAL FINDINGS: Aedes aegypti and Culex quinquefasciatus mosquitoes were fed bloodmeals containing the deletion mutant viruses. Two weeks post-exposure mosquitoes were assayed for infection, dissemination, and transmission. In Ae. aegypti, infection and transmission rates of the NSs deletion virus were similar to wild type virus while dissemination rates were significantly reduced. Infection and dissemination rates for the NSm deletion virus were lower compared to wild type. Virus lacking both NSs and NSm failed to infect Ae. aegypti. In Cx. quinquefasciatus, infection rates for viruses lacking NSm or both NSs and NSm were lower than for wild type virus. CONCLUSIONS/SIGNIFICANCE: In both species, deletion of NSm or both NSs and NSm reduced the infection and transmission potential of the virus. Deletion of both NSs and NSm resulted in the highest level of attenuation of virus replication. Deletion of NSm alone was sufficient to nearly abolish infection in Aedes aegypti mosquitoes, indicating an important role for this protein. The double deleted viruses represent an ideal vaccine profile in terms of environmental containment due to lack of ability to efficiently infect and be transmitted by mosquitoes.

Crimean-Congo hemorrhagic fever virus in Hyalommid ticks, northeastern Kenya.

As part of ongoing arbovirus surveillance, we screened ticks obtained from livestock in northeastern Kenya in 2008 to assess the risk for human exposure to tick-borne viruses. Of 1,144 pools of 8,600 Hyalomma spp. ticks screened for Congo-Crimean hemorrhagic fever virus by reverse transcription PCR, 23 pools were infected, demonstrating a potential for human exposure.

Transmission of West Nile virus by Culex quinquefasciatus say infected with Culex Flavivirus Izabal.

BACKGROUND: The natural history and potential impact of mosquito-specific flaviviruses on the transmission efficiency of West Nile virus (WNV) is unknown. The objective of this study was to determine whether or not prior infection with Culex flavivirus (CxFV) Izabal altered the vector competence of Cx. quinquefasciatus Say for transmission of a co-circulating strain of West Nile virus (WNV) from Guatemala. METHODS AND FINDINGS: CxFV-negative Culex quinquefasciatus and those infected with CxFV Izabal by intrathoracic inoculation were administered WNV-infectious blood meals. Infection, dissemination, and transmission of WNV were measured by plaque titration on Vero cells of individual mosquito bodies, legs, or saliva, respectively, two weeks following WNV exposure. Additional groups of Cx. quinquefasciatus were intrathoracically inoculated with WNV alone or WNV+CxFV Izabal simultaneously, and saliva collected nine days post inoculation. Growth of WNV in Aedes albopictus C6/36 cells or Cx. quinquefasciatus was not inhibited by prior infection with CxFV Izabal. There was no significant difference in the vector competence of Cx. quinquefasciatus for WNV between mosquitoes uninfected or infected with CxFV Izabal across multiple WNV blood meal titers and two colonies of Cx. quinquefasciatus (p>0.05). However, significantly more Cx. quinquefasciatus from Honduras that were co-inoculated simultaneously with both viruses transmitted WNV than those inoculated with WNV alone (p = 0.0014). Co-inoculated mosquitoes that transmitted WNV also contained CxFV in their saliva, whereas mosquitoes inoculated with CxFV alone did not contain virus in their saliva. CONCLUSIONS: In the sequential infection experiments, prior infection with CxFV Izabal had no significant impact on WNV replication, infection, dissemination, or transmission by Cx. quinquefasciatus, however WNV transmission was enhanced in the Honduras colony when mosquitoes were inoculated simultaneously with both viruses.

Isolation and characterization of a new mosquito flavivirus, Quang Binh virus, from Vietnam.

In recent years, a number of flaviviruses that replicate only in an arthropod host have been discovered and characterized. We describe here the isolation and characterization of a new mosquito-only flavivirus in this group. The virus was isolated from Culex tritaeniorhyncus mosquitoes collected in Vietnam in 2002 and was found to be genetically different from mosquito flaviviruses described previously. We propose the isolate be named Quang Binh virus.

Kupe virus, a new virus in the family bunyaviridae, genus nairovirus, kenya.

We have previously described isolation and preliminary identification of a virus related to Dugbe virus (DUGV), family Bunyaviridae, genus Nairovirus. Six isolates of the virus were obtained from pools of Amblyomma gemma and Rhipicephalus pulchellus ticks collected from hides of cattle in Nairobi, Kenya, in October 1999. We report results of further characterization of this virus, including growth kinetics in cell culture and full-length genome sequencing and genetic characterization, which show it to be distinct from DUGV. We suggest that this is a new virus in the family Bunyaviridae, genus Nairovirus, and we propose that it be designated Kupe virus.

Entomologic investigations of a chikungunya virus epidemic in the Union of the Comoros, 2005.

From January to April 2005, an epidemic of chikungunya virus (CHIKV) illness occurred in the Union of Comoros. Entomological studies were undertaken during the peak of the outbreak, from March 11 to March 31, aimed at identifying the primary vector(s) involved in transmission so that appropriate public health measures could be implemented. Adult mosquitoes were collected by backpack aspiration and human landing collection in homes and neighborhoods of clinically ill patients. Water-holding containers were inspected for presence of mosquito larvae. Adult mosquitoes were analyzed by RT-PCR and cultivation in cells for the presence of CHIK virus and/or nucleic acid. A total of 2,326 mosquitoes were collected and processed in 199 pools. The collection consisted of 62.8% Aedes aegypti, 25.5% Culex species, and 10.7% Aedes simpsoni complex, Eretmapodites spp and Anopheles spp. Seven mosquito pools were found to be positive for CHIKV RNA and 1 isolate was obtained. The single CHIKV mosquito isolate was from a pool of Aedes aegypti and the minimum infection rate (MIR) for this species was 4.0, suggesting that Ae. aegypti was the principal vector responsible for the outbreak. This was supported by high container (31.1%), household (68%), and Breteau (126) indices, with discarded tires (58.8%) and small cooking and water storage vessels (31.1%) registering the highest container indices.

A single positively selected West Nile viral mutation confers increased virogenesis in American crows.

West Nile virus (WNV), first recognized in North America in 1999, has been responsible for the largest arboviral epiornitic and epidemic of human encephalitis in recorded history. Despite the well-described epidemiological patterns of WNV in North America, the basis for the emergence of WNV-associated avian pathology, particularly in the American crow (AMCR) sentinel species, and the large scale of the North American epidemic and epiornitic is uncertain. We report here that the introduction of a T249P amino acid substitution in the NS3 helicase (found in North American WNV) in a low-virulence strain was sufficient to generate a phenotype highly virulent to AMCRs. Furthermore, comparative sequence analyses of full-length WNV genomes demonstrated that the same site (NS3-249) was subject to adaptive evolution. These phenotypic and evolutionary results provide compelling evidence for the positive selection of a mutation encoding increased viremia potential and virulence in the AMCR sentinel bird species.

Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically inoculated Aedes aegypti mosquitoes.

To determine the importance of dengue 2 virus (DEN2V) envelope (E) protein glycosylation, virus mutants in one or both of the N-linked glycosylation motifs were prepared. We found that while the E2 mutant virus (N153Q) replicated in mammalian and mosquito cells, the E1 (N67Q) and E1/2 (N67Q and N153Q) mutant viruses were unable to grow in mammalian cells. Infection of C6/36 mosquito cells with either the E1 or E1/2 mutants resulted in the introduction of a compensatory mutation, K64N, restoring glycosylation in the area. All mutants replicated similarly in inoculated Aedes aegypti mosquitoes, with no change in their mutations. These results suggest that N-linked glycosylation of the E protein is not necessary for DEN2V replication in mosquitoes, however N-linked glycosylation at amino acid N67 (or nearby N64) is critical for the survival of the virus in either mammalian or insect cell culture.