Confirmation of Zika virus infection through hospital-based sentinel surveillance of acute febrile illness in Uganda, 2014-2017.

Zika virus (ZIKV), transmitted by Aedes species mosquitoes, was first isolated in Uganda in 1947. From February 2014 to October 2017, the Uganda Virus Research Institute, in collaboration with the US Centers for Diseases Control and Prevention, conducted arbovirus surveillance in acute febrile illness (AFI) patients at St Francis hospital in Nkonkonjeru. Three hundred and eighty-four serum samples were collected and tested for IgM antibodies to yellow fever virus (YFV), West Nile virus (WNV), dengue virus (DENV), chikungunya virus (CHIKV) and ZIKV. Of the 384 samples, 5 were positive for ZIKV IgM. Of these five, three were confirmed by plaque reduction neutralization test (PRNT) to be ZIKV infections. Of the remaining two, one was determined to be a non-specific flavivirus infection and one was confirmed to be alphavirus-positive by reverse transcriptase polymerase chain reaction (RT-PCR). This study provides the first evidence of laboratory-confirmed ZIKV infection in Uganda in five decades, and emphasizes the need to enhance sentinel surveillance.

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.

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.

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.

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.

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.

Isolation of arboviruses from mosquitoes collected in northern Vietnam.

In response to recent increases in cases of pediatric encephalitis with unknown etiology in northern Vietnam, surveillance for arbovirus activity was conducted in four provinces surrounding the city of Hanoi during June 2002 and July-August 2004. A total of 20,615 mosquitoes consisting of 19 species in 1,122 pools were processed for virus isolation; virus isolates were obtained from 44 pools. Sagiyama virus (11 isolates), Getah virus (15 isolates), Oya virus (13 isolates), and Akabane virus (4 isolates) were identified by immunofluorescence assay and sequence analysis of reverse transcription-polymerase chain reaction fragments. Surprisingly, no isolates of Japanese encephalitis (JE) virus were obtained. Isolation of Akabane virus, Oya virus, Getah virus, and Sagiyama virus is reported for the first time from Vietnam.

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.

Growth characteristics of ChimeriVax-DEN2 vaccine virus in Aedes aegypti and Aedes albopictus mosquitoes.

The chimeric yellow fever (YF) 17D-dengue type 2 (ChimeriVax-DEN2) vaccine virus developed by Acambis, Inc. (Cambridge, MA) contains the prM and E genes of wild-type (wt) dengue 2 (DEN-2) (strain PUO-218) virus in the YF vaccine virus (strain 17D) backbone. The potential of ChimeriVax-DEN2 virus to infect and be transmitted by Aedes aegypti, the principal DEN and YF virus mosquito vector, and Aedes albopictus, a species that occurs in areas of active transmission of YF and DEN viruses, was evaluated. Mosquitoes were intrathoracically (IT) inoculated with virus or were fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN2 were compared with the wt DEN-2 and YF 17D vaccine viruses. Replication of YF 17D virus is attenuated in cultured Ae. albopictus C6/36 mosquito cells and in Ae. aegypti and Ae. albopictus mosquitoes. Growth of ChimeriVax-DEN2 virus similarly was restricted in C6/36 cells and in mosquitoes. ChimeriVax-DEN2 replicated in 56% of IT inoculated Ae. aegypti, and virus disseminated to head tissue in 36%, with a mean viral titer of 1.8 log10 PFU/mosquito. Of mosquitoes, 16% of Ae. aegypti and 24% of Ae. albopictus were infected 14 days after a blood meal containing ChimeriVax-DEN2, but virus did not disseminate to head tissue. In contrast, DEN-2 replicated in all IT inoculated and orally infected Ae. aegypti (mean titer 5.5 log10 PFU/mosquito), and virus disseminated to head tissue in 95%. Of Ae. albopictus, 84% were infected after a blood meal containing DEN-2 virus; dissemination occurred in 36%. Replication of ChimeriVax-DEN2 virus in mosquitoes corresponded to that of YF 17D vaccine virus, which is restricted in its ability to infect and replicate in mosquitoes. Therefore, transmission of ChimeriVax-DEN2 virus by vector mosquitoes is unlikely.

Analysis of the replication kinetics of the ChimeriVax-DEN 1, 2, 3, 4 tetravalent virus mixture in Aedes aegypti by real-time reverse transcriptase-polymerase chain reaction.

The vector competence of mosquitoes for chimeric viruses being developed as vaccines to protect against dengue (DEN) virus infection were evaluated in a cooperative agreement with Acambis, Inc. Chimeric viruses have been constructed that contain the premembrane (prM) and envelope (E) genes of each of the wild-type (wt) DEN virus serotypes, DEN-1, DEN-2, DEN-3, and DEN-4, in the yellow fever (YF) vaccine virus (strain 17D) YF-VAX backbone. It was previously shown that the replication profile of ChimeriVax-DEN2 virus in Aedes albopictus C6/36 cells and in vivo in Ae. aegypti mosquitoes corresponded to that of YF-VAX virus; replication was restricted in C6/36 cells, and Ae. aegypti were poorly infected via an artificial infectious blood meal. Thus, there is very little risk of transmission by mosquitoes of ChimeriVax-DEN2 vaccine virus through the bite of a mosquito. However, because ChimeriVax-DEN 1, 2, 3, 4 viruses will be administered to humans simultaneously, growth of a mixture of ChimeriVax-DEN 1, 2, 3, 4 viruses was assessed in both C6/36 cells in culture and in the Ae. aegypti mosquito, which is the primary vector of both YF and DEN viruses. Mosquitoes were intrathoracically (IT) inoculated with virus or fed a virus-laden blood meal, and the replication kinetics of ChimeriVax-DEN 1, 2, 3, 4 were compared with the wt DEN and YF-VAX viruses. A quantitative real-time reverse transcriptase-polymerase chain reaction assay was developed as a method to detect and differentiate replication of each of the four ChimeriVax-DEN serotypes in the ChimeriVax-DEN 1, 2, 3, 4 tetravalent mixture. Growth of the chimeric viruses in C6/36 cells and in IT-inoculated Ae. aegypti was lower than that of YF-VAX virus; in previous studies Ae. aegypti was shown to be refractory to infection by YF-VAX virus. The growth rate of each chimeric virus was similar whether it was a single serotype infection, or part of the tetravalent mixture, and no interference by one chimeric virus over another chimeric serotype was observed. ChimeriVax-DEN viruses infected mosquitoes poorly via an infectious blood meal compared with wt DEN viruses. Therefore, it is unlikely that a mosquito feeding on a viremic vaccinee, would become infected with the chimeric viruses. Thus, there is very little potential for transmission by mosquitoes of the ChimeriVax-DEN vaccine viruses.

Vector competence of Brazilian Aedes aegypti and Ae. albopictus for a Brazilian yellow fever virus isolate.

Because the potential urban yellow fever (YF) mosquito vectors Aedes aegypti and Ae. albopictus are at historical highs in Brazil, both in terms of density and geographical range, we assessed the risk of an urban YF epidemic in Brazil. We evaluated and confirmed in a laboratory setting the vector competence of Brazilian Ae. aegypti for a currently circulating strain of YF virus, and investigated the potential for Brazilian Ae. albopictus to transmit YF.

Investigation of the aedes (Stegomyia) flavopictus complex (Diptera: Culicidae) in Japan by sequence analysis of the internal transcribed spacers of ribosomal DNA.

Aedes (Stegomyia) flavopictus Yamada is widely distributed in Japan and Korea. The species comprises three subspecies based on current morphological taxonomy: Aedes flavopictus in the Palearctic region of Japan, Ae. f downsi Bohart & Ingram from Amami and Okinawa Islands, and Ae. f miyarai Tanaka, Mizusawa & Ingram from Ishigaki and Iriomote Islands of the Ryukyu Archipelago. These subspecies designations are based on observations of a combination of several morphological characters, none of which, by itself is diagnostic for discriminating among the three subspecies. To further study the relationships in this group, we examined the nucleic acid sequence divergence in the internal transcribed spacer regions (ITSI and ITS2) of the ribosomal DNA gene array of Ae. flavopictus individuals collected at five sites from three geographic regions in Japan. Analysis of sequence data by distance and maximum parsimony methods produced phylogenetic trees that showed separation of the specimens into three major clades, corresponding to both subspecies and geographic region. These results were consistent with and support the current classification and geographic distribution of the three subspecies.

Polymerase chain reaction assay identifies Culex nigripalpus: part of an assay for molecular identification of the common Culex (Culex) mosquitoes of the eastern United States.

Nucleotide sequence information on internal transcribed spacer (ITS) 1 and ITS 2 regions of the nuclear ribosomal DNA multigene family was used to develop a polymerase chain reaction assay that identifies Culex nigripalpus Theobald. The assay uses species-specific forward and reverse primers for Cx. nigripalpus and can be used along with previously described primers to distinguish among 4 common taxa of Culex (Culex) of the eastern USA with a single thermal cycler program. The assay distinguishes among the 4 taxa Cx. nigripalpus, Cx. restuans Theobald, Cx. salinarius Coquillett, and members of the Cx. pipiens Linnaeus complex. This assay may be used to verify the morphological identification of individual specimens of Culex or to confirm the species composition of mosquito pools.

Temporal abundance, parity, survival rates, and arbovirus isolation of field-collected container-inhabiting mosquitoes in eastern Tennessee.

Surveillance of container-inhabiting mosquitoes was conducted from June 17 through November 9, 1998, at 2 1997 La Crosse virus (LAC) human case sites (Knox and Cocke counties, Tennessee). Mosquitoes were collected weekly with 2 dry ice-baited Centers for Disease Control miniature light traps, 2 omnidirectional Fay traps, and 40 oviposition traps at each site. A total of 8,408 mosquitoes, composed of Ochlerotatus triseriatus (n = 2,095) and Aedes albopictus (n = 6,313), were reared or collected and assayed for virus. The majority of host-seeking Ae. albopictus (n = 567) collected from July through October from both sites were dissected to determine parity status. Monthly parity rates ranged from 0.78 to 0.85 and 0.79 to 0.92 in Knox and Cocke counties, respectively. The high parity rates indicate that this population of Ae. albopictus has a high daily survival rate and may have a high vector potential. The temporal patterns in Ae. albopictus and Oc. triseriatus egg collections from both of the human case sites were significantly correlated, suggesting that the populations fluctuate in a similar manner across the eastern Tennessee region. Although LAC was not isolated from either species, one isolation of a California serogroup virus, most likely a subtype of Jamestown Canyon virus (JC), was recovered from a pool of 50 male Ae. albopictus reared from eggs collected at the Knox County site (minimum field infection rate of 1.89 per 1,000). This is the 1st report of a very closely related JC-like virus in Ae. albopictus and from Tennessee, as well as the 1st time this potential human pathogen has been isolated from transovarially infected field populations of Ae. albopictus.

STUDIES ON THE SPECIES COMPOSITION AND RELATIVE ABUNDANCE OF MOSQUITOES OF MPIGI DISTRICT, CENTRAL UGANDA.

Prediction of arboviral disease outbreaks and planning for appropriate control interventions require knowledge of the mosquito vectors involved. Although mosquito surveys have been conducted in different regions of Uganda since the mid 30's such studies have not been carried out in Mpigi District. In October 2011, we conducted mosquito collections in Mpigi district to determine species composition and relative abundance of the different species. The survey was conducted in four villages, Njeru, Ddela, Kiwumu and Nsumbain Kammengo sub-county, Mpigi district, Uganda. CDC light traps baited with dry ice (carbon dioxide) were used to capture adult mosquitoes. A total of 54,878 mosquitoes comprising 46 species from eight genera were collected. The dominant species at all sites was Coquilletidia (Coquilletidia) fuscopennata Theobald (n=38,059, 69%), followed by Coquillettidia (Coquillettidia) metallica Theobald (n=4,265, 7.8%). The number of species collected varied from 17 in the genus Culex to 1 in the genus Lutzia. Of the 46 species identified, arboviruses had previously been isolated from 28 (60.9%) suggesting a high potential for arboviral transmission and/or maintenance in Mpigi District.

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.

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.