Rebekah C. Kading

Predicting the Mosquito Species and Vertebrate Species Involved in the Theoretical Transmission of Rift Valley Fever Virus in the United States

Rift Valley fever virus (RVFV) is a mosquito-borne virus in the family Bunyaviridiae that has spread throughout continental Africa to Madagascar and the Arabian Peninsula. The establishment of RVFV in North America would have serious consequences for human and animal health in addition to a significant economic impact on the livestock industry. Published and unpublished data on RVFV vector competence, vertebrate host competence, and mosquito feeding patterns from the United States were combined to quantitatively implicate mosquito vectors and vertebrate hosts that may be important to RVFV transmission in the United States. A viremia-vector competence relationship based on published mosquito transmission studies was used to calculate a vertebrate host competence index which was then combined with mosquito blood feeding patterns to approximate the vector and vertebrate amplification fraction, defined as the relative contribution of the mosquito or vertebrate host to pathogen transmission. Results implicate several Aedes spp. mosquitoes and vertebrates in the order Artiodactyla as important hosts for RVFV transmission in the U.S. Moreover, this study identifies critical gaps in knowledge which would be necessary to complete a comprehensive analysis identifying the different contributions of mosquitoes and vertebrates to potential RVFV transmission in the U.S. Future research should focus on (1) the dose-dependent relationship between viremic exposure and the subsequent infectiousness of key mosquito species, (2) evaluation of vertebrate host competence for RVFV among North American mammal species, with particular emphasis on the order Artiodactyla, and (3) identification of areas with a high risk for RVFV introduction so data on local vector and host populations can help generate geographically appropriate amplification fraction estimates.

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.

PREVALENCE OF ANTIBODIES TO ALPHAVIRUSES AND FLAVIVIRUSES IN FREE-RANGING GAME ANIMALS AND NONHUMAN PRIMATES IN THE GREATER CONGO BASIN

Vector-borne and zoonotic pathogens have comprised a significant proportion of the emerging infectious diseases in humans in recent decades. The role of many wildlife species as reservoirs for arthropod-borne viral pathogens is poorly understood. We investigated the exposure history of various African wildlife species from the Congo basin to mosquito-borne flaviviruses and alphaviruses by testing archived serum samples. Sera from 24 African forest buffalo (Syncerus caffer nanus), 34 African elephants (Loxodonta africana), 40 duikers (Cephalophus and Philantomba spp.), 25 mandrills (Mandrillus sphinx), 32 mountain gorillas (Gorilla beringei beringei), five Grauer’s gorillas (Gorilla beringei graueri), two L’Hoest’s monkeys (Cercopithecus lhoesti), two golden monkeys (Cercopithecus kandti), and three chimpanzees (Pan troglodytes) sampled between 1991 and 2009 were tested for antibodies against chikungunya virus (CHIKV), o’nyong-nyong virus (ONNV), West Nile virus (WNV), dengue 2 virus (DENV-2), and yellow fever virus (YFV) by plaque reduction neutralization test. Specific neutralizing antibodies against ONNV were found in African forest buffalo in the Democratic Republic of the Congo (DRC) and Gabon, duikers in the DRC, and mandrills in Gabon, providing novel evidence of enzootic circulation of ONNV in these countries. African forest buffalo in the DRC and Gabon also demonstrated evidence of exposure to CHIKV, WNV, and DENV-2, while mandrills in Gabon were antibody positive for CHIKV, DENV-2, WNV, and YFV. All of the elephants tested had a strong neutralizing antibody response to WNV. We also document results from a survey of gorillas for arboviruses, of which 4/32 (13%) had antibody to an alphavirus or flavivirus. Overall, our results demonstrate a high prevalence of neutralizing antibodies against multiple arboviruses in wildlife in equatorial Africa.

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.

Mosquitoes of Western Uganda

ABSTRACT The mosquito fauna in many areas of western Uganda has never been studied and is currently unknown. One area, Bwamba County, has been previously studied and documented but the species lists have not been updated for >40 yr. This paucity of data makes it difficult to determine which arthropod-borne viruses pose a risk to human or animal populations. Using CO2 baited-light traps, from 2008 through 2010, 67,731 mosquitoes were captured at five locations in western Uganda including Mweya, Sempaya, Maramagambo, Bwindi (BINP), and Kibale (KNP). Overall, 88 mosquito species, 7 subspecies, and 7 species groups in 10 genera were collected. The largest number of species was collected at Sempaya (65 species), followed by Maramagambo (45), Mweya (34), BINP (33), and KNP (22). However, species diversity was highest in BINP (Simpsons Diversity Index 1-D = 0.85), followed by KNP (0.80), Maramagambo (0.79), Sempaya (0.67), and Mweya (0.56). Only six species Aedes (Aedimorphus) cumminsii (Theobald), Aedes (Neomelaniconion) circumluteolus (Theobald), Culex (Culex) antennatus (Becker), Culex (Culex) decens group, Culex (Lutzia) tigripes De Grandpre and De Charmoy, and Culex (Oculeomyia) annulions (Theobald), were collected from all five sites suggesting large differences in species composition among sites. Four species (Aedes (Stegomyia) metallicus (Edwards), Anopheles (Cellia) rivulorum Leeson, Uranotaenia (Uranotaenia) chorleyi (Edwards), and Uranotaenia (Uranotaenia) pallidocephala (Theobald) and one subspecies (Aedes (Stegomyia) aegypti formosus (Walker)) were collected in Bwamba County for the first time. This study represents the first description of the mosquito species composition of Mweya, Maramagambo, BINP, and KNP. A number of morphological variations were noted regarding the postspiracular scales, hind tibia, and sternites that make Culex (Culex) neavei (Theobald) challenging to identify. At least 50 species collected in this study have previously been implicated in the transmission of arboviruses of public health importance suggesting a high potential for maintenance and transmission of a wide variety of arboviruses in western Uganda.

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.

Mosquitoes Used to Draw Blood for Arbovirus Viremia Determinations in Small Vertebrates

Serial samples from the same individuals may be required for certain virological studies, however, some small animals cannot easily be blood-sampled. Therefore, we evaluated the use of Culex quinquefasciatus Say and Aedes albopictus Skuse mosquitoes as “biological syringes” to draw blood for virus titer determinations in small vertebrates. Groups of chicks (Gallus gallus), hamsters (Mesocricetus auratus), and house sparrows (Passer domesticus) were experimentally infected with West Nile virus (WNV) or Highlands J virus (HJV). In general, good correlation was seen between mosquito- and syringe-derived blood samples at titers ≥5.0 log10 pfu/mL serum as compared with titers <5.0 log10 pfu/mL serum for chicks, hamsters, and sparrows. Ninety-two percent (24/26) of sparrows with virus titers >105 pfu/mL serum had mosquito- and syringe-derived titers within one log of each other. Sparrow viremia profiles generated from single mosquito blood meals and syringe were not significantly different (p>0.05). This technique is valuable for assessing the roles of small vertebrates in the ecologies of arboviruses, and could be used in applications beyond virology and infectious diseases, when <10 µL of whole blood is required.

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.

Inactivation of infectious virus and serological detection of virus antigen in Rift Valley fever virus-exposed mosquitoes fixed with paraformaldehyde.

Formaldehyde is routinely used to fix tissues in preparation for pathology studies, however concerns remain that treatment of tissues with cellular fixatives may not entirely inactivate infectious virus particles. This concern is of particular regulatory importance for research involving viruses that are classified as select agents such as Rift Valley fever virus (RVFV). Therefore, the specific aims of this study were to (1) assay RVFV-exposed Aedes aegypti mosquitoes fixed in 4% paraformaldehyde for the presence of infectious RVFV particles at various time points following infection and (2) demonstrate the utility of immunofluorescence assay (IFA) for the detection of RVFV antigen in various tissues of paraformaldehyde-fixed mosquitoes. Mosquitoes were administered an infectious blood meal containing one of two strains of RVFV, harvested at various time points following infection, intrathoracically inoculated with 4% paraformaldehyde, and fixed overnight at 4°C. The infection status of a subset of mosquitoes was verified by IFA on leg tissues prior to fixation, and infectivity of RVFV in fixed mosquito carcasses was determined by Vero cell plaque assay. Paraformaldehyde-fixed mosquitoes harvested 14 days post infection were also paraffin-embedded and sectioned for detection of RVFV antigen to particular tissues by IFA. None of the RVFV-exposed mosquitoes tested by Vero cell plaque assay contained infectious RVFV after fixation. Furthermore, incubation of mosquito sections with trypsin prior to antibody staining is recommended for optimal visualization of RVFV antigen in infected mosquito tissues by IFA.

Host Selection of Potential West Nile Virus Vectors in Puerto Barrios, Guatemala, 2007

The selection of vertebrate hosts by Culex mosquitoes relative to West Nile virus (WNV) transmission in neotropical countries such as Guatemala is not described. This study determined the feeding patterns of Cx. quinquefasciatus and Cx. nigripalpus and estimated the relative contribution of two common and frequently infected wild bird species, Turdus grayi and Quiscalus mexicanus, to WNV transmission. Engorged mosquitoes were collected from rural and urban habitats after the dry and wet seasons in the Department of Izabal in 2007. Host selection by Cx. nigripalpus varied significantly between urban and rural habitats. Both Cx. quinquefasciatus and Cx. nigripalpus fed predominantly on chickens and other domestic animals. Blood meals from wild birds were rare, accounting for 1.1% of blood meals identified from Cx. quinquefasciatus and 6.5% of blood meals from Cx. nigripalpus. Transmission of WNV by these two mosquito species may be dampened by extensive feeding on reservoir-incompetent hosts.