Rosemary Sang

Genetic and phenotypic characterization of the newly described insect flavivirus, Kamiti River virus

Summary. We have described in the accompanying paper by Sang, et al., ([57], Arch Virol 2003, in press) the isolation and identification of a new flavivirus, Kamiti River virus (KRV), from Ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in Central Province, Kenya. Among known flaviviruses, KRV was shown to be most similar to, but genetically and phenotypically distinct from, Cell fusing agent virus (CFAV). KRV was provisionally identified as an insect-only flavivirus that fails to replicate in vertebrate cells or in mice. We report here the further characterization of KRV. Growth in cell culture was compared to that of CFAV; although growth kinetics were similar, KRV did not cause the cell fusion that is characteristic of CFAV infection. The KRV genome was found to be 11,375 nucleotides in length, containing a single open reading frame encoding 10 viral proteins. Likely polyprotein cleavage sites were identified, which were most similar to those of CFAV and were comparable to those of other flaviviruses. Sequence identity with other flaviviruses was low; maximum identity was with CFAV. Possible terminal secondary structures for the 5′ and 3′ non-coding regions (NCR) were similar to those predicted for other flaviviruses. Whereas CFAV was isolated from insect cells in the laboratory, the isolation of KRV demonstrates the presence of an insect-only flavivirus in nature and raises questions regarding potential interactions between this virus and other mosquito-borne viruses in competent vector populations. Additionally, this virus will be an important tool in future studies to determine markers associated with flavivirus host specificity.

Prediction, Assessment of the Rift Valley Fever Activity in East and Southern Africa 2006-2008 and Possible Vector Control Strategies

Historical outbreaks of Rift Valley fever (RVF) since the early 1950s have been associated with cyclical patterns of the El Niño/Southern Oscillation (ENSO) phenomenon, which results in elevated and widespread rainfall over the RVF endemic areas of Africa. Using satellite measurements of global and regional elevated sea surface temperatures, elevated rainfall, and satellite derived-normalized difference vegetation index data, we predicted with lead times of 2-4 months areas where outbreaks of RVF in humans and animals were expected and occurred in the Horn of Africa, Sudan, and Southern Africa at different time periods from September 2006 to March 2008. Predictions were confirmed by entomological field investigations of virus activity and by reported cases of RVF in human and livestock populations. This represents the first series of prospective predictions of RVF outbreaks and provides a baseline for improved early warning, control, response planning, and mitigation into the future.

Rift Valley Fever Virus Epidemic in Kenya, 2006/2007: The Entomologic Investigations

In December 2006, Rift Valley fever (RVF) was diagnosed in humans in Garissa Hospital, Kenya and an outbreak reported affecting 11 districts. Entomologic surveillance was performed in four districts to determine the epidemic/epizootic vectors of RVF virus (RVFV). Approximately 297,000 mosquitoes were collected, 164,626 identified to species, 72,058 sorted into 3,003 pools and tested for RVFV by reverse transcription-polymerase chain reaction. Seventy-seven pools representing 10 species tested positive for RVFV, including Aedes mcintoshi/circumluteolus (26 pools), Aedes ochraceus (23 pools), Mansonia uniformis (15 pools); Culex poicilipes, Culex bitaeniorhynchus (3 pools each); Anopheles squamosus, Mansonia africana (2 pools each); Culex quinquefasciatus, Culex univittatus, Aedes pembaensis (1 pool each). Positive Ae. pembaensis, Cx. univittatus, and Cx. bitaeniorhynchus was a first time observation. Species composition, densities, and infection varied among districts supporting hypothesis that different mosquito species serve as epizootic/epidemic vectors of RVFV in diverse ecologies, creating a complex epidemiologic pattern in East Africa.

An Investigation of a Major Outbreak of Rift Valley Fever in Kenya: 2006–2007

An outbreak of Rift Valley fever (RVF) occurred in Kenya during November 2006 through March 2007. We characterized the magnitude of the outbreak through disease surveillance and serosurveys, and investigated contributing factors to enhance strategies for forecasting to prevent or minimize the impact of future outbreaks. Of 700 suspected cases, 392 met probable or confirmed case definitions; demographic data were available for 340 (87%), including 90 (26.4%) deaths. Male cases were more likely to die than females, Case Fatality Rate Ratio 1.8 (95% Confidence Interval [CI] 1.3-3.8). Serosurveys suggested an attack rate up to 13% of residents in heavily affected areas. Genetic sequencing showed high homology among viruses from this and earlier RVF outbreaks. Case areas were more likely than non-case areas to have soil types that retain surface moisture. The outbreak had a devastatingly high case-fatality rate for hospitalized patients. However, there were up to 180,000 infected mildly ill or asymptomatic people within highly affected areas. Soil type data may add specificity to climate-based forecasting models for RVF.

Evolution of mosquito preference for humans linked to an odorant receptor

Female mosquitoes are major vectors of human disease and the most dangerous are those that preferentially bite humans. A ‘domestic’ form of the mosquito Aedes aegypti has evolved to specialize in biting humans and is the main worldwide vector of dengue, yellow fever, and chikungunya viruses. The domestic form coexists with an ancestral, ‘forest’ form that prefers to bite non-human animals and is found along the coast of Kenya. We collected the two forms, established laboratory colonies, and document striking divergence in preference for human versus non-human animal odour. We further show that the evolution of preference for human odour in domestic mosquitoes is tightly linked to increases in the expression and ligand-sensitivity of the odorant receptor AaegOr4, which we found recognizes a compound present at high levels in human odour. Our results provide a rare example of a gene contributing to behavioural evolution and provide insight into how disease-vectoring mosquitoes came to specialize on humans.

Tracking epidemic Chikungunya virus into the Indian Ocean from East Africa

The largest documented outbreak of Chikungunya virus (CHIKV) disease occurred in the Indian Ocean islands and India during 2004-2007. The magnitude of this outbreak led to speculation that a new variant of the virus had emerged that was either more virulent or more easily transmitted by mosquito vectors. To study this assertion, it is important to know the origin of the virus and how the particular strain circulating during the outbreak is related to other known strains. This study genetically characterized isolates of CHIKV obtained from Mombasa and Lamu Island, Kenya, during 2004, as well as strains from the 2005 outbreak recorded in Comoros. The results of these analyses demonstrated that the virus responsible for the epidemic that spread through the Indian Ocean originated in coastal Kenya during 2004 and that the closest known ancestors are members of the Central/East African clade. Genetic elements that may be responsible for the scope of the outbreak were also identified.

Isolation of a new flavivirus related to Cell fusing agent virus (CFAV) from field-collected flood-water Aedes mosquitoes sampled from a dambo in central Kenya

Summary. Cell fusing agent virus (CFAV) is an RNA insect virus that was isolated from a line of Aedes aegypti mosquito cells and has been assigned to the family Flaviviridae, genus Flavivirus. We report here the first isolation of a CFA-like virus from field-collected mosquitoes. Mosquito larvae and pupae were sampled from flooded dambos in Central Province, Kenya during the short rain season of 1999. Specimens were reared to adults, identified and pooled by species and were tested for the presence of virus. Two virus isolates were obtained from two pools of Aedes macintoshi mosquitoes. The virus isolates replicated only in invertebrate cells in culture and not in vertebrate cells or in mice. The virus isolates did not antigenically cross-react with known arboviruses but were identified to family by reverse-transcriptase polymerase chain reaction (RT-PCR) performed using primers specific to alphaviruses, bunyaviruses and flaviviruses; only the flavivirus-specific primers produced a DNA fragment of the expected size. Nucleic acid sequencing of this fragment showed the two isolates to be nearly identical. Comparison of sequences to the GenBank database using BLAST identified the virus as most closely related to CFAV. Results from cross-neutralization tests suggested that, although the BLAST search indicated homology to CFAV, the virus isolated represented a new insect flavivirus. Detailed characterization of this new virus, described in Crabtree et al. [7], further supports this finding. We propose this new flavivirus be designated Kamiti River virus (KRV). This is the first isolation of a CFA-like virus from field-collected mosquitoes and indicates the presence of this group of viruses in nature.

The 2007 rift valley Fever outbreak in Sudan

Rift Valley fever (RVF) is a neglected, emerging, mosquito-borne disease with severe negative impact on human and animal health and economy. RVF is caused by RVF virus (RVFV) affecting humans and a wide range of animals. The virus is transmitted through bites from mosquitoes and exposure to viremic blood, body fluids, or tissues of infected animals. During 2007 a large RVF outbreak occurred in Sudan with a total of 747 confirmed human cases including 230 deaths (case fatality 30.8%); although it has been estimated 75,000 were infected. It was most severe in White Nile, El Gezira, and Sennar states near to the White Nile and the Blue Nile Rivers. Notably, RVF was not demonstrated in livestock until after the human cases appeared and unfortunately, there are no records or reports of the number of affected animals or deaths. Ideally, animals should serve as sentinels to prevent loss of human life, but the situation here was reversed. Animal contact seemed to be the most dominant risk factor followed by animal products and mosquito bites. The Sudan outbreak followed an unusually heavy rainfall in the country with severe flooding and previous studies on RVF in Sudan suggest that RVFV is endemic in parts of Sudan. An RVF outbreak results in human disease, but also large economic loss with an impact beyond the immediate influence on the directly affected agricultural producers. The outbreak emphasizes the need for collaboration between veterinary and health authorities, entomologists, environmental specialists, and biologists, as the best strategy towards the prevention and control of RVF.

Laboratory diagnosis of Ebola hemorrhagic fever during an outbreak in Yambio, Sudan, 2004.

Between the months of April and June 2004, an Ebola hemorrhagic fever (EHF) outbreak was reported in Yambio county, southern Sudan. Blood samples were collected from a total of 36 patients with suspected EHF and were tested by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G and M antibodies, antigen ELISA, and reverse-transcription polymerase chain reaction (PCR) of a segment of the Ebolavirus (EBOV) polymerase gene. A total of 13 patients were confirmed to be infected with EBOV. In addition, 4 fatal cases were classified as probable cases, because no samples were collected. Another 12 patients were confirmed to have acute measles infection during the same period that EBOV was circulating. Genetic analysis of PCR-positive samples indicated that the virus was similar to but distinct from Sudan EBOV Maleo 1979. In response, case management, social mobilization, and follow-up of contacts were set up as means of surveillance. The outbreak was declared to be over on 7 August 2004.

Infection and Vertical Transmission of Kamiti River Virus in Laboratory Bred Aedes aegypti Mosquitoes

Abstract Kamiti river virus (KRV) is an insect-only Flavivirus that was isolated from field-collected Ae. macintoshi mosquitoes in 1999, and is closely related to cell fusing agent virus. Both of these viruses belong to the family Flaviviridae, which also contains other viruses of medical importance, such as yellow fever virus, West Nile virus and dengue. Because Ae. macintoshi is the only known natural host to KRV, the main objective of this study was to establish the possibility that other mosquito hosts of the virus exist, by determining its ability to infect Ae. aegypti mosquitoes under laboratory conditions. The study also sought to determine the rates of infection and, subsequently, vertical transmission as a possible means of its maintenance and propagation in nature, given that it neither grows in vertebrate cells or mice. The mosquitoes were infected by the virus either as larvae or adults. Virus assay was done by re-isolation in tissue culture and indirect immunofluoresce assay methods. KRV infected Ae. aegypti mosquitoes, with the observed rates as high as 74 to 96 %. The virus was also transmitted vertically in these mosquitoes. Vertical transmission rates of 3.90 % were observed for the 2nd and 3rd ovarian cycles combined. These results suggest that Ae. aegypti mosquitoes are likely to be infected with KRV in nature, and that vertical transmission is the natural means by which it is maintained and propagated in this host, and possibly others.