Vector competence of Kenyan Culex zombaensis and Culex quinquefasciatus mosquitoes for Rift Valley fever virus.

Rift Valley fever (RVF) continues to be a significant problem in Kenya as well as in Egypt, Yemen, and Saudi Arabia. In order to determine the ability of Kenyan mosquitoes to transmit RVF virus (RVFV), we collected mosquitoes in the Lake Naivasha region of Kenya and evaluated them for their potential to transmit RVFV under laboratory conditions. After feeding on a hamster (Mesocricetus auratus) with a viremia of 10(9.7) plaque-forming units of virus/ml of blood, Culex zombaensis were highly susceptible to infection with RVFV, with 89% becoming infected. In contrast, Cx. quinquefasciatus that were fed on the same hamsters were marginally susceptible, with only 20% becoming infected. Differences in percentages of mosquitoes that developed a disseminated infection were equally disparate, with 55% and 8%, for Cx. zombaensis and Cx. quinquefasciatus, respectively. Forty-eight percent of the Cx. zombaensis with a disseminated infection that fed on a susceptible hamster transmitted virus by bite, indicating a moderate salivary gland barrier. However, the presence of a salivary gland barrier could not be determined for Cx. quinquefasciatus because none of the 18 mosquitoes that took a 2nd blood meal had a disseminated infection. These studies illustrate the need to identify the ability of individual mosquito species to transmit RVFV so that correct decisions can be made concerning the application of appropriate control measures during an outbreak.

Application of the pupal/demographic-survey methodology to identify the key container habitats of Aedes aegypti (L.) in Malindi district, Kenya.

The pupal/demographic-survey methodology was evaluated in three coastal areas (one urban, one peri-urban and one rural) of Malindi district, Kenya, in attempts to identify the types of domestic container that are most productive for Aedes aegypti (L.) pupae. The results demonstrated the practicality and consistency of the methodology, as a tool both for identifying and guiding the targeted control of the most productive container habitats, and for determining the mean numbers of pupae/person, as measures of the risk of dengue transmission.Twenty-five types of container were identified indoors and 50 types outdoors. In total, only 4,178 pupae were seen indoors and 795 outdoors. Pupal productivity was dependent on the type, location and volume of the container and the season of the year. Metallic drums and jerricans contributed >70% of the pupae encountered indoors in the wet season whereas, in the rural area, plastic drums contributed 83.7% of all the larvae seen outdoors during the dry season. Container productivity was not consistent during the different surveys. The highest mean numbers of pupae/person (7.61) and of pupae/household (18.12) were recorded in the rural area.

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.

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.

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

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.

The growing threat of arbovirus transmission and outbreaks in Kenya: a review.

OBJECTIVE: To review the trend in arbovirus outbreaks and activity in Kenya in the last ten years. DATA SOURCE: Published reports of past outbreak investigations and more recent data available at the Arbovirology and Viral haemorrhagic fevers reference centre, Centre for Virus Research, Nairobi. STUDY SELECTION: Past and recent outbreaks and active transmission reports of arboviruses of medical importance in Kenya including Yellow fever (YF), Rift Valley Fever (RVF), Dengue and Crimean Congo haemorrhagic fever. SYNTHESIS: Each of the viruses was reviewed providing critical information on classification, incidence, outbreak, and activity in Kenya, mode of transmission, recognition of cases, management and control. CONCLUSION: There is increased frequency of outbreaks and detection of arbovirus activity in humans and vectors in the last ten years including re-emergence of YF virus as a public health concern in Kenya. The importance of recognition of cases and diagnosis (especially in malaria endemic areas) is critical to management and control. Effective countrywide surveillance backed by diagnostic centres is highly recommended.

The effects of a tsetse DNA virus infection on the functions of the male accessory reproductive gland in the host fly Glossina morsitans centralis (Diptera; Glossinidae).

Freshly deposited third instar Glossina morsitans centralis larvae were infected with the tsetse DNA virus by microinjection, and at emergence adult males were separated from the females and fed on rabbit blood every second day for 8 days. A control group treated with sterile saline were handled similarly. They were dissected, and comparative observations made on the appearance and size of the accessory reproductive glands (ARG) in infected and control males. Regularly fed 8-day-old males from infected and control groups were mated to 2-day-old normal females obtained from the insectay. After separation from copula, the females were dissected and the uteri examined for the presence and quality of the spermatophore. The spermathecae were also examined for insemination. ARG tissues from the control and virus infected regularly fed 8-day-old male flies were fixed and processed for electron microscopic studies. The ARGs from control flies were found to be milky in appearance, whereas those from virus-infected flies were transparent in most parts. The ARGs from virus-infected males were significantly smaller in diameters (F = 42.26, p < 0.0001) and shorter (F = 200.4, p < 0. 0001) than those of the controls. Most of the virus-infected males failed to form a complete spermatophore, whereas almost all the controls formed complete spermatophore as observed in the uteri of the female mates (Chi2 = 111.661, p < 0.0001). The infected males that formed partial spermatophores and those that did not form any at all failed to inseminate their female mates. Histological studies of the ARGs revealed some lesions in the epithelial cells characterized by degeneration of cytoplasmic organelles and detachment of the muscle layer from the basal plasma membrane. However, no virus particles were observed in the affected cells.

The effects of a DNA virus infection on the reproductive potential of female tsetse flies, Glossina morsitans centralis and Glossina morsitans morsitans (Diptera: Glossinidae).

Reproductive anomalies associated with the tsetse DNA virus infection in the female tsetse hosts, Glossina morsitans centralis Machado and Glossina morsitans morsitans Westwood, inoculated with the virus during the 3rd instar larval stage were studied and the data compared to those obtained from the control females injected with sterile physiological saline. Virus infected flies had significantly longer first and second pregnancy cycles (P < 0.0001) and produced pupae that were of significantly less weight in milligrams (P < 0.0001) compared to controls. Transmission of the virus to progeny was not absolute and only 21% of G. m. centralis and 48% of G. m. morsitans first progeny flies from infected females developed salivary gland hypertrophy as a result of transmission from mother to progeny. The virus infected females produced significantly fewere pupae compared to the controls during the experimental period (P < 0.00001).