Potential for populations of Aedes j. japonicus to transmit Rift Valley fever virus in the USA.

Aedes japonicus japonicus was introduced into the northeastern USA in 1998 and has since spread to more than 25 states. Because this species has been shown to be a competent laboratory vector of several viruses, readily feeds on large mammals, and has become a pest in several areas, there is concern that it might serve as a vector of Rift Valley fever virus (RVFV) should that virus be introduced into North America. Infection with RVFV causes mortality in > 90% of young domestic ungulates (e.g., calves, kids, and lambs), as well as causing a febrile illness and occasional deaths in humans. Therefore, we evaluated Ae. j. japonicus captured in North Carolina and in Maryland for their ability to serve as potential vectors for RVFV. After feeding on infected adult hamsters, these mosquitoes were tested for infection, dissemination, and the ability to transmit RVFV after incubation at 26 degrees C for 7-28 days. Both the Maryland and North Carolina populations of Ae. j. japonicus were highly efficient laboratory vectors of RVFV, with infection rates > 90% and dissemination rates > 84% for those mosquitoes that fed on hamsters with viremias > or = 10(8.5) plaque-forming units/ml. Thus, Ae. j. japonicus should be targeted for immediate control should RVFV be introduced into an area where this mosquito is now present.

Potential for Canadian mosquitoes to transmit Rift Valley fever virus.

The rapid spread of West Nile viral activity across North America since its discovery in 1999 illustrates the potential for an exotic arbovirus to be introduced and become widely established across North America. Rift Valley fever virus (RVFV) has been responsible for large outbreaks in Africa that have resulted in hundreds of thousands of human infections and major economic disruption due to loss of livestock and to trade restrictions. However, little is known about the potential for mosquitoes in Canada to transmit this virus, should it be introduced into North America. Therefore, we evaluated mosquito species captured near Winnipeg, Manitoba, Canada, for their ability to serve as potential vectors for RVFV. Mosquitoes were exposed to RVFV by allowing them to feed on adult hamsters inoculated the previous day with RVFV. These mosquitoes were tested for infection, dissemination, and the ability to transmit RVFV after incubation at 25 degrees C for 14-18 days. Based on the detection of virus in saliva collected in capillary tubes, individual Culex tarsalis, Aedes sticticus, and Coquillettidia perturbans were able to transmit RVFV under laboratory conditions. These preliminary results suggest that these 3 species may be able to transmit RVFV, should this virus be introduced into Canada.

Vector Competence of Peruvian Mosquitoes for Two Orthobunyaviruses Isolated From Mosquitoes Captured in Peru.

We evaluated the potential for mosquitoes collected in the Amazon Basin, near Iquitos, Peru, to become infected with and transmit Murutucu (MURV) and Itaqui viruses (ITQV) (Order Bunyavirales, Family: Peribunyaviridae, Genus: Orthobunyavirus). Viremia levels in Syrian hamsters peaked 2 d after infection with either virus, and both viruses were highly lethal in hamsters with virtually all hamsters dying prior to 3-d postinfection. For almost all of the mosquito species tested some individuals were susceptible to infection and some developed a disseminated infection after oral exposure to either MURV or ITQV. However, only the Culex species (Culex (Culex) coronator Dyar and Knab [Diptera, Culicidae], Culex (Melanoconian) gnomatos Sallum, Huchings, and Ferreira [Diptera, Culicidae], Culex (Mel.) pedroi Sirivanakarn and Belkin [Diptera, Culicidae], and Culex (Mel.) vomerifer Komp [Diptera, Culicidae]) successfully transmitted virus by bite. However, even among these species, only about 37% of the individuals with a disseminated infection successfully transmitted these viruses, indicating a significant salivary gland barrier. Although little is known about the medical or veterinary importance of many members of the genus Orthobunyavirus, we have demonstrated that Culex spp. (Diptera, Culicidae) could be potential vectors.

Carbon dioxide sensitivity of mosquitoes infected with California encephalitis virus.

Four species of mosquitoes became sensitive to carbon dioxide approximately 3 to 4 days after they received intrathoracic injectins of California encephalitis virus. Aedes melanimon and Aedes dorsalis infected orally with California encephalitis virus also became carbon dioxide-sensitive, but mosquitoes infected transovarially did not. Sensitivity to carbon dioxide was inhibited by antiserum to California encephalitis virus. To our knowledge this is the first report of carbon dioxide sensitivity induced in arthropods by a bunyavirus and the first demonstration of this phenomenon by an arbovirus in its proven vector.

Enhanced arboviral transmission by mosquitoes that concurrently ingested microfilariae.

Infection, dissemination, and transmission of an arbovirus in mosquitoes are enhanced by concurrent ingestion of microfilariae. Ingestion of Rift Valley fever virus alone infected only 64 percent of female Aedes taeniorhynchus. Of these, only 5 percent of refeeding mosquitoes actually transmitted virus. In contrast, ingestion of the same amount of virus from concurrently microfilaremic (Brugia malayi) gerbils resulted in 88 percent infection and 31 percent transmission. Enhanced transmission of virus may be attributed to increased transit of virus across the midgut wall. Endemic filariasis may promote arbovirus transmission in nature.

Susceptibility of Peruvian mosquitoes to eastern equine encephalitis virus.

Mosquitoes were collected in the Amazon Basin, near Iquitos, Peru, and used in experimental studies to evaluate their susceptibility to strains of eastern equine encephalitis virus (EEEV) that were isolated from mosquitoes captured within 20 km of Iquitos. When fed on hamsters or chickens with a viremia of 4105 plaque-forming units (PFU) of EEEV/ml, Culex pedroi Sirivanakarn and Belkin, Aedesfulvus (Wiedemann), Psorophora albigenu (Peryassu), and Psorophoraferox (Von Humboldt) were susceptible to infection, whereas none of the Aedes serratus (Theobald), Culex vomerifer Komp, Culex gnomatos Sallum, Huchings, and Ferreira, Culex portesi Senevet and Abonnenc, or Culex coronator Dyar and Knab became infected, even though they fed on the same viremic blood sources. When these mosquito species fed on animals with viremias of approximately 10(8) PFU/ml, Cx. pedroi, Ae.II (Brazil-Peru) and a lineage III (Argentina-Panama) isolate of EEEV. This study, combined with the repeated isolation of strains of EEEV from Cx. pedroi captured in the Amazon Basin region of Peru, suggests that Cx. pedroi may be the primary enzootic vector of EEEV in this region.

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.

Laboratory transmission of Japanese encephalitis and West Nile viruses by molestus form of Culex pipiens (Diptera: Culicidae) collected in Uzbekistan in 2004.

We evaluated the molestus form of Culex pipiens pipiens (L.) (hereafter referred to as "molestus") captured near Tashkent, Uzbekistan, for their ability to transmit Japanese encephalitis (family Flaviviridae, genus Flavivirus, JEV) and West Nile (family Flaviviridae, genus Flavivirus, WNV) viruses under laboratory conditions. These molestus were highly competent laboratory vectors of WNV, with infection and dissemination rates of 96 and 81%, respectively. Approximately 75% of female molestus that fed after development of a disseminated infection transmitted virus by bite. Therefore, approximately 60% of those molestus taking a second bloodmeal between 16 and 25 d after an infectious bloodmeal would be expected to transmit WNV by bite. In contrast, these molestus were less efficient vectors of JEV, with infection and dissemination rates of 51 and 25%, respectively. In addition, only 33% of individuals with a disseminated infection transmitted JEV by bite, indicating a significant salivary gland barrier. Therefore, only approximately 8% of orally exposed individuals would be expected to transmit JEV by bite if they took a second bloodmeal 16-25 d later. These data indicate that the molestus form of Cx. p. pipiens should be considered a potentially important vector of WNV in Uzbekistan and may become involved in the transmission of JEV, should this virus be introduced into Uzbekistan.

Vector competence of Peruvian mosquitoes (Diptera: Culicidae) for a subtype IIIC virus in the Venezuelan equine encephalomyelitis complex isolated from mosquitoes captured in Peru.

We evaluated mosquitoes collected in the Amazon Basin, near Iquitos, Peru, for their susceptibility to a subtype IIIC strain of the Venezuelan equine encephalomyelitis complex. This virus had been previously isolated from a pool of mixed Culex vomerifer and Cx. gnomatos captured near Iquitos, Peru, in 1997. After feeding on hamsters with viremias of about 10(8) plaque-forming units of virus per ml, Cx. gnomatos was the most efficient vector. Other species, such as Ochlerotatus fulvus and Psorophora cingulata, although highly susceptible to infection, were not efficient laboratory vectors of this virus due to a significant salivary gland barrier. The Cx. (Culex) species, consisting mostly of Cx. (Cux.) coronator, were nearly refractory to subtype IIIC virus and exhibited both midgut infection as well as salivary gland barriers. Additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of subtype IIIC virus are needed to determine the role that these species play in the maintenance and spread of this virus in the Amazon Basin region.

Pathogenesis of Rift Valley fever virus in mosquitoes--tracheal conduits & the basal lamina as an extra-cellular barrier.

Knowledge of the fate of an arbovirus in a mosquito is fundamental to understanding the mosquito's competence to transmit the virus. When a competent mosquito ingests viremic vertebrate blood, virus infects midgut epithelial cells and replicates, then disseminates to other tissues, including salivary glands and/or ovaries. The virus is then transmitted to the next vertebrate host horizontally via bite and/or vertically to the mosquito's offspring. Not all mosquitoes that ingest virus become infected or, if infected, transmit virus. Several "barriers" to arbovirus passage, and ultimately transmission, have been identified in incompetent or partially competent mosquitoes, including, among others, gut escape barriers and salivary gland infection barriers. The extra-cellular basal lamina around the midgut epithelium and the basal lamina that surrounds the salivary glands may act as such barriers. Midgut basal lamina pore sizes are significantly smaller than arboviruses and ultrastructural evidence suggests that midgut tracheae and tracheoles may provide a means for viruses to circumvent this barrier. Further, immunocytochemical evidence indicates the existence of a salivary gland infection barrier in Anopheles stephensi. The basal lamina may prevent access to mosquito cell surface virus receptors and help explain why anopheline mosquitoes are relatively incompetent arbovirus transmitters when compared to culicines.

Isolation of viruses from mosquitoes (Diptera: Culicidae) collected in the Amazon Basin region of Peru.

As part of a comprehensive study on the ecology of arthropod-borne viruses in the Amazon Basin region of Peru, we assayed 539,694 mosquitoes captured in Loreto Department, Peru, for arboviruses. Mosquitoes were captured either by dry ice-baited miniature light traps or with aspirators while mosquitoes were landing on human collectors, identified to species, and later tested on Vero cells for virus. In total, 164 virus isolations were made and included members of the Alphavirus (eastern equine encephalomyelitis, Trocara, Una, Venezuelan equine encephalomyelitis, and western equine encephalomyelitis viruses), Flavivirus (Ilheus and St. Louis encephalitis), and Orthobunyavirus (Caraparu, Itaqui, Mirim, Murutucu, and Wyeomyia viruses) genera. In addition, several viruses distinct from the above-mentioned genera were identified to the serogroup level. Eastern equine encephalomyelitis virus was associated primarily with Culex pedroi Sirivanakarn & Belkin, whereas Venezuelan equine encephalomyelitis virus was associated primarily with Culex gnomatos Sallum, Huchings & Ferreira. Most isolations of Ilheus virus were made from Psorophora ferox (Von Humboldt). Although species of the Culex subgenus Melanoconion accounted for only 45% of the mosquitoes collected, 85% of the virus isolations were made from this subgenus. Knowledge of the viruses that are being transmitted in the Amazon Basin region of Peru will enable the development of more effective diagnostic assays, more efficient and rapid diagnoses of clinical illnesses caused by these pathogens, risk analysis for military/civilian operations, and development of potential disease control measures.

Potential North American vectors of West Nile virus.

The outbreak of disease in the New York area in 1999 due to West Nile (WN) virus was the first evidence of the occurrence of this virus in the Americas. To determine potential vectors, more than 15 mosquito species (including Culex pipiens, Cx. nigripalpus, Cx. quinquefasciatus, Cx. salinarius, Aedes albopictus, Ae. vexans, Ochlerotatus japonicus, Oc. sollicitans, Oc. taeniorhynchus, and Oc. triseriatus) from the eastern United States were evaluated for their ability to serve as vectors for the virus isolated from birds collected during the 1999 outbreak in New York. Mosquitoes were allowed to feed on one- to four-day old chickens that had been inoculated with WN virus 1-3 days previously. The mosquitoes were incubated for 12-15 days at 26 degrees C and then allowed to refeed on susceptible chickens and assayed to determine transmission and infection rates. Several container-breeding species (e.g., Ae. albopictus, Oc. atropalpus, and Oc. japonicus) were highly efficient laboratory vectors of WN virus. The Culex species were intermediate in their susceptibility. However, if a disseminated infection developed, all species were able to transmit WN virus by bite. Factors such as population density, feeding preference, longevity, and season of activity also need to be considered in determining the role these species could play in the transmission of WN virus.

Reduced Rift Valley fever virus infection rates in mosquitoes associated with pledget feedings.

Infection rates were compared in Culex pipiens and Aedes taeniorhynchus after they fed on Rift Valley fever (RVF) viremic hamsters or ingested similar doses of RVF virus from blood-soaked pledgets. Infection rates were significantly lower for mosquitoes that ingested virus from a pledget than for those that ingested similar doses from viremic hamsters. The method used to prevent normal clot formation for the pledget feedings (i.e., defibrination by shaking with glass beads or addition of heparin) did not affect subsequent infection rates. Both inhibition of normal clot formation and freezing of virus after it had last been propagated were associated with significantly reduced infection rates with the pledget feedings. Laboratory studies using artificial feeding techniques may not give reliable estimates of the vector competence of mosquitoes for arboviruses.

Effect of environmental temperature on the vector competence of Aedes taeniorhynchus for Rift Valley fever and Venezuelan equine encephalitis viruses.

Studies were conducted to determine the effect of environmental temperature on the susceptibility of Aedes taeniorhynchus mosquitoes for Venezuelan equine encephalitis (VEE) and Rift Valley fever (RVF) viruses. Mosquitoes reared at low temperature (19 degrees C) were significantly more susceptible to infection with RVF virus (infection rate = 70%, 158 of 226) than were those mosquitoes reared at 26 degrees C (infection rate = 48%, 135 of 280), regardless of the temperature at which mosquitoes were held after exposure to virus (19 or 26 degrees C). Likewise, for Ae. taeniorhynchus exposed to VEE virus, the infection rate in mosquitoes reared at low temperature (97%, 236 of 243) was significantly greater than was that for mosquitoes reared at 26 degrees C (78%, 217 of 280), regardless of the temperature at which mosquitoes were held after exposure to virus (19 or 26 degrees C). Conversely, in mosquitoes infected with either RVF or VEE virus, virus disseminated from the midgut to the hemocoel more rapidly in mosquitoes held at 26 degrees C than in those held at 19 degrees C, regardless of the rearing temperature. Thus, a combination of low larval-rearing temperature and warm adult-holding temperature resulted in the most efficient mosquito transmission of both viruses.

Effect of environmental temperature on the vector competence of Aedes aegypti and Ae. taeniorhynchus for Ockelbo virus.

Studies were conducted to determine the effect of environmental temperature on the ability of Aedes aegypti and Ae. taeniorhynchus to transmit Ockelbo (OCK) virus. Temperatures tested were 10 degrees C, 17 degrees C, 24 degrees C, and a cyclic (10-24 degrees C, mean = 17 degrees C) regimen designed to mimic continuously temperatures to which a mosquito might be exposed in July and August in central Sweden. Both species were highly susceptible to oral infection, with no consistent association between incubation temperature and infection rates. However, dissemination of OCK virus to the hemocoel in infected mosquitoes was directly related both to the dose of virus ingested and to the extrinsic incubation temperature. After greater than or equal to 14 days extrinsic incubation, once a disseminated infection was achieved, transmission by bite to a susceptible chick did not appear to be affected by either the initial dose ingested, the holding temperatures of 17 degrees C, 24 degrees C, nor the cyclic regimen. When re-fed, 93% (68/73) and 82% (67/82) of the disseminated Ae. taeniorhynchus and Ae. aegypti, respectively, transmitted virus. In contrast, when mosquitoes were held at 10 degrees C, fewer disseminated Ae. taeniorhynchus (0/5) and Ae. aegypti (2/6) transmitted virus. There were no significant differences in infection, dissemination, or transmission rates for Ae. taeniorhynchus held at the cyclic temperature regimen vs. those held at a constant 17 degrees C. Environmental temperature affected the vectorial capacity of Ae. aegypti and Ae. taeniorhynchus for OCK virus, with transmission occurring earlier and at a higher rate in mosquitoes held at higher temperatures.

Nonvascular delivery of Rift Valley fever virus by infected mosquitoes.

To determine whether virus-transmitting mosquitoes inoculate infectious particles extravascularly or directly into the vascular system, we permitted mosquitoes infected with Rift Valley fever virus to feed on the distal third of the tails of suckling mice. Amputation of the distal half of the tail within 5 min after their being bitten significantly increased mouse survival as compared with that of mice whose tails remained intact. Even when tails were amputated 10 or more min after mosquito feeding, the median time to death was significantly longer in the group with the amputated tails (53.5 hr) than in those mice with intact tails (46.0 hr). Mouse survival did not correlate with ingestion of blood by the infecting mosquito. We conclude that mosquitoes inoculate virus extravascularly, rather than directly into the vascular system, when feeding on a vertebrate host. Such extravascular delivery of virus by a transmitting mosquito may affect viral pathogenesis, antiviral activity, and vaccine efficacy.

Effect of the developmental stage at infection on the ability of adult Anopheles stephensi to transmit Rift Valley fever virus.

The ability of adult Anopheles stephensi to transmit Rift Valley fever virus was determined for mosquitoes inoculated at selected times during development. None of 109 female An. stephensi inoculated as adults transmitted virus to hamsters. In contrast, 83% (50 of 60) of those inoculated as larvae transmitted virus by bite to hamsters. Transmission rates decreased as the stage of the mosquito at the time of inoculation changed from larva to pupa to adult. Transmission rates for adult mosquitoes inoculated as larvae, as pupae < 4 hr after pupation, as pupae > 24 hr after pupation, or as adults were 83%, 25%, 11%, and 0%, respectively. Viral titers recovered from mosquitoes were similar for all groups tested, regardless of stage at infection (larva, pupa, or adult) or of transmission status (transmitter or nontransmitter). Thus, differences in transmission rates may have been due to site-specific (i.e., salivary gland) replication, rather than a generalized increase in viral replication in mosquitoes inoculated at an earlier age.

Dual host infections: enhanced infectivity of eastern equine encephalitis virus to Aedes mosquitoes mediated by Brugia microfilariae.

When mosquitoes feed on a vertebrate host that is infected concurrently with virus and microfilariae (mf), both pathogens are ingested. If mf penetrate the mosquito midgut, a small portion of the ingested virus may disseminate directly into the mosquito hemocoel. This phenomenon, termed microfilarial enhancement of arboviral transmission, has the potential to enhance the infectivity of arboviruses to mosquitoes. We investigated whether concurrent ingestion of Brugia mf and eastern equine encephalitis virus would enhance the infectivity and subsequent transmissibility of the virus by Aedes mosquitoes. Trials with Ae. triseriatus and B. pahangi mf indicated that microfilarial enhancement was dose dependent. Both a sufficient number of penetrating mf and a sufficient viremia were required for enhancement to occur. Furthermore, studies with B. malayi and three species of Aedes indicated that under comparable conditions of host viremia and microfilaremia, microfilarial enhancement occurred in some mosquito species (i.e., Ae. aegypti and Ae. taeniorhynchus) but not in others (Ae. triseriatus). We suggest that certain key parameters determine whether dual virus/mf host infections will enhance arboviral infectivity to mosquitoes. These include species differences in the capacity of mf to penetrate the mosquito midgut, the amount of virus passing into the hemocoel during mf penetration, and the innate susceptibility of mosquitoes to hemocoelomically introduced virus.

Transmission of Rift Valley fever virus by the sand fly, Phlebotomus duboscqi (Diptera: Psychodidae).

Studies were conducted to determine if the sand fly Phlebotomus duboscqi could serve as a vector of Rift Valley fever (RVF) virus. When 145 P. duboscqi were fed on a hamster with RVF viremia (approximately 10(9) PFU/ml of blood), 72 (50%) became infected. Of 5 with disseminated infections (i.e., virus recovered from their legs) 4 transmitted virus to hamsters by bite. Sand flies were uniformly infected when RVF virus was inoculated by the intrathoracic route, and each of 31 sand flies so inoculated that fed on a hamster transmitted virus. None of 331 progeny of inoculated sand flies or 230 progeny of orally exposed sand flies contained virus. Sand flies could serve as vectors of RVF virus.

Potential for mosquito transmission of attenuated strains of Rift Valley fever virus.

Studies were conducted to determine if two attenuated strains of Rift Valley fever (RVF) virus could be transmitted by Culex pipiens mosquitoes. Both strains (RVF MP 12 and T1) replicated in and were transmitted by female Cx. pipiens after intrathoracic inoculation. Mosquitoes also became infected with and transmitted the RVF MP12 strain after ingesting virus from a blood-soaked cotton pledget. However, because of the low viremias produced in infected animals, it is unlikely that mosquitoes would become infected by feeding on an animal inoculated with either of these viruses. Although both strains were transmitted by mosquitoes after intrathoracic inoculation, there was no evidence of reversion to a virulent virus.