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.

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.

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.

Rift Valley fever virus in the cardia of Culex pipiens: an immunocytochemical and ultrastructural study.

In this paper, we consider the movement of Rift Valley fever (RVF) virus from infected mosquito midgut epithelial cells into the hemocoel as an important factor in the ultimate ability of the insect to transmit the virus. Our results are therefore significant in the context of vector competence. The mosquito Culex pipiens was identified as the primary vector of RVF in an epidemic that occurred in Egypt in the 1970s. On this basis, we have carried out several studies of RVF virus in this mosquito species. In the research reported here, we used immunocytochemical and transmission electron microscopic techniques to study the occurrence of RVF virus in the mosquito cardia and aspects of the histology and ultrastructure of this organ. The cardia is a complex organ consisting of both foregut and midgut tissue and is the location of the foregut-midgut junction. The cardia is of interest because it appears to provide routes of RVF virus egress from the midgut lumen and it is consistently infected in mosquitoes with disseminated infections, making it a potentially important site of viral amplification and an ideal site for studying RVF viral morphogenesis. In orally infected mosquitoes, large numbers of RVF virions were observed budding into the basal labyrinth associated with the outer cardial epithelial cells and into the noncellular matrix associated with the inner cardial epithelial cells and the cells of the intussuscepted foregut. In mosquitoes infected by injection of virus into the hemocoel and then held for different incubation periods, viral antigen was first detected in the cells of the intussuscepted foregut in the cardia and later in the cardial epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Eastern equine encephalitis virus in birds: relative competence of European starlings (Sturnus vulgaris).

To determine whether eastern equine encephalitis (EEE) virus infection in starlings may be more fulminant than in various native candidate reservoir birds, we compared their respective intensities and durations of viremia. Viremias are more intense and longer lasting in starlings than in robins and other birds. Starlings frequently die as their viremia begins to wane; other birds generally survive. Various Aedes as well as Culiseta melanura mosquitoes can acquire EEE viral infection from infected starlings under laboratory conditions. The reservoir competence of a bird is described as the product of infectiousness (proportion of feeding mosquitoes that become infected) and the duration of infectious viremia. Although starlings are not originally native where EEE is enzootic, a starling can infect about three times as many mosquitoes as can a robin.

Vector potential of selected North American mosquito species for Rift Valley fever virus.

Selected North American mosquito species were evaluated as potential vectors of Rift Valley fever virus. Field populations of Aedes canadensis, Ae. cantator, Ae. excrucians, Ae. sollicitans, Ae. taeniorhynchus, Ae. triseriatus, Anopheles bradleyi-crucians, Culex salinarius, Cx. tarsalis, and Cx. territans perorally exposed to 10(6.2)-10(7.2) plaque forming units of Rift Valley fever virus readily became infected. Infection rates ranged from 51% (65/127) for Cx. salinarius to 96% (64/67) for Ae. canadensis. Disseminated infection rates were generally greater at 14 days than at 7 days after the infectious bloodmeal, and, with the exception of An. bradleyi-crucians, they were not significantly different than the pooled rate of 59% for each species tested. Only 5/55 (9%) of the An. bradleyi-crucians developed a disseminated infection. For most of the species, about half of the mosquitoes with a disseminated infection transmitted an infectious dose of virus to hamsters. While all species, with the exception of An. bradleyi-crucians, transmitted virus, Ae. canadensis, Ae. taeniorhynchus, and Cx. tarsalis had the highest vector potential of the species tested. Following inoculation of approximately 10(1.6) plaque forming units of virus, 100% of the mosquitoes of each species became infected. For most species, transmission rates were similar for inoculated individuals and those that developed a disseminated infection following peroral infection. Viral titers of transmitting and nontransmitting-disseminated individuals were similar for all species tested. These data suggest that, if Rift Valley fever virus was introduced into North America, several mosquito species would be capable of transmitting it.

Experimental infection of six species of ixodid ticks with Dugbe virus (family Bunyaviridae, genus Nairovirus).

The vector potential of each of 6 species of colonized North American and African ixodid ticks was assessed by intracoelomic inoculation with Dugbe virus (IbAr 1792, 14th passage in suckling mouse brain) and viral titers were monitored after selected incubation periods. Persistence of Dugbe virus for greater than or equal to 53 days in 5 species (Dermacentor andersoni, D. variabilis, Amblyomma americanum, Rhipicephalus appendiculatus, and R. sanguineus) indicates that infection occurred. Viral titers were significantly higher in female vs. male D. variabilis, R. appendiculatus, and A. americanum after blood feeding. Blood feeding had no significant effect on the viral titers of either female or male R. sanguineus. D. andersoni males also exhibited no significant change in viral titers after blood-feeding, but 100% (20/20) of drop-off females and 96% (24/25) of post-oviposition females (36 days postinoculation) contained no detectable virus even though virus was still found in unfed specimens less than or equal to 124 days postinoculation. Virus was not recovered from greater than 30,000 1st generation progeny (eggs, larvae, nymphs, adults) collected as eggs from inoculated female D. andersoni, D. variabilis, R. sanguineus, and R. appendiculatus 27-51 days postinoculation. R. sanguineus and R. appendiculatus transmitted Dugbe virus to guinea pigs when allowed to feed 1-3 weeks postinoculation.

Transstadial and horizontal transmission of Rift Valley fever virus in Hyalomma truncatum.

We exposed Hyalomma truncatum and Rhipicephalus appendiculatus to Rift Valley fever (RVF) virus in order to assess the possible role of these ticks as enzootic/epizootic RVF vectors. The virus replicated in H. truncatum after intracoelomic inoculation, and a minimum transmission rate of 17% was achieved after 15 days intrinsic incubation. The virus persisted at least 58 days in these ticks. Virus was also shown to pass transstadially from inoculated H. truncatum nymphs to adults, with peak viral titers reaching 10(3.5) plaque-forming units (PFU) in adult males after they were provided with bloodmeals. Virus was recovered from adult females 121 days after they were inoculated as nymphs. Viral titers peaked in inoculated male ticks after dropping off a host (mean titer = 10(4.3) PFU). RVF virus was not detected in pools of eggs and larval progeny from 11 infected female H. truncatum. H. truncatum larvae and nymphs did not become infected after ingesting greater than 10(2.0) PFU while feeding on a RVF viremic hamster. The number of infected specimens declined rapidly after RVF virus was inoculated into R. appendiculatus adults, and virus was undetectable 12 days post-inoculation.

Arbovirus isolations from mosquitoes collected during 1988 in the Senegal River basin.

During August and September 1988, we collected adult mosquitoes from 14 locations in the Senegal River basin to search for evidence of Rift Valley fever (RVF) viral activity one year after the 1987 outbreak, which occurred along the Senegal-Mauritania border. More than 62,000 specimens representing 18 species in seven genera were collected with carbon dioxide-baited, solid-state Army miniature light traps and sheep-baited traps. Twenty virus isolations from Culex, Aedes, and Anopheles mosquitoes were recovered from six locations: Fanaye Diery (11), Bode (four), Matam (two), Diongui (one), Ndialene (one), and Ngoui (one). Species yielding viral isolates were Anopheles pharoensis (eight), Culex tritaeniorhynchus (three), Cx. univitattus gr. (three), Cx. antennatus (two), Cx. poicillipes (two), Ae. hirsutus (one), and An. gambiae (one). Viruses were identified by complement fixation, and virus and plaque-reduction neutralization testing as Ngari (Bunyavirus, Bunyaviridae) (n = 15), Babanki (Alphavirus, Togaviridae) (n = 3), Bagaza (Flavivirus, Flaviviridae) (n = 1), and Bangui (Bunyavirus-like) (n = 1). No evidence of any RVF viral activity in the Senegal River Basin was detected in the mosquitoes tested.

Vector competence of Egyptian mosquitoes for Rift Valley fever virus.

Reintroduction of Rift Valley fever (RVF) into Egypt in 1993 raised concerns about the potential for Egyptian mosquitoes to transmit the virus. We evaluated the ability of Aedes caspius, Culex pipiens, Cx. antennatus, Cx. perexiguus, Cx. poicilipes, and Anopheles pharoensis collected in the Aswan area and Cx. pipiens collected in the Nile Delta to transmit RVF virus. All mosquito species tested were susceptible to RVF virus infection, with An. pharoensis and Ae. caspius being the most sensitive to infection. However, none of 12 An. pharoensis, including 10 with a disseminated infection, transmitted RVF virus by bite. In contrast, nearly all Cx. pipiens (87%, n = 15) and Cx. perexiguus (90%, n = 10) with a disseminated infection transmitted virus. Overall transmission rates for mosquitoes exposed to hamsters with a viremia > or = 10(7) plaque-forming units/ml were Ae. caspius, 20% (n = 5); Cx. pipiens, 7% (n = 102); Cx. antennatus, 7% (n = 30); Cx. perexiguus, 11% (n = 9); and An. pharoensis, 0% (n = 7). Based on abundance, susceptibility to infection, ability to transmit virus, and feeding behavior, Ae. caspius appeared to be the most efficient vector of the Egyptian mosquitoes evaluated. While less susceptible than Ae. caspius, Cx. pipiens, Cx. antennatus, and Cx. perexiguus were also potential vectors during this RVF outbreak in Egypt.

Trocara virus: a newly recognized Alphavirus (Togaviridae) isolated from mosquitoes in the Amazon Basin.

This report describes Trocara virus, a newly recognized member of the genus Alphavirus, that has been isolated from Aedes serratus mosquitoes collected at two widely separated sites in the Amazon Basin. Biological, antigenic and genetic characteristics of the new virus are given. Results of these studies indicate that Trocara virus is the first member of a newly discovered antigenic complex within the family Togaviridae genus Alphavirus. The public health and veterinary importance of Trocara virus is still unknown.

Effect of incubation at overwintering temperatures on the replication of West Nile Virus in New York Culex pipiens (Diptera: Culicidae).

We examined the effect of simulated overwintering temperatures on West Nile (WN) virus replication in Culex pipiens L. derived from mosquitoes collected during the autumn 1999 WN epizootic in New York. The WN virus was a strain isolated from a dead crow also collected during this outbreak. Virus was recovered from most mosquitoes held exclusively at 26 degres C. In contrast, none of the mosquitoes held exclusively at the lower temperatures had detectable infections. When mosquitoes were transferred to 26 degrees C after being held at 10 degrees C for 21-42 d, infection and dissemination rates increased with increased incubation at 26 degrees C. Future studies involving the attempted isolation of WN virus from overwintering mosquitoes may benefit from holding the mosquitoes at 26 degrees C before testing for infectious virus.

Effects of temperature on fecundity and viral replication in Amblyomma cajennense (Arachnida: Ixodidae) infected with Venezuelan equine encephalomyelitis virus.

To assess how temperature affects fecundity and Venezuelan equine encephalomyelitis (VEE) viral replication in VEE-infected Amblyomma cajennense (F.), adult ticks were inoculated intracoelomically with either VEE virus (I-A variant) or diluent. Ticks were held at 19, 26, or 33 degrees C and tested for VEE virus over 20 d. Up to 10 pairs of ticks (1 female:1 male) from each temperature group were allowed to feed on guinea pigs on day 8 after inoculation. Maximum titers did not differ significantly among infected ticks held at 19, 26, or 330 degrees C (P = 0.59), and ticks maintained high viral titers throughout the sampling period. Likewise, there was no significant difference in the number of eggs oviposited between ticks inoculated with virus or diluent and held at the same temperature, or among groups held at different temperatures (P = 0.48). The percentage of viable eggs from virus-inoculated or diluent-inoculated adults also was not significantly different (P > or = 0.14).

Laboratory transmission of Rift Valley fever virus by Phlebotomus duboscqi, Phlebotomus papatasi, Phlebotomus sergenti, and Sergentomyia schwetzi (Diptera: Psychodidae).

We examined the potential for Phlebotomus papatasi (Scopoli), Phlebotomus duboscqi (Neveu-Lemarie), Phlebotomus sergenti (Parrot), and Sergentomyia schwetzi (Adler, Theodor, & Parrot) to transmit Rift Valley fever (RVF) virus. After feeding on hamsters that had been inoculated with RVF virus, P. papatasi, P. sergenti, and S. schwetzi became infected and developed disseminated infections. All P. papatasi and P. duboscqi inoculated with RVF virus developed high-titer infections. In contrast, only 41% of the inoculated S. schwetzi contained detectable virus, and infected individuals contained significantly less virus than the two Phlebotomus species. Although 50% of the inoculated P. duboscqi transmitted RVF virus to hamsters, only 14% of P. papatasi and none of the S. schwetzi transmitted this virus. Additional studies are needed to determine the role of sand flies as vectors of RVF virus.

Vector competence of North American mosquitoes (Diptera: Culicidae) for West Nile virus.

We evaluated the potential for several North American mosquito species to transmit the newly introduced West Nile (WN) virus. Mosquitoes collected in the New York City metropolitan area during the recent WN virus outbreak, at the Assateague Island Wildlife Refuge, VA, or from established colonies were allowed to feed on chickens infected with WN virus isolated from a crow that died during the 1999 outbreak. These mosquitoes were tested approximately 2 wk later to determine infection, dissemination, and transmission rates. Aedes albopictus (Skuse), Aedes atropalpus (Coquillett), and Aedes japonicus (Theobald) were highly susceptible to infection, and nearly all individuals with a disseminated infection transmitted virus by bite. Culex pipiens L. and Aedes sollicitans (Walker) were moderately susceptible. In contrast, Aedes vexans (Meigen), Aedes aegypti (L.), and Aedes taeniorhynchus (Wiedemann) were relatively refractory to infection, but individual mosquitoes inoculated with WN virus did transmit virus by bite. Infected female Cx. pipiens transmitted WN virus to one of 1,618 F1 progeny, indicating the potential for vertical transmission of this virus. In addition to laboratory vector competence, host-feeding preferences, relative abundance, and season of activity also determine the role that these species could play in transmitting WN virus.

Laboratory transmission of Sindbis virus by Aedes albopictus, Ae. aegypti, and Culex pipiens (Diptera: Culicidae).

The susceptibility of Aedes albopictus (Skuse) for Sindbis (SIN) virus was examined in the laboratory. Ae. albopictus, Ae. aegypti (L.), and Culex pipiens (L.) became infected with and subsequently transmitted SIN virus by bite to chicks after feeding on viremic 1-d-old chicks. After ingesting 10(5.3) plaque-forming units (PFU)/ml, Ae. albopictus had the highest transmission rate (30%) of the 3 species. Transmission by Ae. aegypti was less efficient (7%), whereas none of the Cx. pipiens transmitted virus. Transmission rates were higher for Ae. albopictus (53%) and Cx. pipiens (37%) when they fed on chicks with a viremia of 10(8.4) PFU/ml. Ae. aegypti was not tested at this dose. Based on these studies, the increased geographic distribution of Ae. albopictus, and its opportunistic feeding behavior, this species should be considered as a potential bridge vector of SIN virus.

Transmission of Crimean-Congo hemorrhagic fever virus by Hyalomma impeltatum (Acari:Ixodidae) after experimental infection.

We examined the potential for Hyalomma impeltatum Schulze & Schlottke ticks to transmit Crimean-Congo hemorrhagic fever (CCHF) virus. After feeding on 1-d-old mice that had been inoculated with CCHF virus, larval H. impeltatum became infected with and subsequently transmitted CCHF virus transstadially to nymphs. These nymphs transmitted virus horizontally to guinea pigs and transstadially to adults. A minimum of 13% of the adult H. impeltatum, exposed to virus as larvae, transmitted virus by bite to guinea pigs. An enzyme-linked immunosorbent assay detected CCHF viral antigen in 63% of the adult ticks derived from those exposed to this virus as larvae. This tick species should be considered as a potential vector of CCHF virus.

Replication of Crimean-Congo hemorrhagic fever virus in four species of ixodid ticks (Acari) infected experimentally.

The vector potentials of Hyalomma dromedarii Koch, H. impeltatum Schulze & Schlottke, H. truncatum Koch, and Rhipicephalus appendiculatus Neumann for Crimean-Congo hemorrhagic fever (CCHF) virus (IbAr 10200) were evaluated by intracoelomic inoculation. All three Hyalomma species became infected; infection rates ranged between 80 and 100% at 7-14 d after inoculation, and viral titers increased in unfed specimens almost 100 times above inoculation levels within the first week following infection. Only 40% of the R. appendiculatus became infected, and viral titers of unfed specimens increased less than 10 times above inoculation levels. The virus persisted to 153 d in unfed H. impeltatum. Viral titers were significantly higher in female than in male H. dromedarii, H. impeltatum, H. truncatum, and R. appendiculatus after blood feeding. Blood feeding had little effect on the viral titers of male Hyalomma species. However, the percentage of female and male ticks from which virus was recovered was significantly higher from fed ticks compared with unfed ticks. No virological evidence of transovarial transmission was found in greater than 78,000 first-generation progeny (larvae, nymphs, and adults) of inoculated female H. dromedarii, H. impeltatum, H. truncatum, and R. appendiculatus. All species transmitted CCHF virus to guinea pigs when allowed to feed at both 6 and 21 d after inoculation.