David J. Dohm

Vector Competence of North American Mosquitoes (Diptera: Culicidae) for West Nile Virus

Abstract 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 ≈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.

An Update on the Potential of North American Mosquitoes (Diptera: Culicidae) to Transmit West Nile Virus

Abstract Since first discovered in the New York City area in 1999, West Nile virus (WNV) has become established over much of the continental United States and has been responsible for >10,000 cases of severe disease and 400 human fatalities, as well as thousands of fatal infections in horses. To develop appropriate surveillance and control strategies, the identification of which mosquito species are competent vectors and how various factors influence their ability to transmit this virus must be determined. Therefore, we evaluated numerous mosquito species for their ability to transmit WNV under laboratory conditions. This report contains data for several mosquito species not reported previously, as well as a summary of transmission data compiled from previously reported studies. Mosquitoes were allowed to feed on chickens infected with WNV isolated from a crow that died during the 1999 outbreak in New York City. These mosquitoes were tested ≈2 wk later to determine infection, dissemination, and transmission rates. All Culex species tested were competent vectors in the laboratory and varied from highly efficient vectors (e.g., Culex tarsalis Coquillett) to moderately efficient ones (e.g., Culex nigripalpus Theobald). Nearly all of the Culex species tested could serve as efficient enzootic or amplifying vectors for WNV. Several container-breeding Aedes and Ochlerotatus species were highly efficient vectors under laboratory conditions, but because of their feeding preferences, would probably not be involved in the maintenance of WNV in nature. However, they would be potential bridge vectors between the avian–Culex cycle and mammalian hosts. In contrast, most of the surface pool-breeding Aedes and Ochlerotatus species tested were relatively inefficient vectors under laboratory conditions and would probably not play a significant role in transmitting WNV in nature. In determining the potential for a mosquito species to become involved in transmitting WNV, it is necessary to consider not only its laboratory vector competence but also its abundance, host-feeding preference, involvement with other viruses with similar transmission cycles, and whether WNV has been isolated from this species under natural conditions.

Effect of Environmental Temperature on the Ability of Culex pipiens (Diptera: Culicidae) to Transmit West Nile Virus

Abstract Environmental temperature can affect the ability of mosquitoes to transmit an arbovirus. However, results of various studies indicate that these effects are not consistent among viruses or mosquito species, and there is no information available on the effect of environmental temperature on the ability of North American mosquito species to transmit West Nile (WN) virus. We evaluated the effect of incubation temperature (18, 20, 26, or 30°C) on the ability of Culex pipiens L. derived from specimens collected during the outbreak in New York in 1999 to transmit a strain of WN virus obtained from a crow that died during this outbreak. Although mosquitoes fed on the same viremic chickens, infection rates were directly related to subsequent incubation temperatures. In mosquitoes held at 30°C, virus was recovered from nearly all mosquitoes tested, disseminated infections were detected as early as 4 d after the infectious blood meal, and >90% of all mosquitoes had a disseminated infection 12 or more days after the infectious blood meal. In contrast, for mosquitoes held at 18°C, disseminated infections were not detected until 25 d after the infectious blood meal, and even after 28 d, <30% contained a disseminated infection. Results for mosquitoes held at 20 and 26°C were intermediate for both infection and dissemination rates. The effect of environmental temperature should to be considered when evaluating the vector competence of these mosquitoes and modeling risk of WN virus transmission in nature.

Potential North American Vectors of West Nile Virus

Abstract: 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°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.

Potential for North American mosquitoes to transmit Rift Valley fever virus.

ABSTRACT 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 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 North American mosquitoes to transmit this virus should it be introduced into North America. Therefore, we evaluated selected mosquito species from the southeastern United States for their ability to serve as potential vectors for RVFV. Mosquitoes were fed on adult hamsters inoculated 1 day previously with RVFV. These mosquitoes were tested for infection and ability to transmit RVFV after incubation at 26°C for 7–21 days. None of the species tested (Aedes taeniorhynchus, Ae. vexans, Culex erraticus, Cx. nigripalpus, Cx. quinquefasciatus, and Cx. salinarius) were efficient vectors after they fed on hamsters with viremias ranging from 104.1 to 106.9 plaque-forming units (PFU)/ml. However, Ae. taeniorhynchus, Ae. vexans, and Cx. erraticus all developed disseminated infections after they fed on hamsters with viremias between 108.5 and 1010.2 PFU/ml, and both Ae. vexans and Cx. erraticus transmitted RVFV by bite. These studies illustrate the need to identify the ability of individual mosquito species to transmit RVFV so that appropriate decisions can be made concerning the application of control measures during an outbreak.

Experimental vertical transmission of West Nile virus by Culex pipiens (Diptera: Culicidae).

Abstract Despite the detection of West Nile (WN) virus in overwintering Culex pipiens L. in New York in February 2000, the mechanism by which this virus persists throughout the winter to initiate infections in vertebrate hosts and vectors the following spring remains unknown. After a blood meal, parous mosquitoes generally do not survive until spring and gonotrophic dissociation occurs in only a small percentage of the population. To investigate vertical transmission as a means of viral survival during interepizootics, we intrathoracically inoculated Cx. pipiens and Aedes albopictus (Skuse) with WN virus and subsequently tested their F1 progeny for the presence of virus. Among the Cx. pipiens, we recovered virus from two of 1,417 adult progeny that had been reared at 18°C for a minimal filial infection rate (MFIR) of ≈1.4/1,000 and four of 1,873 adult progeny reared at 26°C (MFIR = 2.1/1,000). The mean titer of the positive pools was 105.6 plaque-forming units (PFU)/ml (=105.9 PFU/mosquito for positive mosquitoes) of virus. Overall, the MFIR was ≈1.8/1,000 for Cx. pipiens. Although reports indicate that Ae. albopictus vertically transmit various viruses in the Japanese encephalitis virus complex, we did not detect WN virus in any of > 13,000 F1 progeny of WN virus-inoculated specimens. Female Cx. pipiens that are vertically infected during the late summer season and then survive the winter could serve as a source of WN virus to initiate an infection cycle the following spring.

Experimental Transmission of Eastern Equine Encephalitis Virus by Ochlerotatus j. japonicus (Diptera: Culicidae)

Abstract We evaluated the potential for Ochlerotatus j. japonicus (Theobald), a newly recognized invasive mosquito species in the United States, to transmit eastern equine encephalitis (EEE) virus. Aedes albopictus (Skuse) and Culex pipiens (L.) were similarly tested for comparison. Ochlerotatus j. japonicus and Ae. albopictus became infected and transmitted EEE virus by bite after feeding on young chickens 1 d after they had been inoculated with EEE virus (viremias ranging from 107.0–8.7 plaque-forming units [PFU]/ml of blood). No Cx. pipiens (n = 20) had detectable levels of virus 14 d after feeding on an EEE-virus infected chicken with a viremia of 108.1 PFU per ml of blood. Depending on the viral titer in the donor chicken, infection rates ranged from 55–100% for Oc. j. japonicus and 93–100% for Ae. albopictus. In these two species, dissemination rates were identical to or nearly identical to infection rates. Depending on the viral titer in the blood meal, estimated transmission rates ranged from 15 to 25% for Oc. j. japonicus and 59–63% for Ae. albopictus. Studies of replication of EEE virus in Oc. j. japonicus showed that there was an “eclipse phase” in the first 4 d after an infectious blood meal, that viral titers peak by day 7 at around 105.7 per mosquito, and that virus escaped the mid-gut as soon as 3 d after the infectious blood meal. These data, combined with the opportunistic feeding behavior of Oc. j. japonicus in Asia and the reported expansion of its range in the eastern United States, indicate that it could function as a bridge vector for EEE virus between the enzootic Culiseta melanura (Coquillett)-avian cycle and susceptible mammalian hosts.

Isolation of Viruses from Mosquitoes (Diptera: Culicidae) Collected in the Amazon Basin Region of Peru

Abstract 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.

Vector Competence of Peruvian Mosquitoes (Diptera: Culicidae) for Epizootic and Enzootic Strains of Venezuelan Equine Encephalomyelitis Virus

Abstract Mosquitoes collected in the Amazon Basin, near Iquitos, Peru, were evaluated for their susceptibility to epizootic (IAB and IC) and enzootic (ID and IE) strains of Venezuelan equine encephalomyelitis (VEE) virus. After feeding on hamsters with a viremia of ≈108 plaque-forming units of virus per milliliter, Culex (Melanoconion) gnomatus Sallum, Huchings, & Ferreira, Culex (Melanoconion) vomerifer Komp, and Aedes fulvus (Wiedemann) were highly susceptible to infection with all four subtypes of VEE virus (infection rates ≥87%). Likewise, Psorophora albigenu (Peryassu) and a combination of Mansonia indubitans Dyar & Shannon and Mansonia titillans (Walker) were moderately susceptible to all four strains of VEE virus (infection rates ≥50%). Although Psorophora cingulata (Fabricius) and Coquillettidia venezuelensis (Theobald) were susceptible to infection with each of the VEE strains, these two species were not efficient transmitters of any of the VEE strains, even after intrathoracic inoculation, indicating the presence of a salivary gland barrier in these species. In contrast to the other species tested, both Culex (Melanoconion) pedroi Sirivanakarn & Belkin and Culex (Culex) coronator Dyar & Knab were nearly refractory to each of the strains of VEE virus tested. Although many of the mosquito species found in this region were competent laboratory vectors of VEE virus, additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of VEE virus are needed to incriminate the principal vector species.

Endemic Venezuelan Equine Encephalitis in Northern Peru

Since Venezuelan equine encephalitis virus (VEEV) was isolated in Peru in 1942, >70 isolates have been obtained from mosquitoes, humans, and sylvatic mammals primarily in the Amazon region. To investigate genetic relationships among the Peru VEEV isolates and between the Peru isolates and other VEEV strains, a fragment of the PE2 gene was amplified and analyzed by single-stranded conformation polymorphism. Representatives of seven genotypes underwent sequencing and phylogenetic analysis. The results identified four VEE complex lineages that cocirculate in the Amazon region: subtypes ID (Panama and Colombia/Venezuela genotypes), IIIC, and a new, proposed subtype IIID, which was isolated from a febrile human, mosquitoes, and spiny rats. Both ID lineages and the IIID subtype are associated with febrile human illness. Most of the subtype ID isolates belonged to the Panama genotype, but the Colombia/Venezuela genotype, which is phylogenetically related to epizootic strains, also continues to circulate in the Amazon basin.