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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., (Published by Oxford University Press on behalf of Entomological Society of America 2020.)

We conducted experimental studies to evaluate mosquitoes captured in Paju County, Gyeonggi Province, Republic of Korea, for their ability to transmit West Nile virus (family Flaviviridae, genus Flavivirus, WNV), Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV), and Getah virus (family Togaviridae, genus Alphavirus, GETV) under laboratory conditions. Both Culex pipiens pallens Coquillett and Culex tritaeniorhynchus Giles were highly susceptible to infection with WNV, with infection rates > 65% when allowed to feed on chickens with viremias of approximately 10(7) plaque-forming units (PFU) of virus/ml blood. In contrast, Cx. tritaeniorhynchus were significantly more susceptible to JEV or GETV (infection rate 100%) than were the Cx. p. pallens (infection rate 3% for JEV and 0% for GETV) captured in the same area when allowed to feed on chickens with viremias of approximately 10(5) PFU of virus/ml blood. The detection of JEV in field-collected Cx. tritaeniorhynchus in Gyeonggi Province in 2000 and the demonstrated ability of this species to transmit this virus support the importance of the continued vaccination of Koreans against JEV and indicate a risk of infection for nonvaccinated individuals.

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.

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.

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 approximately 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.

As part of an evaluation of the ecology of arthropod-borne diseases in the Republic of Korea (ROK), we examined 8,765 mosquitoes captured in Paju County, Gyonggi Province, ROK, for the presence of viruses. Mosquitoes were captured in propane lantern/human-baited Shannon traps, Mosquito Magnet traps, or American Biophysics Corporation (East Greenwich, RI) miniature light traps with or without supplemental octenol bait and/or dry ice. Mosquitoes were identified to species, placed in pools of up to 40 mosquitoes each, and tested on Vero cells for the presence of virus. A total of 15 virus isolations were made from 293 pools of mosquitoes. Viruses were identified by reverse transcriptase-polymerase chain reaction and sequencing and consisted of 14 isolations of Japanese encephalitis (JE) virus and one isolation of Getah (GET) virus. All JE isolates were from Culex tritaeniorhynchus Giles, and the isolate of GET was from Aedes vexans (Meigen). The minimum field infection rate for JE in Cx. tritaeniorhynchus was 3.3 per 1,000, whereas the GET virus infection rate for Ae. vexans was 0.2 per 1,000. Isolation of JE and GET indicated that both viruses were actively circulating in northern Gyonggi Province, ROK. The lack of human cases of JE among the Korean population probably is because of an effective government-mandated vaccination program. The reason for no cases among >10,000 United States military and others that reside or train nearby is unknown, but may be related to personnel protection measures (permethrin-impregnated uniforms and use of deet repellent), adult mosquito control, mosquito selection of nonhuman hosts (unpublished data), and the low symptomatic to asymptomatic ratio of disease in adults.

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 degrees C for a minimal filial infection rate (MFIR) of approximately 1.4/1,000 and four of 1,873 adult progeny reared at 26 degrees C (MFIR = 2.1/1,000). The mean titer of the positive pools was 10(5.6) plaque-forming units (PFU)/ml (=10(5.9) PFU/mosquito for positive mosquitoes) of virus. Overall, the MFIR was approximately 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.

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 10(7.0-8.7) 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 10(8.1) 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 10(5.7) 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.

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 degrees 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 degrees 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 degrees 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 degrees 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.

As part of an evaluation of potential vectors of arboviruses during a Rift Valley fever (RVF) outbreak in the Nile Valley of Egypt in August 1993, we collected mosquitoes in villages with known RVF viral activity. Mosquitoes were sorted to species, pooled, and processed for virus isolation both by intracerebral inoculation into suckling mice and by inoculation into cell culture. A total of 33 virus isolates was made from 36,024 mosquitoes. Viruses were initially identified by indirect fluorescent antibody testing and consisted of 30 flaviviruses (all members of the Japanese encephalitis complex, most probably West Nile [WN] virus) and three alphaviruses (all members of western equine encephalitis complex, most probably Sindbis). The identity of selected viruses was confirmed by reverse transcriptase-polymerase chain reaction and sequencing. Culex antennatus (Becker) and Culex perexiguus Theobald accounted for five (17%) and 23 (77%) of the WN virus isolations, respectively. Despite isolation of viruses from 32 pools of mosquitoes (both WN and Sindbis viruses were isolated from a single pool), RVF virus was not isolated from these mosquitoes, even though most of them are known competent vectors collected during an ongoing RVF outbreak. Thus, it should be remembered, that even during a known arbovirus outbreak, other arboviruses may still be circulating and causing disease.

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.

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.

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 approximately 10(8) 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 > or = 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 > or = 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.

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.

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.

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.

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).

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.