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William S Romoser1, Marco Neira Oviedo1, Kriangkrai Lerdthusnee2, Lisa A Patrican3, Michael J Turell4, David J Dohm4, Kenneth J Linthicum5, Charles L Bailey61Department of Biomedical Sciences, College of Osteopathic Medicine, Tropical Disease Institute, Ohio University, Athens, Ohio, USA; 2Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand; 3Infectious Disease Division, National Center for Medical Intelligence, Fort Detrick, Frederick, Maryland, USA; 4Department of Vector Assessment, Virology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland, USA; 5Center for Medical, Agricultural, and Veterinary Entomology, United States Department of Agriculture – Agricultural Research Service, Gainesville, Florida, USA; 6National Center for Biodefense and Infectious Disease, School of Systems Biology, College of Science, George Mason University, Manassas, Virginia, USABackground: Endemic/enzootic maintenance mechanisms like vertical transmission (pathogen passage from infected adults to their offspring) are central in the epidemiology of zoonotic pathogens. In Kenya, Rift Valley fever virus (RVFV) may be maintained by vertical transmission in ground-pool mosquitoes such as Aedes mcintoshi. RVFV can cause serious morbidity and mortality in humans and livestock. Past epidemics/epizootics have occurred in sub-Saharan Africa but, since the late 1970s, RVFV has also appeared in North Africa and the Middle East. Preliminary results revealed RVFV-infected eggs in Ae. mcintoshi after virus injection into the hemocoel after the first of two blood meals, justifying further study.Methods: Mosquitoes were collected from an artificially flooded water-catching depression along a stream in Kenya, shipped live to the USA, and studied using an immunocytochemical method for RVFV-antigen localization in mosquito sections.Results and conclusion: After virus injection into the hemocoel, RVFV-infected reproductive tissues were found, particularly follicular epithelia and oocyte/nurse cells. Ovarian infection from the hemocoel is a crucial step in establishing a vertically transmitting mosquito line. Ovarian follicles originate from germarial cells, primordia located distally in each ovariole, and infection of these cells is expected to be requisite for long-term vertical transmission. However, no germarial cell infection was found, so establishing a new line of vertically transmitting mosquitoes may require two generations. The findings support the hypothesis that Ae. mcintoshi is involved in the endemic maintenance of RVFV by vertical transmission. Detection of distinct pathology in infected eggs raises the possibility of virus-laden eggs being deposited among healthy eggs, thereby providing an exogenous source of infection via ingestion by mosquito larvae and other organisms. This has potentially significant epidemiological implications. Possible modes of entry of virus from the hemocoel into the ovaries and routes by which larvae might become infected by ingesting virus are discussed.Keywords: hemocoel, ovary, vertical transmission, Aedes mcintoshi, arbovirus, antigen positive

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.)

Venezuelan equine encephalitis virus (VEEV) is an important pathogen of medical and veterinary importance in the Americas. In this report, we present the complete genome sequences of five VEEV isolates obtained from pools of Culex (Melanoconion) gnomatos (4) or Culex (Melanoconion) pedroi (1) from Iquitos, Peru. Genetic and phylogenetic analyses showed that all five isolates grouped within the VEEV complex sister to VEEV IIIC and are members of subtype IIID. This is the first report of full-length genomic sequences of VEEV IIID.

Rift Valley fever virus (RVFV), a mosquito-borne virus, 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. Culex pipiens was implicated as the principal vector of the Egyptian outbreak in 1977 that affected about 200,000 people. In the northern USA, Cx. pipiens occurs both as a mix of forms pipiens and molestus (i.e., US Culex pipiens) as well as pure Cx. pipiens form molestus, the latter mostly in underground locations such as sewers and basements. In order to understand the potential risk of spread of RVFV in the USA, we compared their relative abilities to transmit RVFV in the laboratory. After feeding on hamsters with high viremias, >10(9) plaque-forming units (PFU)/ml, both US Cx. pipiens and Cx. pipiens form molestus were highly susceptible to infection (∼80%) and about 20% of each form developed a disseminated infection. In contrast, when fed on a hamster with a moderate viremia, 10(7.5) PFU/ml, US Cx. pipiens were significantly (P < 0.001) more susceptible (84%) than were the pure form molestus (47%). Similarly, dissemination rates were significantly (P  =  0.0261) higher in US Cx. pipiens (34%) than they were in pure Cx. pipiens form molestus (10%). These results underscore differences in vector competence between genetic forms in the Cx. pipiens complex but also indicate that if RVFV were to arrive in the USA, competent vectors abound in the highly urbanized Northeast.

Rift Valley fever (RVF), a disease of ruminants and humans, 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. As indicated by the rapid spread of West Nile viral activity across North America since its discovery in 1999 and the rapid and widespread movement of chikungunya virus from Africa throughout the Indian Ocean Islands to Asia and Europe, an introduced exotic arbovirus can be rapidly and widely established across wide geographical regions. Although RVF virus (RVFV) is normally transmitted by mosquitoes, we wanted to determine the potential for this virus to replicate in 2 of the most globally distributed and common higher flies: house flies, Musca domestica, and stable flies, Stomoxys calcitrans. Neither species supported the replication of RVFV, even after intrathoracic inoculation. However, S. calcitrans was able to mechanically transmit RVFV to susceptible hamsters (Mesocricetus auratus) after probing on infected hamsters with high viral titers. Therefore, S. calcitrans, because of its close association with domestic animals that serve as amplifying hosts of RVFV, should be considered a possible mechanical vector of RVFV, and it may contribute to the rapid spread of a RVF outbreak. Other Stomoxys species present in Africa and elsewhere may also play similar roles.

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 degrees 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 10(4.1) to 10(6.9) 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 10(8.5) and 10(10.2) 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.

In the collection of field materials to test for the presence of arboviruses, samples must be appropriately maintained to detect arboviral nucleic acids. In austere field conditions this is often difficult to achieve because, during routine specimen processing, storage, and shipping viral RNA degradation could result in detection failure. RNA extraction reagents, while used commonly for their intended purpose of stabilizing RNA during the extraction process, have not been assessed fully for their potential to stabilize RNA before extraction. The potential for virus stabilization at varying temperatures and periods of time remains unknown. Accordingly, the ability of buffer AVL (Qiagen, Valencia, CA), an RNA extraction reagent, to stabilize viral suspensions of dengue, Venezuelan equine encephalitis and Rift Valley fever viruses was evaluated. The ability of buffer AVL to stabilize each viral suspension was examined at 32, 20, 4, and -20 degrees C. RNA in samples placed in buffer AVL was stable for at least 48h at 32 degrees C and refrigerating samples prolonged stabilization. Additionally, placing the sample/buffer AVL mixture at either 4 or -20 degrees C stabilized samples for at least 35 days. When combined with the ability of buffer AVL to inactivate viral samples, this provides the ability to collect and handle potentially infectious samples in a safe way that also provides sample stabilization.

As a result of concerns regarding the geographic spread of West Nile virus (WNV) to Central America, we evaluated the potential for Honduran Culex nigripalpus Theobald to transmit this virus. We tested individual mosquitoes captured in Olancho Province, Honduras, in September 2003. Mosquitoes were allowed to feed on 2- to 4- day-old chickens previously inoculated with a New York strain (Crow 397-99) of WNV. Infection rates in Cx. nigripalpus ranged from 81%-96% after feeding on chickens with viremias between 10(6.3) and 10(7.4) plaque-forming units per milliliter. Development of a disseminated infection was directly correlated with holding time after the infectious blood meal as 68% (19/28) of the mosquitoes tested 20 days after the infectious blood meal had a disseminated infection as compared to 38% (15/40) of the mosquitoes tested 14 days after feeding on the same viremic chickens (viremia = 10(6.97.4)). Nearly all (4/5) Cx. nigripalpus with a disseminated infection that fed on susceptible chickens transmitted virus by bite. In addition, 8 (57%) of 14 Cx. nigripalpus with a disseminated infection transmitted virus when tested by a capillary tube feeding assay. Based on its efficiency of viral transmission in this study and its role in the transmission of the closely related St. Louis encephalitis virus in the southeastern United States, Cx. nigripalpus should be considered a potentially important vector of WNV in Honduras and the rest of Central America.

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 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.

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.

Many assume that common methods to extract viral nucleic acids are able to render a sample non-infectious. It may be that inactivation of infectious virus is incomplete during viral nucleic acid extraction methods. Accordingly, two common viral nucleic acid extraction techniques were evaluated for the ability to inactivate high viral titer specimens. In particular, the potential for TRIzol LS Reagent (Invitrogen Corp., Carlsbad, CA) and AVL Buffer (Qiagen, Valencia, CA) were examined to render suspensions of alphaviruses, flaviviruses, filoviruses and a bunyavirus non-infectious to tissue culture assay. The dilution series for both extraction reagents consistently caused cell death through a 100-fold dilution. Except for the DEN subtype 4 positive control, all viruses had titers of at least 10(6)pfu/ml. No plaques were detected in any extraction reagent plus virus combination in this study, therefore, the extraction reagents appeared to inactivate completely each of the high-titer viruses used in this study. These results support the reliance upon either TRIzol LS Reagent or AVL Buffer to render clinical or environmental samples non-infectious, which has implications for the handling and processing of samples under austere field conditions and low level containment.

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.

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.

To evaluate the vector competence of Culex tarsalis Coquillett for West Nile virus (WN), females reared from larvae collected in Huntington Beach, Orange County, CA, were fed on 2-3-day-old chickens previously inoculated with a New York strain (Crow 397-99) of WN. The Cx. tarsalis mosquitoes were efficient laboratory vectors of WN, with estimated transmission rates of 81% and 91% for mosquitoes that ingested 10(6.5) or 10(7.3) plaque-forming units of WN/mL of blood, respectively. Based on efficiency of viral transmission and the role of this species in the transmission of the closely related St. Louis encephalitis virus, Cx. tarsalis should be considered a potentially important vector of WN in the western United States.

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.

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.

To control West Nile virus (WNV), it is necessary to know which mosquitoes are able to transmit this virus. Therefore, we evaluated the WNV vector potential of several North American mosquito species. Culex restuans and Cx. salinarius, two species from which WNV was isolated in New York in 2000, were efficient laboratory vectors. Cx. quinquefasciatus and Cx. nigripalpus from Florida were competent but only moderately efficient vectors. Coquillettidia perturbans was an inefficient laboratory vector. As WNV extends its range, exposure of additional mosquito species may alter its epidemiology.

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.

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.

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.

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.

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.

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.

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.

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)

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).

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.

Several groups of Rift Valley fever (RVF) virus-exposed and unexposed Culex pipiens were allowed differential access to a carbohydrate food source and their survival monitored. When stressed by deprivation of a carbohydrate source, mean survival times of RVF virus-exposed mosquitoes were consistently higher than those of unexposed mosquitoes in each of the carbohydrate-deficient experiments. These differences were statistically significant when mosquitoes were provided 5% sucrose for 24 hours. Mosquitoes that were provided access to a carbohydrate source for 24 h after a bloodmeal and then were denied access survived significantly longer than did those mosquitoes denied access, regardless of their exposure to RVF virus. When not stressed, RVF virus-exposed individuals had slightly higher daily survival rates than did unexposed individuals.

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.

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.

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.

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.