Laboratory transmission of Japanese encephalitis, West Nile, and Getah viruses by mosquitoes (Diptera: Culicidae) collected near Camp Greaves, Gyeonggi Province, Republic of Korea 2003

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.     

Japanese encephalitis virus in culicine mosquitoes (Diptera: Culicidae) collected at Daeseongdong, a village in the demilitarized zone of the Republic of Korea

In total, 22,846 (17,793 culicines and 5,053 Anopheles spp.) female mosquitoes were captured by a Mosquito Magnet trap at Daeseongdong, a small village adjacent to the military demarcation line (center of the demilitarized zone) in northern Gyeonggi Province, Republic of Korea (ROK). Culicine mosquitoes were identified to species, placed in pools of up to 30 mosquitoes each, and screened for flavivirus using a SYBR Green I-based real-time polymerase chain reaction. In total, 51/660 pools positive for flaviviruses and confirmed by DNA sequencing of the NS5 region, were positive for Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV) (50 Culex tritaeniorhynchus Giles and one Culex bitaeniorhynchus Giles). The JEV maximum likelihood estimations (MLEs) (estimated number of viral RNA-positive mosquitoes per 1,000) for Cx. tritaeniorhynchus and Cx. bitaeniorhynchus were 9.7 and 0.9, respectively. This is the first report of a Cx. bitaeniorhynchus positive for JEV in the ROK. JEV is a local civilian and military health threat and a significant concern for nonimmune (unvaccinated) U.S. soldiers, civilians, and family members deployed to the ROK.     

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

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

Vector competence of Australian mosquitoes (Diptera: Culicidae) for Japanese encephalitis virus

Australian mosquitoes were evaluated for their ability to become infected with and transmit a Torres Strait strain of Japanese encephalitis virus. Mosquitoes, which were obtained from either laboratory colonies and collected using Centers for Disease Control and Prevention light traps baited with CO2 and octenol or reared from larvae, were infected by feeding on a blood/sucrose solution containing 10(4.5 +/- 0.1) porcine stable-equine kidney (PS-EK) tissue culture infectious dose50/mosquito of the TS3306 virus strain. After 14 d, infection and transmission rates of 100% and 81%, respectively, were obtained for a southeast Queensland strain of Culex annulirostris Skuse, and 93% and 61%, respectively, for a far north Queensland strain. After 13 or more days, infection and transmission rates of > 90% and > or = 50%, respectively, were obtained for southeast Queensland strains of Culex sitiens Wiedemann and Culex quinquefasciatus Say, and a far north Queensland strain of Culex gelidus Theobald. Although infection rates were > 55%, only 17% of Ochlerotatus vigilax (Skuse) and no Cx. quinquefasciatus, collected from far north Queensland, transmitted virus. North Queensland strains of Aedes aegypti L., Ochlerotatus kochi (D?nitz), and Verrallina funerea (Theobald) were relatively refractory to infection. Vertical transmission was not detected among 673 F1 progeny of Oc. vigilax. Results of the current vector competence study, coupled with high field isolation rates, host feeding patterns and widespread distribution, confirm the status of Cx. annulirostris as the major vector of Japanese encephalitis virus in northern Australia. The relative roles of other species in potential Japanese encephalitis virus transmission cycles in northern Australia are discussed.     

Host feeding pattern of Japanese encephalitis virus vector mosquitoes (Diptera: Culicidae) from Kuttanadu, Kerala, India

Identification of blood meals of vector mosquitoes is an important tool in the epidemiological investigations of vector-borne diseases. The blood meals of three mosquito species involved in the transmission of Japanese encephalitis virus (JEV) from the Kuttanadu area, Kerala, were determined using the agarose gel diffusion technique. A total of 4959 blood smears belonging to Culex (Culex) tritaeniorhynchus Giles (3273), Cx. (Culex) gelidus Theobald (64), Mansonia (Mnd.) indiana Edwards (735) ,and Ma. (Mnd.) uniformis (Theobald) (887) were tested. Cx. tritaeniorhynchus had predominantly fed on bovids (46.4%), and a good proportion (29%) had fed on more than one host. Cx. tritaeniorhynchus was highly zoophagic, and human feeding accounted for only 1.5% of those individuals successfully tested. Cx. gelidus showed bovid feeding at 36% and pig feeding at 12.5%. The test results showed 42.3% Ma. indiana and 12.2% Ma. uniformis had fed on humans. Multiple feeding was observed in Ma. indiana and Ma. uniformis, and most of the double feedings were from bovids and ovids (7.9 and 20.1%, respectively). Pig feeding accounted for 4.8% of the feedings by Cx. tritaeniorhynchus, 5.3% of Ma. indiana, and 6.4% of Ma. uniformis. This study is significant because of the role played by these mosquitoes in the transmission of JEV in the Kuttanadu area of Kerala, India.     

Isolation of arboviruses from mosquitoes (Diptera: Culicidae) collected from the Gulf Plains region of northwest Queensland,Australia

As part of investigations into Japanese encephalitis (JE) virus and related flaviviruses in northern Australia, 153,529 mosquitoes were collected and processed for virus isolation from the Gulf Plains region of northwest Queensland. Collections from within 30 km of each of the townships of Croydon, Normanton and Karumba yielded 3,087 (2.0%), 66,009 (43.0%), and 84,433 (55.0%) mosquitoes, respectively, from which 16 viruses were isolated. Four isolates of Murray Valley encephalitis (MVE), two of Kunjin (KUN), three of Ross River (RR), and one of Sindbis (SIN) viruses were obtained from Culex sitiens subgroup mosquitoes. Molecular identification of the mosquito species composition of these virus positive pools revealed that most isolates were from pools containing mainly Culex annulirostris Skuse and low numbers of Culex palpalis (Taylor). Only three pools, one each of MVE, KUN, and RR, were from mosquitoes identified exclusively as Cx. annulirostris. Other viruses isolated include one Edge Hill virus from Ochlerotatus normanensis (Taylor), an isolate of SIN from Anopheles meraukensis Venhuis, two isolates of RR from Anopheles amictus Edwards, and single isolates of RR from Anopheles bancroftii Giles andAedes lineatopennis (Ludlow). The isolate of RR from Ae. lineatopennis was the first reported from this species. The public health implications of these isolations in the Gulf Plains region are discussed briefly.     

Isolation of Murray Valley encephalitis and Ross River viruses from Aedes normanensis (Diptera: Culicidae) in Western Australia

Of eight viruses isolated from 28,909 female (643 pools) Aedes normanensis collected from the north of Western Australia, two were identified as Murray Valley encephalitis virus and six as Ross River virus. The two isolates of Murray Valley encephalitis virus represent the first confirmed isolations of this virus from an Aedes species. The possible implications of these findings with regard to virus survival mechanisms during the dry season are discussed.     

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

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

Evolutionary relationships among California serogroup viruses (Bunyaviridae) and Aedes mosquitoes (Diptera: Culicidae)

More than 100 published and four unpublished papers recording virus isolations of California (CAL) serogroup viruses from mosquitoes and other arthropods were analyzed, followed by a comparison of a contemporary classification of mosquitoes of the genus Aedes with a contemporary classification of CAL serogroup viruses. Using criteria of five or more total isolations of a given virus from a given species of mosquito, and a minimum infection rate of 1:10,000 or greater (more frequent), Aedes, and to a lesser extent Culiseta, species were found to be the arthropods most frequently involved as vectors. Such a comparison could not explain all virus-vector associations on the basis of coevolution of viruses in Aedes mosquito species, probably because of the crossing over of viruses to other mosquito species through horizontal transmission and because of deficiencies in currently available classifications. Coevolution of host-parasite systems is discussed, as are the inadequacies of contemporary taxonomic treatments of viruses and Aedes mosquitoes. The paper ends with hypothetical reconstructions of virus-mosquito relationships over time and possible routes of dispersal of California serogroup viruses to their present geographical distributions.     

Isolation of Rift Valley fever virus from mosquitoes (Diptera: Culicidae) collected during an outbreak in domestic animals in Kenya.

During an outbreak of Rift Valley fever (RVF) in livestock near Lake Naivasha, Rift Valley Province, Kenya, 61,347 mosquitoes (1,287 pools) collected in CO2-baited light traps yielded seven viral isolates. Five isolates of RVF virus were recovered from 18,831 Culex zombaensis Theobald and one from 14,439 Mansonia africana (Theobald). One isolate of a Bunyamwera group virus was recovered from 1,175 Aedes quasiunivittatus (Theobald).     

Mosquito (Diptera: Culicidae) dispersal: implications for the epidemiology of Japanese and Murray Valley encephalitis viruses in Australia

One hypothesis to explain the southern extension of Japanese encephalitis (JE) virus from Papua New Guinea into the Torres Strait islands in 1995 and to mainland Australia in 1998 is the dispersal of infected mosquitoes, particularly Culex annulirostris Skuse from which JE virus has been isolated repeatedly. To investigate whether this species disperses in this manner, mosquitoes were identified from 368 aerial kite trap collections operated at 50-310 m (altitude) at inland New South Wales between November 1979 to December 1984. Forty samples (9 during daylight and 31 at night) contained mosquitoes, of which 221 could be identified as Culex australicus Dobrotworsky & Drummond (58.8%), Culex annulirostris (21.3%), Anopheles annulipes Walker s.l. (10.4%), Aedes theobaldi (Taylor) (7.2%), Aedes rubrithorax (Macquart) (1.4%), and Aedes sagax (Skuse) (< 0.9%). During the night, mosquitoes were found in 22.6% of the collections at a mean density (+/- SD) of 91.3 +/- 151.7/10(6) m3 of air sampled. During the day, only 3.8% were positive at a mean density 125.3 +/- 152.1. When examined in relation to possible flying time and wind speed, mean +/- SD dispersal distances by day and night were 23.9 +/- 15.3 km and 152.4 +/- 116.3 km, respectively. These data provide circumstantial evidence that aerial carriage southward approximately 200 km from Papua New Guinea to Cape York peninsula is feasible, but that southern dispersal of Murray Valley encephalitis virus infected mosquitoes from tropical to temperate Australia is unlikely.