Efficient Laboratory Vectors Research Articles

The Emergence of Japanese Encephalitis Virus in Australia in 2022: Existing Knowledge of Mosquito Vectors.

In early 2022, the Japanese encephalitis virus (JEV) was identified as the cause of stillborn and mummified piglets in pig farms in southeastern Australia. Human cases and additional pig farms with infected piglets were subsequently identified across a widespread area encompassing four states. To inform surveillance and control programs, we synthesized existing information on Australian vectors of JEV, much of which was generated in response to incursions of JEV into the northern state of Queensland between 1995 and 2005. Members of the Culex sitiens subgroup, particularly Culex annulirostris, should be considered the primary vectors of JEV in Australia, as they yielded >87% of field detections of JEV, were highly efficient laboratory vectors of the virus, readily fed on pigs and birds (the key amplifying hosts of the virus) when they were available, and are widespread and often occur in large populations. Three introduced species, Culex quinquefasciatus, Culex gelidus and Culex tritaeniorhynchus may also serve as vectors, but more information on their geographical distribution, abundance and bionomics in the Australian context is required. Mosquitoes from other genera, such as Aedes and Verrallina, whilst considered relatively poor vectors, could play a regional or supplemental role in transmission, especially facilitating vertical transmission as a virus overwintering mechanism. Additional factors that could impact JEV transmission, including mosquito survival, dispersal and genetics, are also discussed. Possible directions for investigation are provided, especially in the context of the virus emerging in a region with different mosquito fauna and environmental drivers than northern Australia.

Laboratory transmission potential of British mosquitoes for equine arboviruses

BackgroundThere has been no evidence of transmission of mosquito-borne arboviruses of equine or human health concern to date in the UK. However, in recent years there have been a number of outbreaks of viral diseases spread by vectors in Europe. These events, in conjunction with increasing rates of globalisation and climate change, have led to concern over the future risk of mosquito-borne viral disease outbreaks in northern Europe and have highlighted the importance of being prepared for potential disease outbreaks. Here we assess several UK mosquito species for their potential to transmit arboviruses important for both equine and human health, as measured by the presence of viral RNA in saliva at different time points after taking an infective blood meal.ResultsThe following wild-caught British mosquitoes were evaluated for their potential as vectors of zoonotic equine arboviruses: Ochlerotatus detritus for Venezuelan equine encephalitis virus (VEEV) and Ross River virus (RRV), and Culiseta annulata and Culex pipiens for Japanese encephalitis virus (JEV). Production of RNA in saliva was demonstrated at varying efficiencies for all mosquito-virus pairs. Ochlerotatus detritus was more permissive for production of RRV RNA in saliva than VEEV RNA. For RRV, 27.3% of mosquitoes expectorated viral RNA at 7 days post-infection when incubated at 21 °C and 50% at 24 °C. Strikingly, 72% of Cx. pipiens produced JEV RNA in saliva after 21 days at 18 °C. For some mosquito-virus pairs, infection and salivary RNA titres reduced over time, suggesting unstable infection dynamics.ConclusionsThis study adds to the number of Palaearctic mosquito species that demonstrate expectoration of viral RNA, for arboviruses of importance to human and equine health. This work adds to evidence that native mosquito species should be investigated further for their potential to vector zoonotic mosquito-borne arboviral disease of equines in northern Europe. The evidence that Cx. pipiens is potentially an efficient laboratory vector of JEV at temperatures as low as 18 °C warrants further investigation, as this mosquito is abundant in cooler regions of Europe and is considered an important vector for West Nile Virus, which has a comparable transmission ecology.

Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus.

Zika virus (ZIKV) has emerged since 2013 as a significant global human health threat following outbreaks in the Pacific Islands and rapid spread throughout South and Central America. Severe congenital and neurological sequelae have been linked to ZIKV infections. Assessing the ability of common mosquito species to transmit ZIKV and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We compared infection, dissemination, and transmission rates by measuring ZIKV RNA levels in cohorts of mosquitoes that ingested blood meals from type I interferon-deficient mice infected with either a Puerto Rican ZIKV strain from 2015 (PR15), a Brazilian ZIKV strain from 2015 (BR15), or an ancestral Asian-lineage Malaysian ZIKV strain from 1966 (MA66). With PR15, Cx. quinquefasciatus was refractory to infection (0%, N = 42) and Cx. tarsalis was infected at 4% (N = 46). No ZIKV RNA was detected in saliva from either Culex species 14 or 21 days post feeding (dpf). In contrast, Ae. aegypti developed infection rates of 85% (PR15; N = 46), 90% (BR15; N = 20), and 81% (MA66; N = 85) 14 or 15 dpf. Although MA66-infected Ae. aegypti showed higher levels of ZIKV RNA in mosquito bodies and legs, transmission rates were not significantly different across virus strains (P = 0.13, Fisher’s exact test). To confirm infectivity and measure the transmitted ZIKV dose, we enumerated infectious ZIKV in Ae. aegypti saliva using Vero cell plaque assays. The expectorated plaque forming units PFU varied by viral strain: MA66-infected expectorated 13±4 PFU (mean±SE, N = 13) compared to 29±6 PFU for PR15-infected (N = 13) and 35±8 PFU for BR15-infected (N = 6; ANOVA, df = 2, F = 3.8, P = 0.035). These laboratory vector competence results support an emerging consensus that Cx. tarsalis and Cx. quinquefasciatus are not vectors of ZIKV. These results also indicate that Ae. aegypti from California are efficient laboratory vectors of ancestral and contemporary Asian lineage ZIKV.

Potential for Populations ofAedes j. japonicusto Transmit Rift Valley Fever Virus in the USA1

Aedes japonicus japonicus was introduced into the northeastern USA in 1998 and has since spread to more than 25 states. Because this species has been shown to be a competent laboratory vector of several viruses, readily feeds on large mammals, and has become a pest in several areas, there is concern that it might serve as a vector of Rift Valley fever virus (RVFV) should that virus be introduced into North America. Infection with RVFV causes mortality in > 90% of young domestic ungulates (e.g., calves, kids, and lambs), as well as causing a febrile illness and occasional deaths in humans. Therefore, we evaluated Ae. j. japonicus captured in North Carolina and in Maryland for their ability to serve as potential vectors for RVFV. After feeding on infected adult hamsters, these mosquitoes were tested for infection, dissemination, and the ability to transmit RVFV after incubation at 26 degrees C for 7-28 days. Both the Maryland and North Carolina populations of Ae. j. japonicus were highly efficient laboratory vectors of RVFV, with infection rates > 90% and dissemination rates > 84% for those mosquitoes that fed on hamsters with viremias > or = 10(8.5) plaque-forming units/ml. Thus, Ae. j. japonicus should be targeted for immediate control should RVFV be introduced into an area where this mosquito is now present.

Vector Competence of Australian Mosquitoes for Chikungunya Virus

Chikungunya virus (CHIKV) is a globally emerging arbovirus responsible for unprecedented outbreaks in the western Indian Ocean, the Indian subcontinent and Italy. To assess the receptivity of Australia to CHIKV, we exposed 10 Australian mosquito species to a 2006 strain of CHIKV isolated from a viremic traveler from Mauritius. In susceptibility trials, the infectious dose required to infect 50% of the mosquitoes was 10(0.6) cell culture infectious dose (CCID)(50)/mosquito for Aedes procax, 10(1.7) CCID(50)/mosquito for Aedes albopictus, 10(2.1) CCID(50)/mosquito for Aedes vigilax, and 10(2.6) CCID(50)/mosquito for Aedes aegypti and Aedes notoscriptus. When exposed to blood meals containing between 10(3.5) and 10(4.1) CCID(50)/mosquito of CHIKV, infection rates in these five species, plus Coquillettidia linealis, were >or=81%. Subsequent transmission rates ranged between 20% for Ae. notoscriptus and 76% for Ae. vigilax. In contrast, Culex spp. were poor laboratory vectors, with infection and dissemination rates <or=20% and <or=12%, respectively. Although Australia has efficient laboratory vectors, the role a mosquito species plays in potential CHIKV transmission cycles will also depend on its geographical and temporal abundance, longevity, and association with humans.

VECTOR COMPETENCE OF PERUVIAN MOSQUITOES (DIPTERA: CULICIDAE) FOR A SUBTYPE IIIC VIRUS IN THE VENEZUELAN EQUINE ENCEPHALOMYELITIS COMPLEX ISOLATED FROM MOSQUITOES CAPTURED IN PERU

We evaluated mosquitoes collected in the Amazon Basin, near Iquitos, Peru, for their susceptibility to a subtype IIIC strain of the Venezuelan equine encephalomyelitis complex. This virus had been previously isolated from a pool of mixed Culex vomerifer and Cx. gnomatos captured near Iquitos, Peru, in 1997. After feeding on hamsters with viremias of about 10(8) plaque-forming units of virus per ml, Cx. gnomatos was the most efficient vector. Other species, such as Ochlerotatus fulvus and Psorophora cingulata, although highly susceptible to infection, were not efficient laboratory vectors of this virus due to a significant salivary gland barrier. The Cx. (Culex) species, consisting mostly of Cx. (Cux.) coronator, were nearly refractory to subtype IIIC virus and exhibited both midgut infection as well as salivary gland barriers. Additional studies on biting behavior, mosquito population densities, and vertebrate reservoir hosts of subtype IIIC virus are needed to determine the role that these species play in the maintenance and spread of this virus in the Amazon Basin region.

Vector competence of California mosquitoes for West Nile virus.

To identify the mosquito species competent for West Nile virus (WNV) transmission, we evaluated 10 California species that are known vectors of other arboviruses or major pests: Culex tarsalis, Cx. pipiens pipiens, Cx. p. quinquefasciatus, Cx. stigmatosoma, Cx. erythrothorax, Ochlerotatus dorsalis, Oc. melanimon, Oc. sierrensis, Aedes vexans, and Culiseta inornata. All 10 became infected and were able to transmit WNV at some level. Ochlerotatus, Culiseta, and Aedes were low to moderately efficient vectors. They feed primarily on mammals and could play a secondary role in transmission. Oc. sierrensis, a major pest species, and Cx. p. quinquefasciatus from southern California were the least efficient laboratory vectors. Cx. tarsalis, Cx. stigmatosoma, Cx. erythrothorax, and other populations of Cx. pipiens complex were the most efficient laboratory vectors. Culex species are likely to play the primary role in the enzootic maintenance and transmission of WNV in California.

Vector competence of Culex tarsalis from Orange County, California, for West Nile virus.

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.

Vector competence of selected North American Culex and Coquillettidia mosquitoes for West Nile 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.

Potential North American vectors of West Nile virus.

The outbreak of disease in the New York area in 1999 due to West Nile (WN) virus was the first evidence of the occurrence of this virus in the Americas. To determine potential vectors, more than 15 mosquito species (including Culex pipiens, Cx. nigripalpus, Cx. quinquefasciatus, Cx. salinarius, Aedes albopictus, Ae. vexans, Ochlerotatus japonicus, Oc. sollicitans, Oc. taeniorhynchus, and Oc. triseriatus) from the eastern United States were evaluated for their ability to serve as vectors for the virus isolated from birds collected during the 1999 outbreak in New York. Mosquitoes were allowed to feed on one- to four-day old chickens that had been inoculated with WN virus 1-3 days previously. The mosquitoes were incubated for 12-15 days at 26 degrees C and then allowed to refeed on susceptible chickens and assayed to determine transmission and infection rates. Several container-breeding species (e.g., Ae. albopictus, Oc. atropalpus, and Oc. japonicus) were highly efficient laboratory vectors of WN virus. The Culex species were intermediate in their susceptibility. However, if a disseminated infection developed, all species were able to transmit WN virus by bite. Factors such as population density, feeding preference, longevity, and season of activity also need to be considered in determining the role these species could play in the transmission of WN virus.

Ecology of mosquitoes and St. Louis encephalitis virus in the Los Angeles Basin of California, 1987-1990.

St. Louis encephalitis (SLE) virus has become established in the Los Angeles Basin of California, where it most likely is maintained by horizontal transmission among Culex tarsalis Coquillet and passerine birds in park-riparian habitat. Viral transmission also was detected at low levels in residential habitat by the infrequent seroconversion of sentinel chickens. SLE virus activity was documented in all months except March and April. Cx. tarsalis was incriminated as the primary vector, based on elevated field infection rates (0.17 infected females per 1,000 tested), population abundance concomitant in time and space with increased SLE virus activity, vector competence, and vectorial capacity. Culex stigmatosoma Dyar also may be important in the horizontal maintenance of SLE virus because this species was an efficient laboratory vector, fed frequently on passeriform birds, and had field infection rates (0.15 females per 1,000) comparable with Cx. tarsalis. However, difficulty in trapping adult females precluded the assessment of Cx. stigmatosoma because abundance may have been underestimated and comparatively few females were tested for virus. Although Cx. quinquefasciatus Say was the most abundant species in residential habitats (greater than 85% of total catch at CO2 traps) where it occasionally fed on humans (0.6% of total tested), this species was considered to be of secondary importance because field infection rates were low (0.03 females per 1,000) and few females became infected and transmitted SLE virus in laboratory vector competence experiments. Although Culex erythrothorax Dyar fed on humans in marsh habitat (5% of total tested), this species infrequently fed on birds (12%) and was refractory to SLE virus infection in vector competence experiments; therefore, it was not considered a vector of SLE virus. Future SLE virus surveillance and mosquito control programs should be directed toward Cx. tarsalis populations.

Development of Plasmodium vivax in Anopheles superpictus under experimental conditions.

The susceptibility of Anopheles superpictus for Plasmodium vivax was described quantitatively under laboratory conditions. Of the 697 laboratory females studied in 16 groups, 513 (73.5%) females took a bloodmeal, of which 88.4% developed ookinetes on day 1, 56.1% oocysts between days 3 and 11, and 52.5% sporozoites in the salivary gland on days 15 to 63 post-infection. Sporogony was completed in 10-11 days post-infection. There was no difference in the longevity of uninfected and infected females. Infected females survived an average of 30 days (maximum 63 days). Sporozoites survived up to 50 days in the salivary glands without any observable changes in structure and motility. These data indicate that An. superpictus is an efficient laboratory vector of P. vivax and should not be ignored in future entomological field studies.

Morphology of the Microfilaria of Brugia pahangi and of the Larval Stages in the Mosquito

The detailed morphology of the microfilariae of B. pahangi following several methods of preparation is described. Possible features which will allow its rapid differentiation from the microfilariae of B. and of B. patei are the size and proportions of the cephalic space. Among eleven species of Louisiana mosquitoes tested, Anopheles quadrimaculatus, A. crucians, and Psorophora confinnis were susceptible to infection with B. pahangi. Although infective larvae were recovered from all three species, encapsulation of some larvae was seen in A. quadrimaculatus and P. confinnis. As the chemical nature of the encapsulating material is not known, it is suggested that the term chitinization reaction be dropped. In the development of B. pahangi in the mosquito, the first molt occurs between 4 and 5 days and the second molt at about 8 days, after which the larvae are immediately infective. During the first 3 days of development, microfilarial structures are rearranged and the nerve ring is obliterated. Cell multiplication during this period is minimal; cell growth is marked and larvae become much shorter. The developmental period just preceding the first molt is distinguished by rapid cell multiplication, lengthening of the body, and differentiation of internal structures. The cephalic space of the microfilaria does not form the buccal cavity or any other structure of the infective larva but is shed undeveloped at the first molt. It is suggested that the supernumerary nuclei of the terminal caudal appendage of the microfilaria may be the retained polar bodies. The tail of the microfilaria containing the supernumerary chromatin dots is retained throughout the first and second stages of development. If not dislodged by dissection procedures, its presence affords a reliable distinguishing feature for separating the firstand second-stage larvae of B. pahangi from those of other genera, but not of congeneric species. Infective larvae of B. pahangi could not be distinguished from those of B. malayi. The sex of individual larvae can be identified in late second stage by the position of the genital primordium which is at or just behind the esophago-intestinal junction in males, and at the midesophageal level in females. In light of this and other findings, it is suggested that the R1 cell does not form the genital primordium but develops into the intestine, and that the R2-4 cells are incorporated into the rectum. Brugia pahangi (Buckley and Edeson, 1956) Buckley, 1958, is a filarial parasite of the malayi group principally found in carnivores, but also found in a wide range of other types of hosts. Edeson et al. (1960a) were the first to demonstrate that this worm would develop to the adult stage and produce microfilariae in man. An earlier attempt to infect humans (Buckley, 1958a, b) failed to produce a patent Received for publication 12 January 1962. *A portion from a dissertation submitted to the Graduate School of Tulane University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. This investigation was conducted under the sponsorship of the Commission on Parasitic Diseases, Armed Forces Epidemiological Board, and supported in part by the Office of the Surgeon General, Department of the Army. t Present address: Department of Tropical Health, Schools of Public Health and Medicine, American University of Beirut, Beirut, Lebanon. infection (helminthologically) but did result in severe symptoms of disease strikingly similar to those of eosinophilic lung (tropical eosinophilia). The body length and certain other features of the microfilariae of B. pahangi have been described and compared with those of semiperiodic B. by Laing et al. (1960). These authors and Edeson et al. (1960b) found that Armigeres obturbans is an efficient laboratory vector for B. pahangi. Among the other mosquitoes tested Mansonia (Mansonioides) annulatus and Anopheles barbirostris proved to be the best hosts. M. annulatus and M. longipalpus apparently are the natural vectors in Malaya. Published descriptions of the larvae include length and breadth measurements, anal ratios of the various stages, and characteristics of the terminal papillae of the infective stage (Edeson et al., 1960b; Nelson, 1959). The primary purpose of the present study