Vector competence of selected African mosquito (Diptera: Culicidae) species for Rift Valley fever virus

Outbreaks of Rift Valley fever (RVF) in Egypt, Yemen, and Saudi Arabia have indicated the potential for this disease to spread from its enzootic areas in sub-Saharan Africa. Because little is known about the potential for most African mosquito species to transmit RVF virus (family Bunyaviridae, genus Phlebovirus, RVFV), we conducted studies to determine the vector competence of selected African species of mosquitoes for this virus. All eight species tested [Aedes palpalis (Newstead), Aedes mcintoshi Huang, Aedes circumluteolus (Theobald), Aedes calceatus Edwards, Aedes aegypti (L.), Culex antennatus (Becker), Culex pipiens (L.), and Culex quinquefasciatus Say], were susceptible to infection, and all except Ae. calceatus, Ae. aegypti and Cx. quinquefasciatus transmitted RVFV by bite after oral exposure. Estimated transmission rates for mosquitoes that successfully transmitted RVFV by bite ranged from 5% for Ae. mcintoshi to 39% for Ae. palpalis for mosquitoes that fed on a hamster with a viremia > or = 10(8) plaque-forming units of virus/ml. We did not recover RVFV from any of 3,138 progeny of infected female mosquitoes. RVFV is unusual among arboviruses in that it has been isolated in nature from a large number of species and that numerous mosquitoes and other arthropods are able to transmit this virus in the laboratory. The recent introduction and spread of West Nile virus into the Americas and the spread of RVFV to the Arabian Peninsula illustrates the potential for viruses, once enzootic in Africa, to spread to other parts of the world.     

Comparison of the transmission potential of two genetically distinct Sindbis viruses after oral infection of Aedes aegypti (Diptera: Culicidae)

Within mosquitoes, arboviruses encounter barriers to infection and dissemination that are critical determinants of vector competence. The molecular mechanisms responsible for these barriers have yet to be elucidated. The prototype Sindbis (SIN) strain, AR339, and viruses derived from this strain, such as TR339 virus, have limited infection and transmission potential in the medically important arthropod vector, Aedes aegypti (L.). However, the Malaysian SIN virus strain, MRE16, disseminates in nearly 100% of Ae. aegypti 14 d after oral infection. Here, we compare the spatial and temporal infection patterns of MRE16 and TR339 viruses in Ae. aegypti. The results indicate that a midgut escape barrier is primarily responsible for the significantly lower dissemination and transmission potentials observed after oral infection with TR339 virus. MRE16 and TR339 viruses now represent a well-characterized model system for the further study of virus determinants of vector infection, particularly determinants affecting the midgut escape barrier in Ae. aegypti.     

Arbovirus growth inAedes aegypti mosquitoes throughout their viable temperature range

After intrathoracic inoculation of laboratory-bred Aedes aegypti mosquitoes with 3 Yukon isolates of California encephalitis (CE) virus (showshoe hare subtype), Northway (NOR) and Murray Valley encephalitis (MVE) viruses, viral replication was observed following incubation at 13, 21, 35 and 39 degrees C, which constituted the full temperature range of viability of A. aegypti. Rates of viral replication were reduced at low temperatures and accelerated at high temperatures. Virus-specific immunoperoxidase staining of mosquito salivary glands occurred regularly after thoraces attained maximum infectivity levels. At 13 and 21 degrees C, mosquitoes were infected by 10 to 100 times less CE and MVE viruses than mice, but about 10 times more NOR virus was required to infect mosquitoes than mice.     

Potential of ancestral sylvatic dengue-2 viruses to re-emerge

Dengue viruses (DENV) are the most important arboviral pathogens in tropical and subtropical regions throughout the world. DENV transmission includes both a sylvatic, enzootic cycle between nonhuman primates and arboreal mosquitoes of the genus Aedes, and an urban, endemic/epidemic cycle between Aedes aegypti, a mosquito with larval development in peridomestic water containers, and human reservoir hosts. All 4 serotypes of endemic DENV evolved independently from ancestral sylvatic viruses and have become both ecologically and evolutionarily distinct; this process may have involved adaptation to (i) peridomestic mosquito vectors and/or (ii) human reservoir hosts. To test the latter hypothesis, we assessed the ability of sylvatic and endemic DENV-2 strains, representing major genotypes from Southeast Asia, West Africa and the Americas, to replicate in two surrogate human model hosts: monocyte-derived, human dendritic cells (moDCs), and mice engrafted with human hepatoma cells. Although the various DENV-2 strains showed significant inter-strain variation in mean replication titers in both models, no overall difference between sylvatic and endemic strains was detected in either model. Our findings suggest that emergence of endemic DENV strains from ancestral sylvatic strains may not have required adaptation to replicate more efficiently in human reservoir hosts, implying that the potential for re-emergence of sylvatic dengue strains into the endemic cycle is high. The shared replication profiles of the American endemic and sylvatic strains suggest that American strains have maintained or regained the ancestral phenotype.     

Glycosylation of the dengue 2 virus E protein at N67 is critical for virus growth in vitro but not for growth in intrathoracically inoculated Aedes aegypti mosquitoes

To determine the importance of dengue 2 virus (DEN2V) envelope (E) protein glycosylation, virus mutants in one or both of the N-linked glycosylation motifs were prepared. We found that while the E2 mutant virus (N153Q) replicated in mammalian and mosquito cells, the E1 (N67Q) and E1/2 (N67Q and N153Q) mutant viruses were unable to grow in mammalian cells. Infection of C6/36 mosquito cells with either the E1 or E1/2 mutants resulted in the introduction of a compensatory mutation, K64N, restoring glycosylation in the area. All mutants replicated similarly in inoculated Aedes aegypti mosquitoes, with no change in their mutations. These results suggest that N-linked glycosylation of the E protein is not necessary for DEN2V replication in mosquitoes, however N-linked glycosylation at amino acid N67 (or nearby N64) is critical for the survival of the virus in either mammalian or insect cell culture.     

Occurrence and evolutionary significance of a California encephalitis-like virus in Aedes squamiger (Diptera: Culicidae)

More than 12,000 Aedes increpitus Dyar and 4,600 Aedes squamiger (Coquillett) were tested for the presence of arboviruses to test the hypothesis that there is a coevolutionary relationship between Aedes (Ochlerotatus) mosquitoes and California serogroup viruses. Five strains of a California encephalitis-like virus were isolated from adults reared from larvae of Ae. squamiger collected in January 1989 from a coastal salt marsh at Morro Bay, San Luis Obispo County, California. Viruses were isolated in Vero cell cultures and serotyped by cross-neutralization tests. These isolates represent the first arboviruses isolated from this species. On the basis of morphology, Aedes squamiger has been included in the Aedes stimulans group of the subgenus Ochlerotatus. Other species within the Ae. stimulans group are vectors of California (CAL) serogroup viruses elsewhere in North America. Analysis of isozyme variability supports the inclusion of Ae. squamiger in the Ae. stimulans group and suggests that coastal populations of Ae. increpitus are the closest California relatives of Ae. squamiger. Recovery of virus from Ae. squamiger reinforces the relationship between CAL serogroup viruses and Aedes (Ocherlotatus) mosquitoes. However, the failure to isolate virus from large samples of Ae. increpitus from coastal and low elevation inland habitats suggests a complex evolutionary history involving both vertical and horizontal transmission mechanisms.     

Arboviroses in the region of Nosy-Bé, Madagascar. Serologic and entomologic data

Since 1977, the Pasteur Institute of Madagascar has been studying, during six surveys, the arboviruses of Nosy-Be area, in the north-west of Madagascar. 47.2% out of 271 human sera and 11.3% out of 151 sera of Lemurs, tested for antibodies to 16 arboviruses by the haemagglutination inhibition test, are positive. The results show an important prevalence of Flaviviruses. West Nile and Dengue 1 viruses were probably circulating some years before the surveys. Antibodies against Sindbis and Rift Valley Fever viruses, were found only in few subjects. Bunyamwera and California groups of virus are absent. The rate of positive Lemurs is weak, particularly in Lemur macaco macaco. Flaviviruses are the most frequent. 12,262 haematophagous diptera (11,965 Culicidae belonging to 40 species) were caught. Aedes aegypti and Aedes albopictus are both present. Arbovirus isolation attempts from 394 mosquito pools failed; only Mengo virus was isolated from four pools of Eretmapodites quinquevittatus and one pool of Aedes (Skusea) sp.     

Failure of dengue-2 virus antibody to interfere with the isolation of dengue-2 virus from Aedes aegypti (Diptera: Culicidae)

When isolating dengue virus (DEN) from mosquitoes collected in endemic areas, pools may contain both anti-dengue antibodies from freshly engorged females and virus from DEN infected females. To determine if these antibodies may interfere with virus isolation, we simulated the isolation procedure using Aedes aegypti (L.) that we infected with the 16,681 strain of dengue type 2 virus by intrathoracic inoculation. At 7 d postinfection, we allowed females to engorge on immunized or normal mouse blood. Virus in a mixture of anti-dengue-2 antibodies and dengue-2 virus became inactive after incubation at 37 degrees C for 1 h, but remained infective without incubation. Therefore, at ambient conditions antibodies would not interfere with virus isolation from field-collected Ae. aegypti from endemic areas. In addition, DEN antibodies enhanced virus replication when inoculated into Ae. aegypti, but not C6/36 cells. The mechanism for this in vitro antibody enhancement of infection remains unclear.     

Susceptibility of urban and rural populations of Aedes albopictus from São Paulo State, Brazil, to infection by dengue-1 and -2 viruses

In Brazil, dengue viruses (DENV) are transmitted by Aedes aegypti (L.). However, the recent introduction of Aedes albopictus (Skuse) has caused concern, because some populations of this species also are capable of DENV transmission. Ae. albopictus from rural and urban localities of S?o Paulo State, Brazil, were artificially exposed to infection with DENV-1 and -2 to evaluate their susceptibility. The rates of infection (6.2 and 5.7% for DENV-1; 5.3 and 12% for DENV-2) and dissemination (22.2 and 40% for DENV-1; 0 and 20% for DENV-2) were low in both urban and rural populations, with no significant differences detected between these mosquito populations.     

Failure of dengue viruses to replicate in Culex quinquefasciatus (Diptera: Culicidae).

Culex quinquefasciatus (Say) and Aedes aegypti (L.) were parenterally infected with dengue viruses and virus replication was monitored at intervals after infection in each species. Dengue viruses replicated rapidly in Ae. aegypti, reaching a peak titer of 10(6)-10(7) mosquito infectious dose 50 (MID50) per mosquito. In Cx. quinquefasciatus, however, dengue virus replication did not occur. We conclude that this mosquito species is refractory to infection with dengue viruses and, therefore, does not serve as a vector in nature.     

蚊虫抗登革病毒天然免疫效应

埃及伊蚊和白纹伊蚊是传播登革病毒（Dengue viruses）的主要媒介,通过一次感染血餐,登革病毒被蚊摄取,病毒首先感染蚊中肠组织,病毒越过蚊中肠到达蚊其它组织,包括涎腺。当感染病毒的蚊再次叮吸另一健康人血时,将涎腺中病毒传播给下一个人。蚊利用它的天然免疫系统抗登革病毒而使其“不发病”,并能生存传播这种病毒。蚊抗登革病毒天然免疫主要涉及血细胞、Toll、JAK—STAT、RNAi通道等。本文就蚊抗登革病毒天然免疫效应做了综述。     

Rates of infection in, and transmission of, African horse-sickness virus by Aedes aegypti mosquitoes.

Very low infection rates (less than 3%) were obtained when Aedes aegypti mosquitoes ingested blood contained 5.8--6.5 log10 MLD50/0.02 ml African horse sickness virus (AHSV). When A. aegypti mosquitoes were inoculated intrathoracically with virus, however, high infection rates were achieved. Mosquitoes infected by inoculum failed to transmit virus to embryonated hens eggs by bite, and virus could not be detected in membrane or blood when inoculated mosquitoes were allowed to engorge on uninfected blood through a chick skin membrane. It was concluded that the mosquito A. aegypti is unlikely to be an effective vector of AHSV.     

Identification and characterization of prohibitin as a receptor protein mediating DENV-2 entry into insect cells

Dengue is transmitted primarily by mosquitoes of the Aedes genus. Despite a number of studies, no insect dengue virus receptor protein has been clearly identified and characterized. Using a number of separation methodologies and virus overlay protein binding assays we identified a 35 kDa protein that segregated with susceptibility to dengue serotype 2 (DENV-2) infection in two mosquito species and two mosquito cell lines. Mass spectroscopy identified the protein to be prohibitin, a strongly conserved and ubiquitously expressed protein in eukaryotic cells. Antibody mediated inhibition of infection and siRNA mediated knockdown of prohibitin expression significantly reduced infection levels and subsequent virus production in both Aedes aegypti and Aedes albopictus cell lines. Confocal microscopy showed a significant degree of intracellular colocalization between prohibitin and DENV-2 E protein, and coimmunoprecipitation confirmed that prohibitin interacts with dengue E. Prohibitin is the first characterized insect cell expressed dengue virus receptor protein.     

广东省登革热原学和血清学检测

目的 了解１９７８－１９９５年广东省多次发生登革热Ⅰ－Ⅳ型流行的病学原学。方法 在流行区捕捉９种蚊媒，进行病毒分离。结果 阳性率白纹伊蚊为２０．６９％，埃及伊蚊为１８．９５％，致乏库蚊为１０．２９％，其余６种蚊媒为阴性。这三种蚊媒间的病毒分离阳性率无显著性差异。在海南岛和湛江地区，埃及伊蚊是主要的传播媒介，其他的流行区是白纹伊蚊。患者发病３天内病毒分离的阳性率在７３．５８－８２．０５％之间，发病第     

Rapid determination of viral RNA sequences in mosquitoes collected in the field

A method for rapid determination of viral RNA sequences (RDV) was applied to homogenates of Aedes aegypti collected in Thailand in an area in which dengue fever (dengue hemorrhagic fever) is endemic, using the mosquito cell line C6/36. Nucleic acid sequences of dengue virus type 4 and cell fusing agent virus were detected. This RDV method has the potential to become a standard method for detection of both known and newly emerging, unknown mosquito-borne viruses.     

Nineteen nucleotides in the variable region of 3' non-translated region are dispensable for the replication of dengue type 1 virus in vitro

In many flaviruses, first 50-400 nucleotides of 3' non-translated region (3' NTR) exhibit lower conservation level than other regions and are called "variable region". Two dengue type 1 virus (DENV-1) strains, which have 17- and 29-nt deletion in the variable region, were recently isolated from Japanese dengue fever patients. The effect of a small deletion in the 3' NTR was analyzed using two DENV-1 viruses which were prepared from a newly developed infectious cDNA clone. These included a recombinant virus rDENV-1(02-20), without any deletion in 3' NTR, and rDENV-1m10, with 19-nt deletion in the variable region of rDENV-1(02-20). These two viruses were compared for growth kinetics and plaque morphology in Vero, Huh-7 and C6/36 cells. No apparent difference was detected between rDENV-1(02-20) and rDENV-1m10 in replication efficiency and plaque size in these cell lines. The results suggest that the complete variable region of DENV-1 is dispensable for virus replication and propagation in vitro.