Association of human immune response to Aedes aegypti salivary proteins with dengue disease severity.

Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.

Detection of West Nile virus-specific antibodies and nucleic acid in horses and mosquitoes, respectively, in Nuevo Leon State, northern Mexico, 2006-2007.

In the last 5 years, there has been only one reported human case of West Nile virus (WNV) disease in northern Mexico. To determine if the virus was still circulating in this region, equine and entomological surveillance for WNV was conducted in the state of Nuevo Leon in northern Mexico in 2006 and 2007. A total of 203 horses were serologically assayed for antibodies to WNV using an epitope-blocking enzyme-linked immunosorbent assay (bELISA). Seroprevalences for WNV in horses sampled in 2006 and 2007 were 26% and 45%, respectively. Mosquito collections in 2007 produced 7365 specimens representing 15 species. Culex mosquitoes were screened for WNV RNA and other genera (Mansonia, Anopheles, Aedes, Psorophora and Uranotaenia) were screened for flaviviruses using reverse-transcription (RT)-PCR. Two pools consisting of Culex spp. mosquitoes contained WNV RNA. Molecular species identification revealed that neither pool included Culex quinquefasciatus (Say) (Diptera:Culicidae) complex mosquitoes. No evidence of flaviviruses was found in the other mosquito genera examined. These data provide evidence that WNV is currently circulating in northern Mexico and that non-Cx. quinquefasciatus spp. mosquitoes may be participating in the WNV transmission cycle in this region.

Venereal transmission of La Crosse (California encephalitis) arbovirus in Aedes triseriatus mosquitoes.

Veneral transmission of La Crosse virus by males of Aedes triseriatus was demonstrated. La Crosse virus was detected in the bursa of females after induced copulation, and disseminated infection was shown to occur occasionally. Since males of Aedes triseriatus have transovarial filial infection rates similar to those of females and can repeatedly mate, veneral transmission may be an important supplement to other natural endemic maintenance mechanisms.

A G1 glycoprotein epitope of La Crosse virus: a determinant of infection of Aedes triseriatus.

Arthropod-borne viruses (arboviruses) have specific vector-vertebrate host cycles in nature. The molecular basis of restriction of virus replication to a very limited number of vector species is unknown, but the present study suggests that viral attachment proteins are important determinants of vector-virus interactions. The principal vector of La Crosse (LAC) virus is the mosquito Aedes triseriatus, and LAC virus efficiently infects the mosquito when ingested. However, a variant (V22) of LAC virus, which was selected by growing the virus in the presence of a monoclonal antibody, was markedly restricted in its ability to infect Ae. triseriatus when it was ingested. Only 15% of the mosquitoes that ingested V22 became infected and 5% of these developed disseminated infections. In contrast, 89% of the mosquitoes that ingested LAC became infected and 74% developed disseminated infections. When V22 was passed three times in mosquitoes by feeding, a revertant virus, V22M3, was obtained that infected 85% of Ae. triseriatus ingesting this virus. In addition, V22M3 regained the antigenic phenotype and fusion capability of the parent LAC virus. These results suggest that the specificity of LAC virus-vector interactions is markedly influenced by the efficiency of the fusion function of the G1 envelope glycoprotein operating at the midgut level in the arthropod vector.

Evolution of bunyaviruses by genome reassortment in dually infected mosquitoes (Aedes triseriatus).

Aedes triseriatus mosquitoes became dually infected after ingesting two mutants of LaCrosse (LAC) virus simultaneously or after ingesting, by interrupted feeding, the two viruses sequentially within a 2-day period. After 2 weeks of incubation, approximately 25 percent of the vectors contained new virus genotypes as the result of RNA segment reassortment. New viruses were transmitted when the mosquitoes fed on mice. Viruses ingested more than 2 days after the initial infecting virus did not cause superinfection of the mosquito vectors.

Molecular basis of bunyavirus transmission by mosquitoes: role of the middle-sized RNA segment.

In an examination of the molecular basis of oral transmission of bunyaviruses by mosquitoes., La Crosse (LAC), snowshoe hare (SSH), and LAC-SSH reassortant viruses were compared in their ability to be transmitted to laboratory mice by the natural mosquito vector of LAC virus, Aedes triseriatus. Both LAC virus and the reassortment viruses containing the middle-sized (M) segment from the LAC parent were efficiently transmitted. In contrast, SSH virus and reassortment viruses containing the M RNA from the SSH parent were inefficiently transmitted. Thus, the M RNA segment, which codes for the virion glycoproteins, may be a major determinant of oral transmission of bunyaviruses by mosquitoes.

Genetically engineered resistance to dengue-2 virus transmission in mosquitoes.

The control of arthropod-borne virus diseases such as dengue may ultimately require the genetic manipulation of mosquito vectors to disrupt virus transmission to human populations. To reduce the ability of mosquitoes to transmit dengue viruses, a recombinant Sindbis virus was used to transduce female Aedes aegypti with a 567-base antisense RNA targeted to the premembrane coding region of dengue type 2 (DEN-2) virus. The transduced mosquitoes were unable to support replication of DEN-2 virus in their salivary glands and therefore were not able to transmit the virus.

Entomological studies along the Colorado Front Range during a period of intense West Nile virus activity.

To better understand the ecology of West Nile virus transmission in Northern Colorado, field studies were conducted in Larimer and Weld counties from September 2003 through March 2005. During summer studies, 18,540 adult mosquitoes were collected using light traps and gravid traps. West Nile virus RNA was detected in 24 of the 2,140 mosquito pools tested throughout the study area in 2003 and 2004. Culex tarsalis had the highest minimum infection rate (MIR) in both 2003 (MIR = 34.48) and in 2004 (MIR = 8.74). During winter studies, 9,391 adult mosquitoes were collected by aspirator from various overwintering sites including bridges and storm drains. The most frequently collected species was Culex pipiens. West Nile virus was not detected in our overwintering collections. The relationship between spring adult emergence and temperature inside and outside overwintering sites is described. Species composition of collections as well as the spatial and temporal distribution of West Nile virus detections are presented.

Effect of La Crosse virus infection on insemination rates in female Aedes triseriatus (Diptera: Culicidae).

Aedes triseriatus (Say) (Diptera: Culicidae) females orally infected with La Crosse virus after ingesting an infectious bloodmeal were compared for mating efficiency with females that ingested a noninfectious bloodmeal. After 14-d extrinsic incubation to allow for dissemination of the infection, all females were offered a second noninfectious bloodmeal and were placed in cages with age-matched males for 5 d. After 6 d, insemination rates were determined by detection of sperm in the spermathecae. Insemination rates of the La Crosse virus-infected females were significantly greater than in uninfected females.

Quantitative trait loci that control vector competence for dengue-2 virus in the mosquito Aedes aegypti.

Quantitative trait loci (QTL) affecting the ability of the mosquito Aedes aegypti to become infected with dengue-2 virus were mapped in an F(1) intercross. Dengue-susceptible A. aegypti aegypti were crossed with dengue refractory A. aegypti formosus. F(2) offspring were analyzed for midgut infection and escape barriers. In P(1) and F(1) parents and in 207 F(2) individuals, regions of 14 cDNA loci were analyzed with single-strand conformation polymorphism analysis to identify and orient linkage groups with respect to chromosomes I-III. Genotypes were also scored at 57 RAPD-SSCP loci, 5 (TAG)(n) microsatellite loci, and 6 sequence-tagged RAPD loci. Dengue infection phenotypes were scored in 86 F(2) females. Two QTL for a midgut infection barrier were detected with standard and composite interval mapping on chromosomes II and III that accounted for approximately 30% of the phenotypic variance (sigma(2)(p)) in dengue infection and these accounted for 44 and 56%, respectively, of the overall genetic variance (sigma(2)(g)). QTL of minor effect were detected on chromosomes I and III, but these were not detected with composite interval mapping. Evidence for a QTL for midgut escape barrier was detected with standard interval mapping but not with composite interval mapping on chromosome III.

Bunyaviridae--natural history.

Obviously, the family Bunyaviridae is comprised of a large number of epidemiologically diverse viruses. They vary dramatically in their vector and vertebrate host relationships, geographic distributions, and epidemic potential in humans and animals. Public health practitioners, veterinarians, virologists, entomologists, biologists, ecologists, molecular biologists, and other scientists will all benefit from increased study and knowledge of this fascinating group of viruses.

Control of arbovirus diseases: is the vector the weak link?

Arthropod-borne virus (arbovirus) diseases (ABVDs) remain major threats to human health and well-being and, as an epidemiologic group, inflict an unacceptable health and economic burden on humans and animals, including livestock. The developed world has been fortunate to have escaped much of the burden that arboviruses and their arthropod vectors inflict on humans in disease endemic countries, but the introduction and rapid spread of West Nile virus in the Western Hemisphere demonstrated that we can no longer be complacent in the face of these emerging and resurging vector-borne diseases. Unfortunately, as the burdens and threats of ABVDs have increased, the U.S. and international public health capacity to address them has decreased. Vaccines are not available for most of these agents. Previously successful strategies to control ABVDs emphasized vector control, but source reduction and vector control strategies using pesticides have not been sustainable. New insights into vector biology and vector pathogen interactions, and the novel targets that likely will be forthcoming in the vector post-genomics era, provide new targets and opportunities for vector control and disease reduction programs. These findings and approaches must be incorporated into existing strategies if we are to control these important pathogens.

Green fluorescent protein expressed in living mosquitoes--without the requirement of transformation.

Mosquitoes transmit viruses, protozoa and nematodes that are major causes of morbidity and mortality in humans. Details of arthropod anatomy and development, and the replication and development of pathogens in the arthropod vector, have relied upon examination of dissected or histologically processed material. We constructed a double-subgenomic Sindbis (dsSIN) virus expressing green fluorescent protein to demonstrate the potential of this protein for studying pathogen development in living arthropods. We were able to observe dissemination of virus, and furthermore, it was possible to observe components of the nervous system of mosquito larvae in extraordinary detail and record this on video tape. Although green fluorescent protein has been used as a reporter gene in a number of organisms, expression has relied upon transformation of cells or embryos. Transformation technology has limited applicability, thus we have described an alternative system that, due to the broad host range and viral tropisms of dsSIN viruses, may be useful to scientists in a range of disciplines. Green fluorescent protein may also provide a non-lethal selection method for use in transgenic arthropod research.

Bunyavirus-vector interactions.

Recent advances in the genetics and molecular biology of bunyaviruses have been applied to understanding bunyavirus-vector interactions. Such approaches have revealed which virus gene and gene products are important in establishing infections in vectors and in transmission of viruses. However, much more information is required to understand the molecular mechanisms of persistent infections of vectors which are lifelong but apparently exert no untoward effect. In fact, it seems remarkable that LAC viral antigen can be detected in almost every cell in an ovarian follicle, yet no untoward effect on fecundity and no teratology is seen. Similarly the lifelong infection of the vector would seem to provide ample opportunity for bunyavirus evolution by genetic drift and, under the appropriate circumstances, by segment reassortment. The potential for bunyavirus evolution by segment reassortment in vectors certainly exists. For example the Group C viruses in a small forest in Brazil seem to constitute a gene pool, with the 6 viruses related alternately by HI/NT and CF reactions, which assay respectively M RNA and S RNA gene products (Casals and Whitman, 1960; Shope and Causey, 1962). Direct evidence for naturally occurring reassortant bunyaviruses has also been obtained. Oligonucleotide fingerprint analyses of field isolates of LAC virus and members of the Patois serogroup of bunyaviruses have demonstrated that reassortment does occur in nature (El Said et al., 1979; Klimas et al., 1981; Ushijima et al., 1981). Determination of the genotypic frequencies of viruses selected by the biological interactions of viruses and vectors after dual infection and segment reassortment is an important issue. Should a virus result that efficiently interacts with alternate vector species, the virus could be expressed in different circumstances with serious epidemiologic consequences. Dual infection of vectors with different viruses is not unlikely, because many bunyaviruses are sympatric in nature. For example, the Ae. trivittatus-cottontail rabbit and the Ae. triseriatus-squirrel arbovirus cycles are sympatric in the ecotone between their respective grassland and forest ecosystems (LeDuc, 1979). Should a LaCrosse virus variant or reassortant evolve that was efficiently vectored by Ae. trivittatus mosquitoes, significantly more human infections with La Crosse virus would likely occur. Unlike Ae. triseriatus, Ae. trivittatus mosquitoes are not restricted to forested areas and consequently are more likely to encounter and to feed upon humans.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular immunization of mosquito cells to LaCrosse virus using a recombinant Sindbis virus vector.

A cDNA of the small RNA genome segment of La Crosse (LAC) virus was inserted, in an antisense orientation, into a double subgenomic Sindbis (dsSIN) virus expression vector generating pTE/3'2J/ANTI-S (15,000bp). In vitro transcription of the pTE/3'2J/ANTI-S template generated genomic RNA that was electrotransfected into BHK-21 cells to produce virus. Northern blot analysis of RNA isolated from infected Aedes albopictus (C6/36) cells showed that the TE/3'2J/ANTI-S virus produced a subgenomic mRNA of the appropriate size, indicating transcription of the LAC cDNA segment. C6/36 cells were infected with either TE/3'2J/ANTI-S, TE/3'2J (a dsSIN virus with no LAC insert), or wild type Sindbis (SIN, strain AR339) viruses and subsequently challenged with LAC virus. LAC virus titers were determined using a capture antibody ELISA. Mosquito cells infected with TE/3'2J/ANTI-S virus yielded at least 4 log10 TCID50/ml less LAC virus than cells infected with either TE/3'2J or AR339 SIN viruses. The use of the infectious SIN virus expression vectors provides a novel approach for high level cytoplasmic expression of genes or sequences of interest in arthropod cells, and for evaluating strategies for intracellular immunization against arboviruses.

Reassortment of La Crosse and Tahyna bunyaviruses in Aedes triseriatus mosquitoes.

Experiments were conducted to determine if La Crosse (LAC) and Tahyna (TAH) viruses reassort in Aedes triseriatus mosquitoes and to determine the genotypic frequencies of viruses selected by in vivo vector interactions. A molecular hybridization technique was used to analyze progeny viruses. Probes specific for the La Crosse L, M and S segments (pLAC4.16: LAC L RNA; pLAC4.27: LAC M RNA; pLAC4C-26: LAC S RNA) were used to determine the parental origin of the progeny RNA segments. Following infection with a mixture of LAC and TAH viruses, mosquitoes were held for 23 days extrinsic incubation, then assayed for reassortment. Individual progeny viruses were isolated by plaque assay and propagated in BHK-21 cells. Cytoplasmic RNA was extracted from the cells, blotted in triplicate to Nytran, and each blot was hybridized with 32P-labelled pLAC4.16, pLAC4.27 or pLAC4C-26 to determine the parental origin of each RNA segment. High frequency reassortment occurred in these mosquitoes. All of the expected genotypes resulting from a cross of LAC and TAH were obtained from these mosquitoes. Genotypic frequencies of 708 virus isolates from 39 mosquitoes were: LLL, 150 (21%); LLT, 71 (10%); LTL, 39 (5.5%); LTT, 109 (15%); TTT, 259 (36%); TTL, 16 (2.2%); TLT, 55 (7.8%); TLL, 9 (1.2%).

Molecular and genetic ecotoxicologic approaches to aquatic environmental bioreporting.

Molecular and population genetic ecotoxicologic approaches are being developed for the utilization of arthropods as bioreporters of heavy metal mixtures in the environment. The explosion of knowledge in molecular biology, molecular genetics, and biotechnology provides an unparalleled opportunity to use arthropods as bioreporter organisms. Interspecific differences in aquatic arthropod populations have been previously demonstrated in response to heavy metal insult in the Arkansas River (AR) California Gulch Superfund site (CGSS). Population genetic analyses were conducted on the mayfly Baetis tricaudatus. Genetic polymorphisms were detected in polymerase chain reaction amplified 16S mitochondrial rDNA (a selectively neutral gene) of B tricaudatus using single-strand conformation polymorphism analysis. Genetic differences may have resulted from impediments to gene flow in the population caused by mortality arising from exposure to heavy metal mixture pollution. In laboratory studies a candidate metal-responsive mucinlike gene, which is metal and dose specific, has been identified in Chironomus tentans and other potential AR-CGSS bioreporter species. Population genetic analyses using the mucinlike gene may provide insight into the role of this selectable gene in determining the breeding structure of B. tricaudatus in the AR-CGSS and may provide mechanistic insight into determinants of aquatic arthropod response to heavy metal insult. Metal-responsive (MR) genes and regulatory sequences are being isolated, characterized, and assayed for differential gene expression in response to heavy metal mixture pollution in the AR-CGSS. Identified promoter sequences can then be engineered into previously developed MR constructs to provide sensitive in vitro assays for environmental bioreporting of heavy metal mixtures. The results of the population genetic studies are being entered into an AR geographic information system that contains substantial biological, chemical, and geophysical information. Integrated spatial, structural, and temporal analyses of these parameters will provide invaluable information concerning environmental determinants that restrict or promote gene flow in bioreporter populations.

Detection of expressed chloramphenicol acetyltransferase in the saliva of Culex pipiens mosquitoes.

Mosquito salivary glands play an important role in the transmission of arthropod-borne pathogens. The ability to express genes in mosquitoes would be a powerful approach to characterize salivary gland genes, and to reveal important vector determinants of pathogen transmission. Here we report the use of a double subgenomic Sindbis (dsSIN) virus, designated TE/3'2J/CAT, and a packaged Sindbis replicon virus, designated rep5/CAT/26S, to express chloramphenicol acetyltransferase (CAT) protein in the salivary glands and saliva of transduced female Culex pipiens pipiens. Indirect immunofluorescence analysis revealed that salivary glands of these mosquitoes infected with either TE/3'2J/CAT or rep5/CAT/26S virus (4 or 6 days post-infection (p.i.)) were positive for both SIN E1 antigen and CAT protein. Saliva collected from mosquitoes transduced with TE/3'2J/CAT virus contained a unique 25 kDa protein that corresponded to the size of CAT protein. Additionally, CAT activity assays revealed that saliva collected from mosquitoes transduced with either TE/3'2J/CAT or rep5/CAT/26S virus could contain greater than 5.0 x 10(-5) units of CAT enzyme (3.0 x 10(6) CAT trimers).

The expression of chloramphenicol acetyltransferase in Aedes albopictus (C6/36) cells and Aedes triseriatus mosquitoes using a double subgenomic recombinant Sindbis virus.

Genomic RNA was transcribed in vitro from the double subgenomic recombinant Sindbis (SIN) virus expression vector, pTE/3'2J/CAT, and transfected into BHK-21 cells to generate recombinant virus stocks. TE/3'2J/CAT virus was used to infect C6/36 (Aedes albopictus) cells and adult female Aedes triseriatus. When C6/36 cells were infected with TE/3'2J/CAT virus at a multiplicity of infection (MOI) of greater than 20, 100% of the cells expressed CAT. The number of CAT polypeptides expressed per cell at 24 h post infection (pi) was 8.3 x 10(5). Approximately 4.0 log10TCID50 of the TE/3'2J/CAT virus was intrathoracically inoculated into adult female mosquitoes. Titers greater than 6.0 log10TCID50/ml were detected within 4 days pi and declined to less than 4.0 log10TCID50/ml 20 days following inoculation. CAT activity was detected within 2 days (8 x 10(-5) units of CAT/mosquito or 1.4 x 10(10) CAT polypeptides), peaked at day 6 (4 x 10(-3) units of CAT/mosquito or 7.2 x 10(11) CAT polypeptides), and remained at peak levels to day 20. Immunofluorescence and CAT activity assays were used to localize CAT expression in infected mosquitoes and demonstrated that CAT was present in neural, midgut, ovarian, and salivary gland tissues. Alphavirus-based expression vectors should be useful for expressing heterologous genes in mosquito cells as well as adult mosquitoes.

Interference to oral superinfection of Aedes triseriatus infected with La Crosse virus.

Aedes triseriatus orally infected with a temperature-sensitive mutant of La Crosse virus were, at predetermined times post-infection, orally challenged with wild type La Crosse or Tahyna virus. Most mosquitoes challenged with wild type La Crosse virus within 24 hr of ingestion of the temperature-sensitive virus became superinfected. In contrast, the majority of mosquitoes challenged at 72 hr were resistant to superinfection. Mosquitoes challenged at 7 days or thereafter were refractory to superinfection with La Crosse or Tahyna virus. The onset of interference was correlated with virus titer and antigen expression in midgut cells.