An orbivirus of mosquitoes which induces CO2 sensitivity in mosquitoes and is lethal for rabbits.

An orbivirus, JKT-7400, isolated from Culex mosquitoes in Indonesia, replicated to a high titer and induced cytopathic effects in Aedes albopictus cell cultures. The virus produced lethal sensitivity to carbon dioxide in Culex and Aedes mosquitoes as well as in Drosophila melanogaster fruit flies but was not the agent of the hereditary sensitivity to carbon dioxide previously described for Culex quinquefasciatus. When injected intravenously in high doses, JKT-7400 virus was lethal for rabbits, apparently without replicating to a significant extent. It was not pathogenic for adult mice inoculated intravenously or for adult or suckling mice inoculated intracerebrally and intraperitoneally. Unlike an orbivirus isolated from Culex mosquitoes in China, JKT-7400 did not interfere with the replication of Japanese encephalitis virus in mosquitoes.     

Vertical transmission of hepatitis B virus in Culex quinquefasciatus

An investigation of the vertical transmission of hepatitis B virus (HBV) in Culex quinquefasciatus Say revealed the presence of low levels of the virus in adult F1 progeny from the first ovarian cycle of mosquitoes infected by feeding on HBV positive human blood. HBV was not transmitted vertically during the second, third and fourth ovarian cycles nor to the F2 generation. The salivary glands, ovaries and faeces of the F1 generation did not contain detectable levels of HBV. Progeny of female Cx quinquefasciatus mated with F1 males were negative for HBV.     

Identification of super-infected Aedes triseriatus mosquitoes collected as eggs from the field and partial characterization of the infecting La Crosse viruses

Background

La Crosse virus (LACV) is a pathogenic arbovirus that is transovarially transmitted by Aedes triseriatus mosquitoes and overwinters in diapausing eggs. However, previous models predicted transovarial transmission (TOT) to be insufficient to maintain LACV in nature.

Results

To investigate this issue, we reared mosquitoes from field-collected eggs and assayed adults individually for LACV antigen, viral RNA by RT-PCR, and infectious virus. The mosquitoes had three distinct infection phenotypes: 1) super infected (SI+) mosquitoes contained infectious virus, large accumulations of viral antigen and RNA and comprised 17 of 17,825 (0.09%) of assayed mosquitoes, 2) infected mosquitoes (I+) contained no detectable infectious virus, lesser amounts of viral antigen and RNA, and comprised 3.7% of mosquitoes, and 3) non-infected mosquitoes (I-) contained no detectable viral antigen, RNA, or infectious virus and comprised 96.21% of mosquitoes. SI+ mosquitoes were recovered in consecutive years at one field site, suggesting that lineages of TOT stably-infected and geographically isolated Ae. triseriatus exist in nature. Analyses of LACV genomes showed that SI+ isolates are not monophyletic nor phylogenetically distinct and that synonymous substitution rates exceed replacement rates in all genes and isolates. Analysis of singleton versus shared mutations (Fu and Li's F*) revealed that the SI+ LACV M segment, with a large and significant excess of intermediate-frequency alleles, evolves through disruptive selection that maintains SI+ alleles at higher frequencies than the average mutation rate. A QTN in the LACV NSm gene was detected in SI+ mosquitoes, but not in I+ mosquitoes. Four amino acid changes were detected in the LACV NSm gene from SI+ but not I+ mosquitoes from one site, and may condition vector super infection. In contrast to NSm, the NSs sequences of LACV from SI+ and I+ mosquitoes were identical.

Conclusions

SI+ mosquitoes may represent stabilized infections of Ae. triseriatus mosquitoes, which could maintain LACV in nature. A gene-for-gene interaction involving the viral NSm gene and a vector innate immune response gene may condition stabilized infection.     

Analysis of a Dengue Model with Vertical Transmission and Application to the 2014 Dengue Outbreak in Guangdong Province,China

There is evidence showing that vertical transmission of dengue virus exists in Aedes mosquitoes. In this paper, we propose a deterministic dengue model with vertical transmission in mosquitoes by including aquatic mosquitoes (eggs, larvae and pupae), adult mosquitoes (susceptible, exposed and infectious) and human hosts (susceptible, exposed, infectious and recovered). We first analyze the existence and stability of disease-free equilibria, calculate the basic reproduction number and discuss the existence of the disease-endemic equilibrium. Then, we study the impact of vertical transmission of the virus in mosquitoes on the spread dynamics of dengue. We also use the model to simulate the reported infected human data from the 2014 dengue outbreak in Guangdong Province, China, carry out sensitivity analysis of the basic reproduction number in terms of the model parameters, and seek for effective control measures for the transmission of dengue virus.     

Maxillary palps are broad spectrum odorant detectors in Culex quinquefasciatus

A single type of olfactory sensilla on maxillary palps in many species of mosquitoes houses a very sensitive olfactory receptor neuron (ORN) for carbon dioxide reception. We performed extensive single sensillum recordings from this peg sensillum in Culex quinquefasciatus and have characterized the response threshold and kinetics for CO(2) reception, with a detection threshold less than the CO(2) concentration in the atmosphere. This ORN responded in a tonic mode to lower concentrations of CO(2), whereas higher concentrations generated a phasic-tonic mode of action potential firing. Sensillum potentials accurately represented the response magnitude and kinetics of carbon dioxide-elicited excitatory responses. Stimulation of these ORNs with human breath, a complex mixture of mosquito kairomones and up to 4.5% CO(2), elicited excitatory responses that were reliably detected by CO(2)-sensitive ORNs. Another ORN housed in these sensilla responded to 1-octen-3-ol and to various plant-derived compounds, particularly floral and green leaf volatiles. This ORN showed remarkable sensitivity to the natural enantiomer, (R)-(-)-1-octen-3-ol, rivaling pheromone-detecting ORNs in moths. Maximum neuronal response was elicited with a 10 ng dose. A biological, ecological role of maxillary palps in detection of plant- and nectar-related sources is proposed.     

Cytoplasmic incompatibility as a means of controlling Culex pipiens quinquefasciatus mosquito in the islands of the south-western Indian Ocean

The use of the bacterium Wolbachia is an attractive alternative method to control vector populations. In mosquitoes, as in members of the Culex pipiens complex, Wolbachia induces a form of embryonic lethality called cytoplasmic incompatibility, a sperm-egg incompatibility occurring when infected males mate either with uninfected females or with females infected with incompatible Wolbachia strain(s). Here we explore the feasibility of the Incompatible Insect Technique (IIT), a species-specific control approach in which field females are sterilized by inundative releases of incompatible males. We show that the Wolbachia wPip(Is) strain, naturally infecting Cx. p. pipiens mosquitoes from Turkey, is a good candidate to control Cx. p. quinquefasciatus populations on four islands of the south-western Indian Ocean (La Réunion, Mauritius, Grande Glorieuse and Mayotte). The wPip(Is) strain was introduced into the nuclear background of Cx. p. quinquefasciatus mosquitoes from La Réunion, leading to the LR[wPip(Is)] line. Total embryonic lethality was observed in crosses between LR[wPip(Is)] males and all tested field females from the four islands. Interestingly, most crosses involving LR[wPip(Is)] females and field males were also incompatible, which is expected to reduce the impact of any accidental release of LR[wPip(Is)] females. Cage experiments demonstrate that LR[wPip(Is)] males are equally competitive with La Réunion males resulting in demographic crash when LR[wPip(Is)] males were introduced into La Réunion laboratory cages. These results, together with the geographic isolation of the four south-western Indian Ocean islands and their limited land area, support the feasibility of an IIT program using LR[wPip(Is)] males and stimulate the implementation of field tests for a Cx. p. quinquefasciatus control strategy on these islands.     

Arbovirus detection in synanthropic mosquitoes from the Brazilian Amazon and in mosquito saliva using Flinders Technology Associates cards

Although arbovirus transmission and identifying target vectors may provide a baseline for planning disease control strategies, there are many gaps in knowledge regarding these mosquitoes and viral species in urban, rural, or sylvatic habitats in the Brazilian Amazon. Our goal was to screen for dengue, chikungunya, and Zika viruses in synanthropic mosquitoes and with Flinders Technology Associates (FTA) cards using insect saliva. Mosquitoes were caught using ovitraps and aspirators in the city of Porto Velho, Rondônia, Brazil. Honey-baited FTA cards were placed in mosquito cages for 15 days; whole mosquitoes and FTA cards were analysed for viral RNA using RT-qPCR assays. One pool of Aedes aegypti females was found to be infected with the Zika virus and one male mosquito was infected with dengue-4, suggesting natural vertical/venereal transmission. Our study also reported evidence of vertical/venereal transmission of ZIKV in Culex quinquefasciatus males for the first time in the Brazilian Amazon, and the feasibility of using FTA cards to detect arboviruses in the saliva of field-collected mosquitoes. Vertical/venereal transmission of viruses by atypical mosquito species reinforces the need for combined viral and entomological screening in arbovirus surveillance programs.     

The inheritance of thiabendazole resistance in Haemonchus contortus.

Haemonchus contortus worm populations isolated from naturally infected sheep at the Pastoral Research Laboratory, Armidale, N.S.W., were found to contain approximately 20% of worms resistant to a 50 mg/kg dose of thiabendazole. Following 3 generations of selection with 50 mg/kg thiabendazole the number of worms removed by the anthelmintic was too small to detect differences between treated and control groups. After more than 15 generations of selection, matings between males from the selected strain and non-resistant females produced resistant males and females in equal numbers. Thus, thiabendazole resistance does not appear to be sex-linked. A dose--response assay on the F2 adults indicated that worms from female resistant x male non-resistant crosses were more resistant than F2 adults of the reciprocal cross. An in vitro technique that identified thiabendazole-resistant eggs by their ability to hatch in a solution containing thiabendazole and 0.1% NaCl solution was also used to study the inheritance of resistance. F1 eggs had similar LC50's to the resistant parents. F2 and back-cross eggs from an original mating of thiabendazole-resistant females x non-resistant males had a higher LC50 than F2 and back-cross eggs from the reciprocal mating, indicating a degree of matroclinous inheritance of resistance. However, the resistant parents had tolerances to thiabendazole exceeding those of F2. F3 eggs had a resistance distribution that ranged from that of the resistant to the non-resistant parent. No significant deviation from linearity was observed in any of the dose--response lines. These results indicate that thiabendazole resistance in H. contortus worms is inherited as an autosomal and semi-dominant trait.     

Differential modulation of murine host immune response by salivary gland extracts from the mosquitoes Aedes aegypti and Culex quinquefasciatus

Mosquitoes (Diptera: Culicidae) are major vectors of numerous infectious agents. Compounds in mosquito saliva not only facilitate blood-feeding, but may also have an impact upon the immune system of vertebrate hosts. Consequently, the exposure to mosquito saliva may influence pathogen transmission, establishment and disease development. Using two medically important vector mosquitoes, Aedes aegypti (L.) and Culex quinquefasciatus Say, we examined the effects of mosquito saliva on immune cells of host mice. After antigen-specific or non-specific stimulation, murine splenocyte proliferation and production of both Th1 and Th2 cytokines were significantly reduced in the presence of salivary gland extract (SGE) from Ae. aegypti, but not SGE from Cx. quinquefasciatus. T cell populations were highly susceptible to this suppression, showing increased mortality and reduced division rates - judged by flow cytometric analyses. Evidently these two culicine mosquitoes differ in their host immunomodulatory activities.     

Independent Production of Co2 Sensitivity by Nuclear Gene DLY and a Transmissible Agent from Delayed-Recovery DROSOPHILA MELANOGASTER

Flies of stocks designated delayed-recovery by McCrady and Sulerud (1964) remain temporarily paralyzed after exposure to carbon dioxide. This condition is similar to CO2-sensitivity, which occurs in flies infected with the maternally transmitted sigma viruus, but is due, at least in part, to the third chromosome mutant gene Dly. Because earlier work indicated that extracts of delayed-recovery flies could occasionally transmit CO2 sensitivity when injected into resistant recipients, we have tested the possibility that some delayed-recovery stocks contain a sigma-like transmissible virus, in addition to the Dly gene. We found that TDR-orange, a stock derived from the original delayed-recovery line, and temperature-cured populations of the same stock, both contain some agent that is transmissible by injection. TDR-BC3f, a stock derived by backcrossing through the male line to eliminate maternally transmitted factors, does not appear to contain such an infectious agent, but remains sensitive to CO2. These observations lead us to the conclusion that the originally described delayed-recovery stocks harbor an infectious extrachromosomal agent, in addition to possessing the Dly gene, and each is capable of producing a sensitivity to carbon dioxide.     

West Nile Virus: High Transmission Rate in North-Western European Mosquitoes Indicates Its Epidemic Potential and Warrants Increased Surveillance

Background

West Nile virus (WNV) is a highly pathogenic flavivirus transmitted by Culex spp. mosquitoes. In North America (NA), lineage 1 WNV caused the largest outbreak of neuroinvasive disease to date, while a novel pathogenic lineage 2 strain circulates in southern Europe. To estimate WNV lineage 2 epidemic potential it is paramount to know if mosquitoes from currently WNV-free areas can support further spread of this epidemic.

Methodology/Principal Findings

We assessed WNV vector competence of Culex pipiens mosquitoes originating from north-western Europe (NWE) in direct comparison with those from NA. We exposed mosquitoes to infectious blood meals of lineage 1 or 2 WNV and determined the infection and transmission rates. We explored reasons for vector competence differences by comparing intrathoracic injection versus blood meal infection, and we investigated the influence of temperature. We found that NWE mosquitoes are highly competent for both WNV lineages, with transmission rates up to 25%. Compared to NA mosquitoes, transmission rates for lineage 2 WNV were significantly elevated in NWE mosquitoes due to better virus dissemination from the midgut and a shorter extrinsic incubation time. WNV infection rates further increased with temperature increase.

Conclusions/Significance

Our study provides experimental evidence to indicate markedly different risk levels between both continents for lineage 2 WNV transmission and suggests a degree of genotype-genotype specificity in the interaction between virus and vector. Our experiments with varying temperatures explain the current localized WNV activity in southern Europe, yet imply further epidemic spread throughout NWE during periods with favourable climatic conditions. This emphasizes the need for intensified surveillance of virus activity in current WNV disease-free regions and warrants increased awareness in clinics throughout Europe.     

Fine scale spatiotemporal clustering of dengue virus transmission in children and Aedes aegypti in rural Thai villages

Background

Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that support it, we examined relationships between infected humans and Aedes aegypti in Thai villages.

Methodology/Principal Findings

Geographic cluster investigations of 100-meter radius were conducted around DENV-positive and DENV-negative febrile “index” cases (positive and negative clusters, respectively) from a longitudinal cohort study in rural Thailand. Child contacts and Ae. aegypti from cluster houses were assessed for DENV infection. Spatiotemporal, demographic, and entomological parameters were evaluated. In positive clusters, the DENV infection rate among child contacts was 35.3% in index houses, 29.9% in houses within 20 meters, and decreased with distance from the index house to 6.2% in houses 80–100 meters away (p<0.001). Significantly more Ae. aegypti were DENV-infectious (i.e., DENV-positive in head/thorax) in positive clusters (23/1755; 1.3%) than negative clusters (1/1548; 0.1%). In positive clusters, 8.2% of mosquitoes were DENV-infectious in index houses, 4.2% in other houses with DENV-infected children, and 0.4% in houses without infected children (p<0.001). The DENV infection rate in contacts was 47.4% in houses with infectious mosquitoes, 28.7% in other houses in the same cluster, and 10.8% in positive clusters without infectious mosquitoes (p<0.001). Ae. aegypti pupae and adult females were more numerous only in houses containing infectious mosquitoes.

Conclusions/Significance

Human and mosquito infections are positively associated at the level of individual houses and neighboring residences. Certain houses with high transmission risk contribute disproportionately to DENV spread to neighboring houses. Small groups of houses with elevated transmission risk are consistent with over-dispersion of transmission (i.e., at a given point in time, people/mosquitoes from a small portion of houses are responsible for the majority of transmission).     

Activation, orientation and landing of female Culex quinquefasciatus in response to carbon dioxide and odour from human feet: 3-D flight analysis in a wind tunnel

This study investigated the interaction between carbon dioxide (CO(2) ) and human foot odour on activation, upwind orientation and landing of host-seeking female Culex quinquefasciatus (Say) (Diptera: Culicidae) in a wind tunnel. More mosquitoes landed on warmed glass beads coated with foot odour than on clean beads; adding a plume of 4% CO(2) did not influence the proportion of mosquitoes landing. A second experiment used 3-dimensional video tracking to assess flight performance. Activation was more rapid with CO(2) and with CO(2) + foot odour than with clean air or with foot odour alone. Upwind flights were fastest with CO(2) and with clean air, and slowest with foot odour; the CO(2) + foot odour treatment overlapped the previous three treatments in significance. Flight headings tended more towards due upwind with CO(2) and with clean air than with CO(2) + foot odour or with foot odour alone. In both experiments, many mosquitoes flew upwind in clean air. There was little evidence of females changing course upon entering or exiting the CO(2) plume or reacting to foot odour during flight.     

Transovarial Transmission of Chikungunya Virus by Aedes albopictus Mosquitoes Ingesting Microfilariae of Dirofilaria immitis under Laboratory Conditions

Female Aedes albopictus mosquitoes of the Miki strain were experimentally fed on defibrinated sheep blood containing 5× 10⁷ PFU of chikungunya virus and 20,000 microfilariae of Dirofilaria immitis per milliliter. Fully engorged mosquitoes transmitted the virus to a small percentage of the F1 progeny, but females of the F1 generation did not transmit the virus to the F2 progeny. The control mosquitoes that ingested the virus without microfilariae did not transmit the virus to their eggs, larvae, or pupae in the F1 or F2 generations. These results showed that A. albopictus of this strain that concurrently ingested the virus and microfilariae transmitted the virus by the transovarial route under experimental conditions.     

The impact of maturation delay of mosquitoes on the transmission of West Nile virus

We formulate and analyze a delay differential equation model for the transmission of West Nile virus between vector mosquitoes and avian hosts that incorporates maturation delay for mosquitoes. The maturation time from eggs to adult mosquitoes is sensitive to weather conditions, in particular the temperature, and the model allows us to investigate the impact of this maturation time on transmission dynamics of the virus among mosquitoes and birds. Numerical results of the model show that a combination of the maturation time and the vertical transmission of the virus in mosquitoes has substantial influence on the abundance and number of infection peaks of the infectious mosquitoes.     

Microplate adaptation of Gomori's assay for quantitative determination of general esterase activity in single insects

Esterase activity is monitored in mosquitoes and other arthropod species because high levels of these enzymes can be associated with pesticide resistance. In the 1950s, G. Gomori devised a colorimetric method to detect esterase activity based on their capacity to hydrolyze aryl-esters. We modified this method for use in microtiter plates. Mosquito homogenates (Culex quinquefasciatus Say and C. pipiens L.) from strains susceptible and resistant to insecticides were allowed to hydrolyze alpha-naphthyl acetate in the presence of Triton X-100 and a specific acetylcholinesterase inhibitor. The alpha-naphthol product was detected colorimetrically by a diazo-coupling reaction with Fast Garnet GBC salt. Triton X-100 improved the extraction of esterases and maintained the azo compound in solution. The linear range of the method was 2-20 nmoles of alpha-naphthol; this high sensitivity permitted accurate determinations in 1/30 portions of single adult mosquitoes from the strain with the lowest esterase activity. To avoid variations due to changes in temperature and duration of assay, results were normalized to equivalent enzyme activity units obtained in a spectrophotometer at 25 degrees C. Depending on the number of homogenate dilutions required, performance of the assay in microplates allowed the simultaneous analysis of 20-80 samples. Female mosquitoes showed higher enzyme activity than males when expressed in nmoles/min per mosquito, but differences were reduced when results were expressed as specific activity (nmoles/min per mg protein). A mosquito strain resistant to organophosphates due to the presence of high levels of esterases showed about 200 times more esterase activity than a susceptible strain or a strain resistant due to insensitive acetylcholinesterase.     

Attempted mechanical transmission of lumpy skin disease virus by biting insects

The mosquitoes Anopheles stephensi Liston and Culex quinquefasciatus Say (Diptera: Culicidae), the stable fly Stomoxys calcitrans Linnaeus (Diptera: Muscidae) and the biting midge Culicoides nubeculosus Meigen (Diptera: Ceratopogonidae) were allowed to feed on either lumpy skin disease (LSD) infected animals or through a membrane on a bloodmeal containing lumpy skin disease virus (LSDV). These arthropods were then allowed to refeed on susceptible cattle at various intervals after the infective feed. Virus was detected in the insects by polymerase chain reaction immediately after feeding and at sufficiently high titre to enable transmission to occur. However, no transmission of virus from infected to susceptible animals by An. stephensi, S. calcitrans, C. nubeculosus and Cx. quinquefasciatus was observed.     

Horizontal transmission of murine retroviruses.

Both a feral mouse ecotropic virus (WM-E) and Friend ecotropic virus (F-MuLV) were transmitted horizontally among adult mice. Infection resulted in the production of antiviral antibody in the recipients, with no evidence of viremia or clinical disease. However, persistent low-level virus replication was detectable in the spleens of these mice as long as 8 months after initial infection. External secretions, including saliva, semen, and uterine secretions from viremic mice contained high concentrations of infectious virus. Nevertheless, transmission occurred only from viremic males to either males or females. Male-to-male transmission appeared to occur by parenteral inoculation of infectious saliva during fighting behavior. Evidence is presented that infection of females was by the venereal route. Of four mouse strains examined, NFS/N, IRW, and C57L females were all susceptible to venereal infection, whereas AKR mice were not. Since AKR mice are susceptible to infection by WM-E administered parenterally, this resistance appeared to be mediated by local viral interference due to the high-level expression of endogenous Akv gp70 within the female reproductive tract. Although both WM-E and F-MuLV were transmitted from viremic males to females, infection by WM-E was significantly more efficient than that by F-MuLV. This difference correlated with a distinct difference in cellular tropism of WM-E and F-MuLV within the epididymis of viremic males. F-MuLV gp70 was expressed only within stromal elements, whereas WM-E gp70 was seen largely within the epithelial lining cells and luminal contents of the duct. No evidence of virus expression within germ cells was observed. The possible influence of virus expression by epithelial cells of the female reproductive tract on infection of embryos is discussed.     

Major histocompatibility complex-conferred resistance to Theiler's virus-induced demyelinating disease is inherited as a dominant trait in B10 congenic mice.

Intracerebral inoculation of Theiler's murine encephalomyelitis virus into susceptible strains of mice produces chronic demyelinating disease in the central nervous system characterized by persistent viral infection. Immunogenetic data suggest that genes from both major histocompatibility complex (MHC) and non-MHC loci are important in determining susceptibility or resistance to demyelination. The role of the MHC in determining resistance or susceptibility to disease can be interpreted either as the presence of antigen-presenting molecules that confer resistance to viral infection or as the ability of MHC products to contribute to pathogenesis by acting as viral receptors or by mediating immune attack against virally infected cells. These alternatives can be distinguished by determining whether the contribution of the MHC to resistance is inherited as a recessive or dominant trait. Congenic mice with different MHC haplotypes on identical B10 backgrounds were crossed and quantitatively analyzed for demyelination, infectious virus, and local virus antigen production. F1 hybrid progeny derived from resistant B10 (H-2b), B10.D2 (H-2d), or B10.K (H-2k) and susceptible B10.R111 (H-2r), B10.M (H-2f), or B10.BR (H-2k) parental mice exhibited no or minimal demyelination, indicating that on a B10 background, resistance is inherited as a dominant trait. Although infectious virus, as measured by viral plaque assay, was cleared inefficiently from the central nervous systems of resistant F1 hybrid progeny mice, we found a direct correlation between local viral antigen production and demyelination. These data are consistent with our hypothesis that the immunological basis for resistance is determined by efficient presentation of the viral antigen to the immune system, resulting in local virus clearance and absence of subsequent demyelination.