Detection of sequences from a potentially novel strain of cell fusing agent virus in Mexican Stegomyia (Aedes) aegypti mosquitoes

Flaviviruses (FVs) are a very heterogeneous group of viruses that includes viruses capable of infecting insects and/or vertebrates. Different human-disease-causing FVs are disseminated by mosquitoes, and therefore, the search for FV in these insects has recently been proposed in order to evaluate their potential transmission in a given community. An entomological survey was carried out in Colima (the hyperendemic dengue fever transmission zone in Mexico) to collect culicidae in urban and wild areas. No human-pathogenic FVs were found, but sequences related to a potentially novel strain of cell fusing agent virus (CFAV) were detected in Stegomyia (Aedes) aegypti mosquitoes.     

The pathogenesis of Vesicular Stomatitis Virus,serotype Indiana,inAedes aegypti mosquitoes,after imbibition of a viremic blood meal

Summary This study showed that Vesicular Stomatitis Virus (Indiana) in most instances was not capable of replicating inAedes aegypti when imbibed by the mosquitoes on a viremic host. Rapid inactivation of the virus was observed in some cases within 24 hours after imbibition. Attempts to demonstrate virus inactivation by midgut contentsin vitro were not successful.     

Infection with the wMel and wMelPop strains of Wolbachia leads to higher levels of melanization in the hemolymph of Drosophila melanogaster, Drosophila simulans and Aedes aegypti

Introduction of the life-shortening strain of Wolbachia pipientis, wMelPop, into the key dengue vector, Aedes aegypti, and the anti-pathogen effects in Wolbachia-infected hosts highlights the need for more research into its interactions with its original host, Drosophila melanogaster, and the novel mosquito host. The visual difference in darkness between the eggs of wMelPop Wolbachia-infected and uninfected mosquito hosts after egg deposition led to further investigation into melanization levels of the insects. Both D. melanogaster and A. aegypti infected with wMelPop showed increased levels of melanization, especially in females. This result was also seen in D. melanogaster and Drosophila simulans infected with the closely related wMel strain. D. simulans infected with other strains of Wolbachia did not display this difference. HPLC analysis of hemolymph from mosquitoes showed that this difference was not due to dopamine levels in the host as they were no different in wMelPop-infected and control mosquitoes before or after blood feeding.     

Serological relationships of an iridescent virus (type 25) recently isolated from Tipula sp. with two other iridescent viruses (types 2 and 22).

The isolation, purification, and serological properties of a 130-nm iridescent virus (IV), type 25, from Tipula sp. is reported. The polypeptide profile of this isolate was different from that of another small Dipterous iridescent virus-IV22 isolated from Simulium sp. However, these viruses were indistinguishable when compared by serum neutralization, gel immunodiffusion, complement fixation, tube immunoprecipitation, and immune electron microscopy. Iridescent virus type 2 isolated from Sericesthis pruinosa was shown to be distinct from IV22 and IV25 by all of these techniques.     

Replication of Chilo iridescent virus in the cotton boll weevil, Anthonomus grandis, and development of an infectivity assay

Summary.  The boll weevil, Anthonomus grandis Boheman, is a devastating pest of cotton. Chemical pesticides are problematic due to relative lack of target specificity and resistance. Microbial pesticides may provide viable alternatives because of their narrow host range. Chilo iridescent virus (CIV) is the type species for genus Iridovirus, family Iridoviridae: large, icosahedral cytoplasmic viruses containing a double-stranded DNA genome. Earlier work suggested that CIV replicated in the boll weevil; however, efficiency or production of infectious virus was not established. We showed that CIV undergoes a productive cycle in A. grandis. CIV DNA levels in boll weevil pupae increased significantly from 0 to 3 days post infection. Moreover, virogenic stromata and complete virus particles were observed in the cytoplasm by 7 days. An endpoint dilution assay using viral DNA replication as indicator suggested a 10⁵-fold increase in infectious virus titer over 7 days. This is the first such demonstration in larval infections with genus Iridovirus. Our study establishes that CIV undergoes a productive cycle in the boll weevil and provides an important and useful model system for replication at the organismal level. These results have important implications for the potential of CIV and its components in boll weevil control. Received April 12, 2000 Accepted September 25, 2000     

Characterization of piry virus

Summary Piry virus was found to multiply inAedes aegypti mosquitoes and in cell cultures ofAedes aegypti andAedes albopictus. The virus was demonstrated in sections of brain of infected baby mice, and in BHK21 andA. albopictus cell cultures by electron microscopy. In sections of infected cells the virus is seen as bullets measuring 155 × 62 m, budding from membranes of endoplasmic cysterns or marginal cytoplasmic membranes. The virus was propagated in BHK21 cell cultures, concentrated, and purified by ultracentrifugation by pelleting or continuous flow into density gradients. Negatively stained preparations of such concentrates exhibit predominantly spherical and disc-shaped particles. Bullet-shaped particles are rarely found. Such preparations have nevertheless high viral titers and it is suggested that the bullets separate into spheres and discs. The separation appears to be enhanced by storage, by repeated freezing and thawing, and by foaming of the virus suspension. This phenomenon as well as the ultra-structure is being investigated further.Dedicated to Professor Dr. Dr. C.Hallauer on the occasion of his 70th birthday.     

Fatty acid profiles of invertebrate iridescent viruses

Summary Eight invertebrate iridescent viruses (IVs) from diverse host taxa were grown in a common lepidopteran host,Galleria mellonella. The lipid composition of purified virus was assessed by fatty acid methyl esterase (FAME) analysis using a gas-liquid chromatograph. IV fatty acid profiles were markedly different from those of the host tissues. The interrelationships among the IVs did not follow previous serological and genetic findings. We conclude that FAME analysis is not a useful technique for revealing phylogenetic relationships among these viruses.     

Molecular Anatomy of Chilo Iridescent Virus Genome and the Evolution of Viral Genes

Chilo iridescent virus (CIV) or Insect iridescent virus 6 (IIV-6) is the type species of the genus iridovirus, a member of the Iridoviridae family. CIV is highly pathogenic for a variety of insect larvae and this implicates a possible use as a biological insecticide. CIV progeny and assembly occur in the cytoplasm of the infected cell and accumulate in the fatbody of the infected insects. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure and the amino acid sequences of different viral gene products. The elucidation of the coding capacity and strategy of CIV was the first step towards understanding the underlying mechanisms of viral infection, replication and virus-host interaction. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The coding capacity of the CIV genome was determined by the analysis of the complete DNA nucleotide sequence consisting of 212,482bp that represent 468 open reading frames encoding for polypeptides ranging from 40 to 2432 amino acid residues. The analysis of the coding capacity of the CIV genome revealed that 50% (234 ORFs) of all identified ORFs (468 ORFs) were non-overlapping. The identification of several putative viral gene products including a DNA ligase and a viral antibiotic peptide is a powerful tool for the investigation of the phylogenetic relatedness of this evolutionary and ecologically relevant eukaryotic virus.     

Susceptibility of Aedes aegypti (Diptera: Culicidae) to Acanthamoeba polyphaga (Sarcomastigophora: Acanthamoebidae)

To date there is no report on mosquitoes infected with free-living amoebae. For this reason, the aim of this study was to verify if Aedes aegypti could be susceptible to Acanthamoeba polyphaga under laboratory conditions, so trophozoites were offered as a unique food resource for larvae of first instar. The results show that those amoebae are able to infect and colonize the mosquito gut and could be re-isolated of all stages of the mosquito (larvae, pupae, and adults).     

Electroantennogram,flight orientation,and oviposition responses of <Emphasis Type="Italic">Aedes aegypti</Emphasis> to the oviposition pheromone <Emphasis Type="Italic">n</Emphasis>-heneicosane

Oviposition pheromones specifically influence the females of many insects to lay eggs in the sites resulting in more egg deposition. A previous report describes the principal role of n-heneicosane (C₂₁) identified and characterized from the larval cuticle of Aedes aegypti (L.) in attracting the gravid mosquitoes to oviposit in treated substrates among other chemical components. However, the means by which this compound is perceived by the females for oviposition has not been reported. In this study, we have recorded the peripheral olfactory responses from the antenna of Ae. aegypti from 10⁻⁷ g to 10⁻³ g doses of n-heneicosane. The EAG response of female mosquitoes increased in a dose-dependent manner with increasing stimulus strength. In the orientation assay using Y-maze olfactometer, female mosquitoes were attracted to the odor plume of 10⁻⁶ g and 10⁻⁵ g dose, while the higher dose of 10⁻³ g plume enforced repellency to gravid mosquitoes. The response to oviposition substrates by gravid Ae. aegypti females differed across the range of concentrations of n-heneicosane under multiple choice conditions, larger number of eggs were deposited in 10 ppm (10 mg/l) solutions compared to lower and higher concentrations indicating 10 ppm was most attractive. Application of n-heneicosane at 10 ppm in breeding habitats will be a useful method to attract the gravid mosquitoes using ovitraps for surveillance and monitoring. The possible use of this compound in monitoring of mosquito population in endemic areas in relevance to integrated vector management strategies is discussed in detail.     

Dengue-2-virus-interacting polypeptides involved in mosquito cell infection

For the design of effective antiviral strategies, understanding the fundamental steps of the virus life cycle, including virus–host interactions, is essential. We performed a virus overlay protein binding assay followed by proteomics for identification of proteins from membrane fractions of A7 (Aedes aegypti) cells, C6/36 (Aedes albopictus) cells and the midgut brush border membrane fraction of Ae. aegypti mosquito that bind to dengue-2 virus. Actin, ATP synthase β subunit, HSc 70, orisis, prohibitin, tubulin β chain, and vav-1 were identified as dengue-2-virus-binding proteins. Our results suggest that dengue-2 virus exploits an array of housekeeping proteins for its entry in mosquito cells.     

Transmission of Northway and St. Louis encephalitis viruses by arctic mosquitoes

Summary Transmission of a Canadian arctic isolate of Northway virus has been demonstrated after incubation of arcticAedes communis mosquitoes at 13° C for 27 days after intrathoracic injection of 300 plaque forming units of virus. Replication has also been demonstrated after intrathoracic injection of domesticA. aegypti mosquitoes of this virus. Virions of Northway virus, 84–92 nm diameter were morphologically typical of a bunyavirus after propagation in salivary glands ofA. communis or in tissue cultures of baby hamster kidney (BHK-21) cells. An Ontario isolate of St. Louis encephalitis was transmitted by bites ofA. communis after 27 days incubation at 13° C after oral ingestion of 3 or 30 mouse LD₅₀ virus. This mosquito species transmitted virus after 13 to 76 days incubation at 13° C following intrathoracic injection of 3 mouse LD₅₀ or higher virus doses.Supported through the Medical Research Council Canada Grant MT-2811 and the National Research Council Canda Contract 031-604.     

California encephalitis virus transmission by arctic and domestic mosquitoes

Summary A zero passage arctic mosquito isolate of California encephalitis (CE) virus (showshoe hare subtype) was transmitted by wild-caughtAedes communis mosquitoes after 13 days incubation at 13° and 23° C, after 20 days incubation at 13° C, when mosquitoes imbibed 1 mouse LD₅₀ in a blood meal. Transmission occurred after 20 days incubation at 13° and 23° C when mosquitoes were injected intrathoracically with 1 or 0.1 mouse LD₅₀. Virus was also transmitted byA. aegypti 13 days after infection with 100 mouse LD₅₀ by feeding or intrathoracic injection, and incubation at 13° C. Virus antigen was detected in salivary glands of 42 per cent virus-positiveA. communis mosquitoes by direct immunofluorescence, and in 50 per cent or less ofA. aegypti mosquitoes by immunoperoxidase and immunofluorescence, with somewhat greater regularity by the indirect than the direct technique.This work was supported by Operating Grant MT-2811, Medical Research Council, Canada.     

Replication of feline coronaviruses in peripheral blood monocytes

Summary. Feline infectious peritonitis virus (FIPV) (Coronaviridae) causes the most lethal viral infection in cats: FIP. The related feline enteric coronavirus (FECV) causes mild enteritis. Why these feline coronaviruses manifest so differently in vivo is not known. In this study, infection kinetics (titres and antigen expression) of FIPV 79-1146, and FECV 79-1683, were determined in peripheral blood monocytes from 3 donor cats and compared to those in Crandell feline kidney (CrFK) cells. The infection kinetics in monocytes were host dependent. Monocytes from 1 cat were resistant to both FIPV- and FECV-infection. Monocytes from the other 2 cats could initially be infected by both FIPV and FECV but FIPV infection was sustained in monocytes of only one cat. FECV-infection was never sustained and viral production was up to 100 times lower than in FIPV-infected monocytes. In CrFK cells, FIPV and FECV infection kinetics did not differ. In monocytes of a larger cat population (n = 19) the 3 infection patterns were also found. Considering all 22 investigated cats, 3/22 were not susceptible for FIPV and FECV. The rest could be infected with FECV and FIPV but 10/22 cats had monocytes that only sustained FIPV infection and 9/22 sustained neither FIPV nor FECV infection. Both authors contributed equally.     

Are fish paratenic natural hosts of the caiman haemoparasite Hepatozoon caimani?

The susceptibility of two fish and four mosquito species to the Caiman yacare haemoparasite Hepatozoon caimani was experimentally investigated. Mosquitoes belonging to four species (Aedes fluviatilis, Aedes albopictus, Aedes aegypti and Culex quinquefasciatus) were blood-fed on two naturally infected C. yacare from the Central-West Region of Brazil that exhibited distinct levels of parasitaemia: caimans A (11.05 %) and B (1.25 %). None of the engorged A. fluviatilis, A. albopictus or A. aegypti mosquitoes fed on caiman A survived for the duration of the sporogonic cycle; the great majority of the engorged mosquitoes died within 48 h of the blood meal. All A. aegypti fed on caiman B were negative, whereas 91.3 % of dissected C. quinquefasciatus fed on the same caiman contained oocysts. Characid fish—Metynnis sp. and Astyanax sp.—were individually fed with C. quinquefasciatus females previously engorged (21–23 days) on caiman B. No parasite was found in the Astyanax fish. By contrast, 100 % of the Metynnis fish depicted numerous cysts harbouring cystozoites identical to those of H. caimani, even more than 8 months after the ingestion of the infected mosquitoes. The cysts were located near the veins of the liver and, in some cases, close to the tunica intima of these vessels. No inflammatory reaction was observed. Gametocytes were observed in the blood smears of juvenile caimans that had ingested infected fish 9–12 weeks earlier. The potential role of fish as paratenic vertebrate hosts of H. caimani in nature is discussed.     

Intranuclear polypeptides of measles and subacute sclerosing panencephalitis virus-infected cultures

CV-1 cells infected with either measles virus or five different isolates of SSPE virus each manifest a unique overall pattern of intranuclear polypeptides as well as differences in growth and yields of infectious particles. Of the viral structural proteins within the nucleus, both the P and M species most commonly exhibit migration differences on polyacrylamide gels. Each virus also generates a nonstructural protein of 20,000 daltons. Two of the SSPE viruses grow more slowly than the other viruses and produce significantly lower yields of infectious particles. Their nuclear isolates contain the most slowly migrating M protein, as well as two large species of 80,000 and 135,000 daltons. These findings suggest that there are differences in nuclear polypeptides between the viruses, not reflected in virion structural protein composition nor in the infected host cell cytoplasm, and that these changes occur particularly in less productive, more indolent infections.     

Morphological,morphometric, and molecular characterization of <Emphasis Type="Italic">Hepatozoon</Emphasis> spp. (Apicomplexa,Hepatozoidae) from naturally infected <Emphasis Type="Italic">Caudisona durissa terrifica</Emphasis> (Serpentes,Viperidae)

Hepatozoon spp. are the most frequent intracellular protozoa in snakes. Considering the variety of parasites infecting specimens of Caudisona durissa terrifica and the divergent data in literature where only two species, Hepatozoon romani and Hepatozoon capsulata, are described, the aim of this study was to morphologically, morphometrically, and molecularly characterize Hepatozoon spp. from some naturally infected specimens of C. durissa terrifica, and observe changes caused by these protozoa in parasitized erythrocytes. Four snakes were examined. Two of them had two morphological distinct gamonts, while the other two had only one type of gamont. The six distinct gamonts were provisionally named gamonts A, B, C, D, E, and F. Statistical analysis, however, confirmed the existence of only four parasite populations, those which were capable of inducing significant alterations in determined red blood cells variables. Attempts to infect Aedes aegypti and Culex quinquefasciatus mosquitoes were done for each snake specimen. Some mosquitoes became infected and oocysts were recovered and measured. The detection of Hepatozoon DNA was obtained with success but the molecular characterization was unable to differentiate species of the samples, with respect to the fragment studied.     

Host range and symptom variation of pseudorecombinant virus produced by two distinct bipartite geminiviruses

Summary.  Within the whitefly group only the species Bemisia tabaci (Gennadius) is the vector. Most whitefly-transmitted geminiviruses possess bipartite DNA genomes, DNAs A and B. Although they are closely related to each other, the production of viable pseudorecombinants between bipartite geminiviruses by reassortment of infectious cloned components is generally limited to strains of a particular virus. Following exchange of cloned genomic components of Sida golden mosaic virus (SiGMV/Ho_yv) and Abutilon mosaic virus (AbMV), the pseudorecombinant viruses were infectious in various host plants. The symptom type of pseudorecombinant virus was in most cases determined by DNA B. However, in some host plants also DNA A of the pseudorecombinant virus was involved in the symptom phenotype. Received October 29, 1999 Accepted March 1, 2000