We found a new flavivirus that is widespread in Culex pipiens and other Culex mosquitoes in Japan. The virus isolate, named Culex flavivirus (CxFV), multiplied only in mosquito cell lines producing a moderate cytopathic effect, but did not grow in mammalian cells. The CxFV genome is single-stranded RNA, 10,834 nt in length and containing a single open reading frame encoding a polyprotein of 3362 aa with 5' and 3' untranslated regions (UTRs) of 91 and 657 nt, respectively. Phylogenetic analyses revealed that CxFV is closely related to the insect flaviviruses associated with Aedes mosquitoes, Cell fusing agent (CFA) and Kamiti River virus (KRV). The 3' UTR of CxFV contains four tandem repeats, which have sequence similarities to the two direct repeats in the CFA and KRV 3' UTRs. These results suggest that CxFV may be a new group of insect flaviviruses.
Number- and size-controlled macromolecular associations are common in biology with viruses as a typical example. We report here a novel example of artificial viruses, in which the double-helical DNA is coated with 4-nm sized neutral glycocluster nanoparticles (GNPs) with a coating stoichiometry of approximately 2 GNPs per helical pitch (10 base pairs), where GNP arises from micellization of a cone-shaped, quadruple-chain glycocluster amphiphile having eight saccharide moieties with beta-glucoside termini on the calix[4]resorcarene macrocycle. The resulting "glycoviruses" are compactly packed (54 nm in the case of 7040 base-pair plasmid pCMVluc), are well charge-shielded (zeta congruent with approximately 0 mV), and effectively transfect cell cultures without notable cytotoxicity. The use of artificial viral vectors thus allows a new (nonamine/noncationic/nonpolymeric) access to gene delivery, a potential but still tough subject which has been studied extensively over the last 15 years by using viral or amine-based cationic vectors. The remarkable adhesion-manipulation ability of saccharide clusters also provides a strategy of bottom-up construction of nanometric or mesoscopic sizes.
Abstract. An epidemic of enterohemorrhagic colitis caused by Escherichia coli O157:H7 (EHEC-O157) occurred in a nursery school in a rural area of Japan in September 1996. The EHEC-O157 were isolated both from patients and houseflies collected at the school. The flies were suspected to be mechanical vectors of the pathogen. Feeding experiments of EHEC-O157 to houseflies showed that the ingested bacteria were harbored in the intestine of flies and continued to be excreted at least for 3 days after feeding. Scanning electron microscopy showed that a large number of EHEC-O157 adhered to the surface of the housefly mouthparts and actively proliferated in the minute spaces of the labellum. Food masses containing EHEC-O157 in the fly intestine were completely surrounded by a peritrophic membrane during digestion and discharged rapidly. The persistence of bacteria in the intestine and feces is mainly a result of proliferation in the mouthparts and accumulation in the crop. Our results strongly suggest that houseflies are not simple mechanical vectors of EHEC. The epidemiologic potential of houseflies to disseminate EHEC-O157 may be greater than initially suspected.The housefly (Musca domestica vicina) and flies in general are considered to be mechanical vectors of many kinds of pathogens such as bacteria, 1,2 protozoa, 3 viruses, 4 and helminth eggs. 5,6 In recent outbreaks of enterohemorrhagic colitis in nursery schools in Japan, the epidemiologic survey isolated enterohemorrhagic Escherichia coli O157:H7 (EHEC-O157) from both houseflies collected in the school and from patients. The DNA patterns and the type of verotoxin were identical in EHEC-O157 isolated from both flies and patients. 7,8 This result strongly indicated that houseflies in nursery schools disseminated EHEC-O157 to foods, drinks, plates, and utensils, although direct evidence of the transmission has not been clarified. The infective dose for EHEC-O157 is considered to be very low, similar to that of Shigella flexneri or S. dysenteriae. 9,10 There are many qualitative reports of microbial flora in flies, but there are no reports of the number of bacteria that are harbored in the intestine or crop or how long the potential for dissemination of bacteria persists. We investigated the vector potential of houseflies for EHEC-O157 by feeding the flies with bacteria and monitoring them for several days after feeding. We also used scanning electron microscopy to determine whether EHEC-O157 adhered to the surface of housefly mouthparts and transmission electron microscopy to determine whether bacteria proliferated and persisted in the alimentary canal of flies. MATERIALS AND METHODS Experimental feeding of EHEC-O157 to houseflies.Houseflies maintained in our insectary were used. Two strains of EHEC-O157 (verotoxin 1-or verotoxin 1Ϫ and 2Ϫproducing strains) were grown in trypticase soy broth (Becton Dickinson, Cockeysville, MD). The concentration of bacteria was approximately 10 9 colony-forming units (CFU)/ml. Adult (6-8-day old), female flies were allowed to fe...
We previously reported that enterohemorrhagic Escherichia coli O157: H7 (EHEC) proliferates in the mouthparts of the house fly Musca domestica vicina Macquart and are excreted for at least 3 d after ingestion. However, the role of the crop and excretory behavior of the house fly in contamination of human food with EHEC is not known. In the current experiments, EHEC persisted in the crop of house flies for at least 4 d. The number of EHEC in an excreted droplet was approximately 10(4) 1 h after bacterial feeding, > 1.8 x 10(5) 3 h after feeding, and then drastically decreased after 24 h. Excretion is one of the major mechanisms for decreasing number of EHEC in the crop and gut of the house fly. The frequency of excretion by females with developing eggs in their ovary was clearly higher (6.5 min per drop) than for males or females with mature eggs. Minute eosin-sign around a container filled with eosin-supplemented trypticase soy broth might be derived from frequent contact by house fly contaminated mouthparts. These results show that frequent excretion potentially enhances the dissemination of EHEC to foods, particularly during the first 24 h after ingestion of the bacteria.
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