Use of the Sindbis replicon system for expression of LaCrosse virus envelope proteins in mosquito cells

The Sindbis replicon expression system was used to express La Crosse (LAC) virus envelope glycoprotein genes in both mammalian and mosquito cell culture. Replicon expressed LAC proteins had correct molecular mass (Mr) and were antigenically similar to wild type LAC envelope proteins. In addition, LAC G1 and G2 proteins colocalized when expressed from separate constructs in both mammalian and mosquito cells suggesting that they were trafficked through the cell similarly to wild type LAC proteins. A truncated form of the G1 protein was secreted from mosquito cells when expressed alone. The truncated G1 protein was also secreted from mosquito cells when expressed with the G2 protein, but to a lesser extent than when expressed alone, suggesting that the G2 protein sequestered G1 protein intracellularly. The Sindbis replicon system is a powerful tool for the study of LAC virus protein maturation within mosquito cells and mosquitoes.     

Malaria infection of the mosquito Anopheles gambiae activates immune-responsive genes during critical transition stages of the parasite life cycle

Six gene markers have been used to map the progress of the innate immune response of the mosquito vector, Anopheles gambiae, upon infection by the malaria parasite, Plasmodium berghei. In addition to four previously reported genes, the set of markers included NOS (a nitric oxide synthase gene fragment) and ICHIT (a gene encoding two putative chitin-binding domains separated by a polythreonine-rich mucin region). In the midgut, a robust response occurs at 24 h post-infection, at a time when malaria ookinetes traverse the midgut epithelium, but subsides at later phases of malaria development. In contrast, the salivary glands show no significant response at 24 h, but are activated in a prolonged late phase when sporozoites are released from the midgut into the haemolymph and invade the glands, between 10 and 25 days after blood feeding. Furthermore, the abdomen of the mosquito minus the midgut shows significant activation of immune markers, with complex kinetics that are distinct from those of both midgut and salivary glands. The parasite evidently elicits immune responses in multiple tissues of the mosquito, two of which are epithelia that the parasite must traverse to complete its development. The mechanisms of these responses and their significance for malaria transmission are discussed.     

The results of treatment in 100 patients with limited-stage small cell lung cancer

Strain differences in midgut basal lamina thickness, assessed by measurement in transmission electron micrographs, and disseminated infection rates of dengue-1 virus were compared among three laboratory strains of Aedes albopictus (Skuse). Mean basal lamina thickness for the New Orleans and Houston strains were significantly greater than those for the Oahu strain, which exhibits a higher disseminated infection rate than the former two. Although basal lamina thickness among the F1 progeny of reciprocal crosses of the Oahu and Houston strains were intermediate between the parental strains, they were too variable to be useful as markers in genetic studies. Measurements of basal laminae among individuals of the New Orleans strain, with disseminated or nondisseminated infections, failed to demonstrate a role for basal lamina thickness as a modulator of dengue-1 virus dissemination across the midgut epithelium of Ae. albopictus.     

Pathogenesis of ocular cytomegalovirus infection in the immunocompromised host

Balb/C nude and C.B-17 SCID mice were inoculated with salivary gland passaged cytomegalovirus (SG-MCMV) intraperitoneally. Dissemination of the virus in the systemic and ocular tissues was studied by the direct immunofluorescence test, and the virus growth in each tissue was titrated in mouse embryonic fibroblasts. The mode of viral spread was assessed by inhibiting macrophage function by silica and administering polyclonal murine anti-MCMV antibody in the circulation. The virus first reached the eyelid, conjunctiva, and cornea. Subsequently, it spread in the outer ocular muscles and chorioretinal layer. Ocular tissues were involved as part of a generalized infection. Abrogation of macrophage function by silica did not affect the outcome of the viral distribution. Administration of antibody prior to and 3 days after the viral infection prevented virus dissemination. Ocular CMV infection occurred initially at the anterior segment of the eye in an immunocompromised host. Free virus, not macrophage-bound virus, disseminated via the bloodstream.     

Ultrastructure of the dorsal skins of hairless descendants derived from Mexican hairless dogs

Anopheles tessellatus mosquitoes ingested Plasmodium vivax gametocytes in human erythrocytes suspended in rabbit sera with and without anti-mosquito midgut antibodies. When the mosquito bloodmeal contained anti-midgut antibodies, fewer oocysts of P.vivax developed on the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. Complement inactivated serum also reduced the infection rate and load. A second bloodmeal containing anti-midgut antibodies, given 48 or 72 h later, did not enhance the transmission-blocking effect. IgG purified from anti-midgut sera was shown to mediate the transmission-blocking effect.     

Leukocytes in a Plasmodium falciparum-infected blood meal reduce transmission of malaria to Anopheles mosquitoes

Mosquitoes are infected with Plasmodium falciparum by taking a blood meal from a gametocyte carrier. Since a mosquito takes a volume of 1 to 2 microl, a blood meal may contain 1 x 10(4) to 3 x 10(4) leukocytes (WBC). The majority of WBC are composed of neutrophils which may phagocytose and kill developing gametes inside the mosquito midgut. Phagocytosis was measured in vitro by a luminol-dependent chemiluminescence (CL) assay. In the presence of P. falciparum gametes, sera from areas of endemicity had an increased CL response compared to controls. In mosquito membrane feeding experiments some such sera showed a transmission reduction which was related to the presence of viable WBC. The results of this study suggest that phagocytosis of opsonized gametes inside the mosquito midgut occurs and can contribute to a reduction in the transmission of P. falciparum parasites.     

A1 is a constitutive and inducible Bcl-2 homologue in mature human neutrophils

Digestion of blood within the mosquito midgut is mediated primarily by a series of proteases, and several previous studies have described protease activity within homogenates of the midgut of the malaria vector Anopheles stephensi. We have expanded on these previous data by resolving protease isoforms from the midgut as well as the hemolymph of adult An. stephensi mosquitoes via gel electrophoresis and zymography. Using this procedure, we have been able to identify multiple isozymes of trypsin, chymotrypsin, and aminopeptidase. We were able to detect an increase in the intensity of some of these protease bands plus the appearance of new bands 24 hr after mosquitoes had taken a blood meal. Furthermore, we detected 2 endogenous trypsin isozymes within the hemolymph. There was no upregulation of these hemolymph isozymes after a blood meal, thus suggesting that they may not be involved in digestion of the blood meal by the mosquito.     

Vpx is required for dissemination and pathogenesis of SIV(SM) PBj: evidence of macrophage-dependent viral amplification

The viral accessory protein Vpx is required for productive in vitro infection of macrophages by simian immunodeficiency virus from sooty mangabey monkeys (SIV(SM)). To evaluate the roles of Vpx and macrophage infection in vivo, we inoculated pigtailed macaques intravenously or intrarectally with the molecularly cloned, macrophage tropic, acutely pathogenic virus SIV(SM) PBj 6.6, or accessory gene deletion mutants (deltaVpr or deltaVpx) of this virus. Both wild-type and SIV(SM) PBj deltaVpx viruses were readily transmitted across the rectal mucosa. A subsequent 'stepwise' process of local amplification of infection and dissemination was observed for wild-type virus, but not for SIV(SM) PBj deltaVpx, which also showed considerable impairment of the overall kinetics and extent of its replication. In animals co-inoculated with equivalent amounts of wild-type and SIV(SM) Pbj deltaVpx intravenously or intrarectally, the deltaVpx mutant was at a strong competitive disadvantage. Vpx-dependent viral amplification at local sites of initial infection, perhaps through a macrophage-dependent mechanism, may be a prerequisite for efficient dissemination of infection and pathogenic consequences after exposure through either mucosal or intravenous routes.     

Cogan''s syndrome

It has been possible to infect mosquitoes routinely with cultured gametocytes of Plasmodium falciparum since 1980. This has enabled the development of a reliable bio-assay for potential transmission-blocking vaccines and research on the role of specific antibodies from the host on the parasitic stages in the mosquito midgut. After some development and fine-tuning of the assay, it became apparent that the immune responses of the human host, as well as factors from the parasite and the mosquito, determined the final outcome of the mosquito infection. The age of the mosquito, crowding of parasites inside the peritrophic membrane and the quantity and particularly the quality of the gametocytes ingested all influence the chance of successful transmission. Cytokines and/or other mediators of inflammation from the human host can also reduce transmission, probably by promoting phagocytosis of the freshly emerged gametes by leucocytes in the bloodmeal.     

Bacterial population dynamics in three anopheline species: the impact on Plasmodium sporogonic development

The functional role of bacteria in the midgut of adult mosquitoes is unknown. In this study, we examined the population dynamics of midgut bacteria of laboratory reared Anopheles stephensi, An. gambiae, and An. albimanus. Mosquito midguts were dissected under sterile conditions and examined for the presence of bacteria using standard microbiologic techniques. Ninety percent and 73% (n = 30) of newly emerged An. gambiae and An. stephensi, respectively, harbored bacteria. In contrast, only 17% (n = 23) of An. albimanus harbored any bacteria. The bacterial population increased 11-40-fold in the presence of a blood meal, but then decreased to pre-blood meal levels in 3-5 days. Pseudomonas cepacia, Enterobacter agglomerans, and Flavobacterium spp. were found in all three anopheline species. Midgut bacteria were acquired both transtadially and through the sugar meal. Transtadial transmission was demonstrated by successfully passaging Escherichia coli HS5 from the larval to the adult stage. However, midgut bacteria were acquired more efficiently through the sugar meal than through transtadial passage. An increase in midgut bacterial counts after mosquitoes were exposed to a bacteria/sugar suspension significantly reduced oocyst infection rates and densities in Plasmodium falciparum-infected mosquito cohorts. Since bacteria occur naturally in wild mosquitoes, it may be possible to modify anopheline vector competence using introduced or indigenous bacteria.     

Induced immunity against the mosquito Anopheles stephensi Liston (Diptera: Culicidae): effects on mosquito survival and fecundity

Mice were immunised three to five times with extracts of Anopheles stephensi heads, midguts, ovaries or fat bodies. At each immunisation the effects of feeding An. stephensi on the mice was determined, and changes in mosquito longevity and fecundity examined as the immune response developed. Although variability was common between control cages, significant and consistent reductions in mosquito longevity were observed when midguts were used as immunogens. Other extracts caused transient reductions in mortality. Fecundity was reduced significantly in mosquitoes fed upon mice immunised with each extract in at least one experiment. Mosquitoes fed upon fat-body-immunised mice showed delayed egg-laying as well as overall reduction in fecundity. The results confirm the feasibility of targeting mosquito antigens for novel vaccine development, but the "shotgun" approach used probably fails to successfully hit a suitable target antigen with any consistency. The natural variation in mosquito mortality can be countered by rigorous statistical analysis which can identify subtle effects in a very "noisy" experimental system. The midgut is the obvious target organ for anti-mosquito vaccine development and future work will focus on targeting components of this tissue for further immunisations.     

Tissue repair in the iris in experimental autoimmune uveoretinitis

The pathogenesis of infection with the L-strain of rinderpest virus (RPV) in rabbits was investigated. Of several lymphoid tissues examined, those associated with the gut showed the most marked virus growth. The virus titres were maximal 4 days after inoculation but had declined at day 6. The distribution of viral antigen was examined immunohistochemically with the recently established anti-rabbit CD5 monoclonal antibody (MoAb), which is a pan-T-cell marker, and the anti-RPV-nucleoprotein MoAb. The virus antigen was localized in the CD5+ area at the initial stage of infection but spread to all areas of the lymphoid tissues at the later stages. By flow cytometric analysis with both rabbit CD5 and CD4 MoAbs, a decrease of the CD4+ and CD5+ subpopulations was observed in the spleen and mesenteric lymph nodes.     

The contribution of epidemiology to psychosomatic medicine

The G1 glycoprotein of California encephalitis (CE) virus plays a critical role in the infection of mosquito and mammalian cells. We found that CE virus enters baby hamster kidney (BHK-21) and Aedes albopictus (C6/36) cells by the endocytic pathway. Ammonium chloride, a lysosomotropic amine that prevents release of virus from endosomes, inhibited infection of both cell types when added within 10 min after viral adsorption. In addition, infected cells formed polykaryons when the extracellular pH was lowered to 6.3; optimal fusion occurred at pH 5.8 and 6.0 (C6/36 and BHK-21 cells, respectively). Two neutralizing G1 MAba, 6D5.5 and 7D4.5, inhibited low pH-induced syncytia formation without affecting viral attachment, suggesting a role for G1 in viral entry. Since viral fusion proteins have been demonstrated to undergo conformational changes at low pH, acid-induced changes in G1 and G2 were assessed. While both G1 and G2 demonstrated low pH-induced alterations in detergent binding, only G1 displayed an altered protease cleavage pattern at the fusion pH. These results indicate that the G1 protein of CE virus undergoes conformational changes necessary for low pH-mediated entry into both mosquito and mammalian cells.     

Immunohistological localization of regulatory peptides in the midgut of the female mosquito Aedes aegypti

The midgut of the female mosquito Aedes aegypti was studied immunohistologically with antisera to various regulatory peptides. Endocrine cells immunoreactive with antisera to perisulfakinin, RFamide, bovine pancreatic polypeptide, urotensin 1, locustatachykinin 2 and allatostatins A1 and B2 were found in the midgut. Perisulfakinin, RFamide and bovine pancreatic polypeptide all react with the same, about 500 endocrine cells, which were evenly distributed throughout the posterior midgut, with the exception of its most frontal and caudal regions. In addition, these antisera recognized three to five neurons in each ingluvial ganglion and their axons, which ran longitudinally over the anterior midgut, as well as axons innervating the pyloric sphincter. The latter axons appear to be derived from neurons located in the abdominal ganglia. Antisera to two different allatostatins recognized about 70 endocrine cells in the most caudal area of the posterior midgut and axons in the anterior midgut whose cell bodies were probably located in either the brain or the frontal ganglion. Antiserum to locustatachykinin 2 recognized endocrine cells present in the anterior midgut and the most frontal part of the posterior midgut, as well as about 50 cells in the most caudal region of the posterior midgut. Urotensin 1 immunoreactivity was found in endocrine cells in the same region as the perisulfakinin-immunoreactive cells, but no urotensin-immunoreactive axons were found in the midgut. Double labeling experiments showed that the urotensin and perisulfakinin immunoreactivities were located in different cells. Such experiments also showed that the locustatachykinin and allatostatin immunoreactivities in the most caudal area of the posterior midgut were present in different cells. No immunoreactivity was found in the mosquito midgut when using antisera to corazonin, allatropin or leucokinin IV. Since these peptides have either been isolated from, or can reasonably be expected to be present in mosquitoes, it was concluded that these peptides are not present in the mosquito midgut.     

Biochemical analysis of a blood meal-induced Aedes aegypti glutamine synthetase gene

Glutamine synthetase (GS) in the mosquito, Aedes aegypti, is induced in the midgut following a blood meal. Mosquito GS message is detected as soon as 1 h post-blood feeding and remains stable for 18 h. Using a PCR product encoding mosquito GS, a lambda gt10 adult female mosquito cDNA library was screened. A cDNA clone, pCl5A2, encoding the full translation product of mosquito GS was isolated and sequence analyses performed. Mosquito GS cDNA is 2.5 kb in length and its putative translation product shares all the conserved regions characteristic of the GS gene family, including the presumed ATP biding site. Glutamine synthetase activity in the mosquito midgut is highest at 18 h post-blood feeding. Activity can be detected over a broad pH range, from 6.0 to 7.5. Unlike other cellular GS enzymes, mosquito GS is not active in the presence of ATP. Very low dosages (0.05 mM) of L-methionine S-sulfoximine are sufficient to partially inhibit mosquito GS activity. Inhibition of GS disrupts the normal formation of the midgut peritrophic matrix, suggesting that GS enzyme might be involved in the initial pathway of chitin synthesis. The unique expression pattern and inducible nature of the mosquito GS gene make it an interesting candidate for studying promoter function. Additionally, the blood meal activation of the GS gene makes this a potentially valuable tool in mosquito transformation studies.     

Changes in blood sugar and ammonium levels after ligation of the afferent hepatic vessels

Certain aspects of the development of Ehrlichia canis, causative agent of canine ehrlichiosis (tropical canine pancytopenia) in Rhipicephalus sanguineus ticks were studied. It was found that partial feeding of nymphs infected as larvae with E canis was a desirable, if not necessary, preliminary treatment for successful infection of dogs with ground-up ticks. It remains unclear whether feeding increased the number or altered the virulence of ehrlichiae within tick tissues. Ehrlichia canis organisms were detected by immunofluorescent microscopy in the midgut and hemocytes and by electron microscopy in the midgut and salivary glands of partially engorged adult ticks which had been infected as larvae and nymphs. Organisms were not observed in the ovary. Intracytoplasmic inclusions contained 1 to 80 elementary bodies, each provided with 2 distinct membranes. Infection of the midgut and salivary gland was confirmed by injecting homogenates of these tissues into susceptible dogs. Staining of gut smears of partially engorged adult ticks by fluorescein-conjugated anti-E canis antibody was found to be a reliable indicator of the infection.