Wolbachia infections of phlebotomine sand flies (Diptera: Psychodidae).

Old and New World phlebotomine sand fly species were screened for infection with Wolbachia, intracellular bacterial endosymbionts found in many arthropods and filarial nematodes. Of 53 samples representing 15 species, nine samples offour species were found positive for Wolbachia by polymerase chain reaction amplification using primers for the Wolbachia surface protein (wsp) gene. Five of the wsp gene fragments from four species were cloned, sequenced, and used for phylogenetic analysis. These wsp sequences were placed in three different clades within the arthropod associated Wolbachia (groups A and B), suggesting that Wolbacia has infected sand flies on more than one occasion. Two distantly related sand fly species, Lutzomyia (Psanthyromyia) shannoi (Dyar) and Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho), infected with an identical Wolbachia strain suggest a very recent horizontal transmission.

Identification of cutaneous Leishmaniasis vectors, Phlebotomus papatasi and P. duboscqi using random amplified polymorphic DNA.

Phlebotomus papatasi and P. duboscqi are two closely related, morphologically similar sandfly species that are established vectors of zoonotic cutaneous leishmaniasis in the Old World. We have developed a Random Amplified Polymorphic DNA (RAPD) method to find species-specific DNA profiles of these two species. It was found that using a single 10-mer primer a 'species specific' amplification band of about 0.49 kb was produced in all specimens of P. duboscqi while it was absent in P. papatasi. The 0.49 kb diagnostic band was consistently present in both males and females of P. duboscqi. The suitability of this primer for Phlebotomus species identification will help to find the true vector-parasite relationship in the epidemiology of leishmaniasis, particularly in the African countries where both species are prevalent.

A stable triple Wolbachia infection in Drosophila with nearly additive incompatibility effects.

Drosophila simulans strains infected with three different Wolbachia strains were generated by experimental injection of a third symbiont into a naturally double-infected strain. This transfer led to a substantial increase in total Wolbachia density in the host strain. Each of the three symbionts was stably transmitted in the presence of the other two. Triple-infected males were incompatible with double-infected females. No evidence was obtained for interference between modification effects of the different Wolbachia strains in males. Some incompatibility was observed between triple-infected males and females. However, this incompatibility reaction is not a specific property of triple-infected flies, because it was also observed in double-infected strains.

Wolbachia infections are distributed throughout insect somatic and germ line tissues.

Wolbachia are intracellular microorganisms that form maternally-inherited infections within numerous arthropod species. These bacteria have drawn much attention, due in part to the reproductive alterations that they induce in their hosts including cytoplasmic incompatibility (CI), feminization and parthenogenesis. Although Wolbachia's presence within insect reproductive tissues has been well described, relatively few studies have examined the extent to which Wolbachia infects other tissues. We have examined Wolbachia tissue tropism in a number of representative insect hosts by western blot, dot blot hybridization and diagnostic PCR. Results from these studies indicate that Wolbachia are much more widely distributed in host tissues than previously appreciated. Furthermore, the distribution of Wolbachia in somatic tissues varied between different Wolbachia/host associations. Some associations showed Wolbachia disseminated throughout most tissues while others appeared to be much more restricted, being predominantly limited to the reproductive tissues. We discuss the relevance of these infection patterns to the evolution of Wolbachia/host symbioses and to potential applied uses of Wolbachia.

Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis.

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.

Analysis of Wolbachia protein synthesis in Drosophila in vivo.

Intracellular Wolbachia infections are extremely common in arthropods and exert profound control over the reproductive biology of the host. However, very little is known about the underlying molecular mechanisms which mediate these interactions with the host. We examined protein synthesis by Wolbachia in a Drosophila host in vivo by selective metabolic labelling of prokaryotic proteins and subsequent analysis by 1D and 2D gel electrophoresis. Using this method we could identify the major proteins synthesized by Wolbachia in ovaries and testes of flies. Of these proteins the most abundant was of low molecular weight and showed size variation between Wolbachia strains which correlated with the reproductive phenotype they generated in flies. Using the gel systems we employed it was not possible to identify any proteins of Wolbachia origin in the mature sperm cells of infected flies.

In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line.

A continuous cell line, Aa23, was established from eggs of a strain of the Asian tiger mosquito, Aedes albopictus, naturally infected with the intracellular symbiont Wolbachia pipientis. The resulting cell line was shown to be persistently infected with the bacterial endosymbiont. Treatment with antibiotics cured the cells of the infection. In the course of establishing this cell line it was noticed that RFLPs in the PCR products of two Wolbachia genes from the parental mosquitoes were fixed in the infected cell line. This indicates that the mosquito host was naturally superinfected with different Wolbachia strains, whereas the infected cell line derived from these mosquitoes only contained one of the original Wolbachia strains. The development of an in vitro culture system for this fastidious microorganism should facilitate molecular analysis of the reproduction distorting phenotypes it induces in natural arthropod hosts.

Wolbachia pipientis: bacterial density and unidirectional cytoplasmic incompatibility between infected populations of Aedes albopictus.

Unidirectional cytoplasmic incompatibility is seen when certain Wolbachia-infected insect populations are crossed. Two hypotheses might explain this phenomenon: superinfections with mutually incompatible strains of Wolbachia producing incompatibility when crossed to individuals infected with only a single bacterial strain or, alternatively, a bacterial dosage model, with differences in Wolbachia densities responsible for the incompatibility. A quantitative PCR assay was set up as a general method to compare Wolbachia densities between populations. Using this assay in unidirectionally incompatible stocks of the mosquito Aedes albopictus, we have determined that densities are significantly higher in Houston than in the Mauritius and Koh Samui stocks. This is consistent with a dosage model for the observed crossing patterns, but does not rule out the possibility that superinfection is the primary cause of the incompatibility.

Wolbachia superinfections and the expression of cytoplasmic incompatibility.

Strains of Drosophila simulans from Riverside, California (DSR) and Hawaii (DSH) harbour distinct strains of the cytoplasmic incompatibility microorganism Wolbachia, resulting in the expression of bidirectional incompatibility when crossed. D. simulans lines carrying both of these (superinfected) Wolbachia strains were generated by the transfer of infected DSH cytoplasm into DSR embryos by microinjection. The superinfected flies were unidirectionally incompatible with both DSR and DSH individuals. As a result of this pattern, the superinfected state was observed to replace single infections in laboratory populations. The ability of the superinfection to spread was modulated by the production of singly infected offspring from superinfected mothers: strain segregation was observed under crowded larval rearing conditions. An inverse correlation between the penetrance of the cytoplasmic incompatibility phenotype and the degree of larval crowding was also observed. The findings have implications for the evolution of bidirectionally incompatible strains, and lead to the prediction that superinfections should be relatively common in field populations. Evidence for a natural superinfection in the mosquito Aedes albopictus is discussed. The results also have applied significance for the generation of insect lines capable of driving desirable genes into populations already infected with Wolbachia, thus allowing repeated opportunities for population replacement.

Insect densoviruses may be widespread in mosquito cell lines.

A diagnostic PCR assay was designed based on conserved regions of previously sequenced densovirus genomic DNA isolated from mosquitoes. Application of this assay to different insect cell lines resulted in a number of cases of consistent positive amplification of the predicted size fragment. Positive PCR results were subsequently confirmed to correlate with densovirus infection by both electron microscopy and indirect fluorescent antibody test. In each case the nucleotide sequence of the amplified PCR fragments showed high identity to previously reported densoviruses isolated from mosquitoes. Phylogenetic analysis based on these sequences showed that two of these isolates were examples of new densoviruses. These viruses could infect and replicate in mosquitoes when administered orally or parenterally and these infections were largely avirulent. In one virus/mosquito combination vertical transmission to progeny was observed. The frequency with which these viruses were detected would suggest that they may be quite common in insect cell lines.

Replacement of the natural Wolbachia symbiont of Drosophila simulans with a mosquito counterpart.

Inherited rickettsial symbionts of the genus Wolbachia occur commonly in arthropods and have been implicated in the expression of parthenogenesis, feminization and cytoplasmic incompatibility Wolbachia from the Asian tiger mosquito, Aedes albopictus, to replace the natural infection of Drosophila simulans by means of embryonic microinjection techniques. The transferred Wolbachia infection behaves like a natural Drosophila infection with regard to its inheritance, cytoskeleton interactions and ability to induce incompatibility when crossed with uninfected flies. The transinfected flies are bidirectionally incompatible with all other naturally infected strains of Drosophila simulans, however, and as such represent a unique crossing type. The successful transfer of this symbiont between distantly related hosts suggests that it may be possible to introduce this agent experimentally into arthropod species of medical and agricultural importance in order to manipulate natural populations genetically.

Influence of sulfated carbohydrates on the accessibility of CD4 and other CD molecules on the cell surface and implications for human immunodeficiency virus infection.

The modulatory activity of dextran sulfate with a relative molecular mass of 8 and 500 kDa and pentosan polysulfate with a relative molecular mass of 6 kDa on human T cell surface molecules CD2, CD3, CD4, CD8 and HLA-DR was investigated by analytical flow cytometry. The 500-kDa dextran sulfate induces a complete disappearance of the CD4 and a 50% diminution of CD2 immune reactivity on peripheral blood lymphocytes after a 4-h incubation while the low molecular mass polyanions do not. This modulation of the CD4 immune reactivity includes all CD4 epitopes investigated. It does not correlate with the antiviral effect of polyanions against human immunodeficiency virus infection. The interaction of polyanions with the CD4 presentation is temperature dependent and differs between fresh lymphocytes and immortal cell lines. From our data it can be concluded that mechanisms other than cell surface effects are responsible for the antiviral potency of these drugs. Implications for the modes of antiviral action of sulfated carbohydrates are discussed.

Replication of the scrapie agent in hamsters infected intracerebrally confirms the pathogenesis of an amyloid-inducing virosis.

Following intracerebral infection of hamsters with scrapie agent replication started with or without a very short lag phase. Infectivity titres increased exponentially within 35 to 40 days post-infection to a maximum level of 3 x 10(9) LD50 per brain and then remained constant until death. Minimal detectable amounts of scrapie-associated fibrils (SAF) appeared at 42 days and reached high levels 56 days after inoculation. The first clinical symptoms were diagnosed at about 65 days and animals died after 85 to 95 days. These data confirm earlier results in which peripheral infection first revealed agent replication, then SAF formation and finally clinical disease. Unconventional virus diseases, therefore, can best be described as virus-induced, organ-specific amyloidoses.

Scrapie: a virus-induced amyloidosis of the brain.

We have studied the pathogenesis of scrapie in hamsters, in particular the increase of infectivity and the formation of scrapie-associated fibrils in relation to clinical disease. The results of such studies after intraperitoneal or intracerebral infection are consistent with the idea that transmissible spongiform encephalopathies are a type of virus-induced, brain-specific amyloidosis. Therefore, an appropriate name for the class of viruses that cause these diseases might be amyloid-inducing viruses.

Pathogenesis of scrapie: study of the temporal development of clinical symptoms, of infectivity titres and scrapie-associated fibrils in brains of hamsters infected intraperitoneally.

After an intraperitoneal infection of hamsters with scrapie agent, early low and constant titres of about 100 LD50/brain between days 10 to 50 were followed by a dramatic increase to maximum levels of 3 X 10(9) LD50/brain within about 15 days. The plateau of maximum infectivity remained unchanged from day 70 to the time of the first and final signs of disease at 95 and 123 days post-infection, respectively. Scrapie-associated fibrils (SAF) as measured by immunoblotting of SAF protein could not be detected before 79 days post-infection even when a total brain was used for analysis. Subsequently, the concentration of SAF increased gradually by about 100,000-fold until the time of clinical disease. The kinetics suggest a virus-induced amyloidosis of the brain as the cause of disease.

The major protein of SAF is absent from spleen and thus not an essential part of the scrapie agent.

Scrapie-associated fibrils (SAF) play a controversial role in the discussion about the nature of the scrapie agent. In purification experiments SAF can be detected in brains of infected animals but not in spleen samples with similar titers of infectivity. Thus, SAF are not a constituent of the scrapie virus.

Scrapie: concept of a virus-induced amyloidosis of the brain.

After an intraperitoneal infection disease-specific incorporation of [3H]leucine into protein and [3H]uridine into RNA in the brain precede clinical scrapie in hamsters. Onset of both incorporations are the earliest measurable events in the disease. Infectivity and subsequent clinical symptoms appear only after this biochemical activity has ceased. The disease-specific [3H]protein co-purifies with scrapie-associated fibrils (SAF) and infectivity during differential centrifugation and buffer extraction. SDS-PAGE shows that the [3H]protein is not SAF protein but a protein with an apparently higher mol. wt. The [3H]RNA is metabolically stable and separates from SAF and the main portion of infectivity in the last step of the purification. The appearance of SAF-protein is a late event and correlates with severe clinical symptoms. SAF seems to be derived from a brain protein turning over slowly. Our data are consistent with early pre-clinical virus replication. In this case treatment aimed at suppressing virus replication in the clinical phase of the human Creutzfeldt-Jakob disease is unlikely to produce any beneficial effect.