Wolbachia-Based Dengue Virus Inhibition Is Not Tissue-Specific in Aedes aegypti.

BACKGROUND: Dengue fever, caused by the dengue virus (DENV), is now the most common arbovirus transmitted disease globally. One novel approach to control DENV is to use the endosymbiotic bacterium, Wolbachia pipientis, to limit DENV replication inside the primary mosquito vector, Aedes aegypti. Wolbachia that is naturally present in a range of insects reduces the capacity for viruses, bacteria, parasites and fungi to replicate inside insects. Wolbachia's mode of action is not well understood but may involve components of immune activation or competition with pathogens for limited host resources. The strength of Wolbachia-based anti DENV effects appear to correlate with bacterial density in the whole insect and in cell culture. Here we aimed to determine whether particular tissues, especially those with high Wolbachia densities or immune activity, play a greater role in mediating the anti DENV effect. METHODOLOGY/FINDINGS: Ae. aegypti mosquito lines with and without Wolbachia (Wildtype) were orally fed DENV 3 and their viral loads subsequently measured over two time points post infection in the midgut, head, salivary glands, Malpighian tubules, fat body and carcass. We did not find correlations between Wolbachia densities and DENV loads in any tissue, nor with DENV loads in salivary glands, the endpoint of infection. This is in contrast with strong positive correlations between DENV loads in a range of tissues and salivary gland loads for Wildtype mosquitoes. Lastly, there was no evidence of a heightened role for tissues with known immune function including the fat body and the Malpighian tubules in Wolbachia's limitation of DENV. CONCLUSION/SIGNIFICANCE: We conclude that the efficacy of DENV blocking in Wolbachia infected mosquitoes is not reliant on any particular tissue. This work therefore suggests that the mechanism of Wolbachia-based antiviral effects is either systemic or acts locally via processes that are fundamental to diverse cell types. We further conclude that the relationship between DENV blocking and Wolbachia density is not linear in mosquito tissues.

Structural and ultrastructural alterations of Malpighian tubules of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae infected with different Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) recombinant viruses.

Malpighian tubules constitute the main excretion organ of insects. Infection by egt(-) recombinant AcMNPV baculovirus in lepidopteran larvae promotes early degeneration of these structures, which has been correlated with earlier death of the host. However, no trace of viral infection has been detected in that tissue. We constructed two AgMNPV recombinants with the egfp gene under control of the hsp70 promoter, one being egt(-), and used another two recombinants (one egt(-)) containing the lacZ gene. Morphological alterations in the tubules were analyzed by light and electron microscopies. Bioassays were conducted to compare the pathogenicity of recombinants. Results showed progressive presence of marker proteins and tissue degeneration without signals of infection in the tissue. Morphological and bioassay results showed increased pathogenicity for lacZ-containing recombinants compared to the egfp ones; as for egt(-) viruses, we noted higher intensity and earlier onset of alterations. The absence of infection led us to believe that Malpighian tubules degeneration is provoked initially by the death of tracheal cells attached to the tubules and later, by the death of Malpighian tubule cells themselves. Tubule cell death might be due to oncosis and apoptosis, which may be activated by depletion of energy reserves and by accumulation of marker proteins, respectively. Absence of the egt gene may be leading to a higher energetic expense due to molting, thus aggravating tubule cell death, resulting in faster death of host.

Impact of a basement membrane-degrading protease on dissemination and secondary infection of Autographa californica multiple nucleopolyhedrovirus in Heliothis virescens (Fabricus).

ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, that digests components of the basement membrane (BM) during insect metamorphosis. A recombinant baculovirus that expresses ScathL (AcMLF9.ScathL) kills larvae of the tobacco budworm, Heliothis virescens, significantly faster than the wild-type virus and triggers melanization and tissue fragmentation in infected larvae shortly before death. As BMs are a potential barrier to the spread of baculovirus secondary infection to other tissues in the host, this study tested the hypothesis that the rapid death of insects infected with AcMLF9.ScathL was caused by accelerated secondary infection resulting from the degradation of host BMs by ScathL. Viruses expressing catalytically active or inactive ScathL were used to examine the effects of ScathL activity on budded virus release into the haemocoel during infection, the production of polyhedra in infected larvae and the rate of infection of the gut, trachea, haemocytes, fat body and Malpighian tubules. It was concluded that the enhanced insecticidal efficacy of the recombinant baculovirus that expresses ScathL does not result from altered tissue tropism or accelerated systemic infection. Implications for the role of the BM as a barrier to baculovirus dissemination within the host insect are discussed.

Rhipicephalus appendiculatus (Acari: Ixodidae): dynamics of Thogoto virus infection in female ticks during feeding on guinea pigs.

Engorged nymphs (Rhipicephalus appendiculatus) were inoculated parenterally with Thogoto (THO) virus (approximately 1 microl per nymph; 10(6)-10(7) PFU/ml). The adult females which resulted were used as the source of infected ticks for this study. Hemolymph, salivary glands, synganglion, gut, ovary, and Malpighian tubules were collected on each day of the blood meal and titrated for THO virus by plaque assay. The percent of tissues infected with virus was 16% or less on the day of attachment. Percent infection rose for all tissues throughout 6-7 days of feeding, reaching 40-100% infection during the rapid phase of engorgement. For the first 4 days of feeding, virus titer in the synganglion was higher than in salivary glands (means of 6.4-34.7 PFU/synganglion and 1.6-8.8 PFU/salivary gland pair). From days 5-7, virus titer was generally higher in the salivary gland than the synganglion (means of 422, 408, and 817 PFU/gland pair and means of 62, 811, and 9 PFU/synganglion). However, because a salivary gland pair is much heavier than a synganglion, the virus concentration in the synganglion was much higher than in the salivary gland during the slow phase of feeding. During the rapid phase of feeding, the difference in virus titer between the synganglion and salivary gland reduced. This difference between the early and late stages of feeding may explain why a previous study [J. Gen. Virol. 70 (1989) 1093], using immunofluorescence and immuno-gold labelling, failed to detect virus in the salivary gland early in feeding. These data provide evidence to explain that R. appendiculatus can transmit THO virus within 24h of attachment, an important epidemiological finding.