Response to Dengue virus infections altered by cytokine-like substances from mosquito cell cultures

<p>Abstract</p> <p>Background</p> <p>With both shrimp and commercial insects such as honey bees, it is known that stable, persistent viral infections characterized by absence of disease can sometimes shift to overt disease states as a result of various stress triggers and that this can result in serious economic losses. The main research interest of our group is to understand the dynamics of stable viral infections in shrimp and how they can be destabilized by stress. Since there are no continuous cell lines for crustaceans, we have used a C6/36 mosquito cell line infected with Dengue virus to test hypotheses regarding these interactions. As a result, we accidentally discovered two new cytokine-like substances in 5 kDa extracts from supernatant solutions of acutely and persistently infected mosquito cells.</p> <p>Results</p> <p>Naïve C6/36 cells were exposed for 48 h to 5 kDa membrane filtrates prepared from the supernatant medium of stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Subsequent challenge of naïve cells with a virulent stock of Dengue virus 2 (DEN-2) and analysis by confocal immunofluorescence microscopy using anti-DEN-2 antibody revealed a dramatic reduction in the percentage of DEN-2 infected cells when compared to control cells. Similar filtrates prepared from C6/36 cells with acute DEN-2 infections were used to treat stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Confocal immunofluorescence microscopy revealed destabilization in the form of an apoptosis-like response. Proteinase K treatment removed the cell-altering activities indicating that they were caused by small polypeptides similar to those previously reported from insects.</p> <p>Conclusions</p> <p>This is the first report of cytokine-like substances that can alter the responses of mosquito cells to Dengue virus. This simple model system allows detailed molecular studies on insect cytokine production and on cytokine activity in a standard insect cell line.</p

White feces syndrome of shrimp arises from transformation, sloughing and aggregation of hepatopancreatic microvilli into vermiform bodies superficially resembling gregarines.

Accompanying acute hepatopancreatic necrosis disease (AHPND) in cultivated Asian shrimp has been an increasing prevalence of vermiform, gregarine-like bodies within the shrimp hepatopancreas (HP) and midgut. In high quantity they result in white fecal strings and a phenomenon called white feces syndrome (WFS). Light microscopy (LM) of squash mounts and stained smears from fresh HP tissue revealed that the vermiform bodies are almost transparent with widths and diameters proportional to the HP tubule lumens in which they occur. Despite vermiform appearance, they show no cellular structure. At high magnification (LM with 40-100x objectives), they appear to consist of a thin, outer membrane enclosing a complex of thicker, inter-folded membranes. Transmission electron microscopy (TEM) revealed that the outer non-laminar membrane of the vermiform bodies bore no resemblance to a plasma membrane or to the outer layer of any known gregarine, other protozoan or metazoan. Sub-cellular organelles such as mitochondria, nuclei, endoplasmic reticulum and ribosomes were absent. The internal membranes had a tubular sub-structure and occasionally enclosed whole B-cells, sloughed from the HP tubule epithelium. These internal membranes were shown to arise from transformed microvilli that peeled away from HP tubule epithelial cells and then aggregated in the tubule lumen. Stripped of microvilli, the originating cells underwent lysis. By contrast, B-cells remained intact or were sloughed independently and whole from the tubule epithelium. When sometimes engulfed by the aggregated, transformed microvilli (ATM) they could be misinterpreted as cyst-like structures by light microscopy, contributing to gregarine-like appearance. The cause of ATM is currently unknown, but formation by loss of microvilli and subsequent cell lysis indicate that their formation is a pathological process. If sufficiently severe, they may retard shrimp growth and may predispose shrimp to opportunistic pathogens. Thus, the cause of ATM and their relationship (if any) to AHPND should be determined

TEM of an ATM structure containing an enclosed, sloughed and modified B-cell.

<p>Note the single-layer enclosing membrane and the internal complex of sloughed, modified microvilli.</p

Gross signs of white feces syndrome WFS.

<p>(a) Floating, white fecal strings. (b) White fecal strings on a feeding tray. (c) White intestine of affected shrimp. (d) Golden brown intestine of an affected shrimp. (e) Photomicrograph of fecal string contents.</p

ATM aggregation steps in H&E stained HP tissue sections in comparison to true gregarines.

<p>(a) Small, scattered membrane-lie structures in the HP tubule lumen. (b) More extended membranes beginning to aggregate in the tubule lumen. (c) Tighter aggregation of membranes bound by a continuous outer membrane and taking the shape of ATM. (d) Highly condensed ATM in a tubule lumen. (e) Accumulation of many individual ATM at the center of the HP near the midgut junction. (f) True gregarines clustered near the midgut junction and showing prominent nuclei.</p

TEM of unusual electron-dense particles in HP tubule crypts.

<p>(a) Low magnification of electron-dense particles of highly variable shape in the HP tubule lumen between layers of normal microvilli from facing epithelial cells. (b) High magnification of one of the electron-dense particles between the microvilli on the outside surface of an epithelial cell, possibly prior to cell entry. (c) High magnification of electron dense particles inside an epithelial cell with adjacent microvilli on the cell surface undergoing morphological changes. (d) Low magnification of an epithelial cell containing large numbers of electron dense particles and with microvilli in an advanced stage of transformation.</p

Squash mount of vermiform bodies (ATM) in shrimp hepatopancreatic tissue.

<p>(a) Low magnification photomicrograph showing 3 ATM with the central one inside an HP tubule. (b) Higher magnification photomicrograph showing an ATM containing cyst-like structures later found to be sloughed B-cells. (c) High magnification of an ATM stained with Rose Bengal to more clearly reveal its internal membranous structure.</p

HP tissue smear stained with hematoxylin and eosin to reveal ATM.

<p>The photomicrographs clearly show the vermiform morphology and the cyst-like contents inside or free.</p

Semi-thin sections of HP tissue stained with toluidine blue.

<p>(a) Cross section of an HP tubule near the distal end showing densely stained particles in crypts formed by folds of the tubule epihelium and showing aggregated, transformed microvilli (ATM) in the tubule lumen. Note that microvillar layers of all the cells are intact. (b) Cross sections of HP tubules showing sloughed, transformed microvilli. (c) Cross section of an HP tubule showing a modified, sloughed B-cell in the tubule lumen with microvilli scattered over its surface. Also seen are tubule epithelial cells with normal microvilli and transformed mivrovilli, and one cell denuded of microvilli, undergoing lysis. (d) High magnification of clustered ATM at the center of the HP clearly showing an outer membrane enclosing multitudes of folded transformed microvilli. Some also contain enclosed, sloughed B-cells. Note many free transformed microvilli fragments surrounding the ATM.</p

TEM of steps in microvillar transformation and sloughing.

<p>(a) Low magnification of HP tubule epithelial cells showing normal and transformed microvilli and two denuded cells undergoing lysis. Also shown is an early stage in the aggregation of transformed and sloughed microvilli surrounded by an enclosing membrane. (b) Low magnification of HP tubule epithelial cells with transformed microvilli peeling from the cell surface, prior to cell lysis. (c) Higher magnification of the field from (b) clearly showing the difference between normal and transformed microvilli. (d) High magnification of HP tubule epithelial cells showing the tubular nature of transformed and peeled microvillar layers.</p