The mechanism by which astroviruses cause diarrhea remains unknown. Studies utilizing a turkey model of astrovirus pathogenesis have demonstrated that only mild histological changes occur during infection. No increase in cell death or inflammation was observed, nor was blood present in stools of astrovirus-infected turkeys (24
). These observations, which have been supported in human studies (42
), suggest that epithelial destruction or unregulated inflammation is not the cause of astrovirus-induced diarrhea. In the absence of other known causes of diarrhea, we hypothesized that increased intestinal barrier permeability played a role in astrovirus infection.
We have shown that the addition of HAstV-1 to the apical surface of a Caco-2 model intestinal barrier increases permeability. Studies with UV-inactivated virus, resulting in RNA breaks and uracil dimers (45
), and with purified VLPs suggest that replication is not required and that the capsid alone can mediate the increase in permeability. The kinetics of increased permeability were similar between purified VLPs and infectious HAstV-1. Unfortunately, at this time we do not have the tools to determine how the concentration of the VLPs used in these studies correlates with the number of particles (both infectious and empty) in viral inoculum. Studies are under way to develop the methodologies to make these comparisons. Regardless, it is clear that events early in the virus life cycle, specifically binding or entry, mediate the increase in permeability.
Attempts to determine if entry was the key step were unsuccessful. Both monensin and NH4
Cl are known to inhibit viral entry by ≥99% at concentrations of 0.01 and 20 mM, respectively (10
; data not shown); however, treatment of Caco-2 monolayers at these concentrations decreased TER levels to less than 15% of initial values by 12 hpi (data not shown), rendering such experiments ineffective. Thus, the specific trigger for HAstV-1-induced permeability, whether binding alone or binding and entry, requires further exploration.
We next investigated the cellular mechanisms of increased permeability during astrovirus infection. Reports of astrovirus-induced cell death are conflicting; for humans (42
) and in a turkey animal model (24
), no increase in cell death was observed during infection. However, porcine astrovirus is cytolytic in vitro (44
), and HastV-4 and -8 have been shown to cause apoptosis in cell culture (19
). No significant difference in cell death between mock- and HAstV-1-infected cells was observed in our experiments, nor did UV-inactivated virus or VLPs increase cell death. These observations do not necessarily conflict with those of previous reports. We evaluated death through 36 hpi, a time at which a significant increase in permeability is observed. Guix et al. did not observe an increase in apoptosis until 48 hpi (19
). It therefore seems clear that cell death is not the major cause of increased permeability in our system, although it may contribute later in infection.
We examined several cellular proteins responsible for controlling barrier permeability. The TJ complex is composed of transmembrane proteins that seal the intracellular spaces, the most well-characterized of which are occludin (13
) and claudin (25
). These proteins interact with cytosolic adapter and signaling molecules, which subsequently interact with the actin cytoskeleton (28
). In this way, permeability is regulated by external or internal signals; disruptions at many levels of these interactions can result in increased barrier permeability (12
). This phenomenon has been well described for infections by other enteric pathogens, such as rotavirus (20
), enteropathogenic Escherichia coli
), and Clostridium difficile
). We therefore evaluated TJ protein and actin localization during HAstV-1 infection. In cells treated with infectious HAstV-1 or UV-inactivated virus, occludin was disrupted by 24 hpi, corresponding to a dramatic decrease in the number of actin stress fibers. Changes in ZO-1 and claudin were not observed until 36 hpi, suggesting that actin and occludin relocalization occurred first. This is in contrast to findings for mock-treated cells, which exhibited well-developed perijunctional actomyosin rings, stress fibers, and peripheral staining of TJ proteins. Most agents known to increase barrier permeability do so within minutes or a few hours (40
). However, HAstV-1 takes ~20 h. This is not without precedent. Nitric oxide treatment of Caco-2 cells increases barrier permeability by about 12 h (41
), while tumor necrosis factor alpha-increased permeability requires at least 24 h (27
). Studies are under way to determine if the binding/entry of HAstV-1 results in the synthesis of a cellular factor that increases permeability.
What is the benefit of increasing barrier permeability? One reason may be to increase the spread of the virus. Hypothetically, the flux of fluids driven by the increased permeability may increase viral dissemination. Alternatively, disruption of TJs may allow enteric viruses to move from the intestinal lumen into the serosa, where the virus gains access to the bloodstream and the rest of the body. Animal models have demonstrated systemic turkey astrovirus type 2 (24
) and rotavirus (7
) infections supporting this hypothesis. Finally, increased barrier permeability may expose a viral receptor previously sequestered at the basolateral surface or within junctional complexes themselves. This has been demonstrated nicely for type B coxsackieviruses, which utilize the junctional adhesion molecule CAR. Disruption of the TJ exposes CAR, allowing a productive infection to occur (6
). If a similar scheme is required for astrovirus infection, it would be extremely beneficial for structural proteins to increase permeability, as they are produced in abundance and released during infection. HAstV-1 can infect Caco-2 cells from the basal surface (data not shown); however, the efficiency of infection and virus production relative to that of apical infection as well as the ability of the virus to spread basally are currently unknown. Investigations into the role of increased permeability during astrovirus infection and spread are currently under way.
In conclusion, we have demonstrated that astrovirus infection at the apical surface of model intestinal epithelia results in a time-dependent increase in barrier permeability. This increase in permeability is associated with disruption of the TJ protein occludin as well as the actin cytoskeleton and occurs independently of viral replication. This is the first study to demonstrate that astrovirus increases barrier permeability; future studies will focus on further understanding the cellular mechanisms that contribute to these processes both in vitro and in vivo.