The continual public health threat posed by the emergence of novel influenza viruses necessitates the ability to rapidly monitor infection and spread in experimental systems. To analyze real-time infection dynamics, we have created a replication-competent influenza reporter virus suitable for in vivo imaging. The reporter virus encodes the small and bright NanoLuc luciferase whose activity serves as an extremely sensitive readout of viral infection. This virus stably maintains the reporter construct and replicates in culture and in mice with near-native properties. Bioluminescent imaging of the reporter virus permits serial observations of viral load and dissemination in infected animals, even following clearance of a sublethal challenge. We further show that the reporter virus recapitulates known restrictions due to host range and antiviral treatment, suggesting that this technology can be applied to studying emerging influenza viruses and the impact of antiviral interventions on infections in vivo. These results describe a generalizable method to quickly determine the replication and pathogenicity potential of diverse influenza strains in animals.

Transforming growth factor-beta (TGF-β), a multifunctional cytokine regulating several immunologic processes, is expressed by virtually all cells as a biologically inactive molecule termed latent TGF-β (LTGF-β). We have previously shown that TGF-β activity increases during influenza virus infection in mice and suggested that the neuraminidase (NA) protein mediates this activation. In the current study, we determined the mechanism of activation of LTGF-β by NA from the influenza virus A/Gray Teal/Australia/2/1979 by mobility shift and enzyme inhibition assays. We also investigated whether exogenous TGF-β administered via a replication-deficient adenovirus vector provides protection from H5N1 influenza pathogenesis and whether depletion of TGF-β during virus infection increases morbidity in mice. We found that both the influenza and bacterial NA activate LTGF-β by removing sialic acid motifs from LTGF-β, each NA being specific for the sialic acid linkages cleaved. Further, NA likely activates LTGF-β primarily via its enzymatic activity, but proteases might also play a role in this process. Several influenza A virus subtypes (H1N1, H1N2, H3N2, H5N9, H6N1, and H7N3) except the highly pathogenic H5N1 strains activated LTGF-β in vitro and in vivo. Addition of exogenous TGF-β to H5N1 influenza virus–infected mice delayed mortality and reduced viral titers whereas neutralization of TGF-β during H5N1 and pandemic 2009 H1N1 infection increased morbidity. Together, these data show that microbe-associated NAs can directly activate LTGF-β and that TGF-β plays a pivotal role protecting the host from influenza pathogenesis.

Astroviruses are a leading cause of infantile viral gastroenteritis worldwide. Very little is known about the mechanisms of astrovirus-induced diarrhea. One reason for this is the lack of a small-animal model. Recently, we isolated a novel strain of astrovirus (TAstV-2) from turkeys with the emerging infectious disease poult enteritis mortality syndrome. In the present studies, we demonstrate that TAstV-2 causes growth depression, decreased thymus size, and enteric infection in infected turkeys. Infectious TAstV-2 can be recovered from multiple tissues, including the blood, suggesting that there is a viremic stage during infection. In spite of the severe diarrhea, histopathologic changes in the intestine were mild and there was a surprising lack of inflammation. This may be due to the increased activation of the potent immunosuppressive cytokine transforming growth factor beta during astrovirus infection. These studies suggest that the turkey will be a useful small-animal model with which to study astrovirus pathogenesis and immunity.

Astrovirus infection in a variety of species results in an age-dependent diarrhea; however, the means by which astroviruses cause diarrhea remain unknown. Studies of astrovirus-infected humans and turkeys have demonstrated few histological changes and little inflammation during infection, suggesting that intestinal damage or an overzealous immune response is not the primary mediator of astrovirus diarrhea. An alternative contributor to diarrhea is increased intestinal barrier permeability. Here, we demonstrate that astrovirus increases barrier permeability in a Caco-2 cell culture model system following apical infection. Increased permeability correlated with disruption of the tight-junction protein occludin and decreased the number of actin stress fibers in the absence of cell death. Additionally, permeability was increased when monolayers were treated with UV-inactivated virus or purified recombinant human astrovirus serotype 1 capsid in the form of virus-like particles. Together, these results demonstrate that astrovirus-induced permeability occurs independently of viral replication and is modulated by the capsid protein, a property apparently unique to astroviruses. Based on these data, we propose that the capsid contributes to diarrhea in vivo.

Astroviruses are one of the leading causes of diarrhea worldwide. In spite of its impact on human health, little is known about astrovirus pathogenesis. One reason for this may be the lack of a suitable small animal model for infection. In recent years, there has been increasing information on the mechanism of astrovirus-induced disease in mammals (including humans) and birds. This review summarizes our current state of knowledge on astrovirus pathogenesis.