[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: To understand astrovirus biology, it is essential to understand factors associated with its evolution. The current study reports the genomic sequences of nine novel turkey astrovirus (TAstV) type 2-like clinical isolates. This represents, to our knowledge, the largest genomic-length data set available for any one astrovirus type. The comparison of these TAstV sequences suggests that the TAstV species contains multiple subtypes and that recombination events have occurred across the astrovirus genome. In addition, the analysis of the capsid gene demonstrated evidence for both site-specific positive selection and purifying selection.
Journal of Virology 06/2008; 82(10):5099-103. DOI:10.1128/JVI.01993-07 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the simultaneous detection of two enteric viruses of poultry: turkey enteric coronavirus (TCV) and turkey astrovirus (TAstV). PCR primers were designed to conserved regions within the nucleocapsid gene of TCV and to the polymerase gene of TAstV-2. The primer pairs were successfully used in a multiplex RT-PCR to detect nucleic acid of TAstV-2 and TCV. The test was optimized for use with intestines/feces from naturally infected turkeys. The primers were specific and did not amplify other common RNA or DNA avian viruses. The detection limit was determined to be 10 ng of RNA used as starting template. The use of this specific test allows the rapid and early diagnosis of two financially costly viruses affecting the commercial turkey industry.
[Show abstract][Hide abstract] ABSTRACT: Astrovirus is one of the major causes of infant and childhood diarrhea worldwide. Our understanding of astrovirus pathogenesis trails behind our knowledge of its molecular and epidemiologic properties. Using a recently developed small-animal model, we investigated the mechanisms by which astrovirus induces diarrhea and the role of both the adaptive and innate immune responses to turkey astrovirus type-2 (TAstV-2) infection. Astrovirus-infected animals were analyzed for changes in total lymphocyte populations, alterations in CD4(+)/CD8(+) ratios, production of virus-specific antibodies (Abs), and macrophage activation. There were no changes in the numbers of circulating or splenic lymphocytes or in CD4(+)/CD8(+) ratios compared to controls. Additionally, there was only a modest production of virus-specific Abs. However, adherent spleen cells from infected animals produced more nitric oxide (NO) in response to ex vivo stimulation with lipopolysaccharide. In vitro analysis demonstrated that TAstV-2 induced macrophage production of inducible nitric oxide synthase. Studies using NO donors and inhibitors in vivo demonstrated, for the first time, that NO inhibited astrovirus replication. These studies suggest that NO is important in limiting astrovirus replication and are the first, to our knowledge, to describe the potential role of innate immunity in astrovirus infection.
Journal of Virology 03/2004; 78(3):1564-74. DOI:10.1128/JVI.78.3.1564-1574.2004 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Virology 12/2003; 77(21):11798-808. DOI:10.1128/JVI.77.21.11798-11808.2003 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Twenty-one 3-day-old turkey poults from British United Turkeys of America were orally inoculated with a recently characterized astrovirus, TAstV-2, isolated from turkeys with poult enteritis and mortality syndrome. At 1, 2, 3, 4, 5, 7, and 9 days postinfection (dpi), three inoculated birds were euthanatized, and tissues (intestines, spleen, bursa, and thymus) were collected immediately into 10% neutral buffered formalin. Inoculated birds were diarrheic by 3 dpi, and frothy feces persisted throughout the experimental period. Histologically, there was only slight evidence of enteric damage, which was characterized by mild epithelial necrosis, lamina propria infiltrates, minimal villus atrophy, and mild crypt hyperplasia. In situ hybridization, using a negative sense digoxigenin-labeled riboprobe to the capsid gene of TAstV-2, revealed viral RNA in intestinal epithelial cells at the basal margins of the villi, in distal small intestine, and in cecum at 2 dpi, with subsequent extension to epithelium of the large intestine and proximal small intestine (3-5 dpi). Minimal virus remained by 9 dpi.
[Show abstract][Hide abstract] ABSTRACT: A unique turkey astrovirus has been isolated and sequenced. Primers and probes have been developed using the isolated nucleic acid sequence of the astrovirus and a reverse transcriptase PCR has been developed to detect the presence of avian astrovirus in commercial flocks. Furthermore, purified antigenic polypeptides have been synthesized from each of the open reading frames 1a,1b and 2 of the novel virus.