Beverley E. Bauman

CD8− Dendritic Cell Activation Status Plays an Integral Role in Influencing Th2 Response Development

Whether dendritic cells (DC) play a passive or active role in Th2 response induction is poorly understood. In this study, we show that CD8− DC pulsed with Th2-polarizing Ag (soluble egg Ag (SEA)) from Schistosoma mansoni potently stimulate Th2 responses in vivo and in vitro while failing to undergo a conventional maturation process. Thus, in contrast to DC pulsed with the Th1 response inducing Ag Propionebacterium acnes, SEA-exposed DC exhibit a phenotype that is most similar to that of immature DC, failing to up-regulate expression of CD40, CD54, CD80, CD86, or OX40L; producing no detectable IL-4, IL-10, or IL-12; and displaying only a minor increase in MHC class II expression. Importantly, in vitro derived DC exposed to SEA were phenotypically similar to CD8− DC isolated from active S. mansoni infection. By discriminating between different types of pathogen and responding appropriately, CD8− DC play a major role in the decision process to mount either a Th1 or Th2 response.

IL-4 Plays a Crucial Role in Regulating Oxidative Damage in the Liver During Schistosomiasis

Liver enlargement and hepatocyte proliferation, normal responses in wild-type (WT) mice infected with the parasitic helminth Schistosoma mansoni, were found to be severely impaired in infected IL-4−/− mice. Compared with WT mice, increased levels of O2−, NO, and the more highly reactive ONOO− were detected in the liver and produced by lesional cells isolated from liver granulomas of infected IL-4−/− mice. Concurrently, antioxidant defenses in the liver, specifically catalase levels, diminished dramatically during the course of infection in these animals. This contrasted to the situation in infected WT mice, where catalase levels remained as high as those in normal mice. Actual levels of reactive oxygen and nitrogen intermediates in the livers of infected IL-4−/− animals are thus likely to be considerably higher than those in the livers of infected WT mice. To determine whether these changes contributed to the development of the more severe disease that characterizes infection in the IL-4−/− animals, we treated infected IL-4−/− mice with uric acid, a potent scavenger of ONOO−. This resulted in significantly increased hepatocyte proliferation, decreased morbidity, and prolonged survival. Taken together, these data indicate that IL-4 is playing a protective role during schistosomiasis by controlling the tight regulation of the generation of reactive oxygen and nitrogen intermediates in the liver.

Impaired Th2 development and increased mortality during Schistosoma mansoni infection in the absence of CD40/CD154 interaction.

The role of CD40/CD154 interaction during infection has primarily focused on pathogens that drive inflammatory Th1 responses. In this study, we show that CD40/CD154 interaction is a fundamental requirement for Th2 response development to the parasitic helminth Schistosoma mansoni. Compared with infected wild-type mice, greatly reduced levels of Th2-associated cytokines were measured both in vitro and in vivo, and no IgE or IgG1 was detected in infected CD154−/− mice. In the absence of an overt Th2 response, no exaggerated Th1 response was mounted by CD154−/− mice. Infected CD154−/− mice suffered severe morbidity and mortality, even though parasitemias in wild-type and CD154−/− mice did not differ significantly. These data indicate that CD40/CD154 interaction is required to allow development of a Th2-dominated immune response to S. mansoni and support the view that failure to develop such a response can have fatal consequences.

The Avian Coronavirus Infectious Bronchitis Virus Undergoes Direct Low-pH-Dependent Fusion Activation during Entry into Host Cells

ABSTRACT Coronaviruses are the causative agents of respiratory disease in humans and animals, including severe acute respiratory syndrome. Fusion of coronaviruses is generally thought to occur at neutral pH, although there is also evidence for a role of acidic endosomes during entry of a variety of coronaviruses. Therefore, the molecular basis of coronavirus fusion during entry into host cells remains incompletely defined. Here, we examined coronavirus-cell fusion and entry employing the avian coronavirus infectious bronchitis virus (IBV). Virus entry into cells was inhibited by acidotropic bases and by other inhibitors of pH-dependent endocytosis. We carried out fluorescence-dequenching fusion assays of R18-labeled virions and show that for IBV, coronavirus-cell fusion occurs in a low-pH-dependent manner, with a half-maximal rate of fusion occurring at pH 5.5. Fusion was reduced, but still occurred, at lower temperatures (20°C). We observed no effect of inhibitors of endosomal proteases on the fusion event. These data are the first direct measure of virus-cell fusion for any coronavirus and demonstrate that the coronavirus IBV employs a direct, low-pH-dependent virus-cell fusion activation reaction. We further show that IBV was not inactivated, and fusion was unaffected, by prior exposure to pH 5.0 buffer. Virions also showed evidence of reversible conformational changes in their surface proteins, indicating that aspects of the fusion reaction may be reversible in nature.

Heparan Sulfate Is a Selective Attachment Factor for the Avian Coronavirus Infectious Bronchitis Virus Beaudette

Abstract The avian coronavirus infectious bronchitis virus (IBV) strain Beaudette is an embryo-adapted virus that has extended species tropism in cell culture. In order to understand the acquired tropism of the Beaudette strain, we compared the S protein sequences of several IBV strains. The Beaudette strain was found to contain a putative heparan sulfate (HS)-binding site, indicating that the Beaudette virus may use HS as a selective receptor. To ascertain the requirements of cell-surface HS for Beaudette infectivity, we assayed for infectivity in the presence of soluble heparin as a competitor and determined infectivity in mutant cell lines with no HS or glycosaminoglycan expression. Our results indicate that HS plays a role as an attachment factor for IBV, working in concert with other factors like sialic acid to mediate virus binding to cells, and may explain in part the extended tropism of IBV Beaudette.

A monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for identification of infectious bronchitis virus serotypes.

An antigen-capture enzyme-linked immunosorbent assay (C-ELISA) was developed for detection and identification of infectious bronchitis virus (IBV) serotypes Arkansas, Connecticut, and Massachusetts using monoclonal antibodies (MAbs) specific to the S1 glycoprotein of the respective serotype. The assay (designed as a double-antibody sandwich assay) gave the best results when the S1-specific MAb, antigen, and chicken serum were of the same serotype. However, when a group-specific (M glycoprotein-specific) MAb was used for antigen capture, a distinctive pattern of cross-reactivity was observed between the antigens and heterologous chicken sera, suggesting a complex distribution of epitopes on the IBV M glycoproteins. Treatment of antigen with NP40 enhanced the ELISA signal only when the M glycoprotein-specific MAb was used for antigen capture. Although C-ELISA was inconsistent in detecting IBV in chicken tissue homogenates, it was highly effective in detecting the virus in allantoic fluid after the homogenates were given one chicken embryo passage.

The Exacerbating Effect of Infectious Bronchitis Virus Infection on the Infectious Bursal Disease Virus-Induced Suppression of Opsonization by Escherichia coli Antibody in Chickens

Chickens infected with infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV) commonly develop secondary infection of the respiratory tract with Escherichia coli, resulting in significant economic losses. To understand the host factors that may contribute to the E. coli infection, we investigated macrophage-mediated E. coli phagocytosis, intracellular bacterial killing, and development of opsonizing antibody in previously uninfected chickens and in those infected with IBV, IBDV, and IBDV plus IBV. Macrophages from the peripheral blood and the respiratory tracts of chickens infected with IBV or IBDV plus IBV efficiently performed in vitro phagocytosis of E. coli in the presence of positive-control serum (i.e., E. coli antiserum produced in normal chickens). Those macrophages also had adequate bactericidal activity, indicating that IBV and IBDV infections had not affected their phagocytic activity or bactericidal function. The phagocytic activity of macrophages remained unaffected (P < 0.05) when the positive-control serum was replaced with E. coli antiserum produced in chickens infected with IBV alone. However, when E. coli antisera raised in IBDV-infected and, especially, that produced in IBDV plus IBV-infected chickens were supplemented, the percentage of phagocytosis and number of bacteria ingested per phagocyte were significantly (P < 0.05) less. These results indicate that although IBDV alone has the potential to markedly reduce opsonizing ability of antibody, this effect is significantly (P < 0.05) exacerbated by IBV infection.

Feline aminopeptidase N is not a functional receptor for avian infectious bronchitis virus

BackgroundCoronaviruses are an important cause of infectious diseases in humans, including severe acute respiratory syndrome (SARS), and have the continued potential for emergence from animal species. A major factor in the host range of a coronavirus is its receptor utilization on host cells. In many cases, coronavirus-receptor interactions are well understood. However, a notable exception is the receptor utilization by group 3 coronaviruses, including avian infectious bronchitis virus (IBV). Feline aminopeptidase N (fAPN) serves as a functional receptor for most group 1 coronaviruses including feline infectious peritonitis virus (FIPV), canine coronavirus, transmissible gastroenteritis virus (TGEV), and human coronavirus 229E (HCoV-229E). A recent report has also suggested a role for fAPN during IBV entry (Miguel B, Pharr GT, Wang C: The role of feline aminopeptidase N as a receptor for infectious bronchitis virus. Brief review. Arch Virol 2002, 147:2047–2056.ResultsHere we show that, whereas both transient transfection and constitutive expression of fAPN on BHK-21 cells can rescue FIPV and TGEV infection in non-permissive BHK cells, fAPN expression does not rescue infection by the prototype IBV strain Mass41. To account for the previous suggestion that fAPN could serve as an IBV receptor, we show that feline cells can be infected with the prototype strain of IBV (Mass 41), but with low susceptibility compared to primary chick kidney cells. We also show that BHK-21 cells are slightly susceptible to certain IBV strains, including Ark99, Ark_DPI, CA99, and Iowa97 (<0.01% efficiency), but this level of infection is not increased by fAPN expression.ConclusionWe conclude that fAPN is not a functional receptor for IBV, the identity of which is currently under investigation.

Fluorescence dequenching assays of coronavirus fusion.

For all enveloped viruses, a critical event during entry into cells is the fusion of the viral envelope with the membrane of the host cell. 2 Our current understanding of viral fusion has been driven by fundamental problems first solved with influenza hemagglutinin (HA). Whereas the trigger for HA-mediated fusion is the low pH of the endosome, other viruses (e.g., paramyxoviruses and most retroviruses) undergo a receptor-primed fusion with the plasma membrane at neutral pH. In the case of coronaviruses, however, there is little consensus as to whether virus entry and fusion occur following endocytosis or at the plasma membrane. 5 The coronavirus spike protein (S) is a primary determinant of cell tropism and pathogenesis, being responsible (and apparently sufficient) for receptor binding and fusion. The S protein is categorized as a class I fusion protein, based on the presence of characteristic heptad repeats; as such it shows features of the fusion proteins of influenza virus (HA), retroviruses (Env), and paramyxoviruses (F), for which there is extensive characterization at structural and biophysical level. Although class I fusion proteins share similar structural features, they can have quite different biological properties; i.e., they can be triggered for fusion by low pH or by receptor interaction. Receptor-induced conformational changes have been described for several coronaviruses, and the virus has generally been considered to exhibit a neutral or slightly alkaline pH optimum. However, these fusion data are principally based on cell–cell fusion assays with S-expressing cells and may not recapitulate the fusion event that takes place during virus entry. Indeed, despite being considered to be pH-independent for fusion, there is increasing evidence that coronavirus entry is a low pH-dependent process as infection is sensitive to endosome neutralization. A powerful means of analyzing membrane fusion is the application of fluorescence assays. These techniques offer a number of advantages, including high sensitivity, relative ease in obtaining quantitative data, and the possibility of monitoring fusion by

Avian Infectious Bronchitis Virus Enters Cells Via the Endocytic Pathway

Avian infectious bronchitis virus (IBV) is a group III coronavirus that has a major economic impact in the poultry industry. Infected layers often have a large drop in egg production due to impaired ovary and oviduct functions. Clinical manifestations of IBVinfected chickens include respiratory, renal, and reproductive diseases and are often coupled with secondary infections such as airsaculitis and oviduct salpingitis. Although the etiology, pathogenesis, and diagnosis have been described since the 1930s, the molecular mechanism of viral entry remains elusive. Viruses utilize various mechanisms to gain entry into their host cells. Enveloped viruses need to shed the viral envelope in order to release their genomes into target cells and initiate further replication. The process of uncoating typically follows two distinct mechanisms: pH-dependent fusion in the endosome or pH-independent fusion at the cell surface. Many enveloped viruses (e.g., influenza viruses) utilize various endocytic pathways to travel deep into the cell cytoplasm in order to smoothly bypass the cortical cytoskeleton near the cell periphery. The endocytic pathway also provides a suitable acidic environment for the pH-dependent membrane fusion to deliver the viral genome into the cell. 6 In contrast, paramyxoviruses (e.g., Sendai viruses) are well established to enter target cells via pH-independent fusion with the plasma membrane. For coronaviruses, contrasting results have been reported regarding the role of endosomes and low pH activation, and consequently, there is no general consensus regarding the entry mechanism. A major characteristic of IBV infected cells is the formation of large syncytia, which has become a key piece of evidence supporting fusion with the cell plasma membrane at neutral pH during viral entry. In this model, pH-neutral syncytia formation is thought to be a representative model of coronavirus fusion during entry. However, electron microscopy from Patterson et al. clearly shows IBV entering chorioallantoic membrane (CAM) and chick kidney cells via “viroplexis” through cellular engulfment into