No abstract

f Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7 ؊/؊ ) is lethal. Mortality was associated with undetectable levels of alpha/beta interferon (IFN-␣/␤) in serum, ϳ50-and ϳ10-fold increases in levels of IFN-␥ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-␣/␤ receptor-deficient mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF3/7 ؊/؊ mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF3/7 ؊/؊ mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1-and TRIF-dependent pathways were primarily responsible for IFN-␣/␤ induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-␣/␤ responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.

The recent epidemic of the arthritogenic alphavirus, chikungunya virus (CHIKV) has prompted a quest to understand the correlates of protection against virus and disease in order to inform development of new interventions. Herein we highlight the propensity of CHIKV infections to persist long term, both as persistent, steady-state, viraemias in multiple B cell deficient mouse strains, and as persistent RNA (including negative-strand RNA) in wild-type mice. The knockout mouse studies provided evidence for a role for T cells (but not NK cells) in viraemia suppression, and confirmed the role of T cells in arthritis promotion, with vaccine-induced T cells also shown to be arthritogenic in the absence of antibody responses. However, MHC class II-restricted T cells were not required for production of anti-viral IgG2c responses post CHIKV infection. The anti-viral cytokines, TNF and IFNγ, were persistently elevated in persistently infected B and T cell deficient mice, with adoptive transfer of anti-CHIKV antibodies unable to clear permanently the viraemia from these, or B cell deficient, mice. The NOD background increased viraemia and promoted arthritis, with B, T and NK deficient NOD mice showing high-levels of persistent viraemia and ultimately succumbing to encephalitic disease. In wild-type mice persistent CHIKV RNA and negative strand RNA (detected for up to 100 days post infection) was associated with persistence of cellular infiltrates, CHIKV antigen and stimulation of IFNα/β and T cell responses. These studies highlight that, secondary to antibodies, several factors are involved in virus control, and suggest that chronic arthritic disease is a consequence of persistent, replicating and transcriptionally active CHIKV RNA.

Furin, a predominant convertase of the cellular constitutive secretory pathway, is known to be involved in the maturation of a number of growth/differentiation factors, but the mechanisms governing its expression remain elusive. We have previously demonstrated that transforming growth factor (TGF) ␤1, through the activation of Smad transducers, regulates its own converting enzyme, furin, creating a unique activation/regulation loop of potential importance in a variety of cell fate and functions. Here we studied the involvement of the p42/p44 MAPK pathway in such regulation. Using HepG2 cells transfected with fur P1 LUC (luciferase) promoter construct, we observed that forced expression of a dominant negative mutant form of the small G protein p21 ras (RasN17) inhibited TGF␤1-induced fur gene transcription, suggesting the involvement of the p42/p44 MAPK cascade. In addition, TGF␤ induced sustained activation/phosphorylation of endogenous p42/p44 MAPK. Further-more, the role of MAPK cascade in fur gene transcription was highlighted by the use of the MEK1/2 inhibitors, PD98059 or U0126, or co-expression of a p44 antisense construct that repressed the induction of fur promoter transactivation. Conversely, overexpression of a constitutively active form of MEK1 increased unstimulated, TGF␤1-stimulated, and Smad2-stimulated promoter P1 transactivation, and the universal Smad inhibitor, Smad7, inhibited this effect. Activation of Smad2 by MEK1 or TGF␤1 resulted in an enhanced nuclear localization of Smad2, which was inhibited upon blocking MEK1 activity. Our findings clearly show that the activation of the p42/p44 MAPK pathway is involved in fur gene expression and led us to propose a co-operative model whereby TGF␤1-induced receptor activation stimulates not only a Smad pathway but also a parallel p42/p44 MAPK pathway that targets Smad2 for an increased nuclear translocation and enhanced fur gene transactivation. Such an uncovered mechanism may be a key determinant for the regulation of furin in embryogenesis and growth-related physiopathological conditions.

Even though ferrets are one of the principal animal models for influenza pathogenesis, the lack of suitable immunological reagents has so far limited their use in host response studies. Using recently established real-time PCR assays for a panel of ferret cytokines, we analyzed the local ferret immune response to human influenza isolates of the H1N1 and H3N2 subtypes that varied in their virulence. We observed that the severity of clinical signs correlated with gross- and histopathological changes in the lungs and was subtype-independent. Strains causing a mild disease were associated with a strong and rapid innate response and upregulation of IL-8, while severe infections were characterized by a lesser induction of type I and II interferons and strong IL-6 upregulation. These findings suggest that more virulent strains may interfere more efficiently with the host response at early disease stages.

Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.Canine distemper virus (CDV) and the closely related viruses that infect marine mammals have the highest incidence of central nervous system (CNS) complications among viruses of the genus Morbillivirus. Up to 30% of dogs exhibit signs of neurologic involvement during or after CDV infection, and most wild carnivores that succumb to CDV have some evidence of CNS infection (8,18,20,38,51). With 1 in 1,000 cases, the neurovirulent potential of measles virus (MV) is comparatively lower, but CNS complications remain one of the main problems associated with MV infections in countries with an established health care system (32). Because of its relatively high degree of neurovirulence and the availability of a sensitive small animal model, CDV is an ideal candidate to characterize the events involved in morbillivirus neuroinvasion.All morbilliviruses display a strong lymphotropism, which correlates with the presence of their principal receptor, the signaling lymphocytic activation molecule (SLAM, CD150), on a variety of immune cells (6,36,40). In ferrets infected with a virulent CDV strain, more than 50% of circulating peripheral blood mononuclear cells (PBMC) contain infectious virus, and up to 10% of PBMC in MV-infected monkeys are virus positive (46, 53). Because of these findings and the fact that morbilliviruses are highly cell associated, it is thought that the contact between virus and CNS occurs through infected PBMC. Previous studies of typical wild-type isolates like strain A75/17 in dogs further support this theory (38).At the onset of the symptomatic disease around 14 days after intranasal inoculation, infected cells are predominantly detected in the perivascular spaces of the CNS, the choroid plexus, and the ependyma (19,39). Around 3 weeks after infection, CDV-positive glial cells and neurons are found in the white matter, and...

Furin is recognized as being one of the main convertases of the cellular constitutive secretion pathway but the mechanisms regulating its expression are still unknown. We have previously demonstrated that TGFbeta1 up-regulates its own converting enzyme, furin, creating a novel activation/regulation cycle of potential importance in a variety of physiological and pathophysiological conditions. The fur (fes upstream region) gene is regulated via three alternative promoters; P1, P1A, and P1B. To gain insight into the molecular mechanism(s) underlying this up-regulation, we performed transient cell transfections with P1, P1A, and P1B promoter luciferase constructs. Transfection experiments in HepG2 cells revealed that fur P1 promoter is the strongest and the most sensitive to TGFbeta1 stimulation (5 ng/ml) (3.2-fold vs. 2.4-fold for P1A and 2.1-fold for P1B). Cotransfection with either a dominant negative mutant form of Smad2 [Smad2(3SA)] or a known Smad inhibitor [Smad7] inhibit constitutive and TGFbeta1-induced luciferase activity indicating the participation of endogenous Smads. Increased levels of TGFbeta1-induced transcriptional activation of the P1 promoter by overexpression of Smad2 and/or Smad4 is greatly reduced in the presence of Smad2(3SA) and completely inhibited by Smad7, suggesting the participation of endogenous Smad2/Smad4 complexes. Furthermore, the fork-head activin signal transducer (FAST-1), known to interact with Smad2/Smad4 complexes, is a potent stimulator of TGFbeta1-induced transactivation of the fur P1 promoter. Five prime-deletion analysis of this promoter identified the proximal region (between positions -8734 and -7925), as the nucleotide stretch that carries most of the transcriptional activation of fur P1 promoter by Smad2. Overall, the present data demonstrate that Smad2 and Smad4 possibly in complex with FAST-1 or other DNA binding partners participate in the constitutive and inducible transactivation of the fur P1 promoter. This represents the first detailed study of the transcriptional regulation of the fur gene.

In the context of viral infections, autophagy induction can be beneficial or inhibitory. Within the Paramyxoviridae family, only morbilliviruses have been investigated and are reported to induce autophagy. Here we show that morbilliviruses rapidly induce autophagy and require this induction for efficient cell-to-cell spread. Coexpression of both glycoproteins in cells expressing one of the cellular receptors was required for autophagy induction, and LC3 punctum formation, indicative of autophagy, was mainly observed in syncytia. A similar correlation between syncytium formation and autophagy induction was also observed for other paramyxovirus glycoproteins, suggesting that membrane fusion-mediated autophagy may be common among paramyxoviruses and possibly other enveloped viruses.A s obligate intracellular parasites, viruses have evolved to exploit cellular functions, while cells have in turn developed a broad spectrum of antiviral mechanisms. In addition to the wellcharacterized sensors of pathogen-associated molecular patterns (PAMPs) (18), alteration of autophagy activity is increasingly recognized as an early inducer of innate immune activation (36,45,46). Autophagy is a ubiquitous mechanism involved in the maintenance of homeostasis in response to cellular stress by recycling of long-lived proteins and cytoplasmic organelles. Briefly, damaged cellular constituents are sequestered into the autophagosomes, which fuse with lysosomes to transform into autolysosomes (9). Autophagosome formation involves the conjugation of autophagy-related gene 5 (Atg5) and Atg12 by the ubiquitin E1-like enzyme Atg7 and the E2-like enzyme Atg10 (31, 38), followed by the cleavage of the microtubule-associated protein light chain 3 (LC3) in LC3-I and LC3-II (37). LC3 is a major constituent of the autophagosome, and its intracellular localization and conversion from the cytoplasmic form, LC3-I, to its membrane-bound cleavage product, LC3-II, are frequently used to monitor autophagic activity (15,25). In the context of virus infections, autophagy may be either inhibited to interfere with innate immune activation or induced to promote virus replication (22).Morbilliviruses constitute a genus in the Paramyxoviridae family within the order Mononegavirales (5). These viruses initially infect a broad range of immune cells and then spread to epithelial cells. Attachment to the target cell is mediated by the hemagglutinin (H) protein, upon which the fusion (F) protein induces fusion of the viral and cellular membranes (19). The signaling lymphocyte activation molecule (SLAM), which is expressed on the surface of activated T and B lymphocytes, macrophages, and dendritic cells (43), serves as the immune cell receptor for all morbilliviruses (39), while CD46, a regulator of complement activation, is used only by attenuated and certain wild-type measles viruses (MeVs) (47). Recent reports demonstrate that both SLAM and CD46 recruit the vps34/beclin1 autophagic complex, suggesting that morbilliviruses might induce autophagy pathways upon recepto...