Marion Desdouits

Transcytosis of HTLV-1 across a tight human epithelial barrier and infection of subepithelial dendritic cells

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. In addition to blood transfusion and sexual transmission, HTLV-1 is transmitted mainly through prolonged breastfeeding, and such infection represents a major risk for the development of adult T-cell leukemia/lymphoma. Although HTLV-1-infected lymphocytes can be retrieved from maternal milk, the mechanisms of HTLV-1 transmission through the digestive tract remain unknown. In the present study, we assessed HTLV-1 transport across the epithelial barrier using an in vitro model. Our results show that the integrity of the epithelial barrier was maintained during coculture with HTLV-1-infected lymphocytes, because neither morphological nor functional alterations of the cell monolayer were observed. Enterocytes were not susceptible to HTLV-1 infection, but free infectious HTLV-1 virions could cross the epithelial barrier via a transcytosis mechanism. Such virions were able to infect productively human dendritic cells located beneath the epithelial barrier. Our data indicate that HTLV-1 crosses the tight epithelial barrier without disruption or infection of the epithelium to further infect target cells such as dendritic cells. The present study provides the first data pertaining to the mode of HTLV-1 transport across a tight epithelial barrier, as can occur during mother-to-child HTLV-1 transmission during breastfeeding.

Preclinical studies of a modified vaccinia virus Ankara-based HIV candidate vaccine: antigen presentation and antiviral effect.

ABSTRACT Poxvirus-based human immunodeficiency virus (HIV) vaccine candidates are currently under evaluation in preclinical and clinical trials. Modified vaccinia virus Ankara (MVA) vectors have excellent safety and immunogenicity records, but their behavior in human cell cultures remains only partly characterized. We studied here various virological and immunological aspects of the interactions of MVA-HIV, a vaccine candidate developed by the French National Agency for AIDS Research (ANRS), with primary human cells. We report that MVA-HIV infects and drives Gag expression in primary macrophages, dendritic cells (DCs), and epithelial and muscle cells. MVA-HIV-infected DCs matured, efficiently presented Gag, Pol, and Nef antigens, and activated HIV-specific cytotoxic T lymphocytes (CTLs). As expected with this type of vector, infection was cytopathic and led to DC apoptosis. Coculture of MVA-HIV-infected epithelial cells or myotubes with DCs promoted efficient Gag antigen major histocompatibility complex class I (MHC-I) cross-presentation without inducing direct infection and death of DCs. Antigen-presenting cells (APCs) infected with MVA-HIV also activated HIV-specific CD4+ T cells. Moreover, exposure of DCs to MVA-HIV or to MVA-HIV-infected myotubes induced type I interferon (IFN) production and inhibited subsequent HIV replication and transfer to lymphocytes. Altogether, these results show that MVA-HIV promotes efficient MHC-I and MHC-II presentation of HIV antigens by APCs without facilitating HIV replication. Deciphering the immune responses to MVA in culture experiments will help in the design of innovative vaccine strategies.

Prolonged Myalgia in Sindbis Virus Infection: Case Description and In Vitro Infection of Myotubes and Myoblasts

BACKGROUND Sindbis virus (SINV) is a mosquito-borne alphavirus found in Eurasia, Africa, and Oceania. Clinical SINV infection is characterized by febrile rash and arthritis and sometimes prolonged arthralgia and myalgia. The pathophysiological mechanisms of musculoskeletal and rheumatic disease caused by SINV are inadequately understood. METHODS We studied the muscle pathology of SINV infection ex vivo by examining a unique muscle biopsy obtained from a patient with chronic myalgia and arthralgia 6 months after acute SINV infection and assessed potential genetic predisposing factors by determining the human leukocyte antigen (HLA) and complement factor C4 genes and proteins. In addition, we performed in vitro SINV infections of primary human myoblasts and myotubes. RESULTS In the muscle biopsy we found evidence of muscle regeneration due to previous necrotic lesions likely caused by earlier SINV infection. We showed that human myoblasts and myotubes were susceptible in vitro for SINV infection as the cells became immunoreactive for viral antigens and cytopathic effect was observed. The patient was homozygous for HLA-B*35 alleles and heterozygous for HLA-DRB1*01 and HLA-DRB1*03 alleles and had total deficiency of C4B protein. CONCLUSIONS This study provides new insights concerning pathological processes leading to chronic symptoms in SINV infection and demonstrates for the first time the susceptibility of human myogenic cells to SINV infection.

Productive infection of human skeletal muscle cells by pandemic and seasonal influenza A(H1N1) viruses.

Besides the classical respiratory and systemic symptoms, unusual complications of influenza A infection in humans involve the skeletal muscles. Numerous cases of acute myopathy and/or rhabdomyolysis have been reported, particularly following the outbreak of pandemic influenza A(H1N1) in 2009. The pathogenesis of these influenza-associated myopathies (IAM) remains unkown, although the direct infection of muscle cells is suspected. Here, we studied the susceptibility of cultured human primary muscle cells to a 2009 pandemic and a 2008 seasonal influenza A(H1N1) isolate. Using cells from different donors, we found that differentiated muscle cells (i. e. myotubes) were highly susceptible to infection by both influenza A(H1N1) isolates, whereas undifferentiated cells (i. e. myoblasts) were partially resistant. The receptors for influenza viruses, α2-6 and α2-3 linked sialic acids, were detected on the surface of myotubes and myoblasts. Time line of viral nucleoprotein (NP) expression and nuclear export showed that the first steps of the viral replication cycle could take place in muscle cells. Infected myotubes and myoblasts exhibited budding virions and nuclear inclusions as observed by transmission electron microscopy and correlative light and electron microscopy. Myotubes, but not myoblasts, yielded infectious virus progeny that could further infect naive muscle cells after proteolytic treatment. Infection led to a cytopathic effect with the lysis of muscle cells, as characterized by the release of lactate dehydrogenase. The secretion of proinflammatory cytokines by muscle cells was not affected following infection. Our results are compatible with the hypothesis of a direct muscle infection causing rhabdomyolysis in IAM patients.

Genetic characterization of Chikungunya virus in the Central African Republic

Chikungunya virus (CHIKV) is an alphavirus transmitted by the bite of mosquito vectors. Over the past 10 years, the virus has gained mutations that enhance its transmissibility by the Aedes albopictus vector, resulting in massive outbreaks in the Indian Ocean, Asia and Central Africa. Recent introduction of competent A. albopictus vectors into the Central African Republic (CAR) pose a threat of a Chikungunya fever (CHIKF) epidemic in this region. We undertook this study to assess the genetic diversity and background of CHIKV strains isolated in the CAR between 1975 and 1984 and also to estimate the ability of local strains to adapt to A. albopictus. Our results suggest that, local CHIKV strains have a genetic background compatible with quick adaptation to A. albopictus, as previously observed in other Central African countries. Intense surveillance of the human and vector populations is necessary to prevent or anticipate the emergence of a massive CHIKF epidemic in the CAR.

Immunohistochemical and virological features of HTLV-1-associated myosites: a study of 13 patients from West Indies and Africa

Immunohistochemical and virological features of HTLV-1-associated myosites: a study of 13 patients from West Indies and Africa Marion Desdouits, Olivier Cassar, Thierry Maisonobe, Alexandra Desrames, Achille Aouba, Olivier Hermine, Jacqueline Mikol, Marc Polivka, Isabelle Penisson-Besnier, Pascale Marcorelles, Fabien Zagnoli, Thomas Papo, Arnaud Lacour, Zahir Amoura, Julien Haroche, Patrick Cherin, Antonio Texeira, Anne-Sophie Morin, Franck Mortreux, Eric Wattel, Michel Huerre, Marie-Christine Cumont, Huot Khun, Sylviane Bassot, Sandra Martin-Latil, Graham Taylor, Antoine Gessain, Simona Ozden, Pierre-Emmanuel Ceccaldi

Mother-to-child transmission of HTLV-1: in vitro

Human T-Cell Leukemia Virus type 1 (HTLV-1), that infects around 15 million people world wide, is the causative agent of adult T-cell leukemia/lymphoma and HTLV1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). Besides horizontal transmission, HTLV-1 is transmitted vertically mainly through breastfeeding. This maternal transmission via breast milk appears to be the dominant mode of HTLV-1 spread in the high endemic areas, and is correlated with the presence of HTLV-1 infected cells (lymphocytes, epithelial cells...) in the milk of infected mothers.

Mother-to-child transmission of HTLV-1: in vitro study of HTLV-1 passage across a tight human epithelial barrier

Background Besides horizontal transmission, HTLV-1 is transmitted vertically mainly through prolonged breastfeeding. Maternal transmission via breast milk is the dominant mode of HTLV-1 spread in high endemic areas, and is inked to the presence of HTLV-1 infected cells (lymphocytes, epithelial cells..) in the milk. Infection in young childhood by such mean appears to be a major risk factor for development of ATL in adults. Materials and methods We developed an in vitro model of epithelial barrier (Caco-2 human enterocytic cell line) to assess the mode of passage of HTLV-1 through the digestive tract. Integrity of the barrier was checked by ultrastructural approach, measurement of the trans-epithelial resistance (TER), and diffusion of fluorescently labeled molecules. Results When enterocytes were co-cultured with HTLV-1infected lymphocytes, no structural modifications were detected in intercellular tight junctions. Moreover, the functional integrity of the epithelial barrier was maintained since no TER change was detected in the presence of infected lymphocytes, and passage of small fluorescent markers was unaffected. Although enterocytes were not found to be susceptible to HTLV-1 infection, free infectious HTLV-1 virions were detected in the basal compartment, and such a passage was temperaturedependent, suggesting a transcytotic mechanism of passage. When human dendritic cells were added to the basal compartment, they were found to be productively infected by HTLV-1 that had crossed the epithelial barrier.

Complete Genome Sequences of Two Chikungunya Viruses Isolated in the Central African Republic in the 1970s and 1980s

ABSTRACT Some arboviruses threaten human global health with potentially explosive emergence. Analysis of whole-genome sequences of decades-old isolates might contribute to the understanding of the complex dynamics which drive their circulation and emergence. Here, we report the whole-genome sequences of two Chikungunya viruses isolated in the Central African Republic in the 1970s and 1980s.

P19-19. MVA vaccines are efficiently cross-presented by DCs and do not enhance HIV replication in DC/T cell cultures

Results We report that MVA-GagPolNef efficiently infects a panel of primary and immortalized human cells (including macrophages, DCs, B, epithelial cells, muscular cells and fibroblasts), and drives Gag expression in these cells. Infection was cytopathic, and, as expected with this vector, induced apoptosis within 1–2 days of infection. In contrast, primary T cells were more resistant to infection and apoptosis. DCs infected with MVA-GagPolNef (and not with a control MVA vector) presented HIV antigens and activated HIV-specific CD8+ CTLs. The infection of DCs by MVA induced cell maturation, secretion of various cytokines, and was followed by apoptosis. Interestingly, coculture of MVA-GagPolNef-infected epithelial cells with DCs promoted efficient HIV-Gag antigen cross-presentation without inducing DC infection and cell death. Other MVA-infected cells were similarly efficiently cross-presented by DCs to CD8 T cells. APCs exposed to MVA-GagPolNef also activated HIV-specific CD4 T cells, indicating that this vector promotes both MHC-I-restricted crosspresentation and MHC-II-presentation of HIV antigens by DCs. We also examined the impact of MVA-GaPolNef on HIV replication in DC/T cell cocultures. Interestingly, there was no enhancement of HIV transfer and replication when MVA-GagPolNef was incubated with DCs prior to infection with HIV, nor when MVA-infected cells were added in the cocultures.