Revisiting Koch's postulate to determine the plausibility of viral transmission by human milk.

As breastfeeding is of utmost importance for child development and survival, identifying whether breast milk is a route of transmission for human viruses is critical. Based on the principle of Koch's postulate, we propose an analytical framework to determine the plausibility of viral transmission by breast milk. This framework is based on five criteria: viral infection in children receiving breast milk from infected mothers; the presence of virus, viral antigen, or viral genome in the breast milk of infected mothers; the evidence for the virus in breast milk being infectious; the attempts to rule out other transmission modalities; and the reproduction of viral transmission by oral inoculation in an animal model. We searched for evidence in published reports to determine whether the 5 criteria are fulfilled for 16 human viruses that are suspected to be transmissible by breast milk. We considered breast milk transmission is proven if all 5 criteria are fulfilled, as probable if 4 of the 5 criteria are met, as possible if 3 of the 5 criteria are fulfilled, and as unlikely if less than 3 criteria are met. Only five viruses have proven transmission through breast milk: human T-cell lymphotropic virus 1, human immunodeficiency virus, human cytomegalovirus, dengue virus, and Zika virus. The other 11 viruses fulfilled some but not all criteria and were categorized accordingly. Our framework analysis is useful for guiding public health recommendations and for identifying knowledge gaps amenable to original experiments.

Human T-Lymphotropic Virus Type 1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier.

UNLABELLED: Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4(+) T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis. IMPORTANCE: Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear. Here, we show that the viral transactivator Tax increases activated leukocyte cell adhesion molecule (ALCAM/CD166) expression. This molecule facilitates the migration of lymphocytes across the BBB endothelium. Targeting this molecule could be of interest in preventing or reducing the development of HAM/TSP.

Clinical and virological factors associated with gastrointestinal symptoms in patients with acute respiratory infection: a two-year prospective study in general practice medicine.

BACKGROUND: Gastrointestinal (GI) symptoms, such as diarrhea, vomiting, abdominal pain and nausea are not an uncommon manifestation of an acute respiratory infection (ARI). We therefore evaluated clinical and microbiological factors associated with the presence of GI symptoms in patients consulting a general practitioner (GP) for ARI. METHODS: Nasopharyngeal swabs, stool specimens and clinical data from patients presenting to GPs with an ARI were prospectively collected during two winter seasons (2014-2016). Samples were tested by quantitative real-time PCR for 12 respiratory pathogen groups and for 12 enteric pathogens. RESULTS: Two hundred and four of 331 included patients (61.6%) were positive for at least one respiratory pathogen. Sixty-nine stools (20.8%) were positive for at least one pathogen (respiratory and/or enteric). GI symptoms were more likely declared in case of laboratory confirmed-enteric infection (adjusted odds ratio (aOR) = 3.2; 95% confidence interval [CI] [1.2-9.9]; p = 0.02) or human coronavirus (HCoV) infection (aOR = 2.7; [1.2-6.8]; p = 0.02). Consumption of antipyretic medication before the consultation seemed to reduce the risk of developing GI symptoms for patients with laboratory-confirmed influenza (aOR = 0.3; [0.1-0.6]; p = 0.002). CONCLUSIONS: The presence of GI symptoms in ARI patients could not be explained by the detection of respiratory pathogens in stools. However, the detection of enteric pathogens in stool samples could explained by the presence of GI symptoms in some of ARI cases. The biological mechanisms explaining the association between the presence of HCoVs in nasopharynx and GI symptoms need to be explored.

HTLV-1 and host barriers interactions.

HTLV-1 (Human T-cell Lymphotropic Virus Type 1) is a human retrovirus that infects around 10 million people worldwide. It can be transmitted by sexual contact, transfusion of contaminated blood, and from infected mother-to-child during prolonged breastfeeding. The latter involves viral crossing of the digestive tract. HTLV-1 is the etiological agent of both a lymphoproliferative malignancy, Adult T-cell leukemia/lymphoma, and a chronic inflammatory neuromyelopathy, the Tropical Spastic Paraparesis/HTLV-1 Associated Myelopathy (TSP/HAM). TSP/HAM is characterized by HTLV-1-infected lymphocyte infiltration in the central nervous system; these cells cross the blood-brain barrier, an anatomical barrier that normally isolates and protects the central nervous system from blood. In this context, the present review focuses on latest findings and opinions on the interactions of HTLV-1 with the intestinal barrier, as involved in mother-to-child viral transmission, and with the blood-brain barrier, as involved in TSP/HAM pathogenesis.

Rainbow trout (Oncorhynchus mykiss) muscle satellite cells are targets of salmonid alphavirus infection.

Sleeping disease in rainbow trout is characterized by an abnormal swimming behaviour of the fish which stay on their side at the bottom of the tanks. This sign is due to extensive necrosis and atrophy of red skeletal muscle induced by the sleeping disease virus (SDV), also called salmonid alphavirus 2. Infections of humans with arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), are global causes of debilitating musculoskeletal diseases. The mechanisms by which the virus causes these pathologies are poorly understood due to the restrictive availability of animal models capable of reproducing the full spectrum of the disease. Nevertheless, it has been shown that CHIKV exhibits a particular tropism for muscle stem cells also known as satellite cells. Thus, SDV and its host constitute a relevant model to study in details the virus-induced muscle atrophy, the pathophysiological consequences of the infection of a particular cell-type in the skeletal muscle, and the regeneration of the muscle tissue in survivors together with the possible virus persistence. To study a putative SDV tropism for that particular cell type, we established an in vivo and ex vivo rainbow trout model of SDV-induced atrophy of the skeletal muscle. This experimental model allows reproducing the full panel of clinical signs observed during a natural infection since the transmission of the virus is arthropod-borne independent. The virus tropism in the muscle tissue was studied by immunohistochemistry together with the kinetics of the muscle atrophy, and the muscle regeneration post-infection was observed. In parallel, an ex vivo model of SDV infection of rainbow trout satellite cells was developed and virus replication and persistence in that particular cell type was followed up to 73 days post-infection. These results constitute the first observation of a specific SDV tropism for the muscle satellite cells.

Prevalence of gastrointestinal symptoms in patients with influenza, clinical significance, and pathophysiology of human influenza viruses in faecal samples: what do we know?

This review provides for the first time an assessment of the current understanding about the occurrence and the clinical significance of gastrointestinal (GI) symptoms in influenza patients, and their correlation with the presence of human influenza viruses in stools of patients with confirmed influenza virus infection. Studies exploring how human influenza viruses spread to the patient's GI tract after a primary respiratory infection have been summarized. We conducted a systematic search of published peer-reviewed literature up to June 2015 with regard to the above-mentioned aspects, focusing on human influenza viruses (A(H1N1), A(H1N1)pdm09, A(H3N2), and B). Forty-four studies were included in this systematic review and meta-analysis. The pooled prevalence of any digestive symptoms ranged from 30.9% (95% CI, 9.8 to 57.5; I(2) = 97.5%) for A(H1N1)pdm09 to 2.8% (95% CI, 0.6 to 6.5; I(2) = 75.4%) for A(H1N1). The pooled prevalence of influenza viruses in stool was 20.6% (95% CI, 8.9 to 35.5; I(2) = 96.8%), but their correlation with GI symptoms has rarely been explored. The presence of viral RNA in stools because of haematogenous dissemination to organs via infected lymphocytes is likely, but the potential to cause direct intestinal infection and faecal-oral transmission warrants further investigation. This review highlights the gaps in our knowledge, and the high degree of uncertainty about the prevalence and significance of GI symptoms in patients with influenza and their correlation with viral RNA positivity in stool because of the high level of heterogeneity among studies.

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.

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.

Excretion of Cell-Free and Cell-Associated Zika Virus into Breast Milk of Infected Dams and Identification of Antiviral Factors.

Zika virus (ZIKV) is a mosquito-borne RNA virus belonging to the Flavivirus genus of the Flaviviridae family. During the 60 years following its discovery in 1947, ZIKV caused little concern for public health as the associated infection was reported as mostly asymptomatic or inducing mild symptoms. However, since 2013, severe neurological symptoms have been associated with ZIKV infection, compelling the World Health Organization to declare a Public Health Emergency of International Concern. Among those symptoms, neurological birth defects may affect children born to mothers infected during pregnancy. Additionally, during the past 8 years, ZIKV transmission through breastfeeding has repeatedly been suggested in epidemiological studies and demonstrated on a mouse model by our team. To better understand the biological factors controlling ZIKV transmission through breastfeeding, we investigated the nature of the viral entities excreted in the breast milk of infected dams and evaluated viral transmission to breastfed pups. We show that both cell-free and cell-associated virus is excreted into breast milk and that ZIKV is efficiently transmitted to the breastfed pups. Additionally, we studied murine breast milk cell types, and identified a majority of mammary luminal cells. Finally, we investigated the effect on ZIKV infectivity of several breast milk components that are antiviral against different viruses such as lactoferrin (LF) and lactalbumin (LA), or free fatty acids (FFA). We showed no effect of LF and LA, whereas FFA inactivated the virus. These results bring new insight concerning the mechanisms of ZIKV transmission during breastfeeding and identify biological factors modulating it. These elements should be considered in risk assessment of ZIKV mother-to-child transmission.

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

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.

Mother-to-Child Transmission of Arboviruses during Breastfeeding: From Epidemiology to Cellular Mechanisms.

Most viruses use several entry sites and modes of transmission to infect their host (parenteral, sexual, respiratory, oro-fecal, transplacental, transcutaneous, etc.). Some of them are known to be essentially transmitted via arthropod bites (mosquitoes, ticks, phlebotomes, sandflies, etc.), and are thus named arthropod-borne viruses, or arboviruses. During the last decades, several arboviruses have emerged or re-emerged in different countries in the form of notable outbreaks, resulting in a growing interest from scientific and medical communities as well as an increase in epidemiological studies. These studies have highlighted the existence of other modes of transmission. Among them, mother-to-child transmission (MTCT) during breastfeeding was highlighted for the vaccine strain of yellow fever virus (YFV) and Zika virus (ZIKV), and suggested for other arboviruses such as Chikungunya virus (CHIKV), dengue virus (DENV), and West Nile virus (WNV). In this review, we summarize all epidemiological and clinical clues that suggest the existence of breastfeeding as a neglected route for MTCT of arboviruses and we decipher some of the mechanisms that chronologically occur during MTCT via breastfeeding by focusing on ZIKV transmission process.

Zika Virus Requires the Expression of Claudin-7 for Optimal Replication in Human Endothelial Cells.

Zika virus (ZIKV) infection has been associated with a series of neurological pathologies. In patients with ZIKV-induced neurological disorders, the virus is detectable in the central nervous system. Thus, ZIKV is capable of neuroinvasion, presumably through infection of the endothelial cells that constitute the blood-brain barrier (BBB). We demonstrate that susceptibility of BBB endothelial cells to ZIKV infection is modulated by the expression of tight-junction protein claudin-7 (CLDN7). Downregulation of CLDN7 reduced viral RNA yield, viral protein production, and release of infectious viral particles in several endothelial cell types, but not in epithelial cells, indicating that CLDN7 implication in viral infection is cell-type specific. The proviral activity of CLDN7 in endothelial cells is ZIKV-specific since related flaviviruses were not affected by CLDN7 downregulation. Together, our data suggest that CLDN7 facilitates ZIKV infection in endothelial cells at a post-internalization stage and prior to RNA production. Our work contributes to a better understanding of the mechanisms exploited by ZIKV to efficiently infect and replicate in endothelial cells and thus of its ability to cross the BBB.

Alteration of blood-brain barrier integrity by retroviral infection.

The blood-brain barrier (BBB), which forms the interface between the blood and the cerebral parenchyma, has been shown to be disrupted during retroviral-associated neuromyelopathies. Human T Lymphotropic Virus (HTLV-1) Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP) is a slowly progressive neurodegenerative disease associated with BBB breakdown. The BBB is composed of three cell types: endothelial cells, pericytes and astrocytes. Although astrocytes have been shown to be infected by HTLV-1, until now, little was known about the susceptibility of BBB endothelial cells to HTLV-1 infection and the impact of such an infection on BBB function. We first demonstrated that human cerebral endothelial cells express the receptors for HTLV-1 (GLUT-1, Neuropilin-1 and heparan sulfate proteoglycans), both in vitro, in a human cerebral endothelial cell line, and ex vivo, on spinal cord autopsy sections from HAM/TSP and non-infected control cases. In situ hybridization revealed HTLV-1 transcripts associated with the vasculature in HAM/TSP. We were able to confirm that the endothelial cells could be productively infected in vitro by HTLV-1 and that blocking of either HSPGs, Neuropilin 1 or Glut1 inhibits this process. The expression of the tight-junction proteins within the HTLV-1 infected endothelial cells was altered. These cells were no longer able to form a functional barrier, since BBB permeability and lymphocyte passage through the monolayer of endothelial cells were increased. This work constitutes the first report of susceptibility of human cerebral endothelial cells to HTLV-1 infection, with implications for HTLV-1 passage through the BBB and subsequent deregulation of the central nervous system homeostasis. We propose that the susceptibility of cerebral endothelial cells to retroviral infection and subsequent BBB dysfunction is an important aspect of HAM/TSP pathogenesis and should be considered in the design of future therapeutics strategies.

Evidence That Zika Virus Is Transmitted by Breastfeeding to Newborn A129 (Ifnar1 Knock-Out) Mice and Is Able to Infect and Cross a Tight Monolayer of Human Intestinal Epithelial Cells.

Zika virus (ZIKV) belongs to the Flavivirus genus in the Flaviviridae family. Mainly transmitted via mosquito bites (Aedes aegypti, Aedes albopictus), ZIKV has been classified in the large category of arthropod-borne viruses, or arboviruses. However, during the past two outbreaks in French Polynesia (2013-2014) and Latin America (2015-2016), several cases of ZIKV human-to-human transmission were reported, either vertically via transplacental route but also horizontally after sexual intercourse. Interestingly, high viral burdens were detected in the colostrum and breast milk of infected women and mother-to-child transmission of ZIKV during breastfeeding was recently highlighted. In a previous study, we highlighted the implication of the mammary epithelium (blood-milk barrier) in ZIKV infectious particles excretion in breast milk. However, mechanisms of their further transmissibility to the newborn via oral route through contaminated breast milk remain unknown. In this study, we provide the first experimental proof-of-concept of the existence of the breastfeeding as a route for mother-to-child transmission of ZIKV and characterized the neonatal oral transmission in a well-established mouse model of ZIKV infection. From a mechanistical point-of-view, we demonstrated for the first time that ZIKV was able to infect and cross an in vitro model of tight human intestinal epithelium without altering its barrier integrity, permitting us to consider the gut as an entry site for ZIKV after oral exposure. By combining in vitro and in vivo experiments, this study strengthens the plausibility of mother-to-child transmission of ZIKV during breastfeeding and helps to better characterize underlying mechanisms, such as the crossing of the newborn intestinal epithelium by ZIKV. As a consequence, these data could serve as a basis for a reflection about the implementation of measures to prevent ZIKV transmission, while keeping in mind breastfeeding-associated benefits.

Arboviruses and Muscle Disorders: From Disease to Cell Biology.

Infections due to arboviruses (arthropod-borne viruses) have dramatically increased worldwide during the last few years. In humans, symptoms associated with acute infection of most arboviruses are often described as "dengue-like syndrome", including fever, rash, conjunctivitis, arthralgia, and muscular symptoms such as myalgia, myositis, or rhabdomyolysis. In some cases, muscular symptoms may persist over months, especially following flavivirus and alphavirus infections. However, in humans the cellular targets of infection in muscle have been rarely identified. Animal models provide insights to elucidate pathological mechanisms through studying viral tropism, viral-induced inflammation, or potential viral persistence in the muscle compartment. The tropism of arboviruses for muscle cells as well as the viral-induced cytopathic effect and cellular alterations can be confirmed in vitro using cellular models. This review describes the link between muscle alterations and arbovirus infection, and the underlying mechanisms.

Differentiation-dependent susceptibility of human muscle cells to Zika virus infection.

Muscle cells are potential targets of many arboviruses, such as Ross River, Dengue, Sindbis, and chikungunya viruses, that may be involved in the physiopathological course of the infection. During the recent outbreak of Zika virus (ZIKV), myalgia was one of the most frequently reported symptoms. We investigated the susceptibility of human muscle cells to ZIKV infection. Using an in vitro model of human primary myoblasts that can be differentiated into myotubes, we found that myoblasts can be productively infected by ZIKV. In contrast, myotubes were shown to be resistant to ZIKV infection, suggesting a differentiation-dependent susceptibility. Infection was accompanied by a caspase-independent cytopathic effect, associated with paraptosis-like cytoplasmic vacuolization. Proteomic profiling was performed 24h and 48h post-infection in cells infected with two different isolates. Proteome changes indicate that ZIKV infection induces an upregulation of proteins involved in the activation of the Interferon type I pathway, and a downregulation of protein synthesis. This work constitutes the first observation of primary human muscle cells susceptibility to ZIKV infection, and differentiation-dependent restriction of infection from myoblasts to myotubes. Since myoblasts constitute the reservoir of stem cells involved in reparation/regeneration in muscle tissue, the infection of muscle cells and the viral-induced alterations observed here could have consequences in ZIKV infection pathogenesis.

Characterization of reemerging chikungunya virus.

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.

Productive Infection of Mouse Mammary Glands and Human Mammary Epithelial Cells by Zika Virus.

Zika virus (ZIKV) belongs to the large category of arboviruses. Surprisingly, several human-to-human transmissions of ZIKV have been notified, either following sexual intercourse or from the mother to fetus during pregnancy. Importantly, high viral loads have been detected in the human breast milk of infected mothers, and the existence of breastfeeding as a new mode of mother-to-child transmission of ZIKV was recently hypothesized. However, the maternal origin of infectious particles in breast milk is currently unknown. Here, we show that ZIKV disseminates to the mammary glands of infected mice after both systemic and local exposure with differential kinetics. Ex vivo, we demonstrate that primary human mammary epithelial cells were sensitive and permissive to ZIKV infection in this study. Moreover, by using in vitro models, we prove that mammary luminal- and myoepithelial-phenotype cell lines are both able to produce important virus progeny after ZIKV exposure. Our data suggest that the dissemination of ZIKV to the mammary glands and subsequent infection of the mammary epithelium could be one mechanism of viral excretion in human breast milk.

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Risk factors for seasonal influenza virus detection in stools of patients consulting in general practice for acute respiratory infections in France, 2014-2016.

BACKGROUND: Previous studies reported detection of influenza RNA in stools of patients with seasonal influenza infection. While this detection may have a clinical significance, other factors may influence the stool positivity for influenza viruses. OBJECTIVES: The objective of this study was to investigate demographical, clinical, and microbiological factors which could favor the presence of influenza viral RNA in the stools of patients with laboratory-confirmed influenza infection. METHODS: Acute respiratory infection (ARI) patients were enrolled by general practitioners (GP) during two winter seasons (2014-2016). Nasopharyngeal swabs, stool specimens, and clinical data were collected. Samples were tested for 12 respiratory pathogen groups (nasopharyngeal and stool specimens) and for 12 enteric pathogens (stool specimens). RESULTS: Among the 331 patients with ARI enrolled by GP, 114 (34.4%) presented influenza infection. Influenza RNA was detected in stool samples of 21% (24/114) of the 114 stool specimens analyzed. Hospitalization (adjusted odds ratio (aOR) = 7.8 (95% confidence interval (CI)) [1.7-33.7], P = .02), age between 45 and 64 years (aOR = 4.8 [1.7-14.5], P = .01), consumption of raw shellfish and/or mollusks (aOR = 16.7 [3.6-90.9], P = .00), and use of antibiotics (aOR = 6.4 [2.1-19.8], P = .006) or antiviral treatment (aOR = 7.4 [1.9-29], P = .01) were significantly associated with an increased odds of the detection of influenza RNA in stools. Among the 24 stool samples subjected to viral isolation, no one showed virus growth. CONCLUSIONS: These findings will be useful to studies investigating the dissemination route of influenza viruses to gastrointestinal tract.