Sarah Glorieux

Herpes Simplex Virus Type 1 Penetrates the Basement Membrane in Human Nasal Respiratory Mucosa

Background Herpes simplex virus infections are highly prevalent in humans. However, the current therapeutics suffer important drawbacks such as limited results in neonates, increasing occurrence of resistance and impeded treatment of stromal infections. Remarkably, interactions of herpesviruses with human mucosa, the locus of infection, remain poorly understood and the underlying mechanisms in stromal infection remain controversial. Methodology/Principal Findings A human model consisting of nasal respiratory mucosa explants was characterised. Viability and integrity were examined during 96 h of cultivation. HSV1-mucosa interactions were analysed. In particular, we investigated whether HSV1 is able to reach the stroma. Explant viability and integrity remained preserved. HSV1 induced rounding up and loosening of epithelial cells with very few apoptotic and necrotic cells observed. Following 16–24 h of infection, HSV1 penetrated the basement membrane and replicated in the underlying lamina propria. Conclusions/Significance This human explant model can be used to study virus-mucosa interactions and viral mucosal invasion mechanisms. Using this model, our results provide a novel insight into the HSV1 stromal invasion mechanism and for the first time directly demonstrate that HSV1 can penetrate the basement membrane.

Immobilization of Pseudorabies Virus in Porcine Tracheal Respiratory Mucus Revealed by Single Particle Tracking

Pseudorabies virus (PRV) initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated) nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: −31.8±1.5 mV) experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (−49.8±0.6 mV) and positively (36.7±1.1 mV) charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (−9.6±0.8 mV) diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV) was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV was due to complex mucoadhesive interactions including charge interactions rather than size exclusion.

Clinical and virological outcome of an infection with the Belgian equine arteritis virus strain 08P178

Equine viral arteritis (EVA) is an infectious disease with variable clinical outcome. Outbreaks, causing important economic losses, are becoming more frequent. Currently, there is a shortage of pathogenesis studies performed with European strains. In the present study, eight seronegative ponies were experimentally inoculated with the Belgian strain of equine arteritis virus (EAV) 08P178 (EU-1 clade) and monitored daily for clinical signs of EVA. Nasopharyngeal swabs, ocular swabs, bronchoalveolar cells and blood were collected for virological and serological testing. Two ponies were euthanized at 3, 7, 14, and 28 days post infection (DPI). After necropsy, specimens were collected for virus titration and immunofluorescence. EVA symptoms such as fever and lymphadenomegaly were evident from 3 to 10 DPI. Virus was isolated in nasal secretions from 2 to 9 DPI and in bronchoalveolar cells from 3 to 7 DPI. A cell-associated viraemia was detected from 3 to 10 DPI. After replication in the respiratory tract and draining lymph nodes, EAV reached secondary target organs (high virus titers in internal organs sampled at 7 DPI). At 14 DPI, virus titers dropped drastically and, at 28 DPI, only tonsils were positive. Immunofluorescence revealed both individual and clustered EAV-infected cells. Antibodies were detected starting from 7 DPI. It can be concluded that the Belgian strain 08P178 is a European mildly virulent subtype. At present, most European EAV strain infections were thought to run a subclinical course. This study is a proof that mildly virulent European EAV strains do exist in the field.

Diverse microbial interactions with the basement membrane barrier

During primary contact with susceptible hosts, microorganisms face an array of barriers that thwart their invasion process. Passage through the basement membrane (BM), a 50–100-nm-thick crucial barrier underlying epithelia and endothelia, is a prerequisite for successful host invasion. Such passage allows pathogens to reach nerve endings or blood vessels in the stroma and to facilitate spread to internal organs. During evolution, several pathogens have developed different mechanisms to cross this dense matrix of sheet-like proteins. To breach the BM, some microorganisms have developed independent mechanisms, others hijack host cells that are able to transverse the BM (e.g. leukocytes and dendritic cells) and oncogenic microorganisms might even trigger metastatic processes in epithelial cells to penetrate the underlying BM.

Comparative analysis of replication characteristics of BoHV-1 subtypes in bovine respiratory and genital mucosa explants: a phylogenetic enlightenment

In general, members of the Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as a preferential site for primary replication. Bovine herpesvirus type 1 (BoHV-1) may replicate at both sites and cause two major clinical entities designated as infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis/balanoposthitis (IPV/IPB) in cattle. It has been hypothesized that subtype 1.1 invades preferentially the upper respiratory mucosa whereas subtype 1.2 favors replication at the peripheral genital tract. However, some studies are in contrast with this hypothesis. A thorough study of primary replication at both mucosae could elucidate whether or not different BoHV-1 subtypes show differences in mucosa tropism. We established bovine respiratory and genital organ cultures with emphasis on maintenance of tissue morphology and viability during in vitro culture. In a next step, bovine respiratory and genital mucosa explants of the same animals were inoculated with several BoHV-1 subtypes. A quantitative analysis of viral invasion in the mucosa was performed at 0 h, 24 h, 48 h and 72 h post inoculation (pi) by measuring plaque latitude and penetration depth underneath the basement membrane. All BoHV-1 subtypes exhibited a more profound invasion capacity in respiratory tissue compared to that in genital tissue at 24 h pi. However, at 24 h pi plaque latitude was found to be larger in genital tissue compared to respiratory tissue and this for all subtypes. These similar findings among the different subtypes take the edge off the belief of the existence of specific mucosa tropisms of different BoHV-1 subtypes.

Equine alphaherpesviruses (EHV-1 and EHV-4) differ in their efficiency to infect mononuclear cells during early steps of infection in nasal mucosal explants

Equine herpesvirus type 1 (EHV-1) replicates extensively in the epithelium of the upper respiratory tract, after which it can spread throughout the body via a cell-associated viremia in mononuclear leukocytes reaching the pregnant uterus and central nervous system. In a previous study, we were able to mimic the in vivo situation in an in vitro respiratory mucosal explant system. A plaquewise spread of EHV-1 was observed in the epithelial cells, whereas in the connective tissue below the basement membrane (BM), EHV-1-infected mononuclear leukocytes were noticed. Equine herpesvirus type 4 (EHV-4), a close relative of EHV-1, can also cause mild respiratory disease, but a cell-associated viremia in leukocytes is scarce and secondary symptoms are rarely observed. Based on this striking difference in pathogenicity, we aimed to evaluate how EHV-4 behaves in equine mucosal explants. Upon inoculation of equine mucosal explants with the EHV-4 strains VLS 829, EQ(1) 012 and V01-3-13, replication of EHV-4 in epithelial cells was evidenced by the presence of viral plaques in the epithelium. Interestingly, EHV-4-infected mononuclear leukocytes in the connective tissue below the BM were extremely rare and were only present for one of the three strains. The inefficient capacity of EHV-4 to infect mononuclear cells explains in part the rarity of EHV-4-induced viremia, and subsequently, the rarity of EHV-4-induced abortion or EHM.

Herpes Simplex Virus Type 1 Infection Facilitates Invasion of Staphylococcus aureus into the Nasal Mucosa and Nasal Polyp Tissue

Background Staphylococcus aureus (S. aureus) plays an important role in the pathogenesis of severe chronic airway disease, such as nasal polyps. However the mechanisms underlying the initiation of damage and/or invasion of the nasal mucosa by S. aureus are not clearly understood. The aim of this study was to investigate the interaction between S. aureus and herpes simplex virus type 1 (HSV1) in the invasion of the nasal mucosa and nasal polyp tissue. Methodology/Principal Findings Inferior turbinate and nasal polyp samples were cultured and infected with either HSV1 alone, S. aureus alone or a combination of both. Both in turbinate mucosa and nasal polyp tissue, HSV1, with or without S. aureus incubation, led to focal infection of outer epithelial cells within 48 h, and loss or damage of the epithelium and invasion of HSV1 into the lamina propria within 72 h. After pre-infection with HSV1 for 24 h or 48 h, S. aureus was able to pass the basement membrane and invade the mucosa. Epithelial damage scores were significantly higher for HSV1 and S. aureus co-infected explants compared with control explants or S. aureus only-infected explants, and significantly correlated with HSV1-invasion scores. The epithelial damage scores of nasal polyp tissues were significantly higher than those of inferior turbinate tissues upon HSV1 infection. Consequently, invasion scores of HSV1 of nasal polyp tissues were significantly higher than those of inferior turbinate mucosa in the HSV1 and co-infection groups, and invasion scores of S. aureus of nasal polyp tissues were significantly higher than those of inferior turbinate tissues in the co-infection group. Conclusions/Significance HSV1 may lead to a significant damage of the nasal epithelium and consequently may facilitate invasion of S. aureus into the nasal mucosa. Nasal polyp tissue is more susceptible to the invasion of HSV1 and epithelial damage by HSV1 compared with inferior turbinate mucosa.

A trypsin-like serine protease is involved in pseudorabies virus invasion through the basement membrane barrier of porcine nasal respiratory mucosa

Several alphaherpesviruses breach the basement membrane during mucosal invasion. In the present study, the role of proteases in this process was examined. The serine protease-specific inhibitor AEBSF inhibited penetration of the basement membrane by the porcine alphaherpesvirus pseudorabies virus (PRV) by 88.1% without affecting lateral spread. Inhibitors of aspartic-, cysteine-, and metalloproteases did not inhibit viral penetration of the basement membrane. Further analysis using the Soybean Type I-S trypsin inhibitor for the serine protease subcategory of trypsin-like serine proteases resulted in a 96.9% reduction in plaque depth underneath the basement membrane. These data reveal a role of a trypsin-like serine protease in PRV penetration of the basement membrane.

Kinetics of BoHV-1 Dissemination in an in Vitro Culture of Bovine Upper Respiratory Tract Mucosa Explants

Bovine herpesvirus 1 (BoHV-1) is a well-known disease-causing agent in cattle. There is little known detailed information on viral behavior with emphasis on host invasion at primary replication sites such as the mucosa of the upper respiratory tract. Therefore, an in vitro system of bovine upper respiratory tract (bURT) mucosa explants was set up to study BoHV-1 molecular/cellular host-pathogen interactions. We performed a thorough morphometrical analysis (epithelial integrity, basement membrane continuity, and lamina propria integrity) using light microscopy and transmission electron microscopy. We applied a terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) staining as a viability test. Bovine upper respiratory tract mucosa explants were maintained in culture for up to 96 hours without any significant changes in morphometry and viability. Next, bURT mucosa explants were infected with BoHV-1 (Cooper) and collected at 0, 24, 48, and 72 hours postinoculation (p.i.). Using a quantitative analysis system to measure plaque latitude and invasion depth, we assessed dissemination characteristics in relation to elapsed time p.i. and found a plaquewise spread of BoHV-1 across the basement membrane as early as 24h p.i., similar to pseudorabies virus (PRV). Moreover, we observed that BoHV-1 exhibited an increased capacity to invade in proximal tracheal tissues compared to tissues of the deeper part of the nasal septum and ventral conchae. Revealing a more distinct invasion of BoHV-1 in proximal trachea, we can conclude that, in order to study an important aspect of BoHV-1 pathogenesis, the bovine upper respiratory tract mucosa explant model is the best suited model.

Mimicking Herpes Simplex Virus 1 and Herpes Simplex Virus 2 Mucosal Behavior in a Well-Characterized Human Genital Organ Culture

We developed and morphologically characterized a human genital mucosa explant model (endocervix and ectocervix/vagina) to mimic genital herpes infections caused by herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Subsequent analysis of HSV entry receptor expression throughout the menstrual cycle in genital tissues was performed, and the evolution of HSV-1/-2 mucosal spread over time was assessed. Nectin-1 and -2 were expressed in all tissues during the entire menstrual cycle. Herpesvirus entry mediator expression was limited mainly to some connective tissue cells. Both HSV-1 and HSV-2 exhibited a plaque-wise mucosal spread across the basement membrane and induced prominent epithelial syncytia.