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Bovine herpesvirus 1 (BHV-1) infection may lead to conjunctivitis, upper respiratory tract problems, pneumonia, genital disorders and abortion. BHV-1 is able to spread quickly in a plaque-wise manner and invade by breaching the basement membrane (BM) barrier in the respiratory mucosa. BHV-1 Us3, a serine/threonine kinase, induces a dramatic cytoskeletal reorganization and BHV-1 Us9, a tail-anchored membrane protein, is required for axonal transport of viruses in neurons. In this study, we investigated the role of Us3 and Us9 during BHV-1 infection in the respiratory mucosa. First, we constructed and characterized BHV-1 Us3 null, Us9 null and revertant viruses. Then, we analysed the viral replication and plaque size (latitude) in Madin–Darby bovine kidney (MDBK) cells and the respiratory mucosa as well as viral penetration depth underneath the BM of the respiratory mucosa when inoculated with these recombinant viruses. Knockout of Us3 resulted in a 1 log10 reduction in viral titre and plaque size (latitude) in MDBK cells and the trachea mucosa. There were no defects in the cell-to-cell spread observed for BHV-1 Us9 null virus. Both BHV-1 Us3 null and Us9 null viruses showed a significant reduction of plaque penetration underneath the BM; however, penetration was not completely inhibited. In conclusion, the current findings demonstrated that Us3 and Us9 play an important role in the invasion of BHV-1 through the BM of the respiratory mucosa, which shows the way forward for research-based attenuation of viruses in order to make safer and better-performing vaccines.

Avian infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus of poultry that is spread worldwide. ILTV enters its host via the respiratory tract and the eyes. Although ILTV has been known for a long time, the replication characteristics of the virus in the respiratory and conjunctival mucosa are still poorly studied. To study these characteristics, two in vitro explant models were developed. Light microscopy and fluorescent terminal deoxynucleotidyl transferase dUTP nick end-labelling staining were used to evaluate the viability of mucosal explants, which were found to be viable up to the end of the experiment at 96 h of cultivation. The tracheal and conjunctival mucosal explants were inoculated with ILTV and collected at 0, 24, 48 and 72 h post inoculation (p.i.). ILTV spread in a plaque-wise manner in both mucosae. A reproducible quantitative analysis of this mucosal spread was evaluated by measuring plaque numbers, plaque latitude and invasion depth underneath the basement membrane. No major differences in plaque numbers were observed over time. Plaque latitude progressively increased to 70.4 ± 12.9 μm in the trachea and 97.8 ± 9.5 μm in the conjunctiva at 72 h p.i. The virus had difficulty crossing the basement membrane and was first observed only at 48 h p.i. The virus was observed at 72 h p.i. in 56% (trachea) and 74% (conjunctiva) of the plaques. Viability analysis of infected explants indicated that ILTV blocks apoptosis in infected cells of both mucosae but activates apoptosis in bystander cells.

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.

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.

International audience; 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 situation in an 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 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.

In 2008 and 2009 a large number of cases of haemorrhagic diathesis (HD) in neonatal calves were reported in different European countries. In Flanders, 84 cases of neonatal HD in 30 herds were reported in this period. The disease typically affects calves younger than 1 month old from different breed and gender. Prominent clinical signs are cutaneous bleeding, petechiae on all mucosae, melena and often high fever. Early in the disease, the mental state of the animals is uncompromised. The typical haematological finding is pancytopenia, with severe to complete thrombocytopenia being the cause of the increased susceptibility to bleeding. In seven of the affected herds blood samples of calves of the same age group as the clinical case were collected and on six of those farms at least one subclinical case could be identified. Necropsy findings were generalized petechiae, ecchymoses or haemorrhages and variable lymphadenopathy. Histopathology of haemorrhagic lesions revealed multifocal extravasation of red blood cells (haemorrhage) with preservation of tissue architecture and absence of other abnormalities. Total bone marrow aplasia and depletion of all lymphoid tissue was the most prominent finding on histology. Activated macrophages and haemophagocytosis were seen on bone marrow cytology from two live calves. Polymerase chain reaction for bovine viral diarrhoea virus, bluetongue and epizootic haemorrhagic disease virus was negative. Several attempts to isolate a viral agent were unsuccessful.

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.

Recently, a novel virus has been identified among ruminants in Western Europe. This virus, the so-called Schmallenberg virus, belongs to the family Bunyaviridae, genus Orthobunyavirus, serogroup Simbu and is closely related to Akabane, Aino and Shamonda virus. In cattle, acute symptoms in the dam and adult animals generally include high fever, milk drop and diarrhea. More importantly, infection during gestation may lead to abortion, stillbirth and congenital malformations. As all bunyaviruses, Schmallenberg virus also uses vectors for efficient transmission. Closely related viruses causing similar symptoms, such as Akabane and Akabane- like viruses, are mainly transmitted by Culicoides. It is very likely that Schmallenberg virus is transmitted by similar vectors. This review provides an overview of Bunyaviridae, their epidemiology, symptoms, prevention and control. Special emphasis is put on the Simbu serogroup of the Orthobunyavirus genus pointing out the similarities between them and closely related members.

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.

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.

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.

BackgroundHerpes 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 findingsA 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/significanceThis 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.

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 herpesvirus type 1 (EHV-1) is the causative agent of equine herpes myeloencephalopathy, of which outbreaks are reported with increasing frequency throughout North America and Europe. This has resulted in its classification as a potentially emerging disease by the US Department of Agriculture. Recently, it was found that a single nucleotide polymorphism (SNP) in the viral DNA polymerase gene (ORF30) at aa 752 (N→D) is associated with the neurovirulent potential of EHV-1. In the present study, equine respiratory mucosal explants were inoculated with several Belgian isolates typed in their ORF30 as D752 or N752, to evaluate a possible difference in replication in the upper respiratory tract. In addition, to evaluate whether any observed differences could be attributed to the SNP associated with neurovirulence, the experiments were repeated with parental Ab4 (reference neurovirulent strain), parental NY03 (reference non-neurovirulent strain) and their N/D revertant recombinant viruses. The salient findings were that EHV-1 spreads plaquewise in the epithelium, but plaques never cross the basement membrane (BM). However, single EHV-1-infected cells could be observed below the BM at 36 h post-inoculation (p.i.) for all N752 isolates and at 24 h p.i. for all D752 isolates, and were identified as monocytic cells and T lymphocytes. Interestingly, the number of infected cells was two to five times higher for D752 isolates compared with N752 isolates at every time point analysed. Finally, this study showed that equine respiratory explants are a valuable and reproducible model to study EHV-1 neurovirulence in vitro, thereby reducing the need for horses as experimental animals.