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ABSTRACT Alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), and bovine herpesvirus type 1 (BoHV-1), are significant pathogens affecting humans and animals. These viruses penetrate the upper respiratory tract mucosa, yet the mechanisms facilitating this invasion are not fully understood. This study investigates the role of the gE/gI glycoprotein complex and proteases in mucosal invasion by these viruses. Using species-specific respiratory mucosal explants, we observed that the removal of extracellular calcium disrupts epithelial junction integrity, enhancing viral infection across all viruses and suggesting a common mechanism of targeting a basolaterally located receptor. PRV exhibited significantly faster replication and deeper invasion compared to HSV-1 and BoHV-1. The gE glycoprotein was consistently polarized at the basement membrane across all viruses, indicating a critical role in the process of viral entry and subsequent spread through the epithelium. In this context, “infection” refers to the virus’s attachment to its cell-surface receptor, entry into the cell, and completion of the viral life cycle, culminating in the production of progeny virions. Notably, in gE/gI null mutants of PRV and HSV-1, while the infection was not abortive and the viral life cycle was completed, the infection was delayed, and the invasion into the deeper layers of the epithelium and underlying mucosa was significantly reduced. In BoHV-1 mutants, this effect was even more pronounced, with infection restricted to the apical cells, failing to progress to the basal cells. In addition, PRV and HSV-1 invasion involved serine protease activity, unlike BoHV-1, which correlates with its slower invasion pace. Notably, the protease facilitating PRV invasion was identified as a urokinase plasminogen activator (uPA), while the specific protease for HSV-1 remains unidentified. These findings highlight the critical roles of the gE/gI complex and proteases in alphaherpesvirus pathogenesis, offering potential targets for therapeutic intervention.IMPORTANCEHerpes simplex virus type 1 (HSV-1) infections are a worldwide issue. More than three billion people are infected with HSV-1 globally. Although most infections with HSV-1 occur subclinically, severe symptoms and complications are numerous and can be life-threatening. Complications include encephalitis and blindness. Recently, HSV-1 infections have been associated with the development of Alzheimer's Disease. To date, no effective vaccines against HSV-1 are on the market. Pseudorabies virus (PRV) and bovine herpesvirus type 1 (BoHV-1) are two alphaherpesviruses of major veterinary importance. Although efforts have been made to eradicate these viruses from livestock animals, clinical problems still occur, resulting in great economic losses for farmers. It is evident that new insights into the pathogenesis of alphaherpesviruses are needed, to develop effective treatments and novel preventive therapies.

The addition of phosphates to meat products improves the emulsifying and gelling properties of meat proteins, in turn enhancing overall product quality. The current market trend towards additive-free products and the health issues related to phosphate challenge the industry to develop phosphate-free meat products. The aim of this study was to evaluate the potential of seven protein-based ingredients (pea, blood plasma, gelatin, soy, whey, egg, and potato) to remediate quality losses of emulsified meat products (cooked sausages) upon phosphate elimination. First, the intrinsic gelling and emulsifying characteristics of the proteins were assessed. Next, the proteins were added to phosphate-free sausages, of which quality characteristics during production (viscoelastic behavior and emulsion stability) and of the final products (texture, cooking loss, and pH) were screened. Blood plasma and soy were superior in phosphate-free cooked sausages, as no significant differences in hardness, cooking yield, or stability were found compared to phosphate-containing sausages. Egg and pea also improved the previously mentioned quality characteristics of phosphate-free sausages, although to a lesser extent. These insights could not entirely be explained based on the intrinsic gelling and emulsifying capacity of the respective proteins. This indicated the importance of a well-defined standardized meat matrix to determine the potential of alternative proteins in meat products.

Since insects are a promising alternative protein source, the application potential of three insect larvae (Alphitobius diaperinus, Tenebrio molitor and Zophobas morio) for food purposes was explored. To this end, the effect of isothermal heating at 5 different temperatures (70 °C-90 °C) on structure formation in insect batters was studied rheologically. Meat batters (with the same protein content as insect batters), isothermally heated at 70 °C, were also studied for comparison. Cryo-SEM imaging was used to visualize the microstructure of raw and heated insect batters. These images showed that a network was formed in the heated batters, as well as in the raw batters. However, no clear effect of temperature or insect larva on the microstructure was observed. Rheologically, both the heating temperature applied and the insect larva used were shown to have a significant effect on the viscoelastic properties of the insect batters. Generally, batters containing Z. morio larvae showed both higher storage moduli (G') and longer linear viscoelastic regions (LVRs) compared to the other insect larvae, indicating that these larvae had the best structure forming capacities. Furthermore, both G' and the length of the LVR increased with increasing isothermal heating temperature, indicating more structure formation and structure stability in insect batters heated at higher temperatures. Compared to the meat batters, however, the insect larvae were shown to have inferior structure forming capacities. Even at the highest heating temperature (90 °C) the viscoelastic properties of the insect batters only approached those of meat batters heated at 70 °C. Therefore, it was concluded that higher heating temperatures may need to be employed in insect-based food products compared to meat products in order to obtain sufficient structure formation and the desired textural properties., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The aim of this research was to simultaneously study the effect of meat type (chicken breast and leg meat), animal fatty acid composition (selected pork backfats having a low and high degree of saturation, respectively), and isothermal temperature (50, 60, 70, and 80 °C) on the viscoelastic properties of meat batters during and after application of different time-temperature profiles. Gelation of meat proteins contributed most to the viscoelastic properties of meat batters during heating, whereas crystallization of the lipids especially contributed to the viscoelastic properties during the cooling phase. Although the meat type had little effect on the final viscoelastic properties of the meat product, the fatty acid composition had a clear impact on the melting peak area (and therefore solid fat content) of lard, and subsequently on the final viscoelastic properties of meat batters prepared with different types of fats, with higher G' (elastic modulus) values for the most saturated animal fat. The crystallization of the fat clearly transcended the effect of the meat type with regard to G' at the end of the process. With increasing (isothermal) temperature, G' of meat batters increased. Therefore, it could be concluded that the structural properties of heated meat batters mainly depend on the heating temperature and the fatty acid composition, rather than the meat type., Practical Application: Quality characteristics of cooked sausages depend on multiple factors such as the meat and fat type, non-meat ingredients and processing conditions. From this study it could be concluded that the structural properties of cooked sausage batters mainly depend on the heating temperature and the fatty acid composition, rather than the meat type. Because the fatty acid composition of different animal fats differs widely, these results may be a concern for all manufactures of cooked sausages products with regard to the product structure and final texture, keeping in mind that rendered fat was used in this study, which is not common in sausage making., (© 2018 Institute of Food Technologists®.)

The effectiveness of predictive microbiology is limited by the lack of knowledge concerning the influence of food microstructure on microbial dynamics. Therefore, future modelling attempts should be based on experiments in structured food model systems as well as liquid systems. In this study, fish-based model systems with various microstructures were developed, i.e., two liquid systems (with and without xanthan gum), an emulsion, an aqueous gel, and a gelled emulsion. The microstructural effect was isolated by minimising compositional and physico-chemical changes among the different model systems. The systems were suitable for common growth and mild thermal inactivation experiments involving both homogeneous and surface inoculation. Average pH of the model systems was 6.36±0.03 and average a w was 0.988±0.002. The liquid system without xanthan gum behaved like a Newtonian fluid, while the emulsion and the liquid containing xanthan gum exhibited (non-Newtonian) pseudo-plastic behaviour. Both the aqueous gel and gelled emulsion were classified as strong gels, with a hardness of 1.35±0.07N and 1.25±0.05N, respectively. Fat droplet size of the emulsion and gelled emulsion model systems was evenly distributed around 1μm. In general, the set of model systems was proven to be suitable to study the influence of important aspects of food microstructure on microbial dynamics., (Copyright © 2017. Published by Elsevier Ltd.)

This paper provides insights in the isothermal gelation behavior of white and red chicken myofibrillar proteins (CMP) at different temperatures (20 to 80°C) and the underlying aggregation mechanism, allowing understanding of structure formation in poultry products during thermal processing. At low temperatures (20 to 60°C), an increase in aromatic surface hydrophobicity (SoANS) was found, suggesting potential formation of hydrophobic interactions between CMP. At higher temperatures (60 to 80°C), high SoANS and a significant decrease in total sulfhydryl amount (SH-amount) strongly indicate the presence of hydrophobic interactions and disulfide bonding, resulting in aggregation, as suggested by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The elastic modulus G' after 60 min isothermal heating (G'60min) significantly increased at 70 or 80°C, depending on the type of CMP. Differences in G'60min between white and red CMP were rather small at low temperatures (20 to 60°C). However, at 70°C, white CMP reached higher G'60min compared to red CMP, while the opposite was observed at 80°C. Overall, for every temperature studied, SoANS and SH-amount of red CMP were higher compared to white CMP. The differences in G'60min, SoANS, and SH-amount between white and red CMP were probably due to different isoforms. The results may help to steer quality characteristics of poultry products through intelligent choice of processing conditions., (© 2017 Poultry Science Association Inc.)

Phosphate reduction is of important industrial relevance in the manufacturing of emulsified meat products because it may give rise to a healthier product. The effect of seven different phosphate types was tested on the physicochemical and quality characteristics to select the most promising phosphate type for further cooked sausage manufacturing. Next, phosphate mass fraction was gradually reduced. Tetrasodium di- or pyrophosphate (TSPP) and sodium tripolyphosphate (STPP) increased pH, reduced structural properties, resulted in the highest emulsion stability, lowest cooking loss and had little effect on hardness. Based on the viscoelastic properties, a minimum mass fraction of 0.06% TSPP was sufficient to obtain an acceptable quality product. Rheology proved to be a very useful tool to evaluate the quality of meat products, as it gives insight in the structure of the meat product and especially the functional properties of meat proteins. Based on the obtained results, it can be concluded that the current amount of phosphate added to emulsified meat products can be significantly reduced with minimal loss of product quality.

Passage of the basement membrane (BM), which forms a barrier between the epithelium and the underlying lamina propria, represents an important step in the early pathogenesis of different alphaherpesviruses. Rho GTPase signaling plays an important role in transmigration of cells across the BM during physiological and pathological processes. We reported earlier that the US3 protein kinase of the alphaherpesvirus pseudorabies virus (PRV) interferes with Rho GTPase signaling and causes a reorganization of the host cell cytoskeleton, which as a consequence, enhances viral cell-to-cell spread in epithelial cell cultures. Here, using an ex vivo system of porcine nasal respiratory mucosa explants that allows to study PRV invasion through the BM, we found that a PRV strain that lacks US3 expression (ΔUS3 PRV) showed a reduced spread in mucosal epithelium and was virtually unable to breach the BM, in contrast to isogenic wild-type (WT) or US3 rescue PRV strains. Interestingly, addition of IPA3, an inhibitor of p21-activated kinases that blocks the effects of US3 on the cytoskeleton, suppressed the ability of WT PRV to spread across the BM. In addition, artificial suppression of RhoA signaling using CPC3 (cell-permeable C3 transferase) to mimic the effects of US3 on Rho GTPase signaling, significantly increased passage of ΔUS3 PRV through the BM, whereas it did not significantly affect BM passage of WT or US3 rescue PRV. In conclusion, these data indicate that US3 plays an important role in PRV mucosal invasion across the BM, which involves its interference with Rho GTPase signaling. This is the first report describing an alphaherpesvirus protein that drives viral BM passage., Importance: Many viruses, including alphaherpesviruses, primarily replicate in epithelial cells of surface mucosae, such as the respiratory mucosa. Some of these viruses breach the basement membrane underlying these epithelial cells to reach underlying connective tissue and blood vessels and invade the host. Hence, epithelial spread and basement membrane passage represent crucial but still poorly understood early steps in (alphaherpes)virus pathogenesis. Here, using ex vivo porcine respiratory mucosa explants, we show that the conserved US3 protein of the porcine alphaherpesvirus pseudorabies virus (PRV) is critical for passage of PRV across the basement membrane and contributes to efficient viral epithelial spread. In addition, we show that US3-mediated viral epithelial spread and passage across the basement membrane depend at least in part on the ability of this viral protein to modulate cellular Rho GTPase signaling. This is the first report that identifies an alphaherpesvirus protein that drives viral basement membrane passage., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

NOMAD is a spectrometer suite on board ESA's ExoMars trace gas orbiter due for launch in January 2016. NOMAD consists of two infrared channels and one ultraviolet and visible channel allowing the instrument to perform observations quasi-constantly, by taking nadir measurements at dayside and nightside, and during solar occultations. In this paper, the design, manufacturing, and testing of the two infrared channels are described. We focus upon the optical working principle in these channels, where an echelle grating, used as a diffractive element, is combined with an acousto-optical tunable filter, used as a diffraction order sorter.

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., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Staphylococcus aureus is an opportunistic pathogen able to colonize the upper respiratory tract and skin surfaces in mammals. Methicillin-resistant S. aureus ST398 is prevalent in pigs in Europe and North America. However, the mechanism of successful pig colonization by MRSA ST398 is poorly understood. To study MRSA colonization in pigs, an ex vivo model consisting of porcine nasal mucosa explants cultured at an air-liquid interface was evaluated. In cultured mucosa explants from the surfaces of the ventral turbinates and septum of the pig nose no changes in cell morphology and viability were observed up to 72 h. MRSA colonization on the explants was evaluated followed for three MRSA ST398 isolates for 180 minutes. The explants were incubated with 3×10(8) CFU/ml in PBS for 2 h to allow bacteria to adhere to the explants surface. Next the explants were washed and in the first 30 minutes post adhering time, a decline in the number of CFU was observed for all MRSA. Subsequently, the isolates showed either: bacterial growth, no growth, or a further reduction in bacterial numbers. The MRSA were either localized as clusters between the cilia or as single bacteria on the cilia surface. No morphological changes in the epithelium layer were observed during the incubation with MRSA. We conclude that porcine nasal mucosa explants are a valuable ex vivo model to unravel the interaction of MRSA with nasal tissue.

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Equine herpesvirus 1 (EHV1) is a ubiquitous equine alphaherpesvirus that causes respiratory disease, neurological symptoms and abortions. Current vaccines are not fully protective and effective therapeutics are lacking. A-5021 [(1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine], previously shown to possess potent anti-herpetic activity against most human herpesviruses, was evaluated for its potential to inhibit EHV1 replication. In equine embryonic lung (EEL) cells, infected with either a non-neurovirulent (97P70) or a neurovirulent (03P37) EHV1 isolate, A-5021 proved to be about 15-fold more potent than acyclovir in inhibiting viral replication. Moreover, in equine nasal mucosal explants, A-5021 (at 8 and 32μM) was able to completely inhibit viral plaque formation whereas acyclovir did not exert an antiviral effect at these concentrations. Our data demonstrate that A-5021 is a potent inhibitor of EHV1 replication and may have potential for the treatment and/or prophylaxis of infections with this virus., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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.

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.

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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.

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.

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 D(752) or N(752), 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 N(752) isolates and at 24 h p.i. for all D(752) isolates, and were identified as monocytic cells and T lymphocytes. Interestingly, the number of infected cells was two to five times higher for D(752) isolates compared with N(752) 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.

Two major structural elements of a cell are the cytoskeleton and the lipid membranes. Actin and cholesterol are key components of the cytoskeleton and membranes, respectively, and are involved in a plethora of different cellular processes. This review summarizes and discusses the interaction of alphaherpesviruses with actin and cholesterol during different stages of the replication cycle: virus entry, replication and assembly in the nucleus, and virus egress. Elucidating these interactions not only yields novel insights into the biology of these important pathogens, but may also shed new light on cell biological aspects of actin and cholesterol, and lead to novel avenues in the design of antiviral strategies., (Copyright 2010 Elsevier B.V. All rights reserved.)

An in vitro model of the upper respiratory tract of the horse was developed to investigate mechanisms of respiratory diseases. Four tissues of the upper respiratory tract of three horses were collected. Explants were maintained in culture at an air-liquid interface for 96h. At 0, 24, 48, 72 and 96h of cultivation, a morphometric analysis was performed using light microscopy, scanning electron microscopy and transmission electron microscopy. The explants were judged on morphometric changes of epithelium, basement membrane and connective tissue. Viability was evaluated using a fluorescent Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labelling (TUNEL) staining. No significant changes in morphometry and viability of any of the explants were observed during cultivation. Hence, the in vitro model may be useful to study infectious and non-infectious diseases at the level of the equine respiratory tract, with potential application to the development of vaccines and treatments for diseases of the respiratory tract.

Different alphaherpesviruses, including pseudorabies virus (PRV), are able to cross the basement membrane barrier in nasal respiratory epithelium. As a first step in investigating this invasion process, a detailed quantitative analysis system was set up to determine the kinetics of horizontal and vertical virus spread in nasal explants, using the virulent PRV strain 89V87. Plaque latitudes, total depths, depths measured from the basement membrane and volumes were determined at 0, 12, 24 and 36h post inoculation (pi). PRV 89V87 was found to spread in a plaquewise manner and to cross the basement membrane between 12 and 24hpi. During the 1960s-1970s, an increase in PRV virulence has been reported. To analyse potential differences in efficiency of infection and spread for different historical PRV strains, single infected cells and plaques of infected cells were quantified at 12 and 36hpi in nasal mucosa explants for seven European PRV strains, isolated in the 1960s (Becker, NIA1), the 1970s (NS374, NIA3, 75V19) and later (89V87, 00V72). All viruses were used at second passage in cell culture, except for the Becker strain, which had an unknown passage history. Older strains, Becker, NIA1 and/or NS374, showed lower numbers of primary infectious centers, lower capacity to form plaques and/or lower capacity to cross the basement membrane. The observed differences in virus-mucosa interactions may aid in understanding the virulence increase of PRV. The quantitative assay established here will be of use in unravelling the mechanism of alphaherpesvirus-mediated invasion through the basement membrane.

The mucosal surface of the respiratory tract is a common site of entry of many viruses. Molecular and cellular aspects of the interactions of respiratory viruses with the respiratory nasal mucosa are largely unknown. In order to be able to study those interactions in depth, an in vitro model was set up. This model consists of porcine respiratory nasal mucosa explants, cultured at an air-liquid interface. Light microscopy, scanning electron microscopy and transmission electron microscopy, combined with morphometric analysis and a fluorescent Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labelling (TUNEL) staining were used to evaluate the effects of in vitro culture on the integrity and viability of the explants. The explants were maintained in culture for up to 60 h post-sampling without significant morphometric (epithelial thickness, epithelial morphology, thickness of the lamina reticularis, continuity of the lamina densa, relative amounts of collagen and nuclei) changes and changes in viability. The potential to infect the explants was demonstrated for two porcine respiratory viruses of major importance: suid herpesvirus 1 and swine influenza virus H1N1. In conclusion, this in vitro model represents an ideal tool to study interactions between infectious agents and porcine respiratory nasal mucosa.

In the present review, several cell biological and molecular aspects of virus-cell and virus-host (pig) interactions are reviewed for pseudorabies (Aujeszky's disease) virus. Concerning the virus-cell interactions, the complex cascade of events in the virus replication cycle is given together with the different mechanisms of cell-to-cell spread. The pathogenesis of pseudorabies virus infections in pigs is concentrated on the sequence of events in the respiratory tract. Finally, a short overview is given on the control of the disease and eradication of the virus by the combination of marker vaccines and discriminating ELISA.

A new compact spaceborne high-resolution spectrometer developed for the European Space Agency's Venus Express spacecraft is described. It operates in the IR wavelength range of 2.2 to 4.3 microm and measures absorption spectra of minor constituents in the Venusian atmosphere. It uses a novel echelle grating with a groove density of 4 lines/mm in a Littrow configuration in combination with an IR acousto-optic tunable filter for order sorting and an actively cooled HgCdTe focal plane array of 256 by 320 pixels. It is designed to obtain an instrument line profile of 0.2 cm(-1). First results on optical and spectral properties are reported.

Conformational alterations induced by streptomycin in the bacterial ribosome have been investigated using as probes, ethidium bromide, N-[14C]ethylmaleimide and a spin label nitroxide analog of N-ethylmaleimide. 1. The binding of the antibiotic to the ribosome does not affect the reactivity of sulfhydryl groups towards N-ethylmaleimide. 2. The motional freedom of spin labels bound to ribosomal proteins S1 and S18 is increased but it is hardly affected at other labeled sites. This observation suggests that the binding of streptomycin causes a local loosening of the ribosomal structure. 3. Ribosomes are found to bind less ethidium bromide in the presence of streptomycin, which suggests that the binding of streptomycin decreases the degree of organization of ribosomal RNA.