Aspergillus Fumigatus Spore Proteases Alter the Respiratory Mucosa Architecture and Facilitate Equine Herpesvirus 1 Infection.

Numerous Aspergillus fumigatus (Af) airborne spores are inhaled daily by humans and animals due to their ubiquitous presence. The interaction between the spores and the respiratory epithelium, as well as its impact on the epithelial barrier function, remains largely unknown. The epithelial barrier protects the respiratory epithelium against viral infections. However, it can be compromised by environmental contaminants such as pollen, thereby increasing susceptibility to respiratory viral infections, including alphaherpesvirus equine herpesvirus type 1 (EHV-1). To determine whether Af spores disrupt the epithelial integrity and enhance susceptibility to viral infections, equine respiratory mucosal ex vivo explants were pretreated with Af spore diffusate, followed by EHV-1 inoculation. Spore proteases were characterized by zymography and identified using mass spectrometry-based proteomics. Proteases of the serine protease, metalloprotease, and aspartic protease groups were identified. Morphological analysis of hematoxylin-eosin (HE)-stained sections of the explants revealed that Af spores induced the desquamation of epithelial cells and a significant increase in intercellular space at high and low concentrations, respectively. The increase in intercellular space in the epithelium caused by Af spore proteases correlated with an increase in EHV-1 infection. Together, our findings demonstrate that Af spore proteases disrupt epithelial integrity, potentially leading to increased viral infection of the respiratory epithelium.

Cytokine-armed oncolytic herpes simplex viruses: a game-changer in cancer immunotherapy?

Cytokines are small proteins that regulate the growth and functional activity of immune cells, and several have been approved for cancer therapy. Oncolytic viruses are agents that mediate antitumor activity by directly killing tumor cells and inducing immune responses. Talimogene laherparepvec is an oncolytic herpes simplex virus type 1 (oHSV), approved for the treatment of recurrent melanoma, and the virus encodes the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). A significant advantage of oncolytic viruses is the ability to deliver therapeutic payloads to the tumor site that can help drive antitumor immunity. While cytokines are especially interesting as payloads, the optimal cytokine(s) used in oncolytic viruses remains controversial. In this review, we highlight preliminary data with several cytokines and chemokines, including GM-CSF, interleukin 12, FMS-like tyrosine kinase 3 ligand, tumor necrosis factor α, interleukin 2, interleukin 15, interleukin 18, chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 5, chemokine (C-X-C motif) ligand 4, or their combinations, and show how these payloads can further enhance the antitumor immunity of oHSV. A better understanding of cytokine delivery by oHSV can help improve clinical benefit from oncolytic virus immunotherapy in patients with cancer.

Diversity and connectedness of brine shrimp viruses in global hypersaline ecosystems.

Brine shrimp (Artemia) has existed on Earth for 400 million years and has major ecological importance in hypersaline ecosystems. As a crucial live food in aquaculture, brine shrimp cysts have become one of the most important aquatic products traded worldwide. However, our understanding of the biodiversity, prevalence and global connectedness of viruses in brine shrimp is still very limited. A total of 143 batches of brine shrimp (belonging to seven species) cysts were collected from six continents including 21 countries and more than 100 geographic locations worldwide during 1977-2019. In total, 55 novel RNA viruses were identified, which could be assigned to 18 different viral families and related clades. Eleven viruses were dsRNA viruses, 16 were +ssRNA viruses, and 28 were-ssRNA viruses. Phylogenetic analyses of the RNA-directed RNA polymerase (RdRp) showed that brine shrimp viruses were often grouped with viruses isolated from other invertebrates and fungi. Remarkably, most brine shrimp viruses were related to those from different hosts that might feed on brine shrimp or share the same ecological niche. A notable case was the novel brine shrimp noda-like virus 3, which shared 79.25% (RdRp) and 63.88% (capsid proteins) amino acid identity with covert mortality nodavirus (CMNV) that may cause losses in aquaculture. In addition, both virome composition and phylogenetic analyses revealed global connectedness in certain brine shrimp viruses, particularly among Asia and Northern America. This highlights the incredible species diversity of viruses in these ancient species and provides essential data for the prevalence of RNA viruses in the global aquaculture industry. More broadly, these findings provide novel insights into the previously unrecognized RNA virosphere in hypersaline ecosystems worldwide and demonstrate that human activity might have driven the global connectedness of brine shrimp viruses.

Susceptibility of perivenous macrophages to PRRSV-1 subtype 1 LV and PRRSV-1 subtype 3 Lena using a new vein explant model.

Vessel pathology such as increased permeability and blue discoloration is frequently observed with highly pathogenic PRRSV strains. However, data concerning the viral replication in the environment of blood vessels are absent. In the present study, ex vivo models with swine ear and hind leg vein explants were established to study the interaction of PRRSV-1 subtype 1 reference strain LV and highly pathogenic subtype 3 strain Lena with perivenous macrophages. The replication characteristics of these two strains were compared in vein explants by immunofluorescence analysis. The explants maintained a good viability during 48 hours of in vitro culture. We found that CD163-positive macrophages were mainly present around the veins and their number gradually decreased with increasing distance from the veins and longer incubation time. More CD163+Sn- cells than CD163+Sn+ cells (6.6 times more) were observed in the vein explants. The Lena strain demonstrated a higher replication level than the LV strain, with approximately 1.4-fold more infected cells in the surrounding areas of the ear vein and 1.1-fold more infected cells in the leg vein explants at 48 hours post inoculation. In both LV and Lena inoculated vein explants, most infected cells were identified as CD163+Sn+ (> 94%). In this study, an ex vivo vein model was successfully established, and our findings will contribute to a better understanding of the vein pathology during viral infections (e.g., PRRS, classical and African swine fever).

Several Alphaherpesviruses Interact Similarly with the NF-κB Pathway and Suppress NF-κB-Dependent Gene Expression.

Alphaherpesvirus infection is associated with attenuation of different aspects of the host innate immune response that is elicited to confine primary infections at the mucosal epithelia. Here, we report that infection of epithelial cells with several alphaherpesviruses of different species, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), feline alphaherpesvirus 1 (FHV-1), and bovine alphaherpesvirus 1 (BoHV-1) results in the inactivation of the responses driven by the nuclear factor kappa B (NF-κB) pathway, considered a pillar of the innate immune response. The mode to interact with and circumvent NF-κB-driven responses in infected epithelial cells is seemingly conserved in human, feline, and porcine alphaherpesviruses, consisting of a persistent activation of the NF-κB cascade but a potent repression of NF-κB-dependent transcription activity, which relies on replication of viral genomes. However, BoHV-1 apparently deviates from the other investigated members of the taxon in this respect, as BoHV-1-infected epithelial cells do not display the persistent NF-κB activation observed for the other alphaherpesviruses. In conclusion, this study suggests that inhibition of NF-κB transcription activity is a strategy used by several alphaherpesviruses to prevent NF-κB-driven responses in infected epithelial cells. IMPORTANCE The current study provides a side-by-side comparison of the interaction of different alphaherpesviruses with NF-κB, a key and central player in the (proinflammatory) innate host response, in infected nontransformed epithelial cell lines. We report that all studied viruses prevent expression of the hallmark NF-κB-dependent gene IκB, often but not always via similar strategies, pointing to suppression of NF-κB-dependent host gene expression in infected epithelial cells as a common and therefore likely important aspect of alphaherpesviruses.

Changes in intestinal morphology, number of mucus-producing cells and expression of coronavirus receptors APN, DPP4, ACE2 and TMPRSS2 in pigs with aging.

Porcine enteric viral infections cause high morbidity and mortality in young piglets (<3 weeks). Later, these rates decrease with age. This age-dependent infectivity remains largely unexplored. This study investigated the changes in intestinal morphology, number of mucus-producing cells and expression level of coronavirus receptors in three age groups of pigs. Villus height and crypt depth increased with age from 3 days to 3 months in duodenum and ileum but not in mid-jejunum, where the villus height decreased from 580 µm at 3 days to 430 µm at 3 months. Enterocyte length-to-width ratio increased from 3 days to 3 months in all intestinal regions. The number of mucus-producing cells increased with age in the intestinal villi and crypts. The Brunner's glands of the duodenum contained the highest concentration of mucus-producing cells. The expression of coronavirus receptor APN was highest in the small intestinal villi at all ages. DPP4 expression slightly decreased over time in jejunum and ileum; it was highest in the ileal villi of 3-day-old piglets (70.2% of cells). ACE2 and TMPRSS2 positive cells increased with age in jejunal and ileal crypts and were particularly dominant in the ileal crypts (> 45% of cells). Except for the expression of DPP4 in the jejunum and ileum of young pigs, the expression pattern of the selected coronavirus receptors was very different and not correlated with the age-dependent susceptibility to viral infections. In contrast, the number of mucus-producing cells increased over time and may play an essential role in protecting enteric mucosae against intestinal viruses.

Development of immortalized feline respiratory epithelial cells in an air-liquid-interface culture system for feline herpesvirus-1 study.

Feline herpesvirus-1 (FHV-1) is responsible for approximately 50% of diagnosed viral upper respiratory tract disease in cats. The virus infects and replicates in the epithelial cells located in upper respiratory tract. Commercial vaccines do not protect cats from the infection itself or development of latency. Previously, our lab developed a cell culture model using primary feline respiratory epithelial cells (pFRECs) to study respiratory innate immunity to FHV-1 and FHV-1 deletion mutants. However, the numbers of pFRECs that can be obtained per cat is limited. To improve the usage of respiratory epithelial 3D cultures in FHV-1 research, the present study immortalized feline respiratory epithelial cells (iFRECs) and characterized them morphologically and immunologically and evaluated the response to FHV-1 infection. Immortalization was achieved by transduction with Lenti-SV40T and Lenti-HPV E6/E7. Immortalized FRECs could be successfully subcultured for >20 passages, with positive gene expression of SV40T and HPV E6/E7. Immortalized FRECs expressed similar innate immunity-associated genes compared to pFRECs, including genes of Toll-like receptors (TLR1-9), interferon induced genes (OAS1, OAS3, IFI44, IFITM1, IFIT1), chemokines (CCL2, CCL3, CXCL8), pro-inflammatory and regulatory cytokines (IL-6, IL-4, IL-5, IL-12, and IL-18), and antimicrobials (DEFß10, DEFß4B). Finally, FHV-1 inoculation resulted in characteristic cytopathic effects starting at 24 hpi, with more than 80% cells detached and lysed by 72 hpi. Overall FHV-1 growth kinetics in iFRECs resembled the kinetics observed in pFRECs. In conclusion, we demonstrated that iFRECs are a useful tool to study feline respiratory disease including but not limited to FHV-1.

Cluster of differentiation frequency on antigen presenting-cells: The next step to cervical cancer prognosis?

Viruses may transform infected cells into benign or malignant tumors, promoting cell growth and survival via various intracellular pathways. Some oncogenic DNA viruses, such as human papillomaviruses, can lead to squamous intraepithelial lesions and cervical cancer. Furthermore, the early HPV virus'soncoproteins have been attributed to cancer initiation and development and tumor-enhancing action. In addition to viral oncoproteins, antigen-presenting cells (APC) and the number of clusters of differentiation (CD) markers expressed on their surface play an essential role in disease progression or tumorigenesis inhibition. This article discussed the function of CD markers in the interaction between APCs and cancer cells, immune cells' function in the infection process, and finally infected cells' malignancy. We investigated targeting these markers as a novel insight to create a new therapeutic or diagnosis strategy to prevent cervical cancer progression.

Innate antiviral responses in porcine nasal mucosal explants inoculated with influenza A virus are comparable with responses in respiratory tissues after viral infection.

Nasal mucosal explant (NEs) cultured at an air-liquid interface mimics in vivo conditions more accurately than monolayer cultures of respiratory cell linesor primary cells cultured in flat-bottom microtiter wells. NEs might be relevant for studies of host-pathogen interactions and antiviral immune responses after infection with respiratory viruses, including influenza and corona viruses. Pigs are natural hosts for swine influenza A virus (IAV) but are also susceptible to IAV from humans, emphasizing the relevance of porcine NEs in the study of IAV infection. Therefore, we performed fundamental characterization and study of innate antiviral responses in porcine NEs using microfluidic high-throughput quantitative real-time PCR (qPCR) to generate expression profiles of host genes involved in inflammation, apoptosis, and antiviral immune responses in mock inoculated and IAV infected porcine NEs. Handling and culturing of the explants ex vivo had a significant impact on gene expression compared to freshly harvested tissue. Upregulation (2-43 fold) of genes involved in inflammation, including IL1A and IL6, and apoptosis, including FAS and CASP3, and downregulation of genes involved in viral recognition (MDA5 (IFIH1)), interferon response (IFNA), and response to virus (OAS1, IFIT1, MX1) was observed. However, by comparing time-matched mock and virus infected NEs, transcription of viral pattern recognition receptors (RIG-I (DDX58), MDA5 (IFIH1), TLR3) and type I and III interferons (IFNB1, IL28B (IFNL3)) were upregulated 2-16 fold in IAV-infected NEs. Furthermore, several interferon-stimulated genes including MX1, MX2, OAS, OASL, CXCL10, and ISG15 was observed to increase 2-26 fold in response to IAV inoculation. NE expression levels of key genes involved in antiviral responses including IL28B (IFNL3), CXCL10, and OASL was highly comparable to expression levels found in respiratory tissues including nasal mucosa and lung after infection of pigs with the same influenza virus isolate.

Bacterial Toxins from Staphylococcus aureus and Bordetella bronchiseptica Predispose the Horse's Respiratory Tract to Equine Herpesvirus Type 1 Infection.

Respiratory disease in horses is caused by a multifactorial complex of infectious agents and environmental factors. An important pathogen in horses is equine herpesvirus type 1 (EHV-1). During co-evolution with this ancient alphaherpesvirus, the horse's respiratory tract has developed multiple antiviral barriers. However, these barriers can become compromised by environmental threats. Pollens and mycotoxins enhance mucosal susceptibility to EHV-1 by interrupting cell junctions, allowing the virus to reach its basolateral receptor. Whether bacterial toxins also play a role in this impairment has not been studied yet. Here, we evaluated the role of α-hemolysin (Hla) and adenylate cyclase (ACT), toxins derived from the facultative pathogenic bacterium Staphylococcus aureus (S. aureus) and the primary pathogen Bordetella bronchiseptica (B. bronchiseptica), respectively. Equine respiratory mucosal explants were cultured at an air-liquid interface and pretreated with these toxins, prior to EHV-1 inoculation. Morphological analysis of hematoxylin-eosin (HE)-stained sections of the explants revealed a decreased epithelial thickness upon treatment with both toxins. Additionally, the Hla toxin induced detachment of epithelial cells and a partial loss of cilia. These morphological changes were correlated with increased EHV-1 replication in the epithelium, as assessed by immunofluorescent stainings and confocal microscopy. In view of these results, we argue that the ACT and Hla toxins increase the susceptibility of the epithelium to EHV-1 by disrupting the epithelial barrier function. In conclusion, this study is the first to report that bacterial exotoxins increase the horse's sensitivity to EHV-1 infection. Therefore, we propose that horses suffering from infection by S. aureus or B. bronchiseptica may be more susceptible to EHV-1 infection.

Glyceraldehyde-3-Phosphate Dehydrogenase Restricted in Cytoplasmic Location by Viral GP5 Facilitates Porcine Reproductive and Respiratory Syndrome Virus Replication via Its Glycolytic Activity.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important endemic swine pathogens, causing enormous losses in the global swine industry. Commercially available vaccines only partially prevent or counteract the virus infection and correlated losses. PRRSV's replication mechanism has not been well understood. In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was screened to bind with the viral major envelope glycoprotein 5 (GP5) after PRRSV infection. The interacting sites are located within a 13-amino-acid (aa) region (aa 93 to 105) of GP5 and at Lys227 of GAPDH. Interestingly, viral GP5 restricts the translocation of GAPDH from the cytoplasm to the nucleus. Moreover, cytoplasmic GAPDH facilitates PRRSV replication by virtue of its glycolytic activity. The results suggest that PRRSV GP5 restricts GAPDH to the nucleus and exploits its glycolytic activity to stimulate virus replication. The data provide insight into the role of GAPDH in PRRSV replication and reveal a potential target for controlling viral infection. IMPORTANCE PRRSV poses a severe economic threat to the pig industry. PRRSV GP5, the major viral envelope protein, plays an important role in viral infection, pathogenicity, and immunity. However, interactions between GP5 and host proteins have not yet been well studied. Here, we show that GAPDH interacts with GP5 through binding a 13-aa sequence (aa 93 to 105) in GP5, while GP5 interacts with GAPDH at the K277 amino acid residue of GAPDH. We demonstrate that GP5 interacts with GAPDH in the cytoplasm during PPRSV infection, inhibiting GAPDH entry into the nucleus. PRRSV exploits the glycolytic activity of GAPDH to promote viral replication. These results enrich our understanding of PRRSV infection and pathogenesis and open a new avenue for antiviral prevention and PRRSV treatment strategies.

"Don, doff, discard" to "don, doff, decontaminate"-FFR and mask integrity and inactivation of a SARS-CoV-2 surrogate and a norovirus following multiple vaporised hydrogen peroxide-, ultraviolet germicidal irradiation-, and dry heat decontaminations.

BACKGROUND: As the SARS-CoV-2 pandemic accelerates, the supply of personal protective equipment remains under strain. To combat shortages, re-use of surgical masks and filtering facepiece respirators has been recommended. Prior decontamination is paramount to the re-use of these typically single-use only items and, without compromising their integrity, must guarantee inactivation of SARS-CoV-2 and other contaminating pathogens. AIM: We provide information on the effect of time-dependent passive decontamination (infectivity loss over time during room temperature storage in a breathable bag) and evaluate inactivation of a SARS-CoV-2 surrogate and a non-enveloped model virus as well as mask and respirator integrity following active multiple-cycle vaporised hydrogen peroxide (VHP), ultraviolet germicidal irradiation (UVGI), and dry heat (DH) decontamination. METHODS: Masks and respirators, inoculated with infectious porcine respiratory coronavirus or murine norovirus, were submitted to passive decontamination or single or multiple active decontamination cycles; viruses were recovered from sample materials and viral titres were measured via TCID50 assay. In parallel, filtration efficiency tests and breathability tests were performed according to EN standard 14683 and NIOSH regulations. RESULTS AND DISCUSSION: Infectious porcine respiratory coronavirus and murine norovirus remained detectable on masks and respirators up to five and seven days of passive decontamination. Single and multiple cycles of VHP-, UVGI-, and DH were shown to not adversely affect bacterial filtration efficiency of masks. Single- and multiple UVGI did not adversely affect respirator filtration efficiency, while VHP and DH induced a decrease in filtration efficiency after one or three decontamination cycles. Multiple cycles of VHP-, UVGI-, and DH slightly decreased airflow resistance of masks but did not adversely affect respirator breathability. VHP and UVGI efficiently inactivated both viruses after five, DH after three, decontamination cycles, permitting demonstration of a loss of infectivity by more than three orders of magnitude. This multi-disciplinal approach provides important information on how often a given PPE item may be safely reused.

Functional Analysis of Human and Feline Coronavirus Cross-Reactive Antibodies Directed Against the SARS-CoV-2 Fusion Peptide.

To face the continuous emergence of SARS-CoV-2 variants, broadly protective therapeutic antibodies are highly needed. We here focused on the fusion peptide (FP) region of the viral spike antigen since it is highly conserved among alpha- and betacoronaviruses. First, we found that coronavirus cross-reactive antibodies are commonly formed during infection, being omnipresent in sera from COVID-19 patients, in ~50% of pre-pandemic human sera (rich in antibodies against endemic human coronaviruses), and even in feline coronavirus-infected cats. Pepscan analyses demonstrated that a confined N-terminal region of the FP is strongly immunogenic across diverse coronaviruses. Peptide-purified human antibodies targeting this conserved FP epitope exhibited broad binding of alpha- and betacoronaviruses, besides weak and transient SARS-CoV-2 neutralizing activity. Being frequently elicited by coronavirus infection, these FP-binding antibodies might potentially exhibit Fc-mediated effector functions and influence the kinetics or severity of coronavirus infection and disease.

A substantial neutrophilic inflammation as regular part of severe type 2 chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is generally associated with severe type 2 immune reactions in the white population. However, recent findings suggest an additional role for neutrophils in severe type 2 inflammation. OBJECTIVE: This study aimed to characterize the neutrophilic inflammation in CRSwNP and its relation to eosinophilic inflammation in severe type 2 immune reactions. METHODS: The presence and activation of neutrophils and eosinophils was analyzed in CRS without NP and CRSwNP by measuring cell and activation markers via immunohistochemistry, immunofluorescence, Luminex assay, ELISA, UniCAP, fluorescence-activated cell sorting, and PCR. Differential neutrophil migration was assessed via Boyden-chamber assay and neutrophil survival was analyzed via flow cytometry. RESULTS: Both CRS without NP and CRSwNP displayed variable degrees of eosinophilic and neutrophilic inflammation, with a profound neutrophilic infiltration and activation in type 2 CRSwNP, associated with eosinophil extracellular traps cell death and Charcot-Leyden crystals, but independent of IL-17. Neutrophil extracellular traps cell death in CRSwNP was associated with bacterial colonization, however, neutrophils were less prone to undergo neutrophil extracellular traps cell death in the tissue of patients with severe type 2 CRSwNP. Neutrophils did not show increased migration nor survival in the CRSwNP environment in vitro. CONCLUSIONS: This study demonstrated a severe neutrophilic inflammation associated with severe eosinophilic type 2 inflammatory CRSwNP, the role of which needs further study.

Type 2 inflammation in chronic rhinosinusitis without nasal polyps: Another relevant endotype.

BACKGROUND: Chronic rhinosinusitis without nasal polyps (CRSsNP) is mainly considered a type 1 mediated disease. The role and clinical significance of type 2 immune responses in CRSsNP have not been addressed sufficiently; a recent cluster analysis for CRS described the existence of a subgroup of patients with CRSsNP with a type 2 inflammation. OBJECTIVE: We aimed to characterize the underlying type 2 immune response and its clinical significance in patients with CRSsNP. METHODS: A total of 240 patients with CRSsNP were endotyped and subdivided on the basis of expression of marker cytokines. Clinical data such as recurrence, comorbid asthma and allergy, and numbers of blood eosinophils and neutrophils were collected from all patients. A selection of 15 patients was further characterized for the presence of eosinophils, neutrophils, Charcot-Leyden crystals, and eosinophil extracellular traps in the mucosae. RESULTS: A type 2 immune response with increased levels of IL-4, IL-5, eosinophil cationic protein, IgE, and Staphylococcus aureus enterotoxin-specific IgE was observed in 49% of patients with CRSsNP. Those patients showed increased numbers of blood and tissue eosinophils, and they displayed a considerable eosinophilic inflammation associated with eosinophil extracellular trap cell death and Charcot-Leyden crystals. A significantly increased prevalence of recurrence and asthma was observed in patients with type 2 CRSsNP compared with in patients with non-type 2 CRSsNP. However, only 4 of 117 patients with type 2 CRSsNP developed nasal polyps within 12 years. CONCLUSION: This study shows that type 2 immune responses in CRSsNP follow similar patterns but are less pronounced than in chronic rhinosinusitis with nasal polyps. Also CRSsNP with a moderate type 2 immune response showed a considerable eosinophilic inflammation with clinical impact.

Characterization of feline herpesvirus-1 deletion mutants in tissue explant cultures.

Feline herpesvirus-1 (FHV-1) is the primary cause of viral respiratory and ocular disease in cats. While commercial vaccines can provide clinical protection, they do not protect from infection or prevent latency. Moreover, they are not safe for intranasal administration. Our overall objective is to develop a new mucosal vaccine against FHV-1 disease to address these shortcomings. Feline herpesvirus-1 deletion mutants of glycoprotein C (gC-), gE (gE-), US3-encoded serine/threonine protein kinase (PK-), and both gE and thymidine kinase (gE-TK-) were generated by bacterial artificial chromosome (BAC) mutagenesis. Tracheal tissue explants from eight cats were used to compare the pattern of viral infection and associated tissue damage, as well as virus spread through the basement membrane following inoculation with wild-type virus (WT), and gE-, gE-TK-, PK-, and gC- mutants. Tissues were collected at 24, 48, or 72  hours post-inoculation (hpi) followed by immunohistochemistry (IHC) for FHV-1. Histological changes were graded based on the distribution of virus infected cells and the severity of tissue damage. Inoculations with the WT virus resulted in maximal scores at 72 hpi both at a multiplicity of infection (MOI) of 1 and 0.1. Inoculation with the gE- mutant produced scores similar to scores of explants inoculated with the WT virus at 24 and 48 hpi, but scores were significantly decreased at 72 hpi. Explants inoculated with the gE-TK- mutant showed significantly decreased scores at all time points. Further, the majority of explants inoculated with the PK- mutant resulted in scores of zero at all time points, regardless of MOI. Finally, inoculation with WT resulted in significant stromal invasion below the infected epithelium, while stromal invasion was observed in less than 50 % of the samples following inoculation with gE-, gE-TK-, PK-, or gC- mutants and confined closely to the area surrounding the infected epithelium. In conclusion, the gE-TK- and PK- mutants exhibited significantly reduced virulence, tissue damage and spread to the underlying stroma, suggesting that they may be good vaccine candidates for in vivo testing.

Isolation and characterization of a new population of nasal surface macrophages and their susceptibility to PRRSV-1 subtype 1 (LV) and subtype 3 (Lena).

Sialoadhesin (Sn) and CD163 have been recognized as two important mediators for porcine reproductive and respiratory syndrome virus (PRRSV) in host macrophages. Recently, it has been demonstrated that the highly virulent Lena strain has a wider macrophage tropism than the low virulent LV strain in the nasal mucosa. Not only CD163+Sn+ macrophages are infected by Lena but also CD163+Sn- macrophages. This suggests that an alternative receptor exists for binding and internalization of PRRSV Lena in the CD163+Sn- macrophages. Further investigation to find the new entry receptor was hampered by the difficulty of isolating these macrophages from the nasal mucosa. In the present study, a new population of CD163+Sn- cells has been identified that is specifically localized in the nasal lamina propria and can be isolated by an intranasal digestion approach. Isolated nasal cells were characterized using specific cell markers and their susceptibility to two different PRRSV-1 strains (LV and Lena) was tested. Upon digestion, 3.2% (flow cytometry)-6.4% (confocal microscopy) of the nasal cells were identified as CD163+ and all (99.7%) of these CD163+ cells were Sn-. These CD163+Sn- cells, designated as "nasal surface macrophages", showed a 4.9 times higher susceptibility to the Lena strain than to the LV strain. Furthermore, the Lena-inoculated cell cultures showed an upregulation of CD163. These results showed that our new cell isolation system is ideal for the further functional and phenotypical analysis of the new population of nasal surface macrophages and further research on the molecular pathogenesis of PRRSV in the nose.

An Alphaherpesvirus Exploits Antimicrobial ß-Defensins To Initiate Respiratory Tract Infection.

ß-Defensins protect the respiratory tract against the myriad of microbial pathogens entering the airways with each breath. However, this potentially hostile environment is known to serve as a portal of entry for herpesviruses. The lack of suitable respiratory model systems has precluded understanding of how herpesvirus virions overcome the abundant mucosal ß-defensins during host invasion. We demonstrate how a central alphaherpesvirus, equine herpesvirus type 1 (EHV1), actually exploits ß-defensins to invade its host and initiate viral spread. The equine ß-defensins (eBDs) eBD1, -2, and -3 were produced and secreted along the upper respiratory tract. Despite the marked antimicrobial action of eBD2 and -3 against many bacterial and viral pathogens, EHV1 virions were resistant to eBDs through the action of the viral glycoprotein M envelope protein. Pretreatment of EHV1 virions with eBD2 and -3 increased the subsequent infection of rabbit kidney (RK13) cells, which was dependent on viral N-linked glycans. eBD2 and -3 also caused the aggregation of EHV1 virions on the cell surface of RK13 cells. Pretreatment of primary equine respiratory epithelial cells (EREC) with eBD1, -2, and -3 resulted in increased EHV1 virion binding to and infection of these cells. EHV1-infected EREC, in turn, showed an increased production of eBD2 and -3 compared to that seen in mock- and influenza virus-infected EREC. In addition, these eBDs attracted leukocytes, which are essential for EHV1 dissemination and which serve as latent infection reservoirs. These novel mechanisms provide new insights into herpesvirus respiratory tract infection and pathogenesis.IMPORTANCE How herpesviruses circumvent mucosal defenses to promote infection of new hosts through the respiratory tract remains unknown due to a lack of host-specific model systems. We used the alphaherpesvirus equine herpesvirus type 1 (EHV1) and equine respiratory tissues to decipher this key event in general alphaherpesvirus pathogenesis. In contrast to several respiratory viruses and bacteria, EHV1 resisted potent antimicrobial equine ß-defensins (eBDs) eBD2 and eBD3 by the action of glycoprotein M. Instead, eBD2 and -3 facilitated EHV1 particle aggregation and infection of rabbit kidney (RK13) cells. In addition, virion binding to and subsequent infection of respiratory epithelial cells were increased upon preincubation of these cells with eBD1, -2, and -3. Infected cells synthesized eBD2 and -3, promoting further host cell invasion by EHV1. Finally, eBD1, -2, and -3 recruited leukocytes, which are well-known EHV1 dissemination and latency vessels. The exploitation of host innate defenses by herpesviruses during the early phase of host colonization indicates that highly specialized strategies have developed during host-pathogen coevolution.

Porcine rotavirus mainly infects primary porcine enterocytes at the basolateral surface.

Intestinal epithelium functions as a barrier to protect multicellular organisms from the outside world. It consists of epithelial cells closely connected by intercellular junctions, selective gates which control paracellular diffusion of solutes, ions and macromolecules across the epithelium and keep out pathogens. Rotavirus is one of the major enteric viruses causing severe diarrhea in humans and animals. It specifically infects the enterocytes on villi of small intestines. The polarity of rotavirus replication in their target enterocytes and the role of intestinal epithelial integrity were examined in the present study. Treatment with EGTA, a drug that chelates calcium and disrupts the intercellular junctions, (i) significantly enhanced the infection of rotavirus in primary enterocytes, (ii) increased the binding of rotavirus to enterocytes, but (iii) considerably blocked internalization of rotavirus. After internalization, rotavirus was resistant to EGTA treatment. To investigate the polarity of rotavirus infection, the primary enterocytes were cultured in a transwell system and infected with rotavirus at either the apical or the basolateral surface. Rotavirus preferentially infected enterocytes at the basolateral surface. Restriction of infection through apical inoculation was overcome by EGTA treatment. Overall, our findings demonstrate that integrity of the intestinal epithelium is crucial in the host's innate defense against rotavirus infection. In addition, the intercellular receptor is located basolaterally and disruption of intercellular junctions facilitates the binding of rotavirus to their receptor at the basolateral surface.