RESUMO
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.
Assuntos
Genitália Feminina/virologia , Herpesvirus Humano 1/fisiologia , Herpesvirus Humano 2/fisiologia , Mucosa/virologia , Adulto , Idoso , Moléculas de Adesão Celular/biossíntese , Tecido Conjuntivo/virologia , Células Epiteliais/citologia , Células Epiteliais/virologia , Feminino , Perfilação da Expressão Gênica , Células Gigantes/virologia , Humanos , Ciclo Menstrual , Pessoa de Meia-Idade , Nectinas , Técnicas de Cultura de Órgãos/métodos , Receptores Virais/biossínteseRESUMO
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.
Assuntos
Herpesvirus Bovino 1/fisiologia , Rinotraqueíte Infecciosa Bovina/virologia , Traqueia/citologia , Vagina/citologia , Replicação Viral , Animais , Bovinos , Feminino , Imunofluorescência/veterinária , Herpesvirus Bovino 1/genética , Técnicas In Vitro , Rinotraqueíte Infecciosa Bovina/patologia , Microscopia Eletrônica de Varredura/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Mucosa/citologia , Mucosa/virologia , Filogenia , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Traqueia/virologia , Vagina/virologia , Ensaio de Placa Viral/veterináriaRESUMO
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.
Assuntos
Membrana Basal/virologia , Herpesvirus Suídeo 1/fisiologia , Mucosa Nasal/virologia , Pseudorraiva/virologia , Serina Proteases/metabolismo , Doenças dos Suínos/virologia , Animais , Membrana Basal/ultraestrutura , Técnicas In Vitro , Microscopia Confocal/veterinária , Mucosa Nasal/ultraestrutura , Inibidores de Proteases/farmacologia , Inibidores de Serina Proteinase/farmacologia , Sulfonas/farmacologia , Suínos , Ensaio de Placa Viral/veterináriaRESUMO
Equine herpesvirus type 1 (EHV-1) is an alphaherpesvirus related to pseudorabies virus (PRV) and varicella-zoster virus (VZV). This virus is one of the major pathogens affecting horses worldwide. EHV-1 is responsible for respiratory disorders, abortion, neonatal foal death and equine herpes myeloencephalopathy (EHM). Over the last decade, EHV-1 has received growing attention due to the frequent outbreaks of abortions and/or EHM causing serious economical losses to the horse industry worldwide. To date, there are no effective antiviral drugs and current vaccines do not provide full protection against EHV-1-associated diseases. Therefore, there is an urgent need to gain a better understanding of the pathogenesis of EHV-1 in order to develop effective therapies. The main objective of this review is to provide state-of-the-art information on the pathogenesis of EHV-1. We also highlight recent findings on EHV-1 immune evasive strategies at the level of the upper respiratory tract, blood circulation and endothelium of target organs allowing the virus to disseminate undetected in the host. Finally, we discuss novel approaches for drug development based on our current knowledge of the pathogenesis of EHV-1.
RESUMO
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.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Herpesvirus Equídeo 1/crescimento & desenvolvimento , Herpesvirus Equídeo 1/patogenicidade , Cavalos/virologia , Mucosa Nasal/virologia , Fatores de Virulência/genética , Replicação Viral , Animais , Bélgica , DNA Polimerase Dirigida por DNA/fisiologia , Herpesvirus Equídeo 1/isolamento & purificação , Monócitos/virologia , Mutação de Sentido Incorreto , Técnicas de Cultura de Órgãos , Linfócitos T/virologia , Fatores de Tempo , Proteínas Virais/genética , Proteínas Virais/fisiologia , Virulência , Fatores de Virulência/fisiologiaRESUMO
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.
Assuntos
Herpesvirus Bovino 1/fisiologia , Mucosa Respiratória/citologia , Mucosa Respiratória/virologia , Sistema Respiratório/virologia , Animais , Membrana Basal/ultraestrutura , Membrana Basal/virologia , Bovinos , Herpesvirus Bovino 1/ultraestrutura , Marcação In Situ das Extremidades Cortadas , Cinética , Microscopia , Microscopia Eletrônica de Transmissão , Sistema Respiratório/ultraestruturaRESUMO
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.
Assuntos
Fenômenos Fisiológicos Bacterianos , Membrana Basal/microbiologia , Membrana Basal/virologia , Fungos/fisiologia , Interações Hospedeiro-Patógeno , Fenômenos Fisiológicos Virais , Animais , Membrana Basal/metabolismo , Adesão Celular , HumanosRESUMO
Equine herpes virus (EHV)-1 replicates in the epithelial cells of the upper respiratory tract and reaches the lamina propria and bloodstream in infected mononuclear cells. This study evaluated expression of the late viral proteins gB, gC, gD and gM in respiratory epithelial and mononuclear cells using: (1) epithelial-like rabbit kidney cells and peripheral blood mononuclear cells infected with EHV-1 in vitro; (2) an equine ex vivo nasal explant system; and (3) nasal mucosa tissue of ponies infected in vivo. The viral proteins were expressed in all late-infected epithelial cells, whereas expression was not observed in infected leucocytes where proteins gB and gM were expressed in 60-90%, and proteins gC and gD in only 20% of infected cells, respectively. The results indicate that expression of these viral proteins during early-stage EHV-1 infection is highly dependent on the cell type infected.
Assuntos
Células Epiteliais/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/patogenicidade , Doenças dos Cavalos/virologia , Leucócitos Mononucleares/virologia , Proteínas do Envelope Viral/metabolismo , Animais , Células Epiteliais/imunologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/genética , Herpesvirus Equídeo 1/imunologia , Doenças dos Cavalos/imunologia , Cavalos , Rim/imunologia , Rim/virologia , Leucócitos Mononucleares/imunologia , Nariz/imunologia , Nariz/virologia , Coelhos , Proteínas do Envelope Viral/imunologia , VirulênciaRESUMO
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.
Assuntos
Antivirais/farmacologia , Guanina/análogos & derivados , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/efeitos dos fármacos , Doenças dos Cavalos/tratamento farmacológico , Animais , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Guanina/farmacologia , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/fisiologia , Doenças dos Cavalos/virologia , Cavalos , Técnicas In Vitro , Mucosa Nasal/virologia , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/patogenicidade , Herpesvirus Equídeo 4/patogenicidade , Doenças dos Cavalos/virologia , Cavalos/virologia , Animais , Células Epiteliais/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/fisiologia , Herpesvirus Equídeo 4/fisiologia , Doenças dos Cavalos/diagnóstico , Leucócitos Mononucleares/virologia , Mucosa Nasal/virologia , Técnicas de Cultura de Tecidos , Ensaio de Placa Viral/veterinária , Tropismo Viral , Viremia/veterinária , Viremia/virologia , Replicação ViralRESUMO
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.