Establishment of a Three-Dimensional In Vitro Model of Equine Papillomavirus Type 2 Infection.

There is growing evidence that equine papillomavirus type 2 (EcPV2) infection is etiologically associated with the development of genital squamous cell carcinoma (SCC) and precursor lesions in equids. However, the precise mechanisms underlying neoplastic progression remain unknown. To allow the study of EcPV2-induced carcinogenesis, we aimed to establish a primary equine cell culture model of EcPV2 infection. Three-dimensional (3D) raft cultures were generated from equine penile perilesional skin, plaques and SCCs. Using histological, molecular biological and immunohistochemical methods, rafts versus corresponding natural tissue sections were compared with regard to morphology, presence of EcPV2 DNA, presence and location of EcPV2 gene transcripts and expression of epithelial, mesenchymal and tumor/proliferation markers. Raft cultures from perilesional skin harboring only a few EcPV2-positive (EcPV2+) cells accurately recapitulated the differentiation process of normal skin, whilst rafts from EcPV2+ penile plaques were structurally organized but showed early hyperplasia. Rafts from EcPV2+ SCCs exhibited pronounced hyperplasia and marked dysplasia. Raft levels of EcPV2 oncogene transcription (E6/E7) and expression of tumor/proliferation markers p53, Ki67 and MCM7 expression positively correlated with neoplastic progression, again reflecting the natural situation. Three-dimensional raft cultures accurately reflected major features of corresponding ex vivo material, thus constituting a valuable new research model to study EcPV2-induced carcinogenesis.

Hofbauer Cells and COVID-19 in Pregnancy.

CONTEXT.­: SARS-CoV-2 can undergo maternal-fetal transmission, heightening interest in the placental pathology findings from this infection. Transplacental SARS-CoV-2 transmission is typically accompanied by chronic histiocytic intervillositis together with necrosis and positivity of syncytiotrophoblast for SARS-CoV-2. Hofbauer cells are placental macrophages that have been involved in viral diseases, including HIV and Zika virus, but their involvement in SARS-CoV-2 is unknown. OBJECTIVE.­: To determine whether SARS-CoV-2 can extend beyond the syncytiotrophoblast to enter Hofbauer cells, endothelium, and other villous stromal cells in infected placentas of liveborn and stillborn infants. DESIGN.­: Case-based retrospective analysis by 29 perinatal and molecular pathology specialists of placental findings from a preselected cohort of 22 SARS-CoV-2-infected placentas delivered to pregnant women testing positive for SARS-CoV-2 from 7 countries. Molecular pathology methods were used to investigate viral involvement of Hofbauer cells, villous capillary endothelium, syncytiotrophoblast, and other fetal-derived cells. RESULTS.­: Chronic histiocytic intervillositis and trophoblast necrosis were present in all 22 placentas (100%). SARS-CoV-2 was identified in Hofbauer cells from 4 of 22 placentas (18.2%). Villous capillary endothelial staining was positive in 2 of 22 cases (9.1%), both of which also had viral positivity in Hofbauer cells. Syncytiotrophoblast staining occurred in 21 of 22 placentas (95.5%). Hofbauer cell hyperplasia was present in 3 of 22 placentas (13.6%). In the 7 cases having documented transplacental infection of the fetus, 2 (28.6%) occurred in placentas with Hofbauer cell staining positive for SARS-CoV-2. CONCLUSIONS.­: SARS-CoV-2 can extend beyond the trophoblast into the villous stroma, involving Hofbauer cells and capillary endothelial cells, in a small number of infected placentas. Most cases of SARS-CoV-2 transplacental fetal infection occur without Hofbauer cell involvement.

Vascular obliteration because of endothelial and myointimal growth in COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a systemic multi-organ viral illness. Previous studies have found that many patients had a procoagulant state and/or severe hypoxemia with relatively well-preserved lung mechanics. Mechanisms underlying the damage to vascular tissues are not well-elucidated yet. Histological data in COVID-19 patients are still limited and are mainly focused on post-mortem analysis. Given that the skin is affected by COVID-19 and the relative ease of its histological examination, we aimed to examine the histology of skin lesions in COVID-19 patients to better understand the disease's pathology. METHODS: Five skin lesions from COVID-19 adult patients were selected for a deep histological tissue examination. RESULTS: A strong vasculopathic reaction pattern based on prominent vascular endothelial and myointimal cell growth was identified. Endothelial cell distortion generated vascular lumen obliteration and striking erythrocyte and serum extravasation. Significant deposition of C4d and C3 throughout the vascular cell wall was also identified. A regenerative epidermal hyperplasia with tissue structure preservation was also observed. CONCLUSIONS: COVID-19 could comprise an obliterative microangiopathy consisting on endothelial and myointimal growth with complement activation. This mechanism, together with the increased vascular permeability identified, could contribute to obliteration of the vascular lumen and hemorrhage in COVID-19. Thus, anticoagulation by itself could not completely reverse vascular lumen obliteration, with consequent increased risk of hemorrhage. Findings of this study could contribute to a better understanding of physiopathological mechanisms underlying COVID-19 on living patients and could help further studies find potential targets for specific therapeutic interventions in severe cases.

P16 detection in benign, precursor epithelial lesions and carcinomas of head and neck.

BACKGROUND: Human papillomavirus (HPV) had recently been implicated in the pathogenesis of Head and Neck SCCs. The biological role of HPV in benign and pre-cancerous lesions is far less studied. p16 is a widely accepted marker to detect immonohistochemically the presence of HPV. METHODS: We evaluated, immunohistochemically, expression of p16 in 212 specimens: glottis, supraglottis, oropharynx, nasal/paranasal, with various diagnoses: hyperplasia, polyp/nodule, keratosis, papilloma, inverted papilloma, dysplasia, cancer (SCC). Analysis was completed according to location and disease. RESULTS: Hyperplasias/polyps were all negative for p16. A small percentage of papillomas was p16+ regardless of their location (12.5 %), the majority of inverted papillomas were p16+ (78.6 %) and statistically significant (p < 0.04). In carcinomas, 18/59 were p16+ (30.5 %): nasal/paranasal SCCs had a significantly higher percentage of p16+ cancer cells compared to glottis (p = 0.009), while tumours of the supraglottis/oropharynx had an intermediate score for p16+ cells (p = 0.07). Dysplasias were p16+ in 9/64 (14 %) regardless of grading (p = 0.03 compared to carcinomas). CONCLUSION: p16 was highly detected in inverted papillomas and in certain anatomic sites; however, it failed to be traced in benign lesions and only rarely encountered in dysplasias.

Histiocytic hyperplasia with hemophagocytosis and acute alveolar damage in COVID-19 infection.

The spectrum of COVID-19 infection includes acute respiratory distress syndrome (ARDS) and macrophage activation syndrome (MAS), although the histological basis for these disorders has not been thoroughly explored. Post-mortem pulmonary and bone marrow biopsies were performed in 33 patients. Samples were studied with a combination of morphological and immunohistochemical techniques. Bone marrow studies were also performed in three living patients. Bone marrow post-mortem studies showed striking lesions of histiocytic hyperplasia with hemophagocytosis (HHH) in most (16/17) cases. This was also observed in three alive patients, where it mimicked the changes observed in hemophagocytic histiocytosis. Pulmonary changes included a combination of diffuse alveolar damage with fibrinous microthrombi predominantly involving small vessels, in particular the alveolar capillary. These findings were associated with the analytical and clinical symptoms, which helps us understand the respiratory insufficiency and reveal the histological substrate for the macrophage activation syndrome-like exhibited by these patients. Our results confirm that COVID-19 infection triggers a systemic immune-inflammatory disease and allow specific therapies to be proposed.

The Evolving Spectrum of Precursor Lesions of Cervical Adenocarcinomas.

Modern classification schemes divide cervical adenocarcinomas into human papillomavirus (HPV)-associated and HPV-independent types. The precursor lesions of the former are well known and comprise HPV-associated (usual/endocervical) adenocarcinoma in situ (AIS) and the much less common stratified mucin-producing intraepithelial lesion (SMILE). The precursor lesions of HPV-independent cervical adenocarcinomas are much less well known, although postulated precursors of gastric-type adenocarcinoma include atypical lobular endocervical glandular hyperplasia and gastric-type AIS. In this review, we cover HPV-associated and HPV-independent precursor lesions of cervical adenocarcinomas concentrating on diagnostic criteria (morphology and immunophenotype) and differential diagnosis. We propose a uniform terminology and diagnostic criteria for precursor lesions showing intestinal differentiation with goblet cells because this may be a feature of both HPV-associated and HPV-independent AIS.

Human papilloma virus genotypes in dysplasia and epithelial hyperplasia of oral cavity using the luminex xmap technology. A multicenter study

BACKGROUND: Oral cancer associated with high risk (HPV-HR) human papilloma virus (HPV) has been increasing. HPV-HR has been associated with epithelial dysplasia, however, little information exists on its frequency in epithelial hyperplasia lesions. The aim of this study is to compare HPV genotypes in dysplastic and hyperplastic lesions of oral cavity. MATERIAL AND METHODS: Two hundred and fifty oral lesions: 131 dysplasia and 119 hyperplasia from two regions of Colombia were evaluated. One hundred seventy-four coming from urban area and 104 from a high risk population to oral cancer from a rural area. HPV was identified by qPCR and Twenty-four HPVs genotypes were evaluated by Luminex(R) technology. Logistic regressions were performed to establish the associations between HPV infections with oral dysplasia. RESULTS: Twenty-eight percent (70/250) of the samples were positives for any HPV and HPV-HRs were more frequently than low risk HPVs. HPV-16 was the most detected genotype (16%) followed by HPV-31, 53, 18 and 45. HPV, HPV-HRs and HPV-16 were only associated with dysplasia in urban area; OR 3.28 (CI 95% 1.49-7.17), OR 7.94 (CI 95% 2.97-21.2) and OR 5.90 (CI 95% 2.05-17). Individuals in rural area showed more HPV and HPV-HRs infection in hyperplasic lesions than urban population. The majority of HPV+ lesions had multi-type of HPV (52/70) and the urban individuals showed more genotypes than rural population. CONCLUSIONS: HPV-.HRs are frequently found in hyperplastic and dysplastic epithelial lesions. HPV-HRs and HPV-16 were associated with dysplasia in urban population. Rural high risk population and urban population differ in the frequency and variety of HPV genotypes

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The correlation between infections by human papillomavirus types 6/11 and 16/18 and mammary gland hyperplasia with glandular thickening.

The aim of the present study was to investigate the correlation between human papillomavirus (HPV) type 6/11 and 16/18 infections and glandular thickening mammary gland hyperplasia in order to explore methods for preventing glandular thickening mammary gland hyperplasia. A total of 240 patients with glandular thickening mammary gland hyperplasia who were treated by surgery in our hospital from January 2012 to June 2017 were enrolled in the present study. The hyperplastic breast tissue and adjacent normal breast tissue were taken to test HPV type 6/11 and 16/18 infections using conventional PCR and in situ hybridization techniques. The correlations between HPV type 6/11 and 16/18 infections and glandular thickening mammary gland hyperplasia were analyzed using statistical methods of chi-square test. The infection rates of HPV type 6/11 and 16/18 in the hyperplastic breast tissue were 31.95% and 34.91%, respectively and 11.83% and 14.79% in the normal breast tissue, respectively. The differences were statistically significant (all p<0.05). HPV type 6/11 and 16/18 infections may be closely related to the development of glandular thickening mammary gland hyperplasia, and may be one of the causes of glandular thickening mammary gland hyperplasia.

Common variable immunodeficiency syndrome with chronic diarrhoea.

Common variable immunodeficiency syndrome (CVID) is a heterogeneous disorder characterised by diminished levels of IgG, IgA and/or IgM, and recurrent bacterial infections. Sinopulmonary infections are most commonly reported followed by gastrointestinal (GI) infections. GI tract represents the largest immune organ with abundance of lymphoid cells, its involvement can manifest variably ranging from asymptomatic involvement to florid symptoms and signs. Diffuse nodular lymphoid hyperplasia (DNLH) of the GI tract is characterised by numerous small polypoid nodules of variable size in the small intestine, large intestine or both. It is commonly seen in association to immunodeficiency states such as CVID, IgA deficiency and chronic infections due to Giardia lamblia and Helicobacter pylori and cryptosporidiosis. Repetitive antigenic stimulation leads to lymphoid hyperplasia. We herein describe a case of DNLH of the intestine and another case of duodenal cytomegalovirus (CMV) infection associated with CVID.

NOTCH3 contributes to rhinovirus-induced goblet cell hyperplasia in COPD airway epithelial cells.

RATIONALE: Goblet cell hyperplasia (GCH) is one of the cardinal features of chronic obstructive pulmonary disease (COPD) and contributes to airways obstruction. Rhinovirus (RV), which causes acute exacerbations in patients with COPD, also causes prolonged airways obstruction. Previously, we showed that RV enhances mucin gene expression and increases goblet cell number in a COPD mouse model. This study examines whether RV causes sustained GCH in relevant models of COPD. METHODS: Mucociliary-differentiated COPD and normal airway epithelial cell cultures and mice with normal or COPD phenotype were infected with RV or sham and examined for GCH by immunofluorescence and/or mucin gene expression. In some experiments, RV-infected COPD cells and mice with COPD phenotype were treated with γ-secretase inhibitor or interleukin-13 neutralising antibody and assessed for GCH. To determine the contribution of NOTCH1/3 in RV-induced GCH, COPD cells transduced with NOTCH1/3 shRNA were used. RESULTS: RV-infected COPD, but not normal cell cultures, showed sustained GCH and increased mucin genes expression. Microarray analysis indicated increased expression of NOTCH1, NOTCH3 and HEY1 only in RV-infected COPD cells. Blocking NOTCH3, but not NOTCH1, attenuated RV-induced GCH in vitro. Inhibition of NOTCH signalling by γ-secretase inhibitor, but not neutralising antibody to IL-13, abrogated RV-induced GCH and mucin gene expression. CONCLUSIONS: RV induces sustained GCH via NOTCH3 particularly in COPD cells or mice with COPD phenotype. This may be one of the mechanisms that may contribute to RV-induced prolonged airways obstruction in COPD.

Expression profile of microRNA-146a along HPV-induced multistep carcinogenesis: a study in HPV16 transgenic mice.

PURPOSE: Persistent human papillomavirus (HPV) infection is associated with the development of certain types of cancer and the dysregulation of microRNAs has been implicated in HPV-associated carcinogenesis. This is the case of microRNA-146a (miR-146a), which is thought to regulate tumor-associated inflammation. We sought to investigate the expression levels of miR-146a during HPV16-mediated carcinogenesis using skin samples from K14-HPV16 transgenic mice which develop the consecutive phases of the carcinogenesis process. METHODS: Female transgenic (HPV+/-) and wild-type (HPV-/-) mice were sacrificed at 24-26 weeks-old or 28-30 weeks-old. Chest and ear skin samples from HPV+/- and HPV-/- mice were histologically classified and used for microRNA extraction and quantification by qPCR. RESULTS: Chest skin samples from 24 to 26 weeks-old HPV+/- mice presented diffuse epidermal hyperplasia and only 22.5% showed multifocal dysplasia, while at 28-30 weeks-old all (100.0%) HPV+/- animals showed epidermal dysplasia. All HPV+/- ear skin samples showed carcinoma in situ (CIS). MiR-146a expression levels were higher in HPV+/- compared to HPV-/- mice (p = 0.006). There was also an increase in miR-146a expression in dysplastic skin lesions compared with hyperplasic lesions (p = 0.011). Samples showing CIS had a significant decrease in miR-146a expression when compared to samples showing epidermal hyperplasia (p = 0.018) and epidermal dysplasia (p = 0.009). CONCLUSIONS: These results suggest that HPV16 induces the overexpression of miR-146a in the initial stages of carcinogenesis (hyperplasia and dysplasia), whereas decreases its expression at later stages (CIS). Taken together, these data implicate and suggest different roles of miR-146a in HPV-mediated carcinogenesis.

Phloem-limited reoviruses universally induce sieve element hyperplasia and more flexible gateways, providing more channels for their movement in plants.

Virion distribution and ultrastructural changes induced by the infection of maize or rice with four different reoviruses were examined. Rice black streaked dwarf virus (RBSDV, genus Fijivirus), Rice ragged stunt virus (RRSV, genus Oryzavirus), and Rice gall dwarf virus (RGDV, genus Phytoreovirus) were all phloem-limited and caused cellular hyperplasia in the phloem resulting in tumors or vein swelling and modifying the cellular arrangement of sieve elements (SEs). In contrast, virions of Rice dwarf virus (RDV, genus Phytoreovirus) were observed in both phloem and mesophyll and the virus did not cause hyperplasia of SEs. The three phloem-limited reoviruses (but not RDV) all induced more flexible gateways at the SE-SE interfaces, especially the non-sieve plate interfaces. These flexible gateways were also observed for the first time at the cellular interfaces between SE and phloem parenchyma (PP). In plants infected with any of the reoviruses, virus-like particles could be seen within the flexible gateways, suggesting that these gateways may serve as channels for the movement of plant reoviruses with their large virions between SEs or between SEs and PP. SE hyperplasia and the increase in flexible gateways may be a universal strategy for the movement of phloem-limited reoviruses.

Human papillomavirus does not play a role in the Barrett esophagus: a French cohort.

The role of human papillomavirus (HPV) in Barrett's esophagus (BE) has been examined but remains unclear. The purpose of the study is to dispute the connection between HPV and BE in a prospective case-control study. Biopsies were performed above and inside the Barrett's segment for BE patients and in the distal third of the esophagus for control patients for histological interpretation and for virological analysis. Biopsies for virological analysis were placed in a virus transport medium and immediately frozen in liquid nitrogen. Virological analysis involved real-time PCR using the SyBr® green protocol with modified SPF10 general primers. A total of 180 patients (119 control and 61 BE, respectively) were included. In BE patients, 31, 18, and 12 patients had, respectively, no dysplasia, low-grade dysplasia, and high grade dysplasia. Overall, nine were found to be HPV positive: five were control patients and four BE patients. HPV positive status was not associated with BE. No factors were associated with HPV, in particular the degree of BE dysplasia. HPV infection appears unlikely to be significant in the etiology of BE compared with control patients. (ClinicalTrials.gov, Number NCT02549053).

[The detection of the human papilloma virus during hyperplastic processes in the nose, ears and throat]. / Obnaruzhenie virusa papillomy cheloveka pri giperplasticheskikh protsessakh v LOR-organakh.

The objective of the present work was to carry out the virological and histological studies of various neoplastic and hyperplastic processes in the nose, ears, and throat with a view to identifying the presence of human papilloma virus and Epstein-Barr virus. The brush biopsies and remote neoplasms obtained from 18 patients (including 2 children and 16 adults) presenting with various ENT diseases and tumours were available for the virological investigation with the use of the polymerase chain reaction (PCR) and a system MY09-MY11 degenerate primers . The histological study of biopsies and remote neoplasms was carried out by means of conventional light microscopy. The virological and histological studies conducted in parallel confirmed the diagnostic significance of morphological changes at the tissue and cellular levels caused by the human papilloma virus.

Porcine reproductive and respiratory syndrome type 1 viruses induce hypoplasia of erythroid cells and myeloid cell hyperplasia in the bone marrow of experimentally infected piglets independently of the viral load and virulence.

Porcine reproductive and respiratory syndrome viruses (PRRSV) present a wide phenotypic and genetic diversity. Experimental infections have demonstrated viral replication, including highly pathogenic strains (HP-PRRSV), in primary lymphoid organs such as the thymus. However, studies of the bone marrow are scarce but necessary to help elucidate the immunobiology of PRRSV strains of differing virulence. In this study, whereas viral RNA was detected within the bone marrow of animals experimentally infected with both low virulent Lelystad (LV) and 215-06 PRRSV-1 strains and with the highly virulent SU1-bel strain, PRRSV positive cells were only occasionally detected in one SU1-bel infected animal. PRRSV RNA levels were associated to circulating virus with the highest levels detected in LV-infected pigs. At 3 dpi, a decrease in the proportion of haematopoietic tissue and number of erythroid cells in all infected groups was associated with an increase in TUNEL or cleaved caspase 3 labelling and higher counts of myeloid cells compared to control. The expression of IL-1α and IL-6 was elevated at the beginning of the infection in all infected animals. The expression of TNF-α was increased at the end of the study in all infected groups with respect to control. Different PRRSV-1 strains induced, presummably by indirect mechanisms and independently of viral load and strain virulence, moderate and sustained hypoplasia of erythroid cells and myeloid cell hyperplasia at early stages of infection. These changes were paralleled by a peak in the local expression of IL-1α, IL-6 and TNF-α in all infected groups.

Coexistence of age-related EBV-associated follicular hyperplasia and large B-cell EBV+ lymphoma of the elderly. Two distinct features of the same T-cell dysfunction related to senescence?

Age-related EBV-associated lymphoproliferative disorders form a new clinicopathological group. Until recently, this group was associated with diffuse large B-cell lymphoma (DLBCL), characterised by an aggressive clinical presentation and a poor prognosis. Recent findings in Western Caucasian patients, however, suggest that this entity covers a wide spectrum of diseases, ranging from reactive follicular hyperplasia (HR) to DLBCL. We report the case of an 85-year-old Caucasian man showing lymphadenopathy and numerous hypodense lesions of the liver. Examination of a lymph node revealed follicular hyperplasia with EBV expression confined to germinal centres. The patient was treated with Rituximab and subsequently, the lesions of the liver were explored. They showed extensive necrosis and a polymorphic large B-cell population with strong EBV expression. This is the first report to describe age-related EBV-associated follicular hyperplasia at one site coexisting with DLBCL at another. This case warrants undertaking further investigations each time a diagnosis of age-related EBV-HR is associated with extranodal lesions.

BK Polyomavirus Tubulointerstitial Nephritis With Urothelial Hyperplasia in a Kidney Transplant.

Polyomavirus nephropathy is characterized histopathologically by evidence of viral replication and acute tubular injury with interstitial inflammation, tubulitis, and intranuclear inclusions. Polyomavirus nephropathy typically develops in the kidney transplant as a combination of the unique nature of the transplanted tissue and the immunomodulated status of the patient. We present a case in which a patient had lingering BK viremia and declining kidney function following receipt of lung and kidney transplants. A kidney biopsy was performed, which demonstrated BK polyomavirus tubulointerstitial nephritis, resultant cytopathic changes and tubular/ductal injury, associated urothelial hyperplasia with foci of squamous metaplasia, suspected membranous glomerulopathy, and moderate arterial/arteriolar sclerosis. There was also evidence of more proximal nephron viral involvement, with glomerular parietal epithelium infection and injury present. This case shows impressive BK polyomavirus-associated urothelial hyperplasia in the kidney, which to our knowledge has not been previously illustrated in the literature. There have been numerous studies attempting to show the association of polyomaviruses with the development of carcinoma, and this case report is significant because it is an example of viral-induced changes that are concerning and hold potential for malignant transformation.

Chronic and recurrent benign lymphadenopathy without constitutional symptoms associated with human herpesvirus-6B reactivation.

Chronic/recurrent behaviour may be encountered in some distinct atypical or malignant lymphoproliferations, while recurrences are not generally observed in reactive/benign lymphadenopathies. We retrospectively analysed a consecutive series of 486 human immunodeficiency virus-negative adults, who underwent lymphadenectomy. Neoplastic and benign/reactive histopathological pictures were documented in 299 (61·5%) and 187 (38·5%) cases, respectively. Of note, seven of the 111 (6·3%) patients with benign lymphadenopathy without well-defined aetiology, showed chronic/recurrent behaviour, without constitutional symptoms. Enlarged lymph nodes were round in shape and hypoechoic, mimicking lymphoma. Reactive follicular hyperplasia and paracortical expansion were observed. Human herpesvirus (HHV)-6B positive staining in follicular dendritic cells (FDCs) was documented in all seven patients. Serological, molecular and immunological examinations suggested HHV-6B reactivation. Among the remaining 104 cases with reactive lymphoid hyperplasia in the absence of well-known aetiology and without recurrences, positivity for HHV-6B on FDCs was found in three cases, whereas in seven further patients, a scanty positivity was documented in rare, scattered cells in inter-follicular regions. Immunohistochemistry for HHV-6A and HHV-6B was invariably negative on 134 lymph nodes, with either benign pictures with known aetiology or malignant lymphoproliferative disorders, tested as further controls. Future studies are warranted to investigate a potential association between HHV-6B reactivation and chronic/recurrent benign lymphadenopathy.

Evidence for the Use of Multiple Mechanisms by Herpes Simplex Virus-1 R7020 to Inhibit Intimal Hyperplasia.

Intimal hyperplasia (IH) is the primary cause of vein bypass graft failure. The smooth muscle cell (SMC) is a key element of IH as it phenotypically switches from a contractile to a synthetic state which can become pathological. R7020, which is an engineered strain of Herpes Simplex Virus-1, inhibits IH in animal models. Although it has many characteristics which make it a strong candidate for use as a prophylactic agent how it inhibits IH is not well understood. The objective of this study was to identify modes of action used by R7020 to function in blood vessels that may also contribute to its inhibition of IH. The cytopathic effect of R7020 on SMCs was determined in vitro and in a rabbit IH model. In vitro assays with R7020 infected SMCs were used to quantify the effect of dose on the release kinetics of the virus as well as the effects of R7020 on cell viability and the adhesion of peripheral blood mononuclear cells (PBMCs) to SMCs in the absence and presence of tumor necrosis factor alpha (TNF-α). The observed cytopathic effect, which included R7020 positive filopodia that extend from cell to cell and the formation of syncytia, suggests that R7020 remains cell associated after egress and spreads cell to cell instead of by diffusion through the extracellular fluid. This would allow the virus to rapidly infect vascular cells while evading the immune system. The directionality of the filopodia in vivo suggests that the virus preferentially travels from the media towards the intima targeting SMCs that would lead to IH. The formation of syncytia would inhibit SMC proliferation as incorporated cells are not able to multiply. It was also observed that R7020 induced the fusion of PBMCs with syncytia suggesting the virus may limit the effect of macrophages on IH. Furthermore, R7020 inhibited the proliferative effect of TNF-α, an inflammatory cytokine associated with increased IH. Thus, the results of this study suggest that R7020 inhibits IH through multiple mechanisms.