Viral Entry Site Research Articles

Expression of interferons-alpha and -gamma in testicular interstitial tissue and spermatogonia of the rat.

The testis is divided into two compartments: the seminiferous tubules and the interstitial tissue. The latter essentially consists of the blood and lymphatic vessels, testosterone-producing Leydig cells, and testicular macrophages. In the exploration of the testicular antiviral defense system, we initially searched for interferon (IFN) production by the seminiferous tubule cells. The site of virus entry into the testis is probably the interstitial compartment; thus, it is important to know whether and how the cells in this compartment are protected against viral infection. In addition, as germ cell precursors (spermatogonia) are only partially protected by the blood-testis barrier, it was important to explore the antiviral capability of these cells. In this study we searched for IFN production by Leydig cells, testicular macrophages, and spermatogonia after exposure to Sendai virus. We also investigated the effect of viral exposure on testosterone production by Leydig cells. Our results show that spermatogonia do not constitutively express IFNs and give a very poor response to the virus. In contrast, testicular macrophages constitutively produced type I IFNs, and this production was markedly stimulated by Sendai virus. Leydig cells produced twice as much type I IFNs as testicular macrophages after viral exposure, and they were the only cells producing both IFNalpha and -gamma, with these IFNs being dramatically induced/ increased in response to exposure to the virus. Furthermore, incubation of Leydig cells with the Sendai virus stimulated testosterone production. In conclusion, this study further establishes the topography of IFN expression within the testis. This allows us to hypothesize that the potential antiviral system represented by Leydig cells and, to a lesser extent, by macrophages plays a key role in protecting both androgen production and spermatogenesis.

A simplified vaccinologists' vaccinology and the pursuit of a vaccine against AIDS

Vaccinology is the science and engineering of developing vaccines to prevent infectious diseases. Guidelines come from knowledge of pathogenesis and from successful past vaccines. The vaccine enterprise relies on the evolution of appropriate science and technology. Governmental support and industrial participation are key to successful development of new vaccines. A large challenge for vaccinology is a vaccine which protects against AIDS. Though misguided in its first decade, current vaccine research is directed to use of any and all viral antigens and to elicit both cell-mediated and humoral immune responses that are resident, with memory, at the mucosal sites of viral entry. Recent seminal discoveries guiding the future include selective elicitation of both Type 1 and Type 2 immune responses, and prime-boosting using recombinant viral or DNA vectors and expressed antigens. Success in vaccinology depends on simplification of the complex and on iterative processes in a well-defined pathway. The present and future of vaccinology are discussed in depth.

HIV infection of human choroid plexus: a possible mechanism of viral entry into the CNS.

The present study examines the hypothesis that HIV infection of the choroid plexus (CPx) may be an important site of viral entry into the brain. Formalin-fixed, paraffin-embedded CPx was obtained from 25 patients with AIDS and 13 nonAIDS patients and was processed for light microscopy and for immunohistochemical detection of HIV gp41, T and B lymphocytes, monocytes/macrophages and endothelial cells. Eleven of the 13 nonAIDS CPx were normal and 2 contained inflammatory foci of undetermined etiology. The stroma contained T lymphocytes in all and monocytes in 22%; B lymphocytes and HIV antigen were absent. Choroid plexus of the AIDS cases contained opportunistic infections or lymphoma in 12 and inflammatory foci alone in 6; 7 were normal. T lymphocytes were present in 70% and monocytes in 50%. In addition to the stromal localization, monocytes also were present in supra-epithelial regions and within or adjacent to the capillary endothelium. HIV-positive cells in the CPx were found in 11 cases (44%) and in the supra-epithelial area in another 2. Their presence correlated with neither infection nor lymphoma of the CPx or brain. They were situated in the stroma, supra-epithelial region and (rarely) capillary endothelium. Immunohistochemistry on serial sections identified the HIV-infected cells as monocytes, including those by capillary endothelium and in supra-epithelial areas. The study demonstrates that the CPx contains HIV-infected monocytes in almost half of the cases. Their apposition to endothelium suggests hematogenous origin. These results support the hypothesis that HIV encephalitis may develop from CPx infection.

Entry and release of transmissible gastroenteritis coronavirus are restricted to apical surfaces of polarized epithelial cells

The transmissible gastroenteritis coronavirus (TGEV) infects the epithelial cells of the intestinal tract of pigs, resulting in a high mortality rate in piglets. This study shows the interaction of TGEV with a porcine epithelial cell line. To determine the site of viral entry, LLC-PK1 cells were grown on permeable filter supports and infected with TGEV from the apical or basolateral side. Initially after plating, the virus was found to enter the cells from both sides. During further development of cell polarity, however, the entry became restricted to the apical membrane. Viral entry could be blocked by a monoclonal antibody to the viral receptor aminopeptidase N. Confocal laser scanning microscopy showed that this receptor protein was present at both the apical and basolateral plasma membrane domains just after plating of the cells but that it became restricted to the apical plasma membrane during culture. To establish the site of viral release, the viral content of the apical and basolateral media of apically infected LLC-PK1 cells was measured by determining the amount of radioactively labelled viral proteins and infectious viral particles. We found that TGEV was preferentially released from the apical plasma membrane. This conclusion was confirmed by electron microscopy, which demonstrated that newly synthesized viral particles attached to the apical membrane. The results support the idea that the rapid lateral spread of TGEV infection over the intestinal epithelia occurs by the preferential release of virus from infected epithelial cells into the gut lumen followed by efficient infection of nearby cells through the apical domain.

Basis of neurovirulence of avirulent rabies virus variant Av01 with stereotaxic brain inoculation in mice

Av01 is a variant of the challenge virus standard strain of fixed rabies virus that was selected with a neutralizing anti-glycoprotein monoclonal antibody, and has a single amino acid change in the glycoprotein. It is avirulent after both intracerebral and peripheral routes of inoculation in adult mice. In this study, Av01 was found to be neurovirulent with stereotaxic brain inoculation in either the striatum or cerebellum of adult mice. Mice that had been inoculated simultaneously with Av01 by the intracerebral and intrastriatal routes recovered. More infectious virus was present in the brains of mice inoculated intrastriatally than intracerebrally, and more neurons contained rabies virus antigen. However, the topographical distribution of infected neurons was similar with both routes. Serum neutralizing antibodies against rabies virus were produced later and in smaller quantities after intrastriatal inoculation. Av01 is probably neurovirulent after stereotaxic brain inoculation because this route produces both a direct site of viral entry into the central nervous system and a low level of immune stimulation.

Saturable attachment sites for type 3 mammalian reovirus on murine L cells and human HeLa cells.

Attachment of [35S]methionine-labelled mammalian type 3 reovirus to murine L cells and human HeLa cells was studied under equilibrium conditions. Cellular attachment sites could be completely saturated with 35S-labelled reovirus, indicating that specific attachment sites for reovirus are present on the surface of these cells. We calculated that L cells possess about 86000-105000 attachment sites per cell while HeLa cells possess about 126000-147000 sites per cell for type 3 reovirus. Unlabelled reovirus was highly efficient in competing for attachment by 35S-labelled reovirus to the saturable attachment sites of both L and HeLa cells, further indicating the specificity of the interaction. We also found that unlabelled reovirus competed equally well for both binding and internalization of 35S-labelled reovirus into murine L cells, suggesting that the L cell attachment site may serve as a virus entry site. Phospholipase digestion of L cells had no effect on subsequent reovirus attachment, while treatment of L cells with moderate concentrations of bromelain (but not trypsin, proteinase K or pronase) and Vibrio cholerae neuraminidase reproducibly decreased subsequent reovirus attachment. These results and those of others (Epstein et al., 1984, Virology 133, 46-55) suggest that mammalian reoviruses attach to specific cell surface receptors on at least two species of mammalian cells to initiate the infectious cycle.

Disseminated Herpesvirus Hominis 2 Infection Following Drug Overdose

Fatal disseminated herpesvirus hominis type 2 infection developed in a 24-year-old woman with psychiatric disease. The primary site of virus entry appeared to be the upper respiratory tract, although the cervix and anus could have been primary sites. Dissemination of infection was facilitated by steroid therapy. An endotracheal tube may have increased spread to the lower respiratory tract and an intrauterine device may have aided spread to the uterus. Intranuclear inclusion body-bearing cells in the respiratory secretions should alert the clinician to possible herpesvirus disease, local and disseminated, even in the nonimmunecompromised patient.

A Study of Outbreaks of Aujeszky’s Disease in Cattle

Twenty-nine outbreaks of Aujeszky’s disease in cattle, involving 54 animals, were studied virologically and epidemiologically. The outbreaks could be divided into two distinct groups, viz., such with pruritus on the anterior (Group I) and such with pruritus on the posterior part of the body (Group II). Though low, the average number of affected animals per outbreak was twice as high in Group I as in Group II. Besides in the tissue of the central nervous system, virus was demonstrated in the oral, pharyngeal, and nasal mucous membranes of eight out of 12 animals from Group I outbreaks, which was thought to be indicative of respiratory infection, and in the vagina of the three animals with perineal pruritus from which such material was examined. It is concluded that the site of pruritus need not be identical with the site of virus entry. The probable routes of infection in the outbreaks examined will be discussed in detail in a subsequent paper.

Necrotizing vasculitis and ulcerative colitis.

Abstract Objectives Two of the main target tissues of SARS-coronavirus 2 are the oral cavity pharynx-larynx epithelium, the main virus entry site, and the lung epithelium. The virus enters host cells through binding of the Spike protein to ACE2 receptor and subsequent S priming by the TMPRSS2 protease. Herein we aim to assess differences in both ACE2 and TMPRSS2 expression in normal tissues from oral cavity-pharynx-larynx and lung tissues as well as neoplastic tissues from the same histological areas. The information provided in this study may contribute to better understanding of SARS-coronavirus 2 ability to interact with different biological systems and contributes to cumulative knowledge on potential mechanisms to inhibit its diffusion. Materials and Methods The study has been conducted using The Cancer Genome Atlas (TCGA) and the Regina Elena Institute (IRE) databases and validated by experimental model in HNSCC and Lung cancer cells. Data from one COVID19 positive patient who was operated on for HNSCC was also included. We have analyzed 478 tumor samples and 44 normal samples from TCGA HNSCC cohort for whom both miRNA and mRNA sequencing was available. The dataset included 391 HPV- and 85 HPV+ cases, with 331 P53 mutated and 147 P53 wild type cases respectively. 352 out of 478 samples were male and 126 female. In IRE cohort we analyzed 66 tumor samples with matched normal sample for miRNA profiling and 23 tumor\normal matched samples for mRNA profiling. 45 out of 66 tumors from IRE cohort were male and 21 female, 38 were P53 mutated and 27 wild type. Most patients (63 of 66) in IRE cohort were HPV negative. Normalized TCGA HNSCC gene expression and miRNA expression data were obtained from Broad Institute TCGA Genome Data Analysis Center (http://gdac.broadinstitute.org/). mRNA expression data from IRE cohort used in this study has been deposited to NCBI’s Gene Expression Omnibus and is accessible through GEO series accession number GSE107591. In order to inference about potential molecular modulation of TMPRSS2, we also included miRNAs expression for the 66 IRE cohort matched tumor and normal samples from Agilent platform. DNA methylation data for TCGA tumors were obtained from Wanderer (http://maplab.imppc.org/wanderer/). We used miRWalk and miRNet web tools for miRNA-target interaction prediction and pathway enrichment analysis. The correlation and regression analyses as well as the miRNA and gene modulation and the survival analysis were conducted using Matlab R2019. Results TMPRSS2 expression in HNSCC was significantly reduced compared to the normal tissues and had a prognostic value in HNSCC patients. Reduction of TMPRSS2 expression was more evident in women than in men, in TP53 mutated versus wild TP53 tumors as well as in HPV negative patients compared to HPV positive counterparts. Functionally, we assessed the multivariate effect on TMPRSS2 in a single regression model. We observed that all variables had an independent effect on TMPRSS2 in HNSCC patients with HPV negative, TP53 mutated status and with elevated TP53-dependent Myc-target genes associated with low TMPRSS2 expression. Investigation of the molecular modulation of TMPRSS2 in both HNSCC and lung cancers revealed that expression of microRNAs targeting TMPRSS2 anti-correlated in both TCGA and IRE HNSCC datasets, while there was not evidence of TMPRSS2 promoter methylation in both tumor cohorts. Interestingly, the anti-correlation between microRNAs and TMPRSS2 expression was corroborated by testing this association in a SARS-CoV-2 positive HNSCC patient. Conclusions Collectively, these findings suggest that tumoral tissues, herein exemplified by HNSCC and lung cancers might be more resistant to SARS-CoV-2 infection due to reduced expression of TMPRSS2. The protective mechanism might occur, at least partially, through the aberrant activation of TMPRSS2 targeting microRNAs; thereby providing strong evidence on the role of non-coding RNA molecule in host viral infection. These observations may help to better assess the frailty of SARS-CoV-2 positive cancer patients.

Louping-ill encephalitis in C57 black mice

Weaned C57 black mice developed marked neurological symptoms 8 days after intraperitoneal inoculation with high titres of louping-ill virus. The virus was first detected in nervous tissue on day 4 and replication thereafter proceeded at approximately equivalent rates in different regions of the central nervous system. There was no obvious preferential site of virus entry into the brain. Lesions of encephalitis were found in only 25 per cent, of mice with neurological signs. Histological changes were present in the brains of all i.c. inoculated C57 black mice with symptoms.