Evidence for a multistep mechanism for cell-cell fusion by herpes simplex virus with mutations in the syn 3 locus using heparin derivatives during fusion from within

Addition of heparin-Na+ as well as related substances of high and intermediate MW (Arteparon and polyanion SP54) 3 h after infection inhibit fusion from within (FFWI) induced by HSV strains with mutations in the syn 3 locus only. The concentration of heparin-Na+ required to inhibit FFWI is 10-fold higher (1 mg/ml) than that needed to inhibit adsorption. Instead of fusion, cell rounding is observed. The effect is readily reversible. A low MW heparin disaccharide is ineffective. Neomycin, at a concentration of 8 mM, inhibits FFWI induced by all HSV-1 but not HSV-2 strains, whereas adsorption is inhibited at 3 mM. We conclude from our observations that cell-cell fusion (FFWI) induced by syn 3 …

Vaginal infection of mice with HSV type 2 variant ER−: A new animal model for human primary genital HSV type 2 infections

Abstract Studying the pathogenesis of vaginal infections in mice with two variants of Herpes simplex virus type 2 (HSV-2) strain ER we observed that both variants ER+ and ER− caused severe vaginitis but only ER+ invaded the CNS leading to lethal neurological disease. In contrast, mice infected with ER− cleared the virus from the vagina and recovered from infection. ER+ and ER− expressed equal levels of thymidine kinase (TK) indicating a TK-independent difference in neurovirulence. Using the non-neurovirulent variant ER−, we were able to investigate humoral immune responses late after infection. Vaginal infection with ER− suppressed serum antibody formation after a secondary systemic HSV-1 i…

Two mutations in gB-1 and gD-1 of herpes simplex virus type 1 are involved in the "fusion from without" phenotype in different cell types.

Previous studies have shown that certain strains of herpes simplex viruses type 1 (HSV-1) are able to induce “fusion from without” (FFWO) which means no transcription or translation of the viral genome happens. The main determinants for FFWO in BHK cells are mutations in the C-terminal part of gB-1. But single mutations in this part of the genome are not sufficient to transfer the FFWO phenotype also to Vero cells. Here, we report that FFWO of HSV strains indeed need additional mutations in the N-terminal part of gD in order to produce the FFWO phenotype in BHK and Vero cells. By marker transfer we are able to show that loss of mutations in the N-terminal part of gD influences the ability t…

Single amino acid substitutions in the glycoprotein B carboxy terminus influence the fusion from without property of herpes simplex virus type 1.

Syncytial mutations of herpes simplex virus type 1 (HSV-1) strains ANG, ANG path, HFEM, tsB5 and HSZP cause extensive cell fusion and were mapped to the cytoplasmic domain of glycoprotein B (gB), within the syn 3 locus. These strains are so far the only ones which show the phenotype ‘fusion from without’ (FFWO): 60 min after infection with high m.o.i., cells in a tissue culture are fused without transcription and translation of the viral genome. In this report we detected, using the recombinants 27/III and K-7, that an amino acid exchange from Ala to Val at aa position 854 of gB is the main determinant for FFWO activity of strains ANG, ANG path and recombinant K-7. The transfer of this muta…

The prevalence of human papilloma virus (HPV) infections in oral squamous cell carcinomas: a retrospective analysis of 88 patients and literature overview.

In addition to tobacco and alcohol consumption, the two main risk factors for oral squamous cell carcinoma (OSCC), recent studies have revealed infections with human papilloma virus (HPV) as an additional risk factor for OSCC development. In the field of head and neck malignancies, the prevalence of HPV infections in oropharyngeal cancer (OC) ranges in different studies up to 84%. While HPV infection is discussed as an independent risk factor in this region, its distinguished role in carcinogenesis of tumours localized to the oral cavity remains still uncertain. In this study, we analysed the HPV status in 88 consecutive patients with OSCCs localized anterior of the palatoglossal arch who w…

Containment of a Large SARS-CoV-2 Outbreak Among Healthcare Workers in a Pediatric Intensive Care Unit.

Objective Healthcare workers (HCWs) are particularly exposed SARS-CoV-2 because they are critical in preventing viral transmission and treating COVID-19 patients. Within HCWs, personnel of intensive care units (ICUs) are at the forefront of treating patients with a severe course of COVID-19 infection and therefore represent an extremely vulnerable group. Thus, our objective is to contribute to establish means of infection control protecting HCWs in the frontline of the current pandemic. Design An outbreak of SARS-CoV-2 was detected and contained in a pediatric ICU (PICU). The first positive case was identified with a point-of-care diagnostic system on site. Real-time PCR-based testing syste…

Characterization of fusion from without induced by herpes simplex virus

The process of fusion from without (FFWO) induced by herpes simplex virus (HSV) was analyzed by using various inhibitors and compared to fusion from within (FFWI). The fate of certain elements of the cytoskeleton after FFWO was also investigated. Our experiments demonstrate FFWO as a very suitable system for study of early virus-cell interactions. Zn++ ions proved inhibitory for penetration whilst pretreatment of cells with Ca++ ions before infection enhanced FFWO activity. Dissociation of penetration from the fusion process itself was possible by use of Zn++ ions, low pH-treatment and antiserum on the one hand and N-ethylmaleimide and cytochalasin D on the other. Penetration itself needs o…

Cyclosporin A resistance of herpes simplex virus-induced "fusion from within" as a phenotypical marker of mutations in the Syn 3 locus of the glycoprotein B gene.

We here report research in which nine strains of Herpes simplex virus (HSV) with fusing activity were investigated in order to establish precise phenotypical markers of mutations in the carboxy terminus of glycoprotein B (gB). The gene region encoding the carboxy terminus of gB was isolated, then cloned, and finally sequenced. Our investigation showed that seven strains have different mutations in the syn 3 locus. We observed no base difference in the gB gene region encoding the carboxy terminus of gB of two other strains. Strains with a mutation in the carboxy terminus of gB induced fusion from within (FFWI) in the presence of Cyclosporin A (CyA) at a concentration up to 150 µM. There are …

Pathogenesis of HSV-1/2 induced vaginitis/vulvitis of the mouse: dependence of lesions on genetic properties of the virus and analysis of pathohistology

A scoring system for herpes simplex virus (HSV) induced vaginitis/vulvitis in Balb/c mice was delineated from vaginal infections. Four degrees of vaginitis/vulvitis could be distinguished after infection with suitable strains of HSV despite nearly identical replication rates. The time course of replication, inflammation and pathohistology was compared further. Grade 0 was defined by lack of symptoms despite presence of strong replication, which was detectable at days 3-6. Focal necrotic lesions of the epithelial layer were present containing HSV-specific antigens. DNA could be detected by hybridization only in the outer zone of these areas. At day 6 these zones began to be re-epithelialized…

An antigen fragment encompassing the AD2 domains of glycoprotein B from two different strains is sufficient for differentiation of primary vs. recurrent human cytomegalovirus infection by ELISA

Primary human cytomegalovirus (HCMV) infection during pregnancy is a frequent cause of fatal damage in populations with low prevalence of HCMV. Differentiation of primary vs. recurrent HCMV infection is an important issue in prenatal counseling. Antibodies specific for viral glycoproteins become detectable only with considerable delay with relation to HCMV infection or IgG seroconversion. Thus, lack of glycoprotein specific (gp-specific) antibodies can serve as a convenient indicator to identify those pregnant women that bear an elevated risk for HCMV transplacental transmission and fetal sequelae. In the opposite case, presence of gp-specific antibodies virtually excludes HCMV primary infe…

Colonization of adrenal glands and ovaries of mice by HSV-2 variants

HSV-2 strain ER was shown to consist of variants with different pathogenic phenotype: Variant ER+ replicates to high titers in the adrenal glands and the ovaries but much less in the spleen; the testes were not colonized. ER+ migrates to the spinal ganglia and is highly neuroinvasive after i.p. inoculation. Variant ER- replicates 100-1,000 fold less in the adrenal glands and the ovaries, but proceeds to the spinal ganglia without invading the CNS. However, both variants are highly neuropathogenic after direct i.c. injection. We conclude that neuropathogenicity, neuroinvasiveness and the ability to replicate in the adrenal glands as well as ovaries are each determined by different sets of ge…

Differentiation of herpes simplex virus-induced fusion from without and fusion from within by cyclosporin A and compound 48/80.

Treating strains of herpes simplex virus (HSV) in culture with either cyclosporin A or compound 48/80, allowed the strains to be divided into two groups. Group 1 contains the strains ANG and HFEM of HSV-1 and Lux syn (HSV-2) producing fusion from within (FFWI) and fusion from without (FFWO). Cyclosporin A fails to inhibit both types of fusion at concentrations up to 100 microM. Strains ANG and HFEM belong to the syn 3 marker locus group identified for HSV-1. Group 2 contains all other fusion-producing strains of HSV tested so far. Cyclosporin A inhibits FFWI at concentrations as low as 10 to 20 microM. These strains belong to the syn locus marker groups 1, 2, 4 and 5. From the fact that mut…

Relationship between HLA I surface expression and different cytopathic effects produced after herpes simplex virus infection in vitro.

In the present study, we investigated the effects of herpes simplex virus (HSV) infection on the expression of HLA class I antigens and beta 2-microglobulin in human fibroblasts. The mRNA abundance for HLA class I was shown to be strongly reduced after infection with HSV strains either producing cell rounding or fusion from within (FFWI), however, HLA class I expression on the surface of cells is strongly reduced only after appearance of FFWI. Using a ts mutant (ts 78R) or CyA in combination with a fusion from without (FFWO) inducing strain of HSV, this loss of HLA class I antigens is assumed to be correlated to the rearrangement of the cell membrane during the fusion process itself as a la…

Detection of respiratory viral infections in neonates treated for suspicion of nosocomial bacterial sepsis: a feasibility study.

There is a lack of knowledge concerning the frequency and significance of respiratory viral infections that occur in the neonatal intensive care unit. In the present study, all neonates with suspected nosocomial bacterial sepsis were screened for a panel of respiratory viruses. Respiratory viral infections were detected in 10% of these cases. This was comparable with the frequency of a blood-culture-proven sepsis.

Pathogenese und Diagnostik der Cytomegalovirus-Infektion

Viral Infections in Neonates with Suspected Late-Onset Bacterial Sepsis—A Prospective Cohort Study

Objective The aim of our study was to evaluate the occurrence of viral infections in infants with suspected late-onset bacterial sepsis in a neonatal intensive care unit. Methods In a prospective study, infants with suspected late-onset bacterial sepsis underwent viral testing alongside routine blood culture sampling. Using a multiplex reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay, nasopharyngeal aspirates were analyzed for adenovirus, respiratory syncytial virus (RSV), influenza virus A and B, H1N1 virus, parainfluenza virus 1 to 4, metapneumovirus, coronavirus, and picornavirus. Stools were examined for adenovirus, rotavirus, norovirus, and enterovirus.…