Spectroscopic detection and identification of infected cells with herpes viruses

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and Fourier transform infrared (FTIR) microspectroscopy were previously applied for the identification of various biological samples. In the present study, normal cells in culture and cells infected with herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV) were analyzed by MALDI-TOF and FTIR microscopy. Specific spectral biomarkers for rapid and reliable monitoring and identification of infected cells and probably for the discrimination between these viruses were searched. The results show consistent spectral peaks in all examined normal uninfected human fibroblast cells both in MALDI-T0F and FTIR microscopy. In HSV-2- or VZV-infected cells, two unique peaks for each appeared at m/z 5397 and 5813 or at m/z 3501 and 4951, respectively, in MALDI-TOF spectra. In addition, several peaks that appeared in control uninfected cells at the region m/z 13,000-20,000 disappeared completely in all examined infected samples. When these infected cells were examined by FTIR microscopy, a band at 859 cm(-1) in control uninfected cells was significantly shifted to 854 cm(-1) in both HSV2- and VZV-infected cells. In addition, phosphate levels were considerably increased in all infected cells compared to normal uninfected cells. These parameters could be used as a basis for developing a spectral method for the detection and identification of cells infected with herpes viruses.     

Detection of Vero Cells Infected with Herpes Simplex Types 1 and 2 and Varicella Zoster Viruses Using Raman Spectroscopy and Advanced Statistical Methods

Of the eight members of the herpes family of viruses, HSV1, HSV2, and varicella zoster are the most common and are mainly involved in cutaneous disorders. These viruses usually are not life-threatening, but in some cases they might cause serious infections to the eyes and the brain that can lead to blindness and possibly death. An effective drug (acyclovir and its derivatives) is available against these viruses. Therefore, early detection and identification of these viral infections is highly important for an effective treatment. Raman spectroscopy, which has been widely used in the past years in medicine and biology, was used as a powerful spectroscopic tool for the detection and identification of these viral infections in cell culture, due to its sensitivity, rapidity and reliability. Our results showed that it was possible to differentiate, with a 97% identification success rate, the uninfected Vero cells that served as a control, from the Vero cells that were infected with HSV-1, HSV-2, and VZV. For that, linear discriminant analysis (LDA) was performed on the Raman spectra after principal component analysis (PCA) with a leave one out (LOO) approach. Raman spectroscopy in tandem with PCA and LDA enable to differentiate among the different herpes viral infections of Vero cells in time span of few minutes with high accuracy rate. Understanding cell molecular changes due to herpes viral infections using Raman spectroscopy may help in early detection and effective treatment.     

Contribution of vascular endothelial growth factor in the neovascularization process during the pathogenesis of herpetic stromal keratitis

This report analyzes the role of vascular endothelial growth factor (VEGF)-induced angiogenesis in the immunoinflammatory lesion stromal keratitis induced by ocular infection with herpes simplex virus (HSV). Our results show that infection with replication-competent, but not mutant, viruses results in the expression of VEGF mRNA and protein in the cornea. This a rapid event, with VEGF mRNA detectable by 12 h postinfection (p.i.) and proteins detectable by 24 h p.i. VEGF production occurred both in the virus-infected corneal epithelium and in the underlying stroma, in which viral antigens were undetectable. In the stroma, VEGF was produced by inflammatory cells; these initially were predominantly polymorphonuclear leukocytes (PMN), but at later time points both PMN and macrophage-like cells were VEGF producers. In the epithelium, the major site of VEGF-expressing cells in early infection, the infected cells themselves were usually negative for VEGF. Similarly, in vitro infection studies indicated that the cells which produced VEGF were not those which expressed virus. Attesting to the possible role of VEGF-induced angiogenesis in the pathogenesis of herpetic stromal keratitis were experiments showing that VEGF inhibition with mFlt(1-3)-immunoglobulin G diminished angiogenesis and the severity of lesions after HSV infection. These observations are the first to evaluate VEGF-induced angiogenesis in the pathogenesis of stromal keratitis. Our results indicate that the control of angiogenesis represents a useful adjunct to therapy of herpetic ocular disease, an important cause of human blindness.     

单纯疱疹病毒感染细胞膜系统脂质分子流动性的变化

运用荧光偏振技术了解单纯疱疹病毒(HSV)I型Sm44株感染非洲绿猴肾(Vero)细胞后细胞膜系统脂质分子流动性的变化.结果表明,在所观察的感染后的5个时相中(感染后0、7、14、21、28小时),感染早期(感染后0、7小时)细胞膜的流动性变化不大;感染晚期(14至28小时)细胞膜的脂质流动性明显增强,与未感染病毒的对照组细胞有显著差异,这提示HSV感染晚期的装配,成熟与释放是与被感染细胞的生物膜系统密切相关的.本文对运用该生物物理技术研究病毒和宿主细胞相互关系的意义进行了初步讨论.     

Lipid metabolism in experimental virus infections]

The effect of herpes simplex virus (HSV) and influenza virus (IV) on lipid metabolism was studied. In conditions of acute herpetic infection of rabbits we detected typical dyslipidemia, characterized by increased contents of total cholesterol, beta-cholesterol and triglycerides in the absence of trustworthy differences in concentrations of alpha-cholesterol. The use of antiherpetic preparation furavir, on the background of infection, corrected lipid spectrum of the infected animals. Blood lipid disturbances in acute influenza virus infection of mice were not detected. HSV infection of cell culture of human aorta was accompanied by increased accumulation of free lipids in cells. IV infection, in the same conditions of experiment, did not change the contents of intracellular lipids. The obtained data deepen the existing notions of herpetic and influenza infections pathogenesis and may be useful in understanding etiopathogenesis of certain somatic metabolism diseases.     

Isoprinosine in the treatment of herpes virus infections in children with leukaemia and malignant lymphoma

Isoprinosine was given to 15 children with lymphoblastic diseases and 3 adults during infections with herpes viruses - herpes simplex (HSV) and varicella-zoster viruses (VZV). The control group included 7 children who had previously suffered from infections with HSV and VZV without being treated with isoprinosine. The clinical observations showed a significant inhibitory effect of isoprinosine on viral infections, with the duration of the disease being shortened considerably. The authors think isoprinosine treatment to be justified in viral infections, especially in children with neoplastic diseases, because of the immunostimulating action of the drug, rapid clinical improvement and good tolerance of the drug.     

Regulation of viral and cellular RNA turnover in cells infected by eukaryotic viruses including HIV-1.

The following reviews the role of mRNA stability in the regulation of both viral and cellular gene expression in virus-infected cells. Indeed, several eukaryotic viruses, including the human immunodeficiency virus, HIV-1, regulate cellular protein synthesis via such control mechanisms. The following systems will be discussed: (i) the degradation of viral and cellular mRNAs in cells infected by herpes simplex virus (HSV) and advances made using the HSV virion host shutoff mutant; (ii) the degradation of viral and cellular mRNA and ribosomal RNA in cells infected by vaccinia virus and the possible role of the oligoadenylate synthetase-RNase L pathways; (iii) the turnover of RNAs in cells infected by encephalomyocarditis virus, reovirus, and La Crosse virus; and finally (iv) recent studies from our laboratory on the degradation of cellular mRNAs in cells infected by HIV-1.     

Herpes simplex virus-specific antibodies present in tears during herpes keratitis

We examined the specificity and levels of antibodies present in rabbit tears after induced infection of the rabbit cornea. Two strains of herpes simplex virus-1 (HSV) with different patterns of ocular disease were used: RE which produces stromal disease, and F which produces epithelial disease. We found that (i) IgG, IgA, and IgM antibodies were produced, (ii) the number of specific HSV antigens recognized by these antibodies was no significantly different, and (iii) postinfection (PI) timing and concentration of antibodies varied according to the disease pattern of the virus strain. The animals infected with strain F produced high levels of IgG antibodies early PI which remained constant, while IgA and IgM antibodies also increased early PI but declined after Day 16 PI. Animals infected with strain RE showed low levels of IgA and IgM antibodies which remained low. IgG antibodies increased early PI but declined at Day 16 PI. These differences in times of appearance and in amounts of antibodies in tears may be related to the clinical course of the disease. It has been shown that stromal disease has an immunopathologic basis. Inflammation, cellular infiltration of lymphocytes, and plasma cells are seen in the stroma of RE-infected animals, but these are not present in the stroma of F-infected animals. Infectious virus was not isolated from corneal explants taken from animals during the quiescent stage of the disease. The difference in pathogenicity cannot be explained in terms of specificity of tear antibodies. Even though the disease patterns were different, the number and types of HSV polypeptides recognized by both sets of tears was similar. Consequently, we believe that the immunopathology seen in the stromal disease may be due to the anatomical site of HSV antigens, rather than to differences in specificity of tear antibodies.     

Novel spectral method for the study of viral carcinogenesis in vitro

Fourier transform infrared (FTIR) spectroscopy is a unique technique for the laboratory diagnosis of cellular variations based on the characteristic molecular vibrational spectra of the cells. Microscopic FTIR was used to investigate spectral differences between normal and malignant fibroblasts transformed by retrovirus infection. A detailed analysis showed significant differences between cancerous and normal cells. The contents of vital cellular metabolites were significantly lower in the transformed cells than in the normal cells. In an attempt to identify the cellular components responsible for the observed spectral differences between normal and cancerous cells, we found significant differences between DNA of normal and cancerous cells.     

Herpes simplex virus type 1 infection of endothelial, epithelial, and fibroblast cells induces a receptor for C3b

We recently demonstrated that herpes simplex virus type 1 (HSV 1) induces a receptor on human umbilical vein endothelial cells for complement component C3b (C3bR). We assigned this receptor function to HSV 1 viral glycoprotein C (gC) based on several observations: tunicamycin, which prevents glycosylation and expression of N-linked glycoproteins on the surface of infected cells, markedly reduced expression of the C3bR; monoclonal antibodies to HSV 1 gC blocked detection of the C3bR, whereas monoclonal antibodies to other HSV 1 glycoproteins (gB, gD, gE) had no effect; and the MP mutant of HSV 1, which fails to express gC, did not induce C3bR. We now report that HSV 1 induces C3bR on a wide variety of cell types including bovine thoracic aorta and pulmonary artery endothelial cells, human embryonic lung and embryonic foreskin fibroblasts, and human embryonic kidney cells. To date, all cells studied that are permissive to HSV 1 express C3bR, although the pattern of rosetting of C3b-coated erythrocytes varies among the cell strains examined. We also demonstrate that C3bR expression is not a general response of human umbilical vein endothelial cells to injury, because three other viruses (adenovirus 7, measles, and mumps) do not induce C3bR after infection of these cells. Previously we had shown that among herpes simplex viruses, a variety of HSV 1 strains induce C3bR, whereas HSV 2 strains do not. We now demonstrate that other herpes family viruses (CMV and VZV) do not express C3bR. Therefore, C3bR expression appears to be unique for HSV 1 and occurs on a wide variety of cells permissive to this virus.     

Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection.     

Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22.

Very early in infection, herpes simplex virus (HSV) expresses four immediate-early (IE) regulatory proteins, ICP4, ICP0, ICP22, and ICP27. The systematic inactivation of sets of the IE proteins in cis, and the subsequent phenotypic analysis of the resulting mutants, should provide insights into how these proteins function in the HSV life cycle and also into the specific macromolecular events that are altered or perturbed in cells infected with virus strains blocked very early in infection. This approach may also provide a rational basis to assess the efficacy and safety of HSV mutants for use in gene transfer experiments. In this study, we generated and examined the phenotype of an HSV mutant simultaneously mutated in the ICP4, ICP27, and ICP22 genes of HSV. Unlike mutants deficient in ICP4 (d120), ICP4 and ICP27 (d92), and ICP4 and ICP22 (d96), mutants defective in ICP4, ICP27, and ICP22 (d95) were visually much less toxic to Vero and human embryonic lung cells. Cells infected with d95 at a multiplicity of infection of 10 PFU per cell retained a relatively normal morphology and expressed genes from the viral and cellular genomes for at least 3 days postinfection. The other mutant backgrounds were too toxic to allow examination of gene expression past 1 day postinfection. However, when cell survival was measured by the capacity of the infected cells to form colonies, d95 inhibited colony formation similarly to d92. This apparent paradox was reconciled by the observation that host cell DNA synthesis was inhibited in cells infected with d120, d92, d96, and d95. In addition, all of the mutants exhibited pronounced and distinctive alterations in nuclear morphology, as determined by electron microscopy. The appearance of d95-infected cells deviated from that of uninfected cells in that large circular structures formed in the nucleus. d95-infected cells abundantly expressed ICP0, which accumulated in fine punctate structures in the nucleus at early times postinfection and coalesced or grew to the large circular objects that were revealed by electron microscopy. Therefore, while the abundant accumulation of ICPO in the absence of ICP4, ICP22, and ICP27 may allow for prolonged gene expression, cell survival is impaired, in part, as a result of the inhibition of cellular DNA synthesis.     

Oncogenic Transformation of Hamster Cells after Exposure to Herpes Simplex Virus Type 2

THE possibility of a relationship between herpes simplex viruses (HSV) and human cancer has been suggested^1–4 chiefly on the basis of studies of the epidemiology of cervical cancer, but so far it has not been possible to demonstrate that human herpes viruses can induce primary transformation of normal cells. Injection of herpes simplex virus type 1 (ref. 5) or type 2 (ref. 6) into Syrian hamsters rarely leads to the production of a tumour and it has been difficult to demonstrate herpes viral antigens in tumour cells. Human herpes simplex viruses grown in vitro are characterized by the rapidity with which the infected cell is destroyed, so that cell transformation is impossible, but this effect can be mitigated by inactivation of the herpes virus by ultraviolet irradiation. Indeed, this procedure may have the additional advantage that viral infectivity is removed more quickly than the viral transforming potential⁷.     

The varicella-zoster virus-mediated delayed host shutoff: open reading frame 17 has no major function, whereas immediate-early 63 protein represses heterologous gene expression

We reported that varicella-zoster virus (VZV) causes a delayed host shutoff during its replicative cycle. VZV open reading frame 17 (ORF17) is the homologue of the herpes simplex virus (HSV) UL41 gene encoding the virion host shutoff (vhs) protein which is responsible for the shutoff effect observed in HSV-infected cells. In the present study, we demonstrated that ORF17 is expressed as a late protein during the VZV replicative cycle in different infected permissive cell lines which showed a delayed shutoff of cellular RNA. A cell line with stable expression of VZV ORF17 was infected with VZV. In these cells, VZV replication and delayed host shutoff remained unchanged when compared to normal infected cells. ORF17 was not capable of repressing the expression of the beta-gal reporter gene under the control of the human cytomegalovirus immediate-early gene promoter or to inhibit the expression of a CAT reporter gene under the control of the human GAPDH promoter, indicating that ORF17 has no major function in the VZV-mediated delayed host shutoff. To determine whether other viral factors are involved in the host shutoff, a series of cotransfection assays was performed. We found that the immediate-early 63 protein (IE63) was able to downregulate the expression of reporter genes under the control of the two heterologous promoters, indicating that this viral factor can be involved in the VZV-mediated delayed host shutoff. Other factors can be also implicated to modulate the repressing action of IE63 to achieve a precise balance between the viral and cellular gene expression.     

Cytodiagnosis of herpes simplex keratitis by means of an immunoperoxidase technique. A case report

Typical herpes simplex keratitis that developed in a 5-year-old boy was initially diagnosed cytologically in Papanicolaou-stained samples. Subsequently, an immunoperoxidase staining technique was used to identify the specific type of herpes simplex virus (HSV) in the destained cellular samples. The positive staining helped to establish the diagnosis of a type 1 HSV infection, permitting early treatment with acyclovir and subsequent complete recovery from the ocular herpetic infection. Emphasis is placed on the value of the immunoperoxidase technique for the rapid and specific diagnosis of cases of suspected HSV infection.     

Ocular herpes:the pathophysiology,management and treatment of herpetic eye diseases

Herpesviruses are a prominent cause of human viral disease, second only to the cold and influenza viruses. Most herpesvirus infections are mild or asymptomatic. However, when the virus invades the eye, a number of pathologies can develop and its associated sequelae have become a considerable source of ocular morbidity. The most common culprits of herpetic eye disease are the herpes simplex virus(HSV), varicella zoster virus(VZV), and cytomegalovirus(CMV). While primary infection can produce ocular disease, the most destructive manifestations tend to arise from recurrent infection. These recurrent infections can wreck devastating effects and lead to irreversible vision loss accompanied by a decreased quality of life, increased healthcare usage, and significant cost burden. Unfortunately, no method currently exists to eradicate herpesviruses from the body after infection. Treatment and management of herpes-related eye conditions continue to revolve around antiviral drugs, although corticosteroids, interferons, and other newer therapies may also be appropriate depending on the disease presentation. Ultimately, the advent of effective vaccines will be crucial to preventing herpesvirus diseases altogether and cutting the incidence of ocular complications.     

Ocular herpes: the pathophysiology,management and treatment of herpetic eye diseases

Herpesviruses are a prominent cause of human viral disease, second only to the cold and influenza viruses. Most herpesvirus infections are mild or asymptomatic. However, when the virus invades the eye, a number of pathologies can develop and its associated sequelae have become a considerable source of ocular morbidity. The most common culprits of herpetic eye disease are the herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV). While primary infection can produce ocular disease, the most destructive manifestations tend to arise from recurrent infection. These recurrent infections can wreck devastating effects and lead to irreversible vision loss accompanied by a decreased quality of life, increased healthcare usage, and significant cost burden. Unfortunately, no method currently exists to eradicate herpesviruses from the body after infection. Treatment and management of herpes-related eye conditions continue to revolve around antiviral drugs, although corticosteroids, interferons, and other newer therapies may also be appropriate depending on the disease presentation. Ultimately, the advent of effective vaccines will be crucial to preventing herpesvirus diseases altogether and cutting the incidence of ocular complications.