Cytotoxicity in L929 fibroblasts and inhibition of herpes simplex virus type 1 Kupka by estuarine cyanobacteria extracts

The cyanobacteria are known to be a rich source of metabolites with a variety of biological activities in different biological systems. In the present work, the bioactivity of aqueous and organic (methanolic and hexane) crude extracts of cyanobacteria isolated from estuarine ecosystems was studied using different bioassays. The assessment of DNA damage on the SOS gene repair region of mutant PQ37 strain of Escherichia coli was performed. Antiviral activity was evaluated against influenza virus, HRV-2, CVB3 and HSV-1 viruses using crystal violet dye uptake on HeLa, MDCK and GMK cell lines. Cytotoxicity evaluation was performed with L929 fibroblasts by MTT assay. Of a total of 18 cyanobacterial isolates studied, only the crude methanolic extract of LEGE 06078 proved to be genotoxic (IF > 1.5) in a dose-dependent manner and other four were putative candidates to induce DNA damage. Furthermore, the crude aqueous extract of LEGE 07085 showed anti- herpes type 1 activity (IC50 = 174.10 μg dry extract mL−1) while not presenting any cytotoxic activity against GMK cell lines. Of the 54 cyanobacterial extracts tested, only the crude methanolic and hexane ones showed impair on metabolic activity of L929 fibroblasts after long exposure (48–72 h). The inhibition of HSV-1 and the strong cytotoxicity against L929 cells observed emphasizes the importance of evaluating the impact of those estuarine cyanobacteria on aquatic ecosystem and on human health. The data also point out their potential application in HSV-1 treatment and pharmacological interest.

Seroepidemiology of Herpes Simplex virus type 1 and 2 in Western and Southern Switzerland in adults aged 25-74 in 1992-93: a population-based study.

BACKGROUND: Genital herpes is one of the most prevalent sexually-transmitted diseases, and accounts for a substantial morbidity. Genital herpes puts newborns at risk for very severe disease and also increases the risk of horizontal HIV transmission. It thus stands as an important public health problem. The recent availability of type-specific gG-based assays detecting IgG against HSV-1 and HSV-2 allows to establish the prevalence of each subtype. Worldwide, few data have been published regarding the seroprevalence in general populations of HSV-2, the major causative agent for genital herpes, while no data exist regarding the Swiss population. METHODS: To evaluate the prevalence of IgG antibodies against HSV-1 and HSV-2 in Switzerland, we used a population-based serum repository from a health examination survey conducted in the Western and Southern area of Switzerland in 1992-93. A total of 3,120 sera were analysed by type-specific gG-based ELISA and seroprevalence was correlated with available volunteers characteristics by logistic regression. RESULTS: Overall, seroprevalence rates were 80.0 +/- 0.9% (SE, 95% CI: 78.1-81.8) for HSV-1 and 19.3 +/- 0.9% (SE, 95% CI: 17.6-21.1) for HSV-2 in adults 35-64 year old. HSV-1 and HSV-2 seroprevalence increased with age, with a peak HSV-2 seroprevalence in elderly gentlemen, possibly a seroarcheological evidence of sexually transmitted disease epidemics during World War II. Risk factors for HSV-2 infection included female sex, marital status other than married, and size of town of residence larger than 1500 inhabitants. Unexpectedly and conversely to HSV-1, HSV-2 seroprevalence increased with educational level. HSV-2 infection was less prevalent among HSV-1 infected individuals when compared to HSV-1 uninfected individuals. This effect was most apparent among women at high risk for HSV-2 infection. CONCLUSIONS: Our data demonstrate that by the early nineties, HSV-2 had spread quite largely in the Swiss population. However, the epidemiology of HSV-2 in Switzerland presents paradoxical characteristics, e.g. positive correlation with education level, that have not been observed elsewhere.

Seroepidemiology of herpes simplex virus type 1 and 2 in pregnant women in Switzerland: an obstetric clinic based study.

OBJECTIVE: To assess the seroprevalence of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) IgG antibodies and the seroincidence of HSV-1 and HSV-2 infections in pregnant women attending the maternity clinic of the University Hospital Lausanne. STUDY DESIGN: Blood samples from 1030 women were taken at the usual pregnancy visit in the first trimester to assess the prevalence rate of IgG antibodies against HSV-1 and HSV-2 using a type-specific assay. A second blood sample was taken 6-8 weeks postpartum from returning women who were seronegative for HSV-2 or HSV-1 to assess the incidence of seroconversion (primary infection). RESULTS: The seroprevalence rates were 79.4% (95% CI: 76.9-81.9) for HSV-1 and 21.2% (18.7-23.7) for HSV-2 in women 14-46 years old. Type-specific serostatus patterns were as follows: 17.3% HSV-1/-2: +/+, 62.1% HSV-1/-2: +/-, 3.9% HSV-1/-2: -/+, 16.7% HSV-1/-2: -/-. Two hundred and sixty five women (59 of the 212 seronegative for HSV-1 (27.8%) and 265 of the 812 seronegative for HSV-2 (32.6%)) returned to the outpatient clinic for the post-delivery check and a second blood sample was obtained. One HSV-1 seroconversion was detected (HSV-1 seroconversion rate 2.4%/100 patient×year (95% CI: 0.06-13.4)) in a patient who had symptoms compatible with primary genital herpes. No HSV-2 seroconversion was detected (HSV-2 seroconversion rate: 0/100 patient×year (97.5% one-sided CI: 0-2)). CONCLUSION: Compared to a previous population-based study, our study results suggest a rise in the prevalence of HSV-2 among pregnant women in Switzerland. The low incidence of seroconversion detected during pregnancy is consistent with the very low reported incidence of neonatal herpes in Switzerland. CONDENSATION: This study in a public hospital in Western Switzerland suggests an increasing prevalence of HSV-2, but a low incidence of primary infections in women of childbearing age.

Herpes simplex virus type 1 (HSV-1) pathogenesis and HSV gene therapy of experimental autoimmune encephalomyelitis

The aim of this thesis was to develop new herpes simplex virus (HSV) vectors for gene therapy of experimental autoimmune encephalomyelitis (EAE), the principal model of multiple sclerosis (MS), and to study the pathogenesis of wild-type HSV-1 and HSV-1 vectors in vivo. By introducing potential immunomodulatory factors into mice with EAE we strived to develop therapies and possibly find molecules improving recovery from EAE. We aimed at altering the immune response by inducing favorable Th2-type cytokines, thus shifting the immune response from a Th1- or a Th17-response. Our HSV vector expressing interleukin (IL)-5 modulated the cytokine responses, decreased inflammation and alleviated EAE. The use of a novel method, bacterial artificial chromosome (BAC), for engineering recombinant HSV facilitated the construction of a new vector expressing leukemia inhibitory factor (LIF). LIF is a neurotropic cytokine with broad functions in the central nervous system (CNS). LIF promotes oligodendrocyte maturation and decreases demyelination and oligodendrocyte loss. The BAC-derived HSV-LIF vector alleviated the clinical symptoms, induced a higher number of oligodendrocytes and modulated T cell responses. By administering HSV via different infection routes, e.g. peripherally via the nose or eye, or intracranially to the brain, the effect of the immune response on HSV spread at different points of the natural infection route was studied. The intranasal infection was an effective delivery route of HSV to the trigeminal ganglion and CNS, whereas corneal infection displayed limited spread. The corneal and intranasal infections induced different peripheral immune responses, which might explain the observed differences in viral spread.

Mutagenicity of herpes simplex virus type 1 in a shuttle vector

The shuttle vector plasmid pZ189 was used to find the kinds of mutations that are induced by herpes simplex virus type-1 (HSV-1). In cells infected by HSV-1 the frequency of mutation in supF gene, the mutagenesis marker, was increased over background by from two- to seven-fold, reaching 0.14-0.45%. No increase was induced by infection by vaccinia virus under the same conditions. Mutagenesis was an early event, showing a four-fold increase in mutation frequency at only two hours after infection, and peaking at a seven-fold increase at four hours after infection. DNA sequencing and gel electrophoresis analysis were performed on 105 HSV-1 induced mutants and 65 spontaneous mutants and provided the following information: (1) A change in plasmid size was seen in 54% of HSV-1 related mutants, compared with only 37% of spontaneous mutants. (2) Among point mutations, the predominant type was G:C to A:T transition, which accounted for 51% of point mutations in mutants isolated from cells infected with HSV-1, and 32% of point mutations in spontaneous mutants. (3) Deletions of DNA were seen in HSV-1 related mutants at a frequency of 40%, compared with 29% in spontaneous mutants. The HSV-1 related deletions were about half the length of spontaneous mutants and three contained short filler sequences. (4) Fifteen (15%) of HSV-1 induced mutants revealed the altered restriction patterns on agarose gel electrophoresis analysis and were due either to rearrangements of plasmid DNA, and/or to insertion of sequences derived from chromosomal DNA (seven plasmids). No insertions of DNA from HSV-1 were detected. Among spontaneous mutants, only 5 (7.7%) were rearrangements and none had inserted chromosomal DNA. (5) DNA sequence analysis of seven plasmids with inserted chromosomal DNA revealed that four cases had repetitive DNA sequences integrated and the other three were unidentified sequences from the GenBank database. Three repetitive DNA included $\alpha$ satellite, Alu and KpnI family sequences. The other sequence was identified as tRNA-like component. The observed mutations have implications for the mechanism of malignant transformation of cells by HSV-1. ^

Social stress and the reactivation of latent herpes simplex virus type 1

Psychological stress is thought to contribute to reactivation of latent herpes simplex virus (HSV). Although several animal models have been developed in an effort to reproduce different pathogenic aspects of HSV keratitis or labialis, until now, no good animal model existed in which application of a psychological laboratory stressor results in reliable reactivation of the virus. Reported herein, disruption of the social hierarchy within colonies of mice increased aggression among cohorts, activated the hypothalamic-pituitary-adrenal axis, and caused reactivation of latent HSV type 1 in greater than 40% of latently infected animals. However, activation of the hypothalamic-pituitary-adrenal axis using restraint stress did not activate the latent virus. Thus, the use of social stress in mice provides a good model in which to investigate the neuroendocrine mechanisms that underlie behaviorally mediated reactivation of latent herpesviruses.

Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III–box I hairpin with a 3′ single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III–box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III–box I hairpin in partially unwound DNA.

Creation of drug-specific herpes simplex virus type 1 thymidine kinase mutants for gene therapy.

Herpes simplex virus type 1 (HSV-1) thymidine kinase is currently used as a suicide agent in the gene therapy of cancer. This therapy is based on the preferential phosphorylation of nucleoside analogs by tumor cells expressing HSV-1 thymidine kinase. However, the use of HSV-1 thymidine kinase is limited in part by the toxicity of the nucleoside analogs. We have used random sequence mutagenesis to create new HSV-1 thymidine kinases that, compared with wild-type thymidine kinase, render cells much more sensitive to specific nucleoside analogs. A segment of the HSV-1 thymidine kinase gene at the putative nucleoside binding site was substituted with random nucleotide sequences. Mutant enzymes that demonstrate preferential phosphorylation of the nucleoside analogs, ganciclovir or acyclovir, were selected from more than one million Escherichia coli transformants. Among the 426 active mutants we have isolated, 26 demonstrated enhanced sensitivity to ganciclovir, and 54 were more sensitive to acyclovir. Only 6 mutant enzymes displayed sensitivity to both ganciclovir and acyclovir when expressed in E. coli. Analysis of 3 drug-sensitive enzymes demonstrated that 1 produced stable mammalian cell transfectants that are 43-fold more sensitive to ganciclovir and 20-fold more sensitive to acyclovir.

Analysis of contributions of herpes simplex virus type 1 UL43 protein to induction of cell-cell fusion

Purpose: To investigate whether UL43 protein, which is highly conserved in alpha- and gamma herpes viruses, and a non-glycosylated transmembrane protein, is involved in virus entry and virus-induced cell fusion. Methods: Mutagenesis was accomplished by a markerless two-step Red recombination mutagenesis system implemented on the Herpes simplex virus 1 (HSV-1) bacterial artificial chromosome (BAC). Growth properties of HSV-1 UL43 mutants were analyzed using plaque morphology and one-step growth kinetics. SDS-PAGE and Western blot was employed to assay the synthesis of the viral glycoproteins. Virus-penetration was assayed to determine if UL43 protein is required for efficient virus entry. Results: Lack of UL43 expression resulted in significantly reduced plaque sizes of syncytial mutant viruses and inhibited cell fusion induced by gBΔ28 or gKsyn20 (p < 0.05). Deletion of UL43 did not affect overall expression levels of viral glycoproteins gB, gC, gD, and gH on HSV-1(F) BAC infected cell surfaces. Moreover, mutant viruses lacking UL43 gene exhibited slower kinetics of entry into Vero cells than the parental HSV-1(F) BAC. Conclusion: Thus, these results suggest an important role for UL43 protein in mediating virus-induced membrane fusion and efficient entry of virion into target cells.

An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Oris, by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Oris. P1 nuclease sensitivity of Oris and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein–ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein–ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Oris sequences.

Herpes simplex virus type 2 infection in pregnancy: asymptomatic viral excretion at delivery and seroepidemiologic survey of two socioeconomically distinct populations in São Paulo, Brazil

The objective of the present study was to estimate the prevalence of herpes simplex virus type 2 (HSV 2) antibodies in child bearing women of 2 Brazilian populations with different socioeconomic status and to determine the risk of neonatal HSV exposure by means of maternal cultures at the onset of labor. The study was conducted at 2 hospitals: A, serving very low income patients and B, serving middle socioeconomic class. 173 participants from group A and 127 from B answered a questionnaire which showed that the patients had similar ages (27.7 and 26.8 years, respectively) but differed with regard to socioeconomic status, age at first intercourse (18.6 vs 20.6 years), number of sex partners (1.5 vs 1.2) and previous sexually transmitted diseases (15% vs. 1.5%). History of genital herpes was given by 11% of group A participants and by a similar number, 7%, of patients from group B. In addition, 200 serum samples from population A and 455 from B were tested by ELISA for and HSV antibodies and 92% and 86%, respectively, were found to be positive. Sixty seropositive samples from group A and 90 from B were further analyzed by Western blot, which showed the presence of type 2 specific antibodies in 46% and 36%, respectively, suggesting an overall HSV 2 prevalence of 42% in group A and 31% in B. Cervical specimens were obtained for culture from 299 asymptomatic patients of population A and 313 of B. HSV was isolated from one specimen in each group, indicating a 0.3% incidence of asymptomatic viral excretion in both populations. In conclusion, the prevalence of type 2 antibodies in childbearing women was very high, but it did not differ with the socioeconomic status. The risk of HSV perinatal transmission was also similar in the 2 study populations and it was comparable with the data from developed countries. Our findings do not indicate the need of special screening programs for asymptomatic HSV excretion in Brazilian pregnant women.

PREVALENCE OF HERPES SIMPLEX VIRUS TYPE 2 AND RISK FACTORS ASSOCIATED WITH THIS INFECTION IN WOMEN IN SOUTHERN BRAZIL

SUMMARY The herpes simplex virus type 2 (HVS-2) is the most prevalent infection worldwide. It is a cofactor in the acquisition of human immunodeficiency virus (HIV) and the persistence of human papillomavirus (HPV). This study evaluated the prevalence of HSV-2, using the polymerase chain reaction (PCR), and associated factors in patients treated at the Federal University of Rio Grande (FURG) and Basic Health Units (BHU) in Rio Grande, Brazil. The observed prevalence of HSV-2 was 15.6%. Among the 302 women studied, 158 had received assistance in BHU and 144 were treated at FURG. The prevalence of HSV-2 in these groups was 10.8% and 20.8%, respectively, RR 1.9 and p = 0.012. Knowledge about the Pap smear, and the presence of lesions showed no association with HSV-2 infection. Multivariate analysis showed that the variable that most influenced the risk of HSV-2 infection was the presence of HIV infection, with a relative risk of 1.9 and p = 0.04. Discussion: Genital ulcers are an important entry point for HIV, and condom use is an important strategy to reduce transmission of HIV and HSV-2.

In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus

Peptides with broad-spectrum antimicrobial activity, known as antimicrobial peptides, have been isolated from distinct organisms. This paper describes the in vitro evaluation of the cytotoxicity and antiviral activity of nine peptides with different structures and origins against herpes simplex virus type 1, human adenovirus respiratory strain, and rotavirus SA11. Most of the evaluated peptides presented antiviral activity but they were only active near cytotoxic concentrations. Nevertheless, these results seem promising, and further modifications on the peptide's structures may improve their selectivity and reduce their cytotoxicity.

Acute retinal necrosis in primary herpes simplex virus type I infection.

Here we report the case of an immunocompetent 8-year-old child who developed acute retinal necrosis concomitant with a primary herpes simplex virus type I infection. Ocular inflammation changed along with the development of a specific antibody titer in the serum. This evidence suggests that the immune response of the host can significantly modulate the clinical aspect of the ocular infection.

Enhanced anti-tumor effect of a gene gun-delivered DNA vaccine encoding the human papillomavirus type 16 oncoproteins genetically fused to the herpes simplex virus glycoprotein D

Anti-cancer DNA vaccines have attracted growing interest as a simple and non-invasive method for both the treatment and prevention of tumors induced by human papillomaviruses. Nonetheless, the low immunogenicity of parenterally administered vaccines, particularly regarding the activation of cytotoxic CD8+ T cell responses, suggests that further improvements in both vaccine composition and administration routes are still required. In the present study, we report the immune responses and anti-tumor effects of a DNA vaccine (pgD-E7E6E5) expressing three proteins (E7, E6, and E5) of the human papillomavirus type 16 genetically fused to the glycoprotein D of the human herpes simplex virus type 1, which was administered to mice by the intradermal (id) route using a gene gun. A single id dose of pgD-E7E6E5 (2 µg/dose) induced a strong activation of E7-specific interferon-γ (INF-γ)-producing CD8+ T cells and full prophylactic anti-tumor effects in the vaccinated mice. Three vaccine doses inhibited tumor growth in 70% of the mice with established tumors. In addition, a single vaccine dose consisting of the co-administration of pgD-E7E6E5 and the vector encoding interleukin-12 or granulocyte-macrophage colony-stimulating factor further enhanced the therapeutic anti-tumor effects and conferred protection to 60 and 50% of the vaccinated mice, respectively. In conclusion, id administration of pgD-E7E6E5 significantly enhanced the immunogenicity and anti-tumor effects of the DNA vaccine, representing a promising administration route for future clinical trials.