Human lactoferrin but not lysozyme neutralizes HSV-1 and inhibits HSV-1 replication and cell-to-cell spread

The frequent oral shedding of herpes simplex virus type 1 (HSV-1) in the absence of clinical disease suggests that symptomatic HSV-1 recurrences may be inhibited by the mucosal environment. Indeed, saliva has been shown to contain substances with anti-HSV activity. In the current study, we investigated the anti-HSV-1 activity of human lactoferrin (hLf) and lysozyme (hLz), two highly cationic polypeptides of the mucosal innate defence system

Virusvektorit syövän hoidossa

Virusvektorien käyttö syövän hoidossa on nopeasti kehittyvä uusi tutkimusalue. Sekä lisääntymiskyvyttömien että lisääntymiskykyisten onkolyyttisten virusten monet ominaisuudet sopivat hyvin syövän hoitoon. Virusvektorit voidaan myös varustaa sytotoksisilla siirtogeeneillä tai molekyyleillä, jotka vahvistavat elimistön immuunipuolustusta syöpäkudosta kohtaan. Ensimmäiset virusvektoreihin perustuvat hoidot on Euroopassa ja Yhdysvalloissa hyväksytty kliiniseen käyttöön pitkälle edenneen melanooman sekä hoitoresistenttien B-soluleukemioiden ja lymfoomien hoidossa. Sytolyyttiset ja immuunivastetta stimuloivat geenihoidot ovatkin nopeasti tulossa onkologisten hoitomahdollisuuksien joukkoon suomalaisessa terveydenhuollossa. Esittelemme yleisimmät syövän hoidossa käytetyt virusvektorit sekä niiden käyttömahdollisuudet pahanlaatuisten kasvainten hoidossa

Herpes Simplex Virus Seroprevalence among Pregnant Finnish Women and Their Spouses—A Six-Year Follow-Up Cohort Study

The aim was to evaluate the herpes simplex virus (HSV) seroprevalence and seroconversion among 285 pregnant women and their 120 male spouses in Finland during a six-year follow-up (FU) between 1998–2008. We also studied the effect of sexual habits, pregnancy, and other demographic factors on the acquisition of HSV infection. Combined HSV-1 and HSV-2-IgG antibodies were assessed in the first baseline serum samples with an indirect enzyme immunoassay method. The individuals with seronegative or borderline HSV serology at baseline were additionally tested using their latest FU serum sample available. The overall HSV seroprevalence during the FU was 58.9% (168/285) among the women and 53.3% (64/120) among their spouses. The seroconversion rate was 11.4% (15/132) and 12.5% (8/64) among women and their spouses, respectively. Both spouses were HSV seropositive in 39.2% (47/120). To determine the HSV-2 seroprevalence, we also tested all HSV-seropositive participants using HSV-2-specific antigen. HSV-2 seropositivity was detected in 10.9% (44/405) of the participants. The age (p = 0.006) and history of genital warts (p = 0.006) of the women were associated with combined HSV-1 and/or HSV-2 seropositivity, while a younger age was related to HSV seroconversion (p = 0.023). Among the male spouses, HSV seropositivity was associated with the practice of oral sex (p = 0.033). To conclude, women of childbearing age acquire primary HSV infections and the presence of HSV in oral epithelium is common among HSV-seropositive individuals

Native RNA purification method for small RNA molecules based on asymmetrical flow field-flow fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules.Peer reviewe

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

Antisense RNA directed to the human papillomavirus type 16 E7 mRNA from herpes simplex virus type 1 derived vectors is expressed in CaSki cells and downregulates E7 mRNA

<p>Abstract</p> <p>Background</p> <p>Human papillomavirus (HPV) infection is known to be the most important etiologic factor of cervical cancer. There is no HPV specific therapy available for treatment of invasive squamous cell carcinoma of the cervix and its precursor lesions. The present study elucidates the potential to use herpes simplex virus (HSV) derived vectors for expression of antisense RNA to HPV -16 E7 oncogene.</p> <p>Results</p> <p>We have constructed replication competent, nonneuroinvasive HSV-1 vectors, deleted of the γ₁34.5 gene. The vectors express RNA antisense to the first 100 nucleotides of the HPV-16 E7 gene. We assayed the ability of the antisense E7 vectors R5225 (<it>tk</it>-) and R5226 (<it>tk+</it>), to produce antisense RNA, as well as the consequent effects on E7 mRNA and protein levels in HPV-16 positive CaSki cells. Anti-E7 RNA was expressed by both constructs in a dose-dependent manner. Expression of HPV-16 E7 mRNA was downregulated effectively in CaSki cells infected with the <it>tk- </it>recombinant R5225 or with R5226. The <it>tk+ </it>recombinant R5226 was effective in downregulating E7 protein expression.</p> <p>Conclusion</p> <p>We have shown that anti-E7 RNA expressed from an HSV vector could efficiently downregulate HPV-16 E7 mRNA and E7 protein expression in CaSki cells. We conclude that HSV vectors may become a useful tool for gene therapy of HPV infections.</p

Swarms of chemically modified antiviral siRNA targeting herpes simplex virus infection in human corneal epithelial cells

Peer reviewe

Herpes Simplex Virus Type 1 Us3 Gene Deletion Influences Toll-like Receptor Responses in Cultured Monocytic Cells

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors have a key role in innate immune response to microbial infection. The toll-like receptor (TLR) family consists of ten identified human TLRs, of which TLR2 and TLR9 have been shown to initiate innate responses to herpes simplex virus type 1 (HSV-1) and TLR3 has been shown to be involved in defence against severe HSV-1 infections of the central nervous system. However, no significant activation of the TLR3 pathways has been observed in wild type HSV-1 infections. In this work, we have studied the TLR responses and effects on TLR gene expression by HSV-1 with Us3 and ICP4 gene deletions, which also subject infected cells to apoptosis in human monocytic (U937) cell cultures.</p> <p>Results</p> <p>U937 human monocytic cells were infected with the Us3 and ICP4 deletion herpes simplex virus (d120), its parental virus HSV-1 (KOS), the Us3 deletion virus (R7041), its rescue virus (R7306) or wild type HSV-1 (F). The mRNA expression of TLR2, TLR3, TLR4, TLR9 and type I interferons (IFN) were analyzed by quantitative real-time PCR. The intracellular expression of TLR3 and type I IFN inducible myxovirus resistance protein A (MxA) protein as well as the level of apoptosis were analyzed by flow cytometry. We observed that the mRNA expression of TLR3 and type I IFNs were significantly increased in d120, R7041 and HSV-1 (F)-infected U937 cells. Moreover, the intracellular expression of TLR3 and MxA were significantly increased in d120 and R7041-infected cells. We observed activation of IRF-3 in infections with d120 and R7041. The TLR4 mRNA expression level was significantly decreased in d120 and R7041-infected cells but increased in HSV-1 (KOS)-infected cells in comparison with uninfected cells. No significant difference in TLR2 or TLR9 mRNA expression levels was seen. Both the R7041 and d120 viruses were able to induce apoptosis in U937 cell cultures.</p> <p>Conclusion</p> <p>The levels of TLR3 and type I IFN mRNA were increased in d120, R7041 and HSV-1 (F)-infected cells when compared with uninfected cells. Also IRF-3 was activated in cells infected with the Us3 gene deletion viruses d120 and R7041. This is consistent with activation of TLR3 signaling in the cells. The intracellular TLR3 and type I IFN inducible MxA protein levels were increased in d120 and R7041-infected cells but not in cells infected with the corresponding parental or rescue viruses, suggesting that the HSV-1 Us3 gene is involved in control of TLR3 responses in U937 cells.</p