Publications by authors named "Veijo Hukkanen"

46 Publications

Herpes Simplex Virus Type 1 Clinical Isolates Respond to UL29-Targeted siRNA Swarm Treatment Independent of Their Acyclovir Sensitivity.

Viruses 2020 12 13;12(12). Epub 2020 Dec 13.

Institute of Biomedicine, University of Turku, 20520 Turku, Finland.

Acyclovir is the drug of choice for the treatment of herpes simplex virus (HSV) infections. Acyclovir-resistant HSV strains may emerge, especially during long-term drug use, and subsequently cause difficult-to-treat exacerbations. Previously, we set up a novel treatment approach, based on enzymatically synthesized pools of siRNAs, or siRNA swarms. These swarms can cover kilobases-long target sequences, reducing the likelihood of resistance to treatment. Swarms targeting the essential gene of HSV-1 have demonstrated high efficacy against HSV-1 in vitro and in vivo. Here, we assessed the antiviral potential of a UL29 siRNA swarm against circulating strains of HSV-1, in comparison with acyclovir. All circulating strains were sensitive to both antivirals, with the half-maximal inhibitory concentrations (IC) in the range of 350-1911 nM for acyclovir and 0.5-3 nM for the UL29 siRNA swarm. Additionally, we showed that an acyclovir-resistant HSV-1, devoid of thymidine kinase, is highly sensitive to UL29 siRNA treatment (IC 1.0 nM; I 97%). Moreover, the detected minor variations in the RNAi target of the HSV strains had no effect on the potency or efficacy of UL29 siRNA swarm treatment. Our findings support the development of siRNA swarms for the treatment of HSV-1 infections, in order to circumvent any potential acyclovir resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v12121434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764767PMC
December 2020

Enzymatically synthesized 2'-fluoro-modified Dicer-substrate siRNA swarms against herpes simplex virus demonstrate enhanced antiviral efficacy and low cytotoxicity.

Antiviral Res 2020 10 13;182:104916. Epub 2020 Aug 13.

Molecular and Integrative Biosciences Research Programme, Biological and Environmental Sciences, University of Helsinki, Viikinkaari 9, FI-00014, Helsinki, Finland. Electronic address:

Chemical modifications of small interfering (si)RNAs are used to enhance their stability and potency, and to reduce possible off-target effects, including immunogenicity. We have earlier introduced highly effective antiviral siRNA swarms against herpes simplex virus (HSV), targeting 653 bp of the essential UL29 viral gene. Here, we report a method for enzymatic production and antiviral use of 2'-fluoro-modified siRNA swarms. Utilizing the RNA-dependent RNA polymerase from bacteriophage phi6, we produced 2'-F-siRNA swarms containing either all or a fraction of modified adenosine, cytidine or uridine residues in the antisense strand of the UL29 target. The siRNA containing modified pyrimidines demonstrated high resistance to RNase A and the antiviral potency of all the UL29-specific 2'-F-siRNA swarms was 100-fold in comparison with the unmodified counterpart, without additional cytotoxicity. Modest stimulation of innate immunity signaling, including induced expression of both type I and type III interferons, as well as interferon-stimulated gene 54, by 2'-F-cytidine and 2'-F-uridine modified siRNA swarms occurred at early time points after transfection while the 2'-F-adenosine-containing siRNA was similar to the unmodified antiviral siRNA swarm in this respect. The antiviral efficacy of the 2'-F-siRNA swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified siRNAs. The results support further applications of enzymatically produced siRNA molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose C2' position, for further antiviral studies in vitro and in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.antiviral.2020.104916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424292PMC
October 2020

HERQ-9 Is a New Multiplex PCR for Differentiation and Quantification of All Nine Human Herpesviruses.

mSphere 2020 06 24;5(3). Epub 2020 Jun 24.

Department of Virology, University of Helsinki, Helsinki, Finland

Infections with the nine human herpesviruses (HHVs) are globally prevalent and characterized by lifelong persistence. Reactivations can potentially manifest as life-threatening conditions for which the demonstration of viral DNA is essential. In the present study, we developed HERQ-9, a pan-HHV quantitative PCR designed in triplex reactions to differentiate and quantify each of the HHV-DNAs: (i) herpes simplex viruses 1 and 2 and varicella-zoster virus; (ii) Epstein-Barr virus, human cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus; and (iii) HHV-6A, -6B, and -7. The method was validated with prequantified reference standards as well as with mucocutaneous swabs and cerebrospinal fluid, plasma, and tonsillar tissue samples. Our findings highlight the value of multiplexing in the diagnosis of many unsuspected, yet clinically relevant, herpesviruses. In addition, we report here frequent HHV-DNA co-occurrences in clinical samples, including some previously unknown. HERQ-9 exhibited high specificity and sensitivity (LODs of ∼10 to ∼17 copies/reaction), with a dynamic range of 10 to 10 copies/μl. Moreover, it performed accurately in the coamplification of both high- and low-abundance targets in the same reaction. In conclusion, we demonstrated that HERQ-9 is suitable for the diagnosis of a plethora of herpesvirus-related diseases. Besides its significance to clinical management, the method is valuable for the assessment of hitherto-unexplored synergistic effects of herpesvirus coinfections. Furthermore, its high sensitivity enables studies on the human virome, often dealing with minute quantities of persisting HHVs. By adulthood, almost all humans become infected by at least one herpesvirus (HHV). The maladies inflicted by these microbes extend beyond the initial infection, as they remain inside our cells for life and can reactivate, causing severe diseases. The diagnosis of active infection by these ubiquitous pathogens includes the detection of DNA with sensitive and specific assays. We developed the first quantitative PCR assay (HERQ-9) designed to identify and quantify each of the nine human herpesviruses. The simultaneous detection of HHVs in the same sample is important since they may act together to induce life-threatening conditions. Moreover, the high sensitivity of our method is of extreme value for assessment of the effects of these viruses persisting in our body and their long-term consequences on our health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/mSphere.00265-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316487PMC
June 2020

Comparison of Clinically Relevant Oncolytic Virus Platforms for Enhancing T Cell Therapy of Solid Tumors.

Mol Ther Oncolytics 2020 Jun 19;17:47-60. Epub 2020 Mar 19.

Cancer Gene Therapy Group, Translational Immunology Research Program, University of Helsinki, 00290 Helsinki, Finland.

Despite some promising results, the majority of patients do not benefit from T cell therapies, as tumors prevent T cells from entering the tumor, shut down their activity, or downregulate key antigens. Due to their nature and mechanism of action, oncolytic viruses have features that can help overcome many of the barriers currently facing T cell therapies of solid tumors. This study aims to understand how four different oncolytic viruses (adenovirus, vaccinia virus, herpes simplex virus, and reovirus) perform in that task. For that purpose, an immunocompetent tumor model featuring adoptive tumor-infiltrating lymphocyte (TIL) therapy was used. Tumor growth control (p < 0.001) and survival analyses suggest that adenovirus was most effective in enabling T cell therapy. The complete response rate was 62% for TILs + adenovirus versus 17.5% for TILs + PBS. Of note, TIL biodistribution did not explain efficacy differences between viruses. Instead, immunostimulatory shifts in the tumor microenvironment mirrored efficacy results. Overall, the use of oncolytic viruses can improve the utility of T cell therapies, and additional virus engineering by arming with transgenes can provide further antitumor effects. This phenomenon was seen when an unarmed oncolytic adenovirus was compared to Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (TILT-123). A clinical trial is ongoing, where patients receiving TIL treatment also receive TILT-123 (ClinicalTrials.gov: NCT04217473).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omto.2020.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7163046PMC
June 2020

Novel Antiviral Activities of Obatoclax, Emetine, Niclosamide, Brequinar, and Homoharringtonine.

Viruses 2019 10 18;11(10). Epub 2019 Oct 18.

Department of Clinical and Molecular Medicine, Norwegian University of Science and Technology, 7028 Trondheim, Norway.

Viruses are the major causes of acute and chronic infectious diseases in the world. According to the World Health Organization, there is an urgent need for better control of viral diseases. Repurposing existing antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we identified novel activities of obatoclax and emetine against herpes simplex virus type 2 (HSV-2), echovirus 1 (EV1), human metapneumovirus (HMPV) and Rift Valley fever virus (RVFV) in cell cultures. Moreover, we demonstrated novel activities of emetine against influenza A virus (FLUAV), niclosamide against HSV-2, brequinar against human immunodeficiency virus 1 (HIV-1), and homoharringtonine against EV1. Our findings may expand the spectrum of indications of these safe-in-man agents and reinforce the arsenal of available antiviral therapeutics pending the results of further in vitro and in vivo tests.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v11100964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832696PMC
October 2019

Comparison of Herpes Simplex Virus 1 Strains Circulating in Finland Demonstrates the Uncoupling of Whole-Genome Relatedness and Phenotypic Outcomes of Viral Infection.

J Virol 2019 04 3;93(8). Epub 2019 Apr 3.

Department of Biochemistry and Molecular Biology, Center for Infectious Disease Dynamics, and the Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania, USA

A majority of adults in Finland are seropositive carriers of herpes simplex viruses (HSV). Infection occurs at epithelial or mucosal surfaces, after which virions enter innervating nerve endings, eventually establishing lifelong infection in neurons of the sensory or autonomic nervous system. Recent data have highlighted the genetic diversity of HSV-1 strains and demonstrated apparent geographic patterns in strain similarity. Though multiple HSV-1 genomes have been sequenced from Europe to date, there is a lack of sequenced genomes from the Nordic countries. Finland's history includes at least two major waves of human migration, suggesting the potential for diverse viruses to persist in the population. Here, we used HSV-1 clinical isolates from Finland to test the relationship between viral phylogeny, genetic variation, and phenotypic characteristics. We found that Finnish HSV-1 isolates separated into two distinct phylogenetic groups, potentially reflecting historical waves of human (and viral) migration into Finland. Each HSV-1 isolate harbored a distinct set of phenotypes in cell culture, including differences in the amount of virus production, extracellular virus release, and cell-type-specific fitness. Importantly, the phylogenetic clusters were not predictive of any detectable pattern in phenotypic differences, demonstrating that whole-genome relatedness is not a proxy for overall viral phenotype. Instead, we highlight specific gene-level differences that may contribute to observed phenotypic differences, and we note that strains from different phylogenetic groups can contain the same genetic variations. Herpes simplex viruses (HSV) infect a majority of adults. Recent data have highlighted the genetic diversity of HSV-1 strains and demonstrated apparent genomic relatedness between strains from the same geographic regions. We used HSV-1 clinical isolates from Finland to test the relationship between viral genomic and geographic relationships, differences in specific genes, and characteristics of viral infection. We found that viral isolates from Finland separated into two distinct groups of genomic and geographic relatedness, potentially reflecting historical patterns of human and viral migration into Finland. These Finnish HSV-1 isolates had distinct infection characteristics in multiple cell types tested, which were specific to each isolate and did not group according to genomic and geographic relatedness. This demonstrates that HSV-1 strain differences in specific characteristics of infection are set by a combination of host cell type and specific viral gene-level differences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01824-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450105PMC
April 2019

Personalized Cancer Vaccine Platform for Clinically Relevant Oncolytic Enveloped Viruses.

Mol Ther 2018 09 19;26(9):2315-2325. Epub 2018 Jun 19.

Laboratory of Immunovirotherapy, Drug Research Program, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00790 Helsinki, Finland. Electronic address:

The approval of the first oncolytic virus for the treatment of metastatic melanoma and the compiling evidence that the use of oncolytic viruses can enhance cancer immunotherapies targeted against various immune checkpoint proteins has attracted great interest in the field of cancer virotherapy. We have developed a novel platform for clinically relevant enveloped viruses that can direct the virus-induced immune response against tumor antigens. By physically attaching tumor-specific peptides onto the viral envelope of vaccinia virus and herpes simplex virus 1 (HSV-1), we were able to induce a strong T cell-specific immune response toward these tumor antigens. These therapeutic peptides could be attached onto the viral envelope by using a cell-penetrating peptide sequence derived from human immunodeficiency virus Tat N-terminally fused to the tumor-specific peptides or, alternatively, therapeutic peptides could be conjugated with cholesterol for the attachment of the peptides onto the viral envelope. We used two mouse models of melanoma termed B16.OVA and B16-F10 for testing the efficacy of OVA SIINFEKL-peptide-coated viruses and gp100-Trp2-peptide-coated viruses, respectively, and show that by coating the viral envelope with therapeutic peptides, the anti-tumor immunity and the number of tumor-specific CD8 T cells in the tumor microenvironment can be significantly enhanced.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2018.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127500PMC
September 2018

Novel activities of safe-in-human broad-spectrum antiviral agents.

Antiviral Res 2018 06 23;154:174-182. Epub 2018 Apr 23.

Institute of Biomedicine, University of Turku, Turku 20520, Finland. Electronic address:

According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley fever virus. Thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.antiviral.2018.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113852PMC
June 2018

Herpes simplex and human papilloma virus coinfections in oral mucosa of men-A 6-year follow-up study.

J Med Virol 2018 03 20;90(3):564-570. Epub 2017 Oct 20.

Faculty of Medicine, Department of Oral Pathology, Institute of Dentistry, and Medicity Research Laboratory, University of Turku, Turku, Finland.

Herpes simplex virus (HSV) establishes latency in neurons and recurrent infections in oral mucosa. This prospective study analyzes HSV prevalence in oral mucosal brush samples from men with known human papillomavirus (HPV) status. We hypothesized that HSV-1-infection could facilitate HPV persistence as a cofactor. This study was a part of the Finnish Family HPV study accomplished at the University of Turku/Turku University Hospital, Finland. A total of 139 men (mean age 28.6 ± 4.9 years) were enrolled at 36+-weeks of their partner's pregnancy and thereafter followed-up for 6 years. Altogether, 722 samples, extracted from oral brush samples collected at the enrollment timepoint (baseline) and at 2-, 6-, 12-, 24-, 36-month, and 6 years, were available. HSV DNA was analyzed with quantitative PCR. HSV-1 results were compared with the known HPV data. The prevalence of oral HSV-1 shedding varied between 0-7.2% (mean 2.8%) among the men. Mean copy numbers varied between 4 and 550 genome copies/sample. A total of 18 (12.9%) men were found HSV-1-positive at least once, two of them twice. Neither smoking nor oral sex was associated with the oral HSV-1-DNA finding. HPV/HSV-1 co-infection was found in 6 (4.3%) men, all of them having persistent HPV-infection. In conclusion, HSV-1 and its coinfection with HPV in oral mucosa was rare.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jmv.24965DOI Listing
March 2018

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins.

Viruses 2017 09 25;9(10). Epub 2017 Sep 25.

University of Lille, CHU Lille laboratoire de Virologie, EA3610, F-59037 Lille, France.

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v9100271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691623PMC
September 2017

The ERK-1 function is required for HSV-1-mediated G1/S progression in HEP-2 cells and contributes to virus growth.

Sci Rep 2017 08 23;7(1):9176. Epub 2017 Aug 23.

Department of Biological and Environmental Sciences, University of Messina, Viale F. Stagno d'Alcontres 31, 98166, Messina, Italy.

The herpes simplex virus 1 is able to readdress different cellular pathways including cell cycle to facilitate its replication and spread. During infection, the progression of the cell cycle from G1 to S phase makes the cellular replication machinery accessible to viral DNA replication. In this work we established that HSV-1, in asynchronized HEp-2 cells, strictly controls cell cycle progression increasing S-phase population from 9 hours post infection until the end of HSV-1 replication. The G1/S phases progression depends on two important proteins, cyclin E and CDK2. We demonstrate that their phosphorylated status and then their activity during the infection is strongly correlated to viral replication events. In addition, HSV-1 is able to recruit and distribute ERK1/2 proteins in a spatio-temporal fashion, highlighting its downstream regulatory effects on cellular processes. According with this data, using chemical inhibitor U0126 and ERK dominant negative cells we found that the lack of ERK1 activity affects cyclin E protein accumulation, viral gene transcription and percentage of the cells in S phase, during the viral replication. These data suggested a complex interaction between ERK, cell cycle progression and HSV-1 replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-09529-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569015PMC
August 2017

Chromatin organization regulates viral egress dynamics.

Sci Rep 2017 06 16;7(1):3692. Epub 2017 Jun 16.

Department of Biological and Environmental Science, and Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland.

Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walk modelling of herpes simplex virus 1-sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-03630-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473834PMC
June 2017

Topical treatment of herpes simplex virus infection with enzymatically created siRNA swarm.

Antivir Ther 2017 13;22(7):631-637. Epub 2017 Mar 13.

Department of Virology, University of Turku, Turku, Finland.

Background: Herpes simplex virus (HSV) is a common human pathogen. Despite current antivirals, it causes a significant medical burden. Drug resistant strains exist and they are especially prevalent in immunocompromised patients and in HSV eye infections. New treatment modalities are needed.

Methods: BALB/c mice were corneally infected with HSV and subsequently treated with a swarm of enzymatically created, Dicer-substrate small interfering RNA (siRNA) molecules that targeted the HSV gene UL29. Two infection models were used, one in which the infection was predominantly peripheral and another in which it spread to the central nervous system. Mouse survival, as well as viral spread, load, latency and peripheral shedding, was studied.

Results: The anti-HSV-UL29 siRNA swarm alleviated HSV infection symptoms, inhibited viral shedding and replication and had a favourable effect on mouse survival.

Conclusions: Treatment with anti-HSV-UL29 siRNA swarm reduced symptoms and viral spread in HSV infection of mice and also inhibited local viral replication in mouse corneas.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3851/IMP3153DOI Listing
July 2018

Regulation of kynurenine biosynthesis during influenza virus infection.

FEBS J 2017 01 14;284(2):222-236. Epub 2016 Dec 14.

Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Finland.

Influenza A viruses (IAVs) remain serious threats to public health because of the shortage of effective means of control. Developing more effective virus control modalities requires better understanding of virus-host interactions. It has previously been shown that IAV induces the production of kynurenine, which suppresses T-cell responses, enhances pain hypersensitivity and disturbs behaviour in infected animals. However, the regulation of kynurenine biosynthesis during IAV infection remains elusive. Here we showed that IAV infection induced expression of interferons (IFNs), which upregulated production of indoleamine-2,3-dioxygenase (IDO1), which catalysed the kynurenine biosynthesis. Furthermore, IAV attenuated the IDO1 expression and the production of kynurenine through its NS1 protein. Interestingly, inhibition of viral replication prior to IFN induction limited IDO1 expression, while inhibition after did not. Finally, we showed that kynurenine biosynthesis was activated in macrophages in response to other stimuli, such as influenza B virus, herpes simplex virus 1 and 2 as well as bacterial lipopolysaccharides. Thus, the tight regulation of the kynurenine biosynthesis by host cell and, perhaps, pathogen might be a basic signature of a wide range of host-pathogen interactions, which should be taken into account during development of novel antiviral and antibacterial drugs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.13966DOI Listing
January 2017

Interleukin-27 Inhibits Herpes Simplex Virus Type 1 Infection by Activating STAT1 and 3, Interleukin-6, and Chemokines IP-10 and MIG.

J Interferon Cytokine Res 2016 11 12;36(11):617-629. Epub 2016 Sep 12.

1 Department of Virology, University of Turku , Turku, Finland .

Interleukin-27 (IL-27) inhibits the replication of many viruses, but the mechanism differs according to virus and cell type. In this study, we observed that IL-27 expression was upregulated in herpes simplex virus type 1 (HSV-1)-infected SJL/J mice, which led us to further investigate the role of IL-27 in HSV-1 infection using epithelial, glioma, and immunological cells as cell models. We showed that in all studied cell lines, the IL-27 messenger RNA (mRNA) level was upregulated due to the HSV-1 infection. When the cells were primed with IL-27 before the virus infection, the virus release was prevented, indicating an antiviral role of IL-27 in HSV-1 infection. Furthermore, we observed that IL-27 secretion to the culture medium was reduced in infected epithelial and immunological cells, but not in glioma cells. Not surprisingly, HSV-1 induced type I, II, and III interferons regardless of cell line, but IL-27 itself caused varying interferon responses dependent on cell type. However, common to all cell types was the IL-27-stimulated secretion of IL-6 and chemokines IP-10 and MIG. In addition, IL-27 stimulation activated STAT1 and STAT3 in HeLa and T98G cells, suggesting that IL-27 engages the STAT1/3 pathway, which then leads to the upregulation of IL-6, IP-10, and MIG.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/jir.2016.0015DOI Listing
November 2016

The γ134.5 Neurovirulence Gene of Herpes Simplex Virus 1 Modifies the Exosome Secretion Profile in Epithelial Cells.

J Virol 2016 12 14;90(23):10981-10984. Epub 2016 Nov 14.

Department of Virology, University of Turku, Turku, Finland

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01157-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110198PMC
December 2016

Herpes simplex virus 1 induces egress channels through marginalized host chromatin.

Sci Rep 2016 06 28;6:28844. Epub 2016 Jun 28.

University of Jyvaskyla, Department of Biological and Environmental Science and Nanoscience Center, Jyvaskyla, FI-40500, Finland.

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. We used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gaps frequently contained viral nucleocapsids. These results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep28844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378911PMC
June 2016

Inhibition of clinical pathogenic herpes simplex virus 1 strains with enzymatically created siRNA pools.

J Med Virol 2016 12 30;88(12):2196-2205. Epub 2016 May 30.

Department of Virology, University of Turku, Turku, Finland.

Herpes simplex virus (HSV) is a common human pathogen causing severe diseases such as encephalitis, keratitis, and neonatal herpes. There is no vaccine against HSV and the current antiviral chemotherapy fails to treat certain forms of the disease. Here, we evaluated the antiviral activity of enzymatically created small interfering (si)RNA pools against various pathogenic HSV strains as potential candidates for antiviral therapies. Pools of siRNA targeting 0.5-0.8 kbp of essential HSV genes UL54, UL29, or UL27 were enzymatically synthesized. Efficacy of inhibition of each siRNA pool was evaluated against multiple clinical isolates and laboratory wild type HSV-1 strains using three cell lines representing host tissues that support HSV-1 replication: epithelial, ocular, and cells that originated from the nervous system. The siRNA pools targeting UL54, UL29, and UL27, as well as their equimolar mixture, inhibited HSV replication, with the pool targeting UL29 having the most prominent antiviral effect. In contrast, the non-specific control siRNA pool did not have such an effect. Moreover, the UL29 pool elicited only a minimal innate immune response in the HSV-infected cells, thus evidencing the safety of its potential clinical use. These results are promising for the development of a topical RNA interference approach for clinical treatment of HSV infection. J. Med. Virol. 88:2196-2205, 2016. © 2016 Wiley Periodicals, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jmv.24578DOI Listing
December 2016

HSV-1 Infection Modulates the Radioresponse of a HPV16-positive Head and Neck Cancer Cell Line.

Anticancer Res 2016 Feb;36(2):565-74

Department of Oral Pathology, Institute of Dentistry, Faculty of Medicine, University of Turku, Turku, Finland Department of Pathology, Turku University Hospital, Turku, Finland.

Background: The combined effects of Human papillomavirus (HPV) and Herpes simplex type 1 (HSV-1) infections and their effects on cancer cell radioresistance are unexplored.

Materials And Methods: An HPV16-positive hypopharyngeal carcinoma cell line (UD-SCC-2) was infected with wt-HSV-1 at low multiplicity of infection (MOI) and irradiated with 2 Gy at 24 h postinfection. Viability assays and quantitative reverse-transcriptase PCR for HPV16 E6, E7, nuclear factor kappa B1, B-cell CLL/lymphoma 2 (BCL2), and caspases 3, 8 and 9 at 24, and 72 h, as well as immunocytochemistry for BCL2, caspase 3, cyclin E, mouse double minute 2 homolog (MDM2), HSV-1 and Ki-67 were performed at 144 h postirradiation.

Results: At 144 h, cell viability was significantly lowered by irradiation only in uninfected cells. Infection combined with irradiation resulted in increased expression of E6, E7, BCL2 and NF-κB1 at 144 h. Simultaneously, E6 and E7 were down-regulated in non-irradiated infected cells. Irradiation and infection with 0.00001 MOI separately up-regulated caspase 3 but infection with 0.0001 MOI halved its expression in irradiated cells.

Conclusion: HSV-1 infection modulates radioresistance of HPV16-positive hypopharyngeal carcinoma cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
February 2016

Carriage of herpes simplex virus and human papillomavirus in oral mucosa is rare in young women: A long-term prospective follow-up.

J Clin Virol 2015 Sep 8;70:58-62. Epub 2015 Jul 8.

Department of Virology, University of Turku, Turku, Finland.

Background: Following the primary oral infection, herpes simplex virus (HSV) establishes latency in the ganglia of sensory neurons. Episodically induced by stress, HSV is able to cause recurrent infection at the primary infection site, accompanied by virus shedding. The oral shedding of HSV contributes to mother-child-transmission of HSV. Human papillomavirus (HPV) is associated with oral malignancies, and its interaction with oral HSV should be studied further.

Objectives: To analyze the prevalence of HSV-1 and HSV-2-infection in oral mucosal scrapings of women during and after pregnancy and to elucidate the prevalence of HPV and HSV-co-carriage in oral mucosa.

Study Design: A longitudinal cohort study of 304 mothers in the Finnish Family HPV study followed-up for 6 years after pregnancy, with 7 serial samplings. Mothers' oral brush samples were analyzed with quantitative PCR for HSV-1 and -2 DNA and the findings were compared with their HPV DNA status.

Results: Altogether, 2.2% of all 1873 collected epithelial brush samples were HSV-1 DNA positive, while none tested HSV-2 DNA positive. Of the 304 mothers, 11.8% were HSV-1 DNA positive at least once. Most of the women who tested HSV-1 DNA positive before delivery remained HSV-1 DNA positive also after pregnancy. HSV-1 positive women were almost invariably HPV-negative; only four (0.2%) samples were detected with HSV-HPV co-carriage.

Conclusions: This is the first prospective follow-up study on oral HSV shedding and its association with coexistent HPV, analyzed in the same oral mucosal scrapings. HSV and HPV co-carriage is rare in oral mucosa of healthy young mothers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcv.2015.07.006DOI Listing
September 2015

The combined effects of irradiation and herpes simplex virus type 1 infection on an immortal gingival cell line.

Virol J 2014 Jul 8;11:125. Epub 2014 Jul 8.

Institute of Dentistry, Department of Oral Pathology, University of Turku, Lemminkäisenkatu 2, 20520 Turku, Finland.

Background: Oral mucosa is frequently exposed to Herpes simplex virus type 1 (HSV-1) infection and irradiation due to dental radiography. During radiotherapy for oral cancer, the surrounding clinically normal tissues are also irradiated. This prompted us to study the effects of HSV-1 infection and irradiation on viability and apoptosis of oral epithelial cells.

Methods: Immortal gingival keratinocyte (HMK) cells were infected with HSV-1 at a low multiplicity of infection (MOI) and irradiated with 2 Gy 24 hours post infection. The cells were then harvested at 24, 72 and 144 hours post irradiation for viability assays and qRT-PCR analyses for the apoptosis-related genes caspases 3, 8, and 9, bcl-2, NFκB1, and viral gene VP16. Mann-Whitney U-test was used for statistical calculations.

Results: Irradiation improved the cell viability at 144 hours post irradiation (P = 0.05), which was further improved by HSV-1 infection at MOI of 0.00001 (P = 0.05). Simultaneously, the combined effects of infection at MOI of 0.0001 and irradiation resulted in upregulation in NFκB1 (P = 0.05). The combined effects of irradiation and HSV infection also significantly downregulated the expression of caspases 3, 8, and 9 at 144 hours (P = 0.05) whereas caspase 3 and 8 significantly upregulated in non-irradiated, HSV-infected cells as compared to uninfected controls (P = 0.05). Infection with 0.0001 MOI downregulated bcl-2 in non-irradiated cells but was upregulated by 27% after irradiation when compared to non-irradiated infected cells (P = 0.05). Irradiation had no effect on HSV-1 shedding or HSV gene expression at 144 hours.

Conclusions: HSV-1 infection may improve the viability of immortal cells after irradiation. The effect might be related to inhibition of apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1743-422X-11-125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105526PMC
July 2014

Innate responses to small interfering RNA pools inhibiting herpes simplex virus infection in astrocytoid and epithelial cells.

Innate Immun 2015 May 3;21(4):349-57. Epub 2014 Jul 3.

Department of Virology, University of Turku, Turku, Finland.

Herpes simplex virus (HSV) is a human pathogen that can cause severe diseases such as encephalitis, keratitis and neonatal herpes. Control of HSV infection may be achieved by using small interfering (si)RNAs. We have designed and enzymatically produced pools of siRNAs targeting HSV. In addition to the target-specific effects, such siRNAs may induce innate immunity responses that may contribute to antiviral effects. HSV has versatile ways of modulating innate immunity, and it remains unclear whether HSV-specific antiviral treatment would benefit from the potential immunostimulatory effects of siRNAs. To address this, cell lines derived from epithelium and nervous system were studied for innate immunity reactions to HSV infection, to siRNA treatment, and to a combination of treatment and infection. In addition, the outcome of HSV infection was quantitated. We show that innate immunity reactions vary drastically between the cell lines. Moreover, our findings indicate only a minimal relation between the antiviral effect and the treatment-induced innate immunity responses. Thus, the antiviral effect is mainly sequence specific and the inhibition of HSV infection is not ascribed to the slight innate immunity induction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1753425914537921DOI Listing
May 2015

Co-opting the Fanconi anemia genomic stability pathway enables herpesvirus DNA synthesis and productive growth.

Mol Cell 2014 Jul 19;55(1):111-22. Epub 2014 Jun 19.

Department of Microbiology, NYU Cancer Institute, NYU School of Medicine, New York, NY 10016, USA. Electronic address:

DNA damage associated with viral DNA synthesis can result in double-strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi anemia (FA) genomic stability pathway is exploited by herpes simplex virus 1 (HSV-1) to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV-1-infected cells resulted in monoubiquitination of FA effector proteins FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments, and FANCI-D2 interacted with a multisubunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, whereas HSV-1 productive growth was impaired in monoubiquitination-defective FA cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for nonhomologous end-joining (NHEJ). This identifies the FA-pathway as a cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral life cycle.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2014.05.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4376326PMC
July 2014

Herpes simplex virus type 1 genital herpes in young women: current trend in Northern Finland.

Sex Transm Infect 2014 Mar 15;90(2):160. Epub 2014 Jan 15.

Department of Microbiology, University Central Hospital of Oulu, , Oulu, Finland.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/sextrans-2013-051453DOI Listing
March 2014

A herpes simplex virus-derived replicative vector expressing LIF limits experimental demyelinating disease and modulates autoimmunity.

PLoS One 2013 20;8(5):e64200. Epub 2013 May 20.

Department of Virology, University of Turku, Turku, Finland.

Herpes simplex virus type 1 (HSV-1) has properties that can be exploited for the development of gene therapy vectors. The neurotropism of HSV enables delivery of therapeutic genes to the nervous system. Using a bacterial artificial chromosome (BAC), we constructed an HSV-1(17(+))-based replicative vector deleted of the neurovirulence gene γ134.5, and expressing leukemia inhibitory factor (LIF) as a transgene for treatment of experimental autoimmune encephalomyelitis (EAE). EAE is an inducible T-cell mediated autoimmune disease of the central nervous system (CNS) and is used as an animal model for multiple sclerosis. Demyelination and inflammation are hallmarks of both diseases. LIF is a cytokine that has the potential to limit demyelination and oligodendrocyte loss in CNS autoimmune diseases and to affect the T-cell mediated autoimmune response. In this study SJL/J mice, induced for EAE, were treated with a HSV-LIF vector intracranially and the subsequent changes in disease parameters and immune responses during the acute disease were investigated. Replicating HSV-LIF and its DNA were detected in the CNS during the acute infection, and the vector spread to the spinal cord but was non-virulent. The HSV-LIF significantly ameliorated the EAE and contributed to a higher number of oligodendrocytes in the brains when compared to untreated mice. The HSV-LIF therapy also induced favorable changes in the expression of immunoregulatory cytokines and T-cell population markers in the CNS during the acute disease. These data suggest that BAC-derived HSV vectors are suitable for gene therapy of CNS disease and can be used to test the therapeutic potential of immunomodulatory factors for treatment of EAE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064200PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659099PMC
December 2013

[Herpes simplex].

Duodecim 2013 ;129(1):31-40

Helsingin yliopisto, virologian osasto ja HUS, suu- ja leukasairauksien klinikka ja HUSLAB.

Current picture and treatment of herpes simplex virus infections The contraction of primary herpes simplex infection in early childhood is becoming increasingly less frequent. A growing number of new cases of genital herpes are caused by HSV-1. Excretion of virus by carriers of genital herpes often takes place also during the asymptomatic stage and symptomless recurrences of genital herpes may be very brief in duration. Primary infections and reactivations can be treated with anti-herpes medication. Progress in drug treatment has taken place in the treatment of serious infections of the newborn and in the preventive treatment of herpes reactivations, in which fairly large single doses have proven effective.
View Article and Find Full Text PDF

Download full-text PDF

Source
April 2013

Enzymatically produced pools of canonical and Dicer-substrate siRNA molecules display comparable gene silencing and antiviral activities against herpes simplex virus.

PLoS One 2012 30;7(11):e51019. Epub 2012 Nov 30.

Department of Biosciences, University of Helsinki, Helsinki, Finland.

RNA interference (RNAi)-based sequence-specific gene silencing is applied to identify gene function and also possesses great potential for inhibiting virus replication both in animals and plants. Small interfering RNA (siRNA) molecules are the inducers of gene silencing in the RNAi pathway but may also display immunostimulatory activities and promote apoptosis. Canonical siRNAs are 21 nucleotides (nt) in length and are loaded to the RNA Induced Silencing Complex when introduced into the cells, while longer siRNA molecules are first processed by endogenous Dicer and thus termed Dicer-substrate siRNA (DsiRNA). We have applied RNA polymerases from bacteriophages T7 and phi6 to make high-quality double-stranded RNA molecules that are specific for the UL29 gene of herpes simplex virus (HSV). The 653 nt long double-stranded RNA molecules were converted to siRNA and DsiRNA pools using Dicer enzymes originating from human or Giardia intestinalis, producing siRNAs of approximately 21 and 27 nt in length, respectively. Chemically synthesised 21 and 27 nt single-site siRNA targeting the UL29 were used as references. The impact of these siRNAs on cell viability, inflammatory responses, gene silencing, and anti-HSV activity were assayed in cells derived from human nervous system and skin. Both pools and the canonical single-site siRNAs displayed substantial antiviral activity resulting in four orders of magnitude reduction in virus titer. Notably, the pool of DsiRNAs caused lower immunostimulation than the pool of canonical siRNAs, whereas the immunostimulation effect was in relation to the length with the single-site siRNAs. Our results also propose differences in the processivity of the two Dicers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0051019PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511422PMC
May 2013

Obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection.

J Biol Chem 2012 Oct 21;287(42):35324-35332. Epub 2012 Aug 21.

Institute for Molecular Medicine Finland, FIMM, Helsinki FI-00290, Finland. Electronic address:

Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M112.392142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471742PMC
October 2012

Cathepsins are involved in virus-induced cell death in ICP4 and Us3 deletion mutant herpes simplex virus type 1-infected monocytic cells.

J Gen Virol 2011 Jan 29;92(Pt 1):173-80. Epub 2010 Sep 29.

Department of Virology, University of Turku, Kiinamyllynkatu 13, FI-20520 Turku, Finland.

We have studied cell death and its mechanisms in herpes simplex virus type 1 (HSV-1)-infected monocytic cells. The HSV-1 ICP4 and Us3 deletion mutant, d120 caused both apoptosis and necroptosis in d120-infected monocytic cells. At a late time point of infection the number of apoptotic cells was increased significantly in d120-infected cells when compared with uninfected or parental HSV-1 (KOS)-infected cells. Necroptosis inhibitor treatment increased the number of viable cells among the d120-infected cells, indicating that cell death in d120-infected cells was, in part, because of necroptosis. Moreover, lysosomal membrane permeabilization and cathepsin B and H activities were increased significantly in d120-infected cells. Inhibition of cathepsin B and S activities with specific cathepsin inhibitors led to increased cell viability, and inhibition of cathepsin L activity resulted in a decreased number of apoptotic cells. This indicates that cathepsins B, L and S may act as cell-death mediators in d120-infected monocytic cells. In addition, caspase 3 activity was increased significantly in d120-infected cells. However, the caspase 3 inhibitor treatment did not decrease the number of apoptotic cells. In contrast, inhibition of cathepsin L activity by cathepsin L-specific inhibitor clearly decreased caspase 3 activity and the number of apoptotic cells in d120-infected cells. This might suggest that, in d120-infected monocytic cells, cathepsin L activates caspase 3 and thus mediates d120-induced apoptosis. Taken together, these findings suggest that d120-induced cell death is both apoptotic and necroptotic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/vir.0.025080-0DOI Listing
January 2011