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

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.IMPORTANCE 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.

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

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.

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

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 UL29 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 (IC50) 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 (IC50 1.0 nM; Imax 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.

Ketopinic acid and diisoproylideneketogulonic acid as chiral ion-pair selectors in capillary electrophoresis. Enantiomeric impurity analysis of S-timolol and 1R,2S-ephedrine.

1S,4R-(+)-ketopinic acid [(+)-KPA] has been introduced as a chiral selector for the separation of pharmacologically active amines by non-aqueous capillary electrophoresis (NACE). (+)-KPA gave enantioresolution for most of the compounds previously separated by 2R,3S,4R,5S-(-)-2,3:4,6-di-O-isopropylidene-2-keto-L-gulonic acid [(-)-DIKGA], but with a reversed migration order. A complete enantioresolution (Rs=4.2) was obtained for timolol, a compound that could not be resolved using (-)-DIKGA as the selector. Thus, (+)-KPA was evaluated for the enantiomeric purity determination of S-timolol. A method based on pre-concentration by transient isotachophoresis (tITP) provided a limit of detection (LOD) of 0.2% R-timolol in S-timolol samples. Because of the lack of enantioresolution of ephedrine when (+)-KPA was used as the selector, a method with (-)-DIKGA has been developed and validated for determination of the enantiomeric purity of the 1R,2S enantiomer. The method gave good precision and accuracy with an LOD (S/N=3) of 0.033% for the enantiomeric impurity 1S,2R-ephedrine.

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

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.

Occupational hygiene in terms of volatile organic compounds (VOCs) and bioaerosols at two solid waste management plants in Finland.

Factors affecting occupational hygiene were measured at the solid waste transferring plant at Hyvinkää and at the optic separation plant in Hämeenlinna. Measurements consisted of volatile organic compounds (VOCs) and bioaerosols including microbes, dust and endotoxins. The most abundant compounds in both of the plants were aliphatic and aromatic hydrocarbons, esters of carboxylic acids, ketones and terpenes. In terms of odour generation, the most important emissions were acetic acid, 2,3-butanedione, ethyl acetate, alpha-pinene and limonene due to their low threshold odour concentrations. At the optic waste separation plant, limonene occurred at the highest concentration of all single compounds of identified VOCs. The concentration of any single volatile organic compound did not exceed the occupational exposure limit (OEL) concentration. However, 2,3-butanedione as a health risk compound is discussed based on recent scientific findings linking it to lung disease. Microbe and dust concentrations were low at the waste transferring plant. Only endotoxin concentrations may cause health problems; the average concentration inside the plant was 425 EU/m(3) which clearly exceeded the threshold value of 90 EU/m(3). In the wheel loader cabin the endotoxin concentrations were below 1 EU/m(3). High microbial and endotoxin concentrations were measured in the processing hall at the optic waste separation plant. The average concentration of endotoxins was found to be 10,980 EU/m(3), a concentration which may cause health risks. Concentrations of viable fungi were quite high in few measurements in the control room. The most problematic factor was endotoxins whose average measured concentrations was 4853 EU/m(3).