Construction of an autofluorescence interference-free phosphorescence biosensor for the specific detection of TK1 mRNA.

The anti-interference ability of biosensors is critical for detection in biological samples. Fluorescence-based sensors are subject to interference from self-luminescent substances in biological matrices. Therefore, phosphorescent sensors stand out among biosensors due to their lack of self-luminescence background. In this study, a phosphorescent sensor was constructed, which can accurately detect thymidine kinase 1 (TK1) mRNA in biological samples and avoid autofluorescence interference. When there is no target, polydopamine (PDA) is used as the phosphorescence resonance energy transfer (PRET) acceptor to quench the phosphorescence of the persistently luminescent (PL) nanomaterial. When there is a target, the DNA modified by the PL nanomaterial is replaced by the hairpin H and removed away from the PDA, resulting in a rebound in phosphorescence. The phosphorescent sensor exhibits a good linear relationship in the TK1 mRNA concentration range of 0-200 nM, and the detection limit was 1.74 nM. The sensor fabricated in this study can effectively avoid interference from spontaneous fluorescence in complex biological samples, and sensitively and precisely detect TK1 mRNA in serum samples, providing a powerful tool to more accurately detect biomarkers in biological samples.

Therapeutic targeting at genome mutations of liver cancer by the insertion of HSV1 thymidine kinase through Cas9-mediated editing.

BACKGROUND: Liver cancer is one of the most lethal malignancies for humans. The treatment options for advanced-stage liver cancer remain limited. A new treatment is urgently needed to reduce the mortality of the disease. METHODS: In this report, we developed a technology for mutation site insertion of a suicide gene (herpes simplex virus type 1- thymidine kinase) based on type II CRISPR RNA-guided endonuclease Cas9-mediated genome editing to treat liver cancers. RESULTS: We applied the strategy to 3 different mutations: S45P mutation of catenin beta 1, chromosome breakpoint of solute carrier family 45 member 2-alpha-methylacyl-CoA racemase gene fusion, and V235G mutation of SAFB-like transcription modulator. The results showed that the herpes simplex virus type 1-thymidine kinase insertion rate at the S45P mutation site of catenin beta 1 reached 77.8%, while the insertion rates at the breakpoint of solute carrier family 45 member 2 - alpha-methylacyl-CoA racemase gene fusion were 95.1%-98.7%, and the insertion at V235G of SAFB-like transcription modulator was 51.4%. When these targeting reagents were applied to treat mouse spontaneous liver cancer induced by catenin beta 1S45P or solute carrier family 45 member 2-alpha-methylacyl-CoA racemase, the mice experienced reduced tumor burden and increased survival rate. Similar results were also obtained for the xenografted liver cancer model: Significant reduction of tumor volume, reduction of metastasis rate, and improved survival were found in mice treated with the targeting reagent, in comparison with the control-treated groups. CONCLUSIONS: Our studies suggested that mutation targeting may hold promise as a versatile and effective approach to treating liver cancers.

A comprehensive analysis and experimental validation of TK1 in uterine corpus endometrial carcinoma.

Uterine corpus endometrial carcinoma (UCEC) is becoming a main malignant cancer that threaten to women's health. Thymidine kinase 1 (TK1) is considering to be associated with tumorigenesis and development. Nevertheless, the function of TK1 in UCEC is still unclear. Herein, we analyzed the TK1 expression level in pan-cancer and found that TK1 was upregulated in a variety of cancers including UCEC. Patients of UCEC with high expression of TK1 were related to poor outcome. TK1 was also related to clinical stage, histologic grade and lymph node metastasis. Abnormal expression of TK1 in UCEC was related to promoter methylation while gene mutation was not frequent. TK1 and its associated genes appeared to be prominent in cell cycle and DNA replication, according to GO and KEGG analysis. Analysis of immune infiltration revealed a negative correlation between TK1 and CD8 + T cells, macrophages, and dendritic cells. In vitro experiments, TK1 knockdown resulted in the inhibition of proliferation, migration, invasion and EMT in UCEC cell lines.

The role of serum thymidine kinase 1 activity in neoadjuvant-treated HER2-positive breast cancer: biomarker analysis from the Swedish phase II randomized PREDIX HER2 trial.

BACKGROUND: Thymidine kinase 1 (TK1) plays a pivotal role in DNA synthesis and cellular proliferation. TK1 has been studied as a prognostic marker and as an early indicator of treatment response in human epidermal growth factor 2 (HER2)-negative early and metastatic breast cancer (BC). However, the prognostic and predictive value of serial TK1 activity in HER2-positive BC remains unknown. METHODS: In the PREDIX HER2 trial, 197 HER2-positive BC patients were randomized to neoadjuvant trastuzumab, pertuzumab, and docetaxel (DPH) or trastuzumab emtansine (T-DM1), followed by surgery and adjuvant epirubicin and cyclophosphamide. Serum samples were prospectively collected from all participants at multiple timepoints: at baseline, after cycle 1, 2, 4, and 6, at end of adjuvant therapy, annually for a total period of 5 years and/or at the time of recurrence. The associations of sTK1 activity with baseline characteristics, pathologic complete response (pCR), event-free survival (EFS), and disease-free survival (DFS) were evaluated. RESULTS: No association was detected between baseline sTK1 levels and all the baseline clinicopathologic characteristics. An increase of TK1 activity from baseline to cycle 2 was seen in all cases. sTK1 level at baseline, after 2 and 4 cycles was not associated with pCR status. After a median follow-up of 58 months, 23 patients had EFS events. There was no significant effect between baseline or cycle 2 sTK1 activity and time to event. A non-significant trend was noted among patents with residual disease (non-pCR) and high sTK1 activity at the end of treatment visit, indicating a potentially worse long-term prognosis. CONCLUSION: sTK1 activity increased following neoadjuvant therapy for HER2-positive BC but was not associated with patient outcomes or treatment benefit. However, the post-surgery prognostic value in patients that have not attained pCR warrants further investigation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02568839. Registered on 6 October 2015.

In-vitro and in-silico anti-HSV-1 activity of a marine steroid from the jellyfish Cassiopea andromeda venom.

In this study, we investigated the potential in vitro anti-HSV-1 activities of the Cassiopea andromeda jellyfish tentacle extract (TE) and its fractions, as well as computational work on the thymidine kinase (TK) inhibitory activity of the identified secondary metabolites. The LD50, secondary metabolite identification, preparative and analytical chromatography, and in silico TK assessment were performed using the Spearman-Karber, GC-MS, silica gel column chromatography, RP-HPLC, LC-MS, and docking methods, respectively. The antiviral activity of TE and the two purified compounds Ca2 and Ca7 against HSV-1 in Vero cells was evaluated by MTT and RT-PCR assays. The LD50 (IV, mouse) values of TE, Ca2, and Ca7 were 104.0 ± 4, 5120 ± 14, and 197.0 ± 7 (µg/kg), respectively. They exhibited extremely effective antiviral activity against HSV-1. The CC50 and MNTD of TE, Ca2, and Ca7 were (125, 62.5), (25, 12.5), and (50, 3.125) µg/ml, respectively. GC-MS analysis of the tentacle extract revealed seven structurally distinct chemical compositions. Four of the seven compounds had a steroid structure. According to the docking results, all compounds showed binding affinity to the active sites of both thymidine kinase chains. Among them, the steroid compound Pregn-5-ene-3,11-dione, 17,20:20,21 bis [methylenebis(oxy)]-, cyclic 3-(1,2-ethane diyl acetal) (Ca2) exhibited the highest affinity for both enzyme chains, surpassing that of standard acyclovir. In silico data confirmed the experimental results. We conclude that the oxosteroid Ca2 may act as a potent agent against HSV-1.

Analysis of antiviral drug properties of thymidine kinase of herpes B virus using recombinant herpes simplex virus 1.

IMPORTANCE: Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.

Serum thymidine kinase 1 protein concentrations and presence of its autoantibody as biomarkers for screening dogs with malignant tumors.

BACKGROUND: An accurate and easily accessible method for diagnosing malignancies in local veterinary clinics has not yet been established. OBJECTIVES: To investigate the usefulness of serum thymidine kinase 1 (TK1) protein and its autoantibody as tumor biomarkers in dogs. ANIMALS: Serum samples from 1702 dogs were collected from local animal hospitals and referral animal medical centers in South Korea. METHODS: TK1 protein OD value and TK1 autoantibody ratio (TK1 autoantibody OD/total IgG OD) in serum samples of dogs classified into healthy controls, group with nontumor disease, group with benign and group with malignant tumors were measured using lateral flow immunochromatographic assay methods. RESULTS: TK1 autoantibody levels were significantly higher in malignant tumor group (median 0.71) than in healthy controls (median 0.34), group with nontumor disease (median 0.34), and group with benign tumor (median 0.32, Welch t test, P < .0001). They were also significantly different among dogs with carcinomas (median 0.77), hematopoietic tumors (median 0.71), and sarcomas (median 0.56) than in healthy controls (median 0.34, post hoc Games-Howell test, P < .0001). In the receiver operating characteristic curve of TK1 protein, AUC was 0.633 (95% CI: 0.592-0.675, P < .0001). The AUC of TK1 autoantibody ratio was 0.758 (95% CI: 0.723-0.793, P < .0001). CONCLUSIONS AND CLINICAL IMPORTANCE: TK1 autoantibody is a potentially useful biomarker for differentiating between healthy and tumor-bearing dogs, better than TK1 protein measurement. However, both were inadequate when used as single biomarkers for screening dogs to discover occult malignant tumors.

Development of an optimized, non-stem cell line for intranasal delivery of therapeutic cargo to the central nervous system.

Neural stem cells (NSCs) are considered to be valuable candidates for delivering a variety of anti-cancer agents, including oncolytic viruses, to brain tumors. However, owing to the previously reported tumorigenic potential of NSC cell lines after intranasal administration (INA), here we identified the human hepatic stellate cell line LX-2 as a cell type capable of longer resistance to replication of oncolytic adenoviruses (OAVs) as a therapeutic cargo, and that is non-tumorigenic after INA. Our data show that LX-2 cells can longer withstand the OAV XVir-N-31 replication and oncolysis than NSCs. By selecting the highly migratory cell population out of LX-2, an offspring cell line with a higher and more stable capability to migrate was generated. Additionally, as a safety backup, we applied genomic herpes simplex virus thymidine kinase (HSV-TK) integration into LX-2, leading to high vulnerability to ganciclovir (GCV). Histopathological analyses confirmed the absence of neoplasia in the respiratory tracts and brains of immuno-compromised mice 3 months after INA of LX-2 cells. Our data suggest that LX-2 is a novel, robust, and safe cell line for delivering anti-cancer and other therapeutic agents to the brain.

Thymidine Kinase-Independent Click Chemistry DNADetect Probes for DNA Proliferation Assessment in Malaria Parasites.

Metabolic chemical probes are small-molecule reagents that utilize naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation, and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2'-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells; however, it is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein, we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium. Two of these DNADetect probes exhibit robust labeling of replicating asexual intraerythrocytic Plasmodium falciparum parasites, as determined by flow cytometry and fluorescence microscopy using copper-catalyzed azide-alkyne cycloaddition to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.

Quantitative Structure-Activity Relationship in the Series of 5-Ethyluridine, N2-Guanine, and 6-Oxopurine Derivatives with Pronounced Anti-Herpetic Activity.

A quantitative analysis of the relationship between the structure and inhibitory activity against the herpes simplex virus thymidine kinase (HSV-TK) was performed for the series of 5-ethyluridine, N2-guanine, and 6-oxopurines derivatives with pronounced anti-herpetic activity (IC50 = 0.09 ÷ 160,000 µmol/L) using the GUSAR 2019 software. On the basis of the MNA and QNA descriptors and whole-molecule descriptors using the self-consistent regression, 12 statistically significant consensus models for predicting numerical pIC50 values were constructed. These models demonstrated high predictive accuracy for the training and test sets. Molecular fragments of HSV-1 and HSV-2 TK inhibitors that enhance or diminish the anti-herpetic activity are considered. Virtual screening of the ChEMBL database using the developed QSAR models revealed 42 new effective HSV-1 and HSV-2 TK inhibitors. These compounds are promising for further research. The obtained data open up new opportunities for developing novel effective inhibitors of TK.

Transcriptomic analysis reveals impact of gE/gI/TK deletions on host response to PRV infection.

BACKGROUND: Pseudorabies virus (PRV) causes substantial losses in the swine industry worldwide. Attenuated PRV strains with deletions of immunomodulatory genes glycoprotein E (gE), glycoprotein I (gI) and thymidine kinase (TK) are candidate vaccines. However, the effects of gE/gI/TK deletions on PRV-host interactions are not well understood. METHODS: To characterize the impact of gE/gI/TK deletions on host cells, we analyzed and compared the transcriptomes of PK15 cells infected with wild-type PRV (SD2017), PRV with gE/gI/TK deletions (SD2017gE/gI/TK) using RNA-sequencing. RESULTS: The attenuated SD2017gE/gI/TK strain showed increased expression of inflammatory cytokines and pathways related to immunity compared to wild-type PRV. Cell cycle regulation and metabolic pathways were also perturbed. CONCLUSIONS: Deletion of immunomodulatory genes altered PRV interactions with host cells and immune responses. This study provides insights into PRV vaccine design.

TK1 expression influences pathogenicity by cell cycle progression, cellular migration, and cellular survival in HCC 1806 breast cancer cells.

Breast cancer is the most common cancer diagnosis worldwide accounting for 1 out of every 8 cancer diagnoses. The elevated expression of Thymidine Kinase 1 (TK1) is associated with more aggressive tumor grades, including breast cancer. Recent studies indicate that TK1 may be involved in cancer pathogenesis; however, its direct involvement in breast cancer has not been identified. Here, we evaluate potential pathogenic effects of elevated TK1 expression by comparing HCC 1806 to HCC 1806 TK1-knockdown cancer cells (L133). Transcriptomic profiles of HCC 1806 and L133 cells showed cell cycle progression, apoptosis, and invasion as potential pathogenic pathways affected by TK1 expression. Subsequent in-vitro studies confirmed differences between HCC 1806 and L133 cells in cell cycle phase progression, cell survival, and cell migration. Expression comparison of several factors involved in these pathogenic pathways between HCC 1806 and L133 cells identified p21 and AKT3 transcripts were significantly affected by TK1 expression. Creation of a protein-protein interaction map of TK1 and the pathogenic factors we evaluated predict that the majority of factors evaluated either directly or indirectly interact with TK1. Our findings argue that TK1 elevation directly increases HCC 1806 cell pathogenicity and is likely occurring by p21- and AKT3-mediated mechanisms to promote cell cycle arrest, cellular migration, and cellular survival.

Long term survival and abnormal liver fat accumulation in mice with specific thymidine kinase 2 deficiency in liver tissue.

Deficiency in thymidine kinase 2 (TK2) causes mitochondrial DNA depletion. Liver mitochondria are severely affected in Tk2 complete knockout models and have been suggested to play a role in the pathogenesis of the Tk2 knockout phenotype, characterized by loss of hypodermal fat tissue, growth retardation and reduced life span. Here we report a liver specific Tk2 knockout (KO) model to further study mechanisms contributing to the phenotypic changes associated with Tk2 deficiency. Interestingly, the liver specific Tk2 KO mice had a normal life span despite a much lower mtDNA level in liver tissue. Mitochondrial DNA encoded peptide COXI did not differ between the Tk2 KO and control mice. However, the relative liver weight was significantly increased in the male Tk2 KO mouse model. Histology analysis indicated an increased lipid accumulation. We conclude that other enzyme activities can partly compensate Tk2 deficiency to maintain mtDNA at a low but stable level throughout the life span of the liver specific Tk2 KO mice. The lower level of mtDNA was sufficient for survival but led to an abnormal lipid accumulation in liver tissue.

Uso de la determinación transcutánea de co2 en pacientes con déficit de timidina quinasa 2 y costes de su uso en una consulta de ventilación mecánica no invasiva / Use of transcutaneous co2 determination in patients with thymidine kinase 2 deficit and costs of its use in a noninvasive mechanical ventilation consultation

Objetivo: Describir el uso de la capnografía transcutánea en una población adulta y pediátrica de pacientes con déficit de timidina quinasa 2 y hacer un estudio comparativo de costes de una determinación de gasometría arterial y capnografía en la población de nuestra consulta de VMNI. Metodología: Se realizó una anamnesis y unas pruebas funcionales respiratorias para valorar afectación de la musculatura respiratoria y calidad del sueño. Para determinar la hipoventilación, se midió la pCO2 transcutánea en vigilia y/o durante el sueño. Se realizó un estudio económico para comparar el coste de una determinación de ptcCO2 frente a la determinación mediante GSA. El estudio económico se realizó estimando la población total de pacientes que se valoraba en la consulta de VMNI de manera anual. Resultados: 9 pacientes con déficit de TK2 (4 adultos y 5 niños). A 4 pacientes se les realizó una poligrafía respiratoria basal. A la población pediátrica se les realizó un registro continuo de ptcCO2 con pulsioximetria anual. Se realizaron 4 registros con ptcCO2 y VMNI. Elcoste de la determinación de ptCO2 en comparación con la GSA fue de 6,29 euros frente a 5,37 euros. Conclusiones: La medición de la ptcCO2 es útil en la consulta de VMNI para la realización de medidas puntuales en la consulta como para monitorización continua durante el sueño. Con el uso que realizamos en nuestra consulta de la capnografía transcutánea, la determinación puntual de la pCO2 transcutánea es más económica que la realización de la GSA. (AU)

Objective: to describe the use of transcutaneous capnography in an adult and pediatric population of patients with Thymidine inase 2 deficiency and to compare the costs between blood gases by arterial gasometry (BGA) and capnography in our population. Material and methods: an anamnesis, and respiratory functional tests to assess respiratory muscle involvement, sleep quality were performed.To assess the presence of alveolar hypoventilation the determination of transcutaneous pCO2while awake and/or during sleepwas performed. An economic study has been done to compare the cost of a determination of ptcCO2 versus the determination by BGA. Results: 9 patients with TK2 deficiency (4 adults and 5 children). 4 patients underwent baseline respiratory polygraphy. The pediatric patients underwent at least one continuous recording of ptcCO2 with pulse oximetry each year.4 studies of ptcCO2 duringNIV were performed. The cost in the adult population of a punctual determination of pCO2 by BGA was 6,29 euros while for capnography was 5,37 euros. Conclusions: the measurement of ptcCO2 is useful in the consultation of NIV for the realization of specific measurements in the consultation as for continuous monitoring of this parameter. In our practice of transcutaneous capnography, the punctual determination of transcutaneous pCO2 is cheaper than the BGA. (AU)

Fluoxetine enhances the antitumor effect of olfactory ensheathing cell-thymidine kinase/ganciclovir gene therapy in human glioblastoma multiforme cells through upregulation of Connexin43 levels.

Glioblastoma multiforme (GBM) is the most invasive form of primary brain astrocytoma, resulting in poor clinical outcomes. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy is considered a promising strategy for GBM treatment. Since Connexin43 (Cx43) expression is reduced in GBM cells, increasing Cx43 levels could enhance the effectiveness of gene therapy. The present study aims to examine the impact of fluoxetine on HSV-TK/GCV gene therapy in human GBM cells using human olfactory ensheathing cells (OECs) as vectors. The effect of fluoxetine on Cx43 levels was assessed using the western blot technique. GBM-derived astrocytes and OECs-TK were Cocultured, and the effect of fluoxetine on the Antitumor effect of OEC-TK/GCV gene therapy was evaluated using MTT assay and flow cytometry. Our results showed that fluoxetine increased Cx43 levels in OECs and GBM cells and augmented the killing effect of OECs-TK on GBM cells. Western blot data revealed that fluoxetine enhanced the Bax/Bcl2 ratio and the levels of cleaved caspase-3 in the coculture of OECs-TK and GBM cells. Moreover, flow cytometry data indicated that fluoxetine increased the percentage of apoptotic cells in the coculture system. This study suggests that fluoxetine, by upregulating Cx43 levels, could strengthen the Antitumor effect of OEC-TK/GCV gene therapy on GBM cells.

Combined cytotoxic and immune-stimulatory gene therapy using Ad-TK and Ad-Flt3L: Translational developments from rodents to glioma patients.

Gliomas are the most prevalent and devastating primary malignant brain tumors in adults. Despite substantial advances in understanding glioma biology, there have been no regulatory drug approvals in the US since bevacizumab in 2009 and tumor treating fields in 2011. Recent phase III clinical trials have failed to meet their prespecified therapeutic primary endpoints, highlighting the need for novel therapies. The poor prognosis of glioma patients, resistance to chemo-radiotherapy, and the immunosuppressive tumor microenvironment underscore the need for the development of novel therapies. Gene therapy-based immunotherapeutic strategies that couple the ability of the host immune system to specifically kill glioma cells and develop immunological memory have shown remarkable progress. Two adenoviral vectors expressing Ad-HSV1-TK/GCV and Ad-Flt3L have shown promising preclinical data, leading to FDA approval of a non-randomized, phase I open-label, first in human trial to test safety, cytotoxicity, and immune-stimulatory efficiency in high-grade glioma patients (NCT01811992). This review provides a thorough overview of immune-stimulatory gene therapy highlighting recent advancements, potential drawbacks, future directions, and recommendations for future implementation of clinical trials.

Variant Analysis of the Thymidine Kinase and DNA Polymerase Genes of Herpes Simplex Virus in Korea: Frequency of Acyclovir Resistance Mutations.

The thymidine kinase (TK) and DNA polymerase (pol) genes of the herpes simplex viruses type 1 (HSV-1) and type 2 (HSV-2) are two important genes involved in antiviral resistance. We investigated the genetic polymorphisms of the HSV-TK and pol genes in clinical isolates from Korean HSV-infected patients using next-generation sequencing (NGS) for the first time in Korea. A total of 81 HSV-1 and 47 HSV-2 isolates were examined. NGS was used to amplify and sequence the TK and pol genes. Among the 81 HSV-1 isolates, 12 and 17 natural polymorphisms and 9 and 23 polymorphisms of unknown significance in TK and pol were found, respectively. Two HSV-1 isolates (2.5%) exhibited the E257K amino acid substitution in TK, associated with antiviral resistance. Out of 47 HSV-2 isolates, 8 natural polymorphisms were identified in TK, and 9 in pol, with 13 polymorphisms of unknown significance in TK and 10 in pol. No known resistance-related mutations were observed in HSV-2. These findings contribute to our understanding of the genetic variants associated with antiviral resistance in HSV-1 and HSV-2 in Korea, with frequencies of known antiviral resistance-related mutations of 2.5% and 0% in HSV-1 and HSV-2, respectively.

Phylogenetic and Genomic Characterization of Whole Genome Sequences of Ocular Herpes Simplex Virus Type 1 Isolates Identifies Possible Virulence Determinants in Humans.

Purpose: There are limited data on the prevalence and genetic diversity of herpes simplex virus type 1 (HSV-1) virulence genes in ocular isolates. Here, we sequenced 36 HSV-1 ocular isolates, collected by the Bascom Palmer Eye Institute, a university-based eye hospital, from three different ocular anatomical sites (conjunctiva, cornea, and eyelid) and carried out a genomic and phylogenetic analyses. Methods: The PacBio Sequel II long read platform was used for genome sequencing. Phylogenetic analysis and genomic analysis were performed to help better understand genetic variability among common virulence genes in ocular herpetic disease. Results: A phylogenetic network generated using the genome sequences of the 36 Bascom Palmer ocular isolates, plus 174 additional strains showed that ocular isolates do not group together phylogenetically. Analysis of the thymidine kinase and DNA polymerase protein sequences from the Bascom Palmer isolates showed multiple novel single nucleotide polymorphisms, but only one, BP-K14 encoded a known thymidine kinase acyclovir resistance mutation. An analysis of the multiple sequence alignment comprising the 51 total ocular isolates versus 159 nonocular strains detected several possible single nucleotide polymorphisms in HSV-1 genes that were found significantly more often in the ocular isolates. These genes included UL6, gM, VP19c, VHS, gC, VP11/12, and gG. Conclusions: There does not seem to be a specific genetic feature of viruses causing ocular infection. The identification of novel and common recurrent polymorphisms may help to understand the drivers of herpetic pathogenicity and specific factors that may influence the virulence of ocular disease.

Acyclovir-resistant HSV-1 isolates among immunocompromised patients in southern Taiwan: Low prevalence and novel mutations.

Herpes simplex virus type 1 (HSV-1) can establish latency in humans and easily relapse in immunocompromised patients, with significant mortality. Treatment with acyclovir (ACV) can result in the emergence of HSV resistance. A total of 440 frozen HSV-1 isolates collected from 318 patients from January 2014 to July 2019 were obtained from National Cheng Kung University Hospital in southern Taiwan. These 440 isolates were subjected to phenotypic studies for ACV-resistance by initial screening with the plaque reduction assay (PRA) and further validation by the DNA reduction assay (DRA). The ACV-resistant strains were further investigated by Sanger sequencing for the full-length UL23 and UL30 genes, which encode thymidine kinase and DNA polymerase, respectively. Hematological malignancies or hematopoietic stem-cell transplantation patients accounted for 56.9% (124/218) among the immunocompromised patients (218/318) in this study. Repeated sampling for HSV testing was 50% (109/218) in immunocompromised patients. Only 1.38% (3/218) of immunocompromised patients and 0.9% (3/318) of all patients developed ACV-resistant HSV-1 as measured by phenotypic screening assays. It is noteworthy that a novel Y248D mutation in the UL23 gene from an immunocompromised patient was found by both PRA and DRA. In 3D protein predicting analysis, uncharged Y248 was located at an alpha-helix and substituted by negative-charged D248, which may alter the function of viral thymidine kinase. Besides, three unreported mutations related to natural polymorphism were found in virus isolates from two immunocompetent patients, including 683-688 deletion, R227H, and A351D in the UL30 gene. These data show that the prevalence of ACV-resistant HSV-1 among immunocompromised patients in southern Taiwan is low. These results will be helpful for the clinical management and treatment of HSV infections.