Novel immunomodulatory properties of adenosine analogs promote their antiviral activity against SARS-CoV-2.

The COVID-19 pandemic reminded us of the urgent need for new antivirals to control emerging infectious diseases and potential future pandemics. Immunotherapy has revolutionized oncology and could complement the use of antivirals, but its application to infectious diseases remains largely unexplored. Nucleoside analogs are a class of agents widely used as antiviral and anti-neoplastic drugs. Their antiviral activity is generally based on interference with viral nucleic acid replication or transcription. Based on our previous work and computer modeling, we hypothesize that antiviral adenosine analogs, like remdesivir, have previously unrecognized immunomodulatory properties which contribute to their therapeutic activity. In the case of remdesivir, we here show that these properties are due to its metabolite, GS-441524, acting as an Adenosine A2A Receptor antagonist. Our findings support a new rationale for the design of next-generation antiviral agents with dual - immunomodulatory and intrinsic - antiviral properties. These compounds could represent game-changing therapies to control emerging viral diseases and future pandemics.

RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

Serotonin-2 Receptor Agonists Produce Anti-inflammatory Effects through Functionally Selective Mechanisms That Involve the Suppression of Disease-Induced Arginase 1 Expression.

Functional selectivity in the context of serotonin 2A (5-HT2A) receptor agonists is often described as differences psychedelic compounds have in the activation of Gq vs ß-arrestin signaling in the brain and how that may relate to inducing psychoactive and hallucinatory properties with respect to each other. However, the presence of 5-HT2A receptors throughout the body in several cell types, including endothelial, endocrine, and immune-related tissues, suggests that functional selectivity may exist in the periphery as well. Here, we examine functional selectivity between two 5-HT2A receptor agonists of the phenylalkylamine class: (R)-2,5-dimethoxy-4-iodoamphetamine [(R)-DOI] and (R)-2,5-dimethoxy-4-trifluoromethylamphetamine [(R)-DOTFM]. Despite comparable in vitro activity at the 5-HT2A receptor as well as similar behavioral potency, (R)-DOTFM does not exhibit an ability to prevent inflammation or elevated airway hyperresponsiveness (AHR) in an acute murine ovalbumin-induced asthma model as does (R)-DOI. Furthermore, there are distinct differences between protein expression and inflammatory-related gene expression in pulmonary tissues between the two compounds. Using (R)-DOI and (R)-DOTFM as tools, we further elucidated the anti-inflammatory mechanisms underlying the powerful anti-inflammatory effects of certain psychedelics and identified key mechanistic components of the anti-inflammatory effects of psychedelics, including suppression of arginase 1 expression.

Low-Dose Antithymocyte Globulin: A Pragmatic Approach to Treating Stage 2 Type 1 Diabetes.

OBJECTIVE: Low-dose antithymocyte globulin (ATG) (2.5 mg/kg) preserves C-peptide and reduces HbA1c in new-onset stage 3 type 1 diabetes, yet efficacy in delaying progression from stage 2 to stage 3 has not been evaluated. RESEARCH DESIGN AND METHODS: Children (n = 6) aged 5-14 years with stage 2 type 1 diabetes received off-label, low-dose ATG. HbA1c, C-peptide, continuous glucose monitoring, insulin requirements, and side effects were followed for 18-48 months. RESULTS: Three subjects (50%) remained diabetes free after 1.5, 3, and 4 years of follow-up, while three developed stage 3 within 1-2 months after therapy. Eighteen months posttreatment, even disease progressors demonstrated near-normal HbA1c (5.1% [32 mmol/mol], 5.6% [38 mmol/mol], and 5.3% [34 mmol/mol]), time in range (93%, 88%, and 98%), low insulin requirements (0.17, 0.18, and 0.34 units/kg/day), and robust C-peptide 90 min after mixed meal (1.3 ng/dL, 2.3 ng/dL, and 1.4 ng/dL). CONCLUSIONS: These observations support additional prospective studies evaluating ATG in stage 2 type 1 diabetes.

Herpes Simplex Virus-1 Induced Serotonin-Associated Metabolic Pathways Correlate With Severity of Virus- and Inflammation-Associated Ocular Disease.

Herpes simplex virus-associated diseases are a complex interaction between cytolytic viral replication and inflammation. Within the normally avascular and immunoprivileged cornea, HSV ocular infection can result in vision-threatening immune-mediated herpetic keratitis, the leading infectious cause of corneal blindness in the industrialized world. Viral replicative processes are entirely dependent upon numerous cellular biosynthetic and metabolic pathways. Consistent with this premise, HSV infection was shown to profoundly alter gene expression associated with cellular amino acid biosynthetic pathways, including key tryptophan metabolism genes. The essential amino acid tryptophan is crucial for pathogen replication, the generation of host immune responses, and the synthesis of neurotransmitters, such as serotonin. Intriguingly, Tryptophan hydroxylase 2 (TPH2), the neuronal specific rate-limiting enzyme for serotonin synthesis, was the most significantly upregulated gene by HSV in an amino acid metabolism PCR array. Despite the well-defined effects of serotonin in the nervous system, the association of peripheral serotonin in disease-promoting inflammation has only recently begun to be elucidated. Likewise, the impact of serotonin on viral replication and ocular disease is also largely unknown. We therefore examined the effect of HSV-induced serotonin-associated synthesis and transport pathways on HSV-1 replication, as well as the correlation between HSV-induced ocular serotonin levels and disease severity. HSV infection induced expression of the critical serotonin synthesis enzymes TPH-1, TPH-2, and DOPA decarboxylase (DDC), as well as the serotonin transporter, SERT. Concordantly, HSV-infected cells upregulated serotonin synthesis and its intracellular uptake. Increased serotonin synthesis and uptake was shown to influence HSV replication. Exogenous addition of serotonin increased HSV-1 yield, while both TPH-1/2 and SERT pharmacological inhibition reduced viral yield. Congruent with these in vitro findings, rabbits intraocularly infected with HSV-1 exhibited significantly higher aqueous humor serotonin concentrations that positively and strongly correlated with viral load and ocular disease severity. Collectively, our findings indicate that HSV-1 promotes serotonin synthesis and cellular uptake to facilitate viral replication and consequently, serotonin's proinflammatory effects may enhance the development of ocular disease.

Serotonin 5-HT2A receptor activity mediates adipocyte differentiation through control of adipogenic gene expression.

Serotonin 5-HT2 receptors are expressed in many tissues and play important roles in biological processes. Although the 5-HT2A receptor is primarily known for its role in central nervous system, it is also expressed in peripheral tissues. We have found that 5-HT2A receptor antagonists inhibit human subcutaneous primary adipocyte differentiation. We also show that siRNA knockdown of the 5-HT2A receptor blocks differentiation. Using gene expression analysis in combination with receptor antagonists we found that activity of 5-HT2A receptors is necessary very early in the differentiation process to mediate expression of adipogenic genes, including peroxisome proliferator-activated receptor gamma (ppar-γ), adipocyte protein 2 (aP2), adiponectin, and serine/threonine-protein kinase 1 (sgk1). We show here for the first time that 5-HT2A receptor activity is necessary for differentiation of human primary subcutaneous preadipocytes to adipocytes, and that 5-HT2A receptor activity mediates key genes related to adipogenesis during this process. Importantly, this work contributes to a greater understanding of the adipocyte differentiation process, as well as to the role of 5-HT2A receptors in peripheral tissues, and may be relevant to the development of novel therapeutic strategies targeting this receptor for the treatment of obesity related diseases.

A Novel Mutation in Insulin-Like Growth Factor 1 Receptor (c.641-2A>G) Is Associated with Impaired Growth, Hypoglycemia, and Modified Immune Phenotypes.

INTRODUCTION: Insulin-like growth factor 1 receptor (IGF1R) mutations lead to systemic disturbances in growth and glucose homeostasis due to widespread IGF1R expression throughout the body. IGF1R is expressed by innate and adaptive immune cells, facilitating their development and exerting immunomodulatory roles in the periphery. CASE PRESENTATION: We report on a family presenting with a novel heterozygous IGF1R mutation with characterization of the mutation, IGF1R expression, and immune phenotyping. Twin probands presented clinically with short stature and hypoglycemia. Variable phenotypic expression was seen in 2 other family members carrying the IGF1R mutation. The probands were treated with exogenous growth hormone therapy and dietary cornstarch, improving linear growth and reducing hypoglycemic events. IGF1R c.641-2A>G caused abnormal mRNA splicing and premature protein termination. Flow cytometric immunophenotyping demonstrated lower IGF1R on peripheral blood mononuclear cells from IGF1R c.641-2A>G subjects. This alteration was associated with reduced levels of T-helper 17 cells and a higher percentage of T-helper 1 cells compared to controls, suggesting decreased IGF1R expression may affect CD4+ Th-cell lineage commitment. DISCUSSION: Collectively, these data suggest a novel loss-of-function mutation (c.641-2A>G) leads to aberrant mRNA splicing and IGF1R expression resulting in hypoglycemia, growth restriction, and altered immune phenotypes.

Hypercalcemia from hypervitaminosis A in a child with autism.

Objectives Vitamin A is essential for normal cellular physiology and is often taken as a dietary supplement. Hypervitaminosis A can lead to hypercalcemia by increasing osteoclasts and subsequent bone resporption. Dietary supplements including vitamin A are new popular treatment stategies for autism. Case presentation We report a five-year old boy with autism spectrum disorder presenting with severe abdominal pain and bilateral lower extremity pain, who was found to have persistent hypercalcemia due to hypervitaminosis A. The patient ingested over 700 times the recommended intake of Vitamin A per day for age. Retention of vitamin A in the liver and adipose tissue causes toxic levels of retinoids and hypercalcemia. Conclusions Acute treatment included intravenous rehydration, furosemide, and calcitonin. Pamidronate was the definitive treatment for hypercalcemia from hypervitaminosis A due to its osteoclast inhibition and long biologic half-life. Parents should be counseled on risks of toxicity and absence of evidence showing benefits of vitamin A therapy for autism.

Design, validation and evaluation of a SYBR green-based quantitative PCR array for comprehensive analysis of adenovirus type 5 transcriptional patterns.

The adenoviral genome encodes coordinately expressed early and late gene transcriptional units that specify a complex collection of extensively spliced overlapping mRNAs. These complexities confound the generation of compatible, validated and optimized qPCR assays that permit comprehensive evaluation of adenoviral transcription. We have developed and evaluated a compilation of qPCR assays that represent the majority of the human adenovirus 5 (hAdV5) genome and allow for absolute and relative quantification of transcriptional activity. A panel of specific adenovirus gene primer pairs was designed through computational modeling to be compatible under a single reaction condition, precisely amplify spliced transcript products within each gene class, and not result in cellular or viral RNA/DNA background amplification. Primer pairs and reaction conditions were optimized to generate a single amplification product that was specific for its target amplicon with minimal intra-assay variability. The specificity of target amplicons was confirmed by dissociation curve analysis, gel electrophoresis and sequencing. In all, thirty-two primer sets representing specific gene products, as well as, pan early and late gene regions were validated under identical amplification conditions, thereby enabling a comprehensive assessment of adenoviral transcription within a single plate array. In order to generate positive control templates and to facilitate absolute quantification of gene expression, all target amplicons were cloned to create gene target-specific standards. These plasmid amplicon controls demonstrated that the SYBR qPCR assays exhibited optimal amplification efficiencies with a high sensitivity of detection to less than 10 copies and a linear amplification across at least eight orders of magnitude. The effectiveness and utility of the comprehensive adenoviral transcriptional array was assessed by investigating the changes in Ad5Wt gene expression at 72 versus 24 h post infection. Predictably, overall gene expression was globally increased at 72 h post infection; however, levels of E2 and Late transcripts exhibited the greatest increased expression, reflecting their necessity at this time point for genomic replication and virion assembly. Taken together, these data demonstrate that the adenoviral qPCR transcriptional array is a modular, scalable, and cost-effective method to comprehensively and accurately assess hAdV5 gene transcription. This array is broadly applicable to facilitate: adenoviral vector development; assessment of cell complementation of knockout viruses; antiviral mechanism of action evaluation; next-generation sequencing data validation.

Exocrine Pancreas Dysfunction in Type 1 Diabetes.

OBJECTIVE: Type 1 diabetes (T1D) is characterized by autoimmune ß-cell destruction, but exocrine pancreas abnormalities may also play a role in the disease pathophysiology. Herein, we review the current evidence of exocrine damage in T1D and discuss its underlying pathophysiology, clinical evaluation, and treatment. METHOD: Extensive literature search was performed for "type 1 diabetes" and "exocrine dysfunction" on PubMed and Google Scholar databases. RESULTS: T1D pancreata are significantly smaller than controls, both in weight and volume. T cells, dendritic cells, neutrophils, and products of complement activation are seen in T1D exocrine tissues. Exocrine pancreas fibrosis, arteriosclerosis, fatty infiltration, and acinar atrophy are also observed on histology. Pancreatic exocrine insufficiency (PEI) can be assessed through direct exocrine testing, fecal elastase concentration, and measurement of serum exocrine enzymes. The prevalence of PEI in T1D varies by modality and study but is consistently greater than controls. The clinical relevance of PEI in T1D is debatable, as many patients with laboratory evidence of PEI are asymptomatic. However, in PEI-symptomatic patients reported benefits of pancreatic enzyme replacement therapy (PERT) include relief of gastrointestinal symptoms, improved quality of life, better glycemic control, and optimal nutrition. CONCLUSION: Exocrine pancreas abnormalities often occur in T1D. Whether exocrine dysfunction occurs simultaneously with ß-cell destruction, as a result of ß-cell loss, or as a combination of both remains to be definitively answered. In T1D with gastrointestinal complaints, PEI should be evaluated, usually via fecal elastase measurements. PERT is recommended for T1D patients with symptoms and laboratory evidence of PEI. ABBREVIATIONS: AAb+ = autoantibody positive; AAb- = autoantibody negative; FEC = fecal elastase concentration; PEI = pancreatic exocrine insufficiency; PERT = pancreatic enzyme replacement therapy; PP = pancreatic polypep-tide; T1D = type 1 diabetes.

5-HT2 receptor activation alleviates airway inflammation and structural remodeling in a chronic mouse asthma model.

AIMS: Although the bulk of research into the biology of serotonin 5-HT2A receptors has focused on its role in the CNS, selective activation of these receptors in peripheral tissues can produce profound anti-inflammatory effects. We previously demonstrated that the small molecule 5-HT2 receptor agonist (R)-2,5-dimethoxy-4-iodoamphetamine [(R)-DOI] inhibits TNF-α-mediated proinflammatory signaling cascades and inflammation via 5-HT2A receptor activation and prevents the development of, and inflammation associated with, acute allergic asthma in a mouse ovalbumin (OVA) model. Here, we investigated the ability of (R)-DOI to reverse inflammation and symptoms associated with established asthma in a newly developed model of chronic asthma. METHODS: An 18-week ovalbumin challenge period was performed to generate persistent, chronic asthma in BALB/c mice. Four once daily intranasal treatments of (R)-DOI were administered one week after allergen cessation, with respiratory parameters being measured by whole-body plethysmography (WBP). Cytokine and chemokine levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in homogenized lung tissue, bronchoalveolar (BALF) fluid was analyzed for chemokine modulation by multiplex assays, and Periodic Acid-Schiff and Masson's Trichrome staining was performed to determine goblet cell infiltration and overall changes to lung morphology. KEY FINDINGS: 5-HT2 activation via (R)-DOI attenuates elevated airway hyperresponsiveness to methacholine, reduces pulmonary inflammation and mucus production, and reduces airway structural remodeling and collagen deposition by nearly 70%. SIGNIFICANCE: Overall, these data provide support for the therapeutic potential of (R)-DOI and 5-HT2 receptor activation for the treatment of asthma, and identifies (R)-DOI as a novel therapeutic compound against pulmonary fibrosis.

Activation of 5-HT2 Receptors Reduces Inflammation in Vascular Tissue and Cholesterol Levels in High-Fat Diet-Fed Apolipoprotein E Knockout Mice.

Coronary artery disease (CAD) is a progressive cardiovascular syndrome characterized by cholesterol-induced focal arterial lesions that impair oxygen delivery to the heart. As both innate and adaptive immune cells play critical roles in the formation and progression of arterial plaques and endothelial cell dysfunction, CAD is commonly viewed as a chronic inflammatory disorder. Our lab has previously discovered that 5-HT2A receptor activation with the 5-HT2 receptor selective agonist (R)-2,5-dimethoxy-4-iodoamphetamine [(R)-DOI] has potent anti-inflammatory activity in both cell culture and whole animal models. Here we have examined the putative therapeutic effects of (R)-DOI in the ApoE-/- high fat model of cardiovascular disease. Subcutaneously implanted osmotic minipumps were used to infuse sustained low rates (0.15 µg / hr) of (R)-DOI∙HCl to mice fed a high-fat "Western" diet. (R)-DOI treated mice had significant reductions in expression levels of mRNA for inflammatory markers like Il6 in vascular tissue, normalized glucose homeostasis, and reduced circulating cholesterol levels. As cardiovascular disease is a leading cause of death both globally and in the Western world, activation of 5-HT2A receptors at sub-behavioral levels may represent a new strategy to treat inflammation-based cardiovascular disease.

Development and evaluation of a host-targeted antiviral that abrogates herpes simplex virus replication through modulation of arginine-associated metabolic pathways.

Since their inception five decades ago, most antivirals have been engineered to disrupt a single viral protein or process that is essential for viral replication. This approach has limited the overall therapeutic effectiveness and applicability of current antivirals due to restricted viral specificity, a propensity for development of drug resistance, and an inability to control deleterious host-mediated inflammation. As obligate intracellular parasites, viruses are reliant on host metabolism and macromolecular synthesis pathways. Of these biosynthetic processes, many viruses, including Herpes simplex viruses (HSV), are absolutely dependent on the bioavailability of arginine, a non-essential amino acid that is critical for many physiological and pathophysiological processes associated with either facilitating viral replication or progression of disease. To assess if targeting host arginine-associated metabolic pathways would inhibit HSV replication, a pegylated recombinant human Arginase I (peg-ArgI) was generated and its in vitro anti-herpetic activity was evaluated. Cells continuously treated with peg-ArgI for over 48 h exhibited no signs of cytotoxicity or loss of cell viability. The antiviral activity of peg-ArgI displayed a classical dose-response curve with IC50's in the sub-nanomolar range. peg-ArgI potently inhibited HSV-1 and HSV-2 viral replication, infectious virus production, cell-to-cell spread/transmission and virus-mediated cytopathic effects. Not unexpectedly given its host-targeted mechanism of action, peg-ArgI showed similar effectiveness at controlling replication of single and multidrug resistant HSV-1 mutants. These findings illustrate that targeting host arginine-associated metabolic pathways is an effective means of controlling viral replicative processes. Further exploration into the breadth of viruses inhibited by peg-ArgI, as well as the ability of peg-ArgI to suppress arginine-associated virus-mediated pathophysiological disease processes is warranted.

Prodrugs of herpes simplex thymidine kinase inhibitors.

BACKGROUND: Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. METHODS: Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. RESULTS: Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. CONCLUSIONS: Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo.

ERG2 and ERG24 Are Required for Normal Vacuolar Physiology as Well as Candida albicans Pathogenicity in a Murine Model of Disseminated but Not Vaginal Candidiasis.

Several important classes of antifungal agents, including the azoles, act by blocking ergosterol biosynthesis. It was recently reported that the azoles cause massive disruption of the fungal vacuole in the prevalent human pathogen Candida albicans. This is significant because normal vacuolar function is required to support C. albicans pathogenicity. This study examined the impact of the morpholine antifungals, which inhibit later steps of ergosterol biosynthesis, on C. albicans vacuolar integrity. It was found that overexpression of either the ERG2 or ERG24 gene, encoding C-8 sterol isomerase or C-14 sterol reductase, respectively, suppressed C. albicans sensitivity to the morpholines. In addition, both erg2Δ/Δ and erg24Δ/Δ mutants were hypersensitive to the morpholines. These data are consistent with the antifungal activity of the morpholines depending upon the simultaneous inhibition of both Erg2p and Erg24p. The vacuoles within both erg2Δ/Δ and erg24Δ/Δ C. albicans strains exhibited an aberrant morphology and accumulated large quantities of the weak base quinacrine, indicating enhanced vacuolar acidification compared with that of control strains. Both erg mutants exhibited significant defects in polarized hyphal growth and were avirulent in a mouse model of disseminated candidiasis. Surprisingly, in a mouse model of vaginal candidiasis, both mutants colonized mice at high levels and induced a pathogenic response similar to that with the controls. Thus, while targeting Erg2p or Erg24p alone could provide a potentially efficacious therapy for disseminated candidiasis, it may not be an effective strategy to treat vaginal infections. The potential value of drugs targeting these enzymes as adjunctive therapies is discussed.

Inhibition of LSD1 reduces herpesvirus infection, shedding, and recurrence by promoting epigenetic suppression of viral genomes.

Herpesviruses are highly prevalent and maintain lifelong latent reservoirs, thus posing challenges to the control of herpetic disease despite the availability of antiviral pharmaceuticals that target viral DNA replication. The initiation of herpes simplex virus infection and reactivation from latency is dependent on a transcriptional coactivator complex that contains two required histone demethylases, LSD1 (lysine-specific demethylase 1) and a member of the JMJD2 family (Jumonji C domain-containing protein 2). Inhibition of either of these enzymes results in heterochromatic suppression of the viral genome and blocks infection and reactivation in vitro. We demonstrate that viral infection can be epigenetically suppressed in three animal models of herpes simplex virus infection and disease. Treating animals with the monoamine oxidase inhibitor tranylcypromine to inhibit LSD1 suppressed viral lytic infection, subclinical shedding, and reactivation from latency in vivo. This phenotypic suppression was correlated with enhanced epigenetic suppression of the viral genome and suggests that, even during latency, the chromatin state of the virus is dynamic. Therefore, epi-pharmaceuticals may represent a promising approach to treat herpetic diseases.

Development and evaluation of SYBR Green-I based quantitative PCR assays for herpes simplex virus type 1 whole transcriptome analysis.

There is an emerging need for viral gene specific quantitative PCR (qPCR) assays that validate and complement whole transcriptome level technologies, including microarray and next generation sequencing. Therefore, a compilation of qPCR assays that represented the breadth of the entire Herpes simplex virus type 1 (HSV-1) genome were developed and evaluated. SYBR Green-I-based quantitation of each of the 74 HSV-1 lytic genes enabled accurate and reproducible detection of viral genes using a minimal number of reaction conditions. The amplification specificity of these assays for HSV-1 target genes was confirmed by amplicon size and purity determination on agarose gels, melt temperature dissociation curve analysis, and direct DNA sequencing of amplified products. Analysis of representative target genes demonstrated that these assays accurately and reproducibly quantified target gene expression across a wide and linear range of detection. In addition, minimal intra- and inter-assay variability was observed with significant well-to-well and plate-to-plate/assay-to-assay precision. To evaluate the utility of the developed qPCR assay system, kinetic profiles of viral gene expression were determined for an array of representative genes from all HSV-1 transcriptional gene classes. Collectively, these data demonstrate that the compiled optimized qPCR assays is a scalable and cost-effective method to assess HSV-1 gene expression with broad application potential, including investigation of pathogenesis and antiviral therapies. In addition, they can be employed to validate and complement evolving technologies for genome-wide transcriptome analysis.

The antimicrobial agent C31G is effective for therapy for HSV-1 ocular keratitis in the rabbit eye model.

The amphoteric C31G solution contains equimolar alkyl dimethlyglycine and alkyl dimethyl amine oxide buffered with citric acid. C31G acts as a broad spectrum antiviral and an antibacterial. No previous in vivo studies have been done to test C31G in an animal model of HSV-1 ocular keratitis. We assessed the anti-herpetic activity of C31G in the rabbit eye model using three treatment groups: (1) 1% trifluorothymidine (TFT); (2) 0.25% C31G plus 0.5% hydroxypropyl methylcellulose (HPMC); and (3) vehicle, 0.5% HPMC. Scarified rabbit corneas were inoculated with the HSV-1 strain McKrae. On post inoculation (PI) day 3, rabbits were placed in three balanced groups based on slit-lamp examination (SLE) scores. Treatment began on PI day 3, five times a day for five consecutive days. In addition to the daily, masked SLE scoring, the eyes were assessed daily for stromal opacity, scleral inflammation, neovascularization, eyelid inflammation, inflammatory discharge, and epiphora. C31G and TFT were very effective in reducing the lesions and pathogenesis associated with HSV-1 ocular keratitis. The vehicle control scores were significantly higher and did not effectively treat HSV-1 keratitis. C31G has the potential to be used to treat herpetic keratitis as well as other herpetic topical lesions in humans.