HSV-1 DNA Research Articles

MxB is an interferon-induced restriction factor of human herpesviruses

The type I interferon (IFN) system plays an important role in controlling herpesvirus infections, but it is unclear which IFN-mediated effectors interfere with herpesvirus replication. Here we report that human myxovirus resistance protein B (MxB, also designated Mx2) is a potent human herpesvirus restriction factor in the context of IFN. We demonstrate that ectopic MxB expression restricts a range of herpesviruses from the Alphaherpesvirinae and Gammaherpesvirinae, including herpes simplex virus 1 and 2 (HSV-1 and HSV-2), and Kaposi’s sarcoma-associated herpesvirus (KSHV). MxB restriction of HSV-1 and HSV-2 requires GTPase function, in contrast to restriction of lentiviruses. MxB inhibits the delivery of incoming HSV-1 DNA to the nucleus and the appearance of empty capsids, but not the capsid delivery to the cytoplasm or tegument dissociation from the capsid. Our study identifies MxB as a potent pan-herpesvirus restriction factor which blocks the uncoating of viral DNA from the incoming viral capsid.

T69. Continuous Electroencephalogram (CEEG) as a biomarker for progression of Herpes Simplex Virus Encephalitis (HSE)

Introduction HSE is the most common cause of sporadic fatal encephalitis worldwide and remains a devastating disease despite antiviral therapy. Clinically, it is often characterized by fever, headache, seizures and focal neurologic signs. EEG can be an important tool in the diagnosis of HSV. In the acute stage, EEG can show different EEG patterns, commonly including lateralized periodic discharges (LPDs), sharp waves, focal or generalized slowing. As such, EEG can be used to monitor the progression of the disease in patients with treatment refractory HSE. Methods Case report of a patient with HSE who was monitored on CEEG throughout course of her disease. Results A 62-year old female with no history of neurologic disease, who presented with generalized weakness and lethargy for 2 weeks and rapidly deteriorated with encephalopathy, fever, and seizures. Lumbar puncture demonstrated elevated protein and WBC with positive HSV DNA. CEEG initially showed right prefrontal 2-Hz LPD’s. Over the next 48 h, these evolved into focal right prefrontal status epilepticus. After a period of iatrogenic burst suppression, the CEEG showed right frontotemporal LPD’s which evolved into right frontotemporal electrographic seizures. As the CEEG progressed, the right sided seizures appeared to have a more posterior temporal focus. Later, there was additional independent semi-rhythmic 2–4 Hz delta over the left frontotemporal region which was on the ictal-interictal continuum but did not clearly evolve into seizures. As the recording continued, there were left frontal LPD’s more than right-sided ones. The final CEEG demonstrated bilateral independent sharp waves and generalized periodic discharges (GPD’s) with triphasic morphology with resolution of electrographic seizures. As the CEEG revealed evolution of the underlying disease, treatment was escalated with a 21 day course of acyclovir, multiple anti-epileptic agents, including suppressing medications (i.e., midazolam and ketamine), as well as use of plasma exchange and steroids given the refractory seizures. MRI was obtained at three points during hospital stay and confirmed what CEEG heralded. Initially, there was diffusion restriction of the bilateral (right > left) insular cortices and right hippocampus/amygdala. Repeat MRI showed diffusion normalization with T2-weighted and FLAIR hyperintensity in the right > left insula and new involvement of the right temporal lobe. The final MRI demonstrated involvement of the bilateral inferior frontal regions and left temporal lobe, consistent with evolution from the prior studies and with new findings seen on CEEG, suggesting spread of HSE to these regions. Conclusion We report the case of a patient with HSE who was monitored on CEEG and propose that CEEG can be used as a marker for progression of disease and thus may prompt escalation in therapy. While imaging studies may confirm spread of disease, using CEEG may prevent the delay of treatment and lead to more rapid therapy in cases of refractory HSE.

Oral pemphigus.

The involvement of the oral mucosa in patients affected by pemphigus vulgaris (PV), paraneoplastic, IgA pemphigus, and in some cases iatrogenic pemphigus is common and often a prelude to skin lesions. Intraepidermal bullae are caused by acantholysis, induced by IgG autoantibodies directed against the desmosomes and the domain of numerous keratinocytes self-antigens desmogleins (namely cadherins), thus supporting the autoimmune nature of the disease. Apoptosis may contribute to the acantholysis. Oral mucosal lesions are more commonly refractory to treatment compared to skin lesions and have been associated with disease duration, disease location and possibly the presence of HSV DNA in the oral cavity. Recent publications have stressed the positive role of Rituximab in early disease treatment.

A serious case of chikungunya manifesting primarily as meningoencephalitis with MODS in an infant: A case report and challenges

Background : Chikungunya fever is an arboviral disease characterised by triad of fever, rash and polyarthralgia. Chikungunya virus (CHIKV) is transmitted to humans primarily via the bite of an infected Aedes species mosquito. The virus was first identified some 50 years back. The interest in the research of this disease increased after an important epidemiological outbreak in 2005 on the French metropolitan island of La Reunion and in India. In the last few years some of the unknown and varied manifestations of this arthropod borne alphavirus infection were brought into light. In fact, the name â€~chikungunya’ means â€~that which bends up’ in the Makonde language of East Africa and describes the severe arthritic pains of the disease.The clinical symptoms, in most affected individuals are high fever, severe myalgia and prolonged arthralgia, with occasional history of skin rash (petechiae). Neurologic complications were reported occasionally. Methods : Case Report: A 5-month-old infant presented with high grade fever and vomiting’s for 2 days, associated with irritability and 4 episodes of generalised tonic clonic seizures. Clinical examination revealed a toxic look, bulging AF, exaggerated reflexes. CNS and other system examination were normal. Child developed progressive respiratory failure despite escalating respiratory support requiring intubation and ventilation. Her blood reports including Hemogram, liver and renal function text and electrolytes were all within normal limits initially. Her CRP was elevated. Blood and urine cultures were sterile. Test for Dengue, Leptospira, Rickettsia, HSV, JE serologies, Widal test, Malaria were negative. CSF obtained on day 5 of illness showed 3mononuclear cells/mm3, sugar and protein level were 38 mg/ dl and 45.7 mg/dl respectively. Culture of CSF was negative for bacteria, fungi and mycobacteria. Result of a CSF PCR test for HSV DNA, Enterovirus RNA, VZV DNA, Neisseria meningitis DNA, Haemophilus species, Streptococcus pneumonia DNA, Cytomegalo virus DNA, Mycobacterium tuberculosis complex DNA were negative. During the stay in PICU the child developed rash on day 1 of illness which was maculopapular initially then vesicles appeared in perennial & peri-anal region which later spread to trunk and limbs. Subsequently the child had septic shock, DIC, with MODS (Transaminitis, AKI etc.). Inotropic support was optimised accordingly. Serum (MAC-IgM ELISA) collected 7 days after disease onset showed IgM antibodies to chikungunya virus. After initial antibiotic support, she was managed conservatively. She started showing progressive improvement, her inotropes were weaned and stopped, ventilator support was weaned and child was extubated successfully. Her rash subsided, sensorium and oral acceptance improved. At discharge, there were no neurological sequale noted. On follow up, after 3 months, child was asymptomatic. MRI Brain showed no detectable abnormalities. VEP and BERA were normal. Conclusion : This report highlights that, although rare, Chikungunyavirus may show neurotropism with very severe clinical presentation, including in young patients with unremarkable medical history in endemic regions. The virus can cause severe meningoencephalitis like our patient.

Experimental Oral Herpes Simplex Virus-1 (HSV-1) Co-infection in Simian Immunodeficiency Virus (SIV)-Infected Rhesus Macaques.

Herpes simplex virus 1 and 2 (HSV-1/2) similarly initiate infection in mucosal epithelia and establish lifelong neuronal latency. Anogenital HSV-2 infection augments the risk for sexual human immunodeficiency virus (HIV) transmission and is associated with higher HIV viral loads. However, whether oral HSV-1 infection contributes to oral HIV susceptibility, viremia, or oral complications of HIV infection is unknown. Appropriate non-human primate (NHP) models would facilitate this investigation, yet there are no published studies of HSV-1/SIV co-infection in NHPs. Thus, we performed a pilot study for an oral HSV-1 infection model in SIV-infected rhesus macaques to describe the feasibility of the modeling and resultant immunological changes. Three SIV-infected, clinically healthy macaques became HSV-1-infected by inoculation with 4 × 108 pfu HSV-1 McKrae on buccal, tongue, gingiva, and tonsils after gentle abrasion. HSV-1 DNA was shed in oral swabs for up to 21 days, and shedding recurred in association with intra-oral lesions after periods of no shedding during 56 days of follow up. HSV-1 DNA was detected in explant cultures of trigeminal ganglia collected at euthanasia on day 56. In the macaque with lowest baseline SIV viremia, SIV plasma RNA increased following HSV-1 infection. One macaque exhibited an acute pro-inflammatory response, and all three animals experienced T cell activation and mobilization in blood. However, T cell and antibody responses to HSV-1 were low and atypical. Through rigorous assessesments, this study finds that the virulent HSV-1 strain McKrae resulted in a low level HSV-1 infection that elicited modest immune responses and transiently modulated SIV infection.

The effect of hormonal contraception and menstrual cycle timing on genital herpes simplex virus-2 shedding and lesions

Background The effect of female sex hormones on herpes simplex virus (HSV)-2 shedding and lesion frequency is poorly understood. Previous studies suggest that hormonal contraception may increase the frequency of HSV-2 shedding. Methods We studied HSV-2 seropositive women who performed daily genital swabbing for HSV DNA and completed diaries for genital lesions and menses. We used Poisson mixed effects models to determine if HSV detection varied throughout the menstrual cycle, or in response to hormonal contraception. We used the Wilcoxon signed-rank test and rank-sum test to determine if lesion frequency differed by cycle phase or hormonal contraceptive use. Results In 189 women aged 19 to 46 years who collected swabs on 10,715 days and were not using hormonal contraception, HSV-2 DNA was detected on 20.9% of days in the follicular phase and 17.8% of days in the luteal phase (rate ratio, 1.19; 95% confidence interval, 1.03-1.37, P = 0.02). Genital lesions did not differ in the follicular versus luteal phase (12.8% vs. 10.7%, P = 0.07). In analyses of hormonal contraception, including 244 women, HSV-2 DNA was detected on 19.0% of days for women not using hormonal contraception and 18.3% of days for those using hormonal contraception (P = 0.50). Lesions were present on 11.1% of days for women not using hormonal contraception, and 8.7% of days for those using hormonal contraception (P = 0.66). Conclusions In women with genital HSV-2 infection who are not using hormonal contraception, the follicular phase of the cycle may be associated with a higher frequency of HSV-2 shedding compared to the luteal phase. Lesion frequency is similar during the 2 menstrual phases. Hormonal contraception use was not observed to affect genital HSV-2 DNA detection or lesions.

Establishing Models of Herpes Simplex Virus Type 2 Superinfection of Herpes Simplex Virus Type 1 Seropositive Mice to Test The Efficacy of a Novel Vaccine

Abstract Background Multiple subunit vaccines that elicit neutralizing antibodies (nAbs) against the immunodominant HSV-2 glycoproteins D and/or B (gD and gB) were advanced into the clinic after demonstrating protection against disease in animal models. However, although the vaccines elicited nAbs in seronegative and boosted nAb titers in HSV-1 seropositive (HSV-1+) participants, none prevented HSV-2 infection suggesting that nAbs alone are not sufficient. The results also indicate that current animal models are not predictive of clinical trial outcomes. We recently engineered a candidate single cycle vaccine strain deleted in gD (ΔgD-2) and showed that it elicits high titer non-neutralizing Abs that provide complete protection against HSV-1 or HSV-2. The Abs passively protect naïve mice and activate the Fc receptor to induce antibody-dependent cell mediated cytotoxicity (ADCC). We hypothesize that ΔgD-2 will protect HSV-1+ individuals from HSV-2 because it elicits a different type of immune response. To test this hypothesis, we established a model of HSV-2 superinfection in HSV-1+ mice. Methods We infected mice by corneal scarification with serial dilutions of a clinical strain of HSV-1 (Bx31.1) to identify a sublethal dose associated with seroconversion. We then superinfected mice on the skin with HSV-2 and monitored for disease. The presence of virus in dorsal root ganglia (DRG), the site of HSV latency, was determined by quantitative PCR. Results Corneal infection with 10^4 PFU of HSV-1 resulted in disease in 18/29 (62%) mice and 13/18 survived. Seroconversion was documented in 9/13 survivors. Surviving mice were superinfected 2 weeks post-recovery with HSV-2. All of the mice developed signs of disease, but only 2/9 who were HSV-1+ died compared with 4/4 seronegative mice (P = 0.02, Fisher exact test). HSV-2 DNA was detected in the DRG of 12/13 mice. Conclusion Sublethal HSV-1 corneal disease provides partial protection against HSV-2 superinfection and provides a model to test vaccine efficacy. We speculate that superinfection boosts preexisting nAb titers, a response consistent with immune repertoire freeze, but that ΔgD-2, because it elicits ADCC Abs, will overcome repertoire freeze and provide greater protection against HSV-2 superinfection. Disclosures W. Jacobs, X-vax: Grant Investigator, Research grant and Research support. B. C. Herold, X-vax: Grant Investigator, Research grant and Research support.

Maternal Immunization with a Single-Cycle Herpes Simplex Virus (HSV) Candidate Vaccine, ΔgD-2, Protects Neonatal Mice from Lethal Viral Challenge

BackgroundPerinatal HSV is associated with ~60% mortality if untreated and with substantial morbidity even with appropriate therapy. We recently engineered a single-cycle virus deleted in glycoprotein-D (ΔgD-2) that induces high-titer antibodies (Abs) that are non-neutralizing but activate the Fc receptor (FcR) to elicit antibody-dependent cellular cytotoxicity (ADCC). Immunization with ΔgD-2 completely protects adult mice from HSV-1 and HSV-2 disease following vaginal, skin, intraocular, or intranasal challenge and prevents the establishment of latency (ELife, 2014, JCI Insight, 2016). Thus we hypothesize that maternal immunization with ΔgD-2 and/or passive transfer of immune serum will protect neonates from HSV.MethodsFour- to 6-week-old C57Bl/6 female mice were primed and boosted at 3-week intervals with ΔgD-2 or an equal volume of uninfected cell lysates (VD60 cells). Two weeks post-boost, mice were mated and pups were challenged with a lethal dose of HSV-1 (Bx31.1) at day 7 of birth. To differentiate the contribution of transplacental vs. colostrum Abs, mothers were switched at birth. Alternatively, 7-day-old mice born to nonimmunized mothers received a single dose of immune serum (400 μg total Ab) intraperitoneally at time of intranasal challenge.ResultsThirty-eight of 47 (81%) of the pups born to and nursed by ΔgD-2-immunized mothers survived, exhibited little or no signs of disease and were protected from latency as measured by quantifying HSV DNA by PCR in neuronal tissue. In contrast, 12/14 (86%) of pups born to control vaccinated and nursed mice developed neurological signs of disease and died (P < 0.0001, Fisher’s exact test). Survival was associated with increased ADCC Abs in the serum of neonatal mice. In contrast, passive transfer of immune serum, which consistently protects adult mice from infection, did not protect neonates. If newborns born to immunized mice suckled with control mice, protection was partially abrogated (11/19, 58% survival), suggesting that both systemic and mucosal Abs are required for complete protection.ConclusionMaternal vaccination with ΔgD-2 provides significant protection against intranasal neonatal challenge but may require exposure to systemic and mucosal Abs.Disclosures W. R. Jacobs Jr., xvax: Scientific Advisor, Research support; B. C. Herold, X-vax: Grant Investigator, Research grant and Research support

Sustained Lesion and Shedding Rate Reductions in Genital Herpes Patients 24 Months after Immunization with GEN-003, a Genital Herpes Immunotherapy

BackgroundHerpes simplex viruses (HSVs) are the main cause of genital ulcers worldwide. GEN-003 is an investigational genital herpes immunotherapy composed of HSV-2 antigens gD2DTMR and ICP4.2, and the saponin-based adjuvant Matrix-M2TM (MM2). In a Phase 2 dose-ranging study (GEN-003-002), 3 doses of GEN-003 reduced HSV-2 lesion rate (percent of days with genital lesions) and anogenital HSV-2 shedding rate (percent of days with detectable virus). The antiviral effect of GEN-003 persisted to 12 months after the 3-dose vaccination regimen. We report here the results of an extension study to evaluate efficacy and immunogenicity of GEN-003 at 24 months post-vaccination.MethodsGEN-003-002 subjects who received at least 1 dose of GEN-003 (dose groups: 30 or 60 µg of antigens combined with 25, 50 or 75 µg of MM2) were eligible to enroll in the extension study. At 24 months post-vaccination, anogenital swabs were collected twice daily for 28 days for HSV-2 DNA detection by quantitative PCR. During this period, subjects also reported genital herpes lesion data via a daily reporting tool. Blood samples were collected at the end of the swab collection period to evaluate humoral and cellular immune responses. HSV-2 immunoglobulin G (IgG) was measured by ELISA, and HSV-2 neutralizing antibodies were measured by a colorimetric assay. Cellular responses were evaluated in peripheral blood mononuclear cells using an interferon-g/granzyme B Fluorospot assay.Results140 subjects were enrolled. At 24 months, those in the two best-performing GEN-003-002 study groups, 60 µg antigens combined with either 50 or 75 µg MM2 (60/50 and 60/75, respectively), recorded decreased mean viral shedding rates of 58% and 69% below baseline, similar to the 12-month shedding rate reductions, and mean anogenital lesion rates of 77% and 39% below baseline, respectively (Fig 1). In all dose groups, mean IgG titers to ICP4.2 and gD2ΔTMR were sustained from 12 to 24 months. Similarly, mean neutralizing antibody titers did not change significantly from month 12 to 24.ConclusionGEN-003 induces reductions in HSV-2 shedding and genital herpes lesion rates that persist to 24 months following treatment. Humoral immune responses to GEN-003 are maintained at 24 months after immunization.Disclosures T. Heineman, Genocea Biosciences: Employee, Salary. L. K. Mcneil, Genocea Biosciences: Employee, Salary. T. Oliphant, Genocea Biosciences: Consultant, Consulting fee. A. Natenshon, Genocea Biosciences: Employee, Salary. N. Van Wagoner, Genocea Biosciences: Consultant, Research support and Travel support to present at scientific meetings .J. B. Flechtner, Genocea Biosciences: Employee, Salary S. Hetherington, Genocea Biosciences: Employee, Salary/

New simplex Type 1 lytic infection pyridine system derivative as a novel goal against Herpes

The pyridine system derivatives have been studied as antiviral drug new models against HIV infection using bioisosterism as a powerful tool. In the last decades, the number of this work, thienopyridine derivatives were evaluated as an HSV-1 inhibitor to since the elevated number of acyclovir resistance in immunocompromised patients could promote a continuous viral pool increase and disease recurrence. In these substances, thienopyridine derivative with radical nitrate in para position (109) was able to inhibit 99% of HSV-1 replication in Vero cells with EC 50 value of 100 pM, and SI value three times higher than acyclovir. In kinetic enzymatic assay this substance was also able to inhibit about 50% of HSV-1 DNA polymerase but in a non-competitive mechanism a new synergic system to control herpes infections. Preliminary in vivo model studies revealed that substance 109 is toxic when administered in 300 g/mL concentrations. In this work, we concluded that thienopyridine derivatives can be considered a promising new anti-HSV-1 drug and can be used for clinical testing.

A model of genital herpes simplex virus Type 1 infection in Rhesus Macaques.

Although HSV-2 is the major cause of genital lesions, HSV-1 accounts for half of new cases in developed countries. Three healthy SHIV-SF162P3-infected Indian rhesus macaques were inoculated with 4×108 pfu of HSV-1 twice, with the second inoculation performed after the vaginal mucosa was gently abraded with a cytobrush. HSV-1 DNA was detected in vaginal swabs 5days after the second but not the first inoculation in all three macaques. An increase in inflammatory cytokines was detected in the vaginal fluids of the animals with no or intermittent shedding. Higher frequency of blood α4 β7high CD4+ T cells was measured in the animals with consistent and intermitted shedding, while a decrease in the frequency of CD69+ CD4+ T cells was present in all animals. This macaque model of genital HSV-1 could be useful to study the impact of the growing epidemic of genital HSV-1 on HIV infection.

Highly conserved intragenic HSV-2 sequences: Results from next-generation sequencing of HSV-2 UL and US regions from genital swabs collected from 3 continents

IntroductionUnderstanding the variability in circulating herpes simplex virus type 2 (HSV-2) genomic sequences is critical to the development of HSV-2 vaccines. MethodsGenital lesion swabs containing ≥ 107log10 copies HSV DNA collected from Africa, the USA, and South America underwent next-generation sequencing, followed by K-mer based filtering and de novo genomic assembly. Sites of heterogeneity within coding regions in unique long and unique short (UL_US) regions were identified. Phylogenetic trees were created using maximum likelihood reconstruction. ResultsAmong 46 samples from 38 persons, 1468 intragenic base-pair substitutions were identified. The maximum nucleotide distance between strains for concatenated UL_US segments was 0.4%. Phylogeny did not reveal geographic clustering. The most variable proteins had non-synonymous mutations in < 3% of amino acids. ConclusionsUnenriched HSV-2 DNA can undergo next-generation sequencing to identify intragenic variability. The use of clinical swabs for sequencing expands the information that can be gathered directly from these specimens.

Stress Hormones Epinephrine and Corticosterone Selectively Modulate Herpes Simplex Virus 1 (HSV-1) and HSV-2 Productive Infections in Adult Sympathetic, but Not Sensory, Neurons

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect and establish latency in peripheral neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is associated with the exacerbation of clinical symptoms and the induction of recurrences in humans and animal models. The viruses preferentially replicate and establish latency in different subtypes of sensory neurons, as well as in neurons of the autonomic nervous system that are highly responsive to stress hormones. To determine if stress-related hormones modulate productive HSV-1 and HSV-2 infections within sensory and autonomic neurons, we analyzed viral DNA and the production of viral progeny after treatment of primary adult murine neuronal cultures with the stress hormones epinephrine and corticosterone. Both sensory trigeminal ganglion (TG) and sympathetic superior cervical ganglion (SCG) neurons expressed adrenergic receptors (activated by epinephrine) and the glucocorticoid receptor (activated by corticosterone). Productive HSV infection colocalized with these receptors in SCG but not in TG neurons. In productively infected neuronal cultures, epinephrine treatment significantly increased the levels of HSV-1 DNA replication and production of viral progeny in SCG neurons, but no significant differences were found in TG neurons. In contrast, corticosterone significantly decreased the levels of HSV-2 DNA replication and production of viral progeny in SCG neurons but not in TG neurons. Thus, the stress-related hormones epinephrine and corticosterone selectively modulate acute HSV-1 and HSV-2 infections in autonomic, but not sensory, neurons.IMPORTANCE Stress exacerbates acute disease symptoms resulting from HSV-1 and HSV-2 infections and is associated with the appearance of recurrent skin lesions in millions of people. Although stress hormones are thought to impact HSV-1 and HSV-2 through immune system suppression, sensory and autonomic neurons that become infected by HSV-1 and HSV-2 express stress hormone receptors and are responsive to hormone fluctuations. Our results show that autonomic neurons are more responsive to epinephrine and corticosterone than are sensory neurons, demonstrating that the autonomic nervous system plays a substantial role in HSV pathogenesis. Furthermore, these results suggest that stress responses have the potential to differentially impact HSV-1 and HSV-2 so as to produce divergent outcomes of infection.

Worldwide circulation of HSV-2\u2009\xd7\u2009HSV-1 recombinant strains

Homo sapiens harbor two distinct, medically significant species of simplexviruses, herpes simplex virus (HSV)-1 and HSV-2, with estimated divergence 6–8 million years ago (MYA). Unexpectedly, we found that circulating HSV-2 strains can contain HSV-1 DNA segments in three distinct genes. Using over 150 genital swabs from North and South America and Africa, we detected recombinants worldwide. Common, widely distributed gene UL39 genotypes are parsimoniously explained by an initial >457 basepair (bp) HSV-1 × HSV-2 crossover followed by back-recombination to HSV-2. Blocks of >244 and >539 bp of HSV-1 DNA within genes UL29 and UL30, respectively, have reached near fixation, with a minority of strains retaining sequences we posit as ancestral HSV-2. Our data add to previous in vitro and animal work, implying that in vivo cellular co-infection with HSV-1 and HSV-2 yields viable interspecies recombinants in the natural human host.

Investigation on the association between thyroid tumorigeneses and herpesviruses.

Herpesviruses have been associated with various human malignancies and with thyroid autoimmunity. Aiming to investigate the presence of these viruses in thyroid nodules, we analyzed serum and thyroid tissue from 183 patients (83 benign and 100 malignant thyroid nodules). We also obtained 104 normal thyroid tissues extracted from the contralateral lobe of these patients. We used ELISA to screen the serology of all patients and a real-time quantitative PCR to analyze thyroid tissue viral load in antibody-positive patients. In addition, the presence of herpesviruses was tested by histological analysis in 20 EBV-positive tissues using the expression of LMP-1 by immunohistochemistry (IHC) and EBER by in situ hybridization (ISH). There was no evidence of HSV-2 or CMV DNA, but we found EBV DNA sequences in 29 (16%) thyroid tissue samples. We also found 7 positive EBV cases out of 104 normal tissues. Viral load was higher in tumors than in their respective normal tissues (p = 0.0002). ISH analysis revealed EBER expression in 11 out of 20 (52%) EBV-positive tissues, mostly in malignant cases (8/11, 73%). The presence of high EBV copy numbers in thyroid tumors and the expression of EBER only in malignant cases suggest an association between EBV and thyroid malignancies. However, we did not find any association between the presence of EBV and/or its viral load and any clinical or pathological tumor feature. Further studies aiming to clarify the mechanisms of EBV infection in thyroid cells are necessary to support a possible role in the development of thyroid cancer.

Herpes Simplex Virus Encephalitis in Geriatric Patients

Herpes simplex virus encephalitis (HSVE) is a rare, devastating infectious disease of the brain. We reviewed published case series to determine whether epidemiology, clinical characteristics, and prognosis of HSVE differed in geriatric patients. Geriatric patients have a higher incidence of HSVE and a worse prognosis than the rest of the adult population. The gold standard to confirm HSVE diagnosis is to demonstrate HSV DNA in cerebrospinal fluid using polymerase chain reaction. Antiviral treatment with acyclovir prevents HSVE-related morbidity and mortality if started on time. Diagnosis and treatment of HSVE pose specific challenges due to comorbidities and medical conditions presenting with similar signs and symptoms commonly encountered in geriatric medicine. Thus, timely empirical treatment and confirmation of diagnosis in this patient population relies largely on astuteness of the clinician.

Rad51 Degradation: Role in Oncolytic Virus-Poly(ADP-Ribose) Polymerase Inhibitor Combination Therapy in Glioblastoma.

Clinical success of poly(ADP-ribose) polymerase inhibitors (PARP i ) has been limited to repair-deficient cancers and by resistance. Oncolytic herpes simplex viruses (oHSVs) selectively kill cancer cells, irrespective of mutation, and manipulate DNA damage responses (DDR). Here, we explore potential synthetic lethal-like interactions between oHSV and PARP i . The efficacy of combining PARP i , oHSV MG18L, and G47Δ in killing patient-derived glioblastoma stem cells (GSCs) was assessed using cell viability assays and Chou-Talalay synergy analysis. Effects on DDR pathways, apoptosis, and cell cycle after manipulation with pharmacological inhibitors and lentivirus-mediated knockdown or overexpression were examined by immunoblotting and FACS. In vivo efficacy was evaluated in two GSC-derived orthotopic xenograft models (n = 7-8 per group). All statistical tests were two-sided. GSCs are differentially sensitive to PARP i despite uniform inhibition of PARP activity. oHSV sensitized GSCs to PARP i , irrespective of their PARP i sensitivity through selective proteasomal degradation of key DDR proteins; Rad51, mediating the combination effects; and Chk1. Rad51 degradation required HSV DNA replication. This synthetic lethal-like interaction increased DNA damage, apoptosis, and cell death in vitro and in vivo. Combined treatment of mice bearing PARP i -sensitive or -resistant GSC-derived brain tumors greatly extended median survival compared to either agent alone (vs olaparib: P ≤.001; vs MG18L: P = .005; median survival for sensitive of 83 [95% CI = 77 to 86], 94 [95% CI = 75 to 107], 102 [95% CI = 85 to 110], and 131 [95% CI = 108 to 170] days and for resistant of 54 [95% CI = 52 to 58], 56 [95% CI = 52 to 61], 62 [95% CI = 56 to 72], and 75 [95% CI = 64 to 90] days for mock, PARPi, oHSV, and combination, respectively). The unique oHSV property to target multiple components of DDR generates cancer selective sensitivity to PARP i . This combination of oHSV with PARP i is a new anticancer strategy that overcomes the clinical barriers of PARP i resistance and DNA repair proficiency and is applicable not only to glioblastoma, an invariably lethal tumor, but also to other tumor types.

An HSV-2 Trivalent Vaccine Is Immunogenic in Rhesus Macaques and Highly Efficacious in Guinea Pigs.

A genital herpes vaccine is urgently needed to prevent pain and suffering, reduce the incidence of neonatal herpes, and decrease the risk of HIV acquisition and transmission that accompanies genital infection. We evaluated a trivalent HSV-2 subunit antigen vaccine administered with CpG and alum in rhesus macaques and guinea pigs. The vaccine contains glycoproteins C, D and E (gC2, gD2, gE2) to block virus entry by gD2 and immune evasion by gC2 and gE2. In rhesus macaques, the trivalent vaccine induced plasma and mucosa neutralizing antibodies, antibodies that block gC2 and gE2 immune evasion activities, and stimulated CD4 T cell responses. After intravaginal challenge, a self-limited vaginal infection of brief duration was detected by histopathology and immunohistochemistry in naïve, but not in trivalent immunized macaques. Vaccine efficacy was evaluated in female guinea pigs. Animals were mock immunized, or immunized with gD2, the trivalent vaccine or the trivalent vaccine followed by a booster dose of gD2 (trivalent + gD2). The trivalent and trivalent + gD2 groups were 97% and 99% efficacious, respectively in preventing genital lesions and both outperformed gD2 alone. As a marker of transmission risk, vaginal swabs were evaluated daily for HSV-2 DNA and replication competent virus between five and seven weeks after challenge. HSV-2 DNA shedding was reduced in all groups compared with mock. Shedding of replication competent virus occurred on fewer days in the trivalent than gD2 immunized animals while the trivalent + gD2 group had no shedding of replication competent virus. Overall, the trivalent group had genital lesions on < 1% days and shedding of replication competent virus on 0.2% days. The vaccine has outstanding potential for prevention of genital herpes in humans.

A direct quantitative PCR-based measurement of herpes simplex virus susceptibility to antiviral drugs and neutralizing antibodies

Herpes simplex viruses (HSV) are common human pathogens that can cause painful but benign manifestations and recurrent complaints, but can also cause significant morbidity and mortality on infection of the eye or brain and with disseminated infection of an immunosuppressed patient or a neonate. HSV growth inhibition measurement by plaque or yield reduction is a key task in the development of novel antiviral compounds but the manual methods are very labour intensive. The sensitive and specific PCR technology could be an effective method for quantitation of HSV DNA related to virus replication; however the currently described PCR approaches have a major limitation, namely the requirement of purification of DNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. The monitoring of HSV specific antibody titre is essential in vaccination trials and in the improvement of HSV-based oncolytic virotherapy. Usually, conventional cytopathic effect-based and plaque reduction neutralization tests are applied to measure the neutralization titre, but these methods are also time-consuming. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of HSV-2 DNA directly from the infected cells (direct qPCR) and the method was further adapted to measure the titre of HSV specific neutralizing antibody in human sera. The conditions of direct qPCR assay were optimized to measure the antiviral activity of known and novel antiviral substances. Using HSV-2 seronegative and seropositive patients’ sera, the validity of the direct qPCR neutralization test was compared to traditional cytopathic effect-based assay.The direct qPCR method was able to detect the HSV-2 DNA quantitatively between multiplicity of infection 1/64 and 1/4194304, indicating that the dynamic range of the detection was approximately 65,500 fold with high correlation between the biological and technical replicates. As a proof of the adaptability of the method, we applied the direct qPCR for antiviral inhibitory concentration 50 (IC50) measurements of known and novel antiviral compounds. The measured IC50 of acyclovir was ∼0.28μg/ml, similar to the previously published IC50 value. The IC50 of novel antiviral candidates was between 1.6–3.1μg/ml. The direct qPCR-based neutralization titres of HSV positive sera were 1:32–1:64, identical to the neutralization titres determined using a traditional neutralization assay. The negative sera did not inhibit the HSV-2 replication in either of the tests.Our direct qPCR method for the HSV-2 growth determination of antiviral IC50 and neutralization titre is less time-consuming, less subjective and a more accurate alternative to the traditional plaque titration and growth reduction assays.

Association between herpes simplex virus Types 1 and 2 with cardiac myxoma

Most cases of atrial myxoma are sporadic, and the exact etiology is unknown. We examined if herpes simplex virus (HSV)-1 and HSV-2 antigens and/or DNA could be detected in a cohort of Iranian patients with cardiac myxomas. From July 2004 to June 2014, among a total of 36,703 patients undergoing open heart surgeries, consecutive patients with cardiac myxoma who were treated by surgical excision at our center included in this study. Of 73 patients studied, 56% were female with a mean age of 54 years (ranging from 23 to 77 years). Seventy-four myxomas were surgically removed from 73 patients, since one patient had two myxomas which were located on both the right atrium and right ventricle. The materials for this analysis were retrospectively gathered from extracted tumors that stored in a pathology bank of tissue paraffin blocks. The formalin fixed paraffin embedded tissue samples were investigated for HSV genomic DNA by both immunohistochemistry (IHC) and polymerase chain reaction (PCR) analysis. In all 74 cases there was no presence of HSV 1 and HSV 2 infection. This suggests that HSV may not play a role in sporadic cardiac myxomas; however, evidence for such association is currently lacking, and further studies are required to determine such a role.