Herpes Simplex Virus Type 1 DNA Research Articles

HSV-2 Cellular Programming Enables Productive HIV Infection in Dendritic Cells.

Genital herpes is a common sexually transmitted infection caused by herpes simplex virus type 2 (HSV-2). Genital herpes significantly enhances the acquisition and transmission of HIV-1 by creating a microenvironment that supports HIV infection in the host. Dendritic cells (DCs) represent one of the first innate cell types that encounter HIV-1 and HSV-2 in the genital mucosa. HSV-2 infection has been shown to modulate DCs, rendering them more receptive to HIV infection. Here, we investigated the potential mechanisms underlying HSV-2-mediated augmentation of HIV-1 infection. We demonstrated that the presence of HSV-2 enhanced productive HIV-1 infection of DCs and boosted inflammatory and antiviral responses. The HSV-2 augmented HIV-1 infection required intact HSV-2 DNA, but not active HSV-2 DNA replication. Furthermore, the augmented HIV infection of DCs involved the cGAS-STING pathway. Interestingly, we could not see any involvement of TLR2 or TLR3 nor suppression of infection by IFN-β production. The conditioning by HSV-2 in dual exposed DCs decreased protein expression of IFI16, cGAS, STING, and TBK1, which is associated with signaling through the STING pathway. Dual exposure to HSV-2 and HIV-1 gave decreased levels of several HIV-1 restriction factors, especially SAMHD1, TREX1, and APOBEC3G. Activation of the STING pathway in DCs by exposure to both HSV-2 and HIV-1 most likely led to the proteolytic degradation of the HIV-1 restriction factors SAMHD1, TREX1, and APOBEC3G, which should release their normal restriction of HIV infection in DCs. This released their normal restriction of HIV infection in DCs. We showed that HSV-2 reprogramming of cellular signaling pathways and protein expression levels in the DCs provided a setting where HIV-1 can establish a higher productive infection in the DCs. In conclusion, HSV-2 reprogramming opens up DCs for HIV-1 infection and creates a microenvironment favoring HIV-1 transmission.

Psoromic Acid, a Lichen-Derived Molecule, Inhibits the Replication of HSV-1 and HSV-2, and Inactivates HSV-1 DNA Polymerase: Shedding Light on Antiherpetic Properties.

Psoromic acid (PA), a bioactive lichen-derived compound, was investigated for its inhibitory properties against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), along with the inhibitory effect on HSV-1 DNA polymerase, which is a key enzyme that plays an essential role in HSV-1 replication cycle. PA was found to notably inhibit HSV-1 replication (50% inhibitory concentration (IC50): 1.9 μM; selectivity index (SI): 163.2) compared with the standard drug acyclovir (ACV) (IC50: 2.6 μM; SI: 119.2). The combination of PA with ACV has led to potent inhibitory activity against HSV-1 replication (IC50: 1.1 µM; SI: 281.8) compared with that of ACV. Moreover, PA displayed equivalent inhibitory action against HSV-2 replication (50% effective concentration (EC50): 2.7 μM; SI: 114.8) compared with that of ACV (EC50: 2.8 μM; SI: 110.7). The inhibition potency of PA in combination with ACV against HSV-2 replication was also detected (EC50: 1.8 µM; SI: 172.2). Further, PA was observed to effectively inhibit HSV-1 DNA polymerase (as a non-nucleoside inhibitor) with respect to dTTP incorporation in a competitive inhibition mode (half maximal inhibitory concentration (IC50): 0.7 μM; inhibition constant (Ki): 0.3 μM) compared with reference drugs aphidicolin (IC50: 0.8 μM; Ki: 0.4 μM) and ACV triphosphate (ACV-TP) (IC50: 0.9 μM; Ki: 0.5 μM). It is noteworthy that the mechanism by which PA-induced anti-HSV-1 activity was related to its inhibitory action against HSV-1 DNA polymerase. Furthermore, the outcomes of in vitro experiments were authenticated using molecular docking analyses, as the molecular interactions of PA with the active sites of HSV-1 DNA polymerase and HSV-2 protease (an essential enzyme required for HSV-2 replication) were revealed. Since this is a first report on the above-mentioned properties, we can conclude that PA might be a future drug for the treatment of HSV infections as well as a promising lead molecule for further anti-HSV drug design.

Cellular Requirements for Sensing and Elimination of Incoming HSV-1 DNA and Capsids.

Incoming viruses challenge the cell with diverse foreign molecules, which need to be sensed quickly to initiate immune responses and to remove the viral components. In this study, we investigate the cellular requirements for sensing and degradation of incoming viral DNA and capsids during herpes simplex virus type 1 (HSV-1) infections. Using click chemistry labeling of the viral genome, we found that HSV-1 DNA was released from a subset of capsids into the cytosol early in infection. By next-generation sequencing of cyclic GMP-AMP (cGAMP) synthase (cGAS)-bound DNA from HSV-1-infected cells, we show that HSV-1 DNA was bound by the cytosolic DNA sensor cGAS. Activation of cGAS enzymatic activity by viral DNA did not require proteasomal activity, indicating that viral DNA release into the cytosol is not proteasome-dependent. However, induction of interferon (IFN)-β expression was blocked by inhibition of the proteasome, suggesting a contribution of the proteasome to IFN-β induction through the cGAS-stimulator of interferon genes pathway. Viral DNA was cleared from the cytosol within few hours, in a manner dependent on TREX1 and a cGAS-dependent process. Capsid material in the cytoplasm was also degraded rapidly. This was partially blocked by treatment with a proteasome inhibitor. This treatment led to accumulation of DNA-containing viral capsids near the nucleus and reduced nuclear entry of viral DNA. Thus, cells infected with HSV-1 use a panel of mechanisms to eliminate viral DNA and capsids. This represents a barrier for establishment of infection and potentially enables the host to gear the IFN-β response to a level required for antiviral defense without causing immunopathology.

Quadruplex real-time PCR for rapid detection of human alphaherpesviruses

Infections with the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) as well as with the varicella-zoster virus (VZV) may take a serious course. Thus, rapid and reliable detection of these alphaherpesviruses is urgently needed. For this, we established a qualitative quadruplex real-time polymerase chain reaction (PCR) covering HSV-1, HSV-2, VZV and endogenous human glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR was validated with quality assessment samples and pre-characterized clinical samples including swabs, blood and cerebrospinal as well as respiratory fluids. For comparison, nucleic acids (NA) of selected samples were extracted manually and automatically. The protocol takes approx. 90min, starting with the preparation of NA until the report of results. The oligonucleotide and hydrolysis probe sequences specifically detect and distinguish HSV-1 (530nm), HSV-2 (705nm) and VZV (560nm) DNA. The detection limit was estimated with 100-500 copies/ml HSV-1 and HSV-2/VZV, respectively. All quality assessment samples as well as all the patient samples were classified correctly. Parallel detection of GAPDH (670nm) DNA was implemented to demonstrate correct sampling, but was uncertain in case of swabs. To this end, alphaherpesvirus-free human DNA was also added directly into the mastermix to exclude PCR inhibition. The established protocol for parallel detection and differentiation of alphaherpesviruses is fast, highly specific as well as rather sensitive. It will facilitate HSV-1/2 and VZV diagnostics and may be further improved by opening the 670nm channel for a combined extraction and PCR inhibition control.

Viral Genetics Modulate Orolabial Herpes Simplex Virus Type 1 Shedding in Humans.

Orolabial herpes simplex virus type 1 (HSV-1) infection has a wide spectrum of severity in immunocompetent persons. To study the role of viral genotype and host immunity, we characterized oral HSV-1 shedding rates and host cellular response, and genotyped viral strains, in monozygotic (MZ) and dizygotic (DZ) twins. A total of 29 MZ and 22 DZ HSV-1-seropositive twin pairs were evaluated for oral HSV-1 shedding for 60 days. HSV-1 strains from twins were genotyped as identical or different. CD4+ T-cell responses to HSV-1 proteins were studied. The median per person oral HSV shedding rate was 9% of days that a swab was obtained (mean, 10.2% of days). A positive correlation between shedding rates was observed within all twin pairs, and in the MZ and DZ twins. In twin subsets with sufficient HSV-1 DNA to genotype, 15 had the same strain and 14 had different strains. Viral shedding rates were correlated for those with the same but not different strains. The median number of HSV-1 open reading frames recognized per person was 16. The agreement in the CD4+ T-cell response to specific HSV-1 open reading frames was greater between MZ twins than between unrelated persons (P = .002). Viral strain characteristics likely contribute to oral HSV-1 shedding rates.

Genome-wide association study (GWAS) of human host factors influencing viral severity of herpes simplex virus type 2 (HSV-2).

Herpes simplex virus type 2 (HSV-2) is an incurable viral infection with severity ranging from asymptomatic to frequent recurrences. The viral shedding rate has been shown as a reproducible HSV-2 severity endpoint that correlates with lesion rates. We used a genome-wide association study (GWAS) to investigate the role of common human genetic variation in HSV-2 severity. We performed a GWAS on 223 HSV-2-positive participants of European ancestry. Severity was measured by viral shedding rate, as defined by the percent of days PCR+ for HSV-2 DNA over at least 30 days. Analyses were performed under linear regression models, adjusted for age, sex, and ancestry. There were no genome-wide significant (p<5E-08) associations with HSV-2 viral shedding rate. The top non-significant SNP (rs75932292, p=6.77E-08) associated with HSV-2 viral shedding was intergenic, with the nearest known biologically interesting gene (ABCA1) ∼130Kbp downstream. Several other SNPs approaching significance were in or near genes with viral or neurological associations, including 4 SNPs in KIF1B. The current study is the first comprehensive genome-wide investigation of human genetic variation in virologic severity of established HSV-2 infection. However, no significant associations were observed with HSV-2 virologic severity, leaving the exact role of human variation in HSV-2 severity unclear.

Herpes simplex virus type 2 IgG antibodies in sera of umbilical cord as a proxy for placental infection in asymptomatic pregnant women.

Herpes simplex virus type 2 (HSV-2) infection is one of the most prevalent diseases worldwide and is mainly sexually transmitted. When infecting pregnant women, HSV-2 is able to infect the placenta, can reach the fetus, and may affect the fetal development. We sought to determine the prevalence of HSV-2 infection and reactivation in asymptomatic pregnant women, the correlation between IgG in the maternal circulation and cord blood, and the correlation between circulating IgG, placental, and newborn infection (blood cord). Serum samples and placental tissues from pregnant women and umbilical cord blood samples from their newborns were collected. Anti-HSV-2 antibodies were identified by ELISA, and HSV-2 DNA was detected by nested PCR. The seropositivity of IgG in pregnant women was 29.7% and IgM was detected in 1 woman (0.5%). In the umbilical cord of newborns, 33.1% were IgG-positive and IgM was detected in 2 samples (1.5%). A positive correlation between HSV-2 IgG titers in serum from pregnant women and cord blood samples was found (r=.36, P=.001). A difference between the positive and negative placental groups (maternal side) was found in titers of IgG in sera of umbilical cord, which were significantly higher in the positive placental group (P=.004). We describe for the first time that newborns from mothers with HSV-2 placental infection have higher IgG titers in sera of umbilical cord, suggesting IgGs antibodies can be indicative of placental viral infection in asymptomatic women.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

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.

A trivalent subunit antigen glycoprotein vaccine as immunotherapy for genital herpes in the guinea pig genital infection model

ABSTRACTAn estimated 417 million people worldwide ages 15 to 49 are infected with herpes simplex virus type 2 (HSV-2), the most common cause of genital ulcer disease. Some individuals experience frequent recurrences of genital lesions, while others only have subclinical infection, yet all risk transmitting infection to their intimate partners. A vaccine was developed that prevents shingles, which is a recurrent infection caused by varicella-zoster virus (VZV), a closely related member of the Herpesviridae family. The success of the VZV vaccine has stimulated renewed interest in a therapeutic vaccine for genital herpes. We have been evaluating a trivalent subunit antigen vaccine for prevention of genital herpes. Here, we assess the trivalent vaccine as immunotherapy in guinea pigs that were previously infected intravaginally with HSV-2. The trivalent vaccine contains HSV-2 glycoproteins C, D, and E (gC2, gD2, gE2) subunit antigens administered with CpG and alum as adjuvants. We previously demonstrated that antibodies to gD2 neutralize the virus while antibodies to gC2 and gE2 block their immune evasion activities, including evading complement attack and inhibiting activities mediated by the IgG Fc domain, respectively. Here, we demonstrate that the trivalent vaccine significantly boosts ELISA titers and neutralizing antibody titers. The trivalent vaccine reduces the frequency of recurrent genital lesions and vaginal shedding of HSV-2 DNA by approximately 50% and almost totally eliminates vaginal shedding of replication-competent virus, suggesting that the trivalent vaccine is a worthy candidate for immunotherapy of genital herpes.

Replication-Coupled Recruitment of Viral and Cellular Factors to Herpes Simplex Virus Type 1 Replication Forks for the Maintenance and Expression of Viral Genomes.

Herpes simplex virus type 1 (HSV-1) infects over half the human population. Much of the infectious cycle occurs in the nucleus of cells where the virus has evolved mechanisms to manipulate host processes for the production of virus. The genome of HSV-1 is coordinately expressed, maintained, and replicated such that progeny virions are produced within 4–6 hours post infection. In this study, we selectively purify HSV-1 replication forks and associated proteins from virus-infected cells and identify select viral and cellular replication, repair, and transcription factors that associate with viral replication forks. Pulse chase analyses and imaging studies reveal temporal and spatial dynamics between viral replication forks and associated proteins and demonstrate that several DNA repair complexes and key transcription factors are recruited to or near replication forks. Consistent with these observations we show that the initiation of viral DNA replication is sufficient to license late gene transcription. These data provide insight into mechanisms that couple HSV-1 DNA replication with transcription and repair for the coordinated expression and maintenance of the viral genome.

Clinical and corneal microbial profile of infectious keratitis in a high HIV prevalence setting in rural South Africa.

The purpose of this investigation was to determine the clinical and corneal microbial profile of infectious keratitis in a high human immunodeficiency virus (HIV) prevalence setting in rural South Africa. Data in this cross-sectional study were collected from patients presenting with symptoms of infectious keratitis (n = 46) at the ophthalmology outpatient department of three hospitals in rural South Africa. Corneal swabs were tested for herpes simplex virus type 1 (HSV-1) and 2 (HSV-2), varicella zoster virus (VZV) and adenovirus DNA by real-time polymerase chain reaction (PCR) and for bacteria and fungi by culture. Based on clinical history, disease characteristics and laboratory results, 29 (63 %) patients were diagnosed as viral keratitis, including 14 (48 %) viral keratitis cases complicated by bacterial superinfection, and 17 (37 %) as bacterial keratitis. VZV and HSV-1 DNA was detected in 11 (24 %) and 5 (11 %) corneal swabs, respectively. Among clinically defined viral keratitis cases, a negative viral swab was predominantly (93 %) observed in cases with subepithelial inflammation and was significantly associated with an increased duration of symptoms (p = 0.003). The majority of bacteria cultured were Gram-positive (24/35), including Staphylococcus epidermidis and S. aureus. Viral aetiology was significantly associated with a history of herpes zoster ophthalmicus (p < 0.001) and a trend was observed between viral aetiology and HIV infection (p = 0.06). Twenty-one (47 %) keratitis cases were complicated by anterior uveitis, of which 18 (86 %) were HIV-infected cases with viral keratitis. The data implicate a high prevalence of herpetic keratitis, in part complicated by bacterial superinfection and/or uveitis, in HIV-infected individuals presenting with infectious keratitis in rural South Africa.

Facial paralysis induced by ear inoculation of herpes simplex virus in rat.

Bell's palsy is caused by the reactivation of herpes simplex virus type 1 (HSV-1). Using Balb/c mice inoculated with the KOS strain of HSV-1, we previously developed an animal disease model that simulated mild Bell's palsy. The current study developed an animal disease model of more severe facial palsy than that seen in the mouse model. Three-week-old female Wister rats weighing 60-80g were inoculated on the auricle with HSV-1 and acyclovir was administered intraperitoneally to deactivate the infected HSV-1. Instead of HSV-1, phosphate-buffered saline was used for inoculation as a negative control. Quantitative polymerase chain reaction (PCR), behavior testing (blink reflex), electroneuronography, histopathology of the peripheral nerve, and immunohistochemistry of the facial nerve nucleus were evaluated. Facial palsy occurred 3-5 days after virus inoculation, and the severity of the facial palsy progressed for up to 7 days. Quantitative PCR showed an increase in HSV-1 DNA copies in the facial nerve from 24 to 72h, suggesting that HSV-1 infection occurred in the nerve. Electroneuronography values were 33.0±15.3% and 110.0±18.0% in HSV-1-inoculated and control rats, respectively. The histopathology of the peripheral nerve showed demyelination and loss of the facial nerve, and the facial nerve nucleus showed degeneration. Facial palsy developed in Wister rats following inoculation of the KOS strain of HSV-1 onto the auricles. The behavioral, histopathological, and electroneuronography data suggested that the severity of facial palsy was greater in our rats than in animals in the previous mouse disease model.

Frequency of varicella zoster virus DNA in human adrenal glands.

Varicella zoster virus (VZV) becomes latent in ganglionic neurons derived from neural crest cells. Because the adrenal gland also contains medullary chromaffin cells of neural crest origin, we examined human adrenal glands and medullary chromaffin cell tumors (pheochromocytomas) for VZV and herpes simplex virustype 1 (HSV-1). We found VZV, but not HSV-1, DNA in 4/63 (6%) normal adrenal glands. No VZV transcripts or antigens were detected in the 4 VZVDNA-positive samples. No VZV or HSV-1 DNA was found in 21 pheochromocytomas.

False-negative type-specific glycoprotein G antibody responses in STI clinic patients with recurrent HSV-1 or HSV-2 DNA positive genital herpes, The Netherlands

ObjectivesHerpes simplex virus (HSV) type-discriminating antibody tests (glycoprotein G (gG) directed) are used to identify naïve persons and differentiate acute infections from recurrences. We studied test characteristics of three commercially...

Herpes Simplex Virus Type-2 Cervicovaginal Shedding Among Women Living With HIV-1 and Receiving Antiretroviral Therapy in Burkina Faso: An 8-Year Longitudinal Study.

Background. The impact of antiretroviral therapy (ART) on herpes simplex virus type-2 (HSV-2) replication is unclear. The aim of this study was to assess factors associated with cervicovaginal HSV-2 DNA shedding and genital ulcer disease (GUD) in a cohort of women living with human immunodeficiency virus type-1 (HIV-1) in Burkina Faso.Methods. Participants were screened for cervicovaginal HSV-2 DNA, GUD, cervicovaginal and systemic HIV-1 RNA, and reproductive tract infections every 3–6 months over 8 years. Associations with HSV-2 shedding and quantity were examined using random-effects logistic and linear regression, respectively.Results. Of the 236 women with data on HSV-2 shedding, 151 took ART during the study period. Cervicovaginal HSV-2 DNA was detected in 42% of women (99 of 236) in 8.2% of visits (151 of 1848). ART was associated with a reduction in the odds of HSV-2 shedding, which declined for each year of ART use (odds ratio [OR], 0.74; 95% confidence interval [CI], .59–.92). In the multivariable model, the impact of ART was primarily associated with suppression of systemic HIV-1 RNA (adjusted OR, 0.32; 95% CI, .15–.67). A reduction in the odds of GUD was also observed during ART, mainly in those with HIV-1 suppression (adjusted OR, 0.53; 95% CI, .25–1.11).Conclusions. ART is strongly associated with a decrease in cervicovaginal HSV-2 shedding, and the impact was sustained over several years.

Resistance testing of clinical herpes simplex virus type 2 isolates collected over 4 decades

There is only little information about the role of mutations of the thymidine kinase (TK) and DNA polymerase (pol) genes of herpes simplex virus type 2 (HSV-2) for the development of antiviral resistance. In this study, the polymorphism of TK and DNA pol genes was examined in 82 clinical isolates collected routinely between 1973 and 2013. If novel, presently unclear or resistance-related mutations were found, the resistance phenotype against acyclovir (ACV) and foscarnet (FOS) was analyzed. The four novel amino acid changes G150D, A157T, R248W, L342W and the hitherto phenotypically unclear substitution T131M within the TK gene were identified as natural polymorphisms. Within the DNA pol gene, 17 novel substitutions and the to-date unclear change R628C were characterized as part of natural gene polymorphism. Two novel DNA pol mutations were linked to resistance (M910T) and weak susceptibility to ACV (684 insertion ED), respectively. In one isolate, the genomic cause of ACV resistance could not be identified. Phylogenetic analysis including sequences of this study and of the GenBank revealed a hierarchy of mutation clusters in TK displaying G39E as first common mutation step, followed by N78D and L140F. In conclusion, the present findings allow a deeper insight in the variability of HSV-2 TK and DNA pol genes. The most common substitution G39E can be excluded as unique cause of HSV-2 resistance. This study supports once more the importance of phenotypic adjustment of genotypic results to enhance the clinical utility of genotypic findings.

Herpes simplex virus type 2 (HSV-2) genital shedding in HSV-2-/HIV-1-co-infected women receiving effective combination antiretroviral therapy

The dynamics of genital shedding of HSV-2 DNA was assessed in HIV-1-infected women taking combination antiretroviral therapy (cART). HIV-1 RNA, HIV-1 DNA and HSV DNA loads were measured during 12-18 months using frozen plasma, PBMC and cervicovaginal lavage samples from 22 HIV-1-infected women, including 17 women naive for antiretroviral therapy initiating cART and 5 women with virological failure switching to a new regimen. Nineteen (86%) women were HSV-2-seropositive. Among HSV-2-/HIV-1-co-infected women, HIV-1 RNA loads showed a rapid fall from baseline after one month of cART, in parallel in paired plasma and cervicovaginal secretions. In contrast, HIV-1 DNA loads did not show significant variations from baseline up to 18 months of treatment in both systemic and genital compartments. HSV DNA was detected at least once in 12 (63%) of 19 women during follow up: HSV-2 shedding in the genital compartment was observed in 11% of cervicovaginal samples at baseline and in 16% after initiating or switching cART. Cervicovaginal HIV-1 RNA loads were strongly associated with plasma HIV-1 RNA loads over time, but not with cervicovaginal HSV DNA loads. Reactivation of genital HSV-2 replication frequently occurred despite effective cART in HSV-2-/HIV-1-co-infected women. Genital HSV-2 replication under cART does not influence cervicovaginal HIV-1 RNA or DNA shedding.

Mollaret's meningitis

A 48-year-old man presented with a 2 day history of fever, headache, chills, neck stiff ness, and nausea and vomiting. He had a history of two episodes of viral meningitis, which occurred 30 and 13 years before this presentation. An examination confi rmed meningism without focal neurological defi cits. Results of CSF tests showed protein concentration of 120 mg/dL (normal 12–60 mg/dL), normal glucose concentration, red blood cells 26 cells/μL, and white blood cells 327 cells/μL (10% neutrophils, 84% lymphocytes, 6% monocytes). CSF Gram stain and cultures were negative. Diff -Quik stain of CSF showed many large activated monocytes with several deep nuclear clefts visible as so-called cloverleaf nucleus (green arrowhead, fi gure A), footprintshaped nucleus (white arrowhead, fi gure B), and beanshaped nucleus (green arrow, fi gure C), with a background of normal monocytes (black arrowheads, fi gure A, B) and lymphocytes (black arrows, fi gure A, B). The features of these activated monocytes were compatible with those of Mollaret’s cells, and CSF herpes simplex virus type 2 (HSV2) PCR assay was positive. Large degenerated monocytes were present as ghost cells (asterisks, fi gure B), observed in the slide background. A diagnosis of Mollaret’s meningitis was made. The patient was discharged without antimicrobial therapy and recovered completely. Mollaret’s meningitis was fi rst described by the French neurologist Pierre Mollaret in 1944. The disease is characterised by recurrent (at least three episodes), benign (no long-term sequelae) and brief (2–5 day) episodes of aseptic lymphocytic meningitis, alternating with symptom-free interval, mostly caused by HSV2 infection. The disease is usually self-limited, and antiviral therapy is routinely not recommended. It is therefore also known as recurrent benign lymphocytic meningitis. Typically, results of CSF studies show hypercellularity and predominantly lymphocytic pleocytosis with positive HSV2 DNA by PCR assay. Cellular cytomorphological features of CSF charac teristically show diagnostic Mollaret’s cells, which are multiple activated large monocytes with deep nuclear clefts giving rise to various convoluted, eye-catching shapes of nuclei, such as cloverleaf, bean, and footprint patterns. Usually degenerated monocytes known as ghost cells are present, scattered at the background of the slide. Recognition of these cells in the CSF is crucial for a timely and accurate diagnosis because it could prevent extensive and costly diagnostic studies and antimicrobial therapies.

Herpes simplex virus type 1 and Alzheimer's disease: increasing evidence for a major role of the virus.

Herpes simplex virus type 1 (HSV1), when present in brain of carriers of the type 4 allele of the apolipoprotein E gene (APOE), has been implicated as a major factor in Alzheimer’s disease (AD). It is proposed that virus is normally latent in many elderly brains but reactivates periodically (as in the peripheral nervous system) under certain conditions, for example stress, immunosuppression, and peripheral infection, causing cumulative damage and eventually development of AD. Diverse approaches have provided data that explicitly support, directly or indirectly, these concepts. Several have confirmed HSV1 DNA presence in human brains, and the HSV1-APOE-ε4 association in AD. Further, studies on HSV1-infected APOE-transgenic mice have shown that APOE-e4 animals display a greater potential for viral damage. Reactivated HSV1 can cause direct and inflammatory damage, probably involving increased formation of beta amyloid (Aβ) and of AD-like tau (P-tau)—changes found to occur in HSV1-infected cell cultures. Implicating HSV1 further in AD is the discovery that HSV1 DNA is specifically localized in amyloid plaques in AD. Other relevant, harmful effects of infection include the following: dynamic interactions between HSV1 and amyloid precursor protein (APP), which would affect both viral and APP transport; induction of toll-like receptors (TLRs) in HSV1-infected astrocyte cultures, which has been linked to the likely effects of reactivation of the virus in brain. Several epidemiological studies have shown, using serological data, an association between systemic infections and cognitive decline, with HSV1 particularly implicated. Genetic studies too have linked various pathways in AD with those occurring on HSV1 infection. In relation to the potential usage of antivirals to treat AD patients, acyclovir (ACV) is effective in reducing HSV1-induced AD-like changes in cell cultures, and valacyclovir, the bioactive form of ACV, might be most effective if combined with an antiviral that acts by a different mechanism, such as intravenous immunoglobulin (IVIG).