Comparison of herpes simplex virus 1 genomic diversity between adult sexual transmission partners with genital infection.

Herpes simplex virus (HSV) causes chronic infection in the human host, characterized by self-limited episodes of mucosal shedding and lesional disease, with latent infection of neuronal ganglia. The epidemiology of genital herpes has undergone a significant transformation over the past two decades, with the emergence of HSV-1 as a leading cause of first-episode genital herpes in many countries. Though dsDNA viruses are not expected to mutate quickly, it is not yet known to what degree the HSV-1 viral population in a natural host adapts over time, or how often viral population variants are transmitted between hosts. This study provides a comparative genomics analysis for 33 temporally-sampled oral and genital HSV-1 genomes derived from five adult sexual transmission pairs. We found that transmission pairs harbored consensus-level viral genomes with near-complete conservation of nucleotide identity. Examination of within-host minor variants in the viral population revealed both shared and unique patterns of genetic diversity between partners, and between anatomical niches. Additionally, genetic drift was detected from spatiotemporally separated samples in as little as three days. These data expand our prior understanding of the complex interaction between HSV-1 genomics and population dynamics after transmission to new infected persons.

Vertical mother-to-infant transmission of herpes simplex virus 2 is correlated with tropism due to mutations in viral UL13.

Although neonates are commonly exposed to vaginal herpes simplex virus (HSV)-2, neonatal herpes is rare. Therefore, we analyzed paired infant and maternal HSV-2 isolates from two cases of mother-to-infant transmission to identify viral factors contributing to vertical transmission. Sixteen infant isolates with neonatal herpes and 27 genital isolates in their third trimester were included. The infant isolates were significantly more temperature-independent than the maternal isolates. Sequence comparison revealed viral UL13 protein kinase (UL13-PK) mutation in the infant isolates in both cases. In the expanded cohort, infant isolates (5/18) had significantly more UL13-PK mutations than genital isolates (1/29). Isolates within 8 days post-birth (3/4) had a significantly higher frequency of UL13-PK mutation than those after 9 days (2/14), suggesting a close association between UL13-PK mutations and vertical transmission. Elongation factor 1-delta was identified as a target of UL13-PK by proteomic analysis of UL13-PK-positive and -negative HepG2 cells. The mixed infant isolates with the intact and mutated UL13-PK conferred altered cell tropism, temperature independence adapting to fetal temperature, and better growth properties in Vero and hepatoblastoma HepG2 cells than in HSV-2 with intact and mutated UL13-PK alone, indicating that viral UL13-PK mutation is essential for vertical HSV-2 transmission.

Benign recurrent lymphocytic meningitis (Mollaret's meningitis) in Denmark: a nationwide cohort study.

BACKGROUND AND PURPOSE: Data on clinical features and outcomes of benign recurrent lymphocytic meningitis (BRLM) are limited. METHODS: This was a nationwide population-based cohort study of all adults hospitalized for BRLM associated with herpes simplex virus type 2 (HSV-2) at the departments of infectious diseases in Denmark from 2015 to 2020. Patients with single-episode HSV-2 meningitis were included for comparison. RESULTS: Forty-seven patients with BRLM (mean annual incidence 1.2/1,000,000 adults) and 118 with single-episode HSV-2 meningitis were included. The progression risk from HSV-2 meningitis to BRLM was 22% (95% confidence interval [CI] 15%-30%). The proportion of patients with the triad of headache, neck stiffness and photophobia/hyperacusis was similar between BRLM and single-episode HSV-2 meningitis (16/43 [37%] vs. 46/103 [45%]; p = 0.41), whilst the median cerebrospinal fluid leukocyte count was lower in BRLM (221 cells vs. 398 cells; p = 0.02). Unfavourable functional outcomes (Glasgow Outcome Scale score of 1-4) were less frequent in BRLM at all post-discharge follow-up visits. During the study period, 10 (21%) patients with BRLM were hospitalized for an additional recurrence (annual rate 6%, 95% CI 3%-12%). The hazard ratio for an additional recurrence was 3.93 (95% CI 1.02-15.3) for patients with three or more previous episodes of meningitis. CONCLUSIONS: Clinical features of BRLM were similar to those of single-episode HSV-2 meningitis, whilst post-discharge outcomes were more favourable. Patients with three or more previous episodes of meningitis had higher risk of an additional recurrence.

Development of recombinant rotavirus carrying herpes simplex virus 2 glycoprotein D gene based on reverse genetics technology.

Vaccine development for herpes simplex virus 2 (HSV-2) has been attempted, but no vaccines are yet available. A plasmid-based reverse genetics system for Rotavirus (RV), which can cause gastroenteritis, allows the generation of recombinant RV containing foreign genes. In this study, we sought to develop simian RV (SA11) as a vector to express HSV-2 glycoprotein D (gD2) and evaluated its immunogenicity in mice. We generated the recombinant SA11-gD2 virus (rSA11-gD2) and confirmed its ability to express gD2 in vitro. The virus was orally inoculated into suckling BALB/c mice and into 8-week-old mice. Serum IgG and IgA titers against RV and gD2 were measured by ELISA. In the 8-week-old mice inoculated with rSA11-gD2, significant increases in not only antibodies against RV but also IgG against gD2 were demonstrated. In the suckling mice, antibodies against RV were induced, but gD2 antibody was not detected. Diarrhea observed after the first inoculation of rSA11-gD2 in suckling mice was similar to that induced by the parent virus. A gD2 expressing simian RV recombinant, which was orally inoculated, induced IgG against gD2. This strategy holds possibility for genital herpes vaccine development.

External Guide Sequence Effectively Suppresses the Gene Expression and Replication of Herpes Simplex Virus 2.

Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a promising nucleic acid-based gene targeting approach for gene expression knock-down and modulation. The RNase P-EGS strategy is unique as an EGS can be designed to basepair any mRNA sequence and recruit intracellular RNase P for hydrolysis of the target mRNA. In this study, we provide the first direct evidence that the RNase P-based approach effectively blocks the gene expression and replication of herpes simplex virus 2 (HSV-2), the causative agent of genital herpes. We constructed EGSs to target the mRNA encoding HSV-2 single-stranded DNA binding protein ICP8, which is essential for viral DNA genome replication and growth. In HSV-2 infected cells expressing a functional EGS, ICP8 levels were reduced by 85%, and viral growth decreased by 3000 folds. On the contrary, ICP8 expression and viral growth exhibited no substantial differences between cells expressing no EGS and those expressing a disabled EGS with mutations precluding RNase P recognition. The anti-ICP8 EGS is specific in targeting ICP8 because it only affects ICP8 expression but does not affect the expression of the other viral immediate-early and early genes examined. This study shows the effective and specific anti-HSV-2 activity of the RNase P-EGS approach and demonstrates the potential of EGS RNAs for anti-HSV-2 applications.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.

Neuronal miR-138 Represses HSV-2 Lytic Infection by Regulating Viral and Host Genes with Mechanistic Differences from HSV-1.

Herpes simplex virus 2 (HSV-2) establishes latent infection in dorsal root ganglion (DRG) neurons after productive (lytic) infection in peripheral tissues. A neuron-specific microRNA, miR-138, favors HSV-1 latency by repressing viral ICP0 and host Oct-1 and Foxc1 genes, yet the role of miR-138 in HSV-2 infection was unknown. The ICP0 mRNAs of HSV-1, HSV-2, and chimpanzee herpesvirus each have one to two canonical miR-138 binding sites. The sites are 100% conserved in 308 HSV-1 and 300 HSV-2 published sequences of clinical isolates. In cotransfection assays, miR-138 repressed HSV-2 ICP0 expression through the seed region and surrounding interactions that are different from HSV-1. An HSV-2 mutant with disrupted miR-138 binding sites on ICP0 showed increased ICP0 expression in Neuro-2a cells. Photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation confirmed miR-138 binding to HSV-2 ICP0 and identified UL19 and UL20 as additional targets whose expression was repressed by miR-138 during cotransfection. In Neuro-2a cells, transfected miR-138 and its antagomir decreased and increased HSV-2 replication, respectively, and a knockout experiment showed that miR-138's host targets OCT-1 and FOXC1 were important for HSV-2 replication. In primary mouse DRG neurons, both ICP0 and FOXC1 positively regulated HSV-2 replication, but both overexpressed and endogenous miR-138 suppressed HSV-2 replication primarily by repressing ICP0 expression. Thus, miR-138 can suppress HSV-2 neuronal replication through multiple viral and host pathways. These results reveal functional similarities and mechanistic differences in how miR-138 regulates HSV-1 and HSV-2 infection and indicate an evolutionary advantage of using miR-138 to repress lytic infection in neurons. IMPORTANCE HSV-1 and HSV-2 are closely related viruses with major differences. Both viruses establish latency in neurons from which they reactivate to cause disease. A key aspect of HSV latency is repression of productive infection in neurons. Based on previous work with HSV-1, we investigated the role of a neuron-specific microRNA, miR-138, in HSV-2 infection and established it as a repressor of HSV-2 productive infection in neuronal cells. This repression is mediated mainly by targeting viral ICP0 and host Foxc1 mRNAs, but other pathways also contribute. Despite functional conservation of the role of miR-138 between HSV-1 and HSV-2, many molecular mechanisms differ, including how miR-138 represses ICP0 expression and miR-138 targeting of HSV-2 but not HSV-1 UL19 and UL20. To our knowledge, this study provides the first example of host microRNA regulation of HSV-2 infection.

Herpes simplex virus 2 (HSV-2) evolves faster in cell culture than HSV-1 by generating greater genetic diversity.

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue.

Application of VirCapSeq-VERT and BacCapSeq in the diagnosis of presumed and definitive neuroinfectious diseases.

Unbiased high-throughput sequencing (HTS) has enabled new insights into the diversity of agents implicated in central nervous system (CNS) infections. The addition of positive selection capture methods to HTS has enhanced the sensitivity while reducing sequencing costs and the complexity of bioinformatic analysis. Here we report the use of virus capture-based sequencing for vertebrate viruses (VirCapSeq-VERT) and bacterial capture sequencing (BacCapSeq) in investigating CNS infections. Thirty-four samples were categorized: (1) patients with definitive CNS infection by routine testing; (2) patients meeting clinically the Brighton criteria (BC) for meningoencephalitis; (3) patients with presumptive infectious etiology highest on the differential. RNA extracts from cerebrospinal fluid (CSF) were used for VirCapSeq-VERT, and DNA extracts were used for BacCapSeq analysis. Among 8 samples from known CNS infections in group 1, VirCapSeq and BacCapSeq confirmed 3 expected diagnoses (42.8%), were negative in 2 (25%), yielded an alternative result in 1 (11.1%), and did not detect 2 expected negative pathogens. The confirmed cases identified HHV-6, HSV-2, and VZV while the negative samples included JCV and HSV-2. In groups 2 and 3, 11/26 samples (42%) were positive for at least one pathogen; however, 27% of the total samples (7/26) were positive for commensal organisms. No microbial nucleic acids were detected in negative control samples. HTS showed limited promise for pathogen identification in presumed CNS infectious diseases in our small sample. Before conducting larger-scale prospective studies to assess the clinical value of this novel technique, clinicians should understand the benefits and limitations of using this modality.

Evolving etiologies, comorbidities, survival, and costs of care in adult encephalitis.

Encephalitis is a central nervous system disorder, often caused by infectious agents or aberrant immune responses. We investigated causes, comorbidities, costs, and outcomes of encephalitis in a population-based cohort. ICD-10 codes corresponding to encephalitis were used to identify health services records for all adults from 2004 to 2019. Data were cross-validated for identified diagnoses based on laboratory confirmation using univariate and multivariate statistical analyses. We identified persons with a diagnosis of encephalitis and abnormal cerebrospinal fluid (CSF) results (n = 581) in whom viral genome was detected (n = 315) in a population of 3.2 million adults from 2004 to 2019. Viral genome-positive CSF samples included HSV-1 (n = 133), VZV (n = 116), HSV-2 (n = 34), enterovirus (n = 4), EBV (n = 5), and CMV (n = 3) with the remaining viruses included JCV (n = 12) and HHV-6 (n = 1). The mean Charlson Comorbidity Index (2.0) and mortality rate (37.6%) were significantly higher in the CSF viral genome-negative encephalitis group although the mean costs of care were significantly higher for the CSF viral genome-positive group. Cumulative incidence rates showed increased CSF VZV detection in persons with encephalitis, which predominated in persons over 65 years with a higher mean Charlson index. We detected HSV-2 and VZV more frequently in CSF from encephalitis cases with greater material-social deprivation. The mean costs of care were significantly greater for HSV-1 encephalitis group. Encephalitis remains an important cause of neurological disability and death with a viral etiology in 54.2% of affected adults accompanied by substantial costs of care and mortality. Virus-associated encephalitis is evolving with increased VZV detection, especially in older persons.

TRPV4 channel is involved in HSV-2 infection in human vaginal epithelial cells through triggering Ca2+ oscillation.

Herpes simplex virus (HSV) infection induces a rapid and transient increase in intracellular calcium concentration ([Ca2+]i), which plays a critical role in facilitating viral entry. T-type calcium channel blockers and EGTA, a chelate of extracellular Ca2+, suppress HSV-2 infection. But the cellular mechanisms mediating HSV infection-activated Ca2+ signaling have not been completely defined. In this study we investigated whether the TRPV4 channel was involved in HSV-2 infection in human vaginal epithelial cells. We showed that the TRPV4 channel was expressed in human vaginal epithelial cells (VK2/E6E7). Using distinct pharmacological tools, we demonstrated that activation of the TRPV4 channel induced Ca2+ influx, and the TRPV4 channel worked as a Ca2+-permeable channel in VK2/E6E7 cells. We detected a direct interaction between the TRPV4 channel protein and HSV-2 glycoprotein D in the plasma membrane of VK2/E6E7 cells and the vaginal tissues of HSV-2-infected mice as well as in phallic biopsies from genital herpes patients. Pretreatment with specific TRPV4 channel inhibitors, GSK2193874 (1-4 µM) and HC067047 (100 nM), or gene silence of the TRPV4 channel not only suppressed HSV-2 infectivity but also reduced HSV-2-induced cytokine and chemokine generation in VK2/E6E7 cells by blocking Ca2+ influx through TRPV4 channel. These results reveal that the TRPV4 channel works as a Ca2+-permeable channel to facilitate HSV-2 infection in host epithelial cells and suggest that the design and development of novel TRPV4 channel inhibitors may help to treat HSV-2 infections.

Novel mutations in antiviral multiresistant HSV-2 genital lesion: A case report.

HSV-2 antiviral resistance mainly occurs in immunocompromised patients and especially in HIV-positive individuals receiving long-term antiviral treatment. Those situations can be challenging as few alternatives are available for HSV infection management. To describe clinical and virological significance of two novel potential HSV-2 resistance mutations after treating an obese patient with a pseudotumoral genital HSV-related lesion. Consecutive different antiviral treatments were used: valacyclovir (VACV) then foscarnet (FOS) then topical cidofovir (CDV) and finally imiquimod. Under VACV, genotypic resistance testing revealed a novel mutation within viral thymidine kinase (TK, gene UL23) not previously reported but probably accounting for antiviral resistance: W89G, similar to W88R mutation reported in HSV-1 TK, known to be associated with ACV resistance for HSV-1. Under FOS, while initial mutations were still present, a second genotypic resistance testing performed on persisting lesions showed a novel mutation within viral DNA polymerase (DNA pol, gene UL30): C625R. All three antivirals used in this case are small molecules and pharmacokinetics of VACV, FOS, and CDV have not been evaluated in animals and there are very few studies in human. As small molecules are poorly bound to proteins and distribution volume is increased in obese patients, there is risk of underdosage. This mechanism is suspected to be involved in emergence of resistance mutation and further data is needed to adapt, closely to patient profile, antiviral dosage. This report describes a chronic HSV-2 genital lesion, with resistance to current antivirals and novel mutations within viral TK and DNA pol which may confer antiviral resistance.

Detection of human herpesviruses in cerebrospinal fluids collected from patients suspected of neuroinfectious diseases.

The full spectrum of human herpesviruses (HHV)-associated neuroinfectious diseases in immunocompetent adults remains unclear. Hence, we sought to elucidate the epidemiology and clinical features of these diseases. The study subjects were patients over 16 years old suspected of neuroinfectious diseases who underwent spinal tap performed by neurologists in our university hospital between April 2013 and March 2018. The presence of seven HHV DNAs in cerebrospinal fluid (CSF) was determined by real-time PCR. HHV DNAs were detected in 33 (10.2%) of the 322 patients. The most frequently detected herpesvirus was varicella zoster virus (VZV) (19 patients), followed by HHV-6 (four patients), herpes simplex virus (HSV)-1 (three patients), HSV-2 (three patients), and Epstein-Barr virus (two patients). HHV DNAs were detected in CSF collected from patients with various neuroinfectious diseases, including myelitis, peripheral neuritis, encephalitis, and meningitis. All patients with HSV-1 DNA had encephalitis, whereas all patients with HSV-2 DNA had meningitis. Eleven of the 19 patients with VZV DNA had meningitis. Patients with VZV-associated encephalitis (median age, 80 years) were significantly older than non-encephalitis patients (median age, 60.5 years) (P = 0.046). Although post-herpetic neuralgia was observed in seven (54%) of the 13 patients with VZV and without encephalitis, no such neurological sequela was observed in the four encephalitis patients. In conclusion, HHVs were associated with approximately 10% of neuroinfectious diseases in this cohort. VZV was the most common pathogen, probably due to the large number of VZV meningitis patients. In addition, patients with VZV-associated meningitis were significantly younger than patients with VZV-associated encephalitis.

Development of a multiplex droplet digital PCR assay for detection of enterovirus, parechovirus, herpes simplex virus 1 and 2 simultaneously for diagnosis of viral CNS infections.

BACKGROUND: Enterovirus (EV), parechovirus (HPeV), herpes simplex virus 1 and 2 (HSV1/2) are common viruses leading to viral central nervous system (CNS) infections which are increasingly predominant but exhibit deficiency in definite pathogen diagnosis with gold-standard quantitative PCR method. Previous studies have shown that droplet digital PCR (ddPCR) has great potential in pathogen detection and quantification, especially in low concentration samples. METHODS: Targeting four common viruses of EV, HPeV, HSV1, and HSV2 in cerebrospinal fluid (CSF), we developed a multiplex ddPCR assay using probe ratio-based multiplexing strategy, analyzed the performance, and evaluated it in 97 CSF samples collected from patients with suspected viral CNS infections on a two-channel ddPCR detection system. RESULTS: The four viruses were clearly distinguished by their corresponding fluorescence amplitude. The limits of detection for EV, HPeV, HSV1, and HSV2 were 5, 10, 5, and 10 copies per reaction, respectively. The dynamic range was at least four orders of magnitude spanning from 2000 to 2 copies per reaction. The results of 97 tested clinical CSF specimens were identical to those deduced from qPCR/qRT-PCR assays using commercial kits. CONCLUSION: The multiplex ddPCR assay was demonstrated to be an accurate and robust method which could detect EV, HPeV, HSV1, and HSV2 simultaneously. It provides a useful tool for clinical diagnosis and disease monitoring of viral CNS infections.

A novel cocktail therapy based on quintuplet combination of oncolytic herpes simplex virus-2 vectors armed with interleukin-12, interleukin-15, GM-CSF, PD1v, and IL-7 × CCL19 results in enhanced antitumor efficacy.

BACKGROUND: Selectively replicating herpes simplex virus-2 (HSV-2) vector is a promising treatment for cancer therapy. The insertion of multiple transgenes into the viral genome has been performed to improve its oncolytic activity. METHODS: Herein, we simultaneously constructed five "armed" oncolytic viruses (OVs), designated oHSV2-IL12, -IL15, GM-CSF, -PD1v, and IL7 × CCL19. These OVs delete the ICP34.5 and ICP47 genes with the insertion of transgenes into the deleted ICP34.5 locus. The anti-tumor efficacy in vivo was tested in the syngeneic 4T1 and CT26 tumor-bearing mice model. RESULTS: The OVs showed comparable oncolytic capability in vitro. The combination therapy of oHSV2-IL12, -IL15, GM-CSF, -PD1v, and IL7 × CCL19 exhibited the highest tumor inhibition efficacy compared with the treatment of single OV or two OVs combination. CONCLUSIONS: The OVs armed with different transgenes combination therapy also named 5-valent oHSV2 (also called cocktail therapy) might be an effective therapeutic strategy for solid tumors.

Development of a cell-based reporter assay for detection of Human alphaherpesviruses.

Immunosuppressed patients can suffer from Human alphaherpesvirus (HSV) infection with fast evolution, severe atypical symptomatology, and often-fatal outcome. Thus, the development and validation of new methods in vitro and in vivo to promote an early diagnosis and effective treatment of these patients are crucial. Therefore, this work aimed to develop a cell-based reporter assay for the detection of HSV through the transfection of Vero cells with the ICP10 promoter from HSV-2 linked to the pZsGreen1-1 plasmid. The assay was evaluated on Vero cells infected with HSV-1 or HSV-2 and followed by treating them with anti-HSV agents (acyclovir, gallic acid, convallatoxin, and Uncaria sp. extract) or with no anti-HSV activity agents (Passiflora edulis extract and cardenolide derivatives). The GFP expression was increased by both HSV cellular infection, which was detected by flow cytometry and fluorescence microscopy. F2R Zsgreen1-1 cells infection with 200 and 600 PFU/mL of HSV-2 increased the fluorescence intensity, when compared to the controls, by approximately 30% and 60%, respectively. Infection with 100 and 600 PFU/mL of HSV-1 also increased the fluorescence intensity by approximately 20% and 35%, when compared to the controls, respectively. The F2R ZsGreen1-1 system revealed to be an efficient assay, which can be used for clinical diagnosis, antiviral resistance evaluation, HSV cycle studies, and new antiviral drug research.

Detection of Major Human Herpesviruses in Iranian Patients with Suspected Encephalitis Using Multiplex Polymerase Chain Reaction.

BACKGROUND: The Herpesviridae family plays a significant etiological role in central nervous system viral infections during primary infection or reactivation from a latent form. Early detection is crucial because prescribing some antivirals can prevent severe side effects or life-threatening conditions. METHODS: In this study, 251 CSF specimens were collected from patients with clinical suspicion of viral encephalitis in Pars Hospital, Tehran, Iran. DNA was extracted, and a multiplex PCR was designed to investigate the presence of herpes simplex virus-1, herpes simplex virus-2, varicella zoster virus, Epstein-Barr virus, and cytomegalovirus. RESULTS: Overall, 59 cases of the 251 CSF samples were positive for multiplex PCR (23.5%). The most frequent positive findings were EBV and HSV, with a prevalence of 10.3% and 8.7% (5.5% HSV-1 and 3.1% HSV-2), respectively. Four co-infections were also seen in this study. CONCLUSIONS: This multiplex PCR assay detects simultaneously different herpesviruses in CSF samples of patients with suspected encephalitis in 2 rounds of PCR amplification; therefore, it is a reliable and cost-saving diagnostic method for evaluating patients infected with herpesvirus with neurological disorders.

Recent Out-of-Africa Migration of Human Herpes Simplex Viruses.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are ubiquitous human pathogens. Both viruses evolved from simplex viruses infecting African primates and they are thus thought to have left Africa during early human migrations. We analyzed the population structure of HSV-1 and HSV-2 circulating strains. Results indicated that HSV-1 populations have limited geographic structure and the most evident clustering by geography is likely due to recent bottlenecks. For HSV-2, the only level of population structure is accounted for by the so-called "worldwide" and "African" lineages. Analysis of ancestry components and nucleotide diversity, however, did not support the view that the worldwide lineage followed early humans during out-of-Africa dispersal. Although phylogeographic analysis confirmed an African origin for both viruses, molecular dating with a method that corrects for the time-dependent rate phenomenon indicated that HSV-1 and HSV-2 migrated from Africa in relatively recent times. In particular, we estimated that the HSV-2 worldwide lineage left the continent in the 18th century, which corresponds to the height of the transatlantic slave trade, possibly explaining the high prevalence of HSV-2 in the Americas (second highest after Africa). The limited geographic clustering of HSV-1 makes it difficult to date its exit from Africa. The split between the basal clade, containing mostly African sequences, and all other strains was dated at ∼5,000 years ago. Our data do not imply that herpes simplex viruses did not infect early humans but show that the worldwide distribution of circulating strains is the result of relatively recent events.

Subclinical Genital Herpes Shedding in HIV/Herpes Simplex Virus 2-Coinfected Women during Antiretroviral Therapy Is Associated with an Increase in HIV Tissue Reservoirs and Potentially Promotes HIV Evolution.

Antigen (Ag)-specific immune responses to chronic infections, such as herpes simplex virus type 2 (HSV-2) in HIV/HSV-coinfected persons, may sustain HIV tissue reservoirs by promoting T-cell proliferation but are poorly studied in women on antiretroviral therapy (ART). Mixed anogenital swabs and cervical secretions were self-collected by nine HIV/HSV-2-coinfected women during ART for 28 days to establish subclinical HSV DNA shedding rates and detection of HIV RNA by real-time PCR. Typical herpes lesion site biopsy (TLSB) and cervical biopsy specimens were collected at the end of the daily sampling period. Nucleic acids (NA) isolated from biopsy specimens had HIV quantified and HIV envC2-V5 single-genome amplification (SGA) and T-cell receptor (TCR) repertoires assessed. Women had a median CD4 count of 537 cells/µl (IQR: 483 to 741) at enrollment and HIV plasma viral loads of <40 copies/ml. HSV DNA was detected on 12% of days (IQR: 2 to 25%) from anogenital specimens. Frequent subclinical HSV DNA shedding was associated with increased HIV DNA tissue concentrations and increased divergence from the most recent common ancestor (MRCA), an indicator of HIV replication. Distinct predominant TCR clones were detected in cervical and TLSB specimens in a woman with frequent HSV DNA shedding, with mixing of minor variants between her tissues. In contrast, more limited TCR repertoire mixing was observed in two women with less frequent subclinical HSV DNA shedding. Subclinical HSV shedding in HIV/HSV-coinfected women during ART may sustain HIV tissue reservoirs via Ag exposure or HIV replication. This study provides evidence supporting further study of interventions targeting suppression of Ag-specific immune responses as a component of HIV cure strategies.IMPORTANCE Persons with HIV infection are frequently coinfected with chronic herpesviruses, which periodically replicate and produce viable herpes virions, particularly in anogenital and cervical tissues. Persistent protein expression results in proliferation of CD8+ and CD4+ T cells, and the latter could potentially expand and sustain HIV tissue reservoirs. We found HSV genital shedding rates were positively correlated with HIV DNA concentrations and HIV divergence from ancestral sequences in tissues. Our work suggests that immune responses to common coinfections, such as herpesviruses, may sustain HIV tissue reservoirs during suppressive ART, suggesting future cure strategies should study interventions to suppress replication or reactivation of chronic herpes infections.

Phosphoregulation of a Conserved Herpesvirus Tegument Protein by a Virally Encoded Protein Kinase in Viral Pathogenicity and Potential Linkage between Its Evolution and Viral Phylogeny.

Us3 proteins of herpes simplex virus 1 (HSV-1) and HSV-2 are multifunctional serine-threonine protein kinases. Here, we identified an HSV-2 tegument protein, UL7, as a novel physiological substrate of HSV-2 Us3. Mutations in HSV-2 UL7, which precluded Us3 phosphorylation of the viral protein, significantly reduced mortality, viral replication in the vagina, and development of vaginal disease in mice following vaginal infection. These results indicated that Us3 phosphorylation of UL7 in HSV-2 was required for efficient viral replication and pathogenicity in vivo Of note, this phosphorylation was conserved in UL7 of chimpanzee herpesvirus (ChHV), which phylogenetically forms a monophyletic group with HSV-2 and the resurrected last common ancestral UL7 for HSV-2 and ChHV. In contrast, the phosphorylation was not conserved in UL7s of HSV-1, which belongs to a sister clade of the monophyletic group, the resurrected last common ancestor for HSV-1, HSV-2, and ChHV, and other members of the genus Simplexvirus that are phylogenetically close to these viruses. Thus, evolution of Us3 phosphorylation of UL7 coincided with the phylogeny of simplex viruses. Furthermore, artificially induced Us3 phosphorylation of UL7 in HSV-1, in contrast to phosphorylation in HSV-2, had no effect on viral replication and pathogenicity in mice. Our results suggest that HSV-2 and ChHV have acquired and maintained Us3 phosphoregulation of UL7 during their evolution because the phosphoregulation had an impact on viral fitness in vivo, whereas most other simplex viruses have not because the phosphorylation was not necessary for efficient fitness of the viruses in vivoIMPORTANCE It has been hypothesized that the evolution of protein phosphoregulation drives phenotypic diversity across species of organisms, which impacts fitness during their evolution. However, there is a lack of information regarding linkage between the evolution of viral phosphoregulation and the phylogeny of virus species. In this study, we clarified the novel HSV-2 Us3 phosphoregulation of UL7 in infected cells, which is important for viral replication and pathogenicity in vivo We also showed that the evolution of Us3 phosphoregulation of UL7 was linked to the phylogeny of viruses that are phylogenetically close to HSV-2 and to the phosphorylation requirements for the efficient in vivo viral fitness of HSV-2 and HSV-1, which are representative of viruses that have and have not evolved phosphoregulation, respectively. This study reports the first evidence showing that evolution of viral phosphoregulation coincides with phylogeny of virus species and supports the hypothesis regarding the evolution of viral phosphoregulation during viral evolution.