Development and evaluation of topical formulations that contain hydroethanolic extract from Schinus terebinthifolia against HSV-1 infection

Abstract The therapeutic drugs to treat Herpes simplex virus (HSV) infections have toxic side effects and there has been an emergence of drug-resistant strains. Therefore, the search for new treatments for HSV infections is mounting. In the present study, semi-solid formulations containing a crude hydroethanolic extract (CHE) from Schinus terebinthifolia were developed. Skin irritation, cutaneous permeation, and in vivo therapeutic efficacy of the formulations were investigated. Treatment with the ointment formulations did not result in any signs of skin irritation while the emulsions increased the thickness of the epidermis in Swiss mice. The cutaneous permeation test indicated that the CHE incorporated in the formulations permeated through the skin layers and was present in the epidermis and dermis even 3 h after topical application. In vivo antiviral activity in BALB/c mice treated with the CHE ointments was better than those treated with the CHE emulsions and did not significantly differ from an acyclovir-treated group. Taken together, this suggests that the incorporation of CHE in the ointment may be a potential candidate for the alternative topical treatment of herpetic lesions.

Antiviral Active Compounds Derived from Natural Sources against Herpes Simplex Viruses.

Herpes simplex viruses (HSV) are ubiquitously distributed with a seroprevalence ranging up to 95% in the adult population. Refractory viral infections with herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) represent a major global health issue. In particular, the increasing occurrence of resistance to conventional antiviral drugs make the therapy of such infections even more challenging. For instance, the frequent and long-term use of acyclovir and other nucleoside analogues targeting the viral DNA-polymerase enhance the development of resistant viruses. Particularly, the incidental increase of those strains in immunocompromised patients is alarming and represent a major health concern. Alternative treatment concepts are clearly needed. Natural products such as herbal medicines showed antiherpetic activity in vitro and in vivo and proved to be an excellent source for the discovery and isolation of novel antivirals. By this means, numerous plant-derived compounds with antiviral or antimicrobial activity could be isolated. Natural medicines and their ingredients are well-tolerated and could be a good alternative for treating herpes simplex virus infections. This review provides an overview of the recent status of natural sources such as plants, bacteria, fungi, and their ingredients with antiviral activity against herpes simplex viruses. Furthermore, we highlight the most potent herbal medicines and ingredients as promising candidates for clinical investigation and give an overview about the most important drug classes along with their potential antiviral mechanisms. The content of this review is based on articles that were published between 1996 and 2021.

Genomic nucleotide-based distance analysis for delimiting old world monkey derived herpes simplex virus species.

BACKGROUND: Herpes simplex viruses form a genus within the alphaherpesvirus subfamily, with three identified viral species isolated from Old World monkeys (OWM); Macacine alphaherpesvirus 1 (McHV-1; herpes B), Cercopithecine alphaherpesvirus 2 (SA8), and Papiine alphaherpesvirus 2 (PaHV-2; herpes papio). Herpes B is endemic to macaques, while PaHV-2 and SA8 appear endemic to baboons. All three viruses are genetically and antigenically similar, with SA8 and PaHV-2 thought to be avirulent in humans, while herpes B is a biosafety level 4 pathogen. Recently, next-generation sequencing (NGS) has resulted in an increased number of published OWM herpes simplex genomes, allowing an encompassing phylogenetic analysis. RESULTS: In this study, phylogenetic networks, in conjunction with a genome-based genetic distance cutoff method were used to examine 27 OWM monkey herpes simplex isolates. Genome-based genetic distances were calculated, resulting in distances between lion and pig-tailed simplex viruses themselves, and versus herpes B core strains that were higher than those between PaHV-2 and SA8 (approximately 14 and 10% respectively). The species distance cutoff was determined to be 8.94%, with the method recovering separate species status for PaHV-2 and SA8 and showed that lion and pig-tailed simplex viruses (vs core herpes B strains) were well over the distance species cutoff. CONCLUSIONS: We propose designating lion and pig-tailed simplex viruses as separate, individual viral species, and that this may be the first identification of viral cryptic species.

A correlation analysis of HHV infection and its predictive factors in an HIV-seropositive population in Yunnan, China.

Human herpesviruses (HHVs) have a particularly high prevalence in certain high-risk populations and cause increased morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS). Screening and treating subclinical HHV infections reduce human immunodeficiency virus (HIV) infection incidence, disease progression, and transmission. However, there are few studies on HHVs, HIV coinfection rates, and their related risk factors. We aimed to clarify the prevalence of all eight HHVs in peripheral blood samples collected from HIV-positive patients, and explore the association of HHV infection in HIV-positive patients in an HIV-seropositive population in Yunnan. We recruited 121 HIV-positive patients with highly active antiretroviral therapy (HAART) and 45 healthy individuals. All the eight HHVs were detected using polymerase chain reaction and their epidemiological information and clinical data were collected and statistically analyzed. A high prevalence of HHVs (89.3%) was observed in individuals with HIV infections and with herpes simplex virus (HSV)-2 (65.3%), and HSV-1 (59.5%) being the most common. Coinfection with more than two different HHVs was more common in patients with HIV infections receiving HAART (72.7%) than in healthy controls. Older age, being married, higher HIV-1 plasma viral loads, and use of antiviral protease inhibitors were independently correlated with an increased frequency of HHVs, but we found no association with CD4 count, WHO HIV clinical stage, and HIV infection duration. Our findings are of great significance for the prevention of HHV opportunistic infection in patients with AIDS and their clinical treatment.

Reliable Detection of Herpes Simplex Virus Sequence Variation by High-Throughput Resequencing.

High-throughput sequencing (HTS) has resulted in data for a number of herpes simplex virus (HSV) laboratory strains and clinical isolates. The knowledge of these sequences has been critical for investigating viral pathogenicity. However, the assembly of complete herpesviral genomes, including HSV, is complicated due to the existence of large repeat regions and arrays of smaller reiterated sequences that are commonly found in these genomes. In addition, the inherent genetic variation in populations of isolates for viruses and other microorganisms presents an additional challenge to many existing HTS sequence assembly pipelines. Here, we evaluate two approaches for the identification of genetic variants in HSV1 strains using Illumina short read sequencing data. The first, a reference-based approach, identifies variants from reads aligned to a reference sequence and the second, a de novo assembly approach, identifies variants from reads aligned to de novo assembled consensus sequences. Of critical importance for both approaches is the reduction in the number of low complexity regions through the construction of a non-redundant reference genome. We compared variants identified in the two methods. Our results indicate that approximately 85% of variants are identified regardless of the approach. The reference-based approach to variant discovery captures an additional 15% representing variants divergent from the HSV1 reference possibly due to viral passage. Reference-based approaches are significantly less labor-intensive and identify variants across the genome where de novo assembly-based approaches are limited to regions where contigs have been successfully assembled. In addition, regions of poor quality assembly can lead to false variant identification in de novo consensus sequences. For viruses with a well-assembled reference genome, a reference-based approach is recommended.

Characterization and serotype distribution of Aggregatibacter actinomycetemcomitans: Relationship of serotypes to herpesvirus and periodontal status in Indian subjects.

BACKGROUND: The virulence of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) in any individual depends on the type of strain of this bacterium. To our knowledge, there have been no studies reported in Indian subjects about A. actinomycetemcomitans serotype occurrence, co-existence with herpes virus and the possible influence of such co-existence on periodontal pathology. METHODS: Subjects for this study were a subset of a larger study to identify the prevalence of A. actinomycetemcomitans in chronic periodontitis. A total of 63 subjects (12 periodontally healthy and 51 with chronic periodontitis) who were positive for A. actinomycetemcomitans were serotyped for strain-level identification. The presence of Human Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) was tested in subgingival plaque samples by polymerase chain reaction. RESULTS: All five serotypes a to e were detected. Of the samples analyzed 38.09% harbored a single serotype, 36.5% had two serotypes, 6.3% demonstrated three and 4.7% demonstrated four serotypes. None of the samples showed presence of JP2 strain. Serotypes b, c, and e were most frequently identified in these individuals (46.03%, 36.5% and 38.09% respectively). Presence of serotypes b and c and absence of serotype d was associated with increased PD and CAL. Among 63 samples analyzed, 11 samples had CMV, four samples had EBV and nine samples had both these viruses. The PD and CAL were significantly higher (p = 0.04) when a combination of CMV and one of the serotypes was present indicating a pathological role of the coexistence. CONCLUSION: Multiple serotypes are associated with chronic periodontitis in Indians, however, JP2 strains are not detectable in this cohort. Presence of multiple serotypes and a combination of any serotype with herpesvirus is associated with greater severity of the disease.

Genome sequence variation among isolates of monkey B virus (Macacine alphaherpesvirus 1) from captive macaques.

Complete genome sequences of 19 strains of monkey B virus (Macacine alphaherpesvirus 1; BV) isolated from several macaque species were determined. A low level of sequence variation was present among BV isolates from rhesus macaques. Most variation among BV strains isolated from rhesus macaques was located in regions of repetitive or quasi-repetitive sequence. Variation in coding sequences (polypeptides and miRNAs) was minor compared to regions of non-coding sequences. Non-coding sequences in the long and short repeat regions of the genome did however exhibit islands of conserved sequence. Oral and genital isolates from a single monkey were identical in sequence and varied only in the number of iterations of repeat units in several areas of repeats. Sequence variation between BV isolates from different macaque species (different BV genotypes) was much greater and was spread across the entire genome, confirming the existence of different genotypes of BV in different macaque species.

3,19-isopropylideneandrographolide suppresses early gene expression of drug-resistant and wild type herpes simplex viruses.

A diterpenoid lactone, 3,19-isopropylideneandrographolide (IPAD) compound isolated from Andrographis paniculata (Burm. f.) Nees, has been reported to inhibit herpes simplex virus type 1 (HSV-1) infection at the post-entry step. To identify the molecular target of IPAD, this study characterized the inhibitory effect of IPAD on infection of Vero cells by HSV-1, HSV-2 and a drug-resistant (DR) HSV-1 strain ACGr4 (acyclovir-resistant and thymidine kinase (TK)-deficient). Viral production, gene and protein expression were determined using plaque assays, quantitative RT-PCR and western blotting, respectively. The results showed that IPAD inhibited HSV-1, HSV-2 and DR-HSV-1 infections at 6-12 h post-infection, a time that corresponded with E gene expression. IPAD completely suppressed ICP8 transcription and translation as well as DNA replication and gD expression in the three strains tested, while acyclovir suppressed transcription and translation of UL30 and gD of HSV-2, HSV-1, but had no effect on DR-HSV-1. These results showed that IPAD has a different molecular target from acyclovir and might therefore be an alternative drug for HSV-1 and HSV-2 wild types and DR-HSV-1 strains.

Elaboration of a clinical and paraclinical score to estimate the probability of herpes simplex virus encephalitis in patients with febrile, acute neurologic impairment.

Herpes simplex virus (HSV) encephalitis is associated with a high risk of mortality and sequelae, and early diagnosis and treatment in the emergency department are necessary. However, most patients present with non-specific febrile, acute neurologic impairment; this may lead clinicians to overlook the diagnosis of HSV encephalitis. We aimed to identify which data collected in the first hours in a medical setting were associated with the diagnosis of HSV encephalitis. We conducted a multicenter retrospective case-control study in four French public hospitals from 2007 to 2013. The cases were the adult patients who received a confirmed diagnosis of HSV encephalitis. The controls were all the patients who attended the emergency department of Grenoble hospital with a febrile acute neurologic impairment, without HSV detection by polymerase chain reaction (PCR) in the cerebrospinal fluid (CSF), in 2012 and 2013. A multivariable logistic model was elaborated to estimate factors significantly associated with HSV encephalitis. Finally, an HSV probability score was derived from the logistic model. We identified 36 cases and 103 controls. Factors independently associated with HSV encephalitis were the absence of past neurological history (odds ratio [OR] 6.25 [95 % confidence interval (CI): 2.22-16.7]), the occurrence of seizure (OR 8.09 [95 % CI: 2.73-23.94]), a systolic blood pressure ≥140 mmHg (OR 5.11 [95 % CI: 1.77-14.77]), and a C-reactive protein <10 mg/L (OR 9.27 [95 % CI: 2.98-28.88]). An HSV probability score was calculated summing the value attributed to each independent factor. HSV encephalitis diagnosis may benefit from the use of this score based upon some easily accessible data. However, diagnostic evocation and probabilistic treatment must remain the rule.

Herpes simplex virus encephalitis in Peru: a multicentre prospective study.

Herpes simplex virus (HSV) is one of the most commonly identified infectious aetiologies of encephalitis in North America and Europe. The epidemiology of encephalitis beyond these regions, however, is poorly defined. During 2009-2012 we enrolled 313 patients in a multicentre prospective study of encephalitis in Peru, 45 (14·4%) of whom had confirmed HSV infection. Of 38 patients with known HSV type, 84% had HSV-1 and 16% had HSV-2. Patients with HSV infection were significantly more likely to present in the summer months (44·4% vs. 20·0%, P = 0·003) and have nausea (60·0% vs. 39·8%, P = 0·01) and rash (15·6% vs. 5·3%, P = 0·01) compared to patients without HSV infection. These findings highlight differences in the epidemiology and clinical presentation of HSV encephalitis outside of the Northern Hemisphere that warrant further investigation. Furthermore, there is an urgent need for improved HSV diagnostic capacity and availability of intravenous acyclovir in Peru.

Carriage of herpes simplex virus and human papillomavirus in oral mucosa is rare in young women: A long-term prospective follow-up.

BACKGROUND: Following the primary oral infection, herpes simplex virus (HSV) establishes latency in the ganglia of sensory neurons. Episodically induced by stress, HSV is able to cause recurrent infection at the primary infection site, accompanied by virus shedding. The oral shedding of HSV contributes to mother-child-transmission of HSV. Human papillomavirus (HPV) is associated with oral malignancies, and its interaction with oral HSV should be studied further. OBJECTIVES: To analyze the prevalence of HSV-1 and HSV-2-infection in oral mucosal scrapings of women during and after pregnancy and to elucidate the prevalence of HPV and HSV-co-carriage in oral mucosa. STUDY DESIGN: A longitudinal cohort study of 304 mothers in the Finnish Family HPV study followed-up for 6 years after pregnancy, with 7 serial samplings. Mothers' oral brush samples were analyzed with quantitative PCR for HSV-1 and -2 DNA and the findings were compared with their HPV DNA status. RESULTS: Altogether, 2.2% of all 1873 collected epithelial brush samples were HSV-1 DNA positive, while none tested HSV-2 DNA positive. Of the 304 mothers, 11.8% were HSV-1 DNA positive at least once. Most of the women who tested HSV-1 DNA positive before delivery remained HSV-1 DNA positive also after pregnancy. HSV-1 positive women were almost invariably HPV-negative; only four (0.2%) samples were detected with HSV-HPV co-carriage. CONCLUSIONS: This is the first prospective follow-up study on oral HSV shedding and its association with coexistent HPV, analyzed in the same oral mucosal scrapings. HSV and HPV co-carriage is rare in oral mucosa of healthy young mothers.

Detection and differentiation of herpes simplex viruses by use of the viper platform: advantages, limitations, and concerns.

The Viper HSV-Q(x) assay was evaluated for the detection of herpes simplex virus 1 (HSV-1) and HSV-2 in specimens from oral, anogenital, and other miscellaneous sites. The HSV-Q(x) assay was found to be highly sensitive and accurate; however, a gray zone may be required for specimens with values falling between 50 and 800 maximum relative fluorescence units.

Determination of viral load by quantitative real-time PCR in herpes simplex encephalitis patients.

OBJECTIVE: Human herpesviruses cause various acute, subacute, and chronic disorders of the central nervous system and peripheral nervous systems in adults and children. The objective of the present study is to summarize the experience gained with the estimation of viral load in the central nervous system of children and adults with herpes simplex encephalitis (HSE) admitted to a neurological institute at Nagpur, India, by quantitative real-time PCR (qPCR) assay within the past 4 years. METHODS: The qPCR assay was evaluated retrospectively in 242 cerebrospinal fluid (CSF) samples from patients. Evaluation of possible relationships was done between the herpes simplex virus (HSV) DNA concentration in CSF with that of patients' clinical and laboratory manifestations. The prevalence of the type of HSV in the study population was also determined using type-specific real-time PCR analysis. RESULTS AND CONCLUSIONS: Real-time analysis using type-specific primers revealed the presence of predominantly HSV-1 genotype in the study population. The qPCR results show that in patients with higher viral loads in their CSF, a greater number of cases were associated with the presence of lesions in the brain as revealed by computed tomography/magnetic resonance imaging scan. They required acyclovir therapy for a longer duration and had a poorer clinical outcome than the patients with lower viral loads in their CSF.

Acyclovir-resistant herpes simplex encephalitis in a patient treated with anti-tumor necrosis factor-α monoclonal antibodies.

Herpes simplex virus is the most common cause of severe sporadic encephalitis. We report a case of herpes simplex type 1-encephalitis in a 50-year-old woman receiving anti-tumor necrosis factor-α monoclonal antibodies adalimumab. Although she was an acyclovir naïve patient, a mixed viral population (wild-type and acyclovir-resistant bearing a thymidine-kinase mutation) was identified in the cerebrospinal fluid. The virus in cerebrospinal fluid evolved and a second thymidine-kinase mutant virus emerged. Combined foscavir and acyclovir treatment resolved the herpes simplex encephalitis. To our knowledge, this is the first report of acyclovir-resistant herpes simplex encephalitis in a patient treated with adalimumab.

Detection of human herpesviruses (HHVs) in semen of human male infertile patients.

Recently we demonstrated an ectopic expression of the human herpesvirus 1 thymidine kinase (HHV1-TK) gene by functioning of an intrinsic endogenous promoter in the transgenic rat (TG-rat), suggesting that HHV1 infection in humans induces expression of the TK gene with the ectopic promoter in the testis and results in accumulation of HHV1-TK protein, triggering male infertility similar to that in the TG-rat. Hence, in this study, we started to investigate a relationship between infection of herpesvirus and human male infertility. Semen was donated by Chinese male infertile patients (153 men, aged 21-49 years) with informed consent, followed by DNA preparation and analysis by PCR and DNA sequencing. Semen volume, sperm number and density, and sperm motility were examined. DNAs of HHV1, HHV4, HHV5 and HHV6 were confirmed by PCR, electrophoresis and DNA sequencing. Finally, virus DNA was identified in 59 patients (39%). The number of carriers was 39 (25%) for HHV1, 6 (4%) for HHV4, 33 (22%) for HHV5 and 3 (2%) for HHV6, respectively. Moreover, double-infection was found in 22 out of 59 specimens (37%), most of which were double-infection of HHV1 and HHV5 (15 out of 22 carriers). Though slight severity was present in some of the carriers, the relationship between virus infection and sperm impairment was not conclusive. Accordingly, it is essential to examine whether the viral HHV1-TK gene is expressed in the testis of the infertile human HHV carrier.

Genome-wide prediction of vaccine targets for human herpes simplex viruses using Vaxign reverse vaccinology.

Herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2) are the most common infectious agents of humans. No safe and effective HSV vaccines have been licensed. Reverse vaccinology is an emerging and revolutionary vaccine development strategy that starts with the prediction of vaccine targets by informatics analysis of genome sequences. Vaxign (http://www.violinet.org/vaxign) is the first web-based vaccine design program based on reverse vaccinology. In this study, we used Vaxign to analyze 52 herpesvirus genomes, including 3 HSV-1 genomes, one HSV-2 genome, 8 other human herpesvirus genomes, and 40 non-human herpesvirus genomes. The HSV-1 strain 17 genome that contains 77 proteins was used as the seed genome. These 77 proteins are conserved in two other HSV-1 strains (strain F and strain H129). Two envelope glycoproteins gJ and gG do not have orthologs in HSV-2 or 8 other human herpesviruses. Seven HSV-1 proteins (including gJ and gG) do not have orthologs in all 40 non-human herpesviruses. Nineteen proteins are conserved in all human herpesviruses, including capsid scaffold protein UL26.5 (NP_044628.1). As the only HSV-1 protein predicted to be an adhesin, UL26.5 is a promising vaccine target. The MHC Class I and II epitopes were predicted by the Vaxign Vaxitop prediction program and IEDB prediction programs recently installed and incorporated in Vaxign. Our comparative analysis found that the two programs identified largely the same top epitopes but also some positive results predicted from one program might not be positive from another program. Overall, our Vaxign computational prediction provides many promising candidates for rational HSV vaccine development. The method is generic and can also be used to predict other viral vaccine targets.

[Analysis of clinical features of herpes encephalitis].

Herpes encephalitis makes up more than 20% of all viral encephalitises in countries with moderate climate. Over the period of 1971-2008, 112 inpatients with herpes encephalitis have been examined and treated. Outcomes were as follows: 90 (80%) patients recovered and 22 (20%) patients died. The diagnostic criteria were the relation with herpes simplex virus type I (HSV-1) by immunological and clinical data in the presence of clinical symptoms of viral encephalitis for recovered patients and clinical symptoms of acute encephalitis, morphological foci of necrosis in the brain, relation with HSV, the absence of other alternative diagnosis for dead patients. Based on the analysis of 112 patients, the authors present clinical symptoms of herpes encephalitis. The presentation is supported by 2 case reports.

Atopic dermo-respiratory syndrome is a correlate of eczema herpeticum.

BACKGROUND: Factors favoring the emergence of eczema herpeticum (EH) in patients with atopic dermatitis (AD) remain elusive. The aim of this work was to identify changes in clinical and laboratory parameters in acute EH patients, before and after 6 weeks of treatment, as well as differences between AD patients with and without a history of EH. METHODS: A total of 235 adult subjects were included and subdivided into six groups: (i) AD patients with acute EH, (ii) AD patients with history of EH, (iii) AD without EH but with recurrent herpes simplex virus (HSV) infections, (iv) AD without EH or recurrent HSV infections and healthy non-AD controls (v) with and (vi) without recurrent HSV infections. Clinical examination of AD, assessment of atopic status and severity were performed. Total IgE, allergen-specific IgE and differential blood count were analyzed. Clinical diagnosis of acute EH was confirmed by PCR. RESULTS: More male patients with AD were affected by EH than female patients. Acute episodes of EH are characterized by lower levels of lymphocytes and higher levels of monocytes. AD patients with history of EH display higher total IgE serum levels (ADEH(+) HSV(+) vs ADEH(-) HSV(+) , P < 0.001) and higher sensitization profiles and stronger severity of AD (EASI and SCORAD; ADEH(+) HSV(+) vs ADEH(-) HSV(+) , P < 0.001). Concomitant asthma and rhinitis were identified as correlates of EH. CONCLUSION: From these data, we conclude that AD patients with EH display a distinct clinical and biological phenotype.

Etiology of encephalitis in Australia, 1990-2007.

Encephalitis is a clinical syndrome commonly caused by emerging pathogens, which are not under surveillance in Australia. We reviewed rates of hospitalization for patients with encephalitis in Australia's most populous state, New South Wales, from January 1990 through December 2007. Encephalitis was the primary discharge diagnosis for 5,926 hospital admissions; average annual hospitalization rate was 5.2/100,000 population. The most commonly identified pathogen was herpes simplex virus (n = 763, 12.9%). Toxoplasma encephalitis and subacute sclerosing panencephalitis showed notable declines. The average annual encephalitis case-fatality rate (4.6%) and the proportion of patients hospitalized with encephalitis with no identified pathogen (69.8%, range 61.5%-78.7%) were stable during the study period. The nonnotifiable status of encephalitis in Australia and the high proportion of this disease with no known etiology may conceal emergence of novel pathogens. Unexplained encephalitis should be investigated, and encephalitis hospitalizations should be subject to statutory notification in Australia.