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.

Herpes Simplex Virus Type 1 Clinical Isolates Respond to UL29-Targeted siRNA Swarm Treatment Independent of Their Acyclovir Sensitivity.

Acyclovir is the drug of choice for the treatment of herpes simplex virus (HSV) infections. Acyclovir-resistant HSV strains may emerge, especially during long-term drug use, and subsequently cause difficult-to-treat exacerbations. Previously, we set up a novel treatment approach, based on enzymatically synthesized pools of siRNAs, or siRNA swarms. These swarms can cover kilobases-long target sequences, reducing the likelihood of resistance to treatment. Swarms targeting the UL29 essential gene of HSV-1 have demonstrated high efficacy against HSV-1 in vitro and in vivo. Here, we assessed the antiviral potential of a UL29 siRNA swarm against circulating strains of HSV-1, in comparison with acyclovir. All circulating strains were sensitive to both antivirals, with the half-maximal inhibitory concentrations (IC50) in the range of 350-1911 nM for acyclovir and 0.5-3 nM for the UL29 siRNA swarm. Additionally, we showed that an acyclovir-resistant HSV-1, devoid of thymidine kinase, is highly sensitive to UL29 siRNA treatment (IC50 1.0 nM; Imax 97%). Moreover, the detected minor variations in the RNAi target of the HSV strains had no effect on the potency or efficacy of UL29 siRNA swarm treatment. Our findings support the development of siRNA swarms for the treatment of HSV-1 infections, in order to circumvent any potential acyclovir resistance.

Herpes Simplex Virus Mistyping due to HSV-1 × HSV-2 Interspecies Recombination in Viral Gene Encoding Glycoprotein B.

Human herpes simplex viruses (HSV) 1 and 2 are extremely common human pathogens with overlapping disease spectra. Infections due to HSV-1 and HSV-2 are distinguished in clinical settings using sequence-based "typing" assays. Here we describe a case of HSV mistyping caused by a previously undescribed HSV-1 × HSV-2 recombination event in UL27, the HSV gene that encodes glycoprotein B. This is the first documented case of HSV mistyping caused by an HSV-1 × HSV-2 recombination event and the first description of an HSV interspecies recombination event in UL27, which is frequently used as a target for diagnostics and experimental therapeutics. We also review the primer and probe target sequences for a commonly used HSV typing assay from nearly 700 HSV-1 and HSV-2 samples and find that about 4% of HSV-1 samples have a single nucleotide change in at least one of these loci, which could impact assay performance. Our findings illustrate how knowledge of naturally occurring genomic variation in HSV-1 and HSV-2 is essential for the design and interpretation of molecular diagnostics for these viruses.

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.

Novel transcription regulatory sequences and factors of the immune evasion protein ICP47 (US12) of herpes simplex viruses.

BACKGROUND: Herpes simplex virus (HSV) can cause encephalitis. Its infected cell polypeptide 47 (ICP47), encoded by immediate-early gene US12, promotes immune escape. ICP47 was modified in the clinically approved oncolytic HSV (oHSV) T-Vec. However, transcription regulatory sequence (TRS) and transcription regulatory factor (TRF) of HSV US12 are seldom reported. METHODS: Previously, our laboratory isolated a new HSV strain named HSV-1-LXMW from a male patient with oral herpes in Beijing, China. Firstly, the genetic tree was used to analyze its genetic relationship. The US12 TRS and TRF in HSV-1-LXMW were found by using predictive software. Secondly, the further verification by the multi-sequence comparative analysis shown that the upstream DNA sequence of HSV US12 gene contained the conserved region. Finally, the results of literature search shown that the expression of transcription factors was related to the tissue affinity of HSV-1 and HSV-2, so as to increase the new understanding of the transcriptional regulation of HSV biology and oncolytic virus (OVs) therapy. RESULTS: Here we reported the transcriptional regulation region sequence of our new HSV-1-LXMW, and its close relationship with HSV-1-CR38 and HSV-1-17. Importantly we identified eight different kinds of novel TRSs and TRFs of HSV US12 for the first time, and found they are conserved among HSV-1 (c-Rel, Elk-1, Pax-4), HSV-2 (Oct-1, CF2-II, E74A, StuAp) or both HSVs (HNF-4). The TRFs c-Rel and Oct-1 are biologically functional respectively in immune escape and viral replication during HSV infection. CONCLUSIONS: Our findings have important implication to HSV biology, infection, immunity and oHSVs.

Characterization of a Novel Alphaherpesvirus Isolated from the Fruit Bat Pteropus lylei in Vietnam.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

The new scope of virus taxonomy: partitioning the virosphere into 15 hierarchical ranks.

Virus taxonomy emerged as a discipline in the middle of the twentieth century. Traditionally, classification by virus taxonomists has been focussed on the grouping of relatively closely related viruses. However, during the past few years, the International Committee on Taxonomy of Viruses (ICTV) has recognized that the taxonomy it develops can be usefully extended to include the basal evolutionary relationships among distantly related viruses. Consequently, the ICTV has changed its Code to allow a 15-rank classification hierarchy that closely aligns with the Linnaean taxonomic system and may accommodate the entire spectrum of genetic divergence in the virosphere. The current taxonomies of three human pathogens, Ebola virus, severe acute respiratory syndrome coronavirus and herpes simplex virus 1 are used to illustrate the impact of the expanded rank structure. This new rank hierarchy of virus taxonomy will stimulate further research on virus origins and evolution, and vice versa, and could promote crosstalk with the taxonomies of cellular organisms.

Genetic and phenotypic intrastrain variation in herpes simplex virus type 1 Glasgow strain 17 syn+-derived viruses.

The Glasgow s17 syn+ strain of herpes simplex virus 1 (HSV1) is arguably the best characterized strain and has provided the reference sequence for HSV1 genetic studies. Here we show that our original s17 syn+ stock was a mixed population from which we have isolated a minor variant that, unlike other strains in the laboratory, fails to be efficiently released from infected cells and spreads predominantly by direct cell-to-cell transmission. Analysis of other s17-derived viruses that had been isolated elsewhere revealed a number with the same release phenotype. Second-generation sequencing of 8 plaque-purified s17-derived viruses revealed sequences that vary by 50 single-nucleotide polymorphisms (SNPs), including approximately 10 coding SNPs. This compared to interstrain variations of around 800 SNPs in strain Sc16, of which a quarter were coding changes. Amongst the variations found within s17, we identified 13 variants of glycoprotein C within the original stock of virus that were predominantly a consequence of altered homopolymeric runs of C residues. Characterization of seven isolates coding for different forms of gC indicated that all were expressed, despite six of them lacking a transmembrane domain. While the release phenotype did not correlate directly with any of these identified gC variations, further demonstration that nine clinical isolates of HSV1 also fail to spread through extracellular release raises the possibility that propagation in tissue culture had altered the HSV1 s17 transmission phenotype. Hence, the s17 intrastrain variation identified here offers an excellent model for understanding both HSV1 transmission and tissue culture adaptation.

UL23, UL30, and UL5 characterization of HSV1 clinical strains isolated from hematology department patients.

Genotypic diagnosis of HSV drug resistance can be performed routinely in a clinically relevant time. Nevertheless, data about HSV mutations (polymorphism or resistance) is not exhaustive which hinders the interpretation of such tests. The UL23, UL30, and UL5 genes are of greatest interest as these encode, respectively, thymidine kinase, DNA polymerase, and helicase, which, if mutated may affect the effectiveness of acyclovir, foscarnet, cidofovir, and helicase-primase inhibitors. The present study aimed to extensively characterize UL23, UL30, and UL5 genes. A total of 239 clinical HSV1 recovered from patients admitted to the hematology departments of the Lyon teaching hospitals were included in this single-center retrospective study. Drug resistance was evaluated using the neutral red dye-uptake assay, and sequencing using the Sanger method. Additional information on HSV1 natural polymorphism and resistance is now available. Twenty-two amino acid substitutions related to polymorphism were described on UL23 (E43A, L50M, L68R, Q109K, A133V, A136N, S150L, D258N, S263L, P280S, N301S, A316S, M322L, I326V, D330A, D338H, Q342H, T344I, Q349R, V352L, R370W, E371D), and 6 amino acid substitutions on UL30 (G641R, G645D, E649G, G679D, R681L, I966M). Moreover, the UL23 substitution L242P was added to ACV resistance-related mutations. There were 12 substitutions on UL23 (A37S, V70M, S74L, H151N, P154S, P155Q, L159R, E225L, Y248H, Q270R, N303Y, M372I), and 8 on UL5 (L49I, L138V, S173L, A280T, A575V, V600A, A602T, D862N) that remain of unclear significance with regards to drug resistance. The mean (±standard deviation, SD) number of natural polymorphisms in UL23 was 2.53 (±2.55), in UL30 it was 0.83 (±1.02), and in UL5 it was 5.00 (±1.59) There was no association between HSV1 phenotype and the frequency of substitutions. The results reported herein provide valuable new information concerning HSV1 mutations that will assist the interpretation of genotypic assays.

Effect of storage time and temperature on the detection of Pseudomonas aeruginosa, Acanthamoeba and Herpes Simplex Virus from corneal impression membranes.

The effect of storage time and temperature on the recovery of pathogen DNA from polytetrafluorethylene (PTFE) was investigated. PTFE impression membranes were inoculated with Pseudomonas aeruginosa, Herpes Simplex Virus-1 (HSV-1) or Acanthamoeba and stored at -70 °C, -20 °C, +4 °C or +35 °C. PCR was performed on days 0, 1, 2, 3, 7 and months 1, 3 and 10 post-inoculation. We found no reduction in the DNA recovery of any of the studied microorganisms for the first 3 days of storage up to +35 °C. For HSV-1 and P. aeruginosa, storage for 3 months at +35 °C was associated with a significant reduction in DNA recovery (P<0.001), but not at +4 °C, -20 °C or -70 °C for 1 month for P. aeruginosa and for 10 months for HSV-1. Acanthamoeba DNA recovery was not affected by any storage parameters (P=0.203). These results will inform the investigation of microbial keratitis where access to microbiological testing is not readily available.

Genome-Wide Surveillance of Genital Herpes Simplex Virus Type 1 From Multiple Anatomic Sites Over Time.

Here we present genomic and in vitro analyses of temporally separated episodes of herpes simplex virus type 1 (HSV-1) shedding by an HSV-1-seropositive and human immunodeficiency virus (HIV)/HSV-2-seronegative individual who has frequent recurrences of genital HSV-1. Using oligonucleotide enrichment, we compared viral genomes from uncultured swab specimens collected on different days and from distinct genital sites. We found that viral genomes from 7 swab specimens and 3 cultured specimens collected over a 4-month period from the same individual were 98.5% identical. We observed a >2-fold difference in the number of minority variants between swab specimens from lesions, swab specimens from nonlesion sites, and cultured specimens. This virus appeared distinct in its phylogenetic relationship to other strains, and it contained novel coding variations in 21 viral proteins. This included a truncation in the UL11 tegument protein, which is involved in viral egress and spread. Normal immune responses were identified, suggesting that unique viral genomic features may contribute to the recurrent genital infection that this participant experiences.

Ultrasensitive Capture of Human Herpes Simplex Virus Genomes Directly from Clinical Samples Reveals Extraordinarily Limited Evolution in Cell Culture.

Herpes simplex viruses (HSVs) are difficult to sequence due to their large DNA genome, high GC content, and the presence of repeats. To date, most HSV genomes have been recovered from culture isolates, raising concern that these genomes may not accurately represent circulating clinical strains. We report the development and validation of a DNA oligonucleotide hybridization panel to recover nearly complete HSV genomes at abundances up to 50,000-fold lower than previously reported. Using copy number information on herpesvirus and host DNA background via quantitative PCR, we developed a protocol for pooling for cost-effective recovery of more than 50 HSV-1 or HSV-2 genomes per MiSeq run. We demonstrate the ability to recover >99% of the HSV genome at >100× coverage in 72 h at viral loads that allow whole-genome recovery from latently infected ganglia. We also report a new computational pipeline for rapid HSV genome assembly and annotation. Using the above tools and a series of 17 HSV-1-positive clinical swabs sent to our laboratory for viral isolation, we show limited evolution of HSV-1 during viral isolation in human fibroblast cells compared to the original clinical samples. Our data indicate that previous studies using low-passage-number clinical isolates of herpes simplex viruses are reflective of the viral sequences present in the lesion and thus can be used in phylogenetic analyses. We also detect superinfection within a single sample with unrelated HSV-1 strains recovered from separate oral lesions in an immunosuppressed patient during a 2.5-week period, illustrating the power of direct-from-specimen sequencing of HSV.IMPORTANCE Herpes simplex viruses affect more than 4 billion people across the globe, constituting a large burden of disease. Understanding the global diversity of herpes simplex viruses is important for diagnostics and therapeutics as well as cure research and tracking transmission among humans. To date, most HSV genomics has been performed on culture isolates and DNA swabs with high quantities of virus. We describe the development of wet-lab and computational tools that enable the accurate sequencing of near-complete genomes of HSV-1 and HSV-2 directly from clinical specimens at abundances >50,000-fold lower than previously sequenced and at significantly reduced cost. We use these tools to profile circulating HSV-1 strains in the community and illustrate limited changes to the viral genome during the viral isolation process. These techniques enable cost-effective, rapid sequencing of HSV-1 and HSV-2 genomes that will help enable improved detection, surveillance, and control of this human pathogen.

Identification of broadly reactive epitopes targeting major glycoproteins of Herpes simplex virus (HSV) 1 and 2 - An immunoinformatics analysis.

Infections due to both HSV-1 and HSV-2 constitute an enormous health burden worldwide. Development of vaccine against herpes infections is a WHO supported public health priority. The viral glycoproteins have always been the major hotspots for vaccine designing. The present study was aimed to identify the conserved T and B cell epitopes in the major glycoproteins of both HSV-1 and HSV-2 via rigorous computational approaches. Identification of promiscuous T cell epitopes is of utmost importance in vaccine designing as such epitopes are capable of binding to several allelic forms of HLA and could generate effective immune response in the host. The criteria designed for identification of T and B cell epitopes was that it should be conserved in both HSV-1 and 2, promiscuous, have high affinity towards HLA alleles, should be located on the surface of glycoproteins and not be present in the glycosylation sites. This study led to the identification of 17 HLA Class II and 26 HLA Class I T cell epitopes, 9 linear and some conformational B cell epitopes. The identified T cell epitopes were further subjected to molecular docking analysis to analyze their binding patterns. Altogether we have identified 4 most promising regions in glycoproteins (2-gB, 1-gD, 1-gH) of HSV-1 and 2 which are promiscuous to HLA Class II alleles and have overlapping HLA Class I and B cell epitopes, which could be very useful in generating both arms of immune response in the host i.e. adaptive as well as humoral immunity. Further the authors propose the cross-validation of the identified epitopes in experimental settings for confirming their immunogenicity to support the present findings.

HSV-1 clinical isolates with unique in vivo and in vitro phenotypes and insight into genomic differences.

Strain-specific factors contribute in significant but undefined ways to the variable incidence of herpes simplex virus (HSV) recrudescence. Studies that investigate these strain-specific factors are needed. Here, we used qPCR, in vitro assays, and genomic sequencing to identify important relationships between in vitro and clinical phenotypes of unique HSV-1 clinical isolates. Nine HSV-1 isolates from individuals displaying varying reactivation patterns were studied. Isolates associated with frequent recurrent herpes labialis (RHL) (1) displayed higher rates of viral shedding in the oral cavity than those associated with rare RHL and (2) tended to replicate more efficiently at 33 °C than 39 °C. HSV-1 isolates also displayed a more stable phenotype during propagation in U2OS cells than in Vero cells. Draft genome sequences of four isolates and one variant spanning 95.6 to 97.2 % of the genome were achieved, and whole-genome alignment demonstrated that the majority of these isolates clustered with known North American/European isolates. These findings revealed procedures that could help identify unique genotypes and phenotypes associated with HSV-1 isolates, which can be important for determining viral factors critical for regulating HSV-1 reactivation.

Herpesvirus: an underestimated virus.

Herpes simplex virus (HSV) infections are common and widespread; nevertheless, their outcome can be of unpredictable prognosis in neonates and in immunosuppressed patients. Anti-HSV therapy is effective, but the emergence of drug-resistant strains or the drug toxicity that hamper the treatment is of great concern. Vaccine has not yet shown relevant benefit; therefore, palliative prophylactic measures have been adopted to prevent diseases. This short review proposes to present concisely the history of HSV, its taxonomy, physical structure, and replication and to explore the pathogenesis of the infection, clinical manifestations, laboratory diagnosis, treatment, prophylaxis and epidemiology of the diseases.

Natural recombination in alphaherpesviruses: Insights into viral evolution through full genome sequencing and sequence analysis.

Recombination in alphaherpesviruses was first described more than sixty years ago. Since then, different techniques have been used to detect recombination in natural (field) and experimental settings. Over the last ten years, next-generation sequencing (NGS) technologies and bioinformatic analyses have greatly increased the accuracy of recombination detection, particularly in field settings, thus contributing greatly to the study of natural alphaherpesvirus recombination in both human and veterinary medicine. Such studies have highlighted the important role that natural recombination plays in the evolution of many alphaherpesviruses. These studies have also shown that recombination can be a safety concern for attenuated alphaherpesvirus vaccines, particularly in veterinary medicine where such vaccines are used extensively, but also potentially in human medicine where attenuated varicella zoster virus vaccines are in use. This review focuses on the contributions that NGS and sequence analysis have made over the last ten years to our understanding of recombination in mammalian and avian alphaherpesviruses, with particular focus on attenuated live vaccine use.

Type-specific detection of herpes simplex virus type 1 and type 2 using the cobas® HSV 1 and 2 test on the cobas® 4800 platform.

INTRODUCTION: HSV-1 and HSV-2 are among the most common causes of sexually transmitted infections (stis) globally. these infections are strongly associated with increased risk of hiv acquisition and rare, but devastating, neonatal disease. available treatment options can reduce HSV transmission and improve quality of life. accurate diagnosis early in disease can improve patient management. Areas covered: This paper describes the clinical manifestations of HSV infection often used for clinical diagnostic purposes. The paper then describes the evolution of laboratory diagnostic assays. Serology, culture and molecular diagnostics are described since all are currently in use. The features and performance characteristics of the cobas 4800 HSV1 and HSV2 Test (cobas HSV) on the cobas 4800® system (cobas 4800) are described in detail. Expert commentary: Diagnosis of HSV has historically been unreliable or technically difficult, but the availability of molecular assays such as the cobas HSV test for detection and typing of herpes can improve our ability to correctly manage this disease. Utilization of tools such as the cobas HSV assay may help shorten the time to accurate diagnosis and treatment thus potentially reducing the risk of transmission and the global burden of HSV.

Genotyping of herpes simplex virus type 1 by whole-genome sequencing.

A previous phylogenetic analysis based on 32 full-length sequences of herpes simplex virus type 1 (HSV-1) suggested three major phylogenetic groups (phylogroups) with distinct geographic distribution: (1) western strains from Europe and North America, (2) isolates from Asia and one American strain and (3) isolates from Africa only. Here, we sequenced the genomes of additional 10 clinical HSV-1 isolates from Germany, and subsequently compared these sequences to 40 published HSV-1 genomes. The present data demonstrate that HSV-1 is the most diverse human alphaherpesvirus (mean pairwise p-distance of 0.756 %) and confirm the tripartite tree. However, as the German isolates cluster with strains of both phylogroups I and II, it is demonstrated that the latter is also present in Europe and thus is a Eurasian phylogroup. Tree-order scans indicate that HSV-1 evolution is massively influenced by recombination including all investigated strains regardless of the areal distribution of the phylogroups. Numerous recombination events in the evolution of HSV-1 may also influence genotyping as the present HSV-1 genotyping schemes do not yield results consistent with phylogroup classification. Genotyping of HSV-1 is currently based on analyses of intragenic sequence polymorphisms of US2, glycoprotein G (gG, US4) and gI (US7). Each of the 10 German HSV-1 isolates displayed a different US2/gG/gI-genotype combination, but clustered either in phylogroup I or II. In conclusion, the phylogroup concept provides a HSV-1 typing scheme that largely reflects human migration history, whereas the analysis of single-nucleotide polymorphisms fails to render significant biological properties, but allows description of individual genetic traits.

De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies.

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging. To improve the assembly of HHV-1 genomes we have employed a hybrid genome assembly protocol using data from two sequencing technologies: the short-read Roche 454 and the long-read Oxford Nanopore MinION sequencers. We sequenced 18 HHV-1 cell culture-isolated clinical specimens collected from immunocompromised patients undergoing antiviral therapy. The susceptibility of the samples to several antivirals was determined by plaque reduction assay. Hybrid genome assembly resulted in a decrease in the number of contigs in 6 out of 7 samples and an increase in N(G)50 and N(G)75 of all 7 samples sequenced by both technologies. The approach also enhanced the detection of non-canonical contigs including a rearrangement between the unique (UL) and repeat (T/IRL) sequence regions of one sample that was not detectable by assembly of 454 reads alone. We detected several known and novel resistance-associated mutations in UL23 and UL30 genes. Genome-wide genetic variability ranged from <1% to 53% of amino acids in each gene exhibiting at least one substitution within the pool of samples. The UL23 gene had one of the highest genetic variabilities at 35.2% in keeping with its role in development of drug resistance. The assembly of accurate, full-length HHV-1 genomes will be useful in determining genetic determinants of drug resistance, virulence, pathogenesis and viral evolution. The numerous, complex repeat regions of the HHV-1 genome currently remain a barrier towards this goal.

Viral forensic genomics reveals the relatedness of classic herpes simplex virus strains KOS, KOS63, and KOS79.

Herpes simplex virus 1 (HSV-1) is a widespread global pathogen, of which the strain KOS is one of the most extensively studied. Previous sequence studies revealed that KOS does not cluster with other strains of North American geographic origin, but instead clustered with Asian strains. We sequenced a historical isolate of the original KOS strain, called KOS63, along with a separately isolated strain attributed to the same source individual, termed KOS79. Genomic analyses revealed that KOS63 closely resembled other recently sequenced isolates of KOS and was of Asian origin, but that KOS79 was a genetically unrelated strain that clustered in genetic distance analyses with HSV-1 strains of North American/European origin. These data suggest that the human source of KOS63 and KOS79 could have been infected with two genetically unrelated strains of disparate geographic origins. A PCR RFLP test was developed for rapid identification of these strains.