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.

Dermatitis during Spaceflight Associated with HSV-1 Reactivation.

Human alpha herpesviruses herpes simplex virus (HSV-1) and varicella zoster virus (VZV) establish latency in various cranial nerve ganglia and often reactivate in response to stress-associated immune system dysregulation. Reactivation of Epstein Barr virus (EBV), VZV, HSV-1, and cytomegalovirus (CMV) is typically asymptomatic during spaceflight, though live/infectious virus has been recovered and the shedding rate increases with mission duration. The risk of clinical disease, therefore, may increase for astronauts assigned to extended missions (>180 days). Here, we report, for the first time, a case of HSV-1 skin rash (dermatitis) occurring during long-duration spaceflight. The astronaut reported persistent dermatitis during flight, which was treated onboard with oral antihistamines and topical/oral steroids. No HSV-1 DNA was detected in 6-month pre-mission saliva samples, but on flight day 82, a saliva and rash swab both yielded 4.8 copies/ng DNA and 5.3 × 104 copies/ng DNA, respectively. Post-mission saliva samples continued to have a high infectious HSV-1 load (1.67 × 107 copies/ng DNA). HSV-1 from both rash and saliva samples had 99.9% genotype homology. Additional physiological monitoring, including stress biomarkers (cortisol, dehydroepiandrosterone (DHEA), and salivary amylase), immune markers (adaptive regulatory and inflammatory plasma cytokines), and biochemical profile markers, including vitamin/mineral status and bone metabolism, are also presented for this case. These data highlight an atypical presentation of HSV-1 during spaceflight and underscore the importance of viral screening during clinical evaluations of in-flight dermatitis to determine viral etiology and guide treatment.

Functional development of a V3/glycan-specific broadly neutralizing antibody isolated from a case of HIV superinfection.

Stimulating broadly neutralizing antibodies (bnAbs) directly from germline remains a barrier for HIV vaccines. HIV superinfection elicits bnAbs more frequently than single infection, providing clues of how to elicit such responses. We used longitudinal antibody sequencing and structural studies to characterize bnAb development from a superinfection case. BnAb QA013.2 bound initial and superinfecting viral Env, despite its probable naive progenitor only recognizing the superinfecting strain, suggesting both viruses influenced this lineage. A 4.15 Å cryo-EM structure of QA013.2 bound to native-like trimer showed recognition of V3 signatures (N301/N332 and GDIR). QA013.2 relies less on CDRH3 and more on framework and CDRH1 for affinity and breadth compared to other V3/glycan-specific bnAbs. Antigenic profiling revealed that viral escape was achieved by changes in the structurally-defined epitope and by mutations in V1. These results highlight shared and novel properties of QA013.2 relative to other V3/glycan-specific bnAbs in the setting of sequential, diverse antigens.

A diverse collection of B cells responded to HIV infection in infant BG505.

Increasing evidence suggests infants develop unique neutralizing antibody (nAb) responses to HIV compared to adults. Here, we dissected the nAb response of an infant whose virus is in clinical trials as a vaccine immunogen, with a goal of characterizing the broad responses in the infant to this antigen. We isolated 73 nAbs from infant BG505 and identified a large number of clonal families. Twenty-six antibodies neutralized tier 2 viruses-in some cases, viruses from the same clade as BG505, and in others, a different clade, although none showed notable breadth. Several nAbs demonstrated antibody-dependent cellular cytotoxicity activity and targeted the V3 loop. These findings suggest an impressive polyclonal response to HIV infection in infant BG505, adding to the growing evidence that the nAb response to HIV in infants is polyclonal-a desirable vaccine response to a rapidly evolving virus like HIV.

Epitope profiling reveals binding signatures of SARS-CoV-2 immune response in natural infection and cross-reactivity with endemic human CoVs.

A major goal of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine efforts is to elicit antibody responses that confer protection. Mapping the epitope targets of the SARS-CoV-2 antibody response is critical for vaccine design, diagnostics, and development of therapeutics. Here, we develop a pan-coronavirus phage display library to map antibody binding sites at high resolution within the complete viral proteomes of all known human-infecting coronaviruses in patients with mild or moderate/severe coronavirus disease 2019 (COVID-19). We find that the majority of immune responses to SARS-CoV-2 are targeted to the spike protein, nucleocapsid, and ORF1ab and include sites of mutation in current variants of concern. Some epitopes are identified in the majority of samples, while others are rare, and we find variation in the number of epitopes targeted between individuals. We find low levels of SARS-CoV-2 cross-reactivity in individuals with no exposure to the virus and significant cross-reactivity with endemic human coronaviruses (CoVs) in convalescent sera from patients with COVID-19.

Diversity and Function of Maternal HIV-1-Specific Antibodies at the Time of Vertical Transmission.

Infants of HIV-positive mothers can acquire HIV infection by various routes, but even in the absence of antiviral treatment, the majority of these infants do not become infected. There is evidence that maternal antibodies provide some protection from infection, but gestational maternal antibodies have not yet been characterized in detail. One of the most studied vertically infected infants is BG505, as the virus from this infant yielded an Envelope protein that was successfully developed as a stable trimer. Here, we isolated and characterized 39 HIV-specific neutralizing monoclonal antibodies (nAbs) from MG505, the mother of BG505, at a time point just prior to vertical transmission. These nAbs belonged to 21 clonal families and employed a variety of VH genes. Many were specific for the HIV-1 Env V3 loop, and this V3 specificity correlated with measurable antibody-dependent cellular cytotoxicity (ADCC) activity. The isolated nAbs did not recapitulate the full breadth of heterologous or autologous virus neutralization by contemporaneous plasma. Notably, we found that the V3-targeting nAb families neutralized one particular maternal Env variant, even though all tested variants had low V3 sequence diversity and were measurably bound by these nAbs. None of the nAbs neutralized BG505 transmitted virus. Furthermore, the MG505 nAb families were found at relatively low frequencies within the maternal B cell repertoire; all were less than 0.25% of total IgG sequences. Our findings illustrate an example of the diversity of HIV-1 nAbs within one mother, cumulatively resulting in a collection of antibody specificities that can contribute to the transmission bottleneck.IMPORTANCE Mother-to-child-transmission of HIV-1 offers a unique setting in which maternal antibodies both within the mother and passively transferred to the infant are present at the time of viral exposure. Untreated HIV-exposed human infants are infected at a rate of 30 to 40%, meaning that some infants do not get infected despite continued exposure to virus. Since the potential of HIV-specific immune responses to provide protection against HIV is a central goal of HIV vaccine design, understanding the nature of maternal antibodies may provide insights into immune mechanisms of protection. In this study, we isolated and characterized HIV-specific antibodies from the mother of an infant whose transmitted virus has been well studied.

Oligonucleotide Enrichment of HSV-1 Genomic DNA from Clinical Specimens for Use in High-Throughput Sequencing.

To date more than 400 genomes of herpes simplex virus 1 (HSV-1) and the distantly related HSV-2 have been examined using deep sequencing techniques. This powerful approach has been especially useful for revealing the global genetic diversity that exists within and between strains of each virus species. However, most early methods for high-throughput sequencing required the input of abundant viral genomic DNA to enable the successful production of sequencing libraries, and the generation of sufficient short-read sequencing data for de novo genome assembly and similar applications. Therefore, the majority of sequenced HSV strains have been cultured and expanded in vitro prior to genomic analysis, to facilitate isolation of sufficient viral DNA for sequencing-library preparation. Here, we describe an in-solution targeted enrichment procedure for isolating, enriching, and sequencing HSV genomic DNA directly from clinical specimens. When this enrichment technique is combined with traditional sequencing-library preparation procedures, the need for in vitro culturing, expansion, and purification of viral DNA is eliminated. Furthermore, enrichment reduces the large amount of nonviral DNA that is typically present in specimens obtained directly from natural infections, thereby increasing the likelihood of successful viral genome sequencing and assembly. We have used this approach to prepare viral DNA libraries from clinical specimens derived from skin swabs, saliva, blood, and similar sources. We then use these libraries for deep sequencing and successful de novo assembly of the ~152 kb viral genomes, at coverage depths exceeding 100-1000×, for both HSV-1 and HSV-2.

Personalized viral genomic investigation of herpes simplex virus 1 perinatal viremic transmission with dual fatality.

Here we present a personalized viral genomics approach to investigating a rare case of perinatal herpes simplex virus 1 (HSV-1) transmission that ended in death of both mother and neonate. We sought to determine whether the virus involved in this rare case had any unusual features that may have contributed to the dire patient outcome. A pregnant woman with negative HerpeSelect antibody test underwent cesarean section at 30 wk gestation and died the same day. The premature newborn died 5 d later. Both individuals were found postmortem to have positive blood HSV-1 PCR tests. Using oligonucleotide enrichment and deep sequencing, we determined that viral transmission from mother to infant was nearly perfect at the consensus genome level. At the virus population level, 77% of minor variants (MVs) in the mother's blood also appeared on the neonate's skin, of which more than half were disseminated into the neonate's blood. We also detected nonmaternal MVs that arose de novo in the neonate's viral populations. Of note, one de novo MV in the neonate's skin virus induced a nonsynonymous mutation in the UL6 protein, which is a component of the portal that allows DNA entry into new progeny capsids. This case suggests that perinatal viremic HSV-1 transmission includes the majority of genetic diversity from the maternal virus population and that new, nonsynonymous mutations can occur after relatively few rounds of replication. This report expands our understanding of viral transmission in humans and may lead to improved diagnostic strategies for neonatal HSV-1 acquisition.

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.

Differentiated Human SH-SY5Y Cells Provide a Reductionist Model of Herpes Simplex Virus 1 Neurotropism.

Neuron-virus interactions that occur during herpes simplex virus (HSV) infection are not fully understood. Neurons are the site of lifelong latency and are a crucial target for long-term suppressive therapy or viral clearance. A reproducible neuronal model of human origin would facilitate studies of HSV and other neurotropic viruses. Current neuronal models in the herpesvirus field vary widely and have caveats, including incomplete differentiation, nonhuman origins, or the use of dividing cells that have neuropotential but lack neuronal morphology. In this study, we used a robust approach to differentiate human SH-SY5Y neuroblastoma cells over 2.5 weeks, producing a uniform population of mature human neuronal cells. We demonstrate that terminally differentiated SH-SY5Y cells have neuronal morphology and express proteins with subcellular localization indicative of mature neurons. These neuronal cells are able to support a productive HSV-1 infection, with kinetics and overall titers similar to those seen in undifferentiated SH-SY5Y cells and the related SK-N-SH cell line. However, terminally differentiated, neuronal SH-SY5Y cells release significantly less extracellular HSV-1 by 24 h postinfection (hpi), suggesting a unique neuronal response to viral infection. With this model, we are able to distinguish differences in neuronal spread between two strains of HSV-1. We also show expression of the antiviral protein cyclic GMP-AMP synthase (cGAS) in neuronal SH-SY5Y cells, which is the first demonstration of the presence of this protein in nonepithelial cells. These data provide a model for studying neuron-virus interactions at the single-cell level as well as via bulk biochemistry and will be advantageous for the study of neurotropic viruses in vitroIMPORTANCE Herpes simplex virus (HSV) affects millions of people worldwide, causing painful oral and genital lesions, in addition to a multitude of more severe symptoms such as eye disease, neonatal infection, and, in rare cases, encephalitis. Presently, there is no cure available to treat those infected or prevent future transmission. Due to the ability of HSV to cause a persistent, lifelong infection in the peripheral nervous system, the virus remains within the host for life. To better understand the basis of virus-neuron interactions that allow HSV to persist within the host peripheral nervous system, improved neuronal models are required. Here we describe a cost-effective and scalable human neuronal model system that can be used to study many neurotropic viruses, such as HSV, Zika virus, dengue virus, and rabies virus.

ALBA4 modulates its stage-specific interactions and specific mRNA fates during Plasmodium yoelii growth and transmission.

Transmission of the malaria parasite occurs in an unpredictable moment, when a mosquito takes a blood meal. Plasmodium has therefore evolved strategies to prepare for transmission, including translationally repressing and protecting mRNAs needed to establish the infection. However, mechanisms underlying these critical controls are not well understood, including whether Plasmodium changes its translationally repressive complexes and mRNA targets in different stages. Efforts to understand this have been stymied by severe technical limitations due to substantial mosquito contamination of samples. Here using P. yoelii, for the first time we provide a proteomic comparison of a protein complex across asexual blood, sexual and sporozoite stages, along with a transcriptomic comparison of the mRNAs that are affected in these stages. We find that the Apicomplexan-specific ALBA4 RNA-binding protein acts to regulate development of the parasite's transmission stages, and that ALBA4 associates with both stage-specific and stage-independent partners to produce opposing mRNA fates. These efforts expand our understanding and ability to interrogate both sexual and sporozoite transmission stages and the molecular preparations they evolved to perpetuate their infectious cycle.

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line.

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

Rapid genome assembly and comparison decode intrastrain variation in human alphaherpesviruses.

UNLABELLED: Herpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. Here we developed and applied a streamlined new approach for assembly and comparison of large DNA viral genomes such as HSV-1. This viral genome assembly (VirGA) workflow incorporates a combination of de novo assembly, alignment, and annotation strategies to automate the generation of draft genomes for large viruses. We applied this approach to quantify the amount of variation between clonal derivatives of a common parental virus stock. In addition, we examined the genetic basis for syncytial plaque phenotypes displayed by a subset of these strains. In each of the syncytial strains, we found an identical DNA change, affecting one residue in the gB (UL27) fusion protein. Since these identical mutations could have appeared after extensive in vitro passaging, we applied the VirGA sequencing and comparison approach to two clinical HSV-1 strains isolated from the same patient. One of these strains was syncytial upon first culturing; its sequence revealed the same gB mutation. These data provide insight into the extent and origin of genome-wide intrastrain HSV-1 variation and present useful methods for expansion to in vivo patient infection studies. IMPORTANCE: Herpes simplex virus (HSV) infects more than 70% of adults worldwide, causing epithelial lesions and recurrent disease that manifests over a lifetime. Prior work has demonstrated that HSV strains vary from country to country and between individuals. However, the amount of variation within one strain has not been well studied. To address this, we developed a new approach for viral genome assembly (VirGA) and analysis. We used this approach to quantify the amount of variation between sister clones of a common parental virus stock and to determine the basis of a unique fusion phenotype displayed by several variants. These data revealed that while sister clones of one HSV stock are more than 98% identical, these variants harbor enough genetic differences to change their observed characteristics. Comparative genomics approaches will allow us to explore the impacts of viral inter- and intrastrain diversity on drug and vaccine efficacy.