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NASA NTRS2022

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.

Satish K. Mehta↗

NASA NTRSDate not supplied

Dermatitis During Spaceflight Associated With HSV-1 Reactivation- A Case Study

Human alpha herpesviruses herpes simplex virus (HSV-1 or -2) 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 a 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 high infectious HSV-1 load (1.67×107 copies/ng DNA). HSV-1 from both rash and saliva samples had 99.4% 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.

Herpes, Viral Reactivation, Spaceflight, Dermatiti↗

NASA NTRSDate not supplied

Method Development for Multiplex, In-Situ, and Real-Time Detection of Herpesvirus Reactivation in Spaceflight Crews using Nanopore Sequencing

Reactivation of latent herpesviruses in crews onboard the International Space Station (ISS) is a well-established spaceflight-associated phenomenon and has been linked to overall immune stress. Beyond an indicator of an altered immune state, this stress-induced reactivation of viruses such as herpesvirus simplex virus 1 (HSV-1), Epstein-Barr virus (EBV), and Varicella-Zoster virus (VZV) may cause clinical symptoms in crew. There is currently no established protocol for in-flight monitoring, and samples are analyzed post-flight using ground-based assays. A real-time, in-flight method for herpesvirus detection followed by stress-mitigation strategies would be a significant advance. In this study, we developed a real-time assay for the multiplex detection of HSV-1, EBV, and VZV from saliva that could be implemented for in-situ monitoring of ISS crew. This method builds on previously validated spaceflight-compatible portable molecular protocols and platforms, such as the miniPCR thermal cycler and the MinION sequencer. Since a metagenomic approach is not currently permitted for crew samples (NASA policy), we employed multiplexing-ready primers directed toward targeted regions within the HSV-1, EBV, and VZV genomes. We also used primers for the human gene, Statherin (STATH), as an internal control. These primers were validated using spiked-in, positive control HSV-1, EBV, and VZV from virus-negative saliva extracted using the Zymo-Research Quick-DNA/RNA Viral MagBead Kit. The PCR Barcoding Kit was used to prepare DNA libraries that were then sequenced on the MK1C and analyzed against known reference genomes using minimap2. Following validation of this method with spiked saliva samples, suspected herpesvirus-positive clinical saliva samples were successfully tested. Prior to use onboard the ISS, this method will be deployed to an analog environment during overwintering at Palmer Station, Antarctica in 2023. This work represents the successful application of molecular technologies currently onboard the ISS for the real-time monitoring of latent herpesvirus reactivation from saliva samples. This assay, in combination with stress-reduction strategies, holds the potential to manage herpesvirus reactivation in ISS crew, thereby improving health outcomes and overall immunity.

Patrick M. Rydzak↗

NASA NTRS2007

Subclinical Shed of Infectious Varicella zoster Virus in Astronauts

Aerosol borne varicella zoster virus (VZV) enters the nasopharynx and replicates in tonsillar T-cells, resulting in viremia and varicella (chickenpox). Virus then becomes latent in cranial nerve, dorsal root and autonomic nervous system ganglia along the entire neuraxis (1). Decades later, as cell-mediated immunity to VZV declines (4), latent VZV can reactivate to produce zoster (shingles). Infectious VZV is present in patients with varicella or zoster, but shed of infectious virus in the absence of disease has not been shown. We previously detected VZV DNA in saliva of astronauts during and shortly after spaceflight, suggesting stress induced subclinical virus reactivation (3). We show here that VZV DNA as well as infectious virus in present in astronaut saliva. VZV DNA was detected in saliva during and after a 13-day spaceflight in 2 of 3 astronauts (Fig. panel A). Ten days before liftoff, there was a rise in serum anti-VZV antibody in subjects 1 and 2, consistent with virus reactivation. In subject 3, VZV DNA was not detected in saliva, and there was no rise in anti-VZV antibody titer. Subject 3 may have been protected from virus reactivation by having zoster <10 years ago, which provides a boost in cell-medicated immunity to VZV (2). No VZV DNA was detected in astronaut saliva months before spaceflight, or in saliva of 10 age/sex-matched healthy control subjects sampled on alternate days for 3 weeks (88 saliva samples). Saliva taken 2-6 days after landing from all 3 subjects was cultured on human fetal lung cells (Fig. panel B). Infectious VZV was recovered from saliva of subjects 1 and 2 on the second day after landing. Virus specificity was confirmed by antibody staining and DNA analysis which showed it to be VZV of European descent, common in the US (5). Further, both antibody staining and DNA PCR demonstrated that no HSV-1 was detected in any infected culture. This is the first report of infectious VZV shedding in the absence of clinical disease. Spaceflight presents a uniquely stressful environment which includes physical isolation and confinement, anxiety, sleep deprivation, as well as exposure to increased radiation and microgravity. It is interesting that in our study, VZV and not HSV-1 reactivation was detected, since stress-induced HSV-1 reactivation has been reported (6). Future studies are needed to determine the specific inducer of VZV reactivation.

Cohrs, Randall J.↗

NASA NTRS2007

Subclinical Reactivation and Shed of Infectious Varicella Zoster Virus in Saliva of Astronauts

We have previously detected VZV in healthy astronauts both during spaceflight and shortly after landing. Herein, we show that VZV shed in seropositive astronauts is infectious. A total of 40 saliva samples were obtained from each of the 3 astronauts. From each astronaut, 14 samples were taken 109 to 133 days before liftoff, 1 sample was taken every day during 12 days in space, and one sample was taken for 14 consecutive days beginning the second day after landing. Quantitative PCR was used to detect VZV DNA in saliva. None of 42 preflight saliva samples contained VZV DNA. VZV DNA was detected in saliva from 2 of 3 astronauts. In 1 astronaut, 6 of 12 samples obtained during space flight contained 120 to 2,500 copies of VZV DNA per ml; after landing, 1250 copies of VZV DNA were present on day 2, 45 copies on day 3, and 110 copies on day 5. All samples taken 6 to 15 days after touchdown were negative for VZV DNA. In the second astronaut, 5 of 12 samples obtained during space flight contained 18 to 650 copies of VZV DNA per ml; after landing, 560 copies of VZV DNA were present in saliva on day 2, 340 copies on day 4, 45 copies on day 5, and 23 copes on day 6. All samples taken 7 to 15 days after touchdown were negative for VZV DNA. Saliva taken 2 to 6 days after landing from all 3 astronauts was cultured on human fetal lung cells. After one subcultivation, a cytopathic effect developed in cultures inoculated with saliva from the two astronauts whose saliva contained VZV DNA. Both PCR and immunostaining identified the isolates to be VZV and not HSV-1. Importantly, the astronaut in whom no VZV was detected had a history of zoster 9 years earlier. It is possible that a boost in cell-mediated immunity to VZV which is known to develop after zoster protected him from subclinical reactivation. The genotype of the two VZV isolates was determined by VZV ORF22-based PCR/sequencing along with FRET-based PCR assays that target specific nucleotide polymorphisms. Both VZV isolates were found to be the European genotype which also contained a rare MspI restriction enodnuclease site in VZV ORF62 at position 107,252. These findings extend our previous demonstration of VZV DNA in saliva of astronauts by showing that infectious VZV is also present. Thus, like HSV-1 and HSV-2, VZV can reactivate and shed infectious virus in the absence of clinical disease.

Cohrs, Randall J.↗

NASA NTRS1992

Effects of dimethyl prostaglandin A1 on herpes simplex virus and human immunodeficiency virus replication

We have investigated the direct effect of dimethyl prostaglandin A1 (dmPGA1) on the replication of herpes simplex virus (HSV) and human immunodeficiency virus type 1 (HIV-1). dmPGA1 significantly inhibited viral replication in both HSV and HIV infection systems at concentrations of dmPGA1 that did not adversely alter cellular DNA synthesis. The 50% inhibitory concentration (ID50) for several HSV type 1 (HSV-1) strains ranged from 3.8 to 5.6 micrograms/ml for Vero cells and from 4.6 to 7.3 micrograms/ml for human foreskin fibroblasts. The ID50s for two HSV-2 strains varied from 3.8 to 4.5 micrograms/ml for Vero cells; the ID50 was 5.7 micrograms/ml for human foreskin fibroblasts. We found that closely related prostaglandins did not have the same effect on the replication of HSV; dmPGE2 and dmPGA2 caused up to a 60% increase in HSV replication compared with that in untreated virus-infected cells. HIV-1 replication in acutely infected T cells (VB line) and chronically infected macrophages was assessed by quantitative decreases in p24 concentration. The effective ID50s were 2.5 micrograms/ml for VB cells acutely infected with HIV-1 and 5.2 micrograms/m for chronically infected macrophages. dmPGA1 has an unusual broad-spectrum antiviral activity against both HSV and HIV-1 in vitro and offers a new class of potential therapeutic agents for in vivo use.

Non-NASA Center↗

NASA NTRS2010

Latent Virus Reactivation: From Space to Earth

Reactivation of latent viruses is a recognized consequence of decreased immunity. More recently viral reactivation has been identified as an important in vivo indicator of clinically relevant immune changes. Viral reactivation can be determined quickly and easily by the presence of virus in saliva and other body fluids. Real-time polymerase chain reaction (PCR) is a highly sensitive and specific molecular method to detect the presence of specific viral DNA. Studies in astronauts demonstrated that herpes simplex virus type 1(HSV-1), Epstein-Barr Virus (EBV), cytomegalovirus (CMV), and varicella zoster virus (VZV) reactivate at rates above normal during and after spaceflight in response to moderately decreased T-cell immunity. This technology was expanded to patients on Earth beginning with human immune deficiency virus (HIV) immuno-compromised patients. The HIV patients shed EBV in saliva at rates 9-fold higher than observed in astronauts demonstrating that the level of EBV shedding reflects the severity of impaired immunity. Whereas EBV reactivation is not expected to produce serious effects in astronauts on missions of 6 months or less, VZV reactivation in astronauts could produce shingles. Reactivation of live, infectious VZV in astronauts with no symptoms was demonstrated in astronauts during and after spaceflight. We applied our technology to study VZV-induced shingles in patients. In a study of 54 shingles patients, we showed salivary VZV was present in every patient on the day antiviral (acyclovir) treatment was initiated. Pain and skin lesions decreased with antiviral treatment. Corresponding decreases in levels of VZV were also observed and accompanied recovery. Although the level of VZV in shingles patients before the treatment was generally higher than those found in astronauts, lower range of VZV numbers in shingles patients overlapped with astronaut s levels. This suggests a potential risk of shingles to astronauts resulting from reactivation of VZV. In another clinical study of 25 shingles patients, PCR technology detected VZV in the serum and peripheral blood mononuclear cells of all 25 patients demonstrating for the first time that viremia is a common manifestation of herpes shingles.

Mehta, Satish K.↗

NASA NTRS2015

Summer 2015 Internship Abstract

Green fluorescent protein (GFP) visually shows the expression of proteins by fluorescing when exposed to certain wavelengths of light. The GFP in this experiment was used to identify cells actively releasing viruses. The experiment focused on the effect of microgravity on the GFP expression of Akata B-cells infected with Epstein Barr Virus (EBV). Two flasks were prepared with 30 million cells each and two bioreactors were prepared with 50 million cells each. All four cultures were incubated for 16 days and fed every four days. Cellometer readings were taken on the feeding days to find cell size, viability, and GFP expression. In addition, the cells were treated with Propodium monoazide (PMA) and run through real time PCR to determine viral load on the feeding days. On the International Space Station air samples are taken to analyze the bacterial and fungal organisms in the air. The Sartorius Portable Airport is being investigated for potential use on the ISS to analyze for viral content in the air. Multiple samples were taken around Johnson Space Center building 37 and in Clear Lake Pediatric Clinic. The filter used was the gelatin membrane filter and the DNA was extracted directly from the filter. The DNA was then run through real time PCR for Varicella Zoster Virus (VZV) and EBV as well as GAPDH to test for the presence of DNA. The results so far have shown low DNA yield and no positive results for VZV or EBV. Further inquiry involves accurately replicating an atmosphere with high viral load from saliva as would be found on the ISS to run the air sampler in. Another line of research is stress hormones that may be correlated to the reactivation of latent viruses. The stress hormones from saliva samples are analyzed rather than blood samples. The quantity found in saliva shows the quantity of the hormones actually attached to cells and causing a reaction, whereas in the blood the quantity of hormones is the total amount released to cause a reaction. The particular hormones tested for were cortisol, alpha-amylase, and DHEA. The DHEA was very high in the two control samples tested. Regularly, samples came into the lab from local clinics to be tested for various viruses. Saliva, blood, body scrapes, and tears were received from the clinics and then run for VZV, EBV, and Human Simplex Virus 1 (HSV-1) with the results then reported back to the clinician. Blood, saliva, and urine from astronauts were also tested for viruses and logged. In addition, several cell cultures were brought up and grown, including adherent Human Lung Fibroblast (HFL) cells infected with VZV, and Akata B-cells infected with EBV.

Smith, Courtney↗

NASA NTRSDate not supplied

Method Development for In-situ Detection of Latent Herpesvirus DNA from Saliva using Nanopore Sequencing

Research toward latent herpesvirus reactivation has been intensively addressed through Space Shuttle and International Space Station (ISS) investigations. This work has provided the understanding that persistent reactivation of herpesviruses from asymptomatic crew can be detected through viral shedding in saliva, urine, and blood. Occasionally, viral reactivation from the latency stage can pose a threat to crew health (clinical manifestation) before, during, and after flight missions. Furthermore, previous work detailing correlations with immunity indicate that monitoring viral reactivation could be implemented to assess potential immune dysfunction. While in-flight monitoring is desirable, there is no well-established procedure or method for real-time evaluations, and research to date has relied on postflight, ground-based analysis. The development of portable molecular technologies like the miniPCR™ (miniPCR Bio) thermal cycler and the MinION™ sequencer (Oxford Nanopore Technologies) have confirmed that real-time monitoring is possible in extreme and low resource environments. These devices, combined with simple sample preparation methods, have been used to demonstrate bacterial identification onboard the ISS, as well as rapid viral detection in remote locations on Earth. The work here builds upon previous molecular advancements onboard the ISS toward the development and validation of a spaceflight-compatible method for viral detection from crew samples. Several herpesviruses can be detected in saliva, which provides a non-invasive means to collect samples for monitoring. While the basis for this method lies in previous spaceflight investigations, key points for method optimization include DNA extraction from saliva, viral primer selection, and bioinformatic processes for data analysis. To increase viral yield, numerous DNA extraction methods have been evaluated and will be discussed in detail. For initial development and testing, varicella-zoster virus (VZV) is being targeted though open reading frame 51 and 63 (ORF51, ORF63), as the replication origin-binding protein is highly expressed during latency. Optimization of the thermal cycling parameters has resulted in the ability to test the entire process. The full method has been tested with both viral VZV DNA standards and saliva spiked with varying concentrations of VZV. Viral sequence data were mapped to the reference sequence using minimap2. Prior to mapping, DNA sequencing reads were filtered for length and quality, barcodes were removed, and alignment identity calculated. Following further assessments, statistics were compared across multiple sequencing experiments and are being used to determine the success of the protocols. Forward work will include the incorporation of herpes simplex virus 1 (HSV-1) and Epstein-Barr virus (EBV) primers as well as the validation of results to the terrestrial qPCR standard assay. Upon full validation of the developed method, saliva will be collected from 20 healthy subjects and spiked with viral DNA. These samples will be split and assayed with the MinION and standard qPCR assay.

Hang N Nguyen↗