Metagenomic Analysis of Blood Virome in Ischemic Stroke Reveals an Increase in Herpesvirus Transcripts and Host Immune Activation

Metagenomic Analysis of Blood Virome in Ischemic Stroke Reveals an Increase in Herpesvirus Transcripts and Host Immune Activation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Metagenomic Analysis of Blood Virome in Ischemic Stroke Reveals an Increase in Herpesvirus Transcripts and Host Immune Activation Mike Clarke, Sarina Falcione, Roobina Boghozian, Raluca Todoran, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7521090/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Viral infections may play a role in stroke, with potential contributions to thrombosis and vasculopathy. Associations between stroke risk, influenza, and herpesviruses have been identified in epidemiological studies. However, our understanding regarding the full effects of viral interactions with the immune system and host tissues is still emerging. Evaluating the virome in stroke and vascular disease will provide insights that may aid in the prevention and treatment of stroke. In this study we evaluate the human virome in ischemic stroke. Methods Viruses were measured by total RNA sequencing of blood from 37 patients with ischemic stroke and 32 controls of similar age and vascular risk factor status. RNA reads were examined for viral RNAs using a global alignment platform and a database of human virus genomes. The relationship between the quantity of viral RNA and stroke is examined. Host gene expression following stroke is examined in relation to the presence of viral RNAs. Results Viral RNAs were detected in the blood samples of both ischemic stroke and control groups. Viral reads with a prevalence > 3% and raw counts > 2 were from a total of 6 viral families. This included several common human herpesviruses (HHVs), adenoviruses, and papillomaviruses, as well as human pegivirus, respiratory syncytial virus, and human endogenous retrovirus K (HERV-K). Combined, counts from HHVs were higher in stroke compared to control by a fold change of 2.13. Coinfection with multiple HHVs was more common in stroke, with a 1.23 fold increase in the number of detected herpesviruses. Reads from 2 viral genes were increased in stroke, UL95 from cytomegalovirus (CMV), and EBNA2 from Epstein-Barr virus (EBV). Genes associated with stroke, including apolipoprotein E (APOE), C3, platelet-derived growth factor (PDGF), and CXCL2 were differentially expressed in stroke samples which contained high counts of one or both of UL95 and EBNA2. Conclusion Viral RNAs from multiple families can be detected within the human blood virome. HHV transcripts were the most abundant of viral RNAs detected. Among stroke patients, HHV transcripts were more prevalent, with higher counts, and indicated a higher rate of coinfection with multiple HHV species. Expression of the EBV gene EBNA2 and the CMV gene UL95 may relate to changes in immune gene expression following stroke. Further evaluation is needed to determine the effects that the human virome have on stroke risk, immune response to stroke, and long-term outcome. Virome Ischemic Stroke Neuroinflammation Epstein-Barr Virus Cytomegalovirus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Interactions between viral pathogens and host responses throughout life can influence biological processes and may be contributing to risk of stroke. The recent COVID-19 pandemic has brought attention to the long-term health consequences of such interactions, with some studies suggesting a relationship between severe SARS-CoV-2 infection and an increased incidence of ischemic stroke (IS). 1 Previous population studies have also reported an increase in IS incidence following influenza and shingles. 2 , 3 These findings are supported by a reduction in IS incidence among persons vaccinated for influenza or varicella zoster. 4 Several viral pathogens can cause endothelial cell injury, vascular dysfunction or immune system dysregulation, which may contribute to stroke risk. 5 In addition, the accumulation of viral pathogens over time increases “infectious burden”, which has been proposed as a risk factor for stroke. 6 Many viruses are part of the normal human body, comprising a component of the microbiome known as the virome. These include phage viruses that infect bacteria in the gut, as well as viruses that infect human cells in an acute or persistent manner. While many of these viruses are not associated with disease, our understanding regarding the full effects of viral interactions with the immune system and host tissues is still emerging and may lead to identification of viral factors in complex diseases. Recent research has found a link between enteroviruses in the gut virome and the development of islet autoimmunity, a precursor to type 1 diabetes. 7 In addition, Epstein-Barr virus was found to be present in an overwhelming majority of cases of multiple sclerosis. 8 Evaluating the virome in stroke and vascular disease will provide insights that may aid in the prevention and treatment of stroke. In this study, we examined the blood virome in patients with ischemic stroke using RNA-sequencing for detection of viral-encoded RNA. Changes in the virome between stroke and controls are identified. These elements of the virome are then used to examine differential expression of genes within stroke patients which may affect stroke outcome. Methods Patient selection Male and female patients with stroke and control were recruited from 2008 to 2016 from medical centres at the University of Alberta and the University of California at Davis. The protocol was approved by the institutional review boards at participating universities and written informed consent was obtained. Controls were of similar age, sex and vascular risk factors to stroke patients. Patient histories, examinations and evaluations were performed, and stroke diagnoses were made by board-certified neurologists. Stroke assessments included ECG and Holter monitor, carotid Doppler, computed tomographic (CT) brain scan and magnetic resonance imaging, and magnetic resonance arteriogram of extracranial and intracranial vessels or computed tomographic angiography. Sample collection Blood was collected by venipuncture in PAXgene tubes (Pre-AnalytiX, GmbH, Switzerland), which stabilize blood RNA. Blood was collected within the first 36 hours of stroke onset then stored for 2 hours at room temperature before storing frozen at − 80°C. Total RNA was isolated with PAXgene blood RNA isolation kits according to the manufacturer’s protocol. Quantification of RNA was determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and quality assessment was performed by an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). RNA Sequencing Total RNA library preparation was performed using the NuGEN Ovation Universal RNA-Seq system (Tecan Genomics) with ribosomal RNA and globin transcripts depleted using InDA-C (AnyDeplete). Alignment to Human Reference Genome Raw reads were quality controlled with FastQC v0.12.1 (github.com/s-andrews/FastQC). Trimming of adapter sequences and deduplication was performed using fastp v0.23.4 (github.com/OpenGene/fastp). 9 Alignment of total RNA reads from each sample was performed using the human reference genome GRCh38.p14 (ncbi.nlm.nih.gov/datasets) using STAR version 2.7.11a (github.com/alexdobin/STAR). 10 Aligned reads and unaligned reads were both retained, and quality was verified with FastQC. Generation of Viral Genome Database Viral genome assemblies were obtained from the National Center for Biotechnology Information (NCBI) RefSeq and GenBank databases and filtered to only include whole genomes from known human viruses (not including phage viruses) (Supplementary Table 1). To meet the requirements of the STAR alignment program, the annotation files were converted to GTF format, and linearized annotations for circular viral genomes were created using de novo scripts. To adapt our viral genome database for use with global alignment programs, each annotation file had to be screened for genes that cross the first nucleotide position within the sequence file. These genes are present in viral genomes which have a circular form and have sequence endpoints which extend beyond the last nucleotide within the linear sequence file. Because viral sequences are identified based on the sequence location within the annotated reference genome, the annotation files containing these genes are edited to ensure that each transcript position is within the start- and end- points of the linear sequence file (Supplementary Fig. 1). Viral taxonomy information was collected for each virus in the database using taxonomizR (cran.r-project.org/package = taxonomizr). Metadata for each viral assembly was retrieved using NCBI command-line tools (github.com/ncbi/datasets). 11 Alignment to Viral Database Read files that were unaligned to the human reference genome were screened for sequences of low complexity using fastp software. Low complexity reads were filtered out to reduce the occurrence of non-specific alignments. Alignment was performed using the assembled viral genome database and STAR aligner. Intronic regions were specifically omitted from alignment to further reduce the chance of false positive counts. Each virus identified was manually reviewed as a potential source of contamination. Quantification of Host and Viral Reads All read counts were obtained from aligned read files using featureCounts v2.0.6 from the Subread package (subread.sourceforge.net). 12 Human and viral read counts were combined, then normalized to counts-per-million (CPM) using edgeR v4.2.1 (bioinf.wehi.edu.au/edgeR). 13 Viral count distribution was tested for normality using Shapiro-Wilk test. Comparison of viral reads between patient groups used non-parametric t-testing (Wilcoxon). A low-pass filter was applied to reject viral read counts of less than 2 counts per patient. Additionally, any alignment detected in less than 2 patients of the group was rejected. The remaining aligned viral reads were grouped across variants of a single species into their parent genes for gene-level quantification using NCBI Command Tools to match locus tags to gene identifiers. Genome Coverage Analysis NCBI RefSeq reference genomes were selected for herpesvirus species, based on their higher prevalence and total CPM from our initial alignment. CMV (Human herpesvirus 5 strain Merlin, Accession: NC_006273.2) and EBV (Human gammaherpesvirus 4, Accession: NC_007605.1) were selected. Following the protocol already described, another alignment was performed against the selected reference genomes. Mean read coverages of viral genomes was calculated using SAMtools v1.21 ( https://github.com/samtools/samtools) . 14 Coverage plotting was performed using the r package ggcoverage v1.4.0 ( https://github.com/showteeth/ggcoverage) . 15 Differential Gene Expression Analysis in Stroke Stroke patient blood samples were divided into quartiles based on transcript counts from selected herpesvirus species. Quartile distribution was tested against all patient factors for significant correlation. Comparisons were performed between samples which had the highest quartile of one or more selected HHV transcripts, against samples in the lowest quartile. Principal component analysis was used to visualize the variation in host genes between groups using PCAtools v2.16.0 ( https://github.com/kevinblighe/PCAtools ). Differential gene expression (DGE) analysis of human (host) gene counts between groups was performed with edgeR using a general linearized model (GLM). Host genes were filtered by an absolute log fold change (|log 2 FC|) greater than 1, and a corrected p-value (FDR) of less than 0.1. Pathway Analysis and Host Gene Correlation Host genes with significant DGE in response to high selected virus counts were analyzed for pathway enrichment using GO enrichment analysis with the R package clusterProfiler 4.12.6 ( https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html) . 16 Top pathways were identified based on number of pathway genes being differentially expressed, fold change of these genes, and statistical significance. Visualization of pathway data and gene cluster network was performed using the enrichplot package within the clusterProfiler suite. Results Patients There were 37 patients with stroke and 32 controls (Table 1). The mean age was 66.4 years in the stroke patients and 62.5 years in the controls. Among stroke patients, 81% had hypertension, 22% had diabetes, and 44% had hyperlipidemia. In controls, 59% had hypertension, 38% had diabetes and 66% had hyperlipidemia. The etiology of stroke was cardioembolic in 41%, large vessel in 35%, and lacunar small vessel in 24%. Table 1. Cohort of ischemic stroke and control groups, including overview of stroke risk factors, and subtypes of ischemic stroke. Alignment to Human Reference Genome and Viral Database Read data for all patients passed quality control procedures prior to alignment to the human reference genome with a mean Q value (PHRED score) of 37.8. In patient samples, a mean of 81.7 ± 0.6% (SEM) of reads (control: 80.8 ± 0.9%, stroke: 82.6 ± 0.8%) aligned with the human reference genome. Variations based on patient differences in sex and race were also recorded (Supplementary Figure 2). After low-complexity and read length filtering, the remaining full length reads (unaligned reads) were an average of 1.8 ± 0.2% of the total input reads. Of these unaligned reads, 0.04 ± 0.002% were assigned to viral transcripts within our viral database (Figure 1). Alignments to bovine viral diarrhea virus, murine leukemia virus, human immunodeficiency virus 1 and simian immunodeficiency virus were removed as suspected contaminants (Supplementary Table 2). The input reads which aligned with our viral database were a mean total of only 0.001% of the total input. This represents a significant decrease from initial iterations of this protocol which did not include low-complexity filtering. An average 2.2 x 10 5 reads were removed in this process, minimizing the likelihood of alignment to regions in the viral database containing high levels of repeat sequences. Detection of the Blood Virome Total viral CPM among patients had a non-normal distribution (Shapiro-Wilk, p-value = 4.28x10 -13 ). Patient samples yielded a mean of 14.60 CPM (sd = 24.33) of viral RNA. Of the patient factors (age, sex, incidence of stroke, hypertension, diabetes or high cholesterol), only sex corresponded to a significant change in total viral transcript counts. Males had a higher mean CPM when compared to females (fold change = 1.26, p-value = 0.005). Viral reads that met a minimum threshold for detection (prevalence > 3%, raw counts > 2) were from a total of 6 viral families. Orthoherpesviridae was the largest family with 72.51% of detected reads originating from a human herpesvirus (HHV) (Figure 2a). Viruses from 5 other families were detected above threshold quantities, which included 1 human adenovirus, 5 human papillomaviruses, human pegivirus, respiratory syncytial virus, and HERV-K (Figure 2b). HHV species were among the highest in both prevalence and mean CPM among positive patients (positivity) (Figure 2c). Herpesviridae was the only viral family detected with significantly higher mean CPM in stroke as compared to control (fold change = 2.13, p-value = 0.04) (Figure 3). In addition, males were observed with a higher mean HHV CPM as compared to females (Supplementary Figure 3). HHV Coinfection is More Prevalent in Stroke More distinct species of HHV were detected in patients with stroke (mean = 4.24 species, sd = 1.26) as compared to control (mean = 3.44 species, sd = 1.22) (Figure 4a). Of the 7 HHV species detected, the prevalence was higher among patients with stroke for every species with a mean increase in prevalence of 0.12. HHV-6b had the largest prevalence difference (fold change = 1.73) between IS and control. VZV was only detected in 2 samples, both from the IS group (Figure 4b). Coverage Plotting Reveals Differences in Viral Transcription Most genomic regions of the CMV reference genome were detected, with UL48, UL52, UL69, UL95 and UL112 being notable protein-encoding loci. The total coverage of the CMV reference genome among stroke patients was 0.101% of the total genome, which was not a significant change from the control group (0.109%, p-value = 0.4). The mean depth of coverage for the coding region for the gene UL95 among stroke patients was 1.02, a significant increase over the control group (fold change = 1.8, p-value = 0.048) (Figure 5a). Transcripts from the EBV genome were localized primarily to the EBNA2 region. Total mean coverage of the genome in stroke patients was 0.14%, a significant increase over control (fold change = 1.26, p-value = 0.006). For the coding region of the EBNA2 gene, the mean depth of coverage in stroke patients was 4.11, a significant increase over the control group (fold change = 1.28, p-value = 0.048) (Figure 5b). Select CMV and EBV Transcripts are Significantly Increased Among Stroke Patients When comparing transcript counts across patient groups, multiple genes had a higher mean CPM in patients with IS. CMV UL112 was detected only in very low quantities, however, it was almost exclusively found in patients with IS. UL112 was detected in 7 IS patients with a mean CPM of 0.07, and only 1 control with a CPM of 0.05 (Supplementary Figure 5). The CMV gene, UL95 (fold change = 1.32, p-value = 0.04) (Figure 6a), and EBV EBNA2 (fold change = 2.27, p-value = 0.04) (Figure 6b) were both significantly increased in IS. High Viral Transcript Count and Differential Expression of Host Genes in Stroke DGE analysis of host genes in stroke compared 16 samples from the highest quartiles of EBNA2, UL95, or both, with 14 samples from the lowest quartiles. A significant upregulation of 2,865 genes was observed, with 12 genes identified from our panel of genes relating to IS (Figure 7). CXCL2 (mean CPM = 2.8, sd = 2.7), and MMP7 (mean CPM = 1.3, sd = 1.5) were only expressed in the high viral transcript group. APOE (fold change = 18.0, p-value = 0.004), C3 (fold change = 5.8, p-value = 0.002), EGR1 (fold change = 2.2, p-value = 0.009), EPHA2 (fold change = 7.0, p-value = 0.01), MRC1 (fold change = 4.3, p-value = 0.002), PDGFA (fold change = 2.9, p-value = 0.01), PDGFB (fold change = 2.2, p-value = 0.005), and PSEN2 (fold change = 3.0, p-value = 0.004) had significantly increased mean CPM in the high viral transcript group. C6 and FLT4 showed differential expression by DGE analysis but were not significant by standard comparison of mean CPM (Figure 8). GO enrichment analysis revealed 5 immune-related pathways above our threshold (fold enrichment > 1.1, FDR < 0.05) (Figure 9). Discussion Using RNA-seq analysis of the human blood, we identified viral-associated RNAs in patients with stroke. To our knowledge, this is the first study to investigate the human blood virome in the context of ischemic stroke. This preliminary evaluation provides important insight into the blood virome in stroke, and its potential for immune modulation. The Blood Virome The viruses detected in the blood of stroke patients in this study are supported by similar findings in previous human blood virome studies. Acute infections from mastadentoviruses, respiratory-syncytial virus ( Orthopneumovirus hominis) , and Norwalk virus were detected with prevalences consistent with seasonal infection rates. Within the blood DNA virome, viruses of the Anellovirus genus are known to be highly prevalent. 17 – 19 The rates of detection in this RNA-based study confirm the presence of these viruses in the blood but demonstrate relatively low levels of transcription. HERV-K was highly prevalent, but with a large variation in transcript count among the samples. Human endogenous retroviruses are present as provirus within the human genome, comprising around 8% of the total. Expression of these integrated genomes may influence human physiology and disease. Multiple studies have suggested a relationship between HERV-K transcripts and neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS patients, increased expression of HERV-K transcripts has been shown. In addition, neurotoxicity of these transcripts has been demonstrated. 20 Further evaluation regarding endogenous retroviruses, and their neurological effects in patients with stroke will be of interest. In our study, HHVs represented the largest contribution of detected viral RNA. The abundance of HHV viruses and their transcripts in the blood has been observed previously. 18 , 19 The Herpesviridae family was also the only detected viral family to show a significant increase in viral RNA counts in the IS patients. Additionally, the average number of detected HHV species was higher among the IS patients, indicating that concurrent infection or activation from latency of HHVs is more common in this group. This finding supports the proposed “infectious burden” factor in stroke risk, which has previously been demonstrated by longitudinal analysis of individuals with positive serology for multiple HHV infections. 6 Quantification of Viral Transcripts Elevated EBV counts among males and stroke patients was determined to be the result of higher EBNA-2 transcript counts. EBNA-2 is an important regulator for EBV latency and immortalization of host B-cells. 21 Since other transcripts of the EBV genome were not detected in any significant quantity, any differences in viral activity between patient groups cannot be ascertained. Although total CMV counts were not significantly different in any patient group, transcripts from the UL95 gene were observed at higher counts in stroke patients. The CMV gene UL95 encodes a protein product that is required for late viral gene expression. 22 Actively replicating virus may be infecting multiple cell types, including fibroblasts and endothelial cells, but are likely being detected in our blood samples within PBMCs. Both EBV and CMV are able to infect blood cells and can establish latency within hematopoietic progenitor cells. These viral species are therefore likely to have higher representation within blood samples, consistent with the findings of this study. Given the possibility that EBV and CMV transcripts are being detected primarily within PBMCs, cell counts from each cell type should have been taken for each sample, as described in previous studies. 23 In this way, the presence of viral transcripts could be related to the number of potential host cells collected, a key to determining the transcriptional activity within a host-cell. Without this consideration, our data does not rule out the possibility that patients with stroke have higher PBMC counts, and therefore a larger proportion of PBMCs within our samples, yielding higher viral transcript counts. Host Response to Infection Differential gene analysis of IS samples with a high count from one or both of EBV and CMV transcripts, EBNA2 and UL95 respectively, revealed an increased expression of multiple immune-related genes. Although this is not abnormal with viral infection, this increase of immune activity may have an additive effect on inflammation following IS. For example, our results showed an increased expression of the host gene, early growth response 1 (EGR1). EGR1 is increased during lytic replication of EBV but is also a key regulator of oxidative stress and blood-brain barrier integrity in IS. 24 , 25 In fact, blocking of the EGR1 product during stroke has been shown to reduce infarct size in a rat middle cerebral artery occlusion (MCAO) model. 25 The observed upregulation of complement factor genes C3 and C6 are consistent with complement activation in response to infection by either CMV or EBV. 26 , 27 Complement activation also plays a role in neuroinflammation following IS, and altered expression of these factors may result in worse outcomes. 28 Pathway analysis further supports an increase of immune activation with presence of the viral transcripts EBNA2 and UL95. Both EBV and CMV are highly prevalent and establish latent infections in their host. An increase in both viral transcription and immune activity may indicate viral reactivation. In some cases, herpesvirus reactivation may be a consequence of IS and its associated immune suppression. 29 However, multiple herpesviruses, including CMV and EBV may be increasing the risk of stroke during active infection by causing vasculopathy and thrombosis. 5 Further investigation is needed to evaluate the effect of HHV infection on the human immune system and the relationship to stroke risk or outcome. Detection of Viral RNA by RNAseq Our protocol for detection of viral RNA in blood samples was created to provide highly sensitive detection of viral RNA from a large database of viral genomes. Previous NGS-based analyses of the human virome have implemented the local alignment program, NCBI Basic Local Alignment Search Tool (BLAST). A global alignment protocol, one based on alignment algorithms such as STAR, has multiple advantages which make it well suited to high throughput analysis. The first advantage is that global alignment natively supports paired-end read data, allowing for higher accuracy alignments as compared to single-end. Global alignment programs are also splice-aware, meaning that they allow sequence alignment across intronic regions. Although this feature is not relevant for many viral genomes, it has been identified as an important element in accurately detecting viral transcripts from viruses such as human adenoviruses. 30 Finally, our high-throughput analysis of 200 million reads (on average) per patient could be performed without any subsampling of the total input data set to reduce the required computing time, an approach often utilized for BLAST-based protocols. 31 This platform is therefore, scalable to larger datasets, and requires only a modest amount of computing power. Limitations Blood sample collection is accessible and often does not cause disruption to patient care, making it an ideal medium for virome analysis. However, many viral infections, including HHVs have tissue-specific expression profiles. Additional tissue samples such as those from atherosclerotic plaques, blood clots, or peripheral sites of latency, could be analyzed to provide a more complete understanding of viral activity within the body. Total RNA extracted from our blood samples had an average depth of ~ 200 million reads. This represents a significant increase in specificity for viral detection over methods such as qPCR and ELISA. However, further investigation is needed to confirm the presence of active viruses which correspond to the RNA detected in this study. In addition, RNA sequencing will not detect genomes from DNA viruses but must rely on the appearance of viral transcripts from infected host cells. Thus, evaluating viral DNA and RNA in the same samples would be of value. Our protocol did not include internal controls to validate the sequencing and alignment workflow, an example of which would be using spike-in controls, the addition of viral sequences at a known concentration to our samples prior to sequencing, as described in similar studies. 32 In addition to our sequencing approach, detection of viral antigen, or virus-specific host antibodies by ELISA would further verify detection of virus. Other virome studies have extracted viral particles from human tissues through centrifugation and filtration, then reconstituted an active infection using plaque assay. 33 Such an approach provides confirmation of active virus, and could be applied to future studies of the blood virome in the context of stroke. 34 Conclusion This study presents a protocol for screening of the human blood virome in the context of disease. Six virus families were significantly detected in blood samples from patients with stroke and controls, with HHVs representing the most abundant family. An increase in HHV transcripts were associated with enhanced immune activation following IS. The influence of these viruses on stroke risk and outcome are of interest for further investigation. Abbreviations CMV Cytomegalovirus DGE Differential gene expression EBV Epstein-Barr virus HSV Herpes simplex virus HHV-6b Human herpesvirus 6B HHV-7 Human herpesvirus 7 HHV-8 Human herpesvirus 8 IS Ischemic stroke VZV varicella-zoster virus Declarations Ethics Approval and Consent to Participate Written informed consent was obtained for the patients in this study. Ethics approval was granted by review boards at the University of Alberta and the University of California at Davis. Consent for Publication Not applicable. Availability of Data and Materials The datasets used during the current study are available from the corresponding author on reasonable request. Competing Interests None. Funding Glen Jickling is supported by research grants from the Canadian Institutes of Health Research, NIH, University Hospital Foundation, and the Heart and Stroke Foundation of Canada, and Canada Research Chair. Author’s contributions Patient recruitment, sample collection, and sequencing was performed by GJ. Data processing, analysis and manuscript preparation was performed by MC, with assistance from GJ. All authors were involved in the revision of the final manuscript. Acknowledgements Not applicable. References Luo W, Liu X, Bao K, Huang C. Ischemic stroke associated with COVID-19: a systematic review and meta-analysis. J Neurol . 2022;269(4):1731-1740. doi:10.1007/s00415-021-10837-7 Boehme AK, Luna J, Kulick ER, Kamel H, Elkind MSV. Influenza‐like illness as a trigger for ischemic stroke. Ann Clin Transl Neurol . 2018;5(4):456-463. doi:10.1002/acn3.545 Parameswaran GI, Wattengel BA, Chua HC, et al. Increased Stroke Risk Following Herpes Zoster Infection and Protection With Zoster Vaccine. Clinical Infectious Diseases . 2023;76(3):e1335-e1340. doi:10.1093/cid/ciac549 Holodinsky JK, Zerna C, Malo S, Svenson LW, Hill MD. Association between influenza vaccination and risk of stroke in Alberta, Canada: a population-based study. The Lancet Public Health . 2022;7(11):e914-e922. doi:10.1016/S2468-2667(22)00222-5 Clarke M, Falcione S, Boghozian R, et al. Viral Infection and Ischemic Stroke: Emerging Trends and Mechanistic Insights. Journal of the American Heart Association . 2024;0(0):e035892. doi:10.1161/JAHA.124.035892 Elkind MSV, Ramakrishnan P, Moon YP, et al. Infectious burden and risk of stroke: the northern Manhattan study. Arch Neurol . 2010;67(1):33-38. doi:10.1001/archneurol.2009.271 Lindfors K, Lin J, Lee HS, et al. Metagenomics of the faecal virome indicate a cumulative effect of enterovirus and gluten amount on the risk of coeliac disease autoimmunity in genetically at risk children: the TEDDY study. Gut . 2020;69(8):1416-1422. doi:10.1136/gutjnl-2019-319809 Bjornevik K, Cortese M, Healy BC, et al. Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science . 2022;375(6578):296-301. doi:10.1126/science.abj8222 Chen S. Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta . 2023;2(2):e107. doi:10.1002/imt2.107 Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics . 2013;29(1):15-21. doi:10.1093/bioinformatics/bts635 O’Leary NA, Cox E, Holmes JB, et al. Exploring and retrieving sequence and metadata for species across the tree of life with NCBI Datasets. Sci Data . 2024;11(1):732. doi:10.1038/s41597-024-03571-y Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics . 2014;30(7):923-930. doi:10.1093/bioinformatics/btt656 Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics . 2010;26(1):139-140. doi:10.1093/bioinformatics/btp616 Danecek P, Bonfield JK, Liddle J, et al. Twelve years of SAMtools and BCFtools. GigaScience . 2021;10(2):giab008. doi:10.1093/gigascience/giab008 Song Y, Wang J. ggcoverage: an R package to visualize and annotate genome coverage for various NGS data. BMC Bioinformatics . 2023;24(1):309. doi:10.1186/s12859-023-05438-2 Wu T, Hu E, Xu S, et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innovation . 2021;2(3). doi:10.1016/j.xinn.2021.100141 Kumata R, Ito J, Takahashi K, Suzuki T, Sato K. A tissue level atlas of the healthy human virome. BMC Biology . 2020;18(1):55. doi:10.1186/s12915-020-00785-5 Guo J, Huang X, Zhang C, et al. The blood virome of 10,585 individuals from the ChinaMAP. Cell Discov . 2022;8(1):1-4. doi:10.1038/s41421-022-00476-1 Moustafa A, Xie C, Kirkness E, et al. The blood DNA virome in 8,000 humans. PLoS Pathog . 2017;13(3):e1006292. doi:10.1371/journal.ppat.1006292 Li W, Lee MH, Henderson L, et al. Human endogenous retrovirus-K contributes to motor neuron disease. Sci Transl Med . 2015;7(307):307ra153. doi:10.1126/scitranslmed.aac8201 Zimber-Strobl U, Strobl LJ. EBNA2 and Notch signalling in Epstein–Barr virus mediated immortalization of B lymphocytes. Seminars in Cancer Biology . 2001;11(6):423-434. doi:10.1006/scbi.2001.0409 Isomura H, Stinski MF, Murata T, et al. The Human Cytomegalovirus Gene Products Essential for Late Viral Gene Expression Assemble into Prereplication Complexes before Viral DNA Replication▿. J Virol . 2011;85(13):6629-6644. doi:10.1128/JVI.00384-11 Autio A, Kettunen J, Nevalainen T, Kimura B, Hurme M. Herpesviruses and their genetic diversity in the blood virome of healthy individuals: effect of aging. Immun Ageing . 2022;19:15. doi:10.1186/s12979-022-00268-x Heather J, Flower K, Isaac S, Sinclair AJ. The Epstein–Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1. Journal of General Virology . 2009;90(6):1450-1454. doi:10.1099/vir.0.007922-0 Li YY, Guo JH, Liu YQ, Dong JH, Zhu CH. PPARγ Activation-Mediated Egr-1 Inhibition Benefits Against Brain Injury in an Experimental Ischaemic Stroke Model. J Stroke Cerebrovasc Dis . 2020;29(12):105255. doi:10.1016/j.jstrokecerebrovasdis.2020.105255 Spiller OB, Morgan BP. Antibody-Independent Activation of the Classical Complement Pathway by Cytomegalovirus-Infected Fibroblasts. The Journal of Infectious Diseases . 1998;178(6):1597-1603. doi:10.1086/314499 McConnell I, Klein G, Lint TF, Lachmann PJ. Activation of the alternative complement pathway by human B cell lymphoma lines is associated with Epstein-Barr virus transformation of the cells. Eur J Immunol . 1978;8(7):453-458. doi:10.1002/eji.1830080702 Alawieh A, Elvington A, Tomlinson S. Complement in the Homeostatic and Ischemic Brain. Front Immunol . 2015;6. doi:10.3389/fimmu.2015.00417 Fullerton HJ, Hills NK, Wintermark M, et al. Evidence of Varicella Zoster Virus Reactivation in Children With Arterial Ischemic Stroke: Results of the VIPS II Study. Journal of the American Heart Association . 2025;14(6):e039184. doi:10.1161/JAHA.124.039184 Price AM, Steinbock RT, Lauman R, et al. Novel viral splicing events and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts. PLoS Pathog . 2022;18(9):e1010797. doi:10.1371/journal.ppat.1010797 Cheng T, Chin PJ, Cha K, Petrick N, Mikailov M. Profiling the BLAST bioinformatics application for load balancing on high-performance computing clusters. BMC Bioinformatics . 2022;23(1):544. doi:10.1186/s12859-022-05029-7 Van Boheemen S, Van Rijn-Klink AL, Pappas N, et al. Metagenomic Sequencing for Combined Detection of Rna and Dna Viruses in Respiratory Samples from Paediatric Patients . Microbiology; 2018. doi:10.1101/492835 Vehik K, Lynch KF, Wong MC, et al. Prospective virome analyses in young children at increased genetic risk for type 1 diabetes. Nat Med . 2019;25(12):1865-1872. doi:10.1038/s41591-019-0667-0 Smith MS, Bentz GL, Alexander JS, Yurochko AD. Human Cytomegalovirus Induces Monocyte Differentiation and Migration as a Strategy for Dissemination and Persistence. J Virol . 2004;78(9):4444-4453. doi:10.1128/JVI.78.9.4444-4453.2004 Additional Declarations No competing interests reported. Supplementary Files MetagenomicAnalysisofBloodViromeinIschemicStrokeSupplementary.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 Jan, 2026 Reviews received at journal 03 Nov, 2025 Reviewers agreed at journal 31 Oct, 2025 Reviews received at journal 31 Oct, 2025 Reviewers agreed at journal 21 Oct, 2025 Reviewers invited by journal 05 Sep, 2025 Editor assigned by journal 05 Sep, 2025 Submission checks completed at journal 03 Sep, 2025 First submitted to journal 02 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7521090","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":511554538,"identity":"965b8cb3-fd25-4c20-9adb-f6808ff77883","order_by":0,"name":"Mike Clarke","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3PsQrCMBCA4SuFTpWuEUSfQIgUio8TKdSlQ8Glg2hB0MUH6GuI4ORw5UCXPkAmsQhODo4KIlZXJdbNId8QCNzPJQCa9peEgeUBDoBBVRN4JfUE4Jmw6gnHqkl75u/xug6arvT3FMW7kZOYxVmVePmRZ/Nj6Hoy4JTmA8bQcpWrPBkC2hj3VlJwqk0FK6/q1z2T7IbxeJn2z1S7C9ZC27x8S8jGUHAWllsSwTjalnpL+RdqYNBJ81NE6UbUF2R5XWWy9YvihH7LmfUXh2gonOZ2cpCq5APzx3lN0zTt3QPfPFOdmbwVQQAAAABJRU5ErkJggg==","orcid":"","institution":"University of Alberta","correspondingAuthor":true,"prefix":"","firstName":"Mike","middleName":"","lastName":"Clarke","suffix":""},{"id":511554539,"identity":"32b2675c-6f91-448c-bffd-ffbd3549a53b","order_by":1,"name":"Sarina Falcione","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Sarina","middleName":"","lastName":"Falcione","suffix":""},{"id":511554540,"identity":"4001b7d2-524c-49bd-8499-adda98640f92","order_by":2,"name":"Roobina Boghozian","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Roobina","middleName":"","lastName":"Boghozian","suffix":""},{"id":511554541,"identity":"9eb8729a-52b9-4b9e-a352-dae20c0570a0","order_by":3,"name":"Raluca Todoran","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Raluca","middleName":"","lastName":"Todoran","suffix":""},{"id":511554542,"identity":"a74cad56-fc6a-4b1d-9da8-f2b10eb25978","order_by":4,"name":"Yiran Zhang","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Yiran","middleName":"","lastName":"Zhang","suffix":""},{"id":511554543,"identity":"4c3b2e9f-ab36-4dc4-9189-219d398ba6fd","order_by":5,"name":"Maria Guadalupe C. Real","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Guadalupe C.","lastName":"Real","suffix":""},{"id":511554544,"identity":"3a9c16f5-bcdb-4852-b20c-261fdde52d56","order_by":6,"name":"Alexis StPierre","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Alexis","middleName":"","lastName":"StPierre","suffix":""},{"id":511554545,"identity":"a8bf1a8e-d0ce-4afe-9f0a-485fc0c1ccce","order_by":7,"name":"Twinkle Joy","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Twinkle","middleName":"","lastName":"Joy","suffix":""},{"id":511554546,"identity":"954303b9-038c-4240-b36a-c06b5e0a78d1","order_by":8,"name":"Glen C. Jickling","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Glen","middleName":"C.","lastName":"Jickling","suffix":""}],"badges":[],"createdAt":"2025-09-02 21:23:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7521090/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7521090/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91194064,"identity":"5e054884-6ccd-4bfe-b489-7a201879f52e","added_by":"auto","created_at":"2025-09-12 14:50:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114797,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eWorkflow for alignment of viral and host reads. Total RNA reads from patient blood samples were aligned to the GRCH38.p14 reference human genome to distinguish human from non-human RNA. To non-human RNA, a low complexity filter was applied, and remaining reads were aligned to a database comprising all known human viral genomes.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/9b984f58c875b4417d934e1e.png"},{"id":91192320,"identity":"793b4991-5566-4838-8c80-7fb8d4aa23ad","added_by":"auto","created_at":"2025-09-12 14:42:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePresence of viral RNA in stroke patient blood samples. a) Blood virome composition of all samples given by viral family. b) The percentage of stroke patient blood samples positive for viral RNA from a given species. c) The average count per million (CPM) of virus family for each positive sample.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/cea9222e3ec0f3e978d60c93.png"},{"id":91195413,"identity":"86ab7fc0-7b7e-4e63-84fa-956c0b6f7631","added_by":"auto","created_at":"2025-09-12 14:58:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCPM of viral RNA detected in stroke and control blood samples. Viral counts are grouped by viral family.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/b0f7a6b46ad2a55f33ba5f24.png"},{"id":91192322,"identity":"cd3eb744-717f-4f83-9eaf-22c28625d049","added_by":"auto","created_at":"2025-09-12 14:42:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":55985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRepresentation of HHV virus in stroke and controls. a) Differences in number of HHV species detected in ischemic stroke compared to controls and between females and males. b) Prevalence of HHV species in ischemic stroke and control.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/4e3fb3a85274a9483423cee5.png"},{"id":91192327,"identity":"de351ba2-50bc-4926-86e4-2195b4f0e126","added_by":"auto","created_at":"2025-09-12 14:42:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCoverage plots of HHV genomes for control and stroke groups. Each bar represents the mean depth for that region, binned at 500 base pairs. Significantly detected genes are highlighted in grey for a) CMV, and b) EBV.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/82aaef5a9b0e577337223383.png"},{"id":91195414,"identity":"bfa60bce-de40-4423-8eb0-02e7b4b9646b","added_by":"auto","created_at":"2025-09-12 14:58:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79588,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDifferences in blood transcripts for a) CMV and b) EBV in patients with ischemic stroke compared to controls.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/806181b6728cf7b6411e8ebb.png"},{"id":91194071,"identity":"3f920363-6a39-4077-9f59-b5558a4f7326","added_by":"auto","created_at":"2025-09-12 14:50:05","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":297108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eVolcano plot showing differential expression of human blood cell genes in ischemic stroke with relationship to levels of EBNA2 and UL95 viral transcripts. Genes significantly decreased are shown in red, and those that are significantly increased in green. Genes related to immune response in stroke are labelled.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/5553341a5f0edfe7b8918696.png"},{"id":91192331,"identity":"96dd2c95-3922-4209-83a4-4cde6ffa7860","added_by":"auto","created_at":"2025-09-12 14:42:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":87331,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDifferential expression of selected stroke-related genes between high and low levels of viral transcripts EBNA2 and UL95.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/b30a46375abb01a15386822c.png"},{"id":91192343,"identity":"95feed46-97be-42a2-8d04-c622eb0e3794","added_by":"auto","created_at":"2025-09-12 14:42:05","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":30851,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eImmune-related pathways from GO enrichment analysis of differentially expressed genes between high and low levels of viral transcripts EBNA2 and UL95.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/a08542f0d18613d9691e8921.png"},{"id":91198229,"identity":"7090cc02-faf7-4e93-b8bb-5d80c82f4f7f","added_by":"auto","created_at":"2025-09-12 15:14:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1816283,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/e25d3116-9a3e-49bc-ba37-ac56f02f8110.pdf"},{"id":91192323,"identity":"50fd93b9-5003-4140-9016-973900b0b986","added_by":"auto","created_at":"2025-09-12 14:42:05","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":442229,"visible":true,"origin":"","legend":"","description":"","filename":"MetagenomicAnalysisofBloodViromeinIschemicStrokeSupplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7521090/v1/3695535606faeb1b61b1fc9a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metagenomic Analysis of Blood Virome in Ischemic Stroke Reveals an Increase in Herpesvirus Transcripts and Host Immune Activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInteractions between viral pathogens and host responses throughout life can influence biological processes and may be contributing to risk of stroke. The recent COVID-19 pandemic has brought attention to the long-term health consequences of such interactions, with some studies suggesting a relationship between severe SARS-CoV-2 infection and an increased incidence of ischemic stroke (IS).\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Previous population studies have also reported an increase in IS incidence following influenza and shingles.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e These findings are supported by a reduction in IS incidence among persons vaccinated for influenza or varicella zoster.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Several viral pathogens can cause endothelial cell injury, vascular dysfunction or immune system dysregulation, which may contribute to stroke risk.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e In addition, the accumulation of viral pathogens over time increases \u0026ldquo;infectious burden\u0026rdquo;, which has been proposed as a risk factor for stroke.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eMany viruses are part of the normal human body, comprising a component of the microbiome known as the virome. These include phage viruses that infect bacteria in the gut, as well as viruses that infect human cells in an acute or persistent manner. While many of these viruses are not associated with disease, our understanding regarding the full effects of viral interactions with the immune system and host tissues is still emerging and may lead to identification of viral factors in complex diseases. Recent research has found a link between enteroviruses in the gut virome and the development of islet autoimmunity, a precursor to type 1 diabetes.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e In addition, Epstein-Barr virus was found to be present in an overwhelming majority of cases of multiple sclerosis.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Evaluating the virome in stroke and vascular disease will provide insights that may aid in the prevention and treatment of stroke.\u003c/p\u003e\u003cp\u003eIn this study, we examined the blood virome in patients with ischemic stroke using RNA-sequencing for detection of viral-encoded RNA. Changes in the virome between stroke and controls are identified. These elements of the virome are then used to examine differential expression of genes within stroke patients which may affect stroke outcome.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatient selection\u003c/h2\u003e\u003cp\u003eMale and female patients with stroke and control were recruited from 2008 to 2016 from medical centres at the University of Alberta and the University of California at Davis. The protocol was approved by the institutional review boards at participating universities and written informed consent was obtained. Controls were of similar age, sex and vascular risk factors to stroke patients. Patient histories, examinations and evaluations were performed, and stroke diagnoses were made by board-certified neurologists. Stroke assessments included ECG and Holter monitor, carotid Doppler, computed tomographic (CT) brain scan and magnetic resonance imaging, and magnetic resonance arteriogram of extracranial and intracranial vessels or computed tomographic angiography.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample collection\u003c/h3\u003e\n\u003cp\u003eBlood was collected by venipuncture in PAXgene tubes (Pre-AnalytiX, GmbH, Switzerland), which stabilize blood RNA. Blood was collected within the first 36 hours of stroke onset then stored for 2 hours at room temperature before storing frozen at \u0026minus;\u0026thinsp;80\u0026deg;C. Total RNA was isolated with PAXgene blood RNA isolation kits according to the manufacturer\u0026rsquo;s protocol. Quantification of RNA was determined with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and quality assessment was performed by an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA).\u003c/p\u003e\n\u003ch3\u003eRNA Sequencing\u003c/h3\u003e\n\u003cp\u003eTotal RNA library preparation was performed using the NuGEN Ovation Universal RNA-Seq system (Tecan Genomics) with ribosomal RNA and globin transcripts depleted using InDA-C (AnyDeplete).\u003c/p\u003e\n\u003ch3\u003eAlignment to Human Reference Genome\u003c/h3\u003e\n\u003cp\u003eRaw reads were quality controlled with FastQC v0.12.1 (github.com/s-andrews/FastQC). Trimming of adapter sequences and deduplication was performed using fastp v0.23.4 (github.com/OpenGene/fastp).\u003csup\u003e9\u003c/sup\u003e Alignment of total RNA reads from each sample was performed using the human reference genome GRCh38.p14 (ncbi.nlm.nih.gov/datasets) using STAR version 2.7.11a (github.com/alexdobin/STAR).\u003csup\u003e10\u003c/sup\u003e Aligned reads and unaligned reads were both retained, and quality was verified with FastQC.\u003c/p\u003e\n\u003ch3\u003eGeneration of Viral Genome Database\u003c/h3\u003e\n\u003cp\u003eViral genome assemblies were obtained from the National Center for Biotechnology Information (NCBI) RefSeq and GenBank databases and filtered to only include whole genomes from known human viruses (not including phage viruses) (Supplementary Table\u0026nbsp;1). To meet the requirements of the STAR alignment program, the annotation files were converted to GTF format, and linearized annotations for circular viral genomes were created using de novo scripts. To adapt our viral genome database for use with global alignment programs, each annotation file had to be screened for genes that cross the first nucleotide position within the sequence file. These genes are present in viral genomes which have a circular form and have sequence endpoints which extend beyond the last nucleotide within the linear sequence file. Because viral sequences are identified based on the sequence location within the annotated reference genome, the annotation files containing these genes are edited to ensure that each transcript position is within the start- and end- points of the linear sequence file (Supplementary Fig.\u0026nbsp;1). Viral taxonomy information was collected for each virus in the database using taxonomizR (cran.r-project.org/package\u0026thinsp;=\u0026thinsp;taxonomizr). Metadata for each viral assembly was retrieved using NCBI command-line tools (github.com/ncbi/datasets).\u003csup\u003e11\u003c/sup\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAlignment to Viral Database\u003c/h2\u003e\u003cp\u003eRead files that were unaligned to the human reference genome were screened for sequences of low complexity using fastp software. Low complexity reads were filtered out to reduce the occurrence of non-specific alignments. Alignment was performed using the assembled viral genome database and STAR aligner. Intronic regions were specifically omitted from alignment to further reduce the chance of false positive counts. Each virus identified was manually reviewed as a potential source of contamination.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eQuantification of Host and Viral Reads\u003c/h3\u003e\n\u003cp\u003eAll read counts were obtained from aligned read files using featureCounts v2.0.6 from the Subread package (subread.sourceforge.net).\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Human and viral read counts were combined, then normalized to counts-per-million (CPM) using edgeR v4.2.1 (bioinf.wehi.edu.au/edgeR).\u003csup\u003e13\u003c/sup\u003e Viral count distribution was tested for normality using Shapiro-Wilk test. Comparison of viral reads between patient groups used non-parametric t-testing (Wilcoxon). A low-pass filter was applied to reject viral read counts of less than 2 counts per patient. Additionally, any alignment detected in less than 2 patients of the group was rejected. The remaining aligned viral reads were grouped across variants of a single species into their parent genes for gene-level quantification using NCBI Command Tools to match locus tags to gene identifiers.\u003c/p\u003e\n\u003ch3\u003eGenome Coverage Analysis\u003c/h3\u003e\n\u003cp\u003eNCBI RefSeq reference genomes were selected for herpesvirus species, based on their higher prevalence and total CPM from our initial alignment. CMV (Human herpesvirus 5 strain Merlin, Accession: NC_006273.2) and EBV (Human gammaherpesvirus 4, Accession: NC_007605.1) were selected. Following the protocol already described, another alignment was performed against the selected reference genomes. Mean read coverages of viral genomes was calculated using SAMtools v1.21 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/samtools/samtools)\u003c/span\u003e\u003cspan address=\"https://github.com/samtools/samtools)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003csup\u003e14\u003c/sup\u003e Coverage plotting was performed using the r package ggcoverage v1.4.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/showteeth/ggcoverage)\u003c/span\u003e\u003cspan address=\"https://github.com/showteeth/ggcoverage)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003csup\u003e15\u003c/sup\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDifferential Gene Expression Analysis in Stroke\u003c/h2\u003e\u003cp\u003eStroke patient blood samples were divided into quartiles based on transcript counts from selected herpesvirus species. Quartile distribution was tested against all patient factors for significant correlation. Comparisons were performed between samples which had the highest quartile of one or more selected HHV transcripts, against samples in the lowest quartile. Principal component analysis was used to visualize the variation in host genes between groups using PCAtools v2.16.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/kevinblighe/PCAtools\u003c/span\u003e\u003cspan address=\"https://github.com/kevinblighe/PCAtools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Differential gene expression (DGE) analysis of human (host) gene counts between groups was performed with edgeR using a general linearized model (GLM). Host genes were filtered by an absolute log fold change (|log\u003csub\u003e2\u003c/sub\u003eFC|) greater than 1, and a corrected p-value (FDR) of less than 0.1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003ePathway Analysis and Host Gene Correlation\u003c/h2\u003e\u003cp\u003eHost genes with significant DGE in response to high selected virus counts were analyzed for pathway enrichment using GO enrichment analysis with the R package clusterProfiler 4.12.6 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioconductor.org/packages/release/bioc/html/clusterProfiler.html)\u003c/span\u003e\u003cspan address=\"https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html)\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003csup\u003e16\u003c/sup\u003e Top pathways were identified based on number of pathway genes being differentially expressed, fold change of these genes, and statistical significance. Visualization of pathway data and gene cluster network was performed using the enrichplot package within the clusterProfiler suite.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003ch2\u003ePatients\u003c/h2\u003e\n\u003cp\u003eThere were 37 patients with stroke and 32 controls (Table 1). \u0026nbsp;The mean age was 66.4 years in the stroke patients and 62.5 years in the controls. Among stroke patients, 81% had hypertension, 22% had diabetes, and 44% had hyperlipidemia. In controls, 59% had hypertension, 38% had diabetes and 66% had hyperlipidemia. The etiology of stroke was cardioembolic in 41%, large vessel in 35%, and lacunar small vessel in 24%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTable 1. Cohort of ischemic stroke and control groups, including overview of stroke risk factors, and subtypes of ischemic stroke.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cimg 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\" width=\"695\" height=\"199\"\u003e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAlignment to Human Reference Genome and Viral Database\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRead data for all patients passed quality control procedures prior to alignment to the human reference genome with a mean Q value (PHRED score) of 37.8. In patient samples, a mean of 81.7 \u0026plusmn; 0.6% (SEM) of reads (control: 80.8 \u0026plusmn; 0.9%, stroke: 82.6 \u0026plusmn; 0.8%) aligned with the human reference genome. Variations based on patient differences in sex and race were also recorded (Supplementary Figure 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter low-complexity and read length filtering, the remaining full length reads (unaligned reads) were an average of 1.8 \u0026plusmn; 0.2% of the total input reads. Of these unaligned reads, 0.04 \u0026plusmn; 0.002% were assigned to viral transcripts within our viral database (Figure 1). Alignments to bovine viral diarrhea virus, murine leukemia virus, human immunodeficiency virus 1 and simian immunodeficiency virus were removed as suspected contaminants (Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003eThe input reads which aligned with our viral database were a mean total of only 0.001% of the total input. This represents a significant decrease from initial iterations of this protocol which did not include low-complexity filtering. An average 2.2 x 10\u003csup\u003e5\u003c/sup\u003e reads were removed in this process, minimizing the likelihood of alignment to regions in the viral database containing high levels of repeat sequences.\u003c/p\u003e\n\u003ch2\u003eDetection of the Blood Virome\u003c/h2\u003e\n\u003cp\u003eTotal viral CPM among patients had a non-normal distribution\u0026nbsp;\u003cbr\u003e(Shapiro-Wilk, p-value = 4.28x10\u003csup\u003e-13\u003c/sup\u003e). Patient samples yielded a mean of 14.60 CPM (sd = 24.33) of viral RNA. Of the patient factors (age, sex, incidence of stroke, hypertension, diabetes or high cholesterol), only sex corresponded to a significant change in total viral transcript counts. Males had a higher mean CPM when compared to females (fold change = 1.26, p-value = 0.005).\u003c/p\u003e\n\u003cp\u003eViral reads that met a minimum threshold for detection (prevalence \u0026gt; 3%, raw counts \u0026gt; 2) were from a total of 6 viral families. Orthoherpesviridae was the largest family with 72.51% of detected reads originating from a human herpesvirus (HHV) (Figure 2a). Viruses from 5 other families were detected above threshold quantities, which included 1 human adenovirus, 5 human papillomaviruses, human pegivirus, respiratory syncytial virus, and HERV-K (Figure 2b). HHV species were among the highest in both prevalence and mean CPM among positive patients (positivity) (Figure 2c). Herpesviridae was the only viral family detected with significantly higher mean CPM in stroke as compared to control (fold change = 2.13, p-value = 0.04) (Figure 3). In addition, males were observed with a higher mean HHV CPM as compared to females (Supplementary Figure 3).\u003c/p\u003e\n\u003ch2\u003eHHV Coinfection is More Prevalent in Stroke\u003c/h2\u003e\n\u003cp\u003eMore distinct species of HHV were detected in patients with stroke (mean = 4.24 species, sd = 1.26) as compared to control (mean = 3.44 species, sd = 1.22) (Figure 4a). Of the 7 HHV species detected, the prevalence was higher among patients with stroke for every species with a mean increase in prevalence of 0.12. HHV-6b had the largest prevalence difference (fold change = 1.73) between IS and control. VZV was only detected in 2 samples, both from the IS group (Figure 4b).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCoverage Plotting Reveals Differences in Viral Transcription\u003c/h2\u003e\n\u003cp\u003eMost genomic regions of the CMV reference genome were detected, with UL48, UL52, UL69, UL95 and UL112 being notable protein-encoding loci. The total coverage of the CMV reference genome among stroke patients was 0.101% of the total genome, which was not a significant change from the control group (0.109%, p-value = 0.4). The mean depth of coverage for the coding region for the gene UL95 among stroke patients was 1.02, a significant increase over the control group (fold change = 1.8, p-value = 0.048) (Figure 5a).\u003c/p\u003e\n\u003cp\u003eTranscripts from the EBV genome were localized primarily to the EBNA2 region. Total mean coverage of the genome in stroke patients was 0.14%, a significant increase over control (fold change = 1.26, p-value = 0.006). For the coding region of the EBNA2 gene, the mean depth of coverage in stroke patients was 4.11, a significant increase over the control group (fold change = 1.28, p-value = 0.048) (Figure 5b).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eSelect CMV and EBV Transcripts are Significantly Increased Among Stroke Patients\u003c/h2\u003e\n\u003cp\u003eWhen comparing transcript counts across patient groups, multiple genes had a higher mean CPM in patients with IS. CMV UL112 was detected only in very low quantities, however, it was almost exclusively found in patients with IS. UL112 was detected in 7 IS patients with a mean CPM of 0.07, and only 1 control with a CPM of 0.05 (Supplementary Figure 5). The CMV gene, UL95 (fold change = 1.32, p-value = 0.04) (Figure 6a), and EBV EBNA2 (fold change = 2.27, p-value = 0.04) (Figure 6b) were both significantly increased in IS.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eHigh Viral Transcript Count and Differential Expression of Host Genes in Stroke\u003c/h2\u003e\n\u003cp\u003eDGE analysis of host genes in stroke compared 16 samples from the highest quartiles of EBNA2, UL95, or both, with 14 samples from the lowest quartiles. A significant upregulation of 2,865 genes was observed, with 12 genes identified from our panel of genes relating to IS (Figure 7). CXCL2 (mean CPM = 2.8, sd = 2.7), and MMP7 (mean CPM = 1.3, sd = 1.5) were only expressed in the high viral transcript group. APOE (fold change = 18.0, p-value = 0.004), C3 (fold change = 5.8, p-value = 0.002), EGR1 (fold change = 2.2, p-value = 0.009), EPHA2 (fold change = 7.0, p-value = 0.01), MRC1 (fold change = 4.3, p-value = 0.002), PDGFA (fold change = 2.9, p-value = 0.01), PDGFB (fold change = 2.2, p-value = 0.005), and PSEN2 (fold change = 3.0, p-value = 0.004) had significantly increased mean CPM in the high viral transcript group. C6 and FLT4 showed differential expression by DGE analysis but were not significant by standard comparison of mean CPM (Figure 8). GO enrichment analysis revealed 5 immune-related pathways above our threshold (fold enrichment \u0026gt; 1.1, FDR \u0026lt; 0.05) (Figure 9).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUsing RNA-seq analysis of the human blood, we identified viral-associated RNAs in patients with stroke. To our knowledge, this is the first study to investigate the human blood virome in the context of ischemic stroke. This preliminary evaluation provides important insight into the blood virome in stroke, and its potential for immune modulation.\u003c/p\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eThe Blood Virome\u003c/h2\u003e\u003cp\u003eThe viruses detected in the blood of stroke patients in this study are supported by similar findings in previous human blood virome studies. Acute infections from mastadentoviruses, respiratory-syncytial virus (\u003cem\u003eOrthopneumovirus hominis)\u003c/em\u003e, and Norwalk virus were detected with prevalences consistent with seasonal infection rates. Within the blood DNA virome, viruses of the \u003cem\u003eAnellovirus\u003c/em\u003e genus are known to be highly prevalent.\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e The rates of detection in this RNA-based study confirm the presence of these viruses in the blood but demonstrate relatively low levels of transcription. HERV-K was highly prevalent, but with a large variation in transcript count among the samples. Human endogenous retroviruses are present as provirus within the human genome, comprising around 8% of the total. Expression of these integrated genomes may influence human physiology and disease. Multiple studies have suggested a relationship between HERV-K transcripts and neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS patients, increased expression of HERV-K transcripts has been shown. In addition, neurotoxicity of these transcripts has been demonstrated.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Further evaluation regarding endogenous retroviruses, and their neurological effects in patients with stroke will be of interest.\u003c/p\u003e\u003cp\u003eIn our study, HHVs represented the largest contribution of detected viral RNA. The abundance of HHV viruses and their transcripts in the blood has been observed previously.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e The Herpesviridae family was also the only detected viral family to show a significant increase in viral RNA counts in the IS patients. Additionally, the average number of detected HHV species was higher among the IS patients, indicating that concurrent infection or activation from latency of HHVs is more common in this group. This finding supports the proposed \u0026ldquo;infectious burden\u0026rdquo; factor in stroke risk, which has previously been demonstrated by longitudinal analysis of individuals with positive serology for multiple HHV infections.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eQuantification of Viral Transcripts\u003c/h2\u003e\u003cp\u003eElevated EBV counts among males and stroke patients was determined to be the result of higher EBNA-2 transcript counts. EBNA-2 is an important regulator for EBV latency and immortalization of host B-cells.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Since other transcripts of the EBV genome were not detected in any significant quantity, any differences in viral activity between patient groups cannot be ascertained. Although total CMV counts were not significantly different in any patient group, transcripts from the UL95 gene were observed at higher counts in stroke patients. The CMV gene UL95 encodes a protein product that is required for late viral gene expression.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Actively replicating virus may be infecting multiple cell types, including fibroblasts and endothelial cells, but are likely being detected in our blood samples within PBMCs.\u003c/p\u003e\u003cp\u003eBoth EBV and CMV are able to infect blood cells and can establish latency within hematopoietic progenitor cells. These viral species are therefore likely to have higher representation within blood samples, consistent with the findings of this study. Given the possibility that EBV and CMV transcripts are being detected primarily within PBMCs, cell counts from each cell type should have been taken for each sample, as described in previous studies.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e In this way, the presence of viral transcripts could be related to the number of potential host cells collected, a key to determining the transcriptional activity within a host-cell. Without this consideration, our data does not rule out the possibility that patients with stroke have higher PBMC counts, and therefore a larger proportion of PBMCs within our samples, yielding higher viral transcript counts.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eHost Response to Infection\u003c/h2\u003e\u003cp\u003eDifferential gene analysis of IS samples with a high count from one or both of EBV and CMV transcripts, EBNA2 and UL95 respectively, revealed an increased expression of multiple immune-related genes. Although this is not abnormal with viral infection, this increase of immune activity may have an additive effect on inflammation following IS. For example, our results showed an increased expression of the host gene, early growth response 1 (EGR1). EGR1 is increased during lytic replication of EBV but is also a key regulator of oxidative stress and blood-brain barrier integrity in IS.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e In fact, blocking of the EGR1 product during stroke has been shown to reduce infarct size in a rat middle cerebral artery occlusion (MCAO) model.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e The observed upregulation of complement factor genes C3 and C6 are consistent with complement activation in response to infection by either CMV or EBV.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Complement activation also plays a role in neuroinflammation following IS, and altered expression of these factors may result in worse outcomes.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003ePathway analysis further supports an increase of immune activation with presence of the viral transcripts EBNA2 and UL95. Both EBV and CMV are highly prevalent and establish latent infections in their host. An increase in both viral transcription and immune activity may indicate viral reactivation. In some cases, herpesvirus reactivation may be a consequence of IS and its associated immune suppression.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e However, multiple herpesviruses, including CMV and EBV may be increasing the risk of stroke during active infection by causing vasculopathy and thrombosis.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Further investigation is needed to evaluate the effect of HHV infection on the human immune system and the relationship to stroke risk or outcome.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eDetection of Viral RNA by RNAseq\u003c/h2\u003e\u003cp\u003eOur protocol for detection of viral RNA in blood samples was created to provide highly sensitive detection of viral RNA from a large database of viral genomes. Previous NGS-based analyses of the human virome have implemented the local alignment program, NCBI Basic Local Alignment Search Tool (BLAST). A global alignment protocol, one based on alignment algorithms such as STAR, has multiple advantages which make it well suited to high throughput analysis. The first advantage is that global alignment natively supports paired-end read data, allowing for higher accuracy alignments as compared to single-end. Global alignment programs are also splice-aware, meaning that they allow sequence alignment across intronic regions. Although this feature is not relevant for many viral genomes, it has been identified as an important element in accurately detecting viral transcripts from viruses such as human adenoviruses.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Finally, our high-throughput analysis of 200\u0026nbsp;million reads (on average) per patient could be performed without any subsampling of the total input data set to reduce the required computing time, an approach often utilized for BLAST-based protocols.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e This platform is therefore, scalable to larger datasets, and requires only a modest amount of computing power.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eBlood sample collection is accessible and often does not cause disruption to patient care, making it an ideal medium for virome analysis. However, many viral infections, including HHVs have tissue-specific expression profiles. Additional tissue samples such as those from atherosclerotic plaques, blood clots, or peripheral sites of latency, could be analyzed to provide a more complete understanding of viral activity within the body. Total RNA extracted from our blood samples had an average depth of ~\u0026thinsp;200\u0026nbsp;million reads. This represents a significant increase in specificity for viral detection over methods such as qPCR and ELISA. However, further investigation is needed to confirm the presence of active viruses which correspond to the RNA detected in this study. In addition, RNA sequencing will not detect genomes from DNA viruses but must rely on the appearance of viral transcripts from infected host cells. Thus, evaluating viral DNA and RNA in the same samples would be of value.\u003c/p\u003e\u003cp\u003eOur protocol did not include internal controls to validate the sequencing and alignment workflow, an example of which would be using spike-in controls, the addition of viral sequences at a known concentration to our samples prior to sequencing, as described in similar studies.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e In addition to our sequencing approach, detection of viral antigen, or virus-specific host antibodies by ELISA would further verify detection of virus. Other virome studies have extracted viral particles from human tissues through centrifugation and filtration, then reconstituted an active infection using plaque assay.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Such an approach provides confirmation of active virus, and could be applied to future studies of the blood virome in the context of stroke.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study presents a protocol for screening of the human blood virome in the context of disease. Six virus families were significantly detected in blood samples from patients with stroke and controls, with HHVs representing the most abundant family. An increase in HHV transcripts were associated with enhanced immune activation following IS. The influence of these viruses on stroke risk and outcome are of interest for further investigation.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"623\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7115%;\"\u003e\n \u003cp\u003eCMV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.3691%;\"\u003e\n \u003cp\u003eCytomegalovirus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7115%;\"\u003e\n \u003cp\u003eDGE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.3691%;\"\u003e\n \u003cp\u003eDifferential gene expression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7115%;\"\u003e\n \u003cp\u003eEBV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.3691%;\"\u003e\n \u003cp\u003eEpstein-Barr virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7115%;\"\u003e\n \u003cp\u003eHSV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.3691%;\"\u003e\n \u003cp\u003eHerpes simplex virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7115%;\"\u003e\n \u003cp\u003eHHV-6b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.3691%;\"\u003e\n \u003cp\u003eHuman herpesvirus 6B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7115%;\"\u003e\n \u003cp\u003eHHV-7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.3691%;\"\u003e\n \u003cp\u003eHuman herpesvirus 7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7115%;\"\u003e\n \u003cp\u003eHHV-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.3691%;\"\u003e\n \u003cp\u003eHuman herpesvirus 8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7115%;\"\u003e\n \u003cp\u003eIS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.3691%;\"\u003e\n \u003cp\u003eIschemic stroke\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.7115%;\"\u003e\n \u003cp\u003eVZV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 34.3691%;\"\u003e\n \u003cp\u003evaricella-zoster virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch2\u003eEthics Approval and Consent to Participate\u003c/h2\u003e\n\u003cp\u003eWritten informed consent was obtained for the patients in this study. Ethics approval was granted by review boards at the University of Alberta and the University of California at Davis.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eConsent for Publication\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eGlen Jickling is supported by research grants from the Canadian Institutes of Health Research, NIH, University Hospital Foundation, and the Heart and Stroke Foundation of Canada, and Canada Research Chair.\u003c/p\u003e\n\u003ch2\u003eAuthor\u0026rsquo;s contributions\u003c/h2\u003e\n\u003cp\u003ePatient recruitment, sample collection, and sequencing was performed by GJ. Data processing, analysis and manuscript preparation was performed by MC, with assistance from GJ. All authors were involved in the revision of the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLuo W, Liu X, Bao K, Huang C. Ischemic stroke associated with COVID-19: a systematic review and meta-analysis. \u003cem\u003eJ Neurol\u003c/em\u003e. 2022;269(4):1731-1740. doi:10.1007/s00415-021-10837-7\u003c/li\u003e\n\u003cli\u003eBoehme AK, Luna J, Kulick ER, Kamel H, Elkind MSV. Influenza‐like illness as a trigger for ischemic stroke. \u003cem\u003eAnn Clin Transl Neurol\u003c/em\u003e. 2018;5(4):456-463. doi:10.1002/acn3.545\u003c/li\u003e\n\u003cli\u003eParameswaran GI, Wattengel BA, Chua HC, et al. Increased Stroke Risk Following Herpes Zoster Infection and Protection With Zoster Vaccine. \u003cem\u003eClinical Infectious Diseases\u003c/em\u003e. 2023;76(3):e1335-e1340. doi:10.1093/cid/ciac549\u003c/li\u003e\n\u003cli\u003eHolodinsky JK, Zerna C, Malo S, Svenson LW, Hill MD. Association between influenza vaccination and risk of stroke in Alberta, Canada: a population-based study. \u003cem\u003eThe Lancet Public Health\u003c/em\u003e. 2022;7(11):e914-e922. doi:10.1016/S2468-2667(22)00222-5\u003c/li\u003e\n\u003cli\u003eClarke M, Falcione S, Boghozian R, et al. Viral Infection and Ischemic Stroke: Emerging Trends and Mechanistic Insights. \u003cem\u003eJournal of the American Heart Association\u003c/em\u003e. 2024;0(0):e035892. doi:10.1161/JAHA.124.035892\u003c/li\u003e\n\u003cli\u003eElkind MSV, Ramakrishnan P, Moon YP, et al. Infectious burden and risk of stroke: the northern Manhattan study. \u003cem\u003eArch Neurol\u003c/em\u003e. 2010;67(1):33-38. doi:10.1001/archneurol.2009.271\u003c/li\u003e\n\u003cli\u003eLindfors K, Lin J, Lee HS, et al. Metagenomics of the faecal virome indicate a cumulative effect of enterovirus and gluten amount on the risk of coeliac disease autoimmunity in genetically at risk children: the TEDDY study. \u003cem\u003eGut\u003c/em\u003e. 2020;69(8):1416-1422. doi:10.1136/gutjnl-2019-319809\u003c/li\u003e\n\u003cli\u003eBjornevik K, Cortese M, Healy BC, et al. Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. \u003cem\u003eScience\u003c/em\u003e. 2022;375(6578):296-301. doi:10.1126/science.abj8222\u003c/li\u003e\n\u003cli\u003eChen S. Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. \u003cem\u003eiMeta\u003c/em\u003e. 2023;2(2):e107. doi:10.1002/imt2.107\u003c/li\u003e\n\u003cli\u003eDobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. \u003cem\u003eBioinformatics\u003c/em\u003e. 2013;29(1):15-21. doi:10.1093/bioinformatics/bts635\u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Leary NA, Cox E, Holmes JB, et al. Exploring and retrieving sequence and metadata for species across the tree of life with NCBI Datasets. \u003cem\u003eSci Data\u003c/em\u003e. 2024;11(1):732. doi:10.1038/s41597-024-03571-y\u003c/li\u003e\n\u003cli\u003eLiao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. \u003cem\u003eBioinformatics\u003c/em\u003e. 2014;30(7):923-930. doi:10.1093/bioinformatics/btt656\u003c/li\u003e\n\u003cli\u003eRobinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. \u003cem\u003eBioinformatics\u003c/em\u003e. 2010;26(1):139-140. doi:10.1093/bioinformatics/btp616\u003c/li\u003e\n\u003cli\u003eDanecek P, Bonfield JK, Liddle J, et al. Twelve years of SAMtools and BCFtools. \u003cem\u003eGigaScience\u003c/em\u003e. 2021;10(2):giab008. doi:10.1093/gigascience/giab008\u003c/li\u003e\n\u003cli\u003eSong Y, Wang J. ggcoverage: an R package to visualize and annotate genome coverage for various NGS data. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e. 2023;24(1):309. doi:10.1186/s12859-023-05438-2\u003c/li\u003e\n\u003cli\u003eWu T, Hu E, Xu S, et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. \u003cem\u003eInnovation\u003c/em\u003e. 2021;2(3). doi:10.1016/j.xinn.2021.100141\u003c/li\u003e\n\u003cli\u003eKumata R, Ito J, Takahashi K, Suzuki T, Sato K. A tissue level atlas of the healthy human virome. \u003cem\u003eBMC Biology\u003c/em\u003e. 2020;18(1):55. doi:10.1186/s12915-020-00785-5\u003c/li\u003e\n\u003cli\u003eGuo J, Huang X, Zhang C, et al. The blood virome of 10,585 individuals from the ChinaMAP. \u003cem\u003eCell Discov\u003c/em\u003e. 2022;8(1):1-4. doi:10.1038/s41421-022-00476-1\u003c/li\u003e\n\u003cli\u003eMoustafa A, Xie C, Kirkness E, et al. The blood DNA virome in 8,000 humans. \u003cem\u003ePLoS Pathog\u003c/em\u003e. 2017;13(3):e1006292. doi:10.1371/journal.ppat.1006292\u003c/li\u003e\n\u003cli\u003eLi W, Lee MH, Henderson L, et al. Human endogenous retrovirus-K contributes to motor neuron disease. \u003cem\u003eSci Transl Med\u003c/em\u003e. 2015;7(307):307ra153. doi:10.1126/scitranslmed.aac8201\u003c/li\u003e\n\u003cli\u003eZimber-Strobl U, Strobl LJ. EBNA2 and Notch signalling in Epstein\u0026ndash;Barr virus mediated immortalization of B lymphocytes. \u003cem\u003eSeminars in Cancer Biology\u003c/em\u003e. 2001;11(6):423-434. doi:10.1006/scbi.2001.0409\u003c/li\u003e\n\u003cli\u003eIsomura H, Stinski MF, Murata T, et al. The Human Cytomegalovirus Gene Products Essential for Late Viral Gene Expression Assemble into Prereplication Complexes before Viral DNA Replication▿. \u003cem\u003eJ Virol\u003c/em\u003e. 2011;85(13):6629-6644. doi:10.1128/JVI.00384-11\u003c/li\u003e\n\u003cli\u003eAutio A, Kettunen J, Nevalainen T, Kimura B, Hurme M. Herpesviruses and their genetic diversity in the blood virome of healthy individuals: effect of aging. \u003cem\u003eImmun Ageing\u003c/em\u003e. 2022;19:15. doi:10.1186/s12979-022-00268-x\u003c/li\u003e\n\u003cli\u003eHeather J, Flower K, Isaac S, Sinclair AJ. The Epstein\u0026ndash;Barr virus lytic cycle activator Zta interacts with methylated ZRE in the promoter of host target gene egr1. \u003cem\u003eJournal of General Virology\u003c/em\u003e. 2009;90(6):1450-1454. doi:10.1099/vir.0.007922-0\u003c/li\u003e\n\u003cli\u003eLi YY, Guo JH, Liu YQ, Dong JH, Zhu CH. PPAR\u0026gamma; Activation-Mediated Egr-1 Inhibition Benefits Against Brain Injury in an Experimental Ischaemic Stroke Model. \u003cem\u003eJ Stroke Cerebrovasc Dis\u003c/em\u003e. 2020;29(12):105255. doi:10.1016/j.jstrokecerebrovasdis.2020.105255\u003c/li\u003e\n\u003cli\u003eSpiller OB, Morgan BP. Antibody-Independent Activation of the Classical Complement Pathway by Cytomegalovirus-Infected Fibroblasts. \u003cem\u003eThe Journal of Infectious Diseases\u003c/em\u003e. 1998;178(6):1597-1603. doi:10.1086/314499\u003c/li\u003e\n\u003cli\u003eMcConnell I, Klein G, Lint TF, Lachmann PJ. Activation of the alternative complement pathway by human B cell lymphoma lines is associated with Epstein-Barr virus transformation of the cells. \u003cem\u003eEur J Immunol\u003c/em\u003e. 1978;8(7):453-458. doi:10.1002/eji.1830080702\u003c/li\u003e\n\u003cli\u003eAlawieh A, Elvington A, Tomlinson S. Complement in the Homeostatic and Ischemic Brain. \u003cem\u003eFront Immunol\u003c/em\u003e. 2015;6. doi:10.3389/fimmu.2015.00417\u003c/li\u003e\n\u003cli\u003eFullerton HJ, Hills NK, Wintermark M, et al. Evidence of Varicella Zoster Virus Reactivation in Children With Arterial Ischemic Stroke: Results of the VIPS II Study. \u003cem\u003eJournal of the American Heart Association\u003c/em\u003e. 2025;14(6):e039184. doi:10.1161/JAHA.124.039184\u003c/li\u003e\n\u003cli\u003ePrice AM, Steinbock RT, Lauman R, et al. Novel viral splicing events and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts. \u003cem\u003ePLoS Pathog\u003c/em\u003e. 2022;18(9):e1010797. doi:10.1371/journal.ppat.1010797\u003c/li\u003e\n\u003cli\u003eCheng T, Chin PJ, Cha K, Petrick N, Mikailov M. Profiling the BLAST bioinformatics application for load balancing on high-performance computing clusters. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e. 2022;23(1):544. doi:10.1186/s12859-022-05029-7\u003c/li\u003e\n\u003cli\u003eVan Boheemen S, Van Rijn-Klink AL, Pappas N, et al. \u003cem\u003eMetagenomic Sequencing for Combined Detection of Rna and Dna Viruses in Respiratory Samples from Paediatric Patients\u003c/em\u003e. Microbiology; 2018. doi:10.1101/492835\u003c/li\u003e\n\u003cli\u003eVehik K, Lynch KF, Wong MC, et al. Prospective virome analyses in young children at increased genetic risk for type 1 diabetes. \u003cem\u003eNat Med\u003c/em\u003e. 2019;25(12):1865-1872. doi:10.1038/s41591-019-0667-0\u003c/li\u003e\n\u003cli\u003eSmith MS, Bentz GL, Alexander JS, Yurochko AD. Human Cytomegalovirus Induces Monocyte Differentiation and Migration as a Strategy for Dissemination and Persistence. \u003cem\u003eJ Virol\u003c/em\u003e. 2004;78(9):4444-4453. doi:10.1128/JVI.78.9.4444-4453.2004\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genome-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Genome Medicine](https://genomemedicine.biomedcentral.com/)","snPcode":"13073","submissionUrl":"https://submission.springernature.com/new-submission/13073/3","title":"Genome Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Virome, Ischemic Stroke, Neuroinflammation, Epstein-Barr Virus, Cytomegalovirus","lastPublishedDoi":"10.21203/rs.3.rs-7521090/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7521090/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eViral infections may play a role in stroke, with potential contributions to thrombosis and vasculopathy. Associations between stroke risk, influenza, and herpesviruses have been identified in epidemiological studies. However, our understanding regarding the full effects of viral interactions with the immune system and host tissues is still emerging. Evaluating the virome in stroke and vascular disease will provide insights that may aid in the prevention and treatment of stroke. In this study we evaluate the human virome in ischemic stroke.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViruses were measured by total RNA sequencing of blood from 37 patients with ischemic stroke and 32 controls of similar age and vascular risk factor status. RNA reads were examined for viral RNAs using a global alignment platform and a database of human virus genomes. The relationship between the quantity of viral RNA and stroke is examined. Host gene expression following stroke is examined in relation to the presence of viral RNAs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViral RNAs were detected in the blood samples of both ischemic stroke and control groups. Viral reads with a prevalence \u0026gt; 3% and raw counts \u0026gt; 2 were from a total of 6 viral families. This included several common human herpesviruses (HHVs), adenoviruses, and papillomaviruses, as well as human pegivirus, respiratory syncytial virus, and human endogenous retrovirus K (HERV-K). Combined, counts from HHVs were higher in stroke compared to control by a fold change of 2.13. Coinfection with multiple HHVs was more common in stroke, with a 1.23 fold increase in the number of detected herpesviruses. Reads from 2 viral genes were increased in stroke, UL95 from cytomegalovirus (CMV), and EBNA2 from Epstein-Barr virus (EBV). Genes associated with stroke, including apolipoprotein E (APOE), C3, platelet-derived growth factor (PDGF), and CXCL2 were differentially expressed in stroke samples which contained high counts of one or both of UL95 and EBNA2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViral RNAs from multiple families can be detected within the human blood virome. HHV transcripts were the most abundant of viral RNAs detected. Among stroke patients, HHV transcripts were more prevalent, with higher counts, and indicated a higher rate of coinfection with multiple HHV species. Expression of the EBV gene EBNA2 and the CMV gene UL95 may relate to changes in immune gene expression following stroke. Further evaluation is needed to determine the effects that the human virome have on stroke risk, immune response to stroke, and long-term outcome.\u003c/p\u003e","manuscriptTitle":"Metagenomic Analysis of Blood Virome in Ischemic Stroke Reveals an Increase in Herpesvirus Transcripts and Host Immune Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 14:42:00","doi":"10.21203/rs.3.rs-7521090/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-06T17:16:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-03T10:39:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"261567173079110200055765811076976006027","date":"2025-10-31T13:38:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-31T09:16:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258628893836545107172965973688370967930","date":"2025-10-21T19:17:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-05T14:25:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-05T14:10:12+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-03T06:06:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genome Medicine","date":"2025-09-02T21:09:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genome-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Genome Medicine](https://genomemedicine.biomedcentral.com/)","snPcode":"13073","submissionUrl":"https://submission.springernature.com/new-submission/13073/3","title":"Genome Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0e5bd2ef-c9cb-40fb-aacc-36585c0998f6","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T13:10:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 14:42:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7521090","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7521090","identity":"rs-7521090","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}