In the aftermath of the coronavirus disease 2019 (COVID-19) pandemic, we sought to explore the causal association between COVID-19 and 17 prevalent post-COVID-19 syndrome (PCS) symptoms using Mendelian randomisation (MR) methodology.We used 22 extensive genome-wide association study (GWAS) data sets, incorporating genetic variants as instrumental variables. Univariate Mendelian randomisation (UVMR) analyses involved 15 single nucleotide polymorphisms (SNPs) for COVID-19 patients, 33 for hospitalised COVID-19 patients, and 29 for patients with severe respiratory symptoms due to COVID-19. Furthermore, we further used multivariable Mendelian randomisation (MVMR) analyses based on 93 SNPs for COVID-19 patients, 105 for hospitalised COVID-19 patients, and 99 for patients with severe respiratory symptoms due to COVID-19. With these analyses, we aimed to assess the causal associations between varying levels of COVID-19 infection and 17 prevalent PCS symptoms while accounting for the influence of educational and income levels.UVMR analysis identified potential causal effects of COVID-19 genetic susceptibility on myalgia and pain in various regions. Hospitalised COVID-19 was potentially linked to erectile dysfunction and alopecia areata. Very severe respiratory confirmed patients exhibited increased pain and tobacco use. Meanwhile, the MVMR analysis demonstrated a potential causal link between hospitalised COVID-19 and heart arrhythmia, and a protective effect of COVID-19 on tobacco use after adjusting for educational and income levels.Our MR analysis provides compelling evidence of causal associations between genetic susceptibility to COVID-19 and specific PCS symptoms, in which educational and income levels play a mediating role. These findings shed light on PCS pathogenesis and underscore the importance of considering social factors in its management. Tailored interventions and policies are crucial for PCS-affected individuals' well-being. Further research is needed to explore the impact of social determinants on COVID-19 patients and the wider population. The coronavirus disease 2019 (COVID-19) pandemic has had significant consequences beyond its acute phase, with post-COVID-19 Syndrome (PCS) emerging as a significant concern. Approximately 10-30% of COVID-19 survivors may develop PCS, leading to substantial medical and socioeconomic challenges . With thPrevious investigations into PCS have primarily relied on observational studies and narrative reviews. However, the complex and multifaceted nature of PCS, as well as the diversity of treatment interventions required, has hindered the execution of randomised controlled trials (RCTs) . ObservaWe performed a two-sample MR analysis based on data from 22 extensive genome-wide association study (GWAS) datasets as our primary sources for both exposure and outcome data. Our primary objective was to explore the causal relationship between COVID-19 infection and PCS, with a specific focus on 17 prevalent PCS-related symptoms. We based our selection of these symptoms on comprehensive reviews of high-quality literature , supplemTo address potential confounding factors, we applied the MVRM methodology, including educational and income levels as covariates , which provided additional validation for the findings derived from the univariable MR analyses. We performed statistical analyses using the “TwoSampleMR” package (version 0.5.6) and the “MendelianRandomization” package (version 0.7.0) in R, version 4.2.3 .We used publicly accessible GWAS summary statistics as primary data sources, focusing mainly on individuals of European descent to mitigate potential ethnic disparities. We collected GWAS data for COVID-19 patients, hospitalized COVID-19 patients, and patients with severe respiratory symptoms due to COVID-19, along with corresponding population controls, from the COVID-19 Host Genetics Initiative and other sources . AdditioWe identified a comprehensive range of 17 prevalent symptoms that serve as the primary outcome for PCS; they encompassed various manifestations, including malaise and fatigue, exertional dyspnoea, cardiac arrhythmia, disordered eating patterns, myalgia, dorsalgia, arthralgia, pain in various regions , sleeplessness or insomnia, memory loss, depressive symptoms, anxiety disorders, a history of suicide attempts, alcohol consumption, tobacco usage, erectile dysfunction, and alopecia areata. To prevent sample duplication, we obtained GWAS data from the FinnGen database, while we retrieved only shortness of breath data from the UK Biobank, enabling single-sample MR , while P-values ranging from 0.00294 to 0.05 were considered indicative of suggestive evidence for a causal relationship [The primary approach for the two-sample MR analysis was the random-effects inverse-variance weighted (IVW) method, which effectively addresses heterogeneity among genetic instruments and improves the accuracy of estimates . We alsotionship . Sensititionship ,25. For To adjust for potential confounding effects, we performed MVMR analysis while incorporating educational level and income level. The multivariable IVW, multivariable MR-Egger, and multivariable MR-Lasso approaches were utilized . The useIn the UVMR analysis, 15 SNPs  were extracted from GWAS data of COVID-19 patients after removing palindromic SNPs. Hospitalised COVID patients had 33 selected SNPs  while very severe respiratory confirmed patients had 29 . Subsequently, some SNPs were excluded based on outcome factors and outliers  in the MVMR analysis, considering age, completed full-time education, average total household income before tax, and COVID-19 patients. Combining hospitalised COVID with exposure factors resulted in 102 SNPs, while combining very severe respiratory confirmed patients with COVID-19 yielded 96 . Certain SNPs associated with 17 outcomes were removed based on second phenotypes and outliers (Table S3 in the P < 0.05) and pain in various body regions . In the analysis of COVID-19 hospitalised patients as the exposure factor with erectile dysfunction and alopecia areata as the outcome factors, heterogeneity and its causes were identified . Following the removal of these outliers, we determined that COVID-19 hospitalised patients may experience a potentially detrimental impact on erectile dysfunction  and alopecia areata . For very severe respiratory confirmed patients with COVID-19, we found potentially harmful causal effects on pain in various body regions  and tobacco use . Sensitivity analysis did not reveal heterogeneity or pleiotropy, while the leave-one-out analysis also confirmed that individual SNPs did not affect the IVW estimates  = 1.5; 95% confidence interval (CI) = 1.05-2.14, P < 0.05). Notably, the MVMR analysis also showed a causal protective effect of COVID-19 susceptibility on tobacco use, potentially leading to a decrease in its prevalence . This implies that individuals who possess a genetic predisposition to COVID-19 susceptibility may exhibit a diminished likelihood of engaging in tobacco use. The analysis showed no significant heterogeneity or pleiotropy, reinforcing the reliability of the findings  experiencing persistent pain even after an eight-month period of recuperation . This enThe association between COVID-19 and erectile dysfunction has been firmly established, as systemic inflammation and vascular impairment in the penile region are potential underlying factors . AdditioIn contrast, the relationship between COVID-19 infection and tobacco usage is intricate, with conflicting findings reported in various studies due to multiple confounding variables. A study conducted in Finland indicated a noticeable decrease in daily smoking rates from 2018 to 2020, suggesting that the COVID-19 pandemic did not substantially impact the ongoing decline in smoking prevalence . ConversWe proceeded with additional MVMR analysis to examine the correlation between social and psychological factors and PCS and found that education and income acted as mediators in the relationship between COVID-19 and most PCS symptoms. Following adjustments, the causal connections between COVID-19 and symptoms such as myalgia, pain in multiple regions, erectile dysfunction, and alopecia areata were attenuated. Nevertheless, we did observe a causal association between genetic predisposition for hospitalized COVID-19 and arrhythmia. Multiple studies have provided evidence suggesting that SARS-CoV-2 possesses the capability to bind to ACE2 receptors located on cardiovascular cells . AdditioThrough MVMR analysis, we established a causal association between genetic susceptibility to COVID-19 and a diminished propensity for tobacco use. Conversely, the UVMR analysis indicated that those predisposed to severe respiratory symptoms had a higher risk of tobacco use. Socioeconomic factors, like education and income, significantly influenced tobacco usage among COVID-19 patients. Further research should comprehensively analyse the effects of PSC, education, income, and national policies on tobacco usage, while also exploring the underlying mechanisms involved.P < 5 × 10-8). We excluded outliers and then performed UVMR analysis after excluding the second phenotype. Overall, our findings are suggestive of a causal relationships between COVID-19 infections and various PCS symptoms.In using a two-sample MR design to examine the association between three genetic predispositions for COVID-19 and 17 PCS symptoms and adhering to the assumptions inherent to the methodology, we aimed to overcome challenges such as residual confounding and reverse causation commonly encountered in observational studies. We identified strongly associated SNPs for COVID-19 genetic predisposition (A notable strength of our study is the application of MVMR, which considers education and income as mediators influencing PCS through psychological well-being and behavioural interventions . The varP-values from our MR models did not reach a threshold <0.00294. P-values falling between 0.00294 and 0.05 suggest potential associations. but may be susceptible to Type I errors. Applying Bonferroni correction could reduce statistical power, making it harder to identify causal links between COVID-19 and PCS. Lastly, the classification of COVID-19 phenotypes in our study based on disease severity and hospitalisation status lacked specific details on organ involvement, limiting a comprehensive evaluation of COVID-19's varied effects on PCS across different organ systems. Therefore, larger sample sizes are needed for more precise and reliable conclusions [We also acknowledge certain limitations in our study. First, while we used phenoscanner to exclude SNPs with known biological effects on the outcomes, a complete understanding of all SNPs’ functionality remains lacking, making it challenging to fully rule out pleiotropy. However, the effect estimates remained robust across different MR models and sensitivity analyses did not show significant pleiotropy. Second, the clusions .In our MR analysis, we found evidence for the causal influence of genetic predisposition to varying degrees of COVID-19 infection on the heightened susceptibility to certain prevalent PCS. It is possible that several of these symptoms are potentially influenced by factors associated with educational attainment and income level, adding to a novel understanding of PCS pathogenesis and emphasising the significance of incorporating social and psychological determinants, alongside policy modifications, in the comprehensive approach to PCS treatment and management throughout and following the pandemic. Besides prioritising rehabilitation measures and pharmaceutical interventions, it is crucial to conduct further research on the impact of social and psychological factors and policy modifications on both COVID-19 patients and the broader population. Our study could inform the development of tailored interventions and holistic strategies aimed at improving the well-being of individuals affected by PCS.


This review investigates the intricate role of human endogenous retroviruses (HERVs) in cancer development and progression, explicitly focusing on HERV-K (HML-2). This paper sheds light on the latest research advancements and potential treatment strategies by examining the historical context of HERVs and their involvement in critical biological processes such as embryonic development, immune response, and disease progression. This review covers computational modeling for drug-target binding assessment, systems biology modeling for simulating HERV-K viral cargo dynamics, and using antiviral drugs to combat HERV-induced diseases. The findings presented in this review contribute to our understanding of HERV-mediated disease mechanisms and provide insights into future therapeutic approaches. They emphasize why HERV-K holds significant promise as a biomarker and a target.  Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections that were integrated into the human genome millions of years ago . These vEndogenous retroviruses are present as proviruses, the integrated state of retroviral DNA within the host’s germ-line DNA, passed from one generation to another . The intThe presence of endogenous retroviruses in the human genome can positively and negatively affect the host. On the positive side, endogenous retroviruses have been shown to play a role in embryonic development, placenta formation, and immune response regulation. During early human embryogenesis, HERVs are systematically transcribed stage-specific, and their expression serves as a hallmark of cellular identity and potency in early human embryos ,32. ThisOn the negative side, endogenous retroviruses can have pathogenic potential. Unlike most other HERVs, ORFs encoding retroviral proteins remained in HERV-K . Except The downregulation or epigenetic silencing of HML-2 env results in the dissociation of stem cell colonies and enhanced differentiation along neuronal pathways, suggesting the significance of HML-2 regulation in human embryonic development and neural differentiation . A delicHERVs exhibit a range of significant functions. Firstly, they can serve as promoters or enhancers of antiviral genes, generating nucleic acids or proteins that possess viral attributes, triggering the body’s innate immune system. Secondly, they can activate the NF-κB signaling pathway and regulate pathways connected to immune responses . ThirdlyThese viral elements can be found on the cell surface and as virus-like particles. Moreover, they can operate as targets for CD8 cells, innate immune system stimulants, and antiviral gene activity catalysts. This constellation of features renders HERVs an appealing focal point for cancer immunotherapy ,47,48. CThis review aims to provide an overview of the current understanding of how HERV-K (HML-2) affects cancer and outlines the potential avenues for therapeutic interventions.HERV-K, specifically the subtype HML-2, has been implicated in both normal development and potential disturbances  shaping HERV-K (HML-2) has also been associated with autoimmune and inflammatory diseases. Studies have shown that HERV-K (HML-2) transcripts and proteins can activate immune responses, producing inflammatory molecules. This activation may contribute to the development and progression of diseases such as multiple sclerosis (MS), systemic lupus erythematosus, and rheumatoid arthritis ,48. In aThus, HERV-K (HML-2) proviruses can be classified into two sub-types based on the presence or absence of a specific deletion. Type I proviruses express a protein called Np9, while type II proviruses express the Rec protein, which plays a role in RNA transport ,52. HERVThe interaction between HERV and the innate immune system is crucial in maintaining immune homeostasis and preventing viral infections . The innDysregulation of the innate immune response to HERVs can significantly affect autoimmune diseases. If the innate immune system fails to control HERV expression and production of inflammatory cytokines, it can lead to chronic inflammation, tissue damage, and breakdown of self-tolerance. Some HERV env proteins contain an immunosuppressive domain (ISD) that can modulate immune responses and potentially contribute to immune tolerance, leading to the development of autoimmunity . The humOne interesting aspect of HERV-K-specific immune responses is the potential bystander effect. The bystander effect of HERV-K-specific immune responses refers to the possibility that the immune system may target the cells expressing HERV-K proteins and damage the surrounding healthy cells or tissues that do not express HERV-K. This could result in tissue injury, inflammation, or autoimmune reactions. For example, some studies have suggested that HERV-K expression in tumor cells may trigger an immune response that also affects the normal cells in the tumor microenvironment . SimilarThe cross-reactivity of HERV-K-specific antibodies or T cells with other self-antigens is a significant point to consider. Notably, a recent study that uses bamQuery has provided valuable insights into the immunopeptidome, emphasizing the importance of quantifying RNA expression of tumor-specific antigens, including neoantigens, in both malignant and benign tissues . The stuThese observations are particularly relevant when considering HERV-K elements, as they constitute a significant portion of the human genome. While HERV-K elements have been implicated in various diseases, including cancer, the results produced using bamQuery suggest that the transcriptional activity of HERV-K elements may not be confined solely to pathological conditions. Instead, they may be actively expressed in normal tissues, contributing to the pool of potential antigens.This finding underscores the complexity of HERV-K elements and their regulation, as well as the potential implications for immune responses. The presence of HERV-K-specific antigens in normal tissues raises questions about immune tolerance and the potential for cross-reactivity with HERV-K-related immune responses. As we explore the multifaceted roles of HERV-K elements in health and disease, understanding their transcriptional profile in both pathological and non-pathological contexts becomes essential.The bystander effect of HERV-K-specific immune responses is a complex and intriguing topic that requires further investigation. It may have both beneficial and detrimental effects on human health, depending on the context and outcome of the immune response . On one Moreover, we can depend on the suppression mechanisms of HERVs, which are multi-layered defenses aimed at silencing or limiting their expression, as outlined in HERVs contribute to autoimmunity through various mechanisms, including the production of superantigens, molecular mimicry, and DNA hypomethylation. Certain HERVs can produce superantigens that stimulate CD4 T lymphocytes, triggering an immune response associated with autoimmunity . MoleculIt is theorized that beyond the env protein, additional viral proteins stemming from HERV-K (HML-2), including Rec and Np9, could potentially function as oncoproteins or initiate the production of autoantibodies within the host, thereby playing a role in the progression of diseases. Notably, the env protein has been linked to cell fusion within melanoma, culminating in tumorigenesis. Simultaneously, it is implicated in eliciting immune reactions and fostering the advancement of cancer, along with metastasis, particularly in breast cancer ,75. The LTRs bordering HERV-K (HML-2) harbor regulatory elements that can impact the expression of host genes. These LTRs exhibit transcription initiation capabilities more frequently than regular promoters, often resulting in reciprocal up- and downregulation of human genes. The effects of LTRs can be twofold, with the ability to drive the expression of tumor suppressor genes that are commonly silenced in tumors while also promoting tumorigenesis through promoter activation and gene expression alterations . FurtherIn addition, it is crucial to distinguish between active HERV transcription and passive HERV transcription. Passive HERV transcription refers to instances where HERVs are present in a zone of active transcription and are co-transcribed with other genes ,82,83. TPolymorphic integrations of HERV-K (HML-2) within the human genome further contribute to disease susceptibility by influencing viral protein production and regulation of host genes. Specific polymorphic HERV-K (HML-2) loci have been identified with neurologic and immunologic diseases, such as Sjogren’s syndrome, multiple sclerosis, systemic lupus erythematosus, and rheumatoid arthritis . HoweverThe role of HERV-K in immune-related diseases is a complex and evolving area of research. It is important to distinguish between aberrant immunity to HERVs and aberrant expression of HERVs to understand their potential causal roles in different diseases. Aberrant immunity to HERVs refers to the immune system’s response to the presence of HERV antigens. As retroviral elements, HERVs can produce various proteins, including envelope and gag proteins, which can be recognized as foreign by the immune system. In some cases, individuals with certain diseases may have an abnormal or dysregulated immune response to HERV antigens. This immune response can lead to inflammation, autoimmunity, or other immune-related problems. However, it is crucial to note that aberrant immunity to HERVs may not be the sole cause of the disease. It could be just one contributing factor among various genetic, environmental, and epigenetic factors.On the other hand, aberrant expression of HERVs refers to the abnormal activation or upregulation of HERV elements in specific cells or tissues. In certain diseases, such as cancer, autoimmune disorders, and neurological conditions, there is evidence of increased expression of HERVs. The abnormal expression of HERVs can result from various triggers, such as infections, inflammation, or epigenetic changes. This dysregulated expression of HERVs can lead to the production of viral proteins and RNA, which can activate immune responses and contribute to the pathogenesis of the disease.In some cases, aberrant immunity to HERVs and aberrant expression of HERVs may be interconnected. The immune response to the abnormal expression of HERVs can, in turn, contribute to immune dysregulation and inflammation. This creates a complex feedback loop where HERVs and the immune system interact to influence disease development and progression. Therefore, the same immunity does not cause the same disease despite the presence of HERVs, which highlights the multifactorial nature of these diseases. While HERVs may be involved in various immune-related disorders, their impact can vary depending on other genetic and environmental factors, as well as the specific context of each disease. The interplay between HERVs and the immune system is likely influenced by the overall genetic background of the individual, the state of the immune system, and the presence of other pathogens or triggers. Additionally, the location and timing of HERV expression within the body could also contribute to the diversity of disease manifestations. This hypothesis remains our speculation on a feed-forward mechanism of HERV-K reactivation which may well be relevant, and it should be noted that most inflammatory conditions do not leave a lasting HERV-K reactivated state.HERVs play a complex role in immune-related diseases, and the interplay between HERV immunity and expression is likely a contributing factor in the pathogenesis of these diseases. However, it is essential to recognize that HERVs are just one piece of the puzzle, and their impact on disease development and progression is influenced by various other factors. This section explores the role of HERV-K (HML-2) reactivation in viral infections to establish a cohesive understanding of its impact on disease development . One of During HIV infection, the virus produces its regulatory proteins, such as Tat. Tat plays a crucial role in HIV replication by enhancing the transcription of viral genes . HIV infA recent RNA-seq study found that HIV-1 infection in primary CD4+ T cells activates multiple solo-LTRs from the ERV9 lineage, including LTR12C repeats near antiviral genes. These LTR12C elements are enriched in HIV-1-induced ERVs with transcription start sites. Two HIV-1-responsive LTR12C repeats were identified, acting as promoters for guanylate-binding proteins 2 and 5 (GBP2 and GBP5). These specific LTR12C repeats, unique to greater apes, are associated with heightened cytokine responsiveness in antiviral genes, as seen in comparative studies across primate species ,91. The In COVID-19, interferon (IFN) plays a crucial role in the innate immune signaling pathway and host antiviral immunity. The study discussed the activation of human endogenous retroviruses (HERVs), specifically HERV-K (HML-2), with interferon secretion. High expression of HERVs, including HERV-K, has been observed in COVID-19 patients. A study by Guo et al. divided COVID-19 patients into moderate and severe groups and investigated the activation of HERV-K (HML-2) and the expression of interferon-related genes. They observed significant upregulation of HERV-K (HML-2) genes in COVID-19 patients and its association with increased interferon levels, providing insights into the mechanism of interferon production in COVID-19. The study also explored the expression of interferon-related genes in a monkey kidney cell line and suggested using a cell line that can adapt to viral infection and secrete interferon for more effective vaccine production . The finA high-throughput analysis of specific HERV loci in PBMCs was conducted on healthy controls, convalescent individuals, and those retesting positive after recovering from SARS-CoV-2 infection. Differentially expressed HERV loci (deHERV) were identified in individuals exposed to SARS-CoV-2 infection, regardless of the disease’s clinical form. Distinct deHERV loci were found in convalescent individuals and those retesting positive compared to healthy controls. These HERV loci encompassed various HERV groups, including all three classes. This study underscores the connection between HERV expression in PBMCs and the clinical manifestation and prognosis of COVID-19, shedding light on the interplay between HERVs and cellular immunity. It also provides specific transcriptional patterns that may influence COVID-19’s clinical presentation and course of action . The relationship between human T-cell leukemia virus type 1 (HTLV-1) and HERV-K (HML-2) is of interest in understanding HTLV-1-associated diseases. While HTLV-1 typically remains asymptomatic in most infected individuals, some develop severe conditions like adult T-cell leukemia/lymphoma (ALT) and HTLV-1 HAM/TSP. The mechanisms underlying these diseases remain unclear, but HTLV-1 infection leads to inflammation and neurological symptoms by accumulating infected T cells in the CNS. Damage to uninfected cells may occur due to bystander effects or cross-reactivity of CD8+ T cells with self-antigens in neurons. HERV-K (HML-2) is implicated in this context, given the similarities between HAM/TSP and multiple sclerosis, where HERV expression is associated with disease. Recent studies have shown that the HTLV-1-Tax protein induces the transcription of HERVs . ScreeniInterestingly, the expression of HERV-K (HML-2) in hepatitis C virus (HCV) infection has been shown to have implications for liver damage and treatment. The results indicate that factors beyond inflammatory pathways influence HERV-K (HML-2) expression and may be associated with impaired liver function. For instance, increased HERV-K (HML-2) transcript levels are significantly correlated with elevated non-invasive blood markers like ASAT, ALAT, and albumin, which are indicators of liver damage and worsened disease prognosis. They suggested the potential inclusion of reverse transcriptase inhibitors, such as Raltegravir, in HCV antiviral therapy to improve treatment success. These inhibitors have proven effective in treating retroviral diseases like HIV and have shown the ability to inhibit human endogenous retroviruses . SimilarOverall, the activation of endogenous retroviruses, such as HERV-K (HML-2), in the context of viral infections like HIV, HTLV-1, and COVID-19, highlights the intricate relationship between viral pathogens and the human genome. While these viral remnants have the potential to modulate immune responses and contribute to disease pathogenesis, they also represent a fascinating evolutionary record of ancient viral infections. They may offer opportunities for the development of novel therapeutic strategies.Earlier research has indicated that human endogenous retrovirus (HERV) can induce the proliferation of tumor cells and evade apoptosis, representing a significant contributing factor to tumor advancement. HML-2 expression has been detected in various types of tumors, and its presence has been associated with prognostic features and unfavorable outcomes in these cancers . HERV-K HERV-K sequences are highly expressed in teratocarcinoma cells and have been associated with leukemia and germ cell tumors ,110. RecRegarding the functional role of HML-2 in tumor progression, its expression has been associated with disease advancement in various cancer types, suggesting a functional role rather than a mere consequence of carcinogenesis. Breast cancer, pancreatic cancer, leukemia, and teratocarcinoma cells have been discovered to enhance cell proliferation in response to HML-2 proteins such as env and Np9. Inhibiting HML-2 env in breast cancer reduced cell invasion and migration by affecting the Ras/Raf/MEK/ERK signaling cascade. Similarly, cell migration in assays for wound healing was impacted by the modification of Np9 expression in teratocarcinoma cells ,114. FurAdditionally, it has been discovered that the expression of HML-2 is a distinct marker for embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), being quickly muted following differentiation. Due to this property, HML-2 has been investigated as a marker for cancer stem cells (CSCs) or tumor-initiating cells. When melanoma cells were grown in stem-cell media, HML-2 transcription increased, correlated with improved stemness traits and the proliferation of putative CSC populations. HML-2 suppression stopped the CD133+ CSC population from growing . FurtherRecent research in glioblastoma found a pathological expression of HERV-K (HML-2) in both cerebrospinal fluid and tumor tissue. This expression was associated with a cancer stem cell phenotype and poor patient outcomes. The virus was particularly active in neural progenitor-like cells, driving cellular plasticity and maintaining stemness, which are characteristics associated with tumor aggressiveness and resistance to treatment. Inhibiting HERV-K (HML-2) expression with antiretroviral drugs reduced tumor viability and pluripotency, suggesting that it could be a promising therapeutic target to tackle treatment resistance and recurrence in glioblastoma .Similarly, HERV-K (HML-2) influenced tumorigenic characteristics in colorectal cancer. Knocking out the HERV-K env gene using the CRISPR-Cas9 system in colorectal cancer cells significantly reduced cell proliferation, migration, and tumor colonization. This effect was related to the downregulation of nuclear protein-1 (NUPR1), which plays a crucial role in cancer cell proliferation and migration. Inhibition of HERV-K (HML-2) expression decreased reactive oxygen species levels, further affecting cancer cell behavior . In the In renal cell carcinoma, the envelope protein of HERV-K (HERV-K env) was identified as a novel tumor antigen and a prognostic indicator. Its expression was significantly elevated in clear renal cell carcinoma (ccRCC) compared to other subtypes. Patients with higher HERV-K (HML-2) env protein levels had better disease-specific survival rates, suggesting its potential as a prognostic marker in this type of cancer .The findings from these studies highlight the significant role of HERV-K (HML-2) in various types of cancer, influencing critical aspects such as stem cell characteristics, tumorigenic behaviors, and patient prognosis. The reactivation of these endogenous retroviruses presents exciting new opportunities in cancer research, including potential applications in diagnostics, therapeutic targeting, and prognostic indicators. However, to harness the full potential of HERV-K as a clinical tool, further research is essential to comprehensively understand its underlying mechanisms and clinical implications in cancer development and treatment. To optimize HERV-K as a specific target for different malignancies, more in-depth characterization of HERV-K expression patterns in various cancer types is required. In developing therapeutic strategies, it is crucial to consider how HERV-K contributes to normal development and whether its targeting could disrupt essential cellular processes. This requires a thorough understanding of the functional roles of HERV-K in healthy cells and tissues. By carefully assessing its impact on normal physiology, researchers can design therapies explicitly targeting cancer cells while sparing healthy cells. To validate the effectiveness of HERV-K targeting in cancer management, it is imperative to conduct comprehensive in vitro models and robust clinical research. In vitro studies will provide valuable insights into the mechanisms of HERV-K action and its interactions with various signaling pathways, aiding in the development of targeted therapeutics. Clinical trials will be crucial to evaluate the safety, efficacy, and potential side effects of HERV-K-based therapies in real-world settings.Moreover, HERVs hold promise not only as therapeutic targets but also as potential cancer biomarkers. Monitoring HERV-K expression levels in cancer patients could provide valuable diagnostic and prognostic information, facilitating early detection and personalized treatment strategies. HERV-K (HML-2) represents an exciting avenue in cancer research, with potential applications in diagnostics and targeted therapies. However, translating these findings into clinical practice requires extensive research and validation in diverse cancer types. By advancing our understanding of HERV-K biology and its role in cancer, we can unlock the full potential of these endogenous retroviruses as therapeutic targets and biomarkers for improved cancer management. HERV-K (HML-2) is a retrovirus that can encode various enzymes critical for its replication and integration into the human genome. One such enzyme is reverse transcriptase, which converts the viral RNA genome into DNA. This process is crucial for integrating HERV-K (HML-2) DNA into the host cell’s genome, making it a potential target for antiviral drugs . Non-nucRecent studies have revealed the potential of specific antiviral drugs  expression by antiviral drugs represents a potential therapeutic approach for diseases associated with HERV-K activation. Further research is necessary to understand the mechanisms of action and develop selective inhibitors. Identifying and validating effective treatments targeting HERV-K could lead to improved outcomes for patients with autoimmune disorders and HERV-K-associated tumors.The potential role of HERV-K (HML-2) in cancer and autoimmunity makes it an attractive target for innovative therapeutic strategies. While HERV-based therapies are being explored, the definitive evidence of a causal association between HERVs’ presence/expression and the onset/progression of specific disorders remains to be established. A significant gap in current studies is the need for standardized methodologies and robust genomic characterization of individual HERV groups to produce consistent and reliable results . HERV enHERV-K (HML-2) has been identified as a tumor-associated antigen (TAA) expressed on various cancer cells, including breast cancer and melanoma. This makes it an attractive target for developing immunotherapeutic strategies to combat these diseases. CAR-T cell therapy is a groundbreaking approach in oncology, where T cells are genetically engineered to express chimeric antigen receptors (CARs) on their surface. These CARs enable T cells to recognize and target specific antigens in cancer cells. In the context of HERV-K (HML-2), cell-based technologies such as autologous chimeric antigen receptor T (CAR-T) cells have been designed to specifically target HERV-K envelope protein, which is overexpressed on cancer cells but not on normal cells ,156. TheThis approach demonstrates the potential of utilizing HERV-K and other transposable element-derived proteins as tumor-associated antigens (TAAs) in cell-based therapies ,152. FurDespite the promising results, challenges and limitations still need to be addressed in the development of CAR-T cell therapy targeting HERV-K (HML-2). Further research is required to optimize the CAR-T cell design, improve their persistence and efficacy, and overcome potential immunosuppressive factors in the tumor microenvironment. Nevertheless, the development of HERV-K (HML-2)-specific CAR-T-cell therapy represents an exciting avenue for the advancement of cancer immunotherapy and offers hope for improved treatment outcomes for patients with breast cancer, melanoma, and other solid tumors expressing HERV-K (HML-2) . In addition to antibody-based and cell-based therapies, epigenetic therapy has shown promise in upregulating HERV-K expression and stimulating anti-tumor immune responses. Inhibiting DNA methyltransferases (DNMTs) in various cancer types has been shown to activate immune signaling pathways by sensing HERV-derived double-stranded RNA. This activation restores effector T-cell function and prevents tumor immune evasion. The upregulation of HERV-K and other TAAs through epigenetic therapy suggests combining these approaches with anti-HERV-K antibodies or cell therapies for enhanced therapeutic outcomes ,145.Moreover, HERV-K (HML-2) could also be exploited as an anti-cancer vaccine, particularly in tumor types where HERV-K reactivation occurs early in carcinogenesis. Preclinical studies using a modified vaccinia virus expressing HERV-K env have shown promising results in preventing tumor establishment. The development of HERV-K-based vaccines holds potential for cancer immunotherapy and could be further explored in clinical trials . The appComparing HERV-K to other individual antigenic classes explored for tumor immunotherapy, recent studies suggest that immunization with murine ERVs (MelARV) using VLV technology outperformed immunization studies published for neoantigens. Combining the modified VLV with an α-PD1 checkpoint inhibitor (CPI) exhibited excellent curative efficacy against established mouse colorectal tumors ,163. FurFurthermore, advancements in gene-editing technologies like CRISPR/Cas9 have opened up possibilities for targeting HERV-K elements. In preclinical studies, CRISPR/Cas9-mediated knock-out of the HERV-K env gene has shown promising results in inhibiting its transcription and translation. This disruption of HERV-K function has implications for regulating fundamental cellular processes and could pave the way for novel therapeutic approaches in cancer treatment ,167. WhiFinally, we would like to highlight the utilization of antiviral agents for cancer management. Although antiviral agents were initially developed to combat viral infections, they have shown intriguing potential for oncology. Emerging evidence suggests that certain viruses, including human endogenous retroviruses (HERVs), play a role in cancer development and progression ,170. SinTherefore, the investigation of HERV-K (HML-2) as a potential drug target and the development of antivirals for viral infections and cancer treatment offer promising therapeutic opportunities that hold the potential to revolutionize the field of medicine. Extensive research has examined the role of HERV-K, specifically the HML-2 subtype, in cellular physiology and its relationship with disease pathogenesis. Studies that demonstrated an antiviral effect against HML-2 showed a trend for slower progression of the disease, which supports the possible involvement of this virus in the pathogenesis of diseases. Searching for antivirals that either alone or in combination with each other influence the viral cargo of HERV-K (HML-2) can be advantageous to better understand the mechanism of action of this virus and finding treatments in the case of HERV-K (HML-2) reactivation. Regarding cancer treatment, we could seek a combination of antivirals with chemotherapy. We aim to contribute to this understanding by employing a perspective approach for future research that combines in vitro studies and in silico analyses.The detection of viral cargo in microvesicles has important implications for the diagnosis and treatment of diseases. The viral cargo delivered by microvesicles can induce prominent immunomodulation in recipient cells, which may contribute to developing diseases. Therefore, understanding the role of microvesicles in the assembly and transmission of viruses can help identify potential targets for therapeutic intervention. Exploring HERV-K (HML-2) in teratocarcinoma cell lines and microvesicles can help unravel disease mechanisms for improved treatment strategies in several ways. Firstly, by identifying the role of microvesicles in the assembly and transmission of viruses, this research can help identify potential targets for therapeutic intervention. Secondly, researchers can develop new strategies for diagnosing and treating diseases associated with these viruses by understanding how HML-2 elements are captured during the shedding of microvesicles and delivered to recipient cells. Thirdly, by studying the immunomodulatory effects of viral cargo delivered by microvesicles containing HML-2 elements, researchers can gain insights into how these viruses may contribute to disease development and progression. Finally, by better understanding the mechanisms underlying HML-2-induced diseases, researchers can develop more effective treatments targeting specific aspects of viral replication or transmission. The findings of Morozov et al. on HERV-K (HML-2) from teratocarcinoma cell lines hold promise in understanding the mechanisms underlying HERV-K (HML-2)-induced diseases and discovering enhanced treatment approaches. A comprehensive analysis of HML-2 parameters and properties, including structural proteins, viral RNA, and particle morphology, reveals the presence of truncated forms of transmembrane proteins. Furthermore, electron microscopy uncovers the existence of free virions and microvesicles carrying HML-2 elements, capable of eliciting specific immune responses and modulating recipient cells. Sequencing findings highlight distinct HERV-K108 env presence in Tera-1 cells and multiple env sequences in GH cells . The resIn vitro studies on HERV-K (HML-2) from teratocarcinoma cell lines provide insights into virus assembly and transmission ,176. HowDespite limitations, this study offers significant contributions. It comprehensively analyzes HERV-K (HML-2) from teratocarcinoma cell lines, improving our understanding of virus assembly and transmission. Detecting viral cargo in microvesicles provides insights into their role in disease development. We should continue to explore this connection. In silico studies have emerged as a valuable tool in biomedical research, offering a cost-effective and time-efficient approach to exploring various aspects of disease mechanisms and treatment strategies. One area where in silico studies have made significant contributions is in the prediction of efficacy and dose-response of antivirals. Thus, we could bring this to the context of exploring HERV-K (HML-2) mechanisms in cell lines and microvesicles. In silico studies utilize computational models and algorithms to simulate and predict the behavior of biological systems. By leveraging existing knowledge and experimental data, these studies provide insights into the interactions between antiviral compounds and HERV-K, enabling the prediction of drug efficacy and dose-response relationships. This approach offers a cost-effective and efficient alternative to traditional experimental methods, as it reduces the need for extensive laboratory testing. Furthermore, in silico studies can unravel disease mechanisms associated with HERV-K by investigating the molecular interactions between the retrovirus and host cells, simulating the complex biological processes involved in HERV-K activation, replication, and pathogenicity, and identifying critical molecular targets for therapeutic intervention. Inspired by Karlsson et al., we identified a potential approach to explore new treatments using nonlinear mixed-effects modeling for HERV-K (HML-2). This approach can account for cell-to-cell variation in parameter estimation using a hierarchical model that includes fixed and random effects. The fixed effects represent the average behavior of all cells, while the random effects capture the variability between individual cells. Nonlinear mixed-effects modeling has already been applied in real-world single-cell studies. One example is a study on fluorescence recovery after photobleaching (FRAP), where NLME was used to estimate parameters related to protein mobility and binding kinetics in individual cells ,179. AnoBased on the data acquired on the prediction of the effect of different antivirals and dose regimens on the population, we can extrapolate the findings to predict the drug response of different antivirals on the population level. This will involve relating the active drug concentration to in vivo dose equivalents, building physiologically based pharmacokinetic (PBPK) models, and validating the predictions against realistic exposure estimates derived from biomonitoring data.Based on these examples, this approach is allied to other in silico tools for predicting the efficacy and dose-response of antivirals on HERV-K (HML-2) viral cargo. Key methods and techniques used in in silico studies for predicting the impact of antivirals on HERV-K viral cargo are as follows: (1) bioinformatics and genomic analysis, which play a crucial role in understanding the structure and function of HERV-K elements. By utilizing genomic databases and bioinformatics tools, researchers can identify potential drug targets within the HERV-K genome; (2) virtual screening to identify potential drug candidates that can interact with specific viral targets. Using in silico approaches, researchers can screen large libraries of chemical compounds to predict their potential binding affinity and inhibitory effects on viral components. Virtual screening techniques, such as molecular docking and structure-based drug design, enable the identification of promising antiviral candidates that can selectively target HERV-K viral cargo; (3) molecular docking simulations are designed to predict the binding affinity and interaction between antiviral drugs and viral targets. This technique involves computationally modeling the three-dimensional structures of drug molecules and viral proteins or enzymes to assess the likelihood of effective drug-target binding; (4) systems biology modeling, which integrates experimental data and computational algorithms to simulate the dynamics of HERV-K viral cargo and its response to antiviral drug treatments. By developing mathematical models, researchers can predict the impact of drug interventions on HERV-K expression and replication. These models consider various factors such as drug pharmacokinetics, viral kinetics, and host-virus interactions to provide insights into the efficacy and dose-response relationships of antiviral drugs; (5) network analysis approaches can be applied to investigate the interactions between HERV-K elements and cellular components. By constructing and analyzing interaction networks, researchers can identify key nodes or pathways involved in HERV-K expression and assess the potential effects of antiviral drugs on these networks. Network analysis provides a systems-level understanding of the impact of antiviral interventions on HERV-K viral cargo and can guide the identification of critical targets for drug development.Additionally, an essential aspect to consider in bioinformatics methods is the precise reference of HERVs employed. Regardless of the method employed, it is crucial to specify the genomic references and locations of HERVs under investigation. This information is critical for ensuring accurate analysis and interpretation of HERV-related data. Therefore, it is pertinent to discuss the challenge of multimapping, a common issue encountered when dealing with repetitive elements such as HERVs. Multimapping occurs when sequence reads can be mapped to multiple locations in the genome due to sequence similarity, making it challenging to determine the true source of the read. To address multimapping concerns, recent advancements in bioinformatics tools have provided valuable solutions. Notably, Telescope, a computational software tool described in a study by Bendall et al., offers accurate estimation of transposable element expression, including HERVs, with resolution to specific genomic locations. Telescope tackles the issue of fragment assignment uncertainty by employing a Bayesian statistical model to reassign ambiguously mapped fragments to their most probable source transcript. This approach has been demonstrated to be highly effective in characterizing HERV expression within the transcriptomic landscape. The study by Bendall et al. employed Telescope for single locus analysis of HERV expression in various cell types and revealed significant differences in the retrotranscriptome across these cell types . TelescoThe correlation between aberrant HERV-K (HML-2) expression and various diseases suggests its potential as a diagnostic or prognostic biomarker. However, further research is required to establish HERV-K (HML-2) as a causative factor and to identify specific HERV loci associated with particular diseases. Methodological limitations have hindered studies on the role of specific HERV-K (HML-2) loci. Nonetheless, advancements in sequencing methods and bioinformatic tools offer promising prospects for understanding the causation and development of diseases. The literature reviewed strongly suggests that viral infections can trigger HERV-K (HML-2) reactivation and expression, potentially exacerbating disease progression or influencing the immune response. However, this interaction’s exact mechanisms and implications remain to be fully elucidated. Moreover, the active involvement of HML-2 reactivation in tumor progression indicates that it is more than a mere byproduct of carcinogenesis. HML-2 expression is consistently correlated with disease progression and poor outcomes in various tumors. While larger cohort validations are needed, HML-2 mRNA/protein expression and autoantibodies show potential as robust tools for molecular diagnostics. Functionally, HML-2 proteins contribute to multiple cancer hallmarks, including tumor growth, metastasis, immune evasion, and tumor-promoting inflammation, making them attractive targets for personalized treatment of aggressive tumor subsets with high expression. However, challenges remain in HML-2 biology. Poor annotation in human genome assemblies and the repetitive nature of HML-2 transcripts pose obstacles to conventional RNA-seq and bioinformatic analyses. Accurate quantification and representation of HML-2 loci are essential. The lack of comprehensive information on the tertiary structure and functional motifs of HML-2 proteins limits our understanding of their interactions and potential for drug discovery. Moreover, an extensive catalog of TE/HERV transcript and protein expression in healthy and diseased tissues is necessary to definitively unravel their association with diseases and determine their physiological role.Furthermore, as HML-2 mRNAs can stimulate innate immune receptors, efforts should be made to differentiate their effects from their protein products. Overcoming these challenges and advancing our knowledge of HML-2 biology will facilitate the development of improved diagnostic tools, targeted therapies, and a deeper understanding of their contribution to disease processes.


EGFR gene frequently occur, thus hijacking the EGFR signaling pathway to promote tumorigenesis. Non-small cell lung cancer (NSCLC) tumors in particular often contain prevalent and shared EGFR mutations that provide an ideal source for public neoantigens (NeoAg). Studies in both humans and animal models have confirmed the immunogenicity of some of these NeoAg peptides, suggesting that they may constitute viable targets for cancer immunotherapies. Peptide vaccines targeting mutated EGFR have been tested in multiple clinical trials, demonstrating an excellent safety profile and encouraging clinical efficacy. For example, the CDX-110 (rindopepimut) NeoAg peptide vaccine derived from the EGFRvIII deletion mutant in combination with temozolomide and radiotherapy has shown efficacy in treating EGFRvIII-harboring glioblastoma multiforme (GBM) patients undergone surgery in multiple Phase I and II clinical trials. Furthermore, pilot clinical trials that have administered personalized NeoAg peptides for treating advanced-stage NSCLC patients have shown this approach to be a feasible and safe method to increase antitumor immune responses. Amongst the vaccine peptides administered, EGFR mutation-targeting NeoAgs induced the strongest T cell-mediated immune responses in patients and were also associated with objective clinical responses, implying a promising future for NeoAg peptide vaccines for treating NSCLC patients with selected EGFR mutations. The efficacy of NeoAg-targeting peptide vaccines may be further improved by combining with other modalities such as tyrosine kinase or immune checkpoint inhibitor (ICI) therapy, which are currently being tested in animal models and clinical trials. Herein, we review the most current basic and clinical research progress on EGFR-targeted peptide vaccination for the treatment of NSCLC and other solid tumor types.The epidermal growth factor receptor (EGFR) plays crucial roles in several important biological functions such as embryogenesis, epithelial tissue development, and cellular regeneration. However, in multiple solid tumor types overexpression and/or activating mutations of the  Growth factors are hormone-like molecules that can promote cell division and migration but are also involved in tumor growth and metastasis. Dr. Rita Levi-Montalcini discovered the first growth factor, nerve growth factor (NGF), in 1954 . For theEGFR gene located on chromosome 7p12.2. EGFR belongs to the ErbB family comprised of EGFR/ErbB-1, HER2/ErbB-2, HER3/ErbB-3 and HER4/ErbB-4. All members of the ErbB family are transmembrane receptor tyrosine kinases (RTKs) which consist of an extracellular ligand-binding domain, a transmembrane domain, an intracellular tyrosine kinase (TK) domain, and a regulatory tail region containing conserved phosphorylation sites  is a ~170 kDa protein encoded by the on sites  4]. Erb. ErbEGFRWhen the extracellular ligand-binding domain of EGFR is not bound to its ligand, this protein exists as a monomer on the plasma membrane (PM) and the tyrosine residues located in the intracellular TK domain and regulatory tail domain are not phosphorylated ,7,8. In Similar to many other receptors, EGFR is internalized through multiple endocytic pathways with different features that regulate the activity and fate of this receptor ,12. ThisEGFR has seven different known ligands, including EGF, transforming growth factor-alpha (TGF-α), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigene (EPGN) . BindingAlthough the best characterized functions of the EGFR are aforementioned ligand- and kinase-dependent activation, also called the “canonical” EGFR signaling pathway , novel fEGFR gene amplification and/or overexpression, in addition to a variety of activating mutations in different types of human cancers  in the vaccinated group and seven [3%] in the control group), which had not been observed in the previous Phase II trials [The significantly improved PFS and OS compared with historical controls in these Phase II clinical trials encouraged researchers to proceed to a Phase III trial. A double-blind, randomized, international Phase III trial (ACTIV) in patients with newly diagnosed EGFRvIII-expressing glioblastoma was initiated . In the I trials .R) peptide. The patient experienced a remarkable regression of multiple lung tumor nodules after peptide vaccination, but a liver metastasis continued to progress. which forced vaccination to be stopped. Finally, he died of liver metastasis shortly afterward [Peptide vaccines targeting commonly shared EGFR NeoAgs have also been tested in EGFR-mutated NSCLC patients in phase I clinical trials. In 2016, we reported an 85-year-old Asian patient with stage IV lung squamous cell carcinoma that had progressed after 9 cycles of chemotherapy . Their tfterward . Becausefterward ,40.Building on the results of this case report, we initiated a Phase I trial of personalized NeoAg peptide vaccination (PPV) for treating NSCLC patients . Twenty-+ T cell responses against EGFR NeoAg peptides in their peripheral blood during the course of PPV, particularly L858R and T790M.. In addition, we detected enhanced expansion and tumor infiltration of L858R-specific CD8+ T cells in the PBMC of one responding patient after the administration of a vaccine that contained five NeoAg peptides that all encompassed the EGFR L858R mutation [As expected, the vaccination approach showed a very good safety profile. Other than temporary rash, fatigue and/or fever occurring in three patients, no treatment-related adverse events were observed. In the 24 vaccinated patients, median PFS and OS were 6.0 and 8.9 months, respectively. Within 3 to 4 months of their first dose of PPV, 6 of the 24 patients (25%) experienced a partial response (PR) and 1 (4%) attained a complete response (CR), Notably, all seven objective clinical responders were from the EGFR-mutated cohort, including 4 patients that received concurrent TKI + PPV and 3 patients (including the CR) having received PPV alone. Amongst the 7 responding patients, 5 demonstrated specific CD8mutation . To our Combination therapies involving EGFR-targeted peptide vaccines along with other immunotherapies have not yet been reported in murine models or human clinical trials. However, it is hoped that improved clinical outcomes will be achieved when EGFR-targeted peptide vaccines are used in combination with immune checkpoint inhibitor (ICI) therapy, which has already been observed with other NeoAg peptide vaccine approaches.Ott and colleagues reported the results of a Phase Ib trial of personalized NeoAg peptide vaccine (NEO-PV-01) in combination with the PD-1 antibody nivolumab in 82 patients with advanced melanoma, NSCLC or bladder cancer . Nivolum+ T cells and CD8+ T cells. Subsequently, these T cells can specifically recognize and lyse tumor cells harboring these EGFR mutations, achieving a potent and specific anti-tumor response. It is hoped that the expansion and anti-tumor function of these EGFR mutation-derived neoantigen-specific T cells can be augmented and reinvigorated by ICI therapy [Patients with EGFR-mutated NSCLC have generally shown poor responses to ICI therapy, such as antibodies targeting PD-1/PD-L1 ,66,67,68 therapy ,70, therEGFR gene occur frequently in multiple solid tumor types, potentially providing an ideal source of public NeoAgs to target with immunotherapy. Importantly, the immunogenicity of peptides derived from some of these shared mutations has now been validated in vitro and in vivo in animal models and human clinical trials. Furthermore, EGFR NeoAgs appear to act dominantly in the induction of T cell-mediated immunity in patients and were strongly associated with objective clinical responses, supporting the promising future of EGFR NeoAg-targeted peptide vaccines in treating EGFR-mutated NSCLC patients.As an RTK, EGFR governs multiple critical biological functions including embryogenesis, development, and tissue regeneration. Commonly shared activating mutations of the In the future, the efficacy of EGFR-targeting peptide vaccines may be further improved by screening for more immunogenic peptides, introducing more effective adjuvants, or combining with other immunotherapy such as ICIs. The identification of shared NeoAgs also opens the door to other immunotherapeutic approaches such as T cell receptor (TCR)-based therapies. In addition, because the immunosuppressive TME can jeopardize the anti-tumor function of neoantigen-specific T cells on which the efficacy of EGFR-targeting peptide vaccines relies, TME remodeling reagents such as TKIs, metabolism modulators and cytokines that can relieve the immunosuppression within the TME may further enhance the efficacy of these immunotherapies.


Astroviruses (AstVs) can cause of severe infection of the central nervous system (CNS) in immunocompromised individuals. Here, we identified a human AstV of the VA1 genotype, HAstV-NIH, as the cause of fatal encephalitis in an immunocompromised adult. We investigated the cells targeted by AstV, neurophysiological changes, and host responses by analyzing gene expression, protein expression, and cellular morphology in brain tissue from three cases of AstV neurologic disease (AstV-ND). We demonstrate that neurons are the principal cells targeted by AstV in the brain and that the cerebellum and brainstem have the highest burden of infection. Detection of VA1 AstV in interconnected brain structures such as thalamus, deep cerebellar nuclei, Purkinje cells, and pontine nuclei indicates that AstV may spread between connected neurons transsynaptically. We found transcriptional dysregulation of neural functions and disruption of both excitatory and inhibitory synaptic innervation of infected neurons. Importantly, transcriptional dysregulation of neural functions occurred in fatal cases, but not in a patient that survived AstV-ND. We show that the innate, but not adaptive immune response was transcriptionally driving host defense in the brain of immunocompromised patients with AstV-ND. Both transcriptome and molecular pathology studies showed that most of the cellular changes were associated with CNS-intrinsic cells involved in phagocytosis and injury repair , but not CNS-extrinsic cells (T and B cells), suggesting an imbalance of innate and adaptive immune responses to AstV infection in the brain as a result of the underlying immunodeficiencies. These results show that VA1 AstV infection of the brain in immunocompromised humans is associated with imbalanced host defense responses, disruption of neuronal somatodendritic compartments and synapses and increased phagocytic cellular activity. Improved understanding of the response to viral infections of the human CNS may provide clues for how to manipulate these processes to improve outcomes. Immunocompromised individuals are at increased risk of severe viral infection of the central nervous system (CNS) due to a failure to contain infection before it reaches the brain. Here, we identified the VA1-clade astrovirus infection as the cause of fatal encephalitis in an immunocompromised patient and show that it targeted neurons in the brain. Study of brain tissues from three cases of astrovirus encephalitis in immunocompromised patients showed that the disease was associated with impairment of many functions of neurons and disruption of both excitatory and inhibitory synapses that form neuronal connections. Further analyses showed that the early innate, but not later adaptive immune response was driving antiviral host defense response in the brain. Activation of the CNS-resident cells important for phagocytosis, homeostasis, and repair  was evident, with little activity of the adaptive peripheral immune cells (T cells and B cells). These results demonstrate that the pathogenesis and outcome of astrovirus infection in the brain of patients with underlying immunodeficiencies is defined by an imbalance of different arms of the antiviral host defense system and disruption of functions of infected neurons. These findings suggest pathways that can be targeted to improve outcome of viral CNS infections. Infections of the central nervous system (CNS) in humans cause significant morbidity and mortality and are increasing due to spread of pathogens to new locations, emergence of new pathogens, and an increasing number of patients with immunodeficiencies. Primary (genetic) and secondary (acquired) immunodeficiencies  are assoTo date, twelve publications describe 14 human cases of AstV neurological disease (AstV-ND) –24 with Here, we identified HAstV-NIH of the VA1 genotype as the cause of fatal encephalitis in an immunocompromised adult. We analyzed brain tissue from this patient and two other immunocompromised patients with AstV-ND using an integrative approach to study neurophysiological changes and host defense responses to infection at the level of transcriptional regulation, protein expression, and cell morphology/topology/function.Astroviridae . For this, we used the Panther statistical enrichment test (SET) . SET useCompared to a normal age-matched control, transcriptional modulation of chemical synaptic transmission in the brain of the patient with HAstV-NIH shifted below the significance cut-off value  , indicatWe next asked whether the transcriptional dysregulation of neurophysiology was a common feature of human AstV-ND associated with AstVs of the VA1 genotype. Accordingly, we investigated the differential gene expression in the brain samples from three patients with AstV-ND: (i) AstV-ND-1-NIH (infected with HAstV-NIH), (ii) AstV-ND-2-NY (infected with HAstV-PS ), and i. PostmorThe DEGs for each AstV-ND case were determined by comparisons to the appropriate normal controls , followeFunctional analysis of genes that were downregulated in the brain of three patients with AstV-ND showed that virtually every neuronal subcellular compartment was affected in the two fatal cases (AstV-ND-1-NIH and AstV-ND-2-NY), but not in the brain of the patient that survived the infection (AstV-ND-3-France). These included genes encoding both excitatory and inhibitory synapses , neuronal cell bodies, dendrites, and axons  was markedly reduced . This coSince we observed depletion of MAP2 in the somatodendritic profiles that harbored AstV capsid protein by a double fluorescent immunostaining  and our Taken together, these results validate the gene expression data by demonstrating the loss of expression of critical proteins functioning in the synaptic and somatodendritic compartments of neurons. The net impact of AstV infection of neurons was disruption of synaptic and somatodendritic integrity, loss of afferent innervation of infected neurons, and impairment of both excitatory and inhibitory neurotransmission. Impairment of these essential neural functions may have contributed to the fatal outcome in AstV-ND-1-NIH, and possibly in AstV-ND-2-NY.To gain insight into host defense responses to AstV infection in the brain of patients with impaired adaptive immunity, we first performed SET on unnormalized gene expression data from brain samples of AstV-ND-1-NIH and a normal individual without known neurological disease. We focused on three major gene ontology terms critical for host defense to viral infections of the CNS: immune effector processes, innate immune response, and lymphocyte mediated immunity. As expected, these immune processes were relatively suppressed  in the normal brain . In the Next, we determined the upregulated genes in brain tissue from the three cases of AstV-ND  by comparing them with appropriate normal controls . An analTo confirm the transcriptome results at the protein expression and cell morphology/topology/function levels, we examined tissue sections from AstV-ND-1-NIH using immunohistochemistry and evaluated reactive astrocytosis, microglia/macrophage activation and migration, and lymphocytic activation, migration, and infiltration . StaininExpression of glial fibrillary acidic protein, a marker for astrocyte activation associated with neuronal injury, was increased in infected brain tissue along with hypertrophy of the soma of perivascular  and periThe lysosomal-associated membrane protein CD68, a protein marker of activated phagocytic macrophages and microglia, was detected in numerous cells in the perivascular, parenchymal border, intraparenchymal, and perineuronal locations in AstV-ND-1-NIH . NotablyMinimal numbers of CD3+ T cells were detected in leptomeningeal and perivascular locations, with very restricted migration and infiltration of the adjacent perivascular parenchyma . No CD20In this study, we investigated the neuropathogenesis of AstV-ND based on the changes in gene expression in three patients with the disease and confirmed the findings using immunohistochemistry in several regions of the brain of one of these patients. We demonstrate that neurons are the principal cells targeted by a neuropathogenic VA1 AstV in the human brain and localized AstV capsid protein to the neuronal perikarya, dendrites and axons. These findings indicate that AstV, similar to many other neuropathogenic viruses , can sprThe gradient of neuronal astrovirus infection in the human brain intensifies caudally with the major impact on the brainstem, followed by the cerebellum. A similar caudal gradient of neuronal vulnerability to AstV infection has been seen in the CNS of other mammals . NeuropaThe human CNS has barriers to protect itself from an invasion by various pathogens (including viruses) that otherwise would be detrimental to neural function and could be life-threatening. Viremia ensues when virus replication in the peripheral tissue reaches a threshold such that it can no longer be contained, and the virus enters the bloodstream. Viremia facilitates virus spread to various tissues  and increases the probability of virus invasion of the CNS. Indeed, viremia is a major neuropathogenic determinant for viral CNS infection . HoweverUnderlying conditions in patients with AstV-ND in this study included both primary and secondary immunodeficiencies  predominOne limitation of this study is a small sample size, which included brain samples from only three cases of human AstV-ND that we were able to obtain. However, considering that only 14 cases of AstV-ND have been reported to date, with the neuropathological findings available only for 7 single cases  13–24],–24,13–24With the increasing use of immunosuppression for a variety of disorders  predisposing to opportunistic viral infections of the CNS, as well as the emergence or reemergence of neurotropic viruses  ,52 the fConsent for research studies was obtained for use of leftover brain tissue from AstV-ND-3-France from the patient’s parents; brain tissue was obtained postmortem from AstV-ND-1-NIH and AstV-ND-2-NY and as such is not considered human subjects research.http://www.nimh.nih.gov/hbcc). Formalin-fixed paraffin-embedded (FFPE) brain tissue sections from AstV-ND-1-NIH case were used for histopathological analysis (hematoxylin-eosin [H&E] staining), brightfield colorimetric immunohistochemistry, and double immunofluorescent staining. FFPE sections from an age-matched subject without known neurological disease were used as normal controls.For RNA-seq, frozen brain tissue samples from three patients with AstV-ND were used: (i) AstV-ND-1-NIH ; (ii) AstV-ND-2-NY  ; and  and underwent a double cord blood stem cell transplant. He received cyclosporine and mycophenolate mofetil for graft-versus-host disease prophylaxis. He engrafted with 100% donor cells but developed gastrointestinal graft-versus-host disease and was treated with methylprednisolone and budesonide and his cyclosporine and mycophenolate mofetil were continued. One month after transplant, the patient presented with lower extremity sensory changes and weakness with ascending neuropathy, ataxia, unstable gait, and fatigue. An MRI of the brain and cervical spine were unremarkable, and an EMG showed a sensory motor neuropathy. The cerebrospinal fluid (CSF) showed 5 white blood cells/ml, protein 55 mg/dL, glucose 57 mg/dL and was negative for human herpesvirus 6, varicella-zoster virus, herpes simplex virus, JC virus, and cytomegalovirus. Cytology showed atypical lymphocytes. A paraneoplastic panel was negative and oligoclonal bands were not detected. Three months after his neurologic changes began, a PET scan of brain showed a mildly abnormal thalamus with increase uptake, and one week later, an MRI showed a new fluid attenuated inversion recovery hyperintensity with restricted diffusion in the right midbrain, both cerebellar peduncles, and right lateral pons. An EEG showed background slowing, and the CSF showed protein 48 mg/dL, glucose 65 mg/dL, white blood cell count 5 cells/ml with 68% lymphocytes, and many atypical lymphocytes and was again with negative for human herpesvirus 6, JC virus, and cytomegalovirus. He was treated with acyclovir and foscarnet followed by rituximab and solumedrol. He developed progressive mental status changes, weakness, extra-pyramidal signs, and agitation. An EEG showed generalized slowing and an EMG showed a sensorimotor polyneuropathy involving the bilateral lower extremities that was ascending. CSF showed elevated protein and lack of pleocytosis, and he was treated with high dose intravenous immunoglobulin and steroids for presumptive variant Guillain-Barre. A sural nerve biopsy showed nerve damage. He received an additional course of rituximab and died seven months after transplant. At autopsy, the diagnosis was a multifocal leukoencephalopathy of unknown etiology.RNA was isolated from frozen brain tissue sample from AstV-ND-1-NIH case and hybridized to a microarray containing >3000 viral probes for viruses spanning 31 virus families known to infect vertebrates and >19,000 probes for endogenous genes. The virus microarray and bioinformatics analysis were performed as previously described . An averRNA was isolated from frozen brain tissue sample from AstV-ND-1-NIH case and cDNA was generated using a Superscript First-Strand Synthesis System for RT-PCR using random hexamers (Invitrogen). A first round of PCR was performed using primers panAV-F11 (5′-GARTTYGATTGGRCKCGKTAYGA-3′), panAV-F12 (5′-GARTTYGATTGGRCKAGGTAYGA-3′) and panAV-R1 (5′-GGYTTKACCCACATICCRAA-3′), followed by a second round of PCR with primers panAV-F21 (5′-CGKTAYGATGGKACKATICC-3′), panAV-F22 (5′-AGGTAYGATGGKACKATICC-3′) and panAV-R1 as previously described .https://www.ncbi.nlm.nih.gov/nuccore/OP293097).RNA was isolated from frozen brain tissue sample from AstV-ND-1-NIH case and cDNA was generated using a Superscript First-Strand Synthesis System for RT-PCR using random hexamers (Invitrogen). Astrovirus capsid primers, Astrocap for 5- CGC GCG GAT CCA CCA TGG GGG GAT GGT GGT TTG TCA AG -3 and Astrocap rev 5- CGC GCG TCT AGA CTC GGC GTG GCC TCG GCG CAA -3 yielded a 1,227 bp band, which was cloned into pXLE42·V5 tag vector at the Bam HI and XbaI sites. After confirming the astrovirus capsid sequence, the negative strand was used instilling a 19-point bp change for probe creation, identical to the patient’s bp changes. Sections from the AstV-ND-1 patient’s brain and a control human brain were mounted onto microscope slides and hybridized with either astrovirus or actin probes using a QuantiGene (R superscript circularized) ViewRNA ISH Tissue 2-Plex Assay kit (Affymetrix Cat No. QVT0012). The accession number for the HAstV-NIH astrovirus sequence in Genbank is OP293097.2  containing 1 ml Trizol  in a FastPrep FP 120 instrument  at a speed of 6.0 meters per second. Homogenized Trizol lysate was combined 1-Bromo-3-chloropropane , mixed, and centrifuged at 16,000 x g for 15 min at 4°C. RNA containing aqueous phase was collected from each sample and passed through a Qiashredder column  at 21,000 x g for 2 min to homogenize any remaining genomic DNA in the aqueous phase. The aqueous phase was combined with an equal amount of RLT lysis buffer  with 1% beta-mercaptoethanol , and RNA was extracted using Qiagen AllPrep DNA/RNA mini columns . The RNA yield ranged from 860 ng to 12.4 μg. RNA quality was determined by spectrophotometry at 260 nm and 280 nm and by fluorescence capillary assay . RNA integrity numbers ranged from 2.3 to 6.2.A Truseq Stranded mRNA-Seq Sample Preparation Kit (Illumina) was used to synthesize cDNA and generate sequencing ready libraries following the manufacturer’s protocol. Due to RNA degradation and low RNA integrity values, the fragmentation time was reduced from eight to six min. Library quality and size distribution was assessed on a BioAnalyzer DNA 1000 chip (Agilent Technologies), and concentrations were determined using the Kapa SYBR FAST Universal qPCR kit for Illumina sequencing (Roche). Paired-end 75 cycle sequencing was completed using two Mid Output 150 cycle kits on the NextSeq 550 (Illumina).https://cutadapt.readthedocs.io/en/stable/). The remaining reads were then filtered for low quality bases and low quality reads using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). The remaining reads were mapped to the GRCh38 genome, using HISAT2. Reads mapping to genes were counted using htseq-count. Differential expression analysis was performed using the Bioconductor package DESeq2 along with generation of the sample heatmap using the pheatmap package with default parameters  showed the following: (i) AstV-ND-2-NY [brainstem] transcripts clustered with the brainstem from two age-matched normal controls; AstV-ND-3-France [frontal cortex] clustered with the frontal cortex from two age-matched normal controls, validating this approach . AstV-NDhttp://www.htslib.org/doc/samtools-coverage.html).Viral reads were aligned to the reference astrovirus HAstV-VA1/HMO-C-UK1  Accessi was genehttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE201384).The metadata and normalized data files have been deposited in the GEO database (https://biit.cs.ut.ee/gprofiler/page/docs#significance_threhshold).Functional enrichment analyses of gene expression data were performed using the PANTHER statistical enrichment test (SET) and gProfiler as previously described . A multiFFPE brain tissue sections were stained with hematoxylin and eosin (H&E) or processed for immunohistochemistry. Brightfield colorimetric immunohistochemistry was performed using Bond RX (Leica Biosystems) according to manufacturer protocols. Diaminobenzidine was used for colorimetric detection (brown) and hematoxylin was used for counterstaining. The following primary antibodies were used for brightfield colorimetric immunohistochemistry: antibody against astrovirus capsid protein ,17 (rabbBrightfield colorimetric immunohistochemistry and H&E-stained sections were scanned at x40 magnification using the ScanScope AT2 (Leica Biosystems), and ImageScope software was used for digital slide image analysis. Neuronal virus burden was quantified using brightfield colorimetric immunohistochemistry for AstV capsid protein in each brain region of interest (2–6 sections per each) based on the counts of the AstV-positive (AstV+) neurons and their projections. The counts for AstV+ Purkinje cell bodies/dendrites in the cerebellar cortex were normalized to the length (mm) of the Purkinje cell layer (6 folia). The counts for AstV+ neurons in all other brain regions were normalized to the tissue area (mm2) with sampling focused on the gray matter. Mantra Imaging System and InForm software (Akoya Biosciences) were used for multispectral immunofluorescent image acquisition, spectral unmixing, and generation of simulated H&E images based on tissue autofluorescence according to manufacturer protocols. Simulated H&E images based on tissue autofluorescence were used as a tissue topographical reference for immunofluorescent images and to aid in identification of autofluorescent lipofuscin granules in corresponding immunofluorescent images.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigA) Fold change relative to control for probes to virus families hybridizing to RNA from the brain of the patient with AstV-NIH. (B) Detection of the astrovirus RNA in the brain of the patient with AstV-NIH by PCR. (C and D) In situ hybridization signals (magenta-red) in indicated brain tissue samples using the negative strand astrovirus RNA probe (upper panels) or RNA probes for actin as controls (lower panels).((PDF)Click here for additional data file.S2 FigThe circular dendrogram is constructed from the ORF2/capsid precursor protein sequences of 229 astroviruses retrieved from the NCBI protein sequence database (see (PDF)Click here for additional data file.S3 FigThe heatmap of the sample-to-sample distances in the transformed reads count matrix (dseq2). Samples that clustered together are highlighted by the same colors on the right and within the matrix. The pairs of AstV-ND and normal control samples chosen to identify DEGs for this study are shown on the right by arrows connecting relevant samples.(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


The evolutionary development of an organism is associated with increased complexity of the regulatory potential of non-coding RNAs (ncRNAs), which constitute the majority of the transcriptome . The moshttps://www.gencodegenes.org, GENCODE Release (version30), Release date 04.2019], indicating the possibility that antisense-mediated regulation affects a large number of genes  are lncRNAs that are transcribed from the opposite strand of protein coding genes and are complementary to or overlap the protein-coding transcript . NATs arof genes .Khorkova et al. summarizes NAT-dependent post-transcriptional regulation of gene expression in a wide variety of biological mechanisms: Wang et al. explored the role of MEF2C antisense RNA1 (MEF2C-AS1) on sponging miR-592 in cervical cancer. They found that MEF2C-AS1 was downregulated in cervical cancer and may regulate miR-592 and its target, the R-spondin1 axis, to affect cervical cancer cell invasion and migration. Liao et al. investigated the role of the lncRNA, NEAT1, in high glucose-induced hypertrophy of mesangial cells. They found that high glucose leads to STAT3 activation, resulting in downregulation of miR-222-3p by NEAT1, which acts as an miR-sponge. In this manner, NEAT1 limited miR-222-3p binding with CDK1B mRNA. The release of mRNA leads to elevated CDK1B protein levels, resulting in mesangial cell hypertrophy, which can cause diabetic nephropathy.(i) Multiple NATs have been shown to regulate mRNA degradation or translation through sponging endogenous miRNAs. (ii) NATs bind their mRNA partner, thereby modulating its half-life in a miRNA-independent manner. Short complementary regions within the sense RNA:NAT pair may promote intermolecular RNA:RNA interactions . These i(iii) Mammalian-wide interspersed repeat (MIR)-NATs mediate translational repression. NATs with embedded retrotransposon-derived repeats, such as MIRs, termed MIR-NATs, form effector domains that regulate mRNA-ribosome pairing. MIR-NATs overlapping 5′-UTRs head-to-head compete with internal ribosome entry sites (IRESs) for the 40S ribosome subunit and repress translation .(iv) SINEUPs modulate translation initiation. SINEUPs (inverted SINEB2 sequence-mediated upregulating molecules) are encoded antisense to the 5′ end of the target sense mRNA and can enhance its translation without upregulating mRNA levels. The binding domain of SINEUPs is formed by the antisense region overlapping the start codon of the target mRNA and confers specificity to the protein coding transcript. An effector domain at the 3′ end of a SINEUP comprises embedded transposable element sequences, such as inverted short interspersed nuclear element B2, which is capable of upregulating translation by binding activating protein complexes .Pan et al. review the latest developments in the field of lncRNA-encoded micropeptides.(v) Regulation by NAT-encoded mini-proteins. Short ORFs present in some NATs can be translated into peptides that have downstream regulatory functions in processes such as homeostasis regulation, disease pathogenesis, tumor oncogenesis, and development. (vi) Regulation of protein stability. NATs protect target proteins from degradation by sponging/inhibiting the activity of proteins involved in the ubiquitin-proteasome degradation pathway .(vii) Subcellular localization of proteins: NATs can also modulate the subcellular distribution of proteins , althougAs part of this Research Topic, the comprehensive review by Sirvinskas et al. report that increased expression of the antisense lncRNA, CHROMR, was associated with the development of malignancy and poor prognosis in brain glioma patients. However, experimental clarification of the mechanism of CHROMR action is required to account for the prognostic efficacy of the mRNA PRKRA/lncRNA CHROMR ratio that the authors claimed for glioma patients.As part of this Research Topic, Khorkova et al. fully describe these NAT-mediated biological mechanisms and highlight the recent clinical and pre-clinical developments in nucleic acid-based therapeutics that aim to modulate dysregulated ncRNA-based networks. This approach has opened a new set of therapeutic targets that were previously inaccessible to traditional protein-targeted small molecule inhibitors and will enable numerous new therapeutic possibilities. We hope readers of this Research Topic share our excitement in reading these articles that describe the recent advances in Regulatory RNA research.The recent discovery of vast ncRNA-based regulatory networks has revealed that the tissue- and developmental-specific expression of ncRNAs is important for them to be viable indicators of cell physiological state. Dysregulation of these networks has been implicated in diseases, including cancer. lncRNAs, NATs in particular, play a significant role in the networks involved in disease-relevant mechanisms, working either post-transcriptionally, as described above, or transcriptionally and epigenetically. In this Research Topic,