Interferon induced circRNAs escape herpesvirus host shutoff and suppress lytic infection.

To globally profile circRNAs, we employ RNA-Sequencing paired with chimeric junction analysis for alpha-, beta-, and gamma-herpesvirus infection. We find circRNAs are, as a population, resistant to host shutoff. We validate this observation using ectopic expression assays of human and murine herpesvirus endoribonucleases. During lytic infection, four circRNAs are commonly induced across all subfamilies of human herpesviruses, suggesting a shared mechanism of regulation. We test one such mechanism, namely how interferon-stimulation influences circRNA expression. 67 circRNAs are upregulated by either interferon-ß or -γ treatment, with half of these also upregulated during lytic infection. Using gain and loss of function studies we find an interferon-stimulated circRNA, circRELL1, inhibits lytic Herpes Simplex Virus-1 infection. We previously reported circRELL1 inhibits lytic Kaposi sarcoma-associated herpesvirus infection, suggesting a pan-herpesvirus antiviral activity. We propose a two-pronged model in which interferon-stimulated genes may encode both mRNA and circRNA with antiviral activity. This is critical in cases of host shutoff, such as alpha- and gamma-herpesvirus infection, where the mRNA products are degraded but circRNAs escape.

Update on human herpesvirus 7 pathogenesis and clinical aspects as a roadmap for future research.

Human herpesvirus 7 (HHV-7) is a common virus that is associated with various human diseases including febrile syndromes, dermatological lesions, neurological defects, and transplant complications. Still, HHV-7 remains one of the least studied members of all human betaherpesviruses. In addition, HHV-7-related research is mostly confined to case reports, while in vitro or in vivo studies unraveling basic virology, transmission mechanisms, and viral pathogenesis are sparse. Here, we discuss HHV-7-related literature linking clinical syndromes to the viral life cycle, epidemiology, and viral immunopathogenesis. Based on our review, we propose a hypothetical model of HHV-7 pathogenesis inside its host. Furthermore, we identify important knowledge gaps and recommendations for future research to better understand HHV-7 diseases and improve therapeutic interventions.

Aberrant RNA polymerase initiation and processivity on the genome of a herpes simplex virus 1 mutant lacking ICP27.

Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.

Kaposi's sarcoma-associated herpesvirus terminal repeat regulates inducible lytic gene promoters.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140-kb unique coding region flanked by 30-40 copies of a 0.8-kb terminal repeat (TR) sequence. A gene enhancer recruits transcription-related enzymes by having arrays of transcription factor binding sites. Here, we show that KSHV TR possesses transcription regulatory function with latency-associated nuclear antigen (LANA). Cleavage under targets and release using nuclease demonstrated that TR fragments were occupied by LANA-interacting histone-modifying enzymes in naturally infected cells. The TR was enriched with histone H3K27 acetylation (H3K27Ac) and H3K4 tri-methylation (H3K4me3) modifications and also expressed nascent RNAs. The sites of H3K27Ac and H3K4me3 modifications were also conserved in the KSHV unique region among naturally infected primary effusion lymphoma cells. KSHV origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with that of TR. In the Ori-Lyt region, the LANA and LANA-interacting proteins colocalized with an H3K27Ac-modified nucleosome along with paused RNA polymerase II. The KSHV transactivator KSHV replication and transcription activator (K-Rta) recruitment sites franked the LANA-bound nucleosome, and reactivation evicted the LANA-bound nucleosome. Including TR fragments in reporter plasmid enhanced inducible viral gene promoter activities independent of the orientations. In the presence of TR in reporter plasmids, K-Rta transactivation was drastically increased, while LANA acquired the promoter repression function. KSHV TR, therefore, functions as an enhancer for KSHV inducible genes. However, in contrast to cellular enhancers bound by multiple transcription factors, perhaps the KSHV enhancer is predominantly regulated by the LANA nuclear body.IMPORTANCEEnhancers are a crucial regulator of differential gene expression programs. Enhancers are the cis-regulatory sequences determining target genes' spatiotemporal and quantitative expression. Here, we show that Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeats fulfill the enhancer definition for KSHV inducible gene promoters. The KSHV enhancer is occupied by latency-associated nuclear antigen (LANA) and its interacting proteins, such as CHD4. Neighboring terminal repeat (TR) fragments to lytic gene promoters drastically enhanced KSHV replication and transcription activator and LANA transcription regulatory functions. This study, thus, proposes a new latency-lytic switch model in which TR accessibility to the KSHV gene promoters regulates viral inducible gene expression.

Human herpesvirus 6A nuclear matrix protein U37 interacts with heat shock transcription factor 1 and activates the heat shock response.

Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This process is mediated by a conserved viral heterodimeric complex designated the nuclear egress complex, which consists of the nuclear matrix protein and the nuclear membrane protein. In addition to its essential roles during nuclear egress, the nuclear matrix protein has been shown to interact with intracellular signaling pathway molecules including NF-κB and IFN-ß to affect viral or cellular gene expression. The human herpesvirus 6A (HHV-6A) U37 gene encodes a nuclear matrix protein, the role of which has not been analyzed. Here, we show that HHV-6A U37 activates the heat shock element promoter and induces the accumulation of the molecular chaperone Hsp90. Mechanistically, HHV-6A U37 interacts with heat shock transcription factor 1 (HSF1) and induces its phosphorylation at Ser-326. We report that pharmacological inhibition of HSF1, Hsp70, or Hsp90 decreases viral protein accumulation and viral replication. Taken together, our results lead us to propose a model in which HHV-6A U37 activates the heat shock response to support viral gene expression and replication. IMPORTANCE Human herpesvirus 6A (HHV-6A) is a dsDNA virus belonging to the Roseolovirus genus within the Betaherpesvirinae subfamily. It is frequently found in patients with neuroinflammatory disease, although its pathogenetic role, if any, awaits elucidation. The heat shock response is important for cell survival under stressful conditions that disrupt homeostasis. Our results indicate that HHV-6A U37 activates the heat shock element promoter and leads to the accumulation of heat shock proteins. Next, we show that the heat shock response is important for viral replication. Overall, our findings provide new insights into the function of HHV-6A U37 in host cell signaling and identify potential cellular targets involved in HHV-6A pathogenesis and replication.

Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins.

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

Shared sequence characteristics identified in non-canonical rearrangements of HSV-1 genomes.

IMPORTANCE: Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells.

Herpes simplex virus 1 immediate early transcription initiation, pause-release, elongation, and termination in the presence and absence of ICP4.

IMPORTANCE: Infection with herpes simplex virus 1 (HSV-1) leads to lifelong infection due to the virus's remarkable ability to control transcription of its own genome, resulting in two transcriptional programs: lytic (highly active) and latent (restricted). The lytic program requires immediate early (IE) proteins to first repress transcription of late viral genes, which then undergo sequential de-repression, leading to a specific sequence of gene expression. Here, we show that the IE ICP4 functions to regulate the cascade by limiting RNA polymerase initiation at immediate early times. However, late viral genes that initiate too early in the absence of ICP4 do not yield mRNA as transcription stalls within gene bodies. It follows that other regulatory steps intercede to prevent elongation of genes at the incorrect time, demonstrating the precise control HSV-1 exerts over its own transcription.

Electron microscopy mapping of the DNA-binding sites of monomeric, dimeric, and multimeric KSHV RTA protein.

IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus associated with several human cancers, typically in patients with compromised immune systems. Herpesviruses establish lifelong infections in hosts in part due to the two phases of infection: the dormant and active phases. Effective antiviral treatments to prevent the production of new viruses are needed to treat KSHV. A detailed microscopy-based investigation of the molecular interactions between viral protein and viral DNA revealed how protein-protein interactions play a role in DNA-binding specificity. This analysis will lead to a more in-depth understanding of KSHV DNA replication and serve as the basis for anti-viral therapies that disrupt and prevent the protein-DNA interactions, thereby decreasing spread to new hosts.

Characterization of the HHV-6B U20 Immunoevasin.

Roseoloviruses (human herpesvirus 6A [HHV-6A], -6B, and -7) infect >90% of the human population during early childhood and are thought to remain latent or persistent throughout the life of the host. As such, these viruses are among the most pervasive and stealthy of all viruses; they must necessarily excel at escaping immune detection throughout the life of the host, and yet, very little is known about how these viruses so successfully escape host defenses. Here, we characterize the expression, trafficking, and posttranslational modifications of the HHV6B U20 gene product, which is encoded within a block of genes unique to the roseoloviruses. HHV-6B U20 trafficked slowly through the secretory system, receiving several posttranslational modifications to its N-linked glycans, indicative of surface-expressed glycoproteins, and eventually reaching the cell surface before being internalized. Interestingly, U20 is also phosphorylated on at least one Ser, Thr, or Tyr residue. These results provide a framework to understand the role(s) of U20 in evading host defenses. IMPORTANCE The roseolovirus U20 proteins are virus-encoded integral membrane glycoproteins possessing class I major histocompatibility complex (MHC)-like folds. Surprisingly, although U20 proteins from HHV-6A and -6B share 92% identity, recent studies ascribe different functions to HHV6A U20 and HHV6B U20. HHV6A U20 was shown to downregulate NKG2D ligands, while HHV6B U20 was shown to inhibit tumor necrosis factor alpha (TNF-α)-induced apoptosis during nonproductive infection with HHV6B (E. Kofod-Olsen, K. Ross-Hansen, M. H. Schleimann, D. K. Jensen, et al., J Virol 86:11483-11492, 2012, https://doi.org/10.1128/jvi.00847-12; A. E. Chaouat, B. Seliger, O. Mandelboim, D. Schmiedel, Front Immunol 12:714799, 2021, https://doi.org/10.3389/fimmu.2021.714799). Here, we have performed cell biological and biochemical characterization of the trafficking, glycosylation, and posttranslational modifications occurring on HHV6B U20.

Reactivation of Epstein-Barr Virus from Latency Involves Increased RNA Polymerase Activity at CTCF Binding Sites on the Viral Genome.

The ability of Epstein-Barr virus (EBV) to switch between latent and lytic infection is key to its long-term persistence, yet the molecular mechanisms behind this switch remain unclear. To investigate transcriptional events during the latent-to-lytic switch, we utilized Precision nuclear Run On followed by deep Sequencing (PRO-Seq) to map cellular RNA polymerase (Pol) activity to single-nucleotide resolution on the host and EBV genome in three different models of EBV latency and reactivation. In latently infected Mutu-I Burkitt lymphoma (BL) cells, Pol activity was enriched at the Qp promoter, the EBER region, and the BHLF1/LF3 transcripts. Upon reactivation with phorbol ester and sodium butyrate, early-phase Pol activity occurred bidirectionally at CTCF sites within the LMP-2A, EBER-1, and RPMS1 loci. PRO-Seq analysis of Akata cells reactivated from latency with anti-IgG and a lymphoblastoid cell line (LCL) reactivated with small molecule C60 showed a similar pattern of early bidirectional transcription initiating around CTCF binding sites, although the specific CTCF sites and viral genes were different for each latency model. The functional importance of CTCF binding, transcription, and reactivation was confirmed using an EBV mutant lacking the LMP-2A CTCF binding site. This virus was unable to reactivate and had disrupted Pol activity at multiple CTCF binding sites relative to the wild-type (WT) virus. Overall, these data suggest that CTCF regulates the viral early transcripts during reactivation from latency. These activities likely help maintain the accessibility of the viral genome to initiate productive replication. IMPORTANCE The ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B cells, and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication. Reactivation occurs once the immediate early (IE) EBV lytic genes are expressed. However, the molecular mechanisms behind the switch from the latent transcriptional program to begin transcription of the IE genes remain unknown. In this study, we mapped RNA Pol positioning and activity during latency and reactivation. Unexpectedly, Pol activity accumulated at distinct regions characteristic of transcription initiation on the EBV genome previously shown to be associated with CTCF. We propose that CTCF binding at these regions retains Pol to maintain a stable latent chromosome conformation and a rapid response to various reactivation signals.

Feline herpesvirus 1 (FHV-1) enters the cell by receptor-mediated endocytosis.

Feline herpesvirus type 1 (FHV-1) is an enveloped dsDNA virus belonging to the Herpesviridae family and is considered one of the two primary viral etiological factors of feline upper respiratory tract disease. In this study, we investigated the entry of FHV-1 into host cells using two models: the AK-D cell line and primary feline skin fibroblasts (FSFs). We employed confocal microscopy, siRNA silencing, and selective inhibitors of various entry pathways. Our observations revealed that the virus enters cells via pH and dynamin-dependent endocytosis, as the infection was significantly inhibited by NH4Cl, bafilomycin A1, dynasore, and mitmab. Additionally, genistein, nystatin, and filipin treatments, siRNA knock-down of caveolin-1, as well as FHV-1 and caveolin-1 colocalization suggest the involvement of caveolin-mediated endocytosis during the entry process. siRNA knock-down of clathrin heavy chain and analysis of virus particle colocalization with clathrin indicated that clathrin-mediated endocytosis also takes part in the primary cells. This is the first study to systematically examine FHV-1 entry into host cells, and for the first time, we describe FHV-1 replication in AK-D and FSFs. IMPORTANCE Feline herpesvirus 1 (FHV-1) is one of the most prevalent viruses in cats, causing feline viral rhinotracheitis, which is responsible for over half of viral upper respiratory diseases in cats and can lead to ocular lesions resulting in loss of sight. Although the available vaccine reduces the severity of the disease, it does not prevent infection or limit virus shedding. Despite the clinical relevance, the entry mechanisms of FHV-1 have not been thoroughly studied. Considering the limitations of commonly used models based on immortalized cells, we sought to verify our findings using primary feline skin fibroblasts, the natural target for infection in cats.

Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism.

The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and the alpha-herpesviruses herpes simplex virus (HSV)-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting the involvement of an immediate early or early (IE/E) viral protein. In support of this possibility, genetic (IE1 mutant) and pharmacologic (cycloheximide) strategies that prevent the expression of IE/E viral proteins also block APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which interferes with viral late protein expression, still permits A3B relocalization. These results combine to indicate that the beta-herpesvirus HCMV uses an RNR-independent, yet phenotypically similar, molecular mechanism to antagonize APOBEC3B. IMPORTANCE Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.

Epstein-Barr virus LMP1 enhances levels of large extracellular vesicle-associated PD-L1.

IMPORTANCE: A growing body of evidence has supported the notion that viruses utilize EVs and associated pathways to incorporate viral products. This allows for the evasion of an immune response while enabling viral spread within the host. Given that viral proteins often elicit strong antigenic peptides that are recognized by T cells, the regulation of the PD-L1 pathway through the overexpression of lEV-associated PD-L1 may serve as a strategy for immune evasion by viruses. The discovery that EBV LMP1 increases the secretion of PD-L1 in larger EVs identifies a new potential target for immune blockade therapy in EBV-associated cancers. Our findings may help to clarify the mechanism of LMP1-mediated enhancement of PD-L1 packaging into lEVs and may lead to the identification of more specific targets for treatment. Additionally, the identification of lEV biomarkers that predict a viral origin of disease could allow for more targeted therapies to be developed.

PCR testing for herpesviruses in aqueous humor samples from patients with and without clinical corneal endothelial graft rejection.

To compare prevalence of positive PCR tests for herpesviruses between patients with and without a history of clinical corneal endothelial allograft rejection (AGR). Retrospective cross-sectional study with two-group comparison. A total of 307 aqueous humor (AH) samples from 235 Patients and 244 eyes who underwent penetrating keratoplasty or Descemet membrane endothelial keratoplasty or had a diagnostic AH aspiration due to clinical AGR between 2019 and 2023 were tested for DNA of herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV). PCR test results were compared between the two groups (with/without AGR). Another sub-analysis examined the results of patients without a history of herpetic keratitis. A total of 8% of eyes with clinical AGR (9/108) had a positive PCR result for one of the herpesviruses (HSV:3, CMV:3, EBV:2, VZV:1). All patients in the group without AGR had negative PCR results for all previous viruses (0/136). The difference was statistically significant (p < 0.001). The sub-analysis of eyes without a history of herpetic keratitis also revealed significantly more positive herpes PCR results (7/87) in eyes with AGR than in eyes without AGR (0/42, p = 0.005). Clinical AGR after keratoplasty shows a significant correlation to viral replication. Herpetic infection and AGR could occur simultaneously and act synergistically. Timely differentiation between active herpetic infection and/or AGR is pivotal for proper treatment and graft preservation.

Unmasking the ongoing challenge of equid herpesvirus- 1 (EHV-1): A comprehensive review.

Equid herpesviruses (EHVs) are a group of highly impactful viral pathogens that affect horses, presenting a substantial risk to the global equine industry. Among these, equid herpesvirus-1 (EHV-1) primarily causes respiratory infections. However, its ability to spread to distant organs can lead to severe consequences such as abortion and neurological diseases. These viruses can enter a dormant phase, with minimal activity, and later reactivate to trigger active infections at any time. Recently, there has been a notable rise in the prevalence of a particularly devastating strains of EHV-1 known as equid herpesviral myeloencephalopathy (EHM). In the light of dynamic nature of EHV-1, this review provides a thorough overview of EHV-1 and explores how advances in viral biology affect the pathophysiology of viral infection. The information presented here is crucial for understanding the dynamics of EHV-1 infections and creating practical plans to stop the virus's global spread among equid populations.

Reactivation of herpesviruses during COVID-19: A systematic review and meta-analysis.

To provide a comprehensive systematic review and meta-analysis regarding the cumulative incidence (incidence proportion) of human herpesvirus (HHV) reactivation among patients with coronavirus disease 2019 (COVID-19), we searched PubMed/MEDLINE, Web of Science, and EMBASE up to 25 September 2022, with no language restrictions. All interventional and observational studies enrolling patients with confirmed COVID-19 and providing data regarding HHV reactivation were included. The random-effects model was used in the meta-analyses. We included information from 32 studies. HHV reactivation was considered a positive polymerase chain reaction result taken at the time of COVID-19 infection. Most of the included patients were severe COVID-19 cases. The pooled cumulative incidence estimate was 38% (95% Confidence Intervals [CI], 28%-50%, I2  = 86%) for herpes simplex virus (HSV), 19% (95% CI, 13%-28%, I2  = 87%) for cytomegalovirus (CMV), 45% (95% CI, 28%-63%, I2  = 96%) for Epstein-Barr virus (EBV), 18% (95% CI, 8%-35%) for human herpesvirus 6 (HHV-6), 44% (95% CI, 32%-56%) for human herpesvirus 7 (HHV-7), and 19% (95% CI, 14%-26%) for human herpesvirus 8 (HHV-8). There was no evidence of funnel plot asymmetry based on visual inspection and Egger's regression test for the results of HSV (p = 0.84), CMV (p = 0.82), and EBV (p = 0.27) reactivation. In conclusion, the identification of HHV reactivation in severe COVID-19 patients is helpful in the management of patients as well as the prevention of complications. Further research is required to elucidate the interaction between HHVs and COVID-19. Systematic review registration: PROSPERO CRD42022321973.

The assessment of microbial infection in children with autism spectrum disorders and genetic folate cycle deficiency.

BACKGROUND: The results of disparate clinical studies indicate abnormally frequent cases of certain microorganisms in children with autism spectrum disorders (ASD). However, these data require clarification and systematization. The study aims to study the structure of the microbial profile in children with ASD and genetic folate cycle deficiency (GFCD) and consider differences in diagnostic approaches for identifying microorganisms of different types. METHODS: The study analyzed medical data from 240 children (187 boys and 63 girls) with GFCD aged 2 to 9 years. The children had clinical manifestations of ASD (the study group, SG). The control group (CG) included 53 clinically healthy children (37 boys and 16 girls) of the same age but without GFCD. Both groups of children were tested on active herpetic infections (HSV-1/2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8), ТТV, Streptococcus pyogenes, Candida albicans, Borrelia burgdorferi, Mycoplasma pneumoniae, Chlamydia pneumoniae, Yersinia enterocolitica, Toxoplasma gondii, congenital CMV neuroinfection and postnatal HSV-1/2 encephalitis. The testing used diagnostic methods specified in PubMed-indexed studies. RESULTS: In the SG, TTV was found in 196 children (82%), HHV-7 - in 172 (72%), HHV-6 - in 162 (68%), EBV - in 153 (64%), Streptococcus pyogenes - in 127 (53%), Candida albicans - in 116 (48%), Borrelia - in 107 (45%), Mycoplasma pneumoniae - in 94 (39%), Chlamydia pneumoniae - in 85 (35%), Yersinia entеrocolitica - in 71 (30%), Toxoplasma gondii - in 54 (23%), congenital CMV neuroinfection - in 26 (11%), and postnatal HSV-1/2 encephalitis - in 11 children (5% of cases) (p < p0.05; Z < Z0.05). In the SG, there was a higher microbial load in older children (p < p0.05; Z < Z0.05). No gender differences were found. CONCLUSIONS: The study described and characterized a specific abnormal microbial spectrum with a predominance of viral opportunistic agents in children with ASD associated with GFCD.

Approaches to Evaluating Necroptosis in Virus-Infected Cells.

The immune system functions to protect the host from pathogens. To counter host defense mechanisms, pathogens have developed unique strategies to evade detection or restrict host immune responses. Programmed cell death is a major contributor to the multiple host responses that help to eliminate infected cells for obligate intracellular pathogens like viruses. Initiation of programmed cell death pathways during the early stages of viral infections is critical for organismal survival as it restricts the virus from replicating and serves to drive antiviral inflammation immune recruitment through the release of damage-associated molecular patterns (DAMPs) from the dying cell. Necroptosis has been implicated as a critical programmed cell death pathway in a diverse set of diseases and pathological conditions including acute viral infections. This cell death pathway occurs when certain host sensors are triggered leading to the downstream induction of mixed-lineage kinase domain-like protein (MLKL). MLKL induction leads to cytoplasmic membrane disruption and subsequent cellular destruction with the release of DAMPs. As the role of this cell death pathway in human disease becomes apparent, methods identifying necroptosis patterns and outcomes will need to be further developed. Here, we discuss advances in our understanding of how viruses counteract necroptosis, methods to quantify the pathway, its effects on viral pathogenesis, and its impact on cellular signaling.

'Getting Better'-Is It a Feasible Strategy of Broad Pan-Antiherpesviral Drug Targeting by Using the Nuclear Egress-Directed Mechanism?

The herpesviral nuclear egress represents an essential step of viral replication efficiency in host cells, as it defines the nucleocytoplasmic release of viral capsids. Due to the size limitation of the nuclear pores, viral nuclear capsids are unable to traverse the nuclear envelope without a destabilization of this natural host-specific barrier. To this end, herpesviruses evolved the regulatory nuclear egress complex (NEC), composed of a heterodimer unit of two conserved viral NEC proteins (core NEC) and a large-size extension of this complex including various viral and cellular NEC-associated proteins (multicomponent NEC). Notably, the NEC harbors the pronounced ability to oligomerize (core NEC hexamers and lattices), to multimerize into higher-order complexes, and, ultimately, to closely interact with the migrating nuclear capsids. Moreover, most, if not all, of these NEC proteins comprise regulatory modifications by phosphorylation, so that the responsible kinases, and additional enzymatic activities, are part of the multicomponent NEC. This sophisticated basis of NEC-specific structural and functional interactions offers a variety of different modes of antiviral interference by pharmacological or nonconventional inhibitors. Since the multifaceted combination of NEC activities represents a highly conserved key regulatory stage of herpesviral replication, it may provide a unique opportunity towards a broad, pan-antiherpesviral mechanism of drug targeting. This review presents an update on chances, challenges, and current achievements in the development of NEC-directed antiherpesviral strategies.