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Various viral proteins are post-translationally modified by SUMO-conjugation during the human adenovirus (HAdV) replication cycle. This modification leads to diverse consequences for target proteins as it influences their intracellular localization or cell transformation capabilities. SUMOylated HAdV proteins include the multifunctional oncoprotein E1B-55K. Our previous research, along with that of others, has demonstrated a substantial influence of yet another adenoviral oncoprotein, E4orf6, on E1B-55K SUMOylation levels. Protein SUMOylation can be reversed by cellular sentrin/SUMO-specific proteases (SENPs). In this study, we investigated the interaction of E1B-55K with cellular SENPs to understand deSUMOylation activities and their consequences for cell transformation mediated by this adenoviral oncoprotein. We show that E1B-55K interacts with and is deSUMOylated by SENP 1, independently of E4orf6. Consistent with these results, we found that SENP 1 prevents E1A/E1B-dependent focus formation in rodent cells. We anticipate these findings to be the groundwork for future studies on adenovirus-host interactions, the mechanisms that underlie E1B-55K SUMOylation, as well as the role of this major adenoviral oncoprotein in HAdV-mediated cell transformation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ip, Fiedler, Gornott, Morische, Bertzbach and Dobner.)

Marek's disease virus (MDV) integrates its genome into the telomeres of host chromosomes and causes fatal lymphomas in chickens. This integration is facilitated by telomeric repeat sequences (TMRs) at the ends of the viral genome, and is crucial for MDV-induced lymphomagenesis. The SB-1 vaccine virus is commonly used in commercial bivalent vaccines against MDV and also contains TMRs at its ends. Here, we demonstrate that SB-1 efficiently integrates its genome into the chromosomes of latently infected T cells. Deletion of the TMRs from the SB-1 genome did not affect virus replication, but severely impaired virus integration and genome maintenance in latently infected T cells and in chickens. Strikingly, the reduced integration and maintenance of latent SB-1 significantly impaired vaccine protection. Taken together, our data revealed that the TMRs facilitate SB-1 integration and that integration and/or maintenance of the latent viral genome is critical for vaccine protection., (© 2024. The Author(s).)

Oncogenic viruses contribute to 15% of global human cancers. To achieve that, virus-encoded oncoproteins deregulate cellular transcription, antagonize common cellular pathways, and thus drive cell transformation. Notably, adenoviruses were the first human viruses proven to induce cancers in diverse animal models. Over the past decades, human adenovirus (HAdV)-mediated oncogenic transformation has been pivotal in deciphering underlying molecular mechanisms. Key adenovirus oncoproteins, encoded in early regions 1 (E1) and 4 (E4), co-ordinate these processes. Among the different adenovirus species, the most extensively studied HAdV-C5 displays lower oncogenicity than HAdV-A12. A complete understanding of the different HAdV-A12 and HAdV-C5 oncoproteins in virus-mediated cell transformation, as summarized here, is relevant for adenovirus research and offers broader insights into viral transformation and oncogenesis., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Marek's disease virus (MDV) vaccines were the first vaccines that protected against cancer. The avirulent turkey herpesvirus (HVT) was widely employed and protected billions of chickens from a deadly MDV infection. It is also among the most common vaccine vectors providing protection against a plethora of pathogens. HVT establishes latency in T-cells, allowing the vaccine virus to persist in the host for life. Intriguingly, the HVT genome contains telomeric repeat arrays (TMRs) at both ends; however, their role in the HVT life cycle remains elusive. We have previously shown that similar TMRs in the MDV genome facilitate its integration into host telomeres, which ensures efficient maintenance of the virus genome during latency and tumorigenesis. In this study, we investigated the role of the TMRs in HVT genome integration, latency, and reactivation in vitro and in vivo. Additionally, we examined HVT infection of feather follicles. We generated an HVT mutant lacking both TMRs (vΔTMR) that efficiently replicated in cell culture. We could demonstrate that wild type HVT integrates at the ends of chromosomes containing the telomeres in T-cells, while integration was severely impaired in the absence of the TMRs. To assess the role of TMRs in vivo, we infected one-day-old chickens with HVT or vΔTMR. vΔTMR loads were significantly reduced in the blood and hardly any virus was transported to the feather follicle epithelium where the virus is commonly shed. Strikingly, latency in the spleen and reactivation of the virus were severely impaired in the absence of the TMRs, indicating that the TMRs are crucial for the establishment of latency and reactivation of HVT. Our findings revealed that the TMRs facilitate integration of the HVT genome into host chromosomes, which ensures efficient persistence in the host, reactivation, and transport of the virus to the skin., Competing Interests: Zoltán Penzes is employed by and received a salary from Ceva Santé Animale. All other authors declare that they have no conflict of interest., (Copyright: © 2024 Denesvre et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Adenoviruses are a group of double-stranded DNA viruses that can mainly cause respiratory, gastrointestinal, and eye infections in humans. In addition, adenoviruses are employed as vector vaccines for combatting viral infections, including SARS-CoV-2, and serve as excellent gene therapy vectors. These viruses have the ability to modulate the host cell machinery to their advantage and trigger significant restructuring of the nuclei of infected cells through the activity of viral proteins. One of those, the adenovirus DNA-binding protein (DBP), is a multifunctional non-structural protein that is integral to the reorganization processes. DBP is encoded in the E2A transcriptional unit and is highly abundant in infected cells. Its activity is unequivocally linked to the formation, structure, and integrity of virus-induced replication compartments, molecular hubs for the regulation of viral processes, and control of the infected cell. DBP also plays key roles in viral DNA replication, transcription, viral gene expression, and even host range specificity. Notably, post-translational modifications of DBP, such as SUMOylation and extensive phosphorylation, regulate its biological functions. DBP was first investigated in the 1970s, pioneering research on viral DNA-binding proteins. In this literature review, we provide an overview of DBP and specifically summarize key findings related to its complex structure, diverse functions, and significant role in the context of viral replication. Finally, we address novel insights and perspectives for future research., Competing Interests: The authors declare no conflict of interest.

Importance: Human adenoviruses (HAdVs) generally cause mild and self-limiting diseases of the upper respiratory and gastrointestinal tracts but pose a serious risk to immunocompromised patients and children. Moreover, they are widely used as vectors for vaccines and vector-based gene therapy approaches. It is therefore vital to thoroughly characterize HAdV gene products and especially HAdV virulence factors. Early region 1B 55 kDa protein (E1B-55K) is a multifunctional HAdV-encoded oncoprotein involved in various viral and cellular pathways that promote viral replication and cell transformation. We analyzed the E1B-55K dependency of SUMOylation, a post-translational protein modification, in infected cells using quantitative proteomics. We found that HAdV increases overall cellular SUMOylation and that this increased SUMOylation can target antiviral cellular pathways that impact HAdV replication. Moreover, we showed that E1B-55K orchestrates the SUMO-dependent degradation of certain cellular antiviral factors. These results once more emphasize the key role of E1B-55K in the regulation of viral and cellular proteins in productive HAdV infections., Competing Interests: The authors declare no conflict of interest.

The adenovirus C5 E1B-55K protein is crucial for viral replication and is expressed early during infection. It can interact with E4orf6 to form a complex that functions as a ubiquitin E3 ligase. This complex targets specific cellular proteins and marks them for ubiquitination and, predominantly, subsequent proteasomal degradation. E1B-55K interacts with various proteins, with p53 being the most extensively studied, although identifying binding sites has been challenging. To explain the diverse range of proteins associated with E1B-55K, we hypothesized that other binding partners might recognize the simple p53 binding motif (xWxxxPx). In silico analyses showed that many known E1B-55K binding proteins possess this amino acid sequence; therefore, we investigated whether other xWxxxPx-containing proteins also bind to E1B-55K. Our findings revealed that many cellular proteins, including ATR, CHK1, USP9, and USP34, co-immunoprecipitate with E1B-55K. During adenovirus infection, several well-characterized E1B-55K binding proteins and newly identified interactors, including CSB, CHK1, and USP9, are degraded in a cullin-dependent manner. Notably, certain binding proteins, such as ATR and USP34, remain undegraded during infection. Structural predictions indicate no conservation of structure around the proposed binding motif, suggesting that the interaction relies on the correct arrangement of tryptophan and proline residues.

The multifunctional adenovirus E1B-55K oncoprotein can induce cell transformation in conjunction with adenovirus E1A gene products. Previous data from transient expression studies and in vitro experiments suggest that these growth-promoting activities correlate with E1B-55K-mediated transcriptional repression of p53-targeted genes. Here, we analyzed genome-wide occupancies and transcriptional consequences of species C5 and A12 E1B-55Ks in transformed mammalian cells by combinatory ChIP and RNA-seq analyses. E1B-55K-mediated repression correlates with tethering of the viral oncoprotein to p53-dependent promoters via DNA-bound p53. Moreover, we found that E1B-55K also interacts with and represses transcription of numerous p53-independent genes through interactions with transcription factors that play central roles in cancer and stress signaling. Our results demonstrate that E1B-55K oncoproteins function as promiscuous transcriptional repressors of both p53-dependent and -independent genes and further support the model that manipulation of cellular transcription is central to adenovirus-induced cell transformation and oncogenesis.

Human telomerase RNA (hTR) is overexpressed in many cancers and protects T cells from apoptosis in a telomerase-independent manner. The most prevalent cancer in the animal kingdom is caused by the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV encodes a viral telomerase RNA (vTR) that plays a crucial role in MDV-induced tumorigenesis and shares all four conserved functional domains with hTR. In this study, we assessed whether hTR drives tumor formation in this natural model of herpesvirus-induced tumorigenesis. Therefore, we replaced vTR with hTR in the genome of a highly oncogenic MDV. Furthermore, we investigated the anti-apoptotic activity of vTR, hTR, and their counterpart in the chicken [chicken telomerase RNA (cTR)]. hTR was efficiently expressed and did not alter replication of the recombinant virus. Despite its conserved structure, hTR did not complement the loss of vTR in virus-induced tumorigenesis. Strikingly, hTR did not inhibit apoptosis in chicken cells, but efficiently inhibited apoptosis in human cells. Inverse host restriction has been observed for vTR and cTR in human cells. Our data revealed that vTR, cTR, and hTR possess conserved but host-specific anti-apoptotic functions that likely contribute to MDV-induced tumorigenesis. IMPORTANCE hTR is overexpressed in many cancers and used as a cancer biomarker. However, the contribution of hTR to tumorigenesis remains elusive. In this study, we assessed the tumor-promoting properties of hTR using a natural virus/host model of herpesvirus-induced tumorigenesis. This avian herpesvirus encodes a telomerase RNA subunit (vTR) that plays a crucial role in viral tumorigenesis and shares all conserved functional domains with hTR. Our data revealed that vTR and cellular TRs of humans and chickens possess host-specific anti-apoptotic functions. This provides important translational insights into therapeutic strategies, as inhibition of apoptosis is crucial for tumorigenesis.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects immune cells and causes a deadly lymphoproliferative disease in chickens. Cytokines and monoclonal antibodies promote the survival of chicken lymphocytes in vitro. Here, we describe protocols for the isolation, maintenance, and efficient MDV infection of primary chicken lymphocytes and lymphocyte cell lines. This facilitates the investigation of key aspects of the MDV life cycle in the primary target cells of viral replication, latency, genome integration, and reactivation. For complete details on the use and execution of this protocol, please refer to Schermuly et al., 1 Bertzbach et al. (2019), 2 and You et al. 3 For a comprehensive background on MDV, please see Osterrieder et al. 4 and Bertzbach et al. (2020). 5 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The human adenovirus species C type 5 (HAdV-C5) early region 4 (E4) encodes several distinct polypeptides, defined as E4orf1 to E4orf6/7 according to the order and arrangement of the corresponding open reading frames (ORFs). All E4 gene products operate through a complex network of interactions with key viral and cellular regulatory proteins involved in transcription, apoptosis, cell cycle control, and DNA repair. Here, we generated a set of virus mutants carrying point mutations in the individual E4 genes. The phenotypic characterizations of these mutants revealed that mutations of these ORFs had no or only moderate effects on virus replication. Even a triple mutant that fails to produce E4orf3, E4orf4, and the yet uncharacterized alternatively spliced E4orf3/4 fusion protein, was replicating to wild type levels. The E4orf3/4 protein consists of the N-terminal 33 amino acid residues from E4orf3 and the C-terminal 28 amino acid residues from E4orf4. Intriguingly, we found that, similar to E4orf3, E4orf3/4 possesses properties that support the E1A/E1B-induced transformation of primary rodent cells. These results identify and functionally characterize E4orf3/4 and conclude that E4orf3/4 is another E4 region protein that is dispensable for virus replication but promotes the E1A/E1B-induced transformation of primary rodent cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

It is well established that human adenoviruses such as species C, types 2 and 5 (HAdV-C2 and HAdV-C5), induce a nearly complete shutoff of host-cell protein synthesis in the infected cell, simultaneously directing very efficient production of viral proteins. Such preferential expression of viral over cellular genes is thought to be controlled by selective nucleocytoplasmic export and translation of viral mRNA. While detailed knowledge of the regulatory mechanisms responsible for the translation of viral mRNA is available, the viral or cellular mechanisms of mRNA biogenesis are not completely understood. To identify parameters that control the differential export of viral and cellular mRNAs, we performed global transcriptome analyses (RNAseq) and monitored temporal nucleocytoplasmic partitioning of viral and cellular mRNAs during HAdV-C5 infection of A549 cells. Our analyses confirmed previously reported features of the viral mRNA expression program, as a clear shift in viral early to late mRNA accumulation was observed upon transition from the early to the late phase of viral replication. The progression into the late phase of infection, however, did not result in abrogation of cellular mRNA export; rather, viral late mRNAs outnumbered viral early and most cellular mRNAs by several orders of magnitude during the late phase, revealing that viral late mRNAs are not selectively exported but outcompete cellular mRNA biogenesis.

The major histocompatibility complex (MHC) is crucial for appropriate immune responses against invading pathogens. Chickens possess a single predominantly-expressed class I molecule with strong associations between disease resistance and MHC haplotype. For Marek's disease virus (MDV) infections of chickens, the MHC haplotype is one of the major determinants of genetic resistance and susceptibility. VALO specific pathogen free (SPF) chickens are widely used in biomedical research and vaccine production. While valuable findings originate from MDV infections of VALO SPF chickens, their MHC haplotypes and associated disease resistance remained elusive. In this study, we used several typing systems to show that VALO SPF chickens possess MHC haplotypes that include B9, B9:02, B15, B19 and B21 at various frequencies. Moreover, we associate the MHC haplotypes to MDV-induced disease and lymphoma formation and found that B15 homozygotes had the lowest tumor incidence while B21 homozygotes had the lowest number of organs with tumors. Finally, we found transmission at variable levels to all contact birds except B15/B21 heterozygotes. These data have immediate implications for the use of VALO SPF chickens and eggs in the life sciences and add another piece to the puzzle of the chicken MHC complex and its role in infections with this oncogenic herpesvirus., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bertzbach, Tregaskes, Martin, Deumer, Huynh, Kheimar, Conradie, Trimpert, Kaufman and Kaufer.)

Circular RNAs (circRNAs) are a recently rediscovered class of functional noncoding RNAs that are involved in gene regulation and cancer development. Next-generation sequencing approaches identified circRNA fragments and sequences underlying circularization events in virus-induced cancers. In the present study, we performed viral circRNA expression analysis and full-length sequencing in infections with Marek's disease virus (MDV), which serves as a model for herpesvirus-induced tumorigenesis. We established inverse PCRs to identify and characterize circRNA expression from the repeat regions of the MDV genome during viral replication, latency, and reactivation. We identified a large variety of viral circRNAs through precise mapping of full-length circular transcripts and detected matching sequences with several viral genes. Hot spots of circRNA expression included the transcriptional unit of the major viral oncogene encoding the Meq protein and the latency-associated transcripts (LATs). Moreover, we performed genome-wide bioinformatic analyses to extract back-splice junctions from lymphoma-derived samples. Using this strategy, we found that circRNAs were abundantly expressed in vivo from the same key virulence genes. Strikingly, the observed back-splice junctions do not follow a unique canonical pattern, compatible with the U2-dependent splicing machinery. Numerous noncanonical junctions were observed in viral circRNA sequences characterized from in vitro and in vivo infections. Given the importance of the genes involved in the transcription of these circRNAs, our study contributes to our understanding and complexity of this deadly pathogen. IMPORTANCE Circular RNAs (circRNAs) were rediscovered in recent years both in physiological and pathological contexts, such as in cancer. Viral circRNAs are encoded by at least two human herpesviruses, the Epstein Barr virus and the Kaposi's Sarcoma-associated herpesvirus, both associated with the development of lymphoma. Marek's disease virus (MDV) is a well-established animal model to study virus-induced lymphoma but circRNA expression has not been reported for MDV yet. Our study provided the first evidence of viral circRNAs that were expressed at key steps of the MDV lifecycle using genome-wide analyses of circRNAs. These circRNAs were primarily found in transcriptional units that corresponded to the major MDV virulence factors. In addition, we established a bioinformatics pipeline that offers a new tool to identify circular RNAs in other herpesviruses. This study on the circRNAs provided important insights into major MDV virulence genes and herpesviruses-mediated gene dysregulation.

Adenoviruses are very efficient high-capacity vaccine vectors and are common gene delivery systems. Despite their extensive use in preclinical models and clinical trials over the past decades, adenoviral vectors still require optimization. To achieve that, more thorough characterizations of adenoviral genes and gene products, as well as pathogen-host interactions, are indispensable. The adenoviral DNA binding protein (DBP) is a key regulatory protein involved in various cellular and viral processes. Here, we show that single amino acid exchange mutations in human adenovirus C5 (HAdV-C5) DBP strongly influence adenoviral replication by altering interaction with the cellular ubiquitination machinery. Specifically, phenotypic analyses of DBP mutants demonstrate that single amino acid substitutions can regulate interactions with the cellular USP7 deubiquitinase, impede viral DNA synthesis, and completely abolish viral late protein expression and progeny production. Importantly, cells infected with the DBP mutant UBM5 consistently lack DBP-positive replication centers (RCs), which are usually formed during the transition from the early to the late phase of infection. Our findings demonstrate that DBP regulates a key step at the onset of the late phase of infection and that this activity is unambiguously linked to the formation and integrity of viral RCs. These data provide the experimental basis for future work that targets DBP and its interference with the formation of viral RCs during productive infection. Consequently, this work will have immediate impact on DNA virus and adenovirus research in general and, potentially, also on safety optimization of existing and development of novel adenoviral vectors and anti-adenoviral compounds. IMPORTANCE To further understand the biology of human adenoviruses (HAdVs) and to optimize HAdVs for use in prophylactic and therapeutic therapies, a thorough understanding of key viral proteins is paramount. As one of the essential HAdV proteins, the DNA binding protein DBP plays important roles in various steps of the viral replication cycle. In this work, we aimed at deciphering the role of single amino acid exchange mutations in the HAdV-C5 DBP on interaction with the cellular deubiquitinase USP7 and regulation of viral replication. We identify interaction with USP7, viral replication center formation, and viral progeny production as potently regulated steps of the viral life cycle that are affected by these few and distinct mutations in DBP.

The multifunctional adenoviral E1B-55K phosphoprotein is a major regulator of viral replication and plays key roles in virus-mediated cell transformation. While much is known about its function in oncogenic cell transformation, the underlying features and exact mechanisms that implicate E1B-55K in the regulation of viral gene expression are less well understood. Therefore, this work aimed to unravel basic intranuclear principles of E1B-55K-regulated viral mRNA biogenesis using wild-type human adenovirus C5 (HAdV-C5) E1B-55K, a virus mutant with abrogated E1B-55K expression, and a mutant that expresses a phosphomimetic E1B-55K. By subnuclear fractionation, mRNA, DNA, and protein analyses as well as luciferase reporter assays, we show that (i) E1B-55K promotes the efficient release of viral late mRNAs from their site of synthesis in viral replication compartments (RCs) to the surrounding nucleoplasm, (ii) E1B-55K modulates the rate of viral gene transcription and splicing in RCs, (iii) E1B-55K participates in the temporal regulation of viral gene expression, (iv) E1B-55K can enhance or repress the expression of viral early and late promoters, and (v) the phosphorylation of E1B-55K regulates the temporal effect of the protein on each of these activities. Together, these data demonstrate that E1B-55K is a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes during HAdV-C5 infection. IMPORTANCE Human adenoviruses are useful models to study basic aspects of gene expression and splicing. Moreover, they are one of the most commonly used viral vectors for clinical applications. However, key aspects of the activities of essential viral proteins that are commonly modified in adenoviral vectors have not been fully described. A prominent example is the multifunctional adenoviral oncoprotein E1B-55K that is known to promote efficient viral genome replication and expression while simultaneously repressing host gene expression and antiviral host responses. Our study combined different quantitative methods to study how E1B-55K promotes viral mRNA biogenesis. The data presented here propose a novel role for E1B-55K as a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes.

The human adenovirus type C5 (HAdV-C5) E1B-55K protein is a multifunctional regulator of HAdV-C5 replication, participating in many processes required for maximal virus production. Its multifunctional properties are primarily regulated by post-translational modifications (PTMs). The most influential E1B-55K PTMs are phosphorylation at highly conserved serine and threonine residues at the C-terminus, and SUMO conjugation to lysines 104 (K104) and 101 (K101) situated in the N-terminal region of the protein, which have been shown to regulate each other. Reversible SUMO conjugation provides a molecular switch that controls key functions of the viral protein, including intracellular trafficking and viral immune evasion. Interestingly, SUMOylation at SUMO conjugation site (SCS) K104 is negatively regulated by another multifunctional HAdV-C5 protein, E4orf6, which is known to form a complex with E1B-55K. To further evaluate the role of E4orf6 in the regulation of SUMO conjugation to E1B-55K, we analyzed different virus mutants expressing E1B-55K proteins with amino acid exchanges in both SCS (K101 and K104) in the presence or absence of E4orf6. We could exclude phosphorylation as factor for E4orf6-mediated reduction of E1B-55K SUMOylation. In fact, we demonstrate that a direct interaction between E1B-55K and E4orf6 is required to reduce E1B-55K SUMOylation. Additionally, we show that an E4orf6-mediated decrease of SUMO conjugation to K101 and K104 result in impaired co-localization of E1B-55K and SUMO in viral replication compartments. These findings indicate that E4orf6 inhibits E1B-55K SUMOylation, which could favor assembly of E4orf6-dependent E3 ubiquitin ligase complexes that are known to degrade a variety of host restriction factors by proteasomal degradation and, thereby, promote viral replication.

Over the past decades, studies on the biology of human adenoviruses (HAdVs) mainly focused on the HAdV prototype species C type 5 (HAdV-C5) and revealed fundamental molecular insights into mechanisms of viral replication and viral cell transformation. Recently, other HAdV species are gaining more and more attention in the field. Reports on large E1B proteins (E1B-55K) from different HAdV species showed that these multifactorial proteins possess strikingly different features along with highly conserved functions. In this work, we identified potential SUMO-conjugation motifs (SCMs) in E1B-55K proteins from HAdV species A to F. Mutational inactivation of these SCMs demonstrated that HAdV E1B-55K proteins are SUMOylated at a single lysine residue that is highly conserved among HAdV species B to E. Moreover, we provide evidence that E1B-55K SUMOylation is a potent regulator of intracellular localization and p53-mediated transcription in most HAdV species. We also identified a lysine residue at position 101 (K101), which is unique to HAdV-C5 E1B-55K and specifically regulates its SUMOylation and nucleo-cytoplasmic shuttling. Our findings reveal important new aspects on HAdV E1B-55K proteins and suggest that different E1B-55K species possess conserved SCMs while their SUMOylation has divergent cellular effects during infection. IMPORTANCE E1B-55K is a multifunctional adenoviral protein and its functions are highly regulated by SUMOylation. Although functional consequences of SUMOylated HAdV-C5 E1B-55K are well studied, we lack information on the effects of SUMOylation on homologous E1B-55K proteins from other HAdV species. Here, we show that SUMOylation is a conserved posttranslational modification in most of the E1B-55K proteins, similar to what we know about HAdV-C5 E1B-55K. Moreover, we identify subcellular localization and regulation of p53-dependent transcription as highly conserved SUMOylation-regulated E1B-55K functions. Thus, our results highlight how HAdV proteins might have evolved in different HAdV species with conserved domains involved in virus replication and differing alternative functions and interactions with the host cell machinery. Future research will link these differences and similarities to the diverse pathogenicity and organ tropism of the different HAdV species.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes a devastating neoplastic disease in chickens. MDV has been shown to integrate its genome into the telomeres of latently infected and tumor cells, which is crucial for efficient tumor formation. Telomeric repeat arrays present at the ends of the MDV genome facilitate this integration into host telomeres; however, the integration mechanism remains poorly understood. Until now, MDV integration could only be investigated qualitatively upon infection of chickens. To shed further light on the integration mechanism, we established a quantitative integration assay using chicken T cell lines, the target cells for MDV latency and transformation. We optimized the infection conditions and assessed the establishment of latency in these T cells. The MDV genome was efficiently maintained over time, and integration was confirmed in these cells by fluorescence in situ hybridization (FISH). To assess the role of the two distinct viral telomeric repeat arrays in the integration process, we tested various knockout mutants in our in vitro integration assay. Efficient genome maintenance and integration was thereby dependent on the presence of the telomeric repeat arrays in the virus genome. Taken together, we developed and validated a novel in vitro integration assay that will shed light on the integration mechanism of this highly oncogenic virus into host telomeres.

Human adenovirus (HAdV) infections cause a wide variety of clinical symptoms, ranging from mild upper respiratory tract disease to lethal outcomes, particularly in immunocompromised individuals. To date, neither widely available vaccines nor approved antiadenoviral compounds are available to efficiently deal with HAdV infections. Thus, there is a need to thoroughly understand HAdV-induced disease, and for the development and preclinical evaluation of HAdV therapeutics and/or vaccines, and consequently for suitable standardizable in vitro systems and animal models. Current animal models to study HAdV pathogenesis, persistence, and tumorigenesis include rodents such as Syrian hamsters, mice, and cotton rats, as well as rabbits. In addition, a few recent studies on other species, such as pigs and tree shrews, reported promising data. These models mimic (aspects of) HAdV-induced pathological changes in humans and, although they are relevant, an ideal HAdV animal model has yet to be developed. This review summarizes the available animal models of HAdV infection with comprehensive descriptions of virus-induced pathogenesis in different animal species. We also elaborate on rodent HAdV animal models and how they contributed to insights into adenovirus-induced cell transformation and cancer.

Marek's disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells., Competing Interests: The authors have declared that no competing interests exist.

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella . Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris , host cells, and the microbiota.

Safe and effective vaccines are urgently needed to stop the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We construct a series of live attenuated vaccine candidates by large-scale recoding of the SARS-CoV-2 genome and assess their safety and efficacy in Syrian hamsters. Animals were vaccinated with a single dose of the respective recoded virus and challenged 21 days later. Two of the tested viruses do not cause clinical symptoms but are highly immunogenic and induce strong protective immunity. Attenuated viruses replicate efficiently in the upper but not in the lower airways, causing only mild pulmonary histopathology. After challenge, hamsters develop no signs of disease and rapidly clear challenge virus: at no time could infectious virus be recovered from the lungs of infected animals. The ease with which attenuated virus candidates can be produced and administered favors their further development as vaccines to combat the ongoing pandemic., Competing Interests: Declaration of interests Freie Universität Berlin and the University of Bern received funding from a commercial partner for research similar to that described in this manuscript., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus of chickens that causes lymphomas in various organs. Most MDV genes are conserved among herpesviruses, while others are unique to MDV and may contribute to pathogenesis and/or tumor formation. High transcript levels of the MDV-specific genes MDV082, RLORF11, and SORF6 were recently detected in lytically infected cells; however, it remained elusive if the respective proteins are expressed and if they play a role in MDV pathogenesis. In this study, we first addressed if these proteins are expressed by inserting FLAG tags at their N or C termini. We could demonstrate that among the three genes tested, MDV082 is the only gene that encodes a protein and is expressed very late in MDV plaques in vitro . To investigate the role of this novel MDV082 protein in MDV pathogenesis, we generated a recombinant virus that lacks expression of the MDV082 protein. Our data revealed that the MDV082 protein contributes to the rapid onset of Marek's disease but is not essential for virus replication, spread, and tumor formation. Taken together, this study sheds light on the expression of MDV-specific genes and unravels the role of the late protein MDV082 in MDV pathogenesis. IMPORTANCE MDV is a highly oncogenic alphaherpesvirus that causes Marek's disease in chickens. The virus causes immense economic losses in the poultry industry due to the high morbidity and mortality, but also the cost of the vaccination. MDV encodes over 100 genes that are involved in various processes of the viral life cycle. Functional characterization of MDV genes is an essential step toward understanding the complex virus life cycle and MDV pathogenesis. Here, we have identified a novel protein encoded by MDV082 and two potential noncoding RNAs (RLORF11 and SORF6). The novel MDV082 protein is not needed for efficient MDV replication and tumor formation. However, our data demonstrate that the MDV082 protein is involved in the rapid onset of Marek's disease.

Marek's disease virus (MDV) is a highly cell-associated oncogenic alphaherpesvirus that causes lymphomas in various organs in chickens. Like other herpesviruses, MDV has a large and complex double-stranded DNA genome. A number of viral transcripts are generated by alternative splicing, a process that drastically extends the coding capacity of the MDV genome. One of the spliced genes encoded by MDV is the viral interleukin 8 (vIL-8), a CXC chemokine that facilitates the recruitment of MDV target cells and thereby plays an important role in MDV pathogenesis and tumorigenesis. We recently identified a novel vIL-8 exon (vIL-8-E3') by RNA-seq; however, it remained elusive whether the protein containing the vIL-8-E3' is expressed and what role it may play in MDV replication and/or pathogenesis. To address these questions, we first generated recombinant MDV harboring a tag that allows identification of the spliced vIL-8-E3' protein, revealing that it is indeed expressed. We subsequently generated knockout viruses and could demonstrate that the vIL-8-E3' protein is dispensable for MDV replication as well as secretion of the functional vIL-8 chemokine. Finally, infection of chickens with this vIL-8-E3' knockout virus revealed that the protein is not important for MDV replication and pathogenesis in vivo. Taken together, our study provides novel insights into the splice forms of the CXC chemokine of this highly oncogenic alphaherpesvirus.

The adenovirus type 5 (HAdV-C5) E1 transcription unit encodes regulatory proteins that are essential for viral replication and transformation. Among these, E1A and E1B-55K act as key multifunctional HAdV-C5 proteins involved in various steps of the viral replication cycle and in virus-induced cell transformation. In this context, HAdV-C5-mediated dysregulations of cellular factors such as the tumor suppressors p53 and pRB have been intensively investigated. However, cellular components of downstream events that could affect infection and viral transformation are widely unknown. We recently observed that cellular FAM111B is highly regulated in an E1A-dependent fashion. Intriguingly, previous reports suggest that FAM111B might play roles in tumorigenesis, but its exact functions are not known to date. Here, we set out to investigate the role of FAM111B in HAdV-C5 infections. We found that (i) FAM111B levels are upregulated early and downregulated late during infection, that (ii) FAM111B expression is differentially regulated, that (iii) FAM111B expression levels depend on the presence of E1B-55K and E4orf6 and that (iv) a FAM111B knockdown increases HAdV-C5 replication. Our data indicate that FAM111B acts as an anti-adenoviral host factor that is involved in host cell defense mechanisms in productive HAdV-C5 infection. Moreover, these findings suggest that FAM111B might play an important role in the host antiviral immune response that is counteracted by HAdV-C5 E1B-55K and E4orf6 oncoproteins.

Viral diseases remain a major concern for animal health and global food production in modern agriculture. In chickens, avian leukosis virus subgroup J (ALV-J) represents an important pathogen that causes severe economic loss. Until now, no vaccine or antiviral drugs are available against ALV-J and strategies to combat this pathogen in commercial flocks are desperately needed. CRISPR/Cas9 targeted genome editing recently facilitated the generation of genetically modified chickens with a mutation of the chicken ALV-J receptor Na + /H + exchanger type 1 (chNHE1). In this study, we provide evidence that this mutation protects a commercial chicken line (NHE1ΔW38) against the virulent ALV-J prototype strain HPRS-103. We demonstrate that replication of HPRS-103 is severely impaired in NHE1ΔW38 birds and that ALV-J-specific antigen is not detected in cloacal swabs at later time points. Consistently, infected NHE1ΔW38 chickens gained more weight compared to their non-transgenic counterparts (NHE1W38). Histopathology revealed that NHE1W38 chickens developed ALV-J typical pathology in various organs, while no pathological lesions were detected in NHE1ΔW38 chickens. Taken together, our data revealed that this mutation can render a commercial chicken line resistant to highly pathogenic ALV-J infection, which could aid in fighting this pathogen and improve animal health in the field.

The SARS-CoV-2 pandemic has caused a yet unresolved global crisis. Effective medical intervention by vaccination or therapy seems to be the only possibility to control the pandemic. In this context, animal models are an indispensable tool for basic and applied research to combat SARS-CoV-2 infection. Here, we established a SARS-CoV-2 infection model in Chinese hamsters suitable for studying pathogenesis of the disease as well as pre-clinical testing of vaccines and therapies. This species of hamster is susceptible to SARS-CoV-2 infection as demonstrated by robust virus replication in the upper and lower respiratory tract accompanied by bronchitis and pneumonia as well as significant body weight loss following infection. The Chinese hamster features advantages compared to the Syrian hamster model, including more pronounced clinical symptoms, its small size, well-characterized genome, transcriptome and translatome data and availability of molecular tools., (© 2020 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans., Competing Interests: The authors have declared that no competing interests exist.

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus of chickens. The MDV genome consists of two unique regions that are both flanked by inverted repeat regions. These repeats harbor several genes involved in virus replication and pathogenesis, but it remains unclear why MDV and other herpesviruses harbor these large sequence duplications. In this study, we set to determine if both copies of these repeat regions are required for MDV replication and pathogenesis. Our results demonstrate that MDV mutants lacking the entire internal repeat region (ΔIR LS ) efficiently replicate and spread from cell-to-cell in vitro However, ΔIR LS replication was severely impaired in infected chickens and the virus caused significantly less frequent disease and tumors compared to the controls. In addition, we also generated recombinant viruses that harbor a deletion of most of the internal repeat region, leaving only short terminal sequences behind (ΔIR LS-HR ). These remaining homologous sequences facilitated rapid restoration of the deleted repeat region, resulting in a virus that caused disease and tumors comparable to the wild type. Therefore, ΔIR LS-HR represents an excellent platform for rapid genetic manipulation of the virus genome in the repeat regions. Taken together, our study demonstrates that MDV requires both copies of the repeats for efficient replication and pathogenesis in its natural host. IMPORTANCE Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that infects chickens and causes losses in the poultry industry of up to $2 billion per year. The virus is also widely used as a model to study alphaherpesvirus pathogenesis and virus-induced tumor development in a natural host. MDV and most other herpesviruses harbor direct or inverted repeats regions in their genome. However, the role of these sequence duplications in MDV remains elusive and has never been investigated in a natural virus-host model for any herpesvirus. Here, we demonstrate that both copies of the repeats are needed for efficient MDV replication and pathogenesis in vivo , while replication was not affected in cell culture. With this, we further dissect herpesvirus genome biology and the role of repeat regions in Marek's disease virus replication and pathogenesis., (Copyright © 2021 American Society for Microbiology.)

Human cytomegalovirus (HCMV) is a human pathogenic herpesvirus associated with a variety of clinical symptoms. Current antiviral therapy is not always effective, so that improved drug classes and drug-targeting strategies are needed. Particularly host-directed antivirals, including pharmaceutical kinase inhibitors (PKIs), may help to overcome problems of drug resistance. Here, we focused on utilizing a selection of clinically relevant PKIs and determined their anticytomegaloviral efficacies. Particularly, PKIs directed to host or viral cyclin-dependent kinases, i.e., abemaciclib, LDC4297 and maribavir, exerted promising profiles against human and murine cytomegaloviruses. The anti-HCMV in vitro activity of the approved anti-cancer drug abemaciclib was confirmed in vivo using our luciferase-based murine cytomegalovirus (MCMV) animal model in immunocompetent mice. To assess drug combinations, we applied the Bliss independence checkerboard and Loewe additivity fixed-dose assays in parallel. Results revealed that (i) both affirmative approaches provided valuable information on anti-CMV drug efficacies and interactions, (ii) the analyzed combinations comprised additive, synergistic or antagonistic drug interactions consistent with the drugs' antiviral mode-of-action, (iii) the selected PKIs, especially LDC4297, showed promising inhibitory profiles, not only against HCMV but also other α-, β- and γ-herpesviruses, and specifically, (iv) the combination treatment with LDC4297 and maribavir revealed a strong synergism against HCMV, which might open doors towards novel clinical options in the near future. Taken together, this study highlights the potential of therapeutic drug combinations of current developmental/preclinical PKIs.

Mass spectrometry imaging (MSI) is a label-free molecular imaging technique allowing an untargeted detection of a broad range of biomolecules and xenobiotics. MSI enables imaging of the spatial distribution of proteins, peptides, lipids and metabolites from a wide range of samples. To date, this technique is commonly applied to tissue sections in cancer diagnostics and biomarker development, but also molecular histology in general. Advances in the methodology and bioinformatics improved the resolution of MS images below the single cell level and increased the flexibility of the workflow. However, MSI-based research in virology is just starting to gain momentum and its full potential has not been exploited yet. In this review, we discuss the main applications of MSI in virology. We review important aspects of matrix-assisted laser desorption/ionization (MALDI) MSI, the most widely used MSI technique in virology. In addition, we summarize relevant literature on MSI studies that aim to unravel virus-host interactions and virus pathogenesis, to elucidate antiviral drug kinetics and to improve current viral disease diagnostics. Collectively, these studies strongly improve our general understanding of virus-induced changes in the proteome, metabolome and metabolite distribution in host tissues of humans, animals and plants upon infection. Furthermore, latest MSI research provided important insights into the drug distribution and distribution kinetics, especially in antiretroviral research. Finally, MSI-based investigations of oncogenic viruses greatly increased our knowledge on tumor mass signatures and facilitated the identification of cancer biomarkers., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Modified-live herpesvirus vaccines are widely used in humans and animals, but field strains can emerge that have a higher virulence and break vaccinal protection. Since the introduction of the first vaccine in the 1970s, Marek's disease virus overcame the vaccine barrier by the acquisition of numerous genomic mutations. However, the evolutionary adaptations in the herpesvirus genome responsible for the vaccine breaks have remained elusive. Here, we demonstrate that point mutations in the multifunctional meq gene acquired during evolution can significantly alter virulence. Defined mutations found in highly virulent strains also allowed the virus to overcome innate cellular responses and vaccinal protection. Concomitantly, the adaptations in meq enhanced virus shedding into the environment, likely providing a selective advantage for the virus. Our study provides the first experimental evidence that few point mutations in a single herpesviral gene result in drastically increased virulence, enhanced shedding, and escape from vaccinal protection., Competing Interests: The authors have declared that no competing interests exist.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has precipitated an unprecedented and yet-unresolved health crisis worldwide. Different mammals are susceptible to SARS-CoV-2; however, few species examined so far develop robust clinical disease that mirrors severe human cases or allows testing of vaccines and drugs under conditions of severe disease. Here, we compare the susceptibilities of three dwarf hamster species (Phodopus spp.) to SARS-CoV-2 and introduce the Roborovski dwarf hamster (P. roborovskii) as a highly susceptible COVID-19 model with consistent and fulminant clinical signs. Particularly, only this species shows SARS-CoV-2-induced severe acute diffuse alveolar damage and hyaline microthrombi in the lungs, changes described in patients who succumbed to the infection but not reproduced in any experimentally infected animal. Based on our findings, we propose the Roborovski dwarf hamster as a valuable model to examine the efficacy and safety of vaccine candidates and therapeutics, particularly for use in highly susceptible individuals., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The emergence of SARS-CoV-2 led to pandemic spread of coronavirus disease 2019 (COVID-19), manifesting with respiratory symptoms and multi-organ dysfunction. Detailed characterization of virus-neutralizing antibodies and target epitopes is needed to understand COVID-19 pathophysiology and guide immunization strategies. Among 598 human monoclonal antibodies (mAbs) from 10 COVID-19 patients, we identified 40 strongly neutralizing mAbs. The most potent mAb, CV07-209, neutralized authentic SARS-CoV-2 with an IC 50 value of 3.1 ng/mL. Crystal structures of two mAbs in complex with the SARS-CoV-2 receptor-binding domain at 2.55 and 2.70 Å revealed a direct block of ACE2 attachment. Interestingly, some of the near-germline SARS-CoV-2-neutralizing mAbs reacted with mammalian self-antigens. Prophylactic and therapeutic application of CV07-209 protected hamsters from SARS-CoV-2 infection, weight loss, and lung pathology. Our results show that non-self-reactive virus-neutralizing mAbs elicited during SARS-CoV-2 infection are a promising therapeutic strategy., Competing Interests: Declaration of Interests Related to this work, the German Center for Neurodegenerative Diseases (DZNE) and Charité-Universitätsmedizin Berlin have filed a patent application on which J.K., S.M.R., H.-C.K., E.S.-S., V.M.C., M.A.M., D.W., L.E.S., and H.P. are named as inventors., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In late 2019, an outbreak of a severe respiratory disease caused by an emerging coronavirus, SARS-CoV-2, resulted in high morbidity and mortality in infected humans. Complete understanding of COVID-19, the multi-faceted disease caused by SARS-CoV-2, requires suitable small animal models, as does the development and evaluation of vaccines and antivirals. Since age-dependent differences of COVID-19 were identified in humans, we compared the course of SARS-CoV-2 infection in young and aged Syrian hamsters. We show that virus replication in the upper and lower respiratory tract was independent of the age of the animals. However, older hamsters exhibited more pronounced and consistent weight loss. In situ hybridization in the lungs identified viral RNA in bronchial epithelium, alveolar epithelial cells type I and II, and macrophages. Histopathology revealed clear age-dependent differences, with young hamsters launching earlier and stronger immune cell influx than aged hamsters. The latter developed conspicuous alveolar and perivascular edema, indicating vascular leakage. In contrast, we observed rapid lung recovery at day 14 after infection only in young hamsters. We propose that comparative assessment in young versus aged hamsters of SARS-CoV-2 vaccines and treatments may yield valuable information, as this small-animal model appears to mirror age-dependent differences in human patients.

Marek's disease virus (MDV) is a highly cell-associated alphaherpesvirus that causes deadly lymphomas in chickens. While vaccination protects against clinical symptoms, MDV field strains can still circulate in vaccinated flocks and continuously evolve towards greater virulence. MDV vaccines do not provide sterilizing immunity, allowing the virus to overcome vaccine protection, and has increased the need for more potent vaccines or alternative interventions. In this study, we addressed if the CRISPR/Cas9 system can protect cells from MDV replication. We first screened a number of guide RNAs (gRNAs) targeting essential MDV genes for their ability to prevent virus replication. Single gRNAs significantly inhibited virus replication, but could result in the emergence of escape mutants. Strikingly, combining two or more gRNAs completely abrogated virus replication and no escape mutants were observed upon serial passaging. Our study provides the first proof-of-concept, demonstrating that the CRISPR/Cas9 system can be efficiently used to block MDV replication. The presented findings lay the foundation for future research to completely protect chickens from this deadly pathogen.

Human cytomegalovirus (HCMV) causes serious and even life-threatening diseases, particularly upon congenital or post-transplant infection. Treatment of HCMV infections with currently available drugs targeting viral enzymes is often limited by severe side effects and the emergence of drug-resistant viruses. To avoid this problem, novel therapeutic options directed to host proteins involved in virus replication are being investigated. Recently, we described the pronounced antiherpesviral activity of the trimeric artesunate derivative TF27 at low nanomolar concentrations in vitro and in vivo. In the present study, we report first data on the prophylactic efficacy of TF27 against human and murine CMV and the oncogenic avian alphaherpesvirus Marek's disease virus (MDV). The main findings of this study are (i) a pronounced activity of the experimental drug TF27 against alpha- and betaherpesviruses in vitro upon prophylactic treatment and (ii) a therapeutic and prophylactic efficacy upon oral treatment in an immunocompetent mouse model. Moreover, our data highlight (iii) the tolerability of orally administered TF27 free of compound-associated adverse events and further confirm (iv) the suitability of cellular factors as primary antiviral targets. Thus, we provide evidence for therapeutic and prophylactic antiherpesviral efficacy of TF27 upon oral treatment in immunocompetent hosts and thereby underline its potential for future antiviral drug development., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2020. Published by Elsevier B.V.)

Genome editing technology provides new possibilities for animal breeding and aid in understanding host-pathogen interactions. In poultry, retroviruses display one of the most difficult pathogens to control by conventional strategies such as vaccinations. Avian leukosis virus subgroup J (ALV-J) is an oncogenic, immunosuppressive retrovirus that causes myeloid leukosis and other tumors in chickens. Severe economic losses caused by ALV-J remain an unsolved problem in many parts of the world due to inefficient eradication strategies and lack of effective vaccines. ALV-J attachment and entry are mediated through the specific receptor, chicken Na + /H + exchanger type 1 (chNHE1). The non-conserved amino acid tryptophan 38 (W38) in chNHE1 is crucial for virus entry, making it a favorable target for the introduction of disease resistance. In this study, we obtained ALV-J-resistance in a commercial chicken line by precise deletion of chNHE1 W38, utilizing the CRISPR/Cas9-system in combination with homology directed repair. The genetic modification completely protected cells from infection with a subgroup J retrovirus. W38 deletion did neither have a negative effect on the development nor on the general health condition of the gene edited chickens. Overall, the generation of ALV-J-resistant birds by precise gene editing demonstrates the immense potential of this approach as an alternative disease control strategy in poultry., (Copyright © 2020 Hellmich, Sid, Lengyel, Flisikowski, Schlickenrieder, Bartsch, Thoma, Bertzbach, Kaufer, Nair, Preisinger and Schusser.)

Marek's disease virus (MDV) infects chickens and causes one of the most frequent cancers in animals. Over 100 years of research on this oncogenic alphaherpesvirus has led to a profound understanding of virus-induced tumor development. Live-attenuated vaccines against MDV were the first that prevented cancer and minimized the losses in the poultry industry. Even though the current gold standard vaccine efficiently protects against clinical disease, the virus continuously evolves towards higher virulence. Emerging field strains were able to overcome the protection provided by the previous two vaccine generations. Research over the last few years revealed important insights into the virus life cycle, cellular tropism, and tumor development that are summarized in this review. In addition, we discuss recent data on the MDV transcriptome, the constant evolution of this highly oncogenic virus towards higher virulence, and future perspectives in MDV research.

In contrast to human influenza viruses that replicate in the respiratory tract and are airborne transmitted, avian viruses also replicate in gut epithelial cells and are transmitted via the fecal-oral route. On this route, the virus is exposed to destructive fluids of the digestive tract, which are acidic and contain the proteases pepsin (gizzard) or chymotrypsin and trypsin (intestine). Only the latter enzyme activates virus by cleaving hemagglutinin (HA) into HA 1 and HA 2 subunits. We mimicked the passage of viruses through the gastrointestinal tract by treating them with digestive fluids from chicken and determined titers and integrity of HA by western-blot. Gizzard fluid completely inactivated virions and degrades HA even at a high dilution, but only if the pH was kept acidic. If the fluid is diluted with neutral buffer (mimicking virus uptake with seawater) particles were more resistant. Virions containing an uncleaved HA were even activated suggesting that gastric juice contains a trypsin-like protease. Undiluted intestinal fluid inactivated particles and destroyed HA, but diluted fluid activated virions. A virus isolated from the duck´s intestine is more tolerant against intestinal fluid compared to fowl plague virus suggesting that the former is better adapted to grow in the intestine. We also demonstrate that influenza viruses replicate to high titers in a novel chicken epithelial gut cell line. While viruses with a monobasic HA cleavage site require addition of trypsin, these cells effectively process HA with a polybasic cleavage site, which could be blocked with an inhibitor of the cellular furin protease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Marek's disease virus (MDV) is an alphaherpesvirus that causes Marek's disease, a malignant lymphoproliferative disease of domestic chickens. While MDV vaccines protect animals from clinical disease, they do not provide sterilizing immunity and allow field strains to circulate and evolve in vaccinated flocks. Therefore, there is a need for improved vaccines and for a better understanding of innate and adaptive immune responses against MDV infections. Interferons (IFNs) play important roles in the innate immune defenses against viruses and induce upregulation of a cellular antiviral state. In this report, we quantified the potent antiviral effect of IFNα and IFNγ against MDV infections in vitro. Moreover, we demonstrate that both cytokines can delay Marek's disease onset and progression in vivo. Additionally, blocking of endogenous IFNα using a specific monoclonal antibody, in turn, accelerated disease. In summary, our data reveal the effects of IFNα and IFNγ on MDV infection and improve our understanding of innate immune responses against this oncogenic virus.

Natural killer (NK) cells are key players in the innate immune response. They kill virus-infected cells and are crucial for the induction of adaptive immune responses. Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that causes deadly T cell lymphomas in chickens. Host resistance to MDV is associated with differences in NK cell responses; however, the exact role of NK cells in the control of MDV remains unknown. In this study, we assessed if MDV can infect NK cells and alter their activation. Surprisingly, we could demonstrate that primary chicken NK cells are very efficiently infected with very virulent RB-1B MDV and the live-attenuated CVI988 vaccine. Flow cytometry analysis revealed that both RB-1B and CVI988 enhance NK cell degranulation and increase interferon gamma (IFNγ) production in vitro. In addition, we could show that the MDV Eco Q-encoded oncogene ( meq ) contributes to the induction of NK cell activation using meq knockout viruses. Taken together, our data revealed for the first time that NK cells are efficiently infectable with MDV and that this oncogenic alphaherpesvirus enhances NK cell degranulation and increased IFNγ production in vitro., Competing Interests: The authors declare no conflict of interest.

Nucleoside analogues have been the cornerstone of clinical treatment of herpesvirus infections since the 1970s. However, severe side effects and emergence of drug resistant viruses raise the need for alternative treatment options. We recently investigated the broad and strong antiherpesviral activity of the optimized artesunate derivative TF27 in vitro. TF27 efficiently inhibited replication of the highly oncogenic Marek's disease virus (MDV), a virus that infects chickens, causes deadly lymphomas and threatens poultry populations worldwide. In this study, we used this natural virus-host model for herpesvirus-induced cancer by infecting chickens with MDV, and evaluated the protective efficacy of TF27 and the nucleoside analogue valganciclovir (VGCV) on virus replication and tumorigenesis. We could demonstrate that both drugs reduced viral load in the blood and prevented tumor development in a large portion of the animals. Antiviral treatment also had a positive impact on body weight gain, while no negative compound-associated side effects were observed. This research provides the first evidence that the artesunate derivative TF27 and VGCV can be used in avian species and that they inhibit MDV replication and tumorigenesis. In addition, our study paves the way for promising approaches in future antiherpesviral drug development., (Copyright © 2019 Elsevier B.V. All rights reserved.)