Viral tegument proteins restrict cGAS-DNA phase separation to mediate immune evasion.

DNA-induced liquid-liquid phase separation of cyclic GMP-AMP synthase (cGAS) triggers a potent response to detect pathogen infection and promote innate immune signaling. Whether and how pathogens manipulate cGAS-DNA condensation to mediate immune evasion is unknown. We report the identification of a structurally related viral tegument protein family, represented by ORF52 and VP22 from gamma- and alpha-herpesvirinae, respectively, that employs a conserved mechanism to restrict cGAS-DNA phase separation. ORF52/VP22 proteins accumulate into, and effectively disrupt, the pre-formed cGAS-DNA condensation both in vitro and in cells. The inhibition process is dependent on DNA-induced liquid-liquid phase separation of the viral protein rather than a direct interaction with cGAS. Moreover, highly abundant ORF52 proteins carried within viral particles are able to target cGAS-DNA phase separation in early infection stage. Our results define ORF52/VP22-type tegument proteins as a family of inhibitors targeting cGAS-DNA phase separation and demonstrate a mechanism for how viruses overcome innate immunity.

Characterization of a Unique Novel LORF3 Protein of Duck Plague Virus and Its Potential Pathogenesis.

Duck plague virus (DPV) is a high-morbidity fowl alphaherpesvirus that causes septicemic lesions in various organs. Most DPV genes are conserved among herpesviruses, while a few are specific to fowl herpesviruses, including the LORF3 gene, for which there is currently no literature describing its biological properties and functions. This study first addressed whether the LORF3 protein is expressed by making specific polyclonal antibodies. We could demonstrate that DPV LORF3 is an early gene and encodes a protein involved in virion assembly, mainly localized in the nucleus of DPV-infected DEF cells. To investigate the role of this novel LORF3 protein in DPV pathogenesis, we generated a recombinant virus that lacks expression of the LORF3 protein. Our data revealed that the LORF3 protein is not essential for viral replication but contributes to DPV replication in vitro and in vivo and promotes duck plague disease morbidity and mortality. Interestingly, deletion of the LORF3 protein abolished thymus atrophy in DPV-vaccinated ducks. In conclusion, this study revealed the expression of avian herpesviruses-specific genes and unraveled the role of the early protein LORF3 in the pathogenesis of DPV. IMPORTANCE DPV is a highly lethal alphaherpesvirus that causes duck plague in birds of the order Anseriformes. The virus has caused huge economic losses to the poultry industry due to high morbidity and mortality and the cost of vaccination. DPV encodes 78 open reading frames (ORFs), and these genes are involved in various processes of the viral life cycle. Functional characterization of DPV genes is important for understanding the complex viral life cycle and DPV pathogenesis. Here, we identified a novel protein encoded by LORF3, and our data suggest that the LORF3 protein is involved in the occurrence and development of duck plague.

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1.

BACKGROUND: Infectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain's gC, TK, gG, gD, and gE genes. RESULTS: The study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/µL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28. CONCLUSIONS: The established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

Duck Enteritis Virus Inhibits the cGAS-STING DNA-Sensing Pathway To Evade the Innate Immune Response.

Cyclic GMP-AMP synthase (cGAS), a key DNA sensor, detects cytosolic viral DNA and activates the adaptor protein stimulator of interferon genes (STING) to initiate interferon (IFN) production and host innate antiviral responses. Duck enteritis virus (DEV) is a duck alphaherpesvirus that causes an acute and contagious disease with high mortality in waterfowl. In the present study, we found that DEV inhibits host innate immune responses during the late phase of viral infection. Furthermore, we screened DEV proteins for their ability to inhibit the cGAS-STING DNA-sensing pathway and identified multiple viral proteins, including UL41, US3, UL28, UL53, and UL24, which block IFN-ß activation through this pathway. The DEV tegument protein UL41, which exhibited the strongest inhibitory effect, selectively downregulated the expression of interferon regulatory factor 7 (IRF7) by reducing its mRNA accumulation, thereby inhibiting the DNA-sensing pathway. Ectopic expression of UL41 markedly reduced viral DNA-triggered IFN-ß production and promoted viral replication, whereas deficiency of UL41 in the context of DEV infection increased the IFN-ß response to DEV and suppressed viral replication. In addition, ectopic expression of IRF7 inhibited the replication of the UL41-deficient virus, whereas IRF7 knockdown facilitated its replication. This study is the first report identifying multiple viral proteins encoded by a duck DNA virus, which inhibit the cGAS-STING DNA-sensing pathway. These findings expand our knowledge of DNA sensing in ducks and reveal a mechanism through which DEV antagonizes the host innate immune response. IMPORTANCE Duck enteritis virus (DEV) is a duck alphaherpesvirus that causes an acute and contagious disease with high mortality, resulting in substantial economic losses in the commercial waterfowl industry. The evasion of DNA-sensing pathway-mediated antiviral innate immunity is essential for the persistent infection and replication of many DNA viruses. However, the mechanisms used by DEV to modulate the DNA-sensing pathway remain poorly understood. In the present study, we found that DEV encodes multiple viral proteins to inhibit the cGAS-STING DNA-sensing pathway. The DEV tegument protein UL41 selectively diminished the accumulation of interferon regulatory factor 7 (IRF7) mRNA, thereby inhibiting the DNA-sensing pathway. Loss of UL41 potently enhanced the IFN-ß response to DEV and impaired viral replication in ducks. These findings provide insights into the host-virus interaction during DEV infection and help develop new live attenuated vaccines against DEV.

The US3 Kinase of Herpes Simplex Virus Phosphorylates the RNA Sensor RIG-I To Suppress Innate Immunity.

Recent studies have demonstrated that the signaling activity of the cytosolic pathogen sensor retinoic acid-inducible gene-I (RIG-I) is modulated by a variety of posttranslational modifications (PTMs) to fine-tune the antiviral type I interferon (IFN) response. Whereas K63-linked ubiquitination of the RIG-I caspase activation and recruitment domains (CARDs) catalyzed by TRIM25 or other E3 ligases activates RIG-I, phosphorylation of RIG-I at S8 and T170 represses RIG-I signal transduction by preventing the TRIM25-RIG-I interaction and subsequent RIG-I ubiquitination. While strategies to suppress RIG-I signaling by interfering with its K63-polyubiquitin-dependent activation have been identified for several viruses, evasion mechanisms that directly promote RIG-I phosphorylation to escape antiviral immunity are unknown. Here, we show that the serine/threonine (Ser/Thr) kinase US3 of herpes simplex virus 1 (HSV-1) binds to RIG-I and phosphorylates RIG-I specifically at S8. US3-mediated phosphorylation suppressed TRIM25-mediated RIG-I ubiquitination, RIG-I-MAVS binding, and type I IFN induction. We constructed a mutant HSV-1 encoding a catalytically-inactive US3 protein (K220A) and found that, in contrast to the parental virus, the US3 mutant HSV-1 was unable to phosphorylate RIG-I at S8 and elicited higher levels of type I IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines in a RIG-I-dependent manner. Finally, we show that this RIG-I evasion mechanism is conserved among the alphaherpesvirus US3 kinase family. Collectively, our study reveals a novel immune evasion mechanism of herpesviruses in which their US3 kinases phosphorylate the sensor RIG-I to keep it in the signaling-repressed state. IMPORTANCE Herpes simplex virus 1 (HSV-1) establishes lifelong latency in the majority of the human population worldwide. HSV-1 occasionally reactivates to produce infectious virus and to facilitate dissemination. While often remaining subclinical, both primary infection and reactivation occasionally cause debilitating eye diseases, which can lead to blindness, as well as life-threatening encephalitis and newborn infections. To identify new therapeutic targets for HSV-1-induced diseases, it is important to understand the HSV-1-host interactions that may influence infection outcome and disease. Our work uncovered direct phosphorylation of the pathogen sensor RIG-I by alphaherpesvirus-encoded kinases as a novel viral immune escape strategy and also underscores the importance of RNA sensors in surveilling DNA virus infection.

Alphaherpesvirus infection in a free-ranging narwhal Monodon monoceros from Arctic Canada.

We report the detection of an alphaherpesvirus infecting an adult female narwhal Monodon monoceros captured live during a tagging project in Tremblay Sound, Nunavut, Canada, in August 2018. The individual had 2 open wounds on the dorsum but appeared in good overall health. A blowhole swab was collected, and subsequent virus isolation was performed using a beluga whale primary cell line. Non-syncytial cytopathic effects were seen, in contrast to syncytial cytopathic effects described for monodontid alphaherpesvirus 1 (MoAHV1) isolates previously recovered from beluga whales Delphinapterus leucas from Alaska, USA, and the Northwest Territories, Canada. Next-generation sequencing was performed on a sequencing library generated from the DNA of the viral isolate and the analysis of the assembled contigs permitted the recovery of 6 genes, conserved in all members of the family Orthoherpesviridae, for downstream genetic and phylogenetic analyses. BLASTN (basic local alignment search tool, searching nucleotide databases using a nucleotide query) analyses of the narwhal herpesvirus conserved genes showed the highest nucleotide identities to MoAHV1, ranging between 88.5 and 96.8%. A maximum likelihood phylogenetic analysis based on concatenation of the 6 conserved herpesviruses amino acid alignments revealed the narwhal herpesvirus (NHV) to be the closest relative to MoAHV1, forming a clade within the subfamily Alphaherpesvirinae, genus Varicellovirus. NHV is the first alphaherpesvirus characterized from a narwhal and represents a new viral species, which we propose to be known as Varicellovirus monodontidalpha2. Further research is needed to determine the prevalence and potential clinical impacts of this alphaherpesvirus infection in narwhals.

First detection of Psittacid alphaherpesvirus 5 and coinfection with beak and feather disease virus in naturally infected captive ringneck parakeets (Psittacula krameri) in Brazil.

This study describes a case report in captive rose-ringed parakeets (Psittacula krameri) that developed clinical signs and eventually died after introducing new birds without quarantine. Bronchopneumonia and airsacculitis with syncytial cells associated with intranuclear inclusion bodies were found. Herpesvirus was detected in lungs and liver by PCR, and a nearly complete genome sequence of a Psittacid alphaherpesvirus 5 was obtained from the lung of a bird. Metagenomic analysis also identified beak and feather disease virus in the same samples. The study also highlights the importance of quarantine for avoiding the introduction of new diseases in captive aviaries.

Alphaherpesvirus Genomics: Past, Present and Future.

Alphaherpesviruses, as large double-stranded DNA viruses, were long considered to be genetically stable and to exist in a homogeneous state. Recently, the proliferation of high-throughput sequencing (HTS) and bioinformatics analysis has expanded our understanding of herpesvirus genomes and the variations found therein. Recent data indicate that herpesviruses exist as diverse populations, both in culture and in vivo, in a manner reminiscent of RNA viruses. In this review, we discuss the past, present, and potential future of alphaherpesvirus genomics, including the technical challenges that face the field. We also review how recent data has enabled genome-wide comparisons of sequence diversity, recombination, allele frequency, and selective pressures, including those introduced by cell culture. While we focus on the human alphaherpesviruses, we draw key insights from related veterinary species and from the beta- and gamma-subfamilies of herpesviruses. Promising technologies and potential future directions for herpesvirus genomics are highlighted as well, including the potential to link viral genetic differences to phenotypic and disease outcomes.

Alphaherpesvirus Latency and Reactivation with a Focus on Herpes Simplex Virus.

We are at an interesting time in the understanding of alpha herpesvirus latency and reactivation and their implications to human disease. Conceptual advances have come from both animal and neuronal culture models. This review focuses on the concept that the tegument protein and viral transactivator VP16 plays a major role in the transition from latency to the lytic cycle. During acute infection, regulation of VP16 transactivation balances spread in the nervous system, establishment of latent infections and virulence. Reactivation is dependent on this transactivator to drive entry into the lytic cycle. In vivo de novo expression of VP16 protein is mediated by sequences conferring pre-immediate early transcription embedded in the normally leaky late promoter. In vitro, alternate mechanisms regulating VP16 expression in the context of latency have come from the SCG neuron culture model and include the concepts that (i) generalized transcriptional derepression of the viral genome and sequestration of VP16 in the cytoplasm for ~48 hours (Phase I) precedes and is required for VP16-dependent reactivation (Phase II); and (ii) a histone methyl/phospho switch during Phase I is required for Phase II reactivation. The challenge to the field is reconciling these data into a unified model of virus reactivation. The task of compiling this review was uncomfortably humbling, as if cataloging the stars in the universe. While not completely dark, our night sky is missing a multitude of studies which are among the many points of light contributing to our field. This article is a focused review in which we discuss from the vantage point of our expertise, just a handful of concepts that have or are emerging. A lookback at some of the pioneering work that grounds our field is also included.

Navigating the Cytoplasm: Delivery of the Alphaherpesvirus Genome to the Nucleus.

Herpesviruses virions are large and complex structures that deliver their genetic content to nuclei upon entering cells. This property is not unusual as many other viruses including the adenoviruses, orthomyxoviruses, papillomaviruses, polyomaviruses, and retroviruses, do likewise. However, the means by which viruses in the alphaherpesvirinae subfamily accomplish this fundamental stage of the infectious cycle is tied to their defining ability to efficiently invade the nervous system. Fusion of the viral envelope with a cell membrane results in the deposition of the capsid, along with an assortment of tegument proteins, into the cytosol. Establishment of infection requires that the capsid traverse the cytosol, dock at a nuclear pore, and inject its genome into the nucleoplasm. Accumulating evidence indicates that the capsid is not the effector of this delivery process, but is instead shepherded by tegument proteins that remain capsid bound. At the same time, tegument proteins that are released from the capsid upon entry act to increase the susceptibility of the cell to the ensuing infection. Mucosal epithelial cells and neurons are both susceptible to alphaherpesvirus infection and, together, provide the niche to which these viruses have adapted. Although much has been revealed about the functions of de novo expressed tegument proteins during the late stages of assembly and egress, this review will specifically address the roles of tegument proteins brought into the cell with the incoming virion, and our current understanding of alphaherpesvirus genome delivery to nuclei.

Differences in Antibody Responses against Chelonid Alphaherpesvirus 5 (ChHV5) Suggest Differences in Virus Biology in ChHV5-Seropositive Green Turtles from Hawaii and ChHV5-Seropositive Green Turtles from Florida.

Fibropapillomatosis (FP) is a tumor disease associated with a herpesvirus (chelonid herpesvirus 5 [ChHV5]) that affects mainly green turtles globally. Understanding the epidemiology of FP has been hampered by a lack of robust serological assays to monitor exposure to ChHV5. This is due in part to an inability to efficiently culture the virus in vitro for neutralization assays. Here, we expressed two glycoproteins (FUS4 and FUS8) from ChHV5 using baculovirus. These proteins were immobilized on enzyme-linked immunosorbent assay plates in their native form and assayed for reactivity to two types of antibodies, full-length 7S IgY and 5.7S IgY, which has a truncated Fc region. Turtles from Florida were uniformly seropositive to ChHV5 regardless of tumor status. In contrast, in turtles from Hawaii, we detected strong antibody reactivity mainly in tumored animals, with a lower antibody response being seen in nontumored animals, including those from areas where FP is enzootic. Turtles from Hawaii actively shedding ChHV5 were more seropositive than nonshedders. In trying to account for differences in the serological responses to ChHV5 between green turtles from Hawaii and green turtles from Florida, we rejected the cross-reactivity of antibodies to other herpesviruses, differences in viral epitopes, or differences in procedure as likely explanations. Rather, behavioral or other differences between green turtles from Hawaii and green turtles from Florida might have led to the emergence of biologically different viral strains. While the strains from turtles in Florida apparently spread independently of tumors, the transmission of the Hawaiian subtype relies heavily on tumor formation.IMPORTANCE Fibropapillomatosis (FP) is a tumor disease associated with chelonid herpesvirus 5 (ChHV5) that is an important cause of mortality in threatened green turtles globally. FP is expanding in Florida and the Caribbean but declining in Hawaii. We show that Hawaiian turtles mount antibodies to ChHV5 mainly in response to tumors, which are the only sites of viral replication, whereas tumored and nontumored Floridian turtles are uniformly seropositive. Tumor viruses that depend on tumors for replication and spread are rare, with the only example being the retrovirus causing walleye dermal sarcoma in fish. The Hawaiian strain of ChHV5 may be the first DNA virus with such an unusual life history. Our findings, along with the fundamental differences in the life histories between Floridian turtles and Hawaiian turtles, may partly explain the differential dynamics of FP between the two regions.

Role of Marek's Disease Virus (MDV)-Encoded US3 Serine/Threonine Protein Kinase in Regulating MDV Meq and Cellular CREB Phosphorylation.

Marek's disease (MD) is a neoplastic disease of chickens caused by Marek's disease virus (MDV), a member of the subfamily Alphaherpesvirinae Like other alphaherpesviruses, MDV encodes a serine/threonine protein kinase, US3. The functions of US3 have been extensively studied in other alphaherpesviruses; however, the biological functions of MDV US3 and its substrates have not been studied in detail. In this study, we investigated potential cellular pathways that are regulated by MDV US3 and identified chicken CREB (chCREB) as a substrate of MDV US3. We show that wild-type MDV US3, but not kinase-dead US3 (US3-K220A), increases CREB phosphorylation, leading to recruitment of phospho-CREB (pCREB) to the promoter of the CREB-responsive gene and activation of CREB target gene expression. Using US3 deletion and US3 kinase-dead recombinant MDV, we identified US3-responsive MDV genes during infection and found that the majority of US3-responsive genes were located in the MDV repeat regions. Chromatin immunoprecipitation sequencing (ChIP-seq) studies determined that some US3-regulated genes colocalized with Meq (an MDV-encoded oncoprotein) recruitment sites. Chromatin immunoprecipitation-PCR (ChIP-PCR) further confirmed Meq binding to the ICP4/LAT region, which is also regulated by US3. Furthermore, biochemical studies demonstrated that MDV US3 interacts with Meq in transfected cells and MDV-infected chicken embryonic fibroblasts in a phosphorylation-dependent manner. Finally, in vitro kinase studies revealed that Meq is a US3 substrate. MDV US3 thus acts as an upstream kinase of the CREB signaling pathway to regulate the transcription function of the CREB/Meq heterodimer, which targets cellular and viral gene expression.IMPORTANCE MDV is a potent oncogenic herpesvirus that induces T-cell lymphoma in infected chickens. Marek's disease continues to have a significant economic impact on the poultry industry worldwide. US3 encoded by alphaherpesviruses is a multifunctional kinase involved in the regulation of various cellular pathways. Using an MDV genome quantitative reverse transcriptase PCR (qRT-PCR) array and chromatin immunoprecipitation, we elucidated the role of MDV US3 in viral and cellular gene regulation. Our results provide insights into how viral kinase regulates host cell signaling pathways to activate both viral and host gene expression. This is an important step toward understanding host-pathogen interaction through activation of signaling cascades.

Characterization of a Novel Alphaherpesvirus Isolated from the Fruit Bat Pteropus lylei in Vietnam.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

Novel herpesvirus discovered in walrus liver.

A walrus (Odobenus rosmarus) born in an aquarium and hand-reared in Japan died at the age of 11 months. The affected animal showed fever and anorexia and had high levels of AST and ALT. Necropsy showed multiple necroses in the liver and adrenal glands and histological examination revealed necrotic lesions of the liver and adrenal cortex, both of which contained intranuclear inclusions. Electron microscopic analysis of the liver sample showed herpesvirus-like particles. High-throughput sequencing analysis of the liver sample and phylogenetic analysis of herpesvirus polymerase genes identified a new virus, Walrus alphaherpesvirus 1 (WaHV-1), which belonged to the subfamily Alphaherpesvirinae and had high homology with Phocid alphaherpesvirus 1. Phylogenetic analysis of the UL30 gene encoding glycoprotein B revealed that WaHV-1 was closely related to a cluster of phocid herpesviruses, including one that caused high mortality rates in harbor seals during past outbreaks. The mother walrus of the dead animal showed evidence of herpesvirus infection in the past and potentially harbored WaHV-1. As a result of hand-rearing, the dead animal might have acquired WaHV-1 from its infected mother and succumbed to WaHV-1 due to lack of maternal IgG, including those that could neutralize WaHV-1.

The Herpesviridae Conserved Multifunctional Infected-Cell Protein 27 (ICP27) Is Important but Not Required for Replication and Oncogenicity of Marek's Disease Alphaherpesvirus.

The Herpesviridae conserved infected-cell protein 27 (ICP27) is essential for cell culture-based replication of most herpesviruses studied. For members of the Alphaherpesvirinae, ICP27 regulates the expression of many viral genes, including expression of pUL44 (gC), pUL47 (VP13/14), and pUL48 (VP16). These three viral proteins are dysregulated during Marek's disease alphaherpesvirus (MDV) replication in cell culture. MDV replicates in a highly cell-associated manner in cell culture, producing little to no infectious virus. In contrast, infectious cell-free MDV is produced in specialized feather follicle epithelial (FFE) cells of infected chickens, in which these three genes are abundantly expressed. This led us to hypothesize that MDV ICP27, encoded by gene UL54, is a defining factor for the dysregulation of gC, pUL47, and pUL48 and, ultimately, ineffective virus production in cell culture. To address ICP27's role in MDV replication, we generated recombinant MDV with ICP27 deleted (vΔ54). Interestingly, vΔ54 replicated, but plaque sizes were significantly reduced compared to those of parental viruses. The reduced cell-to-cell spread was due to ICP27 since plaque sizes were restored in rescued viruses, as well as when vΔ54 was propagated in cells expressing ICP27 in trans In chickens, vΔ54 replicated, induced disease, and was oncogenic but was unable to transmit from chicken to chicken. To our knowledge, this is the first report showing that the Herpesviridae conserved ICP27 protein is dispensable for replication and disease induction in its natural host.IMPORTANCE Marek's disease (MD) is a devastating oncogenic disease that affects the poultry industry and is caused by MD alphaherpesvirus (MDV). Current vaccines block induction of disease but do not block chicken-to-chicken transmission. There is a knowledge gap in our understanding of how MDV spreads from chicken to chicken. We studied the Herpesviridae conserved ICP27 regulatory protein in cell culture and during MDV infection in chickens. We determined that MDV ICP27 is important but not required for replication in both cell culture and chickens. In addition, MDV ICP27 was not required for disease induction or oncogenicity but was required for chicken-to-chicken transmission. This study is important because it addresses the role of ICP27 during infection in the natural host and provides important information for the development of therapies to protect chickens against MD.

Effects of gE/gI deletions on the miRNA expression of PRV-infected PK-15 cells.

Pseudorabies virus (PRV) belongs to the Alphaherpesvirinae subfamily of Herpesviridae. PRV-induced pseudorabies is a highly contagious disease that has caused huge economic losses to the global swine industry. The PRV gE/gI gene deletion vaccine strain (Fa ΔgE/gI strain) constructed from the PRV Fa wild-type strain was shown to have a protective effect against infection. However, the interaction between PRV gE/gI genes and host miRNA needs further exploration, and little is known about the regulatory mechanisms of non-coding RNAs during PRV infection. miRNAs play a key regulatory role in viral infection and immune responses, so we analyzed the differential expression of miRNAs induced by the PRV Fa ΔgE/gI strain and Fa wild-type strain in the PK15 cell line. High-throughput sequencing reads were aligned to known Sus scrofa pre-miRNAs in the miRBase database. Target genes of differentially expressed miRNAs were predicted using the miRGen 3.0 database, then filtered miRNA target genes were subjected to Gene Ontology (GO) analysis and Search Tool for the Retrieval of Interacting Genes/ Proteins (STRING) analysis. Stem-loop quantitative real-time PCR was performed to confirm the accuracy of high-throughput sequencing data. In total, 387, 472, and 490 annotated and novel mature miRNAs were identified from PRV Fa ΔgE/gI strain-infected, Fa wild-type strain-infected, and non-infected PK-15 cells, respectively. Five PRV-encoded miRNAs were also identified. GO analysis showed that target genes of differentially expressed miRNAs in PRV Fa ΔgE/gI strain-infected and Fa wild-type strain-infected PK-15 cells were mainly involved in biological regulation and metabolic processes. STRING analysis showed that immune-related target genes of differentially expressed miRNAs in the Toll-like receptor signaling pathway, B cell receptor signaling pathway, T cell receptor signaling pathway, nuclear factor-κB signaling pathway, and transforming growth factor-ß signaling pathway were interrelated. This is the first report of the small RNA transcriptome in PRV mutant wild-type strain-infected and Fa ΔgE/gI strain-infected porcine cell lines. Our findings will contribute to the prevention and treatment of PRV mutant strains.

Herpesvirus Encephalitis in a Little Blue Penguin (Eudyptula minor).

An 11-day-old little blue penguin (Eudyptula minor) died unexpectedly. Prior to hatching, the egg experienced trauma and resultant defects were repaired. The chick hatched without complication and was clinically normal prior to death. Necropsy revealed congested lungs. Histologic examination showed moderate nonsuppurative encephalitis with focally extensive neuronal necrosis and intranuclear inclusions in neurons within necrotic foci. Herpesvirus DNA was detected in brain tissue with a generic herpesvirus polymerase chain reaction. Sanger sequencing demonstrated 100% and 98% sequence homology to sphenicid alphaherpesvirus 1 and penguin herpesvirus 2, respectively. In situ hybridization demonstrated large amounts of herpesvirus nucleic acid in intranuclear inclusions and neuronal nuclei. Combined histology, polymerase chain reaction, Sanger sequencing, and in situ hybridization results were most consistent with herpesviral encephalitis, most likely caused by sphenicid alphaherpesvirus 1. To our knowledge, this is the first report of a herpesvirus infection causing encephalitis in a penguin and the first report of herpesvirus in this species.

Feline Herpesvirus 1 US3 Blocks the Type I Interferon Signal Pathway by Targeting Interferon Regulatory Factor 3 Dimerization in a Kinase-Independent Manner.

As a prevalent agent in cats, feline herpesvirus 1 (FHV-1) infection contributes to feline respiratory disease and acute and chronic conjunctivitis. FHV-1 can successfully evade the host innate immune response and persist for the lifetime of the cat. Several mechanisms of immune evasion by human herpesviruses have been elucidated, but the mechanism of immune evasion by FHV-1 remains unknown. In this study, we screened for FHV-1 open reading frames (ORFs) responsible for inhibiting the type I interferon (IFN) pathway with an IFN-ß promoter reporter and analysis of IFN-ß mRNA levels in HEK 293T cells and the Crandell-Reese feline kidney (CRFK) cell line, and we identified the Ser/Thr kinase US3 as the most powerful inhibitor. Furthermore, we found that the anti-IFN activity of US3 depended on its N terminus (amino acids 1 to 75) and was independent of its kinase activity. Mechanistically, the ectopic expression of US3 selectively inhibited IFN regulatory factor 3 (IRF3) promoter activation. Furthermore, US3 bound to the IRF association domain (IAD) of IRF3 and prevented IRF3 dimerization. Finally, US3-deleted recombinant FHV-1 and US3-repaired recombinant FHV-1 (rFHV-dUS3 and rFHV-rUS3, respectively) were constructed. Compared with wild-type FHV-1 and rFHV-rUS3, infection with rFHV-dUS3 induced large amounts of IFN-ß in vitro and in vivo More importantly, US3 deletion significantly attenuated virulence, reduced virus shedding, and blocked the invasion of trigeminal ganglia. These results indicate that FHV-1 US3 efficiently inhibits IFN induction by using a novel immune evasion mechanism and that FHV-1 US3 is a potential regulator of neurovirulence.IMPORTANCE Despite widespread vaccination, the prevalence of FHV-1 remains high, suggesting that it can successfully evade the host innate immune response and infect cats. In this study, we screened viral proteins for inhibiting the IFN pathway and identified the Ser/Thr kinase US3 as the most powerful inhibitor. In contrast to other members of the alphaherpesviruses, FHV-1 US3 blocked the host type I IFN pathway in a kinase-independent manner and via binding to the IRF3 IAD and preventing IRF3 dimerization. More importantly, the depletion of US3 attenuated the anti-IFN activity of FHV-1 and prevented efficient viral replication in vitro and in vivo Also, US3 deletion significantly attenuated virulence and blocked the invasion of trigeminal ganglia. We believe that these findings not only will help us to better understand the mechanism of how FHV-1 manipulates the host IFN response but also highlight the potential role of US3 in the establishment of latent infection in vivo.

Human herpesvirus portal proteins: Structure, function, and antiviral prospects.

Herpesviruses (Herpesvirales) and tailed bacteriophages (Caudovirales) package their dsDNA genomes through an evolutionarily conserved mechanism. Much is known about the biochemistry and structural biology of phage portal proteins and the DNA encapsidation (viral genome cleavage and packaging) process. Although not at the same level of detail, studies on HSV-1, CMV, VZV, and HHV-8 have revealed important information on the function and structure of herpesvirus portal proteins. During dsDNA phage and herpesviral genome replication, concatamers of viral dsDNA are cleaved into single length units by a virus-encoded terminase and packaged into preformed procapsids through a channel located at a single capsid vertex (portal). Oligomeric portals are formed by the interaction of identical portal protein monomers. Comparing portal protein primary aa sequences between phage and herpesviruses reveals little to no sequence similarity. In contrast, the secondary and tertiary structures of known portals are remarkable. In all cases, function is highly conserved in that portals are essential for DNA packaging and also play a role in releasing viral genomic DNA during infection. Preclinical studies have described small molecules that target the HSV-1 and VZV portals and prevent viral replication by inhibiting encapsidation. This review summarizes what is known concerning the structure and function of herpesvirus portal proteins primarily based on their conserved bacteriophage counterparts and the potential to develop novel portal-specific DNA encapsidation inhibitors.

Conserved G-Quadruplexes Regulate the Immediate Early Promoters of Human Alphaherpesviruses.

Human Alphaherpesviruses comprise three members, herpes simplex virus (HSV) 1 and 2 and varicella zoster virus (VZV). These viruses are characterized by a lytic cycle in epithelial cells and latency in the nervous system, with lifelong infections that may periodically reactivate and lead to serious complications, especially in immunocompromised patients. The mechanisms that regulate viral transcription have not been fully elucidated, but the master role of the immediate early (IE) genes has been established. G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in many organisms including viruses and that represent attractive antiviral targets. In this work, we investigate the presence, conservation, folding and activity of G-quadruplexes in the IE promoters of the Alphaherpesviruses. Our analysis shows that all IE promoters in the genome of HSV-1, HSV-2 and VZV contain fully conserved G-quadruplex forming sequences. These comprise sequences with long loops and bulges, and thus deviating from the classic G-quadruplex motifs. Moreover, their location is both on the leading and lagging strand and in some instances they contain exuberant G-tracts. Biophysical and biological analysis proved that all sequences actually fold into G-quadruplex under physiological conditions and can be further stabilized by the G-quadruplex ligand BRACO-19, with subsequent impairment of viral IE gene transcription in cells. These results help shed light on the control of viral transcription and indicate new viral targets to design drugs that impair the early steps of Alphaherpesviruses. In addition, they validate the significance of G-quadruplexes in the general regulation of viral cycles.