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.

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.

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.

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.

Evaluation of the Safety and Immunogenicity of Duck-Plague Virus gE Mutants.

Duck plague (DP) is an acute infectious disease in the duck industry. The duck plague virus (DPV) is the pathogen, a subfamily of alphaherpesvirinae. gE is a type I membrane protein that contains three parts: an extracellular domain, a transmembrane domain, and a cytoplasmic domain. gE is the major virulence determinant of α-herpesvirus. However, the functions of the gE extracellular and cytoplasmic domains have not been reported in DPV. In this study, a gE extracellular domain deletion mutant and a gE cytoplasmic domain deletion mutant were constructed from DPV. Virus replication kinetics showed that the growth titers of both the gE ectodomain-deleted mutant virus and the gE cytoplasmic domain-deleted virus in DEFs were lower than that of the parental virus CHv-50. DPV CHv-gEΔET and DPV CHv-gEΔCT were continuously passed to the 20th passage in DEFs and the 10th in ducklings. The mutant virus DNA after passage was extracted for identification. The results showed that the gE ectodomain and gE cytoplasmic domain deletion mutant viruses have good genetic stability. The ducklings in each group (n=10) were inoculated with the same titers of DPV CHv-gEΔET, DPV CHv-gEΔCT, DPV CHv-ΔgE, and parental CHv-50, respectively. Clinical symptoms and serum antibody levels were detected after inoculation. The results showed that the virulence of DPV CHv-gEΔCT to ducklings was reduced compared with parental CHv-50, while the virulence of DPV CHv-gEΔET to ducklings was significantly reduced. 105 TCID50 DPV CHv-gEΔET or DPV CHv-ΔgE can induce ducklings to produce DPV-specific antibodies, protect the ducklings from virulent CHv challenge. Therefore, DPV CHv-gEΔET may serve as a promising vaccine candidate to prevent and control duck plague.

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.

Systematic Determination of Herpesvirus in Free-Ranging Cetaceans Stranded in the Western Mediterranean: Tissue Tropism and Associated Lesions.

The monitoring of herpesvirus infection provides useful information when assessing marine mammals' health. This paper shows the prevalence of herpesvirus infection (80.85%) in 47 cetaceans stranded on the coast of the Valencian Community, Spain. Of the 966 tissues evaluated, 121 tested positive when employing nested-PCR (12.53%). The largest proportion of herpesvirus-positive tissue samples was in the reproductive system, nervous system, and tegument. Herpesvirus was more prevalent in females, juveniles, and calves. More than half the DNA PCR positive tissues contained herpesvirus RNA, indicating the presence of actively replicating virus. This RNA was most frequently found in neonates. Fourteen unique sequences were identified. Most amplified sequences belonged to the Gammaherpesvirinae subfamily, but a greater variation was found in Alphaherpesvirinae sequences. This is the first report of systematic herpesvirus DNA and RNA determination in free-ranging cetaceans. Nine (19.14%) were infected with cetacean morbillivirus and all of them (100%) were coinfected with herpesvirus. Lesions similar to those caused by herpesvirus in other species were observed, mainly in the skin, upper digestive tract, genitalia, and central nervous system. Other lesions were also attributable to concomitant etiologies or were nonspecific. It is necessary to investigate the possible role of herpesvirus infection in those cases.

Does the Zinc Finger Antiviral Protein (ZAP) Shape the Evolution of Herpesvirus Genomes?

An evolutionary arms race occurs between viruses and hosts. Hosts have developed an array of antiviral mechanisms aimed at inhibiting replication and spread of viruses, reducing their fitness, and ultimately minimising pathogenic effects. In turn, viruses have evolved sophisticated counter-measures that mediate evasion of host defence mechanisms. A key aspect of host defences is the ability to differentiate between self and non-self. Previous studies have demonstrated significant suppression of CpG and UpA dinucleotide frequencies in the coding regions of RNA and small DNA viruses. Artificially increasing these dinucleotide frequencies results in a substantial attenuation of virus replication, suggesting dinucleotide bias could facilitate recognition of non-self RNA. The interferon-inducible gene, zinc finger antiviral protein (ZAP) is the host factor responsible for sensing CpG dinucleotides in viral RNA and restricting RNA viruses through direct binding and degradation of the target RNA. Herpesviruses are large DNA viruses that comprise three subfamilies, alpha, beta and gamma, which display divergent CpG dinucleotide patterns within their genomes. ZAP has recently been shown to act as a host restriction factor against human cytomegalovirus (HCMV), a beta-herpesvirus, which in turn evades ZAP detection by suppressing CpG levels in the major immediate-early transcript IE1, one of the first genes expressed by the virus. While suppression of CpG dinucleotides allows evasion of ZAP targeting, synonymous changes in nucleotide composition that cause genome biases, such as low GC content, can cause inefficient gene expression, especially in unspliced transcripts. To maintain compact genomes, the majority of herpesvirus transcripts are unspliced. Here we discuss how the conflicting pressures of ZAP evasion, the need to maintain compact genomes through the use of unspliced transcripts and maintaining efficient gene expression may have shaped the evolution of herpesvirus genomes, leading to characteristic CpG dinucleotide patterns.

Motor Skills: Recruitment of Kinesins, Myosins and Dynein during Assembly and Egress of Alphaherpesviruses.

The alphaherpesviruses are pathogens of the mammalian nervous system. Initial infection is commonly at mucosal epithelia, followed by spread to, and establishment of latency in, the peripheral nervous system. During productive infection, viral gene expression, replication of the dsDNA genome, capsid assembly and genome packaging take place in the infected cell nucleus, after which mature nucleocapsids emerge into the cytoplasm. Capsids must then travel to their site of envelopment at cytoplasmic organelles, and enveloped virions need to reach the cell surface for release and spread. Transport at each of these steps requires movement of alphaherpesvirus particles through a crowded and viscous cytoplasm, and for distances ranging from several microns in epithelial cells, to millimeters or even meters during egress from neurons. To solve this challenging problem alphaherpesviruses, and their assembly intermediates, exploit microtubule- and actin-dependent cellular motors. This review focuses upon the mechanisms used by alphaherpesviruses to recruit kinesin, myosin and dynein motors during assembly and egress.

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.

Differences in clinical severity of respiratory viral infections in hospitalized children.

It is uncertain whether clinical severity of an infection varies by pathogen or by multiple infections. Using hospital-based surveillance in children, we investigate the range of clinical severity for patients singly, multiply, and not infected with a group of commonly circulating viruses in Nha Trang, Vietnam. RT-PCR was performed to detect 13 respiratory viruses in nasopharyngeal samples from enrolled patients. We apply a novel clinical severity score and examine associations with the odds of being severe and differences in raw severity scores. We find no difference in severity between 0-, 1-, and 2-concurrent infections and little differences in severity between specific viruses. We find RSV and HMPV infections to be associated with 2- and 1.5-fold increase in odds of being severe, respectively, and that infection with ADV is consistently associated with lower risk of severity. Clinically, based on the results here, if RSV or HMPV virus is suspected, PCR testing for confirmatory diagnosis and for detection of multiple coinfecting viruses would be fruitful to assess whether a patient's disease course is going to be severe.

DPV UL41 gene encoding protein induces host shutoff activity and affects viral replication.

The virion host shutoff (VHS) protein, encoded by the UL41 gene of herpes simplex virus (HSV), specifically degrades mRNA and induces host shutoff. VHS and its homologs are highly conserved in the Alphaherpesvirinae subfamily. However, the role of the duck plague virus (DPV) UL41 gene is unclear. In this study, we found that the DPV UL41 gene-encoded protein (pUL41) degrades RNA polymerase (pol) II-transcribed translatable RNA and induces protein synthesis shutoff. DPV pUL41 was dispensable for viral replication, but the UL41-deleted mutant virus exhibited a significant viral growth defect and plaque size reduction in Duck embryo fibroblast (DEF) cells. Furthermore, DPV pUL41 regulated viral mRNA accumulation to affect viral DNA replication, release and cell-to-cell spread.

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.

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.

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.

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.

Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties.

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric nuclear egress complex (core NEC). These core NECs serve as hexameric lattice-structured platforms for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina as well as membrane-rearranging functions (multicomponent NEC). The regulation of nuclear egress has been profoundly analyzed for murine and human cytomegaloviruses (CMVs) on a mechanistic basis, followed by the description of core NEC crystal structures, first for HCMV, then HSV-1, PRV and EBV. Interestingly, the highly conserved structural domains of these proteins stand in contrast to a very limited sequence conservation of the key amino acids within core NEC-binding interfaces. Even more surprising, although a high functional consistency was found when regarding the basic role of NECs in nuclear egress, a clear specification was identified regarding the limited, subfamily-spanning binding properties of core NEC pairs and NEC multicomponent proteins. This review summarizes the evolving picture of the relationship between sequence coevolution, structural conservation and properties of NEC interaction, comparing HCMV to α-, ß- and γ-herpesviruses. Since NECs represent substantially important elements of herpesviral replication that are considered as drug-accessible targets, their putative translational use for antiviral strategies is discussed.

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.

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.