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The interface between humans and wildlife is changing and, with it, the potential for pathogen introduction into humans has increased. Avian influenza is a prominent example, with an ongoing outbreak showing the unprecedented expansion of both geographic and host ranges. Research in the field is essential to understand this and other zoonotic threats. Only by monitoring dynamic viral populations and defining their biology in situ can we gather the information needed to ensure effective pandemic preparation.

BACKGROUND: Neuroinvasive herpes simplex-1 (HSV-1) isolates including H129 and McIntyre cross at or near synapses labeling higher-order neurons directly connected to infected cells. H129 spreads predominately in the anterograde direction while McIntyre strains spread only in the retrograde direction. However, it is unknown if neurons are functional once infected with derivatives of H129 or McIntyre. NEW METHOD: We describe a previously unpublished HSV-1 recombinant derived from H129 (HSV-373) expressing mCherry fluorescent reporters and one new McIntyre recombinant (HSV-780) expressing the mCherry fluorophore and demonstrate how infections affect neuron viability. RESULTS AND COMPARISON WITH EXISTING METHODS: Each recombinant virus behaved similarly and spread to the target 4 days post-infection. We tested H129 recombinant infected neurons for neurodegeneration using Fluoro-jade C and found them to be necrotic as a result of viral infection. We performed dual inoculations with both HSV-772 and HSV-780 to identify cells comprising both the anterograde pathway and the retrograde pathway, respectively, of our circuit of study. We examined the presence of postsynaptic marker PSD-95, which plays a role in synaptic plasticity, in HSV-772 infected and in dual-infected rats (HSV-772 and HSV-780). PSD-95 reactivity decreased in HSV-772-infected neurons and dual-infected tissue had no PSD-95 reactivity. CONCLUSIONS: Infection by these new recombinant viruses traced the circuit of interest but functional studies of the cells comprising the pathway were not possible because viral-infected neurons died as a result of necrosis or were stripped of PSD-95 by the time the viral labels reached the target.

Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body.ImportanceNeurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There’s a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.

Waning vaccine-induced immunity coupled with the emergence of SARS-CoV-2 variants has led to increases in breakthrough infections, prompting consideration for vaccine booster doses. Boosters have been reported to be safe and increase SARS-CoV-2-specific neutralizing antibody levels, but how these doses impact the trajectory of the global pandemic and herd immunity is unknown. Information on immunology, epidemiology, and equitable vaccine distribution should be considered when deciding the timing and eligibility for COVID-19 vaccine boosters.

The central nervous system has long been thought to regulate insulin secretion, an essential process in the maintenance of blood glucose levels. However, the anatomical and functional connections between the brain and insulin-producing pancreatic β-cells remain undefined. Here, we describe a functional transneuronal circuit connecting the hypothalamus to β-cells in mice. This circuit originates from a subpopulation of oxytocin neurons in the paraventricular hypothalamic nucleus (PVN(OXT)) and via the sympathetic autonomic branch it reaches the islets of the endocrine pancreas to innervate β-cells. Stimulation of PVN(OXT) neurons rapidly suppresses insulin secretion and causes hyperglycemia. Conversely, silencing of these neurons elevates insulin levels by dysregulating neuronal signaling and secretory pathways in β-cells and induces hypoglycemia. PVN(OXT) neuronal activity is triggered by glucoprivation. Our findings thus reveal that a subset of PVN(OXT) neurons form functional multisynaptic circuits with β-cells in mice to regulate insulin secretion and their function is necessary for the β-cell response to hypoglycemia.

Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy to identify three small latency-associated promoters (LAPs) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404 bp), LAP2 (498 bp), and LAP1_2 (880 bp). They drive chronic transcription of the virus-encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAPs in the CNS for 6 months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1α promoter (1,264 bp). Our data suggest that the LAPs are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration., Graphical Abstract, Recombinant adeno-associated viruses (rAAVs) are used as gene therapy vectors. However, they have a limited payload capacity and lack small, long-lasting gene promoters for single-dose gene therapies. We identified small herpesvirus latency-associated promoters showing stable, pan-neuronal transgene transcription and translation in vivo for 6 months after AAV transduction.

Alzheimer's disease (AD) is a neurodegenerative disease affecting ~50 million people worldwide. To date, there is no cure and current therapies have not been effective in delaying disease progression. Therefore, there is an urgent need for better understanding of the pathogenesis of AD and to rethink possible therapies. Herpes simplex virus type 1 (HSV1) has recently received growing attention for its potential role in sporadic AD. The virus is a ubiquitous human pathogen that infects mucosal epithelia and invades the peripheral nervous system (PNS) of its host to establish a reactivable, latent infection. Upon reactivation, HSV1 spreads back to the epithelium and initiates a new infection, causing epithelial lesions. Occasionally, the virus spreads from the PNS to the brain after reactivation. In this review, we discuss current work on the pathogenesis of AD and summarize research results that support a potential role for HSV1 in the infectious hypothesis of AD. We also highlight recent findings on the neuroinflammatory response, which has been proposed to be the main driving force of AD, starting early in the course of the disease. Relevant rodent models to study neuroinflammation in AD and novel therapeutic approaches are also discussed. Throughout this review, we focus on several aspects of HSV1 pathogenesis, including its primary role as an invader of the PNS, that should be considered in the etiology of AD. We also point out some of the contradictory data and remaining knowledge gaps that require further research to finally fully understand the cause of AD in humans.

Varicella vaccine is a live attenuated varicella‐zoster virus. Varicella vaccine can enter latency and later reactivate as herpes zoster. Pseudorabies virus is another herpesvirus closely related to varicella‐zoster virus. The round trip model for pseudorabies virus explains pathogenesis of herpes zoster from vaccine virus.

Rationale: Leptin treats upper airway obstruction and alveolar hypoventilation in leptin-deficient ob/ob mice. However, obese humans and mice with diet-induced obesity (DIO) are resistant to leptin because of poor permeability of the blood–brain barrier. We propose that intranasal leptin will bypass leptin resistance and treat sleep-disordered breathing in obesity. Objectives: To assess if intranasal leptin can treat obesity hypoventilation and upper airway obstruction during sleep in mice with DIO. Methods: Male C57BL/6J mice were fed with a high-fat diet for 16 weeks. A single dose of leptin (0.4 mg/kg) or BSA (vehicle) were administered intranasally or intraperitoneally, followed by either sleep studies (n = 10) or energy expenditure measurements (n = 10). A subset of mice was treated with leptin daily for 14 days for metabolic outcomes (n = 20). In a separate experiment, retrograde viral tracers were used to examine connections between leptin receptors and respiratory motoneurons. Measurements and Main Results: Acute intranasal, but not intraperitoneal, leptin decreased the number of oxygen desaturation events in REM sleep, and increased ventilation in non-REM and REM sleep, independently of metabolic effects. Chronic intranasal leptin decreased food intake and body weight, whereas intraperitoneal leptin had no effect. Intranasal leptin induced signal transducer and activator of transcription 3 phosphorylation in hypothalamic and medullary centers, whereas intraperitoneal leptin had no effect. Leptin receptor–positive cells were synaptically connected to respiratory motoneurons. Conclusions: In mice with DIO, intranasal leptin bypassed leptin resistance and significantly attenuated sleep-disordered breathing independently of body weight.

Herpes simplex virus 1 (HSV-1) strain McKrae was isolated in 1965 and has been utilized by many laboratories. Three HSV-1 strain McKrae stocks have been sequenced previously, revealing discrepancies in key genes. We sequenced the genome of HSV-1 strain McKrae from James Hill, to better understand the genetic differences between isolates., Herpes simplex virus 1 (HSV-1) strain McKrae was isolated in 1965 and has been utilized by many laboratories. Three HSV-1 strain McKrae stocks have been sequenced previously, revealing discrepancies in key genes. We sequenced the genome of HSV-1 strain McKrae from the laboratory of James M. Hill to better understand the genetic differences between isolates.

The peer-reviewed scientific literature is the bedrock of science. However, scientific publishing is undergoing dramatic changes, which include the expansion of open access, an increased number of for-profit publication houses, and ready availability of preprint manuscripts that have not been peer reviewed. In this opinion article, we discuss the inequities and concerns that these changes have wrought.

Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein., Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally delivered genomes is facilitated by pseudorabies virus (PRV) tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP), and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N terminus (PRV mTurq-VP16) or the C terminus (PRV VP16-mTurq). Live-cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. However, immunofluorescence staining of particles in mid-axons indicated that UL13 might be cotransported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as adeno-associated virus (AAV)-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins. IMPORTANCE Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude the indirect role of UL13 in PRV escape from latency in primary neurons, along with its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.

At times when my students and colleagues ask for career advice, I often pause and think about the vagaries of my own career path.…

Wild elephant populations are declining rapidly due to rampant killing for ivory and body parts, range fragmentation, and human-elephant conflict. Wild and captive elephants are further impacted by viruses, including highly pathogenic elephant endotheliotropic herpesviruses. Moreover, while the rich genetic diversity of the ancient elephant lineage is disappearing, elephants, with their low incidence of cancer, have emerged as a surprising resource in human cancer research for understanding the intrinsic cellular response to DNA damage. However, studies on cellular resistance to transformation and herpesvirus reproduction have been severely limited, in part due to the lack of established elephant cell lines to enable in vitro experiments. This report describes creation of a recombinant plasmid, pAelPyV-1-Tag, derived from a wild isolate of African Elephant Polyomavirus (AelPyV-1), that can be used to create immortalized lines of elephant cells. This isolate was extracted from a trunk nodule biopsy isolated from a wild African elephant, Loxodonta africana, in Botswana. The AelPyV-1 genome contains open-reading frames encoding the canonical large (LTag) and small (STag) tumor antigens. We cloned the entire early region spanning the LTag and overlapping STag genes from this isolate into a high-copy vector to construct a recombinant plasmid, pAelPyV-1-Tag, which effectively transformed primary elephant endothelial cells. We expect that the potential of this reagent to transform elephant primary cells will, at a minimum, facilitate study of elephant-specific herpesviruses.

Alpha-herpesviruses establish a life-long infection in the nervous system of the affected host; while this infection is restricted to peripheral neurons in a healthy host, the reactivated virus can spread within the neuronal circuitry, such as to the brain, in compromised individuals and lead to adverse health outcomes. Pseudorabies virus (PRV), an alpha-herpesvirus, requires the viral protein Us9 to sort virus particles into axons and facilitate neuronal spread. Us9 sorts virus particles by mediating the interaction of virus particles with neuronal transport machinery. Here, we report that Us9-mediated regulation of axonal sorting also depends on the state of neuronal maturation. Specifically, the development of dendrites and axons is accompanied with proteomic changes that influence neuronal processes. Immature superior cervical ganglionic neurons (SCGs) have rudimentary neurites that lack markers of mature axons. Immature SCGs can be infected by PRV, but they show markedly reduced Us9-dependent regulation of sorting, and increased Us9-independent transport of particles into neurites. Mature SCGs have relatively higher abundances of proteins characteristic of vesicle-transport machinery. We also identify Us9-associated neuronal proteins that can contribute to axonal sorting and subsequent anterograde spread of virus particles in axons. We show that SMPD4/nsMase3, a sphingomyelinase abundant in lipid-rafts, associates with Us9 and is a negative regulator of PRV sorting into axons and neuronal spread, a potential antiviral function., Author summary Viral pathogenesis often is age-dependent, with more severe outcomes for infected fetuses and neonates compared to adults. As neurons age and mature, dendrites and axons polarize with distinct functions that affect neurotropic virus replication and neuronal spread of infection. This study investigates how neuronal maturation of peripheral nervous system neurons, the site of alpha-herpesvirus life-long latency and reactivation, affects replication and neuronal spread of pseudorabies virus. Characterization of infected immature and mature primary cultures of superior cervical ganglionic neurons revealed significant differences in protein composition and cellular processes that affected the activity of Us9, a viral protein required for sorting virus particles into axons. We identified neuronal and viral proteins that interact with Us9 in immature and mature neurons. Among these, we demonstrate that SMPD4/nsMase3, a sphingomyelinase critical for membrane organization and neuronal function, regulates PRV neuronal spread by preventing capsid association with Us9-containing membranes, presenting a possible antiviral function.

In vertebrates, the nervous system (NS) is composed of a peripheral collection of neurons (the peripheral nervous system, PNS), a central set found in the brain and spinal cord (the central nervous system, CNS). The NS is protected by rather complicated multi-layer barriers that allow access to nutrients and facilitate contact with the peripheral tissues, but block entry of pathogens and toxins. Virus infections usually begin in peripheral tissues and if these barriers are weakened, they can spread into the PNS and more rarely into the CNS. Most viral infections of the NS are opportunistic or accidental pathogens that gain access via the bloodstream (e.g., HIV and various arboviruses). But a few have evolved to enter the NS efficiently by invading neurons directly and by exploiting neuronal cell biology (e.g., rhabdoviruses and alphaherpesviruses). Most NS infections are devastating and difficult to manage. Remarkably, the alphaherpesviruses establish life-long quiescent infections in the PNS, with rare but often serious CNS pathology. In this review, we will focus on how alphaherpesviruses gain access to and spread in the NS, with particular emphasis on bidirectional transport and spread within and between neurons and neural circuits, which is regulated by complex viral-host protein interactions. Finally, we will describe the wide use of alphaherpesviruses as tools to study nerve connectivity and function in animal models.

Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies., Author summary Alpha-herpesviruses represent a group of large, enveloped DNA viruses that are capable to establish a quiescent (also called latent) but reactivatable form of infection in the peripheral nervous system of their hosts. Following reactivation of latent genomes, virus progeny is formed in the soma of neuronal cells and depend on sorting into the axon for anterograde spread of infection to mucosal sites and potentially new host. We studied two alpha-herpesviruses (the veterinary pathogen pseudorabies virus (PRV) and human herpes simplex virus 1 (HSV-1)) and found viral membrane proteins Us7, Us8, and Us9 form a complex, which is able to recruit kinsin-3 motors. Motor recruitment facilitates axonal sorting and subsequent transport to distal egress sites. Complex formation occurs at the trans-Golgi network and mediates efficiency of axonal sorting and motility characteristics of egressing capsids. We also used an artificial kinesin-3 recruitment system, which allows controlled induction of axonal sorting and transport of virus mutants lacking Us7, Us8, and Us9. Overall, these data contribute to our understanding of anterograde alpha-herpesvirus spread and kinesin-mediated sorting of vesicular axonal cargoes.

Recombinant adeno-associated viral vectors (rAAV) are used as gene therapy vectors to treat central nervous system (CNS) diseases. Despite their safety and broad tropism, important issues need to be corrected such as the limited payload capacity and the lack of small gene promoters providing long-term, pan-neuronal transgene expression in the CNS. Commonly used gene promoters are relatively large and can be repressed a few months after CNS transduction, risking the long-term performance of single-dose gene therapy applications. We used a whole-CNS screening approach based on systemic delivery of AAV-PHP.eB, iDisco+ tissue-clearing and light-sheet microscopy, to identify three small latency-associated promoters (LAP) from the herpesvirus pseudorabies virus (PRV). These promoters are LAP1 (404bp), LAP2 (498bp) and LAP1_2 (880bp). They drive chronic transcription of the virus encoded latency-associated transcript (LAT) during productive and latent phases of PRV infection. We observed stable, pan-neuronal transgene transcription and translation from AAV-LAP in the CNS for six months post AAV transduction. In several CNS areas, the number of cells expressing the transgene was higher for LAP2 than the large conventional EF1α promoter (1264bp). Our data suggests that the LAP are suitable candidates for viral vector-based CNS gene therapies requiring chronic transgene expression after one-time viral-vector administration.

Alphaherpesviruses, including pseudorabies virus (PRV), are neuroinvasive pathogens that establish life-long latency in peripheral ganglia following the initial infection at mucosal surfaces. The establishment of latent infection and the subsequent reactivations during which newly-assembled virions are sorted into and transported anterogradely inside axons to the initial mucosal site of infection, rely on axonal bidirectional transport mediated by microtubule-based motors. Previous studies using cultured peripheral nervous system (PNS) neurons have demonstrated that KIF1A, a kinesin-3 motor, mediates the efficient axonal sorting and transport of newly-assembled PRV virions. In this study, we report that KIF1A, unlike other axonal kinesins, is an intrinsically unstable protein prone to proteasomal degradation. Interestingly, PRV infection of neuronal cells leads not only to a non-specific depletion of KIF1A mRNA, but also to an accelerated proteasomal degradation of KIF1A proteins, leading to a near depletion of KIF1A protein late in infection. Using a series of PRV mutants deficient in axonal sorting and anterograde spread, we identified the PRV US9/gE/gI protein complex as a viral factor facilitating the proteasomal degradation of KIF1A proteins. Moreover, by using compartmented neuronal cultures that fluidically and physically separate axons from cell bodies, we found that the proteasomal degradation of KIF1A occurs in axons during infection. We propose that PRV anterograde sorting complex, gE/gI/US9, recruits KIF1A to viral transport vesicles for axonal sorting and transport, and eventually accelerates the proteasomal degradation of KIF1A in axons.ImportancePseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens herpes simplex virus −1, −2 and varicella zoster virus. Alphaherpesviruses are neuroinvasive pathogens that establish life-long latent infections in the host peripheral nervous system (PNS). Following reactivation from latency, infection spreads from the PNS back via axons to the peripheral mucosal tissues, a process mediated by kinesin motors. Here, we unveil and characterize the underlying mechanisms for a PRV-induced, accelerated degradation of KIF1A, a kinesin-3 motor promoting the sorting and transport of PRV virions in axons. We show that PRV infection disrupts the synthesis of KIF1A, and simultaneously promotes the degradation of intrinsically unstable KIF1A proteins by proteasomes in axons. Our work implies that the timing of motor reduction after reactivation would be critical because progeny particles would have a limited time window for sorting into and transport in axons for further host-to-host spread.

Background Herpes simplex virus (HSV) infection of the neonatal brain causes severe meningoencephalitis and permanent neurologic deficits. However, infants infected with HSV at the time of birth follow different clinical courses. Some infants develop only external infection of the skin, eyes, or mouth (SEM disease), while others develop invasive infection of the central nervous system resulting in encephalitis (CNS disease). The factors that explain this clinical divergence are not well understood. While adults can be predisposed to HSV CNS infection by an innate immune defect, no such host susceptibility has been identified in neonates. Therefore, we have taken a novel approach to determine whether variations in the HSV genome contribute to infection of the neonatal brain. We recently defined the viral genetic diversity among HSV-2 isolates cultured from neonates with a range of clinical presentations. Isolates collected from neonates with CNS disease contained several unique amino acid variations in HSV proteins known to contribute to cell-to-cell spread and neurovirulence in mouse models. Methods To understand the relevance of these findings to neonatal CNS disease, we evaluated CNS disease- and SEM disease-associated neonatal HSV-2 isolates in neurologically-relevant in vitro and in vivo models. Results We found that HSV-2 isolates from neonates with CNS disease, as compared to those collected from neonates with SEM disease, displayed enhanced spread in human neuronally-differentiated SH-SY5Y or LUHMES cells and enhanced retrograde transport in rat neurons cultured in modified Campenot chambers. CNS disease-associated isolates also resulted in increased hind limb paralysis and zosteriform disease in a mouse flank scratch infection model, and increased death in a mouse direct intracerebral injection model of encephalitis. Notably, CNS disease and SEM disease-associated isolates resulted in equivalent outcomes following mouse intraperitoneal injection, suggesting similar systemic virulence. Conclusion These data suggest that virus-mediated differences in neuronal spread and transport may contribute to neurovirulence in neonatal HSV disease. Disclosures All Authors: No reported disclosures

Spinal cord and peripheral nerve injuries require the regeneration of nerve fibers across the lesion site for successful recovery. Providing guidance cues and soluble factors to promote neurite outgrowth and cell survival can enhance repair. The extracellular matrix (ECM) plays a key role in tissue repair by controlling cell adhesion, motility, and growth. In this study, we explored the ability of a mesenchymal ECM to support neurite outgrowth from neurons in the superior cervical ganglia (SCG). Length and morphology of neurites extended on a decellularized fibroblast ECM were compared to those on substrates coated with laminin, a major ECM protein in neural tissue, or fibronectin, the main component of a mesenchymal ECM. Average radial neurite extension was equivalent on laminin and on the decellularized ECM, but contrasted with the shorter, curved neurites observed on the fibronectin substrate. Differences between neurites on fibronectin and on other substrates were confirmed by fast Fourier transform analyses. To control the direction of neurite outgrowth, we developed an ECM with linearly aligned fibril organization by orienting the fibroblasts that deposit the matrix on a polymeric surface micropatterned with a striped chemical interface. Neurites projected from SCGs appeared to reorient in the direction of the pattern. These results highlight the ability of a mesenchymal ECM to enhance neurite extension and to control the directional outgrowth of neurites. This micropatterned decellularized ECM architecture has potential as a regenerative microenvironment for nerve repair.

Alphaherpesviruses such as herpes simplex virus and pseudorabies virus (PRV) are neuroinvasive double-stranded DNA (dsDNA) viruses that establish lifelong latency in peripheral nervous system (PNS) neurons of their native hosts. Following reactivation, infection can spread back to the initial mucosal site of infection or, in rare cases, to the central nervous system, with usually serious outcomes. During entry and egress, viral capsids depend on microtubule-based molecular motors for efficient and fast transport. In axons of PNS neurons, cytoplasmic dynein provides force for retrograde movements toward the soma, and kinesins move cargo in the opposite, anterograde direction. The dynamic properties of virus particles in cells can be imaged by fluorescent protein fusions to the small capsid protein VP26, which are incorporated into capsids. However, single-color fluorescent protein tags fail to distinguish the virus inoculum from progeny. Therefore, we established a dual-color system by growing a recombinant PRV expressing a red fluorescent VP26 fusion (PRV180) on a stable cell line expressing a green VP26 fusion (PK15-mNG-VP26). The resulting dual-color virus preparation (PRV180G) contains capsids tagged with both red and green fluorescent proteins, and 97% of particles contain detectable levels of mNeonGreen (mNG)-tagged VP26. After replication in neuronal cells, all PRV180G progeny exclusively contain monomeric red fluorescent protein (mRFP)-VP26-tagged capsids. We used PRV180G for an analysis of axonal capsid transport dynamics in PNS neurons. Fast dual-color total internal reflection fluorescence (TIRF) microscopy, single-particle tracking, and motility analyses reveal robust, bidirectional capsid motility mediated by cytoplasmic dynein and kinesin during entry, whereas egressing progeny particles are transported exclusively by kinesins. IMPORTANCE Alphaherpesviruses are neuroinvasive viruses that infect the peripheral nervous system (PNS) of infected hosts as an integral part of their life cycle. Establishment of a quiescent or latent infection in PNS neurons is a hallmark of most alphaherpesviruses. Spread of infection to the central nervous system is surprisingly rare in natural hosts but can be fatal. Pseudorabies virus (PRV) is a broad-host-range swine alphaherpesvirus that enters neuronal cells and utilizes intracellular transport processes to establish infection and to spread between cells. By using a virus preparation with fluorescent viral capsids that change color depending on the stage of the infectious cycle, we find that during entry, axons of PNS neurons support robust, bidirectional capsid motility, similar to cellular cargo, toward the cell body. In contrast, progeny particles appear to be transported unidirectionally by kinesin motors toward distal egress sites.

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system causing severe deadly or debilitating disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous firing, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. To address this hypothesis, we used a compartmentalized primary neuron culture system to measure egress and spread of pseudorabies virus (PRV), pharmacological and optogenetics approaches to modulate neuronal firing activity, and a live-cell fluorescence microscopy assay to directly visualize the exocytosis of individual virus particles from infected neurons. Using tetrodotoxin to silence neuronal activity, we observed no inhibition of virus spread, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread. Using a live-cell fluorescence microscopy method to directly measure virus egress from infected neurons, we observed no association between virus particle exocytosis and intracellular Ca2+ signaling. Finally, we observed virus particle exocytosis occurs in association with constitutive secretory Rab GTPases, Rab6a and Rab8a, not Rab proteins that are associated with the Ca2+-regulated secretory pathway in neurons, Rab3a and Rab11a. Therefore, we conclude that alpha herpesvirus egress and spread is independent of neuronal activity and Ca2+ signaling because virus particle exocytosis uses constitutive secretory mechanisms in neurons.Author SummaryAlpha herpesviruses, including important human pathogens Herpes Simplex Virus 1 and 2, and Varicella-Zoster Virus, are among the very few viruses that naturally infect the nervous system. These viruses cause recurrent herpetic and zosteriform lesions, peripheral neuropathies, and deadly or debilitating central nervous system diseases. Many of the molecular and cellular mechanisms of viral egress and spread remain unknown, particularly in the context of specialized neuronal cell biology. Our results indicate that elevated firing activity of infected neurons is not functionally or mechanistically linked to virus egress and spread; therefore, therapies targeting peripheral neuropathic symptoms, elevated neuronal activity, and synaptic vesicle secretory mechanisms are unlikely to affect virus spread in the nervous system.

Pseudorabies virus (PRV) is an alphaherpesvirus that infects the peripheral nervous system (PNS). The natural host of PRV is the swine, but it can infect most mammals, including cattle, rodents, and dogs. In these nonnatural hosts, PRV always causes a severe acute and lethal neuropathy called the “mad itch,” which is uncommon in swine. Thus far, the pathophysiological and immunological processes leading to the development of the neuropathic itch and the death of the animal are unclear. Using a footpad inoculation model, we established that mice inoculated with PRV-Becker (virulent strain) develop a severe pruritus in the foot and become moribund at 82 h postinoculation (hpi). We found necrosis and inflammation with a massive neutrophil infiltration only in the footpad and dorsal root ganglia (DRGs) by hematoxylin and eosin staining. PRV load was detected in the foot, PNS, and central nervous system tissues by quantitative reverse transcription-PCR. Infected mice had elevated plasma levels of proinflammatory cytokines (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) and chemokines (Gro-1 and monocyte chemoattractant protein 1). Significant IL-6 and G-CSF levels were detected in several tissues at 82 hpi. High plasma levels of C-reactive protein confirmed the acute inflammatory response to PRV-Becker infection. Moreover, mice inoculated with PRV-Bartha (attenuated, live vaccine strain) did not develop pruritus at 82 hpi. PRV-Bartha also replicated in the PNS, and the infection spread further in the brain than PRV-Becker. PRV-Bartha infection did not induce the specific and lethal systemic inflammatory response seen with PRV-Becker. Overall, we demonstrated the importance of inflammation in the clinical outcome of PRV infection in mice and provide new insights into the process of PRV-induced neuroinflammation. IMPORTANCE Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens such as herpes simplex virus 1 and varicella-zoster virus (VZV). The natural host of PRV is the swine, but it can infect most mammals. In susceptible animals other than pigs, PRV infection always causes a characteristic lethal pruritus known as the “mad itch.” The role of the immune response in the clinical outcome of PRV infection is still poorly understood. Here, we show that a systemic host inflammatory response is responsible for the severe pruritus and acute death of mice infected with virulent PRV-Becker but not mice infected with attenuated strain PRV-Bartha. In addition, we identified IL-6 and G-CSF as two main cytokines that play crucial roles in the regulation of this process. Our findings give new insights into neuroinflammatory diseases and strengthen further the similarities between VZV and PRV infections at the level of innate immunity.

The brain influences glucose homeostasis, partly by supplemental control over insulin and glucagon secretion. Without this central regulation, diabetes and its complications can ensue. Yet, the neuronal network linking to pancreatic islets has never been fully mapped. Here, we refine this map using pseudorabies virus (PRV) retrograde tracing, indicating that the pancreatic islets are innervated by efferent circuits that emanate from the hypothalamus. We found that the hypothalamic arcuate nucleus (ARC), ventromedial nucleus (VMN), and lateral hypothalamic area (LHA) significantly overlap PRV and the physiological glucose-sensing enzyme glucokinase. Then, experimentally lowering glucose sensing, specifically in the ARC, resulted in glucose intolerance due to deficient insulin secretion and no significant effect in the VMN, but in the LHA it resulted in a lowering of the glucose threshold that improved glucose tolerance and/or improved insulin sensitivity, with an exaggerated counter-regulatory response for glucagon secretion. No significant effect on insulin sensitivity or metabolic homeostasis was noted. Thus, these data reveal novel direct neuronal effects on pancreatic islets and also render a functional validation of the brain-to-islet neuronal map. They also demonstrate that distinct regions of the hypothalamus differentially control insulin and glucagon secretion, potentially in partnership to help maintain glucose homeostasis and guard against hypoglycemia.

Herpesviruses are large DNA viruses that utilize the host nucleus for genome replication as well as capsid assembly. After maturation, these 125 nm large capsid assemblies must cross the nucleoplasm to engage the nuclear envelope and bud into the cytoplasm. Here we summarize our recent findings how this motility is facilitated. We suggest that herpesvirus induced nuclear remodeling allows capsids to move by diffusion in the nucleus and not by motor-dependent transport.

In the nearly two decades since the popularization of green fluorescent protein (GFP), fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

Background Viral transneuronal tracing has become a well established technology used to define the synaptic architecture of polysynaptic neural networks. New Method In this report we define the neuroinvasive profile and reporter expression of a new recombinant of the Bartha strain of pseudorabies virus (PRV). The new recombinant, PRV-290, expresses the mTurquoise2 fluorophor and is designed to complement other isogenic recombinants of Bartha that express different reporters of infection. Results & Comparison with Existing Methods: PRV-290 was injected either alone or in combination with isogenic recombinants of PRV that express enhanced green fluorescent protein (EGFP; PRV-152) or monomeric red fluorescent protein (mRFP; PRV-614). Circuits previously defined using PRV-152 and PRV-614 were used for the analysis. The data demonstrate that PRV-290 is a retrograde transneuronal tracer with temporal kinetics similar to those of its isogenic recombinants. Stable expression of the diffusible mTurquoise2 reporter filled infected neurons, with the extent and intensity of labeling increasing with advancing post inoculation survival. In multiple injection experiments, PRV-290 established productive infections in neurons also replicating PRV-152 and/or PRV-614. This novel demonstration of three recombinants infecting individual neurons represents an important advance in the technology. Conclusion Collectively, these data demonstrate that PRV-290 is a valuable addition to the viral tracer toolbox for transneuronal tracing of neural circuitry.

Pseudorabies virus (PRV), an alphaherpesvirus closely related to Varicella-Zoster virus (VZV) and Herpes simplex type 1 (HSV1) infects mucosa epithelia and the peripheral nervous system (PNS) of its host. We previously demonstrated that PRV infection induces a specific and lethal inflammatory response, contributing to severe neuropathy in mice. So far, the mechanisms that initiate this neuroinflammation remain unknown. Using a mouse footpad inoculation model, we found that PRV infection rapidly and simultaneously induces high G-CSF and IL-6 levels in several mouse tissues, including the footpad, PNS and central nervous system (CNS) tissues. Interestingly, this global increase occurred before PRV had replicated in dorsal root ganglia (DRGs) neurons and also was independent of systemic inflammation. These high G-CSF and IL-6 levels were not caused by neutrophil infiltration in PRV infected tissues, as we did not detect any neutrophils. Efficient PRV replication and spread in the footpad was sufficient to activate DRGs to produce cytokines. Finally, by using knockout mice, we demonstrated that TLR2 and IFN type I play crucial roles in modulating the early neuroinflammatory response and clinical outcome of PRV infection in mice. Overall, these results give new insights into the initiation of virus-induced neuroinflammation during herpesvirus infections., Author summary Herpesviruses are major pathogens worldwide. Pseudorabies virus (PRV) is an alphaherpesvirus related to varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV1). The natural host is the pig, but PRV can infect most mammals. In these non-natural hosts, the virus causes a severe pruritus called the ‘mad itch’. Interestingly, PRV infects the peripheral nervous system (PNS) and induces a specific and lethal inflammatory response in mice, yet little is know about how this neuroinflammatory response is initiated. In this study, we demonstrated for the first time how PNS neurons tightly regulate the inflammatory response during PRV infection and contribute to severe clinical outcome in mice. Our work provides new insights into the process of alphaherpesvirus-induced neuropathies, leading to the development of innovative therapeutic strategies.

Neuroinvasive viruses, such as alpha herpesviruses (αHV) and rabies virus (RABV), initially infect peripheral tissues, followed by invasion of the innervating axon termini. Virus particles must undergo long distance retrograde axonal transport to reach the neuron cell bodies in the peripheral or central nervous system (PNS/CNS). How virus particles hijack the axonal transport machinery and how PNS axons respond to and regulate infection are questions of significant interest. To track individual virus particles, we constructed a recombinant RABV expressing a P-mCherry fusion protein, derived from the virulent CVS-N2c strain. We studied retrograde RABV transport in the presence or absence of interferons (IFN) or protein synthesis inhibitors, both of which were reported previously to restrict axonal transport of αHV particles. Using neurons from rodent superior cervical ganglia grown in tri-chambers, we showed that axonal exposure to type I or type II IFN did not alter retrograde axonal transport of RABV. However, exposure of axons to emetine, a translation elongation inhibitor, blocked axonal RABV transport by a mechanism that was not dependent on protein synthesis inhibition. The minority of RABV particles that still moved retrograde in axons in the presence of emetine, moved with slower velocities and traveled shorter distances. Emetine’s effect was specific to RABV, as transport of cellular vesicles was unchanged. These findings extend our understanding of how neuroinvasion is regulated in axons and point toward a role for emetine as an inhibitory modulator of RABV axonal transport., Author summary Rabies virus (RABV) and alpha herpesviruses (αHV) (e.g. herpes simplex virus) evolved to enter the nervous system efficiently each time they infect a host. In most mammals, RABV reaches the brain, causing a fatal encephalitis. Whereas, αHV remain in the peripheral nervous system in a quiescent but reactivatable state. Despite distinct clinical outcomes, both RABV and αHV must invade axons and repurpose the axon transport machinery to travel long distances toward the neuronal cell bodies where virus replication occurs. How virus particles hijack the transport machinery and how axons respond to and regulate infection are questions of significant interest. We investigated how axonal RABV transport is regulated by exposing axons to interferons or protein synthesis inhibitors, both of which restrict transport of αHV particles. Unlike αHV infection, exposure of isolated axons to interferons has no effect on RABV neuroinvasion. However, RABV transport is blocked by axonal exposure to the translation elongation inhibitor, emetine, via a mechanism that does not depend on protein synthesis inhibition. The effect of emetine is not due to a global inhibition of axon transport because emetine does not limit axonal transport of cellular vesicles. Therefore, emetine may be a novel inhibitory modulator of RABV axonal transport.