Publications

2023

When humans experience a new, devastating viral infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), significant challenges arise. How should individuals as well as societies respond to the situation? One of the primary questions concerns the origin of the SARS-CoV-2 virus that infected and was transmitted efficiently among humans, resulting in a pandemic. At first glance, the question appears straightforward to answer. However, the origin of SARS-CoV-2 has been the topic of substantial debate primarily because we do not have access to some relevant data. At least two major hypotheses have been suggested: a natural origin through zoonosis followed by sustained human-to-human spread or the introduction of a natural virus into humans from a laboratory source. Here, we summarize the scientific evidence that informs this debate to provide our fellow scientists and the public with the tools to join the discussion in a constructive and informed manner. Our goal is to dissect the evidence to make it more accessible to those interested in this important problem. The engagement of a broad representation of scientists is critical to ensure that the public and policy-makers can draw on relevant expertise in navigating this controversy.

When humans experience a new, devastating viral infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), significant challenges arise. How should individuals as well as societies respond to the situation? One of the primary questions concerns the origin of the SARS-CoV-2 virus that infected and was transmitted efficiently among humans, resulting in a pandemic. At first glance, the question appears straightforward to answer. However, the origin of SARS-CoV-2 has been the topic of substantial debate primarily because we do not have access to some relevant data. At least two major hypotheses have been suggested: a natural origin through zoonosis followed by sustained human-to-human spread or the introduction of a natural virus into humans from a laboratory source. Here, we summarize the scientific evidence that informs this debate to provide our fellow scientists and the public with the tools to join the discussion in a constructive and informed manner. Our goal is to dissect the evidence to make it more accessible to those interested in this important problem. The engagement of a broad representation of scientists is critical to ensure that the public and policy-makers can draw on relevant expertise in navigating this controversy.

When humans experience a new, devastating viral infection such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), significant challenges arise. How should individuals as well as societies respond to the situation? One of the primary questions concerns the origin of the SARS-CoV-2 virus that infected and was transmitted efficiently among humans, resulting in a pandemic. At first glance, the question appears straightforward to answer. However, the origin of SARS-CoV-2 has been the topic of substantial debate primarily because we do not have access to some relevant data. At least two major hypotheses have been suggested: a natural origin through zoonosis followed by sustained human-to-human spread or the introduction of a natural virus into humans from a laboratory source. Here, we summarize the scientific evidence that informs this debate to provide our fellow scientists and the public with the tools to join the discussion in a constructive and informed manner. Our goal is to dissect the evidence to make it more accessible to those interested in this important problem. The engagement of a broad representation of scientists is critical to ensure that the public and policy-makers can draw on relevant expertise in navigating this controversy.

Our ability to understand the function of the nervous system is dependent upon defining the connections of its constituent neurons. Development of methods to define connections within neural networks has always been a growth industry in the neurosciences. Transneuronal spread of neurotropic viruses currently represents the best means of defining synaptic connections within neural networks. The method exploits the ability of viruses to invade neurons, replicate, and spread through the intimate synaptic connections that enable communication among neurons. Since the method was first introduced in the 1970s, it has benefited from an increased understanding of the virus life cycle, the function of viral genomes, and the ability to manipulate the viral genome in support of directional spread of virus and the expression of transgenes. In this article, we review these advances in viral tracing technology and the ways in which they may be applied for functional dissection of neural networks. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Retrograde infection of CNS circuits by peripheral injection of virus Basic Protocol 2: Transneuronal analysis by intracerebral injection Alternate Protocol 1: Transneuronal analysis with multiple recombinant strains Alternate Protocol 2: Conditional replication and spread of PRV Alternate Protocol 3: Conditional reporters of PRV infection and spread Alternate Protocol 4: Reporters of neural activity in polysynaptic circuits Support Protocol 1: Growing and titering a PRV viral stock Support Protocol 2: Immunohistochemical processing and detection Support Protocol 3: Dual-immunofluorescence localization.

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 can we gather the information needed to ensure effective pandemic preparation.

2022

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) necessitates that the global scientific community monitor, assess, and respond to the evolving coronavirus disease (COVID-19) pandemic. But the current reactive approach to emerging variants is ill-suited to address the quickly evolving and ever-changing pandemic. To tackle this challenge, investments in pathogen surveillance, systematic variant characterization, and data infrastructure and sharing across public and private sectors will be critical for planning proactive responses to emerging variants. Additionally, an emphasis on incorporating real-time variant identification in point-of-care diagnostics can help inform patient treatment. Active approaches to understand and identify "immunity gaps" can inform design of future vaccines, therapeutics, and diagnostics that will be more resistant to novel variants. Approaches where the scientific community actively plans for and anticipates changes to infectious diseases will result in a more resilient system, capable of adapting to evolving pathogens quickly and effectively.

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 pathway. 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. Neurons 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.

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), and it reaches the islets of the endocrine pancreas via the sympathetic autonomic branch to innervate β cells. Stimulation of PVN 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 neuronal activity is triggered by glucoprivation. Our findings reveal that a subset of PVN 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.

2021

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.

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.

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.
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.

BACKGROUND: The mechanisms by which any upper respiratory virus, including SARS-CoV-2, impairs chemosensory function are not known. COVID-19 is frequently associated with olfactory dysfunction after viral infection, which provides a research opportunity to evaluate the natural course of this neurological finding. Clinical trials and prospective and histological studies of new-onset post-viral olfactory dysfunction have been limited by small sample sizes and a paucity of advanced neuroimaging data and neuropathological samples. Although data from neuropathological specimens are now available, neuroimaging of the olfactory system during the acute phase of infection is still rare due to infection control concerns and critical illness and represents a substantial gap in knowledge. RECENT DEVELOPMENTS: The active replication of SARS-CoV-2 within the brain parenchyma (ie, in neurons and glia) has not been proven. Nevertheless, post-viral olfactory dysfunction can be viewed as a focal neurological deficit in patients with COVID-19. Evidence is also sparse for a direct causal relation between SARS-CoV-2 infection and abnormal brain findings at autopsy, and for trans-synaptic spread of the virus from the olfactory epithelium to the olfactory bulb. Taken together, clinical, radiological, histological, ultrastructural, and molecular data implicate inflammation, with or without infection, in either the olfactory epithelium, the olfactory bulb, or both. This inflammation leads to persistent olfactory deficits in a subset of people who have recovered from COVID-19. Neuroimaging has revealed localised inflammation in intracranial olfactory structures. To date, histopathological, ultrastructural, and molecular evidence does not suggest that SARS-CoV-2 is an obligate neuropathogen. WHERE NEXT?: The prevalence of CNS and olfactory bulb pathosis in patients with COVID-19 is not known. We postulate that, in people who have recovered from COVID-19, a chronic, recrudescent, or permanent olfactory deficit could be prognostic for an increased likelihood of neurological sequelae or neurodegenerative disorders in the long term. An inflammatory stimulus from the nasal olfactory epithelium to the olfactory bulbs and connected brain regions might accelerate pathological processes and symptomatic progression of neurodegenerative disease. Persistent olfactory impairment with or without perceptual distortions (ie, parosmias or phantosmias) after SARS-CoV-2 infection could, therefore, serve as a marker to identify people with an increased long-term risk of neurological disease.

2020

Alphaherpesviruses, including pseudorabies virus (PRV), are neuroinvasive pathogens that establish lifelong latency in peripheral ganglia following the initial infection at mucosal surfaces. The establishment of latent infection and 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. Here 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 nonspecific 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 the 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. Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens herpes simplex viruses 1 and 2 and varicella-zoster virus. Alphaherpesviruses are neuroinvasive pathogens that establish lifelong 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.

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.

Pseudorabies virus (PRV) is an alphaherpesvirus related to varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV1). PRV is the causative agent of Aujeskzy's disease in swine. PRV infects mucosal epithelium and the peripheral nervous system (PNS) of its host where it can establish a quiescent, latent infection. While the natural host of PRV is the swine, a broad spectrum of mammals, including rodents, cats, dogs, and cattle can be infected. Since the nineteenth century, PRV infection is known to cause a severe acute neuropathy, the so called "mad itch" in non-natural hosts, but surprisingly not in swine. In the past, most scientific efforts have been directed to eradicating PRV from pig farms by the use of effective marker vaccines, but little attention has been given to the processes leading to the mad itch. The main objective of this review is to provide state-of-the-art information on the mechanisms governing PRV-induced neuropathic itch in non-natural hosts. We highlight similarities and key differences in the pathogenesis of PRV infections between non-natural hosts and pigs that might explain their distinctive clinical outcomes. Current knowledge on the neurobiology and possible explanations for the unstoppable itch experienced by PRV-infected animals is also reviewed. We summarize recent findings concerning PRV-induced neuroinflammatory responses in mice and address the relevance of this animal model to study other alphaherpesvirus-induced neuropathies, such as those observed for VZV infection.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

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.

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.

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.

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 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 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. Surprisingly, immunofluorescence staining of particles in mid-axons indicated that UL13 might be co-transported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing when 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 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. 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 on the indirect role of UL13 in PRV escape from latency in primary neurons and on 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.

2019

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.

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.

2018

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.

Fluorescent protein fusions to herpesvirus capsids have proven to be a valuable method to study virus particle transport in living cells. Fluorescent protein fusions to the amino terminus of small capsid protein VP26 are the most widely used method to visualize pseudorabies virus (PRV) and herpes simplex virus (HSV) particles in living cells. However, these fusion proteins do not incorporate to full occupancy and have modest effects on virus replication and pathogenesis. Recent cryoelectron microscopy studies have revealed that herpesvirus small capsid proteins bind to capsids via their amino terminus, whereas the carboxy terminus is unstructured and therefore may better tolerate fluorescent protein fusions. Here, we describe a new recombinant PRV expressing a carboxy-terminal VP26-mCherry fusion. Compared to previously characterized viruses expressing amino-terminal fusions, this virus expresses more VP26 fusion protein in infected cells and incorporates more VP26 fusion protein into virus particles, and individual virus particles exhibit brighter red fluorescence. We performed single-particle tracking of fluorescent virus particles in primary neurons to measure anterograde and retrograde axonal transport, demonstrating the usefulness of this novel VP26-mCherry fusion for the study of viral intracellular transport.IMPORTANCE Alphaherpesviruses are among the very few viruses that are adapted to invade the mammalian nervous system. Intracellular transport of virus particles in neurons is important, as this process underlies both mild peripheral nervous system infection and severe spread to the central nervous system. VP26, the small capsid protein of HSV and PRV, was one of the first herpesvirus proteins to be fused to a fluorescent protein. Since then, these capsid-tagged virus mutants have become a powerful tool to visualize and track individual virus particles. Improved capsid tags will facilitate fluorescence microscopy studies of virus particle intracellular transport, as a brighter particle will improve localization accuracy of individual particles and allow for shorter exposure times, reducing phototoxicity and improving the time resolution of particle tracking in live cells.

Alpha herpesviruses are common pathogens of mammals. They establish a productive infection in many cell types, but a life-long latent infection occurs in PNS neurons. A vast majority of the human population has latent HSV-1 infections. Currently, there is no cure to clear latent infections. Even though HSV-1 is among the best studied viral pathogens, regulation of latency and reactivation is not well understood due to several challenges including a lack of animal models that precisely recapitulate latency/reactivation episodes; a difficulty in modeling latency; and a limited understanding of neuronal biology. In this review, we discuss insights gained from latency models with a focus on the neuronal and viral factors that determine the mode of infection.

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 non-natural hosts, PRV always causes a severe acute and lethal neuropathy called the "mad itch", which is uncommon in swine. So far, the pathophysiological and immunological processes leading to the development of the neuropathic itch, and 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 hours post-inoculation (hpi). We found necrosis and inflammation with a massive neutrophil infiltration only in the footpad and DRGs by H&E staining. PRV load was detected in the foot, PNS and CNS tissues by quantitative-RT-PCR. Infected mice had elevated plasma levels of pro-inflammatory cytokines (IL6 and G-CSF) and chemokines (Gro-1 and MCP-1). Significant IL6 and G-CSF levels were detected in several tissues at 82hpi. 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 82hpi. PRV-Bartha also replicated in the PNS, 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.Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens such as herpes simplex virus type 1 (HSV-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 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.

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.

2017

Neuroinvasive herpesviruses have evolved to efficiently infect and establish latency in neurons. The nervous system has limited capability to regenerate, so immune responses therein are carefully regulated to be nondestructive, with dependence on atypical intrinsic and innate defenses. In this article we review studies of some of these noncanonical defense pathways and how herpesvirus gene products counter them, highlighting the contributions that primary neuronal in vitro models have made to our understanding of this field.

Alpha herpesvirus genomes encode the capacity to establish quiescent infections (i.e. latency) in the peripheral nervous system for the life of their hosts. Multiple times during latency, viral genomes can reactivate to start a productive infection, enabling spread of progeny virions to other hosts. Replication of alpha herpesviruses is well studied in cultured cells and many aspects of productive replication have been identified. However, many questions remain concerning how a productive or a quiescent infection is established. While infections in vivo often result in latency, infections of dissociated neuronal cultures in vitro result in a productive infection unless lytic viral replication is suppressed by DNA polymerase inhibitors or interferon. Using primary peripheral nervous system neurons cultured in modified Campenot tri-chambers, we previously reported that reactivateable, quiescent infections by pseudorabies virus (PRV) can be established in the absence of any inhibitor. Such infections were established in cell bodies only when physically isolated axons were infected at a very low multiplicity of infection (MOI). In this report, we developed a complementation assay in compartmented neuronal cultures to investigate host and viral factors in cell bodies that prevent establishment of quiescent infection and promote productive replication of axonally delivered genomes (i.e. escape from silencing). Stimulating protein kinase A (PKA) signaling pathways in isolated cell bodies, or superinfecting cell bodies with either UV-inactivated PRV or viral light particles (LP) promoted escape from genome silencing and prevented establishment of quiescent infection but with different molecular mechanisms. Activation of PKA in cell bodies triggers a slow escape from silencing in a cJun N-terminal kinase (JNK) dependent manner. However, escape from silencing is induced rapidly by infection with UVPRV or LP in a PKA- and JNK-independent manner. We suggest that viral tegument proteins delivered to cell bodies engage multiple signaling pathways that block silencing of viral genomes delivered by low MOI axonal infection.

The mesolimbic dopamine pathway receives inputs from numerous regions of the brain as part of a neural system that detects rewarding stimuli and coordinates a behavioral response. The capacity to simultaneously map and molecularly define the components of this complex multisynaptic circuit would thus advance our understanding of the determinants of motivated behavior. To accomplish this, we have constructed pseudorabies virus (PRV) strains in which viral propagation and fluorophore expression are activated only after exposure to Cre recombinase. Once activated in Cre-expressing neurons, the virus serially labels chains of presynaptic neurons. Dual injection of GFP and mCherry tracing viruses simultaneously illuminates nigrostriatal and mesolimbic circuitry and shows no overlap, demonstrating that PRV transmission is confined to synaptically connected neurons. To molecularly profile mesolimbic dopamine neurons and their presynaptic inputs, we injected Cre-conditional GFP virus into the NAc of (anti-GFP) nanobody-L10 transgenic mice and immunoprecipitated translating ribosomes from neurons infected after retrograde tracing. Analysis of purified RNA revealed an enrichment of transcripts expressed in neurons of the dorsal raphe nuclei and lateral hypothalamus that project to the mesolimbic dopamine circuit. These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuit-directed translating ribosome affinity purification can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits.SIGNIFICANCE STATEMENT The mesolimbic dopamine circuit integrates signals from key brain regions to detect and respond to rewarding stimuli. To further define this complex multisynaptic circuit, we constructed a panel of Cre recombinase-activated pseudorabies viruses (PRVs) that enabled retrograde tracing of neural inputs that terminate on Cre-expressing neurons. Using these viruses and Retro-TRAP (translating ribosome affinity purification), a previously reported molecular profiling method, we developed a novel technique that provides anatomic as well as molecular information about the neural components of polysynaptic circuits. We refer to this new method as PRV-Circuit-TRAP (PRV circuit-directed TRAP). Using it, we have identified major projections to the mesolimbic dopamine circuit from the lateral hypothalamus and dorsal raphe nucleus and defined a discrete subset of transcripts expressed in these projecting neurons, which will allow further characterization of this important pathway. Moreover, the method we report is general and can be applied to the study of other neural circuits.

2016

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.

Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS-STING-TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS-STING-TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome.

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.

Undergraduate Senior Theses