The potential role of herpes simplex virus type 1 and neuroinflammation in the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disease affecting similar to 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

The neuropathic itch caused by pseudorabies virus

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

Characterization of Deoxyribonucleic Acid from Actinomycetes

Deoxyribonucleic acid (DNA) analyses were used to assess on a molecular level, the relationships among representatives of the genera Streptomyces, Nocardia and Mycobacterium. The methods developed in this study have been used for routine analysis of DNA from a large number of actinomycetes and have given reliable and reproducible data. DNA isolated from various actinomycetes was characterized by buoyant density determinations in CsCl from which the mole fraction guanine plus cytosine (GC) content was calculated. All the streptomycete DNA preparations studied had buoyant densities in the range of 1.7287 to 1.7312 g cm-3 which corresponded to GC compositions of 70% to 73% GC respectively. The nocardial DNA preparations tested fell in two groups, one with a GC content in the range of 62 to 64% GC and another in a 68 to 70% GC group. The mycobacterial DNA tested had GC values overlapping those of the nocardial DNA specimens; moreover, mycobacterial DNA exhibited a bimodal clustering of GC values, 64 to 65% GC and 67 to 70% GC. All DNA preparations examined by equilibrium buoyant density centrifugation in CsCl contained a single component with no satellite bands. The method of Warnaar and Cohen for assay of DNA/DNA reassociation on membrane filters was modified for studying reassociation of DNA in high GC organisms, DNA isolated from selected actinomycetes was tested for homology with Streptomyces venezuelae S13 mycelial DNA by direct reassociation experiments . Unlabeled DNA from the various actinomycetes was immobilized on Schleicher and Scheull nitrocellulose B-6 membrane filters and then incubated for 15 to 20 hr at 70 C with 14c-labeled DNA. The measure of relatedness was the relative percentage of renaturation of a denatured test DHA with labeled, dentured homolocous DNA. Unrelated DNA having GC contents of 50 and 70% were included as controls. The streptomycetes studied were relatively homogeneous in that measurable interspecific duplexes were formed between the reference DNA and all streptomycete DNA examined. Significantly, the results also suggested that S. venezuelae S13 was related to the nocardial specimens examined but was not related to the mycobacterial cultures studied. The results agreed generally with prior agar-gel studies on DNA reassociation and with previous classifications. Nucleotide sequence divergence in DNA extracted from streptomycetes and nocardiae was determined by measuring the extent of renaturation at 60 C and 70 C. The use of thermal elution of labeled, renatured duplexes from filters substantiated the existence of a class of nucleotide sesquences which can reassociate at 60 C but cannot reassociate at the more exacting 70 C incubation temperature. The use of exacting incubation conditions (70 C) permitted the formation only of t hose DNA duplexes that exhibited a high degree of thermal stability and hence, closely related to the reference DNA. The non-exacting 60 C incubation allowed those sequences to associate which were distantly related. The ratio of binding at 70 C to the binding at 60 C was designated the Divergence Index (DI). The DI was useful for gauging the presence or absence of closely related genetic material and for determining divergence patterns. The conclusions obtained from this method were corroborated by the much more time consuming thermal elution method. The divergence studies suggested that the streptomycetes contain a wide spectrum of related sequences compared to the reference DNA. Interestingly, the nocardiae examined seemed to have a small but significant amount of conserved nucleotide sequence compared to the S. venezuelae Sl3 reference. During these studies on actinomycete DNA it was realized that DNA from S. venezuelae S13 spores had novel properties. As spores aged the buoyant density in CsCl decreased from 1.727 to 1.707 g cm-3, the midpoint of thermal denaturation (Tm) in 0.1 x SSC increased from 85 to 88.5 C, and the apparent reassociation with mycelial DNA decreased from 100 to 30% . Spore DNA in 5 M NaCl04 had the same Tm as mycelial DNA. Spore DNA (1.707 g cm-3 ) after heat denaturation showed a single band in CsCl (1.722 g cm-3). Spore DNA was resistant to pancreatic deoxyribonuclease I, but became progressively sensitive after treatment with 0.5 M sodium acetate. Chemical nucleotide analysis of spore and mycelial DNA showed no detectable difference in GC content. The aberrant nature of spore DNA was not affected by pronase or ribonuclease. Washing the spores with ethanol and acetone prior to DNA extraction restored the isolated DNA to normal buoyant density and Tm values. When alcohol and chloroform extracts of spores were dried and mixed with authentic DNA preparations, no change in Tm or buoyant density was found. Spore DNA was yellow in color at pH values below 12 and pink in color at values above 12. Spore DNA heated in high salt showed three characteristic peaks in Sephadex G-100 column chromatography, but only one peak was found with comparably treated mycelial DNA . Attempts to characterize these peaks have been inconclusive to date. Chemical analyses of spore DNA showed 15 to 20% Folin positive material, 40 to 50% more phosphorus than mycelial DNA and no detectable sugars. It appeared that whatever was bound to the spore DNA could be partitioned to added DNA. Experiments of this type were successful only if the test DNA was added to freshly disrupted, dehydrated spores with crushed dry ice. Rehydration of spores showed loss of this binding activity. No chromatographically identifiable compounds or characteristic colors were extracted from spore DNA by a number of solvents and conditions including: acetone, ethanol, ethanol-ether, ethyl acetate, butanol, 8 M urea, 5% cold trichloroacetic acid and 1 N KOH at 100 C for 30 min. A crude pigmented fraction was isolated from spores which had similar chemical characteristics as aberrant spore DNA. This pigment could not be demonstrated in mycelia; moreover, pigment production seemed to be directly correlated with the age of the spores. The data suggested that this pigment is probably bound to spore DNA and is responsible for the aberrant characteristics of S. venezuelae S13 spore DNA

Probing the connectivity of neural circuits at single-neuron resolution using high-throughput DNA sequencing

There is growing excitement in determining the complete connectivity diagram of the brain—the "connectome". So far, the complete connectome has been established for only one organism, C. elegans, with 302 neurons connected by about 7000 synapses—and even this was a heroic task, requiring over 50 person-years of labor. Like all current approaches, this reconstruction was based on microscopy. Unfortunately, microscopy is poorly suited to the study of neural connectivity because brains are macroscopic structures, whereas synapses are microscopic. Nevertheless, there are several large-scale projects underway to scale up high-throughput microscopic approaches to the connectome.
Here we present a completely novel method for determining the brain's wiring diagram based on high-throughput DNA sequencing technology, which has not previously been applied in the context of neural connectivity. The appeal of using sequencing is that it is getting faster and cheaper exponentially: it will soon be routine to sequence an entire human genome (~3B nucleotides) within one day for $1000.
Our approach has three main components. First, we express a unique sequence of nucleotides—a DNA "barcode"—in individual neurons. A barcode consisting of a random string of even 30 nucleotides can uniquely label 10^{18} neurons, far more than the number of neurons in a mouse brain (fewer than 100 million). Second, we use a specially engineered transsynaptic virus to transport “host” barcodes from one neuron to synaptically coupled partners; after transsynaptic spread, each neuron contains copies of "invader" barcodes from other synaptically coupled neurons, as well its own "host" barcode. Third, we join pairs of host and invader barcodes into single pieces of DNA suitable for high-throughput sequencing. 
Modern sequencing technology could in principle yield the connectivity diagram of the entire mouse brain. Similar approaches can be applied to Drosophila and C. elegans. 
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Alphaherpesvirus infection of mice primes PNS neurons to an inflammatory state regulated by TLR2 and type I IFN signaling

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

Alpha-Herpesvirus Infection Induces the Formation of Nuclear Actin Filaments

Herpesviruses are large double-stranded DNA viruses that replicate in the nuclei of infected cells. Spatial control of viral replication and assembly in the host nucleus is achieved by the establishment of nuclear compartments that serve to concentrate viral and host factors. How these compartments are established and maintained remains poorly understood. Pseudorabies virus (PRV) is an alpha-herpesvirus often used to study herpesvirus invasion and spread in the nervous system. Here, we report that PRV and herpes simplex virus type 1 infection of neurons results in formation of actin filaments in the nucleus. Filamentous actin is not found in the nucleus of uninfected cells. Nuclear actin filaments appear physically associated with the viral capsids, as shown by serial block-face scanning electron micropscopy and confocal microscopy. Using a green fluorescent protein-tagged viral capsid protein (VP26), we show that nuclear actin filaments form prior to capsid assembly and are required for the efficient formation of viral capsid assembly sites. We find that actin polymerization dynamics (e.g., treadmilling) are not necessary for the formation of these sites. Green fluorescent protein-VP26 foci co-localize with the actin motor myosin V, suggesting that viral capsids travel along nuclear actin filaments using myosin-based directed transport. Viral transcription, but not viral DNA replication, is required for actin filament formation. The finding that infection, by either PRV or herpes simplex virus type 1, results in formation of nuclear actin filaments in neurons, and that PRV infection of an epithelial cell line results in a similar phenotype is evidence that F-actin plays a conserved role in herpesvirus assembly. Our results suggest a mechanism by which assembly domains are organized within infected cells and provide insight into how the viral infectious cycle and host actin cytoskeleton are integrated to promote the infection process

Long-term Cre-mediated retrograde tagging of neurons using a novel recombinant pseudorabies virus

Brain regions contain diverse populations of neurons that project to different long-range targets. The study of these subpopulations in circuit function and behavior requires a toolkit to characterize and manipulate their activity in vivo. We have developed a novel set of reagents based on Pseudorabies Virus (PRV) for efficient and long-term genetic tagging of neurons based on their projection targets. By deleting IE180, the master transcriptional regulator in the PRV genome, we have produced a mutant virus capable of infection and transgene expression in neurons but unable to replicate in or spread from those neurons. IE180-null mutants showed no cytotoxicity, and infected neurons exhibited normal physiological function more than 45 days after infection, indicating the utility of these engineered viruses for chronic experiments. To enable rapid and convenient construction of novel IE180-null recombinants, we engineered a bacterial artificial chromosome (BAC) shuttle-vector system for moving new constructs into the PRV IE180-null genome. Using this system we generated an IE180-null recombinant virus expressing the site-specific recombinase Cre. This Cre-expressing virus (PRV-hSyn-Cre) efficiently and robustly infects neurons in vivo and activates transgene expression from Cre-dependent vectors in local and retrograde projecting populations of neurons in the mouse. We also generated an assortment of recombinant viruses expressing fluorescent proteins (mCherry, EGFP, ECFP). These viruses exhibit long-term labeling of neurons in vitro but transient labeling in vivo. Together these novel IE180-null PRV reagents expand the toolkit for targeted gene expression in the brain, facilitating functional dissection of neuronal circuits in vivo

CRISPR/Cas9-constructed pseudorabies virus mutants reveal the importance of UL13 in alphaherpesvirus escape from genome silencing

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 Delta UL13), (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 DUL13 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

Redaction of sensitive data in the publication of dual use research of concern

Editorial. The publication of scientific information that derives from dual use research of concern (DURC) poses major problems for journals because it brings into conflict the benefits of free access to data and the need to prevent misuse of that information by others. Recently, a group of authors and a major scientific journal addressed the issue of publishing information on a newly discovered, highly lethal toxin that can be delivered to large populations and for which there are no available countermeasures. The journal addressed this conflict by permitting the redaction of information that is normally considered essential for publication. This action establishes a precedent for redaction of sensitive data that also provides an example of responsible scientific publishing. However, this precedent leaves many questions unanswered and suggests a need for a discussion by all stakeholders of scientific information so as to derive normative standards for the publication of DURC