Virulent Pseudorabies Virus Infection Induces a Specific and Lethal Systemic Inflammatory Response in Mice.

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.

Us9-Independent Axonal Sorting and Transport of the Pseudorabies Virus Glycoprotein gM.

Axonal sorting and transport of fully assembled pseudorabies virus (PRV) virions is dependent on the viral protein Us9. Here we identify a Us9-independent mechanism for axonal localization of viral glycoprotein M (gM). We detected gM-mCherry assemblies transporting in the anterograde direction in axons. Furthermore, unlabeled gM, but not glycoprotein B, was detected by Western blotting in isolated axons during Us9-null PRV infection. These results suggest that gM differs from other viral proteins regarding axonal transport properties.

A wide extent of inter-strain diversity in virulent and vaccine strains of alphaherpesviruses.

Alphaherpesviruses are widespread in the human population, and include herpes simplex virus 1 (HSV-1) and 2, and varicella zoster virus (VZV). These viral pathogens cause epithelial lesions, and then infect the nervous system to cause lifelong latency, reactivation, and spread. A related veterinary herpesvirus, pseudorabies (PRV), causes similar disease in livestock that result in significant economic losses. Vaccines developed for VZV and PRV serve as useful models for the development of an HSV-1 vaccine. We present full genome sequence comparisons of the PRV vaccine strain Bartha, and two virulent PRV isolates, Kaplan and Becker. These genome sequences were determined by high-throughput sequencing and assembly, and present new insights into the attenuation of a mammalian alphaherpesvirus vaccine strain. We find many previously unknown coding differences between PRV Bartha and the virulent strains, including changes to the fusion proteins gH and gB, and over forty other viral proteins. Inter-strain variation in PRV protein sequences is much closer to levels previously observed for HSV-1 than for the highly stable VZV proteome. Almost 20% of the PRV genome contains tandem short sequence repeats (SSRs), a class of nucleic acids motifs whose length-variation has been associated with changes in DNA binding site efficiency, transcriptional regulation, and protein interactions. We find SSRs throughout the herpesvirus family, and provide the first global characterization of SSRs in viruses, both within and between strains. We find SSR length variation between different isolates of PRV and HSV-1, which may provide a new mechanism for phenotypic variation between strains. Finally, we detected a small number of polymorphic bases within each plaque-purified PRV strain, and we characterize the effect of passage and plaque-purification on these polymorphisms. These data add to growing evidence that even plaque-purified stocks of stable DNA viruses exhibit limited sequence heterogeneity, which likely seeds future strain evolution.

Making the case: married versus separate models of alphaherpes virus anterograde transport in axons.

Alphaherpesvirus virions infect neurons and are transported in axons for long distance spread within the host nervous system. The assembly state of newly made herpesvirus particles during anterograde transport in axons is an essential question in alphaherpesvirus biology. The structure of the particle has remained both elusive and controversial for the past two decades, with conflicting evidence from EM, immunofluorescence, and live cell imaging studies. Two opposing models have been proposed-the Married and Separate Models. Under the Married Model, infectious virions are assembled in the neuronal cell body before sorting into axons and then traffic inside a transport vesicle. Conversely, the Separate Model postulates that vesicles containing viral membrane proteins are sorted into axons independent of capsids, with final assembly of mature virions occurring at a distant egress site. Recently, a complementary series of studies employing high-resolution EM and live cell fluorescence microscopy have provided evidence consistent with the Married Model, whereas other studies offer evidence supporting the Separate Model. In this review, we compare and discuss the published data and attempt to reconcile divergent findings and interpretations as they relate to these models.

Proteomic characterization of pseudorabies virus extracellular virions.

Pseudorabies virus (PRV), a member of the Alphaherpesvirinae, has a complex multilayered extracellular virion that is structurally conserved among other herpesviruses. PRV virions contain a double-stranded DNA genome within a proteinaceous capsid surrounded by the tegument, a layer of viral and cellular proteins. The envelope layer, which encloses the capsid and tegument, contains viral transmembrane proteins anchored in a phospholipid bilayer. The viral and host proteins contained within virions execute important functions during viral spread and pathogenesis, but a detailed understanding of the composition of PRV virions has been lacking. In this report, we present the first comprehensive proteomic characterization of purified PRV virions by mass spectrometry using two complementary approaches. To exclude proteins present in the extracellular medium that may nonspecifically associate with virions, we also analyzed virions treated with proteinase K and samples prepared from mock-infected cells. Overall, we identified 47 viral proteins associated with PRV virions, 40 of which were previously localized to the capsid, tegument, and envelope layers using traditional biochemical approaches. Additionally, we identified seven viral proteins that were previously undetected in virions, including pUL8, pUL20, pUL32, pUL40 (RR2), pUL42, pUL50 (dUTPase), and Rsp40/ICP22. Furthermore, although we did not enrich for posttranslational modifications, we detected phosphorylation of four virion proteins: pUL26, pUL36, pUL46, and pUL48. Finally, we identified 48 host proteins associated with PRV virions, many of which have known functions in important cellular pathways such as intracellular signaling, mRNA translation and processing, cytoskeletal dynamics, and membrane organization. This analysis extends previous work aimed at determining the composition of herpesvirus virions and provides novel insights critical for understanding the mechanisms underlying PRV entry, assembly, egress, spread, and pathogenesis.

Sequence variability in clinical and laboratory isolates of herpes simplex virus 1 reveals new mutations.

Herpes simplex virus 1 (HSV-1) is a well-adapted human pathogen that can invade the peripheral nervous system and persist there as a lifelong latent infection. Despite their ubiquity, only one natural isolate of HSV-1 (strain 17) has been sequenced. Using Illumina high-throughput sequencing of viral DNA, we obtained the genome sequences of both a laboratory strain (F) and a low-passage clinical isolate (H129). These data demonstrated the extent of interstrain variation across the entire genome of HSV-1 in both coding and noncoding regions. We found many amino acid differences distributed across the proteome of the new strain F sequence and the previously known strain 17, demonstrating the spectrum of variability among wild-type HSV-1 proteins. The clinical isolate, strain H129, displays a unique anterograde spread phenotype for which the causal mutations were completely unknown. We have defined the sequence differences in H129 and propose a number of potentially causal genes, including the neurovirulence protein ICP34.5 (RL1). Further studies will be required to demonstrate which change(s) is sufficient to recapitulate the spread defect of strain H129. Unexpectedly, these data also revealed a frameshift mutation in the UL13 kinase in our strain F isolate, demonstrating how deep genome sequencing can reveal the full complement of background mutations in any given strain, particularly those passaged or plaque purified in a laboratory setting. These data increase our knowledge of sequence variation in large DNA viruses and demonstrate the potential of deep sequencing to yield insight into DNA genome evolution and the variation among different pathogen isolates.

Repair of the UL21 locus in pseudorabies virus Bartha enhances the kinetics of retrograde, transneuronal infection in vitro and in vivo.

The attenuated pseudorabies virus (PRV) strain Bartha contains several characterized mutations that affect its virulence and ability to spread through neural circuits. This strain contains a small genomic deletion that abrogates anterograde spread and is widely used as a retrograde-restricted neural circuit tracer. Previous studies showed that the retrograde-directed spread of PRV Bartha is slower than that of wild-type PRV. We used compartmented neuronal cultures to characterize the retrograde defect and identify the genetic basis of the phenotype. PRV Bartha is not impaired in retrograde axonal transport, but transneuronal spread among neurons is diminished. Repair of the U(L)21 locus with wild-type sequence restored efficient transneuronal spread both in vitro and in vivo. It is likely that mutations in the Bartha U(L)21 gene confer defects that affect infectious particle production, causing a delay in spread to presynaptic neurons and amplification of infection. These events manifest as slower kinetics of retrograde viral spread in a neural circuit.

Comparison of the pseudorabies virus Us9 protein with homologs from other veterinary and human alphaherpesviruses.

Pseudorabies virus (PRV) Us9 is a small, tail-anchored (TA) membrane protein that is essential for axonal sorting of viral structural proteins and is highly conserved among other members of the alphaherpesvirus subfamily. We cloned the Us9 homologs from two human pathogens, varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1), as well as two veterinary pathogens, equine herpesvirus type 1 (EHV-1) and bovine herpesvirus type 1 (BHV-1), and fused them to enhanced green fluorescent protein to examine their subcellular localization and membrane topology. Akin to PRV Us9, all of the Us9 homologs localized to the trans-Golgi network and had a type II membrane topology (typical of TA proteins). Furthermore, we examined whether any of the Us9 homologs could compensate for the loss of PRV Us9 in anterograde, neuron-to-cell spread of infection in a compartmented chamber system. EHV-1 and BHV-1 Us9 were able to fully compensate for the loss of PRV Us9, whereas VZV and HSV-1 Us9 proteins were unable to functionally replace PRV Us9 when they were expressed in a PRV background.

Fusion of enhanced green fluorescent protein to the pseudorabies virus axonal sorting protein Us9 blocks anterograde spread of infection in mammalian neurons.

Pseudorabies virus encodes a membrane protein (Us9) that is essential for the axonal sorting of virus particles within neurons and anterograde spread in the mammalian nervous system. Enhanced green fluorescent protein (GFP)-tagged Us9 mimicked the trafficking properties of the wild-type protein in nonneuronal cells. We constructed a pseudorabies virus strain that expressed Us9-GFP and tested its spread capabilities in the rat visual system and in primary neuronal cultures. We report that Us9-EGFP does not promote anterograde spread of infection and may disrupt packing of viral membrane proteins in lipid rafts, an essential step for Us9-mediated axonal sorting.

The alpha-herpesviruses: molecular pathfinders in nervous system circuits.

Several neuroinvasive viruses can be used to study the mammalian nervous system. In particular, infection by pseudorabies virus (PRV), an alpha-herpesvirus with broad host range, reveals chains of functionally connected neurons in the nervous systems of a variety of mammals. The specificity of PRV trans-neuronal spread has been established in several systems. One attenuated strain, PRV-Bartha, causes a reduced inflammatory response and also spreads only from infected post- to pre-synaptic neurons. We review the basics of PRV tracing and then discuss new developments and novel approaches that have enabled a more detailed understanding of the architecture of the nervous system. As questions and techniques evolve in the field of neuroscience, advances in PRV tracing will certainly follow.

A conserved alpha-herpesvirus protein necessary for axonal localization of viral membrane proteins.

Pseudorabies virus, an alpha-herpesvirus, is capable of infecting the nervous system and spreading between synaptically connected neurons in diverse hosts. At least three viral membrane proteins (gE, gI, and Us9) are necessary for the spread of infection from presynaptic to postsynaptic neurons (anterograde spread) in infected rodents. To understand how these proteins effect anterograde spread between neurons, we analyzed the subcellular localization of viral proteins after infection of cultured rat sympathetic neurons with wild-type or mutant viruses. After Us9-null mutant infections but not gE-null mutant infections, only a subset of the viral structural proteins had entered axons. Surprisingly, capsid and tegument proteins but not viral membrane proteins were detected in axons. The spread of Us9 missense mutants in the rodent nervous system correlated with the amount of viral membrane proteins localized to axons. We conclude that the Us9 membrane protein controls axonal localization of diverse viral membrane proteins but not that of capsid or tegument proteins. The data support a model where virion subassemblies but not complete virions are transported in the axon. Our results provide new insight into the process of virion assembly and exit from neurons that leads to directional spread of herpesviruses in the nervous system.

Innervation of the heart and its central medullary origin defined by viral tracing.

The vagus nerve exerts a profound influence on the heart, regulating the heart rate and rhythm. An extensive vagal innervation of the cardiac ventricles and the central origin and extent of this innervation was demonstrated by transynaptic transport of pseudorabies virus with a virulent and two attenuated pseudorabies viral strains. The neurons that innervate the ventricles are numerous, and their distribution within the nucleus ambiguus and dorsal motor nucleus of the vagus is similar to that of neurons innervating other cardiac targets, such as the sino-atrial node. These data provide a neuroanatomical correlate to the physiological influence of the vagus nerve on ventricular function.

Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector.

DNA isolated from defective and nondefective virions of herpes simplex type 1 (HSV-1) (strain Patton) was digested with restriction endonucleases, and the resulting DNA fragments were inserted in the EK2 coliphage vector lambdagtWES . lambdaB. The recombinant DNA was encapsidated in vitro under P4 maximum containment conditions. These lambda-HSV1 hybrids were purified and amplified, and the DNA was isolated in the P4 facility. DNA, free of viable phage and bacteria, was removed from P4 conditions and analyzed. Represented among the hybrids studied to date are DNA fragments from about 50 percent of the normal HSV-1 genome. The hybrids derived from defective HSV-1 DNA fragments demonstrate the existence of many similar but not identical classes of defective genomes.

Herpes simplex virus type-1 glycoprotein D gene: nucleotide sequence and expression in Escherichia coli.

The protein coding region of the herpes simplex virus type-1 glycoprotein D (gD) gene was mapped, and the nucleotide sequence was determined. The predicted amino acid sequence of the gD polypeptide was found to contain a number of features in common with other virus glycoproteins. Insertion of this protein coding region into a bacterial expressor plasmid enabled synthesis in Escherichia coli of an immunoreactive gD-related polypeptide. The potential of this system for preparation of a type-common herpes simplex virus vaccine is discussed.

Two alpha-herpesvirus strains are transported differentially in the rodent visual system.

Uptake and transneuronal passage of wild-type and attenuated strains of a swine alpha-herpesvirus (pseudorabies [PRV]) were examined in rat visual projections. Both strains of virus infected subpopulations of retinal ganglion cells and passed transneuronally to infect retino-recipient neurons in the forebrain. However, the location of infected forebrain neurons varied with the strain of virus. Intravitreal injection of wild-type virus produced two temporally separated waves of infection that eventually reached all known retino-recipient regions of the central neuraxis. By contrast, the attenuated strain of PRV selectively infected a functionally distinct subset of retinal ganglion cells with restricted central projections. The data indicate that projection-specific groups of ganglion cells are differentially susceptible to the two strains of virus and suggest that this sensitivity may be receptor mediated.

Actin and Rho GTPases in herpesvirus biology.

Viruses have evolved a variety of interactions with host cells to create an optimal niche for viral replication, persistence and spread. The actin cytoskeleton of the host cell and actin-regulating Rho GTPase signaling pathways can be involved in several of these interactions. This review focuses on recent findings on herpesvirus interactions with actin and Rho GTPases during viral entry, replication in the nucleus and egress. Unraveling these often fascinating interactions might also provide additional insights into sometimes poorly known aspects of actin biology (e.g. its role in the nucleus) and in the development of novel antiviral therapies.

An in vitro system to study trans-neuronal spread of pseudorabies virus infection.

The neuronal spread of infection of alpha herpesviruses is controlled by unknown mechanisms. In the natural host, primary infection always leads to invasion of the peripheral nervous system, but rarely results in extensive invasion of the central nervous system. After reactivation of latent infection in the peripheral nervous system, virions are produced and shed from epithelial surfaces, but rarely invade the central nervous system. We have been studying two aspects of the general problem. First, using GFP and mRFP fusion proteins, we have used video confocal microscopy to assess mechanisms that influence spread of pseudorabies (PRV) virion components within axons. Second, and the subject of this report, is the development of a new in vitro cell culture system that enables the study of trans-neuronal spread of infection from neurons to non-neuronal cells similar to what happens after reactivation and spread to epithelial surfaces. We have developed a tissue culture system involving tri-chamber Teflon rings that enables facile analysis of trans-neuronal spread. The system duplicates all the known in vivo correlates of trans-neuronal spread and provides the opportunity to do both quantitative and qualitative assessment of spread of PRV infection from infected neurons to non-neuronal cells.

Definition of neuronal circuitry controlling the activity of phrenic and abdominal motoneurons in the ferret using recombinant strains of pseudorabies virus.

During a number of behaviors, including vomiting and some postural adjustments, activity of both the diaphragm and abdominal muscles increases. Previous transneuronal tracing studies using injection of pseudorabies virus (PRV) into either the diaphragm or rectus abdominis (RA) of the ferret demonstrated that motoneurons innervating these muscles receive inputs from neurons in circumscribed regions of the spinal cord and brainstem, some of which have an overlapping distribution in the magnocellular part of the medullary reticular formation (MRF). This observation raises two possibilities: that two populations of MRF neurons provide independent inputs to inspiratory and expiratory motoneurons or that single MRF neurons have collateralized projections to both groups of motoneurons. The present study sought to distinguish between these prospects. For this purpose, recombinant isogenic strains of PRV were injected into these respiratory muscles in nine ferrets; the strain injected into the diaphragm expressed beta-galactosidase, whereas that injected into RA expressed green fluorescent protein. Immunofluorescence localization of the unique reporters of each virus revealed three populations of infected premotor neurons, two of which expressed only one virus and a third group that contained both viruses. Dual-infected neurons were predominantly located in the magnocellular part of the MRF, but were absent from both the dorsal and ventral respiratory cell groups. These data suggest that coactivation of inspiratory and expiratory muscles during behaviors such as emesis and some postural adjustments can be elicited through collateralized projections from a single group of brainstem neurons located in the MRF.

Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage lambda.

Site-affinity (or saf) mutations change the specificity of prophage insertion. We have isolated a saf mutation of the bacteriophage lambda attachment site by inserting the phage chromosome into and then excising it from a secondary host attachment site. This causes reciprocal exchange of two seven base-pair segments (the overlap regions) that lie within the cores of the two sites. Since the two overlap regions differ from each other in nucleotide sequence, the recombinant sites are mutants. We have determined the effect of overlap region homology on recombination. We found that homology promotes integrative and excisive recombination. This suggests that the two overlap regions interact directly during recombination. The pattern of segregation of the saf mutation during site-specific recombination shows that it lies to the right of the point of genetic exchange about 95% of the time. This is a surprising result because lambda integrative recombination normally occurs by two staggered, reciprocal single-strand exchanges, one at each edge of the overlap region (Mizuuchi et al., 1981). Since saf lies within the overlap region, we might have expected that the point of genetic exchange would occur to the left of saf as often as to the right. We offer two models to account for this. (1) The mutation alters the location of one of the single-strand exchange points. (2) Efficient and strand-specific processing of mismatched base-pairs changes the expected segregation pattern.