AMPure PB bead was added to the samples and they were purified as in case of the barcoded samples. Conditioning and annealing of the Sequencing Primer, the binding of the Polymerase to the libraries, as well as Polymerase-template complex binding to the magnetic beads was done exactly as indicated by the PacBio Very Low Input protocol. The DNA concentrations were set to 0. The total volume from the polymerase binding step was used for MagBead binding. The salt molarity was adjusted for optimal binding by adding WB 0.
The total amount of the MagBead-bound complex was loaded onto the PacBio machine. PCR products were pooled then size selected manually by using 0. Size-selected samples were amplified with KAPA enzyme using the conditions as above. The qualities of the samples were checked on the Agilent Bioanalyzer. The volume of the sequencing primer for the annealing, and the polymerase P5 or P6 for the binding was determined using the PacBio Calculator version 2. The final concentration of this mixture was 0.
The complex 0. The sample complex 0. MagBeads were prepared as follows: The total amount of the MagBead-bound complex was loaded onto the instrument. The adapter sequences were supplied by the kit. Samples were loaded on R9. The method based on cDNA generation. This step was carried out by the double-strand specific ligase of the kit. The sample was purified after ligation using the silica columns. These samples were subjected to the end repair and adapter ligation steps, and then they were loaded on the ONT Flow Cells.
Total RNAs from 12 different time points were mixed together, and then polyA selection was carried out. The concentration of the reverse-transcribed and adapted RNA was measured by using the Qubit 2. Samples were loaded onto the R9. For PA-Seq, mapping was carried out with Bowtie v2 ref.
The ONT's Albacore software v. This basecaller identify the nucleotide sequences directly from raw data. The sequencing reads were mapped with GMAP using the same setting as was described above.
Custom routines were used to acquire the quality information presented in this data descriptor. All sequencing reads were mapped to the KJ All data can be used without restrictions. The provided dataset was primarily produced to discover and determine the complexity and expression dynamic properties of PRV transcriptome.
These aligned files can be further analysed using various bioinformatics program packages, such as bedtools 26 , samtools 27 , or visualized using e.
IGV 28 , Geneious 29 or Artemis Transcriptome-wide survey of pseudorabies virus using next- and third-generation sequencing platforms. Data doi: Aujeszky, A. A contagious disease, not readily distinguishable from rabies, with unknown origin. Veterinarius 12 , — Google Scholar. Pomeranz, L. Molecular biology of pseudorabies virus: Impact on neurovirology and veterinary medicine.
Microbiol Mol Biol Rev. Gene and cancer therapy--pseudorabies virus: a novel research and therapeutic tool? Curr Gene Ther. Article Google Scholar. Yang, M. Retrograde, transneuronal spread of pseudorabies virus in defined neuronal circuitry of the rat brain is facilitated by gE mutations that reduce virulence. Song, C. New developments in tracing neural circuits with herpesviruses.
Virus Res. Genetically timed, activity-sensor and rainbow transsynaptic viral tools. Methods 6 , — Granstedt, A. Calcium imaging of neuronal circuits in vivo using a circuit-tracing pseudorabies virus.
Cold Spring. Protoc , pdb. Card, J. A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits. Zhu, L. Maresch, C. Oral immunization of wild boar and domestic pigs with attenuated live vaccine protects against pseudorabies virus infection. Klingbeil, K. Immunization of pigs with an attenuated pseudorabies virus recombinant expressing the hemagglutinin of pandemic swine origin H1N1 influenza A virus.
Characterization of pseudorabies virus transcriptome by Illumina sequencing. BMC Microbiol. Genome Announc 2 , 14—15 Rhoads, A. PacBio Sequencing and Its Applications. Supplementary material 5 XLSX 14 kb. Abstract Since late , outbreaks of pseudorabies virus PRV have occurred in southern China causing major economic losses to the pig industry. Electronic supplementary material The online version of this article Materials and Methods Samples and Virus Isolation A total of samples were collected from the lungs, lymph nodes, kidney, spleen, and brain of pigs suspected to be infected with PRV from intensive pig farms in eastern China including the Anhui, Fujian, Shandong, and Jiangsu provinces Fig.
Open in a separate window. Table 1 Sequence information of strains isolated in this study. Strain name Origin Year Month No. Bold number remind the difference in geographic associations significantly. Discussion The Bartha-K61 vaccine was imported from Hungary to China and has been widely used since the late s.
Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material 1 PDF kb K, pdf. Compliance with Ethical Standards Conflict of interest The authors declare that they have no conflict of interest. Animal and Human Rights Statement This article does not contain any studies with human participants or animals performed by any of the authors. Footnotes Xiaofeng Zhai and Wen Zhao have contributed equally to this work.
Pseudorabies virus variant in bartha-kvaccinated pigs, China, Emerg Infect Dis. Datamonkey a suite of phylogenetic analysis tools for evolutionary biology. BMC Evol Biol. A heterologous heparin-binding domain can promote functional attachment of a pseudorabies virus gC mutant to cell surfaces. J Virol. The receptor-binding domain of pseudorabies virus glycoprotein C is composed of multiple discrete units that are functionally redundant.
Nucleic Acids Symp Ser. Interspecies transmission, genetic diversity, and evolutionary dynamics of pseudorabies virus. J Infect Dis. Emergence and adaptation of H3N2 canine influenza virus from avian influenza virus: an overlooked role of dogs in interspecies transmission.
Transbound Emerg Dis. Novel pseudorabies virus variant with defects in TK, gE and gI protects growing pigs against lethal challenge.
Glycoproteins gIII and gp50 play dominant roles in the biphasic attachment of pseudorabies virus. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. J Gen Virol. Epidemiological situation of pseudorabies and vaccine application in China. Swine Prod. Not so different after all: a comparison of methods for detecting amino acid sites under selection.
Mol Biol Evol. MEGA7: molecular evolutionary genetics analysis version 7. Origin, genetic diversity, and evolutionary dynamics of novel porcine circovirus 3. Adv Sci. Pathogenicity and genomic characterization of a pseudorabies virus variant isolated from Bartha-Kvaccinated swine population in China. Vet Microbiol. The international committee on taxonomy of viruses. Arch Virol Suppl.
Detecting individual sites subject to episodic diversifying selection. PLoS Genet. The porcine humoral immune response against pseudorabies virus specifically targets attachment sites on glycoprotein gC. Correlating viral phenotypes with phylogeny: accounting for phylogenetic uncertainty. Infect Genet Evol.
Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev. Ernst Mayr: genetics and speciation. Less is more: an adaptive branch-site random effects model for efficient detection of episodic diversifying selection. Genomic characterization of pseudorabies virus strains isolated in Italy. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Epidemiology, genetic recombination, and pathogenesis of coronaviruses.
Trends Microbiol. Epidemiology, evolution, and pathogenesis of H7N9 influenza viruses in five epidemic waves since in China. Control of swine pseudorabies in China: opportunities and limitations. The theory of speciation via the founder principle. Pseudorabies epidemic status and control measures in China. While these two processes are closely linked, recent studies indicate that the addition of tegument proteins to the capsid occurs to some extent in an organized and stepwise fashion.
The outer layer of the herpesvirus tegument is analogous to the matrix of some RNA viruses. Like matrix proteins, some of the outer tegument layer proteins interact with the inner side of the envelope and also with the inner tegument proteins attached to the capsid, joining these substructures together for the second, and final, envelopment process.
The protein-protein interactions within the tegument and between tegument and envelope are likely to drive the secondary envelopment process and may be regulated by phosphorylation Secondary envelopment is most commonly believed to occur in a compartment derived from the trans-Golgi network.
Tegument addition to cytoplasmic capsids is thought to be initiated by the direct interaction of the major capsid protein VP5 UL19 and the UL36 tegument protein, which in turn can interact with another tegument protein, UL37 , , This model is consistent with the observation that the inner tegument layer exhibits icosahedral symmetry Secondary envelopment of herpesviruses occurs on the cytoplasmic face of a specialized compartment within the infected cell derived from the trans -Golgi network The interaction between tegument proteins and envelope could occur either via an interaction with the cytoplasmic domain of envelope proteins , or via direct membrane attachment following fatty acid modification of a tegument protein , Interactions between the tegument and envelope proteins are likely to drive the secondary envelopment of herpesviruses: PRV VP22 interacts directly with the cytoplasmic domains of gM and gE However, there is undoubtedly much redundancy in the interactions between viral proteins required for secondary envelopment.
On the other hand, individual deletions of tegument proteins UL11, UL37, UL47, UL48, and UL51 have all resulted in accumulation of unenveloped cytoplasmic capsids, a finding that suggests a defect in secondary envelopment , , , , The UL20 membrane protein is a component of virions and plays a role in sorting mature enveloped virions from the site of secondary envelopment to be released from the cell Light or L-particles so called because they are lighter than virions in Ficoll gradients are noninfectious structures produced during herpesvirus infection reviewed in reference L-particles contain tegument and envelope proteins but no capsid or genomic DNA.
L-particle production probably shares common assembly steps with normal tegument assembly and secondary envelope acquisition, and can still occur under conditions where capsids fail to leave the nucleus L-particles fuse with cells, and may assist in the initiation of the infection cycle by delivering tegument and envelope proteins in addition to those brought by the infectious virus 95 , Support for their biological significance role comes from the finding that structures resembling L-particles are observed in epithelial cells and fibroblasts in the nasal mucosa of pigs after intranasal inoculation with PRV 4.
Many PRV genes can be deleted with little or no phenotype in cultured cells or in animal models of infection. PRV offers the possibility of testing mutants in the natural host. Despite the complications of working with infected animals, many studies confirm the phenotypes found in cultured cells or model animal infections. It is likely that some genes considered to be nonessential perform redundant functions. Recent studies using multiple deletions to probe for functional redundancy have found new or severe phenotypes not seen by the individual mutants often called synthetic lethality.
Triple and quadruple deletion mutant were produced and compared to search for phenotypes hidden by functional redundancy The quadruple deletion could still replicate in tissue culture cells, but formed very small plaques, replicated with delayed kinetics, and produced fold fewer viruses.
In this case, functional redundancy was difficult to define in cultured cells: UL47 and UL48 function independently during cell-cell spread and virus egress, while the simultaneous loss of UL46 and UL49 does not alter the replication of ULnegative or ULnegative PRV mutants. The functional redundancy of structural components in virion maturation is exemplified by the finding that while gM or gE is dispensable for viral growth in cultured cells, a simultaneous deletion of gM and gE cytoplasmic domain produces a synthetic lethal defect in secondary envelopment 44 , Both proteins likely play a role in organizing structural virion components and recruiting them to sites of secondary envelopment.
The cytoplasmic tails of both gE and gM interact with VP22 In addition, PRV gM directs viral envelope proteins to the trans-Golgi apparatus in transfected cells Viral infection of animal cells tends to generate proapoptotic signals that induce cell death and phagocytosis to limit virus replication and spread.
Herpesviruses, like most large DNA viruses, encode proteins that interfere with this apoptotic response reviewed in references 9 and PRV, like HSV-1, is a cytocidal virus that induces rapid and extensive changes in cultured host cells. The cytopathic effects elicited by PRV infection are strain- and cell type-dependent and include inhibition of mitosis, formation of intranuclear inclusions, rounding up of cells, and, occasionally, formation of syncytia reviewed in reference In most cultured cells, the typical apoptotic effects, such as genomic DNA fragmentation or apoptotic-condensed nuclei, are not observed following infection.
Neurons isolated from porcine trigeminal ganglia are more resistant to PRV-induced cell death than neurons isolated from the superior cervical ganglia and other porcine cell types. Trigeminal ganglia neurons survive productive PRV infection longer than other cell types, and this may extend the duration of infectious virus transmission during reactivation from latency US3 is one of the most potent and best characterized antiapoptosis genes in the list.
US3 blocks the lysis of infected fibroblasts by cytotoxic T cells 68 , a block attributed to the US3-dependent inhibition of caspase activation in the target cell. It is generally accepted that the US3 protein kinase prevents apoptosis by direct phosphorylation of target proteins, but the exact details remain unsolved. A recent study highlights the overlap between the substrates of protein kinase A, a cyclic AMP-dependent cellular kinase that can both inhibit and promote apoptosis, and HSV-1 US3, and found US3 expression to result in protein kinase A phosphorylation Whether this signifies an activation of protein kinase A by US3, US3 mimicking protein kinase A antiapoptosis function, or something else is unknown.
PRV infection renders swine testicle cells resistant to apoptosis induced by staurosporine or sorbitol US3 -negative PRV also failed to prevent apoptosis in swine trigeminal ganglia neurons Apoptosis was observed late in US3 -null infected swine testicle cells hours postinfection , suggesting that additional viral proteins may serve to inhibit apoptosis early in infection US3 -negative PRV fails to suppress apoptotic, while US3 transfection experiments in the presence or absence of viral infection show US3 to be responsible for the antiapoptosis activity.
The longer isoform possesses a functional mitochondrial localization sequence within the additional N-terminal 51 amino acids 55 , The localization of US3 seems to be dependent on the isoform examined, the cell type used, and whether US3 is expressed during viral infection or from a transfected plasmid 56 , The long isoform is able to restore antiapoptotic activity to the US3 deletion virus while the short isoform only partially compensates for the deletion of US3 Since the HSV-1 US3 kinase activity is required for its antiapoptotic activity , it is assumed that the same holds true for PRV, though this has yet to be formally tested.
Finally, it is worth noting that US3 from the closely related BHV-1 does not seem to possess any antiapoptotic properties Herpesviruses cause lifelong infection of their hosts, and their persistence and repeated reactivation are facilitated by different immune evasion strategies, including inhibition of complement and antibody function, disruption of immune recognition of infected cells, and viral expression of cytokine-like and chemokine receptor-like molecules reviewed in reference Inhibiting the presentation of virus-derived peptides by MHC class I molecules prevents the recognition and elimination of virus-infected cells by cytotoxic T lymphocytes.
PRV UL However, neither gM, nor any other viral proteins, was required for the gN-dependent TAP inhibition A disruptive insertion in UL A similar disruptive insertion in UL PRV has proven to be a facile model system to investigate the many aspects of alphaherpesvirus biology, providing insights both into the basics of the viral life cycle and the more complex interactions with the host reviewed in reference Areas that benefited particularly from PRV research include the molecular mechanisms of virus attachment, entry, replication, assembly, intracellular trafficking and egress reviewed in references and In addition, various facets of viral pathogenesis are under study, including the mechanisms of neuroinvasion, transneuronal spread, and host immune responses.
The results from these studies have furthered our understanding of these complex phenomena and are bound to yield fascinating new insights. For a number of reasons, PRV is an excellent choice for the study of alphaherpesvirus biology reviewed in reference PRV has a remarkably broad host range, causing a lethal infection in diverse animals, yet poses little to no danger to lab workers. PRV grows well and is easy to manipulate in the laboratory. Purified DNA is infectious and the techniques to replace and manipulate its genes are well established.
Bacterial artificial chromosomes carrying the entire PRV genome have been constructed , Animal experiments can be performed in the natural host, the pig, or in other favored model organisms such as rats, mice, and rabbits.
The laboratory animal models of PRV pathogenesis have provided considerable insight into fundamental problems of herpes biology. These include the chicken embryo eye model 18 , the rat eye model of infection 45 , , the mouse skin flank model 51 , and the mouse nasopharyngeal infection model In the rat eye model, virions are injected into the vitreous humor of the eye where retinal ganglion cells are the first to replicate the virus 64 , The infection spreads to retinorecipient structures of the brain primarily via the optic nerve and the third cranial nerve.
This model has been applied successfully to delineate neural circuitry and to define the PRV gene products required for directional transport. Deletion of any of these genes abolished the anterograde, but not the retrograde, spread of PRV in synaptically connected neurons 47 , 66 , , ,.
Moreover, additional studies demonstrated that gE and gI contribute to virulence, and that their function in virulence was distinct from their function in directional spread , , An alternative model for the study of PRV neurovirulence and neuroinvasion uses chicken embryos A viral inoculum is introduced through a window in the eggshell into the vitreous chamber of the eye at a developmental stage where retinal cells have developed synaptic connections with the optic tectum in the brain.
At this stage of development, the eye is among the largest and most easily accessible tissues. The relatively large yet well-circumscribed target area of the vitreous eye chamber allows consistent, precise primary infections with little to no risk of nonspecific infection of other chick tissues.
The innervation of the eye and eye structures are well known, facilitating analysis of spread. To study a primary PRV infection of an epithelial surface, Brittle et al. In this model, shaved mouse skin flank is scarified and infected with a drop of viral inoculum, such that viral adsorption occurs on the underlying live epithelial tissue. A primary infection in epithelial tissue precedes virus entry into nerve endings of the peripheral nervous system, similar to the course of a natural herpesvirus infection.
In permissive nonnatural hosts, such as the mouse, alphaherpesviruses can spread to the central nervous system. Thus, infection of epithelial tissue, virulence, and neuroinvasion of the peripheral and central nervous systems can all be examined in one animal. Brittle et al. Mice infected with virulent PRV strains self-mutilated their flank regions in response to a virally induced, pruritic stimulus. In addition, these animals died rapidly with virtually no viral particles detectable in the brain by titering assays and immunohistochemical analyses.
There were also no symptoms of central nervous system infection such as behavioral abnormalities. Mice infected with virulent PRV strain Becker, Kaplan, or NIA-3 died at approximately 77 h compared to attenuated vaccine strain Bartha-infected mice, which died at approximately h. Mice infected with viruses lacking US9, gI, or gE died at roughly h, h, and h, respectively.
Interestingly, the longer-lived animals infected with the attenuated strains did not become pruritic or develop skin lesions on the infected flank region, yet developed profound ataxia and tremors associated with hindbrain lesions.
An additional study with a UL54 -null virus found a delay in symptom onset and a lengthened survival time following symptom appearance Prior to death, the severity of pruritus and skin lesions of UL54 -null and wild-type infected mice was indistinguishable.
It has long been assumed that nonnatural hosts of PRV died of classical herpesvirus encephalitis, defined by a massive necrotizing lesion of an expansive region of the brains.
After all, PRV is highly neurotropic and the infected hosts suffered from neurological abnormalities. However, the severe pruritus and relatively shorter time to death by infection with wild-type strains of PRV, coupled with the barely detectable infection of the central nervous system, suggests that the fatal outcome of virulent infection is more a result of the host immune system response, or peripheral nervous system injury, rather than the result of fatal viral encephalitis.
In comparison, the neurological symptoms, central nervous system viremia and long time to death seen after infection with attenuated strains, suggest that the cause of death may be quite different from the virulent strains, possibly a direct result of encephalitis, though no necrotizing brain tissue is observed Over 60 years ago, Sabin used a murine model to study PRV neuroinvasion, and chose an intranasal route of infection to emulate the natural oronasal transmission of PRV in swine Inoculation of the nasal cavity resulted in virally induced neuropathological lesions.
The kinetics and locations of lesion appearance were consistent with a transneuronal spread of PRV from nasal epithelium to synaptically connected higher-order structures in the nervous system.
Lesions were found in neurons of the olfactory system, parasympathetic system, sympathetic nervous system, and primary sensory neurons of the fifth cranial nerve. The route of pseudorabies virus propagation in the mouse nervous system follows intranasal inoculation as described The infection spreads through three neuronal pathways innervating the nasal cavity: i via anterograde spread in the trigeminal circuit, from trigeminal ganglia to spinal trigeminal nucleus, ii via retrograde spread in the sympathetic circuit, from superior cervical ganglion to sympathetic preganglionic neurons in the spinal cord's intermediolateral nucleus, and iii via retrograde spread in the parasympathetic circuit, from pterygopalatine ganglion to the superior salivatory nucleus.
Infected mice exhibit distress symptoms e. Infected neurons could be identified by immunofluorescence with antibodies against viral antigens as well as by the activity of the lacZ reporter gene driven by the endogenous US4 gG promoter. The study further demonstrates that the relative neurovirulence and neuroinvasiveness of the wild-type strain and the US4-null lacZ reporter strain are similar As with the other animal models, a PRV US8 gE -null strain is severely attenuated in the mouse intranasal model, exhibiting reduced virulence and neuroinvasion The infected mice survived longer and only the hunched posture syndrome was apparent.
Infection of first-order neurons innervating the nasal mucosa still occurred in the absence of gE, but retrograde spread within the sympathetic circuit was reduced, while anterograde spread within the trigeminal circuit was completely abolished.
Retrograde spread to second-order neurons in the parasympathetic circuit was not observed for US8 gE -null PRV; however, infection of the first-order neurons of this pathway could not be ascertained. The intranasal model has been used to systematically examine a collection of various gene deletions within PRV 12 - 14 , , , , , ; reviewed in reference Alphaherpesviruses maintain their presence in the host population by infecting and establishing a latent infection in the host peripheral nervous system.
The cycle of infection, latency, and reactivation is critical to viral survival. The broad range of species permissive for PRV neuronal infection in vivo and ex vivo has made PRV invaluable for studying the molecular details of neuronal infection. The study of PRV neuronal infection has enhanced our understanding of virus assembly and long distance transport in axons. The process of viral entry into neurons is thought to be similar to that found for most cultured cells First, the viral envelope fuses with the cell membrane which, in the natural host setting, would be the axon terminal at the periphery of the peripheral nervous system.
Next, the capsids along with the inner tegument proteins are transported retrogradely along the relatively great distance of the axon to the nucleus in the neuron cell soma. Live imaging studies of fluorescent capsids in neurons isolated from embryonic chick dorsal root ganglia have shown that the retrograde transport of capsids in axons is quite fast 1. The capsid transport in axons undoubtedly requires an association with molecular motors, most likely from the dynein family, since capsid movement by diffusion alone would be predicted to take years to reach the neuron soma reviewed in reference Reactivation from latency involves triggering transcription, assembling mature virus, and transmitting infection from the cell body anterogradely through the axon back to epithelial cells at the periphery.
Since capsids travel towards the nucleus after entry and towards the axon terminal during egress, a mechanism must exist to regulate the direction of capsid transport during infection. The direction of transport is presumably determined by the motor complex associated with the capsid: dynein would transport the capsid retrogradely, while kinesins would facilitate anterograde transport.
Smith et al. Thus, the modulation of transport direction, and hence motor association, occurs at the level of the individual capsid, rather than by global regulation of axonal motor transport. Studies of HSV-1 point to a model in which egressing capsids are transported in axons separately from the viral envelope components rather than fully assembled virions , In this model, assembly of mature particles occurs at a distal point along the axon, possibly at the axon terminal reviewed in reference Additional support for this model comes from studies of PRV infection of cultured rat motor neurons identifying viral proteins crucial to the anterograde transport of viral components.
The envelope glycoprotein gE is required for efficient entry of glycoproteins, capsids and some tegument proteins into axons during viral egress Meanwhile, the US9 envelope protein is required for sorting viral glycoproteins, but not capsids or tegument proteins into axons during viral egress Thus, PRV capsids can be transported in axons independently of glycoproteins.
However, entry of capsids into the axon is not sufficient to transmit infection to cells at the distal end of the axon. To better study the neuron-to-cell spread of PRV, Ch'ng and Enquist recently developed a compartmented neuronal culture system wherein cell bodies and axon endings are separated by a physical barrier traversed only by the axons themselves Additionally, the compartment containing the axonal endings is seeded with transformed epithelial cells to allow viral replication.
In this model, the directional spread of PRV can be assessed by infecting the cells in one compartment and measuring the spread of virus through axons to cells in another compartment. In this system, gE, gI, and US9 were all required for transneuronal spread from the neuronal cell body compartment to the epithelial cells in the axon-end compartment. Electron microscopic analyses of PRV-infected neurons identified capsids that are transported within vesicles during anterograde axonal transport rather than directly associated with microtubules as in entry 72 , Since capsids can be transported independently of glycoproteins, the surrounding vesicle probably does not constitute the viral envelope.
The origins and constituents of this transport vesicle remain unknown. Like other herpesviruses, in vitro reactivation of PRV can be induced in explants of trigeminal ganglia or tonsils, or in vivo in pigs or mice 52 , , , Reactivation of latent pseudorabies in pigs is induced by treatment with dexamethasone reviewed in reference 52 or acetylcholine , drugs which influence the host immune and nervous systems, respectively. Reactivation from latency has also been studied using a mouse model.
Because mice are normally unable to survive an acute infection of PRV, establishing a latent infection requires the passive transfer of high-titer neutralizing antibodies prior to viral inoculation Subsequent stimuli can induce reactivation and virus production in trigeminal ganglia explants or nasal cavity of live mice Reactivation was observed in response to mild stress restraint, cold and transportation , as well as acetylcholine and dexamethasone treatment , , , While many viruses can infect the cells of the nervous system, only a handful have the unique ability to spread between synaptically connected chains of neurons: vesicular stomatitis virus , rabies virus reviewed in reference , mouse hepatitis virus 21 , betanodavirus , and alphaherpesviruses reviewed in reference Such neural spread requires that the virus must enter the neuron the first-order neuron and replicate.
The encapsidated viral genome is then transported at or near sites of synaptic contact to a second order neuron where replication takes place again. This property of self-amplification allows the first order neuron to be as intensely labeled as the second and third order neurons. Tracing studies using PRV have been successfully employed in a number of different animal models: pigs the natural host , lambs and sheep , dogs 85 , cats , chicken embryos 18 , ferrets 36 , and other rodents such as rats reviewed in reference , mole rats , mice 51 , gerbils , and hamsters These three properties of pseudorabies virus—transsynaptic spread, self-amplification, and a broad host range—have allowed its use in an extensive number of neuroanatomical studies seeking to define the architecture of multisynaptic pathways reviewed in reference Conventional nonviral tracers that are transported in axons rely on antibody reactivity, radioactivity, enzymatic activity, or fluorescence to label the neurons taking them up reviewed in reference Commonly used conventional tracers include horseradish peroxidase alone or conjugated to wheat germ agglutinin, True Blue, Fast Blue, Fluoro-Gold, latex microspheres coated with fluorescing dyes, subunit B of cholera toxin discussed below , the nontoxic fragment C of tetanus toxin, and dextran amines.
While these tracers can travel within an axon to label distant cell bodies or dendrites, their use in multisynaptic neuronal circuit is limited by most tracers' inability to cross through the synapses from one neuron to the next. Some plant lectin-derived tracers, such as wheat germ agglutinin-horseradish peroxidase, can cross synapses 12 , , , while others, such as Phaseolus vulgaris leucoagglutinin, cannot.
Since propagation inevitably dilutes tracer concentrations and signal intensity, nonamplifiable tracers have limited utility in tracing higher order multisynaptic circuits. In comparison, viral tracers are self-amplifying and do not decrease in signal intensity, whether assessed by the presence of viral antigens or marker genes.
Finally, viral infections can proceed over several synapses to infect multiple segments of a neuronal circuit. PRV can replicate in all central nervous system neurons studied in permissive animals. As explained below, the multisynaptic tracing ability of PRV is heavily influenced by the architecture of the circuitry and the survival time of the infected host. Electron microscopic analyses and tracing studies strongly support the view that PRV spreads in the nervous system primarily by direct cell-cell contact, rather than diffusion of virions through the extracellular space or spread via nonneuronal cells.
Analysis of infected nervous tissue by electron microscopy reveals viral capsids and structural proteins localized at the synapses of infected nervous tissue 63 , However, the strongest evidence that PRV spreads through defined circuitry comes from carefully designed studies of virus transport within the nervous system. Much of this work has been done using the circuitry connecting the ventral musculature of the stomach to the brainstem and higher order structures of the central nervous system Fig.
Three main points are as follows. Stomach injection model. Sagittal view of the rat brain. Innervation of smooth muscle of the ventral stomach. The area boxed in red is magnified to the right.
Motor neurons from the dorsal motor nucleus of the vagus send projections through the vagus nerve to the ventral wall of the stomach. Sensory innervation of the ventral stomach through the left nodose ganglion is shown in pink. Neurons in the dorsal motor nucleus of the vagus exhibit anti-PRV immune reactivity 30 h after stomach injection of wild-type PRV-Becker 64 , PRV travels retrogradely to second-order neurons in the medial nucleus of the solitary tract between 50 and 60 h postinjection and to third-order neurons in the area postrema between 60 and 70 h postinjection.
Labeling of neurons within the left nodose ganglia can be observed by 45 and 50 h postinjection. No cross talk with tongue or esophageal innervation after stomach injection. Injection of PRV-Becker into the ventrolateral musculature of the tongue pathway shown in green results in a very different pattern of infection from injection into the stomach shown in blue After transport through the hypoglossal nerve, PRV immune reactivity can be seen in the hypoglossal nucleus XII 30 h postinjection.
By about 52 h postinjection, PRV infection can be observed in second-order neurons in the spinal trigeminal nucleus pars oralis and pars interpolaris and the ventrolateral brainstem tegumentum and monoaminergic cell groups not shown.
Injection into the smooth muscle of the esophagus shown in orange produced labeling in the dorsal nucleus ambiguus. By 48 hours postinfection, labeling was detected in small bipolar neurons of the nucleus centralis of the medial NTS. The segregation of labeled structures following injection of stomach and esophagus is significant because the axons of these efferent circuits travel together in the vagus nerve, yet PRV infection is absent from the nucleus ambiguous following stomach injection and absent from the dorsal motor nucleus of the vagus followingesophageal injection.
PRV requires an intact circuit for spread in the nervous system. In addition to surgical severance of the left vagus nerve which eliminates PRV transport to the left dorsal motor nucleus of the vagus, further proof that PRV neuronal spread requires intact, synaptically connected neurons is provided by tracing studies that span progressing developmental stages PRV-Bartha immune reactivity in the central nervous system was examined 2.
Rats injected on postnatal day 1 P1 exhibited PRV immune reactivity in the dorsal motor nucleus of the vagus, medial nucleus of the solitary tract, area postrema, and paraventricular nucleus of the hypothalamus by 2. No animals in the P1 group exhibited anti-PRV labeling in the central nucleus of the amygdala, lateral hypothalamic area, bed nucleus of the stria terminalis, insular cortex, or medial prefrontal cortex with the exception of one rat with six labeled neurons in the central nucleus of the amygdala.
Rats injected at later developmental stages exhibit progressively more viral penetrance into the central nervous system; 2. Only rats injected on P8 exhibit infection of neurons in the insular cortex and medial prefrontal cortex. Comparison of anterograde- and retrograde-defective alphaherpesviruses.
In adult rats, stomach injection of PRV-Bartha results in retrograde-only transport of viral infection pathway and PRV-immune reactive structures shown in orange from the dorsal motor nucleus of the vagus to medial nucleus of the solitary tract to area postrema Anti-PRV immunoreactivity 4 to 5 days postinjection does not change after elimination of anterograde transport by surgically severing the axons of pseudounipolar neurons projecting from the nodose ganglia shown in pink; see panel B.
HSV-H has a retrograde spread defect. HSV-H can spread retrogradelyin first-order neurons but only anterograde spread is observed in second-order pathways. Although HSV immune reactive structures appear similar to those infected by PRV after injection into the ventral stomach HSV-H, intact compare with PRV-Bartha , vagal deafferentation illustrated by a red X on the sensory pathway from the nodose ganglia eliminates infection of the medial nucleus of the solitary tract and area postrema HSV-H, vagal deafferentation.
Figure modified from reference with permission of the publisher. After injection into the ventral stomach musculature, transport of the wild-type strain PRV-Becker to structures in the dorsal motor complex of the brainstem Fig. Here, the direction of virus spread is described in relation to the direction of impulse in a circuit, anterograde for spread from presynaptic to postsynaptic neuron, and retrograde for spread from postsynaptic to presynaptic neuron.
Retrograde first-order neuronal transport to the dorsal motor nucleus of the vagus was seen for both PRV-infected and cholera toxin beta subunit-injected stomach Cholera toxin beta subunit was also transported in an anterograde fashion from the stomach to label sensory afferents in the tractus solarius, something not seen in PRV-infected animals, presumably because the animals expired before the viral infection reached these distant structures.
No PRV antigens were ever detected in the adjacent dorsal motor nucleus of the vagus or nucleus of the solitary tract NST complex. Similarly, while injection of virus in the stomach can label the dorsal motor nucleus of the vagus and medial NST mNST , injection of virus into the esophagus led to a pattern of labeling in the nearby nucleus ambiguous but not the dorsal motor nucleus of the vagus or the NST. This is notable because the axons that innervate the esophagus and ventral stomach travel together in the vagus nerve, yet virus labeling of neurons stays specific to the individual circuitries In the stomach infection model, severing the left vagus nerve eliminated virus transport to the left dorsal motor nucleus of the vagus, while transport through the right vagus nerve to the ipsilateral right dorsal motor nucleus of the vagus was unaffected The most compelling evidence that synaptic connections are required for PRV spread come from tracing studies to define the temporal stages of development of forebrain circuitry in the newborn rat Ventral stomach injection of rats with PRV on postnatal day 1 P1 , P4, and P8 allowed a comparison of the circuitry connecting the dorsal motor nucleus of the vagus complex to higher ordered structures in the forebrain Fig.
While axonal projections from the NST to the medial prefrontal cortex and insular cortex are present early in development, PRV only spread from the dorsal motor nucleus of the vagus to these forebrain structures in older rats. The key assumption is that PRV is not transported to brain structures prior to the formation of synaptic connections.
Although few studies have addressed the extent to which PRV transport requires a functional synapse in terms of neurotransmitter release and electrophysiology, recent reports have identified changes in PRV tracing after lesion-induced circuit reorganization 21 , PRV is pantropic, that is, virions infect many different types of cells including neurons and epithelial cells.
Yet, once introduced into a specific neuronal circuit, nonsynaptic spread to peripheral cells in the nervous system is severely limited. Recent studies have examined how and when nonsynaptic spread of PRV is limited in the brain, and whether PRV would be suitable for circuit tracing following intracerebral injection reviewed in reference 8. Particular attention has been paid to the potential for mislabeling of neuronal circuits after intracranial injection of PRV.
Direct PRV injection into brain ventricles, cerebral cavities filled with cerebrospinal fluid, showed that the neurons and the ependymal cells lining the ventricle and the caudal raphe could indeed be infected Immunostaining for viral antigens shows that PRV can infect astrocytes and brain macrophages surrounding infected regions of the central nervous system.
Temporally, infection of astrocytes follows neuronal infection, indicating spread is from neuron to astrocyte However, since astrocytes are susceptible to PRV infection, but not permissive for viral replication, they do not contribute to trans-neuronal spread of the virus 63 , Viral infection does not spread from infected glial cells to nearby nonneuronal cells or to nearby axons outside the circuitry being traced Rather, infection of astroglia is thought to represent an effort of the local intrinsic and innate immune defense to contain the infection 62 , 63 , When injected directly into brain tissue, PRV virions diffuse very little, producing a focal infection site.
PRV could also infect neurons via fibers of passage, axons that traverse but do not synapse on cells at the injection site 74 , Though the potential for leakage into the cerebrospinal fluid exists, PRV performs well as a tracer following intracranial injection Proper experimental design planning for tracing studies should minimize nonspecific labeling, while careful examination of ependymal cells should identify leaks into the cerebrospinal fluid.
Attenuated virus strains possess mutations that reduce virulence. One of the best characterized attenuated PRV strains, PRV-Bartha was isolated after multiple passages of a virulent field isolate in cultured chicken cells and embryos It was used as an effective vaccine against PRV in pigs Though the complete sequence of the PRV-Bartha genome is unknown, molecular and genetic analyses have identified three independent mutations contributing to its reduced virulence: point mutations within UL21 , a signal sequence mutation in the UL44 gC gene , and a 3-kb deletion encompassing US8 gE , US9 and a large portion of US7 gI and US2 , , The attenuated mutants such as PRV-Bartha are favored for tracing studies because they penetrate further into neuronal circuits due to increased host survival time reviewed in reference
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