Pseudorabies virus (PRV) is an alpha herpesvirus of swine causing disease characterized by encephalomyelitis and inflammation of the respiratory tract. Efforts to studying PRV have led to discoveries uncovering the molecular mechanisms of herpesvirus pathogenesis and neuronal invasion. Consequently, PRV can be a powerful tool in exploring the neuroanatomical pathways of the brain, because infection spreads among synaptically connected neurons. PRV infection requires the viral Us9 protein to spread form pre-synaptically connected neurons to post-synaptically connected neurons. Us9 is a membrane protein found in lipid rafts and is required for sorting newly assembled virions into axons. Various studies suggest that sphingolipids, significant lipid raft constituents, and sphingomyelinases are necessary for viral intracellular transport. The host protein, SMPD4, is a neutral sphingomyelinase involved in ceramide production and biogenesis of exosomes. Previous work in this laboratory demonstrated that SMPD4 protein interacts with Us9. SMPD4 was the second most enriched host protein associated with GFP-Us9 in an immunoprecipitation and mass-spectrometry experiment. In this thesis, we studied SMPD4 through a series of immunofluorescence and western blot experiments. We used the neuronal cell line (Neuro2a) to study the function of SMPD4 and Us9 interaction. These cells can be differentiated into central nervous system (CNS) neurons. However, PRV infection in these cells was variable and the production of infection virus was low. Various environmental conditions were explored to optimize PRV infection of these cells. While natural mouse laminin coating induces the growth of two or more neurite-like projections per cell by 25%, it did not affect the percent of Neuro2a cells infected. The infectious cycle is not synchronous in Neuro2a cells, which may reflect the lack of synchrony of the cell cycle. In addition, infected cells appeared to round up and die, perhaps indicating an apoptotic response to infection. We detected SMPD4 using immunofluorescence microscopy. The protein appeared as individual puncta throughout the cell in uninfected and infected Neuro2a cells. Furthermore, we could observe colocalization between SMPD4 and viral capsids.

http://arks.princeton.edu/ark:/88435/dsp01xs55mg12w

Pseudorabies virus (PRV) is a swine alphaherpesvirus that is closely related to human herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). The virus also infects a large number of mammals, including sheep, dogs, and rodents. In these non-natural hosts, infection with a virulent wild-type PRV strain (PRV- Becker), causes a neuropathic itch also known as “mad itch” which is followed by an uncontrolled systemic inflammation, leading to sudden death. Using a mouse footpad inoculation model, it was demonstrated that PRV-Becker infection induces the increased production of two proinflammatory cytokines (IL-6 and G-CSF) in pe- ripheral nervous system (PNS) and central nervous system (CNS) tissues, very early after infection. This suggests that PRV infection activates the nervous system and initiates an early neuroinflammatory response that later escalates into a systemic in- flammation. However, the molecular mechanisms used by PRV to regulate the innate immune responses specifically upon infection of neuronal cells remain unclear. This study aimed to establish an in vitro system that could mimic PRV infection in vivo and help to dissect the mechanisms that initiate this neuroinflammatory response. Using murine neuroblastomas (neuro-2A cells), we showed that all confluent neuro- 2A cells are infected with PRV by 8 hours post inoculation (hpi). We found that the virulent PRV-Becker strain and the attenuated vaccine strain, PRV-Bartha, replicate to the same extent in confluent neuro-2A cells up to 10^5 plaque forming units (PFU) at 48 hpi. Compared to non-infected neuro-2A cells, PRV-Becker- and PRV-Bartha- infected cells show significant decreased IL-6 levels at 48 hpi. Moreover, PRV-Becker- and PRV-Bartha-infected neuro-2A cells do not produce significant amounts of G- CSF and type 1 interferon (IFN). Overall, we demonstrated that PRV infection of neuro-2A cells does not recapitulate the inflammatory cytokine production found after PRV infection of mice.

http://arks.princeton.edu/ark:/88435/dsp01xp68kk261

The VP16 tegument protein of herpes simplex virus 1 (HSV-1) has been shown to have a role in reactivation of latent infection in the peripheral nervous system (PNS), but while it appears to activate viral gene transcription, it is unknown if this protein can also activate neuronal genes. Less research has been done on the VP16 homolog in the related pseudorabies virus (PRV) and any role it may play in activating neuronal genes. By using adeno-associated virus (AAV) vectors that encode either HSV VP16 or PRV VP16 (aka UL48), cultured superior cervical ganglia rat neurons (SCGs) can be transduced and made to express VP16 or UL48 independent of virus infection. Gene expression in SCGs transduced in this manner was compared using RNA-seq and RT-qPCR and it was found that the neuronal gene Jun was enriched in the presence of HSV VP16, Adcyap1 with PRV UL48, and Crem in the presence of both proteins. Subsequent analysis of subcellular localization in AAV vector-transduced and virus-infected SCGs showed that, while localization of Adcyap1 and Jun did not change with or without the presence of the VP16 proteins, Crem was nuclear only in the presence of PRV UL48. It appears that PRV UL48 may be increasing expression of Crem and Adcyap1 but only recruiting Crem to the nucleus for activation of viral gene expression. While the presence of HSV VP16 is connected to enrichment of Crem, that same nuclear localization is not observed, suggesting it may not play the same role in HSV-1 as in PRV.

When infected with the wild-type virus strain of pseudorabies virus (PRV-Becker), mice will exhibit symptoms including a “mad-itch” response, tremors, and weight loss, and eventually die. Interestingly, an attenuated version of PRV (PRV-Bartha) is significantly less virulent. PRV-Bartha elicits less severe symptoms in infected mice and these mice will, on average, live significantly longer. Past rtPCR analysis has shown that these viruses do not replicate beyond the PNS and CNS, yet peripheral organs express elevated levels of pro-inflammatory cytokines and chemokines at the time of PRV-Becker infected animal death. These results have suggested that it is the inflammatory response to PRV as opposed to viral replication in tissues that kills the mice. Currently, little is known about the exact causes of death and of pruritus in the mice when infected with PRV-Becker. It is also unknown why PRV-Bartha is so much less virulent than PRV-Becker. This report characterizes the virulence of the PRV-Becker strain in a murine model by comparing it to the attenuated, PRV-Bartha strain. rtPCR and ELISA analysis conducted in this study shows that PRV-Becker elevates G-CSF levels at early timepoints post infection and a systemic immune response consisting of both G-CSF and IL-6 across organs shortly follows this initial peak of G-CSF. Furthermore, we found that by effectively knocking out type-I IFN signaling in a mouse model, PRV-Bartha induces an immune response more similar to the PRVBecker strain. With IFN signaling absent, PRV-Bartha induces higher levels of G-CSF and viral load in the Dorsal Route Ganglion (DRG). These results promote a model of infection whereby, PRV-Becker is more virulent due to its ability to successfully evade the IFN response. Overall, this study furthers our understanding on the pathogenesis and virulence of PRV and provides potential molecular targets for future therapies.

http://arks.princeton.edu/ark:/88435/dsp01s4655k44z

Recent epidemiological and experimental findings suggest that HSV-1, in particular, might contribute to the pathogenesis of Alzheimer’s disease (AD), although no causal relation has been demonstrated yet. This neurodegenerative disorder is often associated with intracellular and extracellular accumulation of amyloid beta (Aβ), a hallmark of amyloid plaque deposits, from the processing of amyloid precursor protein (APP). However, little if any research has been done on examining the effects of PRV infection on the processing of APP. The aim of this study was to identify and compare the effects of HSV-1 and PRV infection on APP processing in mouse neuroblastoma cells. We found that HSV-1 infection yielded an important C-terminal fragment cleavage product of APP and also induced elevated levels of Aβ intracellularly and extracellularly. Immunofluorescence imaging also showed that HSV induced aggregation of APP to the endomembrane system. These phenomena were not seen with PRV infection. We hypothesize that HSV-1 ICP34.5 may be critical for disruption of this processing pathway and that repeated HSV-1 reactivation could be a risk factor for AD.

Infection by alphaherpesviruses such as varicella-zoster virus (VZV) is a significant cause of neuropathic itch. VZV produces varicella or chickenpox upon primary infection, remains in a latent state in ganglia, and produces herpes zoster (HZ) or shingles if reactivated. Neuron damage from productive infection may lead to lasting pain or itch. In spite of various treatments and several vaccines, postherpetic itch continues to affect HZ patients. PHI is less studied than postherpetic neuralgia, and its mechanisms have not been defined. Pseudorabies virus (PRV), a swine alphaherpesvirus closely related to VZV, produces similar intense itching in non-natural hosts such as mice. Attenuated PRV-Bartha does not induce itch, so comparing PRV-Bartha and wildtype PRV-Becker will reveal the mechanisms of virus-induced neuropathic itch. To establish the mouse hind footpad inoculation model, we must first track how infection spreads and then identify inflammatory mediators responsible for pathology. This study focuses on the first objective; we aim to characterize viral spread and replication throughout the course of PRV-Becker infection. First, we detected PRV antigen in foot, bladder, kidney, and heart with immunohistochemistry at 72 hours post-inoculation (hpi). Using q-PCR to verify these results, we found PRV DNA in foot, dorsal root ganglia (DRG), spinal cord, and brain for both PRV-Becker (82 hpi) and PRV-Bartha (240 hpi). Finally, q-PCR at 24 and 48 hpi showed PRV-Becker replicates in the foot by 24 hpi. Infection spreads to the DRG and spinal cord between 48 and 82 hpi, correlating with development of itch around 66 hpi. Comparing these results with those from PRV-Bartha infection (ongoing experiments) and correlating with cytokine production will increase understanding of virus-induced neuropathic itch.

http://arks.princeton.edu/ark:/88435/dsp014t64gq89m

Viruses are increasingly being used as tracers of neural connections in the nervous systems of animal models. The herpes simplex virus 1 (HSV-1) is a prime target for ongoing research, due to its broad host range, which includes rodents, higher primates and humans. Wild-type HSV-1 can spread in both directions (retrogradely and anterogradely) in a polysynaptic circuit. A strain of HSV-1 known as McIntyre exhibits a unique abrogation of anterograde spread in infected neurons and can only spread in a retrograde fashion. This thesis sought to determine the mechanisms behind this defect, with a specific focus on viral proteins gI and US9. The addition of wild-type gI and US9 proteins led to a significant but incomplete rescue of anterograde spread. Further work must be done to elucidate the effects of glycosylation, the mechanisms of motor recruitment, and the repair of other mutated genes on the spreading abilities of HSV-1 McIntyre. Given that HSV-1 is a chronic human pathogen with very high incidence, understanding its viral spread will be critical to improving antiviral treatments.

http://arks.princeton.edu/ark:/88435/dsp01dn39x4270

The geographical distribution of the four dengue viruses and their primary vector, Aedes aegypti (A. aegypti), has recently undergone a rapid expansion, establishing endemicity in most of the tropics and subtropics. This places a third of the world’s population at risk of dengue infection, making dengue the most important mosquito-borne viral disease today. Since the 1970’s, the incidence of dengue infections has increased drastically and urban dengue hemorrhagic fever (DHF) has emerged as an urgent global health problem. DHF epidemic activity is currently of particular concern in Southeast Asia, where it is one of the leading causes of childhood mortality. This thesis seeks to understand the mechanisms underlying the re-emergence and emergence of dengue and DHF epidemic activity, respectively, and how they direct available and future preventative measures. The historical and epidemiological record suggests that World War II facilitated the worldwide spread of A. aegypti and the four dengue viruses, establishing hyperendemicity in Southeast Asia. This, along with the particular customs and behaviors of the region, presumably stimulated the emergence of DHF epidemic activity in the 1950’s. Until a safe and effective tetravalent vaccine becomes available, the prevention of dengue rests solely on vector control programs. A more thorough understanding of the transmission cycles between A. aegypti dengue viruses and human hots is needed for the development of more successful vector control programs.
http://arks.princeton.edu/ark:/88435/dsp01bz60cz573

Bacteriophage genomes are known to have a "mosaic" structure, which means that a given genome may contain regions highly similar to some phages interspersed with regions highly similar to other phages mixed with unique regions. This paper describes an algorithm to identify individual mosaic segments and quantifies the extent of phage mosaicism in phages that infect a common host, the bacterium M. smegmatis. The results verify previous work referencing the high amounts of phage mosaicism – on average, about half of any given genome of the over 800 in PhagesDB is made up of short mosaic segments that are shared with at least one other phage. Because novel phages are still being discovered frequently, the amount of mosaicism may be even higher. This paper also examines trends in the distribution of mosaic segments based on cluster, date found, and geography, and explores methods to build tree-based phylogenies based on the number of horizontal transfer events among phages.
http://arks.princeton.edu/ark:/88435/dsp015425kd01t