Unique pathogen requires a novel approach to studying virulence
By Seth Palmer
July 29, 2013
Human herpesvirus – commonly known as herpes simplex virus 1 and 2 (HSV-1 and HSV-2) – is like that friend who ends up crashing on your couch and never leaves.
Most of the time he stays out of the way, sort of lurking in the background – relatively innocuous but for the annoying thought of his presence – but occasionally he comes out of the shadows and ends up in the way in your kitchen at mealtime (uninvited and, naturally, wanting a little something for himself); later he interrupts an intimate moment with a perfectly timed and inopportune knock at your bedroom door; and as time drags on, he generally spoils your good times by managing to be everywhere you wish he wouldn’t be at precisely the wrong moments.
But unlike your bum couch-crashing “friend,” you can’t just put herpes out on the street and be done with it.
And worse yet, the Centers for Disease Control and Prevention (CDC) estimate that, in the United States alone, herpes infects over three-quarters of a million (~776,000) new people every year.
“Herpes simplex virus type 1 is fairly ubiquitous around the world,” says Moriah Szpara, an assistant professor of biochemistry and molecular biology at Penn State who was recently recruited from Princeton. "About 70% of all adults have seropositivity, meaning that they have signs of exposure to this virus in their bloodstream, and a fraction of those people suffer cold sores or genital sores -- depending on the site of introduction -- periodically for the rest of their lives. Some people have many reactivations, some people have very few, and nobody really knows why it’s not the same for everyone.”
Beyond being a simple, periodic annoyance, herpes sometimes exhibits hypervirulence, running amok in its host and causing blindness, aseptic meningitis (inflammation of the protective membranes covering the brain and spinal cord), or encephalitis (swelling of the brain) – of which the latter two conditions can be fatal.
In fact, HSV-1 is the leading cause of infectious blindness in the United States and is the main cause of sporadic, fatal encephalitis. Because the drugs currently available to treat HSV only treat the virus’s active, replicating phase and associated symptoms (e.g., oral and genital sores), they do not affect its latency in neurons, which is what allows herpes to infect its host for life.
“In a very small number of cases that we don’t understand the reason for, the virus can progress into the central nervous system and cause encephalitis,” says Szpara, who is also a co-funded member of the Huck Institutes of the Life Sciences and a faculty member of the Bioinformatics and Genomics graduate program. “Because we don’t know why it happens, we can’t predict it and we can’t stop it. It’s good that encephalitis is rare, and most people who have herpes will be fine; but why do those few severe cases occur? We would like to figure that out. Right now, this infection is permanent, and that’s the biggest public health issue surrounding herpes – it’s a dangerous virus, it’s not benign, it’s not friendly, and the infection will never go away. So if you happen to get frequent outbreaks, this will happen for the rest of your life, and we’d really like to be able to fix that.”
Early in her career, Szpara (pronounced like the tiny songbird, but ending with an “ah” instead of an “oh”) is already leading the way in several important areas of human herpesvirus research.
Although a neurobiologist and virologist by training, Szpara has pioneered the use of comparative genomics for analyzing variation in HSV – variation which is key both to understanding the genetic factors that contribute to virulence and ultimately to developing a vaccine or a cure for the virus.
“The way we fix the problems caused by herpes is by understanding what the virus is doing in neurons,” Szpara says. “That thing you see on your skin is a dead-end where the virus is jumping ship to get to the next person. The place where the virus hides is in your neurons, and it doesn’t kill those neurons – it will kill your skin cells, but it uses your neurons as its home base – so that’s where I come in: I’m a virologist and a neurobiologist with a toolbox in comparative genomics. As a neurobiologist, it’s my job to figure out what the weak link is in neurons. And if I can't find it there, then I hope that by using comparative genomics to survey the different viruses circulating out there – since not everybody’s virus is the same – that we’ll find a weak link there. If we could find out what makes one virus worse than another, we might be able to find a way to disable the ones that are really virulent, or maybe we’ll find one that’s really weak that can be used to immunize people. Any of those are possible outcomes.”
Postdoc to present-day
During her postdoctoral work at Princeton, Szpara introduced the use of high-throughput DNA sequencing to her field -- producing the first new HSV genome sequences in two decades. That provided the raw material for the first genome-wide comparative analysis of alpha-herpesviruses (the class of herpesviruses that includes HSV-1 and HSV-2), yielding valuable insights into the diversity of herpesvirus genetic sequences and protein coding ability. Building on this work, Szpara applied the same approaches to studying a related animal herpesvirus, pseudorabies virus (PRV), and discovered several key genomic characteristics contributing to virulence and mutability in PRV. Multiple vaccine strains of PRV, including one recently sequenced by Szpara, serve as a model for HSV vaccine development.
At the same time, Szpara pursued the question of how neurons respond to pathogen infection. In mouse models of HSV-1 and PRV infection, Szpara measured genome-wide transcriptional responses (transcription being the first step in the process of gene expression, which is used by viruses to complete their lifecycles), and discovered over 2,000 significant changes in gene expression. The viral genes that cause those changes in neuronal gene expression can now be targeted with therapeutics for the development of a vaccine or cure.
Now at Penn State, Szpara is focused on finding causes for the range of virulence observed in human herpesvirus – which is crucial to developing a vaccine or cure with maximum efficacy. Szpara is also looking at how HSV infection and latency affect host neurons and neighboring cells, with an eye toward finding intrinsic neuronal features and cellular defense mechanisms that may also provide targets for therapeutics to block the progression of infection.
“There are two sides to what I do,” says Szpara. “One is trying to understand the biology of herpesviruses in neurons, which has not been studied in depth. We know a lot about herpesvirus biology in epithelial cells, but far less about its biology in neurons – not nearly enough to have come up with any therapeutic that is specific to neurons. The other part of what I do is dealing with variation in this virus. What makes the strain that infects Person A different from the one that infects Person B? Why does Person A get cold sores all the time, and Person B gets them maybe once every five years? What’s the difference?”
By better understanding how human herpesvirus works and what differentiates the many strains of the virus, Szpara hopes to be able to identify the specific genetic loci that contribute to virulence. And by transferring the DNA sequence fragments found at those loci between HSV strains – for example, inserting a candidate gene from a more-virulent strain into a strain that is known to be less virulent – Szpara will be able to observe the effects of that gene, in vivo in mouse models and in vitro in cultured neurons, and confirm whether the it increases or reduces virulence.
“In the lab,” Szpara says, “we have mouse neurons that we can culture and use as a proxy for human neurons, and we can use them to look at the cell biology of what the virus is doing and changing in those neurons. We can look at how those neurons respond and we can try to figure out if the response to a really virulent strain is different from the response to a pretty weak herpesvirus. That may be part of the puzzle of why herpesvirus outbreaks are more severe for one person than another.”
Szpara’s lab also uses viral genome sequencing to search for other parts of this puzzle. For this research they collect viral strains from different people, different body sites, or from cases with varying extremes of symptoms. These viruses are studied using Illumina high-throughput sequencing, and bioinformatics tools that detect the differences between strains.
“When we compare those strains to each other,” says Szpara, “we try to understand exactly what in their genetic content caused them to differ in virulence. Once we’ve done that, we can swap the regions that differ and then test the outcome in the neurons we’ve cultured or into an animal model like a mouse. And if we can accurately test and prove those things, then maybe we can find a strain that’s definitively weak – and weak in a way that makes it a good, safe candidate for a vaccine.”
The draw of Penn State
“I came to Penn State,” says Szpara, “because it has the best combination of all the disciplines and resources that I need. I’m working in the Millennium Science Complex (MSC) – a biosafety level-2 building with its own animal facility – which is perfect for my work with HSV and mouse models. I’m part of the Huck Institutes, the Center for Infectious Disease Dynamics (CIDD), the Center for Molecular Investigation of Neurological Disorders (CMIND), and the Center for Systems Genomics – all of which promote and facilitate interdisciplinary and interdepartmental research and collaboration. And I’m surrounded by other people who study every kind of pathogen we know, either in this building or somewhere on this campus: neurobiologists who are very helpful for the work I’m doing culturing neurons; folks doing bioinformatics and genomics work who are extremely valuable to my research with genome sequencing and comparisons; and immunologists, who are really important to my understanding of what happens in pathology in people. Both the physical proximity and the general culture are such that all these people know each other and regularly talk to one another – so here it’s easy for me to work across these disciplines.”
As a co-hire between the Huck Institutes and the Department of Biochemistry and Molecular Biology (BMB), Szpara has access to a number of peers with a variety of specialties focused in both biology and chemistry.
“At the same time that my colleagues in the Huck Institutes span a broad swath of biological expertise," Szpara says, "my colleagues in BMB provide a specifically molecular and biochemical foundation for my work, and they have expertise on whatever we uncover in the search for neurovirulence. The virulence of herpesviruses could be dependent on many factors – whether it’s related to transcriptional regulation via RNA or chromatin, or intra- or inter-cellular signaling, or modification of protein function – all of these are addressed in someone’s particular specialty in BMB.”
Szpara also finds Penn State’s campus system appealing in the possibilities it presents for collaboration – in particular, the connections between faculty at University Park and the College of Medicine in Hershey.
“In addition to everyone here at University Park," says Szpara, "there are folks in Hershey at the College of Medicine who I can collaborate with to study clinical isolates of HSV that have never before been examined in the lab. The fact that the College of Medicine is part of Penn State will facilitate our having an easy and active exchange.”
And in addition to the number and variety of colleagues and potential collaborators, Szpara was impressed by Penn State's technology infrastructure.
“In my decision to come to Penn State," Szpara says, "it's hard to overstate the importance of the people, but this university also has amazing technological resources. As part of my startup, there is an Illumina MiSeq for high-throughput genome sequencing as well as a microarray facility right here in MSC for me to use. And there’s also the Genomics Core Facility, just a few buildings away, which has its own suite of high-throughput sequencing equipment, and they also have the additional capacity to do gene expression analysis. There are important bioinformatics resources here on campus, as well – high-performance computing clusters that I can use, even staff bioinformatics support for training and problem-solving. And the Huck Institutes have their own IT group in place – we don’t have to fight to have good IT support here in our own buildings in addition to what’s already present centrally on campus, because there’s been a prior institutional investment. You don’t want to have to build these resources from scratch if you can help it, and it makes Penn State really appealing that they have invested in all of this and have it already present.”
As she embarks on the admittedly lofty goal of eliminating the public health threat of herpes, Szpara emphasizes the inspiring research environment that she’s found at Penn State:
“It’s all about the people and the infrastructure," she says. "The ‘who’ that’s here and the ‘what’ that’s here are what really matter, and they are what will facilitate the goals of my research.”
Dr. Szpara’s research is supported by the Huck Institutes of the Life Sciences and the Department of Biochemistry and Molecular Biology at Penn State, as well as by grants from the Virus Pathogens Resource (ViPR) and the National Institute of Allergy and Infectious Diseases (NIAID).
Selected publications and additional resources
- Szpara ML, Tafuri YR, Parsons L, Shamim SR, Verstrepen KJ, Legendre M, Enquist LW. A wide extent of inter-strain diversity in virulent and vaccine strains of alphaherpesviruses. (2011) PLoS Pathog 7(10): e1002282.
- Genome browser and bioinformatics analysis site by M. Szpara & P. Koppstein
- Szpara ML, Tafuri YR, Enquist LW. Preparation of viral DNA from nucleocapsids. (2011) J Vis Exp; e3151. Online video.
- Szpara ML, Kobiler O, Enquist LW. A common neuronal response to alphaherpesvirus infection. (2010) J Neuroimmune Pharmacol. Sep; 5(3): 418-27.
- Szpara ML, Parsons L, Enquist LW. Sequence variability in clinical and laboratory isolates of herpes simplex virus 1 reveals new mutations. (2010) J Virol. May; 84(10): 5303-13.
- Data website by M. Szpara & L. Parsons
- Granstedt AE, Szpara ML, Kuhn B, Wang SS, Enquist LW. Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus. (2009) PLoS One. Sep 9; 4 (9): e6923.