Research Projects in Ly’s & Liang’s Labs
How virus-host interactions affect Lassa and Influenza virus
replication, virulence and pathogenesis?
Lassa fever virus
Influenza virus
Lassa Virus Causes Lethal Hemorrhagic Fever
• Severe multisystem syndrome
• Damage to overall vascular system
• Severe symptoms often accompanied by hemorrhages
Arenaviridae
Bunyaviridae
Filoviridae
Flaviridae
Rhabdoviridae
?
2 ambisense
ssRNAs
3 (-)/ambisense
ssRNAs
(-) ssRNA
(+) ssRNA
(-) ssRNA
Lassa (BSL4)
Junin (BSL4)
Machupo (BSL4)
Guanarito (BSL4)
Sabia (BSL4)
Chapare (BSL4)
Lujo (BSL4)
Crimean-Congo H.F.
(BSL4)
Hantavirus (BSL3)
Rift Valley Fever
(BSL3)
Ebola (BSL4)
Marburg
(BSL4)
Kyasanur Forest
Disease (BSL4)
Omsk H.F.
(BSL4)
Yellow Fever
(BSL3)
Dengue (BSL2)
Bas-Congo (BSL4)
Overall Research Objectives & Approaches
• Virulence factors & mechanisms
• Virus-induced immune suppression
• Pathogenesis
proteomics
1. Animal model 2. reverse genetics 3. cell cultures 4. X-ray crystallography 5. Y2H &
Pichinde virus infected guinea pigs
Mass Spect
Hemorrhagic fever-like syndromes in virulent
Pichinde virus-infected guinea pigs
Avirulent
infection
Virulent
infection
liver
stomach
intestine
skin
Pichinde virus: BSL2 arenavirus that is non-pathogenic in humans
Multi-functional roles of Lassa
nucleoprotein in viral RNA synthesis and
host immune suppression
N terminus of Lassa nucleoprotein has a cap binding
pocket required for viral transcription
C
domain
side view
N domain
Cap
binding
pocket
Changjiang Dong, Ph.D., Univ of St Andrews, Scotland
Cap
binding
Qi et al., December 2010, Nature
pocket
Qi et al., December 2010, Nature
Lassa viral minigenome transcription assay
LASV mini-genome with luciferase reporter gene
Luc
5'
+
CMVp
1000
1
Y213A
10
R300A
Y209A
W12A
K309A
W164A
F176A
Y319A
E266A
K253A
wt-15
wt-30
wt-62
w t-125
w t-250
w t-500
vector
10
100
R323A
100
WT
Fold increase of RLuc activity
1000
1
LUC assay
NP
vector
Fold increase of RLuc activity
L
293T transfection
CMVp
Qi et al., December 2010, Nature
C terminus of Lassa nucleoprotein has 3’-5’ exoribonuclease
activity required for innate immune suppression
3’-5’
exoribonuclease
domain
Top view
C
domain
3’-5’ exoribonuclease cavity
N domain
Qi et al., 2010, Nature
Hastie et al., 2011, PNAS
Brunotte et al., 2011, JBC
Hastie et al., 2012, PloS One
Jiang and Huang et al., 2013, JBC, in press
Roles of NP in mediating innate immune suppression and in
enhancing viral replication
Virus-receptor
interaction
Virus replication
Pathogen-associated
molecular patterns
NP
Short dsRNA
Host’s pathogenrecognition receptors
NP
5’pppRN
P P P
A
RIG-1
Long dsRNA, higherorder structure
NP
MDA5
NP
L
Viral RNA
synthesis
IFN-
Qi et al., December 2010, Nature
Summary #1
• Pichinde virus-infected guinea pig as a good surrogate animal model for Lassa
and other hemorrhagic fever diseases.
• The reverse genetics systems of Pichinde virus (BSL2 pathogen) to identify the
virulence determinants and to characterize the virulence mechanisms of Lassa
virus infection.
• Structure-function characterization of Lassa nucleoprotein has revealed
unexpected roles for NP in mediating viral transcription and immune evasion.
Possible collaborative projects
1.
2.
3.
4.
5.
6.
Exploit known 3D structure of Lassa nucleoprotein for rational design of novel
antiviral compounds
Use Lassa minigenome assay to screen for compounds against viral
transcription
Use guinea pig animal model to test compounds against viral hemorrhagic
fevers (coagulopathy, hearing loss, liver hepatitis, congenital viral infection)
Use modified PICV reverse genetics system to deliver transgene into cells
Use modified PICV reverse genetics system as novel vaccine vector platform
Develop antiviral therapeutics targeting virus-host interactions
FDA-approved drugs for influenza
Class
Agents
Brand Name
Approval year
Viral strains
Amantadine
Symmetrel
1966
Flu A
Rimantadine
Flumadine
1993
Zanamivir
(GG167)
Relenza
1999
Flu A/B
Oseltamivir
(GS4104)
Tamiflu
1999
Flu A/B
M2 inhibitors
Flu A
NA inhibitors
Problem: Rapid emergence of drug-resistant strains.
- Develop novel anti-flu drugs
- Use combination of diverse classes of anti-flu drugs
- Targeting host components as an alternative antiviral approach
Virus and host factors involve in influenza replication
Virus
release
Virus
entry
Receptor Tyrosine Kinases
TrKA
Virus
budding
Protein
Maturation
fusion
Genome
Packaging
Protein
translation
uncoating
vRNP
nuclear
export
vRNP
nuclear
import
mRNA
vRNA
cRNA
vRNA
Viral RNA synthesis
NF-κB
signaling
Liang, et al., 2005, J Virol
Regan, et al., 2006, J Virol
Liang et al.,, 2008, J Virol
Kumar et al. 2008, J Virol
Kumar et al. 2011, J Virol
Kumar et al., 2011, AAC
Liang, et al., 2012, PLoS One
TrkA inhibitors strongly inhibit flu virus replication in vitro
A/PR8
A/Aichi x31
7
7
6
6
5
4
3
2
1
5
4
3
2
A9
AG494
5
5
4
3
2
DMSO AG879
A9
AG494
4
3
2
1
1
1
DMSO AG879
B/Victoria
6
Virus yield [log(pfu/ml)]
6
Virus yield [log(pfu/ml)]
7
Virus yield [log(pfu/ml)]
Virus yield [log(pfu/ml)]
A/WSN
8
DMSO AG879
A9
AG494
DMSO AG879
A9
AG494
TrkA inhibitor inhibited flu virus replication in vivo and
protected mice from lethal flu infection
Kumar et al. JVI 2011
Kumar et al., AAC 2011
Possible Collaborations
• Various convenient cell-culture based assays to screen for possible new drug
candidates against different steps of influenza virus replication
• Host signaling pathways (TrkA) play important roles in influenza virus replication and
can serve as alternative anti-flu targets for drug screening.
Other viruses of human and veterinary importance
Avian influenza surveillance and host-pathogen interaction (Drs. Carol Cardona and
Zheng Xing), swine influenza virus surveillance, virus-host interaction, and viral
evolution in response to vaccination (Drs. Sri Sreevatsan, Montse Torremorell, Maxim
Cheeran, Tom Molitor)
Porcine respiratory and reproductive syndrome virus (PPRSV) and circovirus (PCV2):
molecular virology & anti-viral immunity (Dr. Michael Murtaugh)
Avian reoviruses (arthritis, runting, stunting syndrome and malabosorption in chickens and
arthritis and enteritis in turkeys) (Dr. Sagar Goyal)
Emerging phleboviruses (severe fever with thrombocytopenia – SFTS virus) and
Heartland virus (HLV) (Dr. Zheng Xing): host innate immune suppression
Infectious disease ecology: disease transmission and modeling of canine distemper,
rabies, and parvovirus infections of African carnivores (lions) in the Serengeti
ecosystem (Dr. Megan Craft)
Cardona lab major projects
• Phenotyping of influenza A viruses recovered during
surveillance activities in relevant animal hosts.
– Determining host susceptibility, infectious doses
– Measuring host responses to infection
– Goal: which virus or gene segment could contribute to human or
animal disease?
• The host and virus interactions of mixed infections with
orthomyxo- and paramyxo–viruses.
– Interactions in vivo and in vitro
• The development of the chicken gut virome. Following the flow
of the virome from breeders to their progeny through maturity in
a farm environment.
Elucida on of influenza-swine interac ons in popula ons
Goal: develop an understanding of immune responses in emergence of viral variants in the swine popula on and
develop new approaches for protec on against influenza
Drs. Maxim Cheeran, Srinand Sreevatsan, Montsera Torremorel and Tom Molitor
Viral behavior in the
host
Surveillance
• Farm specific profile
• geo distribu on
• vaccina on effects
on strain distribu on
Biological Outputs
1. Understanding dynamics
of gene c change in
mixed infec ons
(compe on versus
viral/strain interference).
2. Popula on immunity.
3. Host-pathogen
interac ons – a systems
analysis using new
genera on methods
Swine Ecosystem
endemic
naïve
Swine Influenza virus
e
vaccinated
unvaccinated
avian
Transmission
Dx Outputs
1. Novel in vivo viral
diagnos cs and strain
typing methods.
2. Biological assays to index
subtype specific host responses
to natural infec ons and vaccines
In popula ons
human
Influenza A viruses
• Strain specific rates
• vaccina on effects
on transmission
• strain emergence
• sequence profil s
• Microevolu on in
host
environment
• Cell-mediated
immunity (CMI)
• Humoral immunity
• Maternal immunity
Host response
Swine influenza research
Drs. Montserrat Torremorell and Srinand Sreevatsan
• Swine influenza transmission and surveillance:
– Models for influenza transmission using the pig as a model:
• Evaluate the effect of immunity (i.e vaccination and passive immunity) on influenza
transmission and dissemination
• Evaluate the effect of immunity on influenza diversity and evolution
• Determine virus genome diversity within and between animals throughout the course
of infection using de novo sequencing techniques
• Evaluate the role of fomites on transmission in experimental settings
• Development of mathematical models for influenza spread in populations
– Assessing subpopulations, routes and methods of influenza transmission under
high risk interspecies transmission settings:
• Assessment of animal subpopulations responsible for maintaining and spreading
influenza
• Determining the role of indirect routes of transmission (aerosols and fomites) to
influenza exposure in animals and people
– Methods to decrease influenza transmission and assessment of influenza
dissemination in populations
Xing, Zheng PhD
Associate Professor
Department of Veterinary
Biomedical Sciences, CVM
Research Interests: Pathogenesis and hostpathogen
interaction of 1) avian influenza virus (AIV) & 2)
bunyaviruses in human and animals
AIV is a zoonotic pathogen that infects birds and humans.
We study host responses and their regulations in infected birds
and humans comparatively. Using genomic and other high
throughput approaches, we study host-virus infections and
identified cellular proteins that are required for viral replication.
We expect to develop antiviral strategies based on the identified
cellular components essential to viral replication.
The bunyavirus we are studying is the one that causes severe
fever with thrombocytopenia syndrome (SFTS) with a high fatality
rate up to 15% in China. A similar virus, Heartland virus (HLV)
has been identified in the Midwestern region of the US. We are
studying the mechanisms how the viral infection suppresses
host innate immunity. We have discovered a unique mechanism
that HLV uses its nonstructural protein (NSs) to modulate host
Collaborations: Promote mutual interest and
Lab Commitment
facilitate problem solving.
Intellectual Property: Translate fundamental
knowledge into useful products and tools.
Advanced Training: Share expertise with
sponsors and end users.
Murtaugh Laboratory
To discover and disseminate knowledge that
enhances animal health; to prepare young scientists
for careers in agriculture, biology, and medicine; to
provide training in genomics and biotechnology.
www.murtaughlab.com
PRRSV Biology
Virion structural characterization
Glycan structural modifications
Viral evolution and diversity
Porcine Immunology
Infectious Disease
Immunity
Anti-viral immunity
PRRSV, PCV2
Early Warning
Biomarkers
Molecular Mechanisms of
Disease Resistance
Mucosal Immunobiology
Functional genomics of mucosal immunity
Differential gene expression in the gut
Opioids in intestinal innate immunity
Kyra Martins,
Scientist
B-cell function
Immunoglobulin diversity
Th1-Th2 paradigm
Jie Zhang,
Visiting Scientist
Cheryl Dvorak,
Research Associate
Xenotransplantation
Molecular mechanisms of engraftment
Pathway identification for stress reduction
Xenopathogen molecular diagnostics
Gene cloning and expression
Protein modification
Bacterial and yeast expression
Protein discovery
Glycan characterization
Michael Murtaugh
Single gene RT-PCR
Transcriptome profiling
Jenny Zhang,
Jr. Scientist
Xiong Wang,
Graduate Student
Marina Figueiredo,
Graduate Student
Immuno-cellular
Methods
Sally Robinson,
Graduate Student
John Schwartz,
Graduate Student
Virus culture
ELISA and ELISPOT
Core Technologies
Quantitative Gene
Expression
Suzanne Stone,
Sr. Lab Tech.
PRRSV disease signatures
Metabolic disorder markers
Protein Engineering
Mass Spectrometry
Diem Ngo,
Sr. Lab Tech.
Proteomics
Biomarker discovery
Functional genomics
Antigen discovery
Sabbatical
Opportunities
Massively Parallel
Sequencing
Agricultural
Biotechnology
Biomedical
Collaborations
Laboratory Outreach
Population genomics
Exon profiling
Evolutionary mechanisms
Advanced Technical
Training
Biotechnology
Workshops for End
Users
Graduate Education in
Molecular Biosciences
Avian reoviruses
Dr. Sagar Goyal
Chicken reovirus is known to cause:
arthritis
runting and stunting syndrome
malabsorption
Turkey enteric reovirus (TERV) is widespread and is
involved in turkey enteritis
Studies on turkey arthritis reovirus (TARV) are at
least 20 years old.
We have isolated TARV from recent outbreaks of
lameness in turkeys and are studying their
pathogenesis in addition to characterizing the
viruses