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Comparative studies of the replication of African and Asian strains of Zika virus

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Comparative studies of the replication of African and Asian strains of Zika virus

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Content Running head: COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

Comparative Studies of the Replication of African and Asian Strains of Zika Virus
Yimin Chen

Presented to the
School of Keck Medicine
Department of Molecular Microbiology and Immunology
UNIVERSITY OF SOUTHERN CALIFORNIA
Master of Science (Molecular Microbiology and Immunology)
The degree conferral date: May 2019
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

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Abstract
Zika virus (ZIKV) is a mosquito-borne virus and can cause severe diseases. Based on
phylogenetic analysis, ZIKV can be classified into African and Asian lineages. Clinical studies
indicated that these two lineages of ZIKV exhibit differences in their pathogenicity. To
understand how these two lineages of ZIKV may be different from each other, I compared the
replication of two strains of ZIKV, MR766 and H/PF/2013, which belong to the African lineage
and the Asian lineage, respectively. These two strains of ZIKV were produced in either Huh7
human hepatoma cells or Vero green monkey kidney cells, and their infectivity and growth
curves were comparatively analyzed in HeLa cervical carcinoma cells, Vero cells and HepG2
human hepatoblastoma cells. I found that these two strains of ZIKV exhibited differences in their
plaque morphologies, cytopathic effects and growth curves. These effects also differed
depending on whether the viruses were prepared in Huh7 cells or Vero cells. In addition, I also
found that these two strains of ZIKV could induce autophagy in their host cells, although with
different induction kinetics, and they could also induce the expression of autophagy-related
genes including Atg4B, Beclin-1, and AKT. Interestingly, the ZIKV NS2B and NS4A proteins,
but not its envelope protein, were found to colocalize with autophagosomes, indicating the
possible involvement of autophagosomes in the replication of ZIKV. My study thus provided
important information for understanding why these two strains of ZIKV differ in their
pathogenicity in patients.

Keywords: Zika virus, plaque assay, viral growth curve, autophagy, autophagosomes, ZikV
NS4A, ZikV NS2B, cytopathic effect (CPE), HeLa cells, Vero cells, Huh7 cells.

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Table of Contents
Abstract ................................................................................................................................. 2
Introduction ........................................................................................................................... 4
Zika virus .......................................................................................................................................... 4
Autophagy ....................................................................................................................................... 5
LC3 ................................................................................................................................................... 8
P62 ................................................................................................................................................... 9
Autophagy associated genes .......................................................................................................... 10
Atg4B ................................................................................................................................................. 10
ATG5, ATG7, and ATG12 .................................................................................................................... 10
ApoE .............................................................................................................................................. 12
Beclin-1 .......................................................................................................................................... 13
AKT ................................................................................................................................................ 14
Material and Methods ........................................................................................................... 14
Viruses ........................................................................................................................................... 14
Cells ............................................................................................................................................... 15
Huh7-GFP-LC3 cell line .................................................................................................................... 15
ZIKV and DEV-2 plaque assay .......................................................................................................... 16
HCV&DEV-2 FF assay (Focus-forming assay, FFA) ............................................................................ 17
Materials: ....................................................................................................................................... 17
Immunofluorescence assay ............................................................................................................ 18
Immunoblotting ............................................................................................................................. 19
Results .................................................................................................................................. 19
Discussion ............................................................................................................................. 31
Conclusions and Future Study ................................................................................................ 40
ACKNOWLEDGEMENTS .......................................................................................................... 42
References ............................................................................................................................ 43
Data ...................................................................................................................................... 46

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

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Comparative Studies of the Replication of African and Asian Strains of Zika Virus
Introduction
Zika virus
Zika virus (ZIKV) is a member of the Flaviviridae family and the Flavivirus genus. Zika
virus mainly causes fever, rash, joint pain, and other mild symptoms. ZIKV spreads very quickly
and causes serious damage. It is an arbovirus (meaning it spreads through certain arthropods) and
is transmitted by mosquitoes. Moreover, it is a small envelope positive-sense single-stranded
RNA virus belonging to the Flaviviridae genus, which is initially translated as a single
polyprotein. It then cleaved post-translationally into 7 nonstructural (NS) proteins (NS1, NS2A,
NS2B, NS3, NS4A, NS4B, and NS5) and 3 structural proteins (C, PrM or M, and E). Six of the
NS proteins (NS2A to NS5) form a replication complex on the cytoplasmic side of the
endoplasmic reticulum membrane (Medin, C., Rothman, A., 2017).
ZIKV has recently reappeared as a human pathogen with the danger of causing an
epidemic. Phylogenetic analysis shows that ZIKV can be divided into two major systems: the
African lineage and the Asian lineage. According to the genebank, (ZIKV) is a flavivirus that
spreads from mosquitoes in Africa and Asia. According to this, I design my studies by using two
strains of ZIKV specifically harvested from African and Asian strains(Jun, S., Wassenaar, T.,
2017). The ZIKV African strain in my study is MR766, and the ZIKV Asian strain is H/PF/2013.
According to the clinical research, African ZIKV lineage strains are more virulent and adults
infected with the ZIKV Asian strain can clear the virus with a healthy immune system. But the
African Strain of ZIKV can lead to acute infection and cause the patient to die. Since 2015, Zika
Virus Asian strain has been prevalent in many countries such as Latin America and South
America. Patients with Zika virus infection showed symptoms of fever, rash and joint pain,
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

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infantile microcephaly, Guillain-Barre Signs, and viral meningitis. Not only that, but Zika virus
infection can also be transmitted through sexual intercourse. Therefore, from the complexity of
the symptoms of viral infection, the currently popular ZIKV Asian strain is completely different
from the ZIKV African strain that was first discovered in 1947, and has become a "new virus." In
my study, I took two typical types from two ZIKV strains to perform the comparison.
Autophagy
Autophagy is a highly conserved, organized catabolic process and an essential cellular
homeostatic process that controls the turnover of damaged cytoplasmic organelles and proteins to
release nutrients during periods of cell stress or starvation to counteract cell death. This process
initiates with a double membrane vesicle called an autophagosome, which fuses with the
lysosome to form an autolysosome, where the engulfed content is degraded by lysosomal
proteases. Cargo degraded by autophagy include organelles, protein, protein aggregates, and
cytoplasmic components. Autophagy is inhibited by nutrients and mTOR and is induced by
stress, HIF, and AMPK. When the autophagosome is fused to a lysosome that provides a
hydrolase, the cargo will degrade. The breakdown products of autophagy are released into the
cytoplasm where they are recycled into metabolic and biosynthetic pathways. A normal
physiological process maintains homeostasis or general function to form new cell formation
through protein degradation and renewal of damaged organelles. During cellular stress, the
autophagy process is enlarged, expanded, and induced.
Autophagy and apoptosis are two processes of programmed cell death (PCD) of cells,
characterized by bulk degradation of long-lived proteins and re-use of organelles. Currently,
autophagy and the molecular regulation mechanism of apoptosis has been well studied
(Shang YY, et al., 2017). The development of autophagy depends on the participation of a series
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

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of autophagy-related proteins (ATGS) and at least 37 ATG proteins have been identified in
mammalian cells, two of which are ubiquitination pathways (ATG8- PE, and ATG12-ATG5-
ATG16L). These two pathways play an important role in the formation of autophagosomes,
which are involved in the initiation, nucleation, extension, closure, maturation, and degradation
of autophagosomes (Mathieu P, Leeanna EH, et al. 2018). In mammals, many related proteins
such as P53, BECLIN-1 (yeast ATG6 homolog), ATG4, ATG5 families are involved in the
molecular regulation of autophagy and apoptosis. The formation of autophagosomes is
dependent on the participation of autophagy-related proteins (ATGs), which are mainly
ubiquitinated by ATG8-PE and ATG12-ATG5 via a phagophore assembly site (Jin, M., &
Klionsky, D., 2013). Among them, phosphatidylethanolamine (PE) of ATG8, or ATG8-PE, is
the only autophagy that remains on the autophagosome membrane before autophagosome
assembly and lysosome fusion to form autolysosome proteins. By detecting the expression level
of ATG8-PE (pro-LC3), it can be used as clear evidence for evaluating the degree of autophagy.
Obviously, under the normal circumstances, autophagy is a self-protection mechanism
that exists in eukaryotic cells, which can induce the cell self-renewal process for the metabolism
and energy, maintenance of cell homeostasis, and also play an important role for regulating cell
survival and death. Once the autophagy function is disordered, the cells gradually reveal their
devil sides followed with tumors and neurodegenerative diseases.
Generally, mammalian autophagy can be divided into the following three types: (1)
Macroautophagy: the process in which soluble macromolecular substances and denatured
organelles in the cytoplasm are encapsulated by the endoplasmic reticulum, mitochondria-
derived monolayer, or the bilayer membrane to form autophagosomes and then fused into
lysosomes. The autophagy lysosomes, with the inclusion of hydrolases, will degrade the
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

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corresponding substrate, and finally, complete the entire autophagy process. (2) Microautophagy:
this process does not form autophagosomes, but the lysosomal membrane itself can directly
encapsulate and phagocytize the substrate that will be degraded in the cell, and then induce the
degradation inside of the lysosome. (3) Molecular chaperone-mediated autophagy (CMA): The
chaperone protein can recognize and bind to the soluble protein with the specific amino acid
sequence, and then transport to the lysosome and be degraded by the hydrolase via the receptor
Lamp2a on the lysosomal membrane. This process has higher selectivity during the process of
protein degradation, which differs from macroautophagy and microautophagy. When the
autophagy occurs, the membrane structure of the endoplasmic reticulum and mitochondria in the
cytoplasm of the cell can form an autophagic precursor with a double-layered cup-shaped
separator membrane; the separation membrane is continuously extended, and then gradually
wrap the secreted wastes to form the closed autophagosome vesicles. When the autophagosomes
and lysosomes fuse to form the autophagic lysosomes, they can degrade their encapsulated
cellular components and recycle the degradation products.
In addition, autophagy can be subdivided into the following five stages. The first stage is
the induction of autophagy, primarily mediated from ATG1 complexes (including ATG1/ULK1,
ATG13, and ATG17/FIP200). At this moment, the rapamycin fails to target the inhibited protein
complex 1 (mTORC1) and the phosphorylation level of ATG13 is decreased. The ATG13-
ATG1-ATG17 complex is forms and initiates the autophagy process. The second stage is the
vesicle nucleation. Vps34 (PI3K)-ATG6 (Beclin-1) complex (containing regulatory protein
kinase Vps15) can act on the nucleation of vesicles and mediate pre-autophagosome structure
(PAS) Formation. This process can also recruit the ATG12-ATG5 and ATG16 multimers as well
as LC3, which can induce the expansion of phagocytic vacuoles. The third stage is the elongation
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phase of autophagosomes, which mainly depends on the binding process of two ubiquitin-based
systems: the combination process of ATG12-ATG5 and the modification process of LC3. The
ATG12-ATG5 and LC3 can interact and adjust together, and both of them require the
participation of ubiquitin-activating enzymes E1 and E2. The maturation stage of
autophagosomes is the fourth phase. Autophagosomes can form autophagosomes by fusion with
lysosomes via the microtubule skeleton under the reaction between the endocytic classification
complex (ESCRT) and monomeric GTPase (Rab S). The lysosomal-associated proteins include
LAMP1, LAMP2, and UVRAG (UV-resistant tumor suppressor genes). The last stage is the lytic
phase of autophagosomes. It is the process of the cleavage of the autophagosome lysosomal
membrane, with the contents that be degraded by the action of lysosomal hydrolase. The amino
acids and proteins that are produced during the degradation process can provide nutrition,
energy, and recycling utilization for the cells.
The synergy of many proteins to form autophagosomes is part of the autophagy process.
Cells use autophagy to break down large proteins, invading pathogens, cellular waste, and toxic
substances. During the autophagy, a key protein called LC3 can attach to the lipid molecule on
the autophagosome membrane. However, without the help of ATG12 and ATG5, LC3 could not
attach to the lipids which indicates that the conjugation of ATG12 and ATG5 is required for
LC3 lipidation in autophagy. Cells can form autophagosomes only if the connection between
these two molecules can be established.
LC3
After the formation of LC3 precursor(pro-LC3-1, ATG8), ATG4 is processed into
cytosolic soluble LC3-I, associated with ATG7 and ATG3. The liposoluble LC3-PE (LC3-II)
will be formed by covalent cross-linking of phosphatidylethanolamine (PE), which is also
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

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involved in the membrane extension process. Moreover, LC3-II is commonly used as a
biomarker for autophagy formation and a crucial multi-signal regulatory protein that localized on
the autophagic membranes. LC3 synthesis is followed by the premise LC3 (pro-LC3, ATG8);
then the C-terminus is hydrolyzed by ATG4 immediately to leave a segment of the polypeptide
and expose the glycine to form the LC3-I followed by the cytosolic distribution. During the
autophagy, LC3-I will covalently bond to phosphatidylethanolamine with the activation of ATG7
and ATG12-ATG5-ATG16L (similar to the ubiquitin modification process) to form the LC3-II,
which can bind to the autophagosomal membrane. Since LC-II is modified by
phosphatidylethanolamine to cause a change in charge, the electron mobility becomes faster and
appears to have a smaller molecular weight than LC3-I in the SDS-PAGE.
P62
P62 is a well-known autophagy protein, also known as SQSTM1 protein, which plays a
critical role in the autophagy and apoptosis in tumor cells. It is a ubiquitin-binding scaffold
protein that colocalizes with ubiquitinated protein aggregates in many neurodegenerative
diseases and liver protein diseases. It includes 4 domains, namely PB1, TB, LIR, and UBA. The
LIR domain is responsible for binding to the autophagy receptor protein Atg8/LC3. The UBA
domain is capable of recruiting ubiquitinated proteins, especially when the protein is exposed to
oxidants and proteasome inhibitors. As an autophagy-specific substrate, p62 can interact with
LC3 to infiltrate into autophagosomes and be efficiently degraded. Therefore, the expression
level of p62 can also be used to monitor the level of autophagy. However, autophagy is not the
only way to degrade p62. It is hard to determine the autophagic state of the cell based on just the
expression level of p62 protein. The p62 protein recognizes toxic cellular waste and then clears
the waste with an isolation process called self-feeding or autophagy. The lack of autophagy
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results in the accumulation of p62, which is detrimental to hepatocytes because it will induce a
cellular stress response to induce the diseases.
Autophagy associated genes
Atg4B
ATG4, a cysteine protease of the C54 family, plays a crucial role in the ATG8 (pro-LC3)
junction system. ATG4 can cleave the C-terminal arginine of ATG8 to expose the C-terminal
glycine residue for covalent attachment to PE to form ATG8-PE anchored to the autophagic
membrane. Next, ATG4 can also deliberate ATG8-PE to fuse autophagosomes with lysosomes
and get recruited to the autophagosomal membrane for membrane elongation. Therefore, the
process of autophagy can be regulated by regulating ATG4-mediated delipidation of ATG8-PE.
Members of the ATG4 family play a very important role in the process of lipidation and
delipidation of ATG8 family members to form autophagosomes. ATG4 has only one member in
yeast, and its lack of function will block the progression of autophagy. In mammalian cells,
ATG4 has four family members: ATG4A, ATG4B, ATG4C, and ATG4D. ATG4B is the most
widely studied member of the ATG4 family and has enzymatic activity against ATG8 family
members (LC3 family and GABARAP family). It has been reported in a paper that ATG4B plays
an important role in the development of tumors. ATG4B is considered to be an oncogene that
promotes the growth of colon cancer cells and is independent of autophagy regulation.
ATG5, ATG7, and ATG12
Atg5 is a required protein for autophagy specificity for the formation of autophagosomes,
a lysosomal catabolic pathway for organelles and proteins. Moreover, ATG5 is present in most
eukaryotes and relatively conserved, playing an important role in the formation of autophagic
vacuoles (AV) (Ma T, Li J, 2015). As it is, in yeast, in the early stage of autophagic formation
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(Preautophagosome), the complex formed by ATG12-ATG5-ATG16L will bind to the
Preautophagosome’s outer membrane. This kind of combination, on one hand, promotes the
expansion of autophagic vacuoles from the vesicle-like and cup-like structures and gradually
develop into semi-annular and cyclic structures (Chen ZH, Cao JF, et al. 2014). On the other
hand, the combination between the ATG5 complex and the autophagic membrane can promote
the recruitment of LC3 (Atg8) to autophagic vacuoles (Ritu Malhotra, James P Warne, et al.
2014). The orientation of the ATG5 complex on the membrane determines the direction of
curvature of the membrane, which extends in a reverse direction of the ATG5 complex. When
the autophagic vacuole of the bilayer membrane structure is about to form a circular closed
structure, the ATG5 complex is detached from the membrane, leaving the membrane-bound
LC3-II (ATG8-PE) to localize on the membrane of the autophagic vacuoles. Therefore, the
amount of LC3-II is measured by the number of autophagic vacuoles. When autophagy occurs in
mammalian cells, the content of LC3 in the cells and the conversion between the LC3-I and
LC3-II are significantly increased (H H Lin, S-M Lin, et al. 2014). Thus, by detecting changes in
the content of LC3-II in cells, it is convenient to judge the state of the cells and determine
whether autophagy is induced or inhibited (Klionsky, D., Abdelmohsen, K, et al. 2016).
ATG12-ATG5 and LC3 are two kinds of typical biomarker proteins on the
autophagosome membrane. The autophagy phenomenon can be judged by western blot detection
of the kinases of the marker proteins. However, since ATG12-ATG5 is only present on the
bilayer membrane in the early stage of autophagy, and LC3 is continuously cleaved and
degraded during autophagy, it is difficult to judge whether autophagy is activated or inhibited.
Basically, the most common way to confirm autophagy is the corresponding of the P62 and LC3-
II/LC3-I. ATG7 also mediates the formation of the ATG5-ATG12-ATG16 complex (which can
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be associated with LC3-II), which is extremely critical for the formation of autophagosomes. The
ubiquitin-like attachment of Atg12 and Atg5 should co-react with the Atg7 and Atg10, which has
similar functions to the E1 and E2 enzymes. Moreover, ATG5 can also act as a pro-apoptotic
molecule targeting mitochondria. If there is a slight damage of DNA, ATG5 will be transferred
to the nucleus and interact with surviving. As it is, ATG5 is known to be regulated by a variety
of stress-induced transcription factors and protein kinases.
ATG12 can be activated by ATG7, transported by ATG10 and bound to ATG5, and
combined with ATG16 to generate a multimeric complex of ATG12-ATG5-ATG16. This
complex localizes to the outer membrane surface of the pre-autophagosome structure and is
involved in the expansion of the pre-autophagy membrane. After formation of LC3
precursor(pro-LC3, also known as ATG8), ATG4 will process into cytosolic soluble LC3-I, with
the association of ATG7 and ATG3. The phosphatidylethanolamine (PE) is covalently linked to
form liposoluble LC3-PE (LC3-II), which is involved in the membrane extension process. The
LC3-II is commonly used as a biomarker for autophagy formation and is also a critical multi-
signal regulatory protein that localizes on autophagic membranes.
ApoE
Apolipoprotein E (ApoE) is a polymorphic protein involved in the transformation and
metabolism of lipoproteins. Moreover, as one of the human apolipoproteins, a component of
lipoprotein granules, it is involved in the synthesis, secretion, processing, and metabolism of
lipoproteins and plays an important role in blood lipid metabolism. Its genes regulate many
biological functions and are involved in the pathogenesis of many diseases. ApoE is a ligand for
LDL receptor, a ligand for hepatocyte CM remnant receptor, and is closely related to lipoprotein
metabolism. ApoE has polymorphism, which determines individual blood lipid level. and is
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closely related to the development of atherosclerosis; ApoE is involved in the activation of
lipohydrolase and participates in immune regulation and regeneration of nerve tissue. Human
ApoE is mainly synthesized in the liver and brain tissues, and also has the ability to be
synthesized in other tissues including monocytes (including macrophages). Human adrenal and
ovarian granulosa cells can also synthesize ApoE.
Beclin-1
Since the mammalian BECLIN-1 gene was cloned in 1998 (Liang XH, Kleeman LK, et
al., 1998), the research on ATG6 (yeast)/BECLIN-1 (mammal) has been intensively studied.
Liang et al. first confirmed that BECLIN-1 is an autophagy protein and tumor suppressor
molecule, which plays a critical role in the autophagy and tumor suppression ((Liang XH,
Kleeman LK, et al., 1998), And Rohatgi et al. demonstrated that BECLIN-1 plays an essential
role in the regulation of autophagy as part of the PtdIns3KC3 1 complex in yeast, like
ATG6/Vps30 in yeast. Role (R A Rohatgi, J Janusis, et al., 2015), Yazdankhah et al. used
affinity chromatography and pristine analysis to confirm the important role of ATG14 and
Rubicon in the formation of autophagosomes. ATG14 is one of the major components of the
PtdIns3KC3 1 complex, also known as ATG14L or Barker (BECLIN- 1-associated autophagy-
related key regulator), which promotes the formation of autophagosomes, while Rubicon acts as
an inhibitor (M Yazdankhah, et al., 2014). They found more important proteins in mammals and
yeast that make up the BECLIN-1 complex, such as the UV radiation resistance-associated gene
(UVRAG) in mammals (Itakura et al., 2008), homologous to yeast in Vps38, and Ambra1
protein, binding to BECLIN-1 and Vps34 proteins, in developing embryos Promote autophagy in
the fetal nervous system (Ryan C. Russell, et al., 2013), which may also be associated to the
diseases induced from ZikV Asian strain.
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BECLIN-1 also plays a critical role in the regulation of autophagy and apoptosis. Under
the normal condition, the BH3 structure of BECLIN-1 protein and the apoptosis inhibitory
protein B-Cell Lymphoma Protein 2 (Bcl-2) Or Bcl-XL (Bcl-2-like protein) binding can inhibit
the occurrence of autophagy. However, if the cell survival environment changes abnormally,
such as lack of growth factors or starvation, Bcl-2 or Bcl-XL will disaggregate from BECLIN- 1,
it will induce the occurrence of autophagy (Ting Sun, et al., 2015). On the other hand, Caspase-3,
Caspase-7, and Caspase-8 were activated and cleaved the 37 kDa and 35 kDa BECLIN-1-C
protein fragments at the TDVD133 and DQLD149 sites of BECLIN-1 respectively. The
BECLIN-1 protein loses the ability for inducing autophagy, localizes to mitochondria, promotes
the release of CytC in mitochondria, and induces apoptosis (Huang, X., et al., 2014).
AKT
Protein kinase B, or Akt, also known as PKB or Rac, plays a critical role in cell survival
and apoptosis. The growth and survival factors such as insulin can induce the Akt signaling
pathway. Thr308 of Akt can be phosphorylated by PDK1. Activated AKT can regulate cell
function via phosphorylating downstream factors such as various enzymes, kinases, and
transcription factors. AKT exerts anti-apoptotic effects through various downstream pathways to
phosphorylate the target proteins. ATK can activate IkB kinase (IKKα) to cause the degradation
of the inhibitor IκB for the NF-κB and release NF-κB from the cytoplasm for the nuclear
translocation and induces its target gene to promote cell survival.
Material and Methods
Viruses
Zika virus strains/isolates MR766 (Rhesus/1947/Uganda), Zika virus strains/isolates
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

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H/PF/2013, Dengue virus type 2.
Cells
BHK-21, Vero, Huh7.5.1, Huh7, Huh7-GFP-LC3. Cell lines were maintained in
Dulbecco's Modified Eagle Medium (DMEM), which BHK-21 and Vero cell lines are
supplemented with 1% 100x Antibiotic-Antimycotic and 5% fetal bovine serum (Invitrogen);
Huh7.5.1 cell line is supplemented with 1% non-essential Amino Acids, 10% fetal bovine serum
(Invitrogen) and 1% 100x Antibiotic-Antimycotic; Huh7 cell line is supplemented with 1% non-
essential amino acids, 10% fetal bovine serum (Invitrogen) and 1% 100x Antibiotic-
Antimycotic; Huh7-GFP-LC3 cell line is supplemented with 1% 100x Antibiotic-Antimycotic,
1% Geneticin G418 and 10% fetal bovine serum (Invitrogen); Hela cell line is supplemented
with 1% 100x penicillin-streptomycin, 5% fetal bovine serum (Invitrogen); HepG2 cell line is
supplemented with 1% non-essential amino acids, 10% fetal bovine serum (Invitrogen) and 1%
100x Antibiotic-Antimycotic, and the dishes for maintaining the HepG2 cell line should be pre-
coated and incubated in PBS with 0.2% collagen in 36.6°C with 5.2% CO
2
for one hour.
Huh7-GFP-LC3 cell line
Huh7-GFP-LC3 is a Huh7 hepatoma cell line that stably expressed the green fluorescent
protein (GFP)-LC3 fusion protein. When the autophagy did not occur, with the transfected
endogenous LC3 and green fluorescent protein GFP-labeled LC3 cells, green fluorescence
showed a diffuse state in the cytosol, which was randomly distributed without the specific
shape. If the autophagy occurred, a large number of autophagosomes will be produced by
the cell. while the endogenous LC3 or exogenous GFP-LC3 appear as a bright green spot on
the autophagosome membrane, to confirm the autophagy state from the cell.
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ZIKV and DEV-2 plaque assay
Passaging and counting 1.7 million cells into the six-well dish with 1.5ml media for each
well.
As to the Zika virus plague assay, Vero cells were plated the day before infection at 3 x
10
5
cells/well. As to the Dengue type 2 virus, BHK-21 cells were plated two days before
infection at 2.5 x 10
5
cells/well.
At the infection section, using FBS wash serum first. Adding 1ml serum-free DMEM for each
well. Six different 10-fold dilutions of purified virus were spread onto monolayers of Vero cells
or BHK-21 cells at 37°C for 3h to initiate binding to cells. then medium containing virus
particles was replaced with 4ml overlaid media. Cells were maintained at 36.6°C in 5.2% CO
2
.
As to ZIKV, I maintained the dishes for 4-5 days. As to Dengue Virus, I maintained them for 2
days.
The size of the plaque was determined by comparing the measured area of a plaque to that of a
well in the 6-well using the formula: the number of plaque per well = (lowest plaque number
well x dilution fold + second lowest plague well x dilution fold) x 5 ÷ 2. Resultant plaques were
photographed using an iPhone 7 plus camera.
Protocol for the overlaid media (cover gel):
A part: 2xDMEM; 4% of FBS; 4mM of L-glutamine; 2mM of Sodium Pyruvate; 5mM HEPES
buffer; 2x antibiotic antimitotic.
B part: 10g of Methylcellulose (CPS 4000) in 500ml ddH
2
O. Sterilized for one liquid cycle.
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Mixed B part with A part.
Fixation section: adding 10% Formaldehyde in PBS for one hour.
Staining section: 1% crystal violet in 20% methyl alcohol for one hour.
HCV&DEV-2 FF assay (Focus-forming assay, FFA)
It is an alternative method for determining viral titer is an FFA. The FFA is analogous to
a plaque assay, except that it uses peroxidase immunostaining to identify foci of infected cells,
rather than plaques. The FFA is a faster assay, requiring an overnight incubation compared to a
4-day incubation for a normal (ZIKV) plaque assay. It is also higher-throughput, being
performed in 8-well chamber slide rather than 6-well tissue plates. Moreover, it may be
especially useful for virus strains that do not form clear plaques. Two disadvantages of the FFA
are that it requires specific antibodies to detect HCV/DEV-2-infected cells, which may be
confounding when considering diverse virus strains or mutant viruses, and it is hard to count
under the microscope.
Materials:
Vero cells; Serum-free DMEM; Acetone; Phosphate-buffered saline(PBS); antibodies;
Dapi buffer; 6-well chamber slide; automated cell counter.
Processes ：
1. Seed Vero cells at 4 x 104 cells/well (by using the automated cell counter) in 8-well
chamber slide.
2. Aseptically peel back tray overlay.
3. Remove the media chamber cover. Fill the chamber with 0.4 ml of medium and cell
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

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suspension in the well. Replace cover.
4. Incubate for 24 hours.
5. Remove slides from the incubator and discard medium.
6. To detach slide from media chamber, grip end of the slide with one hand. Gently squeeze
both ends of media chamber toward the center lifting chamber as gasket release.
7. Fix by using the acetone about 1 minus.
8. Stain with the antibodies. The gasket may be used as a reservoir for reagent incubation.
9. Gasket removal. Insert tip of a thin-bladed spatula or similar tool under gasket at one
corner. Without stretching or tearing the gasket, smoothly lift it away from the slide.
10. Coverslip, using the Dapi to stain the nuclear.
11. Using the microscope to scanning the sample and count the active cells’ amount.
Immunofluorescence assay
In order to confirm the induction of autophagy by HCV, ZIKV, and DEV-2, I performed
fluorescence microscopy. Huh7-GFP-LC3 cells were plated on gelatin-coated glass coverslips
the day before infection into 6cm plates at 1 x 106 cells/plate.
After 36h-pi, the cells were washed for 2 times with 1X phosphate buffered saline (PBS).
After wash, cells were fixed in pre-cooled acetone for 1 minute at room temperature and then
metabolized with 0.2% Triton-X 100 for 10 minutes. The ZIKV envelope protein was detected
using a 1:200 dilutions of the primary antibody isolated and 1:50 of the Alexa Fluor goat-anti-
rabbit or rabbit-anti-mouse antibodies. Images were captured by a Zeiss LSM 700 laser scanning
confocal microscope.
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

19
Immunoblotting
Cells were grown in 10cm plates and lysates were prepared with RIPA buffer (50 mM
Tris- HCl [pH 7.4]; 1 mM EDTA; 1 mM sodium orthovanadate; 0.25% sodium deoxycholate;
150 mM NaCl; protease inhibitor cocktail (Sigma); 1% NP-40) with protein s inhibitor, which is
a vitamin K-dependent plasma glycoprotein synthesized in the liver. The insoluble material was
precipitated by brief centrifugation. The protein concentration of lysates was determined by BCA
protein assay (Thermo Scientific). Lysates containing equal amounts of protein were loaded onto
8% or 15% SDS-PAGE gels (specific for the LC3 protein detection) and transferred to a
nitrocellulose membrane (LI-COR, Lincoln, NE), blocked with 5% milk for 1 hour in RT, and
incubated with the primary antibody overnight at 4 °C. Membranes were blocked in 5% milk,
followed by incubation with primary antibodies at 1:1000 dilutions. Membranes were washed
three times with 1X TBST buffer which containing 0.05% Tween20 (v/v), incubated with
secondary antibodies for 1 hour in RT, and repeated the washing steps to remove unbound
antibody then scanning under the machine.
Results
The plaque morphology between two Zika virus strains that were derived from two
different cell lines (Vero and Huh7 cell line) were different.
In order to get the accurate MOI (Multiplicity of Infection), I used plaque assay followed
by six 10-fold serial dilutions to define the viral titer. During the plaque assay, a confluent
monolayer of host Vero cells were infected with a lytic ZIKV of an unknown concentration that
had been serially diluted to a countable range. When the plates were incubated, the initially
infected Vero cells will release viral progeny. Overlaid media prevented newly released viruses
from spreading to neighboring cells. Consequently, each infectious particle produced a circular
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

20
zone of infected cells called a plaque. Finally, the plaque became large enough to be visible to
the naked eye. 1% Crystal Violet was used to reinforce the contrast between the living cells and
the plaques. The morphology of the plaque between the two ZIKV strains is different. The
plaque of the African Strain MR766, which derived from the Vero cell line, had a circular shape
and was larger than the plaque of the Asian Strain H/PF/2013 (Fig. 1). By performing the
infection, I realized that when I harvested the viruses from different cell lines, the plaque
morphology was different. So I hypothesized that the two ZIKV strains derived from the Huh7
cell line may have different mechanisms and pathology compared to the ZIKV strains derived
from the normal Vero cell line. The Zika virus derived from the Huh7 cell line has a relatively
smaller plaque than the plaque of the Zika virus derived from the Vero cell line. The size of the
plaque is proportional to the efficiency of adsorption, the length of the latent period, and the
burst size of the phage. On the contrary, the morphology of Dengue virus plaques have rough
circles compared to the Zika virus plaque, and the plaque sizes of the Dengue virus were similar
to plaque sizes of the Zika virus African strain MR766. Plaque morphology differed dramatically
depending on growth conditions and viral species. Plaque size, border definition, clarity, and
distribution should all be noted as they can provide valuable information on the growth and
virulence factors of the virus in question.
The CPE (Cytopathic Effect) among the Huh7 cell line, HeLa cell line and HepG2 cell line
from two strains of ZIKV that were derived from two sources (Vero cell and Huh7 cell)
separately.
According to the plaque morphology, I made a second hypothesis that the process of
ZIKV infection may depend on the specific cell line. I designed the experiments into three parts.
In the first part, I used two Zika virus strains that were derived from two different cell lines
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

21
(Vero and Huh7 cell line) to infect the Huh7 cell line and to compare the CPE (Cytopathic
Effect) (Fig. 4). At 6h-pi, the Huh7 cell morphology was the simple squamous epithelium.
However for the ZIKV MR766 African strain derived from Vero cell line, the CPE for infecting
Huh7 cell line shows that the growth of cells slowed down after 24h-pi. At 48h-pi, the cells
shrunk, linearized, and almost 70% surface of cells exhibited numerous filopodia. At 72h-pi,
almost 80% of the cells were dead. On the contrary, for the ZIKV MR766 African strain derived
from Huh7 cell line, the CPE for infecting the Huh7 cell line shows that the growth of cells
slowed down after 24h-pi. Regardless, the growth of the cells were still faster than the cells
infected from the ZIKV MR766 African strain derived from the Vero cell line. Moreover, at 48h-
pi, most of cells remained healthy, and only 10% of cells started to shrink without linearization.
At 72h-pi, almost every cell shrunk and became small circles with vacuolization instead of
dying. On the another side, for the ZIKV H/PF/2013 Asian strain derived from Vero cell line, the
CPE for infecting the Huh7 cell line showed that the growth of cells slowed down after 24h-pi
which was similar to the Vero cell-derived ZIKV MR766 African strain. However, the cells
infected by the Vero cell-derived ZIKV H/PF/2013 Asian strain started to exhibit numerous
filopodia since 24h-pi. Because the ZIKV MR766 African strain derived from Vero cell line is
not an acute virus, the cell’s conditions at 48h-pi were similar to their conditions at 24h-pi, and
only 10% of the cells were dead. At 72h-pi, almost 30% of cells were dead and almost all the
cells started to shrink and became small and circlular with vacuolization instead of dying.
However, for the Huh7 cell-derived ZIKV H/PF/2013 Asian strain, the infection process became
fast. At 24h-pi, the cells maintained the same density as the control. However, at 48h-pi, the CPE
became 60%. One-third of the cells died and the remaining two-thirds of the cells shrunk and
grew filopodia-like tentacles and became spindle-shaped with long protrusions. At 72h-pi, 60%
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

22
of the cells were dead and most cells vacuolized and became little circles. The CPE was almost
90%, which was similar to the cells infected by Vero cell-derived ZIKV MR766 African strain.
In conclusion, two strains of ZIKV derived from Huh7 cell line and Vero cell line have the
reverse cytopathic effects. As it is, the Huh7 cell-derived ZIKV MR766 African strain has
similar CPE to the ZIKV H/PF/2013 African strain derived from Vero cell line. The Vero cell-
derived ZIKV MR766 African strain has similar CPE to the Huh7 cell-derived ZIKV H/PF/2013
African strain.
In the second part, I used both two strains of Huh7 cell-derived ZIKV to infect the HeLa
cell line (Fig. 5). It is obvious that both two strains of ZIKV inhibited the cells’ proliferation rate
and the CPE of HeLa cells are similar. The HeLa cells became spindle-shaped with long
filaments at 24h-pi. And at 72h-pi, both cell lines that were infected from the African strain and
the Asian strain of Zika Virus' CPE were more than 98%. And almost 70% of cells were dead.
The cells that were infected by the ZIKV MR766 Asian strain grew more filopodia and were
more spindle-shaped with long protrusions than the cells that were infected from the ZIKV
H/PF/2013 Asian strain.
In the third part, I used both two strains of ZIKV derived from two cell lines (Huh7 and
Vero cell line) to infect the HepG2 cell line (MOI=1), which is the cell line similar to Huh7 but
different in p53 expression. HepG2 cells carry wild-type p53, whereas Huh7 cells have null and
point mutations at p53 codon 220 respectively. The CPE in Fig. 6 shows that HepG2 cells
infected by both Asian and African strains derived from Vero cells inhibited a higher inhibitory
effect on cell proliferation at 24h-pi and induced a higher degree of CPE at 48h-pi than HepG2
cells that were infected by both Asian and African strains derived from Huh7 cells. Moreover,
for the HepG2 cells that were infected from two strains of Vero cell-derived ZIKV and the Huh7
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

23
cell-derived ZIKV African strain, the CPE was almost 90% at 48h-pi and almost 70% of the cells
were dead. At 72h-pi, the CPE was 100% and the almost all the cells were detached and dead.
On the contrary, for the HepG2 cells that were infected from both African strains of ZIKV
derived from Huh7 cells, the CPE was just 50% and 30% of the cells were dead at 48h-pi. And at
72h-pi, the CPE was 90% but only 60% of the cells were detached and dead. To be more clear,
when I compared the CPE between the cells infected by Huh7 cell-derived ZIKV MR766
African strain with the cells infected from Huh7 cell-derived ZIKV MR766 African strain, the
infection rate became different at 48h-pi. Compared to the cells infected from the Huh7 cell-
derived ZIKV MR766 African strain, the CPE for the cells infected from the Vero derived ZIKV
MR766 African strain was extremely obvious. However, for the cells infected by the ZIKV
H/PF/2013 Asian strain that was derived from the Huh7 cell line and the Vero cell line, the CPE
was similar.
The growth curve of the virus between two ZIKV strains is different.
Since there were dramatic differences in plaque morphology and CPE between the two
strains of ZIKV, I raised the third hypothesis that the viral titer might be different based on
different time lines. After infecting the Huh7 cell line with ZIKV, I aliquoted one-milliliter of
medium of the cell culture media every 24 hours for 3 days. Then I used plaque assay to quantify
the viral titer in the media I collected. Following the data, I calculated and quantified the virus
growth curve. Fig. 7A reveals that the viral titer of the Vero cell-derived ZIKV MR766 African
strain peaked at 24h-pi and started to decrease after 24h-pi. However, the viral titer of the Vero
cell-derived ZIKV H/PF/2013 Asian strain in Fig. 7B continued to increase at 72h-pi and
reached a higher viral titer than the ZIKV MR766 African strain. The highest viral titer of the
ZIKV H/PF/2013 Asian strain was 8 fold higher than the highest viral titer of the ZIKV
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

24
H/PF/2013 Asian strain. And at 72h-pi, the viral titer of the ZIKV H/PF/2013 Asian strain was
2.8 log value higher than the ZIKV H/PF/2013 Asian strain (Fig. 7C). However, when I did same
quantification for the viral titer of the two strains of Huh7 cell-derived ZIKV, the data was
totally different. Both the viral titers of the ZIKV H/PF/2013 Asian strain and the ZIKV MR766
African strain reached the peak at 36h-pi and started to decrease after 36h-pi. At 72h-pi, the viral
titers were close to zero. At 36h-pi, the viral titer of the ZIKV H/PF/2013 Asian strain was
higher than the ZIKV MR766 African strain.
Differential effects of Vero cell-derived African and Asian strains of ZIKV on autophagy
associated proteins in Human hematoma 7 cells.
Following the previous studies, I collected the cell lysates and performed western blotting
assay for the Human hepatoma cells, HeLa cells, and HepG2 cells to check the amount of protein
specifically related to the autophagy field. In Fig. 8, it is obvious that there are significant
differences between Vero cell-derived ZIKV MR766 African strain and ZIKV H/PF/2013 Asian
strain at three-time points (Fig. 8). For the Vero cell-derived ZIKV MR766 African strain, the
P62 protein displayed a radical reduction at 48h-pi, and a drastic decrease in the LC3 upper band
(LC3-I) and a huge induction in the LC3 lower band (LC3-II) seen by SDS-
PAGE/immunoblotting analysis, suggesting that the Vero cell-derived ZIKV MR766 African
strain can induce autophagy significantly at 48h-pi. On the contrary, for the Vero cell-derived
ZIKV H/PF/2013 Asian strain, P62 displayed a radical reduction at 72h-pi, and a drastic decrease
in the LC3 upper band (LC3-I) and a huge induction in the LC3 lower band (LC3-II), indicating
that the Vero cell-derived ZIKV H/PF/2013 Asian strain can increase the autophagy dramatically
at 72h-pi. Moreover for the autophagy-related gene family member ATG4B, the ATG4B protein
induced significantly for the Vero cell-derived ZIKV MR766 African strain at 24h-pi, but a very
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

25
weak band was detected from the Vero cell-derived ZIKV MR766 African strain and it started to
significantly induce at 48h-pi. However, another autophagy-related gene family member ATG7
remained the same for both the Vero cell-derived ZIKV MR766 African strain and the Vero cell-
derived ZIKV H/PF/2013 Asian strain. Moreover, since all the cells were infected by Zika virus,
I used ZIKV envelop protein as the biomarker for the expression of infection rate. Fig. 4 shows
that for the Vero cell-derived ZIKV MR766 African strain, the ZIKV envelop protein induced
dramatically at 24h-pi and continually increased till 72h-pi. In addition, for the Vero cell-derived
ZIKV H/PF/2013 Asian strain, the ZIKV envelop protein induced dramatically at 48h-pi and
continually increased till 72h-pi. In summation, Vero cell-derived ZIKV MR766 African strain
induced P62, the ratio of LC3-II/LC3-I, ATG4B, and ZIKV envelope earlier than the Vero cell-
derived ZIKV H/PF/2013 Asian strain. In addition, the ATG7 remained the same during the
infection process followed by three time points from both Vero cell-derived ZIKV MR766
African strain and ZIKV H/PF/2013 Asian strain infection.
Differential effects of Huh7 cell-derived African and Asian strains of ZIKV on autophagy
associated proteins in Human hematoma 7 cells.
According to the differences shown in the CPE and plaque assay results, I tried to analyze
another source of ZIKV for the infection of the same Human hematoma 7 cells, which are Huh7
cell-derived ZIKV MR766 African strain and ZIKV H/PF/2013 Asian strain (Fig. 9). It was
shown that the P62 protein exhibited reduction for the Huh7 cell-derived ZIKV MR766 African
strain at 60h-pi, and almost faded away at 74h-pi. However, the P62 protein expressed reduction
at both 48h-pi and 74h-pi but was induced again at 60h-pi for the Huh7 cell-derived ZIKV
H/PF/2013 Asian strain. Meanwhile, the autophagy biomarker LC3 upper band dramatically
decreased with dramatic accumulation of the LC3 lower band LC3-II at 60h-pi for the Huh7 cell-
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

26
derived ZIKV MR766 African strain. For the Huh7 cell-derived ZIKV H/PF/2013 Asian strain,
the transformation between LC3-I and LC3-II was induced at 24h-pi. The autophagy-related
gene ATG4B started to induce at 36h-pi and dramatically enhance at 48h-pi for the Huh7 cell-
derived ZIKV MR766 African strain. Meanwhile, the ATG4B protein began to increase at 48h-
pi for the Huh7 cell-derived ZIKV H/PF/2013 Asian strain. Beyond that, the autophagy-related
genes ATG5, ATG12, and the AKT gene were induced for both Huh7 cell-derived ZIKV
H/PF/2013 Asian strain and ZIKV MR766 African strain at 12h-pi. However for the Huh7 cell-
derived ZIKV MR766 African strain, the ATG5 exhibited reduction at 60h-pi. Simultaneously,
the ATG5, ATG12, and AKT protein were reduced at both 36h-pi and 74h-pi and re-induced at
60h-pi for the Huh7 cell-derived ZIKV H/PF/2013 Asian strain. In addition, the Beclin-1 gene
started to accumulate at 48h-pi for the Huh7 cell-derived ZIKV MR766 African strain and was
induced at 24h-pi for the Huh7 cell-derived ZIKV H/PF/2013 Asian strain. Lastly, I scanned the
ZIKV Envelop protein to check the infection rate of ZIKV virus. The western blotting analysis
reveals that the ZIKV envelope band rose up at 24h-pi for both Huh7 cell-derived ZIKV MR766
African strain and ZIKV H/PF/2013 Asian strain.
Differential effects of Huh7 cell-derived African and Asian strains of ZIKV on autophagy
associated proteins in HeLa cells.
Beause the HeLa cell line defies the normal mechanisms of senescence by acquiring
certain mutations and carries a diploid number of chromosomes (46, which is a specific mutant
cell line), I used Huh7 cell-derived ZIKV MR766 African strain and ZIKV H/PF/2013 Asian
strain to infect HeLa cells and collected the cell lysates for western blotting assay (Fig. 6). Fig. 6
indicates that the ratio of LC3-II/LC3-I was continually increased after 24h-pi for both the Huh7
cell-derived ZIKV MR766 African strain and the ZIKV H/PF/2013 Asian strain. Moreover, the
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

27
expression of P62 also presents a continuing decrease for both Huh7 cell-derived ZIKV MR766
African strain and ZIKV H/PF/2013 Asian strain which indicates that autophagy was induced.
However, the time points for P62’s dramatic decrease was different between the two strains of
ZIKV. For Huh7 cell-derived ZIKV MR766 African strain, the P62 decreased dramatically at
60h-pi. However, the Huh7 cell-derived ZIKV H/PF/2013 Asian strain presented rapid reduction
of P62 protein at 48h-pi. For the autophagy-related gene ATG4B and the critical autophagy
regulation protein Beclin-1, the reaction between two strains of Huh7 cell-derived was similar.
The ATG4B and Beclin-1 were rapidly induced at 24h-pi for both Huh7 cell-derived ZIKV
MR766 African strain and ZIKV H/PF/2013 Asian strain, and the bands almost disappeared after
48h-pi. On the contrary, the autophagy-related gene ATG7 kept the same expression level for
both strains of ZIKV, which indicates that ATG5 may not be associated to the infection process
of Huh7 derived ZIKV in HeLa cell line. Meanwhile, the ATG7 gene expression also showed
significant differences between the two strains of Huh7 derived ZIKV. For the Huh7 cell-derived
ZIKV MR766 African strain, the ATG7 band maintains the same expression till 60h-pi. On the
contrary, the ATG7 band was induced at 24h-pi, slightly decreased at 48h-pi, and then kept equal
until 60h-pi for the Huh7 cell-derived ZIKV H/PF/2013 Asian strain. In addition to this, the
Protein Kinase B (PKB), also known as Akt, continually decreased and dramatically reduced at
48h-pi for both strains of Huh7 derived ZIKV. Also, I used the ZIKV envelope protein to check
the infection. However, there is a significant difference between the two strains of Huh7 derived
ZIKV in HeLa cell line according to the ZIKV envelope band expressions, which revealed the
huge difference compared to the Huh7 cell infection. At 24h-pi, the ZIKV envelope band rapidly
accumulated and dramatically reduced at 48h-pi until 60h-pi, which suggests that the ZIKV was
almost absent at 48h-pi for the Huh7 derived ZIKV MR766 African strain. However, for the
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

28
Huh7 cell-derived ZIKV H/PF/2013 Asian strain, the ZIKV envelope band was heavily induced
at 24h-pi and continually increased until 60h-pi, which indicates that the Huh7 cell-derived
ZIKV H/PF/2013 Asian strain continually remained in the HeLa cells till 60h-pi. In summary,
both Huh7 cell-derived ZIKV MR766 African strain and ZIKV H/PF/2013 Asian strain strongly
induced the ATG4B, ATG5, ATG7, ATG12 and ZIKV envelope at 24h-pi which is impressive
and also suggests that at 23h-pi, the infection may induce some specific autophagy associated
signal pathways.
Both strains of ZIKV can induce the autophagosome at very early time points during the
infection.
Following my western blot data which shows that both strains of ZIKV can induce the
autophagy, I checked for autophagy using immunofluorescent assay to identify the autophagy
puncta (autophagosomes). I used Huh7-GFP- LC3 cell lines, which can automatically release
green fluorescence when autophagy is induced. In Fig. 11A, the green bright spots represent
autophagy puncta (i.e., autophagosomes). At 6h-pi, both strains of ZIKV induced GFP-LC3
puncta in the parental Huh7-GFP- LC3 cells.
The NS4A protein of ZIKV was co-localized with autophagosomes, whereas the ZIKV envelope
protein can not co-localize with autophagosomes.
After confirming that autophagosomes can be induced by both strains of ZIKV, I chose
36h-pi as the best time point to check the autophagy puncta with ZIKV proteins followed by the
data from western blotting (between 24h-pi and 48h-pi, the ZIKV proteins was dramatically
induced). The NS4A is a non-structural protein 4A which induces host endoplasmic reticulum
membrane rearrangements, which leads to the formation of virus-induced membranous vesicles
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

29
hosting the dsRNA and polymerase and functions as a replication complex. Moreover, it has
been proved that Zika Virus NS4A and NS4B proteins deregulate Akt-mTOR signaling in
human fetal neural stem cells to induce autophagy (Liang Q, Luo Z, et al. 2016). NS4A may be
associated with autophagosomes or autolysosomes for the regulation of viral RNA replication.
With this idea, I used the ZIKV NS4A antibodies for the immunofluorescent assay on Huh7-
GFP-LC3 cell lines to check whether or not it was co-localized with autophagosomes. In Fig.
11B, the green spots represent the autophagosomes, and the red fluorescence represents ZIKV
NS4A. The cells with red fluorescence are indicative of positive cells that were infected
successfully by the ZIKV. And the immunofluorescences suggest that the NS4A protein of Huh7
cell line derived ZIKV H/PF/2013 Asian strain was co-localized with autophagosomes.
Meanwhile, the negative cell could not induce the autophagosomes as much as positive cells.
Similarly, in Fig.11C, the immunofluorescences of Vero cell line derived ZIKV H/PF/2013
Asian strain also revealed the co-localization between the NS4A and autophagosomes. In
addition, I analyzed the Vero cell-derived ZIKV MR766 African strain (Fig. 12A) and Huh7
cell-derived ZIKV MR766 African strain (Fig. 12B) for comparison and the results were similar.
Although the co-localization was as not as efficient as the co-localization of the ZIKV
H/PF/2013 Asian strain, the immunofluorescences also confirmed an extremely high percentage
of co-localization in the Vero cell-derived ZIKV MR766 African strain. Nevertheless, the
percentage of co-localization between ZIKV NS4A and autophagosomes in Vero cell-derived
ZIKV MR766 African strain was not as higher than the ZIKV H/PF/2013 Asian strain and the
Vero cell-derived ZIKV MR766 African strain. There was around 50%-70% of co-localization.
To be more precise, I also performed immunofluorescence assay for co-localization of NS1
protein of Dengue virus type 2 with autophagosomes, which has already been proven to co-
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

30
localize with autophagosomes in another paper (Heaton, N., 2010). In Fig. 13B, the red
fluorescence represents Dengue virus NS1 protein and the green spots represent GFP-LC3
puncta, which confirms the previous finding of the paper. I used it as the positive control for my
results to confirm my new funding for the NS4A co-localization. In addition to this, I screened
the ZIKV envelope protein with autophagy puncta in the immunofluorescence assay. However,
the results have shown that there is no co-localization between the autophagosomes and ZIKV
envelope protein. To enhance the results of my research, I used the HCV Core protein with the
Huh7-GPF-LC3 cell line as the negative control, which had been confirmed that it was not co-
localized with the autophagosomes. In Fig. 14A, the red fluorescence represents the HCV Core
protein, and the green spots represent the GPF-LC3. Little to no co-localization was revealed on
the immunofluorescence images, which suggests that my results are consistent.
The NS2B protein of ZIKV H/PF/2013 Asian strain was co-localized with autophagosomes
but not for the ZIKV MR766 African strain.
In addition to the ZIKV NS4B, I also tried to detect NS2B and check for co-localization
using immunofluorescence assay. It is interesting that the results between the ZIKV H/PF/2013
Asian strain and the ZIKV MR766 African strain were different. As it is, in Fig. 13A, the red
fluorescence represents ZIKV NS2B, and the green spots represent autophagy puncta. The
merged image revealed an extremely high co-localization rate between ZIKV NS2B and
autophagosomes, which indicates that the ZIKV RNA may replicate on the autophagosomes and
that the NS2B protein may associate with this process on the cell membrane. However, when I
checked the immunofluorescence image for the ZIKV MR766 African strain, the result was
entirely different. In Fig. 13B, the green bright spots represent GFP-LC3 and the red
fluorescence represents ZIKV NS2B. It is obvious that the autophagosomes did not co-localize
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

31
with the ZIKV NS2B for the Vero cell-derived ZIKV MR766 African Strain, which indicates
that the NS2B in ZIKV MR766 African strain may not participate in the autophagosomal
induction process.
Discussion
In this work, I compared the ZIKV MR766 African strain with the ZIKV H/PF/2013
Asian strain by performing experiments that included four parts: plaque morphology and viral
titer for the infection rate, the percentage of cytopathic effect (CPE) from the cell line, kinetic
differences of the expression among the autophagy associated proteins, and immunofluorescence
analysis for the specific proteins and autophagosomes. In order to be more clear, I used plaque
assay to confirm the MOI for the following experiments. It is a very critical procedure in
virology to measure the viral titer, to determine the concentration of virus in a sample, and to
immediately evaluate the active virus and antiviral substances through the including of discrete
plaques cell society. The plaque assay is very essential for studying viral genetics and the
virulence according to the morphology of the plaque. In addition, the overlaid media can limit
cellular infection to the instantly encompassing monolayer, confine viral spread, forestalling
aimless infection through the fluid development medium, permitting the development of discrete
countable foci and consequent plaque arrangement so that the plague can indicate the monolayer
infection process. Some viruses produce smooth circle plaque such as the ZIKV as shown in Fig.
1. However, some viruses produce rough plaque such as the DenV in Fig. 2A, which indicates
that the spread of the new viruses to neighboring cells was not equal. As it is, plaque
morphology can be dramatically different under differing growth conditions and between viral
species. The size of the plaque is proportional to the efficiency of adsorption, the length of the
latent period, and the burst size of the phage. In this study, I compared two ZIKV strains’ plaque
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

32
and found that the ZIKV MR766 African strain can create larger plaque size than the ZIKV
H/PF/2013 Asian strain, which indicates that the ZIKV MR766 African strain is an acute virus
strain compare to the ZIKV H/PF/2013 Asian strain. The ZIKV MR766 African strain can infect
neighboring cells and release new virion faster than the ZIKV H/PF/2013 Asian strain. The
ZIKV MR766 African strain’s RNA replication may be faster than the ZIKV H/PF/2013 Asian
strain, promoting faster viral multiplication. Following this finding, I continued to check the
CPE between two strains of ZIKV. As it is, the ZIKV MR766 African strain performed an acute
and higher CPE than the ZIKV H/PF/2013 Asian strain, which confirmed the clinical symptoms
in the human beings and the plaque morphology of the plaque assay. Meanwhile, I lysed the cells
and determined the viral titer to create a curve. The data continually confirmed the conjecture.
The viral titer of ZIKV MR766 African strain increased rapidly in the first 24h-pi, and then
decreased rapidly, which indicated that the ZIKV MR766 African strain is an acute virus but
unstable. Without the host and nutrition, the virus will quickly lose its activation. On the
contrary, the ZIKV H/PF/2013 Asian strain is not as acute as ZIKV MR766 African strain. It did
not get a high titer within 24h-pi, but the titer continually increased till 72h-pi. Surprisingly, the
ZIKV H/PF/2013 Asian strain viral titer become almost one log value higher than the ZIKV
MR766 African strain, which indicates that the ZIKV H/PF/2013 Asian strain is more stable than
the ZIKV MR766 African strain and can maintain positive activity for a longer time.
Additionally, the culture media became acidic; the medium became yellow and the PH value was
decreased. The elevation of the pH accelerated cell movement and led to the contraction of the
cytoplasm, while lowering of the pH slowed and finally paused all cell activity, causing apparent
gelation of the protoplasm. It is suggested that local pH changes in the microenvironment of a
cell's surface may be a significant factor in controlling cell behavior in culture, which may also
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

33
affect the survival of the virus. If the virus is kept in the same medium without changing the
medium, the environment will become acidic and the virus will lack nutrition. In order to control
the variance, I think I should re-do the experiments by consistently refreshing the media daily or
adding sufficient media before infection. Meanwhile, I found an interesting result from the
plaque assay. The ZIKV H/PF/2013 Asian strain cannot reveal the obvious CPE from the Vero
cell line. However, when I infected the Huh7 cell line with the ZIKV H/PF/2013 Asian strain,
the CPE was dramatically increased. According to these results, I try to figure out the factors that
may be associated with this.
Vero cells (kidney epithelial cells extracted from an African green monkey) are
interferon-deficient, which means that they do not secrete interferon alpha or beta when infected
by a virus. Because of its interferon-deficiency, Vero cells can derive the Zika virus stably
without creating any mutations. people always harvest the ZIKV by using the Vero cell. When I
used the Vero cell line-derived ZIKV to infect the Huh7 cell line, I found that there was
extremely high CPE from the ZIKV H/PF/2013 Asian strain-infected cells. This result differs
from the Vero cell line, which had a lower amount of CPE. In the Vero cell line, it was very hard
to detect more than 2 million pfu/ml for the ZIKV H/PF/2013 Asian strain, and almost no CPE
when I derived the virus at day 5. However, in the Huh7 cell line infected by the ZIKV
H/PF/2013 Asian strain, I saw a very high percentage of CPE. Moreover, I found that the ZIKV
H/PF/2013 Asian strain showed severe symptoms in the next generation, which may associate
with the human sources cells instead of the monkey cells. According to these interesting facts, I
start to the hypothesize that the different sources of ZIKV may affect the virus genetic features,
and that different ZIKV strains may have different reactions depending on their sources of
origin. According to my previous studies, I chose the Huh7 cell line and the Vero cell line as two
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

34
sources to derive the virus and selected the first generation virus to perform experiments with the
lowest possibility for mutation. The plaques created by both strains of Huh7 cell-derived ZIKV
were dramatically smaller than Vero cell-derived ZIKV at day four, which indicated that the
infection rate for the Huh7 cell-derived ZIKV may become slower. The infection efficiency may
have been reduced, or the viruses may have remained inside the cells for a longer period of time
which caused slower cell death. To further examine this idea, I used the two strains of Huh7 cell-
derived ZIKV to infect the same Huh7 cell line. Surprisingly, the CPE caused by the two strains
of Huh7 cell-derived ZIKV presented reverse outcomes compared to the CPE caused by Vero
cell-derived ZIKV. The Huh7 cell-derived ZIKV H/PF/2013 Asian strain had similar CPE to the
Vero cell-derived ZIKV MR766 Asian strain, and the Huh7 cell-derived ZIKV MR766 Asian
strain was similar to the Vero cell-derived ZIKV H/PF/2013 Asian strain. The contrasting data
highly confirmed my hypothesis and also indicated that the pathology of ZIKV derived from
Huh7 cells may have some mutations, which may have caused the infection rate and the virus
titer to become reversed. To be more logical, I continued to aliquot the media of infectious Huh7
cells to analyze the viral titer and create the growth curve of ZIKV. Likewise, the Huh7 derived
ZIKV, especially ZIKV H/PF/2013 Asian strain, is more unstable than the Vero cell-derived
ZIKV. At 36h-pi, both strains of Huh7 derived ZIKV increased dramatically, peaked, and then
rapidly decreased. This indicated that the infection rate specific for the Huh7 derived ZIKV
H/PF/2013 Asian strain was induced. However, it becomes more unstable than the Vero derived
ZIKV H/PF/2013 Asian strain, which suggests that the Huh7 cell line may create some mutation
for the next generation of ZIKV H/PF/2013 Asian strain for the virus pathology and the infection
process. As it is, for the African strain of Zika virus, the extracellular virus may degrade(die).
For the Asian strain of Zika virus, the extracellular virus can keep growing and release around 8
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

35
times more virion than the African strain. The African strain of Zika virus may accumulate in
some structures of the Huh7 cells such as the ER, which may cause an acute destructive power.
According to the specific different responses from the two strains of ZIKV, I tried to
compare the two strains of ZIKV by infecting three typical cell lines. The first one is the Huh7
cell line, a human hepatoblastoma cell line which is already involved with my previous research.
The second one is the HepG2 cell line, an immortalized cell line consisting of human liver
carcinoma cells that were derived from well-differentiated hepatocellular carcinoma. The
differences in P53 expression is very critical to viral research as it determines a lot of
characteristics with regards to cell proliferation and tumor behavior (if doing xenografts). In this
way, HepG2 cells carry wild-type p53, whereas Huh7 cells have null and point mutations at p53
codon 220 respectively. I chose the HepG2 cell line to directly compare with the Huh7 cell line.
The third cell line is the HeLa cell line, "immortal" cells that were derived from cervical cancer
cells from a patient in 1951, which defy the normal mechanisms of aging by gaining certain
mutations. This cell line has abnormally higher proliferating rates compared to other cancer cell
lines. There is also a small amount of p53 mRNA expression in HeLa cells. Moreover, the HeLa
cells have a total of 76 to 80 chromosomes, which are different from other normal cells (46
chromosomes in total). Telomerase allows the addition of sequences at the ends of chromosomes
and is induced during HeLa cell division. Thus, DNA will not be destroyed and cells will not die.
Although some cancer cells have active telomerase, it may be particularly effective in HeLa
cells. To be more clear, I compared the CPE of the infectious HeLa cell line and with the CPE of
the HepG2 cell line. The results of infectious HeLa cell line revealed that the infection rate
between two strains of Huh7 cell-derived ZIKV were relatively different. In contrast to the Huh7
cells infected by Huh7 cell-derived ZIKV, the African strain kept an acute infection rate and had
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

36
a higher fatality rate. The Huh7 cell-derived ZIKV H/PF/2013 Asian strain is relatively mild.
Meanwhile, for HepG2 cell line, both strains of Vero cell-derived ZIKV showed similar CPE
followed by three different timelines, but the Huh7 cell-derived ZIKV MR766 African strain had
a relatively lower CPE than the Huh7 cell-derived ZIKV H/PF/2013 Asian strain. The cell
proliferation for the HepG2 cell line was inhibited at 24h-pi by both strains of Vero cell-derived
ZIKV compared to the Huh7 cell-derived ZIKV. This indicates that the source of viral derivation
may induce some mutants inside the ZIKV to specifically affect cell proliferation. Moreover, the
contrasting results of the infectious Huh7 cell and the HepG2 cell line indicate that the P53 may
be a mediator in the infection process of the ZIKV.
To be more certain, I lysed the previous infectious cells to test the kinetic differences for
the expression of several autophagy-related proteins. In the Huh7 cell line, the reduction of P62
associated with LC3-II/LC3-I indicates that autophagy was induced by both strains of Vero
derived ZIKV, and the ZIKV MR766 African strain can induce autophagy faster (24h earlier)
than the ZIKV H/PF/2013 Asian strain. Moreover, the ATG4B protein was induced dramatically
24h earlier to the P62 and the ratio of LC3-II/LC3-I for both strains of ZIKV, which indicates
that the ATG4B will associate in the formation of LC3-I and is involved in the processed into
cytosolic soluble LC3-I, which is associated with ATG7 and the liposoluble LC3-PE (LC3-II)
will be formed by covalent cross-linking of phosphatidylethanolamine (PE) for the membrane
extension process. However, the ATG7 did not change during the infection process for both
strains of Vero cell-derived ZIKV, which may suggest that ZIKV may use the Atg7-independent
pathway to induce the LC3-I conjugate the PE to generate LC3-II. The ubiquitin-like attachment
of Atg12 and Atg5 should co-react with the Atg7, which has similar functions to the E1 and E2
enzymes. Moreover, the Atg5-Atg12 heterodimer was induced rapidly by ZIKV MR766 African
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

37
strian, which will lead to the formation of phagophores. The ubiquitination pathway ATG12-
ATG5 plays an important role in the formation of autophagosomes and is involved in the
initiation, nucleation, extension, closure, maturation, and degradation of autophagosomes. The
results indicate that ZIKV MR766 African strain can induce the autophagosomes at the
beginning of the infection, which has also been confirmed in my immunofluorescence
experiments. The ATG12-ATG5 is not only present on the bilayer membrane in the early stage
of autophagy but also continually involved in the other phages of autophagy. However, the
reduction at 72h-pi of Atg5-Atg12 heterodimer for the ZIKV H/PF/2013 Asian strain specifically
indicates that after 72h-pi, the autophagosomes induced from ZIKV H/PF/2013 Asian strain may
be digested in the autolysosomes. It was indicated that the ATG12-ATG5 is only present on the
bilayer membrane in the early stage of autophagy, and LC3 is continuously cleaved and
degraded during autophagy when the cells infected by the ZIKV H/PF/2013 Asian strain.
However, for the Huh7 cell-derived ZIKV, the infectious Huh7 cell line revealed
dramatically different expression results. It is interesting that the time points of induction of P62
and LC-II/LC-I is reversed to the Vero cell-derived ZIKV. The African stain induced the
autophagy later than the Asian strain. All the autophagy-related genes were induced in different
ways, which may indicate that unlike the brief co-relationship between the ATG4B and the
autophagy induction time points, the Huh7 cell-derived ZIKV was not induced in the same way.
This indicates that the ATG4B may be a participant in the formation of autophagosomes and
extension phase of autophagy as well as cutting ATG8 to form LC3-I. In addition, the Beclin-1
genes revealed a very close relationship with the formation of LC3-II. For both strains of Huh7
cell-derived ZIKV, Beclin-1 was induced dramatically at the time that the LC3-II was rapidly
induced, which may indicate that the Bcl-2 or Bcl-XL may disaggregate from BECLIN-1 and
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

38
BECLIN-1 may associate with LC3-II formation to induce the occurrence of autophagy.
Furthermore, almost all the ATG5, ATG12, AKT were reduced when LC3-II was dramatically
accumulated, specifically to the Huh7 cell-derived ZIKV MR766 African strain. This may
indicate that all of these proteins are involved in autophagy and are associated with induction
time points. On the contrary, for the Huh7 cell-derived ZIKV H/PF/2013 Asian strain, the ATG5,
ATG12, and AKT proteins did not show connections with the process of LC3-II formation, but
all of them were induced at 60h-pi time points. This was dramatically different to the Huh7 cell-
derived ZIKV MR766 African strain. It may indicate that at the 60h-pi, the cells infected with
Huh7 cell-derived ZIKV H/PF/2013 Asian strain may induce some enzymes inside the cell that
induced ATG5, ATG12, AKT protein simultaneously. Especially, the Akt protein presented a
dramatically interesting results. It is very likely to the HCV in Liu’s paper (Liu, Z., Tian, Y.,
2012). The AKT signaling pathway plays an important role in cell growth and metabolism. The
Huh7 cell-derived ZIKV H/PF/2013 Asian strain may transiently activates the AKT pathway. the
interaction between ZIKV and some co-receptors probably triggered the activation of AKT. The
activation of AKT by ZIKV may important for ZIKV infectivity. Furthermore, the treatment of
cells with the AKT inhibitor AKT-V prior to ZIKV infection may inhibit the ZIKV infection.
However, the reason is not yet clear and requires more experiments to confirm.
For HeLa cell, the results were dramatically different between the Huh7 cell-derived
ZIKV MR766 African strain and the ZIKV H/PF/2013 Asian strain. The autophagy induction
time from the two strains of ZIKV was similar, but the ZIKV H/PF/2013 Asian strain induced
autophagy earlier than the African strain. Moreover, at 24h-pi time point, the ATG4B, BECLIN-
1, and ATG12 were all induced dramatically in both strains of ZIKV and then continually
decreased. For ATG7, the ZIKV MR766 African strain seems to remain at the same level.
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

39
However, it was induced after 24h-pi from the ZIKV H/PF/2013 Asian strain. There were only
two identical parts between two strains of ZIKV, the first being ATG5, which continued to
remain an equal level. The second one is the AKT protein, which continually decreased after
24h-pi. These results suggest that in both strains of ZIKV, the ATG5 may participate in the
combination of the autophagic membrane to promote the recruitment of LC3 (Atg8) to
autophagic vacuoles. It is also supposed that just ATG12 will bind to the preautophagosome’s
outer membrane to promote the expansion of autophagic vacuoles from the vesicle-like and cup-
like structures and gradually develop into semi-annular and cyclic structures in the ZIKV
infected HeLa cell. Moreover, during the formation of autophagosomes, while Rubicon acts as
an inhibitor, there may have some important proteins to make up the BECLIN-1 complex and
induce BECLIN-1 at 24h-pi. However, the accurate process remains unknown and needs more
supporting experiments.
In the final part, NS4A (a non-structural protein 4A) can induce endoplasmic
reticulum membrane rearrangement in the host, resulting in the formation of viral-induced
membrane vesicles of host dsRNA and polymerase. It also performs as a replication complex
which may regulate the ATPase activity of the serine protease NS3 chain helicase region. Dr.
Liang’s paper (Liang, Q, et al., 2016) already confirmed that the ZIKV NS4A and NS4B can
interact together to regulate the AKT-mTOR signaling to induce autophagy inside human fetal
neural stem cells. I start to consider that NS4A and NS2B may be associated with virus
replication for inducing autophagy. My immunofluorescence study results revealed that NS4A
co-localized with autophagosomes for both sources and two strains of ZIKV. Since the
autophagosome is a double membrane structure, the ZIKV RNA may replicate on the membrane
of autophagosomes, or even to the autolysosomes. In addition, only the ZIKV H/PF/2013 Asian
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

40
strain showed the co-localization between the NS2B and the autophagosomes instead of the
ZIKV African strain, which may indicate that the virus replication pathology may have different
signaling pathway within two strains of ZIKV. The ZIKV may induce autophagy to enhance its
own replication. The analysis of proteins associated with ZIKV-induced autophagosomes by
proteomics led to the identification of ZIKV nonstructural proteins as well as proteins involved
in membrane trafficking.
Conclusions and Future Study
It is confirmed that ZIKV can induce autophagosomes by enhancing RNA replication.
The different proteins associated with autophagy have different reactions for the different strains
of ZIKV. In order to gain a complete understanding of the difference between the two strains of
Zika virus, I plan on continuing to performing experiments by knocking-out/knocking-down the
autophagy associated gene and by testing the mechanisms of autophagy signal pathway that are
specific to the different cell lines and different sources of ZIKV. The ATG4B, BECLIN-1 and
the AKT protein all presented a dramatically interesting results. The Huh7 cell-derived ZIKV
H/PF/2013 Asian strain may transiently activates the AKT pathway. The interaction between
ZIKV and some co-receptors probably triggered the activation of AKT. The activation of AKT
by ZIKV may important for ZIKV infectivity. In the future, I will try to perform the treatment of
cells with the AKT inhibitor AKT-V prior to ZIKV infection to test whether or not the it can
inhibit the ZIKV infection. Also, I will test the RNA to check the ATG4B as will. If the ATG4B
did not connected with the RNA replication, it would be more interesting in the cell autophagy
metabolism pathway.
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

41
Moreover, since I only performed my research in vitro, I plan to perform some
experiments in vivo by using some specific gene mutant mice. In addition, the ZIKV
nonstructural proteins NS4B and NS2B are involved in membrane trafficking. The association of
NS4A and NS2B with ZIKV-induced autophagosomes was confirmed by immunofluorescence
microscopy. According to my studies, I will try to detect more proteins associated with lipid rafts
to identify whether there is an association of lipid rafts with ZIKV-induced autophagosomes. I
will also continue to purify the autophagosomes to confirm my previous hypothesis.

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

42
ACKNOWLEDGEMENTS
Foremost, I would like to express my special appreciation to Professor Jing-Hsiung
James Ou for offering me the opportunity to study in his laboratory. His advice on research as
well as my Ph.D application have been priceless. I would also like to express my gratitude to my
thesis committee members Professor Jing-Hsiung James Ou, Professor Weiming Yuan, Professor
Chengyu Liang for their encouragement and insightful comments and questions.
My sincere thanks also go to the senior Postdoc Kenny Tsai, Jay Yeon Kim and Yiyang
Lee for everything not limited to technique training, experiment designing and troubleshooting. I
would also appreciate our lab manager Cecilia Cho for the grammar checking and review of the
thesis.
Last but not the least, I would like to thank to my parents: Qun Liu and Yue Chen, for
supporting my master’s study economically and spiritually.

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Data
A 200µl x 10
-1
200µl x 10
-2
200µl x 10
-3
200µl x 10
-4

B 200µl x 10
-1
200µl x 10
-2
200µl x 10
-3
200µl x 10
-4

Fig. 1 Comparing among the plaque morphology of Vero cell derived ZIKV MR766 African
strain and ZIKV H/PF/2013 Asian strain with four different dilutions. A. ZIVK H/PF/2013
(Asian) strain at 4 day-pi (virus derived from Vero cell line). The purple areas are the alive Vero
cells stained with crystal violet, and the white bright spots were empty area which considering as
one virus ideally. each infectious particle produced a circular zone of infected cells called a
plaque, which is the white bright spots in the picture. Finally, the plaque became large enough to
be visible to the naked eye. 1% Crystal Violet was used to reinforce the contrast between the
living cells and the plaques. Only viruses that cause visible damage of cells can be assayed in
this way. B. ZIKV MR766 (African) strain at 4 day-pi (virus derived from Vero cell line).
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

47
Plaque morphology can be dramatically different under differing growth conditions and between
viral species. The dilution was a serial ten-fold dilution. The size of the plaque is proportional to
the efficiency of adsorption, the length of the latent period, and the burst size of the phage. The
size of the plaque was determined by comparing the measured area of a plaque to that of a well
in the 6-well using the formula: the number of plaque per well = (lowest plaque number well x
dilution fold + second lowest plague well x dilution fold) x 5 ÷ 2. The plaque of the African
Strain MR766, which derived from the Vero cell line, had a circular shape and was larger than
the plaque of the Asian Strain H/PF/2013.

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

48
A B
200µl x 10
-2
200µl x 10
-3
200µl x 10
-2
200µl x 10
-3

C D
200µl x 10
-2
200µl x 10
-3
200µl x 10
-2
200µl x 10
-3

Fig. 2 Comparing among the plaque morphology of the Vero cell derived ZIKV and Huh7 cell
derived ZIKV. A. ZIKV MR766 (African) strain at 4 day-pi (virus derived from Huh7 cell line)
B. ZIKV MR766 (African) strain at 4 day-pi (virus derived from Vero cell line) C. ZIKV
H/PF/2013 (Asian) strain at 4 day-pi (virus derived from Huh7 cell line) D. ZIKV H/PF/2013
(Asian) strain at 4 day-pi (virus derived from Vero cell line). The plaque morphology of the
Huh7 cell derived ZIKV were relatively smaller than the Vero cell derived ZIKV.

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

49
A B
200µl x 10
-2
200µl x 10
-3
200µl x 10
-2
200µl x 10
-3

Fig 3. Comparing among the plaque morphology between DEV-2 and ZIKV. A. DEV-2 at 4 day-
pi (virus derived from BHK-21cell line) B. ZIKV MR766 (African) strain at 4 day-pi (virus
derived from Vero cell line). The plaque morphology of the DEV-2 is rough and irregular, but
smooth and rounded from the ZIKV.

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

50
A
Huh7 6h MOCK (20x) H/PF/2013 huh7 6h (20x) H/PF/2013 vero 6h (20x) MR766 huh7 6h (20x) MR766 vero 6h (20x)

B
Huh7 24h MOCK (20x) H/PF/2013 huh7 24h (20x) H/PF/2013 vero 24h (20x) MR766 huh7 24h (20x) MR766 vero 24h (20x)

C
Huh7 48h MOCK (20x) H/PF/2013 huh7 48h (20x) H/PF/2013 vero 48h (20x) MR766 huh7 48h (20x) MR766 vero 48h (20x)

D
Huh7 72h MOCK (20x) H/PF/2013 huh7 72h (20x) H/PF/2013 vero 72h (20x) MR766 huh7 72h (20x) MR766 vero 72h (20x)

Fig 4. The CPE (cytopatic effects) of two strains of Huh7 cell-derived ZIKV and Vero cell-
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

51
derived ZIKV infected the Huh7 cell line followed by 3 different time points. A. The different
CPE among the MOCK Huh7 cells, the Huh7 cell-derived ZIKV infected Huh7 cells and the
Vero cell-derived ZIKV infected Huh7 cells at 6h-pi. B. The different CPE among the MOCK
huh7 cells, the Huh7 cell-derived ZIKV infected Huh7 cells and the Vero cell-derived Huh7 cells
at 24h-pi. C. The different CPE among the MOCK huh7 cells, the Huh7 cell-derived ZIKV
infected Huh7 cells and the Vero cell-derived ZIKV infected Huh7 cells at 48h-pi. D. The
different CPE among the MOCK huh7 cells, the Huh7 cell-derived ZIKV infected Huh7 cells
and the Vero cell-derived ZIKV infected Huh7 cells at 72h-pi. The CPE caused by the two
strains of Huh7 cell-derived ZIKV presented reverse outcomes compared to the CPE caused by
Vero cell-derived ZIKV. The Huh7 cell-derived ZIKV H/PF/2013 Asian strain had similar CPE
to the Vero cell-derived ZIKV MR766 Asian strain, and the Huh7 cell-derived ZIKV MR766
Asian strain was similar to the Vero cell-derived ZIKV H/PF/2013 Asian strain.

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

52
A
Hela cell MOCK 24h (20x) H/PF/2013 huh7 24h (20x) H/PF/2013 huh7 48h (20x) H/PF/2013 huh7 72h (20x)

B
Hela cell MOCK 24h (20x) MR766 huh7 24h (20x) MR766 huh7 48h (20x) MR766 huh7 72h (20x)

Fig. 5 The CPE of HeLa cell line infected with two strains of Huh7 cell-derived ZIKV. A. The
different CPE for the MOCK HeLa cells and the Huh7 cell-derived ZIKV H/PF/2013 Asian
strain infected HeLa cells at 24h-pi, 48h-pi and 72h-pi. B. The different CPE for the MOCK
HeLa cells and the Huh7 cell-derived ZIKV MR766 African strain infected HeLa cell at 24h-pi,
48h-pi and 72h-pi. The results of infectious HeLa cell line revealed that the infection rate
between two strains of Huh7 cell-derived ZIKV were relatively different. In contrast to the Huh7
cells infected by Huh7 cell-derived ZIKV, the African strain kept an acute infection rate and had
a higher fatality rate. And the Huh7 cell-derived ZIKV H/PF/2013 Asian strain is relatively mild.

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

53
A
HepG2 cell MOCK 24h (20x) MR766 huh7 24h (20x) MR766 24h (20x) H/PF/2013 huh7 24h (20x) H/PF/2013 24h (20x)

B
HepG2 cell MOCK 48h (20x) MR766 huh7 48h (20x) MR766 48h (20x) H/PF/2013 huh7 48h (20x) H/PF/2013 48h (20x)

C
HepG2 cell MOCK 72h (20x) MR766 huh7 72h (20x) MR766 72h (20x) H/PF/2013 huh7 72h (20x) H/PF/2013 72h (20x)

Fig. 6 Two strains of ZIKV derived from Huh7 cell line and Vero cell line infected the HepG2
cell line followed by 3 different time points. A. The different CPE among the MOCK HepG2
cells, the Huh7 cell-derived ZIKV infected HepG2 cells and the Vero cell-derived ZIKV infected
HepG2 cells at 24h-pi. B. The different CPE among the MOCK HepG2 cells, the Huh7 cell-
derived ZIKV infected HepG2 cells and the Vero cell-derived ZIKV infected HepG2 cells at
48h-pi. C. The different CPE among the MOCK HepG2 cells, the Huh7 cell-derived ZIKV
infected HepG2 cells and the Vero cell-derived ZIKV infected HepG2 cells at 72h-pi. Both
strains of Vero cell-derived ZIKV showed similar CPE followed by three different timelines, but
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

54
the Huh7 cell-derived ZIKV MR766 African strain had a relatively lower CPE than the Huh7
cell-derived ZIKV H/PF/2013 Asian strain. The cell proliferation for the HepG2 cell line was
inhibited at 24h-pi by both strains of Vero cell-derived ZIKV compared to the Huh7 cell-derived
ZIKV.

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

55
A B

C D

Fig 7. A. The growth curve of ZIKV MR766 African strain (derived from Vero cell line) (log
value) with the standard deviation. B. The growth curve of ZIKV H/PF/2013 Asian strain
(derived from Vero cell line) (log value) with the standard deviation. C. The growth curve
between two ZIKV strains (derived from Vero cell line) (log value) with the standard deviation
D. The growth curve between two ZIKV strains (derived from Huh7 cell line) (log value) with
the standard deviation.
0
1
2
3
4
5
6
7
0h 24h 48h 72h
VIRAL TITER (LOG)
INFECTED HOURS
The growth curve of Vero cell-
derived ZIKV MR766 African Strain
MR766 DATA 1 MR766 DATA 2
0
1
2
3
4
5
6
7
8
9
0h 24h 48h 72h
VIRAL TITER (LOG)
INFECTED HOURS
The growth curve of Vero cell-derived
ZIKV H/PF/2013 Asian Strain
H/PF/2013 DATA 1 H/PF/2013 DATA2
0
2
4
6
8
0h 24h 48h 72h
VIRAL TITER (LOG)
INFECTED HOURS
The growth curve of two Strains of
Two Strians of Vero Cell-derived ZIKV
MR766 H/PF/2013
0
2
4
6
8
0h 12h 24h 36h 48h 60h 72h
VIRAL TITER (LOG)
INFECTED HOURS
The growth curve of two strains of
Two Straisn of Huh7 Cell-derived ZIKV
MR766 H/PF/2013
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

56

Fig 8. Differential effects of Vero cell-derived African and Asian strains of ZIKV on autophagy
associated proteins in Human hematoma 7 cells. All the proteins were detected in the same
nitrocellulose membrane. Two strains of Vero cell-derived ZIKV were infected in Huh7 cell line
with same MOI (MOI=1) and compared side by side.
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

57

Fig 9. Differential effects of Huh7 cell-derived African and Asian strains of ZIKV on autophagy
associated proteins in Human hematoma 7 cells. All the proteins were detected in the same
nitrocellulose membrane. Two strains of Vero cell-derived ZIKV were infected in Huh7 cell line
with same MOI (MOI=1) and compared side by side.
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

58

Fig 10. Differential effects of Huh7 cell-derived African and Asian strains of ZIKV on
autophagy associated proteins in HeLa cells. All the proteins were detected in the same
nitrocellulose membrane. Two strains of Vero cell-derived ZIKV were infected in Huh7 cell line
with same MOI (MOI=1) and compared side by side.

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

59
A H/PF/2013 Huh7-GFP-LC3 MR766 Huh7-GFP-LC3 MOCK

B Huh7-GFP-LC3 ZIKV MR766 NS4A Merge MOCK

C Huh7-GFP-LC3 ZIKV H/PF/2013 NS4A

Merge MOCK

Fig. 11. The immunofluorescence assay for the two sources of ZIKV H/PF/2013 Asian strains
and the Huh7-GFP-LC3 cell line. Immunofluorescence microscopy of autophagosomes (GFP-
LC3), NS4A(red), and nuclei (blue). A. Both two strains of ZikV can induce the GFP-LC3
COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

60
puncta (i.e., autophagosomes) (green) at 6h-pi during the infection. The MOCK image is the cell
treated with same medium without infected with the ZIKV. B. The NS4A protein of Huh7 cell-
derived ZIKV H/PF/2013 Asian strain was co-localized with autophagosomes (Yellow) at 36h-pi
(MOI=1). However, the negative cells can not induce the GFP-LC3 puncta (i.e.,
autophagosomes) in the Merge image. C. The NS4A protein of Vero cell-derived ZIKV
H/PF/2013 Asian strain was co-localized with autophagosomes at 36h-pi (MOI=1).

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

61
A Huh7-GFP-LC3 ZIKV MR766 NS4A Merge

B Huh7-GFP-LC3 Z IKV MR766 NS4A Merge

Fig. 12. The immunofluorescence assay for the two sources of ZIKV MR766 African strains and
the Huh7-GFP-LC3 cell line. Immunofluorescence microscopy of autophagosomes (GFP-LC3),
NS4A(red), and nuclei (blue). A. The NS4A protein of Huh7 cell-derived ZIKV MR766 African
strain was highly co-localized with autophagosomes (Yellow) at 36h-pi (MOI=1). Whereas, the
negative cells can not induce the GFP-LC3 puncta (i.e., autophagosomes) in the Merge image. B.
The NS4A protein of Vero cell-derived ZIKV MR766 African strain was highly co-localized
with autophagosomes at 36h-pi (MOI=1). In addition, the negative cells can not induce the GFP-
LC3 puncta (i.e., autophagosomes) in the Merge image.

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

62
A Huh7-GFP-LC3 ZIKV H/PF/2013 NS2B

Merge MOCK

B Huh7-GFP-LC3 ZIKV MR766 NS2B Merge MOCK

Fig. 13. The immunofluorescence assay for two strains of ZIKV with the Huh7-GFP-LC3 cell
line. Immunofluorescence microscopy of autophagosomes (GFP-LC3), NS2B(red), and nuclei
(blue). A. The NS2B protein of ZIKV H/PF/2013 African strain was highly co-localized with
autophagosomes (Yellow) at 36h-pi (MOI=1), and the negative cells can not induce the GFP-
LC3 puncta (i.e., autophagosomes) in the Merge image. B. The NS2B protein of ZIKV MR766
African strain did not show the co-localization between the GFP-LC3 puncta at 36h-pi (MOI=1).

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

63
A Huh7-GFP-LC3 ZIKV H/PF/2013 E Merge MOCK

B Huh-GFP-LC3 ZikV MR766 E Merge

Fig. 14. The immunofluorescence assay for two strains of ZIKV with the Huh7-GFP-LC3 cell
line. Immunofluorescence microscopy of autophagosomes (GFP-LC3), ZIKV Envelop(red), and
nuclei (blue). A. The Envelop protein of ZIKV H/PF/2013 African strain did not show the co-
localization between the GFP-LC3 puncta at 36h-pi (MOI=1) B. The Envelop protein of ZIKV
MR766 African strain did not co-localized with the GFP-LC3 puncta at 36h-pi (MOI=1).

COMPARATIVE STUDIES OF TWO STRAINS OF ZIKA VIRUS

64
A Huh7-GFP-LC3 DEV-2 NS1 Merge MOCK

B Huh7-GFP-LC3 HCV core Merge MOCK

Fig 15. The negative control and the positive control from DEV-2 and HCV to enhance the
ZIKV results. A. DEV-2(derived from Huh7 cell line) NS1 protein was highly co-localized with
the autophagosomes (GFP-LC3) at 36h-pi NS1 (MOI=1) (positive control) B. HCV (derived
from Huh7 cell line) Core protein shows no co-localization with the autophagosomes (GFP-LC3)
at 36h-pi (MOI=1) (negative control).

Abstract (if available)

Abstract Zika virus (ZIKV) is a mosquito-borne virus and can cause severe diseases. Based on phylogenetic analysis, ZIKV can be classified into African and Asian lineages. Clinical studies indicated that these two lineages of ZIKV exhibit differences in their pathogenicity. To understand how these two lineages of ZIKV may be different from each other, I compared the replication of two strains of ZIKV, MR766, and H/PF/2013, which belong to the African lineage and the Asian lineage, respectively. These two strains of ZIKV were produced in either Huh7 human hepatoma cells or Vero green monkey kidney cells, and their infectivity and growth curves were comparatively analyzed in HeLa cervical carcinoma cells, Vero cells, and HepG2 human hepatoblastoma cells. I found that these two strains of ZIKV exhibited differences in their plaque morphologies, cytopathic effects and growth curves. These effects also differed depending on whether the viruses were prepared in Huh7 cells or Vero cells. In addition, I also found that these two strains of ZIKV could induce autophagy in their host cells, although with different induction kinetics, and they could also induce the expression of autophagy-related genes including Atg4B, Beclin-1, and AKT. Interestingly, the ZIKV NS2B and NS4A proteins, but not its envelope protein, were found to colocalize with autophagosomes, indicating the possible involvement of autophagosomes in the replication of ZIKV. My study thus provided important information for understanding why these two strains of ZIKV differ in their pathogenicity in patients.

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Asset Metadata

Creator Chen, Yimin (author)

Core Title Comparative studies of the replication of African and Asian strains of Zika virus

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 05/01/2019

Defense Date 05/01/2019

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag autophagosomes,autophagy,cytopathic effect (CPE),HeLa cells,Huh7 cells,OAI-PMH Harvest,plaque assay,Vero cells,viral growth curve,Zika virus,ZikV NS2B,ZikV NS4A

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ou, James (committee chair), Liang, Chengyu (committee member), Yuan, Weiming (committee member)

Creator Email jadeair818@gmail.com,yiminc@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c89-165274

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Document Type Thesis

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Rights Chen, Yimin

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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