Design a Short Primer Sequence for PCR Amplification

The assignment content discusses the polymerase chain reaction (PCR), agarose gel electrophoresis, and CRISPR/Cas9 technology. PCR is a laboratory technique used to amplify specific DNA sequences by denaturing, annealing, and extending primers. Agarose gel electrophoresis is a method for separating and visualizing nucleic acids, where DNA fragments migrate through an agarose gel according to their size and are stained with ethidium bromide. CRISPR/Cas9 technology is a tool used for editing genes by introducing double-stranded breaks in the DNA sequence, which can be repaired by error-prone processes, resulting in insertions/deletions that may disrupt gene function.

Abstract:
Dengue is a vector-borne-disease. It is transmitted to human by mosquito bite. Dengue is one of the most important tropical
and sub-tropical diseases but is now spreading globally as a result of climate change, urbanization and international travel.
Hence, there is a pressing need to control the spread of Dengue virus and to treat Dengue diseases. To be able to infect the
host Dengue virus must recognize host cell surface receptor(s) using virus-encoded envelope protein. Therefore,
identification of virus receptor is very important in understanding the infection process and the development of anti-virals
and vaccine. However, not too much is known about Dengue receptors in the insect and human hosts. All putative Dengue
receptors to date are poorly characterized. The aim of project is to identify and characterize bona fide Dengue receptor using
a range of stringent molecular and cellular techniques. Using a state-of-the art CRISPR genome editing tool we have
successfully knockout a putative Dengue receptor from a mosquito cell line. CRISPR is a recent, very powerful and yet simple
and versatile genome editing tool to silence specific genes in the genome. The CRISPR/Cas9 system was discovered in
bacteria/Archaea as a defence mechanism against phage (virus) infections. It has now been repurposed as a gene editing tool
in various organisms throughout the tree of life
Dengue Fever:
Dengue virus is a single-stranded RNA virus of the Flavivirus genus and the Flaviviridae family. Dengue is a mosquito-borne
viral disease that has rapidly spread in all regions of WHO in recent years. Dengue virus is transmitted by female mosquitoes
mainly of the species Aedes aegypti and, to a lesser extent, Ae. albopictus. This mosquito also transmits chikungunya, yellow
fever and Zika infection. Dengue is widespread throughout the tropics, with local variations in risk influenced by rainfall,
temperature and unplanned rapid urbanization.
Severe dengue (also known as Dengue Haemorrhagic Fever) was first recognized in the 1950s during dengue epidemics in
the Philippines and Thailand. Today, severe dengue affects most Asian and Latin American countries and has become a
leading cause of hospitalization and death among children and adults in these regions.
There are four distinct, but closely related, serotypes of the virus that cause dengue (DEN-1, DEN-2, DEN-3 and DEN-4).
Recovery from infection by one provides lifelong immunity against that particular serotype. However, cross-immunity to the
other serotypes after recovery is only partial and temporary. Subsequent infections by other serotypes increase the risk of
developing severe dengue.
Lifecycle:
Dengue virus is maintained in the urban lifecycle as it is transmitted from mosquito to human to mosquito. The primary
mosquito vector of dengue virus is Aedes aegypti .However, Aedes albopictus also transmits the virus (Brooks, Carroll, Butel,
Morse, & Mietzner, 2010). Dengue virus is transmitted from human to mosquito when a female mosquito feeds on a viremic
human. The incubation period within the mosquito lasts 8-12 days and consists of the virus spreading systemically from the
mid-gut. After this period of time, the virus can be transmitted to another human during any point in the remainder of the
mosquito’s life (WHO,TDR, 2009).
Global burden of dengue:
The incidence of dengue has grown dramatically around the world in recent decades. The actual numbers of dengue cases
are underreported and many cases are misclassified. One recent estimate indicates 390 million dengue infections per year
(95% credible interval 284–528 million), of which 96 million (67–136 million) manifest clinically (with any severity of
disease).1 Another study, of the prevalence of dengue, estimates that 3.9 billion people, in 128 countries, are at risk of
infection with dengue viruses.2

Member States in 3 WHO regions regularly report the annual number of cases.. The number of cases reported increased
from 2.2 million in 2010 to 3.2 million in 2015. Although the full global burden of the disease is uncertain, the initiation of
activities to record all dengue cases partly explains the sharp increase in the number of cases reported in recent years.
Other features of the disease include its epidemiological patterns, including hyper-endemicity of multiple dengue virus
serotypes in many countries and the alarming impact on both human health and the global and national economies.
Before 1970, only 9 countries had experienced severe dengue epidemics. The disease is now endemic in more than 100
countries in the WHO regions of Africa, the Americas, the Eastern Mediterranean, South-East Asia and the Western Pacific.
The America, South-East Asia and Western Pacific regions are the most seriously affected.
Cases across the Americas, South-East Asia and Western Pacific exceeded 1.2 million in 2008 and over 3.2 million in 2015
(based on official data submitted by Member States). Recently the number of reported cases has continued to increase. In
2015, 2.35 million cases of dengue were reported in the Americas alone, of which 10 200 cases were diagnosed as severe
dengue causing 1181 deaths.
Not only is the number of cases increasing as the disease spreads to new areas, but explosive outbreaks are occurring. The
threat of a possible outbreak of dengue fever now exists in Europe as local transmission was reported for the first time in
France and Croatia in 2010 and imported cases were detected in 3 other European countries. In 2012, an outbreak of dengue
on the Madeira islands of Portugal resulted in over 2 000 cases and imported cases were detected in mainland Portugal and
10 other countries in Europe. Among travellers returning from low- and middle-income countries, dengue is the second most
diagnosed cause of fever after malaria.
In 2013, cases have occurred in Florida (United States of America) and Yunnan province of China. Dengue also continues to
affect several South American countries, notably Costa Rica, Honduras and Mexico. In Asia, Singapore has reported an
increase in cases after a lapse of several years and outbreaks have also been reported in Laos. In 2014, trends indicate
increases in the number of cases in the People's Republic of China, the Cook Islands, Fiji, Malaysia and Vanuatu, with Dengue
Type 3 (DEN 3) affecting the Pacific Island countries after a lapse of over 10 years. Dengue was also reported in Japan after a
lapse of over 70 years.
In 2015, Delhi, India, recorded its worst outbreak since 2006 with over 15 000 cases. The Island of Hawaii, United States of
America, was affected by an outbreak with 181 cases reported in 2015 and ongoing transmission in 2016. The Pacific island
countries of Fiji, Tonga and French Polynesia have continued to record cases. The year 2016 was characterized by large
dengue outbreaks worldwide. The Region of the Americas region reported more than 2.38 million cases in 2016, where Brazil
alone contributed slightly less than 1.5 million cases, approximately 3 times higher than in 2014. 1032 dengue deaths were
also reported in the region. The Western Pacific Region reported more than 375 000 suspected cases of dengue in 2016, of
which the Philippines reported 176 411 and Malaysia 100 028 cases, representing a similar burden to the previous year for
both countries. The Solomon Islands declared an outbreak with more than 7000 suspected. In the African Region, Burkina
Faso reported a localized outbreak of dengue with 1061 probable cases.
In 2017 (as of Epidemiological Week 11), the Region of Americas have reported 50 172 cases of dengue fever, a reduction as
compared with corresponding periods in previous years. The Western Pacific Region has reported dengue outbreaks in
several Member States in the Pacific, as well as the circulation of DENV-1 and DENV-2 serotypes.
Transmission:
The Aedes aegypti mosquito is the primary vector of dengue. The virus is transmitted to humans through the bites of
infected female mosquitoes. After virus incubation for 4–10 days, an infected mosquito is capable of transmitting the virus
for the rest of its life. Infected symptomatic or asymptomatic humans are the main carriers and multipliers of the virus,
serving as a source of the virus for uninfected mosquitoes. Patients who are already infected with the dengue virus can

transmit the infection (for 4–5 days; maximum 12) via Aedes mosquitoes after their first symptoms appear. The Aedes
aegypti mosquito lives in urban habitats and breeds mostly in man-made containers. Unlike other mosquitoes Ae. aegypti is a
day-time feeder; its peak biting periods are early in the morning and in the evening before dusk. Female Ae. aegypti bites
multiple people during each feeding period. Aedes albopictus, a secondary dengue vector in Asia, has spread to North
America and more than 25 countries in the European Region, largely due to the international trade in used tyres (a breeding
habitat) and other goods (e.g. lucky bamboo). Ae. albopictus is highly adaptive and, therefore, can survive in cooler
temperate regions of Europe. Its spread is due to its tolerance to temperatures below freezing, hibernation, and ability to
shelter in microhabitats.
Manifestations:
Dengue virus infections can manifest in different ways. Those infected may be asymptomatic, or have manifestations
consistent with classic dengue fever, or dengue hemorrhagic fever with or without dengue shock syndrome (Table 1). The
WHO divides the course of dengue illness into 3 phases: febrile, critical, and recovery. After an incubation period of 4-10
days, the febrile phase of dengue fever begins. There is a sudden onset of high-grade fever, and patients often experience
headache, retro-orbital pain, myalgias, arthralgias, and facial flushing. Many complain of nausea, vomiting, and loss of
appetite. Less commonly, sore throat, injected pharynx, and conjunctivitis are noted. There may be mild hemorrhagic
manifestations such as petechiae, epistaxis and gingival bleeding and rarely gastrointestinal and vaginal bleeding. Also during
this time, there may be hepatomegaly and a steady decrease in the white blood cell count. The febrile phase lasts 2-7 days.
At this point, it cannot be determined which cases will become severe dengue fever (WHO, 1997; WHO, TDR, 2009).
The critical phase begins around days 3-7 when the temperature decreases to and remains at 37.5-38C or less. During this
time, there may be an increase in capillary permeability leading to an increase in hematocrit (WHO, TDR, 2009). An increase
in capillary permeability and plasma leakage is considered dengue hemorrhagic fever (WHO, 1997). One point to note is that
before plasma leakage occurs, there is a steady drop in total WBC count and a rapid drop in platelet count. If there is no
increase in capillary permeability, the patient improves and is considered to have had non-severe dengue infection. Severe
dengue occurs with the manifestation of at least one of the following: plasma leakage with or without shock, severe
bleeding, or severe organ impairment (WHO, TDR, 2009). A chest X-ray or an abdominal ultrasound may be useful in
identifying cases of severe dengue, as those with an increase in capillary permeability may develop pleural effusion and
ascites (WHO, TDr, 2009). In addition to labs showing leucopenia and thrombocytopenia, there will be hemoconcentration as
demonstrated by an elevation in hematocrit (WHO, 1997).
Dengue shock syndrome occurs when excessive amounts of plasma are leaked into the extravascular space. This may occur
around day 4-5 or when the fever drops (WHO, TDR, 2009). Patients will exhibit signs of circulatory failure including cool,
blotchy, and edematous skin, circumoral cyanosis, tachycardia, weak pulse, and a narrowing pulse pressure (WHO,1997). It is
important to note that the diastolic blood pressure rises as the systolic blood pressure remains the same. This can be easily
overlooked if the systolic blood pressure is within the normal range. However, the narrowing of the pulse pressure is a
warning sign of shock and the patient needs prompt and adequate care. Shock is defined by a pulse pressure of less than or
equal to 20mm Hg (WHO, TDR, 2009). If shock is treated, recovery can take place over 2-3 days (WHO, 1997). Multiple organ
failure, metabolic acidosis, and disseminated intravascular coagulation can occur if shock is not recognized and treated
aggressively. Lastly, severe hemorrhages and death may occur (WHO, TDR, 2009).
With proper monitoring and management, the recovery phase will commence consisting of resorption of the extravascular
fluid within 48-72 hours. Symptoms improve and the patient returns to hemodynamic stability. The hematocrit, WBC count,
and platelet count reach normal levels (WHO, TDR, 2009).
The prognosis of dengue fever is good. There is the potential for the sequelae of prolonged fatigue and depression in some
cases. In DHF, the case fatality rate is less than 1% (WHO, 1997). Rare but severe complications of dengue fever that can
occur even without plasma leakage and shock are hepatitis, encephalitis, myocarditis (WHO, TDR, 2009). CNS manifestations

of convulsions, spasticity, altered consciousness, and transient paralysis have been seen in some cases. Acute renal failure
and hemolytic uremic syndrome are other rare findings (WHO, 1997).
Treatment:
The clinical course of dengue virus infection varies, and therefore, treatment is determined individually depending on a
patient’s status. The most important aspects of treating a patient with DF are to recognize early signs of plasma leakage and
to begin fluid therapy. A healthcare provider (HCP) must also recognize dengue shock syndrome and aggressively address the
issues of shock, bleeding, and organ impairment (WHO, TDR, 2009).
The decision to send a patient with DF home can be made if the patient is able to maintain adequate levels of fluid intake
and output. The patient must also have stable hematocrit levels and show no warning signs of severe dengue. A treatment
plan consists of fever control and drinking plenty of fluids containing electrolytes and sugar. NSAIDs are contraindicated due
to the potential for hemorrhagic manifestations. Patients must meet with their HCP on a daily basis to be assessed for signs
of illness progression. It is essential for HCPs to educate their patients on warning signs that necessitate prompt medical
attention. These warning signs include shortness of breath, a fast pulse, severe abdominal pain, persistent vomiting,
jaundice, cool and clammy extremities, lethargy, irritability, convulsions, significant bleeding (i.e. coffee-ground emesis or
black stools), and no urine output for 4-6 hours (WHO, TDR, 2009).
Patients may be admitted if warning signs are present, if there are co-existing conditions, or if they do not have a caregiver
at home or means of transportation to a hospital should they experience warning signs. Pregnant women as well as infants
with dengue virus infection should also be admitted. For a patient with warning signs, first the hematocrit must be measured
and then IV fluids should be aggressively administered. The patient’s status and hematocrit levels must be reevaluated and
IV infusion rates may be adjusted accordingly. Vital signs and peripheral perfusion should be monitored until the patient has
advanced to the recovery phase. Urine output, blood glucose, and organ function should also be monitored. In a patient who
is admitted without warnings signs of severe dengue, IV fluid therapy should only be started if the patient cannot tolerate
oral fluids. HCPs should watch for warning signs of severe dengue and measure the patient’s temperature, fluid intake and
urine output, hematocrit and WBC and platelet counts (WHO, TDR, 2009).
The last category of treatment is for those in the critical phase of dengue fever. Patients in the critical phase need
emergency hospitalization. Those in this category have one or more of the following manifestations: dengue shock and/or
fluid accumulation leading to respiratory distress, severe hemorrhage, and severe organ impairment. IV fluid resuscitation is
essential and usually is the only intervention necessary for treatment of this phase. The goals of fluid resuscitation are to
improve central and peripheral circulation and organ perfusion. If a patient is in shock, IV fluid resuscitation should be
started and the patient must be monitored closely. If there is no improvement, the hematocrit must be measured. If the
hematocrit is still high, a second bolus of fluids should be given. In a patient with shock refractory to treatment, a hematocrit
that is lower than the initial reference hematocrit is indicative of bleeding. In this instance, a blood transfusion is needed
immediately (WHO, TDR, 2009).
Immunization:
In late 2015 and early 2016, the first dengue vaccine, Dengvaxia (CYD-TDV) by Sanofi Pasteur, was registered in several
countries for use in individuals 9-45 years of age living in endemic areas. WHO recommends that countries should consider
introduction of the dengue vaccine CYD-TDV only in geographic settings (national or subnational) where epidemiological data
indicate a high burden of disease. Other tetravalent live-attenuated vaccines are under development in phase III clinical
trials, and other vaccine candidates (based on subunit, DNA and purified inactivated virus platforms) are at earlier stages of
clinical development. WHO provides technical advice and guidance to countries and private partners to support vaccine
research and evaluation.

Dengue receptors:
There are four dengue virus serotypes, i.e., types 1–4. Neutralizing antibodies are specifically induced by initial infection with
each virus serotype, and lifelong immunity is established for the same type of virus. In most cases of initial infection, the host
develops dengue fever, which has a mild prognosis. Cross neutralizing antibodies against different serotypes disappear
within a short period, and a virus of another serotype may cause reinfection (secondary infection). When secondary infection
by a different serotypes occurs, the immune complexes of the viruses with cross-reactive antibodies produced during
primary infection enhance viral infection mediated through Fcγ receptor dependent incorporation of the virus into host cells.
There is a strong possibility that such a progressive response contributes to more severe manifestations, such as DHF and
dengue shock syndrome.
Dengue virus is an enveloped virus about 50 nm in diameter. The envelope glycoprotein (E protein) is present on the viral
membrane. E protein is a functional protein molecule that binds to receptors on the host cell membrane; it is also a major
antigen against host protective immunity, which induces neutralizing antibody. E protein is divided into three functional
domains, termed domains I, II, and III. Domain I, the hinge region, is linked to the two other functional domains. The high
mobility of this region is responsible for the changes in structure of E protein due to variations in external pH. Domain II has a
hydrophobic-rich peptide sequence featuring the membrane fusion activity and contributes to E protein dimerization.
Domain III is thought to be involved in the binding to receptor molecules present on the host cell membrane. During viral
infection, the adsorption of viral particles is initiated by binding of E protein to receptor molecules present on the host cell
membrane. Subsequently, the adsorbed viruses are taken into the cell by endocytosis. The pH decreases inside endosomes
formed by fusion with lysosomes, and the viral membrane is fused with the endosomal membrane mediated through the
action of the E protein fusion peptide. Eventually, the nucleocapsid enters the cytoplasm, and the virus genome is released
into the cytoplasm.
Direct interaction of the virus with host receptor molecule(s) is crucial for virus propagation and the pathological progression
of dengue diseases. Elucidation of the molecular mechanisms underlying interaction of dengue virus with its receptor(s) in
humans and mosquitoes is essential for an understanding of dengue pathology. To date, many candidate molecules have
been proposed as dengue receptors.
DENGUE VIRUS RECEPTORS IN MAMMALIAN CELLS:
Halstead et al. first demonstrated that dengue virus infection in human peripheral blood leukocytes was enhanced by the
presence of non-neutralizing antibody. This enhancement was mediated through Fcγ receptors expressed on leukocytes.
These findings indicated that Fc receptor-mediated entry is involved in secondary infection, particularly infection with a
serotype different from that involved in primary infection. With regard to primary infection and initial contact of the virus
with host cells, investigations to identify receptor molecules have been performed in mammalian cells. Table 1 presents a
summary of dengue virus receptors in mammalian cells proposed in previous studies. The candidate molecules can be
categorized into four major groups. First, carbohydrate molecules such as sulfated glycosaminoglycans (GAGs) and
glycosphingolipid GSL) are thought to act as co-receptor molecules, which enhance the efficiency of virus entry. Among the
sulfated GAGs, heparan sulfate is indispensable for virus adsorption to the host cells. Another type of carbohydrate molecule
has recently been reported to contribute to virus attachment; neolactotetraosylceramide (nLc4Cer), a GSL without sulfation,
may also be a co-receptor on the host cells. Native and (semi)synthetic forms of carbohydrate compounds derived from GAG
and GSL structures successfully inhibited DENV infection of different cell types. These findings strongly suggest that
carbohydrate molecules in the extracellular matrix are positively involved in DENV propagation in target cells. Second,
carbohydratebinding proteins, termed lectins, expressed on dendritic cells (DCs) and macrophages under the human skin are
involved in initial contact of DENV introduced by mosquito bite. Among these lectins, dendritic cell-specific intercellular
adhesion molecule-3-grabbing non-integrin (DCSIGN) has been best characterized in virus-DC interaction. Cryoelectron
microscopic analysis demonstrated that recombinant lectin protein binds directly to N-glycans at position of 67 of E protein

expressed on viral particles. DC-SIGN-mediated entry allows DENV to propagate in DC, meaning that DC is the primary target
for DENV. A recent study showed that another lectin, mannose receptor, contributes to the entry of DENV into macrophages.
Taken together, the above observations indicate that carbohydrate recognition events are associated with DENV propagation
in the human body. Third, factors related to protein folding, such as heat shock proteins and chaperones, may also be
involved in the interaction of DENV serotype 2 (DENV-2) and host cells. It has been reported that a single serotype, DENV-2,
bound these molecules. Fourth, independent studies showed that other proteins, including high-affinity laminin receptor,
CD14-associated protein, and uncharacterized proteins, may also be involved in DENV—host cell interaction. Some of these
proteins reported to date may be identical with regard to properties, such as molecular mass. Some of the proposed
receptors are commonly recognized by different serotypes of DENV, while others seem to interact specifically with a certain
serotype of DENV. These findings strongly suggest that DENV binds multiple molecules that may form complexes on host
cells, and that DENV uses specific combinations of receptor candidates to enter different types of cell. However, the nature
of cellular receptors and molecular mechanisms for dengue virus entry has not yet been fully elucidated.
DENGUE VIRUS RECEPTORS IN MOSQUITO CELLS:
Table 2 summarizes the major receptor molecules proposed in previous studies. Regarding DENV vector, mosquitoes, the
best characterized DENV receptor candidate is prohibitin, a 35 kDa protein. This protein was identified as a DENV-2 ligand by
VOPBA followed by mass spectroscopy analyses using C6/36 cells (a cell lineage derived from the larval stage of A.
albopictus), CCL-125 cells (an A. aegypti derived cell line) and A. aegypti adult mosquitoes. Cell treatment with anti-prohibitin
antibodies as well as the silencing of its mRNA resulted in inhibition of DENV replication. Also, prohibitin specific interaction
with DENV E proteinwas demonstrated by immunoprecipitation and colocalization microscopy experiments, reinforcing its
role as DENV receptor (Kuadkitkan et al., 2010).
Two glycoproteins of 40 and 45 kDa able to bind DENV-4 were also identified on the surface of C6/36 cells (Salas-Benito and
Angel 1997). Antibodies against these molecules prevented virus binding to the cells, reinforcing their role as virus receptors.
Both proteinswere present inmosquito tissues that are recognized as permissive to DENV infection, such as midgut, ovary
and salivary glands, in the different stages of A. aegypti life cycle (Yazi Mendoza et al., 2002). Moreover, specific binding of
gp45 to recombinant E protein was confirmed by affinity chromatography(Reyes-del Valle and del Angel 2004). After several
attempts to elucidate gp45 identity, it was found that this protein is recognized by antibodies against HSP90, which were
already implicated in interaction between DENV and mammalian cells (Chen et al., 1999; Reyes-Del Valle et al., 2005). After
heat shock, it relocates to the cell surface and this relocation correlates with an increase in virus binding, but not with an
enhancement in virus replication, suggesting that gp45 may act as an attachment factor rather being an entry receptor.
Studies on a DENV-binding protein of 50 kDa, which was also identified using VOPBA, revealed that this protein was
recognized by the antibody against the human high-affinity laminin (Sakoonwatanyoo, Boonsanay and Smith 2006), a protein
already proposed to be the DENV-1 receptor in hepatic cells (Thepparit et al., 2004) (see the previous section). However,
inmosquito cells the anti-laminin receptor antibody or soluble laminin only inhibited the replication of DENV serotypes 3 and
4, suggesting that in mosquitoes the laminin receptor could participate in the recognition of DENV-3 and 4, but not of DENV-
1 or 2. The authors also suggested that the previous identified gp45 (Salas-Benito and Angel 1997) and the laminin receptor
could be the same protein.
Two additional proteins of 67 and 80 kDa (named R67 and R80)were shown to bind all four serotypes of DENV (Munoz et al.,
1998). These molecules were purified by affinity chromatography from A. aegypti midguts and from C6/36 cells, and
antibodies against them were able to inhibit DENV binding and infection of C6/36 cells, suggesting that they may act as
receptors for DENV in mosquitoes (Mercado-Curiel et al., 2006). R67 has subsequently been correlated to mosquito strain
susceptibility to DENV infection (Mercado-Curiel, Black and Munoz Mde 2008). Its amount and distribution along the
mosquito midgut were proportional to vector competence and DENV infection, respectively. Afterwards, proteomic analysis
of a fraction of A. aegypti midgut extracts subjected to affinity chromatography with the recombinant E protein or DENV

particles allowed the identification of enolase as a 67 kDa protein, which would be the R67 protein (MunozMde et al., 2013).
Interestingly, we recently found that DENV infection induces enolase secretion by hepatic cells and that the levels of this
protein are increased in the plasma of dengue patients (Higa et al., 2014). Since α-enolase binds plasminogen and modulates
its activation, it is plausible to speculate the association of the increase in α-enolase secretion by infected hepatic cells with
the haemostatic dysfunction observed in dengue patients, including the promotion of fibrinolysis and vascular permeability
alterations.Whether DENV-binding properties of enolase found in mosquito cells is also relevant in humans still needs
investigation.
DENV transmission occurs through a mosquito bite, with virus transfer to the human host via vector saliva. Indeed, DENV
replicates intensely in mosquito salivary glands, and the identification of the virus receptor in this tissue is of great interest.
VOPBA was also used to search DENV receptor in mosquito salivary glands (Cao-Lormeau 2009). These studies revealed that
four proteins (with 77, 58, 54 and 37 kDa) from salivary gland extracts (SGE) of A. aegypti were able to bind to the four DENV
serotypes, and five A. polynesiensis SGE proteins (with 67, 56, 54, 50 and 48 kDa)were able to bind to DENV-1 and DENV-4,
but the identity of these proteins remains unknown.
Entry pathways:
For most of the cells studied so far, including C6/36, HeLa, A549, Huh7, HepG2 and BS-C-1 cells, DENV-2 internalization
occurs via the clathrin-dependent endocytosis (Krishnan et al., 2007; Acosta et al., 2008; Mosso et al., 2008; van der Schaar
et al., 2008; Acosta, Castilla and Damonte 2009; Ang et al., 2010). In Vero cells, however, entry pathway is only dynamin
dependent, occurring through a non-classical endocytic pathway, independent on clathrin, caveolae or lipid rafts (Acosta et
al., 2009).
For C6/36 cells, all four DENV serotypes enter through clathrin-dependent endocytosis (Acosta, Castilla and Damonte 2008,
2011; Mosso et al., 2008). DENV-2 cell trafficking involves actin filaments but not microtubules, suggesting that DENV
infection does not require virus transport from early to late endosomes (Acosta et al., 2008). A similar profile of virus fusion
on early endosomes was also found for HeLa cells (Krishnan et al.,2007). On the other hand, although DENV-2 internalization
also occurs via clathrin-mediated endocytosis in the green monkey kidney cells BS-C-1, single-particle tracking analysis has
shown that the majority of DENV particles are transported from early endosomes to late endosomes, where virus is retained
for about 5 min prior to membrane fusion (van der Schaar et al., 2008; Smit et al., 2011).
Regarding hepatic cells, small interfering RNAs (siRNA) silencing experiments revealed that all DENV serotypes enter Huh7
cells through clathrin-mediated endocytosis, and that DENV-2 requires early to late endosome trafficking (Ang et al.,2010).
Clathrin-mediated-endocytosis dependence of DENV entry in HepG2 cellswas demonstrated by siRNA silencing of target
genes as well (Alhoot, Wang and Sekaran 2012). Macropinocytosis was also identified as one of the DENV entry pathways in
these cells, although clathrin-mediated endocytosis was the predominant entry route (Suksanpaisan, Susantad and Smith
2009). Recognition of PS in viral envelope by TIM and TAM receptors seems to be involved in DENV entry through
macropinocytosis (Meertens et al., 2012). Indeed, the structure of DENV at 37◦C, the physiological temperature of
mammalian cells, exposes patches of viral membrane (Fibriansah et al., 2013; Zhang et al., 2013a), which contains PS
(Meertens et al., 2012). This mechanism was also reported for other viruses (Mercer and Helenius 2008; Jemielity et al.,
2013), and since exposure of PS is a signal for apoptosis, these viruses, including DENV, would use of apoptotic mimicry to
infect cells.
Interestingly, DENV entry pathway can also be altered depending on the cellular system used for virus propagation. For
instance, DENV grown in C6/36 cells enters into Vero cells via the non-classical clathrin-independent pathway, but the same

virus serially propagated in Vero cells uses a clathrin-mediated endocytic pathway (Acosta et al., 2014). In addition, virus
serially propagated in Vero cells showed reduced affinity to cellular heparan sulfate when compared to C6/36 cell-
propagated virus (Acosta et al., 2014), which may result from mutations in E protein that enhance its positive net charge (Lee
et al., 2006; Prestwood et al., 2008; Anez et al., 2009). However, it is still unclear whether this would explain the differences
in the endocytic routes used by insect and mammalian cell-derived viral particles. DENV E protein glycosylation itself also
seems to be important for virus tropism, since mutations in the E protein glycosylation sites did not affect virus growth in
insect cells, but impaired virus infectivity and spread in mammalian cells (Bryant et al., 2007; Mondotte et al., 2007).
The Prohibitins:
Prohibitin 1, (Phb1) has a molecular mass of ~30 kDa and is also known as B cell receptor associated protein-32 (BAP32),
whereas a related protein, prohibitin 2 (Phb2), sometimes referred to as prohibitone, B-cell receptor associated protein-37
(BAP37) or repressor of estrogen receptor action (REA) has a mass of ~37 kDa. For the purpose of this review we will use the
nomenclature of Phb1 and Phb2. The prohibitin name is derived from an historical perspective and probably only relates to
one of the many physiological roles of these proteins. APhb1 cDNA was first isolated by differential hybridization to RNA
from normal versus regenerating rat liver and consequently Phb1 was proposed to be an inhibitor of cellular proliferation,
hence the name prohibitin. The corresponding mRNA when microinjected into normal human diploid fibroblasts attenuated
DNA synthesis. However it was subsequently shown that this effect was attributable to the 3’ untranslated region of the
Phb1 mRNA rather than the coding region of the cDNA. More recently Phb1 protein has been shown to be present in the
nucleus and to interact with transcription factors important in cell cycle progression. Phb2 also interacts with nuclear
transcription factors. REA was studied for several years as an inhibitor of estrogen receptor action and when the cDNA was
eventually cloned, it turned out to have a sequence identical to Phb2.
Tissue Culture Methods:
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable
artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or
mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been
already established.
Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under
the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the
cells have to be sub cultured (i.e.,passaged) by transferring them to a new vessel with fresh growth medium to
provide more room for continued growth.
After the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived from
primary cultures have a limited life span, and as they are passaged, cells with the highest growth capacity
predominate, resulting in a degree of genotypic and phenotypic uniformity in the population.
If a subpopulation of a cell line is positively selected from the culture by cloning or some other method, this cell
line becomes a cell strain. A cell strain often acquires additional genetic changes subsequent to the initiation of the
parent line.
Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a
genetically determined event known as senescence; these cell lines are known as finite. However, some cell lines
become immortal through a process called transformation, which can occur spontaneously or can be chemically or

virally induced. When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it
becomes a continuous cell line.
Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured
invariably consists of a suitable vessel containing a substrate or medium that supplies the essential nutrients
(amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (O2, CO2), and regulates
the physicochemical environment (pH, osmotic pressure, temperature). Most cells are anchorage dependent and
must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others
can be grown floating in the culture medium (suspension culture).
If surpluses of cells are available from sub culturing, they should be treated with the appropriate protective agent
(e.g., DMSO or glycerol) and stored at temperatures below –130°C (cryopreservation) until they are needed. Cells
in culture can be divided in to three basic categories based on their shape and appearance (i.e., morphology).
Cloning of Cells
A population of identical molecules (genes), cells or organisms, all of which are derived from the same parent by asexual
means, is known as a clone1. The process of producing genetically similar molecules, cells, or organisms from a common
precursor by asexual reproduction in vitro or in vivo is termed cloning. (“in vitro in Latin means “in glass”).
Cloning Techniques
Different techniques are used for cloning genes, cells and organisms.
Microbial Cloning
Genetically altered or modified microbial cells can be duplicated very easily and we can get millions of cloned cells in a few
days. These microbial strains are used for a number of purposes.
a. To produce useful compounds such as enzymes and vitamins on large scale.
b. They can be engineered to produce pharmaceutically useful products like human insulin, interferon, human growth hormone
and viral vaccines example: E. coli.
Molecular Cloning is one way of studying the specific proteins involved in cell division. A gene contains the
instructions for how to make a protein. By mutating a gene, the protein’s shape, size and function could all be
affected. Mutating a gene changes its instructions. Once a mutated gene is created and incorporated into a cell’s
DNA, the cell replicates, creating many cells containing the mutant gene. The cells with the changed gene can then
be compared to normal cells.
Below are the steps involved to both make a mutant gene and incorporate it into the DNA of a human cell:
1-Chemically "cut" the gene you want to study from the DNA strand
2-Attach target gene to a small, circular piece of DNA.Together, this is called a plasmid, which serves as the vehicle
for transporting the gene.
3-Put the plasmid into an E. coli cell (or another type of bacteria). As each E. coli cell divides, each new cell
contains a copy of the plasmid containing the gene.
4-Grow a lot of E. coli cells

5-Once your E. coli population has reached your desired number of cells, break apart the E. Coli cells using a
chemical that dissolves the cell wall.
6-Filter the mixture of broken E. coli cells and collect only the plasmids containing the gene.
7-Put the plasmids into human cells. The type of cell varies depending on the research.
8-Over time, the plasmid will be incorporated into the host cell DNA and the new gene will change the proteins
produced.
9-Observe physical changes between the cells with the plasmid and those without.
Cell Passage:
Cell passaging is a technique that enables an individual to keep cells alive and growing under cultured conditions
for extended periods of time. Cells should be passed when they are 90%-100% confluent.
Procedure for Passaging Cells
1. Warm media in 28C water-bath.
2. Check cells in culture multiwell plates under microscope to confirm that the cells are 90%-100%
confluent.
3. Clean hood with ethanol.
4. Sterilize all materials, bottles, etc. which are loaded into the hood. Spray hands with ethanol. Jars of liquid
need to be sprayed with ethanol. Sterile pipets may be placed in the hood directly. Automatic pipetters
should enter the hood WITHOUT sterilization.
5. Spray hands with ethanol.
6. Using the Automatic pipetters, transfer 2ml of the media to the new multiwell plates.
7. Discard 500 μl of the media that in cultured wells (previous one)out.
8. Using the scraper, detach the cells from the bottom of the wells.
9. Check cells under microscope to confirm that cells are detached from the surface.
10. Pipet 100 μlof detached cells to new wells with medium.
11. Pipet the remaining quantity of detached cells to 1.5 ml centrifuge tube and label it to lysate and extract
DNA (purification).
DNA extraction:
The aim from extraction is to release nucleic acid from the cell for use in other procedures and must be free from
contamination with protein, carbohydrate, lipids or other nucleic acids and this leads to used pure nucleic acids for
testing.
Preparing Lysates–Mini Kit
Mammalian Cells Lysate
Use the following protocol to prepare lysate from mammalian cells.
1. Set a water bath or heat block at 55°C.

2. For adherent cells (≤5 × 106 cells), remove the growth medium from theculture plate and harvest
cells by trypisinization or a method of choice.For suspension cells (≤5 × 106 cells), harvest cells
and centrifuge the cells at 250×g for 5 minutes to pellet cells. Remove the growth medium.
3. Resuspend the cells from Step 2 in 200 μL PBS.
4. Add 20 μL Proteinase K (supplied with the kit) to the sample.
5. Add 20 μL RNase A (supplied with the kit) to the sample, mix well by briefvortexing, and incubate
at room temperature for 2 minutes.
6. Add 200 μLPureLinkR Genomic Lysis/Binding Buffer and mix well byvortexing to obtain a
homogenous solution.
7. Incubate at 55°C for 10 minutes to promote protein digestion.
8. Add 200 μL 96–100% ethanol to the lysate. Mix well by vortexing for5 seconds to yield a
homogenous solution.
9. Proceed immediately to Binding DNA.
Purification Procedure Using Spin Columns:
Binding DNA
1. Remove a PureLinkR Spin Column in a Collection Tube from the package.
2. Add the lysate (~640 μL) prepared with PureLinkRGenomic Lysis/Binding Buffer and ethanol to the
PureLinkR Spin Column.
3. Centrifuge the column at 10,000 ×g for 1 minute at room temperature.
4. Discard the collection tube and place the spin column into a clean PureLinkRCollection Tube
supplied with the kit.
5. Proceed to Washing DNA.
Washing DNA
1. Add 500 μL Wash Buffer 1 prepared with ethanol to the column.
2. Centrifuge column at room temperature at 10,000 ×g for 1 minute.
3. Discard the collection tube and place the spin column into a clean PureLinkRcollection tube
supplied with the kit.
4. Add 500 μL Wash Buffer 2 prepared with ethanol to the column.
5. Centrifuge the column at maximum speed for 3 minutes at room temperature. And Discard
collection tube.
6. Proceed to Eluting DNA.
Eluting DNA
1. Place the spin column in a sterile 1.5-mL microcentrifuge tube.
2. Add 25–200 μL of PureLinkR Genomic Elution Buffer to the column
3. Incubate at room temperature for 1 minute. Centrifuge the column atmaximum speed for 1 minute
at room temperature. The tube contains purifiedgenomic DNA.
4. To recover more DNA, perform a second elution step using the same elutionbuffer volume as first
elution in another sterile, 1.5-mL microcentrifuge tube.
5. Centrifuge the column at maximum speed for 1.5 minutes at roomtemperature.The tube contains
purified DNA. Remove and discard the column.
Principle of PCR
The PCR involves the primer mediated enzymatic amplification of DNA. PCR is based on using the ability of DNA
polymerase to synthesize new strand of DNA complementary to the offered template strand. Primer is needed
because DNA polymerase can add a nucleotide only onto a preexisting 3′-OH group to add the first nucleotide.
DNA polymerase then elongate its 3 end by adding more nucleotides to generate an extended region of double
stranded DNA.

Components of PCR
The PCR reaction requires the following components:
1.DNA Template : The double stranded DNA (dsDNA) of interest, separated from the sample.
2.DNA Polymerase : Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures
(98°), and can function at a temperature optimum of about 70°C.
3.Oligonucleotide primers : Short pieces of single stranded DNA (often 20-30 base pairs) which are
complementary to the 3’ ends of the sense and anti-sense strands of the target sequence.
4.Deoxynucleotide triphosphates : Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide
the energy for polymerization and the building blocks for DNA synthesis.
5.Buffer system : Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and
renaturation; also important for polymerase activity, stability and fidelity.
Procedure of PCR
All the PCR components are mixed together and are taken through series of 3 major cyclic reactions conducted in
an automated, self-contained thermocycler machine.
1.Denaturation :This step involves heating the reaction mixture to 94°C for 15-30 seconds. During this, the double
stranded DNA is denatured to single strands due to breakage in weak hydrogen bonds.
2.Annealing : The reaction temperature is rapidly lowered to 54-60°C for 20-40 seconds. This allows the primers to
bind (anneal) to their complementary sequence in the template DNA.
3.Elongation : Also known at extension, this step usually occurs at 72-80°C (most commonly 72°C). In this step,
the polymerase enzyme sequentially adds bases to the 3′ each primer, extending the DNA sequence in the 5′ to 3′
direction. Under optimal conditions, DNA polymerase will add about 1,000 bp/minute. With one cycle, a single
segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These
two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies
are generated and the number of copies of the template is increased exponentially.
Agarose Gel Electrophoresis:
Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA. (Kryndushkin et al., 2003).
Nucleic acid molecules are size separated by the aid of an electric field where negatively charged molecules migrate toward
anode (positive) pole. The migration flow is determined solely by the molecular weight where small weight molecules
migrate faster than larger ones (Sambrook & Russel 2001). In addition to size separation, nucleic acid fractionation using
agarose gel electrophoresis can be an initial step for further purification of a band of interest. Extension of the technique
includes excising the desired “band” from a stained gel viewed with a UV transilluminator (Sharp et al., 1973).
In order to visualize nucleic acid molecules in agarose gels, ethidium bromide or SYBR Green are commonly used dyes.
Illumination of the agarose gels with 300-nm UV light is subsequently used for visualizing the stained nucleic acids.

Throughout this chapter, the common methods for staining and visualization of DNA are described in details. Agarose gel
electrophoresis provides multiple advantages that make it widely popular. For example, nucleic acids are not chemically
altered during the size separation process and agarose gels can easily be viewed and handled. Furthermore, samples can be
recovered and extracted from the gels easily for further studies. Still another advantage is that the resulting gel could be
stored in a plastic bag and refrigerated after the experiment, there may be limits. Depending on buffer during
electrophoresis in order to generate a suitable electric current and to reduce the heat generated by electric current can be
considered as limitations of electrophoretic techniques (Sharp et al., 1973; Boffey, 1984; Lodge et al. 2007).
Preparing and running standard agarose DNA gels:
Several electrophoresis buffers can be used for fractionating nucleic acid such as, Trisacetate-EDTA (TAE) or Tris-borate-EDTA
(TBE) (Sharp et al., 1973; Boffey, 1984; Lodge et al., 2007). TAE gel buffer systems are more convenient than TBE systems, if
post-separation methods are the ultimate goal of running a gel (Rapley, 2000). For gel preparation, agarose powder
electrophoresis grade is mixed with electrophoresis buffer to the desired concentrations (usually with a range of 0,5-2%)
then heated in a microwave oven until completely dissolved. Ethidium bromide is usually added to the gel at concentration
of 0.5 ug/ml for nucleic acid visualization. The mixture is cooled to 600C and poured into the casting tray for solidification.
Immediately after the gel solidification, the comb is removed. The gel is kept in its plastic during electrophoresis and PCR
product mixed with loading dye is placed in the wells. As nucleic acids are negatively charged, wells should be placed towards
the negative electrode. At the same time, ethidium bromide migrates in the reverse direction, meets and couples with DNA
fragments. DNA fragments are visualized by staining with ethidium bromide when adequate migration has occurred. Then,
this fluorescent dye intercalates between bases of DNA and RNA (Corley, 2005). Linear DNA fragments migrate through
agarose gels with a velocity that is inversely proportional to the log10 of their molecular weight (Sambrook & Russel, 2001).
Circular forms of plasmids migrate in agarose gels differently compared to linear DNA of the same size. Typically, uncut
plasmids will migrate faster than the same plasmid when linearized (Sambrook & Russel, 2001).
CRISPR/cas9 technology:
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins function as an adaptive,
small-RNA-based immune system that protects prokaryotes from infectious viruses and plasmids. The CRISPR loci consist of
an array of short repetitive sequences (30–40 bp) separated by equally short spacer sequences. Many spacer sequences
match the genomes of viruses and plasmids of bacteria and archaea. This observation led to the hypothesis that CRISPR
systems protect prokaryotes from infection by these genetic elements. The bioinformatics predictions were first tested by
two experimental studies that showed that CRISPR loci prevent viral and plasmid infection.
CRISPR-Cas immunity develops in three phases. First, in the adaptation phase, Cas proteins integrate short regions of the
invader’s viral or plasmid genome into the CRISPR array as new spacers. Reviews of this phenomenon have been published
elsewhere. Second, the CRISPR array is transcribed and processed to generate small CRISPR RNAs (crRNAs) that contain a full
or partial spacer sequence (the crRNA biogenesis phase. During the third phase, known as targeting, processed crRNAs
associate with Cas nucleases to guide the ribonucleoprotein complex to the target sequence. Cleavage of the target
sequence, also known as a protospacer, results in both the destruction of the invader’s genome and immunity. Variations of
this immune mechanism characterize each of the three major types of prokaryotic CRISPR immune systems, which are
grouped according to cas gene conservation and operon organization
The principle of CRISPR/Cas9-mediated gene disruption is a single guide RNA (sgRNA), consisting of a crRNA sequence that is
specific to the DNA target, and a tracrRNA sequence that interacts with the Cas9 protein (1), binds to a recombinant form of
Cas9 protein that has DNA endonuclease activity (2). The resulting complex will cause target-specific double-stranded DNA

cleavage (3). The cleavage site will be repaired by the non-homologous end joining (NHEJ) DNA repair pathway, an error-
prone process that may result in insertions/deletions (INDELs) that may disrupt gene function (4).