BL222 Medical Microbiology

Fall 2017
Exam 3 (Chapters 12-15) Take Home Portion

This exam must be uploaded to Canvas as a Google Doc no later than Friday, September
4th at 11:30 AM. Due to the generous time allotted for completion, any exam received
after this deadline is subject to a 50% grading penalty. 40 points.

Please completely and directly answer the following questions as a work group. In class
notes, supplemental postings to Canvas, and your textbook are excellent resources to help
get you started, but you will need to perform outside research to provide quality answers
for most of these questions. Your outside sources must be:
Scholarly and credible; this includes other textbooks, journal articles, content from
reputable organizations (CDC, WHO, Pasteur Institute, etc.). Use of non-credible sources
will be factored into your grade.
Properly cited via in-text citations and a formatted reference page (APA or Council
of Science Editors format preferred). No direct quotes are permitted in your answers;
all material should be appropriately paraphrased.

Please feel free to consult with me at any time if you have any questions about guidelines,
formatting, or clarification of material. Given the nature of this exam, I am unable to tell you
ahead of time if you have enough material to answer the question, if your answer is correct,
etc. I also strongly suggest that you develop a teamwork contract as soon as possible
to help you fairly divide the workload across your group (provided on next page).

Please note that plagiarism will not be tolerated in this assignment. Your answers will be
submitted to TurnItIn.com and evaluated for similarity to source material and to material
submitted by other students. Suspected instances of plagiarism will be reported to
Academic Affairs and will result in severe grading penalties and loss of groupwork
privileges.

IMPORTANT NOTE ON GRADING: Demonstration of group effort and peer Team

Member Performance Evaluations will be taken into consideration for individual
exam grades. In order to track individual effort, each group must complete the
Teamwork Contract on the next page and perform all work within the Google Doc. In
order to earn the privilege of the group exam score, each student must 1) fulfill all
obligations as listed on the Teamwork Contract and 2) demonstrate a level of effort
expected for students in a 200-level course. Failure to contribute equally to the
group effort will result in grading penalties and loss of groupwork privileges.
Teamwork Contract (developed in part from Team Dynamics by Kolb and Francoeur)

Mission Statement:
Who is your point person to contact instructor, submit assignments, etc.?
Alexandra Barbera

When, where, and how will your team meet?

We will meet when all team members are available.
We will communicate through a group text message.
We will meet in an area accessible to everyone.
This area will be isolated and free of distractions.

Will there be a common agenda for meetings?

The group meeting are set up to allow us to come together and run ideas off of one
another. Our goal in these meetings is just to simply finish the group exam to the
best of our abilities. Our members believe that coming together helps us write and
efficiently answer questions.

What is expected of each member?

Each member is expected to participate and attend any scheduled meetings.
Each member expects respect out of one another.
Each member is expected to help other members that may be struggling with their
personal objective.

How will your team manage conflict?

We will talk out the conflict as a group and if the conflict cannot be resolved, we will
go to the instructor as a group to come to a conclusion.

List some key strengths/preferences of each member:

Alex- Very helpful when citing and finding applicable sources. Likes to talk things
over with other members to find the bigger picture. Is able to find connections
between sources very easily.
Hannah- A skilled writer and is a very good proofreader to fix and help other
members of the group with what they are trying to say. Helpful in keeping the group
on task and explaining what in depth articles are trying to say.
Victoria- Excellent in transferring ideas into complete thoughts and transferring
those thoughts onto paper. Very willing to help out other group members.
Bryce- Great at interpreting scholarly articles and finding useful information. Very
good at piecing bits of information together to apply to different topics and ideas.
Good public speaker.
How will you ensure members are meeting their personal objectives?
We will ensure the members are meeting their personal objectives by meeting as a
group and discussing progress.

1. The following items pertain to the structure, infection cycle, and pathogenicity of viruses;
we will be exploring hepatitis C, a viral infection that continues to spread worldwide at a rate of 3
million new infections per year. In order to answer this question, you will need to perform
scholarly research at NCBI PubMed. For best results, check the free full text filter button to
ensure you have access to your research.
a. (2 point) How is hepatitis C classified?
When trying to classify a virus there are certain phenotypic characteristics to look
at. These include the viruses morphology, nucleic acid type, mode of replication, host
organism, and the type of disease that this particular virus causes. When looking at the
morphology of hepatitis C (HCV) from the outside to the inside it is encased in a lipid
envelope bilayer covered in specific envelope glycoproteins (1). The glycoproteins that
are on hepatitis Cs envelope are E1 and E2 (2). Within this envelope is the
nucleocapsid layer that houses small basic proteins (2). Finally, inside of this layer is a
single-stranded RNA that contains this viruses entire genome (2). Based on the
Baltimore system of classification, HCV belongs to group IV which means not only does
it contain ssRNA (+) but that it makes a (-) strand that is complementary to its (+) strand
(3). The (-) strand is what is transcribed into mRNA and then translated into proteins (3).
Hepatitis C belongs to the family Flaviviruses and the genus hepacivirus (4). When
looking at HCVs specific host ranges it shows how difficult it is to study this disease
since it can only replicate in humans and chimpanzees (5). This virus is able to
effectively replicate inside of human and chimpanzee livers (6). Hepatitis C is one of the
major causes of chronic hepatitis, or liver inflammation, cirrhosis, or scarring of the liver,
and contributes to the development of liver cancer (2). All of these different
characteristics allow microbiologists to effectively classify hepatitis C amongst other
viruses.

b. (3 points) What is the mechanism of entry hepatitis C into host cells? In your answer,
include any putative (known to exist) host cell receptor molecules, if known

Hepatitis C entry into the host cell deals with lipids and their metabolism (7).
HCVs process of entry into the cell is very slow because several mechanisms are
involved in the uptake of the virus (7). The virus has a lipid envelope similar to that of low
density lipoproteins, but the structure is slightly different (8). Low-density lipoviral
particles are lipid particles that contain a viral RNA strand have a larger diameter and
are proposed to assemble in the endoplasmic reticulum, as compared to free circulating
lipoproteins (8). The cellular uptake of the virus is initiated by glycosaminoglycans, such
as heparin sulfate, and lipoprotein receptors on the host cell (7, 9). There are two
envelope proteins that may participate in the binding in the membrane fusion process,
E1 and E2, but their exact role is not yet clarified in this process. (7). Once the virus is
bound to the host cell surface, members of the tetraspanin protein family, CD81, serve
as an E2-binding protein (9). This protein is a receptor molecule that forms a complex
with the virus (9). CD81 is necessary, but acting alone is not sufficient enough for the
uptake of the virus into the host cell. Scavenger receptor class B type I, SR-BI
interaction is also needed (7). The expression level of SR-BI regulates the host cells
infectivity of HCV by interacting with both the LDLs and the soluble E2 protein (9). The
next step is the translocation of the virus complex to tight junctions (7). The tight junction
proteins Claudin-1 and Occludin act as cofactors and lead to receptor-mediated
 endocytosis (9). Once the endosome is within the host cell, it is acidified in the
cytoplasm and the nucleocapsid is released (9).

c. (2 points) What types of host cells are infected by hepatitis C?

When HCV is within the body, it attaches to hepatocytes with proteins and lipids
within HCV. The scavenger receptor associated with HCV infection is known as a
scavenger receptor class B (SR-B1) (8). SR-B1s main function within the hepatocyte
cell is to bring in lipoproteins (10). HCV is masked with the lipoproteins that surround its
outer layer and is then brought inside the hepatocyte (11). HCV then enters the host
hepatocyte cells plasma membrane and completely emerges itself within the cell. The
protein coat that surrounds HCV that withholds the viral RNA is released inside the
hepatocyte. HCV then takes over the hepatocytes ribosomes. However, in a study
published in 2011, there was evidence that liver cells are only one type of cell that is
targeted by HCV. This study found HCV replication in B cells, T cells, monocytes,
macrophages, Kupffer cells, and dendrocytes (12). In vitro studies have proven the
replication of HCV in cells other than hepatocytes. This would suggest that the biology of
HCV would need to be studied further to gain a better understanding of how to treat it.

d. (2 points each) Discuss the clinical significance and role in the viral life cycle of the
following hepatitis C viral structures:

i. E1
E1 is a glycoprotein found in the envelope of hepatitis C that fuses with
the protein E2 to form a heterodimer (4). The E1E2 complexs role in the life
cycle of HCV is allowing the virus to bind to and gain entrance into the host cell
(4). There is not as much information known about E1 as E2 but it is believed
that E1 has an important function in the fusion of the virus to the membranes
within the cytoplasm (4). E1 is clinically significant because, as some researchers
at the American Society for Microbiology have found, it is possible for certain
monoclonal antibodies to target envelope glycoproteins, like E1 and E2, and
destroy the studied isolates of HCV even though they were diverse genetically
(13).
ii. E2
E2 is a very similar protein to E1 (4). E2 is also a HCV envelope
glycoprotein that is involved in the fusion and entry of the virus to the host cell, by
anchoring the virus to the membrane. When DArienzo et. al. attached E1E2
complexes from patients with HCV to in vitro lentivirus particles and introduced
these particles to human antibodies that specifically target envelope
glycoproteins, the antibodies neutralized the complex (13). This is important in a
clinical setting because this means that the glycoproteins can be a potential
target for antiviral treatment.
 iii. P7
P7 is an integral membrane protein that is believed to act as an ion
channel (4). Functionality tests with chimpanzee HCV showed that if p7 is not
functional or not present in the virus, then the transinfection of the virus between
liver cells in the host is lowered (4). This is relevant clinically because if we can
suppress the activity of HCV in the liver, the health of the patient would be
increased. There has been some research that shows long-alkyl-chain
derivatives of iminosugars, that have deoxynojirimycin or
deoxygalactonojirimycin, could be used to inhibit p7 (14).

iv. NS2
NS2 is a viral transmembrane protein that is not glycosylated (4). Its role
in the HCV lifecycle is the membrane association with the hosts endoplasmic
reticulum (4). NS2 is most seen associated with NS3 because together they act
as a protease (4). This protein has shown an ability to interact with the effector of
apoptosis in the liver but researchers are still not sure what this has to do with
the life cycle of HCV (4). A study of the molecules quercetin and naringenin
showed that these ligands can bind to the binding pocket section of NS2
protease (15). This information is clinically relevant because if these ligands are
bound to this pocket, then NS3 cannot bind to NS2 and the protease complex
cannot form and cannot associate with the ER (15).

v. NS4B
NS4B is another integral membrane protein of Hepatitis C. Its main role in
the viral life cycle is to aid in the association of the virus to the host membrane
(4). It is very important for HCV viability because it anchors the replication
complex to the membrane, alters the activity of RNA replicase, inhibits the
syntheses of the host cell, transforms fibroblast cell lines, and controlling the
induction of interleukin (4). Certain amino acids that are essential for the function
of this protein are greatly conserved which makes them a very attractive target
for antiviral medication and vaccinations (16).

vi. NS5A
NS5A is a phosphorylated zinc-metalloprotein in HCV that is necessary
for regulating cellular pathways and viral replication because it is needed to
assemble the replication complex and in the localization of membranes around
the host nucleus (4). When the site that zinc binds to on this protein was
mutated, HCV RNA replication was inhibited (4). The drug BMS790052 has been
shown to achieve this inhibition but resistance has already been evident so this
metalloprotein may no longer be an effective target for future drugs (17).

vii. NS5B
 NS5B is a membrane protein in hepatitis C that is anchored to the viral
tail (4). Its function is to target the functional protein domain of the host, on the
cytoplasmic side of the endoplasmic reticulum, after translation (4). Creation of
the replicase complex and completion of RNA-binding are not possible without
NS5B (17). The clinical relevance of this protein is that cyclophilin inhibitors have
been shown to inhibit the ability of viral RNA polymerase to incorporate into the
replicase complex, which restricts RNA replication (17). Studies have shown that
the combination of the drugs ribavirin, Debio-025, and a pegylated interferon
have been effective in lowering the levels of hepatitis C in the blood (17).

e. (3 points) How do hepatitis C virions spread from one cell to another in the human body?

In order to follow the spread of hepatitis C from cell to cell, we need to investigate
the creation of the initial virus inside of the host cell. Once successfully inside of the host
cell the viral RNA takes control of the host cells ribosomes where it then begins the
construction of viral reproduction (11). Once these ribosomes are taken over they are
then used to create a template strand of RNA that can then be used to create multiple
copies. When inside the cell the Hepatitis C RNA stops all normal functions inside of the
cell except the reproduction process (11). It is suggested that this process is allowed to
continue because it increases the amount of cells producing the HCV disease (11).
When there is a substantial amount of RNA transcriptase the virus RNA then produces
the complementary strand to complete full viral RNA copies in the thousands (11). The
viral RNA then tells the ribosomes to create the protein capsid that encloses the RNA.
These capsid proteins, better known as capsomeres, attract one another and eventually
build a spherical shell around the HCVs RNA (11). This layer is referred to as the
nucleocapsid. This freshly created virus then travels to the plasma membrane of the host
cell where it attaches. Once attached the virus buds encircling the virus in a lipid coat
while simultaneously releasing it outside the cell. This process will continue until the host
cell dies from exhaustion. Once outside the cell this newly formed virus may then attach
to another functional liver cell to begin this process once again (11). When outside the
host cell the virus must dodge the hosts immune system and other environmental
factors in order to make it to its next target.

2. (4 points) Provide a two paragraph summary of what is currently known about the innate
immune response to Hepatitis C.

Hepatitis C virus can be eliminated from the host if caught early enough. Innate immune
responses are the immediate immune action that starts as soon as the invading
pathogen enters the body (3). After the pathogen is first introduced to the body, a quick
and effective response of the innate immune system is induced. This immune response
needs to be efficient because of the delayed response of the adaptive immune system,
and the body needs to effectively clear the HCV from the body before it becomes chronic
(18). The HCVs genome is very small allowing for rapid replication and if the innate is
not capable of controlling the virus, the late arriving adaptive immune system would not
be able to handle the mass amounts of infected cells. After the virus enter the
hepatocyte and the cell starts to replicate, the HCVs double stranded RNA
intermediates presents a pathogen associated molecular pattern (PAMP) (18). PAMP is
sensed by the innate immune system by the toll-like receptor (TLR) dependent pathway
and cytosolic pathway (18). It is sensed by the binding of RNA to RNA helicases retinoic
acid inducible gene-1 (RIG-1) and melanoma differentiation antigen 5 (Mda5) (18).
Macrophages and dendritic cells sense the invading pathogen by the pathways and
consequently, starts the production of IFNs and cytokines to signal other immune cells
(18, 3). Some people have a better innate immune system than others and this is
because of over-expression of interferon induced genes and interferon stimulated genes
(18). People with an excess of interferon induced genes would produce more
interferons. Interferons (IFNs) are low-molecular mass cytokines that are produced in the
body as a response to an intracellular infection (3). It is the interferons (I, II, and III) in
the innate immune system that is responsible for the production of natural killer cells
(18). The interferons then signal the JAK-STAT signaling pathway to transmit information
from the cell surface to the nucleus (18). Type I interferons deals with antiviral properties
and synthesize proteins on cells that have not been infected (3). Type II interferons help
to bridge the innate and adaptive immune system by sending out natural killer cells as
well as B-cells. Type III interferons are produced by most, but not all, cells and they track
epithelial cell receptors (19).
Natural killer cells (NK) are the most commonly found WBC in the liver (18). NKs
greatly assist the bodys immune system in controlling viruses and tumor cells. This
cells job is to detect if there are any problems occuring inside of the cell. When trying to
find infected host cells the NK cells look for targets that do not carrying the major
histocompatibility complex (MHC) all is fine flag. This flag is taken down once the cell
senses that there is something wrong. It does this by using NOD receptors, which are
intracellular sensors that detect pathogen-associated molecular patterns that are brought
in through endocytosis. This is the pathway of entry used by HCV to enter host cells.
With the MHC stage 1 molecule being taken down NK cells are then able to recognize
the infected host cell. Once this is cell is recognized the NK cell can granulize and attack
this cell and the surrounding hepatocytes. Although the innate immune system works
hard to eliminate pathogens from the body by natural killer cells and INFs, HCV
developed mechanisms to escape the immune responses. HCV can hide during innate
immunity by cleaving and inactivating two significant signaling molecules that react to
PAMPs which induce IFNs (18). The enzyme NS3/4A protease is responsible for this
cleaving and inactivation (18). Other mechanisms that block the innate immune system
include that of blocking ISG protein translation, Jak-STAT signaling, and ISG induction
(18). The virus can also hide from the adaptive immune system by mutations that help
the virus avoid recognition by antibodies (18). Overall, the innate immune system has a
small antiviral effect despite many ISGs but many other reasons for why the innate
immune system fails early on is still unknown(19).
3. (5 points) Hepatitis C transmission in healthcare settings is a growing concern, as several
outbreaks can be traced to improper decontamination protocols. How long is Hepatitis C viable
on inanimate surfaces, and what methods of disinfection work best against this virus?

About 20% to 50% of hepatitis C virus infections can be accounted for by hospital
based transmission (20). Hepatitis C requires high-level disinfectants to inactivate the
pathogen that may reside on medical devices (21). Standard protocol is to treat every
patient as if they have a bloodborne pathogen, but some healthcare facilities fail to abide
by these protocol and only use the disinfection procedure if the patient is known or
suspected to be infected with such pathogens (21). Ethylene oxide sterilization, EtO,
was seen to be implemented in those hospitals that altered their disinfection procedure,
but is not routinely used on endoscopic instruments due to its long processing time (21).
When the medical devices are not disinfected and sterilized properly, pathogens get left
behind and outbreaks can occur. The Center for Disease Control states that the
Hepatitis C virus can survive at room temperature on environmental surfaces for up to
three weeks (22); however, other studies have been shown that HCV has the potential to
remain infectious for up to six weeks (20, 23). Yale researchers found that virus can hold
its infectivity at room temperature to four degrees Celsius for six weeks (20, 23).
Supporting evidence for the viruss prolonged infectivity stems from its lipid envelope
(23). The lipid envelope protects the virus from dehydration by resisting drying out (23).
A possibility of the viability of HCV is though the vehicle transmission by inanimate
objects (23). This is supported because the majority of patients who contracted HCV in a
hospital setting did not have surgery or any invasive procedures (23). Surgery or
invasive procedures could have been an explanation for potential blood to blood contact,
but fomites, or inanimate objects, that were not properly sterilized could carry and
spread the virus by means of hospital equipment.
There are several methods of disinfection that work against the virus. Killing or
remove viruses from a surface requires high level or intermediate level disinfectants (3).
Examples of these levels of disinfectants are ethylene oxide, glutaraldehyde and
formaldehyde, and phelolytics and halogens, respectively (3). Low level disinfectants
have the possibility to kill or remove viruses but may not be entirely effective, so their
use is not recommended for complete removal (3). These disinfectants include that of
alcohols and quaternary ammonium compounds (3). When cleaning patient-care
devices, cleaning the devices right after use with water and detergent agents in order to
remove organic residue is needed before high-level disinfectant techniques (21). High-
level disinfection techniques should be followed by a sterile water rinse to eliminate the
possibility of patient side-effects from the high-level detergent (21). According to the
CDC, cleaning surfaces with at least 2% glutaraldehyde for 20 to 90 minutes should
prevent transmission of HCV (21). Another method discussed by the CDC completely
eliminates HCV from endoscopes using glutaraldehyde for three to five minutes (21).
Phenol and chlorine compounds used showed that phenolic compounds inhibited the
binding and replication of HCV while chlorine compounds were ineffective (21).
 Commercially, researchers found that bleach, CaviCide and alcohol, ethanol, reduces
the viruss ability to infect when used at the recommended levels and did not reduce
when the antiseptics were diluted (20). A study was conducted comparing disinfecting
methods of Goldmann tonometer tips, an instrument used to measure intraocular
pressure, using common cleaning agents (24). The experiment concluded that dry gauze
wipes left behind 95.65% of HCV, isopropyl alcohol 5-second wipes left behind 88.91%,
a cold water wash left behind 4.78%, providone iodine 10% 5-second wipes left 0.72%,
3% hydrogen peroxide soak with a cold water wash left 0.07% of the virus and 70%
isoporpyl alcohol soak with a cold water wash only left 0.02% of HCV behind (24).

4. Recently, a new drug called Harvoni (sofosbuvir/ledipasvir) was approved by the FDA for
successful treatment of hepatitis C. This new treatment can cure a patient of chronic hepatitis C
infection, which was previously impossible with older methods of treatment. In order to answer
this question, you will need to perform scholarly research at NCBI PubMed. For best results,
check the free full text filter button to ensure you have access to your research.

a. (4 points) What is the mechanism of action of Harvoni?

Harvoni falls into the category of direct antiviral agents or DAAs (25). Harvoni is
the first combination drug to treat HCV. It is formed from the combination of sofosbuvir
and ledipasvir. This combination drug is the first form of drug therapy that was not used
with interferons or ribavirin (25). Going along with that this too is a combination drug that
focuses on inhibiting HCVs ability to move intracellularly. Sofosbuvir is a nucleotide
prodrug, which means that it must be activated inside the body. This drug must go
through intracellular metabolism in the liver to form active uridine analog triphosphate
(26). This drug has been proven to be effective against HCV genotypes 1-4 (26).
Sofosbuvir works by inhibiting HCVs NS5B RNA-dependent polymerase. This then
causes a termination in the translation process not allowing HCV to fully construct
proteins from its RNA strands. Ledipasvir, the second drug found in Harvoni, is another
inhibitor of genotypes 1a, 1b, 4a, and 5a with a lesser effect on genotypes 2a and 3a
(26). The exact mechanism of this drug is unknown when looking at recent research but
it is theorized that this drug works by inhibiting hyperphosphorylation of NS5A (26). This
phosphorylated zinc-metalloprotein is suspected to be used for viral replication and
regulation of cellular pathways (4). These inhibitory drugs create a synergistic effect
obstructing both nonstructural proteins NS5B and NS5A.

b. (3 points) Many healthcare providers and public health officials are concerned about
Harvoni due to the high cost of treatment ($94,000 for a full course of treatment). Some
insurance providers are unwilling to cover the cost of this treatment unless a patient is facing
certain death due to the viral infection. As a group, discuss this issue in light of your future
career as a healthcare provider.
 As 3 aspiring physician assistants and a nurse, ethics will undoubtedly play a
major role in our future careers. Each one of us bears the responsibility to care for any
patient we encounter. In the Physician Assistants Code of Ethics, it states that each
Physician Assistants primary responsibility is the health, welfare, and safety of the
patient (27). In the Nursing Code of Ethics, it states that each nurse must advocate for
the best interest of the patient (28). Denying anyone who has HCV this treatment unless
they are facing certain death would be unethical. Arguably, it would be even more costly
and risky for the patient involved. We must inform our patients on what treatment we
believe is the most effective for their condition based on our education. However, as of
this moment, the best treatment is essentially unavailable to a significant population of
people suffering from HCV. This would leave us as the healthcare provider in a dilemma.
One course of action is for us to come together as providers and inform the insurance
company of the importance the use, especially the early use, that this drug has on the
lives of our patients in an attempt to have it more easily covered. Another option is to
fund more research on a more affordable treatment for hepatitis C.
In 2015, Gilead announced that they would start to produce a generic form of
Harvoni with a handful of Indian companies (29). In the 101 developing countries that
these companies would be able to target, 103 million people are living with HCV (29).
This would significantly reduce the costs of treatment for anyone who was seeking out a
cure. For Americans, they would still be scouring for ways to cover the $94,000 it would
cost to be cured. For the course of that treatment, it would be over $1,000 per pill. With
the ease of practically getting whatever you could want off the internet, Americans
sought out these generic pills. With a simple google search, we found that we had the
ability to place an order for the generic Harvoni. The twelve week treatment for the
generic version costs only $1495 (30). Obviously this is the more cost effective treatment
plan considering a single pill for the regular version is nearly the same price. In all reality,
each of us will encounter situations where we know a patient requires a specific medical
treatment, but is unable to have that treatment due to cost or insurance issues. Morally,
each of us know this should not be a factor in the treatment of another human being.
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