Favourite Virus Assignment: Parvovirus
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Transcript of Favourite Virus Assignment: Parvovirus
Favourite Virus Assignment
Samantha Bray
BIOL 4904 – Virology
December 1st, 2015
Q1-1: What is your favourite virus?
My favourite virus is Parvovirus. This assignment will be answered mainly in the context
of canine parvovirus. If information about canine parvovirus could not be found the question will
be answered in the context of other parvoviruses such as adeno-associated virus, human
parvovirus B19, and minute virus of mice parvovirus (MVMp).
Q1-2: What type of genome does your virus use?
Canine parvovirus genome consists of a roughly 5000-nucleotide long linear strand of
either positive sense or negative sense single stranded DNA as their genome. However, the
negative sense stand may be favoured for packaging.1
Q1-3: Using http://viralzone.expasy.org/ (or similar resource), find a virion diagram of
your virus and explain its structure.
Figure 1. Parvovirus virion structure shows general capsid arrangement with T=1
symmetry and a ssDNA viral genome.2
Parvovirus has a spherical, icosahedral, self-assembling capsid that consists of 60 copies
of two types of capsid proteins, VP1 and VP2. The capsid has symmetry of T=1 and it encloses
the single stranded DNA genome. CPV is very small and is only 22 nm in diameter.3
Q2-1: What capsid structure does your virus use?
Parvovirus has an icosahedral capsid structure with a T=1 symmetry.3
Q2-2: Is your virus enveloped?
Parvovirus is a non-enveloped virus and is therefore called a naked virus.4
Q2-3: What is the typical ratio of infectious virus to viral particles produced during
infection with your virus?
When KB cells are infected with adeno-associated virus (AAV) they produce many
classes of particles that differed in the buoyancy is CsCl. They formed AAV particles that had
densities of 1.450 g/cm3, 1.410 g/cm3, 1.350 g/cm3, and 1.320 g/cm3. The only AAV particles
that were infectious had a density of 1.450 g/cm3 and 1.410 g/cm3 and they contained standard
full-length genomes. The AAV particles that had a density of 1.350 g/cm3 and 1.320 g/cm3
contained deleted or snap-back genomes and were therefore non infectious.5 Although I was
unable to find the typical ratio of infectious virus to viral particles I was able to find the particle
to infectivity ratio. For the 1.450 g/cm3 infectious particle is 20-100 fold higher than the 1.410
infectious particles that has a particle to infectivity ratio of less than 100:1.6
Q2-4: What host proteins are found in your virion?
The parvovirus virion is an extremely small virus therefore it carries genes for what is
absolutely necessary. Parvovirus encodes only what is absolutely necessary. They encode for
about 2-4 capsid proteins, mainly making VP1 and VP2 as well as NS1 and NS2 non-structural
protein.7
Q3-1: With the aid of the side diagram, explain each basic step of the viral replication
cycle of your virus. Indicate what steps apply to your virus (3A, 3B, 3C and 5A, 5B).
Step 1: Attachment to host receptors.8
Step 2a: Does not apply
Step 3a: Does not apply – parvovirus does not fuse with plasma membrane.
Step 3b: Mediates clathrin-coated endocytosis.
Step 3c: Virion penetrates host endosomal plasma membrane via pH drop to ~6.0 causing
permeabilization to gain entrance into cytoplasm.8
Step 4: Virion transported to nucleus via microtubule system. ssDNA penetrates into nucleus and
is converted to dsDNA by cellular polymerase. dsDNA is transcribed to give viral mRNAs, and
genomic DNA
Step 5a and Step 5b: Do not apply – virion exits through cell lysis.
Step 6: New virions are released into extracellular medium until they encounter a new target
cell.8
Q3-2: What type(s) of cellular receptors does your virus use for viral entry?
In dogs, parvovirus binds to host transferrin receptors to mediate viral entry through
dynamin dependent clathrin-coated endocytic vesicles.9
Q3-3: Find a research article figure depicting the replication of your virus over time.
Explain.
Essentially, the researchers inoculated CPV-2b strain into canine A-72 cell line that was
maintained in Dulbecco Minimal Essential Medium with 5% FCS in order to asses the kinetics
of canine parvovirus mRNA and viral DNA production during the course of infection. They took
samples of A-72 supernatants and criolysates at 24, 48, 72, and 86 hours post infection. They
extracted the RNA and DNA from the supernatants and criolysates with extraction kits. They
extracted RNA extracted from mock-infected A-72 cells as negative controls. They found that
they were able to detect viral NS2 mRNAs in the criolysate as early as 24 hours and at 38 hours
in the supernatant. They were able to detect DNA at 0 hours in the criolysate and 24 hours post
infection in the supernatant. Both viral mRNA and DNA loads increased in all of the trials.10
Q4-1: What type of polymerase replicates the genome of your virus? Explain how this
affects viral evolution. What are the main forces contributing to genomic variation in the
family of your virus?
Host cell DNA polymerase is responsible for replicating Parvovirus single stranded DNA
genome through a rolling hairpin mechanism. Parvovirus is able to improve its genetic economy
by utilizing host cell DNA polymerase. This affects viral evolution making a more efficient virus
that does not need to waste resources encoding their own viral polymerase.11 The main forces
contributing to genomic variation is recombination of NS1 genes and VP2 genes as well as the
fact that parvovirus even though it is a DNA virus has mutation rates similar to that of RNA
viruses.12
Q4-2: Find your favourite virus in the ICTV website taxonomy tree. Is your virus a type
species? Has the classification changed since the 2005 release?
My virus is not from an assigned order, but it is from Family Parvoviridae, Subfamily
Parvovirinae, Genus Parvovirus and Species Canine Parvovirus. The classification has not
changed since 2002. 13
Q5-1: Research another Bacteriophage. Make a table using 6 criteria describing the
similarities and differences between T7 and this virus.
Table 1. Similarities and differences between Bacteriophage lambda and Bacteriophage T7.
Criteria Bacteriophage lambda14 T7 Bacteriophage14
Capsid
Structure
Non-enveloped,
Icosahedral (T=7).
Non-enveloped
Icosahedral (T=7).
Genome Linear double stranded DNA with
cohesive ends (single stranded
extensions on either end).
Circular single stranded DNA.
Genome
Expression
Sequential Expression, early
genes, middle genes then late
genes. 14
Sequential Expression, early genes,
middle genes then late genes.
Genome Entry
Into Cell
Transcription via RNA
polymerase pulls the genome out
of the virion into the cell.
Requires mannose permease, a sugar
transport system. Is poorly
understood.
Viral Genome
Integration into
Host Genome
Not observed. Integrates viral genome into host
genome creating what is called a
lambda lysogen, which is capable of
turning off all host gene expression.14
Lytic or
Lysogenic
Lytic only. Capable of lytic or lysogenic cycles.
Q5-2: Describe a therapeutic use for phages in humans.
Oncotis, or ear infections are often hard to treat. Researchers created a bacteriophage
cocktail that consisted of five podoviruses and one myovirus. The viruses they selected
specifically target bacteria that are often found to cause ear infections. Using less than 1 million
phages, which is a relatively low dose, they were able to effectively treat the oncotic and provide
their patients with therapy.15
Q6-1: Research another Archaeovirus. Make a table using 6 criteria describing the
similarities and differences between SSV-1 and this virus.
Table 2. Similarities and differences between Sulfolobus Spindled Shaped Virus 1 and
Sulfolobus islandicus filamentous virus.
Criteria Sulfolobus Spindle Shaped Virus 1
(SSV-1)
Sulfolobus islandicus filamentous virus
(SIFV)
Genome Circular supercoiled dsDNA16 Linear dsDNA with modified terminal
end structures.17
Envelope Yes. Yes.
Viral
Genome
Integration
Into Host
Cell
Genome
Yes, carries integrase along in the
virion.16
No, does not carry integrase along in the
virion.17
Host Infects Crenarchaeota that are
hyperthermophilic, liking temperatures
above 80 C.
Infects Crenarchaeota that live in hot
springs.
Virion
Structure
Lemon shaped with short tail fibres at
one end.
Filamentous/rod shaped and each end has
three terminal tails.
Adsorption
Strategy
Virus attaches to host cells using their
tail fibres.18
Virus attaches to host cells using their
terminal tails as claws that fold upon
interaction.18
Q6-2: Does your favourite virus co-replicate with a satellite virus?
Dependoparvoviridae such as adeno-associated virus 1 and 2 (AAV-1, AAV-2) are able
to function as satellite viruses that do not have negative effects on the virus it co-replicates with.
AAV-1 and AAV-2 adeno-associated viruses have replication cycles that depends on the
coinfection with adenovirus or herpesvirus.19
Q6-3: Could your virus be inhibited by the natural or a synthetic CRISPR system?
Exhaustive literature research has resulted in no findings for inhibition of parvovirus by a
natural or synthetic CRISPR system. In a very general comparison however some researchers
have been able to inhibit the replication of other ssDNA viruses although they infect plants not
animals. Specifically they inhibited replication of beet severe curly top virus with
sgRNACRISPR-Cas9 through the introduction of mutations at target sequences.20
Q7-1: Research two other ssRNA+ viruses from different families. Make a table using 6
criteria describing the similarities and differences between Poliovirus and these viruses.
Table 3. Similarities and differences between Poliovirus, Cucumber Mosaic Virus, and
SARS (positive sense single stranded RNA viruses).
Criteria Poliovirus
(Family
Picornavirodae) 22
Cucumber Mosaic Virus
(Family Bromoviridae) 21
SARS (Family
Coronoviridae) 22
Virion
Structure
Non-enveloped,
Icosahedral capsid
(T=1), Small (30 nm).
Icosahedral capsid (T=3),
non-enveloped, Small (29
nm).
Very large (120-160 nm),
Sphere, enveloped,
studded with clubbed
spikes, Helical
nucleocapsid.
Genome
(all are
+ssRNA)
8 kb in length, 5’ end has
a viral protein (VPg),
and a polyadenylated
segment at the 3’ end.
9 kb in length, Segmented
genome into 3 RNA
molecules, 5’ end is capped
and 3’ end has tRNA like
structure.21
27-32 kb in length has a
5’ terminal cap and a
polyadenylated 3’ tail.
Pathology Poliomyelitis (paralysis). Sometimes is
asymptomatic,
Catastrophic Crop Loss
(because it infects plants)
In humans causes severe
acute respiratory illness,
fever, pneumonia,
hypoxemia, and death.
Fun Facts Translated all expressed
genes into a single
polyprotein that gets
cleaved into functional
units by viral encoded
proteinases. 22
The segmented genome is
not all packaged into a
single virion rather it is
packaged into many.
Allows a large genome to
be packaged into a small
particle.
Has the largest known
RNA genome that
encodes for over 20
proteins.
Viral
Entry
Virus binds to cell with
CD155 poliovirus
receptor mediating
causing a conformational
change that creates a
pore through the host
cell membrane allowing
the genome into the cell.
Virus enters through
natural wounds in the plant,
and then uses cell to cell
spread through junctions
such as plasmodesmata to
infect other cells.21
Spike protein binds to
host cell receptor
mediating fusion of
envelope with the host
cell membrane. 22
Proteins Encodes for 3-4 capsid
proteins, 1-3 proteinases,
and 6-8 proteins
associated with
replication.
Encodes for a genome
capping enzyme, a RNA
polymerase, a silencing
suppressor, movement and
capsid proteins.
Encodes for
approximately 12-16
replication proteins, 3-4
envelope proteins, one
nucleocapsid protein, and
4-6 other proteins.
Q8-1: Research two other -ssRNA viruses from different families. Make a table using 6
criteria describing the similarities and differences between Pneumoviruses and these
viruses.
Table 4. Similarities and differences between Pneumoviruses, Rabies Virus, and Ebola
Virus (negative sense single stranded RNA viruses).
Comparison
Criteria
Pneumoviruses 23 Rabies Virus 24 Ebola Virus 25
Taxonomy Order: Mononegavirales
Family: Paramyxoviridae
Genus: Pneumovirus
Order:
Mononegavirales
Family: Rhabdoviridae
Genus: Lyssavirus
Order: Mononegavirales
Family: Filoviridae
Genus: Ebolavirus
Clinical
Symptoms
Respiratory tract infection
Inflammation of the
brain (encephalitis). 24
Hemorrhagic fever
Replication
Strategy
Virus binds to host cell
with HN glycoprotein and
fuses with host cell
plasma membrane
releasing nucleocapsid
into cytoplasm. Genome
replicated by sequential
transcription.
Virus binds to host cell
with G glycoproteins,
mediates clathrin-
mediated endocytosis.
Virus fuses with
endosomal membrane
releasing nucleocapsid
into cytoplasm.
Genome replication
sequential transcription.
Virus binds to host cell
with GP glycoprotein
mediating pinocytosis
uptake of virus, virus
fuses with vesicle
membrane releasing
nucleocapsid into
cytoplasm. Genome
replication by sequential
transcription
Genome Linear -ssRNA, 15 kb,
encodes for 11 proteins
(NS1, NS2, N, P, M, SH,
G, F, M2, L)
Linear -ssRNA, 11 kb,
encodes for 5 proteins,
plus 4 more via
alternative initiation.
(N, M, G L, and P1 –
and through alternative
initiation P2, P3, P4,
and P5).
Linear -ssRNA, 18-19kb,
encodes for 7 proteins
(NP, vp35 vp40, GP, L,
vp30, vp24)
Viral
Transmission
Coughing releases viruses
hanging on respiratory
droplets that get inhaled
by others. 23
Transmission is
zoonotic, and through
animal bites.
Zoonotic, Person to
person infection is
through contact with
infectious body fluids.25
Virion
Structure
Enveloped and spherical
shaped virus.
Enveloped and bullet
shaped virus.
Enveloped and
filamentous shaped.
Q9-1: Identify a virus that can undergo genome reassortment. Draw a diagram that
explains which genome segment exchanges are most likely to promote virulence in a new
reassortant strain of this virus.
Influenza viruses have an ssRNA genome and are classified into types A, B and C
depending on core proteins. Type A viruses are further subdivided according to their envelope
glycoproteins with haemagglutinin (HA) or neuraminidase (NA) activity. The use of NA and HA
for classifying strains shows that these segments are important for the virus. Thus showing that
genome segment exchanges in these regions are important. Influenza virus can undergo genome
segment exchanges that cause major changes in the type A influenza HA and NA surface
antigens. This major reassortment event can promote virulence and may cause large pandemic
outbreaks. HA genome segment exchanges promote virulence because HA is the major antigenic
target of neutralizing antibodies. The host will have neutralizing antibodies that are specific for
the HA antigen and specific for the last strain of influenza encountered. The protective effect of
neutralizing antibodies by one strain of influenza may be reduced, lost, or ineffective against the
new strain of Influenza. This is why new strains are able to cause large pandemic outbreaks.26
Figure 2. Reassortment event with two strains of Influenza. Influenza A and Influenza B.
HA and NA gene segments reassortment leads to increased virulence in Influenza Strain C.
Original artwork by: Samantha Bray
Q9-2: Explain what components of reovirus are recognized by the innate immune system.
What is the cellular sensor responsible for initiating this signalling cascade?
Rotavirus viral genomic dsRNA acts as a PAMP for TLR 3, TLR 7 and TLR9.
Endosomal TLR3 recognizes dsRNA and activates downstream signalling to induce the
production of NFκB, IRF3 and IRF 7 that activate the transcription of Type 1 interferons.
dsRNA can also be recognized in the cytoplasm by PKR or RLRs (RIG-I and MDA5). PKR
responds to dsRNA and through some unknown mechanism promotes the secretion of IFN-β.
Most viral dsRNA is sequestered in viroplasm however in an imperfect process some may be left
in the cytoplasm.27
Q10-1: Research one other small DNA virus from a different family. Make a table using 6
criteria describing the similarities and differences between Parvovirus and this virus.
Table 5. Similarities and differences between parvovirus and nanovirus (small DNA
viruses).
Criteria Parvovirus 28 Nanovirus 29
Virion
Structure
Non-enveloped capsid, round
icosahedral T=1 symmetry made of
only 60 identical capsid proteins. 18-26
nm
Non-enveloped, round capsid with
T=1 icosahedral symmetry. 18-19 nm
Genome
Structure
Linear ssDNA, 4-6 kb genome with
terminal hairpin ends that promote
replication.
6-8 1 kb circular segments of ssDNA
genome, approximately 4 satellite
genome segments that encode
accessory replication proteins. Each
circular segment encodes only one
protein.
Genome
Replication
Strategy
Rolling hairpin mechanism to make
dsDNA transcription templates and
ssDNA genomes to package into
virions. 28
Rolling circle replication to produce
ssDNA genome that can be packaged
into virions, converted to dsDNA
transcriptional templates, or
transported out of nucleus via
movement proteins and into neighbour
cells via plasmodesmata. 29
Associated
Symptoms
Canine parvovirus causes
gastrointestinal upset, and GI tract
damage mainly in puppies. Human B19
Parvovirus causes Fifth disease (cold-
like symptoms, fever, headache, rash,
and skin lesions. 30
Growth stunting, plant death, plant
tissue necrosis.
Host Humans (B19 type), Canines (CPV2
type), and others.
Legume plants.
Transmission Transmission by respiratory/oral
droplets or fecal-to-oral.
Aphid vectors transmit viruses
between plants.
Q11-1: Human papillomaviruses also can transform cells. Using a diagram, explain the
differences and similarities in cell signalling pathways that contribute to oncogenesis
during polyomavirus and papillomavirus infections.
Polyomaviruses have large T antigen. Large T antigen induces separation of pRb protein
from E2F allowing transcription of cellular genes and entry into the S phase of cell cycle. T
antigen binds p53 that blocks the cell cycle and p53-mediated apoptosis. T antigen also binds
p300 and controls the levels of transcription of cellular genes. Cell transformation and tumour
formation occur when parts of viral DNA is integrated into non-permissive host cell genome.
The cell can then express viral early genes expressed (T antigen) that turn on the cell cycle and
block apoptosis, leading to increased growth in the cell and its progeny cells. Oncogenic cells do
not produce virions, only early viral proteins are express. 31
Papillomaviruses have E7 protein that binds to pRb. Like polyomaviruses T-antigen,
papillomavirus E7 protein induces pRb separation from E2F promoting DNA synthesis through
entry into the S-phase of the cell cycle. Unlike T-antigen, E7 protein can induce ubiquitin-
mediated proteolytic degradation of pRb. Papillomaviruses use a different protein (oncogene) E6
to stimulate p53 protein, unlike polyomaviruses that use only 1 viral oncogene (large T antigen)
to stimulate both pRb and p53. E6 binds p53 protein causing its proteosomal degradation. E6
binds PDZ domain on proteins inducing cell proliferation control in later malignancy. These
outcomes from E6 and E7 stimulation predispose the cell to cancer and tumour formation.
Similar to polyomavirus transformed cells, papillomavirus transformed cells are also unable to
prohibit viral replication.31
Q11-2: Find a product sheet for an SV40 transformed cell line on the American Type
Culture Collection (ATCC) website: http://www.atcc.org/
Figure 3. Product sheets for SV40 transformed endothelial cell isolated from axillary
lymphnode/vascular epithelium from Mus musculus.
Q12-1: Do other large DNA viruses other than the Herpesviridae exhibit viral latency?
Give an example or counter-example.
There is likely a large DNA virus other then Herpesviridae that exhibits viral latency,
however it is not common based on the fact that I have searched exhaustively through many
articles trying to find one. I think the reason for this lack of research on other potential latent
DNA viruses is because the majority of funding goes towards studying Herpesvirus and
retrovirus latency because many people are infected with these viruses. Thus, other large DNA
viruses that exhibit latency they are ignored.
The other major virus that exhibits viral latency is HIV retrovirus that has an RNA
genome. It has a clinical latency period that lasts an average of about 10 years (or more if on
antiretroviral drugs). During this time HIV replicates at low levels causing no symptoms. After
the latency period reactivation of the virus leading to AIDS.32
Q12-2: Larry has a cold sore on Valentine’s Day – this makes him sad. There exists a
negative cultural stereotype surrounding Herpesviridae. Make Larry feel better by
explaining why this stigma is unjustified considering the prevalence of these viruses in
society and current therapies.
Larry should not be sad because the stigma that surrounds Herpes virus is unjustified
because essentially everyone gets infected by Herpes virus at one point in his or her life and
becomes latent carriers. For example, the seroprevelence of HSV-1 is 50-70% in healthy adults.
Meaning that 50-70% of healthy adults have pathogen in their blood serum. Herpes virus is so
common that the seroprevelence of VZV is 85-95% in adults.
Treatment/management of the viral infection is through the use of oral or intravenous
drugs such valacyclovir, acyclovir and famcyclovir. When taken daily the antiviral drugs reduce
severity and length of breakouts, and prevent viral spread.33
Q13-1: What are the key viral enzymes of a retrovirus and how do they promote viral
replication?
The key viral enzymes in retroviruses are reverse transcriptase, integrase, and protease.
Generally, without these key enzymes retroviruses cannot replicate in their host cell. Reverse
transcriptase transcribes the viral RNA genome into a double stranded DNA genome promoting
integration into the host’s double stranded DNA genome. Integrase is the enzyme responsible for
integrating the transcribed proviral dsDNA genome into the host genome where it can then be
replicated by along with the host genome. Protease becomes activated when virions are
assembled and start exocytosing from the cell. It is responsible for cleaving the Gag/Pol viral
proteins into functioning structural (capsid, matrix, nucleocapsid) and enzymatic (protease,
integrase, reverse transcriptase) proteins. Without protease, these Gag/Pol proteins would not be
able to reassemble into infectious mature virion.34
Q13-2: What are the major differences between HIV and HTLV-1 infection? Make a
table comparing at least 6 main points.
Table 6. Major differences between HIV and HTLV-1 retroviruses.
Criteria HIV HTLV-1
Replication
Levels
Extremely high levels of active
replication.35
Minimal levels of active replication
once infection is established.35
Gene Expression Encodes 3 accessory proteins Vif,
Vpr and Vpu. Spliced mRNA
encodes Rev, Tat, and Nef.36
Spliced mRNA encodes Rex and Tax
(transcriptional activator) and 4
accessory proteins made through
alternative splicing (p27, p12, p13,
p30). HBZ encoded through antisense
transcription.36
Protein that
escorts unspliced
mRNA from cell
nucleus.
Rev protein.36 Rex protein.36
Associated
Diseases
AIDS.37 Adult T-cell Lymphoma.37
Clinical Signs It is characterized by a significant
decrease and loss of activity in
CD4+ T cells.37
It is characterized by a significantly
increased white blood cell count that
is mostly made up of leukemic T
cells.37
Acute Symptoms Rash, small appetite, fever, Normally Asymptomatic.37
headache, fatigue, malaise, a
general unwell feeling, sore
throat, and night sweats.38
Q14-1: What accessory proteins does your virus encode?
Human B19 parvovirus encodes the NS1 protein. The NS1 protein is a nonstructural
regulatory accessory protein.39
Q14-2: What do your viral accessory proteins do and how were they discovered (described
the experimental approach, not the history of the discovery)
They used human hematopoietic cell lines K562, Raji, and THP-1 as transfectants that
are able to produce the parvoviral NS1 protein upon induction with a bacterial lactose
repressor/operating system. These three transfectant cell lines produced interleukin 6 (IL-6),
which was detected by analyzing cellular supernatant by ELISA. The researches were able to
show that a NF-κB binding site in the IL-6 promoter region mediates the NS1 effect. This means
that regulatory NS1 protein functions as a trans-acting transcriptional activator on the IL-6
promoter. The researchers are using this data as a support for the relationship between human
B19parvovirus and the polyclonal activation of B cells in rheumatoid arthritis, which in turn
means that the NS1 protein plays an indirect regulatory role in the pathogenesis of human B19
associated arthritic disease.39
Q15-1: Write a 200 word summary of the documentary, The Lazarus Effect.
http://www.youtube.com/watch?v=HrJN1_axMEM
The Lazarus Effect is a heart-wrenching documentary about antiretroviral treatment of
HIV in Africa. It aims to show the effects that HIV and AIDS has on the people infected as well
as their loved ones. It scrutinizes the drastic amount of un-necessary loss/deaths of human life
caused by HIV that could be controlled very easily with the help of ARVs. The documentary
highlights the struggles that HIV positive patients go through. For example, many have a hard
time adhering to taking the ARV’s because it is a time-dependent, life long process that is
expensive and inaccessible for many. The Lazurus Effect shows the impact of HIV on mothers,
children, people that have to life with HIV.
The Lazarus Effect highlights the social relations that contribute to poor HIV control in
Africa, putting the blame on embarrassment, and lack of support. This documentary shows the
progress of making HIV, something people tend to be embarrassed about, into something that
people recognize, are aware about, and can talked about. The documentary shows the hard work
of people committed to combatting the problem of HIV, the progress of Africa getting free
ARVs for a small proportion of the people infected, as well as support groups for people that are
infected with AIDS.
Q15-2: What are the major differences between HIV diagnosis and treatments in North
America versus Africa?
In Africa, the main form of HIV treatment is the use of ARVs. Despite genetic
differences or differences in disease progression people infected with HIV in Africa are all given
basically the same treatment. In America ARVs are usually not even considered as a treatment
option. In North America the standard treatment is called HAART (highly active antiretroviral
therapy) that uses a minimum of 3 antiretroviral drugs in combination to suppress HIV
replication. These drugs are customized to the patient depending on a variety of factors such as
patients viral load, strain of HIV, CD4+ T cell count, disease symptoms and other factors. 40
Q16-1: Could your favourite virus endogenate into the human genome? Why or why not?
Due to exhaustive literature search, to date, canine parvovirus cannot endogenate into the
human genome. However, researchers have shown using PCR analysis that canine parvovirus
endogenated into chromosome 5 of the rat genome. Endogenated Parvovirus (EnPV) amino acid
sequences in the rat genome were most similar to sequences of canine parvovirus. EnPV has
amino acid sequences that are share 65%-75% of the same amino acid sequence as the capsid
and non-structural proteins of CPV.41
Q16-2: What are the major differences between exogenous and endogenous retroviruses?
List 5 main points of comparison.
Table 7. Major differences between exogenous retroviruses and endogenous retroviruses.
Comparison
Criteria
Exogenous Viruses 42 Endogenous Viruses 43
Genome
Integration
Genome integrates as proviral
DNA. (not into germ-line cells).
Genome is integrated into the germ-line.
Is virus
active?
Proviral DNA is active and Integrated genome has many mutations
so that most ERVs are rendered inactive.
(Usually loses env ORF).
Genome
Characteristic
Static Genome42 Dynamic Genome: retrotransposition
Cell
Replication
Limited to only the cells that get
infected by the virus or cells that
replicate from a infected cell
(provirus). 42
ERV is replicated in all cells of the body
(because its in germ-line cells) 43
Lifecycles Retrovirus infects cell, RT
transcribes viral RNA into DNA it
moves to nucleus and integrates via
integrase and replicates with the
cell to make genomic RNA and
viral mRNA that can be translated
to make proteins and then packaged
into virions with genomic RNA.44
Retrotransposition: integrated DNA
transcribed into RNA template then RT
into DNA and reintegrated into a new
genomic site.
Complementation in trans: Endogenated
RV can replicate forming a virion in
trans that can be incorporated into new
progeny virions.
Reinfection: Viral replication carries out
like normal retroviral replication to make
mature virions that go on to re-infect the
cell.44
Q17-1: Can your favourite virus be infected with a virophage?
Due to exhaustive literature search I conclude that Canine parvovirus cannot be infected
by virophages to our knowledge. However, Dependoparvoviridae such as adeno-associated virus
1 and 2 (AAV-1, AAV-2) can function as satellite viruses that do not have negative effects on
the virus it co-replicates with. AAV-1 and AAV-2 adeno-associated viruses have replication
cycles that depends on the coinfection with adenovirus or herpesvirus.45
My hypothesis for why Parvoviridae cannot be infected by virophages is because of their
size. Parvoviridae are some of the smallest known viruses, 18-26 nm in diameter46 and the
sputnik virophage is 50 nm.47 This comparison shows that Sputnik is larger than Parvovirus.
Thus virophages would have to be extremely small in order to infect the extremely small
parvovirus.
Q17-2: What are the major differences and similarities between viruses, viroids and
satellite viruses?
Table 8. Similarities and differences between viruses, viriods and satellite viruses.
Comparison Viruses 49 Viriods 48 Satellite Viruses 50
Genome Encode
Proteins
Yes, encodes capsid
proteins
No Yes, encodes capsid
proteins
Genome RNA or DNA RNA RNA
Genome
Encoded
Polymerase
Yes No No
Causes of
Disease
Viremia,
blocking/modulation
of host immune
defenses.
Small interfering RNAs Modulation of Helper
Virus
Viriods are naked, circular, ssRNA molecules that do not encode any proteins or viral
mRNA and replicates autonomously relying on host proteins (RNA polymerase). Viriods mainly
infect plants and cause disease with small interfering RNAs that silence host genes.48
Viruses may be naked, they may have circular or linear genomes, their genome can be
DNA or RNA. They infect both plants and animals. They do encode viral proteins that makes up
its capsid structure and viral mRNAs that. Viruses sometimes rely on the host for viral
replication but can also encode its own proteins that promote viral replication. Viruses cause
many diseases through many mechanisms including viremia and blocking of host immune
defenses, etc.49
Satellite viruses replicate in the presence of a helper virus and are able to modulate
(increase severity or decrease severity) the symptoms caused by the helper virus. They do not
encode their own RNA polymerases for replication. They replicate along with the helper virus
and modulate (increase/decreases) the amount of viral RNA to increase or decrease the severity
of infection by the helper virus. Satellite viruses are able to encode their own capsid proteins that
encapsidate their RNA genome.50
Q17-3: Explain the ecological implications of virophages.
Virophages negatively affect virus replication/viral load in a host, thus virophages act in a
positive way for the host. Thus they decrease virus abundance, and increase host abundance.51
An example of this is the Organic Lake Virophage. The Organic Lake Virophage infects
phycodnaviruses infected cells (algae) and impairs the replication of phycodnaviruses and thus
helps the algae. Thus, according to researches, the introduction of Organic Lake Virus increases
the frequency of algal blooms by reducing algal mortality due to phycodnavirus infection.52
Q18-1: Is infection with your favourite virus associated with changes in the expression
prion proteins?
Through exhaustive literature searching it can be concluded that infection with
parvovirus has not yet been associated with changes in the expression of prion proteins. In one
study researchers wanted to determine if addition of random peptide sequences would modulate
the formation of protein kinase resistant PrP (PrPres) from PrP. To do this researchers used a
random sequence (DGAVQPDGGQPAVRNER) from canine parvovirus. Upon addition of the
peptide PrP was not modulated to form PrPres indicating that the canine parvovirus sequence does
not modulate or change the expression of prion proteins.53
Q18-2: What are the major differences and similarities between viruses and prions?
Viruses have nucleic acid genomes, are enclosed in a proteinaceous capsid and
sometimes have an envelope structure, in comparison prions do not have nucleic acid genomes,
rather they are only composed of proteinaceous infectious agents. Viral proteins are typically
virally encoded whereas prion proteins are host encoded. Viral genomes are infectious agents,
are infectious agents. Viruses usually require host proteins, or their own virally encoded
replication proteins to replicate themselves, whereas prions can replicate themselves without.
Both viruses and prions are able to cause pathologic effects in humans.54
Q18-3: What scientific arguments are made against the prion hypothesis?
One scientific argument against the prion hypothesis is that a small virus could be
responsible for transmission of prion disease by infection neurons in the brain causing changes in
the cell leading to conformational change in PrPc to PrPSc. This goes against the prion hypothesis
that states prion disease results from increased stability or the aggregation of mutated PrPSc.54
Q19-1: What is the route of entry of your favourite virus?
CPV infection is initially acquired through oral/nasal contact with feces that contain
infections virions. CPV enters the lymphoid tissue in the oropharynx by binding to the transferrin
receptor then disseminates hematogeneously into the rest of the body.55
Q19-2: What cell, tissue and species tropism does your favourite virus exhibit?
Canine parvovirus has the ability to bind to the transferrin receptor on host cell surfaces.
This receptor binding interaction is what determines CPVs specificity for dog cells that express
the transferrin.56 According to Merck Veterinary Manual CPV prefers to infect tissues that have
cells that are rapidly dividing, such as the intestinal epithelium, lymphopoietic tissue, as well as
the bone marrow.57
Q19-3: What is the clinical presentation of an infection with your favourite virus?
Dogs infected with Canine parvovirus often stop eating, have a fever, are lethargic,
vomiting, and have severe diarrhoea leading to dehydration, and sometimes death. (A) CPV
causes myocarditis and gastric enteritis leading to bloody diarrhea.58
Q19-4: Elaborate on a mechanism of viral pathogenesis caused by your favourite virus.
In the intestine, CPV infects and destroys active, dividing enterocytes that line the
intestinal crypts. The destruction of enterocytes causes intestinal lesions to form. A dog that has
many lesions leads to mucosal surface collapse, mucosal and serosal haemorrhaging,
malabsorption and maldigestion, as well as bloody diarrhea.59
Q20-1: Tell a story of viral pathogenesis in a 200-word paragraph
Transmission of Ebola is usually introduced into new hosts through contact with
infectious bodily fluids with open wounds or mucosal membranes. Ebola virus has GP that can
either be secreted or attached to Ebola viruses cell surface via a transmembrane domain. Ebola
GP binds to and facilitates entry into epithelial cells (lining blood vessels), monocytes and
macrophages. The exposure to Ebola virus or the damage caused by Ebola virus causes the
release of pro-inflammatory cytokines (TNF a, IL-6, IL-8) that are associated with inflammation,
fever, and damaged vasculature. The secreted GP inhibits neutrophil activation. Ebola replicates
at high rates, and during infection both host and viral proteins contribute to viral pathogenesis.
During infection there is an increase in IFN-y, IFN-a, IL-2, IL-10, and TNF-a.60 These pro-
inflammatory cytokines are associated with systemic inflammatory syndrome, and often lead to
fatality. TNF-a may even contribute to the necrosis, epithelial cell leakiness leading to the
characteristic haemorrhagic symptoms. The products released from necrotic cells also stimulate
further release of pro-inflammatory cytokines contributing to further pathogenesis. Chronic, fatal
infections are typically characterized by necrosis in multiple tissues such as the liver and spleen.
Ebola virus also impairs the adaptive immune response by impairing the function of dendritic
cells and by causing lymphocyte apoptosis. Loss of adaptive immune response may inhibit
antibody production against the virus impairing some hosts (but not all) from fighting off the
virus.61
Q21-1: Tell a story of an emerging virus and its contribution to viral pathogenesis in a 200-
word paragraph.
Hendravirus (HeV) is a newly emerging negative sense single stranded RNA virus that
mainly infects horses. It was first observed in 1994 and is found only in Australia. It is postulated
that HeV originally spread to horses via direct contact with bodily fluids from a flying fox.62
HeV was first recognized in 1994 as a virus that spreads horse to horse via direct contact with
infectious bodily fluids, making it seem as if HeV had limited infectivity to horses. In 2011, a
dog living on a property with horses become infected with HeV showing the virus is not so
limited.64 In horses HeV has a vascular tissue tropism preference that causes a lot of HeVs
signature respiratory disease with symptoms such as epithelial edema, large syncytial cell
formation, haemorrhage, destroying of alveolar epithelium and macrophages. HeV also is also
neurotropic, causing neural necrosis and gliosis.64 In horses these virally associated diseases lead
death in horses 70% of the time.62 In humans HeV causes mild flu like symptoms or respiratory
and neurologic disease that is often fatal.63 HeV in humans has a 57% case fatality rate.64 Horses
infected that recover will always be carries of HeV as they exhibit dormancy thus in Australia
they have laws that enforce euthanization of the animal.62
Q22-1: What PRRs can detect your favourite virus?
Researchers were able to demonstrate that oncolytic parvovirus H-1PV is detected by
TLR3 and TLR9 in human embryonic kidney cells. TLR3 and TLR9 stimulation led to activation
of NFκB. Researchers were also able to show that human dendritic cells when infected with H-
1PV induce both NFκB activation and TNF-α production. One thing important to note about this
study is the fact that these results are seen in vitro and may not reflect real PRR stimulation in
vivo.65
Q22-2: Name an ISG is turned-on in response to infection with your favourite virus. What
transcription factor or IFN regulates the expression of this gene?
Researchers used mouse embryonic fibroblasts (MEFs) and infected them with
parvovirus minute virus of mice. Upon infection a Type I interferon (IFN) response is turned on.
The IFN then binds to membrane bound receptors and turns on the JAK/STAT interferon-
signaling pathway. The pathway involves the phosphorylation of STAT1 and STAT2 transcription
factors as well as the activation and the up regulation of ISGs that encode for PKR, STAT1,
STAT2, and 2′-5′-OAS.66
Q22-3: Name a viral restriction factor that limits the replication of your favourite virus.
Explain its mechanism of action.
The Adeno-associated virus (AAV) is a member of Parvoviridae. It requires co-infection
with a helper adenovirus and carries out replication in the nucleus where they form a nuclear
replication center. The researchers transfected some cells with APOBEC3G, some with
APOBEC3A, and some with C106S then infected the cells with wild type AAV and adenovirus.
They found that only APOBEC3A viral restriction factor was able to inhibit the formation of the
nuclear viral replication centres. They then analyzed why APOBEC3A was the only restriction
factor out of the ones used to restrict viral replication. Using immunofluorescence techniques
they found that it was because APOBEC3A was found in both the cytoplasm and nucleus,
whereas APOBEC3G and C106S were only found in the cytoplasm. Although researchers are
unsure of the mechanism one belief is that APOBEC3A directly binds to single-stranded viral
DNA inhibiting the completion of DNA synthesis.67
Q22-4: Does your virus employ viral evasion strategies to avoid the innate and intrinsic immune
response? Elaborate on one specific mechanism.
Yes. Parvovirus migrates to the nucleus via the cellular microtubule system before
uncoating their ssDNA genome. This allows parvovirus to avoid detection of the genome by
innate DNA sensors such as AIM2. 68
Q23-1: Identify an innate immune evasion mechanism carried out by your favourite virus.
Write a 200-word summary of the mode of action, along with an original diagram.
Murine parvovirus minute virus of mice induces an IFN response in mouse embryonic
fibroblasts although not a great enough response to induce an antiviral state. MVMp can block is
known to block the innate immune response. MVMp infected cells down-regulates the secretion
of IFN and IL-6 from Poly (I:C) stimulation. One mechanism used by MVMp to avoid detection
by innate immune sensors is trafficking to the nucleus. The MVM genome coordinates the
movement of the virion from the cytosol to the nucleus. The virion only uncoats its genome one
it has reached the host cell nucleus during the host cells entry into the S-phase. This virion
trafficking avoids MVMp genome detection from cytosolic DNA sensors such as DAI,
LRRFIP1, IFI16 and ISD that could detect viral genome in the cytosol and mount an antiviral
response against them. Another mechanism used by parvovirus to evade host innate immune
response is the interference with interferon stimulating genes that has antiviral activity. The
researchers postulated that MVMp interferes with ISG activity that restricts viral replication.
Although the exact inhibition mechanism is still unknown, it is indicated that MVMp infected
cells block the stimulation of poly (I:C) which results in the down regulation of IL-6 and IFN
antiviral cytokine expression.69
Figure 4. Cell infected with MVMp that avoids recognition of host innate immune system
by blockage of Poly I:C stimulation as well as migration and decapsidation at the nucleus
to avoid cytoplasmic DNA detection. Original Artwork by: Samantha Bray
Q24-1: Describe the adaptive immune response against your favourite virus?
The main adaptive immune response used against parvovirus is the production of
neutralizing antibodies by B cells.70 Parvovirus B19 produces IgM and IgG antibodies. IgM
antibodies are produced first within the first or second week of infection then declines. IgG
antibodies are then produced to provide long-term adaptive immunity. IgG levels increase during
active infection and levels off as the infection subsides. Individuals that are exposed to
parvovirus B19 will have some IgG in their circulation for the rest of their life.71
Q24-2: What T cell subsets are activated during an infection by your favourite virus?
During infection by human parvovirus B19 CD8+ T cells become activated. These CD8+
T cells are increased and sustained over many months during resolution of acute parvovirus B19
infection.72 Human B19 parvovirus also activate CD4+ T helper cell responses for the production
of IFN-γ.73 Human B19 parvovirus activates Th1 and Th2 helper T cell responses.74
Q24-3: Does your favourite virus encode a virokine or other viral proteins that mimic host
molecules? Explain their role in immune evasion.
Human parvovirus B19 has regions in their viral structural protein VP1 and VP2 that are
homologous to GATA1 a transcription factor that plays a key roll in hematopoietic development,
megakaryopoiesis, steady-state erythropoiesis and red blood cell expansion during anemia.
Additionally, peptides in VP1 were identified that are similar to human cytokeratin. Molecular
mimicry is one of the ways by which parvovirus can induce the production of cross reactive
autoantibodies that contribute to auto immune disease.75
Q25-1: Describe how viral escape mutants affect the replication and spread of your
favourite virus?
Historically feline panleukopenia virus is a parvovirus that used to only infects cats. It is
hypothesized that FPV mutated, and gained the ability to infect canine cells. The ability of FPV
to infect canine cells is associated with the virus’s ability to bind the host cell transferrin
receptor. In the past, FPV could only infect feline cells. However, researchers found that two
mutations in the FPV virus allowed spread of virus from a feline host to a canine host. The two
mutations at residue 93 from Lys to Asn and residue 323 from Asp to Asn allowed FPV to bind
the canine transferrin receptor, gaining the ability to infect a canine host.76
Q25-2: How does your favourite virus influence cellular apoptotic pathways?
To access how viral NS1 influences cellular apoptotic pathways researchers used HeLa
cells and transfected them with pcDNA.CPV2.NS1, some were left uninfected as controls. They
used flow cytometry and real time PCR techniques to analyze the activation of caspase-3, Bcl2,
and the p53 apoptotic pathway. The researchers found that caspase-3 was activated and
upregulated in the cells that were transfected with pcDNA.CPV2.NS1 whereas Bcl2 and p53
were not activated. In short this study found that increased apoptosis in CPV2 infected cells is
caspase-3 dependent and p53 independent.77
Q25-3: Identify and explain one additional viral evasion strategy performed by your
favourite virus that was not covered in this course.
Human parvovirus B19 has a NS1 protein that is a transcriptional activation that
modulates STAT signalling. B19 NSI expression leads to the activation and dimerization of
STAT3, the STAT3 dimers translocate to the nucleus. The activation of STAT3 is associated
with the up regulation of antiviral genes such IL-10Rα, IL-2Rα and IFN receptor proteins (IFNG
and IFNAR). B19 NSI expression suppresses promoter activity of SOCS1 and SOCS3. STAT3
suppression followed by PIAS3 activation is a viral mechanism that promotes viral persistence
and chronic inflammation. Viral B19 NS1 is hypothesized to blocks the phosphorylation and
activation of STAT1 by suppressing TYK2 (a Janus kinase) which contributes to reduced Type 1
antiviral IFN response.78
Q26-1: Is there a vaccine against your favourite virus? If so, describe.
Yes there is a vaccine called Nobivac ® Canine 1-DAPPV that is widely used in dogs
to protect against canine parvovirus as well as adenovirus type 1 (hepatitis), adenovirus type 2 (respiratory disease), parainfluenza and canine distemper. It is a combined modified live virus vaccine and is given as a subcutaneous injection. It can be given to healthy dogs starting at the age of 6 weeks and is given in 2-4 week intervals until the puppy is 12 weeks old. It must be boostered annually to stay effective.79
Q26-2: Is there antiviral regimens against your favourite virus? If so, describe.
Yes there are antiviral regimens against canine parvovirus infection in dogs. Once
infected the main goals of treatment are restoration of fluid, electrolyte and metabolic
abnormalities as well as the prevention of secondary bacterial infections that occur when bacteria
translocate across intestinal epithelium that was disrupted by the virus. Most dogs are put on IV
fluids to correct fluid levels. Antibiotics such as ampicillin or gentamicin are given to combat
bacterial translocation across intestinal epithelium. Antiemetic therapy is given to stop vomiting,
which also combats water loss (dehydration). Dogs with parvovirus infection suffer from
extreme diarrhea, however it is not appropriate to give infected dogs antidiarrheals because it
could cause retention of intestinal contents may increase the risk of bacterial translocation across
the gut. Generally after infection that dog is low on many nutrients, so after vomiting has
stopped the dog should be fed and watered. Oseltamivir can be given to infected dogs to reduce
the amount of weight loss and to increase the white blood cell count (in respect to dogs not given
Oseltamivir). With appropriate supportive care, which in turn helps the dogs immune system
fight against parvovirus infection, 68%–92% of dogs with CPV enteritis will survive.80
Q27-1: With a partner from BIOL4904, decide who has the most pathogenic virus and
justify your answer in terms of transmission, viral replication, viral pathology and clinical
presentation. You must present information comparing and contrasting both viruses. 300-
word minimum.
Compared to Human parvovirus B19, SARS coronavirus is more pathogenic to humans.
SARS Coronavirus spreads between hosts through direct contact with respiratory droplets in
aerosol form, through the fecal-oral route, or through contaminated surfaces.81 Similar to SARS,
Human parvovirus B19 can also be spread by respiratory secretions, but not through the fecal-
oral route.82 Human parvovirus B19 replicates only in human erythroid progenitor cells and it
typically replicates in the nucleus of the cell until virion levels are high enough to cause cell
lysis.83 SARS Coronavirus replicates in host epithelial cells that line the lungs and intestines.
Similar to parvovirus B19 SARS virions also replicate in the host cells until the level of viruses
is so high that it causes the cell to lyse releasing thousands of virions.84 Human B19 parvovirus
only causes mild symptoms called Fifth disease.82 Initial symptoms include fever, runny nose,
and headache, it then progresses into a face and body rash forming a characteristic “slapped
cheek rash” that is common in children.82 Parvovirus B19 may also cause painful swollen joints.
The infection resolves itself usually without treatment and only progresses into more serious
complications in people that have weakened immune systems.82 unlike parvovirus B19, SARS
usually requires treatment or else the infection will persist. SARS symptoms include cough,
breathing difficulties, fever, chills/shaking, head and muscle aches.81 SARS infection had a death
rate of over 50% in people over the age of 65. The death rate from SARS was 9 to 12% of those
diagnosed. In people over age 65, the death rate was higher than 50%. Throughout history SARS
has caused more pathogenesis especially in the 2003 outbreak that caused +8000 deaths
worldwide according to the World Health Organization.85 Human parvovirus B19 infection
rarely causes death in adults, but it has been associated with causes fetal deaths.86
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