Viruses

Question 1

What is the difference between + and - RNA?

Answer 1

+ RNA cann be immediately translated by the host cell → can be less infectious though
-RNA is complementary to mRNA , must be converted to + RNA via RNA polymerase before translation

Question 2

What is ambisense?

Answer 2

Ambisense means that the virus resembles a negative sense RNA virus however they can also translate genes from the positive strand

Question 3

What are the differences between Envelopped and non-envelopped viruses?

Answer 3

Enveloped Viruses
Have lipid-bilayer membranes
Impermeable barrier between their genome and the enviro
Ex HIV, inluenza

Non Enveloped Viruses
No membranes
No barrier, tightly packe protein shell to exclude nucleases or other source of gentic damage
Ex, polio virus, papillomavirus

Question 4

What is the main constraint for viral structure?

Answer 4

The information that is needed to build and assemble the virus cannot exhaust the genetic capacity of the packaged genome → highly symmetrical

Question 5

What are the steps that initiate entry into the host cells for viruses?

Answer 5

Induced conformational changes→ receptors, membrane fusion, penetration
Transmission of signal through membrane prepares cell for invasion
Induction of endocytic pathway

Question 6

Do Viruses affect every type of cell?

Answer 6

No they only infect cells that they can bind to

Question 7

What location of the cell are DNA viruses transported to? RNA viruses?

Answer 7

DNA viruses are transported to the nucleus (they need nucleus materials)
RNA viruses are translated in the cytosol so can be put either in the nucleus or the cytosol

Question 8

Explain the 2 ways that RNA viruses can rplicate their genomes.

Answer 8

RNA-dependent RNA synthesis (RNA replication)

RNA-dependent DNA synthesis (reverse transcription) followed by DNA replication and transcription (ex retroviruses)

Question 9

What is the genetic component that all viruses need?

Answer 9

mRNA needed early in infection to make essential viral proteins (positive sense)

Question 10

What are some differences for dsDNA and ssDNA?

Answer 10

dsDNA only needs cellular RNA polymerase II to make mRNA while ssDNA needs cellular DNA polymerase and RNA polymerase II ot make mRNA (extra step)

Question 11

What are the steps to obtain mrNA form +ssRNA? From - ssRNA?

Answer 11

+ssRNA uses its own Virion RTase to make dsDNA then the hosts Cellular RNA pol II to get mRNA
-ssRNA can use either its own enzymes (Virion RdRp) or Cellular RNA polII to amke mRNA

Question 12

Why are there lots of mutations in viruses and why is that beneficial?

Answer 12

Polymerases that catalyze RNA replication and reverse transcription have minimal proofreading activities
RNA dependent RNA polymerases (RdRp) and reverse transcriptases have error rate that is three orders of magnitude higher than DNA dependent DNA polymerases, allowing for a quicker evolution , but also creates a upper limit on genome size

Question 13

Describe the virus assembly process of a non-enveloped virus

Answer 13

Structural proteins must be retained at the subcellular domain where assembly occurs
MAcromolecules assemble into empty capsids
Viral DNA is inserted into the structure via a packaging sequences at one end of the genome
Precursor core proteins are also packaged into the empty capsid along with DNA
Proteolytic cleavage of the precursor protein by proteinase yields the mature (infectious) virion

Question 14

How are proteins biosynthesized and excreted in viruses?

Answer 14

Rely on eukaryotic cells for the biosynthesis of macromolecules, have similar targeting signals. They travel through the secretory pathway of the cell.
Associate with Er → transported across via a proteinaceous pore → transported to Golgi complex → plasma membrane unless they contain amino acids motifs that localize to them to another location
Intracellular transport happens in coasted vesicles
Protein destined for the nucleus contain nuclear localization signals → to use nuclear pore machinery, other may contain nuclear export signal

Question 15

Describe the 3 mthods of acquiring a lipid bilayer from the host cell for enveloped virus

Answer 15

Proteins are transported into plasma membrane and capsid assembled and development occur simultaneously
Capsid assembly occurs int eh cytoplasma and then the virus buds from the plasma membrane and gets its envelope
Capsid assembly occurs in the cytoplasm and gets its envelope from theER and is exported via exocytosis

Question 16

What are the bilayers that viruses use?

Answer 16

Glycoprotiens are transported to their preferred lipid bilayer (ER or plasma membrane)
Viral capsid develops elsewhere in the cells, nucleus acids are packaged int the capdi and transported in endosomal vesicles to the area where they envelopment happens
Plasma membrane→ viral budding
ER→ virion is transported to the plasma membrane via the transport vesicle

Question 17

Why is it difficult to detect and recover foodborne enteric viruses?

Answer 17

They do not multiply in the food system, die off during storage and preservation
Excreted in high umbers in human feces
Cannot multiply outside of the body , killed by pasteruization, heat , irraditation

Question 18

How is poliovirus prevented

Answer 18

Eradicated except for endemic areas of Afghanistan, Nigeria and Pakistan
Milk and milk products pasteurized to deactivate th virus

Question 19

What are the symptoms of polio virus?

Answer 19

Invades nervous systems, total paralysis in a matter of hours, initial symptoms are fever, fatigue, headache, vomintg, stiffness of neck and pain in the limbs

Question 20

What are the 3 serotypes of Poliovirus?

Answer 20

Based on different capsid proteins
PV1 - most common serotype sin nature
, pakistana dn afghanistan
PV2 likely eradicated
PV3 has not been detected since 2012, likely eradicated

Question 21

What is the genome of poliovirus?

Answer 21

Poly protein encodes 11 mature viral proteins
5’ non-translated region NTR has low functional domains- the cloverleaf and the internal ribosome entry site (IRES) - covalently linked to viral protein VPg
Cloverleaf regulates RNA replication + initiation of translation
IRES mediated translation of viral mRNA
3’ end is polyAdenylated
N to C contains structural P1, non-structural P2 and P3 proteins that are released from the polypeptide chain by proteolytic processing mediated byi virally encoded proteinases

Question 22

What is the life cycle of poliovirus?

Answer 22

Infection starts with the binding of polio virus to cell surface receptor
Uncoating of viral RNA is mediated by receptor dependent destabilization of virus capsid
Cleaving of viral protein VPg is performed by cellular phosphodiesterase
Translation of viral RNA occurs by cap-independent (IRES) mediated) mechanism (3)
mRNA is translated as one long polypeptide, process proteoytic yield mature structural and non-structural proteins (4)
The positive sense RNA serves as a template for complementary negative strand synthesis, thereby producing a double standed RNA (5)
Inititation of many positive strand from a single negative strand produces the partially single stranded replicaive intermediate (6)
Newly synthesize positive sense RNA molecules can serve as templates for translation(7) or associate with capsid precursors to undergo encapsidation and induce the maturation cleavege of VP0 (8)
Lysis of the infected cells results in release of infectious progeny virions (9)

Question 23

HOw is poliovirus assembled?

Answer 23

P1 precursor protein is cleaved to give VP1, VP3 and VP0
They are assembled into 5S monomers and then into 14S pentamers
12 of these pentamers associate directly with genomic RNA being transcribed by membrane boudn complexes to assemble an immature 150S provirion
VP0 further lceaved to yield VP2 and VP4 → mature virion

Question 24

What are symptoms of Hepatitis A Virus?

Answer 24

yellow eyes, abdominal pain and pale stools

Affects the liver, 8 weeks

Question 25

What is a common vector?

Answer 25

Undercooked contaminated seafood

Question 26

What is the treatment of Hep A?

Answer 26

Rest, medication, aute liver→ transplan

Question 27

What are the genetic groups?

Answer 27

One serotype and seven different genetic groups Human ones 1-3 Six subtypes 1A,1B,2A,2B,3A3B The non-humna primate genotype shave been numbered Iv-VI

Question 28

What is the genome of Hep A?

Answer 28

+ssRNA HAV encodes a single polyprotein that major capid proteins are coded for at the N-terminal The remainder of hte polyprotein encodes a series of nonstructural proteins required for HAV RNA replication A small proteins, VPg, is covalently linked to the 5’ end of the genomic RNA and this serves as the protein primer for RNA synthesis

Question 29

How does Hep A replicate?

Answer 29

Most Of the virus appears to be produced in the liver and reaches the intestinal contents by secretion from infected hepatocytes via biliary system

Question 30

What is the cellular lifecycle of Hep A?

Answer 30

The virus enters the hepatocytes via an interaction with cellular receptor (a) Uncoating of the viral particle and realse of the positive sense RNA genome into cell (b) Internal ribsome entry site within the 5’ nontranslated segment of the genome mediated cap independent translation of the viral polyprotein © Polyprotein undegoes co and post translationa processing from viral protease(d) Nonstructural viral proteins assemble into a membrane boudn RNA replicase, bidn the 3’ end of genomic RNA and commence synthesis of a negative strand copy of viral genome (e) Negative strand copy of the genome used as template for synthesis of multiple new copies of genomic positive strand RNA (f) SOme of new synthesized positive sense RNA is recycled for further RNA synthesis or translation (g) Other positive-strand RNA molecules are packaged into new viral particles formed by assembly of the structural proteins, followed by final cleave of the VP1-2A precursor and the amturation cleavage of the VP4/VP2 junction (h) HAV secreted by the cell across the apical membrane of hte hpatocyte→ passed into the bil and small intestine

Question 31

Explain the Virus load of Hep A.

Answer 31

Fecal shedding of the virus reaches its maximu just before the onset of hepatocellular injury at which point the individual is most infectious Accompanied by extended viremia which roughly parallels the sehdding of virus in the feces, lower magnitude Increased levesl of serum alanine aminiotransferase (ALT) activity is indicative of hepatocellular injury Towards end, anti-HAV IgM and IgG antibodies are produced igG is responsible for lasting immunity towards HAV igM is short lived but its presence used to diagnose the illness

Question 32

What is ta method of detecting Hep A in foods?

Answer 32

Food is collected and suspended in a glycine buffer and mized for 15 minutes at room temperature, the virus will become suspended in the buffer The supernatant from this mix (virus) is centrifuged at 170000g for 60 mins, virus collects at bottom Supernatant can be discarded Pellet is resuspended in buffer and added to a QIAshredder Breaks viral capsid and releases RNA RNA is precipitates and purified and can be ues din a RT-qPCR reaction to detect its presence

Question 33

What are common ways that norovirus is spread?

Answer 33

GRoup setting, fall and winter, contagious form the moment they feel ill to three days after. Direct Treatment is rest and medications Main symptoms: diarrhea, vomitng, nausea, stomach cramps

Question 34

What are the genogroups of norovirus (G1-G5)?

Answer 34

``` 5 genogroups ( G1-G5) Further into genetic clusters or genotypes GII most prevalent in humans I,II, IV infect humans III bovine V mice ```

Question 35

What is the genome of norovirus like?

Answer 35

5’ end of Open Redaing Frame 1 (ORF1) is transcribed as single nonstructural polyprotein Cleaved into six mature products by virally encoded protease (NS6 or Pro) Other nonstructural proteins include NS1/2, NS3 (NTPase, NS4 (p22), NS5 (VPg) and NS7 (RNA dependent RNA polymerase RdRp ORF2 encodes the capse proteins referred to as VP1, this protein can be divided into shell and protruding domains P domain further subdivided into P1 stalk and P2 domain with arches ORF3 encodes the minor structural protein VP2 ORF4 (mouse NoV) encodes a newly defined proteins called virulence factor 1 or VF1 NoV genomes covalentyly linked ot VPg at their5’ ends and polyadenylated at their3’ ends

Question 36

Explain the lifecycle of NoV.

Answer 36

NoV attaches to the cell surface using various CHO attachment factors, no sufficient to mediate entry and binding to an unidentified protein receptor is thought to be requires (1,2) Entry (3) and uncoating (4) proceed through undefined pathways Viral genome is translated through interactions with VPg at 5’ end of genome ORF1 polyprotein is co and post translationally cleaved by the viral protease NS6 (6) The replication complex is formed by recruitment of cellular membranes to theperinuclear region of the cells through interactions in part with NS1/2 and NS 4 (7) Genome replication occurs via negative strand intermediate and genomic and subgenomic RNA are generated by the viral RdRp (NS7) using both de novo and Pg dependent mechanisms of RNA synthesis (8) THe erpelicated genomes are translate (within the rpelciation complex) or packaged into the capsid, VP1(9) The virion then assembles and exits (10) this may happen by cellular apoptosis, but unclear

Question 37

What are methods ot detect Nov?

Answer 37

The major barrier with NoV is hte lack of cell culture cultivation systems- 2016 found out it can be cultured in human bile added to enterocyte cell culture Sampes can be tested for NoV in buffer and washed into it PCR or RT-qPCR used to detect the presence of NoV Newer methods are WGS

Question 38

What is the Genome of Rotavirus?

Answer 38

RNA IV +ssRNA Rotavirus has segmented linear ds RNA genome Contains 11 segments which code for 12 viral proteins Viral mRNA contain 5’ cap but lack polyA tail Have 3’ consensus sequence UGACC that is conserved in all segments - terminates translation Individual co-infected with 2 roatviruses, can undergo mixing and lead to genetic reassortment

Question 39

What are the symptoms of Rotavirus?

Answer 39

Same as norovirus

Question 40

What is the structure of Rotavirus?

Answer 40

External surface is made of hte VP7 glycoprotein and embedded with the VP4 spike attachment protein Intermediate VP6 layer and thin VP2 core shell Ordered portion of viral ds RNA that line ethe VP2 shell are shown in gold Viral polymerase complexes PCs consisting o a single subunit each of the viral RNA dependen RNA polymerase VP1 and RNA capping enzyme VP3 are attached to the inner surface of the VP2 shell

Question 41

How are Genes expressed in rotavirus?

Answer 41

dsRNA genomes is never completely uncoated to prevent activation of antiviral state by the cell in response of ds RNA The viral RNA-dependent RNA polymerase VP1 synthesizes a capped mRNA form each dsRNA segment This capped mRNA is translocated to the cell cytoplasm→ translated NSP3 may act as a translation enhance for viral gene expression Most segments are generally monocistronic, however, segment 11 of rotaviruses encode a second protein

Question 42

What is the replication cycle of Rotavirus?

Answer 42

Replication occurs in host-cell cytoplasm The viral VP4 protein attaches to the hsot cell and mediated endocytosis of the virus Virus is partially uncoated in the endosomes (lost the VP4-7 outermost layer) → penetrates the cytoplasm via permeabilization of the host endosomal membrane Transcription of the dsRNA is done by viral polymerase and occurs inside the double layered particles so that dsRNA is never exposed to the cytoplasm The + RNA extruded into the cytoplasm and served as template for viral protein synthesis Progeny cores with replicase activity are produced in cytoplasmic virplasms (virus factories) Late transcription occurs in progeny cores Progeny cores coated with VP6 forming immature virions Progeny cores coated with VP6 (immature) bud across ER and acquire a transient liipdmembrane which is modified with ER viral glycoproteins NSP4 and VP7 Envelope articles also contain VP4 As the particles move to the interior of the ER the lipid membrane is lost, while the virus surface proteins VP4 and VP7 rearrange to form the otuermost virus protein layer, forming the mature infectious triple-layered particles Mature virions are released presumably following cell death and associated breakdown of host plasma membrane

Question 43

Explain the two proposed models of rotavirus assembly.

Answer 43

Concerted MOdel: 11 segments of rotavirus dsRNA are bound by VP1 and VP3 These viral protein/dsRNA complexes undergo assortment via gene-specific interaction smong the RNA molecules VP2 shell then assembles around the assorted complexes to create a full packaged and replication competent core Core-filling model, VP2 shell containing internally tethered viral proteins but lacking nucleic acid assembles