virology-2008 - Recently Updated Pages

Assignments: MCQs

arorar — Mon, 21 Apr 2008 14:09:07 CDT

How this will work. The rules:
1) For each lecture, I will award 0.5 bonus marks for each of 4 MCQs.
2) Each student can earn a maximum of 2.0 bonus marks for MCQs.
3) A student can only earn 0.5 bonus marks for any 1 lecture
4) The MCQs must be reasonable challenging questions.
5) Below, there are 6 slots available for each lecture. If a slot is available, you can put in your answer. If I accept a question for 0.5 marks, I will change the color to red. Occasionally, I will delete the worst answers. While there are less than 4 (the max) red answers, you can edit your own answers to try and improve them (make them worthy of a mark).
6) Underline the correct answer.
7) Your questions must be substantially different from other questions listed.

MCQs for lectures 1

EXAMPLE Name: Bloggs, Joe Q. Which of the following is not true? a) Viruses can infect fungi b) Viruses do not encode rRNA c) Viruses cannot be bigger than 200nm d) Viruses can be neutralized by Ab e) Viruses are not sensitive to streptomycin	Leave blank
Name: Marsters,Candace Q: Which is true of viruses? a) They have low mutation rates due to the small size of their genomes b) They are able to replicate outside the host cell c) Viruses consisting of (+) sense RNA must first be transcribed before it can be translated d) Their life cycles are highly regulated at all stages e) Their genomes often contain stretches of non-coding DNA/RNA	Name: Madalena, Candice Q. What is not part of the definition of a virus? a)obligate intracellular parasite b)living organism c)sub microscopic d)don't have ribosomes e)exist in two phases: intra and extra cellular
Name: Butterworth, Carly Q: Based on your knowledge of Viroids, which of the possible base pair lengths below would be indicative of a Viroid Genome: a) 40 nucleotide bases b) 300 nucleotide bases c) 2000 nucleotide bases d) 10,000 nucleotide bases (10 kb) (HIV) e) 800,000 nucleotide bases (800 kb) (Mimivirus)
Name: Love, Erin Q. Viruses are grouped based on all of the following except: a) genome structure (n.a. sequence) b) morphology c) host type d) temperature required for survival e) causitive disease

MCQs for lecture 2

Name: Ghassemi, Omid Q: Why are there so few anti-viral drugs available? a) Because most viruses are too small to be targeted by drugs b) Because viruses replicate inside host cells c) Drugs often causes severe side affects since viruses use host machinery d) Viruses quickly adapt and produce proteins which degrade some drugs e) A and B f) A, B and C g) A, B, C and D h) B and C
Name: Erin Love Q. What is the basis for the Hemagglutination assay? a) RBCs bind glycosylated residues only on viable virus surfaces and aggregate b) RBCs bind phosphate groups on all viral RNA/DNA and aggregate c) RBCs directly bind each other only in the presence of viable viruses and aggregate d) RBCs bind glycosylated residues on all virus surfaces and aggregate e) RBCs directly bind each other in the presence of all viruses and aggregate
Name: Madalena, Candice Q. Hershey and Chase a) is an experiment that uses radiolabeling to analyze the assembly of viruses b) discovered that some viruses use RNA for the coding material in their genome. c) an experimental procedure for detecting if a specific protein exists in a sample d) an experimental procedure for detecing specific DNA sequences in a sample e) discovered that DNA is the genetic material

MCQs for lecture 3

Name: Butterworth, Carly Why were the dry scabs of small pox lesions used in variolation effective at protecting uneffected individuals from acquiring the disease? a. the virus was inactivated b. antibodies could still target antigens on the viruses c. low inocculum d. unusual route of infection e. all of the above
Name: Erin Love Q. Which of the following is true about the assembly process of TMV? a) protein subunits require the TMV RNA to properly assemble b) subunits at the ends of the rods are least stable c) each subunit binds 6 nucleotide phosphates d) subunits have a higher affinity for RNA than for each other e) A and B
Name: Madalena, Candice Q. A viruses' symmetry is considered rotational rather than reflective because: a) the 60 subunits are only identical in T=1 classified viruses b) The protein subunit tails link under the surface in a disordely fashion c) There are proteins within the virus that can't be seen from the surface ex. VP4 protein d) The chirality of the amino acids e) of the plateaus and canyons on the surface

MCQs for lecture 4

Name: Najafi-Germi, Tina Q. Which of the following is not true about the hemagglutination assay? a) red blood cells will bind to glycosylated regions on virus particles b) a sample with no virus will produce a button at bottom of well c) a sample with virus will produce red blood cell aggregates d) quantifies virus e) measures virus viability
Name: Madalena, Candice Q. When a cell has become infected with a lytic virus it has the ability to get rid of the virus without lysing itself by: a)budding b)secreting the virus via signal proteins c)Attacking the virus with hydrolytic enzymes d)Sequestering the virus by engulfing it in a vacoule e)C and D
Name: Kulai, Tasha Q. Which of the following allow for the detection of viable viruses? a) Immunoprecipitation b) Hemagglutination inhibition assay c) Immunostaining d) MAGI assay e) None of the above

MCQs for lecture 5

Student #0332527 Q: After infecting a suspension of E. coli cells with virus particles, you collect a sample every hour and run the samples on an SDS-PAGE gel. After staining the gel, you notice certain protein bands present at earlier time-points that disappear after 3 hours. These protein bands likely contain: a) early stage viral proteins b) late stage viral proteins c) replicative proteins d) viral DNA
Student: Marsters, Candace Q: In the above growth curve of both intracellular and extracellular viral particles from Western equine encephalitis and Adenovirus type 5, it can be seen that Western equine encephalitis virus has on average 10^1 more extracellular virus particles than intracellular compared to Adenovirus which has 10^3 more intracellular viral particles than extracellular. Knowing that both viruses can infect the same host but Western equine encephalitis virus is enveloped while Adenovirus is not, what reasonable conclusion(s) could be deduced from comparing this data? a) Western equine encephalitiis virus is more infectious than adenovirus b) Upon direct comparison of eclipse and latent periods, it can be seen that Western equine encephalitis virus may infect a host faster c) Particle release is such that Western equine virus may bud from host cell while Adenovirus may clump together within host cells d) a & c e) Nothing can be concluded from comparison of this data
Name: Kulai, Tasha Q: The polio genome encodes a polyprotein precursor. Genome organization is such that viral capsid proteins are translated first and proteases and proteins involved in RNA synthesis are translated last. Why is this genomic organization effective? a) It makes equal amounts of all proteins, thereby providing the vast amount of replication machinery and capsid proteins necessary for replication and assembly. b) It allows equal amounts of protein to be made early, and by subsequent early translational termination, allows only capsid proteins to be made. c) It shortens the length of the eclipse period. d) It ensures many capsid proteins and few replicases are made as ribosomes typically fall off the transcript before reaching the replicase. e) This genomic organization is NOT effective. Too many capsid proteins are made unnecessarily early in the infection cycle.

MCQs for lecture 6

Student #0729017 You have a suspension of 10^10 pfu of virus. You add an antibody then plate it and find that three plaques have formed. What is the most likely explanation? a) the antibody was non-neutralizing b) some of the virus had mutations in the antigenic site c) the antibody was not specific to the virus d) the antibody was neutralizing
Name: Najafi-Germi, Tina Q. You are studying the assembly of the Tobacco mosaic virus. To determine the different subunit interactions between the proteins, you set-up an experiment with various conditions. What do you expect to see at a pH 8.0 and low ionic strength (0.1M)? a) single helix b) disk structures c) monomeric proteins d) crystal structures e) stacked disk rods

MCQs for lecture 7

MCQs for lecture 8

MCQs for lecture 9

Name: Martinson, Geoff
Virulence may be defined as:
a) the ability of a virus to cause disease
b) the presence of disease causing proteins within a virus
c) the severity of disease caused by a virus
d) how well a virus survives in a host

MCQs for lecture 10

Student 0729017 In Rhabdoviruses, the order of structural genes from 3' to 5' on the (-) strand is: a) Nucleocapsid,Large Proteins, Phosphorylated Proteins, Matrix proteins, Glycprotein Spikes b)Nucleocapsid Proteins, Phosphorylated Proteins, Matrix Proteins, Glycoprotein Spikes,Large proteins c) Large proteins, Glycoprotein Spikes, Matrix Proteins, Phosphorylated Proteins, Nucleocapsid Proteins d)Nucleocapsid Proteins, Phosphorylated Proteins, Glycoprotein Spikes, Matrix Proteins, Large Proteins e) Large Proteins, Matrix Proteins, Glycoprotein Spikes, Phosphorylated Proteins, NucleocapsidProteins

MCQs for lecture 11

MCQs for lecture 12

MCQs for lecture 13

Student # 0328305 Q. For potatoe spindle tuber viriod to infect a host, it: a) requires a helper virus for replication b) requires a helper virus to make coat proteins c) alters its helper virus' virulence d) none of the above e) two of the above f) all of the above

MCQs for lecture 14

Name: Roarke Copeland Q. A satellite virus and its helper virus must have similar: a) Promoter sequences b) Capsid structures c) Promoter secondary structures d) Polymerase sequences
Student # 0328305 Q. What is "maturation protein A" required for? a) it acts as a repressor of the replicase gene b) it binds the 5' end of the coat protein gene to allow for replicase protein translation c) it is required for attachment to E.coli pilus and entry of RNA d) it is required for attachment to E.coli pilus and entry of DNA

MCQs for lecture 15

Student # 0328305
Q. Which of the following viruses does NOT have an envelope:
a) falvivirus
b) tobacco mosaic virus
c) coronavirus
d) rubivirus

MCQs for lecture 16

Name: Roarke Copeland Q. What most likely keeps the number of genetic mutations down in coronavirus (very large, 30kb RNA genome)? a) Low number of replications b) An accurate polymerase c) Slower replication d) A proofreading mechanism
Student # 0328305 Q. Viruses that contain N, P, M, G, and L mRNA refer to those viruses with a ______ genome: a) + ss RNA b) - ss RNA c) ds RNA d) small DNA e) large DNA

MCQs for lecture 17

Student # 0328305 Q. If the MOI for influenza virus is high and we use gamma-ray radiation to try to kill the virus, what happens to the virus? a) the virus obtains multiple mutations in its genome and is destroyed b) inactivated segments of the viral genome accumulate and may lead to cancer c) complementation between inactivated segments of the genome occurs, allowing for recombination between segments d) complementation between inactivation segments of the genome occurs, allowing for reassortment of segments

MCQs for lecture 18

Student # 0328305 Q. Which of the following statements about ATLL, is false? a) it infects CD4+ cells b) it infects CD8 cells c) it is transmitted in a similar way as HIV d) it may cause lymphomas of lymph nodes

MCQs for lecture 19

MCQs for lecture20

MCQs for lecture 21

Student # 0328305 Q. Which of the following statements regarding the viral infectivity factor, Vpr, is correct? a) it affects HIV release b) it affects HTLV release c) it increases HIV infectivity d) it increases HTLV infectivity e) it accumulates in the cytoplasm f) it binds nuclear pores g) it causes G1 phase arrest h) one of the above i) two of the above

MCQs for lecture 22

MCQs for lecture 23

MCQs for lecture 24

Chikungunya

oksanan — Wed, 02 Apr 2008 17:29:24 CDT

The chikungunya threat: an ecologic and evolutionary perspective

Oral presentation:
Wiki presentation: Oksana Nemirovsky

Start writing your report here

Chikungunya Virus (CHIKV) Background

mosquito-borne
enveloped alphavirus
originated in West Africa, then spread to Asia and India
clinical symptoms:

unique to CHIKV: painful arthralgia (joint pain) lasting for months

resulting in contorted posture

similar to Dengue fever: fever, nausea and rash.

An alphavirus replication and genetics

virus replication results in apoptosis of vertebrate cells
replication requires viral proteins to interact with host cell nucleus of vertebrate cells
virus can be found in muscular satellite cells for many months, as a reservoir of lasting infection
alphaviruses experience host-dependent balance between mutation rate and evolutionary constrains
genetic variations of CHKV could be grouped in three main clusters:

Western Africa (originator)
Asian, Central,
Southern and East Africa.

Human CHIKV infection characteristics in Africa

Transmission to humans mainly in rural areas with eventual spread to urban areas
Infection is often misdiagnosed and asymptomatic,
persistent immunity exists in population due to frequent infections with other zoonotics: diseases transmitted by other animals

less immunity observed when spread to new locations

moderate impact on public health,

since priority is given to more life threatening diseases by health authorities

Characteristics of CHIKV as an emerging arbovirus ( disease transmitted by arthropods)
1 host diversity is greater in native ecosystem, than in recently colonized urban areas
hosts: human, mosquitoes, birds, monkeys, rodents and squirrels.
2 human migration from rural parts of Africa is responsible for rapid long-distance dispersal of virus
3 vector change could occur in urban ecosystem

Prevention

Decrease contact with diseased vectors through mosquito control
Insect repellents with substances like DEET, Icaridin, and PMD (a substance derived from the lemon eucalyptus tree).
Wearing bite-proof long sleeved garments.
Securing screens on windows and doors to keep mosquitoes out of the house.

Treatment

There is no vaccine currently available.
A serological test for this virus is available
Chloroquine might be a good possible treatment for the symptoms of disease, as well as an antiviral agent

has been used in the treatment and prevention of malaria.
mildly suppresses the immune system: it is used in some autoimmune disorders, such as rheumatoid arthritis

Infected individual should limit further exposure to mosquitoes,
Heavy exercise should be avoided, but mild exercise is actually beneficial.

Conclusion

Primary source of re-emergence is in the rural ecosystems
Human activities are the main cause of viral emergencies

migration, spread and settlement of virus into urban ecosystems
amplification of virus through farming

Important to understand local diverse ecosystems to prevent spread of disease

References

Chevillon C., Briant L., Renaud F., and Devaux C. The chikungunya threat: and ecological and evolutionary perspective (2007). Trends in Microbiology Vol. 16, No. 2.
Wikipedia

Unusual Bonding in Viral Capsids

erinjbmlove — Wed, 02 Apr 2008 12:17:19 CDT

Uploaded by Erin Love

Bonding in Capsids

Most bonds in capsids are non-covalent
- Allows for quick rejection of incorrect structures

Some bonds are isopeptide or disulfide
-Allows virus to have thinner walls and larger internal volume

Isopeptide Bonds

Model: Enterobacteria phage HK97

- dsDNA

- tailed

- 420 structural proteins (Gp5)

- 12 portal proteins (Gp3)

- 60 hexamers and 12 pentamers

- Shell formed through multi-step pathway (moves from spherical to icosahedral shape)

- Conformational change in Gp5 protein

- N-arm and E-loop move

- isopeptide bond forms between Lys and Asn in different subunits (autocatalysis?)

Disulfide Cross-linking

Model: respiratory enteric orphan virus (Reovirus)

- dsRNA

- Non-enveloped animal virus

- T-13 shell composed of an outer and an inner capsid

- S-S between Cys residues in outer capsid subunits

disulphide cross-linking is common in enveloped viruses and less common in non-enveloped viruses
- examples:

- Reovirus

- Papovavirus

- Simian virus 40

- picornavirus

- parvovirus

- adenovirus

Summary

Most bonds in capsids are non-covalent

Unusual bonding:

- Isopeptide bonds (Model: Phage HK97)‏

- Disulfide bonds (Model: Reovirus)‏

References

Conway, J.F., et al. (1995). Proteolytic and conformational control of virus capsid maturation: The bacteriophage HK97 system. JMB. 253 (86-99).

Odegard, A.L., et al. (2003). Disulfide bonding among micro1 trimers in mammalian reovirus outer capsid: a late and reversible step in virion morphogenesis. JVI. 77:9 (5389-5400).

Rader, A.J., Vlad, D.H., Bahar, I. (2005). Maturation Dynamics of Bacteriophage HK97 Capsid. Structure 13, 413-421.

Wikoff, W.R., Liljas, L., Duda, R.L., Tsuruta, H., Hendrix, R.W., and Johnson, J.E. (2000). Topologically linked protein rings in the bacteriophage HK97 capsid. Science 289, 2129-2133.

FIGURES:

http://www.ccbb.pitt.edu/research/bahar_lab/Viral_Dynamics/

http://www.purdue.edu/UNS/html4ever/031215.Baker.reovirus.html

http://johnsonlab.scripps.edu/resources/collaborations/collaborations.php

http://www.biologie.uni-hamburg.de/b-online/e17/17d.htm

http://www.brooklyn.cuny.edu/bc/ahp/LAD/C4b/C4b_proteinShape.html

http://pathmicro.med.sc.edu/mhunt/RNA-HO.htm

http://en.wikipedia.org/wiki/Reovirus

http://www.pitt.edu/~duda/HK97.html

http://www.cosmosmagazine.com/system/files/

‏

Martinson: HIV Tat protein

the_geoffrey — Wed, 02 Apr 2008 01:04:36 CDT

HIV Tat Protein: It's not all bad
Oral & Wiki presentation: Martinson, Geoff

Trans-Activator of Transcription

-Tat stimulates HIV-1 transcription through a positive feedback loop; Tat is spliced from basal levels of RNA synthesis, ships back into the nucleus and recognizes DNA

-Binds the TAR element of DNA, which is a stem-loop structure; a conformation change is induced

-Elongation; affects processivity of RNA Pol2 complex so that full-length viral transcripts are made; without Tat, transcription terminates early

...it's not all bad

-HIV-1 Tat protein is able to mediate transmembrane delivery of fused cargo

Tat Delivery Service

-Cargo is fused (covalently linked) to Tat (small molecule drugs, DNA, oligopeptides, iron particles, b-gal) can cross the plasma membrane of most cells

-Traverses almost all cell types; new transporters (guanidinium-rich transporters) have been shown to even enter through the skin

Protein Transduction Domain

-The PTD is the region conveying cell penetrating properties to the Tat protein

-It's a small (9 amino acids) stretch of basic residues, with the sequence: RKKRRQRRR

-Key to uptake is the array of guanidinium ions of arginine residues

-Took Tat 9-mer and changed each residue one at a time; uptake was compared relative to original Tat 9-mer:

truncated versions (deletions of R less effective than deletions of L); clue as to where to direct studies

substitute (uncharged) alanine for a residue

arginine 9-mer worked better than Tat 9-mer

Positively charged arginines interact with anionic membrane elements through hydrogen bonding

-Driving force is decrease in free energy associated with binding membrane

-Membrane elements: phospholipids, fatty acids, proteins, or heparan sulfate proteoglycans

Tat-cargo enters through endocytosis: clathrin-mediated; caveolin-mediated; macropinocytosis

-No definite mechanism of entry

-Non-endocytotic pathway in which vessels directly diffuse across membrane; relies on membrane potential (studies have suggested that ATP is required in the Tat-peptide traversion of a membrane)

-Mechanism for entry appears to be dependent on the cargo being transported and the cell being entered; important when considering drug delivery

Noteworthy: transduction involves no disruption of the plasma membrane; does not bring in non-linked molecules into/out of the cell

Applications: Cancer Therapy

-The ability of Tat to deliver cargo to most cell types with high specificity makes it a good candidate for cancer therapy

-Current chemotherapies:

show low specificity of target; affect normal cells as well as tumor cells

poorly transported into certain tissues

may be actively excluded by multi-drug resistant cells

Key: initiate tumor suppressor function in cells or turn on apoptosis

-Deliver cell cycle inhibitors to induce apoptosis in tumors

Tat-p53 peptide

-p53 tumor suppressor gene:

activates apoptotic stage

mutated in many tumors; increases cancer cells' resistance to therapy

HDM2 overexpressed in tumors with wild-type p53

-Tat-p53 fusions injected at tumor sites were able to restore p53 function

-p53 accumulates in all cells, though p53 was only activated in cancer cells (specificity); resulted in significant reduction in tumor growth by activating cell arrest cycle (apoptosis)

Future for HIV-1 Tat protein

-Guanidinium-Rich Transporters (GRT) based on the Tat PTD:

focus is on guanidinium ion of arginine (Arg 9-mer)

stereochemistry of chain is modified to increase uptake

able to enter cells and even tissue (ex. skin or lung)

-Uptake is better than Tat

References

-Bayer P, Kraft M, Ejchart A, Westendorp M, Frank R, Rosch P. 1995. Structural studies of HIV-1 Tat protein. J. Mol. Biol. v247, p.529-535

-Brooks, H., Lebleu, B., and E. Vives. 2005. Tat peptide-mediated cellular delivery: back to basics. Ad. Drug Del. Rev. v57, p. 559-577.

-Flint, S.J., Enquist, L.W., Racaniello, V.R., and A.M. Skalka. 2004. Principles of Virology, 2nd Edition. Pp. 269-275. ASM Press, Washington, D.C.

-http://en.wikipedia.org/wiki/Image:Cancer_requires_multiple_mutations_from_NIH.png

-http://reginadailyphotoblog.blogspot.com/2007/11/canada-post.html

-http://www.yourpartybypost.co.uk/acatalog/unicorn.html

-The National Science Foundation. 2005. http://www.nsf.gov/news/overviews/biology/interact01.jsp.

-Wadia, J.S., and S.F. Dowdy. 2004. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Ad. Drug Del. Rev. v57, p. 579-596.

-Wender, P.A., et al. 2008. The design of guanidinium-rich transporters and their internalization mechanisms. Ad. Drug Del. Rev. v60, p. 452-472.

The WIKI

cupton — Sun, 30 Mar 2008 18:24:35 CDT

Welcome to the MICR402 wiki!

This site will be used by students to post projects to the WWW.

You can find the course www site herehi!

Here's some feed from an interesting microbiology blog.

wiki is our central meeting place to plan a

Madalena/Randsalu: Avian and Swine Influenza: our current understanding of the zoonotic risk

CandiceM — Wed, 26 Mar 2008 13:16:31 CDT

Background on Influenza A

Enveloped

RNA virus

Genome is present in 8 separate segments

Classified into subtypes based on the antigenic properties of HA and NA

16 HA (HA1-HA16)

9 NA (N1-N9)

Genetically unstable in the sense that the segements can combine to form alternate serotypes

Fear

The introduction of avian or swine influenza into the human population would set the stage for a pandemic

Based on the notion that humans have no immunity and the virus is transmissible from person to person

It is rare that an influenza virus is transmissible between species

Avian Influenza Virus (AIV)

Highly pathogenic forms: H5 and H7

Spread between birds mainly through infected feces

H5N1 has been circulating in Asia since 1996 – causing many birds to be slaughtered in HK in 2007

More virulent in domestic birds than wild birds

Swine Influenza Virus (SIV)

3 types: H1N1, H3N2, and H1N2

Differ based on geographical location

Most are reassortants of human, avian, and swine virus genes

Pigs are susceptible to both human and avian influenza virus and may act as a ‘mixing vessel’ for those viruses

Transmitted through contact with respiratory secretions and through air

Human Influenza Virus

Subtypes established:H1, H2, H3, N1 and N2

Transmission via respiratory routes

Spread between continents rapid

Humans have receptors for both human and avian influenza viruses, which predominate in the upper and lower respiratory tracts respectively

Are swine an intermediate host for the transmission of influenza from birds to humans?

HA subtypes normally restricted to birds can cross the species barrier to pigs

Only H1N1 that has crossed from wild birds to swine has become established whereas the others have disappeared

the genes of avian viruses may persist after reassortment with one or more influenza viruses endemic in pigs

the trachea of pigs contains receptors for both avian and human influenza viruses

AIVs can transmit to humans without the pig as an intermediary and that genetic reassortment can also occur in humans

limited knowledge of the extent of replication of the Asian H5N1 virus in pigs, it is difficult to estimate whether there is a real chance for reassortment of this virus in a pig.

Barriers

lack of suitable receptors on the host cell

Even if an influenza virus succeeds to enter the cell of a new host, it must successfully coopt host cell processes to replicate there.

Recent research has shown that reassortment in itself is insufficient for the generation of a pandemic influenza virus and that additional genetic changes are likely required

Conclusion

Other factors that will influence influenza virus transmission between species are non-specific immune mechanisms, the routes of virus dissemination/excretion by the “donor” host, the extent of contact between donor and “recipient” host and the influenza immune status of the new host

a strong barrier exists to infection of pigs with influenza viruses from other species, and that major genetic changes are required for consistent pig-to-pig transmission of such viruses

Madalena

CandiceM — Wed, 26 Mar 2008 13:06:41 CDT

There is no abstract available for this page revision.

The Relationship Between Rubella Vaccination and Type 1 Diabetes

scottyb52 — Mon, 24 Mar 2008 14:28:31 CDT

Oral presentation: Beischer, Andrew
Wiki presentation: Bell, Scott

Type 1 Diabetes
An autoimmune disease that is a result of the destruction of insulin producing b-cells of the pancreas
There is a genetic predisposition to Type 1 Diabetes (T1D) through specific alleles of MHC class II.
The particular MCH II strongly associated with T1D are HLA-DR and HLA-DQ (Especially DR3 and DR4)
Evidence for
Environmental Agents
Studies on identical twins have shown that the concordance rate for the disease only approcahes 40%. This suggests that even though genetic predisposition is a pre-requisite environmental factors such as viruses, diet, and beta-cell toxins may be involved in the progression and/or injtation of beta-cell destruction leading to insulin dependant diabetes (IDDM)
Seasonal Variation and epidemiologic data support data that infectious agents play an important role in beginning the autoimmune process
There is a high frequency of children with congenital rubella that develop IDDM.
The most common side effects of congenital defects are cataracts, heart disease, sensorineural deafness, and mental retardation
Monoclonal Antibody to Rubella Virus Capsid
Recgonizes a beta-cell antigen. (1993)
The initial finding of this work was done by immuno-blotting b-cell extracts of both rat and human islets.
An anti-rubella capsid monoclonal antibody as well as sera from NOD (non-obese diabetic) mice and newly diagnosed T1D patiets recgonized the same 52 kDa b-cell protein.
This finding suggests that molecular mimicry due to rubella exposure could sensitize host to b-cell damage.
Cross Reactive Rubella Virus and Glutamic Acid Carboxylase (GAD) (65 and 67) Proteins
Determinants Recgonized by T Cells of Patients with T1D.
Tested the cellular response of T-cells from 60 T1D patients using whole rubella virus, various rubella envelope and coat proteins
Over 80% of the tested subjects had positive cellular responses to the whole rubella virus.
Two rubella proteins had cellular responses over 70% in T1D patients and all T1D CRS patients (there were only 3 of these patients)
There have been 15 GAD proteins known to be T1D associated auto-antigens prior to these findings.
These 15 GAD proteins were screened for cellular responses from T-Cells of the 60 T1D Patients
Four GAD (65 and 67) peptide specific T-cell clones were constructed.
From these clones two or more responded to the rubella peptides in a cytotoxicity assay
Using sequence analysis they determined that the GAD and the rubella epitopes shared the same motif.
This motif is also shared with the minimal binding motifs of HLA-DR3 and DR4.
Conclusion
From these findings a vaccination or infection of rubella could be a triggering factor to developing T1D by boosting autoantigenic T-cells.
This would cause a cellular auto-immune response against the β-cells leading to T1D

The End

Rubella Vaccination and It's Relationship to Diabetes.

scottyb52 — Mon, 24 Mar 2008 13:58:43 CDT

Replace this with the Title of your Project

Oral presentation: Simpson, Homer
Wiki presentation: Simpson, Bart

Start writing your report here:

Auger:Is the term 'prokaryote' obsolete?

LittleMeagan — Sun, 16 Mar 2008 21:55:55 CDT

Is the term 'prokaryote' obsolete?

Oral presentation: Auger, Meagan
Wiki presentation: Auger, Meagan
Is the Term “Prokaryote” Obsolete?
By Meagan Auger

Pace, NR. The molecular tree of life changes how we see, teach microbial diversity. Microbe. 2008. 3(1):15-20.
In science, what is taught in the textbook is not always reflective of actual scientific data.
In the author’s opinion, the concept “prokaryote” is obsolete.
How did this term arise?

· Initial classification of organisms in the 19th century
-didn’t include microbes because of lack of morphological characteristics
-Few properties visible under the light microscope led to inaccurate and misleading classifications
· In the 1960’s and 1970’s the advent of genome sequencing led us to more relevant classifications…
· Cloning-lets us get around not being able to grow some microbes in culture
· sequence of the small rRNA used to derive evolutionary relationships
-ubiquitous, conserved, and the technology was available
· Led to the discovery of archaebacteria/Archaea
· Horizontal transfer of genes→not indicative of all relationships
· The Three Domains
· Bacteria, Archaea and Eukarya
· The common ancestor was a bacterium
· However, the domains are not derived from each other…
· Archaea and Eukarya are more closely related to each other than to Bacteria
· Pro= no, karya=nucleus, suggests a lack
· Can’t tell if referring to common ancestor, Bacteria, or Archaeaa
· The three domains aren’t related
· Author believes most students think…
1. All eukarya are specifically related.
2. All prokarya are specifically related.
3. Eukarya emerge from Prokarya.

· Precepts 2 and 3 are false…
· This is important because language influences how we conceptualize science
· We need to be able to classify things accurately!
· So, we should not use the term prokaryote.
· Use the term “microbe” to refer to anything small, and then specify if Bacteria or Archaea

Gill:Norovirus Background, Disease Patterns and Evolution

gkgill — Fri, 14 Mar 2008 00:11:30 CDT

Norovirus

Oral presentation: Muhammad Ali and Rhian Green
Wiki presentation: Gurjit Gill
Background:

A.K.A
o Norwalk virus
Shape and Structure
o non-enveloped
o round o icosahedral symmetry
o capsid approx. 35-39 nm
Genome
o (+)SS RNA HIGHLY INFECTIOUS
o 7.5 kb
o Poly A stretch at 3' end o encodes 3 open reading frames § VP1-major structural protein § VP2-minor capsid protein o Major capsid protein has a highly variable P2 domain o P2 domain binds histo-blood group antigens (HBGAs) on intestinal epithelial cells for infection o virus replicates inside intestinal epithelial cells ·
Classification
o 5 different genogroups (GI, GII, GIII, GIV and GV)
o Genogroups I, II (19 genotypes) and IV infect humans
o GII.4 main infectious strain o Family: Caliciviridae
Diseases
o Acute gastroenteritis
o Flu-like symptoms: nausea, vomiting, diarrhea, abdominal pain, etc..
o Can be lethal in children, elderly people or immunocompromised individuals
o Some individuals are more susceptible to infection

Individuals encoding FUT2 (α-fucosyltransferase) enzyme are secretor-positive

this means that their blood group can be indentified in their saliva
HBGAs are not expressed in those that are secretor-negative (encode defective FUT2 enzyme) and therefore the virus cannot dock or perhaps enter
Individuals of O blood group at greater risk

Prevention o Thorough cooking of food

can withstand temperatures up to 60°C

o Properly washing hands may reduce virus transmission

BUT because virus has no lipid envelope, less susceptible to detergents and alcohols · chlorine-based disinfectants remove the virus

o Early detection in food by RT-PCR

when surveillance is occurring by FDA or other government agencies

Vaccine
o No vaccine

expensive
many strains and a new strain emerges approximately every year

o Some individuals are naturally resistant to norovirus
o Some individuals do not develop immunity after exposure to the virus

Disease Patterns and Evolution
o Noroviruses account for 6% of all infectious intestinal diseases in England, and 11% in the Netherlands
o 23 million cases of Norovirus are confirmed each year in the United States
o Responsible for 90 % of epidemic non-bacterial outbreaks of gastroenteritis around the world
Reasons for Large Impact ·
o Large human reservoir for infection
o primary host = humans
o Very low infectious dose~100 Virus Particles
o Many routes of transmission

Person-person
Fecal-Oral Route
Food-borne o Water-borne

o Majority of outbreaks occur in hospitals and residential care facilities
o Majority of cases occur during winter months

Lower temperature
Less UV could ease transmission

could also be related to prevalence of disease in northern europe

o Evidence for Evolution

New strains are emerging more frequently than in previous years

assists in escaping the effects of protective herd immunity

Pre-epidemic serum binds poorly to post epidemic virus-like particles

Study from: Lindesmith LC, Donaldson EF, LoBue AD, Cannon JL, Zheng DP, et al. (2008) Mechanisms of GII.4 norovirus persistence in human populations. PLoS Med 5(2): e31. doi:10.1371/journal. pmed.0050031 o Methods

Obtained 176 viral capsid amino acid and nucleic acid sequences for GII.4 strains of norovirus isolated from viral outbreaks between 1987-2005.
Performed in silico BLAST and ClustalX alignment analysis between the capsid sequences.

o Findings

Most highly variable region was P2 subdomain · capsid protein with most surface exposure
This region encodes for an HBGA carbohydrate binding domain

o Significance

Amino acid mutations of the P2 HBGA carbohydrate binding domain allows noroviruses to bind more strongly and with higher efficiency to a greater number of HBGA carbohydrate molecules on the mucosal surfaces of the gut
Escape of host immune system
Causes faster and more persistent infections

o Conclusion

Norovirus GII.4 strains persist by evolving novel carbohydrate-binding domains over time in response to immune-driven selection and by antigenic drift in the receptor-binding regions of the P2 subdomain.

References:

Norovirus, Wikipedia, The free encyclopedia http://en.wikipedia.org/wiki/Norovirus

Lindesmith L, Moe C, Marionneau S, et al (2003). "Human susceptibility and resistance to Norwalk virus infection". Nat. Med. 9 (5): 548–53

Lindesmith L, Moe C, Lependu J, Frelinger JA, Treanor J, Baric RS (2005). "Cellular and humoral immunity following Snow Mountain virus challenge

Huang P, Farkas T, Marionneau S, Zhong W, Ruvoën-Clouet N, Morrow AL, Altaye M, Pickering LK, Newburg DS, LePendu J, Jiang X (2003). "Noroviruses bind to human ABO, Lewis, and secretor histo-blood group antigens: identification of 4 distinct strain-specific patterns". J. Infect. Dis. 188 (1): 19–31

Lindesmith LC, Donaldson EF, LoBue AD, Cannon JL, Zheng DP, et al. (2008) Mechanisms of GII.4 norovirus persistence in human populations. PLoS Med 5(2): e31. doi:10.1371/journal. pmed.0050031

Lopman B, Zambon M, Brown DW. (2008) The Evolution of Norovirus, The “Gastric Flu”. PLos Med 5(2): e42. doi:10.1371/journal.pmed.0050042

Lopman BA, Adak GK, Reacher MH, Brown DWG. (2003) Two epidemiologic patterns of Norovirus outbreaks: surveillance in England and Wales, 1992-2000. Emerg Infect Dis. 9: (1) http://www.cdc.gov/ncidod/EID/vol9no1/020175.htm

Siebenga, JJ., et al. (2007) Epochal Evolution of GGII.4 Norovirus Capsid Proteins from 1995 to 2006. J Virol. 81(18): 9932–9941. Published online 2007 July 3. doi: 10.1128/JVI.00674-07. Copyright © 2007, American Society for Microbiology. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2045401

Assignments: Projects

cupton — Thu, 13 Mar 2008 18:37:17 CDT

If 2 students are assigned to a project, both do the research then one gives the oral ppt presentation (O) and the other puts the information on the WIKI (W).
Students doing solo projects will be provided with a ppt presentation to work from, and will also summarize the material on the WIKI.

Name	Title	Due date
McEwen, Jason	Molecular scale	Mon Jan 14th
Scarrow, Brittany	Cell surface	Mon Jan 14th
Watson-Hurthig, Adam	Cryo-EM	Thurs Jan 17th
Laugesen, Anne	Speed of trans/cription/lation	Thurs Jan 17th
Miles, Sarah	Phage panning	Mon Jan 21st
Liem, Matthew	Classification	Mon Jan 21st
Cann, Melissa	Quantitation RNA oral	Thurs Jan 24th
Chan, Connor	Quantitation RNA wiki	Thurs Jan 31st
Ranns, Blair	Quantitation DNA/prot oral	Thurs Jan 24th
Hughes, Shevaun	Virus detection oral	Mon Jan 28th
Jervis, Drew	Virus detection wiki	Mon Feb 4th
Razavi, Seyed	Quantitation DNA/prot wiki	Thurs Jan 31st
Copeland, Roake	Dengue, background oral	Thurs Jan 31st
Dacho, Kori	Dengue, background wiki	Thurs Feb 7th
Fraser, Shane	Dengue, virus oral	Thurs Jan 31st
Effa, Emily	Dengue, virus wiki	Thurs Feb 7th
Manson, Marita	Dengue, future oral	Thurs Jan 31st
Marren, Tessa	Dengue, future wiki	Thurs Feb 7th
Ehman, Dylan	Gene therapy oral	Mon Feb 4th
Kulai, Tasha	Gene therapy wiki	Mon Feb 11th
Oosten, Heather	Centrifugation oral	Mon Feb 11th
Orr, Lindsay	Centrifugation wiki	Mon Feb 18th
Witze, Tanya	Ebola, background oral	Thurs Feb 14th
Wolfe, Dave	Ebola, background wiki	Thurs Feb 21st
Moore, Eric	Ebola, background oral	Thurs Feb 14th
Murphy, Lisa	Ebola, background wiki	Thurs Feb 21st
Love, Erin	Capsid structure	Mon Feb 25th
Kremer, Jon	Polyoma oral	Thurs Feb 28th
Kroeker, Jon	Polyoma, wiki	Thurs, Mar 6th
Scott, Heidi	Extinction, oral	Mon, Mar 3rd
Wong, Emily	Extinction, wiki	Mon Mar 10th
Auger, Meagan	Tree of Life	Mon Mar 10th (wiki 1 week later)
Ali, Muhammad	Norovirus background, oral	Thurs, Mar 6th
Green, Rhian	Norovirus evolution, oral	Thurs, Mar 6th
Gill, Gurjit	Norovirus evolution, wiki	Thurs, Mar 13th
Beischer, Andrew	Rubella, oral	Thurs, Mar 13th
Bell, Scott	Rubella, wiki	Thurs, Mar 20th
Madalena, Candice	Flu, Oral	Thurs, Mar 20th
Randsalu, Dash	Flu, wiki	Thurs, Mar 27th
Auger, Meagan	HIV-Tat, oral	Thurs, Mar 27th
Martinson, Geoff	HIV-Tat, wiki	Thurs, Apr 3rd
Church, Jessica	Chikungunya, oral	Thurs, Mar 27th
Nemirovsky, Oksana	Chikungunya, wiki	Thurs, Apr 3rd

DNA/Protein Quantification

cupton — Tue, 11 Mar 2008 11:05:13 CDT

Oral Presentation: Blair Ranns
Wiki Presentation: Seyed Morteza Razavi

DNA/Protein Quantification

DNA Quantification
- Southern Blotting
- PCR
- Agarose Gel Electrophoresis
Protein Quantification
- Western Blotting
- SDS-PAGE

Southern blotting;
- Is used to locate a particular sequence of DNA within a complex mixture (eg. a gene in a genome!)
- The amount of DNA needed depends on size and specific activity of the probe
- Shorter probes = More specificity
- Under optimal conditions can expect to detect about 0.1pg of DNA
- Usually the gel is loaded with 10ul of the sample containing DNA

PCR;
- In a reaction tube you have the followings;
o PCR buffer, Mg2+, 3' and 5' primers, DNA polymerase, RNAse (Sometimes), ddH2O, DNA sample
- The volume of the reaction mixture is typically between 15-100ul
- Typically only 10ul of this mixture is ran on an agarose gel
- Sensitivity depends on many things; o How pure your sample is
o Accuracy of the primers
o Number of cycles
o DNA polymerase used
- Typically Taq polymerase is used, which can detect targets of about 5kb
- Maximum target size in PCR is about 40kb

Western Blot;
- Can detect one protein in a mixture of proteins
- Gives you information about the size of the protein
- Typically on a gel one loads 10ul of sample
- This gives a sensitivity of 1ng/band

Gels

-SDS PAGE;
- Separation of proteins based on electrophoretic mobility
- Consists of Stacking gel (Concentrate proteins) and Resolving gel (Separates proteins)
- Sensitivity depends on type of staining used
o Coomassie is about 100ng of protein
o Silver staining is 1000 fold more sensitive

- Agarose gel electrophoresis
- Separation of DNA fragments
- Normally used in the range of 100bp to 20kbp
- Different concentration of agarose is used for fragments with different sizes.
- Usually 10ul of sample is loaded

References

Biochemica, 1995, “Answers to the Questions Most Commonly Asked of Our Technical Services Scientists, No 2. URL: http://www.roche-applied-science.com/PROD_INF/BIOCHEMI/app10-11.pdf. Retrievd, Jan 21/ 2008.
David L. Nelson, and Michael M. Cox. “Lehninger: Principles of Biochemistry”. 2005. http://www.cygnustechnologies.com/Western%20BLot%20vs%20ELISA.pdf http://biology.ucsd.edu/labs/aroian/protocols/sds-page.htm http://lifesciences.asu.edu/resources/mamajis/southern/southern.html http://lifesciences.asu.edu/resources/mamajis/western/western.html
Wikipedia

Wong: Infectious Diseases and Extinction Risk in Wild Mammals

ewong — Sun, 09 Mar 2008 18:24:33 CDT

Infectious Diseases and Extinction Risk in Wild Mammals

Amy B. Pedersen, Kate E. Jones, Charles L. Nunn, and Sonia Altizer

Oral presentation: Scott, Heidi
Wiki presentation: Wong, Emily

Which mammals are most at risk for extinction from infectious diseases?
Infectious diseases are being recognized as an emerging cause of population declines and extinctions in endangered mammals. Using the IUCN Red List of Threatened and Endangered Species and data on hosts threatened by infectious diseases, it was found that the majority (88%) of mammals threatened by parasites are from two taxonomic orders:
1) Carnivora

Species from the families Canidae and Felidae make up the majority of the endangered animals

2) Artiodactyla (any hoofed mammal with an even number of toes; e.g. cattle, deer, camels, pigs, sheep, and goats)

Species from the families Bovidae and Suidae make up the majority of the endangered animals

Which parasites are causing population declines in endangered mammals?
Out of the 31 parasites that were identified as causing declines in threatened mammals, 41% of them are viruses (Table 1). Viruses are more likely to threaten mammals because of their high mutation rates and their ability to infect a wide range of host species, including domesticated animals. Over 70% of the identified viruses are ssRNA viruses, which have a much higher mutation rate than DNA viruses due to the absence of DNA polymerase and its repair mechanisms.

Table 1. Parasites identified as causing population declines or reduced host fitness in mammals listed on the IUCN Red List as threatened by pathogens

Figure 2. Taxonomic distribution of six major groups of parasites reported as threatening (a) artiodactyls and (b) carnivores.

Disease in wild mammals is linked to domesticated animals:
The majority of mammals threatened by parasites (carnivores and artiodactyls) include all major groups of domesticated livestock and companion animals. Due to the high population density and global distribution of domesticated animals, they can serve as reservoirs of infectious disease for wild mammals. It is thought that the close phylogenetic relationships with domesticated mammals increases the risk of cross-species transmission to related wildlife species and may be a key factor in parasite-mediated declines. 96% of all parasites listed as threatening wild mammals were also reported to infect domesticated carnivores or livestock, with close contact being the most common mode of parasite transmission (75%).

Examples of parasite-driven population declines:
1) Canine distemper virus in Serengeti lions, black-footed ferrets, African wild-dogs

Family: Paramyxoviridae, genus: Morbillivirus; (-) ssRNA
Can be spread through the air and through contact with infected bodily fluids, including food and water contaminated with these fluids
Symptoms: fever, respiratory problems, encephalitis, thickened footpads

2) Rinderpest virus in African bovids

Family: Paramyxoviridae, genus: Morbillivirus; (-) ssRNA
Spread mainly by direct contact and by drinking contaminated water; can also be transmitted by air
Symptoms: fever, nasal and eye discharges, irregular erosions in the mouth, the lining of the nose, and the genital tract

3) Phocine distemper virus in harbor seals

Family: Paramyxoviridae, genus: Morbillivirus; (-) ssRNA
Spreads through close contact by inhalation of the virus, or through contact with secretions such as mucus or feces
Symptoms: labored breathing, fever, problems with nervous system

4.) Rabies virus in African wild-dogs

Family: Rhabdoviridae, genus: Lyssavirus; (-) ssRNA
Usually spread by a bite from an infected animal
Symptoms: encephalitis, slight or partial paralysis, cerebral dysfunction, production of large quantities of saliva coupled with an inability to swallow

5.) Ebola virus in African apes

Family: Filoviridae; (-) ssRNA
Spread by direct contact with infected bodily fluids
Symptoms: high fever, severe weakness, organ damage due to disseminated systemic necrosis, hemorrhaging

Prevention strategies:
1) Vaccination of domesticated animal reservoirs

Previous campaign saw the decline of rinderpest virus in wild African bovids after widespread vaccination of domesticated cattle
Current campaign in the Serengeti ecosystem will immunize domesticated dogs against rabies, canine distemper, and parvovirus to reduce disease outbreaks in lions, African wild dogs, and bat-eared foxes

2) Minimizing close contact between wild and domesticated mammals

Using physical barriers like fences
Limiting temporal overlap in the use of water resources and grazing habitats

References:
1.) Pedersen, A. B., K. E. Jones, C. L. Nunn, and S. Altizer. 2007. Infectious diseases and extinction risk in wild mammals. Conservation Biology 21:1269-1279.
2.) http://en.wikipedia.org/wiki/RNA_virus
3.) http://www.marvistavet.com/html/body_canine_distemper.html
4.) http://en.wikipedia.org/wiki/Canine_distemper_virus
5.) http://www.iucn.org/en/news/archive/2007/09/12_pr_redlist.htm
6.) http://en.wikipedia.org/wiki/Rabies
7.) http://en.wikipedia.org/wiki/Phocine_distemper_virus
8.) http://en.wikipedia.org/wiki/Ebola
9.) http://www.defra.gov.uk/wildlife-countryside/ewd/seals/pdv.htm
10.) http://biomarker.cdc.go.kr:8080/pathogen/pathogen_view_en.jsp?pclass=2&id=44
11.) http://www.nature.com/nature/journal/v418/n6900/images/418810a-i1.0.jpg 12.) Daszak, P., A. A. Cunningham, and A. D. Hyatt. 2000. Emerging infectious diseases of wildlife—threats to biodiversity and human health. Science 287:443–449.

Kroeker: Polyomavirus facts

jonkro4 — Wed, 05 Mar 2008 21:40:47 CST

Polyomavirus in Human Merkel Cell Carcinoma

Oral presentation: Kremer, Hayden
Wiki presentation: Kroeker, Jon

Introduction
Family: Polyomaviridae
dsDNA, 5387 base pairs, circular genome
small (40-50nm diameter), icosahedral shape, no lipoprotein envelope
oncogenic (tumor-causing) polyoma means multiple (poly-) tumors (-oma)

Merkel Cell Carcinoma
- Rare but aggressive human skin cancer that typically affects the elderly and immunosuppressed (a feature that was suggestive of an infectious origin)
- Merkel Cell Polyomavirus was found in 8/10 Merkel Cell Carcinomas (5/59 control tissues and 4/25 skin control samples contained MCV)

History
- Discovered by a husband and wife team by analyzing nearly 400,000 mRNA sequences from four samples of tumor tissue using a technique called digital transcriptome subtraction
Pathogenicity

Transmission
- Prior to genome replication, the processes of viral attachment, entry and uncoating occur
- Cellular receptors are unknown but attachment is mediated by viral protien 1 (VP1) (proven by preventing attachment with anit-VP1 antibodies)
- Endocytosed and transported to nucleus where they replicate (able to use host machinery)
- Have two distinct phases, early (non-structural proteins are formed) and late gene expression (structural proteins are formed)
- Exit by budding out or when the cell lyses due to cytotoxicity of viruses in infected cell

Pathogenicity
- Large T-antigen regulates viral life cycle. To do this must modulate cell signalling pathways in host cell. To do this large T-antigen inhibits tumor supressing genes p53 and members of retinoblastoma (pRB) family. Also stimulates growth pathways by binding cellular DNA, ATPase-helicase, DNA polymerase α association, and binding of transcription factors (this causes oncogenic transformation)
- Small T-antigen activates several cellular pathways which stimulate cell proliferation such as mitogen-activated protein kinase (MAPK) pathway and stress-activated protein kinase (SAPK) pathway

Diagnostic Tests
- Use a hameagglutination inhibition to find if there are any corresponding antibodies to virus to see if there has been a past infection
- Use PCR to amplify polyomavirus DNA- used for detection and to determine sub-type
- Urine cytology: virus causes shedding of infected cells in urinary tract; the urine will contain virions or viral DNA
- Quantification of the viral load in both urine and blood: PCR
- Renal biopsy: renal cells are examined under light microscope for virus inclusion of nucleus, also cell lysis and virus partials in extra cellular fluid. Also measured by PCR

Treatment
- Same as those used to treat other skin cancers

References
Martyn K. Whitea, Jennifer Gordona, Krzysztof Reissa, Luis Del Vallea, Sidney Croula, Antonio Giordanob, Armine Darbinyana, Kamel Khalili, Human polyomaviruses and brain tumors. Brain Research Reviews 50 (2005) 69 – 85
Kelley WL, The Tyt common exon of simian virus 40, JC, and BK polyomavirus T antigens can functionally replace the J-domain of the Escherichia coli DnaJ molecular chaperone. Biochemistry Vol. 94, pp. 3679–3684, April 1997
Cinthia B. Drachenberg, Hans H. Hirsch , Emilio Ramos, John C. Papadimitriou, Polyomavirus disease in renal transplantation Review of pathological findings and diagnostic method, Human Pathology (2005) 36, 1245– 1255
Nickeleit V, Hirsch HH, Binet IF, et al. Polyomavirus infection of renal allograft recipients: from latent infection to manifest disease. J Am Soc Nephrol 1999;10:1080 Randhawa
PS, Vats A, Zygmunt D, et al. Quantification of viral DNA in renal allograft tissue from patients with BK virus nephropathy. Transplantation 2002;74:485- 8.
Allander T, Andreasson K, Gupta S, et al (2007). Identification of a third human polyomavirus . J. Virol. 81 (8): 4130–6. doi:10.1128/JVI.00028-07. PMID 17287263. Retrieved on 2008-01-18.
Feng et al., Clonal Integration of a Polyomavirus in Human Merkel Cell Carcinoma. Science 17 January 2008

Orr:Centrifugation Techniques

cupton — Fri, 29 Feb 2008 10:41:22 CST

Centrifugation Techniques

Oral presentation: Oosten, Heather
Wiki presentation: Orr, Lindsay

Centrifugation Techniques
Equilibrium Density Gradient Centrifugation

Sedimentation Centrifugation
By Heather Oosten and Lindsay Orr
Centrifugation
Centrifugation is the process of separating material by a centrifugal force: GRAVITY
Amount of acceleration applied to the sample in the centrifuge is given in multiples of g (g=standard acceleration at the earth’s surface due to gravity). This is a way of standardizing between different centrifuges that have variable diameter size. (Two machines, with the same rpm, will result in different acceleration, if they have a difference in diameter).
g is a product of angular velocity and radius; therefore, it is related to rpm.
Equilibrium Density Gradient Centrifugation aka isopycnography
Used to separate particles of different intrinsic densities. Called a "static" system: the experimental objects come to rest at points in the tube at which they are in density equilibrium with the surrounding solvent at that point

Step 1
Make a gradient in a centrifuge tube. You can use sucrose, cesium chloride, or cesium sulfate just to name a few solutes – there are different ways of creating a gradient.
The solvent at the bottom of the tube must have a density greater than any of the experimental objects in the tube

Step 2
The particles are spun ~30,000 rpm for days until they reach their density equilibria creating a band. According to their density, the particles migrate to their isopycnotic points where they float in equilibrium.

Step 3
Remove the band suspected to be the one you are looking for by collection
Sediment Centrifugation
Definition of sedimentation: "process by which particles suspended in fluid settle to the bottom"

This is a technique that separates particles on the basis of their effective size. It is
a dynamic situation (particles start at the top, and continuously settle through a gradient)

Step 1
Choose a solute that is low in density but high in viscosity eg. Sucrose
And add the experimental particles to the top of the gradient in the centrifuge tube

Step 2
Spin the centrifuge tube at ~20 000 rpm, for up to 20 hrs
Note: If spun for too long, all particles will eventually settle at the bottom, and will no longer be separated.

Step 3

If spun correctly, particles will separate to different density zones along the gradient.
Position depends upon particle size, shape, and density.

Sediment Centrifugation: Theoretical Basis

A high Surface:Mass ratio results in slower settlement rates, due to increasing drag
The shape of a particle will also influence drag
Particles will settle through the gradient at rates proportional to the square root of their molecular weights (S^2/s^2 = MW/mw).
The Svedberg unit, S, is a unit of time (10^-13 seconds) You can compare the relative settlement rates of objects differing only in size; in other words, it predicts the "behavior" of a given particle put in the centrfuge. This is valuable in order to determine how long to spin your molecules, how fast, and at what level in the gradient you should collect your desired particle.
Bigger particles have a bigger Svedberg unit.
References
http://www.science-projects.com/GradCent.htm
http://pubs.acs.org/cgi-bin/abstract.cgi/jacsat/1951/73/i04/f-pdf/f_ja01148a508.pdf?sessid=6006l3 http://www.steve.gb.com/images/science/density_gradient_er.png http://abacus.bates.edu/~ganderso/biology/resources/centrifugation.html http://homepage.usask.ca/~paa151/centrifugation.htm

Wolfe: Biologically Contained Ebola Virus

DaveWolfe — Thu, 21 Feb 2008 00:01:44 CST

Generation of biologically contained Ebola viruses Peter Halfmann*, Jin Hyun Kim*, Hideki Ebihara†‡, Takeshi Noda†, Gabriele Neumann*, Heinz Feldmann‡, and Yoshihiro Kawaoka

Problems working with wild type Ebola virus

Extremely infectious filovirus (potential biological weapon)
Highly lethal (Zaire strain ~90% fatal)
Analysis must be preformed within level 4 biosafety labs (BSL-4)
Not able to work with complete virus which may effect research results
Expense of BSL-4 laboratory and associated lab work

Purpose of biologically contained Ebola Virus

To safely study the Ebola virus life cycle, morphology, and growth properties using virus which highly resembles the wild-type, but lacks a key transcriptional factor. Allowing research outside of a BSL-4 laboratory on potential anti-viral drugs and vaccine development.

Ebola Genome

Nonsegmented, negative-sense RNA genome ~19kb in length Encodes seven structural proteins

NP – Nucleoprotein

Viral Protein 35 (VP35) – RNA replication machinery component

Viral Protein 30 (VP30) – essential transcription factor (activator)

L – RNA-dependant RNA polymerase

Viral Protein 40 (VP40) – matrix protein critical to viral budding

Viral Protein 24 (VP24) – secondary matrix protein involved in nucleocapsid formation

GP – Surface glycoprotein involved in attachment and entry

Figure 1: Diagram of Ebola virus and some of the genomic structural protein subunits

Methods for producing biologically contained Ebola Virus

cDNA clone of Zaire Ebola virus created
replace ORF for VP30 with neomycin (neo) through PCR
insert into full-length Ebola cDNA construct through unique SalI and SacI restriction sites (See Figure 2 below)
- New contruct named EbolaΔVP30-neo virus

Figure 2: Schematic diagram of EbolaΔVP30 constructs

Amplification of EbolaΔVP30-neo virus

Establish stable vero E6 cell line (VeroVP30)
- Cotransfectting Vero cells with two protein expression plasmids
  - VP30 (pCAG-VP30)
  - Puromycin (pPur)
- VP30 expression in clones determined through flow cytometry using antibodies for VP30

Figure 3: Characterization of EbolaΔVP30-neo virus. Expression of Ebola Virus antigens within infected VeroVP30 cells. Confluent VeroVP30 cells (Left) or wildtype Vero cells (Right) were infected with EbolaΔVP30-neo. Seven days later, cells were fixed and an immunostaining assay with an antibody to Ebola Virus VP40 protein was performed. The formation of plaques in the VeroVP30 cell monolayer (Left), but not in monolayers of wild-type Vero cells (Right), illustrates that EbolaΔVP30-neo virus is biologically contained.

Figure 4: Transmission electron microscopy (TEM) image for the morphology of Ebola Viruses budding from infected cells. Vero cells infected with wild type Ebola Virus (Left) and VeroVP30 cells infected with EbolaΔVP30-neo virus(Right). No significant differences in morphology or efficiency of budding between wild-type Ebola virus and EbolaΔVP30-neo virus.

EbolaΔVP30-neo virus grows in VeroVP30 cells but not in wild type Vero cells. Indicates genetic stability with no recombination, while expressing similar morphology and growth rates to wild type Ebola virus.This indicates the virus is biologically contained to cells expressing VP30 and genetically stable (See figure 5 below).

Figure 5: Replication kinetics of wild-type Ebola virus and EbolaΔVP30-neo virus. VeroVP30 cells (Upper) and wild-type Vero cells (Lower) were infected with Ebola virus or EbolaΔVP30-neo at a high multiplicity of infection (m.o.i.) of 1.0 (Left) or a low m.o.i. of 0.01 (Right). Supernatants were harvested every 24 h after infection for 6 days. Viral titers of the respective viruses were determined by infecting confluent VeroVP30 cells or wild-type Vero cells with 10-fold dilutions of the supernatants and subsequent immunostaining. Virus titers for EbolaΔVP30-neo virus (filled squares) and wild-type Ebola virus (open circles) were comparable in VeroVP30 cells (Upper). In wild-type Vero cells (Lower), no replication was detected for EbolaΔVP30-neo virus (filled squares).

Conclusions:

Production of EbolaΔVP30-neo virus is a viable method for obtaining biologically contained Ebola virus. EbolaΔVP30-neo is genomically stable, replicates to high titre levels comparable to wild type, and has indistinguishable cellular morphology.

References:

Halfmann, P, Hyun Kim, J, Ebihara, H, Noda, T, Neumann, G, Feldmann, H, and Kawaoka, Y. (2008). Generation of biologically contained Ebola viruses.PNAS 105:4 p.1129-1133.

http://www.pnas.org/cgi/content/abstract/105/4/1129

http://jvi.asm.org/cgi/content/full/77/5/3334

http://webs.wichita.edu/mschneegurt/biol103/lecture17/Ebola_EM.jpg

http://biomarker.cdc.go.kr:8080/pathogen/pathogen_view_en.jsp?pclass=2&id=44

http://www.nature.com/nm/journal/v6/n12/full/nm1200_1322a.html

The Ebola Virus

ermurphy — Wed, 20 Feb 2008 04:05:08 CST

Oral presentation: Moore, Eric
Wiki presentation: Murphy, Lisa

1. Introduction

Filoviridae family, very similar to Marburg viruses

~19kb Negative-Sense RNA Genome

Length: ~1000nm, diameter: ~80nm

Enveloped filamentous nucleocapsid

Level IV Pathogen

5confirmed strains:

3 have caused disease in humans
New species recently discovered in Uganda outbreak

2. Pathogenicity

7 gene products.

Single viral glycoprotein suspected as cause of observed cytotoxicity

Deceased patients have high levels of inflammatory cytokines, including:

IFN-γ, IFN-α, IL-2, IL-10, TNF- α

Transmission:

direct contact with blood, secretions, organs, etc. of infected individual or animal

Symptoms and Diagnosis:

After Incubation period of 2-21 days

Sudden fever, aching, weakness followed by vomiting, diarrhoea, rash, loss of kidney and liver function

Can ultimately lead to internal and external bleeding

Strain diagnosis requires antigen or RNA detection

Treatment:

Mainly for dehydration
No vaccines or antivirals exist although both in the works

3. Outbreaks

Human:

First

1976 - Sudan/Zaire (now the Democratic Republic of the Congo, or DRC)

Named after the Ebola River Valley in Zaire

318 infected

88% death rate

More recently:

August 2007 - DRC

373 infected

45% death rate

November 2007 - Uganda

93 infected

24% death rate

Natural outbreaks concentrated to central Africa:Republic of Congo, Gabon, Côte d'Ivoire,DRC, Sudan, Uganda

Small Accidental Outbreaks in Asia, Europe and N.A.

Death rates of 25-90% of infected (textbook), 50-90% of clinically ill (WHO)

Total infected/total killed as of June 2005: 1880/1301 (~70%)

Other Primates:

Outbreaks have been devastating for African primates!

Est. 5000 gorillas died between 2002-2003 in Gabon and RC!
90-95% death rate

Natural Host?

WHO and CDC websites still say it is unknown
Initially evidence primarily epidemiological
Increasing evidence points to 3 species of fruit bats as possible carriers:

Hypsignathus monstrosus, Epomops franqueti, Myonycteris torquata

Recent massive capture studies found bats had:

Anti-Ebola IgG

Viral RNAdetected in kidney and liver
None contained both (RNA and antibody)

Bats compete for food with apes then eaten by humans in the affected areas.

4. References:

http://en.wikipedia.org/wiki/Ebola

Principles of Virology: Molecular Biology, Pathogenesis, and Control of Animal Viruses, 2nd Edition. Flint, S.J., Enquist, L.W., Ranciello, V.R., Skalka, A.M. ASM Press (2003). pp. 369,850, 862.

http://www.who.int/csr/disease/ebola/en/

http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/ebola.htmEbola

Yang, Z-y., Duckers, H.J., Sullivan, N.J., Sanchez, A., Nable, E.G., Nable, G.J. Identification of Ebola virus glycoprotein as main viral determinant of vascular cell cytoxicity and injury. Nature Medicine, 6(2000) p.886-889.

Villinger, F., Rollin,P.E., Brar, S.S., Chikkala, N.F., Winter, J., Sundstrom, J.B., Swanepoel, R., Ansari, A.A., Peters, C.J. Markedly elevated levels of interferon (IFN)-gamma, INF-alpha, interleukin(IL)-2, IL-10, and tumor necorsis factor-alpha associated with fatal Ebola virus infection. Journal of Infectious Diseases. 179(1999) p.188-191.

http://www.cdc.gov/ncidod/dvrd/spb/mnpages/dispages/ebola/ebolatable.htm

Bermejo, M., Rodriguez-Teijero, J.D., Illera, G., Barroso, A., Vila, C., Walsh, P.D. Ebola Outbreak Killed 5000 Gorillas. Science 314(2006) p.1564.

Pourrut, X., Kumulungui, B., Wittmann, T., Moussavou, G., Délicat, A., Yana, P., Nkoghe, D., Gonzalez, J-P., Leroy, E.M.. The natural history of Ebola virus in Africa. Microbes and Infection 7(2005) p.1005-1014.

Leroy, E.M., Kumulungui, B., Pourrut, X., Rouquet, P., Hassanin, A., Yana, P., Délicat, A., Pawesk, J.T., Gonzalez, J-P., Swanepoel, R. Fruit bats as resevoirs of Ebola virus. Nature, 438(2005) p.575- 576.

http://noorderlicht.vpro.nl/artikelen/25193751/

Orr Centrifugation

cupton — Tue, 19 Feb 2008 18:22:42 CST

Centrifugation Techniques

Oral presentation: Oosten, Heather
Wiki presentation: Orr, Lindsay

Kulai: Gene Therapy

tkulai — Mon, 11 Feb 2008 02:39:00 CST

Gene Therapy

Oral presentation: Ehman, Dylan
Wiki presentation: Kulai, Tasha

What is gene therapy?

Inserting genetic data into cells, thereby modifying them
Typically, "normal" gene inserted to replace nonfunctional gene

Prevent or treat genetic diseases such as Severe Combined Immunodeficiency (SCID) and hemophilia

The basic idea

Target cell has defective gene, resulting in a nonfunctional protein and a disease phenotype.
Take a virus and remove its genome.
Isolate or construct the functional gene in vitro.
Encapsidate the therapeutic gene in the chosen viral vector.
Infect defective cells with therapeutic virus.
Viral vector inserts therapeutic gene into cell.
Cell expresses functional gene and disease alleviated.

You can see a video of this process here.

Approaches to modify defective genes

Normal gene randomly inserted into genome to replace defective gene
Homologous recombination to replace defective gene with normal gene
Modify gene regulation

Possible vectors

Viruses – built in machinery for gene delivery
Naked DNA & electroporation
Liposomes & Micelles
Virosomes – liposomes with viral membrane proteins

Types of viral vectors

A. Retroviruses

Integrate genes directly into host chromosome
Introduced genes passed on through replication
Integration is random so may disrupt normal function, can cause cancer

B. Herpes Simplex

Specifically targets nervous system
Possible cancer and Parkinson's treatments

C. Adenoviruses

Introduced gene is free in nucleus – not passed on
Requires repeat treatments as not replicated
First gene therapy cancer treatment

D. Adeno-Associated Viruses

Low immunogenicity (appears restricted to neutralizing Abs)
Integrate to specific site on chromosome

Successes

1993 – Gene therapy on stem cells used to treat SCID (bubble boy)
2002 – Sickle Cell treated in mice
2005 – Deafness cured in guinea pigs
2006 – Myeloid cell disorder (i.e., myeloid leukemia) treated
2006 – Retargeted T cells treat melanoma
2007 – Trials to treat genetic blindness
2008 – Chronic pain treated in rats

Hurdles

First trial in 1990 – few results since
Overcoming immune response
Viral toxicity
Tumor inducing – 3 of 20 patients in a study developed leukemia
Many genetic disorders arise from multiple genes
Ethics

Example: Cystic fibrosis

1 in 3900 Americans
Most common reason is deletion of a single Phe
Prevents production of channel protein, causing mucus build up
Chronic infection arises
Life expectancy: 36.8 years

Current strategies:

Defective gene has been cloned and proved able to replace absent protein
Only ~15% of protein function required for prevention of CF
Adeno-associated viruses with cloned protein introduced via aerosol to lungs show promise but still ineffective over long term
Current strategies involve non-viral vectors
Journal reviews predict viable gene therapy cure within 10 years

References

Chirmule, N, Propert, K, Magosin, S et al. (1999) Immune responses to adenovirus and adeno-associated virus in humans. Gene therapy 6: 1574-1583.

Gene Therapy. < archi-mees.de/en/archimedes_work/3_d_animationfilm/archimedes_gene_therapy.html> Archimedes Movingscience. Accessed 27/01/2008.

Gene Therapy. <ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml> Human Genome Project Information. Accessed 27/01/2008.

Griesenbach, U, Geddes, DM, and Alton, EWFW. (2004) Gene therapy for cystic fibrosis: an example for lung gene therapy. Gene therapy 11: S43-S50.

Griesenbach, U, Geddes, DM, and Alton, EWFW. (2006) Gene therapy progress and prospects: cystic fibrosis. Gene therapy 13: 1061-1067.

Targeted Gene Therapy Provides Relief For Chronic Pain, Study Shows. <sciencedaily.com/releases/2008/01/080122110054.htm> Science Daily. Accessed 27/01/2008.

Name: Martinson, Geoff Virulence may be defined as: a) the ability of a virus to cause disease b) the presence of disease causing proteins within a virus c) the severity of disease caused by a virus d) how well a virus survives in a host