Unit 2

Average size range

10nm- 400nm

Latent Persistent Viral Infections

Last a long time, they are able to remain without replicating in a noninfectious state. Remains in certain neurons without ability to spread or transmit to someone. Virus can be activated later on. (chickenpox to zoster/shingles)

Envelope composition

Matrix proteins, they fill region between capsid and envelope. A virus with a membrane is called an enveloped virion. Without a membrane is a non-eveloped virion or a naked virion. The envelope is acquired from a host cell.

Burst size

the # of new virions released from each lysed bacterial cell.

Burst time

the period of time required to complete the entire process (attatchment to release)

Uncoating

the removal of a viral capsid within a host cell. Some viruses are uncoated within vesicles by cellular enzymes, where others are uncoated by enzymes within the cell's cytosol.

Nucleic acid types

viral genome. Some have double stranded DNA or RNA or single stranded.

Slow Infections

(HIV) Slow to develop actual disease (AIDS). HIV infection gets transmissible agents to multiply without resulting in disease symptoms until they cause a fatal infection. Always infectious.

Chronic Infections

(hepatitus B or C) takes a long time for symptoms to display. Always infectious virions and are always being released.

Why is it so difficult to treat viral diseases?

-they have no metabolic processes of their own -A virus may choose to reside in a part of our body that our own White Blood Cells do not want to attack -it is hard to treat viral infections because virus change their makeup, they mutate every in a minute - they can generate resistance pretty easily -without killing the cell, you can not really kill the virus

Capsid morphology & composition

A capsid surrounds a virion and its nucleic acid core and it is composed of proteinaceous subunits called capsomeres

Understand how the acid-fast staining procedure works

Able to distinguish a small group of gram positive bacteria that have extra acids, waxes and glycolipids in their cell walls. Not decolorized by acid alcohol. Important for finding two important diseases: tuberculosis (bacterium= mycobacterium) and leprosy/Hanson's Disease (bacterium= M. Leprae)

understand how a capsule "negative" stain works (as done in lab)

Acid dyes are repulsed by the negative charges on the surface of cells and therefore do not stain the. The negative stains stain the background and leave cells colorless. A counterstain is added to color the cells but will leave the capsule (outside the cell) as the only unstained structure on the slide

Outline the process of Gram Staining, including: The reagents used (in order)

Aseptic techniques: Reagents in order: Crystal Violet for 30 sec (primary stain), rinse, Gram's Iodine for 60 sec (mordant), Ethyl Alcohol until run off is clear (descolorized), Safranin for 30 sec (counterstain), rinse

How would one culture Animal viruses?

Can be cultured on other animals- rats, mice rabbits etc. or in embryonated chicken eggs

How would one culture bacteriophage?

Bacteriaphage would have to be cultured inside a suitable host cell Bacteria can be maintained in either liquid cultures or on agar plates.

Membrane Fusion (animal virus entry)

Common- Membrane bound viruses: the viral envelope and the host cell membrane fuse, releasing the capsid into the cell and cytoplasm and leaving the envelope gylcoporteins as part of the cell membrane.

Understand the structural and biochemical reasons for the color of each type

Crystal violet is the primary stain and will color all cells purple- negative or postie. Gram's Iodine is a mordant which will bind to a dye and make it less soluble, after using Iodine both cells will remain purple. The ethanol acts a decolorizing agent, which breaks down the thin cell wall of Gram-negative cells, allowing the stain and mordant to be washed away; these cells are now colorless. Gram-positive cells, with their thicker cell walls, remain purple. Safranin is the counterstain and it provides a contrasting color to the primary stain. but it is masked by the darker purple already in Gram-positive cells. Gram-negative cells appear pink because safarnin passed through thin cell wall.

What are viroids?

Differ from viruses by lacking proteins. RNA does not code for proteins. Only a pathogen in plants. Resistant to proteases and nucleases. Replicates autonomously

What is similar and what is different about the way animal viruses attach to their host cells (when compared to bacteriophage)?

Difference is that there is a presence of envelopes of animal cells and they lack a cell wall. there are 3 different types of entrys based on whether its a naked virus or membrane bound virus. Uncoating of the capsid occurs after entry. similar because they have the same 5 basic steps in their replication. 1. Attatchment: animal viruses lack both tails and tail fibers, instead rely on their glycoprotein spikes or other attatchment molecules on their capsids or envelopes 2. Entry By direct penetration, membrane fusion, and endocytosis 3. Synthesis: process depends on what type of nucleic acid is involved (DNA, RNA, double stranded or single stranded, coding or noncoding stand) 4. Assmebly: DNA viruses enter nucleus while RNA viruses are replicated in cytoplasm. 5. Release: Enveloped viruses: by budding- the host cell remains intact. The viral capsid is released with cytoplasmic membrane of host, leaving as a enveloped virion. Naked viruses leave via exocytosis or lysis of host cell.

prophage (Bacteriophage)

In inactive bacteriophage (goes into lysogenic cycle). Remains inactive by coking for a protein that suppresses prophage genes.

Specialized Transduction

Only certain host sequences are transferred (along with phage DNA). -Bacterial DNA attatched -Phage + bacterial DNA packaged into new phage - Phage then transferes bacterial DNA to a new bacterium - The transferred bacterial DNA is very specific to the insertion region of the prophage - temperate phage replication -important for transferring genes encoding for certain bacterial toxins.

Describe the proposed mechanism by which prions "replicate."

Prions PrP "replicate" by changing the shape of cellular PrP to become Prion PrP in their shape. Prions do not code for new prion due to their lack of nucleic acids, instead they convert cellular PrP into prions.

Describe how prions can be destroyed/inactivated outside the body

Prions cannot be destroyed by boiling, alcohol, acid, standard autoclaving methods, or radiation.

Describe the chemical composition of prions.

Pure protein, no nucleic acid. Cellular PrP- a-helices which is normal. and Prion PrP in shape of B-pleated sheets (more flattened shape) which is disease causing. Prions cannot be killed by boiling, alcohol, acid or extreme conditions.

Direct Penetration (animal virus entry)

Rare- for naked viruses. A viral capsid attatches and sinks into the cytoplasmic membrane, creating a pore through which the genome alone enters the cell

Generalized Transduction & benefits

This is when phages mistakenly incorporate remaining fragments of bacterial DNA that are about the same length as phage DNA during synthesis. The phage with the bacterial DNA is called a transducing phage and can end up injecting its DNA into a recipient host cell. Donor DNA is incorporated into recipients chromosome by recombination. Not limited to a particular DNA sequence. Benefits bacteria because in the end the reciepient host cell is simply receiving Donor DNA of bacteria, not the genome of an actual bacteriophage.

Why viruses are not usually considered living.

Viruses are argued not to be a living organism because they lack all the characteristics of life: growth, self-reproduction, responsiveness, and the ability to metabolize all within structures called cells. Viruses require a host cell to replicate and that is why they are not considered living.

Discuss the general role of viruses in causing cancers. Name two cancer-related viruses and the specific human cancer associated with each.

Viruses have a link with protooncogenes, genes that play a role in cell division. Activity of oncogenes can cause cancer. Some viruses carry copies of oncogenes as part of their genomes, promote oncogenes already present in the host, and others interfere with normal tumor repression when they insert into repressor genes. Human Papillomaviruz (HPV) causes cervical cancer Human herpesvirus B causes Kaposi's Sarcoma (bloodvessels)

What two questions need to be considered when determining how animal viruses are synthesized inside the host cell? You don't need to know the various synthesis processes (pp. 394-398) for each type of nucleic acid, but understand why their processes can't all be the same.

Whether it is DNA or RNA ? Whether it is double stranded or single stranded?

Latency of Animal Viruses

a process in which viruses remain dormant in cells- called latent viruses or proviruses. May be prolonged for years with no viral activity, signs, or symptoms

Acute Viral Infections

comes on rather quickly with signs and symptoms. It does not last very long

Describe at least two proposed methods for treatment of prion infections (once the prion has entered the body). This comes from Pruisiner's article.

drug developers might be able to design a compound that would bind to a central pocket that could be formed by the four helices. So bound, the drug would stabilize these helices and prevent their conversion into beta sheets. Or also completely shut down the production of cellular PrPs so that no proteins can be transformed into prion PrP

Describe the steps the HIV life cycle.

1. Attatchment: Using gp120 HIV attatches to 4 kinds of cells- helper T cells, cells of macrophage lineage, muscle cells, and dendrite cells. 2. Entry: Using gp41, the viral envelope fuses with the cell's cytpplasmic membrane. And intact capsid of HIV enters the cell. Uncoats to release its ssRNA genome from capsid to cytoplasm. 3. Synthesis of DNA: Reverse transcriptase (carried in the capsid) synthesizes double-stranded DNA (dsDNA) using HIV's ssRNA as a template 4. Integration: The dsDNA made by reverse transcriptase enters the nucleus and inserts into a human chromosome. Integration accomplished by virus enzyme called integrase. Integrated virus is now know as a provirus; it remains a part of the cell for life. 5. Synthesis of RNA and polypeptides: Transcribes and replicates the integrated HIV genes to produce genomic RNA and messenger RNA. Ribosomes translate the mRNA to make viral polypeptides- attatchment proteins, integrase, reverse transcriptase, and capsomeres. 6. Release: 2 RNA, polypeptides and 2 tRNA bud from the host's cytoplasmic membrane. HIV is immature. 7. Assembly, which is completed outside of the host cell via the action of protease. It cleaves the large polypeptide to release reverse transcriptase and capsomeres- allows final maturation of virulent HIV.

Name 3 diseases caused by prions.

Fatal Familial Insomnia (human) . All cases are inherited mutations in the PrP gene . Usually strikes people age 36 to 61 . Disruption of sleep/wake cycle leads to coma, then death BSE or BoviChronic Wasting Disease (deer, elk) . Infectious disease in wild deer and elk primarily in the western United States . Drooling, difficulty swallowing, weight lossne Spongiform Encephalopathy (cattle) . Also known as "Mad Cow Disease" because infected animals act strangely and can be aggressive . Spread rapidly through Britain by rendering infected animals into cattle feed

Color of Gram-positives and Gram-negatives after each step of the process

Gram positve: after crystal violet it will be violet and will stay violet after complete staining because peptidoglycan is thick and crystal violet cannot be washed out through the mordant, decolorizer or the counterstain.

lysogenic conversion (bacteriophage)

In the lysogenic cycle when replication occurs, each daughter cell is infected withthe virus. The lysogenic phages can change the phenotype of a bacterium, from a harmless form into a pathogen.

lab reagents for Capsule Negative Stain

Mix India Ink with bacteria and spread out, air dry and heat fix. Add crystal violet to cover entire smear. Use dH20 to rinse. Blot dry.

Endocytosis (animal virus entry)

Naked viruses and membrane bound viruses: common. some viruses enter host cells by triggering endocytosis- cytoplasmic membrane of host engulfs virus. The capsid must be removed before the viruses can continue to replicate- called uncoating.

How are enveloped animal viruses released from the host cell? How are naked animal viruses released?

Release: Enveloped viruses: by budding- the host cell remains intact. The viral capsid is released with cytoplasmic membrane of host, leaving as a enveloped virion. Naked viruses leave via exocytosis (exit without acquisition of an envelope) or lysis of host cell (death of cell).

Describe the steps/diagram the lytic replication cycle in T4 phage. (Bacteriophage)

Replication cycle of virus usually results in death of lysis of the host. 1. Attatchement: of the virion to the host cell. Attatchment of virus' tail fibers and chemical attraction & fit with complementary receptor proteins on the surgace of the host's cell wall. 2. Entry of the virion or its genome into the host cell by using lysozyme (protein enzyme) to weaken the peptidoglycan of cell wall. Empty capsd is left outside of host cell. 3. Synthesis of new nucleic acids and viral proteins by the host cell's DNA, enzymes and ribosomes by degrading the bacterial DNA into its constituent nucleotides. 4. Assembly of new virions within the host cell. Spontaneous process to form mature virions. 5. Release of new virons from the host cell. Lysozyme completes work on the cell wall and disintegrates to let virions out.

Lysogenic Replication

Some bacteriophages have a modified replication cycle in which infected host cells grow and reproduce normally for many generations before thy lyse. 1. Regular attatchment 2. Regular entry but phage T4 does not take control and the host cell's DNA is not destroyed. (prophage) 3. (Lysogenic Cycle) The prophage is inserted into the DNA of the bacterial chromosome unlike in the lytic cycle. 4. Replication occurs continuously. Each time the cell replicates its infected chromosome, the prophage is also replicated. All daughter cells are infected with the virus and can remain a part of bacterial chromosomes for generations. 5. At some later time a progphage can reenter the lytic phase by recombination or some genetic event. Called induction: when the prophage is taken off from the host chromosome 6. Regular Synthesis 7. Regular Assemly 8. Regular Release.

Reagents for Acid- Fast procedure

Stain placed over boiling water to help the stain penetrate the wall of the acid-fast bacteria. Add Carbol Fuchsin (5min). All cells will stain with bright pink stain. Rinse. Decolorization performed with Acid Alcohol, which will cause carbol fuchsin to leave the cell walls of all non-acid-fast cells. The smear is Counterstained with methylene blue, this will stain the non-acid-fast bacteria a light blue color. Acid-fast bacteria will remain bright pink.

Describe the method used in lab to stain endospores, including reagents in the order used, plus the appearance of cell, endospore and background.

The Schaeffer-Fultn endospore stain uses heat to drive the primary stain, malachite green, into the endospore. After cooling, the slide is decolorized with water and counterstained with safranin. Results in green-stained endospores and red-colored vegetative cells.

induction. (Bacteriophage)

The process where a prophage is removed from the host chromosome to go back into the lytic cycle.