virology ch. 1-5

List the steps of the viral infective cycle:

attachment; entry; uncoating; biosynthesis (transcription/translation); replication; maturation (assembly); and release/exit of a virus. Attachment: of a virion to a cell; Entry into the cell;Transcription of virus genes into the messenger RNA; Translation of virus's mRNA's into virus proteins; Genome replication; Assembly of proteins and genomes into genome virions; Exit of the virions from the cell

Helical virions have precise lengths. What determines the length of a helical virion?

length of the nucleic acid.

What are some of the protective mechanisms to viral infections in plants? How are most plant viruses delivered into a cell?

Plant are immobile and are surrounded by a thick cell (cellulose) walls; most plant viruses are carried across these barriers by vectors.

Define occlusion bodies. What is their origin? How do they protect the viral particles?

Protein crystals coating virus particle. Baculoviridae. By occluding them in protein crystals.

Be able to describe the basics of differential and density gradient centrifugation.

differential uses partial purification of virion while density gradient centrifugation uses purifications of virions.

What are the 7 types of viral genome structures that have been identified? (Fig. 3.1)

ss Linear=parovivruses; ds Linear=Poxviruses; ss Circular=Phage x174; ds circular=baculoviruses; ss, linear=tobacco mosaic virus; ds linear = reoviruses; ss circular=Hepatitis delta virus

For the following human diseases

what is the portal of entry? Influenza; HIV; Polio; Rabies; Herpes, Influenza; mucous membrane; HIV; mucous membrane and general urinary parental; Polio; fecal; Rabies; skin or parental bite; Herpes; skin

What is a Virus

a very small non-cellular parasite of cell

Even though the size of icosahedral capsids can vary widely an icosahedron has three defining characteristics that are invariable. What are these characteristics?

20 faces equilateral triangle; 12 vertices from 5 triangles; 30 edges from edge of 2 triangles; 5-fold 3-fold 2-fold axes

How do the following vectors transmit virus particles to plant tissue? Aphids; nematodes; fungi

Aphids- They feed by injecting the contents of the cell wall. Thus removing contents of the plant cells and introducing virions into the cell. Different types of nematodes transmit different shaped viruses; Nematodes- transmit viruses by piercing the root cells and ingesting their contents thus introducing the virus to the plant.; Different types of nematodes transmit different shaped viruses; Fungi: Can infect potatoes and are transmitted by virions taking development into fungal spores. These fungal spores germinate on to a new host which becomes infected not only with the fungus but the virus also.

What is the current working definition of a virus? What are differences between viruses and typical living cells? Why are viruses generally considered to be "nonliving" until they infect a host cell?

Like living organisms; viruses contain genetic instructions. However; they lack the machinery needed to carry out these instructions. On their own; they are inert chemicals and cannot perform any life functions. However; if they enter a living host cell; they can use the cell's machinery to replicate. When the viruses are outside of a host they are considered a virion. Virions are inert and can be described as non-living. Virions are not living they are waiting to infect a cell once they infect a host cell they become a living cell.

Define: provirus; vector; arbovirus; transovarial; vertical vs. horizontal transmission

Provirus: is a virus genome that is integrated into the DNA of a host cell. Vector: An organism that does not cause disease but spreads infections by conveying pathogens from one host to another. Arbovirus: The transmission of viruses from arthropods (flea,ticks,mosqiotos). Transovarial: Transmission to the next generation within the egg. Vertical transmission: A virus that can be transmitted from parent to members of the next generation. Horizontal transmission: Viruses transmitted from host to host for example kissing or sexual intercourse and viruses transmitted during vectors.

How do viruses with very small genomes efficiently adapt and replicate? What are the selective advantages and disadvantages to a segmented genome?

Smallest known: ssRNA virus: 1239 bp ;Smaller genomes are highly efficient with few gene products overlapping ORFs and multi-functionality of proteins; Advantage: Sometimes genomes are segmented among more than one virion (more common in RNA viruses including influenza) ; Disadvantages: they need efficient mechanism to ensure that each virion packages a full set of segments. For viruses with the segments packaged in separate virions at least one of each virion category must enter a cell in order to initiate an infection

Most DNA viral genomes must enter the nucleus. How is this accomplished?

by motor protein

What is a plaque and how does it form? What is a PFU? How does a quantitative plaque assay work?

plaques are clearings on a lawn of cells representing an infection that has spread from one cell to surrounding ones; it is formed from viruses. Each plaque corresponds to a single virus; can be expressed as a plaque-forming unit (PFU) ; *using cell grown in petri dishes; identified via plaques that form in the cell culture (either bacterial or eukaryotic cells)

For each of the following viruses know the basic mechanism by which the viral particle attaches to the host cell: polio; adenovirus; influenza; HIV

polio-CD155-VP1; adenovirus-; influenza-sialic acid containing glycoprotein-hemagglutinin; HIV-CD4-gp120

Know the general characteristics of a virus

such as: few (if any) enzymes; no ATP- or protein-generating mechanism; do not provide their own amino acids or nucleotides., Biotechnology: source of enzymes for use in molecular biology; vectors for gene delivery into animal cells. Obligatory intracellular parasites. Require living host cells to multiply . They have few (if any) of their own enzymes ;No ribosomes ;No ATP-generating mechanism ;mulitply inside host cells using the host's synthesizing machinery and building blocks;Do not provide their own amino acids; nucleosides or ATP

Know and understand the purposes of modifications at the ends of viral genomes including: proteins 5' methylation 3' polyA tails and tRNA-like structures.

-proteins at 5' end (may be primers for initiation of genome synthesis) -RNA genomes may have 5'-methylated nucleotide cap -RNA genomes may have a poly(A) tail at 3' end -ssRNA may have tRNA-like structure at 3' end to initiate synthesis

Review the innate and adaptive immune systems and the 3 lines of defense that a virus must evade in order to replicate in a new host.

1. Physical and Chemical Barriers (Innate Immunity) -Physical barriers: Skin mucous urine hair. Chemical barriers: Saliva Acidity Sebum gastric juice. 2. Nonspecific Resistance (Innate Immunity)-Phagocytic cells inflammation and fever. 3. Specific Resistance (Acquired Immunity)- This system relies on antigens which are specific substances found in foreign microbes. Most antigens are proteins that serve as the stimulus to produce an immune response. The term "antigen" comes from ANTI-body Generating substances.

What are the functions of the capsid in a virion? Racaniello said in the online lecture that capsids are metastable. What does this mean?

Capsid in a virion is considered protection of the genome. Metastable-protect viral genome outside cell

What are the roles of co-receptors? Discuss the example of the HIV particle receptor and co-receptor proteins.

Co receptors is a secondary surface molecule to help the virion attach to the plasma membrane. For example gp120 attaches to CD4+ receptor and CCR5 or CXCR4 co-receptors gp41 fusion protein mediates fusion of viral envelope with the plasma membrane

Know some of the defining characteristics of animal virus envelopes. Where does the envelope come from in viral particles? What type of viruses are always enveloped?

Helical and a large number if icosahedral viruses. Influenza viruses and herpesviruses.

What are the types of symmetrical structures seen in capsids? What type of capsid does the measles virus have? Influenza? HIV? Ebola?

Helix; Icosahedron; rod; cone ;Measles- Helical; influenza-Enveloped helical virus ; HIV-Rod shaped ; Ebola- helical

Understand the metric units of measurement that are used for microorganisms (especially mm; µm; nm) and be able to relate them to one another.

Herpes Virus 127nm 1.27X10^-5 cm and 1.27X10^-7 M Large Balloon 1.27 X10^9 nm, 127 cm, 1.27M Earth 1.27 X 10^16nm, 1.27 X10^9cm, 1.27 x107 M

What types of proteins are associated with viral envelopes? What are some functions of viral envelope proteins?

Integral membrane (N-or-O glycosylated). Function- fusing virion membrane to a cell membrane during infection process.

What is the definition of "Minimum Infective Dose?" What is a test that can be performed to estimate this number?

Minimum amount of virus that will cause infection in a host. The test that can be preformed is the Infective Assay a TCID50.

Are all viral nucleic acids released from the capsid/nucleocapsid during entry? How does the virion/genome behave after entry into the cell? Different genome types are sorted to different places inside the cell. What is the role of the cellular cytoskeleton in virus entry and sorting?

No; they are not some viruses contain matrix proteins that are attached to the nucleocapsid to the envelope. After entry the viral genome may have to be delivered to a particular destination such as the nucleus. The cellular skeleckton provides support and acting as tracks for the transport of materials to particular sites on the cell. Motor proteins also help with organizing movement closer to the nucleus.

Label the main features of a "One-step growth curve." How would an in vitro and in vivo one-step curve be expected to differ? Be able to label stages of a one-step viral growth curve and how each relates to the events in viral replication. How does a viral growth curve differ from a bacterial growth curve?

One-step growth curve: used to demonstrate and quantify multiplication of viruses in a host cell

What can viral whole genome sequencing reveal to researchers? Of the 4 genome types (ssDNA; dsDNA; ssRNA; dsRNA) which type tends to have the largest genome?

largest; dsDNA virus over 1000kbp

Prior to TEM viruses were identified by size as a "filterable infectious agent." What are flaws in this concept and why is this definition no longer used?

"Filterable agents" are by definition less than 0.2 µm (200 nm) so most viral particles are very tiny--less than 200 nm so were not visualized until development of transmission electron microscopy. Units for viral measurements are usually nm (10-9 m). -Known viral particles range from 20 nm to 1000 nm in diameter/length. There are viruses now days that are smaller then what they believed was very tiny.

What is the most well-studied famous "immortal" cell line? From whom were the cells obtained?

* HeLa cells (from cancer patient Henrietta Lacks) have been the primary cells used in the past 60 years

Are all virus particles infectious? Name a method used to measure infective virus particles (the "particle to PFU ratio").

* No; Quantitative assay

Define: Subunit; Capsid; Nucleocapsid; Envelope; Capsomere; Spikes/Knobs

* Nucleic acid—DNA or RNA; can be single- or double-stranded; linear or circular* Capsid—protein coat made of capsomeres (subunits) surrounding the nucleic acid* Envelope—lipid protein and carbohydrate coating on some viruses (may be "borrowed" from host cell)*Capsomere: Are built from several identical protein molecules that are clealy constructed from discrete structures.*Spikes/Knobs: projection from outer surface on some viruses made of carbohydrate/ protein.*Nucleocapsid: Protein that coats the Virus.*Subunit: capsomeres

Describe how animal viruses are cultured in the laboratory. Why are continuous/immortal cell lines of more practical use than primary cell lines? What are some of the difficulties in maintaining cell cultures in the laboratory? What often happens after several "passages" of plaque purification?

* Primary cell culture lines * Tissues are treated with enzymes to separate cells for a primary cell culture * Virally infected cells are detected via cytopathic effect (CPE) or other method and subcultured * Problem: Primary cell lines die out after a few generations * Continuous cell lines * Transformed or cancerous cells are "immortalized"

Besides the primary sequence of an RNA genome how can an RNA viral genome convey information during the infective cycle? What is secondary and tertiary structure as it refers to RNA?

-Base pairing in complementary sequences may base pair to form secondary and tertiary structures -Secondary structures may further fold into tertiary structural shapes that interact with host cell proteins transcription factors or other factors to direct viral replication. -Thus nucleic acid sequence and structure can convey information and/or control gene expression

What is the average size range of a viral particle? How does this compare to the size of the typical: eukaryotic cell; prokaryotic cell; ribosome; alpha helix of a protein?

20nm to 1000nm. Eukaryotic cell; 100-5000nm; prokaryotic cell; 10000-100000nm; Ribosome; 20nm; alpha helix; 0.15nm

What are the minimum number of subunits per icosahedral face? If capsid proteins are not larger than 20-60 kDa; how are larger virions constructed?

3; constructed from 20 faces??

Describe the ways that animal viruses attach to and enter into a host cell. Differentiate receptor-mediated endocytosis and fusion depending on the type of viral particle.

After binding to receptors animal become irreversible viruses must cross the plasma membrane to gain entry to the host cell. Many viruses such as adenovirus and vesicular stomatitis virus are taken into the cell by clathrin mediated endocytosis where molecules of clathrin accumulate on inner side of plasma membrane at the site where the virions attached. The clathrin forces bend around virion forming a pit which is pinched off to form a clathrin coated vesicle.

What does fluorescence microscopy allow researchers to do? What can fluorescent tagging (eg with GFP) enable researchers to investigate?

Allows the distribution of the protein in infected cells to be monitored. It allows the investigators to monitor the expression of hundreds of thousands of genes.

What is a complex virion? What type of virus is usually a complex virion? What type of head does a bacteriophage have?

Bacteriophage; Head: Isosahedral symmetry

What experimental methods can be used to determine a specific cell receptor for a virus?

Blockage of virus binding by monoclonal antibodies against a specific cell surface protein ; Soluble analogues of the cell receptor block virus binding; Expressing the gene for the cell receptor in resistant cells causes susceptibility to infection

How is membrane fusion in an enveloped virion regulated?

By specific glycoprotein ; Conformational changes mediated by virus binding to a receptor (HIV; herpes) and Low pH occurring after endocytosis and fusion with a lysosome (influenza)

How does a naked icosahedral virion attach to cell receptors? An enveloped virion?

Capsid protein. Glycoprotein. The plasma membrane flows around the virion, more receptors bind, and eventually the virion is completely enclosed in the membrane and pinches off as an endosome. However, part of the virus is not yet in the cytoplasm. For enveloped viruses it may occur in fusion of the virion envelope with the plasma membrane or endocytosis followed by fusion of the virion envelope with the endosome membrane.

What are two meanings of "titer"? What assays can be used to measure titer?

Concentration of infective virus in a specimen or preparation ; Concentration of antibodies against an antigen (e.g. a virus); agglutination test can measure the antibody titer

What is cytopathic effect? Provide an example. Define: inclusion body; syncytium; virion; host range

Cytopathic effects (CPE) are visible effects of viral infection in a cell; ex. bluetongue virus; Stopping cell synthesis (protein production/mitosis); Causing cell lysosomes to release enzymes; Creating inclusion bodies in the cell cytoplasm; Host Range: -The spectrum of host cells a virus can infect: -Most viruses have a vary narrow host range and can only infect a specific type of cell in one host; Virion- complete fully developed viral particleSyncytium: Multinucleated giant cell formed by the fusion of mem branes of a number of individual cells. inclusion body- are nuclear or cytoplasmic aggregates of stable substances usually proteins.

Understand the repeated sequences found at the end of genomes including Direct Terminal Repeats (DTRs) and Inverted Terminal Repeats (ITRs). In a ssRNA or ssDNA virus what happens if there are ITRs at the ends of the genome?

DTR- is the direct terminal repeat. Which the sequence that is known is repeated. ITR-Inverted terminal repeats. The ITR repeats the complementary sequence. In ssRNA and ssDNA the sequence would replicate the inverted complementary strand of the virus.

Know the contributions that each of the following people made to the understanding of viruses: Dimitri Ivanovski; Martinus Beijerink; Walter Reedp; Wendell Stanley;

Dimitri Ivanovski and Martinus Beijerinck- extracts from diseased plants was found in the liquid filtrate; Beijerinck-* Originally called "virus" (Latin for "poison") ; Walter Reed- Yellow Fever. Wendell Stanley- first isolated and visualized the Tobacco Mosaic Virus (TMV) in 1935.

List and define the primary mechanisms of transmission of viruses.

Direct transmission: This is Horizontal vs Vetical transmission where the virus can be passed by generation which is vertical and the virus can be passed by direct contact which is horizontal. Environmental transmission: Viruses can be moved over long distances in a variety of ways. Vector transmission: Where a vector acquires a virus when it feeds on an infected host and subsequently transmits the virus to one or more host. Ex Arthropods

How common are envelopes in bacteriophages? Plant viruses?

Extremely rare inBacteriophage and less common in plant viruses

Define: ID50; LD50;TCID50. What assays can be used to determine TCID50?

ID50: infectious dose for 50% of a sample population-Measures virulence of a microbe/virus ; LD50: lethal dose for 50% of a sample population -Measures potency of a toxin/virus ; TCID50: The dose that infects 50% of inoculated tissue cultures.-The quantal assays can be used to determine the TCID50

What are some features of the typical cell receptor glycoproteins? How is it similar to an immunoglobulin (Ig)?

Immunoglobulin (Ig)-like domains ; Glycoprotein cell-receptors often have Ig-like domains; Disulfide bonds stabilize these domains and in each receptor the site that the virus binds to its located in the outer domain. ;Sometimes called the "IgSF": Immunoglobulin Superfamily

Differentiate between mechanical and biological/circulative transmission of viruses via arthropod vectors.

Mechanical: arthropod carries virus on feet; body; mouthparts; or wings; Circulative: virus crosses gut wall and enters circulation eventually being secreted into the saliva before being transmitted to a new host

Be able to state some applications in medicine or biotechnology that use viruses.

Medical Applications: Anti-bacterial agents: treat bacterial infections in humans ;Oncolytic viruses: directed chemotherapy ;Genetic therapy: vectors for delivery of non-mutated gene into stem cells of patient with genetic diseases

What are the 3 main portals of entry for vertebrate/animal transmission of viruses?

Mucous membranes- (respiratory gastro-intestinal and genitourinary tracts) (#1 line of defense); Skin- (usually broken skin; lesions) (#1 line of defense); Parenteral route -(Inanimate vectors eg. needles); Deposited directly into tissues when barriers are penetrated; # 2 line of defense: inflammation; fever; phagocytes; #3 line of defense: humoral and cellular immunity

Differentiate between susceptible; permissive; and resistant host cells in the context of viral infectivity. What is the only type of cell that can intake a viral particle and replicate it? What are some of the factors that determine whether a cell will be susceptible and/or permissive?

Permissive is when a cell allows replication and a susceptible must have appropriate receptors for the virion. Resistant host cells can be permissive for viruses only if the virus can overcome its defenses. A susceptible and permissive cell is the only cell that has the requirements to for a virus to replicate. Based on if the virus has the proteins ex (transcription factors and enzymes) for the virion to replicate is susceptible while permissive only allows the virus to replicate doesn't necessarily have the correct receptors.

What is the purpose of a spike/projection/knob at capsid vertices? How many capsid vertex spikes per virion are there?

Purpose of projections (spikes): *Attachment to host cell *Delivery of viral genome into host cell*12 vertices

What are some ways that viruses replicate and are transmitted via the mosquito vector?

Some viruses replicate in one or more tissues and organs of their vectors; thus many viruses can replicate in vertebrates and invertebrates.

What is the difference between structural and non-structural proteins in a virus? What is one enzyme that an RNA virus must have as a structural protein? What about a retrovirus such as HIV?

Structural proteins are those proteins that are components of the infective virion. Some of the structural functions can include capsid (viral protection) it can help attach to the host cell essential enzymes primers for replication the ion channels in membranes and it can also interfere with the host cell immune response. A non-structural protein is synthesized by the virus in infected cell but are not considered a virion components. HIV is considered a structural virus??????

What types of bonds are generally formed between virions and cell receptors? Are these bonds reversible or irreversible? Do covalent bonds form?

The types of bonds are hydrogen bonds; van der waal forces; and ionic attractions. At this stage the attachment is reversible and the virion may deattach and covelant bonds are not formed between virions and cell receptors.

Know the basics of and purpose for each of the following applications: Transmission Electron Microscopy; Cryo-electron microscopy; X-ray crystallography; SDS-PAGE; 2d Electrophoresis; ELISA; hybridization; PCR; Real Time PCR; RT-PCR.

Transmission Electron Microscopy-Detection of Virions; Cryo-electron microscopy-Transmission of electron microscopy with computer processing of multiple images; X-ray crystallography-Virions (or other Macromolecules) are crystallized Analysis of diffraction pattern reveals relative positions of molecule/atom Shows a 3D structure; SDS-PAGE-Used in estimating the molecular weight of proteins; 2d Electrophoresis-Separation of miniviruses. The proteins were first separated by isoelectric focusing and then by SDS-Page; ELISA-Detection of viral antigens; Detects antibodies in sample; Real Time PCR-A technique used to determine the number of copies of a specific nucleic acid in a sample; Hybridization-Virus genomes or virus messengers RNAs may be detected using sequence specific DNA probes carrying appropriate labels. Some of the labels are used for antibody detection can be used to label the probes. PCR RT-PCR. - In vitro technique for amplifying the data in RNA sequence first by copying the RNA to DNA by using reverse transcriptase. The DNA is then amplified by a PCR

Uncoating must occur in order for viral genome to be released into the host cell. Where might this occur?

Uncoating occurs for an animal virus in the cytoplasm at the nuclear pore or in the nucleus.

How were viruses originally discovered? Who discovered them? Who first used the term "virus"?

extracts from diseased plants was found in the liquid filtrate. Dimitri Ivanovski and Martinus Beijerinck. Originally called "virus" (Latin for "poison") by Beijerinck

Where are fusion proteins found? What are the main features of fusion proteins? Why must the fusion peptide be hidden until a trigger and conformational change exposes it?

found in virion envelope. Some feature is Gp41 and hemagglutination which is found in measles and influenza viruses and bind strongly to red blood cells and cause them to clump. The fusion will not be able to take place unless the sequence lies hidden and to protect the energy state of the fusion protein.

Prior to tissue cell culture how were viruses traditionally propagated?

in living animal and in embryonated eggs

Understand the roles of cell receptors for viruses. What are the "normal" functions of these receptors? Why must viruses attach to receptors to enter cells

the function of these receptors includes: chemokine and growth factor recognition; cell-to-cell contact; and adhesion. A virion recognizes a highly specific portion of a host cell receptor (usually mediated by a glycoprotein);; A second surface molecule (co-receptor) may be required in order to infect the cell; Binding of the first receptor may cause a conformational change in the virion that enables binding to the co-receptor

What are two triggers that can result in the fusion/insertion sequence being exposed in a fusion protein?

the trigger is a low ph; The other trigger can be acid triggered fusion

How do viruses "survive" to the next generation?

transmission to a new host