Infectious Diseases

Whats the difference between +ve and -Ve sence RNA

+ve: AUG binding site present at start of strand (5' end). therefoe polymerase can bind and replicate -ve: binding site at end of strand therefore cannot be translated (Polymerase 5'>3')

Classification by morphology

- structurally similar can cause very different diseases -poliovirus and norovirus (a calicivirus) have naked icosahedral symmetry but.. >Poliovirus causes poliomyelitis: fever, fatigue, headache, vomiting, stiffness in the neck, and pain in the limbs. In some cases it can also cause muscle weakness. In a small proportion of cases, the disease causes paralysis, which is often permanent. >Norovirus (Norwalk virus) causes acute gastroenteritis (vomiting, diarrhoea)

Stages of the single burst experiment/one step growth curve

-. Eclipse stage >virus particles have broken down after penetrating cells, releasing their genomes as a prerequisite to replication and therefore cannot be detected by the plaque assay >Phage takes over host biosynthesis machinery to produce phage specific mRNAs and proteins >Host protein synthesis is stopped by viral degradation of host DNA, and interference of host transcription and translation. >T4 uses host RNA polymerase to synthesise and phage early mRNA is made within two min. It also uses host nucleotides to replicate its DNA and host ribosomes, enzymes and amino acids to synthesize its enzymes and proteins. Orderly expression of phage directed macromolecular synthesis. >no complete phages are found . -Intracellular Accumulation Phase and Maturation (synthesis) > nucleic acid and structural proteins assemble and infectious phage particles accumulate within the cell. -Latent stage >time before the first new extracellular virus particles appear, ~ 20-25 min for most bacteriophages >approx 40 min after the cells were infected, the curves for the total number of virus particles and for extracellular virus merge because the infected cells have lysed and released any intracellular phage particles by this time -The yield (i.e. number) of particles produced per infected cell can be calculated from the overall rise in phage titre -Shown by plotting number of plaque-forming units (p.f.u.) per bacterial cell against time.

What are flaviviruses

->70 enveloped RNA viruses which cause serious disease in animals and human -Arthropod-borne viruses, transmitted via mosquitoes or ticks -Zika, west nile, Dengue, Hepatitis C, Bovine, diarrhea

Phage life cycle

-Adsorption & Penetration:> Specific receptors e.g. lamB >Injection >Entry via bacterial feature e.g. pilin -Transcription& Translation: >Expression of viral RNA & production of viral proteins -Replication: >Terminal redundancy >Rolling circle >Lysogeny - Assembly & Release: > Follows a pathway >Encodes agents of lysis -Divisions for convenience, all overlap -separate assembly pathways for the head & tail sections of the particle, which come together at a late stage

Phage life cycle: Absorption and penetration

-Adsorption of phage to bacterial wall >Mediated by tail fibres for T4 or by analogous structure on phages lacking tail fibres and it is reversible(weak bond) >Attach to specific receptors on the bacterial cell (host specificity determined by tail fibres on phage) >bacterial receptor varies for different bacteria: Proteins/ LPS/ Pili / Lipoprotein >Phage l uses lamB protein, M13 uses F-pilin (encoded by the F factor). -Penetration >phage T4 irreversible binding causing contraction of the sheath (hollow tail fibre pushed through bacterial envelope) >without contractile sheaths, other mechanisms (enzymes which degrade)

Phage life cycle: Assembly & Release

-Assembly of components often via sub-structures, e.g. pro-head. (maturation and accumulation) -Incorporation of nucleic acid is mostly specific (when a mistake is made bacterial DNA is packaged and a "transducing" phage is produced) -Assembly is active and can involve catalytic and bystander proteins (e.g. scaffold proteins). -Release is also active and many phage encode holins which disrupt cytoplasm membrane and lysozyme (lysin) which degrades the cell wall. Some phage (e.g. M13) extrude from the cell without causing lysis

How does the flavivirus replicate

-Asymmetric replication -RNA-dependent RNA polymerase (RdRp) NS5 copies complementary (-)RNA from genomic (+)RNA -(-)RNA serves as a template for the synthesis of new (+)RNA -Semi-conservative replication leads to 10-fold more (+)RNA than (-)RNA • template for further replication • mRNA for protein synthesis • precursors for viral ncRNA e.g. sfRNA (subgenomic flavivirus RNA) • packaged into virions -Replication takes place in ER derived vesicles - "the membranous web" >feature of flavivirus infected cells, induced by NS4, acts to hid RNA from innate immune triggers and to concentrate virus components for replication and assembly

What is the Hershey-Chase experiment

-Bacteriophage T2 was propagated in Escherichia coli cells which had been 'labelled' either >35S, incorporated into sulphur-containing amino acids in proteins >32P, incorporated into nucleic acids (which do not contain any sulphur) -Attachement allowed then homogenized briefly in a blender which (did not destroy the bacterial cells but knock the phage coats off the outside of the cells). -Analysis of the radioactive content in the cell pellets and culture supernatant (containing the empty phage coats) showed that most of the radioactivity in the 35S-labelled particles remained in the supernatant, while in the 32P-labelled particles, most of the radiolabel had entered the cells -Conclusion: The DNA genome (32P labelled) of the bacteriophage had entered the cells and initiated the infection

What are the uses of baculoviruses

-Biopesticides (not common as expensive and need to be reapplied -Expression of recombinant proteins in insect cell culture

Prevention/Treatment of flaviviruses

-Control of arthropod vectors >Insecticides (chemical & biological) >Insect repellent & mosquito nets -Vaccines >Tick borne encephalitis vaccine (inactivated) >Japanese encephalitis vaccine (inactivated) >Yellow fever vaccine (live attenuated) >Dengue vaccine available in certain countries in 2016 (but only partially effective) >vaccines for WestNileV and HCV in development -Antivirals >Rational drug design targeting non-structural proteins

What are the main ways to classify viruses

-Disease -serology -Host eg: bacteria -Morphology -Genome/replication strategy

The 'Single-Burst' Experiment or 'One-Step Growth Curve' - what is it and who conducted it

-Ellis and Delbruck (1939) -Showed 1.Initiation of infection 2.Replication and expression of the virus genome 3.Release of mature virions from the infected cell -Process >Bacteriophages were added to a culture of rapidly growing bacteria >After a few minutes, the culture was diluted, preventing further interaction between the phage particles and the cells >Repeated samples of the culture were taken at short intervals and analysed for bacterial cells by plating onto agar plates and for phage particles by plating onto lawns of bacteria >there is a stepwise increase in the concentration of phage particles with time, each increase in phage concentration representing one replicative cycle of the virus.

Picornavirus members and genera

-Enterovirus >Poliovirus >EV71 (Human enterovirus 71) >Coxsackievirus B3 -Hepatovirus >Hepatitis A virus -Cardiovirus >Enchephalomyocarditis virus (EMC) -Rhinovirus >Human Rhinovirus -Aphthovirus >Non-human viruses

Vaccines for Picornaviruses

-Foot and Mouth disease vaccines (Inactivated) -Hepatitis A vaccine (Inactivated) -Poliovirus vaccines - must contain all 3 serotypes to confer protection (PV1, PV2 & PV3) >Inactivated (Salk) vaccine (IPV) - incapable of replication >Live attenuated (Sabin) vaccine (oral polio vaccine OPV) - replicates but does not cause disease >Both effective BUT risk of circulating vaccine-derived poliovirus (cVDPV) outbreaks in under immunised populations from OPV as attenuated virus may replicate for years and acquire mutations that cause paralysis.

What allows entry of baculoviruses into cells?

-GP64 recptors present on the surface of peplomers (Enveloped surface projections)

Classification by host

-Genetically similar can infect same host but cause different disease -Polio and rhino virus >Picornaviridae familyicosahedral (+ve sense ssRNA genomes) >poliovirus causes poliomyelitis and replicates in the gut, rhinovirus causes common cold and replicates in upper respiratory tract (URT) -Some viruses infect multiple hosts (Foot and mouth = cattle, sheep, pigs)

How are bacteriophage classified

-Host range -Immunological relationship -Morphology -Enveloped/non-enveloped -Baltimore Classifaction

most common Ig Antibodies

-IgG is most common type in serum -IgA dimer is second most common type in serum, particularly important for mucosal immunity (secretions - tears, saliva, colostrum, mucus)

What are the differences in flavivirus structure when immature and mature

-Immature (intracellular) >60 nm (larger) >60 projections (rough surface) >Resistant to acidic pH >Each spike =3 (E/M dimer) " >E points away from membrane >Fusion peptide is exposed -Mature (extracellular) >50 nm (Smaller) >Smooth surface >Restructures at acidic pH >Herringbone - E/E dimer >E lays on the membrane >Fusion peptide is buried and covered by prM

What are the order of genes transcribed during baculovirus replication

-Immediate-early > Transcribed by host RNA polymerase >Do not require virus encoded transregulators for expression >Involved in establishing infection -Delayed-early (= intermediate) >Often require transregulators, produced from immediate-early gene translation >Involved in manipulating host cell and replication of virus genome -Late (6 - 24 hpi) >Transcribed by virus RNA polymerase >Transition from early to late characterised by shut-down of host DNA replication and translation >Nucleocapsids produced, budded viruses disseminate throughout host -Very late (18 - 72 hpi) >Transcribed by virus RNA polymerase >The most expressed very late genes in AcMNPV are those encoding polyhedrin and P10 >Advanced stage of infection, virions occluded in polyhedron, viral proteases liquefy the host and degrade chitinous exoskeleton, dissemination of occluded progeny

How do baculoviruses reorganise the nuclear structure

-Induction of virogenic stroma (subnuclear compartments)

Where can baculoviruses be isolated?

-Invertebrates eg: Insects, Arachnids, Crustaceans

Bactriophage λ Lysogenic Cycle.

-Lambda integrates via specific sites on the viral and E.coli chromosomes using integration enzymes encoded by the phage: -Attachment sites : >AttB (B-O-B) and AttP(P-O-P) >Int protein and HIF (host integration factor-bacteria) bind AttP >Holliday Junction formed >Intasome >integrase and Excisionase

Chronic infection example

-MI3 > infect E.coli >Forms turbid plaques (slows growth) >Circular ssDNA genome, 6.4kb, 10 genes >infects through F-pilus >uses host machinery to generate dsDNA, compatermers roll up DNA (Circulisation) allowing fast replication.

How does baculovirus display work?

-Modifying gp64 on baculovirus surface to display desired protei on its surface -Used to produce antibodies for vaccination

Bacteriophage

-Obligate parasites that multiply inside bacteria making use of some or all of the hosts biosynthesis machinery -Models for animal cell viruses, gene transfer in bacteria, medical applications (Phage typing, prophylaxsis) -20 types -3 Groups: Tpahge, Helical, Icosahedral

Two stage life cycle of baculovirus

-Occluded virus ingested by insect > Virus in OB -Polyhedra Occlusion body dissolves in gut (alkali pH), releasing nuleocaspid -nucleocaspid transported into nucleus, infecting cell > Newly formed nucleocaspid buds from nucleus into the cytoplasm and out of the host cell -Dissemination into the next cell >nucleus takes up nucleocaspid, polyhedra formed -Polyhedra released to infect more cells -insect dies from infection and disintegrates >releasing occluded virus into plant where it can infect new hosts

Lysogeny

-Phage genome integrates at specific site into host chromosome -The phage is still present in the cell as an integrated copy of the genome termed the prophage -Under certain conditions, such as UV irradiation, it becomes active and resumes a lytic growth cycle

Assembly and release of the baculovirus

-Production of budding virus and polyhedral -budded virus released during early infection via budding >Nucleocaspid gains envelope -Occlusion derived virus (ODV) is released later via cell lysis >Proteins involved in release include Cathepsin (general protease) and Chitinase (dissolves insect skeleton) -Budding virus Is not stable in environment but stable enough to infect cells in host -Occlusion derived is not released straight away to allow budding virus to infect more cells to generate more occlusion derived particles

Baculovirus replication

-Replication occurs as a combination of the rolling circle mechanism and recombination -Occurs within the nucleus -Transcription is phased (temporal control) into early, intermediate, late and very late -Encode novel DNA-dependent RNA polymerase >transcription of late and very late genes -Virus assembly and inclusion in the polyhedron also occur in the nucleus

Functions of viral proteins

-Replication of the virus genome -Packaging the genome into the capsid -Alteration of structure and/or function of host cell

Bacteriophage replication stratergies

-Rolling circle >cicular ssDNA genomes nicked by endonuckeases >free 3' end extended by DNA polymerase (5'>3') >replication continues until 1 or more copies are produced, multiple copies may result chained together as a concatemer >bacteriophage lambda, M13. -Terminal redundancy >Phage genome integrates at specific site into host chromosome The phage is still present in the cell as an integrated copy of the genome termed the prophage >Under certain conditions, such as UV irradiation, it becomes active and resumes a lytic growth cycle

picornavirus structure

-Size: 20 - 30 nm (pico = small) -Capsid symmetry: Icosahedral, T=3 -Non-enveloped -Genome: (+) sense ssRNA, non-segmented (Baltimore Group IV) >5' VPg >3' polyA >10 genes >7 - 8 kb length -Contains 5' and 3' non-translated regions (NTR's) also known as UTR's which contain highly ordered RNA structures >5' is NOT CAPPED! Small protein of 22 aa attached to 5' end - VPg >3' is polyadenylated -Single open reading frame >cleaved during translation into mature components >Divided into three sections -P1: structural proteins = capsid proteins -P2 & P3: non-structural proteins = proteins involved in replication

What does virus multiplication depend on?

-Synthesis of virus gene products eg: virus proteins -Function of viral proteins

Naked helical virus example

-Tobacco mosaic virus -Nucleic acid is encapsidated by a helical array of a single protein to form a protective shell -No known animal viruses

What is lambda repressor switch and how does it work?

-Two genes serve as the molecular switch -Lambda repressor protein (Cl): activates the lysogenic pathway -Cro protein (control of repressor operator): depending on the expression levels activates the lytic pathway -Lysogens are immune to "super infection" by more phage as they are full of repressor -environmental stimuli e.g. UV light indirectly inactivate the cI repressor (via activation of bacterial RecA protein) which activates lytic virus replication

Structure of baculoviruses

-Two phenotypes >Occlusion derived virus (ODV): virions occluded by protein bodies or crystals >Budded virus (BV): virions enveloped (from host), pleomorphic, rod-shaped,

complex viruses example

-Variola virus (smallpox) -Contain more than 100 virus encoded proteins -Wrapped by the endoplasmic reticulum to acquire two layers of membrane, not simply budding from cell surface as in simple enveloped viruses.

Types of phage life cycles

-Virulent (lytic) phage >Phage infect specific host >transcription/translation of phage proteins and replication of phage genome,assembly of progeny phage, released by lysis of the host cell. >Causes Clear plaques. >e.g. T-phage, ViI, Twort, MS2, ϕ6, ϕX174 -Chronic infection >As virulent but phage are released by extrusion from the host cell membrane (not-lethal, but slows growth). >Turbid (cloudy) plaques. >e.g. M13 and other filamentous phage -Temperate phage > lytic infection >certain conditions, lysogenic cycle. >Integrate their genome into the host chromosome, Passive replication of the phage genome (forms prophage) with bacterial genome, all bacterial daughter cells get a copy. >Environmental signals trigger expression of phage proteins and entry into lytic cycle. >e.g. Bacteriophage λ

Classification by disease

-Viruses that cause disease may be genetically very different -Respiratory tract infections: >rhinoviruses (+ve sense ssRNA) or influenza virus (-ve sense, segmented RNA) -Hepatitis: >hepatitis A virus (+ve sense ssRNA) or hepatitis B virus (circular DNA) or hepatitis C virus (+ve sense ssRNA) -non disease causing: TT virus (infects 80% healthy population, asymptomatic (ssDNA genome))

benefits of being a lysogen

-adaptive response as the choice of pathway depends on growth conditions -Good growth of host = opportunity to infect more bacteria so lytic growth is preferred -Poor growth of host = wait for better times (likely to be limited new hosts available for progeny) so lysogenic growth is preferred -Damage to host (e.g. UV) = induction of lytic growth to save bacteriophage

Classification by serology

-diagnostic identification of antibodies in blood serum -Antibodies specific to one type of virus rarely give protection against other viruses unless very closely related (Even then not always the case) -many serotypes e.g. rhinovirus

Baltimore Class 7

-dsDNA wit RNA intermediate. eg:Hepadnavirus and Hepatitis B -On maturation reverse transcriptase. -Viral caspids contain dsDNA as a partially circular genome (NOT covalently closed) -On infection, repair gapped genome followed by transcription.

Baltimore Class 3

-dsRNA -genome is split into parts (Rotaviruses have up to 12 segments per particle) -RNA polymerase produces ss(+ve) RNA which the follows central dogma. -

What can be observed in a baculovirus in a stable environment

-formation of a thick protein shell called occlusion body (OB) -No occlusion body = no cell-cell infection

How Picornavruses effect host cells

-host cell is 'manipulated' to direct high levels of virus production >50,000 UNITS PER CELL -Switching oof of host cell transcription and translation by either viral protease 2A, 3C and/or L depending on virus >Poliovirus and Rhinovirus 2A protein and FMDV L protease cleaves eIF4G (part of the CAP binding complex) (no longer bind to CAP structures) >3C cleaves TATA binding protein (TBP) to switch off transcription from RNA polymerase II (polII) promoters >In infected cells the nuclear pore complex proteins Nup153 and p62 get cleaved effecting localisation of host cell proteins

Enveloped helical virus example

-influenza virus -RNA closely associated with nucleoprotein to form helical structure (ribonucleoprotein (RNP)) -envelope contains host and virus encoded proteins that play role in virus life cycle e.g. adsorption/entry, release - genus orthomyxovirus in the family of Orthomyxoviridae. >ssRNA enveloped viruses with a helical symmetry. >Enveloped particles 80-120nm in diameter. >segmented genome, with 8 RNA fragments (7 for influenza C) >antigens present: haemagglutinin (HA), neuraminidase (NA), nucleocapsid (NA), the matrix (M) and the nucleocapsid proteins. -NP is a type-specific antigen which occurs in 3 forms, A, B and (classification of human influenza viruses). -matrix protein (M protein) surrounds the nucleocapsid and makes up 35-45% of the particle mass. -haemagglutinin (HA) is made up of 2 subunits, HA1 and HA2. HA mediates the attachment of the virus to the cellular receptor. -Neuraminidase molecules are present in lesser quantities in the envelope.

How are monoclonal antibodies produced

-mouse immunised with antigen -spleen cells extracted and fused with immortal B-cell line forming hybridomas -grown and tested for antibody specifity -mass production of antibody

What determines the activation of lytic and lysogenic cycle

-nutritional state of the host cells. -mediated by HfI protease. -At high levels lysis occurs preferentially, cro dominates repressor site -At low levels, associated with a poor medium, lysogeny is established. -Lysogeny is then maintained by CI (lambda repressor) protein, repressing transcription of cro and PR and activates Cl from PRM

How do viruses obtain there envelope

-obtain their envelope by budding through a host cell membrane -virus encoded proteins inserted into the membrane important in adsorption (via host cell receptor) -envelope makes the virus sensitive to detergents

What is the role of P35 in the baculovirus lifecycle

-produced by virus to block apoptotic response of insect cells -triggered by virus DNA replication by inhibiting activation of effector caspases

Why are phage good for lab study compared to viruses

-safe to handle -rapid life cycle -clear phenotype (not too complex) -minimal equipment

Baltimore class 4

-ss(+)RNA -Host cell ribosomes translate it into proteins, proteins produce dsRNA and the RNA polymerase produces the ss(+)RNA -Picornaviridae (e.g. poliovirus, rhinovirus, hepatitis A virus), Caliciviridae (e.g. Norwalk virus / Norovirus), Flaviviridae (e.g. hepatitis C virus), Togaviridae (e.g. rubella virus), Coronaviridae (e.g. SARS) -an be subdivided into two groups: >(a) Viruses with polycistronic mRNA: the genome RNA forms the mRNA and is translated to form a polyprotein product, which is subsequently cleaved to form the mature proteins. >(b) Viruses with complex transcription, for which two rounds of translation (e.g., togavirus) or subgenomic RNAs (e.g., tobamovirus) are necessary to produce the genomic RNA.

Baltimore Class 6

-ss(+)RNA with DNA intermediate. -ss(+)RNA to ss(-)DNA by reverse transcriptase and then to dsDNA. -then produces mRNA to form viral proteins which then produce ss(+)RNA from dsDNA. -integrate DNA into host chromosome every time host cell divides, all progeny cells get a copy of the virus genome (similar to bacteriophage lysogenic cycle) -Human genome is ~8% retroviral DNA! -Human immunodeficiency virus (HIV)

Baltimore Class 5

-ss(-)RNA -Segmented genomes eg: influenza or non segmented eg: measles, encode RdRp -ss(-)RNA to ss(+)RNA which is spliced to mRNA, -Then translated into proteins which help synthesis and package ss(-)RNA. -Genome replication may occur in nucleus or cytoplasm Includes Orthomyxoviridae (e.g. influenza virus), Paramyxoviridae (e.g. measles virus, mumps virus), Rhabdoviridae (e.g. rabies virus) >(a) Nonsegmented genomes (order Mononegvirales), for which the first step in replication is transcription of the (-)sense RNA genome by the virion RNA-dependent RNA polymerase to produce monocistronic mRNAs, which also serve as the template for subsequent genome replication. (Note: Some of these viruses also have an ambisense organization.) >(b) Segmented genomes (Orthomyxoviridae), for which replication occurs in the nucleus, with monocistronic mRNAs for each of the virus genes produced by the virus transcriptase

Baltimore class 2

-ssDNA viruses -Linear, circular or circular multicomponent -Conversation of ssDNA to dsDNa then follows central dogma -Replication occurs in the nucleus, involving the formation of a double-stranded intermediate which serves as a template for the synthesis of single-stranded progeny DNA -Parvoviridae e.g. parvovirus B19 "slapped cheek syndrome" in children, infections in pregnant women can lead to foetal heart failure -Microviridae group of bacteriophage

Three main morphologies of viruses

1. Helical -Tobacco mosaic virus, Bacteriophage M13, Rhabdovirus -may be enveloped so harder to identify 2. Icosahedral - Poliovirus, picornavirus -2/3/5 fold axis 3.Complex -mimivirus, poxvirus

Three characteristics which differ viruses from obligate intracellular parasites

1. No ribosomes ( Uses host) 2.One nucleic acid type 3. Multiply by independent synthesis (synthesis constituent parts which are then assembled to form the new virus particle (Rather than growth and division)).

Stages in virus multiplication

1.Adsorption 2.Penetration 3.Synthesis of virus proteins and replication of genome 4.Maturation (assembly) 5. Release

Standard virus components

1.Must encode or obtain some mechanism of duplicating its genome >Polymerase (RNA or DNA copying enzyme) >Polymerase cofactor(s) - other proteins that help the polymerase find and copy only the virus genome >Nucleoprotein (RNA or DNA binding protein) 2. encode proteins that allow the virus to be released from the cell and transmitted to another cell >Capsid proteins (components of the viral coat) >Assembly factors >Host receptor binding and entry protein 3. encode strings of proteins which can be separated as needed >Proteinase(s) - Protein-cutting enzymes

5 types of Virus morphology

1.Naked icosahedral e.g. poliovirus, adenovirus, hepatitis A virus 2.Naked helical e.g. tobacco mosaic virus, M13 (so far no animal viruses known) 3.Enveloped icosahedral e.g. herpes virus, rubella virus, yellow fever virus 4.Enveloped helical e.g. measles virus, mumps virus, rabies virus, influenza virus 5.Complex e.g. poxvirus

Approximately how many viruses are there and whtas the majority typw

10^31 Bacteriophage

What type of symmetry do icosahedral viruses have?

5:3:2 fold symmetry (Pentagon, triangle, Rhombus)

How does a flavivirus escape from an endosome

>E glycoprotein is a class II fusion protein on the viruses external surface of E protein >is a flat elongated dimer >The E protein has a "spring-loaded" conformation >at neutral extracellular pH the M protein latches on to E to prevent it adopting an open conformation >at acidic pH inside endosome, dimers dissociate and monomers interact with host membrane, then form trimers fusing the host and virus membranes to form a pore >C protein surrounding genome is released and enters into cell

Temporal control of gene expression during phage cycle

>Early gene products prepare the host cell to be exploited by the virus and/or activate intermediate genes >Intermediate gene products ensure that viral replication is preferred over cellular >Late gene products are usually viral structural proteins -ensures that the correct component is present for the relative stage of the life cycle.

Phage life cycle: Transcription, Translation & Replication

>Host protein synthesis stoped >T4 uses host RNA polymerase to synthesise phage early mRNA (two min) >Uses host nucleotides to replicate its DNA and host ribosomes, enzymes and amino acids to synthesize its enzymes and proteins. >Early mRNAs (synthesis) followed by late mRNAs (structural) >Viral gene expression follows an orderly sequence because of modifications of the RNA polymerase. Synthesis of T4 DNA occurs (NO Complete phages found = eclipse period)

what are Viruses

>Obligate intracellular pathogens, incapable of replication outside host cell >Consist of nucleic acid (either DNA or RNA) associated with proteins encoded by the nucleic acid

What is protein eiF4G

>Part of the Cap binding complex >Required to recruit small ribosomal subunit to 5' end of mRNAs >Cleaved form no longer functions - therefore host cell mRNA translation is severely affected. >Shown on gel electrophoresis >Cleaved form of eIF4G is able to function for IRES-dependent translation of viral genome.

Naked icosahedral virus examples

>poliovirus / rhinovirus -capsid composed of 60 copies each of 3 protein subunits (VP1 - VP3), with VP4 internal >caliciviruses -capsid is composed of 180 copies of a single protein (VP1)

What are the conditions to sterilise mist equipment and solutions

Autoclave at 15psi for 15 minutes

Difference between ELISA and Western Blot

ELISA: secondary antibody binds to primary antibody and possesses enzyme which induces a reaction (Horse radish peroxidase changes substrate clear>yellow) Western Blot: Direct binding to antigen or binding of secondary antibody, either posses detector molecule (radioactive, fluorescent)

Enveloped icosahedral virus examples

Herpesvirus Rubella virus

How is picornavirus infection characterised?

Infection is characterized by a rapid host cell 'SHUTOFF' due to cleavage of the cellular protein eIF-4G

Equation for multiplicity of infection

MoI = Plaque forming units (Virons) / Host cell colony forming units (host cells)

What is an Epitope

Sequence/structure on outside of virus which recognises antigens

Picornavirus life cycle

Step 1: Entry 1.Attachment >Receptor binding, affects host specificity > expression or absence of a particular receptor on the surface of a host cell largely determines the tropism of a virus -Endocytosis >Receptor-mediated endocytosis of virus particles into endosomes >Relies on normal formation and internalisation of coated pits >Endosomes fuse with lysosomes - acidification -Uncoating >Conformational change in capsid structure due to receptor interaction and/or acidic environment in endosome >Exposes hydrophobic domains, causes pore to form >vRNA genome enters cytoplasm. 2. Step 2: Translation: >Genome encodes a single polyprotein which after translation is cleaved into the individual proteins >Doesn't have 5' but has internal ribosome entry site (IRES) which bind factors involved in RNA splicing/RNA metabolism >Leads to ribosome recruitment and then translation >Enterovirus & hepatovirus bind IRES group IV and then scan for AUG before translation can occur whilst cardiovirus and aphtovirus undergo initiation on IRES directly on AUG. 3. Cleavage of Polyprotein >The viral protease 2A cleaves the P1 structural region away from the non-structural region >3C protease then cleaves the rest >Precursor proteins play an important role in replication as they often have very different properties to the mature proteins 4. Replication -Replication occurs on membranous vesicles induced in the host cell cytoplasm by viral 2BC protein -VPg is cleaved from genome by host protein > acts as mRNA leading to synthesis of viral proteins -3D polymerase binds VPg -3D polymerase binds RNA loop in genome containing Poly A tract -3D polymerase adds two rUTP bases to VPg -VPg-U-U acts as a primer to initiate both (-) strand replication then (+) strand transcription -newly synthesised RNA's are encapsidated into viral capsids 5. Virus assembly -The capsid is assembled by cleavage of the P1 polyprotein precursor into a protomer consisting of VP0,3,1 -thses join together as first pentamer (x5) and the x12 of these form proviron, enclosing the genome: -cleavage of Vp0 to VP2 + VP4 is final step in mature particle. -Empty caspids are defect of this process. 6. Release -Lysis of infected cell -6hr after infection -10,000 virus particles

Equation for number of virons before dilution

Virons(pfu)/ml = (plaque forming units/dilution factor) x volume plated (ml)

Lysogenic state definition

becoming established as a prophage in the host cell.

Temperate bacteriophage definition

capable of establishing a lysogenic state in a susceptible bacterial host.

Baltimore Class 1

dsDNA -Sub divided >Replicate in host cell nucleus (Host DNA polymerase) eg adenoviridae >cytoplasm (Own DNA polymerase) eg: Poxviridae 1). Virus enters cell, viral mRNA's produced in host by DNA polymerase 2).mRNA translated into proteins by the ribosomes 3).Proteins then replicate viral DNA (Using DNA polymerase from host or their own eg: poxiviruses 4).New copies are packaged forming virons

Host cell translation

mRNA translation uses -5' CAP structure which recruits the cap binding complex (eIF4F) -recruitment of the 40S small ribosomal subunit. -This then scans along until the first AUG is found and the large ribosome (60S) is recruited to form 80S complex -RNA molecules can be circularised via an interaction of PABP (bound to the poly A tail) with eIF4G - stimulating translation

What is Baculovirus classification based on?

morphological variation of the virus structure

Nucleocaspid

nucleic acid genome and protective coat

Prophage state definition

phage genome is mostly repressed and inactive.

What is the structure of flavivirus genome?

• (+) sense RNA genome (Baltimore class IV) • vRNA is infectious and serves as both the genome and the viral messenger RNA • 5' end has a methylated nucleotide cap for canonical cellular translation by host machinery • The 3' terminus is not polyadenylated but forms a loop structure

What are the two genera of baculooviruses

• -Granulovirus (GV) & Nucleopolyhedrovirus (NPV) • NPV have large occlusion body with multiple or singular nucleocaspids within, crystalline structure > OB protein = polyhedron -GV have small occlusion body with a single nuclocaspid, granular structure > OB matrix protein = granulin

Structure of flaviviruses

• 40 -60 nm diameter • Capsid symmetry: Icosahedral, T=3 • Enveloped, with a smooth surface (without projections) >Mature virions contain two virus-encoded membrane proteins (M and E), while immature virions contain a membrane protein precursor (prM) • Genome: (+) sense ssRNA, non-segmented (Baltimore Group IV)

what is a virus like particle and how can it be used in vaccines

• A virus-like particle (VLP) is a correctly assembled capsid that does not contain the infective nucleic acid • VLP is preferable to inactivated whole virus or peptides as cannot cause disease • VLPs can spontaneously assemble in insect cells using Baculovirus Expression System (BEVS) • Cervarix is the first VLP manufactured against Human papillomavirus

How are flaviviruses assembled and released

• Assembly of new virions occurs at the ER • Virions bud from into the ER lumen and acquire envelope with viral glycoproteins E & prM >Heterotrimeric E-prM spikes • Immature virions are transported to the Golgi via secretory pathway progressively decreasing pH to ~pH6 in trans-Golgi network >Conformational change exposes furin cleavage site in prM >M protein" latch" is set, keeping the E fusion protein in flat conformation (Fusion loops are buried and locked in place by M) • Mature particles released by exocytosis >pr portion of prM dissociates on release into the extracellular milleu (neutral pH)

How does cotransfection work with baculoviruses?

• Baculovirus genome is linearised using a restriction endonuclease • Lacking polyhedrin gene and with partial deletion in an essential gene • Linear DNA cannot replicate and re-ligation of the viral DNA would not produce viable virus (essential gene not restored) • Homologous recombination of bacmid DNA with transfer vector containing complete essential gene restores essential gene and circularises genome • Target gene in place of polyhedrin gene, virus can infect insect cells and will express high level of target protein late in infection • products can be isolated and purified

What is BacMam

• Baculovirus genome is modified using a mammalian expression cassette >baculovirus promoters not recognised by mammalian RNA polymerase therefore are non-replicating in mammalian cells >excellent safety profile combined with being well-tolerated by cells • Allows for manipulation of genes

What are the main subfamilies of poxviruses

• Chordopoxvirinae: infect vertebrates >Some of major medical and veterinary importance • Entomopoxvirinae: infect invertebrates

How do flaviviruses enter a cell

• Clathrin dependent receptor mediated endocytosis • Virus particles attach to the host cell via interaction between E protein and host receptor >Specific receptor on host cell unknown • Glucosaminoglycans (GAGs) on host proteoglycans act as attachment factors >concentrate particles on host cell

How many proteins are formed from flavivirus polyprotein cleavage

• Genome encodes a single polyprotein which after translation is cleaved into 3 structural and 7 non-structural proteins • Cleavage is carried out by both host and viral (NS3/2B) proteases on the host ER membrane (furin cleaves prM in the Golgi) • Viruses from the JEV subgroup contain a conserved pseudoknot preceded by a slippery heptanucleotide immediately downstream of the NS1 coding sequence > Induces a ribosomal frame shift which can form stop codons >Allows for structural proteins to be made more than replication proteins

What is the baculovirus expression system

• Method to produce recombinant glycoproteins and membrane proteins • Genetically engineer modified baculovirus genome to express desired gene in place of polyhedrin ('bacmid") • This allows for >high level of protein expression >protein folding and post-translational modifications >Able to scale up larger (Smaller and safer production)

Whats the structure of the RNA-dependant RNA polymerase in flaviviruses

• NS5 - RdRp • NS3- helicase • NS4A - proliferation of the ER • Initiates de novo synthesis of RNA (no primer)

Diseases caused by flaviviruses

• broadly grouped into 1. Viruses that cause vascular leak and haemorrhage >Dengue virus (DENV) and yellow fever virus (YFV) 2. Viruses that cause encephalitis (inflammation of the brain) >West Nile virus (WNV) and Japanese encephalitis virus (JEV) • Relatively few infected individuals develop these severe clinical manifestations, and many are asymptomatic or have an undifferentiated febrile illness

What is sfRNA in flaviviruses

• essential for pathogenicity, and may play a role in inhibiting host antiviral activity • The 3' UTR is important in formation of a subgenomic flavivirus RNA (sfRNA) through genomic RNA degradation by host XRN1

Clinical manifestations of flaviviruses

• ~95% infections asymptomatic or develop undifferentiated febrile illness • ~5% infections lead to severe clinical manifestations, depending on virus these include: >Haemorrhagic fevers >Meningitis >Meningoencephalitis >Encephalitis >Hepatitis >Nephritis