Infectious diseases MCQ

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.

Overview of 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

Baltimore class of Lambda phage

-Baltimore class 1 (dsDNA) -Integrate into the chromosome of E.coli (lysogenic) or replicate extra-chromosomally (lytic) -lambda phage produces clear plaques due to lysis

How are bacteriophage classified

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

Entry of baculoviruses

-Insect ingests virus -Occlusion bodies dissolve in gut releases virus (pH alkaline) -Virus infects cell -Insect dies resulting infection and disintergrates releasing occluded virus

Which bacteriophage have contributed features for genetic manipulation

-Lambda and M13 -Transport fragments of DNA from one bacterial cell to another (Genetic transfer systems) -Cloning vectors for molecular genetics

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.

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

Structural differences in the two baculovirus phenotypes

-Occlusion derived virus (ODV): virions occluded by protein bodies or crystals -Budded virus (BV): virions enveloped; pleomorphic; rod-shaped

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

What is the two stage life cycle of baculovirus lifecycle

-Polyhedra dissolves, fusion to midgut cell -Nucleocaspid transported into nucleus and replicated -Nucleocaspid formed, buds from nucleus to cytoplasm -buds from cyto out of cell -dissemination into next cell -nucleus take up nucleocaspid, polyhedral formed, -polyhedral released by lysis

Baculoviruses

-Two genera: Granulovirus (GV) & Nucleopolyhedrovirus (NPV) >GVs have a granular structure: OB contains a single nucleocapsid; OB matrix protein = granulin >NPVs have a crystalline structure: OB contains multiple nucleocapsids packed singly or in groups; OB protein = polyhedrin -Stable in environment due to formation of a thick protein shell called occlusion body (OB) around the nucleocapsid(s) -Used as biopesticides to control insect pests >Not common as -Also used for expression of recombinant proteins in insect cell culture

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

Spearman karber equation

-Xp=1, Highest log dilution giving all positive response - d, Log dilution factor (Difference between log dilutions) - p, proportion positive at a given dose -Sum of 1-p, Sum values of (1-p) for Xp=1 and all higher dilutions

Baltimore classification of M13

-class 2 -on infection , generates complementary copy of ssDNA which acts as template for replication -RF (replicative form) is double stranded only found inside infected host -Turbid plaques form due to slower bacterial growth rather than lysis

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.

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

What Baltimore class is baculovirus

1

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)

Multiplicity of infection

Average Number of phage per bacterium

Baculovirus life cycle

Entry:Endocytosis -gp64 (phosphoglycoprotein) >Surface of infected cells and on budded virions as a homo-trimer, forming typical peplomer structures >Gp64 is necessary and sufficient for the low pH-activated membrane fusion activity that occurs during endocytosis -Upon acidification of the endosome, the endosomal membrane fuses with the viral envelope, resulting in the release of the nucleocapsid into the cytoplasm, which is then transported towards the nucleus using actin polymerization Replication and assembly -Viral DNA released into nucleus -transcription and replication of viral DNA, and nucleocapsid assembly Exit -Progeny nucleocapsids leave the nucleus acquiring an envelope from the nuclear membranes that is eventually lost. -These nucleocapsids finally travel to the plasma membrane where they bud off acquiring a glycoprotein-rich envelope from the cell membrane to generate BVs

What do bacteriophage serve as models for

GENETIC REGULATION -Mark Ptashne isolated lambda repressor and showed function as key gene expression switch during lambda phage lifecycle (Lysogenic to lytic)

What was the first organism to be fully sequenced and who by

MS2 RNA phage by Walter Fiers 1976

Poisson distribution

P(N) = (m^n x e^-m)/ n! Percentage of cells uninfected

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

Baltimore Class 7

dsDNA with 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.