MULTIPLICATION

1. MULTIPLICATION

2. Multiplication… Remember……. • Viruses are obligate intracellular parasites that can reproduce only within a host cell. • They do not have • Enzymes for metabolism • Do not haveribosomes • Do not have the equipment to make proteins • Viruses use a “lock and key” fit to identify hosts Viruses - when in host cell, they will take control of synthetic and genetic machinery of host cell.

3. LESSON OUTCOME • General steps in multiplication/replication of virus • Multiplication in animal virus - differences between naked and enveloped virus

4. Viral Multiplication – general phases in the life cycle of viruses • Multiplication/Replication cycles involving 5 steps: • Adsorption:the attachment of viruses to host cells • Penetration:entry of virions (or their genome) into host cells • Synthesis:new nucleic acids, capsid proteins, and other viral components- transcription, translation and genome replication • Maturation:assembly of newly synthesized viral components into complete virions • Release:departure (pemergian) of new virions from host cells

5. Animal viruses – general multiplication steps • Adsorption: the attachment of viruses to host cells • Penetration:entry of virions (or their genome) into host cells • Uncoating: to separatenucleic acid from protein coat; - envelop and capsid are dissolved; the nucleic acid is released • Synthesis:new nucleic acids, capsid proteins, and other viral components- transcription, translation and genome replication • Maturation:assembly of newly synthesizedviral components into complete virions • Release:departure of new virions from host cells

6. 1. Adsorption • Invasion begins when the virus encounters a susceptiblehost cell and adsorbsspecifically to receptor sites on the cell membrane • The membranereceptors that viruses attach to are usually glycoproteins the cell requires for its normal function. Ex. - rabies virus  the acetylcholine receptor of nerve cells. - human immunodeficiency virus (HIV or AIDS virus)  CD4 protein on certain white blood cells.

7. Adsorption • The mode of attachment varies between the two general types of viruses (naked and enveloped) • In enveloped virus - influenza virus and HIV, glycoprotein spikes bind to the cell membrane receptors. (Spikes that recognize membrane protein receptor) Enveloped viruses The configuration of the spike has a complementary fit for cell receptors. The process in which the virus lands on the cell and plugs into receptors is termed docking.

8. Adsorption • Naked nucleocapsids (adenovirus, for example) use molecules on their capsids that adhereto cell membrane receptors Protruding molecules - spike (Specific membrane protein involved with cell adhesion) An adenovirus has a naked capsidthat adheres to its host cell by nestling surface molecules on its capsidinto the receptors on the host cell’s membrane. Naked viruses (Attachment sites on surfaces of capsids) Viral recognition of an animal host cell: Rhinoviruses have “canyons” or depressions, in the capsidthat attach to specific membrane proteins on host cell membrane

9. Adsorption • Virus can invade its host cell - through making an exact fit with a specific host molecule. • Host range, may be as i) restrictedas hepatitis B, which infects only liver cells of humans ii) intermediatelike the poliovirus, which infects intestinal and nerve cells of primates (humans, apes, and monkeys) iii)broadas therabies virus, which can infect various cells of all mammals. • Host cells that lack compatible virus receptors areresistant to adsorptionand invasion by that virus – why human liver cells are not infected by the canine hepatitis virus and dog liver cells cannot host the human hepatitis A virus.

10. 2. Penetration/Uncoating • Animal viruses do not have a mechanism for injecting their nucleic acid into host cells – nucleic acid and capsid usually penetrate animal cells. • Penetration -i) endocytosis ii) directfusion of viral envelop with host cell membrane • Endocytosis – the entire virus (including the envelope) is engulfted by the cell – enclosed in a vacuole or vesicle - Most naked viruses enter cell by endocytosis in which virionsare captured by pitlike regions on cell surface – enter the cytoplasm within a membranous vesicle - Enveloped viruses – the envelope fuse with host’s plasma membrane or by endocytosis. In endocytosis the envelope will fuse to vesicle membrane • Uncoating: is a process that releases the viral nucleic acid into cytoplasm. - when enzymes dissolve envelope and capsid, the virus is said to be uncoated. - Naked viruses by proteolytic enzymes, host or virus - Enveloped viruses (poxviruses) by a specific enzyme encoded by viral DNA • Viral entry into the host cell - direct fusion of the viral envelope with the host cell membrane (as in influenza and mumps viruses) - the envelope merges directly with the cell membrane,  release the nucleocapsidinto the cell’s interior.

11. Penetration/Uncoating VIRAL ENTRY INTO HOST CELL

12. 3. Synthesis • The synthetic and replicativephases of animal viruses are highly regulated and extremely complex at the molecular level. Free viral nucleic acid - control over the host’s synthetic and metabolic machinery; depending on the virus genome (DNA or RNA) • The DNA viruses (except poxviruses) enter the host cell’s nucleus and are replicatedin the nucleus, transcription in nucleus • RNA viruses (except retroviruses), are replicated in the cytoplasm, transcription in cytoplasm. • RNA VIRUS REPLICATION AND PROTEIN SYNTHESIS Almost immediately upon entry, the viral nucleic acid alters the genetic expression of the host and instructs it to synthesize the building blocks for new viruses. 1. The RNA of the virus becomes a message for synthesizing viral proteins (translation). - Viruses with positive-sense RNA molecules already contain the correct message for translation into proteins. - Viruses with negative-sense RNA molecules must first be converted into a positive-sense message. 2. Some viruses come equipped with the necessary enzymes for synthesis of viral components; others utilize those of the host. 3. In the next phase, new RNA is synthesized using host nucleotides. Proteins for the capsid, spikes, and viral enzymes are synthesized on the host’s ribosomes using its amino acids.

13. 4. Maturation • Maturation: Once all viral nucleic acid, enzymes, and other proteins have been completely synthesized, assembly of components into complete virions begins. • DNA virus: assembly take place in nucleus • RNA virus: assembly take place in cytoplasm Assembly of Viruses: Host Cell as Factory

14. 5. Release • Release: The release of new virions through a membrane may or may not destroy the host cell. Adenoviruses bud from host cell in a controlled manner (ex. shedding) which does not lyse host cells vs release through lysis – destroy the host cells • To complete the cycle, assembled/matured viruses leave their host in one of two ways. i) Non-enveloped and complex viruses that reach maturation in the cell nucleus or cytoplasm are released when the cell lyses or ruptures. - (cell lysis) ii) Enveloped viruses are released by budding or exocytosisfrom the membranes of the cytoplasm, nucleus, orendoplasmic reticulum? - The nucleocapsid binds to the membrane, which curves completely around it and forms a small pouch. Pinching off the pouch releases the virus with its envelope. Budding of enveloped viruses causes them to be shed gradually, without the sudden destruction of the cell.

15. Release • Regardless of how the virus leaves, most active viral infections are ultimately lethal/deadly to the cell because of accumulated damage. • Lethal damages include a permanent shutdown of metabolism and genetic expression, destruction of cell membrane and organelles, toxicity of virus components, and release of lysosomes. • A fully formed, extracellular virus particle that is virulent (able to establish infection in a host) is called a virion • The number of virions released by infected cells is variable, controlled by factors such as the size of the virus and the health of the host cell. About 3,000 to 4,000 virions are released from a single cell infected with poxviruses, whereas a poliovirus-infected cell can release over 100,000 virions - even a small number of new virions happens to meet another susceptible cell and infect it, the potential for rapid viral proliferation is immense.

16. Release

17. Modes of infection and replication of animal viruses – enveloped virus, DNA genome • The enveloped viruses enter the host cell through • i) endocytosis into host cell cytoplasmic • ii) the fusion of virus envelop with the host’s cell/plasma membrane • Penetration – involves nucleocapsid only • Replication and transcription – takes place in nucleus • Translation in the cytoplasmcapsid and protein are synthesize in cytoplasm • Maturation – assembly of nucleocapsid of new virus particle in nucleus • Some viruses have envelopes that are not derived from the plasma membrane. Herpesvirus has an envelop that is derived from the nuclear membrane. Replication of an enveloped dsDNAanimal virus (e.g. herpesvirus)

18. Synthesis in DNA animal viruses • Synthesis of new genetic material and proteins depends on the viruses • Generally, DNA animal viruses replicate their DNA in host cell nucleus with aid of viral enzymes and synthesize their capsid and other proteins in the cytoplasm with aid of host cell enzymes – typical of adenoviruses, hepadnaviruses, herpesviruses and papovaviruses. • Assembly of nucleocapsid – in nucleus • dsDNA viruses – replication proceeds in a complex series of steps designated as early and latetranscription and translation • Early events – take place before the synthesis of viral DNA and results in production of enzymes and proteins for viral DNA replication • Late events – after the synthesis of viral DNA, results in production of structural proteins needed for building newcapsids.

19. Modes of infection and replication of animal viruses - enveloped virus, RNA genome Nucleic acid synthesis – cytoplasm Assembly of nucleocapsid - cytoplasm General features in the multiplication cycle of an enveloped animal virus. Using an RNA virus (rubella virus), the major events are outlined, although other viruses will vary in exact details of the cycle.

20. Modes of infection and replication of animal viruses – enveloped virus, RNA genome Synthesis in RNA animal viruses • Synthesis in RNA animal viruses takes place in a greater variety of ways than found in DNA viruses: • (+) sense RNA acts as mRNA (e.g. picornaviruses) and viral proteins are synthesize immediately after penetration and uncoating. The nucleus of host cell is not involved. • dsRNA (+) sense are transcribed into ssDNA with help of reverse transcriptase (e.g. retrovirus – HIV) • (-) sense RNA make (+) sense RNA which are mRNA (e.g. measles and influenza) • Nucleic acid replication and assembly of nucleocapsid - cytoplasm

21. Modes of infection and replication of animal viruses – RNA genome • The broadest variety of RNA genomes is found among viruses are those that infect animals. • The genome of class IV can directly serve as mRNA and can be translated into viral protein immediately after infection. • A (-) sense RNA is synthesized as template for replication of more (+) sense RNA

22. MULTIPLICATION (II)

23. ANNOUNCEMENT • PRACTICAL 5: - Each group is required to bring their own sample – as shown in pg 13 in the lab manual. - Each group bring 3 samples: soil, sewage water and chicken faeces

24. LESSON OUTCOME • Bacteriophage – multiplication steps • Lytic cycle and Lysogenic cycle • Virulent, temperate virus, prophage – definition • Different – bacteriophage and animal multiplication how the viruses enter the host cells, release

25. Point of entry for virus: REVISION Point of exit for virus:

26. REMEMBER…. The assembly of newly formed viral particles cytoplasm – eg. Poxvirus, poliovirus Cell nucleus – eg. Human adenovirus nucleocapsids Plasma membrane of host – eg. HIV at the inner surface of host cell’s cell membrane The source to form new viral particles Proteins and glycoproteins– coded by viral genome Envelope lipids and glycoproteins– synthesized by host cell enzymes and are present in the host cell plasma

27. Bacteriophages • Bacteriophages means “eaters of bacteria” The bacteriophages – discovered by Frederick Twort and Felix d’Herelle in 1915 – it first appeared that the bacterial host cells were being eaten by some unseen parasite, hence the name bacteriophage was used. • Most bacteriophages (or phage) contain double-stranded DNA, although single-stranded DNA and RNA types exist as well. • It is known that every bacterial species is parasitized by various specific bacteriophages. • Bacteriophages are of great interest to medical microbiologists because they often make the bacteria they infect more pathogenic for humans. - The most widely studied bacteriophages are those of the intestinal bacterium Escherichia coli— especiallythe T-even phages such as T2 and T4 • The multiplication of T-even bacteriophages -similar stages as the animal viruses described earlier Have been used as a model systems for animal and plant viruses

28. Adsorption • Bacteriophages have specialized structures for attaching to bacterial cell walls – adsorption involve attachmentof specific tail fibers to bacteria’s cell wall • They adsorb to host bacteria using specific receptors on the bacterial surface

29. Penetration • Bacteriophages have a mechanism for injecting their nucleic acid into host cells (nucleic acid and capsid usually penetrate animal cells) • This eliminates the need for uncoating. Only nucleic acid penetrate into host cell. • Penetration of a bacterial cell by a T-even bacteriophage. • After adsorption, the phage plate becomes embedded in the cell wall, and the sheath contracts, pushing the tube through the cell wall and membrane and releasing the nucleic acid into the interior of the cell. • Section through E. coli with attached phages. Note that these phages have injected their nucleic acid through the cell wall and now have empty heads.

30. Synthesis • Entry of the nucleic acid causes the cessation of host cell DNA replication and protein synthesis. Soon the host cell machinery is used for viral replication and synthesis of viral proteins. Maturation • As the host cell produces new phage parts, the parts spontaneously assemble into bacteriophages. • An average-sized Escherichia coli cell can contain up to 200 new phage units at the end of this period.

31. Release • Eventually, the host cell becomes so packed with viruses that it lyses (splits • open) - releasing the mature virions. • The process is hastened by viral enzymes produced late in the infection cycle • that digest the cell envelope, thereby weakening it. Upon release, the virulent • phages can spread to other susceptible bacterial cells and begin a new infection. Involve lysozyme to break the host cell wall

32. Bacteriophages • Bacteriophages can be classified as virulent or temperate • Virulent phage (or lytic phage) –lyse and destroy bacteria they infect • Temperate phage – able to undergo lytic cycleand lysogenic cycle. - temperate phage exhibit lysogeny – the state whereby the DNA of temperate bacteriophages integrate into the host DNA - no replication of new viruses and cell lysis • The host cells are called lysogenic cells • The viral DNA within the bacteria chromosome is called prophage Virulent phage Temperate phage

33. Temperate phages • Undergo adsorption and penetration into the bacterial host but are not replicated or released immediately. - viral DNA enters an inactive prophage state, during which it is inserted into the bacterial chromosome. This viral DNA will be retained by the bacterial cell and copiedduring its normal cell division so that the cell’s progeny will also have the temperate phage DNA. - This condition, in which the host chromosome carries bacteriophage DNA, is termed lysogeny. • Because viral particles are not produced, the bacterial cells carrying temperate phages do not lyse, and host cells appear entirely normal. On occasion, in a process called induction, the prophage in a lysogenic cell will be activated and progress directly into viral replication and the lytic cycle. - Lysogeny is a less deadly form of parasitism than the full lytic cycle and is thought to be an advancement that allows the virus to spread without killing the host. • Because of the intimate association between the genetic material of the virus and host, phages occasionally serve as transporters of bacterial genes from one bacterium to another and consequently can play a profound role in bacterial genetics. This phenomenon, called transduction, is one way that genes for toxin production and drug resistance are transferred between bacteria

34. Reproduction / multiplication of temperate phage Temperate phage – lambda phage Virulent phage – T4 phage Induction: The stimulation of a prophage to initiate a lytic cycle Induction: Due to lack of nutrients for bacterial growth or the presence of chemical toxic to lysogen Prophage: viral DNA within the host genome Lysogen:the bacterium that has combination of temperate phage DNA and host.

35. Replication of a virulent bacteriophage: A virulent phage undergoes a lytic cycle to produce new phage particles within a bacterial cell. Cell lysis releases new phage particles that can infect more bacteria T4 virulent (lytic) phages 5. Release – Lysozyme breaks down the cell wall, allowing viruses to escape – in the process the host cell is lysed  destroy the host 1. Adsorption – chemical attraction, specific protein recognition factors found in tail fibers that bind to specific receptor sites on the host cells. 4. Maturation – T4 head is assembled in host cell cytoplasm from new capsid protein, phage tails from new formed base plates, sheaths and collars. After heads and tails are attached 2. Penetration – lysozyme, weakens the bacterial cell walls – for T4 phages DNA are introduce into the periplasmic space 3. Synthesis – transcription of phage DNA to mRNA, translated on host ribosomes to synthesize capsid proteins and viral enzymes

36. Replication of a temperate bacteriophage: Following adsorption and penetration, the virus undergoes prophage formation • Temperate phages have alternative replication cycle • A productive lytic cycle • A reductive infection, in which the phage remain latent in the host establishing lysogeny • - Lysogeny – occurs when environmental conditions are poor. Allowing survival as a prophage in the host. - lysogen Induction: Due to lack of nutrients for bacterial growth or the presence of chemical toxic to lysogen Lysogenic phages -  phage of E. coli.

37. Relationship between Temperate phages and pathogenicity of host cells • Occasionally phage genes in the bacterial chromosome cause the production of toxins or enzymes that cause pathology in the human. • When a bacterium acquires a new trait from its temperate phage, it is called lysogenic conversion. - The phenomenonwas first discovered in the 1950s in the bacterium that causes diphtheria, Corynebacterium diphtheriae. The diphtheria toxin responsible for the deadly nature of the disease is a bacteriophage product. C. diphtheriae without the phage are harmless. • Other bacteria that are made virulent by their prophages are Vibrio cholerae, the agent of cholera, and Clostridium botulinum, the cause of botulism.

38. Comparison of animal virus and bacteriophage multiplication

39. Animal viruses: Latent viral infections • Latent viral infections: - herpesviruses - herpes simplex virus. These dsDNA viruses that can exhibit a lytic cycle and also able to remain latent within the cells of host. Once activated by a cold, fever, stress or immunosuppression, they replicate resulting in cell lysis. - HIV virus – provirus will become latent until induction whereby HIV virus show AIDS symptom. • Latent infection: - infection of a cell where the replication cycle is not completed, but the virus genome is maintained in the host cell without replicating or causing harm.

40. Phage Growth Growth curve for a bacteriophage: The eclipse phage represents the time after penetration through the biosynthesis of mature phages. The latent period represents the time after penetration through release of mature phages. The number of viruses per infected cell is the viral yield, or burst size

41. Estimation of Phage Numbers • Plaque assay: • Serial dilutions of suspension of phages • Each dilution is inoculated onto a plate containing bacterial lawn • As a result of infection, new phages will lyse the bacteria • After several round of lysis, the bacterial lawn shows clear areas called plaques. • Plaque-forming units (pfu) – counting the no. of plaques X dilution factor = the no. of phages in ml of suspension. plaques