General Virology Lecture 1 PDF

GENERAL VIROLOGY (BS 5th) By Dr. Maimoona Sabir Lecture 1 Introduction  Course Content  History of animal and plant viruses. Classification: structural and functional groups.  Replication of viruses (RNA & DNA/Animal and Bacteriophages).  Principl...

GENERAL VIROLOGY (BS 5th) By Dr. Maimoona Sabir Lecture 1 Introduction  Course Content  History of animal and plant viruses. Classification: structural and functional groups.  Replication of viruses (RNA & DNA/Animal and Bacteriophages).  Principles of viral diagnostic procedures (cultivation and detection)  Tumor and viruses, Virus evolution,  Prion and viroid.  Virus infection of URT, Virus infection of LRT, Viruses causing skin diseases,  Viral infection of blood, gastrointestinal and nervous system  Experiments 1. Detection and quantification of viruses. 2. Hemagglutination Inhibition assay. 3. Chick embryo inoculation. 4. Plaque assay. 5. Transmission electron microscopy 6. Sample preparation for electron microscopy. 7. Isolation and identification of phages from various sources.  Recommended Books Goura-Kudesia and Tim wreghitt 2012, Clinical-Guides-Clinical- Diagnostic-Virology- Cambridge. Jeffrey C. Pommerville Fundamentals of Microbiology, Jones & Bartlett Learning, 2014 GENERAL VIROLOGY (BS 4th ) By Dr. Maimoona Sabir Lecture: History Nature of animal and plant viruses. Classification: structural and functional groups. Replication of viruses (RNA & DNA). Nature of animal and plant viruses Viruses Nobel Laureate Peter Madwar describe a virus “Its Just a piece of bad news wrapped up in protein” Cause many infections of humans, animals, plants, and bacteria Cannot carry out any metabolic pathway Neither grow nor respond to the environment Cannot reproduce independently Obligate intracellular parasites  Discovery of Viruses In 1882, German Agricultural Chemist, Aldof Mayer, was asked by local tobacco growers to study why their tobacco plant were becoming diseased with what Mayer, called it tobacco mosaic disease In the late 19th century Charles Chamberland developed a porcelain filter. This filter was used to study the first documented virus, tobacco mosaic virus. Cont.  Shortly afterwards, Dimitri Ivanovski published experiments showing that crushed leaf extracts of infected tobacco plants were still infectious even after filtering the bacteria from the solution.  Several others scientist documented filterable disease-causing agents, with several independent experiments showing that viruses were different from bacteria, yet they could also cause disease in living organisms Cont. These experiments showed that viruses are orders of magnitudes smaller than bacteria. The term virus was coined by the Dutch microbiologist Martinus Beijerinck (1898). Contagium vivium fluidum (contiginous living fluid) virus= poison In 1898, Foot and mouth disease was suspected as being caused by virus (a highly contagious viral disease of cloven hoofed animal i.e cattle, sheep, deer) Tobacco Mosaic Disease Foot and mouth disease  Cont. Water Reed at Cuba provide evidence linking Yellow fever with a virus, a mosquito born viral disease of human liver and blood. In 1915, English bacteriologist Friederick Twort discover viruses that infected bacteria cells. After two years, French-canadian scientist, Felix d’Herrelle called then Bacteriophages (phage=eat) Class Activity What are the “requirements” for life? Or how do you know if something is alive? Movement, sensitivity, death, complexity, heredity, growth, cellular organization, development, reproduction, regulation Are viruses alive? Class opinion Rest of Science opinion Viruses have simple structural organization Viruses are small, obligate, intracellular particles, that is mostly seen with electron microscope Infect and take over a host cell in order to replicate They lack chemical machinery for generating energy and synthesizing large molecules Viruses must need an appropriate host cell in which they can replicate as a result cause disease.  Viruses: The Basics  A virus is a microscopic “particle” that can infect the cells of a biological organism.  Viruses can only replicate themselves by infecting a host cell and therefore cannot reproduce on their own.  At the most basic level, viruses consist of genetic material contained within a protective protein coat called a capsid. They infect a wide variety of organisms: both eukaryotes and prokaryotes.  A virus that infects bacteria is known as a bacteriophage, often shortened to phage.  The study of viruses is known as virology, and those who study viruses are known as virologists.  The word virus comes from the Latin, poison (syn. venum).  The Nature of Viruses Viral structure - core of nucleic acid surrounded by protein ◦ classified by nature of genomes  Either DNA or RNA  RNA-based viruses – retroviruses (more later) ◦ Lack ribosomes and necessary enzymes for protein synthesis ◦ nearly all form a protein sheath or capsid around their nucleic acid core  Many animal viruses form an envelope around the capsid. Host range - suitable cells for a virus Structure of viruses  Capsid: protein coat of virus particle is called capsid, give shape and symmetry to the virus, protects the viral genome against chemicals and physical agents and other environmental fluctuations  Capsomeres: capsid is subdivided into individual protein subunits called capsomeres(organization of capsomeres yield the viral symmetry)  Nucleocapsid: the capsid with its enclosed genome is called nucleocapsid  Spikes: some viruses have special capsid proteins called spikes or receptor binding proteins, protuding from the surface, helping attach the virus to host cell and facilitate entry into host cell  Nonenveloped: viruses composed only of nucelocapsid is referred as nonenveloped(naked).  Enveloped: Nucleocapsid of many viruses are surrounded by a flexible membrane known as enveloped, composed of lipid and protein like host cell membrane, acquired from host cell during replication, viruses lose their infectivity if the envelope is destroyed.  Envelop is loose fitting structure over the nucleocapsid, so symmetry of capisd may not be apparent  Spikes: protein structure projecting from enveloped.  Matrix: Enveloped viruses contain a layer or two of protein between capsid and envelope called matrix, that hold the nucleocapisd to Enveloped and non enveloped viruses  Size relationship among cells and viruses  Viruses ranges from the very small poliovirus to the much larger smallpox virus.  Viruses are every small in comparison to eukaryotic cell and nucleus.  In 2002 scientist discover giant virus while working with bacterium Legionella in fresh water sample, 750nm in diameter, non enveloped virus.  Can be seen with light microscope and called mimivirus (‘microbe-mimicking virus’)  Linear DNA molecule that contain more than 1000 protein coding genes(DNA & RNA), HIV and influenza virus have around10 genes. Different size of viruses Viral Classifications Viruses are grouped by their shape (a)Nucleocapsid symmetry  Viruses have a host range and tissue specificity Viruses can be classified by their genome (a)Either DNA or RNA (b)RNA-based viruses – retroviruses Viral Classification on the basis of Nucleocapsid  Viruses exist in the form of rod or filament, have helical symmetry e.g rabies and tobacco mosaic virus. Helix is a tightly wound coil resembling a corkscrew or spring.  Viruses have their capsids in the shape of polyhedron with 20 triangular sides, called icosahedral e.g herpes and polio virus  Viruses have capsids with complex pattern, means they have several parts with different shapes. Some bacteriophages have an icosahedral head with collar and tail assembly in the shape of a helical sheath. Poxviruses are brick shaped with filaments. Viral shapes Viral Structure: Viruses are grouped by their shape Viruses have host range and tissue specificity  Viruses can infect almost all cellular organism. A virus host range refers to what organisms(hosts) the virus can infect and is based on virus capsid or envelope structure.  Some viruses have very narrow host range. e.g specific bacteriophages can infect only specific bacterial species. Smallpox only infect humans.  Few viruses have broader host range, as rabies virus infect humans and most warm blooded animals.(bat, dogs etc)  Many viruses infect specific cell type or tissue within multi-cellular organism. This phenomena is called cell/tissue tropism.  Host range for human immunodeficiency virus (HIV) is a human.  HIV primarily infects specific group of white blood cell called T lymphocytes, because the envelope has protein spikes for binding to receptor molecules on these cells. virus does not infect cells in any other organ or tissue such as heart or liver Class activity 1)Identify the role of each structure found on (A) non enveloped (B) enveloped viruses 2)What shapes can viruses have and what structure determined that shape??? 3) How viruses determine host range and tissue tropism???  A taxonomic scheme for all viruses has yet to be adopted universally  Some viruses are named after the disease they caused, e.g poxvirus, measles virus.  Some viruses are named after the location from which they were originally isolated, e.g Ebola and Marburg.  Some are named after the scientist who studied it, Epstein barr virus.  Other are name after morphologically factors- coronaviruses(corona means crown), picornaviruses (pico means small) rna mean ribonucleic acid.  CONT.  A more precise classification system is being devised by the international committee on taxonomy of viruses (ICTV).  Higher order taxa (phyla and classes) have not yet been completely developed.  To date 6 orders are recognized consist of 87 families, each ending with –viridae (herpesviridae).  Viruses have been categories into hundreds of genera, each genus name end with suffix virus (human harpes virus)  DNA Viruses  Eithercontain single stranded (ss) or double stranded(ds) DNA genome with nucleocapsid that are enveloped or non enveloped.  Genomes are replicated by direct DNA-DNA copying using host cell DNA polymerase, which requires that most DNA viruses replicate in host cell’s nucleus, one exception, poxviruses replicate in the host cytoplasm.  Hepadnaviridae (hepatitis B virus) are replicated indirectly through ssRNA intermediate DNA Viruses  RNA Viruses Viruses have RNA genomes consisting of either ssRNA or dsRNA, which are replicated by direct RNA- RNA copying. Nucleocapsid can be enveloped or non enveloped. ssRNA viruses such as picornaviruses and cornaviruses have their RNA genome in the form CONT.  These RNA viruses are referred to as positive stranded (+)because the genome can be directly translated by host ribosomes.  ssRNA viruses such as orthomyxo viruses and paramyxoviruses, have RNA genomes consisting of RNA strands that would be complementary to mRNA, these genomes which can not be directly translated by host ribosomes are referred to as negative strand(-).  Retroviruses are replicated indirectly through dsDNA intermediate(RNA-DNA-RNA) RNA Viruses Baltimore classification system  How DNA viruses differ from RNA viruses RNA virus genome are smaller than DNA virus genome Depends more heavily on host cell proteins and enzymes for replication They tend to be more error prone (mutation) when copying their RNA genome because RNA polymerase lacks the efficient proof reading exhibited by DNA polymerase to correct replication errors. RNA viruses such as flu viruses tend to ‘genetically drift’ evolving more rapidly into new, potentially epidemic strain The Viral Replication Cycle Virus Infection Host cell Infected Host Cell Replicatio n  Viral Replication cycle  The process of viral replication is one of the most remarkable events in nature.  A virus 1) encounters and attaches to the appropriate host cell, 2)invades the host cell a thousand or more times its size. 3) hijacks the metabolism of the cell to produce viral parts that are then 4) assembled.  The best study process of replication is carried out by bacteriophages, these viruses are virulent viruses, mean they lyse the host cell while carrying out lytic cycle of infection.  1. Attachment First step in the replication cycle of virulent phage occurs when phage and bacterial cells randomly collide. Attachments occurs, if site on phage’s tail fibers match with a complementary receptor site on cell envelope of the bacterial cell. Actual Attachment (adsorption) consist of a weak chemical union between phage and receptor site.  2. Penetration Following attachment, the phage DNA must get across the cell envelope The tail of phage release lysozymes, an enzyme that dissolves a portion of the bacterial cell wall. The tail sheath then contracts and tail core then drives through cell wall. As the tip of hollow core reaches the cell membrane below, a protein plug blocking the core is removed and DNA is ejected through the core and into the bacterial cytoplasm.   Biosynthesis  Having entered the cytoplasm, production of new phage genomes and capsid parts begins.  It is important to note that DNA in the T4 phage contains only the genes needed for viral replication.  Therefore as phage genes codes for disruption of host chromosome, phage DNA uses bacterial nucleotides and enzymes to synthesize multiple copies of genome.  mRNA molecule transcribe from phage genes appear in cytoplasm , and biosynthesis of phage enzymes and capsid proteins begins.  The host ribosomes, amino acids and enzymes are all enlisted for biosynthesis.  Assembly or Maturation  Once the phage parts are made, they are assembled into complete virus particles.  The enzymes encoded by viral genes guide the assembly in step by step fashion.  In one area of host cytoplasm, phage heads and tails are assembled from protein subunits., in other area the head are packaged with DNA, and in third area the tails are attached to the heads.  Release For some phages, lysozyme, encoded by bacteriophage genes late in the replicative cycle, degrades the bacterial cell wall. Mature phage particle now burst out from the ruptured bacterial shell and are set free to infect more bacterial cells. The lytic cycle of infection represent a productive infection because many virions are produced.  Lysogenic cycle Some other phages interact with bacterial cells in a slightly different way, called lysogenic cycle. E.g lambda phage also infect e.coli but not enter immediately into lytic cycle of infection. In this cycle phage DNA integrate into the bacterial chromosomes as a prophage. Bacteriophages participating in this cycle is known as temperate phage In this cycle bacterial cell survive the infection and continuous to grow and divide normally. As bacterial cell goes through its cell cycle the prophage is copied and vertically transferred to daughter cell as part of the replicated bacterial chromosomes. Because the prophage remains ‘inactive’ by not lysing the cell. The infection is referred to as a latent (inactive) infection. Animal viral replication often results in a productive infection Like bacteriophages animal viruses also lead often brief but eventful ‘lives’ as they produces more viruses as a result of infection.  Such productive infection have same five steps  Attachment  Penetration  Biosynthesis  Assembly  Release Attachment Animal viruses infect host cells by binding to receptors on the host cell’s plasma membrane. This binding is facilitated by capsid proteins or spikes distributed over the surface of the capsid (e.g adenovirus) or envelope (e.g HIV) Penetration  Some viruses such as HIV and adenoviruses, require a second receptor called co-receptor for viral entry into the cytoplasm.  Viral entry also differ from that of phage, in that animal viruses often are taken into the cytoplasm as intact nucleocapsid. For examples viruses like HIV, the viral envelope fuses with plasma membrane and releases the nucleocapsid into the cytoplasm.  Some other animal viruses like non enveloped adenovirus and enveloped influenza virus, the virion is taken into the cell by endocytosis.  At the attachment site the cell engulfs the virion within a vacuole and brings it into the cytoplasm  Once in the cell regardless of penetration, the capsid disassembles from the genome process called uncoating and genome is transported to the site where transcription or replication will occur.  Biosynthesis  The DNA of a DNA virus supplies the genetic codes for enzymes that synthesis viral parts from available building blocks.  Poxvirus replicate entirely in the host cell cytoplasm.  Most of DNA viruses employ a division of labour: DNA genome are synthesis in the host nucleus and capsid proteins are produce in the cytoplasm.  The proteins are then transported to the nucleus and join with nucleic acid molecules for maturation. Adenoviruses and herpesviruses follow this pattern.  RNA viruses follow a slightly different pattern of biosynthesis. RNA in +ssRNA viruses acts as a messenger RNA, following uncoating the RNA is directly translated into viral protein as genome replication occurs. -ssRNA strand viruses such as the influenza virus uses their RNA as a template to synthesis a complementary (+) strand of RNA, by an enzyme RNA replicase. The synthesized +ss RNA then is used as messenger RNA molecule for protein synthesis as well as the template to form the –ssRNA genome.  Assembly The final assembly for some enveloped viruses is acquisition of an envelope.  In this step envelope proteins (spikes) are synthesized. Release  In final stage the nucleocapsids of some viruses push through the plasma membrane, forcing a portion of the membrane ahead of and around the virion, resulting in an envelope.  Other enveloped viruses like the herpesvirus, fuse with plasma membrane releasing the virion, this process is called budding , need not necessarily kill or damage the cell during release.  The same cannot be said for nonenveloped viruses. They leave the cell when the cell membrane repture, a process that generally  Animal viruses produce latent infection  Unlike RNA viruses that go through productive infection, most of DNA viruses and retroviruses can establish a latent infection, characterize by repression of most viral genes. Thus virus lies ‘dormant’.  e.g some harpes viruses such as harpes simplex virus HSV-1, can generate a productive or latent infection.  In an infected sensory neurons HSV-1 undergoes latency as the viral dsDNA enters the neuron’s cells nucleus and circularizes, no viral particles are produced for months or years until some stress events reactivates the viral dsDNA and new productive infection will be initiated.  Retroviruses, such as HIV also carry out latent infection  How ever in this group of viruses, the virus carries and enzyme called reverse transcriptase, which is used to reverse transcribe its +ssRNA into dsDNA.  The dsDNA then enters the host cell nucleus and like temperate phage DNA inserted into bacterial chromosomes, becomes integrated randomly into the DNA of one chromosome.  This integrated viral genome is referred as a provirus &  Represents a unique and stable association between the viral DNA host genome.  Advantage for virus The advantage for virus is that every time the host cell divides, the provirus will be replicated along with host genome and be present in all progeny cells. In addition as a provirus it is protected from attack by antiretroviral drugs. How every any time the provirus can be reactivated and a productive infection involving biosynthesis and assembly. Formation of Provirus Animal virus infection of a host cell