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Viruses Microbiology 221 Viruses Obligate intracellular parasites They are able to reproduce their life cycle only within the cell of their host They usually have an external capsid composed of proteins Inner core of nucleic acid( dsDNA, ssDNA,dsRNA, and ssRNA) Specificity for the host Classification of viruses According to Baltimore classification, viruses are divided into the following seven classes: dsDNA viruses ssDNA viruses dsRNA viruses (+)-sense ssRNA viruses (-)-sense ssRNA viruses RNA reverse transcribing viruses DNA reverse transcribing viruses where "ds" represents "double strand" and "ss" denotes "single strand". Table of Viruses Viral Capsids Capsids Capsids are made from protein subunits called capsomeres In some viruses, the capsomeres are all the same Geometric shape in others the sub units vary Antigenic spikes Some viruses have molecules inserted into the outer covering of the virus These may be glycoproteins In some cases these serve as a means of attaching to the host cell They are specific for one cell type Capsid Shapes Ebola – Shepherd’s Crook Enveloped Virus Envelopes Lipid composition Acquired from the host when the virus exits the cell Provides a means for viruses to elude the immune system of the host by surrounding itself with the host envelope Enveloped viruses may be more vulnerable to chemical agents(chlorine, hydrogen peroxide, and phenol) They do not survive well on surfaces Viral Life Cycle – Factors Influencing the Life Cycle Nucleic acid Enveloped or naked Shape Host Host Range Primates Vertebrates ( birds) Plants Insects Bacteria Bacteriophages Viruses that infect bacterial cells Genome can be DNA or RNA Bind to specific receptors that are proteins or carbohydrates in the bacterial cell wall Bacteriophage structure Bacteriophages PhiX174 – Spherical Bacteriophage Interesting to study because of its overlapping genes which is a model of efficiency T- 4 Bacteriophage Studied by Luria and Delbruck at Cold Spring Harbor Ds DNA virus 168, 800 base pairs Phage life cycles studied by Luria and Delbruck Filamentous phages Fd Filamentous Circular ss DNA Lies in the middle of the filament Infects through the pilus Create a symbiotic relationship with the host M 13 Used for Genetic engineering experiments Bacteriophages Life Cycles Lytic – They attach and enter the bacterial cell. Complete their life cycle by bursting the bacterial cell – “ lysis” Lysogenic – They attach and enter the bacterial cell. The virus then integrates into the bacterial cell as a “prophage” Process of Infection Attachment Injection Hostile take over( lytic) Integration( lysogenic) – Genetic control Early genes/late genes Replication of nucleic acid Production of viral proteins Assembly Lysis Lytic Life Cycle( Virulent) The viral genome contains a promoter that attaches to host cell RNA Polymerase Early genes code for those proteins that shut down the host, replicate nucleic acid, and code for vital proteins Nuclease genes, are capable of digesting host DNA so that the bases can be used for the production of new virions Late genes code for viral capsid and for those proteins that lead to lysis of the host cell Lytic Cycle Strict control Do not want to lyse the cell prior to the completion of assembly Usually only one virus in a cell at a time Recombination occurs between the two viruses if more than one is present Lysogenic Life Cycle Bacteriophages that do not lyse the bacteria cell are referred to as temperate Lysogenic bacteria contain a copy of the virus which is non infective and is known as the prophage The prophage can remain inactive through many cell division Bacteria can switch between lytic and lysogenic life styles When the host is stressed or damaged by mutagens, this stimulates the prophase to excise itself and Generalized Transduction Any part of bacterial genome can be transferred Occurs during lytic cycle During viral assembly, fragments of host DNA mistakenly packaged into phage head generalized transducing particle Generalized transduction Specialized transduction Lambda Phage Bacteriophage Lambda is a temperate phage meaning that it can undergo either a lytic or lysogenic cycle The phage regulates this cycle genetically through a switch Attachment . Bacteriophage Lambda binds to the target E. coli cell, the tail tip binding to a maltose receptor. The linear phage genome is injected into the cell, and immediately circularises. Transcription starts There are two regulatory viral proteins, CI and Cro CI and Cro compete for the operator promoter sites on the phage DNA When the bacterial host is healthy CI accumulates and the lambda integrates into the bacterial genome and stays in this position Control of lysogeny and lytic cycle Genes needed to establish lysogeny cI yes cII yes cIII yes Genes needed for maintenance of lysogeny cI yes cII no cIII no Insertion sequences Genetic Control of Lytic and Lysogenic Phage Lytic vs. Lysogenic When Cro is low, CI maintains the lysogenic life style When Cro accumulates due to damage of the host DNA or other unsuitable environmental conditions, the virus switches to the lytic life style This activates promoters for phage replication and protein synthesis Serves as a model for understanding viral infectivity and genetic control Integration Integration of bacteriophage lambda requires one phage-encoded protein - Int, which is the integrase - and one bacterial protein - IHF, which is Integration Host Factor. Both of these proteins bind to sites on the P and P' arms of attP to form a complex in which the central conserved 15 bp elements of attP and attB are properly aligned. The integrase enzyme carries out all of the steps of the recombination reaction, which includes a short 7 bp branch migration. Enzymes and Recombination The strand exchange reaction involves staggered cuts that are 6 to 8 bp apart within the recognition sequence. All of the strand cleavage and re-joining reactions proceed through a series of transesterification reactions like those mediated by type I topoisomerases. Excision of bacteriophages Excision of bacteriophage lambda requires two phage- encoded proteins: Int (again!) and Xis, which is an excisionase. It also requires several bacterial proteins. In addition to IHF, a protein called Fis is required. All of these proteins bind to sites on the P and P' arms of attL and attR forming a complex in which the central conserved 15 bp elements of attL and attR are properly aligned to promote excision of the prophage. Lytic Cycle Lytic Lifestyle The 'late early' transcripts -genes for replication of the lambda genome. The lambda genome is replicated in preparation for daughter phage production. Transcription from the R' promoter can now extend to produce mRNA for the lysis and the structural proteins. Structural proteins and phage genomes self assemble into new phage particles. Lytic proteins build sufficiently far in concentration to cause cell lysis, and the mature phage particles escape. Bacteriophage growth curve Plaque Assay Eclipse Period- Penetration through biosynthesis Latent-Spans from penetration up to the point of phage release Lambda and Plaques The plaque produced by Lambda had a different appearance on the Petri Dish. It is considered to be turbid rather than clear The turbidiy is the result of the growth of phage immune lysogens in the plaque The agar surface contains a ratio of about a phage /107 bacteria Plaque assay Methodology Grow bacteria to log phase Prepare dilutions of bacteriophage Mix bacteria with viruses or overlay bacteria with suspension of viral particles Incubate Count infectious particles based upon the number of plaques Plaques are clear areas indicating lysis of infected cells Plaque Assay MOI Average number of phages /bacterium After several lytic cycles the MOI( multiplicity of infection) gets higher due to the release of phage particles Horizontal Transfer of Genes The transfer of genes or blocks of genes ( pathogenicity islands) from bacterium to bacterium or virus to bacteria or virus to virus Has resulted in many changes in microbes that have led to increase in pathogenicity and accumulation of virulence genes, not just resistance Streptococcus pyogenes There are 15 prophages that have been identified in E. coli These prophages belong to the group Siphoridae All but one of these produce a toxin In both strep and staph – the prophage is found at the site of recombination Virulence and Streptococcus pyogenes Streptococcal pyrogenic exotoxins(SPE) contribute to the diverse symptoms of a streptococcal infection. These antigens compare to Staphylococcal antigens of the same type. The A + C genes coding for these toxins were horizontally transferred from strain to strain by a lysogenic bacteriophage. In addition the genes contributed by the phages produce hyaluronidase, mitogenic factor, and leukocyte( WBC) toxins Pathogens with bacteriophages that cause complications Corynebacterium diphtheriae Vibrio cholerae enterotoxogenic E. coli Staphylococcus aureus Clostridium botulinum Staphylococcus aureus and Streptococcus pyogenes: Toxic shock syndrome Toxic shock Parvoviridae Viral family: Parvoviridae single-stranded DNA; naked; polyhedral capsid Size: 18-25nm Examples and diseases: parvoviruses (roseola, fetal death, gastroenteritis; some depend on coinfection with adenoviruses) Papovaviridae double-stranded, DNA; naked; polyhedral capsid Viral family: Papovaviridae; circular dsDNA Size: 40-57nm Examples and diseases: human papilloma viruses (HPV; benign warts and genital warts; genital and rectal cancers) Adenovirus Viral family: Adenoviridae; dsDNA Size: 70-90nm Examples and diseases: adenoviruses (see Fig. 1E) (respiratory infections, gastroenteritis, infectious pinkeye, rashes, meningoencephalitis) Pox Viruses double-stranded, circular DNA; enveloped; complex Viral family: Poxviridae Size: 200-350nm Examples and diseases: smallpox virus (smallpox), vaccinia virus (cowpox), molluscipox virus (molluscum contagiosum-wartlike skin lesions Herpes and Hepadnaviridae double-stranded DNA; enveloped; polyhedral capsid Viral family: Herpesviridae Size: 150-200nm Examples and diseases: herpes simplex 1 virus (HSV-1; most oral herpes; see Fig. 1H), herpes simplex 2 virus (HSV-2; most genital herpes), herpes simplex 6 virus (HSV-6; roseola), varicella-zoster virus (VZV; chickenpox and shingles), Epstein-Barr virus (EBV; infectious mononucleosis and lymphomas), cytomegalovirus (CMV; birth defects and infections of a variety of body systems in immunosuppressed individuals) Viral family: Hepadnaviridae Size: 42nm Examples and diseases: hepatitis B virus (HBV; hepatitis B and liver cancer) Picornaviruses (+)single-stranded RNA; naked; polyhedral capsid Viral family: picornaviridae Size: 28-30nm Examples and diseases: enteroviruses (poliomyelitis), rhinoviruses (most frequent cause of the common cold), Norwalk virus (gastroenteritis), echoviruses (meningitis), hepatitis A virus (HAV; hepatitis A) Togaviruses (+)single-stranded RNA; enveloped; usually a polyhedral capsid Viral family: Togaviridae Size: 60-70nm Examples and diseases: arboviruses (eastern equine encephalitis, western equine encephalitis), rubella virus (German measles) - Strand viruses (-) strand; multiple strands of RNA; enveloped Viral family: Orthomyxoviridae Size: 80-200nm Examples and diseases: influenza viruses A, B, and C (influenza) Viral family: Bunyaviridae Size: 90-120nm Examples and diseases: California encephalitis virus (encephalitis); hantaviruses (Hantavirus pulmonary syndrome, Korean hemorrhagic fever; see Fig. 1G) Viral family: Arenaviridae Size: 50-300nm Examples and diseases: arenaviruses (lymphocytic choriomeningitis, hemorrhagic fevers) HIV produce DNA from (+) single-stranded RNA using reverse transcriptase; enveloped; bulletshaped or polyhedral capsid Viral family: Retroviridae Size: 100-120nm Examples and diseases: HIV-1 and HIV-2 (HIV infection/AIDS; see Fig. 3A); HTLV-1 and HTLV-2 (T-cell leukemia) Key steps in viral infections Adsorption Fusion/Uncoating Hostile Takeover Early Genes for Replication of Virus Late Genes for Proteins and Capsid Lysis Animations of adsorption Flash animation showing adsorption of an enveloped virus. Flash animation showing adsorption of a naked virus. QuickTime movie showing adsorption of an enveloped virus. QuickTime movie showing adsorption of a naked virus. Fusion and uncoating Flash animation showing penetration and uncoating of an enveloped virus entering by fusion. Flash animation showing penetration and uncoating of an enveloped virus entering by endocytosis. QuickTime movie showing penetration and uncoating of an enveloped virus entering by fusion. QuickTime movie showing penetration and uncoating of an enveloped virus entering by endocytosis Animations of Replication Flash animation showing replication of an enveloped virus. Flash animation showing replication of a naked virus. QuickTime movie showing replication of an enveloped virus. QuickTime movie showing replication of a naked virus. Release of Virus Flash animation showing release of a naked virus by cell disintegration. QuickTime movie showing release of a naked virus by cell RNA Virus Global Pandemic Influenza Orthomyxovirus – medium sized enveloped(-) sense RNA – leads to recombination They have a segmented genome Broad host range Whales, swine,birds, and humans Antigens Influenza neuraminidase exists as a mushroomshape projection on the surface of the influenza virus. It has a head consisting of four co-planar and roughly spherical subunits, and a hydrophobic region that is embedded within the interior of the virus' membrane. It is comprised of a single polypeptide chain Hemagglutinin HA binds to an as yet unidentified glycoprotein which is present on the surface of its target cells. This causes the viral particles to stick to the cell's surface. The cell membrane then engulfs the virus and the portion of the membrane that encloses It pinches off to form a new membranebound compartment within the cell called an endosome, which contains the engulfed virus. Avian flu Avian influenza is an infection caused by avian (bird) influenza (flu) viruses. These influenza viruses occur naturally among birds. Wild birds worldwide carry the viruses in their intestines, but usually do not get sick from them. However, avian influenza is very contagious among birds and can make some domesticated birds, including chickens, ducks, and turkeys, very sick and kill them. Infection Infected birds shed influenza virus in their saliva, nasal secretions, and feces. Susceptible birds become infected when they have contact with contaminated secretions or excretions or with surfaces that are contaminated with secretions or excretions from infected birds. Domesticated birds may become infected with avian influenza virus through direct contact with infected waterfowl or other infected poultry, or through contact with surfaces (such as dirt or cages) or materials (such as water or feed) that have been contaminated with the virus. Infection with avian influenza viruses Strains of Avian Flu Hepatitis B Virus Hepatitis B is a DNA Virus of the Hepadnaviridae family of viruses. It replicates within infected liver cells (hepatocytes ). The infectious ("Dane") particle consists of an inner core plus an outer surface coat. Hepatitis B antigens When the virus enters the body of a new host it's initial response, if it's gets past the immune system, is to infect a liver cell. To do this the virus attaches to a liver cells membrane and the core particle enters the liver cell. The core particle then releases it's contents of DNA and DNA polymerase into the liver cell nucleus. From within the cell nucleus the hepatitis B DNA causes the liver cell to produce, via messenger RNA; surface (HBs) proteins, the core (HBc) protein, DNA polymerase, the HBe protein, HBx protein and possibly other as yet undetected proteins and enzymes. HbS Ag Hepatitis B Surface protein(s). (HBsAg) The outer surface coat composed of hepatitis B surface proteins is produced in larger quantities than required for the virus to reproduce. The excess surface proteins clump together into spherical particles of between 17-25nm in diameter but also form rods of variable length. In some cases these particles encapsulate a core particle and produce a complete, and infectious, virus particle that enters the blood stream and can infect other liver cells. The excess spheres, rods and also complete viral particles enter the blood stream in large numbers and are easily detectable. It does however take a while for these proteins to appear. Viral Structure Genes and Gene Products Viral Replication HBV Picornaviruses Polio virus Picornaviruses are non- enveloped. As a consequence, they are resistant to lipid solvents like ether and chloroform which would destroy an enveloped virus. Picornaviruses have an icosahedral capsid. . Poliovirus An enterovirus Enters the body through inhalation of water Used to be from swimming pools Causes encephalitis CNS involvement from mild to severe. Presents with fever, malaise,cnills, and neruological effects Rhinoviruses Picornaviruses replicate in the cell cytoplasm. Picornaviruses can replicate in an enucleated cell (a cell that has had its nucleus removed) because they only need host cell machinery and components that are present in the cytoplasm. They don’t require anything found in the host cell nucleus Echoviruses Picornaviruses have a positive sense RNA genome. The picornavirus genome has the same sense (polarity) as mRNA. The genome alone can use the host cell's machinery to make whatever is needed to replicate the virus. If only genomic RNA is injected into a host cell, it is infectious and the cell produces progeny virions. Rhabdovirus - Rabies Rabies Ss RNA virus Secreted in the saliva of infected animals Transferred by bite or scratch 75,000 cases if rabies occur around theworld in humans Virus travels on axons of motor or sensory neurons Spreads to brain with serious consequences National Rabies Management Program The development of a natural vaccine Research completed at Wistar Institute in Philadelphia, Pa. Vaccine is an oral type placed in natural habitats Food or bait is desirable to many animals such as fox, bear, otter, beaver, and racooons Distribution of Rabies in the United States