Chapter 1

Question?

infectious agents of small size and simple composition that can multiply only in living cells. Viruses are obligate intracellular parasites that are metabolically inert when they are outside their hosts. The virus genome is composed either of DNA or RNA.

Question? No, The Viroids and Prions are smaller than viruses.

Relative Size of Viruses Eye

Capsid: The protein shell, that encloses the nucleic acid genome. Capsomer: Morphological Morphologic units (seen in the electron microscope) making up the capsid. Structural units: Protomer: The basic protein building blocks of the coat. They are usually a collection of more than one nonidentical protein subunit.

Nucleocapsid: The protein-nucleic acid complex representing the packaged form of the viral genome. Envelope: A lipid-containing membrane that surrounds some virus particles. It is acquired during viral maturation by a budding process through a cellular membrane. Spikes: Peplomer: Rod-like proteins projecting from the envelope or surface of a naked virion. Matrix:

Virion : The complete virus particle. In non-enveloped viruses, the virion is identical with the nucleocapsid. In more complex virions (Evneloped viruses), this includes the nucleocapsid plus a surrounding envelope. Defective viruses: virus particle that is functionally deficient in some aspect of replication.

to protect the fragile nucleic acid genome from physical, chemical, or enzymatic damage. The outer surface of the virus is also responsible for recognition of and the first interaction with the host cell. The capsid also has a role to play in initiating infection by delivering the genome in a form in which it can interact with the host cell. Many viral proteins are enzymes or factors that used in viral replication and transcription.

Viruses may be derived from DNA or RNA nucleic acid components of host cells that became able to replicate autonomously and evolve independently. Viruses may be degenerate forms of intracellular parasites Viruses may be the first form of the living material May be they come from another planet !?

Classification of Viruses The following properties have been used as a basis for the classification of viruses: 1.Virion morphology, including size, shape, type of symmetry, presence or absence of peplomers, and presence or absence of membranes. 2.Virus genome properties, including type of nucleic acid (DNA or RNA), size of genome, strandedness (single or double), whether linear or circular, sense (positive, negative, ambisense), segments (number, size), nucleotide sequence, G + C content, and … 3.Physicochemical properties of the virion, including molecular mass, buoyant density, pH stability, thermal stability, and susceptibility to physical and chemical agents, especially ether and detergents.

4.Genome organization and replication, including gene order, strategy of replication, and cellular sites (accumulation of proteins, virion assembly, virion release). 5.Biologic properties, including natural host range, mode of transmission, vector relationships, pathogenicity, tissue tropisms, and pathology. 6.Antigenic properties. 7.Virus protein properties, including number, size, and functional activities of structural and nonstructural proteins, amino acid sequence, and special functional activities (transcriptase, reverse transcriptase, neuraminidase, fusion activities).

 Virus family names have the suffix -viridae  Genus names carry the suffix -virus  In four families (Poxviridae, Herpesviridae, Parvoviridae, Paramyxoviridae), a larger grouping called subfamilies has been defined ( -Virinae)  Virus orders may be used to group virus families that share common characteristics ( -Virales) mononegavirales, Nidovirales, Caudovirales.

ICTV: International Committee on Taxonomy of Viruses By 2005, the International Committee on Taxonomy of Viruses had organized more than 5500 known viruses into 73 families, 9 subfamilies, and 287 genera and 3 order, with hundreds of viruses still unassigned. Currently, 24 families contain viruses that infect humans and animals.

Viroids: Prions: NORMAL PrP C PRION PrP SC

Electron microscopy, cryoelectron microscopy, and x-ray diffraction techniques have made it possible to resolve fine differences in the basic morphology of viruses.

The Shape of Viruses Cubic symmetry (Icosahedral ) DNA viruses RNA viruses

Helical Symmetry In animals, Only RNA viruses TMV

Complex Structure

 Direct Observation in the Electron Microscope  Sedimentation in the Ultracentrifuge  Comparative Measurements

Viral Proteins Viral lipids (Envelope) Sugars Viral Nucleic acids Structural Proteins Enzymes & Factor

Nucleic Acid DNA RNA Double Stranded (DS) Positive Sense Negative Sense RNADNA Single Stranded (SS) Double Stranded (DS) Single Stranded (SS) Virus Genomes

Positive Sense = (+) Sense: Viral RNA acts as an mRNA Within the infected cells… Picornaviridae, Flaviviridae, Togaviridae, Caliciviridae, Coronaviridae, Arteriviridae, Astroviridae, and Retroviridae. Negative Sense = (-) Sense: Viral RNA is complementary to mRNA. Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Bornaviridae.

Group I Like : Adenoviridae, Herpesviridae, Poxviridae, Hepadnaviridae,Papilloma viridae, Polyomavridae Group II : Parvoviridae, Circoviridae Group III: Reoviridae, Birnaviridae Group IV: Picornaviridae, Flaviviridae, Togaviridae, Coronaviridae, Caliciviridae, Astroviridae Group V: Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Arenaviridae, Bonyaviridae, Bornaviridae Group VI: Retroviridae

Heat & Cold Stabilization of Viruses by Salts pH Radiation Photodynamic Inactivation Ether Susceptibility Detergents Formaldehyde Antibiotics & Other Antibacterial Agents

Sterilization: steam under pressure, dry heat, ethylene oxide, and gamma irradiation Vaccine production: use of formaldehyde, β- propiolactone, psoralen + ultraviolet irradiation, or detergents (subunit vaccines) to inactivate the vaccine virus. Surface disinfectants: sodium hypochlorite, glutaraldehyde, formaldehyde, and peracetic acid. Skin disinfectants: include chlorhexidine, 70% ethanol, and iodophores

Attachment

 Rhinovirus: ICAM-1  EBV: CD21=CR 2  Polio: PVR= CD 155  HIV: CD 4  ….. Definition: Susceptibility & Permissivity

Penetration or Engulfment Penetration of the target cell normally occurs a very short time after attachment of the virus to its receptor in the cell membrane. Three main mechanisms are involved in penetration: Three main mechanisms are involved in penetration: Receptor-mediated endocytosis (or Viropexis) Fusion Direct penetration of virus particles across the plasma membrane (or Translocation)

clathrin-independent endocytosis

Endocytosis ( Viropexis)

Fusion

Direct penetration

Uncoating  Uncoating is the physical separation of the viral nucleic acid from the outer structural components of the virion.  often, the genome is released as free nucleic acid. Or rarely, as a nucleocapsid (reoviruses).  Eclipse period:…  Indeed, Viruses are the only infectious agents for which dissolution of the infecting agent is an obligatory step in the replicative pathway.

Expression of Viral Genomes and Synthesis of Viral Components

maturation phase may occur before or after release

Assembly Newly synthesized viral genomes and capsid polypeptides assemble together to form progeny viruses. Icosahedral capsids can condense in the absence of nucleic acid, whereas nucleocapsids of viruses with helical symmetry cannot form without viral RNA. Release  Nonenveloped viruses accumulate in infected cells, and the cells eventually lyse and release the virus particles.  Enveloped viruses mature by a budding process.

1.Electron microscopy 2. Serological assays (ELISA, IF, CF, NT, RIA…) 3.molecular assays: NAT: (PCR, RT-PCR, NASBA, Real Time-PCR, LAMP…) 4.Viral cultivation:  Laboratory Animals  Embryonated chicken egg  Cell culture

1.H2O2: Oxidation agent 2.chromogenic substrates (e.g. TMB, DAB, ABTS) 3.HRP

1) Denaturation 5`3` 5` 94°C 3` 5`

2) Aannealing 3` 5` 3` HO 5`OH 3` 55°C or 60°C or 50°C or …

3) Extention 3` 5` 72°C 5` 3` 5` 3`

94°C 55°C 72°C D DD A A A E E Cycle 1Cycle 2

Taq DNA polymerase: obtained from Thermus Aquaticus archibacteria Pfu (Pyrococcus furiosus,1 in 1.3 million error rate ) Pwo ( Pyrococcus woesei, blunt end ) Vent Tth …

Hosts for Virus Cultivation Laboratory Animals Embryonated Chicken Eggs Cell Culture

Laboratory Animals

Embryonated Chicken Eggs SPF: Specific Pathogen Free

Cell culture Primary Cell Culture Cell Strains (Diploid cell culture) Cell Lines (continuous cell culture)

Primary Cell Culture

Subculture: Passage

Detection of Virus-Infected Cells Development of cytopathic effects (CPE) :  cell lysis or necrosis  giant cell formation ( Syncytia ) Viral Ag detection (serologically) Viral NA detection (Molecular) Hemadsorption Inclusion Body Formation

Negri bodies in a rabies infected cell Inclusion bodies Guarnieri bodies

Recombination Reassortment Genetic reactivation Interference

Translational Strategies in Viruses

Ribosomal Shunting 1.Adenovirus 2.Papillomavirus 3.HBV 4.Sendi virus