Lecture 10: Structure of Viruses PDF

LECTURE 10 Structure of Viruses Ms. Peters  Introduction A virus is an Obligate Intracellular parasite- containing genetic material surrounded by protein. They are acellular; has no cytoplasm, organelles neither can they metabolizes on their own Most viruses rang...

LECTURE 10 Structure of Viruses Ms. Peters  Introduction A virus is an Obligate Intracellular parasite- containing genetic material surrounded by protein. They are acellular; has no cytoplasm, organelles neither can they metabolizes on their own Most viruses range in sizes from 20 – 300 nanometers. Have receptors - binding proteins that allow them to attach to cells Viruses are much smaller than bacteria  Introduction “Filterable agents” – Can pass through filters that can hold back bacteria. Vary widely in size: – Largest – Poxvirus ( 300 nm ) –DNA Virus. – Smallest – Parvovirus ( 20 nm )- DNA Virus They are not readily destroyed by antibiotics Can be observed by an Electron Microscope.  Introduction Viral Shapes: – Spherical -: E.g., Poliovirus – Rod-shaped -: E.g., Tobacco mosaic virus. – Brick - shaped -: E.g., Pox virus – Bullet-shaped -: E.g., Rabies virus  Structure and Morphology Nucleus → Nucleic acid core (genome) + RNA or DNA Capsid - The protein coat surrounding the nucleic acid core  Structure and Morphology Functions of the capsid includes: – Protects nucleic acid from inactivation – Helps to introduce viral genome into host cell Nucleus & Capsid → Nucleocapsid. The outer shell composed of repeating protein subunits blocks that make up the capsid are called capsomers Two types of Capsomers form the Capsid. – Pentagonal capsomers form the vertices. – Hexagonal capsomers form the Sides The polypeptide chains which make up the capsomers are called protomers  Structure and Morphology Protein subunits projecting as spikes on the surface of envelope are called Peplomer Peplomers confers chemical , antigenic & biological properties. They are very susceptible to lipid solvents An envelope may or may not be present. The envelope is derived from the host cell membrane and are lipoproteins in nature The lipid part originates from the host cell origin while the protein from virus.  Structure and Morphology Geometric Shapes (nucleocapsid symmetry): – Helical symmetry – Icosahedral pattern (cubical symmetry ) – Complex Icosahedral – A polygon with 12 Vertices and 20 Facets. The sides are equilateral triangles Helical – The capsomers & nucleic acid are wound together to form a helical or spiral tube Complex - The overall shape of virus is quite variable , but mostly they are spherical, or brick shaped ( Pox-virus ) Structure and Morphology  Viral Replication Steps in viral replication: 1) Recognition of host cell / target cell 2) Attachment of virus particle to the cell surface 3) Penetration into host cells 4)Uncoating of virus of its outer layer and capsid 5) Transcription of mRNA from viral nucleic acid 6) Translation of mRNA into early -Protein synthesis 7) Replication of viral nucleic acid 8) Assembly of virus in the nucleus / cytoplasm: Budding of enveloped Viruses => Lysis & Release of virus  Viral Replication Eclipse phase – From penetration until the appearance of mature daughter virions , Virions cannot be detected inside the host cell. The duration of eclipse phase or replication time is about 3 - 12 hours for most of the viruses  Cultivation of Viruses Virus cannot be cultivated on chemically defined media or artificial culture media which does not contain live cells Viruses can be grown in vivo or in vitro i.e., in cells in an artificial environment such as a test tube or cell culture flask Cultivation of viruses is complex & includes three methods: Animal Inoculation Embryonated Egg Culture Cell Culture or Tissue Culture.  Cultivation of Viruses Animal: Animal virus cultivation is important for: Identification and diagnosis of pathogenic viruses Production of vaccines Basic research studies Herpes simplex virus :Rabbit cornea (Vesicles) Rabies virus :White Mouse (baby/adult): Intracerebral 1 – 3 weeks Encephalitis / Rabies. Dengue virus : Baby White Mouse (1-3 days old) Intracerebral / Subcutaneously 3 – 7 days => Tremor , Paralysis. Polio virus : Monkey Intracutaneously , IM , Intraneural , Intraspinal Paralysis.  Cultivation of Viruses Embryonated Egg The embryo serves as an incubator for viral replication. The location within the embryo is important Chorioallantoic membrane (CAM): 10 – 12 days old embryo. E.g.: Pox virus , HSV. Amniotic cavity : 10 - 11 days old embryo E.g., :Influenza virus , Mumps virus. Allantoic cavity : 10 days embryo. can be propagated in much larger quantities, used for vaccine production eg: Influenza , Rabies vaccine.  Cultivation of Viruses Embryonated Egg Yolk sac : 3 – 8 days old embryo E.g.: Chlamydiae & Ricketssia. Intraembryonal : 8 – 10 days old embryo E.g.: Japanese B encephalitis virus  Cultivation of Viruses Cell or Tissue Culture Used for the isolation , neutralization , production of antigen for serological assay. Three basic types of cell culture : Primary culture :- Made by dispersing cells from freshly removed host tissues , unable to grow for more than a few passage. e.g:- Rhesus monkey kidney cell & Human amnion cell culture. Secondary / Diploid culture :- Diploid cell lines up to 50 passage e.g.,:- Human embryonic lung cell culture. Continuous cell line / Heteroploid cell culture :- Capable of prolonged , Infinite growth , derived from cancerous cells. e.g:- HeLa cell -from cancer of cervix , Hep-2 cell - epithelioma of larynx cell ; Vero cell -Vervet monkey kidney  Detection of Virus Growth in Cell line Cytopathic effect-CPE : Morphological change produced by virus in cell line detected by light inverted microscope. Rounding & ballooning of cell line - HSV. Hemadsorption : Influenza virus ; Mumps virus –hemagglutinins that bind some erythrocytes – Hemagglutination. Interference : Growth of non-CPE virus in cell culture can be detected by subsequent challenge to cell line with known CPE virus. Transformation : Oncogenic viruses – leads to surface growth that appears in a piled-up fashion producing microtumors on cell line. Immunofluorescence : Direct immunofluorescence using specific antibodies is used to detect viral antigens in inoculated cell lines for identification of viruses. Electron Microscopy : To detect viral particle in infected cell lines.  Detection of Virus Growth in Cell line Cytopathic effect-CPE : Unaffected cell line Respiratory syncytial virus-Syncytium or Multinucleated giant cell formation. Hemadsorption  Detection of Virus Growth in Cell line In virus multiplication within infected cells, specific structure called inclusion bodies may be produced. They become far larger than the individual virus particle. The inclusion bodies can be present in:- Nucleus E.g., Herpes virus , Poliovirus , Influenza virus , Yellow fever virus , CMV Cytoplasm E.g., Poxvirus , Rabies virus. Both E.g., Adeno virus & Measles virus  Detection of Virus Growth in Cell line Intra-nuclear Inclusions are mostly found in DNA viruses E.g., CMV Cowdry type A - Variable size, granular appearance. E.g., Yellow Fever Cowdry type B - Circumscribed , multiple. E.g., Polio Intra-cytoplasmic Inclusions are mostly RNA viruses E.g., Guarneri bodies in smallpox, Negri bodies in Rabies. Intra-nuclear & Intra-cytoplasmic Inclusion E.g.,Measles, Adenovirus Intranuclear & Intracytoplasmic , mostly basophilic : Adenovirus. Owl eye appearance ( Intra-nuclear ) – CMV. Cytomegalovirus.  Detection of Virus Growth in Cell line Molecular Diagnosis Recently Molecular methods are used for Rapid viral diagnosis. These include: DNA probe Highly sensitive & specific. Commercially DNA probes are available for detection of CMV, Papillomavirus & Epstein-Barr virus from in clinical specimens. Polymerase Chain Reaction (PCR) This method is useful for diagnosis of HIV, CMV, HSV and Hepatitis- B, C & D Viruses.  Detection of Virus Growth in Cell line Viral Serology Detection of specific viral antibodies in serum of infected patient Methods include: ELISA – Enzyme Linked Immunosorbent Assay - Most used test for many viral infections - commercially available kits e.g., - HIV , Hepatitis B , etc. Hemagglutination inhibition ( HI ) test –used for detection of viruses that agglutinate RBCs of guinea pigs , chickens , human – e.g., Orthomyxoviruses ( Influenza Viruses ). RIA – Radio ImmunoAssay Latex Agglutination Test Western blot.  Bacteriophage Viruses that infect only bacteria. Has a high Host specificity and are able to pass through bacterial filters Structure includes: A polyhedral capsid (head) Nucleic acid Helical sheath Tail fibers Baseplate and pin. Most common habitat is the intestinal tract of animals. Bacteriophage are mostly needed for Bacterial identification Act as carrier of genes from one bacterium to another.  Bacteriophage Depending on method of replication phages can be classified as: Virulent – those that replicates via the lytic pathway Temperate – Those that replicate via both lytic and lysogenic pathway  Bacteriophage Phage Typing Commonly used to sub type a species or genus e.g., Salmonella spp. Vibrio spp. It uses lytic phages inoculated on a lawn of bacteria being studied The phages that can infect the specific bacteria via the lytic pathway produces a zone of clearing (plaque formation) Identification is based on the premise that the ability of different phages to infect bacteria varies between different strains of bacteria. They are compared against a standard scheme of lysis patterns to assign a type to the strain Increasing being replaced by molecular methods such as whole genome sequencing  Bacteriophage Phage Typing Advantages include: It is fast and affordable Can extensively be used for epidemiological surveillance and for investigating outbreaks Gold stand method for the surveillance of S. typhi Disadvantages include: Requires technical expertise Maintenance of the phages is time consuming and expensive Since several phages are required, it is not done at routine diagnostic laboratories  Bacteriophage Phage Typing  Homework Explain in short notes what occurs during each step of viral replication outlined on slide 10 Read up on the immortal cell line of Henrietta Lacks