General Virology PDF

General characteristics of viruses  VIRUSES o Viruses are obligate intracellular parasites o Viruses lack the capacity to make energy or substrates, cannot make their own proteins, and cannot replicate their genome independently of the host cell. o Viruses...

General characteristics of viruses  VIRUSES o Viruses are obligate intracellular parasites o Viruses lack the capacity to make energy or substrates, cannot make their own proteins, and cannot replicate their genome independently of the host cell. o Viruses are small in size < 300 nm Transmission electron microscope (TEM): the image is produced by the electrons transmitted by an ultra-thin specimen Scanning electron microscope (SEM): electrons are reflected point-to-point from the surface of a sample  VIRUS and VIRION The virion (the viral particle) consists of: o a nucleic acid, DNA or RNA (but not both) → genome nucleocapsid o a protective shell of protein → capsid o a membrane (some viruses) → envelope (or pericapsid) o [viral enzymes or other proteins essential for replication] VIRUSES ARE DIFFERENT IN SIZE AND MORPHOLOGY  VIRUSES HAVE DIFFERENT GENOMES DNA viruses - desoxyriboviruses RNA viruses - riboviruses Segmented genome  CENTRAL DOGMA OF MOLECULAR BIOLOGY THE INFORMATION FLOW IN VIRUSES CAN BE REVERSED DNA polymerase – RNA dependent RNA polymerase – RNA dependent  VIRAL STRUCTURE The capsid is a rigid structure made up of proteins. Individual proteins associate into progressively larger units (subunits), which associate into protomers, capsomers and finally in capsid.  VIRAL STRUCTURE The simplest viral structures that can be built stepwise are symmetric and include helical and polyhedral structures. Nonsymmetric capsids are complex forms and are associated with certain bacterial viruses (phages).  VIRAL STRUCTURE The envelope is an external coating to the capsid, composed of lipids and glycoproteins: o the lipid part of the envelope derives from the membrane of the infected cell o the protein part of the envelope is virus-specific and derives from the insertion of viral glycoproteins in the lipid layer. The tegument or matrix is the space between the inner face of the envelope and the nucleocapside. It is usually occupied by virus-specific proteins.  VIRAL REPLICATION The replication cycle of each virus depends on the host cell → cell acts as a factory, providing the substrates, energy, and machinery necessary for the synthesis of viral proteins and replication of the genome. Viral infectious cycle occurs in susceptible and permissive cells: o a susceptible cell has a functional receptor for a given virus – the cell may or may not be able to support viral replication o a permissive cell has the capacity to replicate the virus - it may or may not be susceptible; a semipermissive cell do not support all the steps in viral replication resulting in an inefficient replication If cells are both susceptible and permissive, the infection is PRODUCTIVE If cells are susceptible but not permissive, the infection is ABORTIVE If cells are sensitive but semipermissive, the infection is RESTRICTIVE If cells are both susceptible and permissive but the viral genome is maintained silently in the cell, the infection is LATENT, with possible REACTIVATION  DIAGRAM OF THE INFECTIOUS CYCLE When the nucleic acid is uncoated, infectious virus particles cannot be recovered from the cell - this is the start of the ECLIPSE phase - which lasts until new infectious virions are formed THE MAJOR STEPS IN VIRAL REPLICATION ARE THE SAME FOR ALL VIRUSES  VIRAL REPLICATION Steps 1-2: Recognition of target cell and attachment to the target cell The initial contact between the virion and the host cell is the result of random collisions. In order for a collision to result in an efficient attack, it is necessary that an anti-receptor at the surface of the virion collides with a receptor (and a co-receptor) on the cell membrane. The binding of the viral anti-receptor to cellular receptor determines which cells can be infected by a virus, defining the host range and the tissue tropism. cell types which a particular virus cells and tissues of a host that support is able to infect growth of a particular virus The anti-receptors are capsid proteins in naked viruses, and glycoproteins in the enveloped viruses. The receptors and co-receptors are proteins or carbohydrates on glycoproteins or glycolipids on host cell membrane. The co-receptors are as important as receptors for the biological process of viral entry. HIV ENTRY IN THE HOST CELL  VIRAL REPLICATION Step 3: Penetration o non-enveloped viruses may cross plasma membrane directly (pore-mediated penetration) or enter the cell by receptor-mediated endocytosis o enveloped viruses fuse their membranes with cellular membranes to deliver the nucleocapsid or genome directly into the cytoplasm, or enter the cell by receptor-mediated endocytosis and, then fusion occurs in the endosome.  VIRAL REPLICATION Step 4: Uncoating o the uncoating program involves a stepwise process with a final step → the release of the genome from a protective, confining capsid structure o The details of uncoating are highly variable depending on the nature of the virus and the cell, and they are not completely understood Step 5: Macromolecular synthesis o replication phase of the genome by using cellular or viral enzymes → many replicative strategies are used o transcription to mRNA, and virus protein synthesis: non-structural (functional) and structural viral proteins are synthesized Viruses depend on the host cell for energy supply and low molecular weight precursors necessary for the synthesis of viral macromolecules, as well as for the supply of structures (ribosomes) and of enzymes necessary for protein synthesis VIRAL REPLICATIVE STRATEGIES Baltimore system  VIRAL REPLICATION Step 6: Assembly o new viral particles are assembled from the genome copies and viral proteins: newly synthesized capsid proteins come together to form capsomers, which interact with other capsomers to form the full-sized capsid. Some viruses build the capsid around the viral genome Some viruses first assemble an “empty” capsid and then the genome inside is packaged.  VIRAL REPLICATION Step 7: Exit/release o cytolysis: the newly formed virions accumulate in the assembly site, forming clusters. Viruses are released through cell lysis. o budding: most enveloped virions escape from the infected cells through cellular membranes (plasma membrane, nuclear membrane, endoplasmic reticulum membrane), thus acquiring their envelopes. RELEASE OF A VIRUS BY BUDDING VIRAL CYTOPATHOGENESIS  VIRAL INFECTION CAUSES CYTOPATHIC EFFECT OF CELLS Any detectable changes in the host cell due to viral infection is known as a CYTOPATHIC EFFECT (CPE). CPE may consist of cell rounding, swelling or shrinking, detachment from the surface, cytoplasmic or nuclear inclusion, syncytium formation and others Nuclear and/or cytoplasm inclusion Syncytia formation VIRAL INFECTION CAUSES CYTOPATHIC EFFECT OF CELLS Cytomegaly Uninfected cells Infected cells Cell rounding Uninfected cells Infected cells  VIRAL CULTURES Viruses are obligate intracellular parasites that require a host cell (susceptible and permissive) for the replication. They can be grown in: o Embryonated eggs → still used for the growth of virus for some vaccines o Experimental animals → rarely used in clinical laboratories for the purpose of isolating viruses o Tissue culture cells → constitute the only system for virus isolation  TISSUE CULTURE CELLS Basic medium: Culture conditions: 37°C, in a humidified atmosphere containing 5% CO2 FBS supplies growth Antibiotics prevent promoting factors bacteria contamination Tissue flasks Well culture tissue plates  TISSUE CULTURE CELLS Primary cell cultures are obtained by dissociating specific animal organs with trypsin or collagenase. The cells are then grown as monolayers (fibroblast, epithelial) or in suspension (lymphocyte, erythrocyte) in artificial media supplemented with bovine serum or another source of growth factors. Primary cultures usually have a limited life span because of the contact inhibition and because they undergo senescence. Primary cells can be dissociated with trypsin, diluted, and allowed to grow into new monolayers (passed) to become secondary cell cultures. The process for obtaining primary cells is labor-intensive and time-consuming  TISSUE CULTURE CELLS Secondary cell cultures refer to cultures formed after subculturing of primary cell cultures. They contain a single cell type, they are a more homogeneous cell population. Continuous cell lines derived from transformed cells or tumors, they are often able to be subcultured many times or even grown indefinitely (in which case they are called immortal). TISSUE CULTURE CELLS  DIAGNOSIS OF VIRAL INFECTION o Culture-based systems slow time-consuming labor-intensive poorly sensitive o Electron microscopy expensive not sensitive enough HIV Virus Ebola HBV  DIAGNOSIS OF VIRAL INFECTION o Molecular methods Detection of viral nucleic acids by Polymerase Chain Reaction SCHEMATIC DRAWING OF A COMPLETE PCR CYCLE  DIAGNOSIS OF VIRAL INFECTION o Serology: detection of virus-specific antibodies by immunoenzymatic tests ELISA (enzyme-linked immunosorbent assay)  ANTIVIRAL DRUGS Viruses are obligate intracellular parasites that use the host cell's biosynthetic machinery and enzymes for replication → difficult to inhibit viral replication without being toxic to the host! Most antiviral drugs are targeted toward viral-encoded enzymes or structures of the virus that are important for replication. Some antiviral drugs are actually stimulators of the host innate immune protective responses (interferons). Unlike antibacterial drugs, the activity of most antiviral drugs is limited to a specific virus (narrow spectrum of activity). As occurred with antibacterial drugs, resistance to antiviral drugs is becoming a problem, because of the high rate of mutation for viruses and the long-term treatment of some patients. ANTIVIRAL DRUGS  ANTIVIRAL DRUGS Viral attachment and entry o Docosanol is a saturated 22-carbon aliphatic alcohol; it inhibits fusion between the plasma membrane and the herpes simplex virus envelope, thereby preventing viral entry into cells and subsequent viral activity and replication. Docosanol is used topically in the treatment of recurrent herpes simplex labialis episodes.  ANTIVIRAL DRUGS Virion uncoating o Amantadine, rimantadine only have activity against influenza A. Both drugs block the influx of H+ through the M2 proton channel inhibiting the uncoating and release of the ribonucleoproteins into the cytoplasm.  ANTIVIRAL DRUGS Viral replication (DNA-RNA replication) o Nucleoside analogs are nucleoside with modifications of the base, sugar, or both → they become incorporated into the viral genome leading to the termination of the viral genome chain, causing errors in replication (mutation) or transcription (inactive mRNA and proteins) NUCLEOSIDE ANALOGS  ANTIVIRAL DRUGS Viral assembly o Saquinavir, ritonavir, and indinavir are inhibitors of the HIV proteases The HIV proteases are essential to the assembly of virions, and the production of infectious virions. Inhibitors of HIV proteases are blocking proteolytic cleavage of protein precursors that are necessary for the production of infectious virions  ANTIVIRAL DRUGS Viral release o Zanamivir and oseltamivir are neuroaminidase inhibitors that prevent new viral particles release (influenzaviruses A and B) from the infected cells.  INTERFERONS (IFNs) a group of signaling proteins produced by the body’s cells as a defensive response to viruses. They are important modulators of the immune system. genetically engineered recombinant interferons are being used to treat some viral infections  HOST DEFENSES AGAINST VIRAL INFECTION The antiviral immune response can be divided into an early, nonspecific phase involving innate immune mechanisms, followed by a later, antigen-specific phase involving adaptive immunity by T and B cells.  INNATE RESPONSES INTERFERONS are the body’s first active defense against a viral infection, an “early warning system” inducing an antiviral state. TYPE I INTERFERONS  TYPE I INTERFERONS 2’,5’- oligoadenylate Protein kinase R synthetase  TYPE I INTERFERONS IFN-I induces protection of cells for a relatively short period of time and represents the innate immunity against viruses. At the same time IFN-I stimulate adaptive immune response against viral antigens by stimulation of antigen presentation in dendritic cells and by stimulation of B- and T-cells. This results in enhanced production of antigen specific antibodies and cytotoxic-T cells (for long term protection)