Introduction to Viruses (Part 2) PDF

Introduction to Viruses (Part 2) Khristine Kaith S. Lloren, DVM, MSc College of Veterinary Medicine and Agricultural Sciences De La Salle Araneta University Structure of Viruses Virion A fully assembled infective virus Fundamental component is a nucleoprotein cor...

Introduction to Viruses (Part 2) Khristine Kaith S. Lloren, DVM, MSc College of Veterinary Medicine and Agricultural Sciences De La Salle Araneta University Structure of Viruses Virion A fully assembled infective virus Fundamental component is a nucleoprotein core with the ability to infect host cells and replicate in them, thus ensuring continued survival Capsid Shell of proteins that encloses the genome of vertebrate viruses Nucleocapsid is used to describe the packaged form of the genome in the capsid Two types of capsid symmetry: Icosahedral Helical Envelope In many types of viruses the nucleocapsid is covered by an envelope composed of a lipid bilayer and associated glycoproteins It is acquired when the nucleocapsid buds through a cellular membrane, usually the plasma membrane The glycoproteins are associated with binding to receptors on host cells, membrane fusion, uncoating of the virion and destruction of receptors on host cells Envelope Epitopes on envelope glycoproteins are often important for inducing protective immune responses in infected animals Capsomere Used to describe features such as protrusions seen on the surface of virus particles in electron micrographs Baltimore Classifications Based on the nature of the genome and the pathways of mRNA synthesis 6 Classes -> 7 Replication of Viruses Replication of viruses Viruses can multiply only in host cells They utilize host cell organelles, enzymes and other macromolecules for replication Effect of viral replication (changes in cellular metabolism to cytolysis) In infected cell cultures, the duration of the cycle may range from six to 40 hours. Virus replication stages 1. Attachment to a surface receptor on a susceptible host cell 2. Entry into the cell 3. Uncoating of viral nucleic acid 4. Replication of viral nucleic acid and synthesis of virus-encoded proteins 5. Assembly of newly-formed virus particles and release from host cell 1. Attachment Initial virus-cell interaction is a random event which related to the number of virus particles present and the availability of appropriate receptor molecules Virus-cell interaction determines both host range and tissue tropism of viral species Some viruses have more than one type of ligand molecule and may bind to several cell surface receptors 1. Attachment In some viruses, individual virions can detach and adsorb to another cell in orthomyxoviruses and paramyxoviruses, the detachment is mediated by viral neuraminidase, a receptor-destroying enzyme 2. Entry into the cell Virus uptake or penetration is an energy-dependent process Receptor-mediated endocytosis Clathrin Fusion of viral envelope with the plasma membrane 3. Uncoating The process whereby the viral genome is released in a form suitable for transcription In case of enveloped viruses, in which the nucleocapsid is discharged directly into the cytoplasm, transcription can usually proceed without complete uncoating Some viruses which replicate in the cell nucleus, uncoating may be completed at the nuclear pores 4. Synthesis of Viral Proteins It is the central event in replication of viruses Requires the production of viral mRNA For DNA viruses, which replicate in the nucleus, can avail of host cell transcriptases to synthesize viral mRNA Some viruses utilize their own enzymes to generate mRNA 4. Protein Synthesis Membrane proteins and glycoproteins are synthesized on membrane-bound ribosomes Soluble proteins such as enzymes are synthesized on ribosomes free in the cytoplasm 4. Protein Synthesis Most viral proteins undergo post-translational modification including proteolytic cleavage, phosphorylation and glycosylation In glycolysation, sugar side-chains are added to viral proteins in a programmed manner as the proteins are being transferred from the RER to Golgi apparatus in preparation to final assembly and release from the cell 5. Assembly and Release of Virion The assemble and release of enveloped and non- enveloped viruses are distinct Non-enveloped viruses of animals have icosahedral structure These non-enveloped viruses are usually released following cellular disintegration 5. Assembly and Release of Virion Assembly: Picornaviruses, Reoviruses – occurs in the cytoplasm of the cell Parvoviruses, Adenoviruses and Papovaviruses – assembled in the nucleus Picornavirus Non-enveloped Single-stranded RNA virus 5. Assembly and Release of Virion In enveloped viruses Final step in the process of virion assembly involves acquisition of an envelope by budding from cell membranes Prior to budding, cell membranes are modified by the insertion of virus-specified transmembrane glycoproteins, which aggregate in patches in the plasma membrane 5. Assembly and Release of Virion In enveloped viruses Many are non-cytopathic and may be associated with persistent infections But togaviruses, paramyxoviruses and rhabdoviruses are cytolytic Rhabdovirus Enveloped Single-stranded RNA virus 5. Assembly and Release of Virion In enveloped viruses Flaviviruses, coronaviruses, arteriviruses and bunyaviruses acquire their envelopes inside cells by budding through the membranes of the RER or the Golgi apparatus and then transported in vesicles to the cell surface where the vesicle fuses with the plasma membrane releasing the virion by exocytosis 5. Assembly and Release of Virion In enveloped viruses Herpesvirus are unique Replicate in the nucleus They bud through the inner lamella of the nuclear membrane and accumulate in the space between inner and outer lamellae, in the cisternae of the ER and in cytoplasmic vesicles Released by exocytosis or by cytolysis Herpesvirus Enveloped Double-stranded DNA virus 5. Assembly and Release of Virion In enveloped viruses Poxviruses Assemble and release is a complex process taking several hours Following assembly, virus particles move out of the assembly area and become enveloped in a double membrane derived from the trans golgi network At the periphery of the cell, fusion with the plasma membrane results in loss of the outer layer of the double membrane and release of extracellular enveloped virus Laboratory Diagnosis of Viral Infections Laboratory Diagnosis of Viral Infections Viral diseases of animals can be diagnosed on basis of clinical signs together with postmortem finding and histopathological changes Surveillance is also an important aspect of management Confirmation often requires special laboratory procedures Collections of samples: Oropharynx or nasopharyngeal aspirates – suitable for respiratory diseases Virus particles shed in feces – for enteric viral disease Cells of the buffy coat – viraemia Specimens for virus isolation should be collected into transport medium (consists of buffered isotonic saline with bovine albumin), refrigerated and sent to the laboratory without delay (should be frozen at -70C if deliver is delayed) Detection of virus, viral antigens or nucleic acid: 1. Isolation of live virus Using cell culture, fertile eggs or experimental animals A sensitive procedure when cultural conditions are optimal for a particular virus Number of blind passages may be required before a virus becomes adapted to a particular cell line Detection of virus, viral antigens or nucleic acid: 2. Electron microscopy This method can be used to recognize mixed viral infections and detect viruses which cannot be grown in vitro Has limitations (e.g. large numbers of viral particles must be present in clinical samples) Detection of virus, viral antigens or nucleic acid: 3. Immunofluorescence and Immunohistochemistry Antiviral antibodies labelled with fluorochromes can be used to detect viral antigens in clinical specimens Can be used to demonstrate virus or viral antigen in specimens Detection of virus, viral 4. Immunodiffusion antigens or nucleic acid: Carried out in agar Placing a fluid sample containing the virus under test in a well in the agar opposite a well containing antiserum Fluids diffuse out of the wells, a line of precipitate forms if the sample under test contains viral antigen  Detection of virus, viral antigens or nucleic acid: 5. Hemagglutination and hemadsorption Viruses such as Orthomyxoviridae, paramyxoviridae, adenoviridae and parvoviridae and togaviridae can interact with erythrocytes of many animal species causing hemagglutination Hemadsorption is used to describe binding of erythrocytes to cells infected with hemagglutinating viruses Detection of virus, viral antigens or nucleic acid: 6. Polymerase chain reaction (PCR) In vitro method is based on the amplification of a particular nucleic acid sequence using a thermostable DNA polymerase and primers Detection of virus, viral antigens or nucleic acid: 7. Diagnostic serology Enzyme-linked immunosorbent assay Serum neutralization test Hemagglutination inhibition test Complement fixation tests Western Blotting