Introduction to Virology PDF

Introduction to Virology Course Instructional Objectives MCRO 2.1 The student will identify and explain the components of a typically virus, including the five basic types of viral symmetry and symmetry are seen in ‘classic’ virus particles. MCRO 2.2. The student will describe the receptors and VA...

Introduction to Virology Course Instructional Objectives MCRO 2.1 The student will identify and explain the components of a typically virus, including the five basic types of viral symmetry and symmetry are seen in ‘classic’ virus particles. MCRO 2.2. The student will describe the receptors and VAP found on a naked and enveloped virus & virus family, including their function. MCRO 2.3. The student will explain the viral life cycle including the role of T-cell activation and phagocytosis of the human immune system. MCRO 2.4 The student will identify the classification of DNA vs RNA viruses, include their common name and family name. MCRO 2.5. The student will understand the basics of serology, viral culture, cytology and the molecular techniques used to identify virus in the lab. Khan Academy: Viral structure & classification MCRO 2.1 Components of a Virus Viral Component Role in Viral Life Cycle Example Encodes all the information necessary to produce new progeny virions DNA or RNA Protein “shell” that contains/packages viral nucleic acid, protecting it between infections; may contain VAP Icosahedral, helical, or (rarely) complex Structural proteins Proteins which form the capsid, package the genome, and/or are attachment proteins Matrix, nucleocapsid, VP1-VP4 (reovirus), hexon and fiber (adenovirus), gp41/120 (HIV), HA (influenza), F (measles) Non-structural proteins Proteins which are required for replication, for Polymerase, helicase, protease assembly, or which facilitate disease (flu N), transcription factors, progression immunomodulatory factors Envelope Lipid bilayer which is an anchoring surface for viral attachment proteins, facilitates penetration of the host cell membrane Nucleic acid Capsid Red strings Small inner green and blue spheres Capsid + outer green stalks and large blue spheres Black beads Orange coat MCRO 2.1 Viral Capsids and Symmetry • Most viruses have either icosahedral or helical capsid structure with or without an envelope • The others have: – Complex symmetry – No symmetry • Capsid or nucleocapsid = procapsid AND viral genome http://pathmicro.med.sc.edu/mhunt/intro-vir.htm MCRO 2.1 “Classic” Viral Particles Adenovirus Coronavirus Figure 47-1 – Medical Microbiology, 9th ed. 2021 Rhabdovirus (rabies) Figure 42-1 – Medical Microbiology, 9th ed. 2021 Poxvirus Murray, Rosenthal, and Pfaller – Medical Microbiology, Figure 50-1, 9th ed. 2021 Filovirus (Ebolavirus) Figure 50-4 – Medical Microbiology, 9th ed. 2021 Figure 44-1 – Medical Microbiology, 9th ed. 2021 MCRO 2.1 More Images of Icosahedral Capsids The construction of a spherically (icosahedral) shaped virus similarly involves the packing together of many identical subunits, but, in this case, the subunits are placed on the surface of a geometric solid called an icosahedron. Because the icosahedron belongs to the symmetry group that crystallographers refer to as cubic (not the cube shape), spherically shaped viruses are said to have cubic symmetry, generally known as icosahedral capsid. Read: Sherris & Ryan Chapter 6 Citation: Chapter 6 Viruses—Basic Concepts, Ryan KJ. Sherris & Ryan's Medical Microbiology, 8th Edition; 2022. Available at: https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260922934 Accessed: January 15, 2024 Copyright © 2024 McGraw-Hill Education. All rights reserved MCRO 2.1 Overview: Chapter 6 § Viruses are the smallest form of replicating intracellular microorganisms that are comprised of sets of genes either DNA (DNA viruses) or RNA (RNA viruses) packaged in a protein coat, capsid (naked capsid viruses) or in a nucleocapsid/capsid, and an outer lipid bilayer envelope (enveloped viruses). § Viruses have spikes on their outer surface that bind to the receptors on host cells and antibodies generated against the spikes neutralize the virus. § Viruses are dependent upon host structural components and metabolic functions. § DNA viruses replicate in the nucleus by using host RNA polymerase for transcription and either host or viral DNA polymerase for replication (exception are poxviruses that replicate in the cytoplasm). § RNA viruses replicate in the cytoplasm using its own viral RNA-dependent RNA polymerase for both transcription and replication (exception are influenza viruses and retroviruses that replicate in the nucleus). § Naked capsid viruses are assembled inside the cell and released upon cell death, whereas enveloped viruses acquire lipid bilayer membrane mainly from plasma membrane and in some cases from nuclear or cytoplasmic membranes. Overview: Chapter 6 § Viral-infected cells may result in cell death and tissue damage (pathology) generally seen in acute infections § A viral infection can persist in hosts (humans) causing a chronic or latent infection with little or no pathologic changes in target cells or tissues. § Since most viruses use their own enzymes (RNA or DNA polymerases) which could be a target for antivirals, they are prone to genetic changes due to lack of proofreading ability of these enzymes. § The major genetic changes that viruses undergo are mutation and recombination that allow viruses to escape the immune response and cause damage or persist in the host. § During viral latency, viral genome persists in host and may not be eliminated by antiviral drugs. § It is difficult to develop strategies to eliminate latent viral infections by antiviral drugs. Naked vs Enveloped Viruses • The basic structure of all viruses places the nucleic acid genome (DNA or RNA) on the inside of a protein shell called a capsid; these viruses have a defined external capsid and are referred to as naked capsid viruses. • The genomes of enveloped viruses form a protein complex and a structure called a nucleocapsid, which is often surrounded by a matrix protein that serves as a bridge between the nucleocapsid and the inside of the viral membrane or envelope Read : Sherris & Ryan Chapter 6 Citation: Chapter 6 Viruses—Basic Concepts, Ryan KJ. Sherris & Ryan's Medical Microbiology, 8th Edition; 2022. Available at: https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260922934 Accessed: January 15, 2024 Copyright © 2024 McGraw-Hill Education. All rights reserved MCRO 2.2 Enveloped Viruses The viral envelope lipid layer membrane contains virus-encoded glycoproteins called “spikes” or “peplomers” or “viral envelope proteins.” § Some enveloped viruses also have capsids between nucleocapsid and matrix protein. § Protein or glycoprotein structures called spikes, which often protrude from the surface of virus particles, are involved in the initial contact with receptor on host cells Read : Sherris & Ryan Chapter 6 Citation: Chapter 6 Viruses—Basic Concepts, Ryan KJ. Sherris & Ryan's Medical Microbiology, 8th Edition; 2022. Available at: https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260922934 Accessed: January 15, 2024 Copyright © 2024 McGraw-Hill Education. All rights reserved MCRO 2.2 Viruses and Receptors • Entry of virus mediated by binding of viral attachment protein or VAP to specific receptor on host cell – VAP generally a protein – Host receptor can be protein or carbohydrate • Location of VAP – Naked viruses – VAP is part of capsid structure – Enveloped viruses – VAP is inserted into viral envelope • Entry may require interactions between a 2nd set of viral and host molecules. Provides: – Increased affinity – Increased specificity • Second host molecule = co-receptor Herpes simplex virus and parvovirus B19 – see notes section for relevant virology characteristics MCRO 2.2 Viruses and Receptors FIGURE LEGEND – Herpes simplex virus (HSV) contains on its surface five glycoproteins that are used for its entry into host cells, gB, gC, gD, gH and gL. gB and gC mediate the initial attachment of virus particles to heparansulphate moieties on host cell-surface proteoglycans. gB binding to paired immunoglobulin-like type 2 receptor- (PILR) and gD binding to herpesvirus entry mediator (HVEM), nectin-1, nectin-2 or 3-Osulphotransferase-modified heparan sulphate trigger membrane fusion, which is mediated by gB and the gH–gL heterodimer, and release of the viral nucleocapsid into the host-cell cytoplasm. Viral-gene transcription occurs following the release of viral DNA into the cell nucleus. http://www.nature.com/nri/journal/v8/n11/full/nri2434.html MCRO 2.2 Viruses and Receptors FIGURE LEGEND – A model for parvovirus B19 binding and entry into primary human erythroid cells. Mature human RBCs, which express high levels of P antigen receptor, allow virus binding but not viral entry because they lack the α5β1 integrin coreceptor (A), whereas erythroid progenitor cells, which express both P antigen receptor and α5β1 integrin coreceptor, are permissive for parvovirus B19 entry (B). MCRO 2.2 http://www.bloodjournal.org/content/102/12/3927?sso-checked=true Classification Basics The International Committee for Taxonomy of Viruses (ICTV) considered various properties, including virions, genome, proteins, envelope, replication, and physical and biologic properties. § Virus families are designated with the suffix, -viridae (as in Herpesviridae) § Virus subfamilies with suffix -virinae (Herpesvirinae) § Virus genera with suffix -virus (Herpesvirus) § Virus species designated by a virus type (herpes simplex virus 1). MCRO 2.2 VAP and Cell Receptor Pairs Virus Family Structural Characteristics VAP Receptor Cell tropism HIV-1 Retroviridae ssRNA, positive sense, enveloped gp120 CD4 T cells, macs EBV Herpesviridae dsDNA, icosahedral, enveloped gp350, gp220 CD21 (CR2) B cells Rabies Rhabdoviridae ssRNA, negative sense, enveloped G protein Acetylcholine receptor Neurons and muscle Influenza Orthomyxoviridae ssRNA, negative sense, segmented, enveloped HA Sialic acid Respiratory epithelial cells Rhinovirus Picornaviridae ssRNA, positive sense, icosahedral, non-enveloped VP1,2,3 complex ICAM-1 Epithelial cells SARS-CoV-1 and SARS-CoV-2 Coronaviridae ssRNA, positive sense, enveloped Spike (E2) protein ACE2 (and other proteins) Many different cell types Adenovirus Adenoviridae dsDNA, icosahedral, nonenveloped Fiber protein CAR Many different cell types Parvovirus B19 Parvoviridae ssDNA, icosahedral, nonenveloped VP1,2 Erythrocyte P Ag (globoside) Erythroid precursor cells MCRO 2.2 & 2.4 Viral Life Cycle Khan Academy: Viral Replication MCRO 2.3 Reminder: Transcription vs Translation MCRO 2.3 Reminder: Transcription vs Translation MCRO 2.3 Entry by Direct Fusion Entry by direct fusion: § Some enveloped viruses enter cells by direct fusion mechanism. § Viral envelope proteins (spikes) bind to the receptors on the host cell followed by fusion of the viral envelope with the plasma membrane of the host cells, which is promoted by one of the viral envelope spikes (F protein of RSV and Gp41 of HIV). § After fusion, the nucleocapsid complex is released in the cytoplasm. § This mode of virus entry is seen in enveloped viruses such as paramyxoviruses, herpesviruses, and some retroviruses (HIV). Read : Sherris & Ryan Chapter 6 Citation: Chapter 6 Viruses—Basic Concepts, Ryan KJ. Sherris & Ryan's Medical Microbiology, 8th Edition; 2022. Available at: https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260922934 Accessed: January 15, 2024 Copyright © 2024 McGraw-Hill Education. All rights reserved MCRO 2.3 Viral Life Cycle/Replication Read : Sherris & Ryan Chapter 6 Citation: Chapter 6 Viruses—Basic Concepts, Ryan KJ. Sherris & Ryan's Medical Microbiology, 8th Edition; 2022. Available at: https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260922934 Accessed: January 15, 2024 Copyright © 2024 McGraw-Hill Education. All rights reserved MCRO 2.3 Viral Life Cycle/Replication Viral Life Cell includes: § Attachment § Penetration § Uncoating § Synthetic phase (transcription, translation, replication § Assembly § Release Read : Sherris & Ryan Chapter 6 MCRO 2.3 Viral Life Cycle: Viral Release/Budding Viral release by budding. § Human enveloped viruses acquire lipid bilayer membrane by budding generally from the plasma membrane. § Viral spikes are expressed on the cell surface followed by synthesis of matrix protein that associates near the plasma membrane where viral spikes are present. § The matrix protein attracts the assembled nucleocapsid (genome + nucleoprotein) near the plasma membrane expressing viral spikes followed by envelope membrane wrapping and release of the virus particle Read : Sherris & Ryan Chapter 6 Citation: Chapter 6 Viruses—Basic Concepts, Ryan KJ. Sherris & Ryan's Medical Microbiology, 8th Edition; 2022. Available at: https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260922934 Accessed: January 15, 2024 Copyright © 2024 McGraw-Hill Education. All rights reserved MCRO 2.3 Classification of DNA Viruses STUDY AID! DNA viruses are HHAPPPPY! • H = Hepadnaviridae • H = Herpesviridae • A = Adenoviridae • P = Parvoviridae • P = Poxviridae • P = Papillomaviridae • Py = Polyomaviridae STUDY AID! All DNA viruses have double-stranded genomes EXCEPT Parvoviridae. http://pathmicro.med.sc.edu/mhunt/intro-vir.htm MCRO 2.4 Classification of RNA Viruses • Multiple combinations of genome and capsid structure • Some patterns: – All RNA viruses are singlestranded except for Reoviridae. – All negative sense viruses are enveloped and have helical capsid structure. – All non-enveloped viruses have icosahedral capsid structure. In “genome-speak”, ds = double-stranded and ss = single-stranded. MCRO 2.4 Viral Infection and the Immune System: Overview Viral pathogenesis involves complex interactions between viruses and hosts comprising of transmission, replication, dissemination, immune response, and pathology to produce disease in humans. § Viruses have found several routes to enter and spread in the host and find a target cell/tissue where they can replicate efficiently and cause cytopathic effects to damage the tissue. § In some cases, the immune system is successful in eliminating the virus, whereas in other cases, viruses avoid elimination by the immune system and persist in the host. § While in several cases, the disease is caused by direct viral lysis of the infected cells, in other cases, the disease is immune-mediated such as immune complexes, cytotoxic CD8 T cells, and cytokines. • cytokines: any of a number of substances, such as interferon, interleukin, and growth factors, which are secreted by certain cells of the immune system and have an effect on other cells. § Many DNA viruses and some RNA viruses transform cells causing oncogenesis. § Host factors and defenses play important roles in viral pathogenesis: note that the same virus may cause a mild disease in some hosts and a severe disease in other hosts. § Innate and adaptive immune responses are critical to eliminate or control viral infections in hosts. § Several viral infections cause immune suppression, including a risk of opportunistic and superinfections. § Immunocompromised hosts are vulnerable to many viral diseases. Vaccination is the key to provide protection in the population. Read: Sherris & Ryan Chapter 7 MCRO 2.3 Immunity: Review Week 1 pptx Innate Defenses = non-specific defenses § Ability to resist damaging organisms and toxins § Barriers (skin/gastric acid) § Cells (neutrophils & macrophages) § Chemicals (lysozyme, complement) § Processes (fever, phagocytosis, inflammation) Acquired Immunity = specific defenses § Humoral (antibody-mediated) ----> circulating antibodies § Cellular (cell-mediated) ----> activated T cells MCRO 1.4 Viral Infection and the Immune System Read: Sherris & Ryan Chapter 7 MCRO 2.3 Viral Infection and the Immune System § Viruses cause disease when they breach the host’s primary physical and natural protective barriers; evade local, tissue, and immune defenses; spread in the body; and destroy cells either directly or via bystander immune and inflammatory responses. § Viral pathogenesis comprises of several stages: (1) Transmission and entry of the virus into the host: including food and water, aerosol, respiratory, gastrointestinal, break in the skin, via mucosal or blood, insect or animal bite, and urogenital, anal or sexual routes (2) Spread in the host: Viruses generally multiply at the site of entry to establish infection in the host and can also spread through the host by infecting neighboring cells and/or reach the blood stream (systemic infection) (3) Tropism: capability of viruses to infect a discrete population of cells within an organ. • Cellular or tissue tropism is most often determined by the specific interaction of viral surface proteins (spikes) and cellular receptors on the host cells • Viruses such as HIV use a receptor (CD4) and coreceptor (CCR5 or CXCR4) • Different viruses may use the same receptor on host cells Read: Sherris & Ryan Chapter 7 MCRO 2.3 Viral Infection and the Immune System (4) Virulence and cytopathogenicity: the relative ability of a virus to cause disease. Viral virulence is, basically, the degree of pathogenicity of a virus. • A virus may be of high or low virulence for a particular host. • Different strains of the same virus may differ in the degree of pathogenicity (5) Patterns of viral infection and disease: Not every viral infection results in a disease. Infection involves multiplication of the virus in the host, whereas disease represents a clinically apparent response. • Infections are much more common than disease; unapparent infections are termed subclinical, and the individual is referred to as a carrier (6) Host factors: including immune status, genetic background, age, and nutrition, play important roles in determining the outcome of viral infection. Several innate immune responses (interferons α and β, natural killer (NK) cells, mucociliary responses, and others) and adaptive immune responses (antibody and T-cell responses) influence the outcome of viral infections. • Individuals with weak immune systems or those who are immunocompromised or immunosuppressed often have more severe outcomes. Details of immune responses to infection are described in Chapter 2. Read: Sherris & Ryan Chapter 7 MCRO 2.3 Viral Infection and the Immune System (7) Host defense: The two major types of host defenses are nonspecific (innate) and specific (adaptive) immune responses. • The innate immune response includes interferons, natural killer (NK) cells, macrophages (phagocytosis), mucociliary clearance, and fever. o Interferons are host-encoded proteins that provide the first line of defense against viral infections • The adaptive immune response involves humoral immunity (B cells) and cell-mediated immunity (T cells) o cytotoxic T lymphocytes (CTL) destroy virus-infected cells o after viral infection, the first specific immune response is T-cell mediated in which CD8 T cells recognize viral antigen presented by class I MHC and kill virus-infected cells by secreting perforins and granzymes causing apoptosis of the viral-infected host cell (8) Virus-induced immunopathology: even when the host’s working immune system cannot stop a viral infection leading to disease • This could be true in viral infections in which a large number of cells are infected in an individual • Recent emerging SASR-CoV-2 (COVID-19) pandemic causing multiorgan diseases involves viral load and excessive cytokine release induced damage. Read: Sherris & Ryan Chapter 7 MCRO 2.3 Laboratory Detection of Viral Infection u Serology: identification of antibodies from a peripheral blood sample u Viral culture: use of cell culture to grow viruses in-vitro u Cytology/Histology: Study of cell morphology – identification of nuclear changes in a stained cell preparation, for example u Molecular techniques: A variety of molecular biological techniques can be employed to detect viral genome, for example u Variations of PCR, polymerase chain reaction MCRO 2.5 Growth & Assays of Viruses The growth of human viruses requires that the host cells be cultivated in the laboratory. § To prepare cells for growth in vitro, a tissue is removed from an animal, and the cells are disaggregated using the proteolytic enzyme trypsin. § The cell suspension is seeded into a plastic Petri dish in a medium containing a complex mixture of amino acids, vitamins, minerals, and sugars. § In addition to these nutritional factors, the growth of animal cells requires components present in animal serum. § This method of growing cells is referred to as tissue culture, and the initial cell population is called a primary culture. § The cells attach to the bottom of the plastic dish and remain attached as they divide and eventually cover the surface of the dish. § When the culture becomes crowded, the cells generally cease dividing and enter a resting state. § Propagation can be continued by removing the cells from the primary culture plate using trypsin and reseeding a new plate. § Cells taken from a normal (as opposed to cancerous) tissue cannot usually be propagated in this manner indefinitely. § Permanent cell lines are useful for growing viruses Read : Sherris & Ryan Chapter 6 Citation: Chapter 4 Principles of Laboratory Diagnosis of Infectious Diseases, Ryan KJ. Sherris & Ryan's Medical Microbiology, 8th Edition; 2022. Available at: https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260922674 Accessed: January 15, 2024 Copyright © 2024 McGraw-Hill Education. All rights reserved MCRO 2.5 Growth & Assays of Viruses Viruses are quantitated by a method called the plaque assay. § Briefly, viruses are mixed with cells on a Petri plate so that each infectious particle gives rise to a zone of lysed or dead cells called a plaque. From the number of plaques on the plate, the titer of infectious particles in the lysate is calculated. Virus titers are expressed as the number of plaque-forming units per milliliter (pfu/mL). Read: Sherris & Ryan Chapter 6 Citation: Chapter 4 Principles of Laboratory Diagnosis of Infectious Diseases, Ryan KJ. Sherris & Ryan's Medical Microbiology, 8th Edition; 2022. Available at: https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260922674 Accessed: January 15, 2024 Copyright © 2024 McGraw-Hill Education. All rights reserved MCRO 2.5 PCR Diagnostic applications of the polymerase chain reaction (PCR). A. A clinical specimen (eg, pus, tissue) contains DNA from many sources as well as the chromosome of the organism of interest. If the DNA strands are separated (denatured), the PCR primers can bind to their target sequences in the specimen itself. B. Amplification of the target sequence by PCR. (1) The target sequence is shown in its native state. (2) The DNA is denatured, allowing the primers to bind where they find the homologous sequence. (3) In the presence of the special DNA polymerase, new DNA is synthesized from both strands in the region between the primers. (4-6) Additional cycles are added by temperature control of the polymerase with each new sequence acting as the template for another. The DNA doubles with each cycle. After 25 to 30 cycles, enough DNA is present to analyze diagnostically. C. Internal probe. The amplified target sequence is shown. A probe can be designed to bind to a sequence located between (internal to) the primers. D. Analysis of PCR amplified DNA. (1) The amplified sequence can be cloned into a plasmid vector. In this form, a variety of molecular manipulations or sequencing may be carried out. (2) Direct hybridizations usually make use of an internal probe. The example shows three specimens, each of which went through steps A and B. After amplification, each was bound to a separate spot on a filter (dot blot). The filter is then reacted with the internal probe to detect the PCR-amplified DNA. The result shows that only the middle specimen contained the target sequence. (3) The amplified DNA may be detected directly by agarose gel electrophoresis. The example shows detection of amplified fragments in two of three lanes on the gel. (4) The sensitivity of detection may be increased by use of the internal probe after Southern transfer. The example shows detection of a third fragment of the same size that was not seen on the original gel because the amount of DNA was too small. Read: Sherris & Ryan Chapter 4, Figure 4-9 Citation: Chapter 4 Principles of Laboratory Diagnosis of Infectious Diseases, Ryan KJ. Sherris & Ryan's Medical Microbiology, 8th Edition; 2022. Available at: https://accessmedicine.mhmedical.com/content.aspx?bookid=3107&sectionid=260922674 Accessed: January 15, 2024 Copyright © 2024 McGraw-Hill Education. All rights reserved MCRO 2.5