The Nature of Viruses PDF

THE NATURE OF VIRUSES  VIRUSES ARE NOT CELLS. They are nucleic acid molecules that can invade cells and replicate w/in them (no metabolism of their own).  The Latin word virus means “poisonous fluid”. Previously referred to as “contagium vivum fluidum” meaning contagious...

THE NATURE OF VIRUSES  VIRUSES ARE NOT CELLS. They are nucleic acid molecules that can invade cells and replicate w/in them (no metabolism of their own).  The Latin word virus means “poisonous fluid”. Previously referred to as “contagium vivum fluidum” meaning contagious fluid.  The terms “living” and “non-living” are not applicable to viruses; it is preferable to refer them as being FUNCTIONALLY ACTIVE OR INACTIVE (rather than alive or dead).  Very few or no enzymes of their own for metabolism (lack enzymes for CHON synthesis and ATP generation)  Outside the host cell, the virus particle is also known as a VIRION. The virion is metabolically inert. Nature of Viruses Viruses are heterogeneous class of agents. They vary in size and morphology, complexity, host range, and how they affect their hosts; but certain characteristics are shared by them: 1. They consist of a genome (either RNA or DNA; not both), surrounded by a protective protein shell. 2. They multiply only in living cells. They are dependent on the host’s energy-yielding and CHON-synthesizing apparatus. They are parasites at the genetic level (OBLIGATORY INTRACELLULAR PARASITES). 3. Their multiplication cycle includes, as an initial step, the separation of their genomes from their protective shells. 4. One virus can replicate to produce hundreds of progeny viruses, whereas one cell divides to produce only two daughter cells. COMPARISON BETWEEN VIRUSES AND BACTERIA Typical Rickettsias/ Viruses Points of Comparison Bacteria Chlamydias Intracellular parasite No Yes Yes Plasma membrane Yes Yes No Binary fission Yes Yes No Pass through bacteriological filters No No/Yes Yes Possess both DNA and RNA Yes Yes No ATP-generating metabolism Yes Yes/No No Ribosomes Yes Yes No Sensitive to antibiotics Yes Yes No Sensitive to interferon (antiviral CHON) No No Yes COMPARISON BETWEEN VIRUSES AND CELLS PROPERTY VIRUS CELL Type of nucleic acid Proteins Lipoprotein membrane Ribosomes Mitochondria Enzymes Multiplication by binary fission or mitosis Host Range of Viruses  Spectrum of host cells that they can infect:  Invertebrates & vertebrates,  plants,  protists,  fungi and  bacteria  Most viruses are able to infect specific types of cells of only one host species (host specific).  BACTERIOPHAGES OR PHAGES – viruses that infect bacteria  Host range is determined by the virus’s requirements for its:  specific attachment to the host cell (receptor sites)  Bacteria – cell wall, fimbriae, flagella  Animals – plasma membrane  the availability w/in the host of cellular factors for multiplication Viral Size  Determined with electron microscopy  Sizes range from: 20 – 1000 nm in length TAXONOMY VIRAL STRUCTURE CAPSID MORPHOLOGY ATYPICAL VIRUSLIKE AGENTS VIRAL REPLICATION TOPIC SUMMARY  Taxonomy  Atypical Viruslike Agents  Family  Subfamily  Defective viruses  Genus  Viroids  Species  Pseudovirons  Viral Structure/Components  Prions  Viral nucleic acids (DNA/RNA)  Viral capsid, nucleocapsid, and envelope  Viral Replication  Viral proteins  Lytic cycle  Lysogenic cycle  Viral Capsid Morphology  Helical  Icosahedral/polyhedral  Complex TAXONOMY  According to International Committee on Taxonomy of Viruses (ICTV) 1996; grouping of viruses into families is based on: 1. Nucleic acid type 2. Strategy for replication 3. Morphology  Suffix:  Virus – indicates name of genus  Virinae – indicates name of subfamily  Viridae – indicates name of family  Examples:  Family Herpesviridae, genus Simplexvirus  Family Hepadnaviridae, genus Hepadnavirus/Hepatitis B Virus  Table 13.2 pg 392-393 Microbiology 9th Ed by Tortora, Funke & Case  FAMILY (“-viridae”) Herpesviridae SUBFAMILY (“-virinae”) Alphaherpesvirinae GENUS (“-virus”) Herpes simplex virus FAMILY Paramyxoviridae GENUS Morbillivirus Species Measles virus VIRAL STRUCTURE  Viral nucleic acids  Viral capsid  Viral proteins  Viral envelope 1. Viral Nucleic Acids  A virus can have either DNA or RNA (never both)  Can be single-stranded(ss) or double-stranded (ds) DNA or RNA  The most common forms of viral genomes are ssRNA and dsDNA but other forms may occur:  ssDNA – Parvovirus B19  dsRNA – rotavirus  The DNA Is always a single molecule; the RNA can exist either as a single molecule or in several pieces (segmented), i.e. influenza virus and rotavirus have segmented genomes.  Almost all viruses contain only a single copy of their genome (haploid), EXCEPT for retroviruses, who have 2 copies of their RNA genome (diploid/dimer).  Depending on the virus, the NA can be  Linear  Circular  Segmented (only for RNA)  Total amount of NA varies from a few thousand nucleotides (pairs) to as many as 250,000 nucleotides  Function: viral genomes can encode a simple message or encode hundreds of enzymes and structural proteins DNA VIRUSES RNA VIRUSES Hepadnaviridae Arenaviridae Herpesviridae Astroviridae Adenoviridae Bunyaviridae Papovaviridae Caliciviridae Parvoviridae Coronaviridae Poxviridae Filoviridae Flaviviridae Orthomyxoviridae DNA viruses are “HAPPy”. Paramyxoviridae Picornaviridae Reoviridae Retroviridae Rhabdoviridae Togaviridae RNA Viruses classified into 4 groups based on their strategy for synthesizing mRNA 1. Positive (+) stranded or Sense Strand RNA  ssRNA viruses w/ a positive polarity (same base sequence as an mRNA)  They use their RNA genome directly as mRNA  When it enters a host cell, the viral RNA can be immediately translated by host’s ribosomes into CHON  (+) stranded RNA/mRNA ------ translation------CHON synthesis 2. Negative (-) stranded or Antisense Strand RNA  ssRNA viruses w/ a negative polarity (base sequence is complementary to mRNA)  When it enters a host cell, they are NOT able to begin translation immediately; they must first be transcribed into a positive stranded RNA by an enzyme in their capsid, RNA-DEPENDENT RNA POLYMERASE  Human cells do not have this enzyme, so (-) stranded viruses must carry their own enzyme  (-) stranded RNA ---transcription into mRNA-----translation-----CHON synthesis 3. Double-stranded RNA  dsRNA viruses with negative polarity i.e., reoviruses (Rotavirus)  This viruses also carry their own RNA polymerase  dsRNA ---transcription into mRNA-----translation-----CHON synthesis 4. Retroviruses  ssRNA viruses with negative polarity that are transcribed into dsDNA by the enzyme reverse transcriptase (RNA- dependent DNA polymerase)  The DNA copy is then transcribed into viral mRNA by the host cell RNA polymerase (polymerase II)  Reverse transcriptase synthesizes a complementary DNA from an RNA template  RNA of retrovirus---reverse transcription---DNA---transcription---mRNA--- translation---synthesis  Examples: HIV, HTLV DNA Viruses  Unlike RNA, DNA CANNOT BE TRANSLATED DIRECTLY INTO PROTEINS – it must first be transcribed into mRNA – then, transcribed into structural proteins and enzymes  Every DNA virus has both a (-) and a (+) strand.  The (+) strand refers to the strand that is read; used as a template for transcription into mRNA  The (-) strand is ignored  DNA ---transcription---mRNA---translation---CHONS and enzymes High-Yield Facts on DNA Viruses  All DNA viruses are double stranded except Parvovirus.  All DNA viruses have linear DNA, except  papovavirus and  hepadnaviruses, which are circular  All DNA viruses grow in the host nucleus, except poxviruses.  All DNA viruses are naked viruses, except for:  herpesviruses,  poxviruses, and  hepadnaviruses  All DNA viruses have icosahedral capsids. High-Yield Facts on RNA Viruses  All RNA viruses are single stranded,  RNA viruses grow in the host except reoviruses. cytoplasm, except influenza and  Segmented RNA viruses include retroviruses (cytoplasm and nucleus) orthomyxoviruses and reoviruses.  All RNA viruses are enveloped, except  Positive sense RNA include: for  Caliciviridae  Caliciviruses  Coronaviridae  Picornavirues  Flaviviridae  Reoviruses  Picornaviridae  All RNA viruses have helical capsids,  Retroviridae except:  Picornaviruses  Reoviruses  Togaviruses 2. Viral Capsid and Envelope  Nucleic acid of a virus is protected by a protein coat called CAPSID which are made up Functions of the Capsid: protein subunits called CAPSOMERS OR CAPSOMERES. NUCLEOCAPSID refers 1. Protects the viral genome to the nucleic acid and capsid. 2. Receptor site to initiate infection  ENVELOPE – covers the nucleocapsid; 3. Stimulus for Ab production combination of lipids, proteins and carbohydrates; acquired by budding through 4. Site of antigenic determinants the cell’s nuclear or cytoplasmic membrane  SPIKES – CHO-CHON (glycoprotein) complexes that project from the envelope’s surface; means of attachment; means of identification  Viruses which are not covered by an envelope are known as NAKED/NONENVELOPED VIRUSES - covered only by a capsid Capsid Morphology Capsid morphology can be a basis for classifying viruses. These morphologies can be observed using electron microscopy. 1. Enveloped helical 2. Naked icosahedral/ naked polyhedral 3. Enveloped icosahedral/enveloped polyhedral 4. Complex viruses HELICAL VIRUSES  Resemble long rods that may be rigid or flexible  Viral NA is found w/in a hollow cylindrical capsid that has a helical structure  All human viruses w/ helical nucleocapsid are ENVELOPED.  Examples: Ebola virus, rabies virus, RNA viruses  ICOSAHEDRAL/POLYHEDRAL VIRUSES  Many animal, plant and bacterial viruses are polyhedral/icosahedral or near-spherical.  Icosahedral viruses are either NAKED or ENVELOPED.  The capsid is in the shape of an icosahedron (a regular polyhedron w/ 20 triangular faces and 12 corners(capsomeres). The capsomeres of each face form an equilateral triangle  Examples:  Adenoviruses, poliovirus (naked icosahedral)  Herpesviruses, retroviruses (enveloped icosahedral) 20 triangular faces and 12 corners  COMPLEX VIRUSES  w/ complicated structures like bacterial viruses (bacteriophages)  Capsids contain attached (additional) structures  How is it complicated?  Capsid head is polyhedral and tail sheath is helical  Examples: bacteriophages, poxviruses (no capsids but w/ several coats around the NA) Identify the capsid morphology 3. Viral Proteins  Viral proteins serve several important functions:  Capsid proteins protect the viral genome and mediate the attachment of the virus to the host cell receptors –this determines species and organ specificity.  Outer viral proteins induce neutralizing Abs from the host cell and activate cytotoxic T-cells to kill virus-infected host cells.  Some internal viral proteins are structural (capsid), whereas others are enyzmes like polymerases. The internal viral proteins vary depending on the virus:  RNA viruses may have attached RNA polymerase  DNA viruses may have attached DNA polymerase  Some viruses also produce proteins that are “superantigens” like EBV and CMV. ATYPICAL VIRUSLIKE AGENTS 1. Defective viruses 2. Pseudovirions 1. How are they different 3. Viroids from the regular 4. Prions viruses? 2. What are the diseases associated with prions? VIRAL MULTIPLICATION Topic Summary  Multiplication of Bacteriophages  Lytic Cycle  Lysogenic Cycle  Multiplication of Animal Viruses MULTIPLICATION OF BACTERIOPHAGES  For a virus to multiply, it must invade a host cell and take over the host’s metabolic machinery  Bacteriophages can multiply in 2 ways: 1. Lytic cycle – ends with the death of the host cell 2. Lysogenic cycle – host cell remains alive Lytic Cycle 1. Attachment 2. Penetration 3. Biosynthesis 4. Maturation 5. Release 1. Attachment (Adsorption)  After chance collision b/n bacteria and virus, an attachment site on the virus attaches to a complementary receptor on the bacterial cell  Fibers at the tail’s end (virus) attach to receptor sites on the cell wall (bacteria)  Attachment is a specific binding between viral capsid proteins and specific receptors on the host cellular surface. This specificity determines the host range of a virus. 2. Penetration  Bacteriophage injects its nucleic acid into the bacterium acting like a hypodermic syringe, injecting its NA into the bacterial cell.  Bacteria have strong cell walls that a virus must breach to infect the cell. Tail releases an enzyme, PHAGE LYSOZYME, w/c breaks down a portion of the bacterial cell wall; while the viral capsid remains outside. 3. Biosynthesis  Once inside the host, the viral genome takes-over the metabolism of the host, synthesizing viral NA and proteins  The viral NA is transcribed and translated and the various viral proteins, either enzymes or structural components, proceed to manufacture everything needed to make new viruses.  ECLIPSE PERIOD – time when only separate components (DNA & CHON) can be detected; infective virions are not yet present.  In many phages the entire life cycle from infection to lysis takes only 20 to 40 minutes.  RNA viruses use their own RNA replicase enzymes to create copies of their genomes. 4. Maturation (Assembly)  The phage pieces or parts are assembled to create complete viral particles (virions).  Step when NA is packaged up into capsids; bacteriophage NA and capsids are assembled into complete virions.  The head and tail are separately assembled, and the head is filled with phage NA and attached to the tail.  In viruses such as HIV, assembly occurs after the virus has been released from the host cell. 5. Release  Final stage of viral replication when the virions are released from the host cell (about 50 – 1000 escape from the cell)  The plasma membrane breaks open to release the virions due to LYSOZYME w/c is encoded by the phage’s gene. This is synthesized w/in the cell and causes the bacterial cell to break down (lysis).  The newly released bacteriophages infect other cells and the viral cycle is repeated.  Enveloped viruses (e.g., HIV) typically are released from the host cell by budding. During this process the virus acquires its envelope, which is a modified piece of the host's plasma membrane. Summary: LYTIC CYCLE Step Event Attachment/ The phage attaches to a protein or polysaccharide molecule Adsorption (receptor) on the surface of the bacterial cell Penetration The phage injects its DNA into the bacterial cell; the capsid remains on the outer surface of the cell Biosynthesis Phage genes are expressed, resulting in the production of phage pieces/parts (phage DNA and phage proteins) Assembly The phage genes/parts are assembled to create complete phages Release The complete phages escape from the bacterial cell by lysis of the plasma membrane  LYSOGENIC CYCLE (Bacteriophage Lambda)  LYSOGENIC PHAGES (TEMPERATE PHAGES) do not cause lysis and death of host cell when they multiply but may proceed to lytic cycle  They are capable of incorporating their DNA into the host cell’s DNA to begin the lysogenic cycle  The phage remains latent (inactive)  The infected cells are known as lysogenic cells. 3 Important Results in Lysogeny 1. Lysogenic cells are immune to reinfection by the same phage 2. Phage conversion – host cell may exhibit new properties Examples:  C. diphtheriae can only produce a toxin when it carries a lysogenic phage  Only Streptococci carrying a lysogenic phage can cause Scarlet Fever 3. Makes specialized transduction – certain bacterial genes are transferred from one bacterium to another using a bacteriophage Lysogenic Cycle (page 399 Tortora) 1. Phage attaches to host cell and injects DNA 2. Phage DNA circulizes and enters lytic cycle or lysogenic cycle 3. Phage DNA integrates w/in the bacterial chromosome by recombination, becoming a PROPHAGE (phage in w/c all that remains inside it is DNA) 4. Lysogenic bacterium reproduces normally (multiple cell divisions) 5. Occasionally, the prophage may excise from the bacterial chromosome by another recombination event, initiating a lytic cycle. Multiplication of Animal Viruses 1. Attachment 2. Entry 3. Uncoating 4. Biosynthesis of DNA Viruses/RNA Viruses 5. Maturation 6. Release Steps in the Multiplication of Animal Viruses Step Event Attachment/ Virus attaches to a protein or polysaccharide molecule (receptor) on the surface of the host cell Adsorption Penetration The entire virus enters the host cell, in some cases because it was phagocytized by the cell Uncoating Viral nucleic acid escapes from the capsid Biosynthesis Viral genes are expressed, resulting in the production of pieces/parts of viruses (viral DNA & viral proteins); synthesis of early proteins for genome replication; synthesis of late proteins for structural components of the virion Assembly Viral pieces/parts are assembled to create complex virions Release Complete virions escape from the host cell by lysis or budding BACTERIOPHAGES AND ANIMAL VIRAL MULTIPLICATION COMPARED Stage Bacteriophages Animal Viruses Attachment Tail fibers attach to cell wall Attachment sites are plasma proteins membrane proteins and glycoproteins Entry Viral DNA injected into host cell Capsid enters by endocytosis or fusion Uncoating Not required Enzymatic removal of capsid proteins Biosynthesis In cytoplasm In nucleus (DNA viruses) or cytoplasm (RNA viruses) Chronic infection Lysogeny Latency; slow viral infections; cancer Release Host cell lysed Enveloped viruses bud out; nonenveloped viruses rupture plasma membrane TOPIC SUMMARY  Mode of Transmission  Pathogenicity  Antiviral agents  Prevention and Control  Viral Vaccines Transmission Transmitted from person to person by:  respiratory  fecal-oral  sexual contact  trauma/injection w/ contaminated objects  tissue transplants (blood transfusion)  arthropod or animal bites  gestation (transplacental)  Once introduced into a host, the virus infects susceptible cells, frequently in the URT. Local infection leads to VIREMIA.  Symptomatic disease follows and CMI(cell mediated immunity) mechanisms halt continued viral replication (OVERT)  Tissues my be damaged by lytic viruses or by immunopatholgic mechanisms (directed against the virus but may harm surrounding tissues)  Some may be LATENT and reactivation may occur accompanying immune suppression ----recurrence of symptomatic disease  AUTOIMMUNE PATHOGENESIS – when pathogenic viruses stimulate an immune rxn that cross reacts w/ related human tissue, resulting in damage to host function. This typically results after the acute viral infection has resolved.  Example: Ab produced in measles cross reacts w/ CNS tissues causing post-infectious encephalitis ----subacute sclerosing panencephalitis  Rare viral infections promote transformation or immortalization of host cells resulting to uncontrolled cell growth – ONCOGENIC VIRUSES  Papilloma (wart viruses) could give rise to cervical CA  Oncoviruses causing leukemia and tumors in animals  HOST DEFENSES  Nonspecific Defenses  Specific Defenses 1. Interferons 1. Active immunity 2. Natural Killer Cells 2. Passive immunity 3. Phagocytosis 3. Herd immunity 4. α – Defensins 5. Apolipoprotein B RNA-Editing Enzyme (APOBEC3G) 6. Fever 7. Mucociliary clearance Factors that modify host defenses NONSPECIFIC DEFENSES Properties/Functions 1. Interferons Inhibits viral replication by degrading viral mRNA, they induce the synthesis of a ribonuclease w/c cleaves viral mRNA but not host cell mRNA Alpha and beta interferons are more potent than gamma interferons. Interferons act by binding to a receptor on the cell surface. They do not enter the cell and have no effect on extracellular viruses. 2. Natural Killer cells They are called “natural” killer cells because they are active even without previous exposure to a virus. They are a type of T lymphocytes but do not have antigen receptors. They kill virus-infected cells by secreting perforins and granzymes w/c cause apoptosis of infected cells. 3. Phagocytosis Fixed macrophages of the RES and alveolar macrophages limit viral infections. 4. α-defensins Family of positively charged polypeptides w/ antiviral activity. They interfere w/ HIV binding to the CXCR4 receptor and block entry of the virus into the cell. This explains why some HIV pxs are long- term “non-progressors”. NONSPECIFIC DEFENSES Properties/Functions 5. APOBEC3G An enzyme that causes hypermutation in retroviral DNA by deaminating cytosines in both mRNA and retroviral DNA, thereby inactivating these molecules and reducing infectivity of HIV HIV defends itself by producing Vif (viral infectivity protein), counteracting the effects of this enzyme. 6. Fever Elevated body temperature may: 1. Directly inactivate the virus, particularly enveloped viruses, which are more heat-sensitive 2. Inhibit viral replication 7. Mucociliary Ciliated epithelial cells of the URT clearance 8. Factors that modify 1. Age host defenses 2. Increased corticosteroid levels – lysis of lymphocytes, decreased recruitment of monocytes, inhibition of interferon prodn, and stabilization of lysosomes 3. Malnutrition - leads to decreased Ig prodn and phagocytosis; and reduced skin and mucous membrane integrity SPECIFIC DEFENSES Properties/Functions 1. Active immunity Effected by antibodies and cytotoxic T cells. Elicited either by exposure to the virus or by immunization. How does Ab inhibit viruses? 1. Neutralization - binds to the proteins on the outer surface of the virus, thus, preventing attachment and uncoating 2. Lysis of virus-infected cells with the help of a complement – Ab binds w/ a complement on the cell surface and the complement enzymatically degrades the cell membrane How does a T-Lymphocyte lyse virus-infected cells? 1. Releasing perforins w/c make holes in the cell membrane 2. Releasing proteolytic enzymes (granzymes) 3. Causing apoptosis (programmed cell death) 2. Passive immunity Administration of preformed antibodies. Passive immunity is effective IMMEDIATELY whereas it takes active immunity 7-10 days in the primary immune response (but has shorter duration than active immunity). 3. Herd immunity Immunized individuals are incapable of transmitting the virus. ANTIVIRAL AGENTS VIRUS MODE OF ACTION TARGET DRUGS CMV Nucleoside analog Viral DNA Ganciclovir HIV Nucleoside analog Viral DNA Efavirenz Nucleotide analog Viral DNA Tenofovir disropxil fumarate Nonnucleoside analog Reverse transcriptase Emtricitabine Protease inhibitor Viral protease Fusion inhibitor Virus, host cell membrane HSV/VZV Nucleoside analog Viral DNA Acyclovir Pyrophosphate analog DNA polymerase Foscarnet HBV Nucleoside analog Reverse transciptase Lamivudine Nucleotide analog DNA polymerase Adefovir dipivoxil Influenza A Inhibit penetration and Host cell membrane Amantadine, imantadine uncoating of virus Influenza A and B Prevent release of virus Neuraminidase inhibitors Zanamivir, oseltamivir RSV Inhibit expression of viral mRNA Viral mRNA Ribavirin and CHON synthesis HCV Inhibit expression of viral mRNA ; Viral mRNA or neighboring host Ribavirin plus interferon-alpha increase resistance to virus cells Picornaviruses Inhibit attachment and uncoating Binds to virus Pleconaril of virus SPECIMEN SELECTION AND COLLECTION General Principles  Specimen collection depends on the:  Specific disease syndrome  Viral etiologies suspected  Time of year  In general, it is optimal to collect the specimen as early in the course of the disease as possible. The viral titer is highest during the first 4 days after the onset of symptoms. Exceptions are enterovirus, adenovirus and cytomegalovirus because of prolonged shedding of the virus.  It is best to sample the infected site directly. Exception is certain viral infections of the CNS, when it may be recommended to culture the stool or throat instead of CSF (since virus is shed in stool or throat).  Viral transport medium contains:  Buffered saline  Protein stabilizer  Antibiotics (penicillin, vancomycin, bacitracin, streptomycin and amphotericin B  Swabs shd be cotton, rayon or Dacron on plastic shafts because wood is toxic to viruses.  Calcium alginate may bind and inactivate the virus (inactivates HSV); charcoal is also toxic to several viruses.  If there is delay in processing:  Specimens shd be held at 4deg C, NOT FROZEN since this can destroy infectivity very quickly (only recommended when a prolonged delay of 24hours is anticipated)  When freezing is required or unavoidable, specimens shd be “snap frozen” to at least -70deg C and transported in dry ice or w/ liquid nitrogen Throat, Nasopharyngeal Swab or Aspirate  Nasopharyngeal aspirates are SUPERIOR, in general, to throat or nasopharyngeal swabs  Throat swabs are acceptable for recovering:  Enteroviruses  Adenoviruses  HSV  Nasopharyngeal swabs/aspirate  RSV  Influenza  Parainfluenza  Nasal specimen - rhinovirus Bronchial and Bronchoalveolar Washes  Wash and lavage fluid collected are excellent specimens for the detection of viruses that infect the LRT, especially influenza and adenoviruses Rectal Swabs and Stool Specimens  For detecting  Rotavirus  Enteric adenoviruses (serotypes 40 and 41)  Enteroviruses  Many agents of viral gastroenteritis do not grow in cell culture, they are detected in electron microscopy  In general, stool specimens are preferable to rectal swabs  Rectal swab – insert a swab 3-5cm into rectum to obtain feces  10ml of fresh stool (or from diaper) is sufficient for detection from infants Urine  For detecting:  CMV  Mumps  Rubella  Measles  Polyomaviruses  Adenoviruses  Requires 2-3 specimens for optimum recovery  Best specimen – 10ml first morning urine sample Skin and Mucous Membrane Lesions  For the detection of HSV, VZV, CMV, pox viruses (rare) fr vesicular lesions;  difficult detection once lesions are encrusted or ulcerated Sterile Body Fluids other than Blood  CSF, pericardial and pleural fluids may contain:  Enteroviruses  HSV  VZV  Influenza viruses  CMV  Collected by physician  Blood  For the detection of:  CMV  HSV  VZV  Enteroviruses  Adenoviruses (occasional)  5-10ml of anticoagulated blood is needed  For CMV – heparinized, citrated, EDTA anticoagulated blood is acceptable Bone Marrow  Should be added with anticoagulant (heparin, citrate, EDTA)  Collected by aspiration  for the detection of Parvovirus B19 Tissue  For the detection of viruses commonly infecting the  Lung: CMV, Influenza, Adenovirus, sin nombre virus (Hantavirus)  Brain: HSV  GIT: CMV  Collected during surgery Serum for Ab Testing  Acute and convalescent serum samples may be needed to detect Ab to specific viruses  Acute specimens should be collected ASAP after the appearance of symptoms  Convalescent specimen is collected a minimum of 2-3 weeks after the acute specimen  3-5ml serum is required in both cases Additional readings:  Page 723 Bailey and Scott  Pages 739-740 Bailey and Scott  Pages 741-749 Bailey and Scott LABORATORY DIAGNOSIS Detection, Cultivation and Identification TOPIC SUMMARY DETECTION & IDENTIFICATION CULTIVATION 1. Cytological or Histological Examination 1. Living animals 2. Electron Microscopy 2. Embryonated eggs 3. Virus Isolation in Cell or Tissue Culture 3. Cell cultures 4. Shell Vial Centrifugation-Enhanced (SVCE) Virus Detection 5. Direct Detection of Viral Ags or Genes 6. Target Gene Amplification (PCR) 7. Serological Detection of Viral Antibodies CULTIVATION OF VIRUSES  Viruses must be provided w/ living cells instead of chemical media for their cultivation in-vitro.  Cultivation in the lab:  Living animals  Embryonated eggs  Cell cultures  1.) In living animals  Animals used: mice, rabbits and guinea pigs  Test animal is inoculated w/ the specimen  Animal is observed for signs of the disease  Or killed so that infected tissues can be examined for the virus’ presence  2.) In embryonated eggs  A hole is drilled in the shell of the embryonated egg  Viral suspension is injected into the egg’s fluid  Viral growth is signaled by the:  death of the embryo  cell damage  formation of lesions on the egg’s membranes 3.) In cell cultures a) Plaque method b) Cell lines PLAQUE METHOD  Cell deterioration –detected and counted in similar manner as plaques and reported as PFU/ml (plaque- forming units)  Growth is seen after 2-12 days of Each plaque is produced by a single incubation virus particle; Ideal for bacteriophages The plaque method: 1. Virus, bacteria, and agar mixed, plated and incubated. 2. After replication the virus lyses the bacteria, forming plaques, or clear zones. 3. Each plaque is assumed to come from a single viral particle. 4. The titer of the virus is given in plaque forming units (PFU). CELL LINES Viruses may be grown in:  Primary cell lines – from tissue slices; last only for few generations;  diploid cell lines – from human embryos; can be maintained for 100 generations (applications);  for culturing viruses that require human host  Used to culture rabies virus for a rabies vaccine called “diploid culture vaccine”  Continuous cell lines – used in routine viral growth in the lab  Transformed (cancerous) cells that can be maintained for an indefinite number of generations- IMMORTAL CELL LINES  Eg – HeLa cell line (from a female CA px who died in 1951) Normal cell Transformed cell DETECTION & IDENTIFICATION 1. Cytological or Histological Examination 2. Electron Microscopy 3. Virus Isolation in Cell or Tissue Culture 4. Shell Vial Centrifugation-Enhanced (SVCE) Virus Detection 5. Direct Detection of Viral Antigens or Genes 6. Target Gene Amplification (PCR) 7. Serological Detection of Viral Antibodies 1. Cytological or Histological Examination  Requires properly fixed and stained cells from specimens  Presence of the following will aid in viral dx:  Multinucleated cells  Giant cells  Cytoplasmic or nuclear inclusions  DNA viruses -intranuclear inclusions OWL’S EYE APPEARANCE -  RNA viruses – cytoplasmic inclusions Cells infected with CMV are enlarged and have inclusion  In general, cytological exam is insensitive bodies and non-specific  2. Electron Microscopy  Negative staining technique:  Specimen is placed on a carbon-coated grid  Stained w/ K phosphotungstate or uranylacetate  Stain surrounds the virus and the electron beam highlights the virus  Virus is seen as a light structure against a dark background  Electron mx is the only way to detect:  Astroviruses  Caliciviruses Norwalk virus  Coronaviruses 3. Virus Isolation in Cell or Tissue Culture  Cell culture is the GOLD STANDARD for viral identification  PRINCIPLE:  Cell cultures are animal or human cells grown in vitro that have lost their differentiation  Cells are grown as a single layer on the internal surface of glass/plastic containers to form a cell monolayer  Once a primary cell culture is subcultivated, it is known as a CELL LINE  These cells are sensitive to the effects of viruses and after inoculation and incubation w/ the specimen, they are examined for CYTOPATHOGENIC/CYTOPATHIC EFFECTS (CPE) Methods of Cell Cultures 1. Traditional cell cultures 2. SVCE (shell vial centrifugation-enhanced)cultures 3. Multiwell microplate cultures 3 Types of Cell Cultures 1. Primary cell lines 2. Diploid cell lines 3. Heteroploid or Immortal cell lines Traditional cell cultures Traditional cell cultures SVCE (shell vial centrifugation-enhanced)cultures Multiwell microplate cultures 1. Primary Cell Cultures  Cells grown have the same karyotype (appearance of chromosomes) and chromosome number as the original tissue.  Cells are derived directly from parent tissue; examples are: Human Embryonic Kidney (HEK) Rabbit Kidney (RK) Primary Monkey Kidney (PMK) Rhesus Monkey Kidney (RMK) Cynomolgus Monkey Kidney (CMK) African Green Monkey Kidney (AGMK)  Procedure  Tissue is minced, treated w/ proteolytic enzyme, filtered and added to a nutrient medium  Cells are then transferred to a container w/ flat surface, w/c permits the cell to multiply, forming a monolayer 2. Diploid Cell Lines  75% of cells have the same karyotype as the original tissue  Diploid cell lines can retain the diploid karyotype for approx 20-50 subcultures before losing their viability  Examples are: WI-38 derived from human embryonic lung MRC-5 derived from human embryonic lung Human diploid fibroblasts (HDFs) – derived from human kidney or lung fibroblasts 3. Heteroploid or Immortal Cell Lines  Cell lines with less than 75% normal cells; more than 25% of the cells have an abnormal karyotype  Derived from malignant tissue or other transformed/ cancer cells  These cells can undergo continuous or unlimited subcultures in vitro  Examples are: HeLa (Henrietta Lacks) derived from human cervical carcinoma Hep-2 derived from carcinoma of the human larynx KB derived from nasopharyngeal carcinoma A-549 derived from human lung carcinoma Vero derived from AGMK Cell Culture Indication/s: sensitive to Primary cell lines Influenza viruses, parainfluenza viruses, mumps viruses, enteroviruses and adenoviruses Diploid cell lines (HDFs) HSV, VZV, CMV, adenovirus, rhinovirus Heteroploid cell lines HSV, enteroviruses and adenoviruses  Incubation Conditions:  35-37deg C for 2 weeks - recovery of most viruses  For respiratory viruses, optimal recovery is @ 33deg C for 2 weeks  HSV - 35-37 deg C for 5-7 days  CMV – 35-37 deg C for 21 days Cytopathic Effects (CPE)  CPE – visible changes in virus-infected cells  CPE is observed using light or phase-contrast mx under LPO  CPE type depends on the infecting virus and type of cell line used  Examples of CPE (may be seen in individual cells or in entire monolayer)  Rounding  Clumping  Vacuolation  Granulation  Giant multinucleated cells  Cell fusion  Syncytial formation  Cell lysis Viral growth can cause cytopathic effects in the cell culture. Cytopathic effects can be so characteristic of individual viruses that they can often be used to identify viruses. Syncytia - Multinuclear Cell Cytoplasmic vacuolation in monocytes Rounding of cells Blood smear showing multiple basophilic inclusion bodies in neutrophils Syncytial epithelial cells and acidophilic intranuclear inclusion bodies at mucous membrane of the trachea  Viruses such as influenza, parainfluenza and mumps that produce little or no detectable cellular changes, can be identified through: HEMADSORPTION  Attachment of rbcs to the surface of virus-infected cells.  Limited to viruses with a hemagglutinin protein on their envelope  Example: Influenza A and Influenza B, Hemadsorption - macrophages infected by parainfluenza virus, mumps virus the African Swine Fever virus 4. Shell Vial Centrifugation-Enhanced (SVCE) Virus Detection  Centrifugation of the specimen onto virus-sensitive cells that are grown on cover slips at the bottom of shell vials  After incubation, the cells are fixed, and a fluorescent-labeled monoclonal or polyclonal Ab specific for viral Ags is added  Detection of the Ag-Ab binding is observed through fluorescence mx  ADVANTAGE: produces a more rapid result than conventional cell culture since  Incubation time can be decreased to 1-5 days  Detection time is shortened because viral gene products or Ags are detected rather than CPE  SVCE was originally developed to detect CMV early Ags but is now used to also detect HSV, adenovirus, VZV, influenza A and B, and RSV 5. Direct Detection of Viral Antigens or Genes  Identification of virus through detection of specific Ags present in the patient’s infected cells Methods: 1. Direct Fluorescence Antibody (DFA) - Specimen is fixed onto a slide and stained w/ a virus-specific fluorescein-labeled monoclonal or polyclonal Ab 2. Enzyme Linked Immunosorbent Assay (ELISA) – an “antigen capture” technique in which viral Ag is captured through complexing with specific antibodies ; ELISA would detect color change, indicating a positive result. Adv: rapid, producing same-day results 3. Latex Agglutination – latex beads are coated w/ Abs to the virus, and agglutination indicates a positive reaction Latex Agglutination A microtiter plate using enzyme-linked immunosorbent assay (ELISA). A color change from clear to yellow indicates a test positive for the pathogen 6. Target Gene Amplification (Polymerase Chain Reaction)  Identification of virus through detection of viral DNA or RNA in specimens  PCR is available in gene probe kits (HSV, CMV)  Adv: increased sensitivity due to amplifying one viral genome into several genomes; the viral genome can be amplified 1 million times or GENE PROBE KIT more 7. Serological Detection of Viral Antibodies  An indirect indicator that infection has occurred in the px’s past  Serological methods are useful when:  The virus fails to grow in cell cultures  Viral Ag, NA probe, amplification methods are unavailable  The virus is in a site that cannot be readily cultured (i.e. brain tissue)  Appearance of virus-specific immunoglobulins:  IgM - will begin to appear w/in 1-2 weeks of primary viral infection; IgM levels peak in 3-6 wks of infection and drop to undetectable levels in 2-3 months  IgG - 2 days after appearance of IgM; IgG levels peak in 4-12 wks and remain for several months; some may remain for years or for life Paired sera are recommended for serological detection  Acute-phase specimen (first specimen)  Collected when the clinical signs first appear  Convalescent-phase specimen (second specimen):  collected 2-3 weeks later, depending on the virus Points to Consider:  Traditionally, a four-fold increase in Ab titer indicates a seropositive reaction and strongly supports a dx of current infection  False positive and false negative rxns are important considerations in serological dx  Ab detection is NOT useful in the dx of chronic or recurrent viral infections such as HIV and CMV (Abs may be produced indefinitely in such pxs and do not necessarily indicate a current infection) VIRAL VACCINES VACCINES  Prevention of viral diseases can be achieved by the use of vaccines that induce ACTIVE IMMUNITY or the administration of preformed antibody that provides PASSIVE IMMUNITY. ACTIVE IMMUNITY Active immunity is resistance induced after contact with antigens. Active immunity can be elicited by vaccines containing: 1. Live, attenuated (weakened) viruses 2. Killed virus 3. Purified protein subunits 1. Vaccine containing live virus  Pathogenicity of the virus has been attenuated (weakened)  Virus multiplies in the host producing a prolonged antigenic stimulus  T cell response is activated  Both IgA and IgG are stimulated when given in the natural route 2. Vaccine containing killed virus  Usually given IM do not stimulate a cytotoxic T-cell response (no replication – no stimulation).  Disadvantages – shorter duration of protection, only IgG is produced  Advantages over live vaccine: they do not revert to virulent form and are more heat- stable. 3. Vaccine containing purified protein subunits  Hepatitis B vaccine contain purified viral proteins (envelope protein) often called as subunits.  No viral replication occurs in these vaccines since the viral genome is not included. ACTIVE IMMUNITY In general, live vaccines are preferable to killed vaccines: 1. They induce a higher titer of antibody and hence giving a longer-lasting protection 2. They induce a broader range of antibody: IgA and IgG, not just IgG 3. They activate cytotoxic T cells, which kill virus-infected cells Concerns of the use of Live Vaccines 1. They are composed of attenuated viral mutants 3. A second virus could contaminate the which can revert to virulence. vaccine.  The reversion may occur either during vaccine  Contaminating virus may exist in the cell production or in the immunized person. cultures used to prepare the vaccine ( for both live and killed vaccines).  Only polio vaccine has had problems w/ revertants; measles, mumps, rubella and varicella vaccines have not.  Example of this happened in the preparation of the killed polio vaccine. In 1960, a live simian vacuolating viirus 40 (SV40) was  Even if the virus does not revert, it can still cause a present in the monkey kidney cell cultures. It disease in a host with reduced immunity. For this reason, live viral vaccines SHOULD NOT be given to was resistant to the formaldehyde used to immunocompromised patients or to pregnant kill/inactivate the main virus. women because the fetus may become infected.  SV40 causes sarcoma in a variety of rodents, fortunately no cancer developed in 2. The live vaccine can be excreted by the immunized individuals. immunized person.  It is advantageous if the spread of the virus Special Note: successfully immunizes others, as occurs w/ polio vaccine, however, a problem may occur if a virulent Vaccines grown in chick embryos like influenza, revertant spreads to a susceptible person. Rare cases measles, mumps, and yellow fever, SHOULD NOT BE of paralytic polio have occurred via this route. GIVEN to those who have had an anaphylactic reaction to eggs. CURRENT VIRAL VACCINES USAGE VACCINE CLASSIFICATION COMMON Measles Live Mumps Live Rubella (german measles) Live Varicella (chickenpox) Live Polio Live and killed influenza Live and killed (purified subunits) Hepatitis A Killed Hepatitis B Subunit (HBs Ag only) Rabies Killed SPECIAL SITUATIONS Used when travelling in endemic areas Yellow fever Live Used when travelling in endemic areas Japanese encephalitis Killed For “first responders” of medical emergencies Adenovirus Live (military, medics, ER staff) For “first responders” of medical emergencies Smallpox Live (military, medics, ER staff) CHARACTERISTICS OF LIVE AND KILLED VIRAL VACCINES Characteristic Live Vaccine Killed Vaccine Duration of immunity Longer Shorter Effectiveness of protection Greater Lower Immunoglobulin produced IgA and IgG IgG Cell-mediated immunity produced Yes Weakly or none Reversion to virulence Possible No Stability at RT Low High Excretion of vaccine virus and Possible No transmission to nonimmune contacts PASSIVE IMMUNITY  Passive immunity is provided by the administration of preformed antibody in preparations called immune globulins.  The preformed antibodies (immune globulins) are sourced either from humans or animals whose serum has a high titer of antibody.  Passive immunity also occur naturally when IgG is transferred from mother to the fetus across the placenta and when IgA is transferred from the mother to the newborn in colostrum.  The main advantage of passive immunity is that it provides IMMEDIATE PROTECTION! PASSIVE IMMUNITY  Examples: 1. Rabies immune globulin (RIG) – prevention of rabies 2. Hepatitis B immune globulin (HBIG) – prevention of hepatitis B 3. Varicella – zoster immune globulin (VZIG) – prevention of disseminated zoster to individuals who have been exposed to the virus and who are immunocompromised. 4. Vaccinia immune globulins (VIG) – used to treat some of the complications of the smallpox vaccination. PASSIVE-ACTIVE IMMUNITY  Passive-active immunity is induced by giving both immune globulins to provide immediate protection and a vaccine to provide long – term protection. Examples: 1. RABIES  Rabies immune globulin (RIG) is used in the prevention of rabies in people who may have been exposed to the virus (bite from a rabid dog).  RIG is administered by injecting into the tissue at the bite site and the remainder is given IM.  After RIG, the px will be given with a vaccine containing killed rabies virus from human diploid cells.  RIG and the rabies vaccine should be given at different sites to prevent neutralization. PASSIVE-ACTIVE IMMUNITY Examples: 2. HEPATITIS B  Hepatitis immune globulin (HBIG) is used in the prevention of hepatitis B in people who have been exposed to the virus.  HBIG contains a high titer of antibody to HBV and is obtained from humans to avoid hypersensitivity reactions.  After HBIG, the px is given with hepatitis B vaccine (recombinant vaccine containing only the virus envelope – HBs Ag).  HBIG and hepatitis B vaccine should be given at different sites to prevent neutralization.