An Introduction to Viruses

Transcription

1

2

3 An Introduction to Viruses Lecturer Dr Ashraf Khasawneh Department of Biomedical Sciences Virus infections are Universal.

4 Introduction to Virology A virus is an obligate intracellular parasite containing genetic material surrounded by protein Virus particles can only be observed by an electron microscope 3 Introduction to Virology Recognizing the shape, size, and structure of different viruses is critical to the study of disease Viruses have an inner core of nucleic acid surrounded by protein coat known as an envelope Most viruses range in sizes from nanometers 4

5 Viral Properties Viruses are inert (nucleoprotein ) filterable Agents Viruses are obligate intracellular parasites Viruses cannot make energy or proteins independent of a host cell Viral genome are RNA or DNA but not both. Viruses have a naked capsid or envelope with attached proteins Viruses do not have the genetic capability to multiply by division. Viruses are non-living entities 5 Virus vs. cells Property Viruses Cells Type of nucleic acid DNA or RNA DNA and RNA Proteins Few Many Lipoprotein membrane Enveloped Cell membrane present in present in some viruses all cells Ribosomes Absent Present Mitochondria Absent Present in eukaryotic cells Enzymes None or few Many Multiplication by binary fission No Yes (most cells) 6

6 Viruses are Ultramicroscopic Koneman et al. Color Atlas and Textbook of Microbiology 5th Ed The size of viruses 8

7 VIRAL STRUCTURE SOME TERMINOLOGY virus particle = virion protein which coats the genome = capsid capsid usually symmetrical capsid + genome = nucleocapsid may have an envelope 9 The complete infectious unit of virus particle Structurally mature, extracellular virus particles. Virion 10

8 Viral Structure - Overview Nucleic acid Capsid Nucleocapsid Envelope protein Membrane protein Spike protein Viral envelope** Fig 1. Schematic overview of the structure of animal viruses ** does not exist in all viruses 11 Distinguishing characteristics of viruses Obligate intracellular parasites Extreme genetic simplicity Contain DNA or RNA Replication involves disassembly and reassembly Replicate by "one-step growth 12

9 Naming viruses No taxa above Family (no kingdom, phylum, etc) Classified based on structures, size, nucleic acids, host species, target cells. 19 families of animal viruses (6 DNA, 13 RNA) Family name ends in viridae Subfamily ends in virinae Genus name ends in virus Species Example Family Herpesviridae Subfamily - Herpesvirinae Genus Simplex virus Common name herpes virus (Herpes simplex virus I (HSV-I) Disease fever blisters, cold sores 13 How are viruses named? Based on: - the disease they cause poliovirus, rabies virus - the type of disease murine leukemia virus - geographic locations Sendai virus, Coxsackie virus - their discovers Epstein-Barr virus - how they were originally thought to be contracted dengue virus ( evil spirit ), influenza virus (the influence of bad air) - combinations of the above Rous Sarcoma virus 14

10 Virus particle = virion 15 5 BASIC TYPES OF VIRAL STRUCTURE icosahedral nucleocapsid nucleocapsid lipid bilayer ICOSAHEDRAL ENVELOPED ICOSAHEDRAL helical nucleocapsid nucleocapsid COMPLEX lipid bilayer glycoprotein spikes = peplomers HELICAL ENVELOPED HELICAL 16

11 Viral Structure Varies in size, shape and symmetry 3 types of capsid symmetry: Cubic (icosahedral) Has 20 faces, each an equilateral triangle. Eg. adenovirus Helical Protein binds around DNA/RNA in a helical fashion eg. Coronavirus Complex Is neither cubic nor helical eg. poxvirus 17 VIRAL STRUCTURE (virion) 1. Protect genome during passage from one cell to another 2. Aid in entry process 3. Package enzymes for early steps of infection 18

12 Morphological types CAPSID STRUCTURE 1. Helical capsid Rod-shaped capsomers Coil around hollow center Nucleic acid is kept inside wound-up within tube (Helix ) 19 Morphological types Helical capsid surrounds DNA like hollow tube Ex: Influenza, measles, rabies (enveloped)

13 Helical symmetry 21 Helical symmetry How to assemble 22

14 Helical symmetry In 1955, Fraenkel, Conrat, and Williams demonstrated that tobacco mosaic virus (TMV) spontaneously formed when mixtures of purified coat protein and its genomic RNA were incubated together. TMV, a filamentous virus 23 Morphological types 2. icosahedral PROTOME R 20-sided with 12 corners Vary in the number of capsomers Each capsomer may be made of 1 or several proteins Some are enveloped

15 25 26

16 Icosahedral capsids a) Crystallographic structure of a simple icosahedral virus. b) The axes of symmetry 27 Cubic or icosahedral symmetry 28

17 ICOSAHEDRAL SYMMETRY 29 ICOSAHEDRAL SYMMETRY 30

18 ICOSAHEDRAL SYMMETRY 31 ICOSAHEDRAL SYMMETRY 32

19 Adenovirus 33 Adenovirus 34

20 Enveloped helical virus Enveloped icosahedral virus 35 Helical California Encephalitis Virus Coronavirus Hantavirus Influenza Virus (Flu Virus) Measles Virus ( Rubeola) Mumps Virus Para influenza Virus Rabies Virus Respiratory Syncytial Virus(RSV) 36

21 Icosahedral Adeno-associated Virus (AAV) Adenovirus B19 Coxsackievirus - A Coxsackievirus - B Cytomegalovirus (CMV) Eastern Equine Encephalitis Virus (EEEV) Echovirus Epstein-Barr Virus (EBV) Hepatitis A Virus (HAV) Hepatitis B Virus (HBV) Hepatitis C Virus (HCV) Hepatitis Delta Virus (HDV) Hepatitis E Virus (HEV) Herpes Simplex Virus 1 (HHV1) Herpes Simplex Virus 2 (HHV2) Human Immunodeficiency Virus (HIV) Human T-lymphotrophic Virus (HTLV) Norwalk Virus Papilloma Virus (HPV) Polio virus Rhinovirus Rubella Virus Saint Louis Encephalitis Virus Varicella-Zoster Virus (HHV3) Western Equine Encephalitis Virus (WEEV) Yellow Fever Virus 37 Complex viruses Have additional or special structures Examples: Poxviruses lack normal capsid instead, layers of lipoprotiens and fibrils on surface surface view cross section 38

22 A bacteriophage A bacteriophage is any one of a number of viruses that infect bacteria. They do this by injecting genetic material, which they carry enclosed in an outer protein capsid. The genetic material can be ssrna, dsrna, ssdna, or dsdna ('ss-' or 'ds-' prefix denotes single-strand or double-strand) along with either circular or linear arrangement. 39 Phage - viruses have a polyhedral head, helical tail and fibers for attachment.

23 Classification of viruses Nucleic acid Capsid Presence of envelope Replication strategy 41 RNA or DNA segmented or non-segmented linear or circular CLASSIFICATION NUCLEIC ACID single-stranded or double-stranded if single-stranded RNA is genome mrna (+) sense or complementary to mrna (-) sense 42

24 ENVELOPE OBTAINED BY BUDDING THROUGH A CELLULAR MEMBRANE (except poxviruses) POSSIBILITY OF EXITING CELL WITHOUT KILLING IT CONTAINS AT LEAST ONE VIRALLY CODED PROTEIN ATTACHMENT PROTEIN LOSS OF ENVELOPE RESULTS IN LOSS OF INFECTIVITY 43 Properties of naked viruses Stable in hostile environment Not damaged by drying, acid, detergent, and heat Released by lysis of host cells Can sustain in dry environment Can infect the GI tract and survive the acid and bile Can spread easily via hands, dust, fomites, etc Can stay dry and still retain infectivity Neutralizing mucosal and systemic antibodies are needed to control the establishment of infection

25 Naked viruses( Non Enveloped ) Adeno-associated Virus (AAV) Adenovirus B19 Coxsackievirus - A Coxsackievirus - B Echovirus Hepatitis A Virus (HAV) Hepatitis E Virus (HEV) Norwalk Virus The Baltimore classification system Based on genetic contents and replication strategies of viruses. According to the Baltimore classification, viruses are divided into the following seven classes: 1. dsdna viruses 2. ssdna viruses 3. dsrna viruses 4. (+) sense ssrna viruses (codes directly for protein) 5. (-) sense ssrna viruses 6. RNA reverse transcribing viruses 7. DNA reverse transcribing viruses where "ds" represents "double strand" and "ss" denotes "single strand". 46

26 Virus Classification - the Baltimore classification All viruses must produce mrna, or (+) sense RNA A complementary strand of nucleic acid is ( ) sense The Baltimore classification has + RNA as its central point Its principles are fundamental to an understanding of virus classification and genome replication, but it is rarely used as a classification system in its own right 47 Viral genome strategies dsdna (herpes, papova, adeno, pox) ssdna (parvo) dsrna (reo, rota) ssrna (+) (picorna, toga, flavi, corona) ssrna (-) (rhabdo, paramyxo, orthomyxo, bunya, filo) ssrna (+/-) (arena, bunya) ssrna (+RTase) (retro, lenti) 48

27 Sub-viral agents Satellites Contain nucleic acid Depend on co-infection with a helper virus May be encapsidated (satellite virus) Mostly in plants, can be human e.g. hepatitis delta virus If nucleic acid only = virusoid Viroids Unencapsidated, small circular ssrna molecules that replicate autonomously Only in plants, e.g. potato spindle tuber viroid Depend on host cell polii for replication, no protein or mrna Prions No nucleic acid Infectious protein e.g. BSE 49 Viroids & Prions Viroids ss RNA genome and the smallest known pathogens. Affects plants Prions Infectious particles that are entirely protein. No nucleic acid Highly heat resistant Animal disease that affects nervous tissue Affects nervous tissue and results in Bovine spongiform encepahltits (BSE) mad cow disease, scrapie in sheep kuru & Creutzfeld-Jakob Disease (CJD) in humans 50

28 Viroids Viroids are small ( nt), circular RNA molecules with a rod-like secondary structure which possess no capsid or envelope which are associated with certain plant diseases. Their replication strategy like that of viruses - they are obligate intracellular parasites. Viroids do not encode any proteins and unlike satellites they are not dependent on the presence of another virus Viroid replication Viroids utilize cellular RNA polymerases for their replication Replication is performed by rolling circle mechanism The resulting long RNA molecule is cut in pieces and ligated either autocatalytically or by cellular factors (depending on a viroid) So in a sense, at least some viroids are ribozymes...

29 Examples of plants, infected with various viroids Hepatitis virus a chimeric molecule, half viroid, half satellite Viroid like properties - Rod-like RNA molecule - Rolling circle replication - Self-cleaving activty Satellite like properties - Encodes a protein, which is necessary both for encapsidation and replication - Dependent on presence another virus HBV - Genome larger than for viroids (1640 nt)

30 Prions Prions are rather ill-defined infectious agents believed to consist of a single type of protein molecule with no nucleic acid component. Confusion arises from the fact that the prion protein & the gene which encodes it are also found in normal 'uninfected' cells. These agents are associated with diseases such as Creutzfeldt-Jakob disease in humans, scrapie in sheep & bovine spongiform encephalopathy (BSE) in cattle. Prions Prions are proteinaceous transmissible pathogens responsible for a series of fatal neurodegenerative diseases (in humans, Creutzfeld-Jakob disease and kuru, in animals, bovine spongioform encephalopathy) A prion (proteinaceous infectious particle, analogy for virion) is a type of infectious agent that does not carry the genetic information in nucleid acid! Prions are proteins with the pathological conformation that are believed to infect and propagate the conformational changes of the native proteins into the the abnormally srtructured form

31 Disease name Natural host Prion name PrP isoform Scrapie Sheep, goat Scrapie prion OvPrP Sc Transmissible mink encephalopathy (TME) Chronic wasting disease (CWD) Bovine spongioform encephalopathy (BSE) Feline spongioform encephalopathy (FSE) Exotic unguale encephalopathy (EUE) Mink TME prion MkPrP Sc Elk, mule deer CWD prion MDePrP Sc Cattle BSE prion BovPrP Sc Cat FSE prion FePrP Sc Greater kudu, nyala EUE prion NyaPrP Sc Kuru Human Kuru prion HuPrP Sc Creutzfeldt-Jakob disease (CJD) Gerstmann-Straussler- Scheinker syndrome (GSS) Fatal familial insomnia (FFI) Human CJD prion HuPrP Sc Human GSS prion HuPrP Sc Human FFI prion HuPrP Sc Prion diseases: rare neurodegenerative disorders (one person per million) 1. Sporadic (85 %) In the sixth or seventh decade, rapidly progressive (death in less than a year) Creutzfeldt-Jakob disease (CJD) 2. Familial (inherited-15%) Mutations in the PrP gene that favour the transition from the cellular form to the pathological form of PrP Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia (FFI) 3. Transmissible (rare; a source of great concern) Propagation of kuru disease in New Guinea natives (ritualistic cannibalism) Recently, it has been discovered that BSE had been transmitted to humans in Europe after consumption of infected beef, producing a variant of the CJD called vcjd

32 Transmissible spongioform encephalopathy (TSE)=prion disease A group of progressive conditions that affect the brain and nervous system of humans and animals and are transmitted by prions The pathology: vacuolar degeneration, neuronal loss, astrocytosis and amyloid plaque formation The clinical signs: loss of motor functions (lack of coordination, ataxia, involuntary jerking movements), personality changes, depression, insomnia, confusion, memory problems, dementia, progressive tonic paralysis, death Definitive diagnostic test: biopsy of brain tissue (histopathological examination and immunostaining for PrP Sc) There is no cure α-helix Conformational change β-sheet Normal protein (folded structure) Disease-associated protein (misfolded structure) Aggregation Gain of toxic activity Loss of biological function

33 PrP C The normal protein is called PrP C (for cellular) is a transmembrane glycoprotein (neurons, lymphocytes); its function is unknown; it binds Cu 2+ (regulation its homeostasis) has dominant secundary structure α- helix is easily soluble is monomeric and easily digested by proteases is encoded by a gene designated PRNP located on the chromosome 20 PrP Sc The abnormal, disease-producing protein is called PrP Sc (for scrapie) has the same amino acid sequence (primary structure) has dominant secundary structure β- sheets is insoluble is multimeric and resistant to digestion by proteases When PrP Sc comes in contact with PrP C, it converts the PrP C into more of itself These molecules bind to each other forming aggregates Molecular models of the structure of: PrP C PrP Sc Predominantly α-helix (3) β-sheets (40%), α-helix (30%)

34 Replication cycle The presence of an initial PrP Sc : exogenous (infectious forms) or endogenous (inherited or sporadic forms) This first prion will initiate PrP Sc accumulation by sequentially converting PrP C molecules into PrP Sc in replication cycle PrP Sc molecules aggregate Summary The prions are proteins that carry information for self-reproduction (contradict the central dogma of modern biology) The prions are expressed in cells of healthy humans and animals; their abnormal conformations (PrP Sc ) are insoluble, resistent to digestion and aggregate The PrP Sc attacks the native prion PrP C, changes its conformation into an abnormal form and causes an exponential production of insoluble proteins; they aggregate and form the fibrillar structure Prion disease are rare fatal degenerative disorders; a portion of them can be transmitted; this mechanism is not clear (e.g. transmision of BSE to human) One part of the prion protein can cause apoptosis, or programmed cell death Prions induce no immune reactions within the human

35 DNA VIRUSES DOUBLE STRANDED SINGLE STRANDED NON-ENVELOPED COMPLEX ENVELOPED ENVELOPED NON-ENVELOPED PARVOVIRIDAE POXVIRIDAE HERPESVIRIDAE HEPADNAVIRIDAE CIRCULAR LINEAR PAPILLOMAVIRIDAE POLYOMAVIRIDAE (formerly grouped together as the PAPOVAVIRIDAE) ADENOVIRIDAE 65 DNA viruses From Principles of Virology Flint et al ASM Press 66

36 RNA VIRUSES SINGLE STRANDED positive sense SINGLE STRANDED negative sense DOUBLE STRANDED ENVELOPED NONENVELOPED ENVELOPED NONENVELOPED ICOSAHEDRAL HELICAL ICOSAHEDRAL HELICAL ICOSAHEDRAL FLAVIVIRIDAE TOGAVIRIDAE RETROVIRIDAE CORONAVIRIDAE PICORNAVIRIDAE CALICIVIRIDAE ASTROVIRIDAE ORTHOMYXOVIRIDAE PARAMYXOVIRIDAE RHABDOVIRIDAE FILOVIRIDAE BUNYAVIRIDAE ARENAVIRIDAE REOVIRIDAE 67 NA viruses From Principles of Virology Flint et al ASM Press 68

37 Dr.T.V.Rao MD 70

38 BASIC STEPS IN VIRAL LIFE CYCLE ADSORPTION PENETRATION UNCOATING AND ECLIPSE SYNTHESIS OF VIRAL NUCLEIC ACID AND PROTEIN ASSEMBLY RELEASE 71

39 Viral life cycle 1 Viral replication terminology Plaque forming unit (pfu): measure of the number of particles capable of forming plaques per unit volume, such as virus particles Baculovirus plaques Zones of clearing (plaques) are generated by infection of insect (Sf9) cells with individual baculovirus particles. Uninfected Sf9 cells surrounding the plaque 2 are stained pink with neutral red.

40 PFU: plaque forming unit 211 plaques 1 PFU = 1 plaque = 1 bacterial phage Count : plaque # x Dilution x volume (ml) = PFU/ml Ex: 211 x 10 7 x 1 = 2.11 x 10 9 PFU/ml Viral replication terminology Multiplicity of infection (MOI): ratio of infectious agents (e.g. phage or virus) to infection targets Eclipse phase: period during which the input virus becomes uncoated; 10-12h Synthetic phase: time during which new virus particles are assembled; 4-6h Latent period: no extracellular virus can be detected Burst size: amount of infectious virus produced, per infected cell ; 10-10,000

41 One-step virus growth curve 5 The Replication Cycle Virus replication can be divided into eight arbitrary stages. Regardless of their hosts, all viruses must undergo each of these stages in some form to complete their replication cycle. Not all the steps described here are detectable as distinct stages for all viruses.

42 ATTACHMENT Click after each step to view process PENETRATION UNCOATING HOST FUNCTIONS VIRAL LIFE CYCLE REPLICATION ASSEMBLY (MATURATION) Transcription Translation RELEASE 7 MULTIPLICATION Host Cell Cytoplasm Cell membrane 1. Adsorption. The virus attaches to its host cell by specific binding of its spikes to cell receptors. 2 Receptors Spikes 1 Life cycle Animal virus 2. Penetration. The virus is engulfed into a vesicle and its envelope is uncoated, thereby freeing the viral RNA into the cell cytoplasm. Nucleus RNA 3 3. Duplication/Synthesis. Under the control of viral genes, the cell synthesizes the basic components of new viruses: RNA molecules, capsomers, spikes. New RNA New spikes New capsomers 4 5. Release. Enveloped viruses bud off of the membrane, carrying away an envelope with the spikes. This complete virus or virion is ready to infect another cell Assembly. Viral spike proteins are inserted into the cell membrane for the viral envelope; nucleocapsid is formed from RNA and capsomers. 8

43 Attachment Virus attachment consists of specific binding of a virusattachment protein (or 'antireceptor') to a cellular receptor molecule. Target receptor molecules on cell surfaces may be proteins (usually glycoproteins), or the carbohydrate residues present on glycoproteins or glycolipids. Some complex viruses (e.g. poxviruses, herpesviruses) use more than one receptor and have alternative routes of uptake into cells. Adsorption Enveloped With prominent spikes Naked; with capsid spikes Host range: the collection of hosts that an organism can utilize as a partner Cellular (tissue) tropism: the cells and tissues of a host which support growth of a particular virus

44 Coreceptor: CCR5 CRCX4 Virus Receptors Many examples of virus receptors are now known. Schematic representation of some virus receptors - arrows indicate virus attachment site: Polio HIV Corona Rhino Rhino Influenza Reovirus Rotavirus Echo Corona PVR CD4 MHV ICAM-1 VLA-2 LDL Aminopeptidase N Sialic acid

45 HA: Hemagglutinine How does an animal virus infect its host? Examples of Animal Virus Entry

46 Influenza Virus Receptor Binding The influenza haemagglutinin protein is one of two types of glycoprotein spike on the surface of influenza virus particles, the other type being the neuraminidase protein. Each haemagglutinin spike is composed of a trimer of three molecules, while the neuraminidase spike consists of a tetramer. The haemagglutinin spikes are responsible for binding the influenza virus receptor, which is sialic acid (N-acetyl neuraminic acid). As a result, there is little cell-type specificity imposed by this receptor interaction and therefore influenza viruses bind to a wide variety of different cell types. Influenza Virus Receptor Binding

47 Multiple Receptors In some cases, interactions with more than one protein are required for virus entry - neither protein alone is a functional receptor. Adenovirus receptor-binding is a two stage process involving an initial interaction of the virion fibre protein with a range of cellular receptors, including MHC class I molecule and the coxsackievirus-adenovirus receptor (CAR). Another virion protein, the penton base, then binds to the integrin family of cell surface heterodimers allowing internalization of the particle via receptor-mediated endocytosis. The primary receptor for HIV is the T cell antigen, CD4. These are Several members of a family of proteins known as b- chemokine receptors play a role in the entry of HIV into cells, and their distribution may be the primary control for the tropism of HIV for different cell types (lymphocytes, macrophages, etc). Penetration Penetration of the target cell normally occurs a very short time after attachment of the virus to its receptor in the cell membrane. Unlike attachment, cell penetration is generally an energy-dependent process, i.e. the cell must be metabolically active for this to occur. Three main mechanisms are involved.

48 Translocation 1) Translocation of the entire virus particle across the cytoplasmic membrane of the cell. This process is relatively rare among viruses and is poorly understood. It is mediated by proteins in the virus capsid and specific membrane receptors. Endocytosis 2) Endocytosis of the virus into intracellular vacuoles is probably the most common mechanism. Does not require any specific virus proteins (other than those utilized for receptor binding) but relies on the formation and internalization of coated pits at the cell membrane. Receptor-mediated endocytosis is an efficient process for taking up and concentrating extracellular macromolecules.

49 Fusion 3) Fusion of the virus envelope with the cell membrane, either directly at the cell surface or in a cytoplasmic vesicle. Fusion requires the presence of a fusion protein in the virus envelope which promotes joining of the cell and virus membranes, resulting in the nucleocapsid being deposited directly in the cytoplasm. There are two types of virusdriven membrane fusion: phdependent and phindependent. Fusion Endocytosis Pinocytosis (Viropexis)

50 Uncoating Uncoating is a general term for the events which occur after penetration. Uncoating is one of the stages of virus replication that has been least studied and is relatively poorly understood. The product of uncoating depends on the structure of the virus nucleocapsid. The structure and chemistry of the nucleocapsid determines the subsequent steps in replication.

51 Genome Replication and Gene Expression All viruses can be divided into seven groups - a scheme was first proposed by David Baltimore in Originally, this classification included only six groups, but it has since been extended to include the hepadnaviruses and caulimoviruses. For viruses with RNA genomes in particular, genome replication and the expression of genetic information are inextricably linked, so both are taken into account. The genomes I: Double-stranded DNA. Examples: Adenoviruses, Herpesviruses, Papillomaviruses, Poxiviruses, T4 bacteriophage Some replicate in the nucleus e.g adenoviruses using cellular proteins. Poxviruses replicate in the cytoplasm II: Single-stranded (+)sense DNA. Examples: phage M13, chicken anaemia virus, maize streak virus Replication occurs in the nucleus, involving the formation of a (-)sense strand, which serves as a template for (+)strand RNA and DNA synthesis. III: Double-stranded RNA. Examples: Reoviruses, Rotavirues These viruses have segmented genomes. Each genome segment is transcribed separately to produce monocistronic mrnas. IV: Single-stranded (+)sense RNA Examples: Hepatitis A and C, Small RNA phages, common cold viruses, SARS a) Polycistronic mrna e.g. Picornaviruses; Hepatitis A. Genome RNA = mrna. Means naked RNA is infectious, no virion particle associated polymerase. Translation results in the formation of a polyprotein product, which is subsequently cleaved to form the mature proteins. b) Complex Transcription e.g. Togaviruses. Two or more rounds of translation are necessary to produce the genomic RNA.

52 V: Single-stranded (-)sense RNA. Examples: Influenza viruses, Hantaviruses Must have a virion particle, containing RNA directed RNA polymerase. a) Segmented e.g. Orthomyxoviruses. First step in replication is transcription of the (-)sense RNA genome by the virion RNA-dependent RNA polymerase to produce monocistronic mrnas, which also serve as the template for genome replication. b) Non-segmented e.g. Rhabdoviruses. Replication occurs as above and monocistronic mrnas are produced. VI: Single-stranded (+)sense RNA with DNA intermediate in life-cycle (Retroviruses). Examples: HIV, Avian leukosis virus Genome is (+)sense but unique among viruses in that it is DIPLOID, and does not serve as mrna, but as a template for reverse transcription. VII: Partial double-stranded (gapped) DNA with RNA intermediate (Hepadnaviruses) Example: Hepatitis B This group of viruses also relies on reverse transcription, but unlike the Retroviruses, this occurs inside the virus particle on maturation. On infection of a new cell, the first event to occur is repair of the gapped genome, followed by transcription. The monocistronic mrna problem mrna AAAAAAAA RIBOSOMES PROTEIN AAAAAAAA Make one monocistronic mrna per protein Make a primary transcript and use alternative splicing Make a large protein and then cut it into smaller proteins Include special features in the mrna which enable ribosomes to bind internally 28

53 Class I: Double-stranded DNA This class can be subdivided into two further groups: A)Replication is exclusively nuclear. The replication of these viruses is relatively dependent on cellular factors. B)Replication occurs in cytoplasm (Poxviridae). These viruses have evolved (or acquired) all the necessary factors for transcription and replication of their genomes and are therefore largely independent of the cellular machinery. Class I: Double-stranded DNA Elsevier, 2005.

54 Replication occurs in the nucleus, involving the formation of a double-stranded intermediate which serves as a template for the synthesis of single-stranded progeny DNA. Class II: Single-stranded DNA

55 Class III: Double-stranded RNA These viruses have segmented genomes. Each segment is transcribed separately to produce individual monocistronic mrnas. Class IV: Single-stranded (+)sense RNA These can be subdivided into two groups: Viruses with polycistronic mrna. As with all the viruses in this class, the genome RNA forms the mrna. This is translated to form a polyprotein product, which is subsequently cleaved to form the mature proteins. Viruses with complex transcription. Two rounds of translation (e.g. Togavirus) or subgenomic RNAs (e.g. Tobamovirus) are necessary to produce the genomic RNA.

56 Class IV: Single-stranded (+)sense RNA Class V: Single-stranded ( )sense RNA The genomes of these viruses can be divided into two types: Segmented genomes First step in replication is transcription of the (-)sense RNA genome by the virion RNA-dependent RNA polymerase to produce monocistronic mrnas, which also serve as the template for genome replication. Non-segmented genomes

57 Class V: Single-stranded ( )sense RNA Class VI: Single-stranded (+)sense RNA with a DNA Intermediate Retrovirus genomes are (+)sense RNA but unique in that they are diploid, and do not serve directly as mrna, but as a template for reverse transcription into DNA.

58 Class VII: Double-stranded DNA with RNA Intermediate This group of viruses also relies on reverse transcription. Unlike the retroviruses (class VI), this occurs inside the virus particle during maturation. On infection of a new cell, the first event to occur is repair of the gapped genome, followed by transcription. Class VII: Double-stranded DNA with RNA Intermediate Elsevier, 2005.

59 Downloaded from: StudentConsult (on 15 May :15 AM) 2005 Elsevier All animal RNA viruses code for a Polymerase Positive/negative/double-stranded RNA virus genomes all encode a RNA-depend RNA polymerase. RNA-depend RNA polymerase is associated with negative RNA viruses. Reverse transcriptase is associated with retroviruses. 42

60 Single-strand positive-sense RNA- the virus genome is the virus mrna. 43 Single-strand negative-sense RNAvirus mrna is transcribed from the parental genome. 44

61 Double- stranded segmented RNA- individual virus mrnas are transcribed separately off the parental RNA segments using a transcriptase 45 associated with each segment 46

62 Replication Challenges for DNAViruses Access to nucleus Competing for nucleotides Cell cycle control in eucaryotes - S phase dependent materials for some Viruses (Parvo) Assembly Assembly involves the collection of all the components necessary for the formation of the mature virion at a particular site in the cell. During assembly, the basic structure of the virus particle is formed. The site of assembly depends on the site of replication within the cell and on the mechanism by which the virus is eventually released. in picornaviruses, poxviruses and reoviruses assembly occurs in the cytoplasm in adenoviruses, polyomaviruses and parvoviruses it occurs in the nucleus

63 Maturation Maturation is the stage of the replication-cycle at which the virus becomes infectious. Maturation usually involves structural changes in the virus particle which may result from specific cleavages of capsid proteins conformational changes in proteins. Virus proteases are frequently involved in maturation, although cellular enzymes or a mixture of virus and cellular enzymes are used in some cases. Release Apart from plant viruses which have evolved particular strategies to overcome the structure of plant cell walls, all other viruses escape the cell by one of two mechanisms: For lytic viruses (most non-enveloped viruses), release is a simple process - the infected cell breaks open and releases the virus. Enveloped viruses acquire their lipid membrane as the virus buds out of the cell through the cell membrane or into an intracellular vesicle prior to subsequent release. Virion envelope proteins are picked up during this process as the virus particle is extruded - this process is known as budding.

64 Release by budding Possible consequences to a cell that is infected by a virus: Lytic infections result in the destruction of the host cell; are caused by virulent viruses, which inherently bring about the death of the cells that they infect. When enveloped viruses are formed by budding, the release of the viral particles may be slow and the host cell may not be lysed. Such infections may occur over relatively long periods of time and are thus referred to as persistent infections. Viruses may also cause latent infections. The effect of a latent infection is that there is a delay between the infection by the virus and the appearance of symptoms. Some animal viruses have the potential to change a cell from a normal cell into a tumor cell, the hallmark of which is to grow without restraint. This process is called transformation. 52

65 Pathogenesis of viral infection Lecturer Dr Ashraf Khasawneh Department of Biomedical Sciences Viral epidemiology Endemic: Disease present at fairly low but constant level Epidemic: Infection greater than usually found in a population Pandemic: Infections that are spread worldwide Infectivity: The frequency with which an infection is transmitted when contact between a virus and host occurs Disease index: # persons develop disease/ total infected Virulence: # fatal cases/ total # of cases Incidence: # of new cases within a specific period of time % Prevalence: # of cases of a disease that are present in a particular population at a given time

66 What does a pathogen have to do? Infect (infest) a host Reproduce (replicate) itself Ensure that its progeny are transmitted to another host Virus route of entry 1. Horizontal: (person to person) a) Inhalation- via the respiratory tract ex. RSV, MMR, VZV, Rhinovirus b) Ingestion- via the gastrointestinal tract ex. Hep A, Rota, Astroviruses, Caliciviruses c) Inoculation- through skin abrasions; mucous membranes (e.g. sexual transmission); transfusion; injections (e.g. by doctors or via shared syringes in drug abuse); transplants 2. Vertical : i.e. from mother to fetus a) Transplacental ex. CMV, rubella, HIV b) Delivery ex. Hep B, Hep C, HSV, HIV, HPV c) Breast feeding ex. CMV, Hep B, HIV 3. Zoonotic ( animal to human) a) Animal bite ex. Rabies b) Insect bite ex. Dengue, West Nile c) Animal excreta ex. Hanta, Arena 4

67 Sites of virus entry cilliated epithelium, mucus secretion, lower temperature gastric acid, bile salts Terminology Incubation period: Time between exposure and first symptom Influenza 1-2d Chickenpox 13-17d Common cold 1-3d Mumps 16-20d Bronchiolitis,croup 3-5d Rubella 17-20d Acute respiratory disease 5-7d Mononucleosis 30-50d Dengue 5-8d Hepatitis A 15-40d Herpes simplex 5-8d Hepatitis B d Enteroviruses 6-12d Rabies d poliomyelitis 5-20d Papilloma d Measles 9-12d HIV 1-10y

68 Terminology Communicability: Ability of virus to shed into secretions Localized infection: infection limited to site of entry Disseminated infection: spread throughout the body Primary viremia: site of entry > regional LN > blood Secondary viremia: site of entry > regional LN > blood > organs (liver, spleen) > blood Primary Replication Having gained entry to a potential host, the virus must initiate an infection by entering a susceptible cell. This frequently determines whether the infection will remain localized at the site of entry or spread to become a systemic infection

69 Secondary Replication Occurs in systemic infections when a virus reaches other tissues in which it is capable of replication, e.g. Poliovirus (gut epithelium - neurons in brain & spinal cord) or Lentiviruses (macrophages - CNS + many other tissues). If a virus can be prevented from reaching tissues where secondary replication can occur, generally no disease results. Virus: Rhinoviruses Rotaviruses Papillomavirus es Virus: Enteroviruses Herpesviruses Localized Infections: Primary Replication: U.R.T. Intestinal epithelium Epidermis Systemic Infections: Primary Replication: Secondary Replication: Intestinal epithelium Oropharynx or G.U.tract Lymphoid tissues, C.N.S. Lymphoid cells, C.N.S.

70 Spread Throughout the Host Apart from direct cell-cell contact, there are 2 main mechanisms for spread throughout the host: via the bloodstream via the nervous system via the bloodstream Virus may get into the bloodstream by direct inoculation - e.g. Arthropod vectors, blood transfusion or I.V. drug abuse. The virus may travel free in the plasma (Togaviruses, Enteroviruses), or in association with red cells (Orbiviruses), platelets (HSV), lymphocytes (EBV, CMV) or monocytes (Lentiviruses). Primary viraemia usually proceeds and is necessary for spread to the blood stream, followed by more generalized, higher titre secondary viraemia as the virus reaches other target tissues or replicates directly in blood cells

71 via the nervous system spread to nervous system is preceded by primary viraemia. In some cases, spread occurs directly by contact with neurons at the primary site of infection, in other cases via the bloodstream. Once in peripheral nerves, the virus can spread to the CNS by axonal transport along neurons (classic - HSV). Viruses can cross synaptic junctions since these frequently contain virus receptors, allowing the virus to jump from one cell to another Virulence and cytopathogenicity Virulence: the ability of the virus to cause disease in infected cell Persistent infection Latent infection, lysogeny Chronic infection Permissive cells allow production of virions and/or transformation Virulent viruses Kill target cell and cause disease (productive response) Nonpermissive cells permits cell transformation only Abortive infection no virus replication, early viral proteins cause cell death Cytopathic effect

72 Cytopathic effects- virus-induced damage to cells 1. Changes in size & shape 2. Cytoplasmic inclusion bodies 3. Nuclear inclusion bodies 4. Cells fuse to form multinucleated cells 5. Cell lysis 6. Alter DNA 7. Transform cells into cancerous cells 8. Virokines and viroreceptors: DNA viruses; cell proliferate and avoid host defenses Cytopathic changes in cells

73 17 Patterns of viral infection Inapparent infection( Subclinical infection). Apparent infection: Acute infection Persistent Infection Chronic infections Latent Infection Slow virus infections

74 Patterns of viral infection Acute followed by clearing Chronic Infection Acute followed by persistent infection and virus overproduction Slow chronic infection Chronic Infection Virus can be continuously detected ; mild or no clinical symptoms may be evident.

75 Latent infection The Virus persists in an occult, or cryptic, from most of the time. There will be intermittent flare-ups of clinical disease, Infectious virus can be recovered during flare-ups. Latent virus infections typically persist for the entire life of the host Slow virus infection A prolonged incubation period, lasting months or years, during which virus continues to multiply. Clinical symptoms are usually not evident during the long incubation period.

76 Overall fate of the cell The cell dies in cytocidal infections this may be acute (when infection is brief and selflimiting) or chronic (drawn out, only a few cells infected while the rest proliferate)-cytocidal effect The cell lives in persistent infections this may be productive or nonproductive (refers to whether or not virions are produced) or it may alternate between the two by way of latency and reactivation - Steady state infection Transformation-Integrated infection (Viruses and Tumor) RNA tumor viruses usually transform cells to a malignant phenotype by integrating their own genetic material into the cellular genome and may also produce infectious progeny. Retroviruses: Acute transforming viruses: v-src oncogene mimic cellular genes (proto-oncogene) Insertional mutagenesis: inappropriate expression of a proto-oncogene adjacent to integrated viral genome Transactivating factors: tax gene in HTLV-1; turns on cellular genes causing cellular proliferation DNA tumor virus infections are often cytocidal; thus transformation is associated with abortive or restrictive infections in which few viral genes are expressed. The persistence of at least part of the viral genome within the cell is required for cell transformation. This is accompanied by the continual expression from a number of viral genes. P53: regulates the cell cycle; functions as a tumor suppressor that is involved in preventing cancer. HPV prb: prevent excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide. HPV Apoptosis P53: initiate apoptosis, programmed cell death, if DNA damage proves to be irreparable

77 Types of Viral infections at the cellular level Type Virus production Fate of cell Abortive - No effect Cytolytic + Death Persistent Productive + Senescence Latent - No effect Transforming DNA viruses - Immortalization RNA viruses + Immortalization Mechanisms of viral cytopathogenesis Inhibition of cellular protein synthesis Inhibition and degradation of cellular DNA Alteration of cell membrane structure Glycoprotein insertion Syncytia formation Disruption of cytoskeleton permeability Inclusion bodies Polioviruses, HSV, poxviruses, togaviruses herpesviruses All enveloped viruses HSV, VZ virus, HIV HSV, HIV, RSV Togaviruses, herpesviruses Rabies Toxicity of Virion components Adenovirus fibers

78 Possible consequences to a cell that is infected by a virus Lytic infections: Result in the destruction of the host cell; are caused by virulent viruses, which inherently bring about the death of the cells that they infect. persistent infections: Infections that occur over relatively long periods of time, Where the release of the viral particles may be slow and the host cell may not be lysed. latent infections: Delay between the infection by the virus and the appearance of symptoms. Transformation: Some animal viruses have the potential to change a cell from a normal cell into a tumor cell which grows without restraint