Human Biology 534 Introduction to Virology and Human Retroviruses 26/27 March 2001

1. Human Biology 534 Introduction to Virology and Human Retroviruses 26/27 March 2001 James I. Mullins, Ph.D., Professor and Chairman, Department of Microbiology, jmullins@u.washington.edu Spring Quarter, 2001 Copies of lectures can be downloaded (pdf) from: http://ubik.microbiol.washington.edu/Index.html KEYWORDS are in boldedred

2. Death Rate (per 105) from Infectious Diseases in the US 1900-1996

3. "Virus" is from the Greek meaning for "poison" and was initially described by Edward Jenner in 1798. Recognition of viruses FHow long viruses have been within our midst? 1500 BC: Leg deformities indicative of poliomyelitis, pock marks indicative of smallpox. During the 1800's, all infectious agents were considered to be viruses until Koch developed pure culture techniques which allowed the separation and growth of bacteria. In the late 1800's:Bacteria were purified and established as disease causing agents. It then became possible to distinguish them from the "filterable agents", those able to pass through special filters designed to prevent the passage of bacteria. The first viruses discovered included: Foot and mouth disease (picornavirus), 1898; Yellow fever (flavivirus), 1900; Rous sarcoma virus (oncogenic retrovirus), 1906

4. Over the past 100 years: A newly emerged strain of influenza killed 20 million people in 1918-1919 in the immediate aftermath of World War I (It was the most lethal combatant of the war). A decade later, polio became one of the most feared infections of children and young adults (Franklin D. Roosevelt, the U.S. President throughout the Depression and World War II, had polio). As the last century entered its final 20 years, a new >90% lethal virus, HIV, spread rapidly around the world via body fluid transmission. Viral diseases have played a major role in human history: Over the past 1000 years:Smallpox and measles were brought to North and South America by early European explorers/conquerers. These diseases, for which the native American populations had no acquired (partial) immunity, killed large fractions of the populations, and were a major factor in the decimation of these societies.

5. Over the past 10 years:As the global HIV epidemic continues, sporadic outbreaks of viral diseases spread to humans by non-human hosts, such as Ebola (from chimps?) and Hanta (from rats) raise the concern about future epidemics. Four Corners Virus (Hanta) Ebola

6. Mouse primary spleen cells transduced with a GFP (green fluorescent protein) -retrovirus vector THE DISCIPLINE OF VIROLOGY The study of virology inherently involves a merging together of what has traditionally been thought of as two separate "kinds" of science: basic and applied. We want to figure out how viruses are transmitted, how they replicate, and how the host organism responds. We also want to figure out how to prevent transmission, how to interfere with virus replication, and how we can confer immunity on the host. The "applied" follows from, and is dependent upon, the "basic" in a quite direct way. Virology as it is studied today, is therefore an outgrowth of research in: Infectious diseases - which provides recognition of viral pathogens and their sequelae, and Molecular Biology- because of the usefulness of viruses as probes of cellular metabolic processes and as vectors with potential for gene therapy.

7. WHERE WE STAND IN 2001: PREVENTING • CONTROLING • CURING VIRAL DISEASES Smallpox: effective vaccine; this is the only viral disease that has been wiped out worldwide Measles: effective vaccine since 1963; this disease could be eliminated with a world-wide effort Influenza: effective strain-specific vaccine, but new variant strains emerge periodically Polio: effective vaccine; will soon be the second viral disease wiped out HIV: no vaccine; effective drugs, but they are costly and toxic, plus resistant strains appear. World-wide spread continues via intimate contact. 50 million infected thus far Ebola: no vaccine; important host species unknown (found recently in chimps and rodents); outbreaks controllable because people die quickly and human-human transmission is via blood Hanta: no vaccine; rodent host; easy transmission to humans, but outbreaks controllable We also share the world, and our bodies, with viruses that cause hepatitis, respiratory disease, mononucleosis, diarrhea, genital warts, genital herpes, and some forms of cancer

8. FViruses cannot be grown on sterile media, but require the presence of specific host cells. WHAT IS A VIRUS? Virusesmay be defined as acellular organisms whose genomes consist of nucleic acid, and which obligately replicate inside host cells using host metabolic machinery to different extents, to form a pool of components which assemble into particles called Virions. FA virus differs from a cell in three fundamental ways: i A virus usually has only a single type of nucleic acid serving as its genetic material. This can be single or double stranded DNA or RNA; ii Viruses contain no enzymes of energy metabolism, thus cannot make ATP; iii Viruses do not encode sufficient enzymatic machinery to synthesize their component macromolecules, specifically, no protein synthesis machinery.

9. Viruses are distinguished from other obligate parasites, some of which are even simpler than viruses: MYCOPLASMA: Small bacterium that grows only in complex medium or attached to eucaryotic cells. CHLAMYDIA: Obligate intracellular bacterial parasite which depends on eucaryotic cell for energy. PROTOZOA: Obligate intracellular parasite of eucaryotes that replicate within eucaryotic cells. VIROID: Infectious agents that exist as naked nucleic acid. Found in plants. HEPATITIS DELTA VIRUS (HDV):Viroid-like agent whose replication is dependent upon HBV.

10. PRION (proteinacious infectious agent): Hypothesized identity of the unconventional slow viruses (such as the Kuru and Scrapie agents). No nucleic acid is known to be required for prion function. They are thought by many to consist solely of protein and perhaps lipids.

11. Kuru Scrapie BSE vCJD

13. BSE in Britian CJD, nvCJD in Britian

15. Rotavirus • Virus naming and classification • Usually based on data available at the time of discovery: • i Disease they are associated with, e.g.: • Poxvirus, Hepatitis virus, HIV, measles virus • ii Cytopathology they cause, e.g.: • Respiratory Syncytial virus, Cytomegalovirus • iii Site of isolation, e.g.: • Adenovirus, Enterovirus, Rhinovirus • iv Places discovered or people that • discovered them, e.g.: • Epstein-Barr virus, Rift Valley Fever • v Biochemical features, e.g.: • Retrovirus, Picornavirus, Hepadnavirus

16. Enterically transmitted “Infectious” A E Viral hepatitis F, G, ? Other * NANB Parenterally transmitted “Serum” B C D * 10-20% of cases of presumed viral hepatitis are still not accounted for These naming conventions can lead to confusion later, e.g., viral hepatitis is caused by at least 6 different viruses

17. Viral Hepatitis - An Overview Type of Hepatitis A B C D E Source of feces blood/ blood/ blood/ feces virus blood-derived blood-derived blood-derived body fluids body fluids body fluids Route of fecal-oral percutaneous percutaneous percutaneous fecal-oral transmission permucosal permucosal permucosal Chronic no yes yes yes no infection Prevention pre/post- pre/post- blood donor pre/post- ensure safe exposure exposure screening; exposure drinking immunization immunization risk behavior immunization; water modification risk behavior modification

18. Acute Hepatitis B Virus Infection with Recovery Symptoms HBeAg anti-HBe Total anti-HBc Titer IgM anti-HBc HBsAg anti-HBs HAV Infection 0 4 8 12 16 24 28 32 52 100 20 36 Weeks after Exposure Progression to Chronic HBV Infection Total anti-HAV Symptoms Acute (6 months) Chronic (Years) anti-HBe HBeAg Titer ALT HBsAg Titer Total anti-HBc Fecal HAV IgM anti-HAV IgM anti-HBc 0 4 24 36 12 52 4 5 6 12 0 1 2 3 Months after exposure Weeks after Exposure Years Typical Serological Courses of Infection

19. Chronic Infection Symptomatic Infection Outcome of HBV Infection by Age at Infection 100 100 Symptomatic Infection (%) 80 80 60 60 Chronic Infection (%) 40 40 20 20 0 0 1-6 months 7-12 months Older Children and Adults Birth 1-4 years Age at Infection

20. VirusClassification is now based principally on analysis of the particle: Common morphology: observed by electron microscopy Common serology: antigenic cross-reactivity Related genetic material: form of nucleic acid ssDNA (+ or - strand) dsDNA ssRNA (+ or - strand) dsRNA segmented RNA sequence homology DNA sequence Hybridization

21. Cellular and Viral Genomes Type of GenomeNumber of Gene Equivalents* Human cell (higher eukaryotes) 1,000,000 E. coli (bacteria) 5,000 Poxvirus, Herpesvirus (large DNA virus) 70-200 Influenza, human immunodeficiency virus 8-12 Papilloma, Hepatitis B virus 3-5 Potato spindle tubor "viroid" 1 *The number of average-sized genes assuming that the entire genome encodes sequence information for the synthesis of proteins.

22. Virus Life Cycle Early Phase: i Attachment to and entry of the virion into the host cell; ii Disassembly of the infectious particle; iii Replication of the viral genome; Late Phase: iv Replication of virus structural components; v Reassembly of the replicated pieces into progeny virus particles; vi Release from the host cell.

23. STEPS IN VIRAL REPLICATION 1. Attachment (adsorption) 2. Penetration 3. Uncoating 4. Genome replication 5. Assembly 6. Release

24. ATTACHMENT AND PENETRATION INTO CELLS The first steps in the replication process for all viruses involve the attachment of the virus particle (the virion) onto the cell surface and then the entry of at least a portion of the particle (including the genome) into the cytoplasm. Both of these steps are specific, and are the main reason for the species-specificity of viruses. For all viruses, some aspect of membrane disruption must occur to achieve entry. For enveloped viruses, this almost always involves membrane fusion between the viral envelope and a cell membrane (either the plasma membrane or an internal vesicle membrane). For non-enveloped virions, the viral surface protein(s) usually play a role in causing some very localized membrane disruption. The details of attachment and entry for many viruses are still somewhat unclear, but there are some viruses for which the process has been studied in depth. We will take a look today at three of these: Picornaviruses (poliovirus and "common cold" rhinoviruses), Influenza virus and HIV (retrovirus)

25. Icosahedral virion entry: Picornavirus Poliovirus (and rhinoviruses, which cause about 50% of "colds") have an icosahedral structure which has been highly characterized. The icosahedral surface of the virion consists of 60 copies each of three proteins designated VP1, VP2, and VP3. A groove, or "canyon", in the VP1 structure provides an annulus around each pentameric vertex that provides the specific attachment site to cells. A subsequent interaction of the protein with the cell's plasma membrane leads to the entry of the viral RNA genome into the cytoplasm.

26. Enveloped virion entry: Influenza virus Enveloped viruses gain entry to cells by use of specific viral proteins that have membrane fusion - inducing properties. Influenza virions bind to the cell surface and are endocytosed intact into the cell. The endocytic vesicle then fuses with an acidic vesicle. At pH 5, the hemaglutinin glycoprotein molecules in the influenza envelope undergo a structural transition that causes the amino terminal end of HA2 to flip outward and be exposed to the molecular environment. This highly hydrophobic segment interacts with the vesicle membrane and causes fusion. This fusion event dumps the viral genome into the cell's cytoplasm.

27. Attachment and entry of HIV Studies on HIV over the past 3 years have elucidated its entry process to the degree that it is the best understood of any human virus. The two viral envelope glycoproteins, gp120 and gp41, are responsible for attachment and membrane fusion, respectively. The cell surface glycoprotein CD4 is used as the primary receptor, and one of a few other proteins are used as co-receptors (typically CCR5 and CXCR4).

28. Information Flow in cells: DNA to DNA (Replication) DNA to RNA (Transcription) RNA to Protein (Translation) The Central Dogma

29. Viral Genome Replication Viruses utilize the flow of information of eucaryotic cells, as well as novel pathways, some which violate Central Dogma. The replication strategy of the virus depends on the nature of its genome. Viruses can be classified into seven (arbitrary) groups:

30. I: Double-stranded DNA (Adenoviruses; Herpesviruses; Poxviruses, etc) Some replicate in the nucleus e.g., adenoviruses, using cellular proteins. Poxviruses replicate in the cytoplasm and make their own enzymes for nucleic acid replication. II: Single-stranded (+)sense DNA (Parvoviruses) Replication occurs in the nucleus, involving the formation of a (-) sense strand, which serves as a template for (+) strand RNA and DNA synthesis.

31. III: Double-stranded RNA(Reoviruses; Birnaviruses) These viruses have segmented genomes. Each genome segment is transcribed separately to produce monocistronic mRNAs. IV: Single-stranded (+) sense RNA(Picornaviruses; Togaviruses, etc) 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. V: Single-stranded (-)sense RNA (Orthomyxoviruses, Rhabdoviruses, etc) Have a virion associated RNA directed RNA polymerase. a) Segmented e.g. Orthomyxoviruses. The first step in replication is transcription of the (-) sense RNA genome by the virion RNA polymerase to produce monocistronic mRNAs that serve as the template for genome replication. b) Non-segmented e.g. Rhabdoviruses. Replication occurs as above and monocistronic mRNAs are produced.

32. VI: Single-stranded (+) sense RNA with DNA intermediate in life-cycle (Retroviruses) 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 within the newly infecting virion. VII: Double-stranded DNA with RNA intermediate(Hepadnaviruses) Also rely on reverse transcription, but 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.

33. MATURATION AND RELEASE Maturation proceeds differently for naked, enveloped, and complex viruses Naked icosahedral viruses - Preassembled capsomers are joined to form empty capsids (procapsid) which are the precursors of virions. They are released from infected cells in different ways. Poliovirus is rapidly released, with death and lysis of infected cells. DNA viruses tend to mature in the nucleus tend to accumulate within infected cells over a long period and are released when the cell undergoes autolysis, and in some cases, may be extruded without lysis.

34. Enveloped Viruses - Viral proteins are first associated with the nucleic acid to form the nucleocapsid, which is then surrounded by an envelope. In nucleocapsid formation, the proteins are all synthesized on cytoplasmic polysomes and are rapidly assembled into capsid components. In envelope assembly, virus-specified envelope proteins go directly to the appropriate cell membrane (the plasma membrane, the ER, the Golgi apparatus), displacing host proteins. In contrast, the carbohydrates and the lipids are produced by the host cell. The viral envelope has the lipid constitution of the membrane where its assembly takes place (eg. the plasma membrane for orthomyxoviruses and paramyxoviruses, the nuclear membrane for herpesviruses[on right]). A given virus will differ in its lipids and carbohydrates when grown in different cells, with consequent differences in physical, biological, and antigenic properties.

37. VIRAL PATHOGENESIS Results from: Transmission to a new host Replication and spread within the host (a function of viral tropism) Cell damage and dysfunction (can be mediated by the virus or by immune defense mechanisms) Disease symptoms and abnormal laboratory test values

38. KOCH'S POSTULATES [Proof of etiology for infectious agents] 1) The organism must always be found in the diseased animal but not in healthy ones 2) The organism must be isolated from diseased animals and grown in pure culture away from the animal 3) The organism located in pure culture must initiate and reproduce the disease when reinoculated into susceptible animals 4) The organism should be reisolated from the experimentally infected animals Although not all of these criteria can be met when evaluating the etiology of human disease, and some etiologic agents still cannot be cultured, these general principles guide the establishment of etiology of all infectious agents

39. West Nile virus Transmission: By mosquitoes that have fed on infected birds. In 10-14 days the virus reaches the salivary gland and can be transmitted to humans and animals. Host: At least 17 native bird species, including the house sparrow. Once infected they can pass the virus to mosquitos for up to 5 days. Carrier: Several species can carry the virus, Culex pipiens is most common carrier in the NE US. They take 3-4 blood feedings in their 2-3 weeks life span. Incidental hosts: Humans and horses can be infected but most people do not become sick. Ordinary human contact will not spread the virus.

40. Viral Pathogenesis Viral pathogenesis is an abnormal situation of no value to the virus - the vast majority of virus infections are sub-clinical, i.e. asymptomatic.. For pathogenic viruses, there are a number of critical stages in replication which determine the nature of the disease they produce: 1) Entry into the Host The first stage in any virus infection, irrespective of whether the virus is pathogenic or not. In the case of pathogenic infections, the site of entry can influence the disease symptoms produced. Infection can occur via: * Skin - dead cells, therefore cannot support virus replication. Most viruses which infect via the skin require a breach in the physical integrity of this effective barrier, e.g. cuts or abrasions. Many viruses employ vectors, e.g. ticks, mosquitos or vampire bats to breach the barrier. * Respiratory tract - In contrast to skin, the respiratory tract and all other mucosal surfaces possess sophisticated immune defence mechanisms, as well as non-specific inhibitory mechanisms (cilliated epithelium, mucus secretion, lower temperature) which viruses must overcome. * Gastrointestinal tract - a hostile environment; gastric acid, bile salts, etc * Genitourinary tract - relatively less hostile than the above, but less frequently exposed to extraneous viruses (?) * Conjunctiva - an exposed site and relatively unprotected

41. 2) 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, e.g: Localized Infections: Virus: Primary Replication: Rhinoviruses U.R.T. Rotaviruses Intestinal epithelium Papillomaviruses Epidermis Systemic Infections: Virus: Primary Replication: Secondary Replication: Enteroviruses Intestinal epithelium Lymphoid tissues, C.N.S. Herpesviruses Oropharynx or G.U.tract Lymphoid cells, C.N.S.

42. 3) 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 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 viremia usually proceeds and is necessary for spread to the blood stream, followed by more generalized, higher titer secondary viremia as the virus reaches other target tissues or replicates directly in blood cells. As above, spread to nervous system is preceded by primary viremia. 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.

43. 4) Cell/Tissue Damage Viruses may replicate widely throughout the body without any disease symptoms if they do not cause significant cell damage or death. Retroviruses do not generally cause cell death, being released from the cell by budding rather than by cell lysis, and cause persistent infections, even being passed vertically to offspring if they infect the germ line. Conversely, Picornaviruses cause lysis and death of the cells in which they replicate, leading to fever and increased mucus secretion in the case of Rhinoviruses, paralysis or death (usually due to respiratory failure) for Poliovirus. 5) Cell/Tissue Tropism Tropism- the ability of a virus to replicate in particular cells or tissues - is controlled partly by the route of infection but largely by the interaction of a virus attachment protein (V.A.P.) with a specific receptor molecule on the surface of a cell, and has considerable effect on pathogenesis. Many V.A.P.'s and virus receptors are now known.

44. 6) Persistence vs. Clearance The eventual outcome of any virus infection depends on a balance between two processes: i) Persistence: Long term persistence of virus results from two main mechanisms: a) Regulation of lytic potential The strategy followed is the continued survival of a critical number of virus infected cells - sufficient to continue the infection without killing the host. * For viruses which do not usually kill the cells in which they replicate, this is not usually a problem, hence these viruses tend naturally to cause persistent infections, e.g. Retroviruses. * For viruses which undergo lytic infection, e.g. Herpesviruses, it is necessary to develop mechanisms which restrict virus gene expression, and consequently, cell damage. b) Evasion of immune surveillance - Includes: * antigenic variation * immune tolerance, causing a reduced response to an antigen, may be due to genetic factors, pre-natal infection, molecular mimicry * restricted gene expression * down-regulation of MHC class I expression, resulting in lack of recognition of infected cells e.g. Adenoviruses * down-regulation of accessory molecules involved in immune recognition e.g. LFA-3 and ICAM-1 by EBV. * infection of immunocompromised sites within the body e.g. HSV in sensory ganglia in the CNS * direct infection of the cells of the immune system itself e.g. Herpes viruses, Retroviruses (HIV) - often resulting in immunosuppression.

45. ii) Clearance: 2 mechanisms allow influenza virus to alter its antigenic constitution: Antigenic Drift: The gradual accumulation of mutations (e.g. nucleotide substitutions) in the virus genome which result in subtly altered coding potential and therefore altered antigenicity, and decreased recognition by the immune system. This process occurs in all viruses all the time, but at greatly different rates, e.g. RNA viruses >>> DNA viruses. The immune system constantly adapts by recognition of and response to novel antigenic structures - but is always one step behind. In most cases however, the immune system is eventually able to overwhelm the virus, resulting in clearance. Antigenic Shift: Is a sudden and major change in the antigenicity of a virus due to recombination or reassortment of the virus genome with another genome of a different antigenic type. This process results initially in the failure of the immune system to recognize a new antigenic type.

46. Determinants of cell damage and dysfunction 1. Direct destruction of cells by virus Poliovirus - poliomyelitis Herpes simplex - cold sores Rotavirus - diarrhea HIV - AIDS 2. Immune mediated destruction of virus-infected cells (CTL and Ab) Hepatitis B - hepatitis Dengue - Dengue shock Measles - post-measles encephalitis & SSPE 3. Indirect effector mechanisms Influenza - interleukins and interferons released Rhinovirus - kinin release, vascular changes Respiratory syncytial - IgE Ab-mediated effects Hepatitis B and C - Ab:Ag complex deposition 4. Virus-encoded immune alteration Adenovirus - E3 alters class I MHC expression Vaccinia (and other poxviruses) - TNF analogue Epstein-Barr - Interleukin 10 5. Allow access to other pathogens

47. Subtle effects of viruses 1. Chronic/persistent infection Long term viral growth without disease, more important in immunocompromised hosts Retroviruses, herpesviruses, hepatitis B, adenoviruses 2. Latent infection Lifelong presence of viral genome with potential for reactivation Retroviruses, papillomaviruses, herpesviruses 3. Abortive infection Virus persists with partial replication Measles: subacute sclerosing panencephalitis 4. Oncogenic transformation Retroviruses, hepatitis B, papillomaviruses (5. No viruses produce toxins)

48. Host Cell-Virus Relationships Explanations for the lack of replication in non-permissive cells: • No attachment to cell (no receptor) • Virus can enter but not uncoat • Virus can uncoat but not express genes (mRNA or protein) • Virus can express genes but produce no particles • Progeny particles form but fail to mature • Mature particles accumulate in the cell but are not released • Released viruses are not infectious for other cells

49. LATENCY When virus infection fails to result in an immediate production of progeny. Rather, the virus enters a latent state in which the viral genome may become incorporated into the host's genome or maintained as an extrachromosomal element. Latent viral genomes are passively replicated along with host chromosomes. Not all viral genes remain silent during latency and there may be a perceptible change in the phenotype of the cell. The viral genome may become reactivated, potentially up to decades after the initial infection. Herpes, papilloma, Hepadnaviruses: Latent forms exist as plasmids (nuclear, extrachromosomal element) Virus:Site of latent infection: Herpes simplex - sensory neurons Varicella-zoster - dorsal root ganglion Epstein-Barr - B lymphocytes Papilloma - basal epithelium e.g. Development of "cold sores" by herpes viruses after the host is exposed to large doses of unfiltered sunlight. Retroviruses: Latent forms exist as "proviruses" integrated into the host chromosome HTLV 1 - T cells HIV - CD4+ T cells, macrophages Latency can be associated with tumorigenic transformation of the cell. Infrequently, latent viral nucleic acid may be passed not only to the progeny of the cells within the host, but also through successive generations in the germ line.