Going Viral: Understanding Viruses

Going Viral  NOW LET’S GO VIRAL…. https://www.instagram.com/reel/C2Vs33BO4IU/?igsh=Y3R0dzE1YmhnOW9r https://www.instagram.com/reel/C2OJF1CqDjH/?igsh=MTM3YTJsMzVkeTAwNQ== DID YOU KNOW?  There are an estimated nonillion viruses in the world  Mammals & birds thought to host 1.7 million U...

Going Viral  NOW LET’S GO VIRAL…. https://www.instagram.com/reel/C2Vs33BO4IU/?igsh=Y3R0dzE1YmhnOW9r https://www.instagram.com/reel/C2OJF1CqDjH/?igsh=MTM3YTJsMzVkeTAwNQ== DID YOU KNOW?  There are an estimated nonillion viruses in the world  Mammals & birds thought to host 1.7 million UNDISCOVERED viruses  “Spillover event”- when viruses jump to humans  A little over 219 different viruses known to infect humans (2012)  Viruses in troposphere  WHY DO WE NEED VIRUSES?!  If we eliminated all viruses, we’d probably die in about 2 days  Most aren’t pathogenic to humans  Help with ecosystems, fungi, plants, etc.  Bacteriophages (viruses that infect bacteria) keep bacteria from taking over  By killing bacteria in the oceans, they allow plankton survive & produce oxygen  Oncolytic viruses  CLASS OBJECTIVES  Compare & contrast viruses & prokaryotic & eukaryotic organisms  List the definition, properties, consequences of viral properties  Be able to use basic terminology about viruses  Be able to describe how viruses are structured (capsid, envelope, etc.)  Be able to reference back to table & figures in this lecture as we begin to learn more specifics of each virus  Be able to apply the six stages of the establishment of infectious diseases to viruses  And elaborate on examples of encounter/entry, spread, damage, & three types of outcomes  Aka 4 main routes of entry, vertical vs. horizontal spread, physiological spread, & acute/chronic/latent infection outcome  Provide examples of the body’s intrinsic & extrinsic protective mechanisms  Describe the 6 steps of the viral life cycle and components of each  Describe basic differences between RNA & DNA viruses, positive sense & negative sense, & single stranded & double stranded  VIRUSES ARE NEITHER PRO NOR EUKARYOTIC BECAUSE THEY AREN’T “LIVING” THINGS CANNOT REPRODUCE BY THEMSELVES DEFINITIONS & PROPERTIES OF A VIRUS CONSEQUENCES OF VIRAL PROPERTIES  VIRAL STRUCTURE Knowledge of the structural (size and morphology) and genetic (type and structure of nucleic acid) features of a virus provides insight into how the virus replicates, spreads, and causes disease. Structures of a naked icosahedral capsid virus (top left) & enveloped viruses (bottom) with an icosahedral (left) nucleocapsid or a helical (right) ribonucleocapsid Helical nucleocapsids are always enveloped for human viruses  CAPSID MORPHOLOGY & THE ENVELOPE  Viral nucleic acid surrounded by a capsid (single- or double-layer protein shell)  Nucleic acid + capsid = nucleocapsid  Icosahedral- 20 triangular faces & 12 vertices subunits (soccer ball shape)  Helical- varied subunits, spherical core tight with specific viral capsid proteins  Viral envelope- membrane made of lipids, proteins, and glycoproteins  Nonenveloped (naked)- relatively stable & resistant to temp. changes, acids, proteases, detergents, & drying  Enveloped (not naked)- more fragile & susceptible to heat, acids, detergents & drying. MUST REMAIN WET & generally transmitted in fluids  Virus-specific envelope glycoproteins protrude from outer surface of envelope  i.e. SARS-CoV-2 spike protein  *You may see the term “head and tail” virus morphology, but they infect bacteria, not humans so we won’t learn that as a capsid shape VIRUS PARTICLE= VIRION  CLASSIFICATION Examples:  Structure  Picornavirus- pico “small” + rna “ribonucleic acid”  Togavirus- toga “mantle” (i.e. envelope)  Replication  Retrovirus- retro “reverse”  Location (of body)  Adenovirus- (adenoids)  Reovirus- Respiratory, Enteric, Orphan)  Location (place)  Norwalk virus- Norwalk, Ohio  Coxsackievirus- Coxsackie, New York Reovirus discovered before they knew it caused SPECIFIC disease, therefore they called it an Orphan virus  GLYCOPROTEINS  Most viral glycoproteins have asparagine-linked ( N -linked) carbohydrates and extend through the envelope and away from the surface of the virion  “Spike glycoproteins” (heard of this with Covid?)  Major antigens that elicit protective immunity  Some glycoproteins act as VAPs (Virus Associated Pyramids) that are capable of binging to structures on target cells  If they (VAPs) bind to erythrocytes = hemagglutinins (HAs)  Other glycoproteins include neuraminidase (NA; influenza), Fc receptor & C3b receptor (HSV)  Naked viruses can also express certain glycoproteins, even if they aren’t bound to envelop, just the capsid (i.e. adenoviruses) MAIN GROUPS OF HUMAN VIRUSES REFER TO TABLE PRINT IT OUT.. POST IT EVERYWHERE, VERY HELPFUL. TEST YOUR KNOWLEDGE #1  What are the two capsid shapes?  Icosahedral & helical Encounter Entry WHO REMEMBERS THE Spread SIX STAGES OF Multiplication ESTABLISHMENT OF INFECTIOUS Damage DISEASE?! Outcome  ENCOUNTER & ENTRY  4 Main Routes:  Respiratory  Gastrointestinal  Transcutaneous  Sexual  Exogenous vs. Endogenous Virus  Most viral diseases come from Exo  Endo= reactivation of latent virus from within host cell i.e. HSV, VZV (shingles)  SPREAD & MULTIPLICATION (AKA REPLICATION)  Vertical vs. Horizontal spread (more like how the virus spreads amongst people)  Vertical- fetus infected in-utero  Horizontal- between members of susceptible host population  Physiological spread  Neural- virus spreads through nerves, i.e. HSV, rabies,VZV  Hematogenous- carried in the blood, i.e. CMV, HIV, EBV  High titer in bloodstream=viremia  *Exception, some viruses have multiple pathways for spread, i.e.VSV skin & neural  Incubation Period- the period between exposure to an infection and the appearance of the first symptoms  DAMAGE  Signs & symptoms of viral disease are culmination of a series of interactions between virus & host  Intrinsic protective mechanisms:  Apoptosis  Autophagy (“self ” “eating”)- cellular stress response viral sequestration & degradation in cytoplasmic organelles called autophagosomes  Extrinsic protective mechanisms:  Barriers  Innate immune responses-  Toll like receptors (TLRs)- pattern recognition receptors that identify conserved patterns in pathogens  Interferon- inhibit virus replication indirectly by inducing the expression of cellular proteins that inhibit the protein synthesis machinery  Cell-mediated immunity- NK cells (innate), Cytotoxic T-cells (adaptive)  Adaptive immune response  Neutralizing antibodies (destroy infectivity of virus)  Antibody-dependent cell-mediated cytotoxicity- antibodies lyse virus infected cells OUTCOME  Three types of viral infections:  Acute infection- virus undergoes multiple rounds of replication, results in death of host cell OR host successfully controls virus  Chronic infection- virus particles continue to shed after acute illness, sometimes without host death; host doesn’t successfully control virus  Usually RNA viruses  Latent infection- does not result in production of progeny viruses; host may or may not have “controlled” virus  DNA viruses or retroviruses  Can reactivate  Can cause cellular transformation -> cancer TEST YOUR KNOWLEDGE #2 VIRAL LIFE CYCLE 1. Attachment 2. Entry 3. Uncoating 4. Synthesis of protein & nucleic acid 5. Assembly 6. Release  1. ATTACHMENT & 2. ENTRY & 3. UNCOATING  Attachment: Virus attaches to host cell adhesion receptors on surface  Enveloped viruses: use surface spike proteins  One or more attachment proteins  Naked viruses: surface-exposed capsid regions mediate attachment  Tissue tropism- Bind to specific receptor(s)  Entry: Virion moves along host cell surface where it encounters entry receptors (aka co-receptors)  Membrane fusion: Fuses with host membrane & nucleocapsid is released  OR Endocytosis & virus is uncoated inside cell  Uncoating: virions disassembled. Capsid is removed to make viral genome accessible to the cellular transcription & translation machinery * There are exceptions, but wait until we learn those individual viruses  4. SYNTHESIS OF PROTEIN & NUCLEIC ACID  Depends on if virus is single- or double-stranded & DNA or RNA (we’ll learn more later)  This will be useful for later when we learn the different viruses (so keep it in mind for later/reference)  VIRAL NUCLEIC ACID & “SENSES”  Comprise the viral genome  DNA viruses  DNA viruses move to the nucleus for genome replication (poxviruses are an exception)  Immediate-early and early transcription produces the enzymes and proteins needed for late transcription  Early transcription is followed by genome replication.  Late transcription produces the capsid structural proteins.  Show that assembly results in the formation of the nucleocapsid.  RNA viruses  Most RNA viruses replicate in the cytoplasm (exceptions include influenza and retroviruses). Most are single-stranded.  The RNA virus genome encodes RNA-dependent RNA polymerases as well as the enzymes necessary for viral mRNA processing.  Self-assembly of the genome and structural proteins produces the nucleocapsid.  Classified according to the polarity of their RNA  Positive-sense strand viral genomes act as mRNA for protein synthesis, so translation can begin immediately.  Naked positive-sense viral genomes are infectious.  Negative-sense strand viral genomes do not immediately begin transcription. Like templates for messenger RNA that have to be transcribed into positive RNA Act as a template for synthesis of a positive-sense strand, which is then used for protein synthesis.  Naked negative-sense strand viral genomes are not infectious by themselves. ***Be aware that there are exceptions and complications to this classification system  VIRAL GENETICS  Recombination- Genetic information is exchanged between viruses or a virus and its host.  Reassortment- Viruses with segmented genomes create a hybrid strain.  This can lead to antigenic shift; E.g. H5N1 Bird flu  Complementation- Gene products or functions of a mutated virus are complemented by another virus.  A mutated virus is non-functional until its complement virus arrives and produces a protein or function that is beneficial to both.  E.g. Hepatitis D relies on replicating Hepatitis B to provide its envelope protein.  Phenotypic mixing- A virus genome is partially or completely coated with surface proteins of a different virus.  Be aware that the progeny of this virion will have a protein coat that matches its genome.  6. RELEASE (& M PROTEIN & VIRAL BUDDING)  Release  Nonenveloped- usually when then cell lyses  Lytic virus – burst out of host cell killing it  In contrast to “Lysogenic virus”  Lysogenic virus – integrate into host genome and are replicated when it is replicated; like “hitching a ride” or a “Trojan horse” until conditions trigger it to produce new virus particles  Exocytosis- reverse phagocytosis  Enveloped- budding out of host cell; Some cause apoptosis, some are non-lethal  M-protein- inner surface of envelope & touches nucleocapsid  Stabilize glycoprotein & lipid envelope interaction  Direct viral genome to sites of virus assembly  Help virus budding TEST YOUR KNOWLEDGE #2  What are the 6 steps of viral replication?  Attachment  Entry  Uncoating  Synthesis of protein & nucleic acid  Assembly  Release ALSO A SCIENTIST Rebecca Lee Crumpler, MD (1831-1895) MD from New England Female Medical College  1864- First African-American woman to earn an MD  1883- Published “Book of Medical Discourses,” one of the very first medical publications by an African American  “It may be well to state here that, having been reared by a kind aunt in Pennsylvania, whose usefulness with the sick was continually sought, I early conceived a liking for, and sought every opportunity to relieve the sufferings of others.” Antivirals  TODAY’S CLASS OBJECTIVES  Be able to select appropriate diagnostic tests for viral infections  *no, not specific, just overall tests you can use- rapid antibody, rapid antigen, culture, PCR, serology, ELISA  List ways to prevent infection & describe mechanisms of virucidal agents  Give examples of the way virions can be disrupted  Describe the 9 steps of the detailed viral life cycle & components of each (we’re expanding on the basic overview I introduced during “Going Viral”)  Describe basic differences between RNA & DNA viruses, positive sense & negative sense, & single stranded & double stranded (we’ll discuss this more as we cover the DNA & RNA viruses)  Be able to reference back to table & figures in this lecture as we begin to learn more specifics of each virus  Illustrate the different targets & mechanisms of action of different antiviral drugs  Be able to “prescribe” different antiviral drugs based on mechanisms of action (MOA) & the viruses that can be treated (see handy dandy “Antivirals to Know” chart)  What is the importance of thymidine kinase? HISTORY OF ANTIVIRAL DRUGS APPROVED BETWEEN JANUARY 1959 AND APRIL 2016.  DIAGNOSING VIRAL INFECTIONS  Diagnosing can help you properly treat viral infections  Rapid antibody- Specific antigens used to capture antibody from a patient’s sample  Rapid antigen- Specific antibodies used to capture antigen from a patient’s sample  Enzyme-linked immunosorbent assay (ELISA)- quantify antigen OR antibody in solution  Culture- samples of a virus are placed in different cell cultures which virus being tested for its ability to infect. If cells show changes, known as cytopathic effects, then the culture is positive.  Serology- diagnostic examination of blood serum, esp. regarding immune response to pathogens  PCR- fast, highly accurate way to diagnose certain infectious diseases & genetic changes by finding viral DNA or RNA in a sample  VIRION DISRUPTION  Some antiviral agents don’t have to be “drugs”  Lipids, detergents, acids, etc. can disrupt the surfaces of some viruses (esp. enveloped)  E.g. citric acids disrupt surface proteins of rhinoviruses  Adding citric acids to facial tissues might help prevent rhinovirus transmission?  If the virus is killed, the agent is virucidal WHY SHOULD YOU CARE? *NEW*- SARS-CoV-2 MAIN GROUPS OF HUMAN VIRUSES REFER TO TABLE PRINT IT OUT.. POST IT EVERYWHERE, VERY HELPFUL.  PHASES OF VIRAL REPLICATION  Early phase- virus must recognize an appropriate target cell; attach to the cell; penetrate plasma membrane & be taken up by cell; release (uncoat) its genome into cytoplasm; & (if necessary) deliver genome to nucleus  Late phase- begins with start of genome replication & viral macromolecular synthesis & through viral assembly & release  Eclipse period- the time of synthesizing virus proteins & nucleic acids inside the host cell & ends with appearance of new virions after assembly  Latent period (do NOT confuse with latent infection)- time between injection of viral genome into host cell & host cell lysis. Extracellular infectious virus is not detected; includes eclipse period & ends with the release of new viruses BASIC VIRAL LIFE CYCLE 1. Attachment 2. Entry 3. Uncoating 4. Synthesis of protein & nucleic acid 5. Assembly 6. Release  DETAILED VIRAL REPLICATION 1. Recognition of the target cell 2. Attachment 3. Penetration 4. Uncoating 5. (& 6 & 7) Transcription, Protein synthesis (Translation), & Replication a. While early mRNA & nonstructural protein transcribed (5), the genome can be replicated (7) b. Late mRNA and structural protein synthesis & posttranslational modification of protein (6) 8. Assembly of virus 9. Lysis & release (usually Naked) OR budding & release (usually Enveloped) DRUG TARGETS OF VIRAL LIFE CYCLE  Since viruses use host cells, you must be careful to choose drugs that don’t seriously damage host  Some steps in virus replication differ sufficiently from cellular processes in that they can be inhibited with little or no impact on the host cell  Antiviral drugs = Virustatic only effective against replicating viruses TYPES OF ANTIVIRAL DRUG TARGETS +Enfurvirtide- HIV Protease inhibitors, e.g. Ritonavir, nirmatrelvir (Neuraminidase inhibitors)  1. RECOGNITION OF & 2. ATTACHMENT TO TARGET CELL  Host range- Viruses that bind to receptors expressed on specific cell types may be restricted to certain species (e.g. human, mouse, pangolin, bat)  Tissue tropism- Bind to specific receptor(s)  e.g., neurotropic, lymphotropic 2. Attachment: Virus VAPs/glycoproteins attach to host cell adhesion receptors on surface  Binding of VAPs or structures on surface of virion to receptors on cell initially determines which cells can be infected by a virus  Receptors may be proteins OR carbohydrates OR glycoproteins OR glycolipids  Enveloped viruses: use surface VAPs  Naked viruses: surface-exposed capsid regions, or capsid proteins mediate attachment * There are exceptions, but wait until we learn those individual viruses  PREVENT ATTACHMENT  Examples of agents that prevent viral attachment to the host cell:  Synthetic Neutralizing Antibodies- can bind & neutralize VAPs  Receptor antagonists- type of receptor ligand or drug that blocks/dampens biological response by binding to & blocking a receptor rather than activating  Peptide analogues can block host attachment proteins  Drugs that block HIV co-receptor necessary for entry  E.g. Maraviroc- Blocks HIV co-receptor CCR5  Heparan sulfate & dextran interfere with viral binding on host cells, blocking the attachment of HIV & HSV  Administration of specific antibodies (passive immunization) is oldest form of antiviral therapy (e.g. RegenCoV)  3. PENETRATION & 4. UNCOATING 3. Penetration: Virion moves along host cell surface where it encounters entry receptors (aka co-receptors)  Mechanism of internalization depends on the virion structure & cell type  Naked virus:  Endocytosis (receptor-mediated) & then virus is uncoated inside cell  Viropexis (Picorna, papilloma, & polyoma)- attach to cell & “phagocytized”  Enveloped virus:  Membrane fusion: Fuses with host membrane & nucleocapsid is released inside (3’) 4. Uncoating: virions disassembled. Capsid is removed to make viral genome accessible to the cellular transcription & translation machinery  Genome of DNA viruses (except poxviruses) must be delivered to nucleus  Most RNA viruses remain in cytoplasm PREVENT PENETRATION & UNCOATING  Inhibition of viral fusion proteins  Enfuvirtide blocks HIV fusion  Inhibition of envelope fusion with cell membrane  Docosanol blocks envelope fusion of HSV, etc.  Neutralize vesicle pH, which inhibits viral uncoating  Amantadine & rimantadine against Influenza A  Prevent penetration  Tromantadine prevents HSV penetration  Block receptors to prevent uncoating  Arildone prevent uncoating of picornaviruses by fitting into a cleft in receptor-binding domain of capsid & preventing disassembly of the capsid  5. SYNTHESIS OF PROTEIN & NUCLEIC ACIDS  (& 6 & 7) Macromolecular synthesis & Replication  While early mRNA & nonstructural protein transcribed (5), the genome can be replicated (7)  Late mRNA and structural protein synthesis & posttranslational modification of protein (6)  Depends on if virus is single- or double-stranded & DNA or RNA (we’ll learn more later)  Naked genome of DNA viruses (except poxviruses) & positive-sense RNA viruses (except retro.) are referred to as infectious nucleic acids because they can initiate replication on injection into cell  VIRAL NUCLEIC ACID- PROPERTIES OF DNA VIRUSES  DNA is not transient or labile  Many DNA viruses establish persistent infections (latent INFECTION)  DNA genomes reside in nucleus for replication (except for poxvirus)  Viral DNA resembles host DNA for transcription & replication  Viral genes must interact with host transcriptional machinery (except pox.)  Viral gene transcription is temporally regulated  Early genes encode DNA-binding proteins & enzymes  Late genes encode structural & other proteins  DNA polymerases require a primer to replicate viral genome  Larger DNA viruses encode ways to promote efficient replication of genome  GENOME REPLICATION  Most antiviral drugs are nucleoside analogues  Def: compounds with modifications of the base, sugar, or both  Nucleoside analogues (made up of sugar & base only)  MOA- mimicking their physiological counterparts (endogenous nucleosides) & block cellular division or viral replication by:  Preventing chain elongation, due to absence of a 3′- hydroxyl on the sugar OR  Alter recognition and base pairing, due to a base modification, and induce inactivating mutations  GENOME REPLICATION: NUCLEOSIDE ANALOGUES  Nucleoside analogs must first be phosphorylated to the triphosphate form by viral enzymes (e.g., HSV thymidine kinase), cellular enzymes, or both  e.g. thymidine kinase of HSV phosphorylates acyclovir (ACV), and cellular enzymes apply the rest  Acyclovir is converted to its triphosphate form, which competitively inhibits viral DNA polymerase, incorporates into & terminates growing viral DNA chain, & inactivates viral DNA polymerase.  Therefore, HSV mutants lacking thymidine kinase activity are resistant to ACV GENOME REPLICATION  Nucleoside analogues, continued:  Ribavirin- Purine (in DNA adenine & guanine) nucleoside analogue with broad antiviral spectrum  Works on both RNA & DNA viruses  Inhibits viral replication, promotes hypermutation  Hypermutation of a viral genome by an antiviral drug (like ribavirin) is the equivalent of replacing every fourth letter in an essay with a random letter  Non-nucleoside polymerase inhibitors (polymerase forms DNA & RNA)  Phosphonoformate (PFA)- Pyrophosphate analogs resembling the by-product of the polymerase reaction; functions as a noncompetitive inhibitor of herpesvirus DNA polymerase  VIRAL NUCLEIC ACID- PROPERTIES OF RNA VIRUSES  RNA is labile & transient  Most RNA viruses replicate in cytoplasm (except influenza & retro)  Classified according to polarity of their RNA:  Positive-sense strand- viral genomes act as mRNA for protein synthesis, so translation can begin immediately  Naked + sense viral genomes are infectious nucleic acids  Negative-sense strand- viral genomes do not immediately begin transcription. Act as template for synthesis of + strand, then used for protein synthesis  Genome structure determines mechanism of transcription & replication  All (−) RNA viruses are enveloped, except for (+) RNA genome, & must encode RNA-dependent RNA polymerases for replication  Prone to mutation  5. TRANSCRIPTION & 6. PROTEIN SYNTHESIS  mRNA synthesis is essential for production of virus, NOT a good target for antiviral drugs because it is difficult to inhibit viral RNA synthesis without affecting cellular mRNA synthesis  Antisense oligonucleotides- Small pieces of DNA or RNA that can bind to specific molecules of RNA & blocks ability to make a protein  May be used to block the production of proteins needed for cell growth  Interferons- prevent transcription & protein synthesis  Degradation of viral & cellular mRNA is enhanced & ribosomal assembly is blocked, preventing protein synthesis & viral replication  Discovered by Alick Isaacs & Jean Lindenmann 1957  Proteins synthesized by host cells in response to viruses & other proinflammatory agents (e.g. IFNγ)  Inhibit virus replication indirectly by inducing expression of cellular proteins that inhibit protein synthesis machinery  Pegylated interferon- Attachment of polyethylene glycol to IFN-α (pegylated IFN-α) increases its potency  8. ASSEMBLY & 9. RELEASE OF VIRUS 8. Assembly  Some viral proteins require posttranslational modifications such as phosphorylation, glycosylation, acylation, or sulfation  Matrix proteins (M proteins)- line inside of envelope & facilitate assembly of nucleocapsid into virion in enveloped viruses  Inner surface of envelope & touches nucleocapsid  Stabilize glycoprotein & lipid envelope interaction  Direct viral genome to sites of virus assembly  Help virus budding 9. Release  Nonenveloped- usually when then cell lyses  Exocytosis- reverse phagocytosis  Enveloped- budding out of host cell; Some cause apoptosis, some are non-lethal  8. ASSEMBLY & 9. RELEASE  Protease inhibitors  Inhibitors of HIV protease that fit into the active site of the enzyme  HIV protease is unique & essential to the assembly of virions & production of infectious virions  Ritonavir (navir= “no virus”) & Nirmatrelvir  HIV protease inhibitor drugs that were repurposed & combined & work on SARS-CoV-2 (Paxlovid)  Neuraminidase Inhibitors  Enzyme inhibitors of neuraminidase (Influenza)  In Influenza, neuraminidase enzyme is essential to prevent intracellular & cell-surface aggregation of viral glycoproteins & allow their incorporation into envelope  Oseltamivir, Zanamivir TEST YOUR KNOWLEDGE #2  What type of drug would you prescribe to block viral replication?  Nucleoside analogues ALSO A SCIENTIST Gertrude Elion, (1918-1999) -Never formally was able to get a PhD because of sex bias  Nobel Prize in Physiology or Medicine 1988  Daughter of Lithuanian-Jewish immigrant & a Polish immigrant, who lost all their money during Wallstreet crash of 1929  Worked as a secretary, high school teacher, unpaid chemistry lab technician, food quality supervisor, then Burroughs- Wellcome pharmaceutical company (now GSK)  She developed & helped develop:  Mercaptopurine (Purinethol)- leukemia  Azathioprine (Imuran)- first immunosuppressant  Allopurinol (Zyloprim)- gout  Pyrimethamine (Daraprim)- malaria  Trimethoprim- antibiotic  Acyclovir (Zovirax)- herpes  Nelarabine- cancer ALSO A SCIENTIST Donald Alcendor, PhD -PhD Molecular Virology & Viral Oncology UC Davis  Assistant Professor of Cancer Biology, Meharry Medical College (Meharry Medical College was founded in 1876 in Nashville, Tennessee, to teach medicine to former enslaved Africans and to serve the underserved)  Cytomegalovirus expert for FDA Division of Vaccine Injury & Compensation Program  Consultant & voting member FDA Antiviral Drug Advisory Committee  Cytomegalovirus trafficking of the central nervous system (CNS) with a focus on the Blood-brain and retinal barriers  https://www.nbcnews.com/news/nbcblk/black-scientists-hope- begin-testing-antiviral-drug-coronavirus-two-weeks-n1181101 Naked DNA Viruses TEST YOUR KNOWLEDGE #1  What are the more complex steps of viral replication? 1. Recognition of the target cell 2. Attachment 3. Penetration 4. Uncoating 5. (& 6 & 7) Transcription, Protein synthesis (Translation), & Replication 8. Assembly of virus 9. Lysis & release (usually Naked) OR budding & release (usually Enveloped) MODULE REVIEW NAKED DNA VIRUSES MNEMONIC- PAPPPA! WHY SHOULD YOU CARE? NAKED DNA VIRUSES WHY SHOULD YOU CARE? NAKED DNA VIRUSES  CLASS OBJECTIVES  Be able to differentiate naked & enveloped DNA viruses, and how they differ from RNA viruses in terms of structure, replication, morphology, etc. (on-going & referring back to viral replication)  Be able to compare & contrast naked & enveloped DNA viruses (this lecture & Friday)  Be able to distinguish which viruses are naked DNA viruses and the diseases they cause  Be able to diagnose the naked DNA viruses based on signs, symptoms, diagnostic tests  Be able to prescribe appropriate prevention and treatments including available vaccinations & therapeutics  Useful website: https://www.amboss.com/us/knowledge/General_virology  Yes, the big blue tab at the bottom makes it look like you NEED to sign up for this website, but you DON’T, you can still click the down arrows and access the virus information (although the big blue bar will annoyingly stay at the bottom of the screen)  NAKED (AKA NON-ENVELOPED) DNA VIRUSES Virus Papillomavirus Polyomavirus Parvovirus Adenovirus DNA strand dsDNA dsDNA ssDNA dsDNA DNA shape Circular Circular Linear Linear Capsid shape Icosahedral Icosahedral Icosahedral Icosahedral Important examples Human papillomavirus JC Virus, BK Virus Parvovirus B19 More than 50 serotypes RECOGNITION, ATTACHMENT & PENETRATION & UNCOATING  Recognition & Attach (bind) to their specific receptors  Changes surface proteins to allow virus to enter  Naked viruses: surface-exposed capsid regions, or capsid proteins mediate attachment  Penetration  Endocytosis & then virus is uncoated inside cell  Viropexis (Picorna, papilloma, & polyoma)- attach to cell & “phagocytized”  Uncoating/Nuclear entry- virions disassembled. Capsid is removed  Nucleocapsid of DNA viruses (except pox) is delivered to the nucleus where uncoating occurs  degradation by viral or host enzymes or by simple dissociation  REPLICATION  These DNA viruses replicate in nucleus of host:  Double-stranded DNA (dsDNA):  Papillomavirus, Polyomavirus, Adenovirus do NOT have reverse transcriptase  Replication: viral sense DNA strand (5’-3’) is transcribed to mRNA using host cell's RNA polymerase → viral proteins are formed  Protein synthesis: host or viral (adenovirus codes its own) DNA polymerase replicates dsDNA  Single-stranded DNA (ssDNA): only Parvoviridae  Replication: Host cell DNA polymerase converts ssDNA viral genome to dsDNA → Host DNA polymerase creates progeny ssDNA  Protein synthesis: Sense DNA strand (5’-3’) serves as template for mRNA (using host cell's RNA polymerase) → viral proteins are formed  Purified nucleic acids of dsDNA viruses are infectious-perpetuation of viral life cycle without intact virions  ASSEMBLY & RELEASE  Assembly  Progeny viral DNA, early & late proteins, & capsid  Release  Usually lysis of the cell since they are not enveloped TEST YOUR KNOWLEDGE #1  How do DNA viruses differ from RNA viruses? (Besides the obvious DNA & RNA)  Nuclear entry does not occur in most* RNA viruses  What is the exception? WARNING: THERE WILL BE SOME GRAPHIC IMAGES IN TODAY’S LECTURE  PAPILLOMAVIRUSES OVERVIEW 20-30 million people infected in US 3 million new infections/year Human papillomaviruses subclassified into types based on DNA sequence HPV 1-4 cause majority of common cutaneous warts HPV 6 & 11 typically cause genital warts (condyloma) Can exist on inanimate objects & cause infection  SIGNS & SYMPTOMS- PAPILLOMAVIRUS  Warts  Risk factors:  Cutaneous (common, plantar, flat)  # of sexual partners  Anogenital (condyloma)  Weakened immune system  Oropharynx (respiratory  Damaged skin papillomatosis)- impaired laryngeal  Personal contact function & may compromise airway  Age  Cancer  Diagnostic tests  Acetic acid test- lesions turn white  Biopsy  Pap smear  All followed by PCR test HPV WARTS PICTURES  PREVENTION & TREATMENT- PAPILLOMAVIRUS  Gardasil 9 vaccine  Approved for use in males & females aged 9-45 to protect against cervical cancer & genital warts  Two doses at least 6 months apart (3 doses if over 15 years old)  Regular pap smears  Salicylic acid & trichloroacetic acid- treat warts  Podofilox-prescription that destroys genital wart tissue  Surgical or cryotherapy removal in doctor’s office  POLYOMAVIRUS OVERVIEW- BK & JC VIRUSES  JC Virus (John Cunningham- cancer patient)  Causes progressive multifocal leukoencephalopathy (PML)  Progressive damage of brain white matter  Harmless unless weakened immune system  BK Virus  Feels like common cold, then latent infection  Usually contract it as a child  Harmless unless weakened immune system  Typically associated with patients who had kidney infection  To remember the diseases associated with polyomaviruses, think: “JC virus leads to a Junky Cerebrum (PML) and BK virus leads to Bad Kidneys (nephropathy in immunocompromised patients).” THE FOOD AND DRUG ADMINISTRATION (FDA) HAS GRANTED FAST TRACK DESIGNATION TO ANTIBKV, AN INVESTIGATIONAL ANTIBODY THERAPEUTIC THAT TARGETS BK POLYOMAVIRUS (BKV) INFECTION IN KIDNEY TRANSPLANT RECIPIENTS.  PARVOVIRUS OVERVIEW- ONLY B19 INFECTS HUMANS  Fifth Disease  Symptoms appear ~14 days after infection  Bright red rash appears on both cheeks- “Slapped Cheek disease.”  Rash then appears on trunk, then arms & legs, swollen & painful joints  Subsides in about 2 weeks  Serious complications- chronic anemia  ADENOVIRUS OVERVIEW *IV ribavirin given for SEVERE adenovirus in infants, children, and immunocompromised  SIGNS & SYMPTOMS- ADENOVIRUS  Symptoms depend on disease  “Pink eye” & “Swimming pool conjunctivitis”  Common flu-like symptoms  Fever  Sore throat  Acute gastroenteritis  Pneumonia  PREVENTION & TREATMENT- ADENOVIRUS  No specific treatment, & no approved antivirals  Most are mild & need medications to relieve symptoms  Over-the-counter pain meds or fever reducers  Maintain proper chlorination in swimming pools  First identified outbreak in military personal on base  Military more at risk- close quarters & stress  Vaccine exists for the military- two oral tablets  Contains live adenovirus Type 4 and Type 7 & will prevent most illnesses by these serotypes  TEST YOUR KNOWLEDGE #2  Iva, a 19-year-old biologically female patient presents to your office for her annual pap smear. As her doctor, you are aware she is sexually active, has had 2 previous partners, and uses condoms. However, you find an abnormal pap test (positive, which means abnormal or unusual cells), and you suspect HPV, so you perform a PCR and discover you were right. 1. Which strain typically causes condyloma? 2. If she uses condoms every time, how did she potentially contract this? 3. What is something you could have done as her doctor to prevent this from happening? ALSO A SCIENTIST Eva Ramón Gallegos, PhD  Scientist, professor & researcher at the Escuela Nacional de Ciencias Biológicas of the Instituto Politécnico Nacional in Mexico  With Elizabeth Maldonado (+20 YEARS of work), they developed a cheap & efficient way to detect HPV in women… 98% positive predictive value (who remembers what this is?!)  Determined photodynamic therapy is effective in eliminating HPV-16 & HPV-18 Enveloped DNA Viruses  MODULE REVIEW *We won’t cover Hepadnaviruses today, during Hepatitis lecture later WHY SHOULD YOU CARE? ENVELOPED DNA VIRUSES HSV-2 estimation 2012, ASM WHY YOU SHOULD CARE CLASS OBJECTIVES  Be able to differentiate naked & enveloped DNA viruses, and how they differ from RNA viruses in terms of structure, replication, morphology, etc. (ongoing)  Be able to compare & contrast naked & enveloped DNA viruses (last class & today)  Be able to distinguish which viruses are enveloped DNA viruses and the diseases they cause  Be able to diagnose the enveloped DNA viruses based on signs, symptoms, diagnostic tests  Be able to prescribe appropriate prevention and treatments including available vaccinations & therapeutics  What is the herpesvirus not naturally found in human hosts that can cause disease?  Useful website: https://www.amboss.com/us/knowledge/General_virology ENVELOPED DNA VIRUSES Virus Family Herpesviridae Poxviridae DNA Strand dsDNA dsDNA DNA Shape Linear Linear Capsid shape icosahedral Nucleosome Important HSV1, HSV2, VZV, EBV, Smallpox, Examples CMV Vaccinia VIRAL LIFE CYCLE- ATTACHMENT & ENTRY & UNCOATING  Attach & bind to their specific receptors  Changes surface proteins to allow virus to enter  Entry  Fusion- Viral envelope fuses with the host cell membrane causing the virus to enter  (Remember- Depends on pH: neutral pH= fusion occurs at cell surface, acidic pH= fusion occurs in endosome… aka endocytosis)  Endocytosis  https://www.youtube.com/watch?v=D9OtJU3F6eQ  Uncoating/Nuclear entry  Nucleocapsid of DNA viruses is usually delivered to the nucleus where uncoating occurs  Degradation by viral or host enzymes or by simple dissociation  REPLICATION, ASSEMBLY, & RELEASE  These DNA viruses replicate in the nucleus of the host cell  Double-stranded DNA (dsDNA):  Without reverse transcriptase: viral negative DNA strand is transcribed to mRNA using host cell's RNA polymerase → viral proteins and progeny dsDNA are formed  https://www.youtube.com/watch?v=fH1zS7hlW54 More detail than what is necessary for this class  Remember lytic means burst  Latent infection most commonly occurs in nerve cells  Assembly  Progeny viral DNA, early & late proteins, & capsid  Release  Envelope tends to bud out, keeping host cell alive, or exocytosis  *HSV can perform cell lysis though* TEST YOUR KNOWLEDGE #1  What are the 5 Herpesviridae we’re going to learn today? 1. Herpes Simplex Virus 1 (HSV1) 2. Herpes Simplex Virus 2 (HSV2) 3. Epstein-Barr Virus (EBV) 4. Cytomegalovirus (CMV) 5. Varicella Zoster Virus (VZV) WARNING: THERE WILL BE SOME GRAPHIC IMAGES IN TODAY’S LECTURE *sorry Karen ONLY *8* HUMAN HERPESVIRUS *EXCEPTION: MACACINE ALPHAHERPESVIRUS-1 AKA HERPES B VIRUS **ALL HERPESVIRUSES CAN CAUSE LATENT INFECTION IN SPECIFIC TISSUE ALPHAHERPESVIRUSES HERPES SIMPLEX 1 (HSV1), HERPES SIMPLEX VIRUS II (HSV2), VARICELLA ZOSTER VIRUS (VZV)  PATHOGENESIS OF PRIMARY & RECURRENT HSV & VZV  HSV 1 & HSV 2 are closely related viruses  Can be asymptomatic  Distinguishable by PCR  HSV 1 above the waist, HSV 2 below the waist*  *Exception, we’re seeing a rise in vice versa because oral sex was believed to be “safe” Signs & Symptoms: Genital Pain or itching.You may experience pain and tenderness in your genital area until the infection clears. Small red bumps or tiny white blisters. These may appear a few days to a few weeks after infection Ulcers Scabs Orofacial Tingling and itching. Many people feel itching, burning or tingling around the lips for a day or so before a small, hard, painful spot appears and blisters erupt Blisters. Small fluid-filled blisters typically erupt along the border of your lips. Oozing and crusting  MUCOCUTANEOUS LESIONS CAUSED BY HSV A. Primary gingivostomatitis with multiple eroded lesions B. Recurrent infection with lesions on the vermilion border of the lip and beyond C. Multiple grouped erosions on an erythematous base on the penis D. Lesions on the uterine cervix. TRIGGERS  SKIN LESIONS CAUSED BY VZV  A. Chickenpox lesions on the back of a child (primary disease)  B. Zoster in a child who presented with 6 days of eye pain and a rash in the distribution of the ophthalmic branch of the trigeminal nerve  C. A linear eruption of zoster that could be mistaken for poison ivy  D. Zoster around the trunk in an immunocompromised patient  DIAGNOSIS, TREATMENT, & PREVENTION  Diagnosis  Usually PCR of skin scrapings  Prevention  Safe-sex education & condoms & dental dams with HSV1 & HSV2  *Vaccine??? TBD. See: Also a Scientist  Live, attenuated VZV vaccine prevents chickenpox in normal & some immunologically impaired children  *Considerations: immunodeficient children not candidates for vaccine, but can be given human immunoglobulin after exposure  Treatment  Acyclovir- Nucleoside analogue, Oral pill  Penciclovir- Nucleoside analogue, Topical application BETA- & GAMMAHERPESVIRUSES  CYTOMEGALOVIRUS INFECTION (ESP. CONGENITAL)  from the Greek cyto-, "cell," + megalo-, "large"  ~50-80% of adults in US have had a CMV infection by age 40  Permanent, Latent  Infects epithelial cells, monocytes/macrophages, T cells.  Can be transmitted in bodily fluids (urine, tears, saliva, blood, semen, cervical secretions, breast milk) even in patients with dormant infectious; albeit not highly contagious  Usually harmless, unless pregnant or immunocompromised A. Infant with severe congenital CMV with stigmata of disease including petechial rash, microcephaly, jaundice, and abnormal posture of the upper extremities secondary to central nervous system (CNS) damage. B.Computerized tomographic image of an infant with CNS damage secondary to congenital CMV with severe periventricular calcifications (white signal lining ventricles) and ventriculomegaly.  EPSTEIN-BARR VIRUS (EBV) AKA “KISSING DISEASE”  One of the most-common human viruses  Spread through bodily fluids  Kissing  Sharing drinks & food, cups, utensils, toothbrushes  Having contact with drooled-on toys  Can cause infectious mononucleosis (“mono”)  Usually asymptomatic in children, mild, illness in teens & adults. Mono can last several months. Watch for splenomegaly  Can affect central nervous system & cause encephalitis, meningitis, & Guillain-Barre syndrome (Rare- Autoimmune & attacks nerves)  Linked to autoimmune diseases (fibromyalgia) & cancer  Hodgkin’s Lymphoma- Usually upper body. Lymphatic system. Most treatable  Burkitt’s lymphoma (Non-Hodgkin’s)- Anywhere in body. Lymphatic system. Rare & aggressive.  PREVENTION & TREATMENT  Diagnostics  PCR  Antibody tests in EBV, but not always reliable because it might not differentiate current or past infection  Prevention*  Prophylactic antiviral therapy- before organ transplant to prevent viral reactivation due to compromised immune system  Preemptive antiviral therapy- administering antivirals after monitoring viral loads. Start to rise? Antiviral given  Treatment  CMV-  Nucleoside analogues- Inhibits viral DNA-dependent polymerase.  Phosphonoformate aka non-nucleoside inhibitor (Foscarnet)- inhibit Genome Replication. Inhibits viral DNA polymerase.  EBV-  Nucleoside analogues (acyclovir)- but less effective against post-transplant lymphoproliferative disorders seen in EBV  TEST YOUR KNOWLEDGE #2  You were so fascinated by the discussion on Tulane Primate center and all the cool research they’re doing there, that you decide to spend a summer at their summer research program (yes, it’s paid, yes this is an #ad, no I didn’t get paid for it, yes, I’m trying to distract you with unnecessary information).You’re curious about the angry monkey face and want to see it for yourself, so on your first day, you go to the cages and make faces at a female Indian Macaca mulatta. But oh no! A technician left the cage unlocked and the primate bites you!  What herpesvirus should you be tested for after the bite?  POXVIRUSES  Have nucleosome instead of capsid  Contains DNA & surrounded by its own membrane  Unlike other DNA viruses do NOT require access to the host nucleus  Replicate in cytoplasm within compartments termed “viral factories”  POXVIRUSES  Both smallpox & monkeypox spread via bodily fluids, respiratory droplets, skin-skin contact, fomites (bedding & clothing)  Smallpox (Variola Virus)  Eradicated in 1980 due to vaccines  Last natural outbreak in the US in 1949  Monkeypox  Monkeypox causes lymph nodes to swell (lymphadenopathy) while smallpox does not SMALLPOX VACCINE *It’s gross, but it’s from 1955…. Science has come a looooooooooooooooooooooooooooooooooooooooong way since then. ALSO A SCIENTIST- HSV 2 VACCINE Lesia Dropulic, MD -Staff Clinician, Laboratory of Infectious Diseases, Medical Virology Section, National Institute of Allergy & Infectious Disease  HSV529 vaccine- HSV2 vaccine candidate  https://academic.oup.com/jid/article/220/6/990/5486075  https://clinicaltrials.gov/ct2/show/NCT01915212  Phase 1: A phase of research to describe clinical trials that focus on the safety of a drug. They are usually conducted with healthy volunteers, and the goal is to determine the drug's most frequent and serious adverse events and, often, how the drug is broken down and excreted by the body. These trials usually involve a small number of participants.