Antivirals - Microbiology Block 2 - Past Paper PDF

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 age...

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