[MIC LEC - LE 2] 01 - Basic Virology (V1).pdf

MICROBIOLOGY | TRANS 01 LE Introduction to Virology PREPARED BY DR. MARGARITA V. EDUARDO |...

MICROBIOLOGY | TRANS 01 LE Introduction to Virology PREPARED BY DR. MARGARITA V. EDUARDO | 09/13/2024 | Version 1 02 OUTLINE Electron microscope I. General Characteristics of V. Viral Replication Cycle → Uses a beam of electrons to magnify the image of Viruses A. Attachment viruses A. Size of Cells vs. Virion B. Penetration B. RNA Viruses C. Uncoating C. DNA Viruses D. Synthesis D. Other General E. Assembly Characteristics F. Release II. Taxonomy G.Viral Growth Curve III.Viral Structures VI. Pattern of Viral Infections and A. Virion Cytopathogenicity B. Envelope A. Abortive Infection Figure 1. (L) RNA Viruses; (R) DNA Viruses C. Spikes B. Lytic Infection D. Capsid C. Persistent Infection A. SIZE OF CELLS VS VIRION E. Viral Proteins & their VII. Basic Types of Cultures Table 1. Size of Cells vs. Virion Functions VIII. Method of Detection of Size of Cells Virions F. Genome Viruses IV. Baltimore Classification A. Development of Cytopathic Prokaryotic RNA and DNA A. Group I: dsDNA Event →1-10 μm → 20-30 nm B. Group II: ssDNA B. Viral Growth in Chick Bacteria → Able to pass through filters C. Group III: dsRNA Embryo Eukaryotic Filtration is not enough to destroy D. Group IV: ssRNA C. Adsorption of RBC to →10-100 μm viruses present in water E. Group V: ss(-)RNA Infected Cells Fungi, → You’ll have to add more F. Group VI: ss(+)RNA IX. Classification of Human procedures to destroy the virus Human (Retrovirus) Viruses G.Group VII: dsDNA X. References B.RNA VIRUSES (Retrovirus) XI. Formative quiz Table 2. RNA Virus XII. Appendix dsRNA Reoviridae, Picobirnavirus Must Lecturer Book Previous Youtube Double stranded RNA → Can measure up to 100 nm ❗️ Know 💬 📖 📋 Trans 🔺 Video Orthomyxoviridae: Flu virus Rhabdoviridae: Rabies virus SUMMARY OF ABBREVIATIONS ssRNA (-) Paramyxoviridae: Measles dsDNA double stranded DNA Single stranded negative Arenaviridae ssDNA single stranded DNA strand RNA Bunyaviridae dsRNA double stranded RNA Bornaviridae ssRNA single stranded RNA Filoviridae RT Retrovirus ssRNA (RT) Retroviridae IFN Interferon Single RNA retrovirus → Example: HIV and HTLV ICTV International Committee on Taxonomy of Viruses Hepeviridae HTLV Human T-lymphotropic Virus Caliciviridae HBV Hepatitis B Virus Picornaviridae HbAg Hepatitis B Antigen ssRNA (+) Coronaviridae CPE Cytopathic Effects Single stranded positive → Example: SARS virus RSV Respiratory Syncytial Virus strand RNA Astroviridae HSV Herpes Simplex Virus Togaviridae CAM Chorio-allantoic Membrane Inoculation Flaviviridae LEARNING OBJECTIVES C.DNA VIRUSES ✔ Apply the properties of a virion as to how they are Table 3. DNA Virus classified Adenoviridae ✔ Discuss the steps of viral replication with their important Poxviridae structures dsDNA Herpesviridae ✔ Correlate the effects of viruses on host cells to their lab Double stranded DNA Polyomaviridae diagnosis Papillomaviridae I. GENERAL CHARACTERISTICS OF VIRUSES Asfarviridae Acellular ssDNA (+) Parvoviridae → DON’T have cell organelles & metabolic processes Single stranded DNA → Example: Parvovirus which is required by living cells (i.e. prokaryotes and Hepadnaviridae eukaryotes) dsDNA (RT) → Hepatitis B virus → Not considered as living things Submicroscopic D. OTHER GENERAL CHARACTERISTICS → Can’t be demonstrated under compound microscope No genes to encode protein synthesis → Optical microscope uses visible light to magnify images No genes to produce energy and membrane biosynthesis → Used for gram-staining and other staining methods Obligate intracellular parasites LE 2 TG 9 | R.J. Gonzalez, K. Guinto, J. Gueterrez, TE | M.K. Gonzalez AVPAA | J. Gonzaga PAGE 1 of 14 TRANS 1 D. Haquihaca, J. Huan VPAA | A. Escolano MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo → Not capable of self replication when they are outside of Table 4. 5 Rank Taxonomy Structure Given by the ICTV 💬living cells Similar to atypical bacteria (i.e. Rickettsia, Mycoplasma, and Chlamydia) TAXA Order virales EXAMPLES Nidovirales Herpesvirales Mono- negavirales Resistant to antibiotics Family Coronaviridae Herpesviridae Paramyxo- Inhibited in replicating to produce progeny by antiviral viridae viridae agent and interferon (IFN) Subfamily Coronavirinae Alpha- Paramyxo- → Interferons are proteins that are part of our natural virinae herpesviridae virinae Genus Betacorona Varicella virus Morbillivirus defenses virus virus → “Interfere” with viruses to keep them from multiplying Species SARS Cov2 Varicella Zoster Measles Virus → Interfere by signaling the neighboring host cell that a generally virus 💬 virus is coming Interferon is activated by the IFN gene → When IFN is released outside, this signals the (disease) virus The vast majority of virology and microbiology specialty journals request that manuscript authors follow the official neighboring cell to produce antiviral protein to destroy ICTV taxonomy, but unfortunately it is rarely enforced viral particles III. VIRAL STRUCTURES A. VIRION Entire infectious unit Complete viral particle consisting either: 2 parts or 3 parts Naked Nucleocapsid Virion (2 parts) → Capsid: covering → Nucleic acid (genome): inside the capsid Enveloped Virion (3 parts) → Capsid → Nucleic acid (genome) → Envelope: protruding spike is found on its surface Figure 2. Antiviral action of Interferon (IFN) II. TAXONOMY Names are established in 1966 by the International Committee on Taxonomy of Viruses (ICTV) ICTV was given the task of developing a single universal taxonomic scheme for all viruses infecting: → Animals (vertebrates, invertebrates, and protozoa) → Plants (higher plants and algae) Figure 4. Viral Structures. (L) Naked Nucleocapsid Virion; (R) 💬 → Fungi, bacteria, and archaea Enveloped Virion The only taxa used in classifying viruses: Order, Suborder, B. ENVELOPE Family, Subfamily, Genus, Subgenus, and Species Figure 5. Envelope Phospholipid bilayer derived from the host cell membrane Produced during budding & release All enveloped viruses derived their envelope from host cell Figure 3. Taxa used in classifying viruses membrane: → EXCEPT: envelope of Herpes which is from nuclear membrane → Table 5. Naked DNA and Naked RNA Naked DNA Naked RNA Parvoviridae Picornaviridae Papillomaviridae Reoviridae Polyomaviridae Caliciviridae Adenoviridae Hepeviridae MICROBIOLOGY Introduction to Virology PAGE 2 of 14  MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo Table 6. Enveloped vs. Naked Viruses → CCR5 or CXCR4 acts as co-receptors, which helps with Attributes Enveloped viruses Naked RNA the attachment Injurious agents Sensitive Resistant to ether (ether, alcohol, (labile easily and other D. CAPSID detergent & dry destroyed) environmental Protein coat that encloses the genome 3 kinds of symmetry: heat) Mode of transmission Direct contact e.g. HIV 💬 factors Feco-oral route e.g.Poliovirus → Icosahedral (cubic) ▪ Polyhedral or cubic w/ 20 flat sides Reaction to Can NOT withstand Can withstand ▪ E.g. Mostly DNA and (+) sense RNA gastric acidity ▪ Icosahedral naked virus: no envelope Inserted viral proteins (peplomers) important in: ▪ Icosahedral enveloped virus: inside envelope → Attachment (e.g. spike, hemagglutinin, neuraminidase) → Helical → Induction of protective immunity (HA for flu vaccine) ▪ Spiral or cylindrical-shaped forming a helix ▪ E.g. Enveloped (-) sense RNA ▪ Helical naked virus: coiled nucleocapsid ▪ Helical enveloped virus: inside the envelope → Complex ▪ Lacks the symmetry feats of common viral capsids ▪ E.g. Poxvirus (w/ dumbbell shaped plank by bilateral bodies inside) and Rabies virus (bullet-shaped) Figure 6. Influenza A infects host cell 💬 Hemagglutinin is used for attachment → A receptor site is present in RBC through which 💬 hemagglutinin will attach itself In the host cell the specific receptor for Hemagglutinin is Figure 9. The 3 kinds of Symmetry 💬 the Glycan chain There are specific receptor attachment for each virus E. VIRAL PROTEINS & THEIR FUNCTIONS Structural proteins → Attachment to host cell receptor Enzymes (e.g. DNA/RNA polymerase) Matrix protein → Interaction between nucleocapsid & envelop Antigenic variants → Serotyping Capsid → Provide structural symmetry for the virus particle Protein in the envelope → Protein the viral genome against inactivation of nucleases Figure 7. SARS-CoV 1 and SARS-CoV 2 The simplest viruses contain only enough RNA or DNA to 💬Spike protein attaches to the ACE2 receptor or the encode four proteins The most complex can encode 100-200 proteins TMPRSS2 (co-receptor) C. SPIKES GLYCOPROTEIN IN THE SPIKES Protrude from the viral surface/envelope E.g. SARS-CoV, HIV Involved in specific binding to receptors in the host cell Kaposi’s Sarcoma-associated Herpes Virus (KSHV) / membrane to facilitate viral entry 💬Herpesvirus 8 💬💬 Made up of glycoproteins (e.g. gp120 of HIV) Viral Receptor (glycoprotein spikes): gB, gHgL Host Receptor (in humans): CD98 Very important for tissue/viral tropism for a virus to gain entry to host cell → its glycoprotein spikes must interact w/ host cell receptor Figure 8. HIV virus 💬 gp120 attaches to the CD4+ (host cell receptor) → Patients with HIV have an decreased CD4+ MICROBIOLOGY Introduction to Virology PAGE 3 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo Figure 13. Matrix Proteins ANTIGENIC (SEROTYPING) VARIANTS Figure 10. KSHV/Herpesvirus 8 Some showing different antigens Exhibit specific activities → e.g. Hepatitis B Virus (HBV) (Group VII: dsDNA) → Example: ▪ HBsAg (Hepatitis B surface Antigen) ▪ Influenza hemagglutinin of spikes → where RBCs ▪ HBcAg (Hepatitis B core Antigen) attach → produce agglutination ▪ HBeAg ▪ Used to detect influenza virus in the serum of → Enable us to identify antibodies produced by the patient patients based on the antigen present Figure 11. Influenza virus HA ENZYMES Figure 14. Hepatitis B Virus: Antigenic Variants Essential for initiation of the viral replicative cycle when the Function: virion enters the cell → Determine the antigenic characteristics of the virus → RNA polymerase → Host immune response is directed against antigenic ▪ Responsible for copying DNA sequence into an RNA determinants of proteins or glycoproteins sequence during the process of transcription. ▪ Antibodies present helps in determination if a person → Reverse transcriptase is immune, has chronic, or acute hepatitis ▪ Present in retroviruses ▪ e.g. HIV, HTLV (Baltimore classification VI) and HBV OTHER FUNCTIONS OF VIRAL PROTEINS (Baltimore classification VII) Provide structural symmetry for the virus particle (CAPSID) Protect the viral genome against inactivation of nucleases Figure 15. Capsomeres (blue) Capsomeres: Finite number of protein subunits that surround the capsid F. GENOME STRANDS Linear strands → Single: Picornavirus, Parvovirus → Double: Poxvirus Circular strands Figure 12. Retrovirus infection and reverse transcription → Single: Hepatitis D virus (RNA) MATRIX PROTEIN → Double: Papillomavirus (DNA) Found between the envelope and the nucleocapsid. May have enzymatic activities and/or biologic functions related to infection. MICROBIOLOGY Introduction to Virology PAGE 4 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo ▪ dsRNA − Reoviridae ▪ ssRNA, segmented genomes − Orthomyxoviridae, Bunyaviridae, Arenaviridae ▪ Circular RNA − Delta virus RNA Genome → Under the Baltimore Classification Group V → Negative (-) strand RNA viruses ▪ Must transcribe the (-) to (+) strand ▪ Brings its own RNA-dependent polymerase for replication Figure 16. Segmented Genome Strands POLARITY Segmented strands (more than one segment) Based on the reading frame of mRNA which is (+) sense → Orthomyxoviridae POSITIVE SENSE (+) ▪ Influenza virus Same polarity as mRNA ▪ With 8 segments Maybe used for early protein synthesis → Arenaviridae RNA viruses with (+) sense genome are infectious ▪ Lassa fever virus → Functions as mRNA within the infected cell and can be ▪ 2 segments immediately translated by host cell → Bunyaviridae ▪ Positive sense RNA genome is translated by the host ▪ Hanta fever virus ribosome → the RNA genome is used directly as ▪ 3 segments mRNA to translate viral protein → Baltimore Classification Group IV → Examples: Picornavirus, Togavirus, Coronavirus Figure 19. RNA with (+) strand using their RNA genome directly as mRNA Figure 17. Linear and Circular Genome Strands ❗️ MNEMONIC (for Segmented Genomes) NEGATIVE SENSE (-) Complementary to or antisense of mRNA RNA viruses with segmented strands Does not encode mRNA B-O-A-R Carry virion-associated RNA-dependent RNA → Bunyaviruses (e.g. Hanta fever virus) polymerase to produce initial mRNA → Orthomyxoviruses (e.g. Influenza virus) RNA molecule is NOT infectious → Arenavirus (e.g. Lassa fever virus) → Example: Rhabdovirus & Orthomyxovirus → Reovirus (e.g. Rotavirus) An mRNA must be transcribed by using the negative strand as template RNA-dependent RNA polymerase → Transcribes the (-) strand into a (+) viral mRNA → Eventually translating it to a viral protein Figure 18. Single and Double RNA/DNA Genome Strands DNA viruses → Most have linear, double stranded DNA, EXCEPT: ▪ ssDNA − Parvoviridae ▪ Circular DNA − Papilloviridae, Polyomaviridae, Hepadnaviridae RNA viruses Figure 20. RNA with (-) strand as template for transcription of → Most have linear, single stranded RNA, EXCEPT: (+) viral mRNA, to be translated into viral proteins MICROBIOLOGY Introduction to Virology PAGE 5 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo AMBISENSE (+) AND (-) ❗→ DNABCViruses is composed of 7 groups RNA segment has BOTH (+) and (-) in the same strand → Segmented RNA strand mostly regions are (-) and ▪ Group I: dsDNA – DNA (+/-) some (+) ▪ Group II: ssDNA – DNA (+) → e.g. Arenaviridae (Lassa fever virus) and → RNA Viruses Bunyaviridae (Hanta fever virus) ▪ Group III: dsRNA – RNA (+/-) ▪ Group IV: ssRNA (+) – RNA (+) ▪ Group V: ssRNA (-) – RNA (-) → Retroviruses ▪ Group VI: ssRNA – RNA (+) ▪ Group VII: dsDNA – DNA (+/-) All viruses must direct the synthesis of mRNA to produce proteins All viral protein synthesis is completely dependent upon the translational machinery of the cell GROUP I: dsDNA Genome has the same polarity as the host cell they are infecting Figure 21. Ambisense The flow of information follows a conventional pathway: → dsDNA → mRNA → viral protein GENOME REPLICATION dsDNA with viral DNA: Transcription starts with information from the DNA strand → DNA-dependent RNA polymerase producing the is copied to encode a new molecule of RNA which will mRNA directly from the positive polarity → (+) mRNA → serve as the blueprint translated to produce protein → Performed by RNA polymerase which uses the DNA → Requires a DNA-dependent DNA polymerase from strand as the template either the virus or the host cell → copies the (+) and (-) Translation - protein synthesis strand → dsDNA with Viral DNA → The mRNA uses the host cellular machinery Eventually, viral progeny is produced ❗️MNEMONIC (Group I: dsDNA viruses) Viruses belonging to Group I are “HAPPy”: → Herpesviridae (e.g. HSV1&2 etc.) → Adenoviridae (e.g. Mastadenovirus that causes colds) → Papillomaviridae (e.g. HPV 1&2 etc.) → Poxviridae (e.g. Smallpox etc.) → Polyomaviridae (BK and JC virus) Figure 22. Genome Replication IV. BALTIMORE CLASSIFICATION (BC) Figure 23. Baltimore Classification of Viruses In 1971, David Baltimore founded the ”Baltimore’s Figure 24. Group I dsDNA genome replication Classification”, which classifies viruses into different GROUP II: ssDNA (+) families based on their type of genome and method of This group only has one family known as Parvoviridae replication → Example: Parvovirus B19 - cause of 5th disease Baltimore Classification (BC) Since it is (+) stranded it follows a conventional pathway: → Will remain highly useful for teaching purposes → DNA → mRNA → protein → Unlikely to be useful as top ranks in official virus → Viral genome manufactured first forming a double taxonomy stranded replicative form → With a DNA-dependent RNA polymerase, it copies the DNA negative strand producing the positive viral mRNA and translate it into viral protein at the host ribosome MICROBIOLOGY Introduction to Virology PAGE 6 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo → Requires a DNA-dependent DNA polymerase from → Togaviridae (Rubella etc) either the virus or the host cell to copy the (-) DNA → Filoviridae (Dengue etc) strand to produce a single stranded positive DNA viral genome Figure 25. Group II ssDNA genome replication Figure 27. Group IV ss(+)RNA genome replication GROUP III: dsRNA (+/-) GROUP V: ssRNA (-) It is a double stranded RNA RNA is negative strand There is a positive strand but the positive strand here → Negative RNA will be used as template CANNOT be used as mRNa because the positive strand → Some will have to carry their own RNA polymerase to here is hydrogen bonded to the negative strand. It needs make viral mRNA then through translation, viral protein its own viral RNA polymerase is produced Viral RNA-dependent RNA polymerase serves as: → Since the genome of minus-stranded RNA viruses → transcriptase to transcribe mRNA CANNOT be used as mRNA, the virus must carry and → replicase to replicate the RNA genome RNA-dependent RNA-polymerase within its capsid Only one family: Reoviridae (rotavirus) → When viral genomes are needed, the plus stranded → Rotavirus - can cause diarrhea in children RNAs are used as template to make minus stranded → non-enveloped RNA → triple layered capsid → segmented RNA genome ❗️ MNEMONIC (for Group V) (Always Bring Polymerase Or Fail Replication) (-) STRAND RNA VIRUS EXAMPLE Arenaviruses Lassa Fever Virus Bunyaviruses Hanta Fever Virus Paramyxoviruses Mumps & Measles Virus Orthomyxoviruses Influenza Virus Filoviruses Ebola & Marburg Virus Rhabdoviruses Rabies Virus Delta Virus - not a family but is under group V ssRNA (-) Figure 26. Group III dsRNA genome replication GROUP IV: ssRNA (+) Positive single RNA → Can use their genome directly as mRNA with translation by the host ribosome occurring as soon as the unsegmented viral genome gains entry into the cell → One of the viral genes expressed yields and RNA-dependent RNA-polymerase (or RNA replicase), which creates ss(-)RNA from the plus-stranded genome → minus-stranded RNA can be used as template for Figure 28. Group V ss(-)RNA genome replication ss(+)RNA, which can be used as mRNA or as genomes GROUP VI: ssRNA (+) (Retrovirus) for the newly forming virus Why are they called “reverse” or “retrovirus”? Viruses belonging to this group: → RNA converted to DNA; not DNA to RNA → Caliciviridae (Norwalk virus) → via Reverse Transcriptase → Coronaviridae (SARS, MERS) RNA positive strand → Hepeviridae (Hepatitis E) Despite the fact that the retroviral genome is composed of → Picornaviridae (Polio etc) ss(+)RNA, it is NOT used as mRNA MICROBIOLOGY Introduction to Virology PAGE 7 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo The virus uses reverse transcriptase to synthesize a piece of ssDNA complementary to the viral RNA genome The RNA is then converted to dsDNA by reverse transcriptase (RNA-dependent DNA polymerase) The DNA is spliced into the host cell genome for subsequent transcription and translation using the enzyme integrase Examples: HIV and HTLV Figure 31. Retrovirus infection and reverse transcription Figure 29. Group VI ss(+)RNA genome replication SUMMARY OF BALTIMORE CLASSIFICATION GROUP VII: dsDNA (+/-) (Retrovirus) Double stranded DNA Family: Hepadnaviridae (Hepatitis B) The dsDNA genome is gapped, and subsequently filled in to form a closed circle serving as a template for production of viral mRNA → Single stranded in one part Despite having double stranded DNA genome they replicate via a ssRNA intermediate To reproduce the genome, RNA is reverse transcribed back to DNA Figure 32. Summary of Baltimore Classification All viruses must direct the synthesis of mRNA to produce proteins All viral protein synthesis is completely dependent up the translational machinery of the cell V. VIRAL REPLICATION CYCLE Six Steps: ”APUSAR” → Attachment → Penetration → Uncoating → Synthesis → Assembly → Release Figure 29. Group VII dsDNA genome replication SUMMARY OF RETROVIRUSES Table 7. Summary of Retroviruses Type VI: Type VII: ssRNA (+) dsDNA (+/-) Family Retroviridae Hepadnaviridae Virus HIV,HTLV Hepatitis B Structures Enveloped, Enveloped, icosahedral icosahedral Site of Replication nucleus nucleus Figure 33. Steps in Viral Replication MICROBIOLOGY Introduction to Virology PAGE 8 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo A. ATTACHMENT/ABSORPTION D. SYNTHESIS Viral proteins on the capsid or phospholipid envelope Involves in the replication of viral genome and protein interact with specific receptors on the host cellular surface synthesis through transcription or translation Specificity determines the host range (tropism) of a virus DNA viruses replicates in the nucleus Examples: SARS and HIV → Except: poxviruses RNA viruses replicates in the cytoplasm → Except: ▪ orthomyxoviruses (influenza) ▪ retroviruses (HIV, HTLV, Hep B) Figure 37. Synthesis Involves the production of nucleic acid and protein polymers Figure 34. SARS-COV/HIV Viral transcription leads to the synthesis of messenger B. PENETRATION RNA (mRNA) which encodes viral proteins. The process by which the viruses enter the host cell → Early proteins - non structural elements such as Two methods enzymes → Direct fusion with the host cell membrane (some with → Late Proteins - structural components (ex. envelope, enveloped viruses) microprotein) E. ASSEMBLY The process by which structural proteins, genomes and viral enzymes are assembled into virus particles Enclosing the viral genome in protein coat (capsid) F. RELEASE “Last step” Released through: → Budding - creating enveloped viruses → Lysis - most naked viruses ▪ Apoptosis - release by lysis of naked viruses results in host cell death G. VIRAL GROWTH CURVE Phase 0: Entry Phase 1: Decline → Virus decreases in number but continues to function Figure 35. Direct fusion Phase 2: Eclipse Period → Viropexis (receptor mediated endocytosis) - several → no virus is detectable inside the cell enveloped and all naked → viral component synthesis occurs → “Virus preparing by synthesizing their proteins” Phase 3: Exponential/Rise Period → “Period where they replicate exponentially” → progeny virus increases exponentially for a period of time (8-72hrs) Phase 4: Latent Period → No additional increase in virus yield occurs → May yield of 100-10,000 virions per cell → Can be intracellular or extracellular ▪ when released extracellularly, they can find other Figure 36. Direct fusion host cell to infect Phase 5: Cytoplasmic Effect C. UNCOATING → Mark derangement of cell function leading to lysis and Degradation of the capsid by viral or host enzymes thus cell death releasing the nucleic acid → Virus replicate and produce progeny Start of ECLIPSE PHASE → cell death and cytopathic effects (CPE) → period when the infectious particles can’t be recovered → Inhibition of cellular protein and nitric acid (NA) synthesis MICROBIOLOGY Introduction to Virology PAGE 9 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo → Latent Infection ▪ viral genetic materials remain in the host cell. ▪ virus has been incorporated in the host genome but may be activated in the later time ▪ E.g. V2 virus (HSV3) − They are dormant in the nerve ganglia and reactivated in the later time VII. BASIC TYPES OF CULTURES Primary → Used for vaccine production Figure 38. Viral Growth Curve → Vaccine production, very expensive VI. PATTERN OF VIRAL INFECTIONS & Semicontinuous CYTOPATHOGENICITY → Transferred from primary cell line A. ABORTIVE INFECTION → Vaccine Production The host cell is non-permissive Continuous → No replication → Production of serologic antigens. Source of cells for → No virions produced neutralizing antibody assays → Defective infecting virus (Hep D) → HeLa culture: Derived from the tissues of Henrietta ▪ Example: Hepatitis D (Delta RNA virus) Lacks − In the absence of Hep B, it cannot replicate o It is called virozoid or satellite o replication is incompetent − If inside Hep B, it can replicate B. LYTIC INFECTION Virus replicate and produce progeny Cell death and CPE Inhibition of cellular protein and NA synthesis Evidence to establish viral infection → Thickening (swelling) → Presence of inclusion bodies → Syncytial bodies ( cell fusion of infected host cell) Figure 40. Steps in culturing Primary cell line, Semicontinuous cell line, and Continuous cell line; Transferring primary cell line to another medium, it becomes semi continuous cell line Cell Line Origin Viruses Primary Rhesus Monkey Poliovirus type 1, (e.g. LLMCK2) kidney enterovirus, rhinovirus, poxvirus group Semicontinous African green RSV, measles, HSV, (e.g. CV-1) monkey kidney VZV fibroblast Continuous Human lung Adenovirus, HSV, (e.g. A549) carcinoma Influenza, Measles, Derived from (HeLa, Vero, Mumps, Parainfluenza, cancer or other Hep2, LC-MCK1, poliovirus, respiratory immortalized cells MDCK) syncytial virus (RSV), rotavirus, varicella zoster virus (VZV), metapneumovirus (MPV) Table 8. Types of Culture for Viruses Figure 39. Cause of Lytic Infection VIII. METHOD OF DETECTION C. PERSISTENT INFECTION Methods of detection of viruses in infected host cells: Small number of virus particles are produced with little or → Adsorption of RBC to infected cells no CPE → Detection of virus-specific nucleic acid (e.g. PCR for Infected cells survive the effects of viral replication COVID-19) 3 types: → Development of cytopathic event (CPE) → Viral transformation → Viral growth in chick embryo ▪ viral infection or gene product induces unregulated A. ADSORPTION OF RBC TO INFECTED CELLS cellular growth forming tumors in the host caused by Some viruses have receptors for RBC oncogenic viruses Hemadsorption → Chronic Infection → cells acquire the ability to stick to mammalian red blood ▪ low level of viral production without immune cells clearance ( e.g. Hep B or Hep C) MICROBIOLOGY Introduction to Virology PAGE 10 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo Figure 41. Example of cells positive for hemadsorption Figure 45. Syntial Formation. Cell in the middle is infected with virus and fuses with neighboring cells forming a syncytium Figure 42. Presence of hemadsorption in mumps virus Figure 46. Syncytium formation in RSV and Measles infection BALLOONING OF CELLS Original shape of the cell is lost, and the virally-infected cells become round and ballooned Example: Enterovirus 71, HSV Figure 43. Test for hemagglutination; (Bottom) Hemagglutination test of serially diluted samples. Sample number 4 is negative for all solutions, no more virus that has grown Figure 47. Ballooning of cells B. DETECTION OF VIRUS SPECIFIC NUCLEIC ACID D. VIRAL GROWTH IN EMBRYONATED CHICK EMBRYO Polymerase Chain Reaction (PCR) You grow the virus on a live chick DNA is denatured, annealed, and extended Detect the presence: Example: Covid 19 Virus → Pocks on CAM (growth at the allantoic membrane) → Hemagglutination (used for influenza) → Inclusion Bodies Figure 44. PCR testing C. DEVELOPMENT OF CYTOPATHIC EVENT (CPE) SYNCYTIAL FORMATION Figure 48. Parts of a chick embryo Infected cell fuses with neighboring cells to form a syncytium Amniotic/Allantoic Inoculation Multinucleated, enlarged cell → Inject 0.2 ml of the virus to the amniotic fluid Normal cells appear separate while syncytial cells are → Pull the syringe to the area of allantoic fluid and inject fused. 0.2 ml Example: RSV, Measles virus MICROBIOLOGY Introduction to Virology PAGE 11 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo → ”You can test for influenza using allantoin by extracting the allantoin and testing it for hemagglutination. Positive growth results to hemagglutination” Figure 49. Allantoic inoculation Chorio-allantoic Membrane Inoculation (CAM) → You make a hole and then you drop the specimen and after a few days you may see pocks formation Figure 50. Pocks in chick embryo IX. CLASSIFICATION OF HUMAN VIRUSES Figure 52. RNA Viruses X. REVIEW QUESTIONS 1. Viruses are cellular Figure 51. Summary of Baltimore Classification a. True b. False 2. All enveloped viruses derived their envelope from host cell membrane a. True b. False 3. Hepatitis B falls under which of the following under Baltimore Classification a. Group I b. Group IV c. Group VII 4. Viral replication occurs during the latency stage of infection. a. True b. False 5. Which among these viruses produces DNA by reverse transcription, with DNA as template? Figure 53. DNA Viruses a. HIV b. Hepatitis B c. Poliovirus d. Smallpox MICROBIOLOGY Introduction to Virology PAGE 12 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo 6. What is the family of the largest DNA virus? a. Poxviridae b. Papillomaviridae c. Herpesviridae d. Adenoviridae 7. Which of the following is NOT a basic property included in viral classification? a. Viral morphology b. Viral replication method c. Presence or absence of an envelope d. Presence or absence of a capsid 8. T/F: There is no limit to the number of virions that can be present inside a cell a. True b. False 9. What do you call the coalescent, large, multinucleated cell that arises from viral infection? a. Syncytium b. Langhans giant cell c. Epithelioid cell 10. Which of the following is a retrovirus? a. Rotavirus b. Marburg virus c. HIV ANS: 1. B. They are acellular. Do not have cell organelles & metabolic processes which is required by living cells 2. B. Except for Herpes, they are derived from nuclear membrane 3. C. Group VII dsDNA (Retrovirus) 4. B. Viral Replication occurs during Phase 2 5. B. Hepatitis B Virus is the only DNA virus that utilizes reverse transcriptase (RT). 6. A. Poxviridae is the family of the largest and most complex DNA virus. It is part of the enveloped viruses and is classified as Group I in the Baltimore Classification. 7. D. A capsid (protein coat surrounding the DNA/RNA) is present in ALL viruses; has 3 kinds of symmetry: icosahedral, helical, complex 8. B. In phase 4: Latent/Plateau of the viral growth curve; virions are limited to 100-10,000 per cell. No additional increase in yield of the virus can occur. 9. A. In a syncytium, infected cells fuse with neighboring cells, forming a nucleated giant cell 10. C. Rotavirus is a reovirus, marburg virus is a filovirus XI. REFERENCES 2026 Trans 2025 Trans Asynchronous Lecture prepared by Dr. Eduardo 7th Edition, Jawetz, Melnick, & Adelberg's Medical Microbiology. 28th ed. Pp 411-434 MICROBIOLOGY Introduction to Virology PAGE 13 of 14 MICROBIOLOGY | LE 2 Introduction to Virology | Dr. Margarita V. Eduardo XII. APPENDIX XII. Classification of DNA Human Virus Table 9. DNA Viruses VIRION TYPE FAMILY EXAMPLES BC GROUP Enveloped Icosahedral Hepadnaviridae Hepatitis B Grp 7 Herpesviridae HSV1, HSV2, HSV3 (Varicella virus), HSV4 (Epstein Barr virus), Grp 1 HSV5 (CMV) HSV6 (Roseolo virus - agent of 6th dis), HSV8 (Kaposi sarcoma associated virus) Naked Icosahedral Adenoviridae Mastadenovirus (cold) used as viral vector Grp 1 Papillomaviridae Human papillomavirus Polyomaviridae BK virus, JC virus, Markell cell carcinoma virus Parvoviridae Parvovirus B19 (agent of 5th dis) Grp 2 Enveloped Complex Poxviridae Variola major (smallpox), vaccinia (cowpox), Molluscom Grp 1 contagiosum Table 10. RNA Viruses VIRION TYPE FAMILY EXAMPLES BC GROUP Naked Icosahedral Caliciviridae Norwalk Grp 4 Hepeviridae Hepeviridae Hep E Grp 4 Reoviridae Rotavirus Grp 3 Picornaviridae Polio, Entero, Rhino, Echo, Cocksakie, Hep A Grp 4 Enveloped Icosahedral Togaviridae Rubella, Chikungunya, WEE, Grp 4 Retroviridae HIV, HTLV Grp 6 Flaviviridae Yellow Fever, Dengue, Jap Encephalitis, SLE, Hep C Grp 4 Enveloped Helical Coronaviridae SARS Cov 1, MERS, SARS Cov 2 Grp 4 Bunyaviridae Hanta Grp 5 Arenaviridae Lassa Delta Virus Hep D (Not a family) Paramyxoviridae Paramyxo, Mumps, Measles, RSV Rhabdoviridae Rabies Orthomyxoviridae Influenza Filoviridae Ebola Marburg : MICROBIOLOGY Introduction to Virology PAGE 14 of 14