Viral Structure & Classification (Introduction to )

VERSION 1 Objectives

❖ Distinguish the viruses from other microorganisms. ❖ General characteristics of viruses. ❖ Structure & symmetry of viruses. ❖ Classification of viruses. ❖ Steps of replication. ❖ Laboratory diagnosis of viral infections.

Any future corrections Colour index: will be in the editing file, so please check it ! "#$% &'()*+,-+. - Grey: Extra info & explanation. frequently. - Purple: only in girl’s slides. - Green: Only in boy’s slides. Scan the code - Or click here Properties of Microorganisms

Characteristic Parasite Fungi Virus

Cell Yes Yes Yes No

Type of nucleus Eukaryotic Eukaryotic Prokaryotic -----

Both DNA & Both DNA & Both DNA & DNA or RNA Nucleic acid RNA RNA RNA

Ribosomes Present Present Present Absent

Mitochondria Present Present Absent Absent

Mitosis 0r Binary fission Special (one viral particle produces Replication Mitosis (Bacteria=Binary fission) budding millions of viruses) Characteristics of Viruses

Acellular Obligate intracellular organisms 1 Viruses are not cells. 3 It cannot reproduce outside No or mitochondria, so 1), 2) synthesis, and 3)energy production is entirely reliant on host cell machinery (intracellular resources).

Tiny particles Replicate different from cells 2 Can only be seen via E.M 4 One virus can produce millions - Size: 20-300 nm of/many viruses. - Unit of viruses: nm (10-9) - ~1000 smaller than bacteria) Viral Structure

Viral Structure

1- Viral genome 2- 3- Envelope Internal core of viral Protective protein coat Some viruses have genome that surrounds the nucleic lipoprotein membrane. (containing DNA or RNA). acid. 1-Viral Genome (genetic material)

DNA Viruses RNA Viruses (Deoxyribonucleic acid) (Ribonucleic acid)

All are double stranded All are single stranded (ex: ) Except: Parvoviruses (single stranded) Except: Reoviruses (double stranded)

Number of molecules: Single Molecule (Haploid) ● Single (Haploid) Meaning: one molecule of double stranded DNA ● Double (Diploid ex: Retroviruses) ● Multiple pieces of RNA (ex: Reovirus and )

Polarity/Sense: After infection, viral genome: ● (+) polarity/sense acts directly as mRNA (directly translated into protein). - ● (-) polarity/sense must first be transcribed to mRNA then can be translated into protein. ○ (-)polarity viruses contain virus-specific for (transcriptase) called: RNA dependent RNA polymerase.

All viruses are haploid (have a single copy of genetic material) Except: Retroviruses ex: HIV (Diploid: have two copies of the same genetic material) 2- Capsid (Protein Coat)

❖ A protein coat that surrounds the viral genome. ❖ Made up of subunits called capsomeres. ❖ Nucleocapsid = nucleic acid (genome) + capsid ❖ Arrangement of capsomers in capsid gives viruses their symmetry.

Protects the nucleic acid Note: Function All Viruses have a capsid but not all have a nuclear envelope Facilitates its entry into the cell (for non-enveloped viruses)

Note: know shapes only (no need to Symmetry: memorize examples). Based on arrangement of capsomeres

Cubic (Icosahedral) Helical Complex 20 triangles that form an icosahedron, spherical in shape

A.Non-enveloped: B.Enveloped: Elongated Pleomorphic Adenovirus Herpesvirus Filoviruses Influenza v. Poxviruses 3- Envelope (Found in some viruses only)

❖ Enveloped viruses are more sensitive to harsh environmental factors such as :

heat and dryness (Non-enveloped viruses are more resistant).

❖ Envelope is acquired when the virus is released from infected cell (process is called budding).

❖ Envelope is derived from host cell membrane. ➢ Except: envelope of Herpes Viruses which is derived from host nuclear membrane.

❖ Envelope is a lipoprotein membrane ➢ from host membrane ➢ Protein/glycoprotein is virus-specific (from the virus itself) ❖ Glycoproteins:

➢ attaches to cell receptor (mediate the attachment of virus to host cell) ➢ Virus-specific: specific glycoproteins attach to specific cell receptors ➢ Glycoproteins are usually in the form of spike-like projections Viral

Internal Viral Proteins Outer Viral Proteins

➢ Structural proteins: ➢ glycoproteins on envelope: Capsid proteins (capsomere) are considered internal proteins in enveloped viruses. - Mediate attachment to specific receptors on the host cell. (specific glycoprotein binds to specific cell receptor) ➢ Non-structural proteins (enzymes):

- transcriptase : RNA dependent RNA - Induce neutralizing . (act as polymerase (an enzyme that catalyzes the replication of RNA from an ; stimulates to produce RNA template) antibodies. is in all single stranded RNA viruses with negative polarity - Are a target of antibodies - enzyme (an enzyme used to generate complementary DNA from an RNA template) is in Retroviruses and HBV ( Viruses) . Classification of Viruses

Single Stranded 1. Number of Strands Double Stranded

DNA

2. Type of Nucleic Acid** RNA 3. Polarity of genome:

Positive Negative

Icosahedral, cubic Classification of Viruses 4. Type of Symmetry Helical

Complex

Present 5. Presence or Absence of the envelope Absent

Medically Important Viruses

1-DNA

Single stranded Double stranded Notice that in DNA Non-enveloped Enveloped Non-enveloped viruses, there is no helical symmetry Icosahedral Complex Icosahedral Icosahedral And that there is Adenoviridae no complex symmetry in RNA viruses

2-RNA

Single stranded Double stranded

Non-enveloped Negative strand Positive strand

Enveloped Enveloped Non-enveloped Icosahedral

Helical Coronaviridae Helical Icosahedral Filoviridae Hepeviridae Icosahedral Retroviridae Virus Replication Steps (it is recommended to watch both videos)

Attachment site: 1-Adsorption ❖ Glycoprotein (enveloped) (Attachment) ❖ Folding in the capsid proteins (non-enveloped)

There are two methods: 2-Penetration ❖ Fusion (enveloped). fuses with host cell membrane and enters the cell. (Entry) ❖ (enveloped: viruses fuse with endosome membrane),(non-enveloped: virus pores or lyses endosome membrane).

❖ Removal of capsid 3-Uncoating ❖ Release of viral genome in: (for RNA viruses) or nucleus (for DNA viruses).

❖ Positive Single Stranded RNA viruses, their single strand of RNA acts directly as mRNA (Direct) which will be translated (with help of the cell’s ribosomes) to proteins & enzymes. 4-Synthesis of Viral (NO NEED for transcription, positive single stranded RNA is translated directly to make viral proteins) components ❖ Negative RNA viruses, on the other hand, their RNA must be transcribed into mRNA first by RNA dependent RNA polymerase (mRNA, viral proteins, N.A.) which is then translated to make viral proteins (Indirect). ❖ Viral genome is replicated in both cases.

❖ (Nucleic acid + Viral proteins= Virions) 5-Assembly Nucleic acid and viral proteins assemble to make virions (an entire viral particle). Millions of virions are produced.

❖ Enveloped viruses: undergo budding and get their membrane either from the: 6-Release 1)cell membrane (in RNA viruses) or 2)nuclear membrane (in DNA viruses e.g herpes virus). ❖ Non-enveloped viruses: virus or ruptures from the cell membrane (cell is damaged). Microscopic Examination Cell Culture

Isolation of virus and diagnosis

Viral Laboratory Antigen/ Diagnosis of Detection Viral Infections Routinely used

Molecular Method Serological Most expensive, not routinely Tests used Routinely used, for diagnosis Microscopic Examination

Light Microscopy Electron Microscopy

- Shows histological appearance and - Shows size and morphology of the virus (to see virus itself). (to see the effect of the virus on the cells not the virus itself) - Expensive, so used in research and new viruses E.g. inclusion bodies. E.g. - Diagnosis of viral GE (gastroenteritis) such as rota & adenovirus. - Diagnosis of skin lesions caused by herpes or poxvirus.

- It is replaced by antigen detection & molecular tests. Owl’s eye (CMV) Virus cultivation 1st step: Cell culture ❖ -Tells whether or not there is infection through cytopathic effect. It is not a Methods of Virus Cultivation definitive diagnosis.

1. Laboratory animals (effectiveness of ) 2nd step: further investigation to identify through other tests like: -Immunofluorescence 2. Embryonated eggs (to develop vaccine) -ELIZA 3. Cell culture (isolation of virus) -Molecular testing

Purpose: isolation of virus (DNA or RNA), new virus diagnosis. Cell Culture: Used to see: cytopathic effect (not virus or type of virus)

1 or 2 subpassages Primary cell Problems regarding cell culture: (lasts a very short period which is why it is difficult to maintain) culture - Long incubation (5 days or more), it can be solved with rapid culture technique. Diploid cell culture 20 to 50 subpassages (semi-continuous) (lasts for a few weeks) - Sensitivity is variable.

- Susceptible to bacterial contamination.

Continuous Indefinite (cancerous cell line) - Some viruses don’t grow in cell culture cell line (HCV - virus) Rapid cell culture technique:

● A modification of cell culture called Shell vial assay ● Based on immunofluorescent detection of viral ● 1-3 days Immunofluorescence

Detection of Viral Growth 1. Cytopathic effects: the affected cell will appear round, shrink and aggregated (in a group). Syncytium (giant multinucleated cell) and loses adherence. Basis of cell culture.

Uninfected CC Cell rounding Syncytium 2. Immunofluorescence (IF) basis of Shell vial assay

3. Other methods. -Used specifically for viruses to detect: -Examples of Serological tests: ● Antigen ● Immunofluorescence Serological Tests ● Antibody ● ELIZA

Antibody and Antigen Detection Antigen detection (Indirect indication of (Direct indication of viral infection) viral infection) It is a test that looks for (antibodies) from a sample to determine the availability of (antigen).

Immunofluorescence (IF) Test Sample Virus

Nasopharyngeal aspirate Influenza V Enzyme- linked immunosorbent IF assay (ELISA) Skin scrapings HSV

Techniques Faeces Rotavirus ELISA Only for antibody detection: Blood HBV(HBsAg) Complement fixation test (CFT) Immunofluorescence ELIZA Molecular Test (Enzyme-Linked Immunosorbent Assay)

Uses fluorescent microscope and antibodies Same principle as IF. Uses Polymerase Chain Reaction labeled with fluorescent dye Uses spectrometer and antibodies labeled - Amplifies viral genome and nucleic acid Look through a fluorescent microscope: with enzymes instead of fluorescent dye. -Used for diagnosis as a confirmatory test . ● ● If green fluorescent: positive result Yellow: positive result -It is the only way to monitor a patient’s ● ● No fluorescence: negative result Colorless: negative result response to treatment by measuring /concentration. ❖ Direct IF: antigen detection ● Direct: antigen detection. ● Add antibody labeled with fluorescent dye to Indirect: antibody detection. cells on slide.

❖ Indirect IF: antibody detection Add secondary-antibodies labeled with fluorescent dye to patient serum.

The coloured cells indicate reactivity MCQs

1- Viral symmetry is determined by: 4-Which of the following is directly translated into protein inside the host cell? a) Viral envelope b) Viral Capsid a) Positive ddDNA c) Viral genome b) Negative ssDNA d) Viral Enzyme c) Positive ssRNA d) Negative ddDNA 2-Which of the following is true about viruses? 5-Which of the following is not an internal ? a) They have either DNA or RNA a) Transcriptase b) They reproduce by binary fission b) Reverse transcriptase c) All of them have an envelope c) Capsid proteins in enveloped viruses d) They are made up of one cell d) Glycoproteins

3-All viruses are haploid except: 6-Viruses are considered obligate parasites because

they?

a) Influenza 6-c

b) Parvoviruses 5-d

a) Are acellular 4-c

c) Reoviruses b) Are tiny particles 3-d

d) Retroviruses c) Need a host to complete their life cycle 2-a

d) None of the above 1-b MCQs

7-To see the virus which method is used? 10-Which of the following is used to monitor a patient’s response to treatment by measuring a) Light microscope viral load? b) Cell Culture c) a) Complement Fixation Test d) Fluorescence b) Immunofluorescence c) Polymerase Chain Reaction 8-Which if the following is a virus cultivation technique d) ELIZA used to isolate the virus? e)None of the above

a ) ELIZA 11-which of the following is not a structural b) Embryonated eggs classification of viruses? c) Cell cultures d) Laboratory animals a) complex e) Polymerase Chain Reaction b) vibro c) helical 9-Which is not an example of a serological test? d) isocahedral

a) Complement Fixation Test 12- The presence of which of the following is a

direct indication of a viral infection: b 12-

b) Immunofluorescence b 11-

a) Antibody

c) ELIZA c 10-

b) Antigen

d) FISH d 9-

e)None of the above c) ribosomes c 8-

d) endotoxins c 7- e) chemotaxins T & F SAQ

Slide 5 1. All viruses can be grown in cell cultures. ( ) 1- List the three main viral structure components.

2. Complement fIxation test is used for antigen

detection. ( ) 2- What is a nucleocapsid? Slide 7 3. Immunofluorescence uses antibodies labelled with enzymes. ( ) 4. Molecular tests are used to amplify viral 3- What are the functions of viral ? Slide 7 genome and nucleic acids. ( )

5. Primary cell cultures can last for several 4- What are the types of symmetry that viruses exhibit? Slide 8 weeks. ( ) 6. Electron Microscopy shows cytopathic effect

and histological appearance. ( ) 5- What are the 5 criteria for , and Slide 11

7. Non-enveloped viruses leave the host cell by which criteria is only present in RNA viruses? 10-F

lysing it. ( ) F 9-

8- T 8-

8. produces millions of viruses T 7-

6- What is an example of viruses that contain an Slide 9

from one virus. ( ) F 6-

envelope that is derived from the host’s nuclear

9. Transcriptase enzyme is found in retroviruses. membrane? F 5-

( ) T 4-

10. Capsids are derived from host membrane. ( ) 3-F

7- What are the problems regarding cell culture? Slide 16 F 2-

1- F 1- Team Leaders

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Team Members

- Sadem Al Zayed - Abdulaziz Alderaywsh - Sarah Alqahtani - Sultan Alqahtani - Noura Alshathri - Faisal Alomri - Ghadah Alsuwailem - Munib Alkhateeb - Shahad Almezel - Abdulaziz Alomar - Noura Alsalem - Muhannad Alomar - Sumo Alzeer - Renad Alhomaidi - Raghad Albarrak Contact us through: [email protected] - Reema Alowerdi