VIROLOGY_INTRO_by_Mr._Patrick_year_1_new_[Autosaved]_newest-1.pptx

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1. VIROLOGY 1 Lecture slide Mr. P atrick

2. Definitions Virus : Genetic elements that can replicate independent of a cells chromosome but not independent of the cells themselves Virion : Mature infectious form of genetic material that is typically extracellular Envelope : covering possessed mostly by animal viruses that surrounds the polyhedral or helical nucleocapsid .

3. Definition of a virus Sub macroscopic entity consisting of a single nucleic acid surrounded by a protein coat and capable of replicating only within the living cell of bacteria, animal or plants Obligate Intracellular parasitic

4. Virus Living Cell RNA or DNA core (center), protein coat ( capsid ) Copies itself only inside host cell--REPLICATION DNA or RNA NO NO NO NO Cell membrane, cytoplasm, genetic material, organelles Asexual or Sexual DNA and RNA YES—Multicellular Organisms YES YES YES Structure Reproduction Genetic Material Growth and Development Response to Environment Change over time Obtain and Use Energy

5. Viruses vs Bacteria Key difference bacteria viruses Definition They are unicellular organisms found in most habitats on earth. They are non-living particles consist of genetic material (RNA or DNA) enclosed by a protein core. They are organic structures that interact with living organisms. Where do they grow? They grow in harsh conditions such as a deep portion of the crust of the earth, radioactive waste, and acidic hot springs. They are infectious agents that need a host to replicate. classification They are classified according to their morphology: Cocci – spherical-shaped bacteria Vibrio – comma-shaped bacteria Bacillus – rod-shaped bacteria Spirilla – spiral-shaped bacteria Spirochaetes – tightly coiled bacteria They are classified according to core content, presence of outer envelope, the structure of capsid, and the way mRNA is produced.

6. Viruses vs Bacteria cont Key differences bacteria viruses infectivity They infect all life forms. They can infect all forms of life including bacteria and archaea . visualization They are visualized under a  light microscope . They are visualized by electron microscope Reproduction requirement bacteria can grow and reproduce without a host. Viruses need a host to reproduce as they replicate inside the host. size They are large in size (around 1000 nm).Various shapes and arrangements of Bacterial cells They are small in size (20 to 400 nm)

7. Viruses vs Bacteria cont … Key difference Bacteria Viruses Cell wall composition The cell wall contains peptidoglycan/lipopolysaccharide. They don’t have a cell wall but has a protein coat Number of cells They are unicellular They do not have cells. Cellular structure Bacteria are cells and are prokaryotic in nature (displaying characteristics of a living organism) They are not cells and exist as DNA or RNA particles and enveloped within a protein shell.See :  Difference between RNA and DNA Ribosomes They contain ribosomes. They donot contain ribosomes. Genetic material They contain a single circular chromosome. They have strands of DNA/RNA

8. Viruses vs Bacteria cont … Key Difference Bacteria Viruses Metabolism They metabolize within the cell. Metabolism is not present in a viral particle Reproduction The reproduction process takes place through the process of binary fission and conjugation. They reproduce by invading the host cell and create copies of genetic materials/proteins. They destroy the host cell and release new particles. Possession cellular Machinery They possess cellular machinery. They donot possess cellular machinery. Beneficial They can be categorized as harmful or beneficial. Some bacteria are considered good or healthy, especially those found in the gut. They are harmful, but some are used for genetic engineering purposes Ability to infect/Nature of infection   They cause localized infections. They cause systemic infections.

9. Viruses vs Bacteria cont … Key Differences Bacteria Viruses treatment They are treated and managed using antibiotics. Treated with antivirals Most have no treatment but supportive care They can be prevented using vaccines. Symptoms/clinical manifestations The symptoms are confined to a particular infected area of the body as well as the type of bacteria that causes it. Typical clinical manifestations include the following: Swelling Pain Redness Nausea and vomiting Fever Typical symptoms of viral infections are: Respiratory symptoms like cough and cold Sneezing Tiredness Diarrhea Nausea and vomiting

10. Viruses vs Bacteria cont … Key Differences Bacteria Viruses Characteristics They are single-celled organisms. Some of them grow as independent single cells while some are multi-cellular fruiting bodies and play an important role in the life cycle. They do not have complex organelles in the cell.  They have an internal organization but don’t have a plasma membrane-like other living cells. Bacterial cells have ribosomes (spherical units) where protein assembles from amino acids using the data encoded in the DNA ribosome. They may only contain DNA or RNA. They can’t contain both. They reproduce at a tremendous amount, but they need a living host to replicate. Many viruses have the ability to mutate. Viruses are like parasites; they use the host cell’s metabolic machinery. They alone cannot grow and divide, they would need a host cell to produce and assemble their viral components

11. Viruses vs Bacteria cont Key Differences Bacteria Viruses Transmission Exposure to body fluids Close contact with an infectious person Touching contaminated surfaces Mother to child transmission during childbirth Contact with infected animals/carriers Exposure to body fluids Close contact with an infectious person Touching contaminated surfaces Mother to child transmission during childbirth Contact with infected animals/carriers Examples Bacillus Coccus Vibrio cholera Rickettsia Staphylococcus aureus Helicobacter bacteria Streptococcus pneumoni Hepatitis A Virus Papillomavirus Ebola Virus Hanta Virus Rotavirus SARS- CoV SARS-CoV-2   Zika Virus Nairovirus

12. Viral Structure We are going to construct a virus beginning from the 1 Nucleic Acid DNA or RNA 2 Capsid (Helical or Icosahedral symmetry) 3 Nucleocapsid (Nucleic acid + Capsid) 4 Envelope

13. Basic virus structure Capsid protein Nucleocapsid Naked capsid virus DNA RNA or = + Nucleocapsid Lipid membrane, glycoproteins Enveloped virus +

14. Nucleic Acid Spike Projections Protein Capsid Lipid Envelope Virion Associated Polymerase

15. 1. Nucleic acid (Structure of Virus) Virus are classified as having either DNA or RNA but never both The Nucleic acid can be single stranded or double stranded, linear, or looped Nuclei acid sequences can encode a simple message or encode hundreds of enzymes and structural proteins

16. Viral genomes Viruses can either have 1. DNA as genetic material This may be single stranded linear or circular Double stranded linear or circular 2. RNA as genetic material This may be single stranded linear or circular Double stranded linear or circular

17. Viral genome 3.Both DNA and RNA but at different stages of their reproductive cycle E.g. (see classification of viruses) Retroviruses contain RNA in virion stage but replicate through a DNA intermediate Human hepatitis B virus contains DNA in virion stage but has RNA intermediate during replication

18. Viral genome All viruses use the host cells translational machinery mRNA which must be generated and is translated on the hosts ribosomes DNA viruses use hosts own RNA polymerase to transcribe mRNA directly from viral DNA

19. 2. The viral capsid Is the protein coat that surrounds nucleic acid The capsid is composed of structural units called capsomers , which are the structural protein units that make up the capsid (aggregates of viral-specific polypeptides .) The capsid has a symmetry that is classified as helical, icosahedral (a 20-sided polygon), or complex and is used as a criterion for viral classification . Nucleocapsid : The nucleic acid plus the capsid Functions Of The Capsid :

20. 2. The viral capsid cont … As protection of the viral genome As the site of receptors necessary for naked viruses to initiate infection As the stimulus for antibody production As the site of antigenic determinants important in some serologic tests

21. I cosahedral capsid Take 1 or more polypeptide chains and organize them into a globular protein subunit. This will be the building block of our structure and is called a capsomer . Arrange the capsomers into an equilateral triangle . Place 20 triangles together to form an icosahedron fig 1. Package the DNA or RNA inside the icosahedral

22. Icosahedral capsid Fig 1

23. Structure of icosahedral unenveloped virus

24. Capsid symmetry Icosahedral Helical Naked capsid Enveloped Lipid Glycoprotein Matrix

25. Helical Symmetry In helical symmetry the protein capsomers are bound to RNA (always RNA because only RNA viruses have helical symmetry) and coiled into a helical nucleoprotein capsid. Most of these assume a spherical shape except for the rhabdoviruses ( rabiesvirus ), which have a bullet-shaped capsid.

26. Helical Symmetry

27. 4. The viral envelope S urrounds the nucleocapsid of enveloped viruses. I s composed of viral-specific glycoproteins and host-cell derived lipids and lipoproteins. C ontains molecules that are necessary for enveloped viruses to initiate infection, act as a stimulus for antibody production, and serve as antigens in serologic tests. I s the basis of either sensitivity of a virus.

28. Properties of enveloped viruses Envelope is sensitive to Drying Heat Detergents Acid Consequences Must stay wet during transmission Transmission in large droplets and secretions Cannot survive in the gastrointestinal tract Do not need to kill cells in order to spread May require both a humoral and a cellular immune response Adapted from Murray, P.R. Rosenthal K.S., Pfaller, M.A. (2005) Medical Microbiology, 5 th edition, Elsevier Mosby, Philadelphia, PA Box 6-5

29. Properties of naked capsid viruses Capsid is resistant to Drying Heat Detergents Acids Proteases Adapted from Murray, P.R. Rosenthal K.S., Pfaller, M.A. (2005) Medical Microbiology, 5 th edition, Elsevier Mosby, Philadelphia, PA , Box 6-4

30. Properties of naked capsid viruses Consequences Can survive in the gastrointestinal tract Retain infectivity on drying Survive well on environmental surfaces Spread easily via fomites Must kill host cells for release of mature virus particles Humoral antibody response may be sufficient to neutralize infection Adapted from Murray, P.R. Rosenthal K.S., Pfaller, M.A. (2005) Medical Microbiology, 5 th edition, Elsevier Mosby, Philadelphia, PA , Box 6-4

31. 4. Spikes Penetrating the membrane are additional proteins that determine the specificity of the virus to host cells. These are called spikes Not all viruses posses the spikes, Spike determine viral virulence and antigenic properties of the viruses,

32. Classification of viruses Viruses are classified according to their 1. Nucleic Acid: a. Type of Nucleic acid: DNA, RNA b. Double vs Single Stranded c. Single or segmented pieces of nucleic acid d. Positive (+) or negative (-) Standed RNA

33. Classification of virus cont …. 2. Capsid a. icosahedral b. Helical 3. Envelope a. Naked b. Enveloped 4. Size a. the diameter of the helical capsid b. The number of capsomers in icosahedral capsid

34. RNA Viruses There are Three types of RNA (+) Stranded (-) Stranded RNA of retrovirus

35. Positive (+) Stranded Means the virus is just like a messenger RNA ( mRA ) When a positive (+) stranded RNA virus enters a host cell, its RNA can immediately be translated by the host's ribosomes into protein. The + Stranded means the RNA is just like a mRNA

36. Negative stranded When negative (-) stranded RNA viruses enter a cell, they are not able to begin translation immediately . They must first be transcribed into a positive (+) strand of RNA (like mRNA). To do this, negative (-) stranded RNA viruses must carry, in their capsid, an enzyme called RNA-dependent RNA polymerase, which will carry out the transcription of the negative (-) strand into positive (+). Human cells do not have an RNA dependent RNA Polymerase, so Negative stranded virus must carry their own

37. DNA Virus Unlike RNA, DNA cannot be translated directly into proteins. It must first be transcribed into mRNA , with subsequent translation of the mRNA into structural proteins and enzymes

38. Every DNA virus has both a negative (-) strand and a positive (+) strand. Here is the confusing part: The positive (+) strand refers to the strand that is read, while the negative (-) strand is ignored. Unlike positive (+) stranded RNA, which is translated directly into proteins, the positive (+) stranded DNA is used as a template for transcription into mRNA.

39. Classification of Human Viruses Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Table 2.1

40. Complex capsid These capsid are neither purely helical nor purely icosahedral, and may poses extra structures such as protein tails or comlex outer wall

41. membrane as they leave There may be various glycoproteins embedded in their cell membranes. Viruses that do not have membranes are referred to as naked or nonenveloped . Those with membranes are referred to as enveloped.

42. Baltimore classification Strands(negative polarity). 6- ss-RNA viruses associated with the enzyme reverse transcriptase. Viruses were divided into six groups based on the their nucleic acid and m-RNA production. 1- ds-DNA viruses. 2- ss -DNA viruses. 3- ds- RNA viruses. 4- ss -RNA viruses with positive strands( positive polarity). 5- ss -RNA viruses with negative

43. Viral Replication Viruses can not replicate on their own. They need a suitable host cell they can attack The steps in viral replication is as below

44. Steps in virus replications 1- Adsorption (attachment). 2- Penetration. 3- uncoating. 4- Replication of the viral genome. 5- Transcription of the viral genome into m-RNA. 6- Translation of m-RNA into viral proteins. 7- protein synthesis, 8- Viral assembly .

45. Steps in virus replication 1-Adsorption (attachment ). Viruses must recognize and bind to specific cellular receptors on the surface of the infected cell via particular glycoproteins. 2-Penetration. A- Enveloped viruses that has the ability to form syncytia ( multi-nucleated giant cell ) enter the cell through fusion of the viral envelope with cell plasma membrane( eg . Paramyo and herpes viruses ). 2- The remaining enveloped viruses enter the cell through endocytosis.

46. Entry of enveloped viruses, fusion of the viral envelope.

47. VIRAL REPLICATION

48. Steps in virus replication B- Unenveloped viruses enter the cell either by endocytosis ( endosome lyses as with adenoviruses) or by forming a pore in the membrane of the cell. The viral RNA is then released inside the cell (picornaviruses).

49. Endocytosis Endocytosis involves invagination of the cell membrane to form vesicles in the cell cytoplasm. Infected viruses are then engulfed inside these vesicles. Each vesicle fuses with a lysosome to form lysosomal vesicle. The viral envelope fuses with lysosomal membrane and the viral nucleocapsid is expelled into the cytoplasm.

50. Endocytosis.

51. Steps in virus replication 3- Uncoating. Release of the viral genome from its protective capsid to enable the viral nucleic acid to replicate. 4- Transcription. Synthesis of m-RNA. 5-Translation. The viral genome is translated using cell ribosomes into structural and non-structural proteins.

52. Viral Replication 6- Replication of the viral nucleic acid. 7-Assembly. New virus genomes and proteins are assembled to form new virus particles. 8-Release. Enveloped viruses are released by budding from the infected cells. Unenveloped viruses are released by rupture of the infected cells.

53. Release of enveloped viruses by budding

54. DNA VIRUS These are some times reffered to as HHAPPPY viruses Herpesviridae (Herpes simplex, varicella-zoster , Cytomegalovirus Epstein – Barr virus) Hepadnaviridae (Hepatitis B virus) Adenoviridae (adenovirus) Papovaviridae (Papilloma virus, Polymavirus ) Parvoviridae ( B19) Poxviridae (Small pox, Vaccinia , Molluscum contagiosum ) Most DNA virus are double stranded, show icosahedral symmetry and replicate in the nucleus W where DNA customarily replicate

55. DNA viruses cont … Two DNA Viruses break these rules 1. Parvoviridae I t has single DNA Strand S imple virus 2. Poxviridae Double stranded DNA Complex DNA coding for Hundreds o f proteins replicates in the cytoplasm

56. DNA Viruses Three of the DNA viruses have envelopes Ie Herpes, Hepadna , Pox Three are Naked Ie ( A woman must be naked for the PAP smear exam. PApova Adeno PArvo

57. RNA Viruses RNA viruses are generally opposite of DNA viruses Most are Single Stranded ( Half are + stranded, Half negative stranded Replicate in the cytoplasm

58. RNA Viruses Togaviriadae Orthomyxoviridae Coronaviridae Paramyxoviridae Retroviridae Rhabdoviridae Piconaviridae Bunya Calici viridae Arenaviridae Reoviridae Flaviridae

59. RNA Viruses Cont.. Exceptions 1. Reoviridae are Double stranded 2. Three are non enveloped: Picorna, Calici and Reoviridae 3. Five have Icosahedral symmetry; Reo, Picorna, Toga, Flavi, Calici (Rhabdo has Helical Symmetry 4. Two undergo replication in the Nucleus : Retro, Orthomyxo

60. RNA Viruses positive sense (+stranded) Caliciviridae (Norwalk agent) Picornaviridae ( Coxsackie virus, Echo virus, Hepatitis A virus, Polio viruses, Rhino viruses Flaviviridae (Dengue virus, Hepatitis C virus, St Louis encephalitis virus, yellow fever virus Togaviridae Eastern, Western, and Venezuelan equine encephalo-myelitis virus , Rubella Retroviridae (HIV, Leukemia virus, Sarcoma viruses)

61. RNA viruses Negative Sense/Stranded Parammyxoviridae (Mumps virus, Measles, virus, Parainfluenza virus, Respiratory syncytial virus) Rhadoviridae ( Rabies virus, Vesicular Stomatitis virus Filoviridae (Ebola virus, Marburg virus) Orthomyxoviridae (Influenza viruses) Bunyaviridae (California encephalitis virus, Hantavirus) Delta virus (Hepatitis delta virus) Coronaviridae ( corona viruses,

62. Viruses that Infect Bacteria Bacteriophage All bacteriophages are composed of a nucleic acid molecule that is surrounded by a protein structure. Most contain dsDNA Often make the bacteria they infect more pathogenic for humans

63. B acteriophages structure Icosahedral capsid head containing DNA Central tube surrounded by a sheath Collar Base plate Tail pins Fibers

64. B acteriophages structure Similar stages as animal viruses Adsorb to host bacteria The nucleic acid penetrates the host after being injected through a rigid tube inserted through the bacterial membrane and wall Entry of the nucleic acid causes the cessation of host cell DNA replication and protein synthesis The host cell machinery is then used for viral replication and synthesis of viral proteins As the host cell produces new parts, they spontaneously assemble

65. Figure 6.17

66. Figure 6.18

67. Figure 6.19

68. Bacteriophage types Two types/ categorizes of bacteriophage ie  lytic and lysogenic.  Virulent bacteriophages – Lytic cycle (cytoplasmic viral replication) In This bacteriophage type, the bacteriophage injects its genetic material into a host cell, which allows this genetic material to replicate, producing many new phages. Once the host cell is filled with new bacteriophages, the host cell raptures from within, releasing the newly formed phages.

69. Bacteriophage types cont …. Temperate bacteriophages – Lysogenic cycle The lysogenc cycle is one where a phage infuses its generic material into a host, but instead of rapidly replicating, this generic material finds its way to the host’s genetic material and infuses itself with it, becoming a prophage . It becomes part of the host’s genetic material and when the host cell divides, the temperate phage genetic material also undergoes a replication process . Lysogenic bacteriophages remain in this symbiotic state; in which it causes no harm to the host cell but rather quietly uses the resources provided by the host cell.

70. Lysogenic bacteriophage cont …. However, when a lysogenic bacteriophage feels under a certain amount of pressure, or its survival is placed at risk, it has the ability to switch from the lysogenic cycle to the lytic cycle, which results in rapid replication of newly formed phages which burst out of the host cell.

71. Figure 6.20

72. Techniques in cultivating and identifying animal viruses viruses require living cells as their medium In vivo- laboratory –bred animal and embryonic birds tissues In vitro –cells or tissue culture methods As viruses are intracellular obligatory parasites, they always need living cells for their growth. They cannot be grown on any artificial media . There are three methods employed for the cultivation of animal viruses

73. Diagnosis of viruses/methods for virus diagnosis Virus isolation, cultivation and detection is done in three methods ie ; Animal inoculation Embryonated eggs or chick embryo method. Tissue culture or cell culture . b. Virus detection; this is done by Observing for cytopathic effect (CPE) Fluorescent Antibody Techniques FAT) Heamaglutination (HA)

74. Techniques in Cultivating and Identifying Animal Viruses Primary purposes of viral cultivation To isolate and identify viruses in clinical specimens To prepare viruses for vaccines To do detailed research on viral structure, multiplication cycles, genetics, and effects on host cells

75. 1. Animal Inoculation Laboratory animals play essential roles in studies of viral pathogens Susceptible experimental animals like Mice, Monkey, Rabbits, Guinea Pigs etc. are used for the cultivation of viruses. Virus sample to be cultivated should injected into the experimental animal. It is important to select specific host animal for particular viruses . The animals should be healthy

76. Embryonate Eggs or Chick embryo method Good pasture (1931) was the first who used hen’s embryonated egg for the cultivation of viruses. Embryonated egg provides several sites for the cultivation of viruses. eg 1 . Chorio - allantoic membrane 2 . Allantoic cavity 3 . Amniotic cavity 4 . Yolk sac

77. Embryonate Eggs or Chick embryo method 5 . Embryo Different site is used for growth of different viruses. Eg . 1. Chorio-allantoic membrance is used for the cultivation of pox virus. 2. Allantoic cavity is employed for the Influenza virus . There are several advantages, chick embryos are packed in their shells and have natural resistant against bacterial contamination. Chick embryo method is cheaper and easy to handle.

78. Figure 6.21

79. Fig :Chick embryo method for cultivation of animal viruses

80. Tissue culture Steinhardt and colleagues (1913), was the first who used bits of tissue or organ for the cultivation of viruses. Now advance techniques are develop in Tissue culture . Three types of tissue cultures are available. 1.Organ culture : Small bits of organs are used for the cultivation of virus . 2.Explants culture : Fragments of minced tissue can be grown as ‘explants’ embedded in plasma clots.

81. Tissue culture 3.Cell culture: This is most common method for viral cultivation and growth of viruses. Different types of cell cultures are used for different viruses. General method is as follows Tissue like Monkey Kidney, Rabbit Kidney is taken and treated with proteolytic enzymes such as Trypsin and by mechanical shaking, tissues are dissociated into the component cells.

82. Method of cell culture cont. Trypsin, the proteolytic enzyme digest the binding material that binds the cells together in a tissue and results into free cells. He La cells (i.e. human cells from cervical cancer region) are also commonly used cell system for the cultivation of viruses 2. After treatment with trypsin, cells are washed, counted and suspended in the growth medium. 3 .

83. Method of cell culture cont. 3. Growth medium constitutes all those essential elements required for the growth of cell viz. essential amino acids, vitamins, salts, glucose, bicarbonates (buffer) with atmosphere containing 5% CO2and supplemented with 5% calf serum. 4. Antibiotics are added into the growth medium to prevent bacterial contaminants. Some indicators like phenol red, neutral red etc. are added into the growth medium, Change in indicator colourin growth medium, indicates the growth of virus in cell culture. In such growth medium cells divides and multiply. Then these .

84. Method of cell culture cont. In such growth medium cells divides and multiply. Then these cells are dispensed in bottle or petri-plates. On incubation, cells divides to form a confluent monolayer sheet of cells occur within a week. Various types of cell cultures used for virus cultivation are-

85. Types of cell culture Primary cells ; for example monkey kidney cells Secondary cells ; for example human embryonic kidney or skin fibroblasts. Continuous cells ; for example vero cells, hela cells and hep 2 cells, BHK  

86. Primary cell culture: Derived form normal cells ( eg . Monkey kidney tissue). The fresh monolayer of the cell is referred as Primary culture. They are capable of only limited growth in culture and cannot e maintained in serial culture. Primary cell cultures are used during cultivation of viruses for vaccine production.

87. Disadvantages of Primary Cells Some primary cells have got passive antibodies which will prevent viruses from growing. They are very expensive and often difficult to obtain reliable supply.

88. 2. Diploid cell strains: These are cells of a single type that retain the original diploid chromosome number and karyotype(appearance of two sets of chromosome) during serial sub cultivation for a limited number of times. They are also employed for the production of viral vaccine .

89. 3. Continuous cell lines: These are the cells of single type, usually derived from cancer cells, that are capable of continuous serial cultivation indefinitely. Eg . Human cancer cells Viz. He La, Hep – 2 and KB lines have been used for many years. These cell lines are stored in the cold (-700c) for use when necessary. The karyotypeof these cells is aneuploid (a variable multiple of the haploid chromosome number .) Continuous cell lines are now widely useful in cultivating many types of viruses.

90. Figure 6.22

91. Using Cell (Tissue) Culture Techniques Most viruses are propagated in some sort of cell culture The cultures must be developed and maintained Animal cell cultures are grown in sterile chambers with special media Cultured cells grow in the form of a monolayer Primary or continuous

92. Virus detection Viral growth in cell culture can be detected by the following methods a . Cytopathic effect (CPE) Due to the viral growth, morphology of cultured cell change, these changes can be readily observed under microscope. These morphological changers in cell culture is called Cytopathic effect (CPE) and viruses causing CPE are called cytopathogenicvirus .’ Eg . Adenovirus cause large granular clumps in cell culture .

93. Viral detection cont. b. Fluorescence Antibody Technique (FAT ): In this technique, cell from virus infected cultures can be stained by fluorescent conjugated antigen. Fluorescent dye such as fluorescein isothioacynate and rhodamine are generally used to tag with antibodies. FAT is very useful in testing for rabies virus in clinical specimen within few hours with 100% accuracy.

94. c . Haemagglutination : Haemagglutinationis the phenomenon of clumping of RBCs. Viruses such as mumps, measles and influenza can able to agglutinate the RBCs. their presence can be indicated by addition of guinea pig erythrocytes to the culture. If the viruses are cultivated in the cell culture, the erythrocytes will adsorb onto the cell surface also called ‘ haemadsorption Viral detection cont.

95. CPE

96. FLURESCENT ANTIBODY TECHNIQUE

97. HEAMAGLUTINATION

98. Treatment of Animal Viral Infections Because they are not bacteria, antibiotics are ineffective Antiviral drugs block virus replication by targeting one of the steps in the viral life cycle Interferon shows potential for treating and preventing viral infections Vaccines stimulate immunity

99. Introduction to Vaccination Definition of a vaccine A vaccine is a substance that is introduced into the body to prevent infection or to control disease due to a certain pathogen, which is a disease-causing organism, such as a virus, bacteria or parasite. The vaccine “teaches” the body how to defend itself against the pathogen by creating an immune response against the specific pathogen.

100. Definition of a vaccine cont. A vaccine is also defined as a substance that is used to stimulate the body's immune response against a specific pathogen A vaccine is usually best effective when introduced prior in to the living organism prior to the organism being infected by the pathogen such that the body builds immunity against the pseudo-organism Vaccines are usually administered through needle injections, but some can be administered by mouth or sprayed into the nose.

101. Vaccination Vs immunization definition Vaccination:   The act of introducing a vaccine into the body to produce protection from a specific disease. Immunization:   A process by which a person becomes protected against a disease through vaccination. This term is often used interchangeably with vaccination or inoculation .

102. Vaccine types The main types of vaccines that act in different ways are: Live-attenuated vaccines Inactivated vaccines Subunit , recombinant, conjugate, and polysaccharide vaccines Toxoid  vaccines mRNA  vaccines Viral vector vaccines T here is a risk of side effects with all vaccines, but some are less likely to cause side effects than others.t

103. Live-attenuated vaccines These are live version of the germ or virus that causes disease but in weakened state (attenuated) modified in a way that the pathogen is not able to cause disease itself but can produce a robust immune response. Although the pathogen is alive, it is a weakened version that does not cause any symptoms of infection as it is unable to reproduce once it is in the body . Typically , live vaccines lead to a stronger, more prolonged and robust immune response in comparison to inactivated vaccines

104. Live-attenuated vaccines Live-attenuated vaccines can be made to create immunity against viruses or bacteria, but they are more commonly used for viruses. Live-attenuated vaccines trigger an immune response that is similar to what would occur during a natural infection, but the person is not able to pass on the virus to other people and will not become ill with the disease the virus causes.

105. The immunological mechanism of live-attenuated vaccines usually involves a broad immune response including the involvement of CD4+ & CD8+ T lymphocytes (T-cells) as well as antibodies against the pathogen (produced by B-cells). Live-attenuated vaccines usually result in long-term (potentially life-long) protection without requiring additional doses in adulthood .

106. Advantages of live-attenuated vaccines over some other forms of vaccines include the production of a robust, strong antibody and cell-mediated immune response, long-lasting immunity, with a relatively quick onset of action . Disadvantages include include production, maintenance and transport considerations due to the use of live pathogens and the fact that because they are live pathogens it could lead to issues in immune-compromised individuals

107. Considerations and limitations of live-attenuated vaccines Whilst live-attenuated vaccines lead to strong, robust and long-term immunity to viruses (and some bacteria) in most healthy individuals, there may be cases where such vaccines may not be appropriate. Specifically, individuals with compromised immune systems – either due to chemotherapy, HIV infection, or primary disorders of immunodeficiency should not be given live-attenuated vaccines .

108. Limitations cont.. Compared to inactivated vaccines, live-attenuated vaccines also need to be carefully prepared, stored, transported and administered. These normally need to be kept uninterruptedly at cold temperatures, and this may present a challenge to more remote areas of the world or where such facilities do not exist . In summary, live-attenuated vaccines are highly effective and safe vaccines used in the prevention of a variety of viral diseases

109. Examples of live-attenuated vaccines The most common live-attenuated vaccines include : Live-attenuated Influenza (flu) vaccines (LAIV) Measles, Mumps & Rubella (MMR) Polio – nearing global eradication due to mass vaccination Smallpox – officially eradicated due to mass vaccination Chickenpox Yellow Fever Japanese encephalitis Shingles Rotavirus

110. Inactivated vaccine/ Killed vaccine A n inactivated vaccine is a vaccine consisting of virus particles, bacteria, or other pathogens that have been grown in culture and then killed to destroy disease-producing capacity; i n contrast, live vaccines use pathogens that are still alive. Inactivated vaccines usually don't provide immunity (protection) that's as strong as live vaccines. So you may need several doses over time (booster shots) in order to get ongoing immunity against diseases.

111. Advantages of killed vaccines Inactivated pathogens are more stable than live pathogens. Increased stability facilitates the storage and transport of inactivated vaccines . Unlike  live attenuated vaccines , inactivated vaccines cannot revert to a  virulent  form and cause disease. For example, there have been rare instances of the live attenuated form of poliovirus present in the oral  polio vaccine (OPV ) becoming virulent, leading to the inactivated polio vaccine (IPV) replacing OPV in many countries with controlled wild-type polio transmission .

112. Unlike live attenuated vaccines, inactivated vaccines do not replicate and are not contraindicated for  immune-compromised i ndividuals.

113. Disadvantages Inactivated vaccines have a reduced ability to produce a robust immune response for long-lasting immunity when compared to live attenuated vaccines.   Boosters are often required to produce and maintain protective immunity . Pathogens must be cultured and inactivated for the creation of killed whole-organism vaccines. This process slows down vaccine production when compared to  genetic vaccines .

114. Examples include: Viral : Injected polio vaccine (Salk vaccine) Hepatitis A vaccine Rabies vaccine Most influenza vaccines Tick-borne encephalitis vaccine Some  COVID 19 vaccines :  CoronaVac ,  Covaxin ,  QazVac ,  Sinopharm BIBPP   Sinopharm WIBP, TURKOVAC,  CoviVac

115. Bacterial : Injected typhoid vaccine Cholera vaccine Plague vaccine Pertussis vaccine

116. Subunit, recombinant, polysaccharide, and conjugate vaccines Subunit, recombinant, polysaccharide, and conjugate vaccines  use specific pieces of the pathogen —like its protein, sugar, or capsid (a casing around the germ). Because these vaccines use only specific pieces of the pathogen, they give a very strong immune response that's targeted to key parts of the pathogen

117. E xamples of recombinant BCG ( rBCG ) expressing foreign antigens from diverse pathogens have been described, such as  malaria, tuberculosis (TB), HIV, leishmania , pertussis

118. Toxoid vaccines Toxoid vaccines  use a toxin (harmful product) made by the pathogen that causes a disease . They create immunity to the parts of the pathogen that cause a disease instead of the germ itself. That means the immune response is targeted to the toxin instead of the whole germ Toxoid vaccines  use toxoids (as antigens) to induce an immune response in protecting against diseases caused by toxins secreted by specific  pathogen Toxoid vaccines contain a toxin or chemical made by the bacteria or virus. They make you immune to the harmful effects of the infection, instead of to the infection itself. Examples are the  diphtheria and tetanus vaccines .

119. mRNA Vaccines To trigger an immune response, many vaccines put a weakened or inactivated pathogen into our bodies. Not mRNA vaccines. Instead,  mRNA vaccines use mRNA created in a laboratory to teach our cells how to make a protein—or even just a piece of a protein—that triggers an immune response inside our bodies. This immune response, which produces antibodies, is what helps protect us from getting sick from that pathogen in the future.

120. mRNA Vaccines continued After vaccination, the mRNA will enter the muscle cells. Once inside, they use the cells’ machinery to produce a harmless piece of what is called the spike protein. The spike protein is found on the surface of the virus After the protein piece is made, our cells break down the mRNA and remove it, leaving the body as waste .

121. mRNA Vaccines continued Next , our cells display the spike protein piece on their surface. Our  immune system recognizes that the protein does not belong there. At the end of the process, our bodies have learned how to help protect against future infection with the virus that causes COVID-19.

122. mRNA Vaccines continued The benefit is that people get this protection from a vaccine, without ever having to risk the potentially serious consequences of getting sick with COVID-19. Any  side effects from getting the vaccine are normal signs the body is building protection.

123. mRNA Vaccines continued This triggers our immune system to produce antibodies and activate other immune cells to fight off what it thinks is an infection. This is what your body might do if you got sick with COVID-19.

124. Viral vector vaccines Viral vector vaccines use a “vector virus,” which is a harmless, modified version of a virus that does not cause COVID-19 . Viral vector COVID-19 vaccines use a modified version of a different virus (a vector virus) to deliver important instructions to our cells .

125. Viral vector vaccines The vector virus delivers important instructions to our cells on how to recognize and fight the virus that causes COVID-19. Like all vaccines, viral vector vaccines benefit people who get vaccinated by giving them  protection against diseases like COVID-19  without them having to risk the potentially serious consequences of getting sick. J ohnson and Johnson’s Janssen COVID-19 vaccine is a viral vector vaccine

126. How Viral Vector COVID-19 Vaccines Work Viral vector COVID-19 vaccines use a modified version of a different virus (a vector virus) to deliver important instructions to our cells. First, viral vector COVID-19 vaccines are given in a muscle in the upper arm. The vector virus in the vaccine is  not  the virus that causes COVID-19, but a different, harmless virus.

127. How Viral Vector COVID-19 Vaccines Work It enters the muscle cells and uses the cells’ machinery to produce  a harmless piece  of what is called a spike protein. The spike protein is found on the surface of the virus that causes COVID-19. After the spike protein piece is made, our cells break down the vector virus and remove it .

128. How Viral Vector COVID-19 Vaccines Work Next , our cells display the spike protein on their surface. Our  immune system  recognizes that the spike protein does not belong there. This triggers our immune system to produce antibodies and activate other immune cells to fight off what it is an infection. This response is similar to what your body does if you get sick with COVID-19, but is temporary .

129. How Viral Vector COVID-19 Vaccines Work At the end of the immune building process, our bodies have learned how to help protect against future infection with the virus that causes COVID-19. People get this protection from a vaccine, without ever having to risk the potentially serious consequences of getting sick with COVID-19. Any  side effects from getting the vaccine are normal signs the body is building protection.

130. Epidemiology of viral disease Epidemiology is the study of the distribution, the dynamics, and the determinants of diseases in populations. The risk of virus infection and/or clinical disease is determined by characteristics both of the virus, and the levels of innate and acquired resistance in the community.

131. Epidemiology of viral disease Virus transmission is affected by behavioral, environmental, and ecological factors. Knowledge of these factors contributes to evidence-based policy decisions as to how best to control and prevent virus diseases. Terminologies:   Viral shedding, chain, mode and routes of transmission, portal of entry and exit, reseviour

132. Transmission ,terminology Mode of infection How an infectious agent, also called a pathogen, can be transferred from one person, object, or animal, to another . Chain of Infection The chain of infection refers to the series of events that result in a new person (also called a susceptible host) becoming infected with an infectious agent . In epidemiology, a triad model called the chain of infection states that infectious diseases occur because of the interaction between an infectious agent, a host, and their environment.

133. Terminology in Transmission Fomite A  fomite is an object or surface that is capable of transmitting disease and infectious agents.  Fomites  can also be referred to as passive vectors. Fomites can include pens, phones, work surfaces, countertops, tabletops, handrails, and doorknobs. Portal of Entry A portal of entry is how an infectious agent enters a susceptible host. For the pathogen to multiply, the portal of entry has to provide access to tissues. Portals of entry are often the same as the portal of exit in the disease host. This is seen with the influenza virus. Flu exits through the respiratory tract in the host and enters through the respiratory tract in the susceptible host. Other portals of entry include through the skin, mucous membranes, and blood.

134. Terminology in transmission Portal of Exit A portal of exit refers to the path through which an infectious agent leaves a host. The portal of exit usually matches where an infectious agent is found inside a person’s body. For instance, flu viruses leave through the respiratory tract, enterovirus 70, a cause of hemorrhagic conjunctivitis, leaves through secretions from the eyes, and the mite that causes scabies uses skin-to-skin contact as its portal of exit.

135. Terminology in transmission cont. Reservoir In epidemiology, people, animals, objects, or environments carrying an infectious agent are called reservoirs. This is where the infectious agent lives, grows and proliferates. Sexually transmitted diseases, skin conditions, and respiratory illnesses are all found in human reservoirs. However, humans may not always show signs of infection or illness. These types of people are called asymptomatic or passive carriers. Those who can transmit infectious agents before they experience symptoms of infection themselves are called incubatory carriers.

136. Convalescent carriers are people who have experienced illness because of an infectious agent and are still able to transmit it to others. Chronic carriers are people who are capable of transmitting infections to others months or years after they first become infected. Symptomatic carriers are less likely to spread disease, as they are aware of the risks they pose to other people. Asymptomatic carriers are less likely to be careful about who or what they come into contact with, and as such can spread disease unknowingly.

137. . Humans can also be infected by animal reservoirs. Zoonotic diseases are diseases that can be spread from animals to humans under natural conditions and include rabies, anthrax, and SARS . Susceptible Host The susceptible host is the final stage in the chain of infection. There is a complex range of parameters that determine who may be a susceptible host for a certain infectious disease. For instance, a person’s genetic makeup may make them more or less susceptible to disease. They may have specific immunity to a disease due to antibodies from previous infections or vaccination. Socioeconomic factors may also determine how likely it is that someone can become a susceptible host for an infectious disease.

138. The Different Modes of Transmission Contact Contact is the most frequent mode of transmission of health care associated infections and can be divided into: direct and indirect. • Direct: involves direct body surface to body surface contact and physical transfer of microorganism between an infected or colonized person to another person by touch . • Indirect: involves contact between a person and a contaminated object. This is often a result of unclean hands contaminating an object or environment. The microorganism remains on this surface to be picked up by the next person who touches it

139. Direct contact takes place through skin-to-skin contact, as well as kissing and sexual intercourse. However, direct contact does not only refer to contact between humans. Direct contact with contaminated soil is also possible as well as through contact with fomites. Infection through respiratory droplets is a form of direct contact, such as through sneezing, coughing, or talking.

140. Mode of transmission cont.. Direct contact Direct contact

141. Indirect contact prevention

142. 2. Droplets Transmission occurs when droplets containing microorganisms generated during coughing, sneezing and talking are propelled through the air. These microorganisms land on another person, entering that new person’s system through contact with his/her conjunctivae, nasal mucosa or mouth. These microorganisms are relatively large and travel only short distances (up to 6 feet/2 metres ). However these infected droplets may linger on surfaces for long periods of time, so these surfaces (within the range of the coughing/sneezing person) will need additional cleaning. For this reason there may be both Droplet and Contact Precautions required at the same time s s show above

143. Examples of microorganisms that are spread by droplet transmission are: Influenza , C olds , R espiratory syncytial virus (RSV) and some organisms causing pneumonia.

144. 3. Droplet Transmission Droplet transmission can occur when a person comes within 1 meter of an infected person. Infectious diseases can be spread through respiratory droplets released into the air when a person coughs or sneezes at this close proximity . Respiratory droplets can enter the body through the mucosal membranes of the body, and so a susceptible host is at risk of contracting an infectious disease if respiratory droplets come into contact with a susceptible host’s mouth, nose, or eyes.

145. 4. Common Vehicle Common Vehicle Applies to microorganisms that are transmitted by contaminated items such as food, water, medications, medical devices and equipment. To inhibit the transmission of microorganisms by this mode: • Clean patient equipment between uses with different patients • Handle, store and prepare food properly • Careful store and draw up doses of medication from multidose medication vials .

146. 3. Airborne transmission Airborne transmission of infectious agents occurs either by: • Airborne droplet nuclei (small particles of 5 mm or smaller in size) • Dust particles containing infectious agents. Microorganisms carried in this manner remain suspended in the air for long periods of time and can be dispersed widely by air currents. Because of this, there is risk that all the air in a room may be contaminated Some examples of microorganisms that are transmitted by the airborne route are: M. tuberculosis, rubeola , varicella and hantaviruses.

147. ROUTE OF TRANSMISSION OF VIRUSES By direct contact e.g. infection with viruses which are released on the surface of the skin. Viruses which can be transmitted sexually are herpes viruses, HIV and hepatitis B virus. By ingestion of viruses such as enteroviruses , rotaviruses and hepatitis A virus . By inhaling viruses in airborne droplets or dust particles e.g. Influenza viruses, measles virus, adenoviruses, rhinoviruses. By contact with an article such as a floor mat contaminated with a virus that causes eye infection, By a mother infecting her child during pregnancy or birth e.g. cytomegalovirus, rubella virus and HIV. By the bite of an infected mosquito, sandfly and tick e.g. arboviruses . By the bite of an animal host e.g. rabies virus.

148. ROUTE OF TRANSMISSION OF VIRUSES By the man coming into contact with vegetation, food or articles that have been contaminated with the excretions of infected animals especially rodents eg By the direct transfer of viruses from one person to another especially highly infectious viruses such as ebola , Marburg and Lassa fever viruses.

149. Factors that influence the transmission and spread of viral diseases Lack of clean piped water supplies. Inadequate facilities for the disposal of faeces . Poor personal and domestic hygiene. Overcrowding greatly assists in the spread of droplet infections. Dry windy weather helps to spread viruses carried in dust particles. Existence and closeness of animal hosts. The length of time a virus is excreted. The number of carriers in the community. Malnutrition and existing poor health especially when caused by diseases which lead to immune-suppression.

150. Factors cont ………. Unavailability of essential vaccines due to their high cost or lack of cold storage facilities. Houses those are poorly constructed and rodent-infested. The number of different vertebrate and vector hosts.  

151. Viral infection effects on Cells/ cytocidal Cells that support viral replication are called permissive. Infections of permissive cells are usually productive because infectious progeny virus is produced. Most productive infections are called cytocidal ( cytolytic ) because they kill the host cell. Infections of nonpermissive cells yield no infectious progeny virus and are called abortive When the complete repertoire of virus genes necessary for virus replication is not transcribed and translated into functional products the infection is referred to as restrictive ..

152. Viral infection effects on Cells/ cytocidal In persistent and in some transforming infections (oncogenic), viral nucleic acid may remain in specific host cells indefinitely; progeny virus may or may not be produced 1.Cytocidal Infections Infection by cytocidal viruses is usually associated with changes in cell morphology, in cell physiology and sequential biosynthetic events. Many of these changes are necessary for efficient virus replication.

153. a . Morphologic Effects : The changes in cell morphology caused by infecting virus are called cytopathic effects (CPE). Common examples are rounding of the infected cell, fusion with adjacent cells to form a syncytia ( polykaryocytes ), and the appearance of nuclear or cytoplasmic inclusion bodies. Inclusion bodies may represent either altered host cell structures or accumulations of viral components .

154. b . Effects on Cell Physiology :   The interaction of virus with the cell membrane and/or subsequent events , may change the physiological parameters of infected cells, including movement of ions, formation of secondary messengers, and activation cascades leading to altered cellular activities c. Effects on Cell Biochemistry : Many viruses inhibit the synthesis of host cell macromolecules, including DNA, RNA, and protein. Viruses may also change cellular transcriptional activity, and protein-protein interactions, promoting efficient production of progeny virus. For some viruses, specific cellular biochemical functions may be stimulated in order to enhance virus replication .

155. d . Genotoxic Effects : Following virus infection, breakage, fragmentation, rearrangement and/or changes in the number of chromosomes may occur . e. Biologic Effects : Virus-specified proteins may alter the cell's antigenic or immune properties, shape, and growth characteristics.

156. 2. Persistent Infections Some viruses evolved the ability to remain in specific cells for long periods of time. These infections include: latent chronic , and slow virus infections . The type of persistent infection usually influences the extent of cellular changes.

157. a . Latent Infection : Latent infections are characterized by restricted expression of the episomal or integrated virus genome. The viral genomic product(s) are associated with few, if any, changes in the latently infected cell . b. Chronic Infection : The cellular effects of chronic infection are usually the same as those of acute cytocidal infections, except that production of progeny may be slower, intermittent or limited to a few cells. The long-term cellular changes may result in severe disease, immune suppression or may trigger immune responses to damaged, or undamaged cells or tissues.

158. c. Slow Infection : This type of virus-cell interaction is characterized by a prolonged incubation period, without significant morphological and physiological changes of infected cells. A slow progression of cellular injury may take years and is followed by extensive cellular injury and disease .

159. 3. T ransforming Infections (oncogenic ) DNA or RNA tumor viruses may mediate multiple changes that convert a normal cell into a malignant one. RNA tumor viruses usually transform cells to a malignant phenotype by integrating their own genetic material into the cellular genome and may also produce infectious progeny. DNA tumor virus infections are often cytocidal ; thus transformation is associated with abortive or restrictive infections in which few viral genes are expressed .

160. Stages of Transformation : Transformation involves at least two processes: first, the cell gains the capacity for unlimited cell division (immortalization), and second, the immortalized cells acquire additional heritable genetic changes by which the cell is able to produce a tumor in an appropriate host . Mechanisms of Oncogenic Transformation : There are two general patterns by which cell transformation may be accomplished: The tumor virus may introduce and express a so-called transforming gene in the cells or

161. The tumor virus may alter the expression and (or) coding capacity of preexisting cellular genes. After development of a malignant phenotype the relevant segment(s) of the viral genome may or may not be retained in the transformed cells, depending on the mechanism of transformation. These mechanisms are not mutually exclusive, and both may occur in the same cell.

162. Human embryo skin muscle cells were infected with human cytomegalovirus and stained at selected times to demonstrate (A) uninfected cells, (B) late virus cytopathic effects (nuclear inclusions, cell enlargement), (C) cell degeneration, and (D) a focus of infected cells in a cell monolayer (i.e., a plaque), hematoxylin and eosin stain. (A, × 255; B, × 900; C, × 225; D, × 20.)

163. Formation of multinucleated cells The figure represents the cytopathology of measles virus-induced syncytia.

164. Chromosomal aberrations resulting from cytomegalovirus infection of human peripheral blood lymphocytes

165. Clinical Virology Systematic virology/ medically important viruses

166. Site of viral infection

167. Viruses of Medical Importance Virus Family Genome Virus Disease Parvoviridae Ss Parvovirus B19 fifth disease, aplastic crisis Polyomaviridae Ds JC virus progressive multifocal leukoencephalopathy BK virus BK nephropathy in renal transplant patients Merkel cell virus Merkel cell skin carcinoma Papillomaviridae Ds Some human papillomavirus ( eg . Type 16, 18) cervical cancer, penile cancer and oral cancer Adenoviridae Ds Adenoviruses (>50 types) acute respiratory diseases, conjunctivitis, gastroenteritis (type 40 & 41) Poxviridae ds Smallpox virus ( variola ), Vaccinia virus, Molluscum contagiosum virus (MCV), Skin lesions (smallpox, vaccinia, molluscum contagiosum )

168. DNA Viruses That Are Pathogens of Human Diseases Virus Family Genome Virus Diseases Herpesviridae ds Herpes simplex virus 1 & 2 Oral and genital lesions Varicella-zoster virus Chickenpox, shingles Epstein-Barr virus Infectious mononucleosis, associated with human neoplasms ( Burkitt’s lymphoma, Nasopharyngeal carcinoma) Cytomegalovirus can be life-threatening for the immunocompromised Human herpesvirus type 6 & 7 Roseola infantuma (children) Kaposi sarcoma-associated sarcoma virus

169. DNA Viruses That Are Pathogens of Human Diseases Virus Family Genome Virus Diseases Hepadnaviridae ds-RT Hepatitis B virus acute and chronic hepatitis B, associated with hepatocellular carcinoma