Virology.pptx virus classification nand methods slides

virology is the study of virus and also describes the types of virus discuss and classification

1. Virology Dr. Shujaat Ali ( RPh, PhD ) Assistant Professor Virology

2. Introduction: Virus is a Latin word derived from Venom which means “Poison”. S mall infectious agent which caused infection in humans, animals, plants and bacteria . Viruses contain DNA or RNA and a protein coat (Capsid with Capsomeres) that protects the genetic material. Some are enclosed by an envelope Minuscule, Acellular. Obligate intracellular parasites (need living host cells to replicate)

3. Introduction: Cannot carry out any metabolic pathway (No ATP generation system, No ribosomes for protein systhesis ) Neither grow nor respond to the environment. Cannot reproduce independently Some viruses have spikes Most viruses infect only specific types of cells in one host

4. The Discovery of Virus: Edward Jenner first introduced the term “Virus” to microbiology. Virus was first discovered by the Russian microbiologist “Dmitri Ivanosky ” in 1890 and named “Tobacco Mosaic Virus”. In 1935  Wendell Stanley  crystallized tobacco mosaic virus (TMV); an  accomplishment  for which he was awarded a share of the 1946 Nobel Prize in Chemistry

5. Since Dmitri Ivanovsky 's 1892 discovered that the agent which caused tobacco mosaic disease was filterable. He obtained bacteria free filterate from ground up infected plants and placed on the healthy leaves of tobacco. He found that filterate produced the disease in healthy plant. After that the presence of similar filter passing , ultramicroscopic agents was seen in the victims of many disease including foot and mouth disease ( herpes and voricella infections) and yellow fever as well. In 1935 stanley then purified these filterable agent by crystallization and found that causative agent have only nucleic acid and protein in their structure. Hence the agents are simply described as “virus” The Discovery of Virus:

6. Structure of Virus

7. Size of a Virus: Viral size is determined by Electron Microscopy. Most of the viruses are smaller than bacteria but larger viruses (e.g. vaccinia virus) are same in size to very small bacteria (mycoplasmas, rickettsias , chalamydias ) Viruses range from 20nm-1000nm in length.

8. Size of a Virus: “ Microbiology, an Introduction ” by tortora

9. Structure of a Virus: A V irion is a complete, fully developed, infectious viral particle composed of nucleic acid and surrounded by a protein coat outside a host cell. Viruses are classified by their nucleic acid and by difference in the structures of their coat. Thus,  viral nucleic acids  can be DNA or RNA, double-stranded or single-stranded, monopartite or multipartite, linear or circular, as short as 2 kb or up to 2500 kb long.

10. Capsid of a Virus: The nucleic acid of virus is protected by a protein coat called the capsid The structure of capsid is determined by the nucleic acid and the mass of virus Capsid is composed of protein subunits called capsomeres Capsomere may be composed of a single or several types of proteins The arrangement of capsomere is characteristic of particular type of virus

11. Envelope of a Virus: In some viruses, capsid is covered by an envelope consisting of combination of lipids, proteins and carbohydrates. Viruses that lack enveloped are known as non-enveloped or naked viruses . All of the negative stranded RNA viruses are enveloped. It is acquired during the replication in the host cell and is unique to each type of the virus.

12. Envelope of a Virus: The envelop protects the nucleic acid from nuclease enzyme in biological fluids and promotes attachment to host The structural components of envelope remains biologically active only in aqueous medium and are destroyed in acidic medium. Therefore most of the enveloped viruses are usually transmitted through body fluids (blood, respiratory droplets, tissue exudates etc ).

13. Envelope of a Virus:

14. Spikes of a Virus: Viruses may or may not have spikes Spikes are carbohydrate-protein complexes that projects from the surface of the envelope It helps attachment to the host cells. Spikes causes clumping of RBCs. Influenza virus ( haemaglutination )

15. Spikes of a Virus:

16. General Morphology of Virus

17. Viruses are classified on the basis of capsid architecture Electron microscopy and X-Ray Crystallography reveals the shapes of a capsid On the basis of Capsid structure: General Morphology of Virus Helical Viruses Polyhedral Viruses Enveloped Viruses Complex Viruses

18. Helical Virus: The viral genome is found within hollow cylindrical capsid that has a helical structure. Rod like appearance May be rigid or flexible Examples are: Rabies Virus Ebola Virus

19. Polyhedral Virus: The polyhedral virus appears as many sided appearance The virus consists of capsids in the shape of an icosahedron (regular polyhedron with 20 triangular faces 12 corners ) The Capsomere of each face forms an equilateral triangle. Example=Adenovirus

20. Enveloped Virus: The helical or polyhedral virus covered with envelope are called enveloped viruses. Enveloped Helical Viruses i.e. Influenza Virus Enveloped Polyhedral Viruses i.e. Herpes Simplex Virus Enveloped Viruses are roughly spherical.

21. Complex Virus: Bacterial Viruses (Bacteriophage) These viruses possess a capsid that is neither purely helical nor purely icosahedral, and that may possess extra structures such as protein tails or a complex outer wall.

22. Complex Virus: They have a complex structure consisting of an icosahedral head bound to a helical tail, which may have a hexagona l base plate with protruding protein tail fibres . This tail structure acts like a molecular syringe, attaching to the bacterial host and then injecting the viral genome into the cell .

23. Complex Virus:

24. Taxonomy of a Virus: Viral species: A group of viruses sharing the same genetic information and ecological niche (host). Thus Common names are used for viral species e.g. Human Immunodeficiency Virus (HIV). Subspecies are designated by a number e.g. HIV-I The suffix –virus is used for genus names Family name ends in –viridae E.g. Family Herpesviridae , Genus Simplexvirus .

25. Virus Families Families of Viruses that affect human are 1) Single-stranded DNA, Nonenveloped viruses Parvoviridae Human parvovirus Fifth disease Anemia in i mm un o c o m p r o m i s e d patients

26. Virus Families Fifth disease , also  called   Erythema infectiosum , is a mild viral illness that most commonly affects children. It is  called fifth disease  because it is the  fifth  of the five viral rash  diseases  of childhood (the other four being measles, rubella, chicken pox and roseola ).

27. Virus Families 2 ) Double-stranded DNA, Nonenveloped viruses Adenoviridae Mast adenovirus Respiratory infections in human Tumors in animals P apovaviridae Papilloma virus cause tumours

28. Virus Families Adenoviridae Adenoviruses are a group of common viruses that infect the lining of your eyes, airways and lungs, intestines, urinary tract, and nervous system. They're common causes of fever, coughs, sore throats, diarrhea, and pink eye. Infections happen in children more often than in adults, but anyone can get them.

29. Virus Families Papillomavirus , also spelled  papilloma virus , any of a subgroup of  viruses  belonging to the family  Papillomaviridae  that infect  birds  and  mammals , causing  warts  ( papillomas ) and other  benign   tumours , as well as malignant  cancers  of the genital tract and the  uterine cervix  in humans. They are small polygonal viruses containing circular double-stranded  DNA  (deoxyribonucleic acid).

30. Virus Families 3) Double-stranded DNA, E nveloped viruses Poxviridae Orthropoxvirus Smallpox and cowpox Hepadnaviridae Hepadnavirus Hepatitis B, Liver tumor

31. Virus Families 3) Double-stranded DNA, E nveloped viruses Herpesviridae Simplexvirus (HHV1 and HHV 2) Varicellavirus (HHV 3) Lymphocryptovirus (HHV 4) Cytomegalovirus (HHV 5) Roseolovirus (HHV 6) HHV 7 Kaposi's sarcoma (HHV 8 ) (Fever, Blisters, ChickenPox , Burkitt’s lymphoma)

32. Virus Families Kaposi's sarcoma (HHV 8 ) a disease in which cancer cells are found in the skin or mucous membranes that line the gastrointestinal (GI) tract, from mouth to anus, including the stomach and intestines . Burkitt’s lymphoma is an aggressive non-Hodgkin B-cell lymphoma .

33. Virus Families Non-Hodgkin  B-cell lymphoma D iffuse large B-cell lymphoma is the most common type of non-Hodgkin lymphoma. It grows quickly in the lymph nodes and often the spleen, liver, bone marrow, or other organs are also affected. Signs and symptoms of diffuse large B-cell lymphoma may include fever, drenching night sweats, and weight loss.

34. Virus Families 4 ) Single-stranded RNA, + strand Nonenveloped viruses Picornaviridae (largest groups of the viral classification) Enterovirus Rhinovirus (common cold) Hepatitis A virus Caliciviridae Hepatitis E virus Norovirus (Norwalk agent) causes gastroenteritis.

35. Virus Families Norovirus infection can cause the sudden onset of severe vomiting and diarrhea. The virus is highly contagious and commonly spread through food or water that is contaminated during preparation or through contaminated surfaces. You can also be infected through close contact with an infected person. Diarrhea, stomach pain and vomiting typically begin 12 to 48 hours after exposure. 

36. Virus Families 5) Single-stranded RNA, + strand E nveloped viruses Togaviridae Alpha virus Rubivirus (rubella virus) Flaviviridae Hepatitis C virus Flavivirus Arbovirus

37. Virus Families 5) Single-stranded RNA, + strand E nveloped viruses Coronaviridae Coronavirus Upper respiratory tract infections

38. Virus Families 6 ) Single-stranded RNA, - strand, one RNA Strand Rhabdoviridae Vesiculo virus Lyssavirus (rabies virus ) Filoviridae Filovirus (Ebola Virus)

39. Virus Families 6 ) Single-stranded RNA, - strand, one RNA Strand Paramyxoviridae Paramyxovirus (mumps virus) Morbillivirus (measles virus ) Deltaviridae Hepatitis D virus

40. Virus Families 7) Single-stranded RNA, - strand, multiple RNA Strands Orthomyxoviridae Influenzavirus (A, B and C) Enveloped Spikes can agglutinate RBCs. Bunyaviridae Hantavirus Haemorrhagic fever

41. Virus Families 7) Single-stranded RNA, - strand, multiple RNA Strands Arenaviridae Arenavirus VEE ( Venezuelan equine encephalitis ) and Lassa Fever ( viral hemorrhagic fever)

42. Virus Families 8 ) Single-stranded RNA, p roduce DNA Retroviridae Lentivirus (HIV) Oncoviruses (Leukemia)

43. Virus Families 9) Double-stranded RNA, Nonenveloped Reoviridae Reovirus (Also known as Respiratory ) The virus may also be referred to as enteric cytopathic bovine orphan virus (ECBO). It is endemic in cattle populations worldwide, and although normally fairly nonpathogenic, it can cause reproductive, respiratory, or enteric disease – particularly when the animal is concurrently infected with another pathogen. Rotavirus ( Mild respiratory infections and gastroenteritis) Colorado tick fever

44. Note: There are no viruses known with circular dsRNA genomes

45. The process of viral replication is one of the most remarkable events in nature. A virus invades a living host cell a thousand or more times its size, hijacks the metabolism of the cell to produce copies of itself, and often destroys the host cell when new virions are released. Viral Replication and Its Control

46. N egative-sense strand  of DNA is equivalent to the template strand, whereas the , Positive-sense strand is the non-template strand whose nucleotide sequence is equivalent to the sequence of the mRNA transcript.

47. One of the best studied processes of replication is that carried out by bacteriophages of the T-even group (T for “type”). Bacteriophages T2, T4, and T6 are in this group. They are large, complex, naked DNA virions with the characteristic head and tail of bacteriophages . Viral Replication and Its Control

48. Viral Replication and Its Control

49. 2. Penetration . Following attachment, the tail of the phage releases lysozyme , an enzyme that dissolves a portion of the bacterial cell wall. The tail sheath then contracts and the tail core drives through the cell wall. As the tip of the core reaches the cell membrane below, the DNA is ejected through the hollow tail core and on through the cell membrane into the bacterial cytoplasm. The ejection process takes less than two seconds and the capsid remains outside. Viral Replication and Its Control

50. 3. Biosynthesis. Having entered the cytoplasm, production of new phage genomes and capsid parts begins. As phage genes code for the disruption of the host chromosome, the phage DNA uses bacterial nucleotides and enzymes to synthesize multiple copies of its genome. Messenger RNA molecules transcribed from phage DNA appear in the cytoplasm, and the biosynthesis of phage enzymes and capsid proteins begins. Bacterial ribosomes , amino acids, and enzymes are all enlisted for biosynthesis. Because viral capsids are repeating units of capsomeres , a relatively simple genetic code can be used over and over. Viral Replication and Its Control

51. 4. Maturation. Once the phage parts are made, they are assembled into complete virus particles. The enzymes encoded by viral genes guide the assembly in step-by-step fashion. In one area of the host cytoplasm, phage headsand tails are assembled from protein subunits; in another area, the heads are packaged withDNA ; and in a third area, the tails are attached to the heads.

52. 5. Release. Mature phage particles now burst out from the ruptured bacterial shell. For some phages, lysozyme , encoded by the bacteriophage genes late in the replicative cycle, degrades the bacterial cell wall. The mature bacteriophages are set free to infect more bacterial cells.

53. ANIMAL VIRUS REPLICATION

54. It is a specific binding between viral capsid proteins and specific receptors on the host cellular membrane. This specificity determines the host range of a virus. For example, HIV infects a limited range of human leucocytes . This is because its surface protein, gp120 on CD4 receptor cells. This mechanism has evolved to favors those viruses that infect only cells in which they are capable of replication. Attachment to the receptor can induce the viral envelope protein to undergo changes that results in the fusion of viral and cellular membranes with the help of spikes. Attachment

55. Virions enter the host cell through receptor-mediated endocytosis or membrane fusion . This is often called viral entry . In some case the viral envelope fuses with the plasma membrane and release the nucleocapsid into the cytoplasm and in other case the whole enveloped virion enter the cell. Penetration

56. It is a process in which the viral capsid is removed: This may be by degradation by viral enzymes or host enzymes or by simple dissociation; the end-result is the releasing of the viral genomic nucleic acid. Uncoating

57. Now this process diverges once again because some viruses contain DNA and some contains RNA. The DNA of DNA viruses supplies the genetic codes for enzymes that can synthesize viral parts. RNA virus follows a slightly different pattern. The RNA can be act as m RNA molecule and immediately supplying the code for protein synthesis such virus is said to have sense and called positive stranded RNA virus or sense virus (e.g polio virus). Replication

58. In the other RNA viruses however the RNA is used as a template to synthesize a complementary strand of RNA. The original strand is said to have antisense and virus is said to be antisense virus or negative strand RNA virus ( e.g Measles virus). Usually the RNA polymerase enzyme is present in the virus to synthesize the complementary strand.

59. Other RNA virus called retro virus has a particularly interesting method of replication. Retro virus carry their own enzyme called reverse transcriptase. The enzyme use the viral RNA as a template to synthesize single –stranded DNA ( the term reverse transcription). Once formed the DNA serves as a template to form a complementry DNA strand. The viral RNA Is then destroyed, and the two DNA strands twist around each other to form a double helix.

60. The DNA now migrates to a cell nucleus and integrates into one of the host cell`s chromosomes, where it is known as provirus from this position the DNA encoded the new retroviruses. Whenever the host divides, the viral genome is also replicated. The viral genome is mostly silent within the host; however, at some point, the provirus may give rise to active virus, which may release from the host cells. Enveloped viruses (e.g., HIV) typically are released from the host cell by budding . During this process the virus acquires its envelope, which is a modified piece of the host's plasma or other, internal membrane.

61. Entry by direct fusion

62. Fig. 19-7 Capsid RNA Envelope (with glycoproteins) Capsid and viral genome enter the cell HOST CELL Viral genome (RNA) Template mRNA ER Glyco- proteins Capsid proteins Copy of genome (RNA) New virus

63. Fig. 19-8a Glycoprotein Reverse transcriptase HIV RNA (two identical strands) Capsid Viral envelope HOST CELL Reverse transcriptase Viral RNA RNA-DNA hybrid DNA NUCLEUS Provirus Chromosomal DNA RNA genome for the next viral generation mRNA New virus

64. Fig. 19-8b HIV Membrane of white blood cell HIV entering a cell 0.25 µm New HIV leaving a cell

65. SUNARYATI VIRAL LIFE CYCLE ATTACHMENT PENETRATION HOST FUNCTIONS ASSEMBLY (MATURATION) Transcription REPLICATION RELEASE UNCOATING Translation MULTIPLICATION Click after each step to view process

66. 1. Attachment 2. Penetration. 3. and 4. Biosynthesis and Maturation. Animal Virus Replication Often Results in a Productive Infection

67. Viruses may be inactivated by many of the physical and chemical agents. Few methods for inactivating viruses are described below: Formaldehyde reacts with free amino groups on viral genome and modify it and prevent its replication. Heat alters structure of viral proteins and nucleic acids, causing them to unfold and denature. INACTIVATION OF VIRUSES :

68. c) Ultraviolet light inactivates viruses by stimulating adjacent thymine or cytosine bases on DNA molecules to bind together and form pairs called dimers . The dimers twist the molecule out of shape , and distorted viral genome cannot replicate . d) X-rays cause breaks in the sugar phosphate backbone of nucleic acid. e) Lipids solvents such as ether, chloroform and detergents, all of which dissolve the lipid in the envelope of viruses and inactivate them .

69. f ) Heavy metal compounds such as mercury and silver derivatives inactivate viruses.

70. Viruses are obligate intracellular parasites so they depend on host for their survival. They cannot be grown in non-living culture media or on agar plates alone, they must require living cells to support their replication. CULTIVATION OF VIRUSES

71. The primary purpose of virus cultivation is: To isolate and identify viruses in clinical samples. To do research on viral structure, replication, genetics and effects on host cell. To prepare viruses for vaccine production. purpose of virus cultivation

72. Following techniques are commonly used for Cultivation of viruses : Animal Inoculation Inoculation into embryonated egg Cell Culture Techniques of cultivation:

73. Viruses which are not cultivated in embryonated egg and tissue culture are cultivated in laboratory animals such as mice, guinea pig, hamster, rabbits and primates are used. The selected animals should be healthy and free from any communicable diseases. Suckling mice(less than 48 hours old) are most commonly used. Suckling mice are susceptible to toga virus and coxsackie virues ( Picronavirus ), which are inoculated by intra-cerebral and intra-nasal route. 1)Animal Inoculation

74. Viruses can also be inoculated by intraperitoneal and subcutaneous route. After inoculation, virus multiply in host and develops disease. The animals are observed for symptoms of disease and death. Then the virus is isolated and purified from the tissue of these animals. Live inoculation was first used on human volunteers for the study of yellow fever virus. 1)Animal Inoculation

75. A dvantages of Animal Inoculation Diagnosis, Pathogenesis and clinical symptoms are determined. Production of antibodies can be identified. Primary isolation of certain viruses. Mice provide a reliable model for studying viral replication. Used for the study of immune responses, epidemiology and oncogenesis .

76. Disadvantages of Animal Inoculation Expensive and difficulties in maintenance of animals. Difficulty in choosing of animals for particular virus Some human viruses cannot be grown in animals,or can be grown but do not cause disease. Mice do not provide models for vaccine development. It will lead to generation of escape mutants Issues related to animal welfare systems.

77. 2. Inoculation into embryonated egg

78. Good pasture in 1931 first used the embryonated hen’s egg for the cultivation of virus. The process of cultivation of viruses in embryonated eggs depends on the type of egg which is used. Viruses are inoculated into chick embryo of 7-12 days old. For inoculation, eggs are first prepared for cultivation, the shell surface is first disinfected with iodine and penetrated with a small sterile drill. After inoculation, the opening is sealed with gelatin or paraffin and incubated at 36°c for 2-3 days. After incubation, the egg is broken and virus is isolated from tissue of egg. 2. Inoculation into embryonated egg

79. Viral growth and multiplication in the egg embryo is indicated by the death of the embryo, by embryo cell damage, or by the formation of typical pocks or lesions on the egg membranes Viruses can be cultivated in various parts of egg like chorioallantoic membrane, allantoic cavity, amniotic sac and yolk sac.

80. Advantages of Inoculation into embryonated egg Widely used method for the isolation of virus and growth. Ideal substrate for the viral growth and replication. Isolation and cultivation of many avian and few mammalian viruses. Cost effective and maintenance is much easier. Less labor is needed. The embryonated eggs are readily available. Sterile  and wide range of tissues and fluids They are free from contaminating bacteria and many latent viruses. Widely used method to grow virus for some vaccine production.

81. Disadvantages of Inoculation into embryonated egg The site of inoculation varies with different virus. That is, each virus have different sites for their growth and replication.

82. There are three types of tissue culture; organ culture, explant culture and cell culture. Organ cultures  are mainly done for highly specialized parasites of certain organs e.g. tracheal ring culture is done for isolation of coronavirus. Explant culture  is rarely done. Cell culture  is mostly used for identification and cultivation of viruses. Cell culture is the process by which cells are grown under controlled conditions. Cells are grown in vitro on glass or a treated plastic surface in a suitable growth medium. At first growth medium, usually balanced salt solution containing all essential nutrients . On incubation the cell divide and spread out on the glass surface. 3. Cell Culture (Tissue Culture)

83. Advantages of cell culture Tissue culture technique is most widely used method for isolation and propagation of viruses from clinical material. Relative ease, broad spectrum, cheaper and sensitivity. Tissue culture techniques have an advantage over embryo culture vaccines in minimizing the possibility of patients developing hypersensitivity to egg albumin.

84. Cultivation of virus

85. Cultivation in embryonated egg Virus injected into appropriate region Method widely used for production of vaccines SUNARYATI

86. SUNARYATI

87. SUNARYATI Cultivation in cell cultures

88. Cytopathic effect - normal monolayer structure is disrupted by viral infection Cell lines developed from embryonic tissue Continuous cell lines (immortal) - HeLa Maintenance of cell culture lines is technically difficult; must be kept free of microbial contamination. SUNARYATI Cultivation in cell culture