PMB 213.VIRUS.docx

**VIRUSES: FROM PHARMACEUTICAL MICROBIOLOGY PERSPECTIVE.(PMB 213)** [Objectives] 1. History on origin of Viruses 2. Reasons for studying viruses 3. Characteristics 4. Definition of viruses: take note of key words 5. **Replication of viruses** 6. Diagram of viruses 7. Classification of...

**VIRUSES: FROM PHARMACEUTICAL MICROBIOLOGY PERSPECTIVE.(PMB 213)** [Objectives] 1. History on origin of Viruses 2. Reasons for studying viruses 3. Characteristics 4. Definition of viruses: take note of key words 5. **Replication of viruses** 6. Diagram of viruses 7. Classification of virus. 8. Types of viruses: 9. Understand of this microorganism differ from other organisms Bacteria, prions and fungi 10. Bacterial and viral interactions 11. Fungus and viral interactions 12. Possible outcomes of Host cell and viral interactions 13. Test used in identifying Virus in and host **tissue culture techniques** **History on origin of Viruses** A prominent Russian botanist Dimitri Ivanovsky published the first report showing that the agent causing tobacco mosaic disease passed through porcelain filters and could not be seen or cultivated. This finding was later proved by a Dutch microbiologist Martinus Beijerinck, that the filterable tobacco mosaic agent (toxin) was a novel type of microorganism that he termed "a contagious living fluid. As a result, three universal theories have been adopted on the origin of viruses. **The first virus hypothesis (Independent entities theory)** In this theory, RNA viruses are thought to have been descendants of the RNA world and the DNA viruses evolved later from RNA. **The second virus hypothesis (Reduction/degenerate/ regressive evolution theory)** This means that cells of unicellular organisms lost their membranes, cell walls and other structures as well as their cytoplasm, hence retaining only the nucleic acid and some vital proteins in order to live in other cells as parasites. **The third virus hypothesis (The escape theory)**. Plasmids and mobile genetic elements are extracellular genetic materials capable of exit and entry into the genomes of other cells. **Definition of viruses** A **virus** is a ultramicroscopic [infectious agent](https://en.wikipedia.org/wiki/Infectious_agent) that replicates only inside the living [cells](https://en.wikipedia.org/wiki/Cell_(biology)) of an [organism](https://en.wikipedia.org/wiki/Organism). Viruses infect all [life forms](https://en.wikipedia.org/wiki/Life_forms), from animals and plants to [microorganisms](https://en.wikipedia.org/wiki/Microorganism), including [bacteria](https://en.wikipedia.org/wiki/Bacteria) and [archaea](https://en.wikipedia.org/wiki/Archaea). Viruses are found in almost every [ecosystem](https://en.wikipedia.org/wiki/Ecosystem) on Earth and are the most numerous type of biological entity **Characteristics** 1. They are all potentially infectious 2. Presence of single nucleic acid 3. Incapability to grow 4. Reproduction from the genetic material OF HOSTonly 5. Absence of enzymes for energy metabolism 6. Absence of ribosomes 7. Absence of information for the production of enzymes in the energy cycle 8. Absence of information for the synthesis of ribosomal proteins 9. Absence of information for the synthesis of ribosomal RNA and soluble tRNA **Differences among viruses, bacteria, fungi and prions.** Fungus vs bacteria vs virus table +-----------------------+-----------------------+-----------------------+ | Feature | Virus | Prion | +=======================+=======================+=======================+ | Disease: | Depend on the virus | Very Long | | | | | | a. Incubation time | Yes | No | | | | | | b. Immune response | Yes | No | | | | | | c. Cytopathologic | Yes | No | | effect | | | | | | | | d. Inflammatory | | | | response | | | +-----------------------+-----------------------+-----------------------+ | Disinfection by: | Yes | No | | | | | | a. Formaldehyde | Most are affected | No | | | | | | b. Heat at 80^o^C | Yes | No | | | | | | c. Ionizing and uv | | | | radiations | | | +-----------------------+-----------------------+-----------------------+ | Presence of Nucleic | Yes | No | | acid | | | +-----------------------+-----------------------+-----------------------+ | Specific Morphology | Yes | No | +-----------------------+-----------------------+-----------------------+ | Disease condition | Not specific | The brain. | +-----------------------+-----------------------+-----------------------+ | Filterable | Yes | Yes | +-----------------------+-----------------------+-----------------------+ **Application of Viruses: the benefits of the study of virus.** 1. viruses in research has helped our understanding of the basics of molecular genetics, such as DNA replication, transcription, RNA processing, translation, protein transport, and immunology. 2. Phages are often used as vectors to genetically modify bacteria. 3. viral therapy uses viruses to genetically modify diseased cells and tissues. Scientists have focused on gene therapy for diseases caused by single-gene defects, such as cystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia. In gene therapy, the correct version of the gene is introduced to human cells by using a viral vector such as the adenovirus 4. gene therapy and in the treatment of cancer. Gene therapy is the insertion of genes into a person's cells and tissues to treat a disease. In the case of a genetic disease, the defective gene is replaced with a working gene. Although the technology is still new, it has been used with some success. 5. Oncolytic viruses are viruses that lyse and kill cancer cells. Some researchers are hoping to treat some cancers with these viruses. 6. industrial production: expression of heterologous proteins by viruses is used for the production of various pharmaceutical proteins, vaccine antigens and antibodies 7. The use of viruses in materials science and nanotechnology. **Classification of virus.** Basis of Classification The following properties, listed in the order of importance, have been used as a basis for the classification of viruses: 1\. Nucleic acid type: RNA or DNA, single-stranded or doublestranded i.e. strategy of replication. 2\. Size and morphology including type of symmetry, number of capsomeres and presence of membranes. 3\. **Susceptibility to physical and chemical agents.** 4\. Immunologic properties. 5\. Natural method of transmission 6\. Host, tissue and cell tropism. 7\. Pathology, including inclusion body formation. 8\. **Symptomatology.** **Some aspects of susceptibility to physical and chemical agents**. **Disinfectants.** The viruses are usually more resistant than bacteria to chemical disinfectants. Most viruses are relatively resistant to phenol. The oxidizing agents, such as hydrogen peroxide, potassium permanganate, hypochlorite, and organic iodine compounds, are most active antiviral disinfectants. Formaldehyde and b-propiolactone are also active virucidal agents, which arecommonly used for preparation of killed viral vaccines. The chlorination of drinking water is useful for killing most of the common viruses with exception of hepatitis A and polioviruses. These two viruses are relatively resistant to chlorination. **Temperature.** Most of the viruses with few exceptions are highly heat labile. They are inactivated within seconds at 56°C, within minutes at 37°C, and within days at 4°C.The viruses such as influenza, measles, and mumps are very labile and may survive outside the host only for a few hours. Other viruses, such as polio and hepatitis A, are relatively much stable and may survive for many days, weeks, or even months in the environment.Viruses, such as hepatitis B, show resistance to heating at 60°C for 60 minutes;The viruses are stable at low temperature. They can be stored by freezing at 235°C or 270°C. Lyophilization or freeze-drying is useful for long-term storage of the viruses. The poliovirus is an exception, as it does not withstand freeze-drying. **PH** The viruses usually remain viable in a pH range of 5--9, but are sensitive to extremes of acidity and alkalinity. Rhinoviruses are very susceptible to acidic pH, while enteroviruses are highly resistant. **Lipid solvents** Ether, chloroform, and detergents are active against enveloped viruses but are not active against nonenveloped, naked viruses. **Radiation** The viruses are readily inactivated by sunlight, ultraviolet (UV) radiations, and ionizing radiations. **Some aspect in Symptomatology of viral particles** The oldest classification of viruses is based on the diseases they produce, and this system offers certain conveniences for the Pharmacist and clinician. - Generalized Diseases: Disease in which virus is spread throughout the body via the bloodstream and in which multiple organs are affected. These include smallpox, vaccinia measles, rubella, chickenpox, yellow fever, dengue, enteroviruses, and many others. - Diseases Primarily Affecting Specific Organs: The virus may spread to the organs through the bloodstream, along the peripheral nerves or by other routes. a. Diseases of the nervous system -- poliomyelitis, aseptic meningitis (polio -- coxsackie -- and echoviruses), rabies, arthropod-borne encephalitides, lymphocytic chromeningitis, herpes simplex meningoencephalitis of mumps, measles, vaccinia and "slow" virus infections. b. Diseases of the respiratory tract -- Influenza, parainfluenza, RJ pheumonial and bronchiolitis, adenovirus pharyngitis, common cold (caused by many viruses). c. Localized diseases of the skin or mucous membranes -- Herpes simplex, molluscum contagiosum, warts, harpangina, herpes zoster and others. d. Diseases of the eye-Adenovirus conjunctivitis, New-castle virus conjunctivitis, herpes kerato conjunctivitis and epidemic hemorrhagic conjunctivitis (enterovirus -- 70). e. Diseases of the Liver -- Hepatitis type A (infectious hepatitis) and type B (serum hepatitis), yellow fever. In neonate, enteroviruses, herpes v is used and rubella virus. f. Diseases of the salivary of glands -- mumps and cytomegalovirus. g. Diseases of gastrointestinal tract -- Gastroenteritis A virus and gastroenteritis B virus (rotavirus), poliovirus, Hepatitis A, Hepatitis B. **Replication of virus** Replication of Viruses Viruses can multiply only in active host cells, the replicate cycle of viruses can be divided into number of stages: 1. Attachment to surface receptors on the susceptible host cell. 2. Entry in to the cell. 3. Uncoating of viral nucleic acid. 4. Replication of viral nucleic acid and synthesis of virus encoded proteins. 5. Assembly of newly-formed virus particles and release from host cell. **Attachment to surface receptors on the susceptible host cell**: Virions must first attach to cell surface receptors in order to produce infection, this attachment depend on number of the virus particles present and the availability of appropriate receptors molecules, viruscell interaction determines both the host range and tissue tropism of viral species. Viruses can select a point where they can utilize the wide range of host cell surface proteins as receptors. Some viruses have more than on type of ligand molecules and they may bind to several surface receptors proteins surface receptors in sequential order during attachment. **Entry in to the cell:** Virus penetration in an energy depended process when can occur in number of steps:- a. receptors-mediated endocytosis occur after virus attaches to receptors and particular sites on the plasma membrane, this virus receptors complex is taken in to the cell in specialized vesicles, then by acidification within the vesicles lead to degradation of viral structures. b. In enveloped viruses (Paramyxovirus, Retrovirus and Herpesvirus), involves fusion of the viral envelop with the plasma membrane , this allows release of the nucleocapsid directly into the host cells cytoplasm. c. Direct introduction or translocation of viral genomes into the cytoplasm through channels in the plasma membrane e.g. non-enveloped viruses (Picornavirus). **Uncoating of viral nucleic acid**:- Uncoating means:- the process where by the viral genome is released in a form suitable for transcription. e.g. a. In enveloped viruses: nucleocapsid is discharged directly into the cytoplasm, transcription can usually proceed without complex uncoating b. In non- enveloped virus uncoating may results from lysosomal proteolytic enzymes activity. **Replication of viral nucleic acid and synthesis of virus encoded proteins**: a. replication of DNA virus:- Double stranded DNA viruses (Herpis, Para, Adenovirus) which replicate in the nucleus of the cell have relatively direct replication strategy, the viral DNA in transcripted by cellular DNA-depended RNA polymerase (transcriptase) to form messenger RNA. b. replication of RNA virus:- Double stranded RNA virus (Reo, Birnavirus) transcription occur in the cytoplasm by viral transcriptase, which produced by ribosome of infected cells after viral RNA binding. In positive sense single strand RNA viruses it\'s RNA can act directly as messenger RNA after infection. c. according to viral gene code deferent types of proteins are produced inside infected cells by it\'s ribosomes membrane proteins and glycol proteins are synthesized on membrane bounded ribosomes while soluble proteins including enzymes and synthesized on ribosomes free in cytoplasm. Short specific amino acid sequences, known as sorting sequences facilitate the incorporation of proteins of various cellular location where they are required for metabolic activity. **Assembly of newly-formed virus particles and release from host cell**: Procapsid were formed by incorporation of viral N.A. in structural formed proteins according to the symmetry of original infected virus. Non-enveloped viruses are usually released following cellular disintegration. The assembly of Picorna and Reovirus occur in the cytoplasm of the cell where as Parvo, Adeno and Papova are assembled in nucleus. Enveloped viruses released by budding form infected cell membrane at that time the viral enveloped was formed by cell membrane modification through the insertion of virus-specified transmembrane glycoproteins but some those enveloped viruses like Toga, Paramyxoand Rhabdo viruses are cytolytic in there releases. Flavi, Corona, Artiri and Bunya virus acquire there envelops inside cell by binding through the membrane of the rough endoplasmic reticulum or Golgi apparatus. Herpes viruses which replicate in the nucleus they bud from nuclear membrane. **Bacterial and viral interactions.** Bacterial viruses, or bacteriophages, are viruses that only infect bacteria. Since viruses don't have the organelles needed to replicate their genes, they reproduce within the host cells they infect. There are two ways that bacteriophages can reproduce within the host bacteria: the lytic cycle and the lysogenic cycle. **The Lytic Cycle** The lytic cycle, also referred to as the "reproductive cycle", is a six-stage process in which bacteriophages replicate inside a host cell. The process of reproduction occurs as follows: a. the bacteriophage uses its tail fibers to latch on the surface proteins located on the outside of a bacterial cell. It then contracts its tail, injecting the phage DNA/RNA into the host cell and leaving the empty capsid that remains outside. b. By making use of the organelles in the cell, the phage DNA/RNA takes command and arranges the production of proteins and copies of the genome using the host and viral enzymes. Since each phage copy is made up of head, tail, and tail fibers, three separate types of proteins assemble to create each separate component. c. During the formation of the head, the genome is placed inside the capsid. Lastly, the phage produces genes to make an enzyme, endolysin, that damages the cell wall of the host cell, allowing fluid from the outside. d. The entry of excessive amounts of fluid leads to the swelling of the cell. e. Eventually, the cell lyses and releases all the phage particles that were constructed to further infect new hosts. **The Lysogenic Cycle** The lysogenic cycle is the other form of reproduction used by bacteriophages. The main component that makes the lysogenic cycle different from other forms of replication is that it doesn't necessarily end with the lysis of the host cell. - The process first starts with the bacteriophage latching onto the surface of the host cell and inserting its DNA/RNA inside. - Instead of commandeering the production of more viruses, the DNA/RNA of the phage is combined with a particular section of the bacterial genome. This leads to the integration of phage DNA/RNA into the genome of the bacterium, also known as the prophage. - As the bacterium reproduces normally, the prophage is replicated as well and is transmitted to bacterial daughter cells. - As time passes, a large population of bacteria infected with the prophage is produced. - Occasionally, the prophage exits the chromosome of the bacterium and allows the initiation of the lytic cycle. The process of using both the lysogenic and lytic cycles allows the replication of more viruses in a shorter amount of time.\ \ These knowledge gave birth to gene therapy.The principles of phage therapy have potential applications not only in human medicine, but also in dentistry, veterinary science, food science and agriculture. - An important benefit of phage therapy is derived from the observation that bacteriophages are much more specific than most antibiotics that are in clinical use. - Theoretically, phage therapy is harmless to the eucaryotic host undergoing therapy, and it should not affect the beneficial normal flora of the host. - Phage therapy also has few, if any, side effects, as opposed to drugs, and does not stress the liver. Since phages are self-replicating in their target bacterial cell, a single, small dose is theoretically efficacious. On the other hand, this specificity may also be disadvantageous because a specific phage will only kill a bacterium if it is a match to the specific subspecies. Thus, phage mixtures may be applied to improve the chances of success, or clinical samples can be taken and an appropriate phage identified and grown. - Phages are currently being used therapeutically to treat bacterial infections that do not respond to conventional antibiotics. - They are reported to be especially successful where bacteria have constructed a biofilm composed of a polysaccharide matrix that antibiotics cannot penetrate. **Fungus and viral interaction** - Mycoviruses (also known as mycophages) are viruses that infect fungi. Mycoviruses are viruses that selectively infect fungi. Although the first reports on mycoviruses date from the 1960s **Effects of viral activities on Host cell.** Effects of viral infection on the host cell Viral invasion in the host cell leads to viral replication, causing either destruction (lyses), changed (altered) function, or proliferation of the host cell. The most important effects as follow 1. Cytopathic effects ( CPE) The viral effect causes lyses of the host cells by lytic viruses, which appears as plaques (clear zones) as a result of the lyses of the host cells, that can be seen by naked eye or by light microscope (Bacteriophages) 2. Inclusion body formation Inclusion bodies may be formed during the replication of some viruses in the cytoplasm or nucleus of the host cells. These bodies may be considered to be colonies of mature viroins or any cellular material be produced as a results of viral infection. The presence of these inclusions helps for identification (diagnosis) of some viral infection (i.e. infection by small pox, poliomyelitis. 3. Cell fusion -Cell fusion Some viral infections cause fusion of the infected host cells. This fusion happen when the infection with the some enveloped viruses such as Paramyxoviruses and Herpesviruses. 4. Changing of surface host cells receptors Viral infection changes the cell membrane or cytoplasmic membrane components (protein), by fusion of viral protein with cell membrane protein that causes the induction of the body host to form humoral immunity against specific viruses. 5. Interferon production Interferon is a protein substance, produced and released after viral infection, that cases protection of the non infected cells from the viral reinfection of the same virus or other. **CULTIVATION OF VIRUSES** Isolation and Cultivation of Viruses Viruses are obligate intracellular parasites so they depend on the 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. Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate). The primary purpose of virus cultivation is: (Why we need to cultivate viruses?) a\. To isolate and identify viruses in clinical samples. b\. To prepare vaccine and antigen c\. To study viral expression and replication (viral structure, genetics). d\. To study the pathogenesis of viral diseases and of viral oncogenesis (effects on a host cell). Cultivation of viruses can be discussed under the following headings: 1\. Inoculation into embryonated egg 2\. Animal Inoculation 3\. Cell Culture **Inoculation into embryonated egg**: 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 37°c for 2-4 days.After incubation, the egg is broken and the virus is isolated from tissue of egg. Viral growth and multiplication in the egg embryo are 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 cavity andYolk sac. Isolation and Cul **Animal Inoculation**.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.Mice are still the most widely used animals in virology. Mice can be inoculated through several routes, i.e. intracerebral, subcutaneous, intraperitoneal, and intranasal.  - 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 the 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 mediumAt first growth medium, a usually balanced salt solution containing 13 amino acids, sugar, proteins, salts, calf serum, buffer, antibiotics, and phenol red are taken and the host tissue or cell is inoculated. 4. On incubation, the cell divide and spread out on the glass surface to form a confluent monolayer 5. Incubation of cell culture tubes in a stationary rack can be used in place of a roller drum. To counteract the pH decrease, a bicarbonate buffering system is used in the culture medium to keep the cells at physiologic pH (pH 7.2). Phenol red a pH indicator that is red at physiologic, yellow at acidic and purple at alkaline once inoculated with the specimen, cell cultures are incubated for (1-4) weeks depending on the viruses suspected. Periodically the cells are inspected microscopically for the presence of the virus, indicated by areas of dead or dying cells called cytopathic effect (CPE). There are three basic types of cell cultures: - Primary cell cultures:These are normal cells derived from animal or human cells.They are able to grow only for limited time and cannot be maintained in serial culture (5-10 passage in culture). They are used for the primary isolation of viruses and production of the vaccine.Examples: - Monkey kidney cell culture, - Human amnion cell culture, - Heterogeneous population of cells. - Human embryonic lung strain, - Rhesus embryo cell strain, - Homogeneous population of cells. Continuous cell lines (Heteroploid cultures): They are derived from cancer cells. They can be serially cultured indefinitely so named as continuous cell lines. They can be maintained either by serial subculture or by storing in deep freeze at -70°c.Due to derivation from cancer cells, they are not useful for vaccine production, while its importance lies in the identification and cultivation of the virus. Examples: - HeLa (Human Carcinoma of cervix cell line), - HEP-2 (Human Epithelioma of larynx cell line), - Vero (Vervet monkey) kidney cell lines, BHK-21 (Baby Hamster Kidney Illustration of in-vitro culture techniques for spermatogenesis \...