MicroPara Virus Notes (Sir Bong) PDF

5 virus in 1 bacteria TOPIC 1 The Virus Disclaimer: The contents of this note are all taken from the internet. The source of the information is attached in every topic. The purpose...

5 virus in 1 bacteria TOPIC 1 The Virus Disclaimer: The contents of this note are all taken from the internet. The source of the information is attached in every topic. The purpose of compiling this note is solely for academic undertakings and not for any form of businesses. This note is intended for nursing students only and not for outside use. Copying this note outside is not permitted. Please do not reproduce nor resend this note. I. Importance of Studying the Virus Source: Understanding Virus by Shubhrata R. Mishra, 2011 Used in Phage typing bacteria (Salmonella specie’s) Source of enzymes (reverse transcriptase and RNA polymerase) simple, no covering, no nucleus has capsid to Used as Pesticides (baculoviruses and myxoma virus to control insect pests) enclose the core or Anti-bacterial agents (phages used to treat human infection-antibiotic resistant genetic material bacteria) reverse transcriptase Anti-cancer agents (genitically modified strains of herpes simplex virus and vaccine enzyme fpr PMAT virus used to infect and destroy tumor cells Gene vectors for protein production (baculoviruses and adenovirusesused as vectors to take genes into animal cells growing in culture, then inserted into cells gene encoding useful proteins such as vaccine component for mass production of protein) Gene vectors for treatment of genetic diseases (retroviruses non-mutated copy of the mutated genes are used as vector to introduce into their stem cells like the case of bubble syndrome) II. What is a Virus? Small obligate intracellular parasites (cannot multiply unless it invades a specific host cell and influences the cell metabolic machinery) Ultramicroscopic size from 20 nm to 450 nm Not cellular in nature Made of the covering called capsid and viral envelope (envelope CHON-not found in all, membrane CHON and spike CHON) and the central core nucleic acid molecules, matrix CHON enzyme (not found in all) The genome is either DNA or RNA in a single or double stranded form. Positive sense viral RNA can immediately be translated into CHON by the host cell unlike the negative sense RNA that needs to be converted to positive sense RNA by an RNA polymerase before translation Are active only if inside the host cells Covered by capsid that encloses nucleic acid core Non-enveloped viruses can be more resistant to changes in temperature, pH, and some disinfectants than are enveloped viruses. Attachment to host cell is with high specificity Many viruses attach to their host cells to facilitate penetration of the cell membrane, allowing their replication inside the cell. Many viruses use some sort of glycoprotein to attach to their host cells via molecules on the cell called viral receptors. The shape of a viral coat has implications on how a virus infects a host. Does not have enzyme to carry metabolic process Does not have machinery to synthesize protein virus is isog bec. If it causes permanent alteration of the host cell genetic material it causes cancer Oncoviruses are mammalian viruses causing tumor like the papillomaviruses and Epstein-Barr virus Are microorganisms that can causes cytopathic changes like cell lysis, DNA alteration, forming multinucleated cells and damage to nuclear and cytoplasmic inclusion bodies Viroids are ssRNA genome; smallest known pathogen Prions are infectious particles that are protein, no nucleic acid, highly heat resistant, affects nervous system of animals (like madcow disease, scrapie in sheep and Creutzfeldt-Jakob disease) Bacteriophage are viruses that infect bacteria Source: https://www.britannica.com/summary/virus Source: https://thebiologynotes.com/virus-classification-on-the-basis-of-morphology- and-replication/  Source: https://lsc.org/news-and-social/news/covid-19-words-to-know III. Structure, Shape and Size of the virus? A. Parts of a Virus and its function: Source: https://www.britannica.com/science/virus/Size-and-shape 1. The nucleic acid Encodes the genetic information for the synthesis of all proteins. Come in a variety of forms, including single-stranded or double-stranded DNA or RNA. All double-stranded DNA viruses consist of a single large molecule, Most double-stranded RNA viruses have segmented genomes, with each segment usually representing a single gene that encodes the information for synthesizing a single protein. Viruses with single-stranded genomic DNA are usually small, with limited genetic information. Some single-stranded DNA viruses are composed of two populations of virions, each consisting of complementary single- stranded DNA of polarity opposite to that of the other. The nucleic acids of virions are arranged into genomes.  The virions of most plant viruses and many animal and bacterial viruses are composed of single-stranded RNA. In most of these viruses, the genomic RNA is termed a positive strand because the genomic RNA acts as mRNA for direct synthesis (translation) of viral protein. Several large families of animal viruses, and one that includes both plant and animal viruses (the Rhabdoviridae), however, contain genomic single-stranded RNA, termed a negative strand, which is complementary to mRNA. All of these negative-strand RNA viruses have an enzyme, called an RNA-dependent RNA polymerase (transcriptase), which must first catalyze the synthesis of complementary mRNA from the virion genomic RNA before viral protein synthesis can occur. A distinctive large family of single-stranded RNA viruses is called Retroviridae; the RNA of these viruses is positive, but the viruses are equipped with an enzyme, called a reverse transcriptase, that copies the single-stranded RNA to form double-stranded DNA. 2. The protein capsid hep b or a forever with u The protein capsid provides the second major criterion for the classification of viruses. The capsid surrounds the virus and is composed of a finite number of protein subunits known as capsomeres, which usually associate with, or are found close to, the virion nucleic acid. Under the right environmental conditions, viral RNA and protein molecules in liquid suspension will assemble themselves into a perfectly formed and fully infectious virus. The length of the helical virus capsid is determined by the length of the nucleic acid molecule, which is the framework for the assembly of the capsid protein. The various helical viruses have different lengths and widths, depending on the size of the nucleic acid as well as on the mass and shape of the protein molecule. 3. Lipoprotein Envelope Surrounding viruses of either helical or icosahedral symmetry are lipoprotein envelopes, unit membranes of two lipid layers interspersed with protein molecules (lipoprotein bilayer). protects innercore Composed of phospholipids and neutral lipids (largely cholesterol) of virus derived from cell membranes during the process known as budding. All proteins of the cell membrane, however, are replaced by proteins of viral origin during budding.  All the viral envelope lipids originate from the cell, their relative proportions vary from those in the cell membrane because the viral proteins select only certain lipids during budding. Associated with the virion membrane are “integral” glycoproteins, which completely traverse the lipid bilayer, and “peripheral” matrix proteins, which line the inner surface. The glycoproteins contain regions of amino acids that, in the first step of what are the viral infection, recognize host-cell receptors. amino acids? Matrix proteins appear to function in the selection of regions of the cell membrane to be used for the viral membrane, as well as to aid the virion in entering cells. B. Shapes and Sizes of a virus Sources: 1. https://courses.lumenlearning.com/boundless-microbiology/chapter/structure- of-viruses/ 2. https://www.britannica.com/science/virus/The-protein-capsid A. Shapes: Shapes of viruses are predominantly of two kinds: a. rods, or filaments, so called because of the linear array of the nucleic acid and the protein subunits; and b. spheres, which are actually 20-sided (icosahedral) polygons. Most plant viruses are small and are either filaments or polygons, as are many bacterial viruses. Other shapes: 1. Helical : ▪ are composed of a single type of capsomer stacked around a central axis to form a helical structure, which may have a central cavity, or hollow tube. ▪ Some of these helical viruses form rigid rods, while others form flexible rods, depending on the properties of the assembled proteins. 2. Icosahedral : ▪ Most animal viruses are icosahedral or near-spherical with icosahedral symmetry. ▪ Most, if not all, of the polygonal viruses are icosahedral; like a geodesic dome, they are formed by equilateral triangles, in this case 20. ▪ Each triangle is composed of protein subunits (capsomeres), often in the form of hexons (multiples of six) that are the building blocks of the capsid. ▪ There are 12 vertices (the apical junctions of these 20 triangles), each comprising a penton (five subunits). ▪ These icosahedral virions have three axes of fivefold, threefold, and twofold rotational symmetry. Certain icosahedral viruses, usually those that are more complex,  contain internal proteins adhering to the nucleic acid that are not accessible at the surface of the virions. 3. Prolate: ▪ an isosahedron elongated along one axis and is a common arrangement of the heads of bacteriophages. 4. Envelope: ▪ a modified form of one of the cell membranes, either the outer membrane surrounding an infected host cell or internal membranes such as nuclear membrane or endoplasmic reticulum, that form an outer lipid bilayer known as a viral envelope. 5. Complex: ▪ These viruses possess a capsid that is neither purely helical nor purely icosahedral, and that may posses extra structures such as protein tails or a complex outer wall. Source: https://www.123rf.com/photo_148363373 B. Sizes of the virus The amount and arrangement of the proteins and nucleic acid of viruses determine their size and shape. The genomes of Mimiviruses and Pandoraviruses, which are some of the largest known viruses, range from 1 to 2.5 Mb (1 Mb = 1,000,000 base pairs of DNA). The smallest animal viruses belong to the families Parvoviridae and Picornaviridae and measure about 20 nm and about 30 nm in diameter, respectively. Most viruses vary in diameter from 20 nanometres (nm; 0.0000008 inch) to 250–400 nm; the largest, however, measure about 500 nm in diameter and are about 700–1,000 nm in length. Only the largest and most complex viruses can be seen under the light microscope at the highest resolution.  Polygonal viruses vary greatly in size, from 20 to 150 nm in diameter, essentially proportional to the size of the nucleic acid molecule coiled up inside the virion. Source: https://viralzone.expasy.org/5216 not all viruses have IV. What are the different kinds of the Virus? capsules Sources: 1. https://courses.lumenlearning.com/boundless-microbiology/chapter/structure-of-viruses/ 2. https://www.britannica.com/science/virus/Disease 3. https://nofisunthi.blogspot.com/2018/04/understanding-viruses-worksheet-answers.html The criteria used for classifying viruses into families and genera are primarily based on three structural considerations: (1) Type and size of their nucleic acid, (2) The shape and size of the capsids, and (3) The presence of a lipid envelope, derived from the host cell, surrounding the viral nucleocapsid. A. Based on the protein capsid: 1. Single (or segmented) linear nucleic acid molecule with two free ends is essentially completely extended or somewhat coiled (a helix) and looks like a long, extended rod-like structure 2. Those in which the nucleic acid, which may or may not be a covalently closed circle, is wound tightly into a condensed configuration, like a ball of yarn and looks like a symmetrical polygon. capsomeres- basic unit of capsid greater the no.- thicker capsid ->the virulent the virus  B. Based on the number of capsomeres is a basis for taxonomic classification of these virus families. C. Based on the type of Genes 1. DNA Viruses: C. directs the host cell’s replication proteins to synthesize new copies of the viral genome and to transcribe and translate that genome into viral proteins D. cause human diseases, such as chickenpox, hepatitis B, and some venereal diseases, like herpes and genital warts. 2. RNA Viruses: To replicate their genomes in the host cell, the RNA viruses encode enzymes that can replicate RNA into DNA, which cannot be done by the host cell. These RNA polymerase enzymes are more likely to make copying errors than DNA polymerases and, therefore, often make mistakes during transcription. For this reason, mutations in RNA viruses occur more frequently than in DNA viruses. This causes them to change and adapt more rapidly to their host. Human diseases caused by RNA viruses include hepatitis C, measles, and rabies. D. Based on Baltimore Classification: Ss-single stranded DNA or RNA; ds-double stranded DNA or RNA (+) RNA is the one which can function as mRNA for the synthesis of proteins. (-) RNA cannot function as mRNA. Group Gene Type Mode of mRNA Production Example of Virus I dsDNA mRNA is transcribed directly from the DNA Herpes simplex template Virus (HSV) II ssDNA DNA is converted to double stranded from Parvovirus before RNA is transcribed III dsRNA mRNA is transcribed from the RNA genome Rotavirus IV ssRNA+ Genome functions as mRNA Common Cold (pirconavirus) V ssRNA- mRNA is transcribed from the RNA genome Rhabdovirus which makes it VI ssRNA-RT Reverse transcriptase makes DNA from HIV powerful despite RNA genome; DNA is then incorporated in single stranded the host genome,mRNA is transcribed from the incorporated DNA VII dsDNA-RT The viral genome is double stranded DNA, Hepatitis B virus but viral DNA is replicated through an RNA intermediate V. Viral Reservoir Sources: 1. doi: https://doi.org/10.1371/journal.pbio.3000217.t003 2. https://journals.plos.org/plosbiology/article/figure?id=10.1371/journal.pbio.3000217.t003 3. https://www.cdc.gov/csels/dsepd/ss1978/lesson1/section10.html  Reservoir Disease Rabies virus Bat Rabies Ebola virus Little collared-fruit Bat Ebola virus plus SARS-Cov Chinese-horse shoe Bat Severe Acute Respiratory Syndrome MERS-CoV Egyptian tomb bat Middle-East Respiratory Syndrome Nipah/Hendra Flying Foxes Encephaliitis Arena viruses Rodents Filoviridae viruses Poorly understood Hantaviridae Rodents Coronaviridae Bats MERS, SARS Paramyxoviridae Bats Chikungunya, encephalitis Togaviridae Monkey, bird, horse Flaviviridae Vector: mosquito,bats Dengue, Hepatitis-C Orthomyxoviridae Birds Influenza A, B, C Retroviridae Primates HIV 1, 2 and Human T-lymphotropic Virus Measles, mumps, No reservoir; need large Acute self-limiting infection—lifelong rubella, polioa, population with immunity hepatitis Aa, continuing chain of enterovirusesa, c, transmission dengue Respiratory No reservoir; Acute self-limiting infection— RSV syncytial virus, reinfections occur, virus immunity more short-lived rotavirusb, can survive in smaller- influenzab, sized population coronaviruses, rhinovirusesc Herpes simplex, Human reservoir; Persistent infection-intermittent varicella-zoster, infected individuals can replication +/– shedding CMV, EBV, other provide lifelong source herpesviruses of virus HIV, HBV, HCV, Human reservoir; Persistent infection—continuous CAUSE LEUKEMIA HTLV-1, human infected individuals can replication papillomavirus provide lifelong source of virus A. Human reservoirs. Includes the sexually transmitted diseases, measles, mumps, streptococcal infection, and many respiratory pathogens. Humans were the only reservoir for the smallpox virus, naturally occurring smallpox was eradicated after the last human case was identified and isolated. Human reservoirs may or may not show the effects of illness. Asymptomatic or passive or healthy carriers are those who never experience symptoms despite being infected. Incubatory carriers are those who can transmit the agent during the incubation period before clinical illness begins. Convalescent carriers are those who have recovered from their illness but remain capable of transmitting to others.  Chronic carriers are those who continue to harbor a pathogen such as hepatitis B virus or Salmonella Typhi, the causative agent of typhoid fever, for months or even years after their initial infection. Carriers commonly transmit disease because they do not realize they are infected, and consequently take no special precautions to prevent transmission. Symptomatic persons who are aware of their illness, on the other hand, may be less likely to transmit infection because they are either too sick to be out and about, take precautions to reduce transmission, or receive treatment that limits the disease. B. Animal reservoirs. Transmitted from animal to animal, with humans as incidental hosts. The term zoonosis refers to an infectious disease that is transmissible under natural conditions from vertebrate animals to humans. Long recognized zoonotic diseases include brucellosis (cows and pigs), anthrax IF INFECTED (sheep), plague (rodents), trichinellosis/trichinosis (swine), tularemia (rabbits), FROM ANIMALS and rabies (bats, raccoons, dogs, and other mammals). Zoonoses newly emergent in North America include West Nile encephalitis (birds), and monkeypox (prairie dogs). Many newly recognized infectious diseases in humans, including HIV/AIDS, Ebola infection and SARS, are thought to have emerged from animal hosts, although those hosts have not yet been identified. C. Environmental reservoirs. Plants, soil, and water in the environment are also reservoirs for some infectious agents. Many fungal agents, such as those that cause histoplasmosis, live and multiply in the soil. Outbreaks of Legionnaires disease are often traced to water supplies in cooling towers and evaporative condensers, reservoirs for the causative organism Legionella pneumophila. VI. Viral Transmissions Source: 1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150207/figure/f0010/ 2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150207/ A. Human Transmission: Transmission cycles require virus entry into the body, replication, and shedding with subsequent spread to another host. Shedding of virus usually occurs from one of the body openings or surfaces also involved in the entry of viruses. With localized infections the same body openings are involved in both entry and exit ; in generalized infections a greater variety of modes of shedding is recognized, and some viruses are shed from multiple sites, for example, hepatitis B virus, HIV, and cytomegalovirus in semen, cervical secretions, milk, and saliva. The amount of virus shed in an excretion or secretion is important in relation to transmission.  Very low concentrations may be irrelevant unless very large volumes of infected material are transferred; on the other hand some viruses occur in such high concentrations that a minute quantity of material, for example, less than 1 μl, can transmit infection. Virus transmission may be horizontal or vertical; Most transmission is horizontal, that is, between individuals within the population at risk. 1. Respiratory or Salivary Influenza, measles, rhinoviruses, coronaviruses Respiratory transmission is a very efficient way to quickly infect a large number of contacts and spread virus globally (compare influenza with HIV). Two mechanisms: 1. Inhalation of aerosol Aerosols are created especially by sneezing and coughing, less by talking. Large droplets (>10 μm) soon sink to the floor, while small droplets can spread further but dry out and virus becomes inactivated; Respiratory infections is affected by increased virus survival in cooler temperatures, 2. Contaminated objects. Infected respiratory secretions contaminate tissues, environmental surfaces and objects, hands. Shaking hands, handling objects etc. provides a pathway for virus to the nose and mouth of a new person 2. Fecal-oral Enteroviruses, rotaviruses Enteric viruses are shed in the feces and vomit, and the more voluminous the fluid output the greater is the environmental contamination caused. These viruses tend to be hardier and able to survive environmental conditions longer outside the body than the enveloped respiratory viruses. Two epidemiological patterns are seen: 1. A point source outbreak occurs when many people ingest contaminated food or water, for example at weddings or other functions. This particularly occurs with salads, uncooked shellfish, or through drinking unsafe river water or well water contaminated with sewage, and 2. Person-to-person spread by the fecal–oral route is more gradual and occurs more efficiently in households without running water, hand- washing facilities, or toilets, and where there is poverty and a lack of education. 3. Sexual contact Herpes simplex 2, papillomavirus, HIV Many viruses can be found in semen or vaginal secretions.  Sexual transmission of virus infections by mucosal contact is efficient because the virus is kept moist and does not need to survive long outside the body; Sexual transmission is enhanced when there is a greater number of consecutive partners, when concurrent genital mucosal tears or intercurrent infections (e.g., ulcerating STDs) are present, and when the male involved is not circumcised. The most important examples are HIV, HBV, human papillomavirus, and herpes simplex type 2, although other herpesviruses, hepatitis B, and HTLV I are also sexually transmitted with ease. 4. Blood-borne HIV, HBV, HCV, Hepatitis B,C and D, HTLV Highly sensitive tests, for example polymerase chain reaction (PCR), are often used because of the extra risk caused by the large volume of blood transfused and the usual compromised health of recipients. Blood is the usual source from which arthropods (e.g., mosquitoes, ticks, sandflies) acquire viruses during the course of taking a blood meal. Less commonly, some arthropods (e.g., horseflies, other biting flies) transmit viruses passively by contamination of their mouth parts and interrupted blood feeding on multiple hosts. Despite this, in most instances blood-borne viruses are not shed from the unbroken skin of an infected person, and transmission by normal skin contact is negligible. the ff.diseases can be 5. Mother to Child transmitted from mother to child HBV, HIV, Rubella, CMV, HSV, Enteroviruses except Three situations where this occurs namely 1. Via the integration of proviral DNA directly into the DNA of the germline of gametes and fertilized eggs, 2. Transplacental spread during pregnancy, and 3. Perinatal or postnatal spread via saliva, milk, or other secretions. Vertical transmission of a virus may be lethal to the fetus and cause abortion, it may be associated with congenital disease or congenital abnormalities, or it may cause a sub-clinical infection. In the case of HIV and hepatitis B, vertical transmission introduces infection to a new generation of infants who are then capable of transmitting infection to succeeding birth cohorts for many years to come. B. Arthropod transmission The most complex of all virus transmission modes. Arbovirus (arthropod-borne virus) refers to a virus whose life cycle involves alternating replication stages in a vertebrate host and in a blood- feeding arthropod (usually mosquitoes or ticks). The arthropod vector acquires virus by feeding on the blood of a viremic animal or person. The ingested virus replicates, initially in the arthropod gut and then in the salivary gland, over several days (the extrinsic incubation period); this period varies with different viruses and is influenced by ambient temperature.  Virions in the salivary secretions of the vector are injected into new vertebrate hosts when the arthropods subsequently take a blood meal. In addition to the above, Arthropod transmission may occur mechanically by contamination of the insect’s biting parts (“flying pin”). Infected vertebrates usually recover rapidly, eliminate the virus, and develop a lasting immunity to reinfection. The arthropod must: 1. be easily infected even when feeding on vertebrate host with a low titered viremia (called a low infection threshold), 2. be supportive of virus replication to a titer sufficient to infect its next vertebrate victim, 3. be able to deliver virus from the productive infection in its salivary gland into its saliva and then into its vertebrate host’s blood/tissues, and 4. be able to continue this sequence for its lifespan without adverse pathological effects of the infection. C. Nosocomial transmission Refers to transmission while a person is in a hospital or clinic Iatrogenic transmission refers to transmission “by the hand of the doctor.” The lethal Ebola virus outbreak in Zaire in 1976, is a classic example of an iatrogenic and nosocomial infection. More common examples of nosocomial virus infections spreading by the respiratory route are chickenpox, influenza, and respiratory syncytial virus infections in hospital settings. Hepatitis B and C viruses, and to a lesser extent HIV, can also be transmitted by doctors, dentists, acupuncturists, tattooists, etc., Needle stick and similar injuries transmission. Pseudomonas aeruginosa transmissions VII. Virus Pathogenicity Source: https://www.atsu.edu/faculty/chamberlain/website/tritzmed/LECTS/MECHANIS.HTM Virulence of viruses is not well defined. lytic? Factors contributing to the virulence of the virus are: A. Ability to enter the cell B. Ability to grow within the cell C. Ability to combat host defense mechanisms D. Ability to produce temporary or permanent damage in the host via: 1. Cell lysis 2. Production of toxic substances 3. Cell transformation 4. Induction of formation of substances which are not specified by the viral genome, but are apparently cellular products normally not produced by the cell. 5. Induction of structural alterations in the host cell a. Nuclear (including chromosomal) b. Cytoplasmic Common Portal of Entry: directly at trauma sites or insect bite, but mostly via mucous membranes of the respiratory and alimentary tracts.  To initiate infection, virus particles must first survive on these mucous-covered membranes in the presence of viral and non-viral commensals. To replicate, the virus must enter host cells either in the mucous membrane itself or through the surface membrane. Replication in mucous membrane cells can produce the disease effects directly as in respiratory diseases, but sometimes it provides a staging post for subsequent damaging replication in another site, e.g., polio virus replicates first in the alimentary tract cells and ultimately in anterior horn cells of the spinal cord. The host anti-viral defense mechanisms include: low ph kills virus A. Non-specific host defense mechanisms 1. Humoral factors: Low pH of inflammatory exudates; Enzymes; Mucous; Virocidins 2. Cellular factors: Nucleases; Proteases; Interferon B. Specific host defense mechanisms 1. Antibody 2. Activated phagocytes The ability of a virus to replicate in a particular cell depends on inherent features of the cell as well as the virus. control reverse transcriptase to rapidly grow HIV Virus Stages of Replication: A. Attachment read more B. Penetration on C. Uncoating replication D. Provision of energy and precursors of low-molecular-weight compounds E. Nucleic acid and protein synthesis F. Assembly G. Release VIII. Host Responses to Viral Infection Source: https://www.atsu.edu/faculty/chamberlain/website/tritzmed/LECTS/MECHANIS.HTM virus- rashes 1. Cell lysis May occur due to a physical internal pressure exerted by the multiplying virus. The cell becomes filled with virus and merely bursts. This is common with bacterial viruses, but not with animal viruses. With animal viruses, cell lysis is usually the result of one of four types of allergic reactions: 1. Type I. IgE antibodies fixed to mast cells react with the complete virus or with viral components, triggering release of histamine and activation of slow reacting substance (SRS-A) and eosinophil chemotactic factor (ECF-A). These act on blood vessels, smooth muscle and secreting glands to give the typical anaphylactic type reaction. 2. Type II. IgG and/or IgM antibodies are involved in this reaction. The effects can be of two types:  (a) The virus (or viral component) - complement - antibody complex is fixed to a cell, usually an erythrocyte or leukocyte or platelet, resulting in complement-dependent cell lysis. This is the pathogenic mechanism in many viral diseases where anemia is one of the clinical manifestations. (b) A virus component, commonly the capsid protein, is expressed on the surface of the infected cell. Antibody and complement bind to this infected cell and cause a lysis of that cell. This is thought to be the major mechanism of viral-induced cell lysis. 3. Type III. IgG and/or IgM antibodies form complexes with viral antigen and complement, generating neutrophil chemotactic factors, with resultant local tissue inflammation and destruction. Although much rarer, some viral diseases may result in a generalized rather than localized tissue destruction. This type of disease is a multi-system complement-dependent vasculitis in which immune complexes are deposited along the endothelial surfaces of blood vessels, stimulating inflammation and vascular wall damage. 4. The III reactions are known as Arthus-type reactions. The classical symptoms of this type of hypersensitivity are edema, polymorphonuclear leukocyte infiltration and hemorrhage. These are followed by secondary necrosis which reaches a maximum in 8-24 hours. This type of hypersensitivity is due to precipitating antibody only, and requires a large amount of antibody. The antibody is not fixed to the tissues. Histamine does not duplicate the reaction and antihistamines do not suppress the reaction. 5. Type IV. This type of allergic reaction does NOT involve antibody. Sensitized T-lymphocytes react directly with viral antigen, usually that antigen expressed on the surface of an infected cell, producing inflammation through the action of lymphokines. This leads to lysis of the infected cell. This is a delayed-type hypersensitivity which results in the Zinkernagel-Dougherty phenomenon. This is probably the second most common allergic reaction to viruses. 2. Production of toxic substances During the course of virus replication, many viral components as well as by- products of viral replication accumulate in the cell. These are often cytotoxic (e.g., Vaccinia virus in HeLa cells). The molecular mechanism of these toxins is not known in most cases. Gross morphological defects due to viral toxic substances: 1. Cytotoxicity of preformed viral parts. e.g., Sendoi virus, Newcastle disease virus, measles virus and SV5 produce rapid polykaryocytosis (fusion of chromosomes). 2. Herpes virus components produce syncytia (multi-nucleated protoplasmic mass, seemingly an aggregation of numerous cells without a regular cell memorize red outline). substances as it 3. Penton of adenovirus causes host cell rounding and cell detachment from explains the process of glass. death 4. A double-stranded RNA from enterovirus causes rapid death, without the production of infectious virus, of cells susceptible and unsusceptible to enterovirus infection. 5. The fiber antigen of the adenovirus capsid inhibits RNA, DNA and protein synthesis.  6. Large quantities of some viruses, such as influenza virus and poxviruses, cause rapid toxic effects in some animals. 3. Cell transformation They either multiply in a normal manner and are eventually released from the cell, or they may be dormant in the cell and eventually transform the cell into a malignant cell. It is believed that the transformation process involves the integration of viral nucleic acid into the host chromosome. When this happens, the cell achieves certain characteristics of malignant cells. 4. Suppression of the immune mechanisms Since many viruses are known to replicate in cells of the lymphoreticular system, it is possible that these viruses can affect the immune system. Viruses or virus-like particles have been found in the thymus, lymph nodes, spleen, bone marrow, stem cells, plasma cells, lymphocytes, macrophages, monocytes, polymorphonuclear leukocytes and Kupffer cells. The nature and extent of the immunologic alteration depends on the organ or cell type infected and the species of virus causing the infection. A. Humoral Immunity (a) In animal model systems some viruses can cause a depression of the low count synthesis of immunoglobulins of the IgM and IgG classes. Although CD4- HIV human leukemia has not yet been shown to be of viral etiology, the positive analogy with animal systems is strengthened by the fact that human leukemia victims do have a reduced ability to synthesize immunoglobulins. (b) Viruses which do not produce leukemia but infect lymphoid tissue also decrease the immune response of the host decreasing antibody (IgM and IgG) response to a variety of antigens. Depression of the immune response is greatest in adults, temporary in neonates and absent in chronic virus infections. (c) Both leukemia and lymphoma viruses also decrease the ability of an animal to undergo anaphylaxis. This is thought to be due to a reduced synthesis of IgE. Theories on how viruses depress immune function: (a) Viruses alter the uptake and processing of antigens. (b) Viruses depress cellular protein (antibody) synthesis. (c) Viruses destroy antibody-producing cells. (d) Viruses increase immunoglobulin catabolism. B. Cellular immunity (a) Both leukemia viruses and non-leukemia viruses can either prevent or ameliorate homograft rejection across weak histocompatibility barriers. (b) Many viruses promote the growth of tumors which would normally be rejected by the host's cellular immune mechanisms.  (c) More relevant to human medicine is the fact that infection with measles virus, influenza virus, chickenpox virus, polio virus or rubella virus causes a depression of delayed hypersensitivity as measured by skin reaction to tuberculin. The major theory explaining these phenomena relates the reduced cellular immunity to a depressed ability to undergo lymphocyte blast transformation. C. Reticuloendothelial system and phagocytosis Infection of human polymorphonuclear leukocytes with mumps virus, influenza virus or Coxsackie virus decreases the ability of these cells to engulf bacteria. 5. Induction of non-normal host-specified products Virus-infected cells, at times, will produce compounds coded for by the host DNA, but which are not normally produced by the host. These are often cytotoxic at relatively high concentrations. Other host compounds which are normally found in low concentration may be produced in higher concentration during a virus infection. Some virus-induced products release autolytic enzymes from the cells own lysosomes. 6. Induction of structural alterations in the host cell: Cytoplasmic changes Small non-enveloped RNA viruses produce a large eosinophilic mass which displaces the nucleus. There is a generalized increase in basophilia. The cytoplasm appears to bubble at the cell periphery. Myxoviruses (influenza, fowl, plague) cause cytoplasmic vacuolization, contraction and degeneration. "Buds" appear on cell surface. Myxoviruses (mumps, NDV) cause eosinophilia and Feulgen-negative cytoplasmic inclusions. Reovirus and measles virus cause eosinophilia. Poxviruses cause formation of Feulgen-positive cytoplasmic inclusions which contain virions. Herpesvirus causes vacuolization. Nuclear changes Pyknosis (nucleus pushed to eccentric position in cell); e.g., small non- enveloped RNA viruses, influenza virus, fowl plague virus, mumps virus, NDV. Nuclear inclusion (bodies in the nucleus); e.g., herpesvirus, adenovirus. Margination and coarsening of chromatin; e.g. herpesvirus, poxvirus. Polykaryocytosis (many nuclei in the same cytoplasmic field); e.g., herpesvirus and measles virus. Formation of chromosomal bridges. e.g. herpesvirus and polyoma virus. Formation of chromosomal breaks. If both chromatids are broken, the break is complete. If only one chromatid is broken, the break is partial. A second important characteristic that has been used in the classification of chromosomal breaks is dependent on whether or not healing or reunion has occurred in the broken ends. If no reunion has occurred, then there is  a gap or a terminal deletion. If reunion occurs in other than the original position, then a structural rearrangement is the result. Structural rearrangements of chromosome: Defects in the mitotic apparatus (alteration of the spindle and mitotic mechanism). These alterations produce changes in chromosome number and are of three types: 1. Changes in spindle mechanism. This is seen in virus-induced syncytia, where various nuclear groups exhibit some degree of mitotic syndromy. These synchronized metaphase plates share common spindles and polar groups, and by virtue of this become rearranged in various geometric shapes. During anaphase, chromosomes in the various metaphase plates that are sharing the same pole come together at this pole, producing new chromosomal rearrangements and changes in chromosome number in each nuclear group. 2. Changes in mitotic mechanism. This is seen in virus-induced persistence of nucleoli during mitosis. The end result is a change in chromosome number. Normally, nucleoli disappear during mitosis and then reappear at telophase. However, in cells treated with inhibitors of DNA synthesis or infected with certain viruses, the nucleolus is visible during mitosis. The importance of the persistent nucleolus is that the nucleolus is formed at specific areas of chromosomes, the nucleolus organizer, and then it persists, it joins together and two chromatids of these chromosomes and produces separation difficulties during anaphase, which may result in nondisjunction. 3. Induction of mitotic delay or mitotic inhibition. This is a frequently observed phenomenon in acute virus infections of cells in cultures, although it appears to be a non-specific phenomenon. Membrane changes The human cell membrane is a dynamic structure continually changing in lipid and protein content during normal cellular growth and division. Viral infection of the cell often results in viral protein being incorporated into this membrane. There is also limited evidence suggesting that the lipid content is altered. These changes can lead to production of antibodies against the cell membrane and lysis of this membrane as previously discussed. Example of Viral Pathogenicity important Source: Mechanism of Microbial Disease by N. Engleberg, Victor Dirita and Terence S. Dermody Group of Viruses Tissue Damages Picornaviruses and Coronaviruses Lytic viruses are enteroviruses like polio virus, Infect GIT coxsackievirus, echovirus, rhinovirus, Replicates in the intestine and can be shed in the stool hepatitis virus, kobovirusesy weeks to months after infection It may replicates in the neurons of the gray matter it will causes flaccid paralysisile rashes  May cause aseptic meningitis, myocarditis, pleuritic-lung, herpangina-pharynx, febr Symptoms depends on: size of the viral inoculation, concentration of virus in the blood, virulence of the individual virus strain, level of antibodies, efficiency of the innate immune response, comorbidities, Paramyxoviruses; measles, mumps, The envelope contains viral integral membrane glycoproteins that are determinants virulence In measles, it form giant cell in the respi-epithelium causes cought, coryza and conjunctivitis Rhabdo virus Causes lesions in the brain with the presence of negri bodies in the cytoplasm Cause fever, headache, difficulty in swallowing, paresthesia, increased muscle tone, hypersalivation, paralysis and hydrophobia The envelope contains glycoprotein –G, matrix protein(M), helical ribonucleoprotein(RNP), nucleoprotein (N-protein) Influenza virus Possess hemagglutinin(HA) and neuramidase (NA) Colds, pharyngitis, bronchiolitis Rotavirusis, noroviruses Causes acute gastro-enteritis With two outer capsid protein, a hemagglutinin (VP4), and glycoprotein (VP7) Causes shortening and atrophy of the villi, denuded villi, mononuclear cell infiltration of lamina propia, destructof the mature absorptive cells HIV RNA viruses with reverse transcriptase to that allow them to use RNA as template to make DNA HIV infection of CD4 T-lymphocytes result in the eventual destruction of these cells disabling the immune system and allowing opportunistic infection AIDS causes diarrhea, oral candidiasis, fever weight loss Adenoviruses The capsomeres two types of protein the pentons and hexons Causes common colds, chills, headache, muscle aches, and fever The protein blocks the production of MHC class class 1 mRNA, the glycoprotein prevents the transport of newly synthesized MHC class1 protein Human Papillomaviruses Replicates in the squamous epithelial cells Not lytic virus Causes local infection like warts in the hand, feet and genital Contain tightly packed proteins calledL1 and L2 and single copy of circular viral genome which are  transported in keratinocyte with hundreds to thousands capsids Herpes Simplex Virus/Varicella- Causes oral and genital herpes, occasionaly with Zoster Virus encephalitis; VZV causes chicken pox and shingles HSV and VZV both destroy the epitheilial cell in which they replicate in the skin and mucus membrane causing vesicular lesions that rupture, leaving a shallow gray white ulcer on an erythematous base. The viral envelope with glycoprotein bud with the cytoplasmic vacuoles derived from the golgi apparatus and transport to the cell surface via exocytosis, they immediately attach to the adjacent cell causing cell to cell spread. Recurrence is due to activation of virus in the neurons Cytomegalovirus and Epstein-Barr Infect the epithelial cell of the salivary gland, that will virus cause persistent infection and viral shedding Can block intrinsic cellular defense and interfere with cellular immune response because of its big genes infectious Exhibit broad cellular tropism capable of infecting most cells Hepatitis Virus Damaged the NK and cytotoxic T-cell and continuous recurrent hepa infection cycle of low-level damage leading to liver cirrhosis and causes carcinoma hepatocellular carcinoma IX. Viral Diseases: MEMORIZE Disease Virus Involved Encephalitis Meningitis JC virus, measles, LCM virus, Arbovirus, rabies Common cold Rhino virus, Parainfluenza virus, Respiratory syncytial virus Eye infection Herpes simple virus, adenovirus, cytomegalovirus Pharyngitis Adenovirus, Eipstein-barr virus, cytomegalovirus Gingivostomatitis Herapes simplex 1 Parotitis Mumps virus Pneumonia Influenza Virus A and B, adenovirus, SARS coronavirus Cardiovascular Coxsackie B virus Hepatitis Hepatititis A,B, C, D and E Myelitis Polio virus, HTLV-1 Skin infection Varicella-zoster virus, Human herpes virus, smallpox, Molluscum contagiosum, Human papillomavirus, Parvovirus B19, Rubella, measles, Coxsackie A virus Sxuallt Transmitted Herpes simplex virus type 2, human papillomavirus, HIV Diseases Gastroenteritis Adenovirus, Rotavirus, Norovirus, Astrovirus, Corobavirus what is rotavirus Pancreatitis Coxsackie B Virus  SELF-READING VIRAL Infections: Source: https://courses.lumenlearning.com/microbiology/chapter/the-viral-life-cycle/ Persistent Infections Persistent infection occurs when a virus is not completely cleared from the system of the host but stays in certain tissues or organs of the infected person. The virus may remain silent or undergo productive infection without seriously harming or killing the host. Mechanisms of persistent infection may involve the regulation of the viral or host gene expressions or the alteration of the host immune response. The two primary categories of persistent infections are latent infection and chronic infection. Examples of viruses that cause latent infections include herpes simplex virus (oral and genital herpes), varicella- zoster virus (chickenpox and shingles), and Epstein-Barr virus (mononucleosis). Hepatitis C virus and HIV are two examples of viruses that cause long-term chronic infections. Latent Infection There are viruses that are capable of remaining hidden or dormant inside the cell in a process called latency. These types of viruses are known as latent viruses and may cause latent infections. Viruses capable of latency may initially cause an acute infection before becoming dormant. For example, the varicella-zoster virus infects many cells throughout the body and causes chickenpox, characterized by a rash of blisters covering the skin. About 10 to 12 days postinfection, the disease resolves and the virus goes dormant, living within nerve-cell ganglia for years. During this time, the virus does not kill the nerve cells or continue replicating. It is not clear why the virus stops replicating within the nerve cells and expresses few viral proteins but, in some cases, typically after many years of dormancy, the virus is reactivated and causes a new disease called shingles (Figure 7). Whereas chickenpox affects many areas throughout the body, shingles is a nerve cell-specific disease emerging from the ganglia in which the virus was dormant. Latent viruses may remain dormant by existing as circular viral genome molecules outside of the host chromosome. Others become proviruses by integrating into the host genome. During dormancy, viruses do not cause any symptoms of disease and may be difficult to detect. A patient may be unaware that he or she is carrying the virus unless a viral diagnostic test has been performed. Chronic Infection A chronic infection is a disease with symptoms that are recurrent or persistent over a long time. Some viral infections can be chronic if the body is unable to eliminate the virus. HIV is an example of a virus that produces a chronic infection, often after a long period of latency. Once a person becomes infected with HIV, the virus can be detected in tissues continuously thereafter, but untreated patients often experience no symptoms for years. However, the virus maintains chronic persistence through several mechanisms that interfere with immune function, including preventing expression of viral antigens on the surface of infected cells, altering immune cells themselves, restricting expression of viral genes, and rapidly changing viral antigens through mutation.  Eventually, the damage to the immune system results in progression of the disease leading to acquired immunodeficiency syndrome (AIDS). The various mechanisms that HIV uses to avoid being cleared by the immune system are also used by other chronically infecting viruses, including the hepatitis C virus. X. Life Cycle of a Virus A. Life Cycle (Source: https://courses.lumenlearning.com/microbiology/chapter/the-viral-life- cycle/) The life cycle of bacteriophages has been a good model for understanding how viruses affect the cells they infect, since similar processes have been observed for eukaryotic viruses, which can cause immediate death of the cell or establish a latent or chronic infection. All viruses depend on cells for reproduction and metabolic processes. By themselves, viruses do not encode for all of the enzymes necessary for viral replication. But within a host cell, a virus can command cellular machinery to produce more viral particles. Bacteriophages replicate only in the cytoplasm, since prokaryotic cells do not have a nucleus or organelles. In eukaryotic cells, most DNA viruses can replicate inside the nucleus, with an exception observed in the large DNA viruses, such as the poxviruses, that can replicate in the cytoplasm. RNA viruses that infect animal cells often replicate in the cytoplasm. Virulent phages typically lead to the death of the cell through cell lysis. Temperate phages, on the other hand, can become part of a host chromosome and are replicated with the cell genome until such time as they are induced to make newly assembled viruses, or progeny viruses. A. Life Cycle of Bacteriophage 1. Lytic Cycle- the bacteriophage takes over the cell, reproduces new phages, and destroys the cell. 2. Lysogenic Cycle- ▪ During the lysogenic cycle, instead of killing the host, the phage genome integrates into the bacterial chromosome and becomes part of the host. The integrated phage genome is called a prophage. ▪ A bacterial host with a prophage is called a lysogen. The process in which a bacterium is infected by a temperate phage is called lysogeny. It is typical of temperate phages to be latent or inactive within the cell. ▪ As the bacterium replicates its chromosome, it also replicates the phage’s DNA and passes it on to new daughter cells during reproduction. The presence of the phage may alter the phenotype of the bacterium, since it can bring in extra genes (e.g., toxin genes that can increase bacterial virulence). This change in the host phenotype is called lysogenic conversion or phage conversion. 3. Transduction  ▪ Occurs when a bacteriophage transfers bacterial DNA from one bacterium to another during sequential infections. ▪ There are two types of transduction: generalized and specialized transduction. Generalized transduction occurs when a random piece of bacterial chromosomal DNA is transferred by the phage during the lytic cycle. Specialized transduction occurs at the end of the lysogenic cycle, when the prophage is excised and the bacteriophage enters the lytic cycle. B. Life Cycle of Viruses with Animal Hosts Source: https://courses.lumenlearning.com/microbiology/chapter/the-viral-life-cycle/) After binding to host receptors, animal viruses enter through endocytosis (engulfment by the host cell) or through membrane fusion (viral envelope with the host cell membrane). Many viruses are host specific, meaning they only infect a certain type of host; and most viruses only infect certain types of cells within tissues. This specificity is called a tissue tropism. Examples of this are demonstrated by the poliovirus, which exhibits tropism for the tissues of the brain and spinal cord, or the influenza virus, which has a primary tropism for the respiratory tract. Steps of influenza infection.: 1. Attachment when the influenza virus becomes attached to a target epithelial cell. This image shows a spherical virus binding to the surface of a host cell. 2. Penetration when the cell engulfs the virus by endocytosis; this shows the virus within a vacuole. 3. Uncoating when the viral contents are released; the image shows the virus being released from the vacuole. 4. Biosynthesis when the viral RNA enters the nucleus where it is replicated by RNA polymerase. Step 5. Assembly when the new phage particles are assembled. Step 6 is release when new viral particles are made and released into the extracellular fluid. The cell, which is not killed in the process continues to make new viruses. READ MORE OF THE PROCESS  Animal viruses do not always express their genes using the normal flow of genetic information— from DNA to RNA to protein. Some viruses have a dsDNA genome like cellular organisms and can follow the normal flow. However, others may have ssDNA, dsRNA, or ssRNA genomes. The nature of the genome determines how the genome is replicated and expressed as viral proteins. If a genome is ssDNA, host enzymes will be used to synthesize a second strand that is complementary to the genome strand, thus producing dsDNA. The dsDNA can now be replicated, transcribed, and translated similar to host DNA. If the viral genome is RNA, a different mechanism must be used. There are three types of RNA genome: dsRNA, positive (+) single-strand (+ssRNA) or negative (−) single-strand RNA (−ssRNA). If a virus has a +ssRNA genome, it can be translated directly to make viral proteins. Viral genomic +ssRNA acts like cellular mRNA. If a virus contains a −ssRNA genome, the host ribosomes cannot translate it until the −ssRNA is replicated into +ssRNA by viral RNA-dependent RNA polymerase (RdRP) (see Figure 5). The RdRP is brought in by the virus and can be used to make +ssRNA from the original −ssRNA genome. The RdRP is also an important enzyme for the replication of dsRNA viruses, because it uses the negative strand of the double-stranded genome as a template to create +ssRNA. The newly synthesized +ssRNA copies can then be translated by cellular ribosomes. Source: https://www.news-medical.net/news/20201208/Amilorides-reduce-SARS-CoV-2-viral-replication-in- vitro.aspx XI. Antiviral Drugs Source: https://www.britannica.com/science/antiviral-drug/Anti-HIV-drugs 1. Anti-herpesvirus drugs Herpesvirus is the DNA-containing virus that causes such diseases as genital herpes, chickenpox, retinitis, and infectious mononucleosis. After the viral particle attaches to the cell membrane and uncoats, the viral DNA is transferred to the nucleus and transcribed into viral mRNA for the viral proteins. Drugs that are effective against herpesviruses interfere with DNA replication. The nucleoside analogs (acyclovir and ganciclovir) actually mimic the normal nucleoside and block the viral DNA polymerase enzyme, which is important in the formation of DNA. All the nucleoside analogs must be activated by addition of a phosphate group before they have antiviral activity. Some of the agents (acyclovir) are activated by a viral enzyme, so they are specific for the cells that contain viral particles. Other agents (idoxuridine) are activated by cellular enzymes, so these have less specificity. Non-nucleoside inhibitors of herpesvirus replication include foscarnet, which directly inhibits the viral DNA polymerase and thus blocks formation of new viral DNA. 2. Anti-influenza drugs Influenza is caused by two groups of RNA-containing viruses, influenza A and influenza B. When the RNA is released into the cell, it is directly replicated and also is used to make protein to form new viral particles. Amantadine and rimantadine are oral drugs that can be used for the prevention and treatment of influenza A, but they have no effect against influenza B viruses. The action of amantadine is to block uncoating of the virus within the cell and thus prevent the release of viral RNA into the host cell. Zanamivir, peramivir, and oseltamivir are active against both influenza A and influenza B. Zanamivir is given by inhalation only, peramivir is given intravenously, and oseltamivir can be given orally. These drugs are inhibitors of neuraminidase, a glycoprotein on the surface of the influenza virus. Inhibition of neuraminidase activity decreases the release of virus from infected cells, increases the formation of viral aggregates, and decreases the spread of the virus through the body. If taken within 30 hours of the onset of influenza, both drugs can shorten the duration of the illness. Covid-19 Drugs: 1. Favipiravir- acts as a substrate for the RNA-dependent RNA-polymerase (RdRp) enzyme, which is mistaken by the enzyme as a purine nucleotide, incorporated in the viral RNA strand, preventing further extension, 3. Anti-HIV drugs Human immunodeficiency virus (HIV), the virus that causes AIDS, is a retrovirus.  Retroviruses like HIV, contains reverse transcriptase, an enzyme that converts viral RNA into DNA. This DNA is integrated into the DNA of the host cell, where it replicates. Reverse transcriptase (RT) inhibitors work by blocking the action of reverse transcriptase. There are two groups of RT inhibitors. Nucleoside RT inhibitors (e.g., zidovudine, didanosine, zalcitabine, lamivudine, and stavudine) must be phosphorylated to become active. These drugs mimic the normal nucleosides and block reverse transcriptase. Because the different nucleoside RT inhibitors mimic different purines and pyrimidines, use of two of the drugs in this group is more effective than one alone. The second group of RT inhibitors are the non-nucleoside inhibitors (e.g., delaviridine, efanvirenz, and nevirapine), which do not require activation and, because they act through a different mechanism, exhibit a synergistic inhibition of HIV replication when used with the nucleoside RT inhibitors. Protease inhibitors (e.g., ritonavir, saquinavir, and indinavir) block the spread of HIV to uninfected cells by inhibiting the viral enzymes involved in the synthesis of new viral particles. Because they act at a different point in the life cycle of HIV, use of a protease inhibitor with an RT inhibitor suppresses replication better than either drug alone. 4. Anti-RSV drugs Respiratory syncytial virus (RSV) causes a potentially fatal lower respiratory disease in children. The only pharmacological therapy available for treatment of the infection is the nucleoside analogue ribavirin, which can be administered orally, parenterally, or by inhalation. Ribavirin must also be activated by phosphorylation in order to be effective. An injectable humanized monoclonal antibody is available for prevention of RSV infection in high-risk infants and children. It provides passive immunity and must by given by intramuscular injection once a month during RSV season. 5. Interferons 6. Interferons represent a group of nonspecific antiviral proteins produced by host cells in response to viral infections as well as in response to the injection of double-stranded RNA, some protozoal and bacterial components, and other chemical substances. Interferon results in the production of a protein that prevents the synthesis of viral components from the viral nucleic acid template. The interferons are of interest because they have broad-spectrum antiviral activity and because they inhibit the growth of cancer tissue. However, the use of interferon is limited by adverse effects, a relative lack of efficacy, and the requirement for local or intravenous administration. - END -