Virology Lectures PDF

MIDTERMS PART I. INTRODUCTION BRIEF HISTORY 1892 IWANOWSKI A Russian scientist who demonstrated the first known virus that caused Tobacco Mosaic Disease 1898 LOEFFLER & FROSCH Demonstrated that the causative agent of Foot and Mout...

MIDTERMS PART I. INTRODUCTION BRIEF HISTORY 1892 IWANOWSKI A Russian scientist who demonstrated the first known virus that caused Tobacco Mosaic Disease 1898 LOEFFLER & FROSCH Demonstrated that the causative agent of Foot and Mouth Disease of cattle can readily pass bacteria-proof filters and could not be seen with the microscope. 1922 SANARELLI Discovered Myxomatosis (Contagious Rabbit Tumor) Bacteriophage was described as a parasite of bacterial cells, causing them to swell and burst. 1935 STANLEY Extracted crystalline nucleoprotein from tobacco plants with mosaic disease. He initiated the investigation of viral nature. TERMS VIRIONS Complete infective virus particle. Identical to the nucleocapsid CAPSID Protein shell that encloses the nucleic acid core (genome) NUCLEOCAPSID The capsid together with the enclosed nucleic acid STRUCTURE UNITS The basic units of similar structure in the capsid. May be polypeptides CAPSOMERES Morphologic units seen on the surfaces of isometric virus particle Represents clusters of structure units PRIMARY NUCLEIC ACID STRUCTURE Spatial arrangement of the complete nucleic acid chain e.g. single or double stranded circular or linear branched or unidirectional TERTIARY NUCLEIC ACID Fine spatial detail in the helix such as super coiling breakage STRUCTURE points, deletions, gaps, catenation, and regions, of strand separation ENVELOPE The outer coat some viruses acquire as they penetrate or are budded from the nuclear or cytoplasmic membrane. It always contains altered host cell membrane components. PEPLOMERS Morphologic units composed of structural units embedded in the envelope COMPLEMENTATION A general term to describe situations were mixed infections result in enhanced yields of one or both viruses in the mixture TRANSLATION The mechanism by which a particular base sequence in messenger RNA produces a specific amino acid sequence in a protein TRANSCRIPTION The means by which specific information encoded in a nucleic acid chain is transferred to messenger RNA TRANSCAPSIDATION A form of complementation in which two viruses “hybridize”. Example: An adenovirus capsid is spontaneously transferred to SV-40 nucleoids (or DNA) HELPER VIRUS A virus required by a defective virus to complete its replication. It is usually closely related to the defective virus. DEFECTIVE VIRUS Virus particle that is functionally deficient in some aspect of replication, thus require a helper virus. It may interfere with replication of normal virus. PSEUDOVIRUS Normal-appearing particles under an electron microscope which are formed when the capsid sometimes erroneously encloses host nucleic acid. They do not replicate. VIROIDS A class of infectious agents, occurring in plants and perhaps in animals, that are smaller than viruses and consist of a short strand of ribonucleic acid without a capsid. THE BIOLOGICAL NATURE OF VIRUSES In 1966, Lwoff and Tournier made it clear that viruses differ from other living things, including microorganisms by 5 characteristics: 1. Mature virus particles have only one type of nucleic acid, either DNA or RNA, whereas other microorganisms possess both types. 2. Virions are unable to grow or undergo binary fission. 3. Virions make use of the ribosomes of their host cell. 4. Virions are reproduced solely from their nucleic acid. 5. Viruses lack genetic information for the synthesis of essential cellular systems such as that responsible for the production of energy with high potential. Viruses are particulate in nature; thus, they can be: a. filtered out of suspensions with appropriate filters  b. centrifuged out of suspensions with ultracentrifuges Composition of a Viral Particle 1. Nucleic Acid a. DNA viruses - all exist as single molecule, double stranded except Parvovirus b. RNA viruses – all are single stranded, except reoviruses which are double stranded - either single molecule or segmented 2. Other Chemical Components: a. Protein – makes up most of the capsid b. Lipid – phospholipid, glycolipid, neutral fats, fatty acids, fatty aldehydes cholesterol. Phospholipid predominates in the viral envelope c. Carbohydrates – all viruses contain carbohydrates since nucleic acid contains ribose and deoxyribose Properties of Viral Components: 1. Nucleic acid - carries the genetic information for the replication of the virus - the type of nucleic acid is determined thru: a. examination of intact virus or free nucleic acid Enzyme Digestion Test – detects nucleic acid type using free nucleic acid; reliable method for determining the nucleic acid type Uranyl Acetate – specific stain for DNA. It has no affinity for RNA. b. examination nucleic acid strand – determined by fixing smears of purified virus with an alcohol fixation followed by staining with acridine orange Double stranded viruses – stain red in fluorescent microscope 2. Protein - primarily exist as component of capsid Important functions of viral protein: a. Determine the antigenicity of the virus and are very much involved in immunogenic process. This function is important in vaccine production. b. Determine the relatedness of viruses. This function is important in diagnosis. c. Protect the viral genome against inactivation by nucleases in tissues. d. Participate in adsorption of the virus particle to a susceptible cell.  e. Serve as they provide structural symmetry to the virus. 3. Lipids - are found in viruses that have an envelope - ether sensitive or chloroform sensitive Pox virus is the only virus family whose envelope is ether resistant but chloroform sensitive. - In general, lipids are added to the virus particles as it matures or buds through the cell or nuclear membrane - Lipids in virus is very difficult to distinguish from the host cell lipids. 4. Carbohydrates - exist with lipid in the viral envelope - appear in the form of glycoproteins which are important parts of viral antigenic determinants - synthesis is controlled by the viral and host cell genome PHYSICAL AND CHEMICAL CHARACTERISTICS OF VIRUSES Effects of Heat and Cold - Most viruses are inactivated by heating at 56oC for 30 minutes. - The ideal way to preserve viruses is to: a. store at –60oC or lower b. store under dry ice refrigeration  Virus preparations stored in this way must be tightly closed to prevent liberation of CO2. This compound will lower ph to acidic condition wherein viruses are sensitive to. c. lyophilization - Viruses are preserved in dry state for long period of time at 4oC. - Although some heat-resistant viruses withstand this condition, some titers are lost. - Enveloped viruses are less stable than those without envelope. They do not withstand repeated freezing and thawing. Dimethyl Sulfoxide (DMSO) in concentrations above 5% provides enveloped viruses with greater stability when maintained at very low temperature. Inactivation by Vital Dyes Toluidine Blue, Neutral Red and Acridine Orange are dyes that can penetrate many viruses to varying degrees.  They can inactivate viruses, except polio virus) once they combine with nucleic acid in the presence of light. - Adenovirus and Reovirus are moderately susceptible. - Herpesvirus and Vaccinia are readily susceptible. - Poliovirus is susceptible to photodynamic inactivation. Those dyes penetrate the nucleic acid in the absence of light. Effect of Ph Most viruses are stable at ph 5-9, except FMD virus which are inactivated at ph 6.0. Stabilization by Salts  Polio virus – can be stabilized by molar concentrations of salt  Picornavirus and Reovirus – can be stabilized by 1M magnesium chloride  Orthomyxovirus and paramyxovirus - are stabilized at 1M magnesium sulfate  Herpes simplex virus - can be stabilized at 1M sodium sulfate Antibiotic Sensitivity Rifampicin – only antibiotic with viral effects; active against poxvirus, presumably acting against the RNA polymerase which is essential to pox replication Chemical Inactivants o Inorganic solvents such as ether and chloroform readily inactivate enveloped viruses. o Phenol and hexylresorcinols are excellent protein denaturants. They strip protein from some viruses and release infectious nucleic acid o Formaldehyde is an alkalyting agent employed for vaccine use. o Ethelene oxide is another alkalyting agent which is an effective virucide in a humid atmosphere o Acetylethylenemine is effective inactivant for FMD virus vaccine. o Organic iodine compounds are relatively ineffective against viruses because small amounts of organic matter rapidly deplete the active iodine. CLASSIFICATION AND NOMENCLATURE OF VIRUSES ICTV – International Committee on Taxonomy of Viruses  established at the 9th International Congress of Microbiology  responsible for classification and nomenclature of viruses Intervirology – Official journal of the virology section of the International Association of Microbiological Societies  Publish detailed characterization data of virus groups Viruses have been separated into the following, based on their physical, chemical, and biological characteristics: a. Families – names end in idae b. Genera – names end with the word virus  - members share certain common characteristics Bases in Classifying Viruses: 1. The type of nucleic acid - DNA Virus or RNA Virus 2. The number of strands of nucleic acid and their physical construction - single or double stranded; linear or circular 3. Polarity of viral genome RNA viruses in which the viral genome can be used directly as mRNA are “positive stranded”. Those by which a transcript has first to be made are termed “negative stranded”. 4. The symmetry of the nucleocapsid - helical, icosahedral, or complex 5. The presence or absence of a lipid envelope – enveloped virus or unenveloped virus 6. The site of replication - nucleus or cytoplasm 7. The virus tissue affinity Ex.: neurotropic- has affinity to nerve tissues; example: rabies virus; dermatropic – has affinity to skin tissues; example: pox virus Nomenclature of Viruses: 1. Based on the type of the disease they cause/produce Example: Pox virus Herpes virus 2. Based on acronyms Example: Picorna Virus – from the words “pico” which means small and RNA 3. Based on morphological features Example: Corona Virus - appears like crown or spike Over-all morphology: a.Icosahedral – polio and adeno viruses b.Helical – rabies virus c.Enveloped – influenza virus d.Complex – poxvirus, vesicular stomatitis virus 4. Based on the placed where they were first discovered 5. Based on the name of the discoverer REPLICATION OF VIRUSES Steps: 1. Adsorption/Attachment 2. Penetration 3. Uncoating 4. Biosynthesis/Eclipse Phase a. Transcription  b. Translation 5. Assembly 6. Maturation and Release/Egression 1. Adsorption/Attachment - An interaction between a virus and the host cell at the surface of host cell. - The first step in the establishment of infection for the production of viral progeny. - This is mediated by electrostatic charges and receptors on the surface of the cells. 2. Penetration – can only occur by endocytosis, fusion and translocation 3. Uncoating – the process of removing the envelope and/or capsid for the release of the viral nucleic acid - primarily accomplished by cellular enzymes or by intra-virion enzymes that are activated by adsorption and penetration 4. Biosynthesis/Eclipse Phase – phase of viral reproduction in which nucleic acid is replicated, transcribed, and translated into proteins a. Transcription - the synthesis of RNA from a DNA template where the code in the DNA is converted into a complementary RNA code a. Capping – the addition of a methyl group on the 5’ end of the transcript b. Adenylation – the addition of 50-200 adenine base to the 3’ end of the transcript c. Methylation – the addition of methyl groups to some adenine bases d. Splicing – creation of new RNA’s b. Translation - the synthesis of protein from an mRNA template where the code in the mRNA is converted into an amino acid sequence in a protein: Early proteins – enzymes, inhibitors Late proteins – structural proteins Post translational processing- proteins migrate to the site (nucleus or cytoplasm) of viral assembly without modification Nucleic acid replication – occurs at the same time as production of structural proteins  o occurs in the nucleus of the host cell for all DNA viruses except Poxvirus and Irido virus o occurs in the cytoplasm of the host cell for all RNA viruses except Orthomyxo viruses and Retro viruses 5. Assembly – The process by which the viral components are assembled Example: individual capsomeres form together to form capsid the site of assembly is unique to individual virus families nucleocapsid will assemble where nucleic acid is located Example: DNA viruses assemble in the nucleus except Pox Virus and Irido virus which assemble in the cytoplasm. RNA viruses assemble in the cytoplasm with the exception of Orthomyxoviruses and Retroviruses which assemble in the nucleus and then migrate into the cytoplasm. 6. Maturation and Release/Egression - Maturation for viruses generally takes place at the site of nucleic acid replication within the host cell. Maturation of DNA viruses occurs in the nucleus. Maturation of RNA viruses occurs in the cytoplasm. - Release/Egress of mature infectious virions occur by two methods in an animal system. These methods are determined by: i. the replication cycle of the virus ii. the presence or absence of an envelope Methods of Release: 1. Cell lysis – occur for non-enveloped viruses e.g.: Picorna virus - rapid Parvo virus – slow 2. Budding – occur with enveloped viruses - there is invagination of the virus from the cellular membrane into the extracellular space THE MECHANISMS AND CONSEQUENCES OF VIRAL INFECTION - Viruses are completely dependent on the living cell for their survival and replication. - The alterations caused by viruses in cells are regulated by the cell-virus relationship: a. Some viruses produce little or no alteration in the biochemical mechanisms of the cell. There is no adverse effect on each other. b. Some viruses have severe effect. It results to pathological changes. Disease - the alteration of the state of the body or of some of its organs that interrupts or disturbs proper performance of bodily functions Infection- a condition when living agents enter an animal’s body and set-up a disturbance of function in any part Contagious disease- one that can be transmitted from one individual to another by direct or indirect contact ***All contagious diseases are also infectious, but infectious diseases are not necessarily contagious*** Superinfection- refers to a fresh invasion or re-infection by a pathogen in an animal that already has as existing infection Mixed Infection- more than one species of organism is present; sometimes referred to as secondary infection. - example: shipping fever, kennel cough Properties of Pathogenic Organisms Virulence - refers to the disease-producing power of a virus, the degree of pathogenicity within a group or species - usually involves a set of morphologic, biochemical, and functional traits that must be present together for successful infection and disease production Pathogenicity - is the quality or state of being pathogenic, the potential ability to produce disease Consequences of Viral Infection in Host Cells 1. Viruses can cause cell damage which leads to cell destruction. - There are morphological alterations of the infected cell. 2. Formation of virus-specific structures called inclusion bodies  - They become larger than the viral particle. - Inclusion bodies maybe located in:  Cytoplasm for pox virus  Nucleus for herpes virus  Both in nucleus and cytoplasm for measles virus - The presence of Inclusion Bodies may be of considerable diagnostic aid. 3. In case of enveloped viruses, the release of viral particles is by budding process which is slow, and the host cell is not lysed. The cell may remain alive and continue to produce virus for long period of time. This happens in persistent infection. 4. Latent infection of the host - there is a delay of infection by the virus and the appearance of symptoms is also delayed. 5. Transformation of cells - the normal cell is changed to cancer cell - transformed cells are characterized by loss of contact inhibition, altered morphology and indefinite growth, reduced serum requirement and antigenic alteration in cell surfaces. Sources of Infection 1. Direct or immediate contact with a diseased individual - involves actual contact - ex: rabies, AIDS 2. Contact through fomites - contact of inanimate objects that serve to carry infections from one animal to another - ex: via surgical instruments, waterers, feeding troughs 3. Contact with disease carriers - more serious sources of infection than the diseased 4. Infection from the environment 5. Infection from food and water Examples: a) streams, rivers, and water holes b) silage c) offals and other feeds 6. Air-borne infections - common in the viral respiratory diseases, especially in animals kept close together, e.g., overcrowding 7. Infections from blood sucking arthropods - infectious agents may replicate inside the vector’s cell - some agents such as horsefly carry the microorganisms mechanically 8. Iatrogenic Infections - medical or surgical interferences may inadvertently result in introduction of disease-producing viruses into an infected host - examples: a) intramammary infusion of an antibiotic  b) passing of stomach tube or urinary catheter c) dehorning/debeaking 9. Nosocomial Infections - acquired during an animal’s hospitalization - majority are caused by antibiotic-resistant organisms that survive in hospital environment - important hot factors in epizootiology are: a. diminished resistance to infection due to disease or surgery b. lowered colonization resistance of GIT due to antibiotic administration 10. Endogenous infections from normal microflora - appropriate circumstances provide normally nonpathogenic organisms with the opportunity to cause disease Fates of Infecting Organisms 1. Destroyed by the host tissue - either rapidly & destroyed or undergo initial growth and multiplication in the tissues but organisms are destroyed after a short time; no extensive infection occurs 2. Eliminated in the secretion and excretions of the host e.g.- pus, droplets, feces, urine, discharges from the reproductive tract 3. Destroyed with the carcass- if the disease is fatal to the host improper disposal of dead animals can result in the serious outbreak of the disease 4. Microorganisms and host reach impasse microorganisms are unable to cause serious damage to the host and yet the host is unable to eliminate the organisms Carrier- individual that discharge virulent organisms with their excretions, although apparently normal Viral Pathogenesis 1. Portals of entry and exits a. Skin - e.g., Pox Virus, Bovine Papilloma Virus - virus enters via skin abrasions, trauma, arthropod bites or by accidental inoculation b. Respiratory tract - virus enters via inhalation, sheds in the nasal secretion and expelled by coughing and sneezing (Ex. SARS-CoV-2 virus) c. Alimentary tract - virus is ingested and expelled in the fecal material  - e.g., enterovirus, reovirus, Rota virus, corona virus, FMD, etc. d. Eye - virus infects the eye and excreted via the eye thru the ocular secretions - e.g., adenovirus e. Genito-urinary tract - virus enters via venereal transfer and causes intra-utero infection and found in fetuses and placenta - e.g., Hog cholera, Porcine parvovirus - some viruses are excreted in the urine f. Other Routes Saliva – FMD Milk – FMD Semen – FMD, Swine Parvo Dried blood – FMD Meat products – Swine Vesicular Disease Via food, water, dust & litter – HC & FMD Mechanism of Spread in the Body: Infection → Body surfaces (portals of entry) ↓ Lymph Node ↓ Blood (primary viremia) ↓ Bone Marrow ↓ Liver ↓  Spleen ↓ Blood Vessels ↓ Blood (secondary viremia) Inflammatory Response of Host to Viruses -The inflammation that accompanies viral infections is secondary to primary cellular alterations. Inflammatory Responses:  Viral infection – infiltration of mononuclear cells, including macrophages, lymphocytes, and plasma cells  Bacterial infection- polymorphonuclear leukocytes predominate in lesions of acute bacterial diseases - There is edema. - In rabies, the neuronal cells are destroyed and there is no inflammatory response. - Viral infection of cells causes chromosome damage, little breakage, fragmentation, and rearrangement of chromosomes. - Viral infections are recognized by a number of non-specific constitutional disturbances including: a. fever c. anorexia e. headache b. myalgia d. malaise These signs are attributed to a number of factors such as: i. absorption of degranulation product from the injured cell ii. viral toxicity iii. degree of viremia - Death due to viral infection is usually due to: 1. vascular shock 2. viral toxicity 3. functional failure of vital organs ANTIVIRAL CHEMOTHERAPY Points of Action of Antivirals in the Virus’ Life Cycle: 1. Binding to the free virus particle.  2. Interference with virus adsorption or attachment to the receptor binding site of the cell. 3. Inhibition of virus uncoating and release of nucleic acid. 4. Inhibition of viral nucleic acid transcription and translation. 5. Interference with cellular processing of viral polypeptides 6. Prevention of virus budding. Deterrents to the effective treatments of viral disease: 1. Strict parasitic relationship of the virus and its host cell - Virus depends on the metabolism of the host cells for its replication - Majority of virus inhibitors act against cellular processes - A useful virus inhibitor must prevent completion of the viral growth cycle in the infected cell without causing lethal damage of the uninfected cells. - Desired effect can be achieved by a compound that acts directly on the component of the virus or on a viral product that is essential for successful viral replication 2. Nature and pathogenesis of viral diseases - Many viral diseases may be recognized too late for effective treatment with a virus inhibitor. Antiviral Agents and Mode of Action 1. Analogues of ribonucleosides and deoxyribonucleosides - Inhibits RNA and DNA synthesis - bloxuridine and trifluorothymidine - vidarabine - acyclover - ganciclover - zidovudine - ribavirin 2. Amantidine and Rimantidine - interferes viral replication by blocking penetration; no effect on adsorption - used effectively in prophylaxis. - little of no effect therapeutically, during the course the disease - for members of Ortho and Paramyxo groups, Pseudorabies virus, etc. 3. Other Antiviral Agents a. Isatin-B-Thiosemicarbazone (IBT) -inhibits Poxviruses -interferes with synthesis of late proteins so that no progeny of virus particles are formed b. 2-hydroxybenzylbenzimidazole and Guanidine  - against Picorna, Polio, and FMD Viruses - interfere with replication by blocking the synthesis of viral RNA polymerase, thus preventing formation of viral protein and viral RNA - No effect when administered after synthesis of RNA polymerase c. Rafampicin and related derivatives -prevents initiation of transcription by binding to the viral RNA polymerase but does not bind to animal RNA polymerase d. Arildone and Rhodanine -inhibits uncoating of Picorna viruses e. Actinomycin D -inhibits replication of DNA viruses and some RNA viruses f. Disodium phosphonoacetate -promising, stable, nontoxic antiviral drug that selectively inhibits DNA- dependent DNA polymerase essential for DNA replication -specific for viral enzyme of several herpesviruses with little effect on cellular DNA synthesis LABORATORY DIAGNOSIS OF VIRAL INFECTIONS A. ISOLATION OF VIRUSES Purposes: 1. To identify a virus of concern in a herd program 2. To point out a possible public health problem (e.g., Rabies) 3. To establish a viral etiology of a disease 4. To determine the immunological type of a given virus when epidemics occur (e.g.FMD) 5. To pinpoint the exact agent when serologic test methods fail - When agent shares common antigen with other viruses - Knowledge of the signs produced by each virus is important to establish a primary disease agent Collection of Clinical Material a) Time of Collection - acute stage of illness is the ideal time to collect specimens for virus isolation - immediately after death is also ideal  - viruses isolated from a dead specimen which has been dead for several hours may not reflect the true cause of the disease b) Materials to be Collected - depend on the nature of diseases - blood, nasal swabs, nasopharyngeal swabs, feces, urine, pus, vesicular fluid, spinal fluid, biopsy specimen, autopsy specimens - Virus is usually excreted in the nasal or pharyngeal secretions of animals in acute stages of respiratory illnesses. - Pox lesion contains viruses in the fluid and scabs. - Viremic animals contain viruses in their blood. - Diseases involving CNS carries the virus in the blood, nerves or in the brain. - All body excretions contain virus during the acute stage of the illness. c) Manner of Collection All specimens should be taken with sterile instruments in a sterile manner. If there is a need to collect tissue sample from different sites, separate sterile instruments must be used for procuring each tissue sample. Handling of Clinical Materials One who handles tissue samples suspected with viral diseases must know the biophysical and biochemical properties of viruses. Example, viruses are heat sensitive and acid sensitive. i. for fresh tissues freeze the tissue sample at –60oC or lower immediately or at -20oC until dry ice is obtained ii. for shipment:  place the specimen in a wide-mouthed thermos jug or polysterene  insulated carton and fill with dry ice or with 50% glycerol iii. for small pcs of tissues, fecal materials of mucus – place in tight, sterile, screw-cap vial with 50% glycerol  store at 6oC  if stored in dry ice, vial should be airtight because the gaseous phase of dry ice is CO2 which changes the ph of fluids and inactivate ph-labile viruses Preparation of Clinical Materials Before Viral Isolation Substrates for Viral Isolation: 1. susceptible animals 2. cell cultures 3. embryonated hen’s egg  - Fluid specimens can be inoculated directly or after dilution with a buffered solution (pH 7.2-7.6) - Solid tissues must be prepared into suspensions. Coarse particles may tend to plug to inoculating equipment so they must be centrifuged at 2,000 rpm for 10 minutes to remove them from suspensions. Removal of other Microbes: 1. Chemical Means a. Ether – bactericidal at 10-15% for 1-2 hours b. Penicillin (100IU/ml) - Streptomycin (100g/ml) is employed media. It can also be given to test material as pre-treatment to eliminate contaminating agents. c. Proflavine dye – 10-4 M for 1 hour at ph 9 at 37oC. However, this must be removed by resins before inoculation. - It has little or no effect on Enteroviruses and Rhinoviruses. - It photosensitizes bacterial and fungal contaminants when exposed to light. 2. Mechanical means a. Ultrafiltration – using earthenware, porcelain and asbestos filters b. Differential Centrifugation - convenient and excellent method 18,000 rpm for 20 min – precipitates bacteria 40,000 rpm for 60 min – precipitates virus 1. Isolation of Viruses in Embryonated Hen’s Egg a. Yolk sac inoculation - for large elementary-body agents - 5 to 7 days old, fertilized chicken eggs - smaller viruses can also be inoculated by yolk sac route, but they invade the embryo and multiply in the body tissues rather than in the yolk sac b. Chorioallantoic cavity inoculation - used when viruses causing respiratory infection are suspected - e.g.: Influenza virus NCD virus - those viruses that grow readily in the entodermal cells of the chorioallantoic sac wall of 8-11 day old embryonated hen’s eggs are liberated into the chorioallantoic fluid - also used in encephalomyelitis viruses and the mumps virus c. Chorioallantoic membrane inoculation - This method is applicable in 7–10-day old embryo.  - Pox virus produces lesions that are discrete and can be counted while Canine Distemper virus produces lesion that is less clearly defined. d. Amniotic sac inoculation - Used principally for the isolation of influenza virus from throat washings - Also used for the isolation of the encephalomyelitis viruses - applicable to 7-15 days of age chicken embryo e. Intravenous inoculation - Applicable to 12–14-day old embryos - Used for adoption and replication of some strains of foot and mouth disease virus. 2. Isolation of Viruses in Tissue Culture - Most widely used methods for isolation of viruses from clinical materials. - The evidence of viral replications are the following:  cytopathic effect  viral interference  hemadsorption  hemagglutination  presence of fluorescent antigen  presence of complement-fixing antigen 2 approaches: 1. Monolayer Cell Culture- single layer cells attach to a glass or plastic surface 2. Suspended Organ or Tissue Culture - whole organ or parts of the organ are grown and maintained - It is quite difficult because it is necessary to consider the cell, its nutritional requirements, its susceptibility to viruses, as well as the virus itself - However, some viruses such as Corona virus replicates only in organ tissues 5 Basic Methods: 1. Suspended Cell Method - An old method but still employed for production of vaccines for FMD 2. Plasma Clot Cell Method - A film of plasma (chicken plasma) is layered into a glass surface and induced to clot by the addition of embryo extract. 3. Monolayer Cell Culture - Uses microtiter sterile plastic assay plates  - It is useful in titration test, neutralization test and studying virus growth in cell 4. Direct Cell Culture - same procedure as monolayer cell culture - commonly used in cells taken from biopsy and autopsy of diseased animals 5. Organ Culture - uses culture derived from embryonic respiratory tissues 3. Isolation of Viruses in Live Animals There is a need for proper selection of a susceptible host. Natural host are best animal to be used but uneasy. Suckling animals are preferred because they develop recognizable infection than older animals. Laboratory animals can be utilized to substitute the natural host. Inoculation route must be directed into the organ or tissue where virus produces lesions in the natural host. Example: In encephalitic animal, brain materials may be taken and be inoculated directly into the mice through intracerebral inoculation. a. Intracerebral inoculation- used to isolate neurotropic viruses. It requires 30 days observation time. Mice, guinea pigs and rabbits are commonly used. Control animals are necessary for comparison of results. b. Intranasal inoculation – intended for pneumotropic viruses. Mouse is the animal of choice. It requires 15 days observation time. c. Intraperitoneal inoculation – preferred in isolating Chlamydial and Rickettsia organisms. Guinea pigs is the animal of choice. B. SEROLOGIC DIAGNOSIS OF VIRAL DISEASES 2 Indispensable Elements: 1. antigen 2. antibody Methods: 1. Serum Neutralization 2. Complement Fixation 3. ELISA 4. HA-HI 5. Others – Hemadsorption-Inhibition, Precipitation, Agglutination, Immunodiffusion, Radioimmunoassay, Indirect Hemagglutination, Flocculation Test Methods for quick and accurate diagnosis:  1. FAT 2. ELISA –used for ag-ab systems that can be adapted to automated procedure. 3. FVPT –fluorescent virus precipitin test Preparation 1. Collect blood samples in sterile sealed units, preferably B-D vacutainer. 2. Allow to clot at room temperature for a few hours before refrigeration. 3. Decant the serum from the clot into a sterile centrifuge tube. 4. Centrifuge serum sample at 2,500 rpm for 20-30 min. 5. Remove serum and place in a sterile vial without disturbing RBC. 6. Label the sample with source and date obtained. 7. Freeze at –10 to –20oC until the analysis. If it requires shipment, ship it under refrigeration Note: Do not freeze the blood sample before the serum is harvested. C. DIAGNOSIS OF VIRAL DISEASES BY ELECTRON MICROSCOPY Diagnosis of viruses using electron microscopy is not effective in detecting small concentrations of virus. Proper detection of viruses depends on the concentration of viruses: 1,000,000 viruses/ml –just detectable 10,000,000 viruses/ml- greater degree of detection 1,000,000,000 viruses/ml – permits rapid diagnosis One who examines viruses in electron microscopy must have enough knowledge of the viral morphology. Immune Microscopy Technique – has enhanced sensitivity and accuracy - viral antiserums are mixed with the suspect viral specimen. Positive result reveals virus particles that bind together by antibody, producing clumps PART II. DNA VIRUSES A. FAMILY PARVOVIRIDAE Parvovirus 3 Genera: 1. Parvovirus 2. Dependovirus 3. Densovirus  GENUS PARVOVIRUS Contain linear, single-stranded DNA Nonenveloped; do not contain lipids Cube-shaped - 18 to 26 nm in diameter - contains 32 capsomeres in a nucleocapsid. Each capsomere is 3-4 nm, arranged in icosahedral symmetry Resistant to many environmental and chemical agents - Stable at wide ph range (3-9) - Stable at wide temperature range (withstand 56oC for 60 minutes) - Resistant to lipid solvents and disinfectants Susceptible to ultraviolet light and inactivated by Aldehydes, beta propiolactone, hydroxylamine, and oxidizing agents such as Na hypochlorite During replication, they do not code for enzymes required. They only rely on the enzymes in the host cell to code their enzymes. Transcription of DNA occurs in the nucleus. mRNA is transported in the cytoplasm where synthesis of protein occurs. Then the proteins are transported back to the nucleus where assembly and maturation occurs. Complete growth cycle occurs in 20- 24 hours. Produce Cowdry type A intracellular inclusion bodies that thickens the nuclear membrane. It appears hollow/clear nonstaining area around the nucleolus and inside nuclear membrane. Significant pathogens: 1. Feline parvovirus or Feline Panleukopenia Virus 2. Canine Parvovirus 3. Porcine Parvovirus 4. Mink Enteritis Virus – biological variant of Feline Parvovirus 5. Others: Bovine Parvovirus, Goose Parvovirus, Gastroenteritis Virus of Humans DISEASES CAUSED BY PARVOVIRUSES 1. Feline Panleukopenia (FP, FPL) Caused by Feline Panleukopenia Virus Highly contagious viral disease of domestic and exotic cats. Most devastating disease of cats known in unvaccinated populations. Characterized by sudden onset depression fever dehydration anorexia leukopenia vomiting often high mortality diarrhea Incubation period is usually 4-5 days. Forms of the disease: a. Peracute Form shows sign of acute poisoning with 100% mortality and subnormal temperature. b. Acute or Typical Form - sudden onset of clinical signs  - 40oC or higher - severe depression and complete anorexia - severe vomiting with fetid diarrhea and blood casts on feces that may lead to severe dehydration and electrolytes imbalance. - 25-90% mortality - Characteristic attitudes: 1. hunched with head between paws and hang over a water or food disk 2. third eyelid is prominent 3. abdomen is tender with pain upon palpation 4. mesenteric nodes are enlarged, and GIT contains excess gas and fluids c. Subacute or mild form – no clinical signs but pregnant cats will abort, fetal resorption or deliver mummified or stillbirths d. Neonatal form – involves kittens infected during late gestation or the first few days after birth - sudden death without sign or develop ataxia at ambulatory age (2-3 weeks) e. Chronic form – confusing with leukemia virus infection Properties of Feline Panleukopenia Virus (FPV) - small, 20-24 nm - single stranded DNA - susceptible to 1:32 or stronger dilution of commercial Na hypochlorite (5-6% or as household bleach) Epizootiology –worldwide Pathogenesis – depends on the state of mitotic activity of various tissues. It requires actively dividing cell to complete its replicative cycle. Oral route ↓ Lymphoid tissues of oral pharynx (primary infection) ↓ Regional lymph node - viremic within 24 hours after ingestion ↓ Overall, the body – epithelia crypt cells of ileum and jejunum; cytolytic replication destroys epithelial lining of crypt, becomes ballooned, blunted villi - uterus and fetus of pregnant queen - other tissues with rapidly dividing cells: thymus, bone marrow, lymph node Immunity - rapid and solid response  - recovered cats from natural infection are immuned for life Diagnosis - based on the history, clinical signs plus leukopenia which occurs in all infected cats - confirmed by viral isolation, identification, or viral antigen with immunoflourescence tests, serologic tests or pathological changes Materials/specimens: 1. swab (pharyngeal or rectal) – place in a viral transport medium and refrigerate. Submit to laboratory. 2. tissues (spleen, thymus, ileum or mesenteric lymph nodes) 3. serum - serologic tests; ELISA requires paired serum: one during the acute phase and another one 2 weeks after Gross Pathological Changes: 1. Evidence of dehydration – sticky dry tissues, sunken dry eyes 2. Small Intestines: a) hose-like appearance, turgid, edematous and dilated. b) petechial hemorrhages or hyperemia on serosal surfaces 3. Scanty and watery feces which is yellowish gray with fetid odor 4. Hemorrhagic mesenteric lymph nodes 5. Gross reduction of cerebellum size Treatment Objective: to keep the animal survive for 5-7 days. Management: 1. give symptomatic treatment a. Vomiting and diarrhea leading to dehydration - restore fluid and electrolyte balance by: i. preventing loss of fluid –antiemetic or anticholinergic ii. replacing lost fluid b. Coagulation disturbance – i. heparin ii. whole blood transfusion c. Ataxia – no treatment in case of neonatal from 2. prevent secondary bacterial infection - give broad spectrum antibiotic parenterally until gastroenteritis is controlled. Then follow up orally 5 days thereafter. 3. give supportive treatment ex: good nutrition –while food is withheld until gastroenteritis is controlled, give supplements. After controlling gastroenteritis, give baby food in small amount but frequent.  Prevention Immunize at 8-9 weeks old as initial dose then give booster dose 4 weeks after the first dose 2. Canine Parvovirus Infection Caused by Canine Parvovirus Synonyms are: CPV-1 CPV-2- most pathogenic type Minute Virus of Canines (MVC). Number 1 disease that affects dogs Severity varies subclinical, acute, severe or fatal Properties of CPV – same with FPL virus Two main syndromes: a. Acute Enteritis –with similar signs to cats with FPL - Signs are severe in pups. Fluid stool is light gray or yellow gray with or without hemorrhages - Temperature is 40-40.5oC - Shock-like or sudden death of puppies – as early as 2 days after the onset of illness. - Leukopenia- 500 to 2,000/dl is common at peak of illness b. Myocardial Disease - common to un-immune dogs, characterized by necrosis of myofibrils - 2 forms: i. characterized by sudden death (acute). Puppies less than 8 weeks old are healthy appearing. 70% of the puppies in the litter is affected. They show sign of respiratory distress and attempt to vomit. Cardiac arrythmias are present. ii. affects 30% of the litter characterized by subacute heart failure, exercise intolerance and subclinical arrythmias. Epizootiology and Pathogenesis: - CPV-2 - shed by carrier dogs in feces periodically - endemic once initial episode occurs - Sporadic in unvaccinated pups after losing passive immunity - Also infect other members of the canid family: coyotes, wolf, and red foxes - Transmitted indirectly via fecal-oral route and directly via dog-to-dog contact.  Mouth ↓ Tonsils → spread in the lymphatics (primary viremia) ↓ all mitotic cells in the body: day 1-thymus day 2 to 3-germ centers of the lymph nodes day 3 to 4- intestines lesions:  necrosis of crypt epithelium of jejunum and ileum  loss of epith’l cells and dilation of the remaining crypts  presence of inclusion bodies in the epithelial cells  In advance cases: epithelium regenerates; inflammatory cells infiltrate in lamina propia Fecal excretion of CPV:  3 days after infection  peak at 4 to 7 days  decreased after 7 days; then there will be appearance of viral neutralizing antibody in serum Immunity - rapid and effective humoral response - recovered dogs are solidly protected for long periods Diagnosis - Enteritis: a) clinical signs b) hematological results – leukopenia, usually lymphopenia c) microscopic lesions d) confirmatory: laboratory findings: ▪ viral isolation ▪ seroconversion of ab titers to vs electron microscopy - Myocardial Ds: a) clinical signs b) laboratory findings:  echocardiography, ultrasonic  cardiography or radiography  clinical chemistry  - increased enzyme activities that suggest heart muscle involvement: i. AST-Aspartate Aminotransaminase ii. SGOT- Serum Glutamic Oxaloacetic Transaminase iii. LDH –Lactic Dehydrogenase iv. CPK-Creatinine Phosphokinase Treatment - extensive fluid and supportive therapy to allow sufficient time for an effective antibody response - hyperimmune serum treatment during the first 4 days after infection increases recovery rate - antimicrobial treatment may harm normal flora of the intestine. It cannot replace fluid treatment - there is no effective treatment in myocardial disease Prevention 1. Immunization – 16 to 18 weeks because vaccine is interfered by maternal antibody 2 vaccines available: ▪ modified live CPV vaccine ▪ inactivated CPV vaccine 2. Disinfection – using commercial bleach or aldehyde preparation 3. Control of animal movement 4. Good husbandry practices 3. Bovine Parvoviral Infection - caused by Bovine Parvo Virus, a ubiquitous enteric virus of cattle - characterized by: a. subclinical infection in calves b. fetal infection and reproductive failure of pregnant cows - In calf:  the incubation period is 1-2 days  diarrhea is the main sign; watery then become mucoid  fever at 41oC; some becomes stunted, others recover without side effects  conjunctivitis  respiratory disease - In pregnant cows: ▪ abortion Property of virus similar with FPV Pathogenesis -cattle is the only known host  -affects jejunum. Ileum and cecum; also in spleen, adrenals, LN, thymus and heart muscle Diagnosis -clinical diagnosis is not possible 1. lab isolation of virus 2. serologic confirmation 3. electron microscopy 4. Porcine Parvovirus Infection - caused by Porcine Parvovirus - characterized by: a) subclinical infection in adult and young b) severe reproductive failure in pregnant sows and gilts Subclinical infection:  postnatal oronasal transmission  virus replicate to high titers in tissues containing rapidly dividing cells, significant in lymphoid tissues  do not involve intestinal crypt, unlike other parvo viruses  exhibit only transient leukopenia with no harmful effects  usually goes unnoticed - Maternal reproductive failure is the only significant disease produced ▪ transplacental transmission ▪ embryonic death (mummification) ▪ fetal resorption - No evidence of reproductive disease in boar Properties Of the Virus - cubic symmetry with 32 capsomeres on virion - virions are released through cytolysis - it hemagglutinates erythrocytes - uses guinea pigs in assay Pathogenesis IP=10 to 14 days Stage of infection Effect 30 days embryo Resorption 56 days embryo Death of fetus 30-70 days embryo Death and mummification 70 days and older Survive, occasional neonatal death & stillbirth Immunity - long-lasting (lifetime for recovered sow)  Prevention and Control 1. Immunization  Inactivated vaccine – 2 doses, 2-4 weeks apart at 4-8 months (before breeding of gilt/sow)  Modified live-virus –single dose before breeding 2. Disinfection of facilities – Na hypochlorite 5. Aleutian Disease in Mink - caused by Aleutian enteritis Virus - an immunological disorder caused by persistent parvovirus infection - Characterized by:  persistent viremia  pronounced plasmacytosis  hypergammaglobulinemia  progressive immune complexes - Incubation Period is long – few to many months - Ocular lesions –due to deposition of immune complex ▪ uveitis ▪ iridocyclitis with cellular infiltration of the limbus - same properties with Porcine Parvo virus FAMILY PAPOVAVIRIDAE - small (42-55 nm) - non-enveloped - double-stranded DNA 2 genera: 1. Papilloma Virus Small (50-55 nm) Produces papillomatosis (warts) in animals and humans 2. Polyoma Virus Small (42-45 nm) Non-pathogenic for most part of their natural host. It can produce tumors in newborn rodents GENUS PAPILLOMA VIRUS - Double stranded cyclic DNA - Icosahedral virions - Capsid is composed of 72 capsomeres, with skew arrangement Resistance: a) environmental conditions (acid, heat) b) ether Viral Replication:  - do not readily reproduced in cell cultures - replication and assembly of the virions occur in the nucleus - Have predilection on epithelial cells - Infection of cells causes transformation of cells that may result to tumor or wart formation. Infection is species-specific and restricted to certain defined epithelial tissues. - Principal viruses in the genus are oncogenic, especially in newborn or young animals. DISEASES CAUSED BY PAPILLOMA VIRUSES Bovine Papillomatosis Caused by Bovine Papilloma Virus Synonyms: Fibropapillomatosis/ Infectious Papilloma/ Warts Common viral disease of the skin of young cattle Manifested by benign tumors or warts in: ▪ alimentary tract, larynx, urinary bladder, and eye ▪ teats and udders of cows ▪ penis of bulls ▪ skin in young stocks less than 2 yrs old - most common Characteristic of the Disease: - IP is 3 to 8 weeks or longer - Papillomas – common warts; small nodular epidermal growths that may develop slowly for time or grow rapidly into dry, horny, whitish, and cauliflower-like masses o Finally, it will fall off because of dry necrosis on their bases - Skin wart – most frequently occur in head, particularly in the region around the eyes. It also occurs at the sides of the neck. Other parts of the body are less commonly affected o It generally regresses spontaneously without causing any harm. - Warts in udders and teats of cows or penis of bulls - will interfere milking and breeding because usually they are pedunculated and large. They are also persistent. Properties of the Virus: There are 6 serotypes: 1. BPV-1- subgroup A and produces fibropapillomas 2. BPV-2 -do- 3. BPV-5 -do- 4. BPV-3- subgroup B and produces true epithelial papillomas 5. BPV-4 -do- 6. BPV-6 -do- Epizootiology and Pathogenesis Transmission:  direct –by contact of infected animals  indirect – abrasion from contaminated inanimate objects   predilection: – at the basal cells of the epithelium. In the process of replication, these viruses cause excessive growth of the epithelium to form characteristic benign tumor or wart. Immunity  Develops after several months, then skin lesions regress spontaneously.  Once cattle eliminate skin warts, they appear resistant to re-infection.  Teat warts increase with age Diagnosis  made from the clinical signs  confirmed by biopsy and histopathological exam Treatment  Generally, it does not require treatment because the virus is self-limiting. However, if it interferes breeding or milking, the following may be done: a) cryosurgery b) give autogenous vaccine intradermally to affected cattle at 1.5 to 2.0 ml once a week for 3 weeks Prevention and Control  Vaccination – using commercial wart vaccine is beneficial in some outbreaks - In some cases, it produces little or no protection Good Management Practices 1. eliminate sharp objects 2. do not interchange halters, ropes, and brushes 3. segregate affected cattle 4. disinfect contaminated objects Equine Papillomatosis - Caused by Equine Papilloma Virus - Affects skin of horse and mule - Appear most commonly on the nose and around the lips as small, elevated, horny masses that vary in number from few to several hundreds. If there are few of them, they may be larger than 1 cm. - Cannot be transmitted to other species of animals - Natural infection produces solid immunity - Congenital papillomas may occur if there is infection from uterus Caprine and Ovine Papillomatosis - Caused by Caprine and Ovine Papilloma Virus - Goats – warts rarely occur in the skin, teats, and udder - They are spread by milking. - Sheep – lesions occur in face, legs or on hairy skin Canine Papillomatosis - caused by Canine Papilloma Virus - Tumors are found around the lips and in the mouths. They cause serious inconvenience and interfere mastication. - Highly contagious to entire kennel. Also infects coyotes Signs: - reluctant to eat - halitosis from necrotizing tumors - bouts of hemorrhage from the mouth due to trauma - ropy oral discharge Treatment - disappears 1-5 months without treatment - If masses interfere with mastication, swallowing and/or breathing, surgical removal is indicated. - Cryosurgery may prevent the spread of virus to new sites. Prevention - Segregation of infected dogs from susceptible dogs Rabbit Papillomatosis 2 types: a. Rabbit Cutaneous Papilloma  caused by Cotton Tail Rabbit Papilloma Virus  known as Shope Papilloma  occurs on skin, never found on the mucous membranes of the mouth  Sites of predilection: o sides of neck o shoulders o abdomen o inside of thighs b. Oral Papilloma  Caused by Oral Papilloma Virus  occurs in oral mucosal, never found in skin  found under the surface of: o tongue o gums o floor of mouth Immunity - The two types of viruses are not related to each other immunologically. One cannot produce immunity for each other. Genital Papillomatosis of Pigs - Caused by Porcine Papilloma Virus - a non-enveloped DNA virus that causes transmissible genital papilloma in swine - Affects genital regions of boars Cleaning and Disinfection: - Papillomaviruses survive well in the environment and remain infective following exposure to lipid solvents and detergents, low pH, and high temperatures Transmission - Transmission of SPV is sexual and occurs through natural servicing or artificial insemination of gilt/ sow by an infected boar Diagnosis - Can be isolated in porcine kidney cell lines - Polymerase chain reaction can be used to amplify the virus Immunity - Disease caused by SPV is limited and resolves after a matter of weeks, once a sufficient anti-viral - Immune response has developed No vaccine available for SPV Prevention and Control - To prevent transmission of SPV, semen can be screened before a boar is used for breeding Gaps in Preparedness - There is little known about SPV. Although it seems to be a mild, self-limiting disease, more research is necessary to understand the potential impact of the infection on the reproductive health of swine herds. - Studies are also needed to determine the best practices for SPV disinfection FAMILY ADENOVIRIDAE I. Introduction  Historical Background Wallace Rowe et al. (1953) observed that explant cultures of human adenoids degenerated spontaneously. After thorough investigations he and his team successfully isolated a new virus that they named “adenovirus”. The follow year, Cabasso et al. (1954) found that the causative agent of infectious canine hepatitis was an adenovirus. Most of these viruses were observed highly host-specific, collected from humans and many other mammals and birds, usually from the upper respiratory tract, but sometimes from feces.  The virus was discovered on cell cultures of tonsils and adenoidal tissue from children. Hence, until now called the “Adenovirus”.  Many produces subclinical intestinal infection, subclinical respiratory infections, however, few produce severe systemic disease.   Taxonomy Family ADENOVIRIDAE Two genera: Mast adenovirus Aviadenovirus  Serotypes 40 serotypes of human adenovirus, divided into 4 subgroups in accordance to their agglutinating ability in rhesus monkey and rat blood cells. Mammals: 2 canine, 16 simian, 9 bovine, 1 equine, and 2 mouse adenovirus serotypes. Birds: 11 fowl, 4 turkey, and 3 goose  Description Virions: Double stranded 23,000 daltons Isometric, nonenveloped particles have icosahedral symmetry Each penton carries filamentous fiber 70-90 nm diameter, with 252 capsomeres- each 7nm in diameter.  Resistant to solvent (e.g., ether); but inactivated by many disinfectants  Density of 1.34 g/mL in cesium chloride  Mammalian antigens differ from avian antigen  In monolayer cultures, form grapelike cluster  Lightly host-specifity  Generally, causes ocular and respiratory disease  Many produces mild or subclinical respiratory diseases.  Some produces mild or subclinical intestinal infection  Few produce severe systemic diseases  Contains double – stranded DNA  Non enveloped  Icosahedra symmetry  70 to 90 nm  252 capsomeres, 7nm each o Pentons – 12 vertex capsomeres that are antigenically distinct from other  240 capsomeres (hexons). Each penton carries a filamentous  Projection of fiber.  Resistant to ether but can be inactivated by many disinfectants  Viral assembly takes place in the nucleus of the cell, where IB are seen.  Oncogenic – under certain conditions - produces cytopathological pattern in monolayer cell cultures with marked rounding of cells that form aggregates in grapelike clusters DISEASES OF ADENOVIRUS Genus Mastadenuvirus 1. Infectious Canine Hepatitis 2. Canine Adenovirus 2 Infection  3. Bovine Adenovirus Infection 4. Equine Adenovirus Infection 5. Porcine Adenovirus Infection 6. Ovine Adenovirus Infection I. Characteristics of Infectious Canine Hepatitis - Caused by Canine Adenovirus Virus 1 (CAV-1), the first canine adenovirus officially recognized Syn. Canine adenovirus I infection, fox encephalitis, hepatitis contagiosa canis, and Rubarth’s disease - a highly contagious diseases of domestic and wild canids caused by Canine Adenovirus type 1 (CAV- 1). - was described as an acute disease of young dogs with severe hepatitis, edema of the gallbladder, tonsillitis, multifocal vasculitis, and hemorrhage. - most infections mild; unvaccinated dogs suffer severe systemic disease, mainly the liver. routine vaccination was the mitigating method for this previously common disease - Does not infect humans - Acute disease of young dogs (newly weaned puppies) with severe hepatitis, edema of gallbladder, tonsillitis, multifocal vasculitis and hemorrhage - Less effect on respiratory tissues Characteristic: Per acute Course/Form  Dogs become moribund within hours of the first signs of clinical illness  Resembles poisoning 30 Typical Case/ Form  Biphasic febrile response (39.4-40.7oC)  Apathetic  In appetent/anorectic  Shows intense thirst  Sometimes exhibit edema of head, neck & lower portion of abdomen  Vomiting & diarrhea are common  Painful abdomen  Leucopoenia and early febrile state  Transient opacity of cornea (BLUE EYE) due to edema & anterior uveitis. Afghan Hound has predilection to eye lesions.  Mucous membranes are usually pale and petechial or ecchymotic hemorrhages may appear on the gums or other mucous membranes.  albuminuria Encephalitic form - occurs among wild foxes  Loss of appetite is noted a day or 2 before other signs  Violent convulsions - initial sign  Lethargy with common watery nasal and eye discharge   Feces is soft & filled with mucus- sometimes with profuse diarrhea w/ blood streaks  Rapid course- 1 hour or usually less than 24 hours  Few cases last for 3 days. Properties of Virus Resistance: a. Survives well when frozen or dried b. Survives between ph 3-9 at room temperature c. Ether & chloroform resistant d. Survives for days in 0.5% phenol Susceptibility: a. 0.2% formal in at 24 hrs b. 50oc after 150 minutes c. 60o c in 3-5 minutes Cultivation - Successfully cultivated in roller tube cultures of dog kidney CAV-1 host species: Domestic and Wild canines Clinical signs Domestic pop. - Frequent in puppies - Loss of appetite (1-2 days) - Weight loss - Abdominal pain - Petechiae of oral mucosa - Violent convulsions - Lethargic, wanders aimlessly and blindly interrupted with convulsions - Watery eyes and nasal discharge (common) - Transient opacity of the cornea, “blue eyes” (due to edema and anterior uveitis)- result to photophobia - Feces is soft & filled with mucus. - Hepatomegaly - Acute inflammation of the tonsils - Albuminuria Clinical Signs in Wild pop. - ICH disease in Vulpes spp. - Transmittable to dogs - Occurs short as 1 hour and usually less than 24 hours, leads to death - Intermittent convulsions - Paralysis of limbs - Little lost weight due to the rapidity of the course Viral Properties of CAV-1 - Icosahedrons with 252 subunits-capsomeres (typical for adenovirus)  - Diameter: 75-80 nm - Double Stranded - Survives when dried or frozen - Inactivated in 24 hours by 0.2 percent phenol - Inactivated in at 50°C after 150 minutes/60°C in 3-5 minutes - Ether & chloroform resistant - Survives pH 3 and 9 at room temperature - Hemagglutinates chicken red blood cells at 4°C and pH 7.5 to 8 - Hemagglutinates rat and human type O blood cells at pH 6.5 to 7.5 Epizootiology - CAV-1 transmits by “direct contact” from infected and susceptible dogs via urine- contaminated environment/contaminated fomites e.g., feed and water dishes. - Major source of transmission is URINE - Incubation Period: 4-9 days (IV-2/3 days; SQ- 3/4 days; orally-4/6 days; direct contact-6/9 days) - Unlike canine distemper, ICH is not transmitted by droplet inhalation - Transmitted by: - Direct contact of infected & susceptible dogs - Indirect contact with urine-contaminated environment e.g., fomites - food & water dishes - Transmitted via Oro-nasal exposure - Short course of disease - Progress is more rapid than Canine Distemper (CD) - 10 – 25% mortality Natural Infection ( oro-nasal exposure) ↓ Tonsils (localize & replicate) ↓ Regional Lymph Node ↓ (through lymphatic) Blood stream → liver, kidney main target areas for eye replication and endothelium pathological damage - Tissue injury is caused by cytolysis as virus replicates - Has predilection for endothelial cells; vascular damage results to petechial & ecchymotic hemorrhages as in Disseminated Intravascular Coagulation (DIC) Pathogenesis - Cytolysis: principal pathologic process of in the vascular system - Disseminated intravascular coagulation (DIC) features thrombocytopenia, altered platelet activity, prolonged prothrombin time, depressed factor VII activity, and increased fibrinogen degradation products - Sufficient damage of liver shows hepatic necrosis  - Basic Pathogenesis of ICH 1. CAV-1 localizes and replicates in the tonsils 2. Spreads to regional lymph nodes to the blood 3. Target organ for viral replication and pathologic damage liver, kidney, eye and endothelium of several organs Necropsy Findings - No evidence of emaciation - Liver - somewhat swollen & light in color with stiff capsule; more prominent than normal lobule - Blood vessels and sinusoids – greatly dilated and blood content is increased - Endothelial cells & Kupffer cells – greatly swollen with Cowdry Type inclusion bodies - Gall bladder is thickened - Spleen is enlarged Immunity - CAV-1 protects dogs against itself and CAV-2 - CAV-2 protects dogs against itself and CAV-1 Diagnosis - Presumptive diagnosis base on clinical signs and clinicopathological findings - Virus isolation in cell cultures - Serologic test - Gross and microscopic lesions at necropsy Treatment - No specific antivirus therapy has been proved affective - Fluid therapy in severe cases - IV of fresh whole blood (to counter ICH) - Hypertonic glucose infusions (should be administered to counteract hypoglycemia) Prevention and Control - Inactivated or attenuated CAV-1 vaccines followed by canine distemper vaccines and other vaccines as indicated by manufacturer - CAV-2 vaccines - Vaccinate puppies at 8-10 weeks and 4 weeks later - For wild canids (i.e., fox) immunize individuals using hyperimmune homologous serum with CAV-1 vaccine II. Characteristics of Canine Adenovirus 2 Infection Syn. Canine infectious tracheobronchitis (CIT,CITB), infectious canine laryngotracheitis virus infection, kennel cough, Toronto A26/61 virus infection - Respiratory diseases caused by CAV-2 virus - Most common respiratory pathogens in dogs - Infections are mild but complications occur when mix infections occur with other virus or bacteria  - Chronic yet mortality is low - Usually occurs in kennels, adoption centers, and pet shops - Mild to in apparent respiratory disease of young dogs - Signs include rhinitis, tonsillitis, pharyngitis and Tracheobronchitis - Virus has strict affinity for epithelial cells lining the respiratory tract; it fails to produce hepatitis in dogs - severity of the disease is frequently increased by the presence of Bordetella bronchiseptica - Incubation period is 3-5 days - Starts with mild fever for short time. Then followed by tonsillitis for few days, harsh dry hacking cough for 6–7 days, then fatal pneumonia - Other signs include depressions, dyspnea, anorexia, muscular trembling and serous to mucopurulent nasal discharge Clinical Signs: - Tonsilitis - Dry hacking cough (6-7 days) - Depression - Dyspnea - Anorexia - Muscular trembling - Serous nasal discharge - Dogs with a harsh, dry cough have less lung involvement than dogs with soft, moist, pulsating cough Viral Properties - Typical properties of Adenovirus in the Genus Mastadenovirus but DNA differ from that of CAV-1. However, CAV-2 can cross-react antigenetically with CAV-1. Epizootiology - Maybe conveyed in aerosol transmission - Incubation period: 3-5 days - Viral replication produces severe narcotizing & proliferate bronchitis & brochiolitis - Lesions are confined in the respiratory tract: o Atelectasis and congestion in the lung o Congestion and hemorrhages in the bronchial lymph nodes o Cowdry type A inclusion bodies in bronchial epithelium, alveolar septa and turbinate epithelium - No lesion (no IB, edema or hemorrhage) in liver and gallbladder - No corneal opacities Pathogenesis - Affinity on epithelial cells of respiratory cells - Viral replication produces a severe necrotizing and proliferative bronchitis and bronchiolitis which results to bronchiolitis obliterans - Lesions are restricted in the respiratory tract - Atelectasis and congestion of the lungs - Congestion and hemorrhages of the bronchial lymph nodes  - Proliferative, edematous changes in the lungs after 10 days Diagnosis - CIT can be determined through clinical signs - Laboratory test via Nasal or pharyngeal swab samples or tracheal wash samples for isolation - Identification through cell cultures Treatment - Generally mild, duration of 7-10 days, thus, do not require specific therapy - If no systemic signs, confine in a warm environment and rest is usually sufficient for natural recovery - Suppressing antitussives: to suppress dry, hacking cough o Warm environment and rest – usually sufficient for natural recovery o CNS-suppressing antitussives – suppress dry, hacking cough o Antibiotics – for secondary bacterial infection via aerosol or intratracheal administration, twice a day for 3 days:  Kanamycin sulfate (250 mg)  Gentamycin sulfate (50 mg)  Polymyxin B sulfate (166,666 IU) Prevention and Control - Attenuated and inactivated vaccines - Inclusion of CAV-2 vaccine in canine routine vaccinations - Vaccinate puppies at 8 -10 weeks old (on first visit to veterinarian) and 4 weeks later III. Bovine Adenovirus - In 1962, Morton Klein first isolated two adenoviruses from cattle species during a search for viruses that is responsible in the production of poliovirus antibodies. These viruses we now know as the type 1 and 2 Bovine Adenovirus. - James H. Darbyshire (1986), in England isolated another adenovirus from the conjunctiva of a healthy cow, describing it as a distinct serotype and is now classified as the BAV type 3. - There are nine serotypes recognized. “Others will probably be identified as studies progress”- Hagan, 1988 Characteristics of Bovine Adenovirus Infection - Infects cattle's and produces mild or subclinical respiratory disease. On some occasion, enteric infection. - In United States, type 3, 4, and 5 are usually suspected in the production of the disease. - Speculated to cause abortion and weak newborn calves - affects 2-16 weeks old calves, especially those that are colostrum deprived. - natural disease is enhanced by cold weather and other agents such as viral diarrhea virus Necropsy Findings: - Lung is collapsed and emphysematous – very prominent histologic findings:  proliferative bronchitis with necrosis   bronchiolar occlusion, resulting in alveolar collapse  nuclear inclusion bodies in bronchiolar epithelium, septal cells and bronchial lymph nodes. Clinical Signs: - Tonsilitis - Conjunctivitis - Pneumonia - Pneumoenteritis - Diarrhea - Polyarthritis/weak calf syndrome - BAV Type 5: - Pyrexia - Polyarthritis - Sometimes diarrhea in colostrum-deprived calves Viral Properties of BAV - Similar morphology and size to human and Aviadenovirus - Type 1 and Type 2 agglutinate rat erythrocytes - BAV-3: 25% DNA homology to human adenovirus type 5 - Type 3: Oncogenic Epizootiology - Transmitted via direct contact of infected and susceptible cattles or contaminated excretions. - Aerosol transmission may occur. - Route of entry via mouth or respiratory tract. Pathogenesis - Infection begins in lymphoid tissue of oropharynx/epithelial cells of respiratory tract - Spreads to the intestinal epithelium causing enteritis. - Spreads to respiratory system by systemic circulation to infect lungs causing pneumonia. Diagnosis - Laboratory isolation & ID of virus - Sample nasal exudates, conjunctiva, feces of acutely ill cattles. - Agar- gel immunodiffusion, Serum neutralization, hemagglutination-inhibition (HI) assay, complement fixation, passive haemaglutination, florescent antibody and ELISA test Treatment - No specific treatment. - Antibiotics given to help control secondary infection from bacteria. Prevention - BAV-1 & BAV-2 vaccine combine w/ IBR or BP 3 vaccines are safe and effective against homologous virus. - Good management, health care, and housing  - For now, effective vaccines does not exist. IV. Equine Adenovirus Infection (EAdV) Characteristics of Equine Adenovirus Infections (EAdV) - Common viruses of horses - produces asymptomatic/mild respiratory infections Clinical Signs: - In Neonates:  Fever  Nasal & ocular discharge  Dyspnea - Lesions  Bronchopneumonia w/interstitial pneumonia  Tracheitis  Rhinitis  Conjunctivitis  Cauda Equina Neuritis Viral Properties of CAV-1 - Typical of that adenovirus traits Diagnosis - Serum-neutralization method - Hemagglutination-inhibition methods Treatment – none Epizootiology - Transmitted oral & nasopharyngeal routes Pathogenesis - Little is known due to its self-limited nature Prevention and Control - Experimental studies have been done yet vaccine are not available commercially. V. Porcine Adenovirus Infection (PAV) Characteristics of PAV - First isolated from rectal swab of a 12-day old piglet with diarrhea. Also, from brain of 10-week-old normal pig w/encephalitis. - Widespread virus of swine - Produce asymptomatic infection, may be associated with pneumonia, enteritis, kidney lesions, transplacental infections, and encephalitis. - Currently there are 4 recognized serotypes of Porcine Adenovirus. - Most serotypes are of low pathogenicity - Serotype 4, isolated in pigs with diarrhea and encephalitis. Clinical Signs  - Diarrhea Viral Properties of PAV - Consist of all morphologic properties and biochemical properties of other adenovirus. Diagnosis - Laboratory isolation i.e., cell cultures Treatment – none Epizootiology – IP: 3-4days Pathogenesis - Strains usually replicate in primary cell cultures of embryonic and piglet kidney, rabbit kidney, porcine embryonic lung/porcine embryonic testes. - Villi of terminal jejunum and ileum are shortened w/ destroyed epithelial cells. Prevention and Control – none VI. Ovine Adenovirus Infection Characteristics: - 6 serotypes - Produces mild or unapparent infection e.g., respiratory & intestinal tract - Infection may predispose to more serious infection Clinical Signs: - In lambs, serotype 6 results to fever (4-8 days) - Serotype 4 produced, no clinical signs, however, lesions are present - Mild respiratory distress similar to BAV-2 infections - Lower respiratory tract infection - Subclinical intestinal infections - Lesions  Pnemoenteritis  Interstitial pneumonia Viral Properties - Typical properties of Mastadenovirus in the Adenoviridae family Diagnosis - Laboratory isolation e.g., cell cultures etc. VII. Other Mammalian Adenovirus Infection a. Murine Adenoviruses – occurs in mouse colonirs Eliminated in the urine over extended periods Disseminated pathological lesions: a) brown fat in heart and adrenals b. Opossum Adenovirus – can produce disease that has not been determined c. Simian A- with 16 recognized serotypes  Genus Aviadenovirus Characteristics: Syn. Egg-drop syndrome (EDS), fowl adenovirus infections hemorrhagic enteritis (HE), inclusion body hepatitis (IBH), marble spleen disease (MSD) - Common infectious agents of poultry and other avian species - Various diseases attributed to 12 avian adenovirus serotypes - Mild respiratory diseases - Etiologic agent of Quail Bronchitis and Egg-drop syndrome-1976 (results to soft or thin eggshells/with no eggshells) MSD is characterized by: - pulmonary congestion and edema - splenomegaly - hepatomegaly IBH is characterized by: Sudden increase in mortality of birds 5-7 weeks old - Few birds appear to be sick. - Findings are: a. hemorrhages b. pale and friable livers c. hepatomegaly d. anemia - Inclusion Bodies (IB) are present in hepatocytes EDS – clinical disease of fowl characterized by soft or thin-shelled eggs or eggs with no shell at all - Birds are healthy, sometimes with edematous uterus - Change in Na pump mechanism of epithelial cells in uterine mucosa increases Na concentration and decreases concentration of K, Ca, Mg and glucose in uterine fluid - Incubation Period is 10-24 days - The degenerative epithelial cells contain IB intranuclearly - Causative agent, Adenovirus 127 is antigenically indistinguishable from hemagglutinating adenovirus of ducks which is non-pathogenic to laying hens but protects the birds against subsequent infection from adenovirus 127. HE – caused by HE virus in turkey - a subclinical infection in chicken that produces intranuclear IB  Other conditions attributed to avian adenovirus infection: a. Atrophy of Bursa of Fabriciuos b. Hemorrhagic Aplastic Anemia Syndrome c. Tenosynovitis and leg weakness, where Avian reovirus is also involved Clinical Signs: - Unapparent - Lesions  Splenomegaly  Hepatomegaly  Pulmonary edema  Inclusion body hepatitis  Pneumonia  Hemorrhages  Pale & friable liver - Laboratory Findings:  anemia Viral Properties: - Typical of another adenovirus - Lack a common complement-fixing antigen shared by mast adenovirus - Cythopathic effect in chicken embryo kidney Diagnosis - Restriction endonuclease mapping - Cell cultures - Embryonated eggs - Immunodiffusion test - Microneutralization test - ELISA test A. Turkey Adenovirus Infections Characteristics: - Three classes:  Fowl adenovirus  Turkey adenovirus  Turkey hemorrhagic enteritis virus Cause respiratory illness, marble spleen disease, and enteric disease in turkey poults. manifested by: a. Respiratory Illness – severe in day-old poults b. Hemorrhagic Enteritis - affects young turkeys  show bloody diarrhea   acute death – occur in large number of dark red, bloody clots in intestinal tract, many petechiae in the mucous membrane of small intestine, atrophy of spleen and cecum. c. Marble Spleen Disease – described in young poults and ring- necked pheasant Clinical Signs Viral Properties - Birds die rapidly Avian adenovirus characteristics - Bloody diarrhea - Acute hemorrhagic enteritis FAMILY HERPESVIRIDAE 3 Subfamilies: Subfamily Alphaherpesvirinae - contains herpes viruses characterized by:  variable host range  short replicative cycle  rapid cytolysis of infected cells  produces latent infections primarily in nerve ganglia - 2 genera: a) Simplex virus b) Poikilo virus Subfamily Betaherpesvirinae - comprises herpes viruses with: a) long replicative cycle b) slow progression of infection in cultures &host c) frequent formation of cytomegalic cells d) produces latent infections in secretory glands, lymphoreticular cells, kidneys, and other tissues - 2 genera: Cytomegalo virus Muromegalo virus Subfamily Gammaherpesvirinae - includes viruses characterized by: a) restricted host range b) produces latent infection of lymphoid tissue c) has predilection to T or B lymphocytes 3 genera: Lymphocryptovirus Thetalymphocryptovirus Rhadinovirus Properties of Herpes Viruses - medium-sized enveloped viruses - 4 structural elements of herpes virion:  1. central core containing the nucleic acid which is linear, double stranded DNA 2. icosahedral capsid surrounding the core, containing 162 hollow capsomeres 3. asymmetrical coat/layer surrounding the capsid called tegument 4. outer envelope surrounding the tegument and capsid - Virus-encoded glycoproteins are incorporated into the virion envelope and are visible as "spikes" that project from its surface. Replication: - 12 hours or more for rapidly cytolytic virus  e.g.: Pseudorabies virus - 70 hours or more for slowly replicative virus  e.g., Cytomegalovirus  Transcription and replication of viral DNA as well as the formation of capsid both occur in the nucleus - Protein synthesis and processing occurs in the cytoplasm. Proteins are transmitted back to the nucleus where they are assembled into capsids. - Intranuclear eosinophilic inclusion bodies are formed in infected cells. - Characteristic of the disease varies from inflammation to tumors DISEASES CAUSED BY SUBFAMILY ALPHAHERPESVIRINAE A. Infectious Bovine Rhinotracheitis (IBR) Synonyms: Bovine Coital Exanthema Infectious Bovine Necrotic Rhinotracheitis Red Nose Disease Infectious Pustular Vulvovaginitis (IPV) Necrotic Rhinitis - Caused by Bovine Herpes Virus 1 (BHV-1), also known as IBR virus or IPV The natural host is cattle, but it also infects goats and pigs. Character of the Disease - 3 forms of the disease: a) Respiratory Form - usually occurs in cold months of the year - average mortality rate is 10% - there is fever, depression, upper and lower catarrhal respiratory signs, and conjunctivitis: i. abundant mucopurulent nasal discharge ii. nasal mucous membranes are very congested and shallow ulcers appear necrosis of the wings of nostrils and muzzle occur iii. dyspnea and mouth breathing- nares are clogged by inflammatory exudates iv. fetid breath due to necrosis of nasal mucosal v. accelerated respiratory rate vi. deep bronchial cough b) Reproductive Form - occurs in heifers, dairy cows, and bulls - mortality rate is negligible  - there is depression, inflammatory pustular lesions in vulva and vagina of cows and on prepuce and penis of bulls. - In cows - the vulva swells with sticky exudates - round pustules appear on reddened mucosal in bulls – presence of exudates similar with that of the females there is temporary and permanent impairment of a bull’s inability to mate. c) Encephalitis Form - usually occur in calves with high mortality (50%) but low morbidity - Intranuclear IB occur in astrocytes and neurons Properties of IBR Virus (BHV-1) Resistance: - stable when suspended in culture medium at ph 7.0 - resistant to 20% ethyl ether for 16 hours at 4oC Susceptibility: - inactivated by equal parts in a suspension with:  alcohol  acetone  chloroform Cultivation: - can be cultivated in:  bovine embryo tissue culture cells  bovine kidney cells  pig, dog, sheep, goat. horse and human kidney cells  can be cultivated within 24-48 hours Epizootiology and Pathogen