Mycovirology & Virology Introduction PDF

[MLS 415] Mycology and Virology M3: Introduction to Virology Professor: Joanne Krystine Tago Date: March 11, 2024 INTRODUCTION TO VIROLOGY VIRUS – means poison or noxious agent in Latin VIRION – refers to an entire virus particle Viral Genome: Based on its organization DIFFERENCES VIRUSES BACTERIA FUNGI Not a living organism, but are active No cell ⇒ acellular External structures may be covered by proteins called capsids Genetic material: DNA or RNA (not both) Do not undergo metabolic processes on their own Remain dormant until they attach to a host cell Prokaryotic Genetic material: arranged at the central portion of the structure (anuclear area) → genome is not encased on a membrane No mitochondria but can still produce ATP Has cell wall that is mostly made up of peptidoglycans Has DNA Can live as either aerobic or anaerobic organism Eukaryotic Either single-celled (yeast) or multi-celled (mold) Has cell wall that is made up of chitin and cellulose Genetic material: DNA Can live as either aerobic or anaerobic organism Reproduction: ○ Yeast: budding ○ Mold: hyphal extension or spore formation Commonly seen as mushrooms → ranges from toxic to edible Saprophytes Positive sense – genome is similar to the host cell’s mRNA ○ readable by ribosomes Negative sense – genome is not similar to host cell’s mRNA ○ not readable by ribosomes ○ can travel with RNA polymerase, which helps them to transform from negative sense genome to positive sense genome allowing their material to be read by the ribosomes DNA Viruses Most are double-stranded Except: → Parvovirus RNA Viruses Most are single-stranded Except: → Reovirus Viral Structure Protein coat or Capsid ○ composed of capsomeres ○ symmetry/ shapes ○ capsomeres can assemble into: Helical (Most common), polyhedral, spherical , complex CLASSIFICATION OF VIRUSES DNA VIRUSES ➔ ➔ ➔ ➔ Poxviridae Herpesviridae Adenoviridae Papovaviridae RNA VIRUSES ➔ ➔ ➔ ➔ ➔ ➔ ➔ Picornaviridae FiloCaliciParamyxoTogaOrthomyxoFlavi- ➔ ➔ ➔ ➔ ➔ ➔ ➔ ReoRhabdoBunyaCoronaAenaAstroRetro @mlstranses | 1 Envelope → lipid bilayer consisting of matrix proteins and glycoproteins ○ places viruses into one of 7 groups depending on the: combination of their nucleic acid strandedness (single or double) sense (positive or negative) KEY FEATURES IN BALTIMORE CLASSIFICATION: DNA Viruses are enveloped Except: Parvovirus Adenovirus Papovavirus (Papillomavirus & Polyomavirus) RNA Viruses are enveloped Except: Calicivirus Reovirus Astrovirus Picornavirus Size Measured by nanometer (nm) ○ 1nm = 0.001μm = 1x10-6 mm Cannot be seen using light microscope E/M; vary from 10 – 300 nm Smallest Largest Parvovirus (22 nm) Picornavirus (28 nm) Poxvirus (225 – 300 nm) Paramyxoviridae (150-300 nm) Filoviridae (80x1000 nm) Baltimore Classification Created by David Baltimore Based on how they generate their mRNA Classiﬁcation system that is mostly used today All double stranded genome are (+) and (-) sense All other genomes are only (+) sense ○ except: Group V only (-) sense ○ Group II can package both negative and positive strand into their capsid so they may also be (+) and (-) sense at the same time Group I viruses ○ remember that double stranded DNA can make mRNA directly Group II viruses ○ while single stranded DNA requires conversion to double stranded DNA to make mRNA Group III viruses ○ double stranded RNA can be used as a template to make mRNA in a similar way the double-stranded DNA ○ but is uses an RNA-dependent RNA polymerase instead of a DNA-dependent RNA polymerase Group IV viruses ○ single stranded (+) sense RNA is already a mRNA ○ can be used to make proteins immediately ○ alternatively, it can be copied into its (-) sense to produce more (+) sense mRNA still requires an RNA-dependent RNA polymerase Group V viruses ○ function exactly like the post-conversion Group IV viruses ○ can produce the (+) sense mRNA from the (-) sense genomic RNA strand using RNA-dependent RNA polymerase Group VI and VII viruses ○ carry a reverse transcriptase to produce DNA from an RNA strand ○ can make double stranded DNA, which can be used to produce the mRNA Group VII viruses ○ genome is comprised of a circular, gapped double stranded DNA some of its DNA is single stranded some of its genome contains RNA as a primer to generate the require strand of single stranded RNA that will eventually become the template for the reverse transcriptase @mlstranses | 2 # CHARACTERISTICS I dsDNA mRNA is transcribed directly from the DNA template II ssDNA DNA is converted to double-stranded form before RNA is transcribed III dsRNA mRNA is transcribed from the RNA genome IV ssRNA (+) Genome functions as mRNA Common cold (picornavirus) V ssRNA (-) mRNA is transcribed from the RNA genome Rabies (rhabdovirus) VI ssRNA viruses w/ reverse transcriptase Reverse transcriptase makes DNA from the RNA genome; DNA is then incorporated in the host genome; mRNA is transcribed from the incorporated DNA dsDNA viruses w/ reverse transcriptase The viral genome is double-stranded DNA, but viral DNA is replicated through an RNA intermediate; the RNA may serve directly as mRNA or as a template to make mRNA VII MODE OF mRNA PRODUCTION EXAMPLE Herpes simplex (herpesvirus) Canine parvovirus Rotavirus HIV Hepatitis B virus VIRAL REACTION TO CHEMICAL AND PHYSICAL AGENTS ETHER FORMALDEHYDE Distinguishes enveloped viruses from naked viruses Enveloped viruses are generally sensitive Naked viruses are RESISTANT The envelope of most viruses is derived from the host cell’s cytoplasmic membrane ○ except Herpes viruses since they derive their envelope from the host cell’s nuclear membrane The presence of an envelope confers instability rather than protection ○ enveloped viruses are more sensitive than naked viruses, especially to heating, drying, detergents and lipid solvents Destroys viral infectivity by reacting with nucleic acid SS genomes are inactivated more readily than DS genome ○ because single stranded genome has lesser products to work on Minimal adverse effect on antigenicity of proteins ○ Still able to cause infection Some studies showed that the inactivation of formaldehyde has an effect of the early step of viral replication @mlstranses | 3 reduces the ability of the Polio virus to bind to the receptors; thus, the virus will not be able to gain entry into the host cell ○ abolishes the infectivity of the viral DNA; thus, it limits the virus from infecting only few cells Activity of formaldehyde: creates crosslinks; materials far apart will be pulled together ○ RADIATION Ultraviolet, x-ray, high-energy particles inactivate viruses ○ even naked viruses Ionizing and Non-ionizing ○ The lower the wavelength the higher chances that it causes an effect Infectivity is the most radiosensitive property ○ this property is embedded in the genome Exposure to radiation damages part or components of the genome ○ replication may be a problem HEAT Enveloped viruses rapidly drop in titer at 37°C Icosahedral viruses lose infectivity after several hours at 37°C Viral infectivity is destroyed by heating at 50- 60°C for 30 minutes ○ except HBV and Polyoma viruses HBV is a very resistant pathogen ➔ can survive heating at 60°C even up to 4 hours ➔ can be inactivated by high temperatures such as in autoclaving (121°C) or direct heat ○ Polyoma viruses are also inactivated by high temperature ≥70°C is required to affect thermal inactivation VIRAL REPLICATION Viral Replication → term used to indicate the formation of biological viruses during the infection process inside the target host cell Viruses must ﬁrst penetrate and enter the cell before viral replication can occur Purpose: multiplication and survival ADDITIONAL INFORMATION: (from the video) Viruses are transferred as particles known as virions Once the virions enter the host cell, it disassembles and the viral genome interfere with cellular processes 1st Stage: ATTACHMENT & PENETRATION Virion Attachment: Viruses cannot pass through biological membranes on their own. They utilize membrane proteins to attach to speciﬁc receptors on the surface of host cells. Membrane Proteins Involved: These may include ○ sialic acid-rich glycoproteins, ○ proteoglycans (heparan sulfate), ○ receptors ( LDL or CD4) ○ proteins forming tight junctions such as (occludin and claudin) Entry Mechanisms: Enveloped viruses fuse with the host cell membrane after attachment, releasing their capsid into the host cytoplasm. ○ Non-enveloped viruses may be endocytosed upon receptor binding and transported along cytoskeletal ﬁlaments in the cytoplasm. ○ 2nd Stage: UNCOATING Capsid Disassembly: The viral genome, often RNA in RNA viruses, needs to enter the host cell cytoplasm. ○ The viral capsid is disassembled, triggered by factors like endosomal pH changes, leading to the release of the viral genome. Genome Release: Once released in the cytoplasm, the viral RNA (if required) may undergo transformations, such as mRNA transcription, initiating viral protein translation. ○ Viral mRNA often contains sufﬁcient information to encode multiple proteins, facilitated by speciﬁc folding patterns or internal ribosome entry sites (IRES). 3rd Stage: REPLICATION/ SYNTHESIS Early Protein Synthesis: The ﬁrst viral proteins synthesized are typically involved in replication processes. These proteins are catalytically active and produced in smaller amounts initially. Late Protein Synthesis: Structural proteins necessary for forming new capsids are subsequently produced in larger quantities. 4th Stage: ASSEMBLY Once sufﬁcient viral components, including RNA replication products and structural proteins, are produced, they self-assemble into new virions. 5th Stage: RELEASE (1) Exocytosis: Some viruses exit the host cell through exocytosis, where viral particles travel through the endoplasmic reticulum and Golgi apparatus before being released. ○ Maturation often occurs in different cell compartments, depending on pH. (2) Budding: Viral proteins are incorporated into the host membrane, forming a new envelope for the virion. @mlstranses | 4 ○ Viral components assemble directly at the budding site, which could be the endoplasmic reticulum, Golgi apparatus, or plasma membrane. (3) Cell Lysis: Many non-enveloped viruses are released via cell lysis, where the virus disrupts the host cell's plasma membrane, leading to cell death. ○ DNA Viruses DNA viruses follow a similar replication process but have some variations: ○ Nuclear Import: The viral DNA needs to be delivered into the host nucleus for transcription. Inside the nucleus, viral DNA may undergo transformations before transcription. ○ Assembly and Release: Viral structural proteins are often transported into the nucleus for assembly. ○ Virions then penetrate the nuclear membrane and are released via vesicle formation. Adsorption (Attachment) Binding between viral capsid proteins and speciﬁc receptors on host cell membranes Viral tropism (speciﬁcity) Glycoproteins, proteoglycans, receptors (LDL & CD4), occludins & claudins Attachments triggers changes on the viral envelope proteins or viral surface proteins (in the case of naked viruses) ○ results in the fusion of viral and cellular membranes SYNTHESIS PHASE a.k.a. Viral Entry Virions enter the host cell through: ○ membrane fusion Envelope is left at the cell membrane; which combines w/ the cell membrane Causes change in conformation ○ receptor-mediated endocytosis Due to Cytokines For naked viruses Uncoating Physical separation of nucleic acid from its protein coat Mediated by cellular enzymes Initiated by different triggers such as: ○ endosomal pH ○ presence of cellular enzymes The environment gradually becomes acidic which affects the capsid proteins Virus begins to make copies of itself Inhibition of host cell DNA Early: synthesis of nucleic acids catalytically active and would require only a small amount to be synthesized “early protein” Late: synthesis of structural proteins would require large quantities of structural proteins “large proteins” Location of Viral Genome Replication Nucleus Cytoplasm DNA viruses, except Poxvirus ○ poxviruses are very large (300nm) ○ would not ﬁt into the host cell’s nucleus ○ synthesis occur in the cytoplasm Uncoating occurs in the cytoplasm followed by the transportation of the genome into the nucleus RNA viruses, except for retroviruses & Inﬂuenza virus ASSEMBLY or MATURATION Penetration causes destabilization of capsid proteins, separating capsomeres from one another and exposing the genome Newly formed nucleic acids are enclosed by capsids Maturation: proper orientation of the structures inside the virus ○ in some viruses, it occurs in place as they are assembled within the cell ○ while other viruses may undergo maturation once they are separated from the host cell RELEASE Two Mechanisms: ○ Rupture or lysis ⎼ for naked viruses ○ Budding or exocytosis ⎼ for enveloped viruses For HIV: → it undergoes maturation after release SPECIMEN COLLECTION FOR VIRAL STUDIES Collect specimens as soon as possible after the onset of symptoms ○ the chance of patient recovery is best during the ﬁrst 3 days after onset of symptoms ○ patient recovery is reduced the longer the specimen is collected from the patient @mlstranses | 5 Collect autopsy samples ASAP after death before tissues start decomposing All specimens (except feces) must be collected in a designated sterile container and kept cool on ice or refrigerated (not frozen) Specimen Optimal specimen collection schedule Cerebrospinal ﬂuid (CSF) for most PCR or culture samples, they should be held at 4 deg C and must arrive in the laboratory within 24 hours Observe aseptic technique and proper labeling ○ Volume Within 7 days of onset 1-2 mL Amniotic ﬂuid 3 mL Vitreous ﬂuid (eye) 1 mL Urine Specimen handling instructions Within 2 weeks of onset 5 mL Blood 5-7 mL In yellow ACD tube Bone marrow aspirate 2-5 mL In yellow ACD tube Bronchoalveolar lavage (BAL) 3-5 mL Swabs (nasal, throat, nasopharyngeal, vesicle/ lesion, rectal, eye/conjunctiva, genital) NP: within 5 days VESCL/GEN swabs - as early as possible Swab in M4 Aspirates (nasal, tracheal, sinus, vesicle) 1-2 mL Nasal wash Within 5 days of onset Feces Within 2 weeks of onset 3-5 mL 5 mL Tissue Variable Nasopharyngeal Swab or Aspirate In viral transplant media M4 Aspirates are superior to swabs for recovering viruses Procedure is time-consuming and uncomfortable for the patient For COVID-19 specimens, nasopharyngeal swab is the specimen of choice ○ more convenient Best for isolation of respiratory syncytial virus, inﬂuenza, parainﬂuenza, rhinovirus In container without preservatives In sterile saline or viral transport media Bronchial and Bronchoalveolar Washing Excellent specimens for detection of viruses infecting the lower RT Includes tracheal aspirates and sputum Should NOT be placed in a container with the universal transport media ○ should only be placed on a clean or sterile container E.g. Inﬂuenza virus, adenoviruses Conjunctival Swab Nasal Wash Aspirate 10 mL (for adult) sterile NSS into a sterile syringe or bulb Tilt head back and apply pressure to one nostril Have patient hold breath and quickly “squirt” 5 mL NSS into open nostril Tilt head forward to allow ﬂuid to drain or expel into sterile collection cup Repeat with other nostril, if possible Transfer this ﬂuid to the sterile conical centrifuge tube Evert the lower eyelid and gently rub the conjunctival surface with a mini-tipped ﬂocked swab 5-10 ml fresh diarrheic stools or 5-10g formed or soft stools Rectal swab may be acceptable ○ Insert a swab 3-5 cm into rectum to obtain feces Rota-, enteric adeno-, enteroviruses (do not grow in cell cultures) Stool and Rectal Swab Throat Swab Inﬂamed or purulent areas of the posterior pharynx Swabs are allowed to touch the posterior pharynx at the back and the tonsillar fossae Best for isolation of enteroviruses, adenoviruses, herpes simplex viruses Urine Virus recovery may be increased by processing multiple (2-3) specimens Specimen: at least 10 ml of a clean voided, ﬁrst morning urine CMV, mumps, rubella, measles, polyomavirus, adenoviruses @mlstranses | 6 Vesicular lesions ○ intact, well-deﬁned, and shows characteristic features of the viral infection HSV, Varicella-Zoster virus, Enteroviruses ○ VZV causes Chickenpox and Shingles Usually found in the Dorsal Root Ganglia (clusters of nerve cell bodies located along the spinal cord) These ganglia serve as reservoirs for certain neurotropic viruses; Viral particles can establish latent infections within the DRG, where they remain dormant until reactivation occurs. EDTA is preferred for PCR test Heparinized spx. are not used in PCR Test ○ citrate or heparin may be used if for culture 2 ml specimen is acceptable for pediatric patients Detection is best done by separating and culturing WBCs Polymorphprep (Na metrizoate + dextran) ○ MNs, PMNs are isolated from RBCs in one step ○ Skin and Mucous Membrane Lesions A Tzanck smear is a simple and rapid diagnostic test used to detect viral infections, particularly those caused by HSV and VZV. Bone Marrow Procedure: Scraping cells and ﬂuid from the lesion onto a glass slide. Staining and examining under a microscope for viral cytopathic effects like multinucleated giant cells and inclusion bodies. Interpretation: Presence of multinucleated giant cells and inclusion bodies suggests an active viral infection, It helps differentiate viral infections from other causes of vesicular lesions. Collected by aspiration ○ only done by physicians Sterile tube with EDTA Should be stored and transported at room temperature ○ should be prioritized – bcs BLASTIC cells are sensitive to cold temps Parvovirus B19 – infects children Tissue Collected by biopsy Small pieces of tissue should be placed in Viral Transport Media (VTM) or sterile Phosphate Buffer (PBS) Do NOT add the swab to the transport media Rotaviruses, adenoviruses, caliciviruses, astroviruses, Norwalk virus, and a group of Noroviruses. Serum for Antibody Testing Sterile Body Fluids other than Blood (1) CSF, (2) pericardial, (3) pleural and (4) peritoneal ﬂuids Specimens are collected aseptically by the physician 0.2 mL for PCR test (especially for CSF) 0.5 mL to culture CSF Do NOT dilute in universal transport media Enteroviruses, HSV, inﬂuenza virus, CMV Blood Detect CMV ○ occasionally HSV, VZV, enteroviruses, adenoviruses 3-5 ml anticoagulated blood (EDTA, citrate or heparin) collected in a vacutainer tube Acute specimens ○ collected ASAP after the appearance of the symptoms Convalescent serum ○ 2-3 weeks after the acute serum ○ to check the stability of patient’s immune system 3-5 ml serum SPECIMEN TRANSPORT, STORAGE, PROCESSING FOR VIRAL CULTURE Specimens for Viral Culture Do NOT stand at RT or higher Store in ice & transport to the lab ASAP If delay is unavoidable, should be refrigerated @mlstranses | 7 Storage up to 5 days at 4°C ○ 6 or more days at -20°C or preferably at -70°C Before freezing: dilute or emulsify in viral transport medium Transport blood in anticoagulated sterile tube Clotted blood are unacceptable ○ should be processed ASAP ○ if delayed, hold specimen at 2-8°C ○ processing must occur within 12-24 hours ○ serum may be stored for days at 4°C or for weeks / months at -20°C or below before testing Swabbed samples ○ cotton, rayon, dacron (preferred) Must be emulsiﬁed in viral transport medium before transporting Cytopathic Effect (CPE) Viral Transport Media Fetal Bovine Serum, albumin, or gelatin Antimicrobials (Gentamicin & Amphotericin B) ○ to inhibit contaminants Gentamicin: bacterial contaminants Amphotericin B: fungal contaminants Examples: ○ Stuart, Amies, Leibovitz-Emory ○ Hanks balanced salt solution (HBSS) ○ Eagle’s tissue culture medium ○ Virocult® and ∑-Virocult® METHODS OF DETECTING VIRUSES Cytology and Histology Electron Microscopy Immunodiagnosis Molecular Detection Cell culture Viral Serology CYTOLOGY AND HISTOLOGY Specimens: tissue samples or body ﬂuids Cytology stains: ○ Papanicolaou (Pap) ○ Giemsa stain ○ H&E detects inclusions & syncytia of CMV, Rabies, HPV, MCV Detects: inclusions: structures that are not supposed to be inside tissues or host cells syncytia: giant cell made up of different cells infected by viruses (HSV or chickenpox infections) Can identify: → HSV, Varicella-Zoster inclusion bodies Cellular changes ○ cell death or lysis ○ syncytia formation ○ inclusion body formation ○ transformation of host cells Morphologic changes in the virus-infected host cells Observable on light microscopy Cell Death or Lysis → Human corneal epithelial cells infected with HSV1 ○ Image A (0 hour) exhibits cobblestone appearance with 90% conﬂuence conﬂuence: refer to the spaces in between cells ○ Image B (8 hours) cytopathic effect could be seen with 80-75 conﬂuence ○ Image C (12 hours) – more cells are gone ○ Image D (24 hours) – 50-60% conﬂuence Cellular Changes: Inclusions or Syncytia Negri body inclusion ○ Rabies Giant cell with “owl’s eye” inclusion ○ CMV @mlstranses | 8 Guarnieri bodies ○ Smallpox Warthin-Finkeldey syncytia formation ○ Measles ○ giant syncria made up of about 40% of infected cells Koilocytes ○ HPV ○ squamous cells with vacuolation “halo” around the nucleus ○ Cervical smears – cytoplasm should be clear Cowdry type A ○ Herpes simplex ○ Very large inclusion Henderson-Patterson or Molluscum body ○ Molluscum contagiosum ○ seen inside blister formations ELECTRON MICROSCOPY When using electron microscope: ○ the material to be viewed should be treated with electron dense material like sodium tungstate provides a dark background ○ then add the electron condensed material ○ the material will be mounted with copper grids Transmission Electron Microscope ○ allows visualization of the internal structures of the virus Scanning Electron Microscope ○ allows visualization of the external structure of the virus [LEFT] TEM: Rotaviruses notice the empty virions (inactivated viruses) [RIGHT] SEM: Adenoviruses notice the icosahedral formation TEM & SEM Useful for detecting viruses that do not grow readily in cell cultures Examples ○ gastroenteritis (Rotavirus, Norwalk agent, Adenovirus) ○ encephalitis (Herpes simplex, Measles, JCV) Immune Electron Microscopy ○ speciﬁc antisera is added so viral particles will be agglutinated allowing them to be viewed as groups ○ rapid and accurate morphological diagnosis of viral agents ○ Classical Phase simple add the anti-sera before viewing it under the electron microscope ○ Solid Phase anti-sera is loaded onto the copper grids VIRAL SEROLOGY Indirect evidence of viral infection To evaluate immune status ○ recall acute specimen and convalescence serum To diagnose viral infections if virus cannot be cultivated 1-2 weeks: IgM begin to appear followed a few days later by IgG ○ IgM levels peak in 3-6 weeks and drop to undetectable levels in 2-3 months ○ IgG levels peak in 4-12 weeks and remain for several months, some for life Diagnosis of Active Infection Detection of virus-speciﬁc IgM in acute phase serum sample Detection of a four-fold rise in antibody titer between acute and convalescent sera Detection of Viral Antigens Immunoﬂuorescence Assay ○ a microscopic method that detects and visualizes viral proteins expressed in cells via Ag-Ab reactions ○ used for research rather than diagnostic purposes @mlstranses | 9 Enzyme Immunoassay ○ ELISA uses an enzyme to detect the binding of antigen or antibody such as, (1)horseradish peroxidase, (2)alkaline phosphatase, (3)lactoperoxidase, (4)beta galactosidase Principles involved in ELISA are: Antigen-antibody reaction − presence of antigen or antibody is detected in the sample Enzymatic chemical reaction − rate of formation of antigen-antibody complex is used is used to determine the quantity of either the antigen or antibody involved in the reaction – the enzyme catalyzes the colorless substrate to produce a colored product Signal detection and quantiﬁcation − the intensity of colored product generated by the enzyme and the substrate is detected and measured KEY FEATURES OF ELISA (From the video) Direct ELISA: Involves testing for the presence of an antigen. A known antibody is immobilized or absorbed onto the surface of a microtiter plate well. After rinsing to remove unbound antibodies, the sample suspected of containing the antigen is added. An enzyme-linked antibody speciﬁc to the antigen is introduced. ○ If the antigen is present in the sample, it binds to the immobilized antibody and subsequently to the enzyme-linked antibody. A colorless substrate for the enzyme is added. Development of color indicates the presence of the antigen in the sample. Indirect ELISA: Detects the presence of antibodies in a sample. The antigen to be detected is immobilized onto the surface of a microtiter plate well. After washing away unbound antigens, the serum suspected of containing the antibodies is added. An enzyme-linked antibody, capable of reacting with the constant region of other antibodies (i.e., secondary antibody), is then added. ○ If antibodies speciﬁc to the antigen are present in the serum, they bind to the immobilized antigen and subsequently to the enzyme-linked secondary antibody. A colorless substrate for the enzyme is added. Development of color indicates the presence of antibodies that recognize the antigen in the sample. Western Blot ○ also called Protein Immunoblotting ○ considered the gold standard test for HIV infection conﬁrmation ○ requires technical expertise; time-consuming NO longer recommended by WHO, CDC, APHC due to: Prone to False (+) Indeterminate results (if infection is early) Long TAT ○ a widely accepted analytical technique used to detect speciﬁc proteins in the given sample ○ uses Sodium Dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in the given sample ○ the separated proteins are then transferred or blotted onto a matrix where they are stained with antibodies (used as a probe) speciﬁc to the target protein ○ by analyzing location and intensity of the speciﬁc reaction, expression details of the target proteins in the given cells or tissue homogenate could be obtained KEY FEATURES OF WESTERN BLOT (From the video) The process described is the Western blotting technique, a powerful tool used to detect and identify speciﬁc proteins within a complex mixture. Here's a comprehensive breakdown of the steps involved: (1) Denaturation and Electrophoresis: Proteins within the sample are denatured and coated with sodium dodecyl sulfate (SDS), which imparts a negative charge to the proteins, making them proportional in charge to their mass. The denatured proteins are then separated based on their molecular weight by electrophoresis, where they migrate through a polyacrylamide gel towards the positively charged anode. ○ This separation allows for the visualization of individual protein bands. @mlstranses | 10 (2) Transfer onto a Membrane: After electrophoresis, the separated proteins are transferred from the gel onto a membrane, typically made of materials like nitrocellulose or PVDF (polyvinylidene diﬂuoride). ○ This transfer process, known as "blotting," preserves the spatial arrangement of the separated proteins. (3) Blocking: The membrane is incubated in a blocking solution, often containing milk or bovine serum albumin (BSA). ○ This step prevents nonspeciﬁc binding of antibodies and reduces background noise. (4) Primary Antibody Incubation: The membrane is then incubated with a primary antibody speciﬁc to the target protein. ○ The primary antibody recognizes and binds to a speciﬁc epitope (a unique region) on the target protein. (5) 1st Washing: Unbound primary antibodies are removed by washing the membrane, reducing nonspeciﬁc binding and background noise. (6) Secondary Antibody Incubation: The membrane is incubated with a secondary antibody conjugated to an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase. ○ This secondary antibody speciﬁcally recognizes and binds to the primary antibody. (7) 2nd Washing: Excess unbound secondary antibodies are removed by washing the membrane, further reducing background noise. (8) Detection: The presence of the target protein is visualized through enzymatic activity. The membrane is exposed to a substrate solution containing a chromogenic or chemiluminescent substrate speciﬁc to the enzyme conjugated to the secondary antibody (e.g., HRP). In the presence of the substrate, the enzyme catalyzes a reaction that generates a detectable signal, typically a colored precipitate or luminescent signal, directly proportional to the amount of target protein bound by the primary antibody. MOLECULAR DETECTION Nucleic acid ampliﬁcation tests such as the PCR are widely used nowadays in identiﬁcation of viral agents Polymerase Chain Reaction – process done to amplify genetic materials; takes hours, weeks or months ○ a technique that is widely used in molecular biology and genetics that permits the analysis of any sequence of DNA or RNA ○ ○ allows a speciﬁcally targeted DNA sequence to be copied and/or modiﬁed in predetermined ways this reaction has the potential to amplify one DNA molecule to become over 1 billion molecules in less than 2 hours KEY FEATURES OF PCR (From the video) Polymerase Chain Reaction (PCR) is a widely used molecular biology technique for amplifying speciﬁc segments of DNA. It relies on speciﬁc oligonucleotide primers, nucleotides for DNA polymerization, and a heat-stable DNA polymerase, typically Taq polymerase from the thermophile Thermus aquaticus. Procedure: 1. Denaturation: The reaction mixture is heated to around 95°C for 30 seconds. a. This step causes the separation (denaturation) of the DNA strands, breaking the hydrogen bonds between them. 2. Annealing: The temperature is then lowered to around 55°C for 30 seconds. a. During this step, the speciﬁc oligonucleotide primers anneal or bind to the complementary sequences ﬂanking the target DNA region. 3. Extension: The temperature is raised to around 72°C, which is the optimal temperature for Taq polymerase activity. a. Taq polymerase utilizes the primers as starting points for DNA synthesis, adding nucleotides one at a time to extend the primers and create complementary strands of DNA. 4. Cycle Repeated: The denaturation, annealing, and extension steps constitute one cycle. a. This cycle is typically repeated multiple times, usually around 25 to 30 cycles, to amplify the target DNA exponentially. b. After two cycles, four double-stranded DNA sequences are produced from the original target DNA. 5. Thermocycler: To automate the precise control of temperature changes required for each step of the PCR process, a specialized instrument called a thermocycler is used. a. The thermocycler rapidly cycles through different temperatures, ensuring optimal conditions for denaturation, annealing, and extension. @mlstranses | 11 CELL CULTURE Viruses can be grown in vivo or in vitro IN VIVO: important for the identiﬁcation and diagnosis for pathogenic viruses in clinical specimens, production of vaccines, and basic research studies host sources serves as an incubator for viral replication (e.g. developing embryo or whole animal) the location within the embryo or the host animal is very important ○ tropism: attacks speciﬁc host cells which contains receptors sites for the viruses (e.g. amniotic cavity, chorioallantoic membrane, yolk) IN VITRO: includes bacteriophage grown in the presence of a dense layer of bacteria culture media usually used: 0.7% soft agar for lytic bacteriophages, lysis of bacterial host can be readily observed when there is plaque ○ clear zones where bacterial cells have already lysed Identiﬁcation of viruses detected on cell cultures are based on: ○ the cell type that support viral replication ○ time of detection of CPE ○ morphology of CPE Conventional Cell Culture → cells grow into a layer on bottom of cell culture ﬂasks or dish ○ cells are moistened and nourished by immersing in cell culture media Dulbecco’s Modiﬁed Eagle Medium (comes with bovine serum) ○ incubated for 48 hours at 37°C with 5% CO2 ○ once it has been subcultured in vitro, it becomes a cell line once cells are close together, mitosis is triggered to stop which would results to apoptosis hence, 100% conﬂuence should be avoided by doing subculture Examples: Primary monkey kidney cell (PMK) Human embryonic kidney (HEK) ○ Low-Passage (Diploid) Cell Lines → cell cultures that remain virus-sensitive through 20-50 passages ○ examples: Human diploid ﬁbroblast cells (HDF) derived from human kidney and lung ﬁbroblasts WI-38 and MRC-5 (human embryonic lung) – MRC (Microbiology Research Council) is more common: line 5 or 7 Continuous Cell Lines → cells can be passed and remain sensitive to virus infection indeﬁnitely ○ “immortal cell lines” ○ are usually collected from cancer cells ○ examples: Hep-2 cells HeLa cells − originally cultivated from tumor cells obtained from Henrietta Lacks, who died due to cervical cancer due to HPV (1951) − ﬁrst continuous cell lines produced Kinds of Cell Culture A cell culture becomes a cell line once it has been passed or subcultured Primary Cell Lines – freshly prepared from cells or tissues ○ extracted by: mechanical scrapings mincing (to release the cells) enzymatic method using trypsin (inhibits cells from sticking together) ○ those that have been passed only once or twice since harvesting ○ further passage results in decreased susceptibility to viral infection ○ usually requires a liquid culture medium in a petri dish or tissue/cell culture ﬂask for cells to have a solid surface for attachment of growth ○ has limited lifespan due to contact inhibition Uses of Cell Cultures Virus Quantiﬁcation using Plaque Assay – a standard technique used to determine the concentration of infectious viruses in a sample. Objective: The primary goal of the Plaque Assay is to count the number of viruses present in a speciﬁc volume of a sample, typically measured in terms of infectious dose. @mlstranses | 12 Procedure: 1. Preparation of Cell Culture: A conﬂuent monolayer of host cells, often referred to as "hot cells," is prepared in a petri dish or a cell culture plate. a. These cells are susceptible to the virus being tested. 2. Virus Infection: The host cell monolayer is then infected with the virus at varying dilutions. a. This is done to ensure that the resulting plaques are countable and not too numerous or sparse. 3. Overlay with Semi-Solid Medium: After infection, the cell monolayer is covered with a semi-solid medium such as agar or carboxymethyl cellulose. a. This medium prevents the spread of the virus to neighboring cells, ensuring that each virus infects only a single cell. 4. Plaque Formation: As the infected cells replicate and lyse, they release viral particles that can infect neighboring cells. a. This results in the formation of visible plaques, which are areas of cell death in the monolayer. 5. Plaque Visualization and Counting: The plaques can be visualized either by (1) direct observation or using an (2) optical microscope. a. Plaque formation typically takes 3-14 days, depending on the virus being tested. b. The plaques are manually counted, and the results are used to calculate the number of plaque-forming units (PFU) per sample unit volume. c. Each plaque is assumed to represent one infectious virus particle. Viral Adsorption: Discard medium from each well and wash cells with PBS. Add 500μl of diluted viruses into each well. Incubate viruses and cells at 37°C for 1 hour. Shake the plates every 15 minutes to ensure uniform viral adsorption. Overlay Infected Cells with Agarose: Prepare an agarose solution containing 0.3% agarose and culture medium, with 2% FBS. Autoclave a 3% agarose solution and culture medium, pre-warmed at 45°C. Mix 4mL of 3% agarose solution with 36mL of culture medium, keep at 42°C. Discard medium from each well and wash cells with 500μl PBS. Add 3mL of 0.3% agarose solution into each well. Keep plates at room temperature until agarose overlay solidiﬁes. Incubate the plates at 37°C for 60-84 hours to allow viral plaques to form. Cell Fixation and Staining: Fix cells with a 3.6% formaldehyde solution for 1-24 hours. Remove formaldehyde solution and wash out agarose overlay. Stain cells with 0.5% crystal violet at room temperature for 1 minute. Rinse each well with tap water. Count and analyze viral plaques to determine viral concentration. Fetal Bovine Serum (FBS): FBS is NOT USED as a cell culture medium in the plaque assay due to its interference with virus growth. ○ However, it is sometimes used as an overlay to protect medical technologists from potential infection. Buffer and Staining: Phosphate buffer solution (PBS) is used for washing the cells, formaldehyde for ﬁxation, and crystal violet for staining. KEY PROCEDURE FOR CELL CULTURE (From video) Cell Seeding: Seed cells into a 6-well plate and incubate at 37°C for 24 hours. Ensure cells reach a conﬂuency of 90-100%. Virus Dilution: Soak the virus at 37°C in a water bath. Use culture media without FBS to dilute the virus. Each tube contains 1080μl of culture medium without FBS. Add 120μl of virus into the ﬁrst 2 tubes, mix well. Take 120μl of virus into the next tube and repeat to create 10x serial dilutions. Cytopathic Effect → not just for tissues that has been stained in the histopathology laboratory @mlstranses | 13 [LEFT to RIGHT] RSV and HSV Measles Hemadsorption of RBCs & syncytial formation by mumps virus onto cell sheet surface Quantification of Cell Culture CPE Quantitation Negative Interpretation Uninfected monolayer Equivocal (-/+) Atypical alteration of monolayer involving few cells → might be a need to repeat the steps 1+ 1% - 25% monolayer exhibit CPE 2+ 25%-50% monolayer exhibit CPE 3+ 50%-75% monolayer exhibit CPE 4+ 75%-100% monolayer exhibit CPE @mlstranses | 14 Mycology and Virology | MLS-415 F1: Hepatitis A, B, C, D, & E Professor: Thynee Tago, MSMT Date: April 27, 2024 HEPATITIS A CLASSIFICATION: Genus: Hepatovirus Family: Picornaviridae STRUCTURE: 27 to 32 nm spherical particle Cubic symmetry A linear single stranded RNA 7.5 kb CONTENT: Capsid Viral Protein Genome PEOPLE W/ INCREASED RISK OF HEPATITIS A: Hepa A = Infection of the Liver through ingestion of contaminated Fecal material Natural host → HUMANS Eyes may appear Jaundiced due to it being a liver disease Incubation: 10-50 days; Averaging 25-30 People w/ occupational risk for exposure to Hepa A People in developed countries adopting children from underdeveloped countries People w/ Chronic Liver Diseases Most prominent Sign and Symptom: YELLOWING of the SKIN/SCLERA For some there could be fever, stomach pain, darkened urine and stool is light colored Joint pain may also occur (They feel tired) Children may appear ASYMPTOMATIC; Adults are usually SYMPTOMATIC Symptoms develop and appear 2-7 weeks after infection which may last < 2 months but for some it can reach up to 6 months (the longevity depends on the stability of the immune system) Travels Internationally Men who have Sex with Men (MSM) Users of injection and Non-injection drugs Homeless people People w/ Hepa B/C or with HIV Laboratory Diagnosis Serologic assays: ○ PCR ○ ELISA HAV in Stool – Usually detected 1st ; Basically, 2 weeks before and after the onset of jaundice, HAV can be detected in the stool. Anti-HAV IgM – Usually peaks 2 weeks after the elevation of LIVER ENZYMES Anti-HAV IgG – Usually detected AFTER the onset of disease and could persist for a long time Treatment, Prevention and Control TREATMENT No specific treatment – usually you are advised to rest or have an adequate nutrition; fluids also are needed since the virus itself is self-limiting; Does not usually progress to Chronic Liver Disease unless the patient has an underlying condition on the liver. COPY FOR: ORIT, ANNE CHELSEA R. | 1 VIRUS INACTIVATION Heating food to above 85 deg C for at least 1 minute ○ The longer, the better ○ Also depends on the quality of the food Surface disinfection with Sodium Hypochlorite or Bleach (1:100) VIRUS DESTRUCTION Autoclaving Boiling in water Dry heat (180 deg C for 1 hour) or (160 deg C for 2 hours) Ultraviolet Irradiation (1 min at 11 watts) Treatment with FORMALIN or treatment with CHLORINE (10-15 ppm for 30 mins) ○ Formalin (1:4000) 3 days @ 37 deg C Natural Host → HUMANS Can survive OUTSIDE the body and can remain infectious for at least 7 days Most people with chronic HBV infection are ASYMPTOMATIC and have no evidence of liver disease or injury Average of 90 days or Weeks up to 6 months (could be longer) TRANSMISSION: Sexual Intercourse Sharing of Needles/ Syringes Through birth Contact w/ contaminated blood PREVENTION AND CONTROL Vaccination of Hepatitis A - Best prevention method ○ Specially for Frontliners, Staffing for child care, Food handlers, Military personnel, etc. Practicing Good Hand Hygiene HEPATITIS B Enveloped dsDNA; Icosahedral capsid 3 forms: ○ Spherical (22nm) ○ Tubular/ Filamentous (22-200nm) ○ Dane Particle (42nm) Complete virion (w/ genome) CLASSIFICATION: Genus: Orthohepadnavirus Family: Hepadnaviridae CONTENTS: Hepatitis B surface antigen (HBsAg) Hepatitis B core antigen (HBcAg) Viral DNA genome Clinical Manifestations PEOPLE W/ INCREASED RISK OF HEPATITIS B: Infants born from mothers w/ Hepa B Sex with someone who has HBV MSM Patients that are constantly exposed to needles (Dialysis indiv.) Users of injection and Non-injection drugs Healthcare workers MOST COMMON SYMPTOMS: Jaundice, Loss of Appetite, Nausea and Vomiting, Fever and Fatigue Laboratory Diagnosis & Identification HEPATITIS B ANTIGENS HEPATITIS B ANTIBODY Hepatitis B surface antigen (HBsAg) 1st to appear in the SERUM (once there is Hepatitis B surface antibody (anti-HBs) infxn) Hepatitis B core antigen (HBcAg) CANNOT be detected in the serum but can be detected through LIVER BIOPSY Found inside HEPATOCYTES Total hepatitis B core antibody (anti-HBc) Means that the virus is actively multiplying in the liver (Viral Replication) Perform Immunohistochemistry COPY FOR: ORIT, ANNE CHELSEA R. | 2 Treatment, Prevention and Control TREATMENT There is no medication available Rest, adequate nutrition and fluids Patients with more severe symptoms may need to be hospitalized Take medication indefinitely (under the guidance of the attending physician) ○ To avoid damaging the liver PREVENTION AND CONTROL Getting vaccinated Proper handwashing Never share needles, syringes or even water Follow universal precautions Use birth controls to prevent the spread of sexually transmitted disease Hepatitis B envelope antigen (HBeAg) Appears in the serum Hepatitis B envelope antibody (anti-HBe) Presence indicates INFECTIVITY of the individuals (Viral Infection) Used to identify whether the patient has Acute or Chronic HBV infection Also used to identify immune status of the individual (either susceptible or Immune) Interpretation HEPATITIS C HBsAg (+) Acute infection → (+) for IgM and Total Anti-HBc Chronic infection → (+) ONLY for Total Anti-HBc ○ Meaning only IgG is detected, and the body stopped producing IgM anti-HBc HBsAg (-) Immune due to PREVIOUS INFECTION → (+) for Anti-HBs and Total Anti-HBc ○ Patient has Core Antigen, meaning the virus got into the patient which produced HBcAg Immune due to VACCINATION → (+) ONLY for Anti-HBs ○ Patient has no Core Antigen, meaning the virus did not reach the patient → no HBcAg → no Anti-HBc Susceptible → (-) for BOTH Anti-HBs and Total Anti-HBc Acute Infection Acute Infection w/ (↑) Infectivity Convalescent Immune due to previous infection HBsAg + + + - HBeAg - + - - Anti-HBs - - - + Anti-HBe - - + + CLASSIFICATION: Genus: Hepacivirus Family: Flaviviridae STRUCTURE: Single-stranded RNA genome surrounded by icosahedral capsid with envelope. COPY FOR: ORIT, ANNE CHELSEA R. | 3 Non-cytopathic virus; usually enters the liver cell and undergoes replication simultaneously causing cell necrosis by several mechanisms, like Immune mediated cytolysis and other phenomena such as: ○ Hepatic steatosis, ○ Oxidative stress ○ Insulin Resistance Natural Host → HUMANS TRANSMISSION: Sexual Intercourse Sharing of Needles/ Syringes Through birth Contact w/ contaminated blood Sharing of Toothbrush or Razors for shaving EIA → RIBA → PCR If both positive for EIA and RIBA then proceed to PCR testing PEOPLE W/ INCREASED RISK OF HEPATITIS C: People with HIV People who is in Hemodialysis People who donated/Received blood or from Organ transplants Healthcare and emergency medical and public safety personnel Medtechs (constant exposure to needles) Mucosal exposures Children who are born from mothers with HCV infxn. CLINICAL MANIFESTATIONS People with newly acquired HCV infection usually are ASYMPTOMATIC The average period from exposure to symptom onset is 2-12 weeks Most people with Chronic HCV infection are asymptomatic ○ May also lead to diseases that is not only related to the liver such as: Diabetes Glomerulonephritis Porphyria Cutanea Tarda Non-Hodgkin's Lymphoma SIGNS & SYMPTOMS Loss of appetite Nausea vomiting Joint pain Jaundice Fever fatigue Dark urine Clay-colored stool Abdominal pain CONFIRMATORY TESTING: Qualitative and Quantitative assays for HCV RNA: RT-PCR or Recombinant Immunoblot Assay (RIBA) HCV genotyping Serologic testing Liver Biopsy – to determine the degree of liver damage; chronic HCV TREATMENT PEGylated interferon combined with ribavirin ○ PEG + Interferon + Ribavirin ○ Polyethylene Glycol will serve as a VESICLE/ CONTAINER for the interferon ○ Ribavirin is an antiviral drug Antiviral therapy Telaprevir and boceprevir Sofosbuvir Orthotopic liver transplantation People living with Hepatitis C should: Be vaccinated against hepatitis A and Hepatitis B Avoid alcohol Check with their doctor taking any prescription pills, herbs, supplements or over the counter medications (Medicol; ibuprofen) Be tested for HIV PREVENTION AND CONTROL Safe and appropriate use of healthcare injections; Safe handling and disposal of sharps and waste; Provision of comprehensive harm reduction services to people who inject drugs Testing of donated blood for HBV and HCV (as well as HIV and syphilis) Training of health personnel Prevention of exposure to blood during sex LABORATORY DIAGNOSIS SCREENING TESTS FOR ANTIBODY to HCV (anti-HCV): Enzyme Immunoassay (EIA) ○ In low-risk patients w/ (+) EIA, they will have to undergo confirmatory testing using RIBA COPY FOR: ORIT, ANNE CHELSEA R. | 4 HEPATITIS D CLINICAL MANIFESTATION More severe than the other type of Hepatitis viruses ○ Can run either acute or chronic course 3-7 weeks of incubation period ○ Fatigue ○ Lethargy ○ Nausea ○ Anorexia Symptoms last for about 3-7 days Jaundice occurs in the next phase of symptoms ○ Fatigue and nausea continues ○ Abnormal Serum Bilirubin Level ○ Clay-colored stool ○ Dark urine Left: Hepatitis D virus sans the capsid as it borrows structures from HBsAg, Right: Hepatitis B virus CLASSIFICATION: Genus: Deltavirus (hence the letter D) Family: Kolmioviridae SMALLEST virus known to infect animals, therefore it is not really a full virus, but considered a SATELLITE VIRUS/SUBVIRAL AGENT. Why? ○ Because of its special structure STRUCTURE: Small, Spherical, Single-stranded, enveloped particle with Hepa B surface antigen, and negative-sense RNA molecule Genetic material is wrapped in HBsAg ○ Usually present if Hepatitis B is primarily present (Coexisting infection) PATHOGENESIS The production and transmission of HDV is ENTIRELY DEPENDENT on HBV to provide HBsAg Replicates only in the hepatocytes HDV antigens ○ Small delta antigen – usually produced in the early stages of infection ○ Large delta antigen – produced in the later stages of infection High Risk of Infection: People with Chronic HBV, Injection-drug users, commercial sex workers, Men-having-sex with men ➔ ➔ ➔ ➔ ➔ Transmission: Broken skin Contact with infected blood ◆ E.g. blood transfusion, sharing of syringes, sharp objects, etc. Mother to child (Rare case) (at birth) Unprotected Sexual Intercourse Sharing toothbrushes, razors, etc. Complications HDV is known to occur either as a co-infection or a superinfection Co-infection Superinfection occurs when both HDV and HBV contracted simultaneously Acute HDV & HBV infection occurs when Chronic HBV carriers are infected with HDV Severe acute hepatitis and Chronic Hepatitis D infection in 80% of the cases LABORATORY DIAGNOSIS & IDENTIFICATION The diagnosis of Hepatitis D infection is made following serologic tests for the virus Radioimmunoassay (RIA) & Enzyme Immunoassay (EIA): Detects the total anti-HDV antibodies Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR): To monitor any ongoing HDV infection Detects 10-100 copies of HDV genome in infected serum TREATMENT No specific treatment for HDV PEGylated interferon alpha PREVENTION AND CONTROL Prevention of Hepatitis D virus is based on prevention of Hepatitis B virus Co-infection: the HBV or post exposure prophylaxis can be used to prevent the infection Superinfection: educate chronic HBV carriers about transmission and risky behaviors; proper health infection COPY FOR: ORIT, ANNE CHELSEA R. | 5 HEPATITIS E Other names: Enteric Hepatitis; Self-limiting Hepatitis; Most common cause of Acute Hepatitis ○ Acquired through contaminated water or food that is not properly cooked Replication only occurs in the LIVER ○ Incubation may go from 20-40 days (slow) Reactivation is possible CLASSIFICATION Genus: Hepevirus Species: Hepeviridae STRUCTURE: Small, nonenveloped virus with a single-stranded RNA genome; 4 genotypes Transmission Fecal-Oral; vertical transmission is possible; zoonotic transmission; breastfeeding Disease Hepatitis similar to that caused by hepatitis A virus except for extraordinarily high case fatality rate (10%-20%) among pregnant women Diagnosis Serology Treatment Supportive Prevention Avoid contact with the virus PATHOGENESIS Characteristics Genotype 1 Genotype 2 Genotype 3 Genotype 4 Geographic location Africa and Asia Mexico, West Africa Developed Countries China, Taiwan, Japan Transmission route Waterborne fecal-oral; person-to-person Waterborne fecal-oral Food-borne Food-borne Groups at high risk for infection Young adults Young adults Older Adults (>40 years) and males, Immuno-compromis ed persons Young adults Zoonotic transmission NO YES Chronic infection Occurrence of Outbreaks Common Smaller scale outbreaks YES NO Uncommon Uncommon CLINICAL MANIFESTATIONS When they occur, the signs and symptoms of Hepatitis E are similar to those of other types of acute viral hepatitis and liver injury Fever Fatigue Loss of appetite Nausea Vomiting Abdominal pain Jaundice LABORATORY DIAGNOSIS AND IDENTIFICATION Diagnosis can be confirmed only by testing for the presence of antibody against HEV or HEV RNA Liver function tests ○ Increased levels of serum bilirubin (↑) ○ Increased AST (↑) ○ Increased ALT (↑) CONFIRMATORY TEST: Nucleic acid testing is recommended to confirm positive serology results in areas where HEV is not endemic TREATMENT AND MANAGEMENT Hepatitis E usually resolves on its own without treatment (Typically advised to rest) There is no specific antiviral therapy for acute hepatitis E Patients who do develop fulminant liver failure need liver transplantation PREVENTION & CONTROL Good sanitation COPY FOR: ORIT, ANNE CHELSEA R. | 6 VACCINE No FDA-approved vaccine for hepatitis E is currently available Clean Drinking Water Boiling and Chlorination of Water Avoiding raw pork and venison VIRUS FAMILY NUCLEIC ACID TRANSMISSION INCUBATION (DAYS) Hepatitis A Picornaviridae Single-strand RNA; Nonenveloped Fecal-oral 15-502 Hepatitis B Hepadnavirus Double-strand DNA Parenteral; sex 30-1808 >90% Infants

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