VIROLOGY SUM.pdf

VIROLOGY BALTIMORE CLASSIFICATION Based on viral genome and mechanism of mRNA Genome could be o Double stranded (+/-) o Single stranded (+) (-) o RNA or DNA o Linear, circular, segmented Genome replication can enter target cell v...

VIROLOGY BALTIMORE CLASSIFICATION Based on viral genome and mechanism of mRNA Genome could be o Double stranded (+/-) o Single stranded (+) (-) o RNA or DNA o Linear, circular, segmented Genome replication can enter target cell via: o Directly: +ssRNA can dsDNA also can enter cell directly? o need prior modification ▪ RNA dep RNA pol: synthesis RNA from RNA template ▪ Reverse transcriptase: RNA to DNA 7 classification: CLASS 1: dsDNA Can be incorporated into host cell to be transcribed as mRNA Does not need to bring its own enzyme, it use host RNA polymerase II herpes CLASS 2: ssDNA Replication involves DNA dep DNA pol ssDNA – dsDNA – mRNA need to be dsDNA because RNA pol reads double stranded DNA parovirus CLASS 3: dsRNA replication involves RNA dep RNA pol 2 strands need to separate and use 1 for a template Not infectious because cant directly be translated to mRNA rotavirus CLASS 4: +ssRNA Replication can be direct using ssRNA as mRNA Still need RdRp Also called sense Hepatitis C CLASS 5: -ssRNA Replication need RNA dep RNA pol because ssRNA strand is complementary to mRNA -ssRNA - +ssRNA – mRNA  Not infectious because cant directly be translated to mRNA Influenza & ebola CLASS 6: ssRNA retrovirus Two copies will need RNA dep DNA pol or REVERSE TRANSCRIPTASE HIV CLASS 7: dsDNA retrovirus Replicates using DNA dep DNA pol Hepatitis B VIRUS STRUCTURE Genome, nucleocapsid, envelope, matrix OR tegument, enzyme 1. Nucleic acid or gnome Genetic information for the synthesis of protein Function: o Acts directly as mRNA or template for mRNA o Acts as template to make new generations of genomes dsDNA: single and large dsRNA: segmented, where each segment has its own single gene ssDNA: small ssRNA (+): directly use for synthesis translation of protein ssRNA (-): complementary to mRNA, so will need RdRp for transcription for protein synthesis without the genome, virus will not be infectious 2. Nucleocapsid core Cover the virus genome, enzyme, receptor binding,  Function: protect genome, transmission and infection Composed of protein subunits: capsomeres Highly immunogenic o Have epitope for immune response Made of virus encoded protein o This protein will be the target for vaccination o Generate antibodies against nuclear capsule protein Types of arrangement a. HELICAL ARRANGEMENT o Single type of protein around central axis: only 1 gene is required o segmented linear nucleic acid, coiled o Hollow, rod-shape or filamentous o Protein subunit repeated over and over forming capsid: need less energy to assemble o Tobacco mosaic virus o Can be enveloped or naked, usually RNA b. ICOSAHEDRAL ARRANGEMENT o More prevalent o Packaged with 20 sides of equilateral triangles o Gives spherical appearance o Non enveloped: poliovirus, HPV (released via lysis/cytopathic, more virulent) o Enveloped: herpes, Hep B, HIV (released via budding) 3. Envelope Made of lipid bilayer Some envelope could come from host membrane when formed during budding o Virus can use either outer membrane or internal membrane (ER, nuclear membrane) of host Membrane studded with protein: surface, spikes, peploma o Code for virus and host by bind with the host cell receptor Naked or no envelope virus: nucleocapsid will bind to host Enveloped virus: glycoprotein in lipid bilayer will bind to receptor WAYS OF BUDDING a. Bud from plasma membrane o Matrix will be there to stabilise virus particle o HIV, influenza b. Bud into cytoplasmic vesicles in ER or golgi o Does not need matrix protein o Released by exocytosis o HBC, HCV c. Bud through nuclear membrane o Have teguments protein instead of matrix o HSV 1&2 Characteristic of enveloped virus: o Often glycosylated: have glycoprotein as receptor o Bind to receptors on cell surface o Highly immunogenic: antigenic epitope for immune response Function of envelope o Envelopes virus nucleocapsid: better protection o Transmission and infection o Cause instability because of the added lipid bilayer o Easily dehydrated during transmission: more flu in winter because people huddle, morre moist environment. Sensitive to surroundings  4. Matrix Some have matrix OR tegument Layer between nucleic acid and envelope Made of virus encoded matrix protein Present only in virus that bud in PLASMA MEMBRANE Function o Stabilise nucleocapsid o Coordinate envelopment of nuclear capsid during budding and assembly of virus particle o Specific component for specific virus Ex: HIV, influenza A 5. Tegument Function exactly the same like matrix Made of virus encoded tegument protein Layer between nuclear capsid and envelope Present only in virus that bud in NUCLEAR MEMBRANE Function o Stabilise nucleocapside o Coordinate budding and transport through cytoplasm HSV: herpes 6. Enzyme Contain in the virus nucleocapsid Made of protein Present in virus that CANNOT directly become mRNA (all except +ssRNA, dsDNA) Common enzyme: RNA dependent RNA polymerase Synthesis viral mRNA from RNA template (direct or RdRp) or synthesis viral DNA from RNA template (reverse transcriptase) Transcription of dsDNA genome takes place in cytoplasm because they carry own transcription machinery, that allows them to transcribe genome in cytoplasm DNA dep RNA pol are found in nucleus Function o Necessary for synthesis of viral nucleic acid o Polymerase: make new genomes or mRNA o Integrase: integrates virus DNA to chromosome o Protease: digest virus polyprotein VIRUS INFECTION AND REPLICATION STEPS OF INFECTION (usually happen in the nucleus, then to the cytoplasm then releae) Remember that virus is an intracellular parasite that is metabolically inactive unless its in a host and hijack machinery 1. ATTACHMENT & INTERNALISATION (PENETRATION) & UNCOATING Virus particle must be partially or completely disassembled  Genome must be encapsidated (nucleocapsid) to protect the nucleic acid when transfer to host cell Then inside the cell, protective is unstable so genome is released a. Attachment: o Virus particle encounter host o Attachment in capsid via glycoprotein in the envelope attach to cell receptor on host surface o Receptor help with uncoating, which can aslo be triggered with low pH o Enveloped bind via transmembrane glycoprotein o Nonenveloped virus bind via capsid surface or projections b. Internalisation o Receptor mediated mechanisms o Evade host physical defenses: dead skin, low pH levels, mucous layers, ECM o Naked virus eukaryotic virus enter via DIRECT INJECTION of genome, no fusion o Enveloped eukaryotic virus enter MEMBRANE FUSION or ENDOCYTOSIS c. Uncoating o Release of genome from nucleocapsid o Some uncoat at plasma, some in intracellular 2. EXPRESSION & REPLICATION Make viral RNA o To synthesis viral protein and assembly of genome, genome must be copied into mRNA o RNA virus except retrovirus and +ssRNA use RdRP o Retrovirus and most DNA virus use RNA polymerase 2: produce cellular mRNA Make viral protein o mRNA need to be translated by host machinery a. Expression o Remember baltimore classification o Depends on viral genome o Enzyme is used in this step if necessary (RdRp, reverse transcriptase) o DNA virus use host cell protein and enzyme to replicate viral DNA and transcribe mRNA = protein synthesis b. Replication o Copy the viral genome RNA or DNA o Occurs in different location: ▪ Nucleus: more DNA (HSV, VZV) and less RNA (influenza) ▪ Cytoplasm: more RNA (rhinovirus, HCV) and less DNA (poxyvirus) ▪ Both location: HIV, HBV 3. ASSEMBLY & RELEASE A. Assembly o Produce protein and genome synthesis o Assembly of the nucleocapsid in cytoplasm or nucleus o Package genome and enzyme to nucleocapsid B. Release o Naked virus: release by lysis from the cell or cytopathic (Structural changes in cell): Rhinovirus, poliovirus, HAV o Enveloped virus: release by budding from nucleus via plasma membrane, ER, golgi and release outside cell via exocytosis: HBV, HIV REPLICATION OF RNA VIRUSES KEYPOINTS RNA dep RNA polymerase enzyme catalyse replication of RNA from RNA template o RdRp produce RNA genome and mRNA from RNA templates DNA dependent RNA polymerase catalyse transcription of RNA from DNA template Viral mRNA must be readable by host ribosome DISEASE Both polio and HCV are +ssRNA Genome is translated upon entry to cytoplasm, produce viral protein including Rdrp + strand is copied to a – strand, where its used as a template for the synthesis of additional + strand The new + strand will then be a template for new genomic replication 1. POLIOVIRUS (PICORNAVIRIDAE) Structure o Genome: +ssRNA linear o Non enveloped with icosahedral nucleocapsid o Capsid protein: VP1, VP2, VP3, VP4 o Class 4 baltimore classification Receptor o CD155: immunoglobulin superfamily o virus spike protein VP1 will attach with CD155 o naked virus: bind directly via mediated injection to host cell receptor character o still have RdRP even though +ssRNA can be used directly as mRNA o does it mean it doesn’t carry the RdRp enzym but use hosts (why??) o replicates in cell cytoplasm o released by cell lysis proteins (structural vs non structural) o structural: 4 protein in nucleocapsid VP1, VP2, VP3, VP4: ▪ VP1 VP2 VP3: surface of capsid ▪ VP4: inside capsid with RNA genome ▪ Function: protect RNA and help attach to host receptors,, formulating virion o Non structural protein: RdRp, VPg ▪ expressed inside cell after virus infect the host cell ▪ function: replicates viral genome  ▪ RdRp for replicating virus RNA genome ▪ VPg is linked to 5 end of virus RNA: initiate RNA synthesis replication 1. +ssRNA: virus polyprotein cleaved 2. RdRP create dsRNA 3. RdRp create +ssRNA for actual mRNA & new genome 4. Production of capsid protein VP1-4 5. Packaging of RNA 6. Release via lysis cytopathic 2. HEPATITIS C HCV (FLAVIRIDAE) Structure o +ssRNA o Enveloped icosahedral with core protein © o 45-60nm o Envelope protein: glycoprotein E1 & E2 o Class 4 baltimore classification Receptor o Cellular receptor CD81 Replication 1. E1 and E2 protein bind to receptor CD81 2. Receptor mediated endocytosis + fusion 3. Release nucleocapsid 4. Assemble in cytoplasm 5. Replication: non structural protein change environment, ER, pH, and release genome 6. Genome function as mRNA since its +ssRNA 7. +RNA + RdRp becomes dsRNA + RdRp becomes +RNA for new genomes 8. Bud with the ER and released via exocytosis (non cytopathic) 3. CORONAVIRUS Class 4 baltimore classification Replication  1. Virus bind to receptor and enter via surface fusion 2. Encodes genome to begin translation 3. +ssRNA + RdRp to make -RNA, transcribed and translated, 4. Bud into ER and released via exocytosis 4. INFLUENZA A (ORTHOMYXOVIRIDAE) Structure o -ssRNA genome with 8 segments (can less, not more) o Enveloped with helical nucleocapsid and a matrix o Class 5 baltimore o Protein: Hemagluttinin HA & Neuraminidase NA o Enzyme: RdRp Genome o Linear 8 segments but can code for 10 protein via splicing o (only the one with 8 segment can create infection) o 13600 nt o Enzyme is attached to each segment Receptor o Sialic acid o Respiratory epithelium o HA Hemagluttinin protein binds to sialic acid receptor o neuroamidase will cleave to actually allow the virus to leave cell Characteristic o Difference between bird flu in lung vs human o Alpha 23: lower respiratory tract (lethal, low transmission) o Alpha 26: upper respiratory tract (not lethal, high transmission) Replication o Virus HA protein bind to receptor sialic acid o Enter via receptor mediated endocytosis o Low pH cause fusion of virus envelope and release nucleocapsid o RdRp enzyme is carried by the virus and replicates in nucleus o Enveloped at plasma membrane o Released via budding where neuroamidase cleaves sialic acid from the cell surface to actually exit GENOME -SSRNA 1. RdRp attach to -RNA 2. New +rNA is synthesised 3. RdRp attach to +RNA 4. New -RNA is synthesised 5. Create new genome 5. HIV (RETROVIRIDAE) Structure o +ssRNA with TWO IDENTICAL COPIES o Icosahedron nucleocapsid with matrix o reverse transcriptase protein, integrase (IN), nucleocapsid (NC) o Similar symptoms to flu o Need reverse transcription enzyme o Class 6 baltimore classification  Genome o Envelope protein gp120 and gp41 o Gp120 will bind to cellular receptors CD4 o The GP120 binding make conformational change for Gp41 binds to CCRS and CXCR4 o 3 protein: reverse transcriptase, integrase, protease Receptor o CD4 molecules on T lymphocytes o Infects monocytes and macrophage Replication 1. Virus protein gp120 binds to CD4 and gp41 to CCRS and CXCR4 2. Cause conformational change that expose gp41 to bind 3. Enter via receptor mediated surface fusion 4. 6 helix bundle for fusion as an antiviral drug target 5. Release of nucleocapsid genome to cytoplasm o Replication in cytoplasm and nucleus REVERSE TRANSCRIPTASE (in cytoplasm) Main function: ssRNA to dsDNA 3 steps: i. Enzyme 1: RNA dep DNA pol make 2 copies of +ssRNA to a RNA-DNA hybrid where the new DNA strand is complementary to the RNA strand ii. Enzyme 2: RNase H will cleave and degrade the RNA strand in the RNA-DNA hybrid, leaving a ssDNA behind iii. Enzyme 3: DNA dep DNA pol will use -ssDNA as a template to synthesis a DNA strand, creating a linear dsDNA INTEGRATION (in nucleus) 1. Circularisation of linear DNA: protects it from degradation 2. Cleavage of terminal dinucleotides at 3 prime end of DNA: use the integrase (IN) enzyme 3. Single stranded nick and end join to host DNA: 3 prime end of virus DNA is linked to 5 prime end of host DNA 4. Ligation into host DNA: gaps is filled in, make the virus DNA a part of the host cell DNA 5. Use host RNA polymerase II to create +RNA new genomes and mRNA from the pre dsDNA result of RT Assembly of nucleocapsid, genome packaging, release via budding REPLICATION OF DNA VIRUSES PRIMING OF DNA SYNTHESIS USING DdDp 1. RNA Okazaki fragments 2. DNA hairpin structure 3. protein covalently attached to the 5 prime end STRATEGIES FOR REPLICATION OF COMPLEMENTARY STRAND 1. Strand displacement strategy: adenovirus (protein), parvovirus and poxvirus (DNA hairpin) 2. Replication fork strategy: leading and lagging strand, herpes and human papillomaviruses DISEASE EXAMPLES! 1. ADENOVIRUS** Structure o Linear dsDNA o Naked icosahedral nucleocapsid Characteristic o Backbone for astrazeneca o Clinical infection will attack respiratory and gastroenteritis o 7 species A-G Replication o Bind to receptor and adhesion molecule o Enter via endocytosis o Release nuclear capsid and utilise MICROTUBULES to move into nucleus o Genome will be injected in the nucleus o 3 rounds of gene expression 2. HEPATITIS B HBV Structure o Enveloped virus with icosahedral nucleocapsid o dsDNA (relaxed circular) ▪ relaxed circular means the genome is not entirely a double stranded. ▪ Outside is circular but inside the nucleotide is not complete, ▪ so its not super coiled o have its own enzyme DAN dep DNA pol and reverse transcription o protein: surface antigens of L, M, S characteristic o jaundice or yellow color for clinical symptoms o attack on hepatocytes or liver cell o blood sample could show ▪ normal DNA genome with virions ▪ empty surface antigen particles with HBeAg in blood stream replication o important: relaxed circular rcDNA will become covalently close circular cccDNA o uncoating in cytoplasm o rcDNA becomes cccDNA in nucleus o either goes ▪ transcription and translation ▪ virus DNA integrates with host genome o using host RNA polymerase II: ds cccDNA becomes +ssRNA pregenome o goes outside nucleus and encapsidation o reverse transcription: +ssRNA pregenome to ds RC DNA ▪ Enzyme 1: RNA dep DNA pol use RNA pregenome to create a DNA strand (-ssDNA)  ▪ Enzyme 2: RNase H degrades the RNA strand of the pregenome, leaving a RNA primer for synthesising +DNA strand ▪ Enzyme 3: DNA dep DNA pol syntheses +ssDNA strand using the -ssDNA strand as a template o Release through ER REVERSE TRANSCRIPTASE OF HIV AND HBV Both replicate using RT that happens in cytoplasm HBV happens inside nucleocapsid, HIV happens outside nucleocapsid HBV use RT when its dsDNA and ready to exit cell HIV use RT when its dsDNA but just when it gets incorporated to cell 3. HERPESVIRIDAE FAMILY (HSV, HHV1, VZV) Structure o dsDNA o enveloped icosahedral nucleocapsid with 6 protein o tegument protein 10-20 o double the size of influenza Receptor o HSV: heparan sulphate proteoglycan HSPG o VZV: insulin degrading enzyme IDE replication o enter via surface fusion, exit via budding at nucleus, ER, exocytose o HSV envelope bind with HSPG o VZV glycoprotein E gE binds with IDE o Nucleocapsid + tegument enter cytoplasm and transported by microtubule (dynein meidated) o Enter nucleus o Produce 3 mRNA classes ▪ Immediate early mRNA (IE): essential control for function ▪ Early mRNA (E) ▪ Late mRNA (L) o Produce 3 protein classes ▪ Regulatory IE ▪ Nonstructural E ▪ Structural L: essential for virus protein assembly o DNA replication via ROLLING CIRCLE ▪ Linear dsDNA genome is circularised by DNA ligase into a cccDNA ▪ Enzyme cleaves in the circular DNA, open the outer circle of the dsDNA ▪ Use internal circle as template for leading strand, displacing original strand ▪ Release leading strand and lagging strand forming a dsDNA long molecule o Virus assemble and bud with nuclear membrane and fuse to cytoplasm  o Release via exocytosis VIRUS HOST INTERACTION 1. TRANSMISSION a) Respiratory: influenza A, measles b) Enteric: poliovirus, hep a, rotavirus, adenovirus c) Blood borne: HBV, HCV, HIV, Ebola d) Sexually transmitted: HIV, HBV, HSV, HPV, Ebola e) Skin or direct contact: Ebola, HSV f) Zoonotic (DIRECT transmission) o Monkeys: HIV o Dogs: rabies o Bats: hendra o Civets and bats: SARS o Birds: influenza A g) Arbovirus (part of zoonotic but INDIRECT) o Animal – arthropod – human o All 3 host must have receptor o Mosquito, tick o Zika, dengue, wet nile, ross river, murray valley 2. REPLICATION INSIDE HOST Cause cell destruction or dysfunction Spread to other cells by blood and lymph Type of infection o Localised: spread adjoining cells nearby  o Systemic: spread to other cells via blood lymph 3. VIRUS HOST INTERACTION (antiviral immunity) Type of infection o Transient: infection is controlled (influenza A, polio, measles) o Persistent infection: unable to clear or control (HSV, HIV, HBV, HCV, HPV) ▪ Effectiveness is determined by ▪ Rate of virus replication and presentation of antigens ▪ Size of infecting virus dose ▪ Route of infection ▪ Age of host ▪ Evasion of the immune response Successful control need innate and adaptive: control spread + eliminate infected cells Type of immune response o Innate: early, rapid, limited, non specific. Aim to SUPPRESS infection o Adaptive: slow, specific, memory. Aim to CLEAR infection ▪ Humoral: B cell and antibody ▪ Cell mediated: cytotoxic T cell Examples: o transient and localised: influenza A o transient and systemic: measles and poliovirus o persistent and localised: herpes simplex, HBV, HCV o persistent and systemic: VZV, HIV HERPES SIMPLEX PERSISTENT EX lytic cycle: epithelial layer, immune system attack and clear infection virus migrate and hide in ganglions where T cell cannot target latent cycle: virus enter sensory nerve endings waiting for reactivation VARICELLA ZOSTER VSV PERSISTENT EX respiratory infection local: reside in ganglion systemic infection but reactivation is local systemic: skin rash and chicken pox reactivation: shingles clinical presentation o primary replication in lymph nodes o secondary in liver, spleen o flu like symptoms + rash control: varivax vaccine HIV PERSISTENT EX immune suppression leads to AIDS (when multiple disease starts to occur)  long symptoms like flu target cells: CD4 5 cell, macrophage, dendrites, monocytes humoral and cell mediated immune response persistent: virus titre will decrease but virus stay inside CD4 cell (irreversible), then over years CD4 decrease and HIV RNA copies increase. CD4 cell number starts to drop due to o Direct killing infected cells through fusion of CD4t cell cause SYNCYTIA o Synctia: process of multiple host cell fuse to become a big cell with multiple nuclei ▪ Gp120 on surface of HIV bind to CD4 receptor in T cell, then virus fuse with cell membrane and virus enter to infect. But Gp120 can also bind to CD4 receptor on nearby healthy CD4 T cell, so it fuse again together forming a big cell. ▪ Synctia allow HIV to spread directly from one cell to another without being expose to extracellular environment o Kill infected CD4 T cell by CD8 t cell o Threshold could take 8 years Treatment: ART antiviral that target integrase, protease, reverse transcriptase protein MEASLES PARAMYXOVIRIDAE -ssRNA Envelope protein F. H (fusion and hemagglutinin) Enter via surface fusion, RdRp, assemble at plasma, bud Infect respiratory through receptor CD150 (macrophage, T and B cell) & receptor nectin 4 (epithelial cell) Target lymphocyte: drain antigen to lymph node then attack T and B cell Clinical presentation o 8-10 incubation o Dissemination to lymph and endothelial cells o Primary viremia: lymphnode o Secondary viremia: spread to body surface, rash, itchiness Treatment: MMR vaccination or anti itch lotion POLIOVIRUS Fecal oral transmission: infect intestine Asymptomatic infection Replicates in pharynx, GIT, released in feces Spread to lymph node and can spread to CNS Spread to CNS: paralysis, polio muscle atrophy, aseptic meningitis (Attack motor neuron) Controlled by IPV (inactivated via injection) or OPV (live attenuated orally) vaccination Treatment: iron lung chamber to help with respiratory paralysis because neuron attacked, muscle weak LOCALISED INFECTION EXAMPLES HEPATITIS B Local: Infect liver cells Transmission via blood or body fluid Clinical infection: o Adult: transient o Children: persistent (cause chronic liver disease, cirrhosis, cancer or liver failure) Prevention: HbsAg, yeast Treatment: nucleoside analogue antiviral Cancer: hepatocellular carcinoma Stage o Cirrhosis and hepatocellular carcinomar: late stage  o Immune attack hepatocytes, deposition of collagen and scarring in liver that cause FIBROSIS o Fibrous tissue block circulation HEPATITIS C Localisied, transient, persistent Transmission via direct exposur to blood Attack liver via receptor CD81 Transient 25, peristent 75% Treatment: direct acting antiviral to stop virus replication Prevention: no vaccine Cancer: hepatocellular carcinoma Treatment: interferon DETECTION VIA PATTERN RECOGNITION RECEPTOR (PRR) Innate immune system Induce IFN 1 o Induce cellular resistance o Inhibit viral replication o Impede viral dissemination o Macrophage, natural killer, fibroblast report to produce IFN 1 1. Cytosolic receptor Within cytoplasm: for intracellular pathogens Have both DNA and RNA (MDAs, RIG-1) sensor: IRF 3, NF B How to distinguish pathogenic RNA and DNA o Cap: RNA lack methyl cap but show triphosphate o dsRNA in cytoplasm: should be single, not double o dsDNA in cytoplasm: DNA should be in nucleus or mitochondria 2. Toll like receptor attach in cell surface or endosome aim for extracellular pathogens and recognise PAMPs expressed by specialised cell: macrophage, dendrites, neutrophil TLR 1,2,4,5,6,10,11,12,13 o TLR 3: dsRNA o TLR 7 TLR 8: ssRNA o TLR 9: DNA How to distinguish nucleic acid o Location of TLR o TLR binding to nucleic acid o RNA double strand or single strand EPIDEMIOLOGY OF VIRUS INFECTION Factors that promot this emergence: 1. Virus factors o Host o Cellular receptors o Mutations: changes in the genetic makeup of viruses that allow them to infect and adapt to growth in new hosts ▪ changes in the genetic makeup of viruses that allow them to infect and adapt to growth in new host. ▪ Reassortment: when virus have segmented genome, reassortment and become new virus within the same virus family/species o Different strands from the sam virus ▪ Virus mutate o Poor proof reading of virus polymerase enzyme during the RdRp, etc o If we have mutation in the same virus: ANTIGENIC DRIFT (different version of surface glycoprotein, different globular head) o Allow infecion of previously immun hosts ▪ Genetic reassoortment = dramatic change o In virus with SEGMENTED genome, the segment are reassorted (random packaging of RNA during assembly) o This create a NEW antigenetcially distinc virus: ANTIGENIC SHIFT ▪ These 2 allow adaptation of virus to the human host ▪ Ex: pigs get infected with human flu and bird flu, new virus will have some protein that have both glycoprotein 2. Virus transmission o Primary transmission of animal virus to humans o Secondary transmission within human population (human to human) 3. Host factors o Ecological changes ▪ Human enter new area: deforestation, cave ▪ Increase interaction of anima hosts and human o Environmental changes ▪ Climate, global warming: mosquito breeding ▪ Increased opportunities for arbovirus transmission o Demographic consideration: ▪ Air travel, migration: rapid spread o Change in human behavior ▪ Sexual or medical practices ▪ Sporting events DISEASES 1. SARS COV Families: alpha, gamma, delta Replication o Virus SARS CoV 1 & 2 attach via ACE 2 o Enter via surface fusion o Release genome o Make mRNA using RdRp o Replicate in cytoplasm and assmble helical nucleocapsid o Bud to ER and release via exocytosis Host: bat, civet, camel Transmission: respiratory and fecal oral 2. INFLUENZA A Host: specificity defined by receptor Zoonotic: human, swine, bird, cows, dog, cats, seals Receptor human o sialic acid linked glycan o A2,6 linkage: upper respiratory tract o A2,3 linkage: lower respiratory tract Receptor avian  o A2,3 linkage in intestine and trachea Receptor pigs o Mixed vessel so have both receptors for bird and human flu o H1N1: easy spread, rare fatality, upper resp o H5N1: slow spread, more fatality, low resp Subtypes of virus o 18 subtypes of hemagluttinin, 16 in birds o 11 subtypes of neuraminidase o So total could have 144 possible subtype ▪ Human: H1N1, H3N2, ph1n1 ▪ Swine: H1N1, H3N2, H3N1, H1N2 ▪ Avian: all Antigenic variation (what makes influenza hard to control) a. DRIFT o Mutation within same virus subtype o Affect surface glycoprotein o Seasonal flu o RdRp does not proof read, changes in ORF, changes in protein b. SHIFT o Pigs that have both receptor for A26 and A23 combined o Reassortment of genome produce new virus o Mixture of surface antigens o pandemic Controlled: yearly vaccination Ttreatment: inhibitor neuraminidase to stop virus to be cleaved and exit 3. NIPAH & HENDRA paramyxoviridae zoonosis hendra: similar to measles CONTROL OF VIRUS INFECTION BEFORE INFECTION Prevent transmission o Epidemiology: surveillance and vector control o Public education and control measures o Reduce rate of transmission Establish protective immunity o Passive immunisation with antibodies o Active immunisation with vaccine VACCINATION Protect individual o Prime immune response o Rapid secondary immune response o Train WBC without showing clinical symptoms Protect community o Herd immunity o Not all members need, but high rate needed TYPES 1. Live attenuated virus Poliovirus OPV, rotavirus, measles, VZV Grow virus in cultured cells, force to mutate and replicate in cell line  In human o Replicate poorly o Does not cause disease Concern o Reversion to wild type risk to immunodeficient recipient Advantage o Cheap, natural route o Single dose o Good humoral and cell mediated o Long duration Disadvantage o Contamination o Expensive to store o Side effect because live virus o Reversion to virulence via mutation 2. Inactivated virus Poliovirus IPV, HAV, influenza Advantage o Stable o Safe: virus cannot replicate, no reversion cos dead Disadvantage o More expensive o More humoral than cell mediated o Short live, need multiple dose, need adjuvant 3. recombinant subunit HBV, HPV papillomavirus Advantage o Reduction and quality control o Non toxic o Safe Disadvantage o Less immunogenic cos don’t use whole virus o Need adjuvant and booster o Not good with cell mediated immunity 4. DNA or RNA vacc Virus gene is clone in plasmid vectors or as mRNA mRNA code for spike protein, inject to muscle to function as mRNA pfizer for COVID 5. Live attenuated recombinant virus adenovirus and fowlpoxvirus astrazeneca for COVID spike protein incorporated to genome DNA to become part of genome replication ANTIVIRAL DRUG small molecule chemotherapeutic agent interfere with target replication target enzyme: does not allow RT, etc  does not protect against viral infection DIAGNOSIS OF VIRUS INFECTION SAMPLE COLLECTION SAMPLE TRANSPORT depend on clinical presentation, risk factor, test ensure not damaged or inactivate type 4 or -80 degrees. Dry ice -50 or -60, Liquid test depends on: nitrogen o current infection: virus genome NEVER -20 degrees: lots of ice crystal o previous infection: serology Avoid freeze thaw repetition ex for hiv sample Enveloped virus more susceptible o blood to test for virus, antibodies o heparin blood to test for CD4 level BLOOD BORNE VIRUS INFECTION Transmitted via exposure to infected blood Replicate in liver and release to blood stream Virus that circulate in blood: VIREMIA Samples could be taken from blood or liver biopsy RESPIRATORY VIRUS INFECTION Transmitted via aerosols and inhalation Samples could be taken from respiratory (nasal swab) or blood SEXUALLY TRANSMITTED VIRUS INFECTION Virus present in blood and body secretion Samples could be taken from vesicle fluid or blood (test antibody) DIAGNOSTIC METHOD 1. Grow virus in vitro in cell lines and identify o Need to amplify for virus to grow and could use supernatant o Different virus different cell lines because each virus have their own receptor o Immunostaining: after grow the virus then we can identify viral antigen, or see RNA/DNA 2. Look directly for virus using electron microscopy o Not that specific, so we have to know the structure o Low sensitivity o Cannot different virus species in same family 3. Detection of virus protein: antigen o Antigen is part of pathogen that create the immune response o Immunostaining Can detect single monolayer positive cell virus antigen suing virus specific antibodies coupled to enzyme Ex: horseradish peroxidase o Rapid antigen test Use viral antigen Detect the presence of antigen Swab, have antibody that react, then show signal Take a sample, diffuse, antibodies will capture. Theres a positive control (antugen will diffuse to membrane). Antibody bind to the antigen, showing another line. First line indicate antigen diffuse to membrane, second line indicate theres an antigen: covid that is bound with the antibody o Immunofluorescence  Detect virus antigens using virus specific antibodies coupled to fluorescent tag instead of enzyme Layer of cells, infect with virus, present on infected cells, secondary antibody that is fluorescently conjugated in the microscopy Ex: FITC 4. Detect genome by RT PCR or PCR o Amplification and detection of virus genome o Analysing genome sequence o Quantitative RT PCR for RNA virus, PCR for DNA virus o Sensitive: single copy amplification Need specific primer for each virus 2 primer: upstream and downstream If we know nucleotide, we can know the energy and melting curve o Read out: specific interaction with nucleotides bonds. Know the melting curve and we know the reference for some virus. So we can know if the melting curve in this sample same with the reference for virus A 5. Detect antibodies in serum of infected patient o Serology diagnosis o Epidemiology study Study prevalence of exposure in community Immunisation program, previous disease, history of exposure o Immune status Immunisation program o Previous exposure Patients occupation o How is this done: ELISA (enzyme linked immunosorbent assay) Detect secondary antibody which react against the human antibody o Serological study: