Virology Study Guide

📖 Core Concepts Virology – scientific study of viruses: their structure, classification, replication, host interaction, and disease. Virulence – degree to which a virus causes disease; measured by severity of clinical signs and mortality. Virus classification – two main frameworks: ICTV taxonomy – hierarchical (realm → species) based on morphology, genome, replication, and phylogeny. Baltimore classification – groups viruses into 7 classes according to how they generate messenger RNA. Infectivity vs. genome copy number – infectious particles are measured by plaque‑forming units (PFU) or focus‑forming units (FFU); total viral genomes are measured by PCR‑based viral load. Reverse genetics – creation of infectious virus from cloned cDNA (or plasmids), enabling targeted gene manipulation. Reassortment & recombination – mechanisms that shuffle viral genetic material, generating new strains (e.g., influenza antigenic shift). --- 📌 Must Remember Baltimore Classes dsDNA, 2. ssDNA, 3. dsRNA, 4. (+)ssRNA, 5. (–)ssRNA, 6. ssRNA‑RT, 7. dsDNA‑RT. Plaque assay read‑out – 1 PFU = one infectious virion that creates a visible plaque. ID₅₀ – dose that infects 50 % of a cell/animal population; calculated by Reed‑Muench or Spearman‑Kärber methods. PCR limitation – cannot distinguish infectious from non‑infectious particles; a “test of cure” must wait until nucleic acid clears. Electron microscopy magnification – up to 10⁷×; negative staining gives dark background; cryo‑EM yields near‑atomic resolution. Serological tests – complement fixation, hemagglutination inhibition, neutralization, ELISA (enzyme immunoassay). Lateral flow assay – point‑of‑care strip using monoclonal‑antibody‑tagged gold or colored particles. --- 🔄 Key Processes Plaque assay Infect monolayer cells with serial virus dilutions. Overlay with semi‑solid medium (agarose). Incubate → plaques appear where cells lyse. Count plaques; calculate PFU = (number of plaques × dilution factor) / volume plated. Real‑time quantitative PCR (qPCR) Extract viral nucleic acid. Add primers/probe specific to target gene. Amplify; monitor fluorescence each cycle. Determine Ct (cycle threshold); compare to standard curve → genome copies per mL. Cryo‑EM single‑particle reconstruction Vitrify virus suspension on EM grid. Collect thousands of 2‑D projection images. Align & average particles → compute 3‑D density map → fit atomic models. Reverse genetics for influenza Clone each viral RNA segment into a plasmid under a Pol I promoter. Co‑transfect 8 plasmids plus supporting polymerase‑expressing plasmids into cultured cells. Rescue recombinant virus; screen for desired mutations. Reassortment in segmented viruses Coinfect a cell with two parental strains. During packaging, genome segments are mixed, producing progeny with novel segment combinations. --- 🔍 Key Comparisons Plaque assay vs. Focus‑forming assay Plaque: relies on visible cell lysis; takes days. Focus: uses fluorescent antibodies to detect infected cells before lysis; faster, can be used for viruses that do not form clear plaques. Electron microscopy vs. PCR for detection EM: visualizes intact particles; no amplification bias; cannot identify species without labeling. PCR: highly sensitive, specific to sequence; cannot confirm infectivity. Serological neutralization vs. ELISA Neutralization: functional test (antibodies block infection); measures protective immunity. ELISA: detects binding antibodies or antigens; easier, high‑throughput, but not functional. Cryo‑EM vs. X‑ray crystallography Cryo‑EM: works with native particles, no crystals needed, captures multiple conformations. X‑ray: requires crystals, provides atomic resolution of isolated proteins, limited to static structures. --- ⚠️ Common Misunderstandings “PCR‑positive = infectious” – PCR detects any viral nucleic acid, including degraded fragments; confirm infectivity with culture‑based assays. “All viruses form plaques” – many (e.g., some enveloped viruses) produce diffuse CPE; focus‑forming or TCID₅₀ assays are alternatives. “Negative‑stain EM shows internal structure” – it only outlines the exterior; cryo‑EM is needed for internal detail. “Reassortment only occurs in influenza” – any segmented RNA virus (e.g., rotavirus, bunyavirus) can reassort. --- 🧠 Mental Models / Intuition “RNA → mRNA” shortcut – think of the Baltimore class as the path an RNA genome must take to become mRNA: dsDNA → transcription → mRNA (class I) (+)ssRNA → directly serves as mRNA (class IV) (–)ssRNA → must be transcribed by viral RNA‑dependent RNA polymerase (class V) Infectivity vs. genome count – picture a jar of marbles: PFU are the sparkling marbles that actually roll (infect); PCR counts all marbles, sparkling or not. --- 🚩 Exceptions & Edge Cases Segmented‑genome viruses with non‑reassortant recombination – influenza can also undergo homologous recombination, though rare. High multiplicity of infection (MOI) – at MOI ≫ 1, multiple virions infect the same cell, causing “co‑infection” artifacts in plaque counts. Isothermal amplification (e.g., TMA) – works without thermal cycler but may have lower specificity; useful for field diagnostics. --- 📍 When to Use Which Diagnostic setting Rapid bedside: lateral flow assay. Confirmatory, high‑sensitivity: real‑time qPCR. Morphology or unknown agent: negative‑stain EM. Quantifying infectivity Plaque assay: when virus forms clear plaques. Focus‑forming assay: for viruses with poor CPE. TCID₅₀: when plaque assay impractical. Structural study Cryo‑EM: for whole virions, flexible proteins, or when crystals fail. X‑ray crystallography: for high‑resolution capsid or enzyme structures. --- 👀 Patterns to Recognize Cytopathic effect (CPE) patterns – ballooning cells → herpesvirus; syncytia → paramyxovirus; clearing zones → lytic phage. Serology “rise” – a four‑fold rise in antibody titer between acute and convalescent sera indicates recent infection. PCR Ct values – low Ct ≈ high viral load; abrupt Ct drop after treatment suggests effective viral clearance. Reassortant genome signatures – mixed segment lineages in phylogenetic trees (e.g., HA from one clade, NA from another). --- 🗂️ Exam Traps Confusing PFU with genome copies – exam may ask “viral load” but give a PFU number; remember they are different metrics. Mixing up Baltimore classes – e.g., class VI (ssRNA‑RT) viruses are retroviruses; they first make DNA before mRNA. Assuming all viruses are cultured in eggs – only certain orthomyxoviruses; many animal viruses require cell lines. “Negative‑stain EM proves identity” – without immunogold labeling, EM alone cannot specify species. “ID₅₀ = LD₅₀” – ID₅₀ measures infection; LD₅₀ measures lethal dose; they are not interchangeable.