Concept of Ecological Niche Guide 1

MICRO EXAM 3 SG Ecological niche: combination of circumstances including physical habitat, available nutrients, and interactions w/ other organisms that support the life of an individual of a species  Any circumstance where nutrients, moisture, & necessities are concentrated = potential niche o Living things represent possible niches…  Concentrated nutrients to sustain life, abundance of water, maintained at useful temperature to support chemical reactions, etc.  Life expands to fill available niches Defining Viruses—Virology  Characteristics o Acellular o Obligate intracellular parasites  Can only replicate within a host cell, not in culture media  May not cause disease o Smaller than cellular organisms  Able to pass through filters that would retain bacteria & larger organisms  E. coli = ~2 microns, Herpes simplex = ~120 nm o No code for complete protein synthesis or energy-generating mechanisms  Not related to the tree of life  Able to modify a living cell’s ability to do so o Nucleic acid genome enclosed in a protein package (capsid: protein coat surrounding the viral nucleic acid, protects the viral genome, & aids in transfer between hosts) o Exist extracellularly or intracellularly  When extracellular: inactive—lacking enzymes & unable to reproduce outside of living cells  When intracellular: exist as nucleic acids o No gene that all viruses share  Cannot be classified on basis of nucleotide similarity o Processes of infected cells similar to uninfected cells  Structure o Body types  2 capsid types…  Icosahedral: regular polyhedron w/ 20 equilateral triangular faces o Appear more rounded in nature; 2-fold, 3-fold, & 5-fold symmetry o Efficient way to enclose space o Enclosed space containing the genome  Helical: shaped like a hollow tube of protein walls o Capsid constructed from a single subunit polymerized into a helix o Capsid proteins bind to the genome, which is wound to the inside of the helix  Capsids can be…  Enveloped: capsid covered by a lipid membrane taken up from the host cell o Contains viral proteins, spikes, involved in attachment to host cell o Aids virus in entering cells Surrounded by grease—sensitive to agents that dissolve lipids  Destroyed by soaps & bile salts Nonenveloped (naked): capsid lacking a membrane o No layer exterior to capsid o  Not as sensitive to drying—more stable on surfaces in environment  Difficult to decontaminate Body accessories—aid virus w/ entering cells  Sheath o o  o Tail fibers Genome types  DNA genome viruses  Types o dsDNA—like human genome o ssDNA—genome is not double stranded  RNA genome viruses  Types o dsRNA—like our DNA, only RNA o ssRNA—two types  Positive sense: same as mRNA  Negative sense: opposite of mRNA  Characteristics o Use nucleic acid polymerases not found in uninfected cells, making them vulnerable to drugs that target those polymerases o Polymerases are ‘error-prone’  Mutate often, making them difficult to treat Replication of Viruses (Overview)  Must solve 3 basic problems… o Get into the cell  Virus = large macromolecular object—must enter cell w/o destroying plasma membrane o Make more of the virus  Steps (overlapping)  Uncoating: removing genome from its package  Viral gene expression: expressing the genome—making RNAs & proteins encoded in the viral genome  Genome replication: copying the genome via replication o May occur within nucleus, cytoplasm, or both o Doesn’t require a cell containing a nucleus  Assembly: making new capsids  Genome packaging: putting newly replicated genomes into new capsids o Get out of the cell (virus egress)  May involve destroying the plasma membrane  Viruses exist in two states o Replication inside of cell a virus = genome + RNAs + proteins o When outside of cell a virus = genome encased by a protein shell (virion) Studying Life Cycle of Viruses  Life cycle only observed by infecting host cells  Requirements (3) o Nutrients (EX: glucose) Growth signals  Small proteins—hormones, growth factors o Substrate  Something that cells are able to latch onto (EX: basement membrane) Mechanism o Obtain the virus [from infected individual] o Identify cells permissive for infection  Permissive: able to support all molecular events necessary for replication of the virus  Some viruses rely on macromolecules found only in some cell types o Expose the permissive cells to the virus o  o Measure growth of virus in terms of population increase Measuring Virus Population Growth  Plaque assay o Lysis of host cells observed as plaques on culture of permissive cells o Several dilutions of a sample containing virus particles are plated with appropriate host cells o o When multiplicity of infection is low, each plaque has arisen from the multiplication of a single virion  Infect at high multiplicity of infection to establish a synchronous infection  Multiplicity of infection (M.O.I.): # of infectious virus particles/cell in culture being infected o High multiplicity = M.O.I. ≥ 1 o Low multiplicity = M.O.I. < 1  Synchronous infection: all cells of culture in the same phase of infection at a specific time o Requires an M.O.I. of at least 5 A count of the plaques at such particular dilution is used to estimate the # of virions in the original sample   # of plaques = # of viruses exposed to the cells 20-200 plaques is ideal to count in order to determine virus quantity Single Step Growth Curve: growth curve is plotted on a log scale, because virus growth may increase by several log orders of magnitude  Phases (3) o Eclipse o Productive o Plateau  Taking apart the growth curve o Attachment to host cell  Must occur in order to gain entry into cell  Attachment is accomplished by specific interactions between molecules on the surface of the virion (ligands) & molecules on the surface of the host cell (receptors)  In many cases, the viral surface ligand is a protein  In un-enveloped viruses, receptor binding proteins may be capsid proteins or proteins of accessory structures Whether or not a virus will be taken up depends on if the cell contains its receptor type Viruses have evolved to use host receptors that are always on host cells & are important for normal host cell function Virus entry  Virus genome or entire nucleocapsid enter cytoplasm of host cell  Strategies (3)  Release of viral nucleic acid into the cytoplasm o Hole only needs to be big enough for nucleic acid chain to enter  Fusion of the viral envelope with the host cell’s plasma membrane o Proteins on the virus envelope cause fusion of the envelope & cell membrane o Allows entry of nucleocapsid   o  o o Used by enveloped viruses Entry by endocytosis o Virus mistaken to be harmless o Used by nonenveloped (and some enveloped) viruses Synthesis  Once the virus genome is no longer in its envelope or capsid (or both), it is no longer infectious  The virus seems to ‘disappear’ from the culture—the virus is there, but nothing infectious is measured  Genome expression—virus genomes bust direct synthesis of virus proteins that can…  Ensure genome replication  Build capsids  Put genomes into capsids  Genome expression (synthesis of mRNA) strategies (4)  Viruses w/ DNA genomes are able to utilize cellular machinery of host o 1) ss & dsDNA viruses: dsDNA used as the template for transcription  ssDNA: use cellular DNA polymerase to synthesize second strand  dsDNA: able to depend solely on cellular machinery (DNA polymerase)  Viruses w/ RNA genomes are unable to utilize cellular machinery of host—cellular organisms lack enzymes needed to replicated RNA or to synthesize mRNA from RNA, so virus must carry in enzymes within nucleocapsids or must be synthesized during infection o 2) ssRNA (+) that are retroviruses: genome is reverse transcribed (integrated into  cellular genome) into dsDNA using RNA-dependent DNA polymerase (reverse transcriptase), which then serves as the template for mRNA synthesis  Uses host cellular RNA polymerase to transcribe mRNA o 3) ssRNA (+) that aren’t retroviruses: RDRP used to make antigenomes which then serve as the synthesis of mRNA  RDRP is not brought into cell, is made from translation of viral genome o 4) ssRNA (-): RDRP uses the original genome as temple for synthesis of mRNA  RDRP brought into cell Genome replication (synthesis of genomes) strategies (4)  1) ss & dsDNA viruses: ssDNA genome serves as the template for synthesis of the complementary strand to form dsDNA o ssDNA rolling circle replication, NOT semi-conservative  Many copies from 1 template, copies linked end-to-end  Copies cleaved by endonuclease o dsDNA IS semi-conservative  Includes origin of replication, replication fork, helicases, Okazaki fragments, etc  2) ssRNA (+) that are retroviruses: dsDNA made from ssRNA cellular RNA polymerase used to make full-length transcripts  3) ssRNA (+) that aren’t retroviruses: genome is same sense as mRNA Must first generate antigenome template = complementary to mRNA for mRNA synthesis & is used for full-length genome synthesis 4) ssRNA (-): genome is opposite sense as mRNA o RDRP synthesizes antigenome, which serves template for synthesis of genomes o  o o Assembly  Capsids = self-assembling  Basic approaches (2)  Empty capsid built first, then genome is added inside o Most DNA genome viruses  Capsid built around genome o Most RNA genome viruses Egress (virion release)  Basic approaches (3)  Destroy cell, releasing viruses that have been assembled within o Most non-enveloped viruses  Assemble at plasma membrane—assembly & egress are one in the same o Some enveloped viruses  Assemble at internal membrane to use cellular machinery to ship virus out o Many enveloped viruses Growth Curve Steps Summarized Within Phases  Eclipse: preparation for virus population growth within cell o Attachment o Entry o Genome synthesis o Genome replication  Productive: rapid production of many new virus particles o Assembly o Egress  Plateau: may represent death of all available host cells for viruses that break open host cells for egress OR cellular resources for replication have be exhausted in viruses that egress in less destructive ways HIV Virus  Life cycle o Infects only a small subset of human cells o o o o  Host cells must express both CD4 & CCR5 or CXCR4 cellular receptors  Determines type of disease caused by HIV Enters cell via fusion of its envelope w/ cellular plasma membrane  ssRNA(+) genome retrovirus  2 copies of the genome of the virion are copied into host dsDNA by reverse transcriptase  Reverse transcriptase came packed into the virion upon entry dsDNA transported into cellular nucleus & integrated into host chromosomes  DNA transcribed into full-length RNA copies of genome by host RNA polymerase  Host RNA polymerase guided to viral genome by sequences within genome that bind to transcription activator proteins Full-length genome copies used for:  mRNA to make proteins  Genomes to be packaged Assembly & egress (single step) occur once enough viral proteins are made  Capsid assembles around 2 genomes copies = virion envelope  Virion is released & matures once virus protease cleaves specific proteins  Treatment o Antiviral drugs: target virus-specific processes that do not occur in uninfected cells  Treatment = combination of 3+ drugs  Virus-specific processes:  Membrane fusion inhibitors  Reverse transcriptase inhibitors  Integrase inhibitors  Protease inhibitors o Patients lead normal lives  Life expectancy not as high  No detectable virus  NOT cured  If drug therapy is stopped, the virus returns & disease progression continues  Virus latency Types of Infection (3)  Productive: virus enters a cell containing appropriate nutrients for replication—therefore making more of itself  Abortive: virus enters a cell that is unusable for replication (something wrong w/ virus or cell is not appropriate)— therefore the virus is unable to make more of itself o Genome may hang around for the life of the cell—may have bad effects (cancer) o Not able to reactivate  Latent: virus enters a cell & remains quiescent for a time, but is able to reactive later to make more of itself Bacteriophage Lysogeny  Example of latent infection  Life cycle o Virus genome enters cell & is integrated into host cell genome o Viral genes that encode proteins necessary for replicating genome copies & proteins for making capsids are not expressed = lysogeny  Lysogeny: relationship between phages and its host  Prophage is replicated & inherited by progeny cells o Virus may be reactivated in response to stress  Virus leaves chromosome & viral genomes that encode proteins necessary for replicating genome copies & proteins for making capsids are expressed o Lysogenic stage ends and lytic cycle commences when induction occurs  Induction: conditions arise that cause the prophage to initiate synthesis of phages proteins and to assemble new virions  Lytic cycle: host cell lyses & progeny phages are released Virus Pathogenesis  Pathogenesis: biological mechanism(s) that lead to a disease state o Exposure to most viruses does NOT lead to disease o Disease is the result of interplay between virus, host cells, & host responses  Steps o Virus tropism: particular cell types that viruses infect based on distribution of surface receptors in tissues  Cells that the virus can infect productively (correct receptors, nutrients, etc.)  Helps determine route of exposure o Virus shedding o Portal of entry: tissue or body area where the virus first encounters permissive cells  Not necessarily the same place where the virus entered the body  Common examples = skin, respiratory mucosa, alimentary tract o Primary replication: occurs in permissive cells at the portal of entry o Dissemination: virus spreads from portal of entry to other permissive tissues or cells in the body o Secondary replication: occurs in other tissues after virus dissemination Pathogenesis of HIV & AIDS  Virus tropism o Restricted to cells that express the appropriate receptors  Routes of shedding o Blood, semen/vaginal secretions, breast milk  Route of exposure o 1) Direct exposure of virus to blood stream  Transfusion, needle exchange, sexual contact w/ areas where skin integrity is compromised o 2) Exposure of virus to areas where white blood cells are close to mucosa surface  Areas of inflammation  Portal of entry  Primary replication o WBCs  Dissemination o Movement of virus & infected cells through lymph & blood  Secondary replication (years) o Lymph nodes, blood, spleen, bone marrow, & thymus (where WBCs are concentrated) o Phases (3)  Acute infection (weeks)  Virus replicates in many WBCs & reaches high concentrations in the blood, immune system responds & clears most of the virus o Possible symptoms = swollen lymph nodes o Ends when # of permissive cells begins decreasing  Chronic infection (years) Virus replicates at a low level, gradually depleting population of host cells o Loss of host cells beyond replacement level o Clinical latency—virus is replicating, but there aren’t observable symptoms Clinical AIDS  Virus has depleted host cells so much that the host is now severely immune suppressed —loss of control over HIV, virus levels increase dramatically o Host is unable to control other infections as well   Pathogenesis of Ebola  Zoonosis: disease transferred from a non-human reservoir host (bat) to a human  Virus tropism o Can infect many host cell types (receptors are widely distributed), prefers macrophages & dendritic cells  Immune cells below the surface of the skin, reached via micro skin abrasions  Routes of shedding o Blood, stool, saliva, semen, breast milk  Route of exposure o Exposure of virus to any area where there is access to living cells, especially its preferred host  Portal of entry  Primary replication o Macrophages & dendritic cells  Dissemination o Lymphatic system  Secondary replication o Occurs within lymph nodes (damages immune system), liver (damages liver, production of clotting factors —external bleeding), & adrenal gland (impaired synthesis of steroids  Infection o Incubation (~10 days): no obvious symptoms o Acute symptomatic disease (~6 days): begins with prodrome phase (non-specific symptoms), but o develops into disease-specific symptoms (vomiting, diarrhea, loss of appetite, rash, edema)  May be due to immune response rather than virus replication Death or convalescence Bacterial Host Responses to Phage Infection  Phage: viruses that infect bacteria  Restriction/modification systems o Bacteria encode enzymes that could potentially cut their own DNA  EX: restriction endonuclease, EcoRI—recognizes short sequences (5’-GAATTC-3’ or 3’CTTAAG-5’)  Makes a break in both strands at these sites, which occur every ~4000 base-pairs, meaning EcoRI could cut an E. coli chromosome into 1250 pieces, BUT IT DOESN’T o Enzyme EcoRI modification methylase recognizes the same sequences of DNA Does not make a break, instead it attaches a methyl group to the internal A residues Only present during replication—methylates adenines on newly replicated strands, preventing cutting by the EcoRI endonuclease When the DNA genome of bacteriophage is injected   o  into the cell, its DNA is unmethylated, and hence, unprotected from EcoRI  EcoRI cuts the bacteriophage, preventing productive infection  E. coli uses EcoRI to distinguish bacterial genome from non-self (viral genome) Bacteriophage infection may result in abortive infection Bacterium hold remnants of phage DNA within their chromosome in arrays of clustered regularly interspaced short palindromic repeats (CRISPRs)  Phage DNA integrated between two palindromes o Pre-crRNA: transcribing of CRISPRs results in a set of ‘trophy’ sequences separated by stem-loop structures formed by palindromic sequences  Cut into trophy + stem loop pieces o If the cell is in contact w/ the ‘trophy’ again, the DNA of the infecting phage is recognized by base-pairing w/ the crRNA = “memory” o Cas effector complex cuts the phage DNA  Cas effector complex = RNA that has the CRISPR spacer palindrome connected to an RNA sequence homologous to a gene you want to change + protein  Causes a double-stranded break Understanding How Viruses “Cause” Disease  Koch’s postulates o 1) The suspected pathogen must be found in every case of disease & not be found in healthy individuals. o 2) The suspected pathogen can be isolated & grown in pure culture. o o o 3) A healthy test subject infected with the suspected pathogen must develop the same signs & symptoms of disease as seen in postulate 1. 4) The pathogen must be re-isolated from the new host & be identical to the pathogen from postulate 2. Virus Discovery & Testing  Discovery & identification pathways o Pathway  Culture  Sample derived from tissue showing characteristic symptoms o EX: respiratory symptoms > nasal swabs or lung sputum samples  Permissive cells may be derived from some tissues that show characteristic symptoms  Examination for cytopathic effect (CPE) o Cytopathic effect (CPE): microscopic or macroscopic degenerative changes/ abnormalities within host cells/tissues caused by virus that are distinct from lysis o Several forms (3)  Cell lysis: cell breaks open & dies  Cell rounding: cells become largely circular (stains darker)  Syncytium formation: fusion of cell membranes  Virus is isolated & amplified in (3) ways: o   o EX: meta-genomics: identification & characterization of SARS-CoV-2    o Limiting dilution: o Plaque isolation: o Amplification on permissive cells: Morphological characterization  Viruses are stained (heavy metal), droplet loaded, & analyzed in TEM  Morphology classification = size, capsid type, enveloped/naked  Allows preliminary classification & guides PCR primer design Genome sequences  Members of a virus family have common genes  PCR primers are designed to amplify these genes & allow sequencing to place that virus within a tree of its relatives Identify genomes for a mass amount of viruses Principle  All viruses make mRNA for protein synthesis  If all mRNA is sequenced within an infected sample, viral + host mRNA is sequenced  Software can be used to throw away host cell sequences, leaving non-host sequences  Software can be used to match non-host sequences to a database of viral genome sequences to identify & characterize viral sequences  Results from infected samples can be compared to uninfected samples, increasing confidence in identification of disease-causing agent Steps  Clinical sample  RNA purification  Library preparation  RNA-sequencing  Filtering reads o 1) Aligning reads to a reference o 2) De novo assembly EX: PCR test for virus genomes—clinical testing for active virus infection  Steps  Clinical sample o Minutes  RNA purification o Minutes  Reverse transcription o ~1 hours  PCR amplification & quantitation of PCR product amount o ~3 hours