Microbiology Exam 3 Review PDF

Microbiology Exam 3 Review ★ Quiz Questions ○ To classify an arthropod as a vector of disease, it must Be shown to become infected when feeding on a host that has an infectious agent present in the blood Associate and feed on host under natural conditio...

Microbiology Exam 3 Review ★ Quiz Questions ○ To classify an arthropod as a vector of disease, it must Be shown to become infected when feeding on a host that has an infectious agent present in the blood Associate and feed on host under natural conditions ○ Which of the following terms describes non-human primates in the YF virus transmission cycle? Enzootic host ○ Which of the following terms describes Aedes aegypti in the YF transmission cycle? Vector Bridge vector ○ Which organisms are part of the sylvatic (enzootic) disease transmission cycle of YF virus? Non human primates Aedes aegypti ○ Which organisms are part of the epidemic transmission cycle of YF virus? Humans Aedes aegypti ○ Which of the following best describes the route of viral infection within the mosquito? Food canal, midgut, hemocoel, saliva ○ Which of the following best describes the role dicer plays in the RNAi pathway? Binds and cuts dsRNA into small pieces ○ Which of the following describes the final step of the RNAi pathway? Any sequence that complementary binds to the ssRNA loaded into the RISC complex will be degraded ○ Which of the following historical events was heavily influenced by YF? Spanish American War Louisiana Purchase ○ YF can be directly transmitted from person to person FALSE ○ SIT vector control strategy relies on the release of sterile ___ mosquitoes into the environment Male ○ The RIDL vector control strategy relies on a dominant lethal gene being expressed during ___ mosquito development Female ○ Which of the following mating pairings would result in cytoplasmic incompatibility in mosquitoes due to a Wolbachia infection? Uninfected female mates with an infected male Infected MALE w/ non-infected FEMALE ○ What flavivirus protein is responsible for receptor binding? E ○ Flavivirus particles bind at the ___ Endosomal membrane ○ What statement best describes the initial flavivirus translation event? 10 viral proteins are first translated together as one polyprotein from one transcript After translation the proteins are cleaved into individual components ○ Flavivirus particles are assembled at the ___ ER ○ A mutation in the YF virus genome occurs that prevents the cleavage of prM in the trans-golgi network. How does this mutation impact the virus? The released particles will not be able to fuse with the endosomal membrane ○ Cyclization motifs in the flavivirus genomic RNA ___ Inhibit translation of genomic RNA and promote replication ( – strand production) ○ Without the cyclization motif of the flavivirus genome ___ Viral polymerase and ribosome risk colliding (and premature abortion of replication and translation) ○ Which cells are typically the first cells to be infected during a dengue virus infection? Langerhan cells ○ What is the PAMP that serves as the initial target site recognized during a dengue virus infection? Carbohydrate attachments present on E and M ○ The jaundice associated with a YF viral infection is a direct result of __ Damage to the hepatocytes due to viral replication Damage to the hepatocytes due to activation of eosinophils ○ Which antibodies are responsible for enhancing disease progression in ADE? Non-neutralizing antibodies ○ What best describes the vaccine format used for the YF virus? Live attenuated vaccine ○ What best describes the vaccine format used for the current limited use for Dengue virus?  Recombinant chimeric vaccine ○ The generation of a vaccine to Dengue virus is problematic because __ A vaccine must be effective against all 4 serotypes ○ The main cause of severe malaria is __ P. falciparum ○ Which of the following is capable of transmitting Plasmodium? Anopheles gambiae Anopheles freeborni Anopheles stevensi ○ Mitochondrial DNA sequencing suggests the P. falciparum most likely evolved from __ A cross-species transmission event from gorillas ○ Immediately after the mosquito bites that transfers Plasmodium into the blood of its human host, ___ first invades the ___ in order to establish replication within ___ Sporozoites Kupffer cells Hepatocytes ○ Which feature on P. falciparum is responsible for binding to erythrocytes? PfEBA175 ○ Which of the statements describes the role of PfEMP1? It is a protein expressed on the surface of the erythrocyte causing it to stick to the endothelial lining of the blood vessel ○ Which of the following is responsible for the maturation of Plasmodium gametes? Drop in temperature from 37C ○ Which of the following are sites of Plasmodium sexual reproduction? Mosquito midgut ○ Which developmental stage of Plasmodium is part of the mosquito developmental cycle? Ookinete Gametocyte ○ Which developmental stage of Plasmodium is present in the mosquito salivary glands? Sporozoite ○ Which developmental stage of Plasmodium is released from the liver schizont? Merozoite ★ Key Points ○ Lecture 14 Define disease vector  Any agent that carries and transmits a pathogen to another living organism Agent for transmission Identify important characteristics for disease transmission Vectors influencing disease transmission differently ○ Sporadic introduction to population ○ Consistent circulation Constantly always carrying pathogen Whenever we have contact with vector, have possibility for infection ○ Vector drives transmission Identify different arthropod vectors (i.e. mosquitoes, flies, fleas, ticks) Hematophagous ○ Blood meals In saliva, there are pathogens present Transmission through bites Species important when looking at vectors of disease ○ Anopheles – Malaria ○ A. aegypti – YF and Dengue flaviviruses Define epidemic vs epizootic vs enzootic cycles of disease transmission Epidemic ○ Increase in incidence/prevalence of infection within a human population Human Aedes aegypti Endemic ○ Stable incidence of of infection within a human population Epizootic ○ Increase in incidence/prevalence of infection within an animal population Enzootic ○ Stable incidence of infection within an animal population Non-human primates Aedes aegypti Describe the role of a bridge vector in transmission cycles of disease. Bridge vector ○ Acts as a link between transmission across different animal hosts (primates & humans) Primates and humans are unable to directly infect each other, but the mosquitoes can carry the virus  Be able to explain simple and complex transmission cycles of diseases based on diagrams. Simple transmission cycle ○ Pathogen directly transmitted from one host to another Infected mosquito bites human The infected blood of a human will then infect other mosquitoes Cycle continues ○ YF and Dengue transmission Epidemic transmission in conjunction with endemic transmission via non-human primates (enzootic host) Complex transmission cycle ○ Pathogen transmitted through multiple intermediate hosts or vectors ○ FOR EEEV Enzootic host – passerine bird Enzootic vector – mosquito Epizootic host – pheasant Epizootic vector – mosquito Dead-end host – humans & horses Do not replicate the EEEV virus enough to keep up with transmission ○ Makes it harder to find the cause/beginning of transmission Describe different factors that impact vector borne disease transmission Climate & weather ○ Standing water – breeding ground ○ Rain or humidity ○ Warmer temperatures – increase life cycles Human environment ○ Sanitation ○ Limited access to healthcare Vector & host population/availability ○ Amount of vectors present in an area ○ Amount of hosts (ex. primates or humans) Increase transmission Travel ○ Infected humans traveling can introduce pathogens to new areas Describe the steps required for vector incrimination  Vector incrimination ○ Vector must associate with & feed on vertebrate host under field conditions Seasons and location of arthropod activity may coincide with host infection ○ Naturally infected vectors must be consistently recovered from the field ○ Vector must be shown to become infected when feeding on a host known to be infected ○ Vector must be shown to transmit pathogen via controlled conditions Compare and contrast the three main virus families transmitted by mosquitoes Flaviviruses ○ ssRNA positive strand ○ Enveloped ○ Icosahedral YF virus, Dengue, West Nile, Zika Alphaviruses ○ ssRNA positive strand ○ Enveloped ○ Icosahedral EEEV Bunyaviruses ○ ssRNA negative strand ○ Enveloped ○ Pleomorphic La Crosse, Rift Valley Describe the mosquito developmental life cycle and how it impacts disease transmission  Blood meal required for egg development ○ Female mosquito transmits infection Eggs are laid on surface of still water Larvae develop aquatically near the surface of the water Compare and contrast the male and female mouthparts and how it impacts disease transmission Anatomy ○ Dipterans – true fly, two-winged insect ○ Mouth parts, antennae, and odor receptors differ between sexes Even what they feed on! Females ○ Feed on nectar and blood Attracted to warmth, CO2, and sweat ○ Mouthparts designed to pierce skin Males ○ Feed on nectar Explain the steps involved in a mosquito bite and how it impacts disease transmission. Mosquito bite ○ Mouth probes until blood vessel is found ○ Saliva is injected Assists with uptake of blood Contains anti clotting factors, vasodilators, and antiplatelet factors ○ Mast cells are stimulated that leads to histamine release Swelling occurs at the site of the bite  Explain the pathway of infection in mosquitoes (where does the virus go from entry to exit) Virus taken up via mouthpiece & food canal Enters the midgut Infects midgut and breaches barrier Transports through the hemocoel to the salivary glands ○ Hemocoel possess a lot of immune functions Infects salivary glands and grows to high titers ○ See the highest amount of replication here ○ An amplifying infection Explain the pattern of infection used within the vector and why this pattern specifically beneficial for a vector of disease Acute ○ High level of replication with rapid resolution (days) Chronic ○ Constant replication at low levels OR high levels with immune evasion Latent ○ High level of replication followed by almost complete acquiescence/dormancy Can reactivate ALLOWS FOR CONSTANT TRANSMISSION Name the three major pathways involved in mosquito immunity RNAi (RNA-interference) – primary antiviral defense Toll Imd Explain how RNAi works to prevent viral infection RNAi (prevents viral infection) ○ Made during replication of the viral genome ○ Dicer recognizes the dsRNA and cuts it up into pieces ○ One strand is loaded into the RISC complex (RNA-induced silencing complex) Argonaute = main component ○ Complementary base-pairing of the RNA strand targets it for destruction By Argonaute associated RNase activity ○ Lecture 15 Explain how yellow fever spread to the Americas  Spread during the forced expatriation (removal from home country) of West Africans through the slave trade ○ Needed more workers because native workers were being decimated by smallpox YF virus endemic in Africa ○ Disease in native Africans are relatively mild HOWEVER, the resistance of African slaves to the disease in comparison to the severe disease in Europeans & Native Americans increased demand for MORE slave workers ○ A cycle The late 18th century of YF epidemics caused government to shut down Explain how yellow fever impacted historical events such as the Louisiana Purchase and the Spanish American War 1801 – France losing Haiti due to YF and revolution ○ Uprising of African workers in France-occupied Haiti Napoleon sends large force of troops to stop uprising 27,000 French troops die of YF, while revolutionaries are not affected ○ Haiti gained independence! 1803 – Louisiana Purchase ○ Napoleon sells the Louisiana Purchase Due to massive losses from Haitian uprising Loss of capital Increased need for resources in Africa & Europe Mid-late 19th century – Outbreaks along Mississippi River 1898 – Spanish-American War ○ YF endemic in Cuba ○ Led to the establishment of US Army YF Commission (1900) Explain how the mechanism of yellow fever transmission was discovered 1878 – ○ Patrick Mason demonstrated transmission parasite causing filariasis by mosquitos Mosquitoes are vectors that transmit infectious agents 1892 –  ○ Ivanowsky found that tobacco mosaic disease was transmitted by an agent smaller than bacteria Agent was virus (but not discovered yet) 1901 – ○ Walter Reed (head of YF Commission in Cuba) demonstrated that the agent was causing YF could pass through filters & was present in the blood of infected individuals Self inoculated using mosquitoes that had bitten infected individuals No symptoms occurred Used mosquitoes that had bitten an infected person 12 days before Got sick and died Concluded transmission caused by mosquitoes Identify the vector of flavivirus infection (Genus species) Aedes aegypti Explain the factors that make a pathogen eradicable Limited host range ○ Only humans or a defined vector Harder to eradicate when there are multiple hosts Mechanisms of transmission intervention (vaccine) Has been eliminated in a smaller geographic area Significant public health burden ○ Many incidences/deaths have affected other circumstances Economy! Makes financial sense to the countries involved Explain why yellow fever is not a good candidate for eradication Has multiple host ranges ○ Enzootic cycle w/ non-human primates Describe the vaccine design and efficacy for yellow fever Vaccine development ○ 1937 by Max Theiler ○ Attenuated by Chick embryo fibroblasts If humans could be vaccinated or eradicate Aedes aegypti, then transmission cycle could be broken ○ No one knew about the sylvatic cycle until the 1960’s  Explain the advantages and disadvantages of using DTT as a control method for yellow fever DDT – chemical spray ○ Began in 1946 for malaria – US was malaria free by 1949 Advantages ○ Helped to reduce cases of YF and malaria Disadvantages ○ Environmental effects on birds and shellfish DDT banned in US in 1972 Stockholm Convention led to global restriction in 2004 Still used in emergency situations Explain the advantages and disadvantages of using SIT to control flaviviruses SIT – sterile insect technique ○ Males exposed to non-lethal levels of radiation or mutation causing chemicals in the germline Males are sterile bc progeny are lethally mutated Explain the advantages and disadvantages of using RIDL to control flaviviruses RIDL – release of insects carrying a dominant allele ○ Mosquitos engineered using transposon-like gene elements where a “lethal” gene is expressed only in females during development No adult females thus no blood feeding Explain the state of yellow fever now and what factors allow the problem to persist YF has now spread to many other countries ○ Inconsistent vaccine coverage Supply constraints & lack of financial resources ○ Urbanization (mosquitoes thrive in densely populated areas) ○ Sylvatic transmission cycle (reservoir in animal populations; enzootic) ○ Increased traveling Describe how Wolbachia infected Aedes is used to control flaviviruses Wolbachia ○ Gram negative bacteria ○ Endosymbiont (live in hosts) Transmitted from mother to child  Causes cytoplasmic incompatibility ○ Embryos die who are from uninfected F mating with infected M If infected F mate with either infected or uninfected M… ○ Produce offspring but have decreased life spans ○ Reduce viral replication and thus lowers TRANSMISSION Identify the regions of the world most at risk for yellow fever infection Africa (West Africa), South America Describe the factors contributing to the re-emergence of yellow fever in sub-Saharan Africa and South America Loss of vector control programs due to banning DDT Intrusion of sylvatic life cycle Failure to immunize at-risk populations (delivering vaccine) Economic & political instability – particularly due to wars ○ Lack of physical access & funds ○ Lecture 16 Describe the hierarchical relationship between Flaviviridae family, Flavivirus genus, and species. 4 genre ○ Flavivirus ○ Pestivirus ○ Hepacivirus  ○ Pegivirus (proposed) All similar in virion morphology, genome organization, & replication strategy Exhibit diverse biological properties & lack serologic cross-reactivity Give examples of the major members of Flavivirus Yellow Fever Dengue (types 1-4) West Nile Tick-borne encephalitis Japanese encephalitis Compare and contrast the different host, diseases, and vectors associated with Flavivirus Yellow Fever ○ Monkeys and humans ○ Hemorrhagic fever Dengue ○ Monkeys and humans ○ Fever, rash, hemorrhagic fever West Nile ○ Birds and humans ○ Fever, rash, arthralgia Tick-borne encephalitis ○ Rodents, birds, and humans ○ Encephalitis Japanese encephalitis ○ Birds, pigs, and humans ○ Encephalitis Describe the structure of the Flavivirus virion Icosahedral virus T = 3 (180 subunits) Lipid envelope Proteins ○ E dimer = responsible for attachment ○ M protein ○ Capsid protein Describe the viral and host proteins involved in flavivirus attachment ATTACHMENT E protein ○ Responsible for attachment and fusion  ○ Binds to cellular receptor Not a specific receptor, but can use Lectins ○ Bind sugars Integrins ○ Connect cells together Broader host cell receptor allows the virus to infect multiple hosts! Describe the class II fusion mechanism used for entry (what is the fusion protein, what does it look like pre and post fusion, where is the fusion peptide located, where does fusion occur, what is the trigger for fusion) ENTRY ○ Receptor-mediated endocytosis (virus taken up into endosome) ○ pH will drop and trigger fusion with endosome Causes conformational change Class II fusion mechanism ○ E protein responsible for fusion Start as a dimer & end as a trimer ○ Pre-fusion E protein is a dimer that lies on the surface of the viral membrane ○ Trigger Low pH within endosome Induces a conformational change & dimer reorganizes into a trimer ○ Post-fusion E protein becomes a trimer and each monomer extends and stabilizes the fusion core → draw endosome and viral membrane together Fusion peptide Piece of the E protein Hydrophobic, so it will bury internally at dimer interface Feature Table (Class I influenza v. Class II dengue) Feature Class I Class II Conformational Metastable fusion Metastable dimer to change during trimer to stable stable fusion trimer fusion fusion trimer  Predominant alpha-helix beta-sheets secondary structure of fusion protein Post-fusion Trimer of hairpins Trimer of hairpins structure w/alpha helix coil composed of beta-structure Maturation to Proteolytic Proteolytic pre-fusion state processing of fusion processing of through – protein companion protein Fusion peptide N-terminal peptide Internal loop at location buried in trimer fusion protein tip – interface capped by dimer interaction Explain how the genome organization allows for immediate translation of a polypeptide + sense genome (mRNA sense) ○ Able to be translated immediately bc it does not have to be transcribed Translation outsourced to host machinery Explain the difference between the initial polypeptide and the mature viral proteins Viral genome ○ Contains a cap ○ Has no poly A tail → poly A tail usually helps with stopping translation INSTEAD, use stem-loops of RNA Polypeptide precursor ○ 1 single polypeptide containing multiple proteins Mature proteins ○ Viral and host proteases will cleave polyprotein to make the multiple mature proteins Explain the problem between translation and replication that arises from being a positive sense genome. + sense RNA will be translated and replicated in the cytoplasm ○ First ribosomes will translate (make polypeptide) from 5’ end ○ At the same time, some polyproteins that have been created will start to replicate on the 3’ end  This can end of CLASHING The mRNA and the + strand transcript Explain how flaviviruses use the circularization motif to resolve this problem. Specific RNA sequences at the ends of the genome contain cyclization motifs that will pair together and form a loop (bring 5’ and 3’ together) ○ Viral replicase host factors stabilize terminal interaction Loops causes either ribosomes or replications to bind and prevent the other from binding ○ For replication, the proteins bind to cyclization motif and prevent ribosome binding – allows replication but NOT translation Identify the viral protein responsible for replication Viral polymerase → NS5 Describe the steps and location of viral assembly and budding ER ○ Recruit newly formed genomes which interact with capsid protein (C) to form viral cores Viral glycoproteins are also recruited and anchor to the ER membrane (E and prM) ○ Immature virion is created through budding of the virus into the ER lumen Golgi ○ Immature virions transported via vesicles to Golgi through secretory pathway ○ prM proteins undergo conformational change & cleaved by furin → creates mature M protein Also results in rearrangement of E protein into mature, infectious form Cell Membrane ○ Mature virions packaged in vesicles and travel to cell membrane Exit via exocytosis Do NOT bud from plasma membrane – happens in ER Explain the difference between viral glycoprotein modifications in the mosquito vector and the human host. In humans = complex sugars are added during glycoprotein modification  ○ Complex sugars allow virion a higher rate of immune evasion & receptor activation In mosquitoes = high mannose sugars are added ○ Simpler sugars allow virion to have efficient replication and transmission Describe the role of prM in viral infection and the importance of the cleavage of prM prM = inhibits premature fusion in the low pH environment of the Golgi ○ (trans-golgi network) Furin protein will cleave the pr peptide to form the mature M protein If the prM is not cleaved, the released particles/virus are unable to fuse with the endosomal membrane ○ Lecture 17 Describe the basic symptoms associated with yellow fever and dengue viruses. YF virus ○ Symptoms First – fever, chills, back pain, nausea, mild hemorrhaging, & nose bleeds Second – short remission (6-24 hrs) Third – symptoms recur w/ more vomiting (black) and jaundice (more toxic stage) Fourth – increased hemorrhaging (bleeding) Death in 20-50% of cases 7-10 days following onset ○ Recovery Prolonged convalescence (recovery) with severe fatigue Takes months Dengue virus ○ Dengue Fever Symptoms First – headache, fever, eye pain, upper back pain, rash (4-6 days after infected) Second – muscle/joint/bone pain, vomiting, weakness, exhaustion Third – possible hemorrhaging (mild) ○ Not lethal but debilitating  ○ Dengue Hemorrhagic Fever & Dengue Shock Syndrome (2nd infections) Symptoms Similar to DF plus vascular leakage ○ Vascular leakage causes drop in BP and “shock” Hemorrhaging can occur internally & externally Recovery Spontaneous OR w/ electrolyte therapy ○ Death in 1-10% of cases Explain steps involved in the initial flavivirus infection and how it spreads, paying close attention to the type of cells involved. Initial infection (epidermal layer) ○ Mosquito probing releases virus into epidermis & bloodstream ○ Infection of Langerhans (immune APC) cells via DC-SIGN recognition of high mannose glycoproteins Mosquito cells add the glycans to receptors ○ Langerhans cells migrate to lymph nodes Have high concentration of macrophages that can be infected by flavivirus (YF or Dengue) ○ Virus released into bloodstream & infect other tissues (VIREMIA) Can be picked up by mosquitoes during a blood meal Glycan addition to proteins (modifications in Golgi) ○ Initial infection dependent on virus coming from mosquito host Adds high mannose additions (allow recognition) COMPARE TO HUMANS ○ Which add complex sugars to virions Explain the steps involved in yellow fever pathogenesis, paying close attention to the type of cells involved and how it is resolved. Kupffer cells infected (liver APCs) → causes infection of hepatocytes (liver cells) ○ Causes cytokine production and signals eosinophils Eosinophils = immune cells associated with parasitic infection/allergic responses  Eosinophil activation causes hepatic degradation & jaundice ○ Major symptom of liver dysfunction ○ Jaundice – caused by leftover bilirubin Describe the yellow fever vaccine design, paying close attention to the type of vaccine format used. Disease resolved via neutralizing antibody response – only get infected once ○ Ab bind to virus – prevent infection – target for uptake by macrophages Vaccine ○ 17D live attenuated virus Derived from Asibi strain by 203 passages in non-primate cells (chicken) 32 amino acid changes – mostly in E protein Loss of virulence in humans Inhibited ability of virus to infect mosquitoes Explain the differences between classic dengue fever and dengue hemorrhagic fever and shock syndrome, both in terms of symptoms, lethality, and cause. Pathogenesis ○ Macrophages are primary site of replication Dengue Fever (LESS SEVERE) ○ Self-limiting infection – resolves on its own Macrophages infected & endothelial cells of small blood vessels ○ Symptoms become apparent 2-7 days post infection Headache, fever, nausea, vomiting, bone pain, weakness, exhaustion ○ Children to appear to suffer less severely than adults Dengue hemorrhagic fever (DHF) – MORE SEVERE ○ Lethality rate of 1-10% ○ Caused by all 4 serotypes ○ Fever, increased hemo-concentration, low platelet count ○ Spontaneous hemorrhaging Define the difference between neutralizing vs non-neutralizing antibodies. Neutralizing antibodies ○ Bind to virus ○ Prevent infection (ex. attachment & fusion of virus) ○ Recruit macrophage & prevent it from getting infected  Non-neutralizing antibodies ○ Aid in amplification of infection ○ Recruit macrophages but do not protect them from getting infected by the virus Explain the role of ADE in dengue virus infection and how it impacts pathogenesis and vaccine design. Antibody dependent enhancement (ADE) ○ Antibodies for one serotype may bind but NOT neutralize another serotype ○ Outer components of virus less conservative antibodies do not cross-react fully ○ DEN-Ab complexes allow uptake by macrophages w/Fc receptors Assista infection increasing kinetics of virus replication & viral load ○ Vascular permeability increased due to inappropriate cytokine release Explain why we have seen an emergence of DHF. Human population growth – increase vector contact Urbanization w/poor infrastructure – favor vector breeding Air travel – allows movement of viremic humans & dissemination of multiple serotypes Hyperendemic areas – increased chances of serial infection ○ ISSUE 4 serotypes of DENV against which to vaccinate Compounded by problem of antibody-dependent enhancement of disease leading to DHF (dengue hemorrhagic fever) Explain the different vaccine design strategies used for dengue virus, focusing on the different vaccine formats and the challenges for each. Subunit vaccines using E glycoprotein from different serotypes ○ Protein expression and purification in both bacteria and eukaryotes HOWEVER does not stimulate robust response Attenuated virus vaccine ○ Passage of Dengue in non-human tissue culture cells – leading to attenuation in humans HOWEVER, concerns about incomplete immune response and issues with production Recombinant noninfectious subviral particles  ○ Expression of just surface glycoproteins in cells – lead to production of virus-like particles that do not contain genetic material (noninfectious) HOWEVER, can produce antibodies but not a complete immune response DNA vaccines ○ DNA only driving production of E protein HOWEVER, problem w/ number of doses needed for sero-conversion & need to express 4 different Dengue proteins Recombinant chimeric viruses ○ Introduction of major antigens from Dengue into attenuated virus backbone (like YF vaccine virus) HOWEVER, Dengvaxia only licensed vaccine (many restrictions) Have to carefully balance response to all 4 serotypes of Dengue ○ Lecture 18 Describe the mechanism of transmission of malaria and how it was in discovered Malaria – “bad air” ○ Caused by Plasmodium parasites Plasmodium spp. thought to co-evolve w/humans P. falciparum more recent human pathogen being introduced between 10-50k years ago Charles Laveran – French physician ○ Observed parasites in RBCs of patients suffering from malaria (late 19th century) Sir Ronald Ross ○ Demonstrated mosquitoes vector of malaria using birds Malaria has played significant roles in wars (US troops in WWII) ○ Numbers increasing due to drug resistance ○ Sporadic imported cases in malaria free countries Describe global distribution of malaria and who is most at risk Sub-Saharan Africa Southeast Asia South Asia Central & South America ○ Children most at risk  Describe the clinical symptoms of malaria Headache Fever Shivering Arthralgia (joint pain) Vomiting Hemolytic anemia Jaundice Hemoglobinuria – oxygen and hemoglobin in urine Retinal damage Convulsions ○ Periodicity to the symptoms ○ Symptoms change and flow SEVERE FORM ○ 6-14 days post infection (w/ Plasmodium falciparum) Enlarged spleen & liver Severe headache Restricted blood flow to brain Renal failure Seizures Opisthotonus (muscle spasm) Uncoordinated eye movement Coma Define apicomplexans (and explain what it has to do with Plasmodium) Phylum of protists ○ Obligate parasites evolved from a free-living photosynthetic ancestor Apicoplasts ○ Organelle derived by an endosymbiotic event ○ Required for fatty acid biosynthesis ○ Non-photosynthetic BUT believed to have evolved from red algae List the five major Plasmodium spp. associated with malaria in humans. P. falciparum P. vivax P. ovale P. malariae P. knowlesi Explain the origin of Plasmodium falciparum Most severe form  ○ Thought to diverge from P. reichenowi Consequence of human-chimpanzee speciation ○ NOWADAYS Came from cross-species transmission event from gorillas From mitochondrial DNA sequencing List the three Anopheles spp. associated with the most transmission. Anopheles is associated with Plasmodium virus transmission ○ 460 characterized species 100 can transmit human malaria 30-40 are predominantly responsible Most responsible for transmission ○ An. gambiae ○ An. stevensi ○ An. punctulatus Explain the general Plasmodium life cycle and how it relates to transmission (next lecture will have much more detail). Programmed developmental cycle Mosquito essential for cycle ○ For TRANSMISSION and DEVELOPMENT Explain the current economic and societal concerns facing malaria Malaria linked with poverty (not symptom but cause) ○ Contributes to high death rates low life expectancy reduction in workforce impairment of cognitive abilities in children ○ High impact on health expenditure Loss of at least $12 billion per year to African Nations ○ Problems can be compounded by substandard or counterfeit drugs Approx. 1/3 drugs in Sub-Saharan Africa and Southeast Asia are counterfeit or substandard ○ Historically, hard to get buy-in from non-malaria nations to assist w/ funding, research, prevention, and treatment ○ Lecture 19 Describe the Plasmodium falciparum genome. 14 chromosomes, 23 MB  Haploid EXCEPT at zygote stage in mosquito Low prop. of spliced mRNAs 60% of genes have no assigned function ○ (only know 40% of functions) Low # of metabolic & amino acid synthesis genes ○ Host cell dependent High proportion of genes involved in immune evasion ○ Co-evolved with humans ○ Prevent host from clearing the parasite from the body Draw a flow chart of the developmental cycle of Plasmodium within both the human and mosquito host. Describe the initial infection within humans, paying to close attention to the developmental form and the importance of the apical complex in movement and invasion. Initial infection ○ 12-24 sporozoites (incoming infectious parasite) injected during blood meal Gliding motility to help with infection Mediated by actin-myosin motors on exterior ○ Push way through blood vessels into liver Requires secretion of proteins from apical complex  Proteins secreted change throughout life cycle ○ Apical complex Polar ring Rhoptries Micronemes Define the sporozoite Infective stage of Plasmodium that is transmitted to humans from mosquitoes ○ Travels to liver cells where it develops further Describe the steps involved in P. falciparum infection of the liver, focusing on the progression of development. Infection of the Liver ○ Move through Kupffer cells to access underlying hepatocytes Kupffer cells – resident liver macrophages that are involved in immune response Infection of Hepatocytes ○ Sporozoites secrete apical complex proteins Help form parasitophorous vacuole Vacuole protects from intracellular attacks ○ Sporozoite undergoes asexual reproduction into merozoites in the liver schizont Schizogony – form of production ○ SPOROZOITE → SCHIZONT → MEROZOITE Describe the role of the parasitophorous vacuole and schizont in development. Parasitophorous vacuole ○ Help to protect from intracellular attacks Schizont ○ Multinucleate form of parasite that develops in hepatic cells From asexual reproduction of sporozoite THEN segments into merozoites Define the merozoite. Developmental form of parasite that infects RBCs ○ Produced by schizogony Describe the steps involved in P. falciparum infection of the red blood cell, focusing on the progression of development. Merozoites are released into bloodstream and bind & enter RBCs Teardrop shape  ○ Interact between sialic acids & peptides on erythrocyte glycophorin A and 175-kd erythrocyte-binding antigen (PfEBA175) on the merozoite Merozoite interacts w/RBC via PfEBA proteins on its surface ○ Reorients and binds at its apical end (smaller side) ○ Forms a junction and secretes proteins that evade RBCs ALSO forms the parasitophorous vacuole for entry Merozoite develops into a trophozoite ○ Secretes proteins into host RBCs ○ PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1) made by parasite & secreted Creates “knobs” on surface of RBCs (severely distorted RBC) PfEMP1 binds RBCs to endothelial cells in blood vessels Define the trophozoite. Activated stage of development where it remodels the RBCs ○ Once RBCs are stuck to the endothelial lining → reduce blood flow, matures BACK into a SCHIZONT ○ Asexual reproduction → produces 16-20 merozoites RBC ruptures and releases many merozoites (to infect more RBCs) Define the gametocyte. Sexually committed merozoites that produce male & female gametes (gametocytes) ○ Critical for infection of mosquito Describe the role of PfEMP1 in infection and pathogenesis. Creates “knobs” on the surface of RBCs ○ Binds to endothelial cells on blood vessel lining & reduce blood flow Describe the initial infection within the mosquito, including what happens to the merozoites and gametocytes. Infection of Mosquito ○ Gametocytes Produced in human bloodstream Do not actively invade RBCs or cause symptoms ○ Pretty much dormant Taken up by mosquito during blood meal & deposited in midgut  Drop in temperature in mosquito (compared to human) causes gametocytes to develop into GAMETES ○ Will become fertilizes and produce zygotes – ONLY DIPLOID FORM ○ Merozoites Also taken up by mosquito, but will not survive in the midgut for very long Define the ookinete. A diploid zygote (mature) that undergoes meiotic nuclear division forming haploid nuclei ○ Develops in midgut 5 hours post blood meal Describe how the ookinete in the midgut progresses to the sporozoite in the salivary glands. Ookinete (midgut) → Sporozoite (salivary glands) ○ Ookinete is formed by the fertilization of male and female gametes Migrates through midgut epithelium & lodges into basal lamina → forms oocyst ○ Oocyst covered in host protein protecting it from the immune system of the mosquito Nuclear division occurs rapidly & center forms Center gives rise to thousands of SPOROZOITES Oocyst maturation takes up to 14 days ○ Sporozoites exit oocyst, swim through hemolymph, and attach to basal lamina of salivary glands Then breach achieving infection Define the oocyst. Capsule-like structure that forms on the basal lamina of the midgut Ookinete → oocyst ○ Undergoes sporogony to produce many sporozoites Takes up to 14 days! Once fully mature, sporozoites burst out and travel to salivary glands to wait until transmission to blood meal ○ Lecture 20 Explain the difficulties inherent in the Plasmodium life cycle that make it difficult for us to mount an immune response.  Immunity to malaria is complicated! ○ Antibodies can be present and still fail to protect from malaria ○ Immunity can build over time BUT not be completely protected Protection not maintained in the absence of repeat infections ○ Immunity may reduce symptom severity but not prevent infection ○ Proteins that seem to be obvious targets fail to initiate immune response ○ Immunity using a specific protein can be protective in some people but not to others Problems the Life Cycle Presents for Antigen Recognition ○ Intracellular – meaning little time outside the host cells Sporozoite → minutes before entering liver Merozoite → minutes before entering RBCs ○ RBCs do not express MHC Contained within vesicle Proteins released but not presented ○ High reproductive rate, so high variation in population Sexual state increases variation/recombination ○ Deliberate variation in proteins 60 variants of PfEMP1 gene Transcriptional switching Mechanisms causing variation in surface proteins Explain the immune response to the sporozoite CSP coats exterior ○ Shed during transmigration through hepatocytes ○ Able to be presented via MHC1 Problems ○ Not much is presented ○ Repeat infections raise CTL response ○ Variation in CSP reduces response efficacy Explain the immune response to the merozoite Merozoite surface proteins can elicit antibody response ○ High degree of variation ○ Shed during infection Problems  ○ Antibody binding to shed protein – reduces effectiveness of response ○ RBCs do not present antigen via MHC1 Explain the role of var gene in pfEMP1 expression and immune evasion and response PfEMP1 ○ Expressed on surface of infected RBCs ○ 60 var genes Only expresses one at a time Via MHC-2 DBL (duffy binding-like) domains – in var gene ○ Variable & bind to different ligands Ligands include molecules on surface of endothelial cells In blood vessels and immune molecules Explain the role of pFEMP1 in pathogenesis Monoallelic expression – one var gene expressed at a time ○ Controlled by epigenetic mechanisms & chromatin modifications ○ Switching of var gene occurs