1
Q
What host receptor does SARS-CoV-2 bind to for cell entry?
A
ACE2 (Angiotensin-Converting Enzyme 2)
2
Q
Which host protein works in synergy with ACE2 to facilitate viral entry?
A
TMPRSS2 (Transmembrane Serine Protease 2)
3
Q
What is the role of TMPRSS2 in SARS-CoV-2 infection?
A
It cleaves/activates the spike (S) protein, enabling membrane fusion and viral entry
4
Q
In which cells is ACE2 highly expressed relevant to COVID-19?
A
Airway epithelial cells and vascular endothelial cells
5
Q
Which viral protein binds directly to the ACE2 receptor?
A
spike (s) protein
6
Q
What is released into the host cell after viral entry?
A
+RNA
7
Q
What happens immediately after SARS-CoV-2 binds to ACE2?
A
Viral entry into the host cell via membrane fusion or endocytosis
8
Q
Why can the SARS-CoV-2 genome be translated immediately?
A
Because it is positive-sense RNA and functions like mRNA
9
Q
Which viral proteins are translated first after entry of covid
A
pp1a and pp1ab polyproteins
10
Q
A
11
Q
what are some likely characteristics of emerging pathogens
A
- likely zoonotic
- likely have a broad host range numerous mammal and non-mammal species
- can be any pathogen group but viruses are overrepresented and ~40% are RNA viruses
- many fall within CDC category C
12
Q
which emerging pathogens were noted by the CDC in 2015 that need immediate research and development
A
- Crimean-Congo haemorrhagic fever
- Filovirus diseases (EVD and Marburg)
- Highly pathogenic emerging Coronaviruses relevant to humans (MERS Co-V and SARS)
- Lassa Fever
- Nipah
- Rift Valley Fever
- Other serious diseases: chikungunya, severe fever with thrombocytopenia syndrome, and zika virus
13
Q
which coronaviruses cause severe disease
A
- SARS-CoV
- MERS-CoV
- SARS-CoV-2
14
Q
which coronaviruses cause mild disease
A
- HKU1
- NL63
- OC43
- 229E
15
Q
describe the structure of SARS-CoV-2
A
- enveloped beta-coronavirus
- viral envelope is coated by spike glycoproteins, envelope, and membrane proteins
- ssRNA genome
16
Q
what other coronaviruses is SARS-CoV-2 similar to
A
- SARS-CoV-1 (80%)
- bat coronavirus RaTG13 (96.2%)
- suggests animal origin
17
Q
how is host cell binding + entry mediated w SARS-CoV-2
A
- by the S protein
- S1 subunit of S protein contains the receptor binding domain that binds to the peptidase domain of ACE2
18
Q
why does SARS-CoV-2 have pandemic potential
A
- higher transmission relative to SARS-CoV-1
- tropism is potentially enhanced for nasal epithelial cells
- recognizes ACE2 from a diversity of animal species (including humans)
- has an intermediate affinity for human ACE2
- highest infectious potential is before/ within first 5 days of symptom onset
19
Q
who is Alina Chan
A
- molecular biologist
- wrote a ny times op-ed about why SARS-CoV-2 prob started in a lab
20
Q
who is Paul Offit
A
- prof of pediatrics
- co-inventor of rotavirus vaccine
- author of over 160 papers on vaccine safety
- wrote a blog post arguing against Chan’s nytimes op-ed
21
Q
what were Chan’s arguments in the nytimes op-ed
A
- SARS-like virus that caused the pandemic emerged in Wuhan, a city where the world’s foremost SARS-like virus research lab is located
- a year before the outbreak, Wuhan had proposed creating viruses w SARS-CoV-2’d defining features
- Wuhan was doing this work under biosafety level 2 conditions, which couldnt have prevented SARS-CoV-2 from escaping
- the hypothesis that it came from an animal at a market in Wuhan isn’t supported by strong evidence
- key evidence that would be expected if it did come from wildlife trade is missing
22
Q
what were Offit’s arguments against chan’s points
A
- bat coronavirus spillover events into humans is common (~70% of human viruses and bacteria have animal origins, and 1 in 40 people in china have antibodies to it)
- Wuhan lab studied a strain called WIV1, which didnt cause disease in people. also no evidence of the researchers sick w COVID prior to the outbreak
- they weren’t woring w SARS-CoV-2. but if they were, level 2 containment wouldve been enough.
also all the early cases happened on the other side of this big river where animal-human spillover is likely, rather than near the Wuhan institute - 2 lineages were detected early, why would there be 2 diff viruses created in a lab and then brought to the market
also they killed at the animals at the market, so there wasn’t evidence to test cause she argued that the evidence was missing - there was evidence of SARS-CoV-2 in places that process animals thatve been slaughtered , and also in other animals that were likely intermediate hosts between bats and humans (like racoons and rats)
23
Q
what are some research areas in SARS-CoV-2 according to the CDC
A
- antiviral research ability of antiviral meds to treat/prevent infection
- vaccine development many now available
- pathogenesis research how viruses can be transmitted to a host, severity of illness, how much is produced, and what organs are affected
- virus stability research how long it can survive under diff conditions
24
Q
A
25
which magazines were both working on the human genome project and which was private/public
- **nature** public
- **science** private
26
who directed the human genome project for nature mag.
Francis Collins
27
who led and run the human genome project for science mag.
- **led** Celera Genomics
- **run** J craig ventor
28
what are the 3 major improvements in NGS
1. Libraries are prepared in a **cell free system** instead of being cloned into vectors that are transformed into bacteria
2. **Millions-to-billions** of sequencing reactions are carried out **in parallel**
instead of running reactions hundreds at a time as in automated Sanger sequencing
3. The sequencing output is **‘read’ directly** without the need to be separated by capillary electrophoresis
29
what are the diff types of clonal amplification in NGS
- emulsion PCR
- bridge amplification PCR
30
describe how illuma works
- clonal population is made via bridge amplification
- polymerase adds 1 nucleotide to every growing strand
- these have reversible terminators (a fluorescent label and a blocking group)
- the machine reads the colour, so they can tell which base was added
- then a chemical removes the blocking group + dye, letting the next cycle occur
31
what gen is illumina
2nd
32
**true/false** PacBio SMRT uses PCR
- **false**
- so it might have less bias cause errors are avoided
33
**true/false** Nanopore uses PCR
falsew
34
describe quality scores
- each base is given a Phred (Q) score
- which represents the probability that an incorrect base call happened at that specific position
- high phred means high confidence
- typically okay w phred 20-30 or higher
35
what is the equation for Phred (Q)
Q= -10 x log10P; *where P is the error probability*
36
what is a finished sequence
- those in which all gaps are closed
- the quality of sequence of the entire genome is raised so that the error rate is **less than 1/10,000 bases**
37
what are toxins
- **molecules produced by living organisms that are poisonous to other species**
- small molecule, peptide, protein toxin
38
how did researchers figure out how peptide toxins are synthesized
- **sequenced the DNA**
- looked at sequences related to AMA1 and PHA1
- similar upstream and downstream portions. middle is diff
39
what does SEB stand for
Staphylococcal Enterotoxin B
40
what is the most common cause of food poisoning in the US
Staphylococcal Enterotoxin B (SEB)
41
how does SEB work
- **cross-links T cells and VB T-cell receptor**
- *T cells are binding sites for major MHC II molecules*
- **activates proinflammatory response**
42
what biological toxins are category A biothreat agents
botulinum neurotoxins
43
what biological toxins are category B biothreat agents
- ricin
- staphylococcal enterotoxin B
- Clostridium perfringens epsilon toxin
44
why is ricin a threat
* No antidote available *kinda have to wait it out n see if you live*
* Castor grows in many places in the world
* Ricin extraction from beans is not difficult
* Ricin purification methods found on www
45
what is the lethal dose of ricin
3-15 μg/kg via injection or inhalation
46
Forensic ricin typing depends on the identification of the ____ and the assessment of the ______
- toxin
- source plants
47
describe the structure of ricin proteins
- **heterodimeric glycoprotein**
- composed of A and B chains
- A is the *business end*. responsible for toxicity + death
- B is responsible for cell entry
48
describe the A chain of ricin
- N-glycosidase
- **alters structure** Removes an adenine residue from the 28S rRNA in the 60S subunit of the ribosome
- leads to the inhibition of protein synthesis
- *toxicity and death will follow*
49
describe the B chain of ricin
- lectin
- Binds to galactose-containing glycolipids as well as cell surface glycoproteins
- facilitating entry of the toxin into cells
50
describe the ricin mechanism of action
- B chain binds to cell surface
- enters the cell by endocytosis in membrane vesicles
- transported to endosomes
- *some* enter golgi apparatus
- here they undergo retrograde transport to the ER
- A and B chains are cleaved in the ER, so theyre separated
- A chains translocate to the cytosol, where theyll find the ribosomes
- they remove an A from position 4324 of 28S rRNA in the 60S ribosomal subunit
- the depurinated rRNA canot bind protein elongation factors now, and protein synthesis stops
- this happens to many ribosomes and the cell will die
51
where does botulinum neurotoxin come from
- **bacteria of the genus clostridium**
- C. botulinum, C. baratii, C. butyricum
52
what are the diff serotypes of Clostridial BoNT, and how do they differ
- **differ based on AA sequence**
- A,B,C,D,E,F,G
53
which of the serotypes of Clostridial BoNT are associated w naturally occurring botulism in humans
- A
- B
- E
- F
54
which of the serotypes of Clostridial BoNT produce botulism in primates when aerosolized
- C
- D
- G
55
which of the serotypes of Clostridial BoNT are food borne botulism usually associated w
- A
- B
- E
- *rarely (<1%)* F
56
describe Group I of C. botulinum
- proteolytic (can digest complex proteins)
- all serotype A
- some serotype B + F
57
describe Group II of C. botulinum
- non proteolytic
- all serotype E
- some serotype B+F
58
describe Group III of C. botulinum
- all serotype C
- all serotype D
59
describe Group IV of C. botulinum
- restricted so far to organisms isolated from an argentinean soil sample
- no clinic reports
- although organisms of this group have been isolated from autopsy specimens
60
what produces neurotoxins of serotype E and F
- C. butyricum
- C. baratii
61
describe serotype C of clostridium
- Avian species causing massive outbreaks in wild waterfowl or domestic flocks
- found in dogs, mink and cattle
- only a single human case
62
describe serotype D of clostridium
- association w cattle
- only 1 human case
63
what serotype of clostridium might be re-named c. argentinense
group IV
64
describe the botulinum neurotoxin structure
- secreted as a 900 kDa protein complex
- this complex is made up of the neurotoxin and NAPS
- the neurotoxin is 150 kDa and made of 2 subunits
- the subunits (heavy and light chains) are linked by a disulfide bond
- the heavy chain is 100 kDa
- light chain is 50 kDa
65
describe the NAPs in the protein complex of BoNT
- **neurotoxin-associated proteins**
- made of hemagglutinins and non-toxin nonhemagglutinins
- they protect the toxin and stabilize it
66
describe the heavy chain of BoNT
- 100 kDa
- much of the sequence variation b/w toxins is in this subunit
- it determines the serotype
67
describe the light chain of BoNT
- 50 kDa
- a zinc endopeptidase
- responsible for the toxicity *but needs H chain to produce the toxic activity in vivo*
68
which BoNT serotypes are known to affect humans
- A
- B
- E
- F
69
what BoNT subunit is the binding/recognition domain
C terminal of H chain
70
what BoNT subunit is the transport/translocation domain
N terminal of H chain
71
what BoNT subunit is the catalytic/enzyme domain
L chain
72
describe how BoNT A works by comparing normal transmitter release and under the influence
- **normal**
- inside the neuron's axon terminal, there are synaptic vesicles filled w acetylcholine
- to release acetylcholine, the vesicle needs help from SNARE proteins
- the SNARE proteins pull the vesicle down to the membrane so it can fuse, and let ACh spill into the synaptic cleft
- ACh then binds to the ACh receptors on the muscle cell
- the muscle then contracts
- **w BoNT**
- BoNT binds to a BoNT receptor on the neuron and enters the cell *the H chain helps w this*
- once inside, the L chain cleaves SNARE proteins (specifically SNAP-25)
- w SNAP-25 cut, the SNARE complex cant form
- without SNARE, the vesicle cant fuse w the membrane
- no fusion, no ACh release, no muscle cell activation
- *note that diff serotypes will cleave diff SNARE proteins, so in this example its SNAP-25, but in other cases itll be diff*
73
what are the diff types of SNARE proteins
- synaptobrevin
- syntaxin
- SNAP-25
74
**true/false** diff toxin serotypes affect diff SNARE proteins
true
75
which serotypes cleave SNAP-25
- A
- C
- E
76
which serotypes cleave syntax
- B
- D
- F
- G
77
which serotypes cleave synaptobrevin
C
78
the decision to use 16s rRNA vs WGS depends on what
- type of data required
- research question
- throughput requirements/ limitations
- cost
79
what can PMI help us w
- validting or refuting alibis
- identifying suspects and witnesses
- reconstructing the death scene
80
what is decomposition
The return of a cadaver to its chemical constituents through chemical decomposition and the action of organisms that consume tissues
81
what influences the decomposition processes
- climate
- weather
- geography
82
what are the stages of decomp
- fresh
- early-decomp
- advanced-decomp
- skeletonization
83
what types of changes happen during the fresh stage of decomp
- *some outwards signs of change butttt*
- cellular and biochemical level *mostly*
- initiated by oxygen deprivation
84
what are the diff elements within the fresh stage of decomp
- algor mortis
- rigor mortis
- liver mortis
85
describe the bacteria during the fresh stage of decomp
- those that dont require a living host
- and those not restricted in growth cause immune system
86
describe insect evidence in the fresh stage of decomp
- blow flies (calliphoridae)
- may lay eggs in protected areas
- like eyes, ears, nose, body, thicker hair
87
describe the bacteria during early decomp
- gut decomposes so bacteria enters in and moves through circulatory system
- metabolizing blood and making black residue
- anaerobic bacteria proliferate and produce gases causing the remains in bloat
- thennn the pressure of the gases forces fluids out *purge*
88
describe the insects during early decomp
- eggs hatch
- adult flies lay more eggs that hatch
- maggot masses travel around the body
- spread digestive enzymes and bacteria
- necrophagous beetles and parasitic insects may also colonize the body now
89
describe insects activity in advanced decomp
- fly activity declines
- coleoptera that like drier substrates may continue
90
describe insect activity during skeletonization
none
91
At 20°C rigor mortis sets in at ______h after death and persists for _____ h
- 2-6 h
- 24-84 h
92
Livor mortis begins between _____h after death and will continue to develop up to _____
- 30 min and 3 h
- 12 h
93
microbes may not be informative for PMI on humans until after ____hours postmortem
48
94
what is currently the most devastating pathogen in canada
- **wheat leaf rust**
- caused by *Puccinia triticina f. sp.
tritici*
95
what does *Puccinia triticina f. sp.
tritici* cause
- **wheat leaf rust**
- $100M per year in damages
- *currently most devastating pathogen in canada*
96
what does *Puccinia striiformis* cause
- **wheat stripe rust**
- loss of 10-90% in wheat varieties
- loss of 5-50% in barely varieties
- *problematic in US and canada*
97
**true/false** *Puccinia striiformis* is a dual threat
true
98
what represents a "potential pathogen of biblical proportions"
- **Wheat stem rust caused by Puccinia graminis f.sp tritici - new races**
- with no cultivated resistance
99
100
how do we collect microbial data from decomposing bodies for regression models
1. collect data from multiple decomposing individuals
2. to start processing: extract DNA
3. PCR amplify taxonomically informative DNA regions:
4. the **16S rRNA** amplicon to survey **bacterial and archeal** populations present in a microbial community
5. the **18S rRNA** amplicon for **eukaryotic** characterizations
6. the internal transcribed spacer (**ITS**) gene region is used for **fungi**
7. give data to AI so they can develop regression models so that later on w unknown PMI bodies, it can predict it
101
102
viruses are ______ parasites
molecular
103
describe the structure of staphylococcus
gram pos
104
what are the steps in viral infection of eukaryotic cell
1. **virion adsorption to the cell surface**
2. **viral penetration of the cell surface** via mmebrane fucion or endocytosis
3. **uncoating of the viral genome** pH changed in endocytotic vesicle and capsid is degraded
4. **primary transcription** *gene expression* usually done asap after entry via their own polymerases
5. **replication of viral genome** viral proteins, the targets for antiviral development
6. **secondary transcription** expression of progeny genomes
7. **packaging of progeny genomes** capsid proteins self-assemble, nucleic acid is inserted
8. **release of progeny virions** enveloped viruses acquire lipid bilayer
105
what is field or street isolate
isolate obtained directly form the natural host, not cycled in cell culture
106
what are the diff vaccine types
- killed
- live attenuated
- subunit (from recombinant proteins)
107
what is the most prevalent type of virus family
dsDNA
108
describe the structure of Poxviridae
- linear dsDNA
- exceptionally large enveloped viruses
-complex viral capsid symmetry
109
describe the structure of Herpesviridae
- linear dsDNA
- icosahedral symmetry
110
what are segmented RNA viruses
**RNA viruses that contain two or more unique segments** of nucleic acid, negative-sense, ambisense or double-stranded
111
describe Flaviviridae
- icosahedral
- +RNA
- Single large polyprotein that is proteolytically cleaved
- primarily spread by insect vectors
112
describe Rhabdoviridae
- -RNA
- protracted time to cross neuromuscular junction *(the only virus where post-exposure immunization is helpful)*
113
describe Filoviridae
-RNA
114
what are some Filoviridae
- ebola
- marburg
- *both risk 4 pathogens*
115
describe Orthomyxoviridae
- -RNA
- Segmented genome
116
how are Enzyme-linked Immunosorbent Assay (ELISA) done
* Method involves capturing virus antigens or virus specific antibodies on a solid surface (e.g., microtitre plate) then exposing these to a substrate that allows detection
* ELISAs specific for IgM and IgG allow distinction of previous exposure from recent infection
* Increase in IgG antibodies 2x to 4 x over 2 weeks indicates seroconversion, diagnostic of active primary infection
* ELISAs to screen populations for immunization efficacy or previous exposure to newly identified virus
117
what are the three elements to traditional epidemiology
1) clinical presentation
2) pathogen identification
3) anecdotal circumstances
118
describe the case of **The State of Louisiana Vs. Richard Schmidt (1994)**
- gastroenterologist
* Gave his ‘ex-mistress’ a “vitamin B shot”
* January 1995 - she was diagnosed as being HIV+
* Her sexual partners for past 8 years were HIV-
* Schmidt admits to affair, but HIV was not his doing
* Warranted search of office uncovered a blood sample *which is odd cause normally they'd send the sample in*
- Two HIV genes were analyzed - gp120 (envelope protein) and RT (reverse transcriptase)
- both RT and gp120 phylogenies revealed HIV in blood sample and victim sample were in sister clads consistent with them being closely related *but no directionality*
- *basically could see theyre related but not for sure which way*
119
is gp120 quickly or slowly evolving
- quickly
- *this is the envelope protein gene*
120
is RT quickly or slowly evolving
- slowly
- *this is the reverse transcriptase gene*
121
what case set the legal precedent for the admissibility of microbial forensics
schmidt case
122
what does heterotrophic mean
they are **unable to produce their own food** and **must absorb it from the environment**
123
describe the basic structural unit of fungi
- *either*
- a chain of filament-like nonmotile cells **hyphae**
- independent **single yeast cell**
124
how many fungi were once located on the US federal government select agent list
- **2 closely related species**
- Coccidioides immitis
- Coccidioides posadasii
- *both caused valley fever, but have since been removed*
125
what type of fungi have been used for bio weapons
- fungal toxins
- *not live*
126
what are some fungi that are resistant to one or more antifungal drugs
- multidrug resistant **Candida auris**
- triazole-resistant **Aspergillus fumigatus**
127
what is rDNA ITS stand for and what is it
- **nuclear ribosomal internal transcribed spacer region**
- a universal DNA barcode marker for fungi *cause names aren't universally the same*
128
hallucinogenic fungi synthesize which controlled substances
- psilocin
- psilocybin
129
how do Psilocybin and other classical hallucinogens exert their psychedelic actions in humans
by activating 5- HT2A serotonin receptors
130
which regions did Nugent and Saville look at
- internal transcribed spacer region of the
rDNA **(ITS-1)**
* 5’portion of the nuclear large ribosomal
subunit of rRNA **(nLSU rRNA or 28S)**
131
in the Nugent and Saville paper, what linkage was suggested
- **between speciation and retention of the ability to produce psychoactive compounds**
- basically there was one specific event that led to psylosibin production, which is why the red ones are diff from the others
132
whats a unique feature of Ophiocordyceps unilateralis
- **zombie ants!!**
- kills and colonizes carpenter ants
- makes them form a sexual fruiting body (stroma) which erupts from the insects head
- it climbs as high as it can be then is killed by the fungus
133
what are some limitations of the rRNA ITS region
1. insufficient variability to delineate the various species in the Aspergillus and Fusarium species complexes
2. heterozygosity of this locus in Rhizopus species
3. problems w the reliability of the ITS sequences deposited in the reference databased
134
what is a fungi on the agricultural bioterrorism watch list
potato wart (Synchytrium endobioticum)
135
what is Chytridiomycosis by Bd
- disease discovered in **1998**
- caused by Batrachochytrium dendrobatidis **(Bd)**
- responsible for global declines/ extinctions of **amphibians**
- it disrupts the skin protective layer so they can't absorb oxygen and regulate electrolytes so they die
136
what is Chytridiomycosis by Bsal
- *emerging* disease discovered in **2013**
- caused by Batrachochytrium salamandrivorans **(Bsal)**
- responsible for global declines/ extinctions of **salamanders and newts** *96% decline in european pops*
- affects their skin, gives them ulcers, messes w fluid and electrolyte balance, and then death
137
what is Candidiasis
thrush
138
what is Candida auris
- a new fungus
- first identified Japan 2009
- has overcome the human thermal barrier
- readily spreads between patients
- resistance has developed to antimycotics
139
what is Puccinia striiformis
- stripe rust
- a Basidiomycete Plant Pathogen
- a problem in US and canada
- dual threat w losses to wheat and barley
140
what is Puccinia triticina f. sp. tritici
- a Basidiomycete Plant Pathogen
- wheat leaf rust
- currently the most devastating pathogen in canada
- can cause $100 million/year in damages
141
what is the danger of Puccinia graminis f. sp. tritici
- new rages (Ug99) have emerged w no cultivated resistance
- a pathogen of "biblical proportions"
- not in north america yet
- 100% loss once it gets to a field
142
when was the last major canadian epidemic
- 1955
- wheat stem rust
- called barberry
- caused hundreds of millions of dollars in losses
143
how does wheat stem rust spread
- rusts secrete effector proteins called (Avr)
- Avr allows it to invade the plant and suppress the host immune system
- wheat varieties have evolved/ been bred w specific resistance genes (Sr)
- the interaction follows the *gene for gene hypothesis* where if Sr gene matches Avr gene, a defense response is triggered
- this often means localized cell death
144
what makes Ug99 so virulent
cause it had mutations in many Avr genes, evading detection by common Sr genes
145
what is done to take caution when working w Ug99
- its a hermetically sealed Level 3 lab
- only used during the winter in case of escape
- wear scrubs, go through 4 doors, negative air pressure, shower before leaving
146
what kind of research is being done in the AAFC research facility in Morden MB on Ug99
- using Sr genes in diff combos to create *multi-gene stacks* of resistance
- ie. combining 3-5 Sr genes into one wheat variety
- this makes it harder for Ug99 to evade detection of alll the Sr genes present
147
what is forensic palynology
the study of pollen and other palynomorphs (dusts) for evidence at a crime scene
148
what are the diff ways to do castor bean genotyping
- **AFLPs** genome sequencing made other techiques preferable
- **SSRs** low diveristy makes these markers less useful
- **chloroplast DNA** lacks discrimination power
- **nuclear SNPs** have good potential
149
Microbial
flashcards
Decks in class (15)
# Cards
-
Intro45
-
Pandemics and Epidemics23
-
Risk Assessment and Societal Infrastructure99
-
Disease and Microbial Forensics Investigations116
-
4: Organisms- Viruses183
-
5: Organisms: Fungi161
-
6: Organisms: Bacteria121
-
9: Food Security & Agricultural Attack109
-
10: Microbiome Analysis103
-
11: Toxin Analysis & Nucleic Acid Analysis153
-
Sequencing from Lab DNA21
-
12: DNA sequencing, genome assembly, DNA diagnostics84
-
13: Emerging Pathogens118
-
Maybe long answers3
-
Important i think149
