Day 2: Coronaviruses, Arboviruses, HIV-1 Flashcards
HC03, 04, 05
1
Q
HC03: Types of viral DNA genomes
A
(+) ssDNA
(-) ssDNA
dsDNA
2
Q
Types of viral RNA genome
A
(+) ssRNA > direct translation
(-) ssRNA > conversion
dsRNA
3
Q
The central dogma of a virus is dependent on the …
A
host machinery
4
Q
DNA virus requirement
A
Needs to travel to the nucleus
> all the parts there to make mRNA from viral DNA
5
Q
(-) ssDNA in host nucleus
A
> directly mRNA made
(-) strand DNA, virus can do a lot
6
Q
dsDNA viruses
A
Have large battery: large DNA
> large viruses
> go into nucleus as well
7
Q
(+) ssDNA viruses in humans
A
do not exist in humans
8
Q
Often, DNA viruses cause … infection
A
chronic
> virus replicates with the host cell
9
Q
Cell favourite for DNA virus
A
Rapid dividing cell
> highly proliferative progenitor cells
> does not want to infect definitely differentiated cells
10
Q
Are RNA viruses often chronic?
A
No, do not integrate into genome
> like coronaviruses
11
Q
(+) ssRNA characteristics
A
- Directly translated to proteins
- faster synthesis
- is the own mRNA
- like coronaviruses
12
Q
(-) ssRNA characteristics
A
- Needs to make mRNA with RNA-dependent RNA polymerase
- Virus needs to carry its own polymerase
> human cells do not have RNA-dependent RNA polymerase
13
Q
dsRNA example
A
rotavirus
14
Q
Where is the cells do the viral RNA reside?
A
In the cytosol > protein synthesis > make mRNA or be the mRNA
- exception: HIV goes to nucleus
15
Q
RNA are … chronic except
A
Not, except HIV, which can get inside nucleus or if it is inside regenerative cells like hepatocytes
16
Q
Which cells preferred by RNA virus: different types
A
- RNA are Acute viruses: Hit and run: infect, spread (through excretion, sneezing and pooping) and infect again
> epithelial cells: lung intestine > secretory cells make mucus etc > specialized and differentiated cells preferred
> acute viruses bind these cells (epithelial cell receptors): cell does not have to divide in order for virus to spread, and close to the excretion sites for more contamination
17
Q
Coronavirus structural proteins
A
- Membrane glycoproteins (M) (on envelope membrane)
- Spike proteins (S) (on the envelope membrane)
- Envelope protein (E)
- Nucleocapsid protein (around the RNA to protect it, form the capsid)
18
Q
Coronavirus genome
A
(+) ssRNA, largest human RNA virus, 30,000 nt
> direct protein synthesis
> S, E, M and N genes on 3’ side of ORF
> at 5’: products which are transcribed first
19
Q
First steps coronavirus after infection
A
Translate 1a and 1b genes at the 5’ of the ORF only by stopping the translation early
> proteins made for replication
> build a protective ‘house’ around the virus
> polyprotein of 1a and 1b made which is cleaved by virus its own proteases: go into cytosol
20
Q
Entry coronaviruses: SARS-CoV-2
A
> Use receptor on differentiated cell, the ACE2
only on cilia epithelial cells ACE2
Entry via receptor mediated endocytosis and fusion with endosome membrane
21
Q
Coronavirus when (+) ssRNA in the cytosol
A
> Make double membrane vesicles to hide from TLRs (Toll like receptors, which look out for pathogens to signal this) inside the cell: protective house made at the ER
ER bulges into double membrane vesicles around the ssRNA > replicase complexes makes more and make (-) ssRNA strands
when enough (-) strands made and (+) strands made from them > make subgenomic messengers
22
Q
Making subgenomic messengers by corona, what is made
A
RNA-dependent RNA polymerase jumps at the (-) strand to make little parts of (+) strands with just one gene for example > for viral replication and release
> subgenomic messengers needed for spike proteins for example and the E, M and N genes (only the non-structural proteins 1a and 1b are made directly from the primary (+) ssRNA)
> make (-) strand from (+) strand
» you want 5’ UTR (for translation initiation) and not 1a and 1b, not needed for replication and release of new virus
23
Q
Why not splicing of the RNA genome of coronaviruses?
A
That is only possible in the nucleus, alternative mechanisms required
24
Q
Coronavirus goal principle of the virus
A
Make new viruses as fast as possible and release before detection and elimination in the host > spread fast
25
Human coronaviruses types
8 types in total, just 5 circulating still
> SARS-CoV-2
> HCoV-HKU1
> HCoV-OC43
> HCoV-NL63
> HCoV-229E
26
Sarbecoviruses
SARS-CoV and SARS-CoV-2
> SARS-CoV is extinct: had a high mortality rate especially at older age > patients isolated fast
>> in China and Hongkong
27
Merbecoviruses
MERS-CoV
> in Saoudi-Arabia, not worldwide as well
> From camels to humans
> Deathly, mostly in patients with underlying conditions: septic shock, acute respiratory distress syndrome and organ failures
> fever, cough, chills, sore throat and rapid progress to pneumonia
> also asymptomatic carriers
> Low prevalence now and regulated
28
Porcine DELTA coronavirus
HKU15
> From sea animals which can infect pigs
> can infect humans
> can be dangerous when becoming more infectious, now already vaccinations
> mild disease in children: cough and abdominal pain and fever
29
Besides SARS-CoV-2, 4 other endemic coronaviruses:
Seasonal coronaviruses: causing a cold
> in humans for hundreds of years
> HCoV- HKU1/OC43/NL63/229E
30
HCoV-229E history
Detected long ago and symptoms related to cold
> can be asymptomatic as well and infectious still
31
First infection with seasonal coronaviruses
For all of them > before age of 6 years old
32
When susceptible for seasonal coronaviruses
After maternal antibodies transferred to the child start to disappear
33
Seasonal coronaviruses acute/chronic
Acute > when following adult humans in study > spikes of antibodies (short infections, peaks)
> viruses disappeared
> half-life of antibodies has to do with it as well
> decrease in levels
34
SARS-CoV-2 progression
At start pandemic: highest mortality, lowest amount of positive tests
> later on, spikes in positive test but not that high peaks in mortality as first
>> eldery most vulnerable
> Omicron appeared: more infectious, less deadly
>> evolution to milder variant with lowered pathogenicity.
>> virus does not want the host to die: more favourable to infect other persons when the host is alive and well and goes into public except for isolation in hospital or quarantaine elsewere
35
HC04: Arboviruses are transmitted through:
Vectors
36
Infections spread on ... to reach large area
Wings: by insects, birds, bats, (and planes)
37
Are blood-sucking insects favourable for the virus as vector?
Yes > get into the bloodstream immediately.
38
Requirement arboviruses
Adaptations to survive in these different host vector species
39
Vector
Animal between human infections
40
Arbovirus meaning
Arbo: arthropod borne > transmission by stinging insects
41
Are arboviruses a family of viruses?
No, just same infection route
42
Two types of arboviruses
- Mosquito-borne: Flaviviridae, Bunyaviridae and Togaviridae
- Tick-borne: Flaviviridae, Bunyaviridae and Reoviridae
43
Almost all mosquito-borne viruses are transmitted by mosquitos of the subfamily ..., mainly genera ... and ...
Culicinae Culex and Aedes (with bend in body)
44
Culicinae Culex transmitted viruses diseases
Mostly derived from birds, most risky for encephalitis
45
Culicinae Aedes transmitted viruses disease
- can cause haemorrhagic disease
- life cycle involves water source: diverse sources even glass of tap water is enough
> remove water sources is important
46
Why can Aedes mosquitos still spread viruses?
Stopped eradication campaigns and human travel
> climate change enables survival in previously inhabitable areas
47
Tiger mosquito and viruses
Culicinae Aedes > Aedes albopictus
> trasmission DENV and CHIKV > can breed in more temperate zones because eggs hatch there due to difference in lipid composition
>> differences in ability to transmit virus exists between local mosquito populations: genetics
>> can survive Dutch winter now due to climate change
> virus replicates in Aedes mosquito as well
> virus needs multiple host proteins: should replicate in mosquito and human cells
48
Transmission Chikungunya virus (CHIKV)
Through Aedes aegypti and Aedes albopictus > both active at daytime
49
Yellow fever virus (YFV) genome and family
Flavivirus
> (+) ssRNA enveloped virus
50
YFV mosquitos and disease
> sting (active) at daytime, protection when sleeping does not work
> continuous protective clothing required
> fatal in 10-20% of cases
> genome, capsid and spike proteins which go through envelope
> make one large polyprotein which is cleaved by protease which is mostly made by the virus itself > releases itself first and then releases other proteins
51
Linear ssRNA of YFV, the ends
3' end of genome not polyadenylated but forms loop structure
5' end has methylated nucleotide cap generated by virus specific methyltransferase
52
Flavivirus life cycle
> enter by receptor-mediated endocytosis
> release nucleocapsid into cytoplasm by fusion with endosomal membrane
> fusion event is triggered by the acidic pH in endosome and mediated by major envelope protein E
>translation, polyprotein processing, viral RNA replication on membranes
> immature virus assembly into ER
> mature, replicate, release
53
Origin YFV and DENV (dengue virus)
Non-human primates > transmission cycles viruses in mosquitos and monkeys
> spillover to human
54
Disease symptoms YFV infection
clinical spectrum
> subclinical infection
> abortive nonspecific febrile illness without jaundice
> Life-threatening disease with fever, jaundice, renal failure and haemorrhage
>> Yellow fever affects all ages, but disease severity and lethality is highest in eldery > onset appears 3-6 days after mosquito bite
55
Live-attenuated vaccine for YFV
17D and 17DD vaccines produce high levels of protection, protective immunity occurs in nearly 100% of individuals within 3-4 weeks after vaccination
>> benefit of vaccination should first be investigated because serious adverse effects particularly when >60y
56
DENV transmission and family
- Transmission by Aedes mosquitos
- Flavivirus
>> enveloped (+) ssRNA
57
Clinical problems DENV infection
- Severe complications through repetitive infections: dengue haemorrhagic fever and dengue shock syndrome
> break-bone fever
> rash, fever, severe pain in deep tissues
58
Proteins made by DENV
Is flavivirus: makes large polyprotein
> from each protein one
> envelope protein at 5'
> More envelope proteins made
> ribosomes do not always make the end of the ORF, then only 5' parts made with those proteins > more E made than polymerase because there is more needed
59
DENV genome encodes >>>
Structural proteins
> Capsid (C)
> Membrane (M)
> Envelope (E)
7 nonstructural proteins
60
Chikungunya virus (CHIKV) genome and family
Enveloped (+) ssRNA virus family Togoviridae
61
Clinical symptoms CHIKV infection
Severe headache, complications at joints, problems with walking
62
How are envelope proteins made by CHIKV and polyprotein made
Via subgenomic messengers
> make a lot of E except of 5' RNA synthesis and polymerase proteins.
> genome is tranlated into non-structural polyprotein which is processed by host and viral proteases, the structural program (polyprotein) is expressed through subgenomic mRNA (jump to the last bit making (+) from the made (-) for replication)
63
Transmission CHIKV
From humans/monkeys to Aedes mosquitos to humans
64
CHIKV disease and vaccine
> no vaccine available
> causes acute febrile polyarthralgia (joint pain) and arthritis
> low mortality
65
Zika virus (ZIKV) threat
Also arbovirus besides YFV, DENV and CHIKV
> transmitted by Aedes mosquitos
66
Symptoms ZIKV
> rash, fatigue, headaches, swollen and painful joints
> can affect the unborn child: microcephaly and other birth defects/
> sporadic disease
> no deaths
> also found in semen, sexual transmission is possible
> threat has increased because mutations
67
Japanese encephalitis virus (JEV)
Arbovirus
> Flavivirus
> leading cause encephalitis in Asia
> severe disease 1 in 250
> amongst survivors: large part have long-term neurologic sequelae
> no specific treatment
> vaccine is available
68
JEV enzootic transmission cycle
Natural cycle
> Culex mosquitos (vector) and waterbirds (reservoir host)
> Incidental (dead-end) hosts humans and horses
>> do not develop sufficient concentrations of JEV in blood to infect feeding mosquitos (low viral load)
Amplification cycle
> Culex mosquitos and Pigs (amplifying hosts)
69
Viral load measurment
Via PCR, check the viremia
70
HC05: Kaposi's sarcomas (KS)
Often seen in AIDS (acquired immune defieciency syndrome) patients
> caused by herpesvirus HHV8 > opportunistic
> Replicated when immuncompromised
71
HIV is a retrovirus, name its characteristics
- RNA virus
- Two copies of the genome per particle
- Enveloped virion
- Non-segmented genome
- Can make DNA from the RNA with reverse transcriptase (1) and insert the DNA into genome host cell in nucleus (2) > impossible to remove when cell alive: 2 exceptional properties
72
Subgenomic messenger mechanism
> RNA pol starts at clustering RNA at the 3' UTR of mRNA and makes copy of all structural proteins and jumps to the 5' UTR > minus strands made with only 5' UTR and structural proteins (growing chain 5>3)
>> specific jump sequence at jumping site
>> RNA-dependent RNA pol will make subgenomic (+) messengers from the (-) strands
> specific for coronaviruses
73
8% of the human genome is residual of retroviruses, which mean they partly ...
drive evolution
74
HIV is from the retrovirus family and in the lentivirus group. What does this entale?
That there is a slow progression from infection to disease (with symptoms) (7-10 years)
> HIV can cause AIDS
75
Inside the HIV particle
- 2 copies of the viral RNA genome ((+) ssRNA) complexed with proteins such as reverse transcriptase and integrase
> these structures are protected by a capsid
76
HIV particle capsid made out of ...
gag proteins
77
Outside of the HIV particle
- Lipid membrane taken from host cell when budding from cell: covers the viral particle
- envelope proteins gp120 (glycoprotein) and gp41 form spike-like structures > sugar groups are bound to proteins
78
Polymerase of HIV
DNA polymerase > makes DNA but is flexible in template > makes first DNA strand from RNA template (then RNA removed by viral RNase H activity) > then make DNA from DNA template > double stranded proviral DNA
>> needs primer to start
>> reverse transcriptase
> RNA polymerase needs RNA template
79
Genome HIV and reverse transcription
gag-pol-env and a primer binding site (pbs)
> also two R (repeats) at outsides and U5 (unique 5' site) or U3 within it
> start at PBS > make bit pbs-U5-R
> translocate to R on 3' end
> make the rest until pbs
> polypurine site before U3 is hybridized > make U3-R-U5-Primer
> translocate to pbs DNA
make the rest
> now: U3, R and U5 at each end!
> proviral DNA is longer than viral RNA with one U3, R and U5
80
The primer for the reverse transcription of HIV genome:
3' of our own tRNAs
81
Life cycle of HIV in the host cell
1 Binding
2 Fusion
3 Uncoating
4 cDNA synthesis
5 Integration
6 Transcription
7 Packaging
8 Budding
9 Release of particle
82
Reverse transcriptase of HIV: how does it become functional
It is already present in the viral particle of HIV
83
Integration HIV
Double strand breaks made > repaired with viral cDNA between it > done by integrase
84
Which enzyme transcribes HIV viral proteins
RNA polymerase 2 in the nucleus
85
Viral RNAs of HIV-1 made by transcription contain splice sites. What happens with it?
Evasion of splicing in nucleus by viral proteins
86
How does viral proviral DNA make it to the nucleus
A pre-integration complex is made around it which can pass the nuclear pores. (unsure how)
87
Viral Rev protein of HIV
Limits the number of integrations per cell by binding to and inhibiting the integrase
> unlimited viral integrations result in death of the cell (is detected easier)
88
Are there introns in HIV RNA?
No
89
Tat protein of HIV
Improve viral protein synthesis by upregulating transcription
> Transcription elongation is stimulated by binding to the TAR loop (early gene product)
> RNA polymerase 2 for transcribing retroviral DNA
90
One fate of provirus of HIV in host genome is the transcription. What is the other possible fate?
Provirus can become latent, for unstance due to CpG methylation of the LTR (promoter)
91
Why is it hard to get rid of HIV
It is silent > the cells do not appear as being infected, but the virus can become active by re-activating transcription of the provirus
92
Battling silent HIV
Induce transcription to expose cells as infected
93
HIV was first identified as a variant of the Human T-lumphotropic virus (HTLV). Is that true?
No: HIV is a lentivirus and HTLV is a delta retrovirus
94
One of the genuses of the retroviridae family is the spumaviruses. What is their main transmission route?
From apes to human
95
Two types of retroviruses: simple and complex
- Simple encodes only gag-pol-env
- Complex additionally encode accessory proteins
96
Complex view of HIV-1 genome
gag > structural proteins
pol > viral enzymes: protease-RT-Integrase
env > envelope protein binds to target cell
important virus specific proteins: tet and rev
less important accessory proteins
97
The general rule is that 1 mRNA encodes 1 protein: cap-scanning-start-stop, how does HIV-1 encode more than 15 proteins from a single RNA?
Multiple open reading frames and code polyproteins
98
Name a polyprotein of HIV-1
pol > Protease, reverse transcriptase and Integrase
Gag > multiple structural proteins
99
Ribosomes tend to fall off: how and what is the result
More made at 5' > more gag made than pol
>> sometimes frameshift needed to get ribosomes into other reading frame
>> capsid proteins needed more
100
Gag and Pol are made as polyproteins, how are they cleaved
First auto-cleavage: release Protease from the pol polyprotein
> than remainder of pol, and gag
> gag and pol made as polyproteins that are packaged in the virion (cleavage after infection)
101
The capsid proteins from gag
MA (matrix) > associated with viral membrane (p17)
CA (capsid) > condenses to cone shaped core (CA)
NC (nucleocapsid) > coats RNA genome
102
Layers around HIV genome in particle
First layer > CA / p24
Second layer > MA / p17
Third payer > viral envelope
103
How is the env protein cleaved?
By a cellular protease, not the HIV Protease
104
HIV-1 Protease
- Cuts itself out of pol polyprotein to release others later
- Inhibited with protease inhibitors
105
HIV-1 RT
- Needs primer with 3' OH termination
- Template: RNA or DNA
- Lacks proofreading: high error rate
- RNase H activity: nuclease specific for RNA in the RNA:DNA hybrids
106
Drugs against RT
NRTI and NNRTIs
> Nucleoside RT inhibitors
> Non-nucleoside RT inhibitors
107
NRTIs
- Resemble nucleosides from primer but lack the 3'OH
> no extension possible: no dsDNA made
- phosphorylation NRTIs upon uptake in cell
- dNTP mimics
> toxicity: cellular polymerases
> resistance: RT catalytic core mutations
- Should have higher affinity for viral polymerase than cellular polymerase
108
NNRTIs
- Discovered by random screening
- Bind hydrophobic pocket on outside of RT
- Resistance: in contact amino acids
109
HIV-1 Integrase
- Endonuclease activity
- Integrated retroviral DNA into host genome (random each chromosome)
- Gets viral dsDNA in proximity of DNA breaks and hosts repair mechanism ligates it in
110
Integrase inhibitors
- Catalytic inhibitors
- Allosteric inhibitors
111
HIV-1 envelope proteins
Encoded by env
> Gp120 and gp41
- Bind host receptor molecules: CD4 + CCR5
- Target for antivirals
- Major target for vaccine strategies
112
Splicing HIV-1 RNA
Full-length HIV viral RNA is spliced and exported from nucleus to be transcribed by host ribosomes in order to generate env and the smaller proteins such as Tat and Rev
> in this way, more env made than pol, even though env is downstream of pol
> splice gag-pol off
113
Disease stages HIV
- Acute infection: massive destruction CD4+ T-cells in mucosal tissues
> cells from blood go to tissues to replace CD4+ T-cells and CD4 decrease in blood
- Chronic phase: chronic infection in tissues
- Acquired Immunodeficiency syndrome (AIDS): almost no CD4+ T-cells: mortality to opportunistic infections which cannot be repressed
114
Prognostic markers HIV
- Viral RNA load in blood (RNA copies/ml)
- CD4 count (cell/ul)
>> this decreases to virtually zero
115
Effects on patients care for HIV
- New approach to patient monitoring: diagnostic improvement: monitor plasma viral load and CD4+ counts
- Pharmocology: development antivirals: by realizing that viral proteins can be targeted even though they are inside the cell
116
Origin of HIV
From African primate species
> but these rarely get AIDS?
> HIV-1 group M, N, O and P are result of separate transmissions of SIV from apes to humans
> M and N are cross-species transmissions from chimpanzees (SIVcpz)
> O and P been transmitted from gorillas (SIVgor)
117
Which HIV-1 group is responsible for pandemic?
The M viruses, the N, O and P groups are rare
118
HIV-2 origin
From transmission SIV in West Africa
119
Almost 50% of HIV diagnoses were ..
Late-stage
120
HIV-1 group M is still adapting to its new host, with which results?
- Becomes more virulent
- Newly infected patients have higher viral load and lower CD4 counts
- Strains are starting to circulate that have escaped from certain HLA-types which used to be protective against AIDS development like HLA-B27 (targets gag epitope)
- HIV strains of today can be less efficiently inhibited with antibodies
