WEEK 1 Flashcards
(66 cards)
1
Q
Blood serum vs blood plasma
A
plasma - anticoagulant is added to whole blood coagulation is prevented causing all components to be remain in suspension.
serum - anticoagulant is not added the clotting factors promote clot formation, using fibrinogen as a clotting agent - removed RBC as a solid mass
2
Q
Tubes for blood samples
A
3
Q
professionalism standards
A
- professionalism
- competence
- behaviour
4
Q
Papillomaviruses
A
- important family of dsDNA viruses
- (PVs) infect humans, mammals and bird species
- distinctive characteristics of papillomaviruses is their genotype - specific host restriction
5
Q
Human papillomaviruses
A
- HPV can be further classified based on tropism
- mucosal types are further characterised by associated with cancer development
- Small, non - enveloped, dsDNA viruses around 55nm in diameter with icosahedral symmetry
- virus capsid also contains structural protein L2 - facilitating packaging of viral genome into nascent virions/ viral entry
6
Q
cutaneous HPV and non- melanoma skin cancer
A
cutaneous HPV and non- melanoma skin cancer
7
Q
low risk mucosal HPV
A
- HPV6 and 11 cause recurrant respiratory papillomatosis
- Cause >90% of genital warts
8
Q
high risk mucosal HPV
A
- high risk HPV types are associated with several cancers
- major drivers of HPV - induced oncogenesis are the E6 and E7 protein
9
Q
HPV life cycle
A
- Hpv infection typically occurs at a site of epithelial abrasion
- life cycle of virus is completely dependant on differentiation of epithelium
10
Q
HPV entry
A
- infect mitotically active keratinocytes in basal membrane of epithelia exposure due to abrasions
- HPV enter epithelial cells via unconventional clathrin- independent endocytic pathway
11
Q
HPV trafficking
A
- Virions enter early endosome system and traffick to late endosomes/ multivesicular bodies (LE/MVB)
12
Q
HPV replication
A
- early viral proteins E1 and E2 co opt host DNA replication machinery to establish basal viral replication
- Productive phase of HPV life cycle requires keratinocyte differentiation in squamous epithelium
13
Q
protein phase
A
- infected basal cell divides, one daughter cell remains in basal layer to continue proliferating while other cells migrate towards suprabasal layers and undergo differentiation
- activation of late viral promotor occurs when cells undergo differentiation allowing viral genome amplification
14
Q
HPV egress
A
- L1 and L2 caspid proteins are produced in upper layers of epithelium
- HPV is non lytic and virions can only be released when reach surface of epithelia
15
Q
HPV E1 and E2
A
- replication of HPV genomes is largely carried out by host cell factors
- viral elements necessary and sufficient for HPV DNA replication include URR and HPV E1 and E2 proteins
- E1 functions as a helicase, unwinding viral replication origin to allow access of cellular replication proteins
- E2 interacts with E1 and helps to bind to viral origin
16
Q
HPV E1^E4
A
Levels of viraal E1^E4 rise as genome amplification begins allowing accumulation of E1^E4 in upper layer of epithelium
17
Q
HPV E5
A
- E5 is a small membrane protein that plays a role in late stages of viral life cycle
- E5 demonstrates weak transforming activity in human keratinocytes
- HPV E5 contain 3 putative transmembrane domains (TMD)
- E5 plays a key role in immune evasion
18
Q
Cervical cancer
A
- 4th common malignancy and 4th leading cause of cancer related deaths in women worldwide
- other risk factors include smoking, weak immune system, birth control pills, multiple sexual partners
19
Q
advanced cervical cancer may include
A
- loss of appetite and weight loss
- fatigue
- pelvic, back loss/ leg pain
- heavy viginal bleeding
20
Q
Head and neck cancer
A
- head neck squamous cell carcinoma (HNSCC) comprises a group of malignancies
- HPV primarily causes cancers in tonsils
- hard to diagnose as symptoms can be mild and mimic less serious conditions
21
Q
Treatment/ prevention
A
Treatments for HPV - associated diseases
- no current specific treatment for HPV infection or HPV associated cancers
Prevention of HPV - associated diseases
- Most HPV infections are spontaneous resolved without intervention
- best treatment option available is prevention
22
Q
HPV vaccines
A
- HPV vaccine are developed using virus like particles (VLPs)
- VLP vaccines induce antibody titters 2-3 orders of magnitude higher than natural infection
- protection not completely type - specific
23
Q
Rabies virus
A
- rhabdoviruses
- 59,000 human deaths/ year
- 99% transmission dogs > humans
24
Q
Timeline - clinical signs and symptoms
A
two forms of human rabies :
- furious (encephalitic)
- paralytic (dumb)
25
symptoms
**prodromal stage**
- headache
- malaise
- fever anorexia
- nausea
- vomiting
**secondary stage**
- hydrophobia
- difficulty swallowing
- agitation
- hallucination
26
diagnostics
**antigen tests**
- direct fluorescent antibody test (FAT)
- rabies tissue culture inoculation test (RTCIT)
- mouse inoculation test (MIT)
- direct rapid immunohistochemical test (dRIT)
- RT-PCR + qPCR
**Serological tests**
- fluorescent antibody neutralisation test (FAVN)
- enzyme linked immunosorbent assay (ELISA)
- pseudotyped virus neutralisation assay
27
Direct fluorescent anti body test
-gold standard test
brain impression smear
- fixed with acetone
fluid containing anti - rabies antibodies with fluorescent tag placed on slides
- fresh tissue should be examined
28
Direct rapid immunohistochemical test (dRIT)
PURPOSE - dRIT is a diagnostic tool used for detecting animal samples
method - uses lebelled monclonal or polyclonal antibodies that bind to rabies virus antigens in infected tissues
speed - typically obtained within an hour, rapid alternative to gold standard (DFA)
29
RTCIT - rabies tissue culture inoculation test
Purpose - laboratory based test used to detect rabies virus in brain tissue by observing viral replication in cultured cells
methods - inoculating mouse neuroblastoma (NA+ cells) or other susceptible cell lines with homogenized brain sample followed by incubation and staining with fluorescent anti-rabies antibodies
detection - after 34-96 hours of incubation, infected cells are examined under a fluorescent microscope
sensitivity - RTCIT more sensitive than direct fluorescent antibody test (FAT) and can detect rabies
use - commonly used as confirmatory test for rabies virus diagnosis
advantages - more sensitive than traditional methods
limitations
requires specialised facilitieis, trained personel
30
mouse inoculation test (MIT)
purpose - traditional laboratory test used to detect live rabies virus by observing disease developmental in inoculated mice
methods -homogenized brain sample from suspected rabid animal is injected intracereberally into labaratory mice
detection - observed up to 28 days for sign of rabies
sensitivity - highly sensitive
use - confirmatory test for rabies diagnosis and for viral strain characterisation
advantages - detect low levels of rabies virus
limitations
- ethical concerns due to animal use
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RT PCR and qPCR
Real time detection of viral RNA
Rapid
closed tube so less chance of contamination
sensitive pan lyssavirus assay
32
SEROLOGY
fluorescent antibody neutralisation test(FAVN)
Purpose - FAVN used to measure rabies vrius neutralising antibodies in serum
methods - serial dilutions of serum sample are incubated with fixed amount of live rabies virus
detection - test measures ability of antibodies in serum to neutralize rabies virus
senstivity - highly senstive
use - required for rabies antibody titrartion in animals travelling to rabies free countries
advantages - provies quantitiave measure of protective immunity
limitations - requires level 3 laboratory
33
What are viral pseudotypes
replication defective virus - removed need for high containment
functional studies of early stage of infection
good correlation to serilogical assays with authentic virus
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diagnostics
safe cheap simple robust
portable battery operated
point of care compatible
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human herpesviruses
- large dsDNA genome rnaginf from 120kb to 250kb and encode between 70 and 200 genes
- herpesviruses are divided into 3 subfamilies, alpha, beta and gamma on basis of biological properties
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HSV- 1
- Icosahedral nuleocaspid 100nm diameter
- enveloped, glycoprotein spikes project
- HSV-1 often acquired in infancy
- mist adults carry virus
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HSV-1 latency in neurone
- replication of virus in peripheral tissue - cells are attacked by immune system
- virus enters neurone and genome is transported to nucleus
- viral DNA stored in nucleus with little gene expression
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HSV-1 primary infection
- extensive lesions affecting mouth and lips
- seen in children and young adults
- gingivostomatitis associated with primary infection of child
- vesicles intensely inflamed on gums and oral mucosa and form ulcers
- takes about 2 weeks to heal and associate with weakness and general malaise
39
clinical disease
primary infection
- often asymptomatic in immunocompetent individuals
- when symptomatic, presents as mononucleosis - like illness
congenital CMV infection
- most severe form in newborns
- occurs placenta when mother has a primary or reactivated infection during pregnancy
- hearing loss, microcephaly, intracranial calcifications
40
Epstein - barr virus
- member of herpes virus family
- Epstein barr virus (HHV4)
- ds DNA genome around 170kb
- membrane enclosed icosahedral
- capsid with glycoprotein spikes
- encodes about 90 gene
41
Transmission of EBV
- passed through saliva person to person
- large adaptive immune repsone to virus B cells and T cells
- passed through breast milk from mother to child
- in resource limited countries primary infection occurs in infants
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combatting herpesviruses
- effective drugs that target viral DNA polymersase : acyclovir (ACV) and variants
- IV, oral , topical use
- ACV uses > 30 years
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acyclovir
- acts by blocking replication of viral genome
- delivery as water soluble prodrug
- circulates throughout the body enters all cells
- converted to active drug by phosphorylation by a viral enzyme
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other antivirals for herpesviruses
helicase / primase inhibitors
- H/P unwinds DNA and synthesizes primers at replication forks
- inhibitor eg AIC316
bicyclic nucleoside analogues
- one for VZV - phosphorylated by viral TK - presumed to inhibit viral polymerase
45
Herpesviruses diagnostics
samples
- swabs
- blood
- CSF
- tissue biopsy
molecular testing
- PCR is gold standard
- detects viral DNA/RNA w high sensitivity
- commonly used for HSV,VZV,EBV, CMV in blood and CSF
46
serology tests
- detects antibodies
used for :
- EBV - monospot test
- CMV - igG/ igM for primary infection or reactivation
- HSV - type specific igG for past exposure
- limitations - past exposure
virus culture and antigen detection
- cell culture- less common
- Direct fluorescent antibody (DFA )testing for HSV/VZV detection
47
hepatitis
- inflammation of the liver
- enlargement or swelling of the liver often caused by viral infection by also by other factors like toxins (alcohol), autoimmune diseases or metabolic disorders
- damage can potentially lead to serious complications like cirrhosis, organ failure or cancer
48
symptoms
- jaundice (yellowing of skin/eyes)
- fatigue and weakness
- abdominal pain
- dark urine and pale stools
- nausea, vomiting and loss of appetite
49
Hepatitis A virus
- causes infectious hepatitis and liver disfunction
- incubation period can vary 10-50 days
- children are asymptomatic (80-95%) in relation to adult 10-25%
- viremia is transient
- virus in stool - 2 weeks before and 2 weeks after jaundice
50
HAV epidemiology
- transmission is mainly faecal- oral route. sexual and intravenous route is not common
- sources of infections - raw oysters, undercooked foods
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HAV pathogenesis
- irus attaches to cellular glycoprotein receptor on hepatocytes
- HAV replication does not result in cytopathology
- IgM appears in blood at onset of symptoms
- IgG confers lifelong, broadly reactive immunity
52
Hepatitis B virus
- causes acute and chronic hepatitis, cirrhosis, liver cancer
- incubation period 45-160 days
- many adults and children with acute HBV infection are asymptomatic
- virus posses reverse transcriptase and acts in later part of replication
- DNA integrates with host cell DNA with help of viral integrase
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HBV epidemiology
transmission is primarily through blood and bodily fluids
- Sexual contact
- sharing needles / syringes
- mother to child transmission during birth
- occupational exposure
- blood transfusions
54
HBV pathogenesis
- reaches hepatocytes and attached by receptor
- not directly cytopathic
55
HBV diagnosis and treatment
sample - blood
primary tests
HBsAg = current infection (acute/ chronic )
Anti - Hbc IgM ( hepatitis B core IgM antibody ) - acute infection
Treatments
- vaccine is available
- acute HBV infection often resolves on its own with supportive care
- Chronic HBV infections requires antiviral treatments to suppress viral replication and reduce liver damage
56
Hepatitis C virus
- blood borne infection
- asymptomatic
- acute infection
- chronic infection - 85% of acute infectious progress to chronic infection
- can lead to fibrosis, cirrhosis , cancer, impaired liver function and ultimately liver failure
57
HCV epidemiology
- 3% of world population has been infected at some time
- transmission : sexual, saliva and organ transplant
58
HCV pathogenesis
Main target :
- hepatocytes
- liver inflammation ause by immune response to viral replication that trigger inflammation within liver
- cirrhosis
- hepatocellular carcinoma
Other organs
- kidney inflammation due to immune complex depostion
- skin disorders such as lichen planus and porphyria cutanea tarda
59
Diagnosis and treatment
- sample - blood
primary tests
anti - HCV antibodies = first line screening for past or current infection
Treatments
- NS3/4A proteases inhibitors
- NS5A inhibitors
- NS5B polymerase inhibiros
- Drugs are used in combination
60
Hepatitis E virus
- transmitted via faecal- oral route (similar to HAV)
- common initial symptoms = mild fever, reduced appetite, nausea/vomiting
61
HEV epidemiology
- most common cause of new hepatitis infections worldwide
- HEV endemic in most equatorial countries leading to sporadic outbreaks
62
HEV pathogenesis
Main targets
- hepatocytes (main functional cells of liver )
- immune response that causes most of cytopathology
63
Diagnosis/ treatment
Diagnosis
- sample - blood
primary tests
- IgM antiHEV - indicates active infection
- IgG antiHEV - indicates past or chronic infection
additional tests
- HEV PCR - immunocompromised individuals
- liver function test
Treatments
- self limiting
- supportive care
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