26 oktober 2020
Bacterial and viral zoönoses, the One Health concept
For human: bacteria and viruses a
re most pathogenic
VIRUSES
- Discovery by Martinus Willem Beijerinck (filter → virus in fluid : infectious agent)
- Virus means poison
- Viruses have been around for a long time
- Every life form has its own virus (even viruses have viruses)
→ bacteriophage → infects bacteria (most dominant type of virus in ecosystem)
- No separate kingdom for viruses: no single prehistoric virus
- Smallest and most abundant biological agents
Classification of viruses:
1) type of genome (single, double stranded | DNA or RNA) 2) symmetry of the particle
- sense : plus strand: encodes directly for protein (translation by host ribosomes)
- antisense : minus strand: needs to be transcribed to the plus strand before
translation
3) possession of an envelope (or not) 4) segmented genome (or not).
- Envelope: less stable
- Envelope: lipid bilayer of your own cell, but viral proteins sticking through layer
Virome = all viruses in the body or in a certain organ (detection by deep sequencing)
BACTERIA:
10 million more bacterial cells in the human body than cells (skin & gut flora)
→ many are beneficial, but a few are pathogenic → infectious diseases
Microorganisms cover the surface of the skin & reside deep in the hair and glands
Gram + : thick peptidoglycan layer
Flagella: long filaments enabling bacteria to move
Transmission:
- Zoonose: infection we get directly from animals (wild or domestic)
Definition: zoonosis is an infectious disease transmitted from animals to humans, sometimes
with the help of a vector
→ Uncooked animal products
→ Contact with live animals through grooming & petting
→ Bites & scratches
→ Contact of dried urine and faeces
→ Traveling of humans & food : flying is a great way to spread pathogens over large
distances
- birds, bats, insects & airplanes
, → ongoing increase in human population → more food from animals
- Between species with the help of an insect vector (fly, mosquito, tick)
- Climate change can contribute to pathogen transmission : expansion of arthropod vectors
One health concept: human and animal diseases should not be seen as separate entities
Biologic response gradient: a microbe does not always cause the same disease in
everyone
Virus discovery and Koch’s postulates
Viromics: the best technique to find unknown viruses
Enrich for viruses and sequence all nucleic acids via NGS
How to enrich for viruses? → VIDISCA:
Virus Discovery cDNA-AFLP
- Low host/bacterial DNA & low in ribosomal RNA
- Size selection (viruses are small) →
centrifugation : in supernatant : viruses
- DNase (virus protect DNA/RNA) → virus
infection → a lot of broken cells in mucus
: open nuclei → you do not want to
sequence that → DNase : get rid of DNA
(viruses have capsid to protect their own
DNA)
- Reverse transcription (ribosomes = the
enemy : look like viruses, small & genetic
material is protected by proteins) →
sequence of ribosomal RNA is known →
use random hexamers in the reverse
transcription that do not anneal to
ribosomal RNA (96 hexamers instead of
4096)
= ENDOH primers
- Sequence all viruses
- High sensitivity/efficiency
Digestion: restriction enzymes that frequently cut & are
not sensitive to methylation (CG)
- MSE1 : recognize TTAA (cuts every +- 250 bp)
Size selection: small ones (not informative), big ones (too big for sequence) → everything
around 250 bp
Ligation: TTAA sequence recognized by restriction enzyme → 3’ overhang (palindrome) →
TA overhang → anneal adaptor (also with TA overhang) & add ligase to ligate 2 fragments
(only with an anchor) → TTAT = dead end : after ligation → stop : in this way only fragments
with the ligated anchor (when there is TTAA again → again digestion by restriction enzyme)
Sequence → you know more or less where the fragments are located at the genome,
because it looks like a virus that is already known
Library: fast, sensitive & use all information
Bacterial and viral zoönoses, the One Health concept
For human: bacteria and viruses a
re most pathogenic
VIRUSES
- Discovery by Martinus Willem Beijerinck (filter → virus in fluid : infectious agent)
- Virus means poison
- Viruses have been around for a long time
- Every life form has its own virus (even viruses have viruses)
→ bacteriophage → infects bacteria (most dominant type of virus in ecosystem)
- No separate kingdom for viruses: no single prehistoric virus
- Smallest and most abundant biological agents
Classification of viruses:
1) type of genome (single, double stranded | DNA or RNA) 2) symmetry of the particle
- sense : plus strand: encodes directly for protein (translation by host ribosomes)
- antisense : minus strand: needs to be transcribed to the plus strand before
translation
3) possession of an envelope (or not) 4) segmented genome (or not).
- Envelope: less stable
- Envelope: lipid bilayer of your own cell, but viral proteins sticking through layer
Virome = all viruses in the body or in a certain organ (detection by deep sequencing)
BACTERIA:
10 million more bacterial cells in the human body than cells (skin & gut flora)
→ many are beneficial, but a few are pathogenic → infectious diseases
Microorganisms cover the surface of the skin & reside deep in the hair and glands
Gram + : thick peptidoglycan layer
Flagella: long filaments enabling bacteria to move
Transmission:
- Zoonose: infection we get directly from animals (wild or domestic)
Definition: zoonosis is an infectious disease transmitted from animals to humans, sometimes
with the help of a vector
→ Uncooked animal products
→ Contact with live animals through grooming & petting
→ Bites & scratches
→ Contact of dried urine and faeces
→ Traveling of humans & food : flying is a great way to spread pathogens over large
distances
- birds, bats, insects & airplanes
, → ongoing increase in human population → more food from animals
- Between species with the help of an insect vector (fly, mosquito, tick)
- Climate change can contribute to pathogen transmission : expansion of arthropod vectors
One health concept: human and animal diseases should not be seen as separate entities
Biologic response gradient: a microbe does not always cause the same disease in
everyone
Virus discovery and Koch’s postulates
Viromics: the best technique to find unknown viruses
Enrich for viruses and sequence all nucleic acids via NGS
How to enrich for viruses? → VIDISCA:
Virus Discovery cDNA-AFLP
- Low host/bacterial DNA & low in ribosomal RNA
- Size selection (viruses are small) →
centrifugation : in supernatant : viruses
- DNase (virus protect DNA/RNA) → virus
infection → a lot of broken cells in mucus
: open nuclei → you do not want to
sequence that → DNase : get rid of DNA
(viruses have capsid to protect their own
DNA)
- Reverse transcription (ribosomes = the
enemy : look like viruses, small & genetic
material is protected by proteins) →
sequence of ribosomal RNA is known →
use random hexamers in the reverse
transcription that do not anneal to
ribosomal RNA (96 hexamers instead of
4096)
= ENDOH primers
- Sequence all viruses
- High sensitivity/efficiency
Digestion: restriction enzymes that frequently cut & are
not sensitive to methylation (CG)
- MSE1 : recognize TTAA (cuts every +- 250 bp)
Size selection: small ones (not informative), big ones (too big for sequence) → everything
around 250 bp
Ligation: TTAA sequence recognized by restriction enzyme → 3’ overhang (palindrome) →
TA overhang → anneal adaptor (also with TA overhang) & add ligase to ligate 2 fragments
(only with an anchor) → TTAT = dead end : after ligation → stop : in this way only fragments
with the ligated anchor (when there is TTAA again → again digestion by restriction enzyme)
Sequence → you know more or less where the fragments are located at the genome,
because it looks like a virus that is already known
Library: fast, sensitive & use all information
