Download cell cultures

Virus detection is (was) TRICKY!
• Large number of viruses,
– often recognized by antibodies (from host serum),
• Large number of viral serotypes,
»
– serotype: same virus, different antisera specificity,
• high sequence homology between relatives,
• infections often involve multiple viruses.
Antigenic Drift
National Institute of Allergy and Infectious Diseases (NIAID)
Antigenic Shift
National Institute of Allergy and Infectious Diseases (NIAID)
Old Identification Techniques…
(but good)
…required isolation, in vitro cultures and
immunological assays,
– antibodies must be produced for each serotype,
...microscopy,
– some viral families can be identified by their capsule
morphology,
• can’t ID specific types of virus.
Identification
• In the early 1980's, it took scientists more
than two years to discover the AIDS virus.
In 1976, it took five months to discover the
Legionnaire's disease bacterium, and it took
about the same length of time in 1993 to
isolate the hanta virus, which caused an
outbreak of respiratory illness in New
Mexico.
NYT: Thursday, April 17, 2003
PCR Revolutionized Virus Identification
• facilitates detection of several
viruses in parallel,
– through multiplexing of type
specific primers,
• through design of degenerate
primers for members of a viral
family.
Requires
knowledge of
each viral
sequence.
Returns nonspecific,
positive data.
Proceedings of the National Academy of
Sciences
September 9, 2002
SARS
Severe
Acute
Respiratory
Syndrome
Time Line I
• ~March 2003: SARS is identified as a potential
world health concern,
• March 18: infected monkey cell culture shows
signs of cell lysis,
• March 21: electron microscopy reveals a
“coronavirus” in the sample,
– antibody confirmation (highest recognition to cat
coronavirus).
Time Line II
• March 22: UCSF DNA Array researcher receives
cultures,
– DeRisi: “We literally begged the CDC. We were salivating.”
• March 23: DeRisi and colleagues determined that the
virus had similarities to coronaviruses that infect birds,
people and cows,
– but was different than any known coronavirus specimen,
• mid-April: UBC and CDC sequence the coronavirus
genome. It is a novel variety.
• Similar virus found in exotic food animals in China*.
The Idea
Make a DNA microarray from
representative sequences from multiple
viral families!
The design:
• 70-nt oligonucleotides based on pre-existing
viral genome sequences,
• about 5 oligos per virus type.
And this helps how??
• one test, multiple (100s) viruses detectable,
• only small amounts of sample required (no culture),
• sensitive detection between relatives,
• less labor and time intensive,
• not testing for a specific candidate or family, so no
inherent bias,
• able to detect unknown viruses.
The Wonders of Technology
• BLAST/N (free Bioinformation),
– establishes nucleotide similarities in/between families,
• RNeasy,
»
– kit for facile isolation of RNA from cells,
• Random PCR,
– amplifies sample “target” DNA without bias (or knowledge),
• HeLa Cells (and other human cell cultures),
– immortalized human cells.
Making the Array
choosing 70-mers Targets
• Download virus sequences from the free
public database,
• Divide each into 70 base pair portions,
– check 70-mers each ~25 bases, up and down
the genome,
– compare these portions with the entire viral
sequence database, looking for highly
conserved regions.
Basic Local Alignment Search Tool
Blast
• sequence similarity finder,
• identifies sequence alignments between a
sequence of interest, and a database of sequences,
– exact matches are scored and are weighed against
single base mismatches, gaps and deletions,
– a score is obtained that provides some measure of the
sequences similarity.
BlastN compares a DNA sequence with a nucleotide database.
link
TTAAAACAGCTCTGGGGTTGCTCCCACCCCAGAGGCCCACGTGGCGGCTAGTAATCTGGTATCAGGTACCTTTGTACGCCTGTTTTATATCCCTTCCCCCGTAACTTTAGAAGCTTATCAAAAGTTCAATAGCAGGGGTACAAGCCAGTACCTCTACGAACAAGCACTTCTGTTTCCCCGGTGAAATCATATAGACTGTACCCACGGTCAAAAGTGATTGATCCGTTATCCGCTT
GAGTACTTCGAGAAGCCTAGTATCGCCTTGGAATCTTCGACGCGTTGCGCTCAACACTCTGCCCCGAGTGTAGCTTAGGCTGATGAGTCTGGGCACTCCCCACCGGCGACGGTGGCCCAGGCTGCGTTGGCGGCCTACCCATGGCTGATGCCGTGGGACGCTAGTTGTGAACAAGGTGTGAAGAGCCTATTGAGCTACTCAAGAGTCCTCCGGCCCCTGAATGCGGCTAATCCTA
ACCACGGAGCAATCGCTCACGACCCAGTGAGTAGGTTGTCGTAATGCGTAAGTCTGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCCTTTATATTCATACTGGCTGCTTATGGTGACAATTTACGAATTGTTACCATATAGCTATTGGATTGGCCACCCAGTGCTGTGCAATATATTTGAGTGCTTCTTTCATAGGTGTTACCAACATCACATTTAAACCACAATAGTCA
GTGCAAATGGGGGCTCAAGTTTCAACCCAAAAGACCGGTGCACACGAGAATCAAAACGTGGCAGCCAATGGATCCACCATTAATTATACTACCATCAACTACTACAAAGACAGCGCGAGTAACTCCGCTACTAGACAAGACCTCTCCCAAGATCCATCAAAATTCACAGAACCGGTTAAGGACTTAATGTTGAAAACAGCACCAGCTTTAAACTCGCCCAACGTGGAAGCATGTG
GGTACAGTGACCGTGTGAGGCAAATCACTTTAGGTAACTCGACCATTACCACACAGGAAGCAGCTAATGCTATTGTTGCTTATGGTGAGTGGCCCACTTACATAAATGACTCAGAAGCTAATCCGGTAGATGCACCCACTGAACCAGACGTTAGTAGCAACCGTTTTTACACCCTGGAATCGGTGTCTTGGAAGACCACTTCAAGGGGATGGTGGTGGAAGCTACCAGATTGTCT
AAAAGATATGGGAATGTTTGGTCAGAATATGTACTATCACTACTTAGGACGCTCTGGTTACACCATTCATGTCCAGTGCAACGCTTCTAAGTTTCACCAAGGGGCGTTAGGAGTTTTCCTGATACCAGAGTTTGTCATGGCTTGCAACACTGAGAGTAAAACATCATATGTTTCATACATCAACGCAAATCCTGGTGAAAGGGGCGGTGAGTTCACGAACACCTACAACCCATCA
AACACTGATGTCAGTGAGGGCAGACAGTTCGCAGCACTGGATTATCTGCTGGGTTCTGGTGTCCTAGCAGGAAACGCTTTCGTATACCCGCACCAGATCATCAATTTGCGCACCAACAACAGTGCAACAATTGTGGTACCATATGTGAACTCGCTCGTGATTGATTGTATGGCAAAACACAATAACTGGGGTATTGTCATATTACCACTGGCACCCTTGGCCTTTGCCGCAACAT
CGTCACCACAGGTGCCTATTACAGTGACCATCGCACCCATGTGTACAGAATTTAATGGGTTGAGAAATATTACCATCCCAGTACATCAAGGGTTGCCAACAATGAACACACCTGGTTCCAATCAATTCCTTACATCTGATGACTTCCAGTCGCCCTGTGCCTTACCTAATTTTGATGTTACTCCACCGATACACATACCCGGGGAAGTGAAGAATATGATGGAACTAGCTGAAAT
TGACACACTGATCCCAATGAACGCAGTGGACGGGAAGGTGAACACAATGGAAATGTATCAAATACCATTGAATGACAATTTGAGCAAGGCACCTATATTCTGTCTATCTTTATCACCTGCTTCTGACAAACGACTGAGTCACACCATGTTGGGTGAAATTCTAAATTATTACACTCATTGGACGGGGTCCATCAGGTTCACCTTTCTATTTTGTGGTAGTATGATGGCCACTGGT
AAACTGCTCCTCAGCTATTCCCCACCAGGAGCTAAACCACCAACCAATCGCAAGGATGCAATGCTAGGTACACACATCATCTGGGACCTAGGGTTACAATCCAGTTGTTCCATGGTTGCACCGTGGATCTCTAACACAGTGTACAGGCGGTGTGCACGTGATGACTTCACTGAGGGCGGGTTTATAACTTGCTTTTATCAAACTAGAATTGTTGTGCCTGCTTCAACCCCCACCA
GTATGTTCATGTTAGGCTTTGTTAGTGCGTGTCCAGATTTCAGTGTCAGACTGCTTAGGGACACTTCCCATATTAGTCAATCAAAACTAATAGCACGCACACAAGGCATTGAAGACCTCATTGACACAGCGATAAAGAATGCCTTAAGAGTGTCCCAACCACTTCGGCCCAGTCAACTGAAGCAACCCAATGGAGTAAATAGCCAGGAGGTGCCAGCTCTAACTGCTGTGGAAAC
AGGAGCATCTGGTCAAGCGATCCCCAGTGATGTGGTGGAAACTAGACACGTGATAAATTACAAAACCAGGTCTGAATCGTGTCTTGAGTCATTCTTTGGGAGAGCTGCGTGTGTCACAATCCTATCTCTGACCAACTCTTCCAAGAGCGGGGAGGAGAAGAAGCATTTCAACATATGGAACATTACATACACCGACACTGTTCAGTTACGCAGAAAATTGGAGTTTTTCACGTAT
TCCAGGTTTGACCTTGAAATGACTTTTGTGTTCACAGAGAATTACCCTAGTACAGCCAGTGGGGAAGTGCGTAACCAATGTGACCAGATCATGTACATTCCACCAGGGGCACCCCGTCCATCATCCTGGGATGACTATACATGGCAATCCTCCTCTAACCCTTCCATCTTTTACATGTATGGAAATGCACCTCCACGGATGTCAATTCCTTACGTGGGGATTGCCAATGCCTATT
CACACTTCTATGATGGCTTTGCACGGGTGCCACTTGAGGGTGAGAATACCGATGCTGGTGACACGTTTTACGGCTTAGTGTCCATAAATGATTTTGGAGTTTTAGCAGTTAGAGCGGTAAACCGCAGTAACCCACACACTATACACACATCTGTGAGAGTGTACATGAAACCAAAACACATTCGGTGTTGGTGCCCCAGACCTCCTCGGGCTGTATTATACAGGGGAGAGGGAGT
GGACATGATATCCAGTGCAATTCAACCTCTGACTAAAGTAGACTCAATTACCACTTTTGGGTTTGGCCACCAGAACAAGGCAGTGTACGTTGCCGGTTACAAGATTTGTAACTACCACCTAGCAACCCCAAGTGATCACTTGAATGCAATTAGTGTGTTATGGGACAGGGATTTAATGGTGGTGGAATCTAGAGCCCAGGGAACTGATACCATTGCCAGGTGTAGTTGCAGGTGT
GGAGTTTACTATTGTGAATCTAGAAGGAAGTACTACCTTGTCACTTTCACTGGCCCAACGTTTCGATTTATGGAAGCAAATGACTACTATCCAGCAAGATACCAGTCTCACATGCTGATAGGGTGCGGATTTGCAGAACCCGGGGACTGTGGTGGGATATTAAGGTGCACTCATGGGGTGATTGGTATCATCACTGCAGGGGGTGAAGGGATAGTAGCCTTTGCTGACATTAGAG
ACCTTTGGGTGTATGAAGAGGAAGCCATGGAACAGGGAATAACAAGTTACATCGAATCCCTCGGTACAGCTTTTGGTGCAGGGTTCACCCACACAATCAGTGAGAAAGTGACTGAGTTGACAACAATGGTCACCAGTACTATCACAGAAAAATTACTGAAAAATTTAGTGAAAATAGTATCGGCTCTAGTGATTGTTGTGAGAAATTATGAGGACACTACCACGATCCTTGCAAC
ACTAGCATTACTTGGGTGTGATATATCTCCTTGGCAATGGTTGAAGAAGAAGGCATGTGACTTACTAGAGATTCCTCATGTGATGCGCCAAGGTGATGGGTGGATGAAGAAATTCACAGAGGCTTGCAATGCAGCTAAAGGGCTTAGATGGGTCAGCAATAAAATTTCCAAGTTTGTAGATTGGTTGAAGTGTAAAATTATCCCAGAAGCTAAGGACAAGGTGGAATTTCTCACC
AAGTTGAAACAGCTAGACATGCTGGAAAATCAAATTGCAACCATCCACCAATCTTGCCCCAGCCAAGAACAACAAGAGATCCTTTTCAACAACGTAAGATGGCTAGCAGTCCAGTCCCGTCGGTTCGCGCCATTATATGCTGTGGAGGCACGCCGGATTAGCAAAATGGAGAGCACAATAAACAATTATATACAGTTCAAGAGCAAACACCGTATTGAGCCAGTATGTATGCTCG
TTCATGGGTCACCAGGGACGGGTAAGGGTATAGCTAGCTCATTGATAGGTAGAGCAATAGCAGAGAGGGAAACCACATCGGTCTACTCAGTGCCACTGGCCCCATCTCACTTTGATGGCTATAAACAACAAGGGTATGATATGGACGACCTAAACCAAAACCCCGATGGTATGGACATGAAACTGTTTTGCCAAATGGTATCAACAGTGGAGTTCATTCCTCCAATGGCCTCACT
AGAGGAGAAGGGTATTTTGTTTACATCTGATTATGTCCTGGCTTCTACCAACTCTCACTCAATAGCACCACCCACAGTAGCTCATAGTGATGCCTTAACCAGACGATTTGCATTTGATGTGGAGGTTTACACGATGTCTGAACATTCAGTCAAAGGCAAACTAAACATGGCCACAGCCACTCAGTTGTGTAAGGATTGTCCAACACCTGCAAATTTCAAAAAGTGTTGCCCTCTC
GTCTGTGGAAAGGCCTTGCAATTAATGGACAGGTACACCAGACAGAGGTTCACTGTGGATGAGATTACCACATTAATCATGAATGAGAAAAACAGAAGGGCCAACATTGGCAATTGTATGGAAGCCTTGTTTCAAGGACCACTGAGGTACAAAGATCTGAAGATTGATGTGAAGACAGTTCCCCCCCCTGAGTGCATCAGTGATTTATTACAAGCAGTGGATTCTCAAGAGGTTA
GGGATTACTGTGAGAAGAAAGGCTGGATCGTTAACGTTACTAGCCAGATACAATTAGAAAGGAACATCAATAGGGCCATGACTATACTCCAAGCTGTTACCACATTTGCAGCAGTCGCAGGAGTAGTGTATGTAATGTACAAACTCTTCGCTGGCCAGCAGGGTGCATACACTGGCTTGCCAAACAAAAAGCCCAATGTCCCTACTATCAGAATTGCTAAAGTCCAGGGGCCAGG
ATTTGACTATGCAGTGGCAATGGCAAAAAGAAACATAGTTACTGCAACCACCACTAAGGGTGAATTTACCATGTTAGGGGTGCATGATAATGTAGCAATATTGCCAACCCATGCCGCTCCAGGAGAAACTATCATTGTTGATGGGAAAGAAGTGGAGATCCTAGATGCCAGAGCCTTAGAAGATCAAGCGGGAACCAACCTTGAGATTACCATTATTACTCTAAAAAGAAATGAG
AAATTTAGAGACATCAGACCACATATTCCCACCCAAATTACTGAAACTAACGATGGAGTGTTGATCGTGAACACTAGTAAGTACCCCAACATGTATGTCCCAGTTGGTGCTGTGACTGAACAGGGATATCTTAATCTCAGTGGACGCCAAACTGCTCGTACTTTAATGTACAACTTTCCAACACGAGCAGGCCAGTGCGGAGGAATCATCACTTGTACTGGCAAAGTCATTGGGA
TGCACGTTGGCGGGAACGGTTCACATGGGTTTGCAGCAGCCCTTAAGCGATCATACTTCACCCAAAATCAGGGCGAAATTCAATGGATGAGGTCATCAAAAGAAGTGGGGTACCCCATTATAAATGCCCCATCCAAGACAAAGTTAGAACCCAGTGCTTTTCACTATGTTTTTGAAGGTGTTAAGGAACCAGCTGTGCTCACTAAAAATGACCCCAGACTAAAAACAGATTTTGA
AGAAGCCATCTTTTCAAAATACGTGGGGAACAAAATTACTGAAGTGGACGAATACATGAAAGAAGCAGTGGATCACTATGCAGGACAGTTAATGTCACTGGATATCAACACAGAACAGATGTGCCTGGAGGATGCCATGTACGGTACTGATGGTCTTGAAGCTCTGGATCTTAGCACTAGTGCTGGATACCCTTATGTTGCAATGGGGAAAAAGAAAAGAGACATTCTAGACAAA
CAGACCAGGGATACTAAAGAGATGCAGAGACTTTTGGACACCTATGGAATCAACCTACCATTAGTCACATACGTGAAAGATGAACTCAGATCAAAGACTAAAGTGGAACAAGGAAAGTCAAGGTTGATTGAAGCTTCCAGTCTTAATGATTCAGTTGCAATGAGAATGGCCTTTGGCAATCTTTACGCAGCTTTCCACAAGAATCCAGGCGTGGTGACAGGATCAGCAGTTGGTT
GTGACCCAGATTTGTTTTGGAGCAAGATACCAGTGCTAATGGAAGAAAAACTCTTCGCTTTTGACTACACAGGGTATGATGCCTCACTCAGCCCTGCTTGGTTTGAAGCTCTTAAAATGGTGTTAGAAAAGATTGGATTCGGCAATAGAGTAGACTACATAGACTACCTGAACCACTCTCACCACCTTTATAAAAACAAAACTTATTGTGTCAAAGGTGGCATGCCATCCGGCTG
CTCTGGCACATCAATTTTCAACTCAATGATTAATAACCTGATCATTAGGACGCTTTTACTGAGAACCTACAAGGGCATAGACTTGGACCACCTAAAAATGATTGCCTATGGTGATGACGTGATAGCTTCCTACCCCCATGAGGTTGACGCTAGTCTCCTAGCCCAATCAGGAAAAGACTATGGACTGACCATGACTCCAGCAGATAAATCAGCAACCTTTGAAACAGTCACATGG
GAGAATGTAACATTCCTGAAAAGATTTTTCAGAGCAGATGAGAAGTATCCATTCCTGGTGCATCCAGTGATGCCAATGAAAGAAATTCACGAATCAATCAGATGGACCAAGGACCCTAGAAACACACAGGATCACGTACGCTCATTGTGCCTATTGGCTTGGCATAACGGCGAAGAAGAATATAATAAATTTTTAGCTAAAATCAGAAGTGTGCCAATCGGAAGAGCTTTATTGC
TCCCAGAGTACTCCACATTGTACCGCCGATGGCTCGATTCATTTTAGTAACCCTACCTCAGTCGGATTCGGATTGGGTTATACTGTTGTAGGGGTAAATTTTTCTTTAATTCGGAG
> 20 Nucleotide Matches
+ Significant
Picking
Oligos
Coxsackie
Homology
Blast
Comparisons
Picking
Oligos
RV
Homology
Blast
Comparisons
Spotting the Probes
• For most of the viral
sequences, the five best
70-mers were chosen,
– plus the anti-sense
sequence (why?),
• These were spotted onto
the solid substrate.
Making the Targets
• Collect nasal lavage (snot),
– or cell cultures,
• Extract RNA (RNeasy kit),
– reverse transcriptase with
degenerate primers,
5’ - GTTTCCCAGTAGTCTCNNNNNNNN - 3’
Degeneracy
primers
5’ - GTTTCCCAGTAGTCTCNNNNNNNN - 3’
…the N(s) can be any base, thus all of the 65,536
possible combinations are made,
– random primes bind to complementary sequences,
– reverse transcriptase builds off the 3’ end,
– some of the primers “face” each other,
…PCR is then performed using...
5’ - GTTTCCCAGTAGTCTC - 3’
Labeling Targets
• Cy3 (uninfected)
green
– people,
– cell lines,
• Fluorescent base
inserted into the
sequence.
• Cy5 (infected)
red
– people,
– cell lines,
• Fluorescent base
inserted into the
sequence.
HeLa Cells?
target sources
• Cervical cancer cells from Henrietta Lacks, 1951,
• Cultured and used worldwide for study.
In this study:
• Infected cell cultures with viruses of interest (KSHV aka
HHV-8, Poliovirus1, rhinoviruses, enteroviruses, hepatitis B
and C, papillomavirus),
• Then infected people with known cold virus,
• simple colds (OK work if you can get it).
Summary
(so far)
Extract RNA
RT/PCR
Label
Similar for DNA Viruses
Raw Data
Positives, and
graphical
depiction.
• Hybridization results. Select targets versus
several probes.
Type-specific ID, and detection of family members (within study).
SO NEATO!
Type-specific ID, and detection of other RVs (outside study).
Let’s get people sick!
Snot from off the street...
At least they didn’t have canine distemper or bovine parainfluenza!
And this helps how??
• one test, multiple (100s) viruses detectable,
• only small amounts of sample required (no culture),
• sensitive detection between relatives,
• less labor and time intensive,
• not testing for a specific candidate or family, so no
inherent bias,
• able to detect unknown viruses.
To Study
• Figure (1a),
– What’s Blast, how was it used,
– What does this figure represent?
• Figure (4a,b,c),
– What does each figure demonstrate,
– How was the result confirmed?
• Hybridization, PCR Primers (degenerate, random),
• rtPCR.
Exam Wednesday
review Monday
•
•
•
•
•
Chromosomal Mutations (specially structural),
DNA Science,
Bacterial Genetics,
Malaria Paper,
Virus Paper,
• Everything else going back to kindergarten.