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,

Requires knowledge of

each viral sequence.

Returns non-specific,

positive data.

• through design of degenerate primers for members of a viral family.

Proceedings of the National Academy of Sciences

September 9, 2002

SARSSevere 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!

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.

• 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.

Making the Arraychoosing 70-mers Targets

Basic Local Alignment Search ToolBlast

• 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.

TTAAAACAGCTCTGGGGTTGCTCCCACCCCAGAGGCCCACGTGGCGGCTAGTAATCTGGTATCAGGTACCTTTGTACGCCTGTTTTATATCCCTTCCCCCGTAACTTTAGAAGCTTATCAAAAGTTCAATAGCAGGGGTACAAGCCAGTACCTCTACGAACAAGCACTTCTGTTTCCCCGGTGAAATCATATAGACTGTACCCACGGTCAAAAGTGATTGATCCGTTATCCGCTTGAGTACTTCGAGAAGCCTAGTATCGCCTTGGAATCTTCGACGCGTTGCGCTCAACACTCTGCCCCGAGTGTAGCTTAGGCTGATGAGTCTGGGCACTCCCCACCGGCGACGGTGGCCCAGGCTGCGTTGGCGGCCTACCCATGGCTGATGCCGTGGGACGCTAGTTGTGAACAAGGTGTGAAGAGCCTATTGAGCTACTCAAGAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACCACGGAGCAATCGCTCACGACCCAGTGAGTAGGTTGTCGTAATGCGTAAGTCTGTGGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCCTTTATATTCATACTGGCTGCTTATGGTGACAATTTACGAATTGTTACCATATAGCTATTGGATTGGCCACCCAGTGCTGTGCAATATATTTGAGTGCTTCTTTCATAGGTGTTACCAACATCACATTTAAACCACAATAGTCAGTGCAAATGGGGGCTCAAGTTTCAACCCAAAAGACCGGTGCACACGAGAATCAAAACGTGGCAGCCAATGGATCCACCATTAATTATACTACCATCAACTACTACAAAGACAGCGCGAGTAACTCCGCTACTAGACAAGACCTCTCCCAAGATCCATCAAAATTCACAGAACCGGTTAAGGACTTAATGTTGAAAACAGCACCAGCTTTAAACTCGCCCAACGTGGAAGCATGTGGGTACAGTGACCGTGTGAGGCAAATCACTTTAGGTAACTCGACCATTACCACACAGGAAGCAGCTAATGCTATTGTTGCTTATGGTGAGTGGCCCACTTACATAAATGACTCAGAAGCTAATCCGGTAGATGCACCCACTGAACCAGACGTTAGTAGCAACCGTTTTTACACCCTGGAATCGGTGTCTTGGAAGACCACTTCAAGGGGATGGTGGTGGAAGCTACCAGATTGTCTAAAAGATATGGGAATGTTTGGTCAGAATATGTACTATCACTACTTAGGACGCTCTGGTTACACCATTCATGTCCAGTGCAACGCTTCTAAGTTTCACCAAGGGGCGTTAGGAGTTTTCCTGATACCAGAGTTTGTCATGGCTTGCAACACTGAGAGTAAAACATCATATGTTTCATACATCAACGCAAATCCTGGTGAAAGGGGCGGTGAGTTCACGAACACCTACAACCCATCAAACACTGATGTCAGTGAGGGCAGACAGTTCGCAGCACTGGATTATCTGCTGGGTTCTGGTGTCCTAGCAGGAAACGCTTTCGTATACCCGCACCAGATCATCAATTTGCGCACCAACAACAGTGCAACAATTGTGGTACCATATGTGAACTCGCTCGTGATTGATTGTATGGCAAAACACAATAACTGGGGTATTGTCATATTACCACTGGCACCCTTGGCCTTTGCCGCAACATCGTCACCACAGGTGCCTATTACAGTGACCATCGCACCCATGTGTACAGAATTTAATGGGTTGAGAAATATTACCATCCCAGTACATCAAGGGTTGCCAACAATGAACACACCTGGTTCCAATCAATTCCTTACATCTGATGACTTCCAGTCGCCCTGTGCCTTACCTAATTTTGATGTTACTCCACCGATACACATACCCGGGGAAGTGAAGAATATGATGGAACTAGCTGAAATTGACACACTGATCCCAATGAACGCAGTGGACGGGAAGGTGAACACAATGGAAATGTATCAAATACCATTGAATGACAATTTGAGCAAGGCACCTATATTCTGTCTATCTTTATCACCTGCTTCTGACAAACGACTGAGTCACACCATGTTGGGTGAAATTCTAAATTATTACACTCATTGGACGGGGTCCATCAGGTTCACCTTTCTATTTTGTGGTAGTATGATGGCCACTGGTAAACTGCTCCTCAGCTATTCCCCACCAGGAGCTAAACCACCAACCAATCGCAAGGATGCAATGCTAGGTACACACATCATCTGGGACCTAGGGTTACAATCCAGTTGTTCCATGGTTGCACCGTGGATCTCTAACACAGTGTACAGGCGGTGTGCACGTGATGACTTCACTGAGGGCGGGTTTATAACTTGCTTTTATCAAACTAGAATTGTTGTGCCTGCTTCAACCCCCACCAGTATGTTCATGTTAGGCTTTGTTAGTGCGTGTCCAGATTTCAGTGTCAGACTGCTTAGGGACACTTCCCATATTAGTCAATCAAAACTAATAGCACGCACACAAGGCATTGAAGACCTCATTGACACAGCGATAAAGAATGCCTTAAGAGTGTCCCAACCACTTCGGCCCAGTCAACTGAAGCAACCCAATGGAGTAAATAGCCAGGAGGTGCCAGCTCTAACTGCTGTGGAAACAGGAGCATCTGGTCAAGCGATCCCCAGTGATGTGGTGGAAACTAGACACGTGATAAATTACAAAACCAGGTCTGAATCGTGTCTTGAGTCATTCTTTGGGAGAGCTGCGTGTGTCACAATCCTATCTCTGACCAACTCTTCCAAGAGCGGGGAGGAGAAGAAGCATTTCAACATATGGAACATTACATACACCGACACTGTTCAGTTACGCAGAAAATTGGAGTTTTTCACGTATTCCAGGTTTGACCTTGAAATGACTTTTGTGTTCACAGAGAATTACCCTAGTACAGCCAGTGGGGAAGTGCGTAACCAATGTGACCAGATCATGTACATTCCACCAGGGGCACCCCGTCCATCATCCTGGGATGACTATACATGGCAATCCTCCTCTAACCCTTCCATCTTTTACATGTATGGAAATGCACCTCCACGGATGTCAATTCCTTACGTGGGGATTGCCAATGCCTATTCACACTTCTATGATGGCTTTGCACGGGTGCCACTTGAGGGTGAGAATACCGATGCTGGTGACACGTTTTACGGCTTAGTGTCCATAAATGATTTTGGAGTTTTAGCAGTTAGAGCGGTAAACCGCAGTAACCCACACACTATACACACATCTGTGAGAGTGTACATGAAACCAAAACACATTCGGTGTTGGTGCCCCAGACCTCCTCGGGCTGTATTATACAGGGGAGAGGGAGTGGACATGATATCCAGTGCAATTCAACCTCTGACTAAAGTAGACTCAATTACCACTTTTGGGTTTGGCCACCAGAACAAGGCAGTGTACGTTGCCGGTTACAAGATTTGTAACTACCACCTAGCAACCCCAAGTGATCACTTGAATGCAATTAGTGTGTTATGGGACAGGGATTTAATGGTGGTGGAATCTAGAGCCCAGGGAACTGATACCATTGCCAGGTGTAGTTGCAGGTGTGGAGTTTACTATTGTGAATCTAGAAGGAAGTACTACCTTGTCACTTTCACTGGCCCAACGTTTCGATTTATGGAAGCAAATGACTACTATCCAGCAAGATACCAGTCTCACATGCTGATAGGGTGCGGATTTGCAGAACCCGGGGACTGTGGTGGGATATTAAGGTGCACTCATGGGGTGATTGGTATCATCACTGCAGGGGGTGAAGGGATAGTAGCCTTTGCTGACATTAGAGACCTTTGGGTGTATGAAGAGGAAGCCATGGAACAGGGAATAACAAGTTACATCGAATCCCTCGGTACAGCTTTTGGTGCAGGGTTCACCCACACAATCAGTGAGAAAGTGACTGAGTTGACAACAATGGTCACCAGTACTATCACAGAAAAATTACTGAAAAATTTAGTGAAAATAGTATCGGCTCTAGTGATTGTTGTGAGAAATTATGAGGACACTACCACGATCCTTGCAACACTAGCATTACTTGGGTGTGATATATCTCCTTGGCAATGGTTGAAGAAGAAGGCATGTGACTTACTAGAGATTCCTCATGTGATGCGCCAAGGTGATGGGTGGATGAAGAAATTCACAGAGGCTTGCAATGCAGCTAAAGGGCTTAGATGGGTCAGCAATAAAATTTCCAAGTTTGTAGATTGGTTGAAGTGTAAAATTATCCCAGAAGCTAAGGACAAGGTGGAATTTCTCACCAAGTTGAAACAGCTAGACATGCTGGAAAATCAAATTGCAACCATCCACCAATCTTGCCCCAGCCAAGAACAACAAGAGATCCTTTTCAACAACGTAAGATGGCTAGCAGTCCAGTCCCGTCGGTTCGCGCCATTATATGCTGTGGAGGCACGCCGGATTAGCAAAATGGAGAGCACAATAAACAATTATATACAGTTCAAGAGCAAACACCGTATTGAGCCAGTATGTATGCTCGTTCATGGGTCACCAGGGACGGGTAAGGGTATAGCTAGCTCATTGATAGGTAGAGCAATAGCAGAGAGGGAAACCACATCGGTCTACTCAGTGCCACTGGCCCCATCTCACTTTGATGGCTATAAACAACAAGGGTATGATATGGACGACCTAAACCAAAACCCCGATGGTATGGACATGAAACTGTTTTGCCAAATGGTATCAACAGTGGAGTTCATTCCTCCAATGGCCTCACTAGAGGAGAAGGGTATTTTGTTTACATCTGATTATGTCCTGGCTTCTACCAACTCTCACTCAATAGCACCACCCACAGTAGCTCATAGTGATGCCTTAACCAGACGATTTGCATTTGATGTGGAGGTTTACACGATGTCTGAACATTCAGTCAAAGGCAAACTAAACATGGCCACAGCCACTCAGTTGTGTAAGGATTGTCCAACACCTGCAAATTTCAAAAAGTGTTGCCCTCTCGTCTGTGGAAAGGCCTTGCAATTAATGGACAGGTACACCAGACAGAGGTTCACTGTGGATGAGATTACCACATTAATCATGAATGAGAAAAACAGAAGGGCCAACATTGGCAATTGTATGGAAGCCTTGTTTCAAGGACCACTGAGGTACAAAGATCTGAAGATTGATGTGAAGACAGTTCCCCCCCCTGAGTGCATCAGTGATTTATTACAAGCAGTGGATTCTCAAGAGGTTAGGGATTACTGTGAGAAGAAAGGCTGGATCGTTAACGTTACTAGCCAGATACAATTAGAAAGGAACATCAATAGGGCCATGACTATACTCCAAGCTGTTACCACATTTGCAGCAGTCGCAGGAGTAGTGTATGTAATGTACAAACTCTTCGCTGGCCAGCAGGGTGCATACACTGGCTTGCCAAACAAAAAGCCCAATGTCCCTACTATCAGAATTGCTAAAGTCCAGGGGCCAGGATTTGACTATGCAGTGGCAATGGCAAAAAGAAACATAGTTACTGCAACCACCACTAAGGGTGAATTTACCATGTTAGGGGTGCATGATAATGTAGCAATATTGCCAACCCATGCCGCTCCAGGAGAAACTATCATTGTTGATGGGAAAGAAGTGGAGATCCTAGATGCCAGAGCCTTAGAAGATCAAGCGGGAACCAACCTTGAGATTACCATTATTACTCTAAAAAGAAATGAGAAATTTAGAGACATCAGACCACATATTCCCACCCAAATTACTGAAACTAACGATGGAGTGTTGATCGTGAACACTAGTAAGTACCCCAACATGTATGTCCCAGTTGGTGCTGTGACTGAACAGGGATATCTTAATCTCAGTGGACGCCAAACTGCTCGTACTTTAATGTACAACTTTCCAACACGAGCAGGCCAGTGCGGAGGAATCATCACTTGTACTGGCAAAGTCATTGGGATGCACGTTGGCGGGAACGGTTCACATGGGTTTGCAGCAGCCCTTAAGCGATCATACTTCACCCAAAATCAGGGCGAAATTCAATGGATGAGGTCATCAAAAGAAGTGGGGTACCCCATTATAAATGCCCCATCCAAGACAAAGTTAGAACCCAGTGCTTTTCACTATGTTTTTGAAGGTGTTAAGGAACCAGCTGTGCTCACTAAAAATGACCCCAGACTAAAAACAGATTTTGAAGAAGCCATCTTTTCAAAATACGTGGGGAACAAAATTACTGAAGTGGACGAATACATGAAAGAAGCAGTGGATCACTATGCAGGACAGTTAATGTCACTGGATATCAACACAGAACAGATGTGCCTGGAGGATGCCATGTACGGTACTGATGGTCTTGAAGCTCTGGATCTTAGCACTAGTGCTGGATACCCTTATGTTGCAATGGGGAAAAAGAAAAGAGACATTCTAGACAAACAGACCAGGGATACTAAAGAGATGCAGAGACTTTTGGACACCTATGGAATCAACCTACCATTAGTCACATACGTGAAAGATGAACTCAGATCAAAGACTAAAGTGGAACAAGGAAAGTCAAGGTTGATTGAAGCTTCCAGTCTTAATGATTCAGTTGCAATGAGAATGGCCTTTGGCAATCTTTACGCAGCTTTCCACAAGAATCCAGGCGTGGTGACAGGATCAGCAGTTGGTTGTGACCCAGATTTGTTTTGGAGCAAGATACCAGTGCTAATGGAAGAAAAACTCTTCGCTTTTGACTACACAGGGTATGATGCCTCACTCAGCCCTGCTTGGTTTGAAGCTCTTAAAATGGTGTTAGAAAAGATTGGATTCGGCAATAGAGTAGACTACATAGACTACCTGAACCACTCTCACCACCTTTATAAAAACAAAACTTATTGTGTCAAAGGTGGCATGCCATCCGGCTGCTCTGGCACATCAATTTTCAACTCAATGATTAATAACCTGATCATTAGGACGCTTTTACTGAGAACCTACAAGGGCATAGACTTGGACCACCTAAAAATGATTGCCTATGGTGATGACGTGATAGCTTCCTACCCCCATGAGGTTGACGCTAGTCTCCTAGCCCAATCAGGAAAAGACTATGGACTGACCATGACTCCAGCAGATAAATCAGCAACCTTTGAAACAGTCACATGGGAGAATGTAACATTCCTGAAAAGATTTTTCAGAGCAGATGAGAAGTATCCATTCCTGGTGCATCCAGTGATGCCAATGAAAGAAATTCACGAATCAATCAGATGGACCAAGGACCCTAGAAACACACAGGATCACGTACGCTCATTGTGCCTATTGGCTTGGCATAACGGCGAAGAAGAATATAATAAATTTTTAGCTAAAATCAGAAGTGTGCCAATCGGAAGAGCTTTATTGCTCCCAGAGTACTCCACATTGTACCGCCGATGGCTCGATTCATTTTAGTAACCCTACCTCAGTCGGATTCGGATTGGGTTATACTGTTGTAGGGGTAAATTTTTCTTTAATTCGGAG

link

> 20 Nucleotide Matches + Significant

CoxsackieHomology

Blast Comparisons

Picking Oligos

RVHomology

Blast Comparisons

Picking Oligos

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’

Degeneracyprimers

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

Exam Wednesdayreview Monday

• Chromosomal Mutations (specially structural),

• DNA Science,

• Bacterial Genetics,

• Malaria Paper,

• Virus Paper,

• Everything else going back to kindergarten.