A Viral Metagenomics Study A Viral Metagenomics Study of Honey Bee Colonyof Honey Bee ColonyCollapse DisorderCollapse Disorder

Participating Experts:W. Ian Lipkin, M.D.Professor of Epidemiology, Neurologyand PathologyMailman School of Public Health andCollege of Physicians and SurgeonsColumbia UniversityNew York, NY

Michael Egholm, Ph.D.Vice President of Research& Development454 Life SciencesBranford, CT

Sean Sanders, Ph.D.Commercial EditorScience/AAAS

Moderator:Sponsored by:

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Viral Metagenomics Webinar“A Viral Metagenomics Study of Honey Bee Colony Collapse

Disorder”

24 October 2007

W. Ian LipkinMailman School of Public Health and College

of Physicians and SurgeonsColumbia University

Michael Egholm454 Life Sciences, A Roche Company

An introduction to surveillance and pathogen discoveryHow and why do infectious diseases emerge and re-emerge

Agenda

Proof of causationKoch’s postulates and beyond

Staging an investigation

What can/cannot be done using with different diagnostic technologies

• Technical challenges

• Strengths and limitations

• Introduction to 454 Sequencing

A bee story…

Future perspectives

A. Fauci, NIAID/NIH

• Microbial adaptation and change

• Host susceptibility to infection(e.g. aging, HIV, cancer, transplantation, stress)

• Population growth and density

• Inadequate/deteriorating public health and regulatory infrastructure

Factors in Emerging/Re-emerging Diseases

Modified from Institute of Medicine

Factors in Emerging/Re-emerging Diseases, continued

Modified from Institute of Medicine

• Urbanization and crowding (humans & animals)

• Changing ecosystems (deforestation, global warming)

• Globalization of travel and trade

Agriculture and World Health

Global Trade in Agricultural Products

United Nations

Koch, 1890

Microbe occurs in every case of a diseaseMicrobe must be specific for that diseaseMicrobe can be isolated, grown in the laboratory and cause disease after inoculation into animals

CAUSATION: IMPLICATION OF MICROBES IN DISEASE

Potential confoundsSome microbes cannot be grown in the laboratory (Koch)Host and environmental factors may influence expression of diseaseLongterm/distant sequelae may obscure relationship to diseaseSuitable animal models may not exist (Koch)

Fredericks and Relman, 1996

Sequences should be present in most cases of a disease at sites of pathology

Few/no sequences should be present in hosts or tissues without diseaseSequences should diminish with resolution of disease and increase with relapse

Sequences should be present prior to the onset of disease

CAUSATION: IMPLICATION OF MICROBES IN DISEASE

Presence of a microbe or its genetic footprints correlate with disease (e.g. animal models, immunological responses consistent with infection, response to treatment, vaccine prophylaxis…)

A Staged Strategy for Pathogen Discovery

MassTag PCR Panels• Respiratory disease • Hemorrhagic fevers • Meningoencephalitides• Poxviruses $15/assay, 96 samples in 6 Hours

GreeneChips• Viral• Pan-pathogen • Respiratory$75/assay, 8 samples in 15 hrs

Shotgun Sequencing$5,000/assay – 1 week

QuantitativeReal-Time PCR

Consensus PCRCloning & Sequencing

Serology (IgM, increase in IgG titer)Pathology (agent distribution)Challenge experiments (reproduce disease)

454 Genome Sequencer FLX Overview

� Instant cloning by emulsion PCR� ~ 400K clonal reads � ~ 250 bp average read length� Less than 8 hours run time� MIDs allow 96 samples/plate� Average individual read accuracy >99.5% � Extremely low substitution error rate of less

than 1:10,000� Low bias from both emPCR and

Sequencing� Consensus de novo assembly >99.99% at

>10X coverage

For details and list of 90+ peer reviewed papers enabled by 454 Sequencing go to www.454.com orwww.roche-applied-science.com

454 Sequencing Overview

Shear DNA and add linkers ‘Emulsion PCR’Clonal amplification

Deposition of beads onto PTP with 1.6M wells.

Sequencing-by-synthesisDetection of PPi release

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Dramatic Cloning Bias by Conventional Sequencing Example 500 kb stretch in Listeria monocytogenes

454 coverage

Reference position (bp)

Courtesy of Drs. Nusbaum and Young of the Broad Ins titute

ABI coverage

Generation of 454 Library from DOP-PCR Product

Bio

PA BBio

Ligation

Fill inBio

Bio

Bio

1) Capture on SA-Beads & Wash2) Alkaline Elution

A B

P

Fragment end Polishing

High Throughput Sequencing

Algorithm for Reductive Analysis of Sequence DataProgram for automated identification of pathogen sequences

Outbreak of Transplant-Associated Encephalitis Identification of a novel arenavirus

Colony Collapse Disorder Profile of an emerging threat to agriculture and economic welfare

Sample preparationEnrichment for pathogen sequences

Primer

Repeat

Trimming FilteringClustering Assembly

Raw Reads

host

ContigsTrimmed Reads Unique Reads

Reductive Analysis of High Throughput Sequencing Data

Contigs and SingletsContigs and Singlets

Candidates withNucleotide Homology

Candidates withNucleotide Homology

BLASTN

Candidates withProtein Homology

Candidates withProtein Homology

BLASTX

viruses

bacteria

fungi

metazoans

parasites

Sequence � TaxonomySequence � Taxonomy

Candidate organism

Thomas BrieseSean ConlanDiana Cox-FosterLiwang CuiAndrew DrysdaleMichael EgholmDennis vanEngelsdorpJay EvansDavid GeiserEddie HolmesMady Hornig

Jeffrey HuiStephen HutchisonAbby KalksteinW. Ian LipkinVince MartinsonGustavo PalaciosJeff PettisNancy MoranPhenix-Lan QuanJan Fredrik SimonsJunhui Zhai

U of Arizona; Columbia; Penn State; USDA,FL Dept of Agriculture; PA Dept of Agriculture; and 454 Life SciencesCox-Foster et al. Science 318:283-287, 2007

Metagenomic Analysis of CCD

To Bee or Not to Bee…Colony Collapse DisorderGlobal pollination of > 90 fruit and vegetable crops$14.6B/year in US alone

Family apoidea

AlfalfaAlmondAppleApricot (some varieties)ArtichokeAvocadoBlackberryBlack locustBlueberryBoysenberryBroadbeansBroccoliBrussels sproutsBuckwheatCabbageCarawayCarrotsCatalpaCauliflowerCeleryCherryChestnutChicoryChinese cabbage

Clover (some varieties)CoconutCoffeeCollardsCorianderCottonCrabappleCranberriesCrownvetchCucumbersCurrantsDewberryDillEggplantEndiveFennelFigsFlaxGarlicGooseberriesGrapes (muscadine)GrapefruitGuarGuava

Holly KaleKenafKiwiKohlrabiLeekLespedeza (bush)Lima beansLoquatsMacadamia nutMaple (red)Mandarin orangeMangoMuskmelonsMustardNectarinesOkra OnionsOrange PapayaParsleyParsnipsPeachesPear

Pepper Persimmon (native)Plums and PrunePumpkinsRadishRapeRaspberryRutabagaSafflower SquashStrawberrySunflowerSweetcloverTangeloTangerineTendergreensTrefoilTurnipsVetchWatermelonsYucca

Colony Collapse Disorder

Collapsed colonies

Absence of adult bees, dead or alive

Presence of capped brood

Presence of food stores, not ransacked

The Clinician in Pathogen DiscoveryWest Nile virus, New York, 1999

Deborah Asnis, MD/Tracy McNamara, DVM

CCD

Diana Cox-Foster

Penn State

Normal

7 CCD operations, 6 colonies/operation, 10 bees/col ony4 Royal jelly samples

Strategy for CCD Project

RNA extractionDNAse I digestionRandom amplification

Metagenomic Sequencing/Bioinformatic Analysis

Closest sequencedrelatives identifiedby BLAST

Raw Reads

35%

33%0%

1%

0%

3%

0%

28%

Order Hymenoptera

Metazoa (Other)

Plants

Eukaryota

Fungi

Bacteria

Viruses

Unclassified

Assembled Contigs

26%

25%

3%5%

1%

8%

2%

30%

Order Hymenoptera

Metazoa (Other)

Plants

Eukaryota

Fungi

Bacteria

Viruses

Unclassified

974351668Total

27732506Unclassified

6530Viruses

2688127Bacteria

24318Fungi

99286Eukaryota

4242Plants

31991419Metazoa (Other)

3368219390

440-

Order Hymenoptera60% rRNA

ReadsContigsClassification

Raw Reads

35%

33%0%

1%

0%

3%

0%

28%

Order Hymenoptera

Metazoa (Other)

Plants

Eukaryota

Fungi

Bacteria

Viruses

Unclassified

Assembled Contigs

26%

25%

3%5%

1%

8%

2%

30%

Order Hymenoptera

Metazoa (Other)

Plants

Eukaryota

Fungi

Bacteria

Viruses

Unclassified

974351668Total

27732506Unclassified

6530Viruses

2688127Bacteria

24318Fungi

99286Eukaryota

4242Plants

31991419Metazoa (Other)

3368219390

440-

Order Hymenoptera60% rRNA

ReadsContigsClassification

35%

Raw Reads

35%

33%0%

1%

0%

3%

0%

28%

Order Hymenoptera

Metazoa (Other)

Plants

Eukaryota

Fungi

Bacteria

Viruses

Unclassified

Assembled Contigs

26%

25%

3%5%

1%

8%

2%

30%

Order Hymenoptera

Metazoa (Other)

Plants

Eukaryota

Fungi

Bacteria

Viruses

Unclassified

974351668Total

27732506Unclassified

6530Viruses

2688127Bacteria

24318Fungi

99286Eukaryota

4242Plants

31991419Metazoa (Other)

3368219390

440-

Order Hymenoptera60% rRNA

ReadsContigsClassification

33%

Raw Reads

35%

33%0%

1%

0%

3%

0%

28%

Order Hymenoptera

Metazoa (Other)

Plants

Eukaryota

Fungi

Bacteria

Viruses

Unclassified

Assembled Contigs

26%

25%

3%5%

1%

8%

2%

30%

Order Hymenoptera

Metazoa (Other)

Plants

Eukaryota

Fungi

Bacteria

Viruses

Unclassified

974351668Total

27732506Unclassified

6530Viruses

2688127Bacteria

24318Fungi

99286Eukaryota

4242Plants

31991419Metazoa (Other)

3368219390

440-

Order Hymenoptera60% rRNA

ReadsContigsClassification

28%

viruses

bacteria

fungi

metazoans

parasites

Varroa destructor mite

Kynetoplastide parasite species

Nosema sp.

Neisseria species

Gamma proteobacteria

Viruses

ssRNA (+)

Dicistroviridae

Cripavirus

Black Queen Virus

Israel acute paralysis virus

Kashmir bee virus

Iflavirus

Sacbrood virus

Deformed wing virus

Chronic bee paralysis virus

Kingdom Taxon (rank) Organism Bacteria Firmicutes (phylum) Lactobacillus sp. 1,2

Uncultured Firmicutes 2 Bacteria Actinobacteria (class) Bifidobacterium sp. 1 Bacteria Alphaproteobacteria (class) Bartonella sp. 1,2

Gluconacetobacter sp. 1,2 Bacteria Betaproteobacteria (class) Simonsiella sp. 1,2 Bacteria Gammaproteobacteria (class) Two uncultured species 1,2 Fungus Entomophthorales (order) Pandora delphacis Fungus Mucorales (order) Mucor spp. Fungus / Microsporidian

Nosematidae (family) Nosema ceranae

Fungus / Microsporidian

Nosematidae (family) Nosema apis

Eukaryota Trypanosomatidae (family) Leishmania/Leptomonas sp. Metazoan Varroidae (family) Varroa destructor Virus (unclassified) Chronic bee paralysis virus* Virus Iflavirus (genus) Sacbrood virus Virus Iflavirus (genus) Deformed wing virus* Virus Dicistroviridae (family) Black queen cell virus Virus Dicistroviridae (family) Kashmir bee virus* Virus Dicistroviridae (family) Acute bee paralysis virus Virus Dicistroviridae (family) Israeli acute paralysis virus of bees*

Bacterial Composition of CCD and Non-CCD Hives

16S rRNA analysis

Number of positive samples (% of samples tested)

Agent CCD

(n=30) Non-CCD

(n=21) Total

(n=51)

Positive Predictive

Value (%)

Sensitivity (%)

Specificity (%)

IAPV 25 (83.3) 1 (4.8) 26 (51.0) 96.1 83.3 95.2

KBV 30 (100) 16 (76.2) 46 (90.2) 65.2 100 23.8

N. apis 27 (90) 10 (47.6) 37 (72.5) 73.0 90.0 52.4

N. ceranae 30 (100) 17 (80.9) 47 (92.1) 63.8 100 19.0

All 4 agents 23 (76.7) 0 (0) 23 (45.0) 100 76.7 100

Value of CCD Candidate Markers

Real time PCR analysis of pools of up to 10 bees fr om 30 CCD and 21 non CCD operations Jan 2004 through May 2007

Work in Progress

Global distribution of IAPV

Challenge experiments in A mellifera with IAPV +/- other potential factors

Phylogenetic analyses of IAPV and KBV

Reverse genetic studies of IAPV

Characterization of other potential pathogens ident ified through metagenomics during the course of the CCD p roject

Improvements in metagenomic technology (sample preparation, throughput)

Future Application of Pathogen Discovery/Surveillance TechnologiesInfectious Causes of Chronic Disease

DiseaseCervical CA

Chronic Hepatitis/Hepatocarcinoma

Lyme Arthritis

Peptic Ulcer Disease

Whipple Disease

Bladder CA

Premature Birth

Cerebral Palsy

CauseHuman papilloma virus

Hepatitis B and C viruses

Borrelia burgdorferi

Helicobacter pylori

Tropheryma whippelii

Schistosoma haematobium

Various

Various

Compelling evidence

Future Application of Pathogen Discovery/Surveillance Technologies

Chronic Diseases Potentially Linked to Infection

DiseaseCardio/Cerebrovascular Disease

Diabetes Mellitus

Obsessive Compulsive Disorder

(PANDAS)

Schizophrenia

Inflammatory Bowel Disease

Idiopathic Pulmonary Fibrosis

Autism

Acute Macular Degeneration

CauseDental/sinus infections

Enteroviruses, other viruses

Group A streptococcus

Prenatal infections

?

?

?

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The future of microbes and mankind will probably unfold as episodes of a suspense thriller that could be entitled Our Wits versus Their Genes. The global scientific and public health communities must confront this reality, not only with wisdom but also with vision and sustained commitment to meet a perpetual challenge.Ó

"The future of microbes and mankind will probably unfold as episodes of a suspense thriller that could be entitled Our Wits versus Their Genes. The global scientific and public health communities must confront this reality, not only with wisdom but also with vision and sustained commitment to meet a perpetual challenge."

Josh Lederberg

A Viral Metagenomics Study of Honey Bee Colony Collapse Disorder

For more information visit: www.genome-sequencing.com or www.454.com

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