Brute force in the clinic

Keen to document exactly which microbes are there, many researchers are using

brute-force 'metagenomic' studies in which they extract microbial DNA from the

body cavity of choice and hurl it all into a sequencing machine.

NGS and virus discovery

Eric Delwart

Blood Systems Research Institute

UCSF Laboratory Medicine

Rapid pace of viral discovery is providing many “orphan

viruses” as candidate pathogens

The discovery curve for human virus species Woolhouse M E et al. Proc. R. Soc. B 2008;275:2111-2115

Some common diseases may be associated

with still unknown viruses

• Gastroenteritis (~50% unexplained in the US)

• Respiratory illness (15-25% unexplained)

• Encephalitis (~60-80% unexplained in CA)

• Hepatitis

• Chronic fatigue syndrome

• Non-polio acute flaccid paralysis

• Auto-immune diseases (diabetes, MS, Kawasaki)

• Cancers

Virus Size R

elat

ive

size

of

viru

ses

and

b

acte

ria

400 nm filters used to purify viral particles

Bacterium (Staphyllococcus aureus)

Chlamydia

Pox virus

Herpes virus

Influenza Virus

Picornavirus

(polio)

Random RT-PCR amplification

RNA DNA

Melt/annneal and extend cDNA with Klenow DNA

polymerase

5’GTCCATGCATGACTCGAGTCNNNNNNNN3’

30-40 PCR rounds

Randomized 3’

5’GTCCATGCATGACTCGAGTC3’

Reverse transcription

Filtration and nuclease enriched viral nucleic acids

Signature tag

Compiling of data

into web-based

format that is

dynamic and

searchable

Raw reads Sequencing Bin and trim

Assembly Blast search

data collecting

and quality

control

Sequence data generation and analysis

Bioinformatics: the perception..

The reality..

Wide variety of viral

genome types have

been “re-discovered”

Viruses detected:

BLASTx E score <10-5

New parvovirus Long contigs

A new highly divergent calicivirus with borderline E score 3x10-4

(23% protein identity)

E=10-10

E=10-5

E=10-20

Highly divergent viruses are not recognized by BLASTx

?

? ?

Borderline recognized E=10-5

Unrecognized E<10-5

Novel animal viral genomes helps identify

highly divergent human virus.

Expansion of viral sequence space by new

species/genus

Recognized E=10-20

Varivax

Other viruses:

None

Detected

104,273

Other viruses:

None

Detected

60,735 PV1

21,251 PV2

59,943 PV3

Oral poliovirus 1,2,3 vacinne Metagenomics is useful tool to detect viral contaminations

Rotarix

Some

Rotavirus

Sequences

(458)

6,344 Porcine Circovirus-1

HEV-B, HEV-C,

Cosavirus,

Adenovirus,

rhinovirus,

Cucumber

mosaic virus

Number mammalian viruses in stool from Pakistani children with

non-polio acute flaccid paralysis

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7

Diarrhea

Healthy

Co-infections in high density farm piglets

Number of mammalian viruses per feces

Num

ber

of pig

lets

Parvoviridae family (ssDNA)

Smallest known viruses, very environmentally resistant.

Common pathogen of cats, dogs, pigs. Vaccination available.

Resistant to heat and solvent/detergent inactivation (problem

for transfusion).

Genera infecting vertebrates:

Parvovirus: Murine minute virus

Erythrovirus: B19

Dependovirus: Adeno-associated virus

Amdovirus: Aleutian mink disease virus

Bocavirus: HBoV, HBoV2-3-4

Partetravirus: PARV4

Unassigned: Bufavirus 1-2

Recognizable by protein similarity (BLASTx)

Too divergent to anneal to microarray based on pre-existing

nucleotide sequences in GenBank

PARV4 typical parvovirus genome structure

PARV4: founder of new

Parvovirus genus (Partetraviridae)

Consensus primer PCR identified PARV4 relatives

PARV4 antibodies in US/Europe limited to blood exposed subjects:

First blood transmitted parvovirus?

Hitch-hiker to blood-borne pandemics of HIV and HCV?

Sharp CP, Lail A, Donfield S, Gomperts ED, Simmonds P. Transfusion. 2012 Jul;52(7):1482-9.

PARV4 sero-conversion following heat treated clotting factors

Is PARV4 a pathogen ?

200 healthy blood donors from Los Angeles: 2% positive

200 patients with symptoms of acute viral infection (HIV RNA

negative MSM and/or IDU): 6% positive

p=0.03

Higher PARV4 prevalence may simply reflect higher general

exposure to blood borne viruses in high risk groups

PARV4 in CSF of two unexplained encephalitis cases India Benjamin LA, Lewthwaite P, Vasanthapuram R, Zhao G, Sharp C, Simmonds P, Wang D,

Solomon T. Emerg Infect Dis. 2011 Aug;17(8):1484-7.

PARV4 in cases of fetal hydrops

Chen MY, Yang SJ, Hung CC. Emerg Infect Dis. 2011 Oct;17(10):1954-6.

Picornaviridae family

Very wide range of symptoms and transmission strategies

(e.g. poliovirus versus rhinoviruses)

2008 only four genera known to infect humans (Enterovirus,

Hepatovirus, Parechovirus, Kobuvirus).

Infects humans

Recent increase of Picornaviridae

genera infecting humans

Salivirus

Frequency of cosavirus and enterovirus detection by RT -nPCR

% RT -PCR positive Cosavirus Enterovirus

Acute flacc id paralys is 49% 76%

Healthy controls 44% 61%

Prevalence of cosavirus and enterovirus in

feces of Pakistani children

Cosaviruses may be second most common

human enteric viral infection.

Genetic diversity (5 species and >30

serotypes) complicates disease

association studies

!

Multiple astroviruses clades

in extensively sampled

mammals

Viral sequences in human plasma pools with Roche 454

28543

3562

1000

1783

6107

183

19

8

3

6

8393

296

2972

0

0

13 x B19V (12079 reads)

1 x PARV4 (3 reads)

1 x Human papillomavirus 27 (1 read)

Recent analysis with Illumina HiSeq also showed:

WU polyomavirus, HERV, lab and extraction column

contaminants.

Many new “orphan” viruses being detected

using metagenomics.

Close homologues of human viruses found in

animals.

Very high rate of viral co-infections in healthy

young humans and animals.

“New” human orphan viruses since 2005

• Parvoviruses:

– B19 erythrovirus (3 genotypes)

– PARV4 (3 genotypes)

– Bocavirus-1/2/3/4 (2 species)

– Bufavirus-1/2 (2 species)

• Picornaviruses:

– Enterovirus/Hepatovirus/Parechovirus/Kobuvirus (5 genera with countless serotypes)

– Cosaviruses-A/B/C/D/E (5 species >33 genotypes)

– Salivirus/Klassevirus (1 species)

– Cardioviruses (1 species ~8 genotypes)

• Astroviruses:

– Human astrovirus (8 genotypes)

– Human astroviruses MLB-1/2/3 (3 genotypes)

– Human astroviruses VA1/2/3/4 (4 genotypes)

• Polyomaviruses:

– BKV/JC -KIPyV -WUPyV

– MCPyV -HPyV6 -HPyV7

– TSPyV -HPyV9 -HMWPyV

DIAGNOSTICS

Target anti-viral

Detect minority drug resistance mutations (HIV/HCV/HBV)

Avoid unneeded antibiotic therapies

Understand transmission networks

BIOLOGICS QUALITY CONTROL

Serum, cell supernatants, mAb.

VETERINARY MEDICINE

Diagnostics

Vaccine

ENVIRONMENTAL QC

Agricultural products (plants-animals-etc)

Sewage

Practical uses for virus NGS (beside virus discovery)

Sequence what?

-Everything (total DNA and RNA of host and virus)

-Enrich viral NA by filtration and nuclease resistance

-Enrich known pathogens NA with micro-array or beads

Issues in NGS for virus detection

Quality controls

-Reduce nucleic acid contamination

-Sensitivity using standards (mixed /ss/ds/circular

DNA/RNA viruses)

-Effect of sample type

(blood/respiratory/feces/biopsies)

-Best method for unbiased amplification.

Bioinformatics

Speed

De novo assembly?

Curated viral database

Report only known viruses? (what %

nucleotide/protein similarity criteria?)

Issues in NGS for virus detection

Blood Systems Research Institute

and UCSF:

Linlin Li

Tung Phan

Lark Coffey

Terry Ng

Xutao Feng

Beatrix Kapuszinsky (Stanford)

Tongling Shan (CAS Shanghai)

Amit Kapoor (Columbia U)

Joe Victoria (Boehringer-Ingelheim)

Morris Jones (USDA)

MANY MANY other collaborators for human, animal and environmental samples

Stanford University

Chunlin Wang

University of Edinburgh

Peter Simmonds

Paul Ehrlich Institut

Sally Baylis

WHO poliovirus eradication

Sohail Zaidi

University of Helsinki

Maria Söderlund-Venermo

NHLBI, USAIDS, and BSRI funding

!

High sero-prevalence Bocavirus sero-reactivity

in European children and adults

HBoV1 HBoV2 HBoV3 HBoV4

Children: 26% 25% 11% 3%

Adults: 59% 34% 15% 2%

Kantola K,et al. J Infect Dis. 2011 Nov;204(9):1403-12.