Insel Riems Jena

Celle Mariensee Braunschweig

Friedrich-Loeffler-Institut

Federal Research Institute for Animal Health

Martin Beer and Thomas C. Mettenleiter

Examples of use of next generation/

whole genome sequencing:

1. Syndromic Surveillance (field veterinarians)

Since August 2011 in North Rhine-Westphalia cases of

drastic milk drop and fever reported in cattle;

symptoms in the Netherlands dominated by diarrhea in cattle

All tests for „classical“ viral infections were negative:

BTV, EHDV, FMDV, BHV-1, MKFV, BVDV, RVFV, BEFV

Metagenomic analysis of 3 pooled blood samples

from diseased dairy cows from Schmallenberg

Discovery of Schmallenberg Virus

What is Metagenomics?

Metagenomics is the application of modern

genomics techniques to the study of

communities of microbial organisms directly

in their natural environments, bypassing

the need for isolation and lab cultivation

of individual species.

Chen K, Pachter L (2005). PLoS Comput Biol 1(2): e24. doi:10.1371/journal.pcbi.0010024

1. Syndromic Surveillance (field veterinarians)

2. Sampling

Host

Metagenomic analysis – Next generation sequencing

Microbiom Host

Virus

Sample Choice

- Body fluid/tissue material?

- Preferably the matrix with the lowest quantity of host nucleic acids (NA)

- The highest possible quantity of pathogen NA

Example

•Cattle genome: 2.97 Gbp

•PCV2 genome: 1768 b

→ mass ratio pCV2:Cattle = 1:1,679,864

cow 441; 213 days in milk (farm 1)

7th September

bo

dy w

eig

ht

milk y

ield

45 kg

22 kg

14 days weight gain

1. Syndromic Surveillance (field veterinarians)

2. Sampling

3. Sequencing

Different technologies

(up to 3 billion reads per run!)

Next generation sequencing (NGS)

- Read: short fragments (50 to 800 bp)

- Contig: larger sequence pieces assembled from reads

1. Syndromic Surveillance (field veterinarians)

2. Sampling

3. Sequencing

4. Sequence Analysis

“Work flow” Prozess

Data-Analysis

RIEMS: Rapid Information Extraction from Metagenomic Sequence Datasets

A novel Orthobunyavirus-Infection in German cattle

-------------------------------------------------- Since August 2011 farmers and veterinarians in North Rhine-Westphalia

(Germany) and The Netherlands have reported clinical disease in cattle and suspected a new introduction of Bluetongue disease. The main clinical signs were fever and a significant drop of milk yield for

several days, in some cases also diarrhea and abortions. Samples were submitted to the German national reference laboratory for bluetongue

disease at the Friedrich-Loeffler-Institut, Insel Riems. Diagnostic analyses excluded BTV, FMDV, BVDV, BHV-1, MCFV or exotic

viruses like EHDV, Rift Valley fever virus or bovine ephemeral fever virus.

Therefore, 3 pooled samples from a farm with acute signs of the disease (fever greater than 40 C and milk drop of less than 50

percent) were investigated using metagenomic analysis with a Genome Sequencer FLX instrument (ROCHE). The analysis yielded 6 sequence

fragments with homology to the L and the M segments of viruses from the genus Orthobunyavirus. Further metagenomic analyses led to the detection of sequences homologous to Orthobunyavirus S segment. The

sequences were related to genomic sequences of Shamonda-, Aino-, and Akabane-virus, viruses which are mainly transmitted by Culicoides

spp.

Promedmail 19.11.2011

1. Syndromic Surveillance (field veterinarians)

2. Sampling

3. Sequencing

5. Diagnostics (PCR)

4. Sequence Analysis

Metagenomics Workflow

Sample !

Next-Generation- Sequencing !

Data analysis!

Confirmation by

qPCR

- 12 positive samples (PCR)

- 6 cattle farms

- 11 adult cattle

- 1 stillborn twin calf

- All in North Rhine-Westphalia

1. Syndromic Surveillance (field veterinarians)

2. Sampling

3. Sequencing

5. Diagnostics (PCR)

4. Sequence Analysis

6. Comparative Information

Saeed et al, JGV 2001

Schmallenberg-Virus

Akabane-Virus: Mild primary infection,

Congenital malformations

Arthrogryposis Credit: Dr. P. Mansell, University of Melbourne

Photo ID: AKA_001

Torticollis and Arthrogryposis Credit: Dr. K. Kawashima, National Institute of Animal Health,

Japan

Photo ID: AKA_002

Clinical symptoms and pathogenesis

Arthrogryposis, torticollis, brain

hypoplasia, brachygnathia

inferior, skoliosis

Pictures: Courtesy of Dr. Brügmann,

LVI Oldenburg

1. Syndromic Surveillance (field veterinarians)

2. Sampling

3. Sequencing

5. Diagnostics (PCR)

4. Sequence Analysis

6. Comparative Information

7. Epidemiology

March 1, 2012

Sept 04, 2012 http://empres-i.fao.org/eipws3g/

EMPRES, 27 September 2013

Zoonotic infection? Serological studies in exposed Humans

No evidence of human infection

• SBV-infections detected only in ruminants

so far

– Cattle

– Sheep

– Goats

– Bison

– Roe deer (antibody detection)

– Red deer (antibody detection)

– Mouflons (antibody detection)

– Alpaca (antibody detection)

Aino/Akabane

Shamonda

Sathuperi

Sango

Sabo

Peaton

Oropouche

Douglas Simbu

Shuni

Schmallenberg

Akabane

First Ad Hoc Group meeting on February 09, 2012

1. Syndromic Surveillance (field veterinarians)

2. Sampling

3. Sequencing

5. Diagnostics (PCR)

4. Sequence Analysis

6. Comparative Information

7. Epidemiology

8. Research

Schmallenberg Virus

- Genome with 3 Segments (S, M, L), negative-stranded RNA

- Transmission by insect vectors (Culicoides spp.)

- Simbu serogroup : mild primary infection, but congenital disorders!

Serum samples

days post infection

0 2 4 6 8 10 12 14

co

pie

s/µ

l

1e+0

1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

R10

R11

R12

R13

R14

oral

contact

re-infected

4-5 days

Short viremia of less than 6 days in naive cattle

No viremia in re-infected or oronasally inoculated or contact cattle

No infection in contact animals detected

Experimental infections and immunity

R10 bis 14: s.c. infection

2 animal orally inoculated

2 animals re-infected

3 Contact animals

Pregnancy in cattle: 9 months

Pregnancy in sheep: 5 months

Zusammenfassung und Ausblick

Orthobunyavirus of Simbu Serogroup detected in Europe

for the first time

Infects domestic and wild ruminants

Acute infections mild, characteristic congenital malformations

Virus isolated

Virus detected in insects (midges)

Animal experiments: viremia 2-5d, non-contagious,

infection elicits protective immunity

No human infection

Vaccines available

Summary

M. Holsteg – LWK NRW

R. Jungblut – UA Arnsberg

M. Brügmann - LVI Oldenburg

Christian Korthase

Moctezuma Reimann, Bianca Hillmann

Sven Sander, Karin Lissek

Patrik Zitzow

Peter Kirkland, Elizabeth Macarthur Agricultural

Institute, Australia

Robert Tesh, UTMB, Galveston, USA

Wim van der Poel, CVI, Netherlands

Brigitte Cay , CODA-CERVA, Belgium

Stephan Zientara, ANSES, France

Marion Koopmans, RIVM, Netherlands

Emiliana Brochi, IZSLER, Italy

Boehringer

Ingelheim

Thank you to all colleagues who participated in this

transparent and friendly international cooperation

Thank you for your attention!

The basic definition of metagenomics is the

analysis of genomic DNA from a whole

community.

Gilbert JA, Dupont CL (2011). Ann Rev Mar Sci 3: 347-71. 10.1146/annurev-marine-120709-142811

What is Metagenomics (1)?

Why Do Metagenomic Pathogen Detection (1)?

Because every causative agent of infectious disease relies on nucleic acids (NAs) both for its genome and gene expression (only known exception so far are transmissible spongiform encephalopathies)

AND

Modern shotgun sequencing methods detect all NAs in a given sample with ± equal probability

Deformation of the vertebral

column, torticollis, brachygnathia

inferior in a calf Pictures: Courtesy of Dr. Martin Peters, SVUA

Arnsberg, Germany

Hydranencephaly

and cerebellar hypoplasia

Example 2

- sequencing RNA isolated from serum from

BVDV persistently infected cattle

- 5% viral reads

Sample Choice

Sample Choice Example 1

-Fish are dying for unknown reason

-Sample from metabolically highly active gill tissue

-DNA virus expected

-Sequencing library from RNA

-229,000 sequencing reads

-2 reads RNA virus (0.00087%)