Next generation sequencing:

Application in research and diagnostics in

veterinary virology

Science&BBQ day 05.09.2018

Claudia Bachofen

Institute of Virology, Vetsuisse faculty, University of Zürich

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• Introduction

• NGS in the veterinary field

• NGS in veterinary virology

• Examples for applications

• Outlook

Next generation sequencing:

Application in research and diagnostics in

veterinary virology

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Introduction

Butanol extraction of pig faeces → negative contrast EM

©Elisabeth Schraner

“Virome refers to the collection of nucleic acids, both RNA and DNA, that make up the viral community

associated with a particular ecosystem”

3Which viruses are present??

Next Generation Sequencing (NGS)

Classical methods (Shotgun cloning, Pan-PCRs,

multi-virus arrays)

• Targeted / semi-targeted

• Limited amount of information

• Selective

• Untargeted

• +/- Unlimited amount of information

• +/- Non-selective

Introduction

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www.genome.gov/sequencingcostsdata.

NGS entersthe market

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NGS in the veterinary field

40% of Swiss veterinary reference labs use NGS 6

▪ High species and sample diversity

▪ Costs…

→ Flexibility in sample preparation and analysis→ For many species not much known about virome in healthy animals

→ Cost-efficient sequencing approach→ Pooling of samples→ Collective sample of group of animals

Advantages:

▪ Identification of all viruses in one test

▪ Fast characterisation of viruses without previous knowledge of the genome.

▪ Analysis of viral diversity within an animal

Challenges:

NGS in veterinary virology

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Relative enrichment for virus particlesDNase

RNase

Sample preparation

NGS in veterinary virology

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→ DVM thesis Jakub Kubacki

Illumina NextSeqbarcoded sequencing

Sample preparation

NGS in veterinary virology

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Virus surveillance

Virus characterisation

Virus identification

NGS for vet virology - applications

Outbreak investigation

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Virus identification

NGS for vet virology - applications

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Which viruses (known/unknown) are present?

Examples:▪ Porcine kobuvirus in diarrheic piglets▪ A novel gemycircularvirus in Swiss waterbuffaloes▪ Torque-Teno virus in a pig with neurological signs

Virus identification

Example: Four months old pig with neurological symptoms

Histopathological changes:

• Brain: Mulitfocal lymphocytes infiltration in grey and white matter

• Spinal cord: Demyelinisation and axonal Wallerian degeneration

Suspicion: Polioencephalomyelitis enzootica suum → Teschen-Talfan disease,

porcine Teschovirus-1 (Enterovirus, Picornaviridae)

No specific diagnostic performed in Switzerland – then!

???

? ?

?

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Result: Torque teno sus virus 1 (TTV1)

Family: Anelloviridae

Circular ssDNA genome, ~3.8 kb, TTV-1 and -2, high diversity

Clinical meaning??

Example: Four months old pig with neurological symptoms

Virus identification

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→ No correlation of TTV-1 prevalence to neurological - but to respiratory signs

Example: Four months old pig with neurological symptoms

Virus identification

→Master project Deborah Peltzer

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Neuro pos Neuro negOverall

→ 42% virus prevalence

*

Example: Four months old pig with neurological symptoms

Virus identification

→Master project Deborah Peltzer

15→ Correlation to age: More virus with increasing age

NGS for vet virology - applications

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Details about identified viruses:Full-length genome sequence, quasispecies diversity

Examples:▪ Influence of cell-culture passaging on BVDV quasispecies diversity▪ Full-genome sequence of a feline calicivirus field strain▪ Full-genome sequence of papillomaviruses from Okapi and Boa

Virus characterisation

Virus characterisation

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Example: Novel Papillomavirus from an Okapi, OjPV-1

- Okapi (Okapia johnstoni) with wart-like skin lesions- Papillomavirus expected (RCA positive)- NGS → de novo assembly- Most closely related (65%) to giraffe papilloma virus

(GcPV-1)

Virus characterisation

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Example: Novel Papillomavirus from an Okapi, OjPV-1

NGS for vet virology - applications

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Identifying viruses in outbreak situations:Known/unknown viruses, molecular tracing

Examples:▪ Porcine bocavirus in organs of poor-doing farrowing sows ▪ Intrahepatic icterus in 2 herds caused by porcine circovirus type 2▪ Hepatitis E virus in Ticino

Outbreak investigation

• 78-y old male, hospitalised in Lugano: Extreme fatigue, severe jaundice and dark urine, recovery after 4 daysStool HEV RT-PCR positive

• Mortadella HEV RT-PCR positive

Example: Molecular tracing of Hepatitis E virus

Outbreak investigation

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• 78-y old male, hospitalised in Lugano: Extreme fatigue, severe jaundice and dark urine, recovery after 4 daysStool HEV positive

• Mortadella HEV RT-PCR positive

→ Sequencing of both using next generation sequencing

Example: Molecular tracing of Hepatitis E virus

Outbreak investigation

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Example: Molecular tracing of Hepatitis E virus

Outbreak investigation

→ The virome of a pork sausage:

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→ Switzerland has its own HEV-3 subgroup: 3s

Example: Molecular tracing of Hepatitis E virus

Outbreak investigation

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NGS for vet virology - applications

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Monitoring the viruses present in a group of animals

Virus surveillance

Virus surveillance

Example: Suitability of chewing rope fluid and collective faeces

→ 2 bays, 17 animals

Collective samples: faeces from floor, chewing rope fluid

Individual animals: faeces, nasal swabs, bronchial lavage, blood

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Viruses detected Faeces Nasal swab BAL Serum Chewing rope Feaces floor

Adeno-associated viruses 13716 25 0 95 256 933

Astrovirus 55018 4 69 317 958 2080

Bocavirus 62614 100 16 54 13566 8493

Enterovirus 6666 0 0 0 - 1884

Influenzavirus 0 0 6 2 - -

Kobuvirus 3922 0 0 0 87 932

Rotavirus C 7 0 0 4 1 9

Sapelovirus 20767 0 7 3 - 685

Sapovirus 409 0 2 0 3 23

stool-associated circular viruses 82974 17 16 13 461 5179

Teschovirus 21 0 0 0 2 -

Torque Tenovirus 0 0 0 1630 - -

Circo-like virus 0 0 23 24 520 -

Feaces-associated gemycirvularvirus 8 5 6 11 129 -

Porcine Cytomegalovirus 0 2 0 114 7857 -

Total number of reads 246122 153 145 2267 23840 20218

Virus surveillance

✓ NGS has worked; combination of chewing rope and faeces ideal✓ Viruses only present in serum and low viral load in BAL are missed

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Example: Suitability of chewing rope fluid and collective faeces

Outlook - research

Intervirome interactions:Virus infection dynamics in newly mixed cow herd

→ DVM thesis Karin Meier

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Intravirome interactions:Commensal viruses in health and disease

→ DVM thesis Charlotte Rickli

Outlook - diagnostics

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Acknowledgments

Mathias AckermannCornel Fraefel

Xaver SidlerRoger Stephan

Titus Sydler, Giulia Rosato

Jakub KubackiKarin MeierCharlotte RickliIsabelle Specker

Julia LechmannMarco SteckMartina Buchser

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