11/29/2016

1

Laura Bergner

Richard Orton, Daniel Streicker

Bats and viruses

• Reservoir of many emerging infectious diseases

– Influenza, rabies, SARS

• Host over 200 viruses in 27 viral families

Luis et al. 2015

11/29/2016

2

Viral diversity in bats

• What viruses are present in bats?

• How is the diversity of bat viruses distributed

geographically and across species?

• What are the ecological and anthropogenic factors

influencing viral diversity in bats?

Studying viruses in bats

• Methods for detecting viruses

– Culture

– Serology

– PCR (polymerase chain reaction)

• Only for specific taxa that have

been previously characterized

11/29/2016

3

Genomic techniques for studying viral

diversity

• Metagenomics

– Sequence-based characterization of taxa in an

environmental or host sample

– “Virome”

• Illumina sequencing

– Massively parallel – millions of sequences per run

– Shotgun sequencing

Genomic techniques for studying viral

diversity

• Advantages

– Viral discovery

– Less biased than other methods

– Possible data on diet, parasites, bacteria, host

genetics?

• Disadvantages

– Many sequences thrown away

– Dark matter

11/29/2016

4

Bat viromes

• Insectivorous bats – mostly dietary plant and

insect viruses

• Frugivorous bats – new mammalian viruses

Donaldson et al. 2010 Baker et al. 2013

Bat viromes

• 40 bat species from different ecological

regions across China

Wu et al. 2016

11/29/2016

5

Why are vampire bats (Desmodus

rotundus) interesting?

Wray et al. 2016

Objectives and expectations

1. Characterize viral diversity in D. rotundus using

metagenomics

2. Investigate ecological and anthropogenic factors influencing

viral diversity in D. rotundus

• Known viruses in D. rotundus: rabies virus, paramyxovirus,

coronavirus, polyomavirus, adenovirus

• Known in other Neotropical bat species: influenza virus

11/29/2016

6

Study sites

−16

−12

−8

−4

−80 −76 −72 −68

Department

Amazonas

Apurimac

Ayacucho

Cajamarca

Cusco

Huanuco

Lima

Loreto

Sampling D. rotundus

• Trap bats at known colonies

• Saliva, fecal, whole blood swabs from up

to 30 bats per site

• Urine swabs from individuals as possible

• Pooled urine samples from some sites

11/29/2016

7

Laboratory pipeline

Illumina sequence data

11/29/2016

8

Viral metagenomic

raw reads

Remove vampire

bat reads

Remove other eukaryote

and prokaryote reads

Remove low quality, low

complexity, duplicate reads

Cleaned reads

Bioinformatic pipeline

Cleaning reads - NextSeq

Step

Raw reads

Trim galore

Prinseq

Vampire bat

PhiX

Ribopicker

Cleaned reads

2.0e+07

1.5e+07

1.0e+07

5.0e+06

0

Re

ad

s

H_EN_RL H_EN_VTM H_UN_VTM SV_EN_RL SV_EN_VTM SV_UN_RL

11/29/2016

9

Cleaning reads - MiSeq

4e+06

3e+06

2e+06

1e+06

0

Re

ad

s

H1 H2 H3 H4 SG1 SV1 SV2 SV3

Viral metagenomic

raw reads

Classify

reads/contigs

Assembly

Remove vampire

bat reads

Remove other eukaryote

and prokaryote reads

Predict ORFs

Remove low quality, low

complexity, duplicate reads

Cleaned reads

Bioinformatic pipeline

Visualize taxa Compare samples

H_EN_VTM

H_EN_RL

H_UN_VTM

SV_EN_RL

SV_EN_VTM

SV_UN_RLPodoviridae

Siphovir idae

Adenoviridae

PCoA of Taxonomy us ing Hellinger : PC 1 (66.3%) vs PC 2 (18.8%)

-1-2-3-4-5-6 PC1 1 2 3 4 5 6

-0.5

-1

PC2

0.5

1

11/29/2016

10

Total sequencing reads

Viruses 0.3 %

Diet?

Parasites?

Microbiome?

Pilot run questions

• In what buffer should we collect metagenomic

samples in the field?

(VTM or RNALater?)

• Will VTM work for vampire bat samples?

– Fetal bovine serum (FBS) may contain cow viruses

• Can we extract samples to enrich for viruses

relative to other taxa?

• What viruses do we find?

11/29/2016

11

Pilot run design

Sample type

Fecal swab

Saliva swab

VTM RNALater

Unenriched

H_UN_VTM

SV_UN_VTM

H_UN_RL

SV_UN_RL

EnrichedEnriched

H_EN_VTM

SV_EN_VTM

H_EN_RL

SV_EN_RL

Same sample, reads vs contigs

Reads Contigs

11/29/2016

12

Fecal swabs – same individuals,

different storage medium

VTM RNALater

Diverse viruses in FBS

Bovine viral diarrhea virus – a common cell culture contaminant

11/29/2016

13

Viruses inconsistent between sample

types, buffer, extraction method

Bacteriophages

Sample (non)clustering based on viral

diversity-1

-0.5

PC2

0.5

1

-6 -5 -4 -3 -2 -1 PC1 1 2 3 4 5

6

11/29/2016

14

Sample type clustering based on viral

diversity-0.02

-0.01

PC2

0.01

0.02

-0.5 PC1 0.5 1

A few viruses in Peruvian vampire bats

(so far…)

• Bat hepevirus – 2 sites

• Circovirus – 2 sites

• Bat mastadenovirus

• Bat adeno-associated virus

• Bat anellovirus

• Herpesvirus – all sites, saliva

11/29/2016

15

Summary

• Metagenomic sequencing method works but

inconsistent viruses detected between buffers

and enrichment methods

• Bacteriophages predominate

• Possibly due to pooled samples and low viral

load

• FBS viruses are present but consistent, so we

can exclude them from VTM samples

Future plans

• Exclude bacteriophages

• More optimization needed for

extraction/pooling

• Replicates

• Process samples by colony for larger set of

sites across Peru

11/29/2016

16

Thank you!Peru

Carlos Tello

Jorge Carrera

Anthony Almeyda

Francisco Espinosa

Dan Becker

Glasgow

Roman Biek

Julio Benavides

John Claxton

Chris Davis

Gavin Wilkie

Ana Filipe

Kath Allan

William Marciel

Alice Broos

Pablo Murcia

Diverse viruses in FBS

BVDV

11/29/2016

17

Adenoviridae

Myoviridae

Podoviridae

Siphoviridae

unclassified Caudovirales

Caudovirales

Herpesviridae

Papillomaviridae

unclassified dsDNA phages

unclassified dsDNA viruses

dsDNA viruses, no RNA stage

Retroviridae

Anelloviridae

Circoviridae

Parvoviridae

ssDNA viruses

Flaviviridae

Picornaviridae

unclassified Picornavirales

Picornavirales

Tombusviridae

ssRNA positive-strand viruses, no DNA stage

Viruses

root

Legend:

H1 H2 H3 H4 SG1 SV1 SV2 SV3

Viral families in similarly treated

samples