Molecular diagnostics have revolutionized disease detection and pathogen characterization. Next-generation sequencing allows for high-throughput analysis of samples and has advanced our understanding of viruses. Deformed wing virus of honeybees exists as a quasispecies with different strains exhibiting varying virulence. Exposure to Varroa mites has increased the prevalence and diversity of DWV strains worldwide over the past few decades, contributing to honeybee population declines. Continued development of molecular techniques will further our knowledge of pathogen evolution and improve disease management.

1. Molecular Diagnostics:
Present and Future
Declan Schroeder
dcschroe@umn.edu
Molecular Virologist
Veterinary Population Medicine
Leman conference
St Paul, 17-18 September 2018

2. 1977 1986 1995 20051998 2007
bp Gbp
THROUGHPUT
20152011
2010

4. Hassan Magboul - YouTube
Yes
Yes
No
No
Can you Do
something
about it ?
Do you
have a
problem?
Then Why
Worry?Sequence !
Then Why Worry

5. Sample Types
Tissue, Tissue Homogenate, Blood/Serum, Feces, surface tissues Swabs, Oral Fluids,
Processing fluids, Food/water/air & environment (abiotic/biotic)
Sequencing
Sanger, Illumina (Novaseq, HiSeq, MiSeq, iSeq, & Miniseq), PacBio & MinION
Decision tree
metagenomics
Nucleic Acid Isolation
DNA RNA
Isolates (pathogen)
genomics
Library PrepPCRBarcoding
Microbiome/ Pathogen discovery
Transcriptomics
(RNAseq)
RT-PCR

6. Genome Assembly
(reference or
de novo)
Comparative Genomics
Bioinformatic Pipelines
(Linux or off-the-shelf)
Decision tree (continued)

7. www.mba.ac.uk

8. Infection Death
(individual/population)
Assymptomatic
Infection Disease free
(individual/population)
Healthy
(individual/population)
The power of molecular diagnostics

9. Wilfert et al. 2016
59% ↓
1947 2005
DWV

10. Infection Death
(individual/population)
Assymptomatic

11. Definition
a virus species is a polythetic class of viruses that
constitute a replicating lineage and occupy a particular
ecological niche from Virus Taxnomy by 8th ICTV Report

12. A quasispecies is a cloud of diverse variants
that are genetically linked through mutation,
interact cooperatively on a functional level,
and collectively contribute to the
characteristics of the population.
Lauring AS, Andino R (2010) Quasispecies Theory and the Behavior of RNA
Viruses. PLoS Pathog 6(7): e1001005. doi:10.1371/journal.ppat.1001005
& master variants

13. Figure 1. RNA viruses exist as a quasispecies.
Lauring AS, Andino R (2010) Quasispecies Theory and the Behavior of RNA Viruses. PLOS Pathogens 6(7): e1001005.
doi:10.1371/journal.ppat.1001005
http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1001005

14. Family Picornaviriales
Genus Iflavirus
Species Deformed Wing Virus Varroa destructor virus 1 Deformed Wing Virus
Strains Deformed wing virus Varroa destructor virus 1
Kakugo virus
Type A Type BVariants Type C

15. *
*
Type B
Type A
Type C
319 Years
181 Years

16. DWV strain diversity
1.E+11
1.E+08
1.E+10
1.E+09
1.E+07
1.E+06
1.E+05
1000
1.E+04
100
NumberofDWVcopiesperbee
*
DWV strain diversity
1.E+11
1.E+08
1.E+10
1.E+09
1.E+07
1.E+06
1.E+05
1000
1.E+04
100
1.E+11
1.E+08
1.E+10
1.E+09
1.E+07
1.E+06
1.E+05
1000
1.E+04
100
NumberofDWVcopiesperbee
*
0 0 0 <1 <2 >3Varroa exposure time (years)
--
Location
Year
DWV prevalence
DWV strain diversity
2009 2009 2009 2009 2010 2009
Kauai Maui Big Is. Big Is. Big Is. Oahu
0 0 0 <1 <2 >3Varroa exposure time (years) 0 0 0 <1 <2 >3Varroa exposure time (years)
--
Location
Year
DWV prevalence
DWV strain diversity
2009 2009 2009 2009 2010 2009
Kauai Maui Big Is. Big Is. Big Is. Oahu
0 0 0 <1 <2 >3Varroa exposure time (years)
--
Location
Year
DWV prevalence
DWV strain diversity
2009 2009 2009 2009 2010 2009
1.E+08
1.E+09
1.E+07
1.E+06
1.E+05
1000
1.E+04
100
NumberofDWVcopiesperbe
*
Kauai Maui Big Is. Big Is. Big Is. Oahu
0 0 0 <1 <2 >3Varroa exposure time (years) 0 0 0 <1 <2 >3Varroa exposure time (years)
--
Location
Year
DWV prevalence
DWV strain diversity
2009 2009 2009 2009 2010 2009
1.E+08
1.E+09
1.E+07
1.E+06
1.E+05
1000
1.E+04
100
1.E+08
1.E+09
1.E+07
1.E+06
1.E+05
1000
1.E+04
100
NumberofDWVcopiesperbe
*
Kauai Maui Big Is. Big Is. Big Is. Oahu
Type A

17. Swindon
Hawaii
No DWV Type C

19. Kevill & Martin

20. Baltimore Classification
BLV
DWV &
PPRSV
EhV &
LdV
SIV

22. Acknowledgements
• Agricultural Research, Education,
Extension and Technology Transfer
Program (AGREETT)
• Minnesota Department of Agriculture