1. • Sampling Gizzard shad (Dorosoma cepedianum) from
Sandusky Bay, Lake Erie
• Tissue harvesting for virological examination
• Culturing assays on Epithelioma papulosum
cyprini (EPC)
• If no CPE after 4-7 days, cells were subcultivated &
observed for further 7 days
• If CPE occurred, VHSv was detected verified by RT-qPCR
• Virus isolation/RNA extraction
• Reverse Transcription into cDNA
• RT-qPCR (SYBR Green)- detection of virus
• Sulforhodamine B colorimetric (SRB) assay for
cytotoxicity screening & cell density
OVERVIEW METHODOLOGY
This study analyzes the occurrence and cellular immune
responses of fishes to Viral Hemorrhagic Septicemia
virus (VHSv). Samples were collected during the spring
of 2016 from several locations in Lake Erie and tested at
the Lake Erie Center (LEC) for the presence of VHSv.
Positive samples were then cultured and examined for
cytopathic effects (CPE) as compared with the VHSv-IVb
isolate (MI03GL) from 2003.
RESEARCH QUESTIONS
1. Is VHSv still present in Lake Erie in 2016?
2. What is level of CPE of the 2016 samples as
compared to the reference VHSv-IVb Great Lakes
isolate?
BACKGROUND
VHSv is a viral fish pathogen found to infect >80 species
across the globe. VHSv-IVb was first reported in the
Great Lakes in 2003, subsequently infecting over 30
species. The genome of VHSv is comprised of single
stranded RNA & replicates entirely in the cytoplasm.
Symptoms of VHSv may include erratic swimming,
bloated abdomen, and external and internal petechial
hemorrhaging. Viral infection mostly occurs during the
spring spawning season & cooler water temperatures.
No detection of the virus had been reported since 2012
in Lake Erie, when two asymptomatic fishes were tested
positive. Furthermore, no further diagnosis of CPE have
been conducted with newer isolates.
CONCLUSIONS
• VHSv-IVb is still present in Lake Erie
• Positives detected in Sandusky Bay
• Fishes were reported as asymptomatic
• VHSv-IVb isolates from 2016 (B9, B13, B18) showed
cytopathic effects
• Cultures infected with the 2016 isolates showed
lower cell death than WT
• 2015 samples failed to grow in culture
• May have deteriorated over time
RESULTS
Kwadwo A. Ofori1,2,4, Megan Niner1,3, Douglas Leaman2, Carol Stepien1,3
1Lake Erie Center, 2Department of Biological Sciences, and 3Department of Environmental Sciences, University of Toledo
4Cheyney University of Pennsylvania
ACKNOWLEDGEMENTS
Funding provided by the NSF REU program #DBI-1461124 to the University of Toledo’s
Lake Erie center, “Undergraduate Research and Mentoring- Using the Lake Erie Sensor
Network to study Land Lake Ecological Linkages”.
The authors would like to thank Dr. Czajkowski, Shelby Edwards, Rachel Lohner, and
Matthew Snyder.
Beach seining for fishes in Maumee Bay of Lake Erie in June of 2016 Gizzard shad displaying typical VHSv symptoms (external hemorrhaging)
during a 2006 outbreak.
Harvesting fish tissues
CELLULAR RESPONSE TO 2016 ISOLATES OF VIRAL HEMORRHAGIC SEPTICEMIA VIRUS (VHSv)
SRB assay results showing CPE levels from three 2016 isolates (B9,
B13, & B18) compared to the original wild type (WT) isolate. Of the
2016 isolates, B13 showed the highest level of cytopathic effect,
followed by B18. B9 had the lowest cytopathic effect on the cell
culture, and all displayed a lower level of cell death as compared
to the WT control isolate.
Not displayed: 2015 H31 & H6 which did not show CPE.
Structure of VHS virus adapted from Stepien et al. 2015. PLoS ONE.
Large plaque formationSmall plaque formations
CPE effect was evaluated by the visual detection of plaque
formation in cell cultures
SYBR Green assay result showing positive results for sample B18
with positive (VHS high & low) & negative controls (actin &
water), run in triplicate.
Two positive isolates had been detected in 2015, but not analyzed
and a total of 19 VHSv-positive fishes were detected in 2016 from
Lake Erie.
B18
Low VHS
High VHS
Water Control
Actin Control
VHSv-IVb Detection
0.0000001
0.000001
0.00001
0.0001
0.001
0.01
0.1
1
WT B9 B13 B18
IC50(M.O.I)
Isolate Designation