IV SIMPOSIO DE SANIDAD E INOCUIDAD CESAIBC Ponencias 19 de Septiembre

1. Herpesviruses of Molluscs
Carolyn Friedman
• Herpesvirus infections have been detected in many mollusc
species in association with mortality outbreaks, including
Crassostrea virginica
C. gigas
Ostrea edulis
O. angasi
O. chilensis
Ruditapes decussatus
R. phillipinarum
Pecten maximus
Haliotis diversicolor supertexa
Haliotis laevigata
Haliotis rubra

2. Global distribution of OsHV
OsHV infections

3. OsHV-1
OsHV-1
• Icosahedral DNA virus, replicates in nucleus
and migrates to cytoplasm (enveloped by
nuclear membrane)
• OsHV-1 in France has been characterized
– Virions have been
purified, described, and sequenced
• The genome is 207 kb
From: Davison et al. 2005
– Sequence comparisons showed that
OsHV-1 is tenuously related to vertebrate
herpesviruses

4. Herpesvirus evolutionary tree
FISH AND TERRESTRIAL
AMPHIBIANS VERTEBRATES OYSTERS

5. Oyster herpesviruses (OsHV)
• Mortalities are typically short in duration and can
reach up to 90% (in larvae) and 40-80% (in seed)
– Mortalities particularly affect small and/or fast growing
seed oysters
• Virus also detected in multiple adult species where
mortality not recorded
• Associated with warm water temperatures and high
densities of animals
– 24-25 C needed for virion replication based on lab
experiments in larvae
– 24-25 C also associated with field mortalities of seed
oysters

6. Possible modes of transmission
• In hatcheries, vertical transmission has been
suggested
• In the field, at least in Tomales Bay, uninfected
seed oysters are outplanted each year
– OsHV has not been detected in any hatcheries or
nurseries to date in the US
– Adult bivalves in the bay may have latent infections
• In lab – cannot transmit to stages older than
larvae

7. OsHV Transmission in Tomales Bay
Mortality Max Mean Dection of OsHV
100 30
90
80 25
70 20
60
50 15
40
30 10
20 5
10
0 0
5/17- 6/4- 6/17- 7/2- 7/16- 7/30 8/13- 8/28- Cum.
6/3 6/16 7/1 7/15 7/29 -8/12 8/27 9/9

8. OsHV Transmission in larvae
100
90
80
70
Percent Survival
60
50
40
30
20
10
0
1 3 5 7 9
Experiment Day

9. OsHV Diagnostic Methods
4d
• Light microscopy
– Nuclear hypertrophy and
chromatin margination
• Cells of the gills, mantle, and velum
(not epithelial cells)
• Signs of viral-induced apoptosis
– Slight or no inflammatory 4b
response around infected cells
– Changes often described in
larvae but not juvenile or adult
oysters

10. OsHV Diagnostic Methods 2
• In situ hybridization
– Section through the
visceral ganglion.
– Labelled cells
(arrowheads) and non-
labelled cells (arrows).
– The DIG-labelled probe
reacts strongly within
the cytoplasm and the
nucleus of nervous cells
(bar=10 um).

11. OsHV Diagnostic Methods 3
• Transmission Electron
Microscopy (TEM)
– Presence of spherical to
polygonal unenveloped
particles ~80 nm in
diameter in nucleus of
infected larvae and spat
– Enveloped virions ~122
nm in cytoplasmic
vesicles, perinuclear
space & extrcellularly

12. OsHV Diagnostic Methods 4

13. OsHV Diagnosis Methods 5
• Multiple conventional and QPCR primer sets have
been designed to amplify regions of the OsHV-1
genome
• PCR allows for both diagnosis of OsHV and the
comparison of possible OsHV variants
A)
UL X US
TRL IRL IRS TRS
B)
C C
A GP B

14. Global distribution of OsHV
OsHV infections PCR as diagnostic tool

15. OsHV research in our lab
• Improving survival of seed oysters in the field
• OsHV transmission in larval and seed oysters
– may include the addition of Vibrio tubiashii in transmission
experiments
• Comparison of global OsHV variants
• Testing histology blocks from early Tomales Bay
mortalities as well as imports into TB from other parts
of the world
• Developed 2 qPCR assays
– One DNA-based and one RNA-based

16. OsHV µ-var 1
• In 2008- high mortality rates of 80% to 100% in
Crassostrea gigas
– Mainly juvenile oysters from May to September
• 75% positive batches for OsHV-1
– ?new biotype of OsHV-1?
• Extracts of field affected oysters  induced
mortalities (80% IM, 40% cohabitation) in spat and
juvenile oysters
– qPCR and TEM confirmed viral infection
• 0.1μm filtration or UV inactivated OsHV µvar

17. OsHV µ-var
• “Both biotypes identified in isolates, OsHV1 and
OsHV1 μVar*, were virulent and generated
mortality with the oyster stages used”
• “the first time that such results trial were obtained”
• “Analysis of various target sequences within viral
genome present in infected batches demonstrated
the presence of polymorphism”  OsHV1 μVar
and patented finding
• From Sauvage et al. 2009

18. Specific questions
Could you tell me what do you do when detect a positive
sample to OsHV-1?
Do you have an estimation about the losses when an
episode of OsHV-1 occurs?
Is there any governmental regulation to avoid dispersion of
OsHV-1?
Do you have a surveillance program for OsHV-1 in oyster
farms or environment?
If yes, how is it?

19. If we detect a new positive sample (ie in a new
location), we repeat our analysis, add sequence
analysis and in situ hybridization to confirm infection
The losses due to a herpes outbreak in Tomales Bay
can certainly be greater than 90%, at least in patches.
Regarding regulations, OsHV is not on our list of
diseases that California regulates. It probably will be
added next time we make changes, but that will be
more than a year from now. However, we still can use
more broad, open-ended regulations to restrict
movement of infected product or seed.

20. .
I was hoping to get the OsHV-1 RT PCR assay going in my
lab this summer and we got most of the way there but have
not had time to set up the proper standards yet for
quantification. I hope to be completely setup by next Spring
to conduct regular testing and do some experiments. Also
want to get OsHV uvar testing. So currently there is no
proper surveillance program for OsHV-1 but I expect there
will be by next year. We will examine oysters from the
growing areas listed above as well as from naturalized C.
gigas in numerous southern California harbors and bays.

21. To my knowledge OsHV has only been detected in Tomales
Bay and Drakes Bay. The growers in these areas will not
send seed to other growing areas. Actually, all of the seed
or larvae that enters the state's growing areas- Carlsbad
Lagoon, Santa Barbara, Morro Bay, Tomales Bay, Drakes
Bay and Humboldt Bay- comes either from Taylor in
Washington (or Hawaii), Coast in Washington (or
Hawaii), Whiskey Creek in Oregon, Hawaiian Shellfish in
Hilo, or from Humboldt Bay to the other growing areas

22. Abalone herpesvirus disease
• Known affected species - to date, primarily
observed in
– Taiwan beginning in 2003, detected 2003-2005
• both subspecies of Haliotis diversicolor (aquatilis and supertexta)
– Australia beginning in December 2005/January
2006
• Haliotis laevegata
• H. rubra
• hybrids of H. laevegata x H. rubra
• Losses typically occur when water temperatures are
<22C and often range from 16-19C

23. The first abalone farm infected with herpes-like virus
Gross observations of ALVD
•Tank water turbid and frothy (above) from regurgitated food
particles and mucus in water in Taiwan
•Rapid onset of mortality in tanks, ponds, or wild populations
• No visible change in abalone feeding habits prior to onset

24. Clinical signs: Holiotis diversicolor
supertexta
Tank water was turbid and bubbly.
Healthy vs. moribund abalone

25. AHLVD: Gross observations
• Affected abalone with clinical signs varying from
none to
– Stiff pedal muscle with darkened lateral mantle
– Increased mucus production reported in many
cases
– And may present swollen, prolapsed mouth with
everted radula in some cases (noted in Australian
abalone species)
– Mortalities typically observed within 3 days of onset
of clinical signs, and dead abalone may remain
adhered to substrata
– Losses often complete within 9-14d

26. AHLVD: Gross Signs Australia

27. AHLVD in Australia
Infected farm (above) and healthy farm (right)

28. AHLVD: Histology 1
• Light microscopic observations
– The main pathological change is ganglioneuritis
with lesions prominent in cerebral and pedal
ganglia
– Lesions characterized by nerve tissue necrosis
accompanied by hemocytosis in some nerve
tissue
• In nerves under mucosa of esophagus and intestine
– No Cowdry type A inclusions were observed
– However neuronal cells may contain marginated
chromatin

29. AHLVD: Histology 2
(Australia + Taiwan)

30. Normal vs GNV Nerves

31. AHLVD: TEM 1
• Transmission electron microscopic (TEM)
observations
– Spherical, enveloped virus (~100nm) with
icosahedral (hexagonal) nucleocapid and dense
core
– Naked virions observed in nucleus and particles
with smooth envelope in cytoplasm
– Negative-contrast electron microscopy also reveals
hexagonal particles with single, smooth envelope
(~100nm)

32. AHLVD: TEM 2
Australia
Taiwan

33. AHLV: experimental transmission
Cumulative mortality in abalone
100
80
Co-habitation
% mortality
60 Fresh virus injected
Frozen virus injected
40 DMEM injected
Untreated
20
0
1 2 3 4 5 6
Days post-exposure to virus

34. AHLV: experimental transmission 2
Cumulative mortality in abalone exposed to virus
infected water
100
80 Co-habitation
100% water
% mortality
60 10% water
1% water
40 0.01% water
0.001% water
20 Untreated control
0
1 3 5 7 9 11 13 15 17 19 21 23
Days post-exposure

35. AHLV: Summary
• Rapid onset of mortalities occur with these disease
leading to high levels of mortality
• Transmission experiments indicate virus is highly
pathogenic
• AHLV spread rapidly in both Taiwan and Australia
including human caused (spread in farms and
processing plants) and nature (water movement)
• Molecular methods will help us better understand the
similarities between the virus in Taiwan and Australia
as well as earlier reports in China

36. Treatment and Control of Viruses
• No treatment available
• Strict Farm and processing plant hygiene
(mainly abalone)
• Health examination prior to importation and
quarantine to assess sub-clinical infections
• How do you think this should be done?