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IV SIMPOSIO DE SANIDAD E INOCUIDAD CESAIBC Ponencias 19 de Septiembre
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Herpesviruses of MolluscsCarolyn Friedman
• Herpesvirus infections have been detected in many molluscspecies 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
Global distribution of OsHV
OsHV infections
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
– Sequence comparisons showed that OsHV-1 is tenuously related to vertebrate herpesviruses
From: Davison et al. 2005
OsHV-1
Herpesvirus evolutionary tree
TERRESTRIAL
VERTEBRATES
FISH AND
AMPHIBIANS OYSTERS
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
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
0102030405060708090
100
5/17-
6/3
6/4-
6/16
6/17-
7/1
7/2-
7/15
7/16-
7/29
7/30
-8/12
8/13-
8/27
8/28-
9/9
Cum.
0
5
10
15
20
25
30Mortality Max Mean Dection of OsHV
OsHV Transmission in Tomales Bay
OsHV Transmission in larvae
0
10
20
30
40
50
60
70
80
90
100
1 3 5 7 9
Experiment Day
Perc
en
t S
urv
ival
OsHV Diagnostic Methods
• 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
response around infected cells
– Changes often described in
larvae but not juvenile or adult
oysters
4d4d
4b4b
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).
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
OsHV Diagnostic Methods 4
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
TRL IRL IRS TRSUL US
X
C CA BGP
A)
B)
TRL IRL IRS TRSUL US
XTRL IRL IRS TRS
UL USX
C CA BGP
C CA B
C CA BGP
A)
B)
Global distribution of OsHV
OsHV infections PCR as diagnostic tool
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
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
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
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?
Specific questions
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.
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
.
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.
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
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
Gross observations of ALVDThe first abalone farm infected with herpes-like virus
•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
Tank water was turbid and bubbly.
Clinical signs: Holiotis diversicolor
supertexta
Healthy vs. moribund abalone
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
AHLVD: Gross Signs Australia
AHLVD in Australia
Infected farm (above) and healthy farm (right)
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
AHLVD: Histology 2(Australia + Taiwan)
Normal vs GNV Nerves
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)
AHLVD: TEM 2
Taiwan
Australia
AHLV: experimental transmission
Cumulative mortality in abalone
0
20
40
60
80
100
1 2 3 4 5 6
Days post-exposure to virus
% m
ort
ality
Co-habitation
Fresh virus injected
Frozen virus injected
DMEM injected
Untreated
AHLV: experimental transmission 2
Cumulative mortality in abalone exposed to virus
infected water
0
20
40
60
80
100
1 3 5 7 9 11 13 15 17 19 21 23
Days post-exposure
% m
ort
ali
ty
Co-habitation
100% water
10% water
1% water
0.01% water
0.001% water
Untreated control
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
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?