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

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OsHV Transmission in Tomales Bay

OsHV Transmission in larvae

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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

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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

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Co-habitation

Fresh virus injected

Frozen virus injected

DMEM injected

Untreated

AHLV: experimental transmission 2

Cumulative mortality in abalone exposed to virus

infected water

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100% water

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1% water

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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?