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
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
Herpesvirus evolutionary tree FISH AND TERRESTRIAL AMPHIBIANS VERTEBRATES 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
OsHV Transmission in Tomales Bay Mortality Max Mean Dection of OsHV100 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
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
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 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
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
Specific questionsCould you tell me what do you do when detect a positivesample to OsHV-1?Do you have an estimation about the losses when anepisode of OsHV-1 occurs?Is there any governmental regulation to avoid dispersion ofOsHV-1?Do you have a surveillance program for OsHV-1 in oysterfarms or environment?If yes, how is it?
If we detect a new positive sample (ie in a newlocation), we repeat our analysis, add sequenceanalysis and in situ hybridization to confirm infectionThe losses due to a herpes outbreak in Tomales Baycan certainly be greater than 90%, at least in patches.Regarding regulations, OsHV is not on our list ofdiseases that California regulates. It probably will beadded next time we make changes, but that will bemore than a year from now. However, we still can usemore broad, open-ended regulations to restrictmovement of infected product or seed.
.I was hoping to get the OsHV-1 RT PCR assay going in mylab this summer and we got most of the way there but havenot had time to set up the proper standards yet forquantification. I hope to be completely setup by next Springto conduct regular testing and do some experiments. Alsowant to get OsHV uvar testing. So currently there is noproper surveillance program for OsHV-1 but I expect therewill be by next year. We will examine oysters from thegrowing 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 TomalesBay and Drakes Bay. The growers in these areas will notsend seed to other growing areas. Actually, all of the seedor larvae that enters the states growing areas- CarlsbadLagoon, Santa Barbara, Morro Bay, Tomales Bay, DrakesBay and Humboldt Bay- comes either from Taylor inWashington (or Hawaii), Coast in Washington (orHawaii), Whiskey Creek in Oregon, Hawaiian Shellfish inHilo, 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
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
Clinical signs: Holiotis diversicolor supertextaTank water was turbid and bubbly. 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 in AustraliaInfected 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: 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)
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
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?