Download Types of nuclear changes

Viral pathogens
Lightner 2011
• DNA and RNA viruses infect marine
species.
• Often specific host (taxonomic) affinity
– But we are seeing some with very broad host
ranges
• E.g. WSSV – infects all aquatic arthropods tested
to date but disease varies among species
• Viruses often (but not always) result in
catastrophic losses – esp in crustaceans
– See OIE-listed diseases
Types of nuclear changes
Viral inclusions/occlusions
•
• Parvovirus and
provisionally in genus
Brevidensovirus
Cowdry Type A
nuclear inclusion
(IHHNV)
• Hypertrophy – enlargement
• Marginated chromatin – chromatin
pushed to margins of nucleus
• Pyc(k)nosis – nuclear condensation
(very basophilic)
Baculovirus penaei • Whispovirus nuclear
intranuclear
inclusions (WSSV)
occlusion bodies
(BP)
• Karyolysis - dissolution of the cell
nucleus with loss of its affinity for basic
stains sometimes occurs normally but
usu. in necrosis
• Karyorrhexis - a degenerative cellular
process involving nuclear fragmentation
and breakup of chromatin into
unstructured granules (still basophilic)
Most images: D. Lightner (2011)
Herpesvirus evolutionary tree
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
FISH AND
AMPHIBIANS
TERRESTRIAL
VERTEBRATES
OYSTERS
1
Global distribution of OsHV
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
OsHV infections
Possible modes of transmission
OsHV Transmission in Tomales Bay
• 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
Mortality
Max
Mean
Dection of OsHV
30
25
20
15
10
5
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 Transmission in larvae
OsHV Diagnostic Methods
• Light microscopy
100
90
– Nuclear hypertrophy and
chromatin margination
80
Percent Survival
100
90
80
70
60
50
40
30
20
10
0
70
60
• Cells of the gills, mantle, and velum
(not epithelial cells)
50
40
• Signs of viral-induced apoptosis
30
20
– Slight or no inflammatory
response around infected cells
10
0
1
3
5
Experiment Day
7
9
– Changes often described in
larvae but not juvenile or adult
oysters
2
OsHV Diagnostic Methods 2
• In situ hybridization
OsHV Diagnostic Methods 3
• Transmission Electron
Microscopy (TEM)
– Section through the
visceral ganglion.
– Labelled cells
(arrowheads) and nonlabelled cells (arrows).
– The DIG-labelled probe
reacts strongly within
the cytoplasm and the
nucleus of nervous cells
(bar=10 um).
– 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
OsHV µ-var 1
• Multiple conventional and qPCR primer sets have
been designed to amplify regions of the OsHV-1
genome
• In 2008- high mortality rates of 80% to 100% in
Crassostrea gigas
• PCR allows for both presumptive diagnosis (proxy)
of OsHV presence and the comparison of possible
OsHV variants
• 75% positive batches for OsHV-1
A)
TRL
X
UL
IRL
US
IRS
TRS
– Mainly juvenile oysters from May to September
– new biotype of OsHV-1 and is now listed by OIE
• Extracts of field affected oysters  induced
mortalities (80% IM, 40% cohabitation) in spat and
juvenile oysters
– qPCR and TEM confirmed viral infection
B)
C
A GP B
C
• 0.1μm filtration or UV inactivated OsHV µvar
• Sauvage et al. 2009
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
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
3
Clinical signs: Holiotis diversicolor
supertexta
AHLVD: Gross Signs Australia
Tank water was turbid and bubbly.
Healthy vs. moribund abalone
AHLVD in Australia
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
Infected farm (above) and healthy farm (right)
AHLVD: Histology 2
(Australia + Taiwan)
– No Cowdry type A inclusions were observed
– However neuronal cells may contain marginated
chromatin
Normal vs GNV Nerves
4
AHLVD: TEM 2
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)
Australia
Taiwan
AHLV: experimental transmission
AHLV: experimental transmission 2
Cumulative mortality in abalone
Cumulative mortality in abalone exposed to virus
infected water
100
100
80
80
Fresh virus injected
Frozen virus injected
DMEM injected
40
Untreated
% mortality
% mortality
Co-habitation
60
Co-habitation
100% water
60
10% water
1% water
0.01% water
40
0.001% water
20
Untreated control
20
0
1
2
3
4
5
6
Days post-exposure to virus
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?
5
Crustacean Diseases
• Although crustaceans may be infected by a variety of pathogens, certain
highly virulent/pathogenic organisms are considered important enough
for international harmonization of detection methods and actions as
follows:
• Diseases of crustaceans notifiable to the OIE:
• Taura syndrome (viral/penaeid shrimp)
• White spot disease (viral/penaeid shrimp and other decapods)
• Yellowhead disease (viral/penaeid shrimp)
• Tetrahedral baculovirosis (Baculovirus penaei) (viral/penaeid shrimp)
• Spherical baculovirosis (Penaeus monodon-type baculovirus)
(viral/penaeid shrimp)
• Infectious hypodermal and haematopoietic necrosis (viral/penaeid
shrimp)
• Crayfish plague (Aphanomyces astaci) (fungal/freshwater crayfish)
• Infectious myonecrosis
• White tail disease
• Baculovirus diseases: Spherical and Tetrahedral
Viral diseases
• Over 50 crustacean viral diseases described
since first discovery of a crab virus in 1960s
(Vago 1966)
• Range from enveloped to non-enveloped
cytoplasmic viruses (a lot in portunid crabs;
see Johnson 1983) to nuclear viruses
• Rod-shaped nuclear viruses are most common
in shrimp culture major impediment to
industry success
• We will focus on the White Spot Virus as an
example in farmed shrimp and Panulirus argus
virus 1 (PaV1) in wild spiny lobsters
• Not notifiable but cool: PaV1 lobster iridovirus disease
White Spot Syndrome Virus (WSSV) 2
White Spot Syndrome Virus (WSSV) 1
• First seen 1992-1993 in northeast Asia (Penaeus
monodon in Thailand) and is currently the most
problematic disease of cultured penaeid shrimp in
Asian countries
• dsDNA rod-shaped virus that causes disease in
stressed individuals  rapid and nearly complete
losses (Whispovirus, in the family Nimaviridae (Mayo
2002a, 2002b)
• November 1995, 1st documented case in Western
Hemisphere.
– Pond-reared P. setiferus from a south Texas farm
– Shrimp packing plants implicated as the source:
major importers and re-processors of shrimp
imported from affected areas of Asia
Differential susceptibility of four penaeid species to White Spot Syndrome
Virus (WSSV). Penaeus vannamei (van) and P. setiferus (set) are highly
susceptible to WSSV, while P. aztacus (azt) and P. duorarum (duo)
appear to be more resistant.
• Natural infections observed in: Penaeus monodon, P.
japonicus
• In laboratory studies, a strain WSSV from Thailand
was found to produce severe, lethal infections in two
other important farmed species: P. vannamei, P.
stylirostris
• WSSV appears unique in its ability to infect most if not
all crustaceans (marine and freshwater) to which it has
been exposed
• Pathogenicity varies among host species as shown by
the graph on the next slide
THE SPREAD OF WHITE SPOT VIRUS
16
14
Number Surviving
12
az t C on t
az t W S S V
d u o C on t
duo W S S V
se t Co nt
se t W S SV
10
8
6
1996
1995 1997
12/1999
1/1999
9/1999
2000 1997
1999
1993
1993
1992
1994 1994
1995
2001
v a n C o nt
v an W S S V
4
2
0
0
1
2
3
4
5
6
7
8
9
10
D ays Po st In itial Exp o su re
W SSV challenge of W estern H em isphere Penaeid Shrim p
6
WSSV or Red Disease Clinical Signs and
Diagnosis
WSSV or Red Disease Diagnosis 1
• Target organ = ectodermal and mesodermal origin: esp
cuticular epithelium and underlying CT
• Acutely affected shrimp:
– Rapid reduction in food consumption
– Lethargic
– In many cases, moribund shrimp have pink to reddishbrown coloration (hence the name "red disease" for the
disease), due to expansion of the cuticular
chromatophores and few if any white spots
• Acute phase
– Loose cuticle with white spots on inner carapace
• May represent abnormal deposits of calcium salts by the
cuticular epidermis But also find virus present.
• Typically occurs after the acute phase
WSBV Diagnosis: DNA virus
•Routine H&E histology: appears
similar to a non-occluded Type C
baculovirus (rod shaped bacilliform
virus = new taxonomy)
Lightner 2000
•In situ hybridization and Dot blot
hybridization with gene probe. PCR with
specific primer pairs for WSSV group.
•Rapid field test: impression smear stain.
Durand et al. 2003, Lighnter et al. 2000
• In order to address concerns over international
trade and expansion of viral infections, Durand et
al. examined the load of WSSV in the head and
tail of infected shrimp using histology, ISH and
real-time PCR
• Histology and ISH indicated ~ levels
• Real-time PCR demonstrated quantitatively that
the head had slightly more WSSV than the tail
– When they examined peeled versus tail meat: virus
load in peeled shell = 55% of the total tail viral load
Lightner et al. 2000
7
Zwart et al. (2010) Demonstrated evolution of the WSSV in
Asia with genome deletions associated with host-pathogen
dynamics in a farm situation
Figure 1. Map of Asia, showing the geographical origins of WSSV isolates used for this study.
PLoS ONE 5(10): e13400. doi:10.1371/journal.pone.0013400
• During the spread of WSSV in farmed shrimp in Asia, the
virus exhibited changes relative to its putative ancestor:
– Biology:
• 1. Higher host mortality,
• 2. Shorter host survival time (faster mortality),
• 3. Higher in-host fitness
– Genotype:
• 1. Significant changes during spread in Asia = two genomic
deletions in the variable regions of two open reading frames
(ORFs = genes): ORF14/15 and ORF23/24
• 2. The total size of genomic deletions measured between the
oldest and the most recent WSSV isolates analyzed as of
2010 are ~15 kbp (~5% of the viral genome)
Table 1. Origins of the Asian WSSV isolates used in this study.
Zwart MP, Dieu BTM, Hemerik L, Vlak JM (2010) Evolutionary Trajectory of White Spot Syndrome Virus (WSSV) Genome Shrinkage during
Spread in Asia. PLoS ONE 5(10): e13400. doi:10.1371/journal.pone.0013400
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013400
Figure 4. Dynamics of WSSV genome shrinkage over time when the virus was introduced into
a new location.
Zwart MP, Dieu BTM, Hemerik L, Vlak JM (2010) Evolutionary Trajectory of White Spot Syndrome Virus (WSSV) Genome Shrinkage during
Spread in Asia. PLoS ONE 5(10): e13400. doi:10.1371/journal.pone.0013400
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013400
Figure 2. Schematic representation of the ORF23/24 variable region of the Asian WSSV
isolates.
Zwart MP, Dieu BTM, Hemerik L, Vlak JM (2010) Evolutionary Trajectory of White Spot Syndrome Virus (WSSV) Genome Shrinkage during
Spread in Asia. PLoS ONE 5(10): e13400. doi:10.1371/journal.pone.0013400
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013400
Figure 5. Kaplan-Meier survival curves for WSSV isolates with different genome sizes:
As genome deletions occur, shrimp mortality rises: virus is more lethal!
Figure 4. Dynamics of WSSV genome shrinkage.
Panel A: the year of first outbreak for the country a WSSV isolate was collected from is
plotted on the x-axis, and the genome size in kpb is plotted on the y-axis. The circles
are data points, whereas the line indicates the model fitted to all the data.
Zwart MP, Dieu BTM, Hemerik L, Vlak JM (2010) Evolutionary Trajectory of White Spot Syndrome Virus (WSSV) Genome Shrinkage during
Spread in Asia. PLoS ONE 5(10): e13400. doi:10.1371/journal.pone.0013400
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013400
Zwart MP, Dieu BTM, Hemerik L, Vlak JM (2010) Evolutionary Trajectory of White Spot Syndrome Virus (WSSV) Genome Shrinkage during
Spread in Asia. PLoS ONE 5(10): e13400. doi:10.1371/journal.pone.0013400
http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013400
8
What does this mean?
Mechanisms for WSSV International
Transfer & Introduction
• The pattern of spread was fast
• Biological and genetic changes within Asia
resulted in increased virulence and
pathogenicity
Imported
Live PLs or Broodstock
• Asia
• Middle East
• Turkey & Greece
• Central & S.America
(after introduction)
• Pattern of deletions within each country
suggest that anthropogenic movements of
animals facilitated spread of WSSV
Interestingly in some cases of WSSV
tolerated infections
Imported
Frozen Shrimp
• United States
• Central America
• Australia
• Spain
Viruses can impact wild populations too
• Populations of shrimp showing clinical signs typically
experience high mortality rates:
• Research by Don Behringer and Jeff
Shields
– Cumulative mortalities reach 100% within 3 to 10 days of the
onset of clinical signs
• Tsai et al. (1999): long-standing WSSV infections in
cultured shrimp without mass mortalities.
– 10% of shrimp had white spots in the carapace and
40% had WSSV-infected branchial cells based on in
situ hybridization tests.
– Unlike most documented WSSV discoveries, these
data suggest low-level, chronic WSSV infections may
persist in low stress culture conditions.
Background
Spiny Lobster Landings
• Panulirus argus
9
8
– Supports largest spiny lobster
fishery
7
6
– Florida Keys and Florida Bay
• $30 million fishery in Florida
Tons
• Fishery in USA
5
4
• $500 million Caribbean-wide
3
• Trap & recreational fishery
2
Commercial Landings
Recreational Landings
2001
1998
1995
1992
1989
1986
1983
1980
1977
1974
1971
1968
1965
1962
1959
1956
0
• Georgia to Brazil
1953
1
– Range
Time (yr)
Photo credit Mark Butler
9
Panulirus argus Virus 1 (PaV1)
• First naturally occurring pathogenic virus in a
lobster – 1991 = initial observation
• Target tissues = hyalinocytes and semigranulocytes (hemocytes), soft CT, some
haematopoietic tissues and fixed phagocytes
(Shields and Behringer 2004)
PaV1 virions:
Unenveloped,
icoashedral,
DNA virus,
nucleocapsid
=182 nm
– Loss of hemocytes and clotting ability, milky
hemolymph, atrophy of storage cells in HP with focal
necrosis and ischemia  chronic wasting
Widespread distribution of PaV1
• Varies with site and can
be as high as 30% but a
mean of ~7%
prevalence 1999-2001
surveys
• Found through out
Keys & Florida Bay
• Highest
prevalence in
smallest juveniles
• Adults can
become infected
Range
0-22%
Range
0-29%
• Adult surveys
• Jul 2001 = 4 of 863
(<1%)
• Oct 2001 = 0 of 668
Visibly infected (%)
Prevalence by size class
• Juvenile surveys
18
16
14
12
10
8
6
4
2
0
**
<20
20-30
30-40
>40
Size class (mm)
N=711
External signs
•
•
•
•
Loss of clotting ability
Lethargy
Morbidity
Carapace discoloration
(yellowing) in advanced
cases
• Solitary behavior
Healthy
Infected
10
PaV1 infects:
• Fixed phagocytes in HP =
primary sites of PaV1
infection
• Hyalinocytes &
semigranulocytes, not
granulocytes
• Connective tissue cells.
• Cells show
– Hypertrophied nucleus
– Nuclear inclusions
– “Waxy” cell membrane
– margination of
chromatin
Histopathology
• Debris and exudates in hemolymph
• Loss of hemocytes
– nuclear hypertrophy,
chromatin margination
– pycnosis, karyorrhexis
– lysis
Atrophy of hepatopancreas in advanced infections
Histological summary
• Cause of death: metabolic wasting
– Cell lysis: hemocytopenia,
– Altered clotting: hemophilia
– Focal necrosis with ischemia
– Atrophy of hepatopancreatic tubules
– Loss of RI cells
PaV1 Impact on host behavior
• Underwater surveys of juvenile lobsters:
– infected lobsters rarely shared shelters with
conspecifics
• < 7% shared dens and > 93% were solitary
• Despite that healthy lobsters generally preferred to
live together
– > 56% shared dens and < 44% were solitary
• Behringer et al. 2006 conducted a study to
examine this trend in the lab and found that
healthy lobsters actively avoided infected
ones
PaV1 Transmission
• Suggests healthy juveniles avoid
infected animals even before they are
infectious thereby limiting transmission
• Infected animals not impacted in
behavior initially and this may serve to
aid in viral transmission
• Transmission via contact, water (up to a
few meters away), cannibalism, and
inoculation
– Former two most likely
11
Butler et al. 2008 PaV1 Transmission
DAO 79:173-182
How is the Virus Transmitted?
Virus conclusions
Contact Transmission Trial
• Viral pathogens are key drivers of farmed penaeid shrimp
and impact early life stages of Pacific oysters
70
• WSSV is unique in its ability to infect a wide range of
crustacean taxa
60
Prevalence (%)
• Healthy lobsters held
with infected lobsters.
• Examined after 80
days.
• Transmission related to
size.
• Typical time to death
within 30-90 days but
some appear to survive
infection, especially
larger individuals
50
• WSSV has been moved via transfers of infected shrimp
40
• Evolution of the virus is evident in farmed situations
resulting in increased pathogenicity and virulence via
viral genome reduction
30
20
10
• Viruses can also impact wild populations (e.g. PaV1)
0
<20
25-35
40-50
Size class (mm)
• Lobsters can detect early PaV1 infection and avoid
infected individuals
• PaV1 is more pathogenic to younger lobsters and infects
a variety of tissues resulting in a wasting syndrome
12