* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Types of nuclear changes
Survey
Document related concepts
Trichinosis wikipedia , lookup
Leptospirosis wikipedia , lookup
Sexually transmitted infection wikipedia , lookup
Oesophagostomum wikipedia , lookup
Cross-species transmission wikipedia , lookup
Eradication of infectious diseases wikipedia , lookup
Human cytomegalovirus wikipedia , lookup
2015–16 Zika virus epidemic wikipedia , lookup
Hepatitis C wikipedia , lookup
Middle East respiratory syndrome wikipedia , lookup
Influenza A virus wikipedia , lookup
Ebola virus disease wikipedia , lookup
West Nile fever wikipedia , lookup
Orthohantavirus wikipedia , lookup
Hepatitis B wikipedia , lookup
Marburg virus disease wikipedia , lookup
Herpes simplex virus wikipedia , lookup
Transcript
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