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White spot disease (WSD) of penaeid shrimp is characterised by
high and rapid mortality accompanied by gross signs in moribund shrimp of white,
initially circular, inclusions or spots in the cuticle, sometimes accompanied by
overall red body coloration. Disease progression is characterised by cessation
of shrimp feeding followed within a few days by the appearance of moribund
shrimp swimming near the surface at the edge of rearing ponds. The cuticular
inclusions range from minute spots to discs several millimetres in diameter,
which may coalesce into larger plates. These are most easily observed by
removing the cuticle over the cephalothorax, scraping away any attached tissue
with the thumbnail and holding the cuticle up to the light. Rapid and severe
pond mortality follows shortly after the first appearance of gross signs.
However, the appearance of white spots in the cuticle is unreliable even for
preliminary diagnosis of WSD as similar inclusions can be produced by high
alkalinity and other environmental conditions and bacterial white spot syndrome. Therefore, preliminary diagnosis must include
histopathological examination.
The causative agent of WSD is
white spot syndrome virus (WSSV) or white spot virus
(WSV), a double-stranded DNA (dsDNA) virus of the
genus whispovirus within the family nimaviridea.
WSSV has a wide host range among crustaceans and is
potentially lethal to most of the commercially cultivated penaeid shrimp
species. Virions are rod-shaped to elliptical with a trilaminar envelope and
they are large (80-120 x 250-380 nm). Negatively stained virions purified from shrimp haemolymph show
unique, tail-like appendages . The virions are generated in
hypertrophied nuclei of infected cells without the production of occlusion
bodies. In initial reports, WSSV was described as a non-occluded baculovirus,
but even while the molecular data were still limited, the preliminary WSSV DNA
sequence analysis (Maeda & Walker, pers. comm., , the
morphological characteristics and the general biological properties of the virus already highlighted its uniqueness. Recent data,
including the genome sequence and phylogenies based on DNA polymerase and
protein kinase, suggest that WSSV is a member of a new virus family. The status of work on WSSV has been reviewed by Lightner, Flegel, Flegel et al. ,
Loh et al., Lo & Kou and Lo
et al..
The size of the WSSV genome has been
differently reported for different isolates: 305107 bp (GenBank Accession
No. AF332093), 292967 bp (GenBank Accession No. AF369029)
and 307287 bp (GenBank Accession No. AF440570) for viruses
isolated from the People's Republic of China, Thailand and Taipei China,
respectively. The sequences of these three isolates are almost identical, with
the size differences being due mostly to several small insertions and one large
(~12 kbp) deletion. For two of these isolates
(from the People's Republic of China and Thailand), the analysis
of the complete WSSV genome has been published. The
following descriptions are based mostly on the WSSV isolate from the People's
Republic of China. The WSSV genome has a total G+C content of
41%. Three per cent of the WSSV genome is made up of nine homologous regions
containing 47 repeated mini-fragments that include direct repeats, atypical
inverted repeat sequences, and imperfect palindromes, while the remaining 97% of
the sequence is unique. A total of 531 putative open reading frames
(ORFs) were identified by sequence analysis, among which 181 ORFs
are likely to encode functional proteins. Thirty-six of these 181 ORFs have been
identified by screening and sequencing a WSSV cDNA library or else have already
been reported to encode functional proteins. Transcription of another 52 ORFs
was confirmed by reverse-transcription polymerase chain reaction
(RT-PCR). For 80% of the putative 181 ORFs there is a potential
polyadenylation site (AAT-AAA) downstream of the
ORF.
Among the 181 annotated ORFs, the proteins encoded by 18
ORFs show 40-68% identity to known proteins from other viruses or organisms or
contain an identifiable functional domain. These proteins include enzymes
involved in nucleic acid metabolism and DNA replication, a collagen-like
protein, and six published WSSV structural proteins. Thirty ORFs have predicted
proteins that show a partial homology (20-39% identity) to known
proteins or contain one or two sequence motifs (as opposed to a
functional domain). The remaining ORFs encode proteins with no homology
to any known proteins or motifs. The uniqueness of this virus means that it is
hard to directly apply other virus infection models to interpret the infection
strategy of WSSV; on the contrary, WSSV's infection strategy must be
investigated ab initio. The continuing thorough study of its functional
genomics and molecular biology are therefore urgently needed to better
understand its nature, replication strategy and the molecular mechanisms of its
pathogenesis, and also to provide useful information for the development of
virus control measures. To date, although much of the sequence of the WSSV
genome is already known, and the predicted sequences for many genes have already
been published, only a few WSSV genes have been studied beyond this sequence
analysis. These include genes encoding the DNA polymerase,
ribonucleotide reductase small and large subunits , structural
proteins, a novel chimeric polypeptide of
cellular-type thymidine kinase and thymidylate kinase, a
protein kinase and a nucleocapsid protein with nuclear
targeting behaviour. Many genes that are important for the
completion of WSSV's infection cycle still remain to be
studied.
WSD outbreaks were first reported from farmed
Penaeus japonicus in Japan in 1993 and
the causative agent was named penaeid rod-shaped DNA virus (PRDV)
or rod-shaped nuclear virus of P. japonicus (RV-PJ) . Later, outbreaks of viral disease with similar gross signs and
caused by similar rod-shaped viruses were reported from elsewhere in Asia and
other names were applied: hypodermal and haematopoietic necrosis baculovirus
(HHNBV) in the People's Republic of China;
white spot baculovirus (WSBV) and PmNOBIII in Taipei China; and systemic ectodermal and mesodermal
baculovirus (SEMBV) or PmNOBII in Thailand.
The virus from the People's Republic of China has also been called Chinese
baculovirus (CBV). Shrimp exhibiting the gross
signs and histopathology of WSD have also been reported from Korea, India, the Philippines
and the USA. During 1999, WSD also had a severe impact on
the shrimp industries of both Central and South America.
Because of similar gross signs, ultrastructure and molecular biology, Lightner
has included these viruses in a single white spot syndrome
baculovirus or white spot syndrome virus (WSSV) complex, which is
referred to here simply as white spot virus or WSSV.
The
histopathology of WSD is distinctive and can be used for diagnosis with moribund
shrimp during outbreaks. These moribund shrimp
exhibit systemic destruction of tissues of ectodermal and mesodermal origin with
many infected cells showing hypertrophied nuclei with lightly to deeply
basophilic central inclusions (haematoxylin and eosin [H& E]
staining) surrounded by marginated chromatin. These begin as Cowdry
type-A inclusions. The inclusions can be seen in rapidly stained squash mounts
of gills or subcuticular tissue or in tissue sections. In
tissue sections, the best tissues for examination are gill tissue and
subcuticular tissue of the stomach or cephalothorax.
Confirmation
of WSD requires more detailed analysis by PCR , antibody-based assays, in-situ DNA
hybridisation, or transmission electron microscopy
(TEM). Using such techniques, it has been shown
that WSSV from captured juvenile and broodstock shrimp or other carriers are
identical or closely related. These
molecular techniques have also been used to confirm infection of more than 40
penaeid and non-penaeid crustacean carriers and, tentatively,
an aquatic insect larval carrier. Some of these carriers have
been shown to transmit WSSV to shrimp in laboratory
tests. Detection of WSSV in carrier shrimp or other crustaceans cannot be
reliably accomplished by histological methods, and more sophisticated techniques
are required.
As WSSV readily infects species of shrimp such as
P. vannamei that are geographically separated from the WSSV endemic area
, it is possible that such shrimp, and especially
specific pathogen free (SPF) lines from controlled breeding
programmes, could be used for WSSV bioassays. Another
approach would be to use continuous cell lines, but unfortunately none is
presently available for the study of shrimp viruses, although WSSV has been
cultivated in primary cell lines from the lymphoid organ of P. vannamei and P. monodon where it was used to
measure WSSV titres and to observe virus replication, respectively. However, the
need to continually prepare cultures from different shrimp or from shrimp of
undefined or uncertain history currently presents problems for test
standardisation. Because of these limitations, it is recommended that detection
of WSSV in the absence of overt signs of disease, as for surveillance or for
certification of broodstock or fry for stocking, should be carried out by PCR,
Western blot analysis, or in-situ DNA hybridisation.
http://www.oie.int/eng/normes/fmanual/A_00048.htm
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