Complete description of virus right from Introduction, description, Virus Classification, Epidemiology, Structure, Replication, Genetic variants, Distribution, Host range, Genomic organisation, Mode of Transmission, Mechanical vectors, Target organs, Disease cycle, Diagnosis, Control and Treatment methods

1. Taura Syndrome Virus
ASHISH P M
M F Sc Scholar (Aquatic Animal Health Management)
ICAR - Central Institute of Fisheries Education,
Mumbai, Maharashtra 400061
email : ashishpm246@gmail.com
Mobile : +918073217468; +919686587839

2. Introduction
Taura syndrome (TS) is one of the more devastating diseases
affecting the shrimp farming industry worldwide
It was first described in Ecuador during the summer in farms near
the mouth of the Taura river in June 1992
Retrospective studies suggests that Taura syndrome might have
occurred on a shrimp farm in Colombia as early as 1990
Between 1992 and 1997, the disease spread to all major regions of
the Americas where white leg shrimp (Litopenaeus vannamei) is
cultured

3. The cause of the disease was initially attributed to fungicide
toxicity resulting from the use of 2 systemic fungicides Tilt® and
Calixin® (Propiconazole) on banana plantation adjacent to the
affected shrimp farms
As analytical data demonstrated propiconazole pesticide in water,
sediments and hepatopancreas tissues of shrimp harvested from
affected farms
However, this was proved wrong when the experiments conducted
in the laboratory failed to induce histological lesions
And the epizootics of pond-reared shrimps have been reported and
confirmed where bananas are not grown and neither of these
chemicals is employed

4. It was demonstrated that the disease could be transmitted by
feeding Taura victims to healthy shrimp in early 1994
Rivers' postulates were fulfilled in 1994 and proved the viral
etiology of the syndrome
Then the virus was named Taura Syndrome Virus, often referred to
as TSV
It was also referred by the name Infectious Cuticular Epithelial
Necrosis Virus (ICENV) by some authors

5. Virus Classification
Group: Group IV ((+)ssRNA)
Order: Picornavirales
Family: Dicistroviridae
Genus: Aparavirus
Species: Taura syndrome virus

6. Description
TSV was first classified as a possible member of the family
Picornaviridae based on biological and physical characteristics
It was later reclassified in the Dicistroviridae family of order
Picornavirales, genus Cripavirus
It has since been reassigned to a second genus in the same family -
the Aparavirus

7. Structure
TSV is a non-enveloped,
icosahedral virus with a diameter
of 32 nm
Single stranded, positive sense
RNA virus and has a genome of
10,205(10.2kb) nucleotides
(excluding the 3' poly-A tail)

8. Replication

9. Replication The nucleus (N)
exhibits a normal
appearance. The
cytoplasmic
organelles, including
Golgi (short arrows),
rough endoplasmic
reticulum (RER, long
arrows), and
mitochondria (Mi)
can be seen in the
perinuclear region.
Also shown in the
cytoplasm are large
electron-dense
inclusion bodies (I),
endocytic vacuoles
(E), and lysosomes
(L). Scale bar = 0.5
μm

10. Ultrastructural changes
in cells at the mid-
stages of an acute
phase infection with
Taura syndrome virus
(TSV). (a) The nucleus
(N) displays a normal
appearance, while the
cytoplasm contains a
large number of rough
endoplasmic reticulum
(RER) and intracellular
inclusion bodies (I)

11. Ultrastructural changes
in cells at the mid- stages
of an acute phase
infection with Taura
syndrome virus (TSV). (b)
Higher magnification of
an equivalent region
shows that the cytosol
contains clusters of
developing small
membranous vesicles
(SMVs), distended
mitochondria (Mi), and
RER that are covered by
small in- vaginations
(arrows) studded with
electron-dense particles.
Scale bars = 0.5 μm
Dis Aquat Org 73: 89–101, 2006

12. Ultrastructural changes
in cells at late stages of
infection with Taura
syndrome virus (TSV). (a)
The pyknotic nucleus (N)
is surrounded by
vesicular distribution of
the nuclear membrane.
The cytoplasm contains a
large number of small
membranous vesicles
(SMVs) and rough
endoplasmic reticulum
(RER) that carry electron-
dense materials (arrow).
(b) Higher magnification
of the rectangular area
in (a), rotated by 90°,
shows SMVs and RER.
Scale bars = 0.5 μm
Dis Aquat Org 73: 89–101, 2006

13. Transmission electron
micrographs of Taura
syndrome virus (TSV) in
an infected cell. At (a)
low and (b) high
magnifications, the
nucleus (N in a) shows
developing pyknosis as
indicated by increased
electron density of the
nucleoplasm, while TSV
particles (arrowhead in
b) are present in the
cytoplasm. Scale bars =
0.5 μm
Dis Aquat Org 73: 89–101, 2006

14. Genome Organisation
Schematic diagram of the genome organization of TSV. Numbers indicate
nucleotide positions. ORFs 1 and 2 are shown as open boxes and UTRs as
a single line. The approximate positions of the BIR-like sequence (BIR),
helicase (H), protease (P) and RNA-dependent RNA polymerase (RdRp)
are indicated.
Journal of General Virology (2002), 83, 915–926.

15. TSV genome is 10205 nucleotides long, excluding the 3’ poly(A)
tail
Two large ORFs were identified in the positive-sense RNA
sequence
ORF1, which ends at nucleotide 6736, encodes a 234 kDa
polyprotein with 2107 amino acids.
ORF2 is in a different frame to ORF1
ORF2 starts from nucleotide 6947 and extends to nucleotide
9982

16. ORF2 encodes a 1011 amino acid protein of approximately 112
kDa
ORFs 1 and 2 represent 92 % of the TSV genome (6320+3029=
9349 nt)
The other 8% consists of non-coding regions or UTRs
The 5’ UTR is 377 nucleotides in length
An intergenic region of 207 nucleotides separates ORFs 1 and 2
The 3’ UTR corresponds to 226 nucleotides, excluding the poly(A)
tail

17. ORF1 - amino acid product
The predicted amino acid product of ORF1 contains sequence motifs
of non-structural proteins that correspond to the conserved motifs
of:
a helicase (NTP-binding protein)
a protease and
an RNA-dependent RNA polymerase (RdRp) (found in viruses
from the picorna-like virus superfamily)

18. ORF2-amino acid product
The deduced amino acid sequence of ORF2 compared to the
protein databases revealed significant alignment with the
structural polyprotein of the insect RNA viruses such as DCV, CrPV,
PSIV, TrV, RhPV, HiPV and BQCV
TSV particles have three major proteins, designated Vp1 to Vp3
(55, 40 and 24 kDa)
Ø Vp1= 55 kDA
Ø Vp2= 40 kDA
Ø Vp3= 24 kDA

19. Non-structural protein- BIR
v In addition to H, P & RdRp, a short amino acid sequence located
in the N-terminal region of ORF1 presented a significant similarity
with a baculovirus IAP repeat (BIR) domain of inhibitor of
apoptosis proteins from double-stranded DNA viruses and from
animals.
v The presence of this BIR-like sequence is the first reported in a
single-stranded RNA virus, but its function is unknown.

20. At least one BIR domain is required for an anti-apoptotic function
in all of the IAP family of proteins, but not all BIR-containing
proteins are necessarily involved in apoptotic regulation
The location of the TSV BIR-like sequence in ORF1 upstream of
the usual non-structural proteins suggests that it may be a protein
that is transcribed early.
Therefore, this protein could have an important function with
respect to the biology and development of TSV.
The intergenic region shows nucleotide sequence similarity
with those of the genus Cricket paralysis-like viruses

21. Genetic variants
In general, RNA viruses exhibit high genetic diversity have high
rates of spontaneous mutation
These very high rates might be due to the lack of proofreading
function of the RNA-dependent RNA polymerase
The VP1 amino acid sequence of TSV was chosen for use in
differentiating among the isolates because of its higher variation
than other major capsid proteins (VP2 and VP3)

22. The phylogenetic analysis of amino
acid sequence revealed three distinct
groups of isolates, which are
associated with their geographic
origins.
Belize
SE Asia
Americas
But as of May 2009, four genetic
clusters are recognized: Belize (TSV-
BZ), America (TSV-HI), Southeast Asia
and Venezuela

23. Reports from the shrimp farming industry have indicated that TSV
from Belize is more virulent than TSV from Hawaii.
Belize isolate Hawaii isolate
First Mortality 2nd day post infection 2-4 days post infection
50 % of the mortality Before day 3 After 4-6 days
Most deaths occured 2-4 days 4-13 days
Cumulative mortality 100% 71-89%

24. Distribution
After the outbreak of TSV in Ecuador in 1992, the disease quickly
spread to other countries in the Americas
Until 1998, it was considered to be a Western Hemisphere virus.
The first Asian outbreak occurred in Taiwan
The wide distribution of the disease has been attributed to the
movement of infected host stocks for aquaculture purposes.
Might be the stable nature of the virus helped in bringing out of
disease from Western Hemisphere to Taiwan during importation

25. By 1999, TSV was introduced into Southeast Asia through infected
stocks of L. vannamei intended for aquaculture and subsequently
spread throughout much of the region
Taura syndrome can spread rapidly when introduced in new areas
A shrimp farmer described the 1995 outbreak in Texas as,
"This thing spread like a forest fire... There was no stopping
it. I just sat there and watched it and in a matter of three days, my
shrimp were gone. Dead!"

26. TSV affected Countries : China, United States (Hawaii & Texas),
Colombia, Ecuador, Peru, Venezuela, Indonesia, Saudi Arabia, South
Korea, Taiwan, Thailand, Belize, Mexico, Costa Rica, Honduras,
Nicaragua

27. Host range
P. vannamei (Adversely affected, Cumulative mortality 50% to
90%)
P. stylirostris and P. setiferus are moderately susceptible
Experimental infections have been successfully produced in P.
setiferus, P. chinensis, P. schmitti, P. vannamei and P. stylirostris
But not in P. duorarum or P. aztecus, which appear to be resistant
to TSV
Another study mentioned that P. monodon and M. rosenbergii
could be infected by TSV although the infected shrimp survived
and showed no clinical signs of infection.

29. Transmission of Disease
Horizontal transmission through cannibalism or by contaminated
water has been demonstrated
Vertical transmission is strongly suspected but has not been
experimentally confirmed.

30. Mechanical vectors
Birds : TSV will remain infectious for up to 48 hours in the faeces of
shrimp eating birds like Sea gulls (wild or captive) and Chicken
(used as laboratory surrogate for shrimp eating birds)
Aquatic insects : The water boatman (Corixidae) an aquatic insect
that feeds on shrimp carcasses in shrimp farm ponds
Larus atricilla (Sea gull) Corixa sp.

31. Target organs
Infects
Cuticular epithelium (or
hypodermis) of the general
exoskeleton
Foregut
Hindgut
Gills
Appendages
Connective tissues
Haematopoietic tissues
Lymphoid organ (LO)
Antennal gland.
Does not infect
Enteric organs (endoderm-
derived hepatopancreas, midgut
and midgut caeca mucosal
epithelia)
Smooth, cardiac, striated muscle
Ventral nerve cord, its branches
and its ganglia

32. Disease cycle
TS often causes high mortality during the first 15 to 40 days of
stocking into shrimp ponds.
Prevalence : 0 to 100%
Mortality : 40 to >90% in cultured populations of PL, juvenile, and
subadult life stages
The course of infection may be acute (5–20 days) to chronic (more
than 120 days) at the pond and farm level
The disease has three distinct phases that sometimes overlap:
Acute (up to 5 to 20 days)
Transition
Chronic (up to 120 days)

33. 1. Acute phase
Clinical signs can occur as early as 7 hours after infection in some
individuals and last for about 4–7 days.
Mortality during this phase can be as high as 95%.
Gross signs
Anorexia, lethargy, erratic swimming
Opacification of the tail musculature
Soft cuticle
Red tail due to the expansion of the red chromatophores
Edges of uropods & pleopods shows focal epithelial necrosis
Large scale mortalities during ecdysis

34. Moribund, juvenile, pond-reared white
shrimp (Penaeus vannamei) peracute
phase.
The shrimp are lethargic, have soft shells,
and a distinct red tail fan
Rough edges of the cuticular epithelium in
the uropods that are suggestive of focal
necrosis of the epithelium at those sites
(arrow)

35. Histopathology
Multifocal areas of necrosis in the cuticular epithelium of the
general body surface, appendages, gills, hindgut, and foregut (the
oesophagus, anterior and posterior chambers of the stomach)
Cells of the subcuticular connective tissues and adjacent striated
muscle fibres basal to affected cuticular epithelium are
occasionally affected
In some severe cases, the antennal gland tubule epithelium is also
destroyed (experimentally found)
Prominent in the multifocal cuticular lesions are conspicuous foci
of affected cells that display an increased eosinophilia of the
cytoplasm and pyknotic or karyorrhectic nuclei

36. Cytoplasmic remnants of necrotic cells are often extremely
abundant and these are generally presented as spherical bodies
(1–20 µm in diameter) that range in staining from eosinophilic to
pale basophilic
These structures, along with pyknotic and karyorrhectic nuclei, give
lesions a characteristic ‘peppered’ or ‘buckshot-riddled’ appearance,
which is considered to be pathognomonic for TS disease when there
is no concurrent necrosis of the parenchymal cells of the LO
tubules
The absence of necrosis of the LO in acute-phase infections with TSV
distinguishes TS disease from acute-phase yellowhead virus
genotype 1 in which similar patterns of necrosis to those induced by
infection with TSV may occur in the cuticular epithelium and gills

37. In TSV-infected tissues, pyknotic or karyorrhectic nuclei give a
positive (for DNA) Feulgen reaction
It distinguishes them from the less basophilic to eosinophilic
cytoplasmic inclusions that do not contain DNA.
The absence of haemocytic infiltration or other signs of a
significant host-inflammatory response distinguishes the acute
phase of infection with TSV from the transitional phase of the
disease

38. Prominent areas of necrosis in the cuticular
epithelium (large arrow)
Adjacent to the focal lesions are normal
looking epithelial cells (small arrow). 300x
Cytoplasmic inclusions and pyknotic and
karyorrhectic nuclei give the lesion a
pathognomonic 'peppered' or 'buckshot-
riddled' appearance.
The peracute nature of the lesion is suggested
by the absence of haemocytes in or near the
lesion

39. Lesion in the cuticular epithelium and
subcutis of the carapace
(Arrow) Nuclear pyknosis and karyorrhexis,
increased cytoplasmic eosinophilia, and an
abundance of variably staining, generally
spherical cytoplasmic inclusions

40. (Uropod) Focal area of necrosis in the
cuticular epithelium evidenced by the
presence of a vacant zone
A few expanded red chromatophores are
also apparent in the subcuticular
connective tissues
Cuticular epithelial cells and subcuticular
connective tissue cells positive for the virus.
The probe does not react with the pyknotic
and karyorrhectic nuclei (arrows) because
virus is solely cytoplasmic

41. 2.Transition/Recovery phase
Gross signs
Cuticular epithelium regeneration and healing and which might be
secondarily infected with bacteria
Randomly distributed, melanized irregular shaped (brownish/black)
lesions on cuticle & tail region
Sites of acute lesions which have progressed onto subsequent stages
of hemocytic inflammation
Moulted shrimps cast off the melanised lesions

42. Histopathology
Typical acute-phase cuticular lesions decline in abundance and
severity and are replaced by conspicuous infiltration and
accumulation of haemocytes at the sites of necrosis.
The masses of haemocytes may become melanised giving rise to
“the irregular black spots” that characterise the transition phase of
the disease.
In H&E sections, such lesions may show erosion of the cuticle,
surface colonisation and invasion of the affected cuticle and
exposed surface haemocytes by Vibrio spp.

43. LO will appear to be normal with H&E staining. However, ISH
results show the diffuse positive signals can be observed within
the walls of the lymphoid organ or within developing LOSs
Formation of thick melanised haemocytic plug at basal cuticular
epithelium to temporarily close the 'wound' from the outside

44. Penaeus vannamei shrimp displaying typical clinical signs of Taura syndrome
disease at the end of the acute phase. Multifocal, melanized lesions on the
thorax and tail are visible (indicated by arrows) in TSV-infected shrimp.

45. • Heavily colonised with masses
of bacteria (B)
• A thick, melanised,
haemocytic 'plug' (H) at
cuticular epithelium

46. 3.Chronic phase
The chronic phase is first seen six days after infection and persist
for at least 12 months under experimental conditions.
This phase is characterized histologically by the absence of acute
lesions and the presence of LOS of successive morphologies
These LOSs are positive by ISH for TSV
A low prevalence of ectopic spheroids can also be observed in
some cases
Diagnosis of the disease during the chronic phase is problematic,
as shrimp do not display any outward signs of the disease and do
not show mortality from the infection
Survivors may become carriers for life.

47. Gross signs
No obvious signs of disease.
Normal feeding.
Less tolerant to environmental stressors than uninfected shrimp.
Histopathology
Numerous prominent LOS, which may remain associated with the
paired LO ,or which may be detach and become ectopic LOS.
Ectopic LOS are found in heart, gill, subcuticular connective tissues,
etc,.

48. LOS in TSV
Study shows the three distinct morphotypes of LOS morphotypes in
TSV (Type A, B & C)
1. Type A : Earliest detecteable, appeared to be evolve from
activated LO tubule phagocytes that had sequestered TSV
2. Type B : contained necrotic cells, TSV-positive by in situ
hybridization for up to 32 wk, persistent, long-term infections
suggested that TSV replication occurred within these LOS
3. Type C : TSV-negative and characterized by cells with condensed
basophilic nuclei, a reduction in overall cell size, and progressive
atrophy leading to degradation without an inflammatory
response, not suitable for diagnosis

49. Pond reared juvenile white shrimp in the chronic or recovery phase of TS.
Multiple melanised foci mark sites of resolving cuticular epithelium necrosis due to
TSV infection

50. Normal-looking LO cords or
tissue is characterised by multiple
layers of sheath cells around a
central haemolymph vessel (Small
arrow)
LOS lack a central vessel and
consist of cells that show
karyomegaly and large
prominent cytoplasmic vacuoles
and other cytoplasmic inclusions
(large arrow)

51. Diagnosis

52. The acute phase of TSV is easily confirmed by any of the several
diagnostic methods currently available.
Transitional and early chronic phase TSV infection is also readily
diagnosed by histology and molecular methods
Whereas, Based on the clinical signs and symptoms of the TS
disease the chronic phase would be difficult to diagnose
As the chronic phase shows no gross lesions diagnosing it
becomes problematic and further it may silently carry the virus for
future infection.
The only organ which can be used for diagnosis in this phase is the
lymphoid organ, i.e,. for the presence of LOSs

53. The presence of the virus is detected by the ISH assay with cDNA
probe for TSV or MAb 1A1.
The LOS give positive reaction to the virus while other target organ
don’t.
ISH and qRT- PCR are the most reliable methods for detecting TSV
during late chronic phase infection. RT-PCR was also reliable if
hemolymph was used as sample source
---( Diseases of aquatic organisms 82(3): Jan 2009)

54. Detection methods
I. ISH vs IHC
IHC result diminishes earlier than the ISH assay.
IHC was generally less sensitive than ISH & sometimes unable to
confirm TSV infection (when tested for 24 week samples)
The infection once disease signs subsides as infection passes
through the transition stage and the virus becomes sequestered in
the LO as infection progresses to the chronic stage

55. II. RT PCR vs qRT PCR
Reverese Trascriptase (RT) PCR and Quantitative Real-Time (qRT)
PCR are being used for detection of the chronic phase infection is
shrimps.
Detection of TSV by RT-PCR was highly dependent on sample
source.
Haemolymph and Pleopod sample are taken for the detection of
RNA
Pleopod sample showed positive in RT PCR till 14 week of post
infection
In other hand the haemolymph showed positive till 60 week of
post infection
However the sensitivity of RT PCR was low than the qPCR

56. It was found that RNA isolated from hemolymph generally
contained 10 to 100 times or more copies of TSV RNA than did
pleopods sampled from the same shrimp
A possible reason for this was that pleopods were more difficult to
process than hemolymph, requiring grinding and less intact RNA
might have resulted
Determining the most appropriate method and sample type to
detect TSV is important in shrimp that survive acute infection
RT-PCR using pleopods as a RNA source or the use of methods such
as IHC or histology alone will not reliably detect the chronic phase
TSV infection

57. qRT-PCR utilizing hemolymph RNA and ISH analysis of tissue
sections containing lymphoid organ were clearly the most reliable
methods for detecting the virus in shrimp for more than 1 year post
infection

58. Control & Treatment
No scientifically confirmed reports of effective vaccination,
chemotherapy and immunostimulation treatments.
Resistance breeding: TSV-resistant domesticated stocks of P.
vannamei and P. stylirostris have been developed and are
commercially available
Some domesticated lines of TSV-resistant P. vannamei (that are
also TSV-free) are in widespread use by the shrimp-farming
industries of the Americas and South-East Asia.
Disinfection of eggs and larvae
General husbandry practices: SPF, SPR, etc.

59. References
Bonami, J.R., Hasson, K.W., Mari, J., Poulos, B.T. and Lightner, D.V., 1997. Taura
syndrome of marine penaeid shrimp: characterization of the viral agent. Journal
of General Virology, 78(2), pp.313-319.
Cheng, L., Lin, W.H., Wang, P.C., Tsai, M.A., Ho, P.Y., Hsu, J.P., Chern, R.S. and Chen,
S.C., 2011. Epidemiology and phylogenetic analysis of Taura syndrome virus in
cultured Pacific white shrimp Litopenaeus vannamei B. in Taiwan. Diseases of
aquatic organisms, 97(1), pp.17-23.
Hasson, K.W., Lightner, D.V., Mari, J., Bonami, J.R., Poulos, B.T., Mohney, L.L.,
Redman, R.M., Brock, J.A., 1999. The geographic distribution of Taura syndrome
virus (TSV) in the Americas: determination by histopathology and in situ
hybridization using TSV-specific cDNA probes. Aquaculture 171, 13–26.
Hasson, K.W., Lightner, D.V., Mohney, L.L., Redman, R.M. and White, B.M., 1999.
Role of lymphoid organ spheroids in chronic Taura syndrome virus (TSV)
infections in Penaeus vannamei. Diseases of Aquatic Organisms, 38(2), pp.93-105.

60. Lightner, D.V., Redman, R.M., Hasson, K.W. and Pantoja, C.R., 1995. Taura
syndrome in Penaeus vannamei (Crustacea: Decapoda): gross signs,
histopathology and ultrastructure. Diseases of aquatic organisms, 21(1), pp.53-59.
Tang, K.F. and Lightner, D.V., 2005. Phylogenetic analysis of Taura syndrome virus
isolates collected between 1993 and 2004 and virulence comparison between
two isolates representing different genetic variants. Virus research, 112(1-2),
pp.69-76.
Manual of Diagnostic Tests for Aquatic Animals (OIE), Chapter 2.2.7. Infection
with Taura syndrome virus
Poulos, B.T., Noble, B.W. and Lightner, D.V., 2008. Comparison of Taura syndrome
virus (TSV) detection methods during chronic-phase infection in Penaeus
vannamei. Diseases of aquatic organisms, 82(3), pp.179-185.