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A natural outbreak of infectious spleen and kidney necrosis virus (ISKNV) threatens wild pearlspot, Etroplus suratensis in Peechi Dam in the Western Ghats biodiversity hotspot, India

Wiley
Transboundary and Emerging Diseases
Authors:
  • at ICAR National Bureau of Fish Genetic Resources

Abstract

A large‐scale mortality of pearlspot, Etroplus suratensis was reported from Peechi Dam, an artificial tropical lake made for irrigation and drinking water supply in Kerala, India during 2018. This dam is located in the premises of Western Ghats, recognized as one of the biodiversity hotspots of the world. The objective of this study was to identify the aetiological agent of this large‐scale mortality of E. suratensis by systematic diagnostic investigation and identification of pathogen. Virus isolation was carried out on a species‐specific pearlspot fin (PSF) cell line. Infected PSF cells showed cytopathic effects (CPE) like cell shrinkage, rounding, enlargement, clustering, and subsequent detachment of cells with a high viral titre of 106⋅95 TCID50 mL−1 at 8 days post inoculation (dpi). Histopathological examination of the fish showed the presence of numerous abnormal enlarged basophilic cells and intracytoplasmic eosinophilic inclusions in the liver. Moreover, transmission electron microscopy (TEM) analysis revealed the presence of large numbers of 125–132 nm viral particles in the spleen tissues. PCR amplification and phylogenetic analysis of the major capsid protein (MCP) gene sequence confirmed that the causative agent was Infectious spleen and kidney necrosis virus (ISKNV) of the genus Megalocytivirus. The experimental infection recorded 86.7±2.7% mortality in the E. suratensis (body weight ‐ 11.01±2.7 g; body length 8.01±2.23 cm) injected with 1 × 104⋅25 TCID50 mL−1 ISKNV per fish. Our detailed investigation provided definitive diagnosis of ISKNV in the severe mass mortality event in wild E. suratensis in Peechi Dam, India, adding one more species to expanding host range of ISKNV infection. The high mortality rate of ISKNV infection in pearlspot suggests the perilous nature of this disease, particularly among the wild fish population. This article is protected by copyright. All rights reserved
Received: 20 August 2021 Revised: 18 February 2022 Accepted: 20 February 2022
DOI: 10.1111/tbed.14494
ORIGINAL ARTICLE
A natural outbreak of infectious spleen and kidney necrosis
virus threatens wild pearlspot, Etroplus suratensis in Peechi Dam
in the Western Ghats biodiversity hotspot, India
Thangaraj Raja Swaminathan1Tina Kollannoor Johny1Sundar Raj Nithianantham1
Arun Sudhagar1Pravata Kumar Pradhan2
Krupesha Sharma Sulumane Ramachandra3Reshma R. Nair1Neeraj Sood2
1Peninsular and Marine Fish Genetic
Resources Centre, ICAR-NBFGR, CMFRI
Campus, Kochi, Kerala, India
2ICAR National Bureau of Fish Genetic
Resources, Lucknow, Uttar Pradesh, India
3ICAR Central Marine Fisheries Research
Institute, Kochi, Kerala, India
Correspondence
Thangaraj Raja Swaminathan, Peninsular and
Marine Fish Genetic Resources Centre,
ICAR-NBFGR, CMFRI Campus, Kochi 682 018,
Kerala, India.
Email: rajathanga@yahoo.co.in
Abstract
A large-scale mortality of pearlspot, Etroplus suratensis was reported from Peechi Dam,
an artificial tropical lake made for irrigation and drinking water supply in Kerala, India
during 2018. This dam is located in the premises of Western Ghats, recognized as
one of the biodiversity hotspots of the world. The objective of this study was to iden-
tify the aetiological agent of this large-scale mortality of E. suratensis by systematic
diagnostic investigation and identification of the pathogen. Virus isolation was carried
out on a species-specific pearlspot fin (PSF) cell line. Infected PSF cells showed cyto-
pathic effects (CPEs) like cell shrinkage, rounding, enlargement, clustering, and subse-
quent detachment of cells with a high viral titre of 10695 TCID50 ml1at 8 days post-
inoculation (dpi). Histopathological examination of the fish showed the presence of
numerous abnormal enlarged basophilic cells and intracytoplasmic eosinophilic inclu-
sions in the liver. Moreover, transmission electron microscopy (TEM) analysis revealed
the presence of large numbers of 125–132 nm viral particles in the spleen tissues.
PCR amplification and phylogenetic analysis of the major capsid protein (MCP) gene
sequence confirmed that the causative agent was infectious spleen and kidney necrosis
virus (ISKNV) of the genus Megalocytivirus. The experimental infection recorded 86.7
±2.7% mortality in the E. suratensis (body weight 11.01 ±2.7 g; body length 8.01 ±
2.23 cm) injected with 1 ×10425 TCID50 ml1ISKNV per fish. Our detailed investiga-
tion provided definitive diagnosis of ISKNV in the severe mass mortality event in wild
E. suratensis in Peechi Dam, India, adding one more species to expanding host range of
ISKNV infection. The high mortality rate of ISKNV infection in pearlspot suggests the
perilous nature of this disease, particularly among the wild fish population.
KEYWORDS
emerging wildlife disease, host range, Etroplus suratensis, infectious spleen and kidney necrosis
virus, Peechi Dam
TransboundEmerg Dis. 2022;1–11. © 2022 Wiley-VCHGmbH 1wileyonlinelibrary.com/journal/tbed
2SWAMINATHAN ET AL.
1INTRODUCTION
India is endowed with four biodiversity hotspots (Western Ghats,
North East Region, Himalayas and Nicobar Islands) with a rich diversity
of fishes. The fish species in these biodiversity-rich regions are under
threat from various natural or anthropogenic impacts or a cascading
combination of both (Jena & Gopalakrishnan, 2012). Amongst these
factors, disease outbreak is an important factor that negatively affects
the population structure and diversity of wild fish. Particularly, trans-
boundary and emerging viral diseases can cause acute mortality among
the wild fish population (Bain et al., 2010; Groocock et al., 2007;Hyatt
et al., 1997; Lovy et al., 2018; Thresher et al., 2018). Apart from biodi-
versity losses, viral infections in the wild can also result in cross-species
transmission (host spill-over) and contribute to an increased fitness of
the viruses in new environments (Price et al., 2017). Such spill-over
infections with complex evolutionary histories were identified among
the viruses belonging to the Iridoviridae family that has a wide range of
hosts such as fish, reptiles, amphibians and insects (˙
Ince et al., 2018;
Price et al., 2017).
Infectious spleen and kidney necrosis virus (ISKNV) is an emerging and
transboundary fish pathogen that causes large-scale mortalities in fish.
ISKNV is a type species that belongs to the family Iridoviridae and genus
Megalocytivirus, while other members of Megalocytivirus are red sea
bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), giant
gourami iridovirus (GGIV), and scale drop disease virus (SDDV) (Zhu
et al., 2021). Amongst these, ISKNV causes both disease with clinical
signs or subclinical infection in about 50 different freshwater, brack-
ish, and marine species, particularly during the summer (Kurita & Naka-
jima, 2012). Since the first report of ISKNV in Chinese mandarin fish,
Siniperca chuatsi (He et al., 2002), ISKNV was reported from Australia
(Goetal.,2006), Korea (Song et al., 2008), Singapore (Jeong et al.,
2008), Malaysia (Subramaniam et al., 2014), Germany (Jung-Schroers
et al., 2016) and Indonesia (Sukenda et al., 2020). Recently, ISKNV was
reported from exotic ornamental fishes with subclinical infection from
India (Girisha et al., 2021). Global live ornamental fish trade without the
compliance of appropriate pathogen screening and quarantine serves
as the major reason for the transboundary spread of ISKNV (Jung-
Schroers et al., 2016; Rimmer et al., 2015).
The genus Etroplus is the only endemic cichlid genus reported from
Indian waters. This genus comprising three species, namely Etroplus
suratensis,Etroplus maculatus and Etroplus canarensis, and all the three
species are native to India. Etroplus suratensis, also known as pearlspot
or green chromide is a euryhaline fish, distributed in the coastal
lagoons, riverine estuaries, and freshwater habitats from the Southern
Karnataka up to Chilka Lake in India and Sri Lanka (Jayaram, 2010).
Owing to its high market value, natural breeding in confined conditions
and amenability to polyculture with other fish species, it is the most
sought-after candidate species for brackish water aquaculture in Ker-
ala, India (Chandrasekar et al., 2014), with reported production of 4858
tonnes (Kerala Inland Fisheries Statistics, 2010). Furthermore, captive
breeding technology of E. suratensis is well established (Padmakumar
et al., 2009) and it was declared as the official state fish of Kerala, India
in 2010 (Padmakumar et al., 2012). Etroplus suratensis is susceptible to
both bacterial and viral pathogens. The bacterial pathogens reported
from E. suratensis are Vibrio fischeri, Vibrio anguillarum, Klebsiella pneu-
moniae, Proteus vulgaris, Pseudomonas stutzeri, Pseudomonas aeruginosa
and Escherichia coli (Dhivya et al., 2015; Ravi et al., 2019). To date, there
is only one published report of a systemic Iridoviral infection in E. mac-
ulatus (Armstrong & Ferguson, 1989). Though pearlspot is relatively
resistant to nervous necrosis virus (NNV), it has been reported to act
as a carrier of NNV that can spread the virus to other susceptible fish
species (Marappan et al., 2019).
In this article, we report an extensive mass mortality of an endemic
fish, E. suratensis in Peechi Dam, an artificial freshwater tropical lake
under the Peechi-Vazhani wildlife sanctuary in the Western Ghats bio-
diversity hotspot. The fish kill event occurred during August 2018 and
fish samples were collected for laboratory analysis. Moreover, subse-
quent samplings were also performed from August 2019 to January
2020 to survey the persistence of infection after the large-scale mor-
tality event. The specific objective of the study was to isolate and char-
acterize the aetiological agent of this large-scale mortality event of E.
suratensis in India. Preliminary characterization of the infection was
analyzed using histopathology and transmission electron microscopy
(TEM) analysis. Subsequently,the pathogen was identified using molec-
ular methods and the infectious nature of the pathogen was confirmed
using animal experiments. A megalocytiviral infection was confirmed
and the virus was identified as ISKNV.
2MATERIALS AND METHODS
2.1 Case history and sampling
A fatal outbreak in E. suratensis was reported from Peechi Dam, Ker-
ala, India in August 2018. Both dead (n=98) and moribund (n=7) fish
(body length 17 ±5.36 cm; body weight 196 ±15.0 g) were collected
from four different sampling locations including landing sites, shore-
line, and from within the waterbody of the dam. The surface water
temperatures were also recorded during the samplings. The details of
the sampling and sampling sites are given in Table S1 and Figure 1.
Except E.suratensis, we did not observe any mortality or clinical signs
in other fish species. Morbid E.suratensis swimming at the banks of
the dam were collected randomly using a scoop net and 14 other fish
species were collected using cast net. Fish samples were transported
to Aquatic Animal Disease Diagnosis Laboratory,PMFGR Centre, ICAR
National Bureau of Fish Genetic Resources, Kochi, Kerala, India. Skin
and gill biopsies were collected for parasitological analysis and inter-
nal organs were taken for bacteriological and virological analyses from
seven moribund E.suratensis and 14 other fish species. Brain, liver, kid-
ney and spleen tissues were collected and stored in 99% ethanol and
L-15 media with 2% FBS for molecular diagnosis and virology, respec-
tively. For histopathological analysis, liver, gills, spleen, intestines and
kidney tissues were fixed in 10% neutral buffered formalin. Kidney
and spleen samples from moribund E.suratensis were fixed in 2.5% glu-
taraldehyde for TEM analysis and only four samples were further pro-
cessed for TEM. Furthermore, a monthly survey was conducted from
the local fisherfolks in the Peechi dam during 7–10 months after the
fish kill event regarding the species distribution in their catch.
SWAMINATHAN ET AL.3
FIGURE 1 Map showing the location of the outbreak, Peechi Dam, Kerala, India. Inset shows zoomed out view of Peechi Dam with borders
indicating watershed boundaries; the boxed area is the location of the infectious spleen and kidney necrosis virus (ISKNV) associated mortality
event in wild pearlspot
2.2 Examination of infected fish
The wet mount preparations of skin and gill biopsies were examined
for the presence of parasites by light microscopy. For bacteriological
analysis, the swabs of brain, liver, kidney and spleen tissues were sep-
arately streaked onto tryptone soya agar (TSA) (HiMedia, India) plates
and incubated at 28C for 24–72 h. Individual bacterial colonies with
unique morphology and growth characteristics were subjected to bio-
chemical and molecular characterization.
For virological analysis, gill, brain, liver, spleen and kidney tissues
were pooled and homogenized in Leibovitz’s15 (L-15) medium sup-
plemented with 1000 IU/ml penicillin, 1000 µg/ml streptomycin, and
2.5 µg/ml amphotericin B (Life Technologies, USA) without foetal
bovine serum (FBS). The tissues homogenates were subjected to three
freeze-thaw cycles for the release of virus particles and were clarified
by centrifugation at 300 ×gfor 30 min at 4C and subsequent filtra-
tion through a 0.22 µm filter (Millipore, Carrigtwohill, Ireland). The fil-
trate (500 µl) was inoculated onto a 70%–80% monolayer of PSF cell
line (Swaminathan et al., 2010)ina25cm
2flask (Nunc, Roskilde, Den-
mark) and incubated at 28C. Following adsorption for 1 h, 5 ml of
maintenance medium (L-15 medium with 2% FBS) was added. The inoc-
ulated cells were regularly monitored for cytopathic effect (CPE) under
an inverted microscope (Nikon, Japan) for up to 15 dpi.
2.3 Histopathology and TEM
The liver, kidney and spleen tissues for histopathology were fixed in
neutral buffered formalin (NBF) at tissue to volume ratio of 1:10 for
24 h. Following the initial fixation, the tissues were rinsed with 70%
alcohol and transferred to fresh NBF. The fixed tissue samples were
dehydrated in ascending grades of alcohol, cleared in chloroform and
embedded in paraffin wax. The paraffin-embedded tissues were sec-
tioned to a thickness of 3–4 µm, mounted on glass slides and stained
with haematoxylin and eosin. The stained sections were examined
for histopathological alternations by light microscopy (Nikon Eclipse
E800).
The kidney and spleen tissues fixed with 2.5% glutaraldehyde were
subjected to another round of fixation with 2.0% osmium tetroxide
(prepared in 0.1 M phosphate buffer) for 1 h at 4C. The tissues were
dehydrated in ascending grades of ethanol and embedded in araldite
CY212. Ultra-thin sections (6070 nm) were made using a microtome
(Leica Ultracut UCT), and mounted onto copper grids stained with
uranyl acetate and alkaline lead citrate. The sections were stained with
saturated uranium acetate and lead citrate solution and the ultrastruc-
tural features of the sections were examined using a transmission elec-
tron microscope (Tecnai T12 Spirit Transmission Electron Microscope)
at 60 kV.
2.4 Polymerase chain reaction and sequence
analysis
DNA and RNA extraction from the liver, spleen, brain and kidney of
the seven moribund E. suratensis and 14 other fish species were per-
formed using DNAeasy Blood and Tissue kit (Qiagen, Hilden, Ger-
many) and Gene JET RNA purification kit (Thermo Scientific, Lithua-
nia), respectively. Verso cDNA kit (Thermo Scientific) was used for
cDNA synthesis. The samples (n=5) were analyzed for the presence
of spring viraemia of carp virus (SVCV) (Stone et al., 2003), cyprinid
4SWAMINATHAN ET AL.
herpesvirus-2 (CyHV-2) (Jeffery et al., 2007), koi herpesvirus (KHV)
(Bercovier et al., 2005), koi ranavirus (KIRV) (George et al., 2015)and
carp edema virus (CEV) (Oyamatsu et al., 1997) using virus-specific
primers. The DNA samples were screened for the presence of RSIV
and ISKNV using the universal primer for Megalocytiviruses (Mishra
et al., 2018). The reaction mixture contained 1X GoTaq flexi buffer
(Promega), 50 pmol each of forward and reverse primers, 1 mM dNTP
mix, 2.5 mM MgCl2, 1.25U of GoTaq DNA Polymerase (Promega) and
50 ng of template DNA. The thermal regime for PCR reaction consisted
of an initial denaturation step of 5 min at 94C, 35 cycles of 94C for
30 s, 55C for 30 s, 72C for 1 min and a final extension of 10 min
at 72C. The PCR products were analyzed on 2% agarose gel contain-
ing ethidium bromide (EtBr) and visualized using a Bio Imaging System
(Bio Rad). The bacterial isolates from the samples were identified by
genomic DNA isolation and PCR amplification of the 16S rDNA using
universal primers. The PCR products were sequenced using Applied
Biosystems 3730 XL capillary sequencer (AgriGenome Labs Pvt Ltd,
Cochin, Kerala) and the sequences thus obtained were compared with
those available from GenBank using online BLAST tools – nucleotide
BLAST (http://www.ncbi.nlm.nih.gov/blast).
Multiple sequence alignment and phylogenetic analyses were con-
ducted using MEGA X (Kumar et al., 2018). Multiple sequence align-
ment was performed using MUSCLE, and evolutionary history was
inferred using the Maximum Composite Likelihood method (Tamura &
Nei, 1993); the robustness of the trees was evaluated using 1000 boot-
strap replicates. The phylogenetic tree was drawn to scale, with branch
lengths in the same units as those of the evolutionary distances.
2.5 Determination of ISKNV replication efficiency
The replication efficiency of ISKNV was determined using pooled tis-
sue homogenates of liver, brain, spleen and kidney tissues in sterile
phosphate-buffered saline (PBS, pH 7.4). The homogenate was clarified
by centrifugation at 7500 x gfor 20 min at 4C and filtration through
0.22 µm filter membranes. Tenfold (101to 1010) serial dilutions of
the positive homogenate were prepared in a growth medium and 100
µl of each dilution was transferred to a 48 well microtiter plate, start-
ing from the highest to the lowest dilution. Further, 100 µl of PSF cells
(1 ×104cells per ml) was dispensed into each well containing the sam-
ple. Ten wells containing 100 µl of cell suspension and 100 µlofL-15
with 2% of FBS served as the control. The plates were incubated at
28C and all the wells were examined daily for the appearance of CPE
for 2 weeks. The 50% tissue culture infective dose (TCID50) was deter-
mined using the method of Reed and Muench (1938).
2.6 Experimental infection
Healthy E. suratensis (body weight 11.01 ±2.7 g; body length 8.01
±2.23 cm) were procured from local farms in Kerala and acclima-
tized in the lab for 15 days in FRP tanks with good aeration, 12 h
light/dark condition, water temperatureof 25 ±2C, and optimal water
quality conditions. Five fish were randomly collected and screened
for ISKNV before the experimental infection. For the experimental
infection study, the fish were divided into six fibre reinforced plas-
tic tanks (500 L capacity) each having 20 fish. The six tanks were
grouped into two as control and infected with three tanks each (trip-
licates in each group). Subsequently, the fish were anaesthetized with
MS-222 (Sigma, USA) at a dose rate of 120 mg/L. Fish in the infected
group were injected intraperitoneally (i/p) with 100 µlof1×10425
TCID50 ml1ISKNV grown on PSF cells and those in the control group
were injected i/p with 100 µl of sterile PBS. Each group was housed
in separate tanks under the same conditions and the fish were mon-
itored daily for clinical signs and mortality. This experiment was con-
ducted and observations were made for a period of 20 days until
there was no further mortality among the experimental animals. The
experimental infection study was carried out according to the guide-
lines of Animal Research: Reporting In Vivo Experiments (ARRIVE;
Kilkenny et al., 2010). All the experimental infection procedures in
this study were examined and approved by ICAR National Bureau
of Fish Genetic Resources (NBFGR) Institute Animal Ethics Commit-
tee (IAEC) vide approval Number G/IAEC/2019/1 dated 4 October
2019. Dead/moribund fish were collected and their spleen and kid-
ney tissues were subjected to PCR analysis to confirm the presence
of ISKNV.
3RESULTS
3.1 Case study
The mass mortality event of E. suratensis was reported in Peechi
Dam, Kerala during August 2018 (Figure 2a). Moreover, mortality
was not observed in the other 14 fish species recorded during sam-
pling, namely Systomus sarana, Ompok malabaricus, Clarias dussum-
ieri, Puntius chola, Garra mullya, Pseudetroplus maculatus, Glossogob-
ius giuris, Devario malabaricus, Rasbora daniconius, Mystus malabricus,
Amblypharyngodon melettinus, Salmostoma clupeoides, Channa striata,
and Oreochromis mossambicus (Figure 2b). The dead fish showed exter-
nal lesions like haemorrhagic patches and erosions on the skin and loss
of scales, and swelling of gills and body (Figure 2c). During necropsy, the
sampled fish showed congestion of the liver with rounded borders and
a mottled appearance, ascites and haemorrhagic kidney (Figure 2d).
The water temperature of the water in the dam was 20–22C. Local
fishers did not report catching any pearlspot in 10 months after the fish
kill event revealed the complete disappearance of pearlspot in Peechi
Dam.
3.2 Parasitological and bacteriological
examination
Only a few numbers of parasites, viz. Dactylogyrus sp. were observed
from the wet mounts of gill biopsies. This parasite was observed only
in six out of 105 sampled fishes (5.7% prevalence). Bacteriological
examination of the infected fish did not reveal the presence of any
significant pathogens (data not shown).
SWAMINATHAN ET AL.5
(a) (b)
(c) (d)
FIGURE 2 Large-scale mortality, Clinical signs and post-mortem lesions of the infected pearlspot, Etroplus suratensis; (a) Dead E. suratensis on
the shoreline of Peechi dam; (b) fish species (n=15) diversity recorded during the sampling at Peechi Dam; (c) infected fish showing haemorrhages
on the surface, blackening of the body surface, and swelling of body and gills; (d) infected fish showing enlargement of the liver and necrotic liver,
haemorrhagic kidney and enlarged gall bladder
3.3 Histopathology and TEM
Histopathology of the liver of the infected pearlspot revealed hepatic
parenchyma with enlarged basophilic cells, presence of eosinophilic
inclusions, and intracytoplasmic eosinophilic inclusions (Figure 3a,b).
Sections of the spleen exhibited increased red pulp and depletion of
white pulp with the presence of enlarged basophilic cells (Figure 3c).
TEM revealed numerous icosahedral viral particles consistent with iri-
dovirus particles with a capsid of 125132 nm in diameter in the cyto-
plasm of spleen cells (Figure 4).
3.4 Virological examination and evaluation of
replication efficiency
At 3 dpi, CPEs were apparent in the PSF cell line inoculated with pooled
tissue homogenate of the infected fish. The morphological changes
included cell shrinkage, rounding, enlargement, clustering and detach-
ment of cells (Figure 5a–d and the cells detached from the monolayer at
10 dpi. The viral titre at 8 dpi was 10695 TCID50 ml1. The clarified cul-
ture supernatant from PSF cells also tested positive for ISKNV during
PCR with universal primers for Megalocytiviruses.
3.5 PCR, sequencing and phylogenetic analysis
All the infected fish tested negative in the diagnostic PCR assay of
CyHV-2, SVCV, KHV, KRIV and CEV. However, DNA extracted from
naturally infected fish, experimentally infected fish and infected PSF
cells yielded 294 bp amplicons in the universal PCR targeting the major
capsid protein of Megalocytiviruses, but not from the 14 other fish
species (Supporting Information Image S1). The amplicon sequences
showed 98.97% and 98.63% identity to MCP sequences of Grouper
iridovirus from Malaysia (JQ253373.1) and Angelfish iridovirus from
Singapore (MK689685.1). The partial MCP sequence of ISKNV from
E. suratensis was deposited in GenBank under the accession number
MZ352928.
Phylogenetic analysis construed significant homology between
ISKNV from E. suratensis and ISKNV isolates from Oscar (pairwise dis-
tance 0.0186), Asian seabass, Angelfish, Marble sleepy goby, Red drum,
Barramundi, African lampeye, Grouper fish, Giant seaperch and Molly
(pairwise distance 0.0209). Red seabream iridovirus (RSIV) and Turbot
reddish body iridovirus (TRBIV) formed distinct clusters in the dendro-
gram. Human alphaherpesvirus, used as an outgroup clustered sepa-
rately from the Megalocytviruses, is included in the analysis (Figure 6).
3.6 Experimental infection
In vivo experimental infection experiments were conducted by inocu-
lating 100 µlof1×104.25 TCID50 ISKNV into healthy pearlspot. The
experimentally infected fish showed clinical signs such as lethargy, loss
of appetite and abnormal swimming behaviour at 3 dpi. The infected
fish showed an enlarged spleen and kidney. Mortality started at 4 dpi in
the infected group and reached a cumulative mortality of 86.7 ±2.7%
by 11 dpi (Figure 7). The control group did not show any mortality or
clinical signs of the disease.
6SWAMINATHAN ET AL.
(a) (b)
(c)
FIGURE 3 Histopathology of liver of Etroplus suratensis infected with infectious spleen and kidney necrosis virus (ISKNV): (a) hepatic
parenchyma showing extensive destruction with haemorrhages (thick arrow) and presence of eosinophilic inclusions (thin arrow); (b) hepatic
parenchyma with intracytoplasmic eosinophilic inclusions (thin arrow); (c) section of spleen showing increased red pulp and depletion of white pulp
with the presence of enlarged basophilic cells (thin arrow)
FIGURE 4 Transmission electron microscopy of ultrathin sections
of spleen from infected pearlspot showing large numbers of
cytoplasmic hexagonal mature virus particles
4DISCUSSION
Our investigation provides the first evidence of ISKNV infection asso-
ciated with large-scale mortality of wild pearlspot, E. suratensis from
Peechi dam, Kerala, India during 2018. Isolation of the virus on PSF
cells, MCP gene-based molecular diagnosis and experimental infection
trials confirmed that the causative agent was ISKNV. The viral agent
was also visualized as 125132 nm particles in the infected tissues.
ISKNV was not detected in other 14 species recorded in the dam (Sup-
porting Information File S1). Though Peechi dam harbours diverse fish
assemblages, the outbreak did not cause any mortality in the 14 fish
species recorded during the sampling. However, the local fishermen
who witnessed the mortality suggested that most pearlspot died in
deeper water and then floated on the surface in a decomposed state,
making collection of moribund fish difficult for testing. The fishing sur-
vey undertaken 7–10 months after the mortality event showed that
the pearlspot population was reduced near to nil in the dam. Previous
experimental reports suggest that Iridoviruses can spread by means of
horizontal transfer through water (Go et al., 2006; Sano et al., 2002).
This suggests the likelihood of waterborne transmission of ISKNV in
pearlspot.
Megalocytivirus is an important viral pathogen in fish with a very
broad host range, often resulting in huge mortality of the infected
fish. ISKNV is one of the most studied type species of genus Megalo-
cytivirus. In the present investigation, we confirmed ISKNV infection
in a new host, E. suratensis in a freshwater reservoir in India. Parasito-
logical and bacteriological examination did not identify the presence
of any significant pathogens. Though Megalocytivirus was considered
an exotic pathogen in India, red sea bream iridovirus disease (RSIVD)
was reported from India during 2019 in cage cultured Asian seabass
in the brackish waters (Girisha et al., 2020). Molecular screening of
the ornamental fish samples during routine aquatic animal disease
surveillance also identified the presence of ISKNV in asymptomatic
SWAMINATHAN ET AL.7
(a) (b)
(c)
FIGURE 5 The cytopathic effect (CPE) in pearlspot fin (PSF) cells following infection with infectious spleen and kidney necrosis virus (ISKNV)
at 28C. (a) Uninfected PSF cells; (b) PSF cells infected with ISKNV showing shrinkage, and rounding at 3 dpi; (c) infected PSF cells at 10 dpi
fish species including Maylandia lombardoi,Cyprinus carpio koi and Pun-
tius titteye (Girisha et al., 2021). Such persistent ISKNV infections were
also reported during the PCR analysis of apparently healthy molly and
angelfish in Maharashtra and Odisha states of India during 2018–2019
(Pattanayak et al., 2020). In the present study, in spite of the wide
host range of ISKNV, apart from E. suratensis other 14 fish species col-
lected from Peechi dam neither showed any clinical sign nor positive for
ISKNV infection. Moreover,the ranching of E. suratensis seeds in Peechi
dam for commercial fisheries could be a possible source of viral entry.
However,further detailed studies are needed to identify the host speci-
ficity and source of viral entry to prevent future disease outbreaks.
Several cell lines including Tilapia heart (TH) (Fraser et al., 1993),
Chinese perch brain (CPB) (Fu et al., 2015), bluegill fry (BF-2) (Ramírez-
Paredes et al., 2021), grunt fin (GF) (Thanasaksiri et al., 2019)and
Mandarin fish fry (MFF-1) (Zhu et al., 2021) were used for the iso-
lation of ISKNV from a number of susceptible fish species. Megalo-
cytivirus AFIV-16 was isolated on the SKF-9 cell line from imported
angelfish Pterophyllum scalare in Australia (Kawato et al., 2020). In our
study, we used a species-specific cell line PSF and observedCPE typical
of ISKNV like cell shrinkage and rounding, cell enlargement and clus-
tering and detachment of cells form the monolayer. The CPEs on PSF
cells were consistent with the changes observed in TH cells upon the
inoculation of spleen and intestine ultrafiltrates from infected gourami.
However, no CPE was recorded in fathead minnow (FHM) cell lines
(Fraser et al., 1993). The high viral titre of 10695 TCID50 ml1obtained
in the present study was similar to the ISKNV titres obtained in man-
darin fish Siniperca chuatsi fry (MFF-1) and Siniperca chuatsi brain (CPB)
cell lines (Dong et al., 2008; Fu et al., 2015).
Further, molecular identification of the viral pathogen was achieved
by the analysis of major capsid protein (MCP), the predominant struc-
tural component of all viruses. As MCP is a late evolving gene and con-
tains highly conserved domains, it permits accurate determination of
virus taxonomy (Lin et al., 2014; Tidona et al., 1998). Diagnostic PCR for
all significant viral pathogens such as SVCV, CyHV-2, KHV, KIRV and
CEV did not yield any amplicons. Screening for ISKNV using the univer-
sal primer for Megalocytivirus (Mishra et al., 2018) produced 294 bp
amplicons. After sequencing and BLAST analysis, ISKNV was identified
to be the aetiological agent of the fatal disease outbreak in green chro-
mide.
Phylogenetic analysis was performed for a detailed understanding
of the genetic relationships between the ISKNV isolate from our study
and Iridovirus isolates from other fish species. The ISKNV sequence
from E. suratensis clustered with the IKSNV sequences from other fish,
suggesting that it belongs to the ISKNV species. The genetic distance
computed using MEGA X revealed close relatedness of our ISKNV iso-
late to ISKNV isolate from Oscar, identified in our lab. Regardless of
the genetic diversity that exists among ISKNV strains present in India,
its detection in wild pearlspot underpins the importance of biosecurity
in aquaculture to evade the probable spreading of the virus in the wild.
Etroplus suratensis is one of the three cichlid species endemic to the
Indian peninsular region. ISKNV is known to infect other exotic cich-
lidssuchasOscar(Astronotus ocellatus), angel fish (Pterophyllum scalare)
and Nile tilapia (Oreochromis niloticus) (Ramírez-Paredes et al., 2021;
Rimmer et al., 2015). Some of these exotic cichlid species are cultured
and traded in India for ornamental and food purposes. Though it is pre-
sumed that exotic and new pathogens are being introduced into the
8SWAMINATHAN ET AL.
FIGURE 6 Phylogenetic tree of major capsid protein (MCP) gene sequence of infectious spleen and kidney necrosis virus (ISKNV). The
evolutionary history was inferred by using the maximum likelihood method and Tamura–Nei model. The tree is drawn to scale, with branch lengths
measured in the number of substitutions per site. Human alphavirus was used as the outgroup
importing country through the live ornamental fish trade, their role in
the translocation and establishment of new pathogens has not received
much attention. The international trade of live freshwater and marine
ornamental fish has been considered as a probable source of Megalo-
cytivirus, particularly, the spread of ISKNV-like viruses (Jeong et al.,
2008; Whittington & Chong, 2007). ISKNV transmission is possible
either through a horizontal route, via pond and tank water and also via
infected fish (He et al., 2002), or through vertical transmission (Jeong
et al., 2008).
In our study, ISKNV was identified as the etiological agent in the
large-scale mortality of E. suratnesis in Peechi Dam, India. With a total of
19,370 reservoirs spreading over 3,153,366 ha, reservoir fisheries con-
stitute a significant proportion of aquaculture resources in India (FAO).
Peechi dam is an artificial tropical lake built across the Manali River,
with a catchment area of approximately 1300 ha in Kerala, located in
the southern part of India. The dam was built for irrigation in the sur-
rounding places and other districts and to meet the drinking water
requirements of the population of neighbouring towns. The Peechi dam
comes under the premises of Peechi-Vazhani wildlife sanctuary in the
Western Ghats., which is considered as one of the regions harbouring
the richest biodiversity in the world. Recently, virus-associated mortal-
ity events were reported for CEV in wild carps from natural and arti-
ficial lakes in the United States and Italy, respectively (Marsella et al.,
2021; Tolo et al., 2021). A fish viral disease outbreak in this sensitive
region may therefore lead to irreversible biodiversity losses among the
endemic wild fish population.
From a fisheries point of view, even though ichthyofaunal diversity
of a tropical reservoir is largely representative of the diversity of the
parent river system, habitat changes as well as stock/species intro-
duction exert significant effects on fish diversity. There is a possibility
that the emerging and or re-emerging pathogens would dangerously
infect native endemic fishes and may endanger the species diversity in
the ecosystem. In the present study, we hypothesize that ISKNV might
have been translocated through the infected pearlspot seeds that were
procured or collected from wild without testing for pathogens during
different ranching programmes and also by illegal and unintentional
release of seeds into the dam water. Though ISKNV is not a zoonotic
pathogen, a viral outbreak of this magnitude mandates periodic screen-
ing of other potential zoonotic pathogens in the fish and water col-
lected from the dam to prevent any disease outbreaks in humans.
SWAMINATHAN ET AL.9
FIGURE 7 Cumulative mortality in pearlspot in experimental infection trials with infectious spleen and kidney necrosis virus (ISKNV)
5CONCLUSION
This study presents the first evidence for ISKNV infection in native wild
E. suratensis population in a biodiversity hotspot region in India, sug-
gesting the host range expansion of this exotic pathogen in India. As
ISKNV is an emerging pathogen with several reported cases of inap-
parent infections, the host range and geographical distribution of this
virus is hitherto unknown. This exerts a potential threat to the indige-
nous fish species in this region. Moreover, this situation necessitates
the screening of ISKNV in both wild and cultured fish during routine
surveillance programs such as the ongoing National surveillance pro-
gramme of aquatic animal diseases (NSPAAD) of India, so as to curb
further transmission of this virus to wild indigenous fishes of India.
Therefore, future research on ISKNV should emphasize on the modes
of transmission and prevalence of this within wild populations, and
the development of management strategies to prevent infection or
disease.
ACKNOWLEDGEMENTS
The authors are thankful to Director General, Indian Council of Agri-
cultural Research, DDG (Fy) Indian Council of Agricultural Research,
New Delhi, India and Director, ICAR-National Bureau of Fish Genetic
Resources, Lucknow, India for their support, encouragement and guid-
ance. This research was carried out under National Surveillance Pro-
gramme for Aquatic Animal Diseases under Pradhan Mantri Matsya
Sampada Yojana (PMMSY) from the Department of Fisheries, Ministry
of Fisheries, Animal Husbandry and Dairying, Government of India (No.
35028/05/2012-Fy (Trade)-Vol II dated 19 June, 2020) and thankfully
acknowledged funding.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ETHICAL STATEMENT
All the experimental infection procedures in this study (Proposal
number: NBFGR/IAEC/2019/0014) were evaluated and approved
by Institute Animal Ethics Committee (IAEC) of ICAR National
Bureau of Fish Genetic Resources (NBFGR) (CPCESA Registration
No: 909/GO/Re/S/05/CPCSEA dated 09.09.2005 and CPCSEA Ref file
No. 25/111/2014-CPCESA dated 05th December 2018) vide approval
Number G/IAEC/2019/1 dated 4 October 2019.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
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How to cite this article: Swaminathan, T. R., Johny, T. K.,
Nithianantham, S. R., Sudhagar, A., Pradhan, P. K., Sulumane
Ramachandra, K. S., Nair, R. R., & Sood, N. (2022). A natural
outbreak of infectious spleen and kidney necrosis virus
(ISKNV) threatens wild pearlspot, Etroplus suratensis in Peechi
Dam in the Western Ghats biodiversity hotspot, India.
Transboundary and Emerging Diseases, 1–11.
https://doi.org/10.1111/tbed.14494
... Pathogens, ranging from viruses and bacteria to parasites and fungi, have evolved sophisticated mechanisms for infecting and exploiting their hosts. Some pathogens, such as infectious spleen and kidney necrosis virus (ISKNV) (Swaminathan et al. 2022), Cyprinid herpesvirus 2 (CyHV-2) (Sahoo et al. 2016), and tilapia lake virus (TiLV) (Behera et al. 2018), are highly virulent and can cause mass mortalities in susceptible fish species. Sometimes parasite, such as myxozoan, may not directly kill their hosts but can weaken them, making them more susceptible to predation, competition, or other stressors (Holzer et al. 2021). ...
... This can have significant ecological consequences, particularly in species with small population sizes or low reproductive rates. In extreme cases, mass mortalities can lead to population crashes or local extinctions, disrupting food webs and ecosystem dynamics (Swaminathan et al. 2022). Even in cases where disease outbreaks do not result in mass mortalities, the effects on individual fish can be profound. ...
... This led to the complete depletion of pearl spot stock in the affected dam, which exaggerated the livelihood of the local fishers dependent on them. Subsequent clinical investigations led to the identification of ISKNV as the causative agent behind the outbreak (Swaminathan et al. 2022). Interestingly, the presence of ISKNV in India had only been reported recently (Girisha et al. 2020a;Swaminathan et al. 2021). ...
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Transboundary diseases pose significant challenges to the conservation of wild fish populations, with implications for ecosystem health and biodiversity. This chapter explores the dynamics, impacts, and conservation implications of transboundary diseases in wild fish populations, providing insights for effective management strategies. The dynamics of transboundary diseases are shaped by interactions between pathogens, fish hosts, and environmental conditions. Factors such as globalization, climate change, and habitat degradation contribute to the spread and emergence of pathogens, leading to disease outbreaks with profound ecological and socio-economic impacts. Transboundary diseases can have devastating effects on fish populations and ecosystems, including mass mortalities, changes in species composition, and disturbances to ecosystem function. These impacts cascade through aquatic ecosystems, affecting other species, trophic levels, and ecological processes. Furthermore, the socio-economic implications of disease outbreaks, including economic losses, food insecurity, and public health risks, underscore the urgency of effective conservation strategies. Conservation strategies for addressing transboundary diseases in wild fish populations require a multifaceted approach that integrates surveillance and monitoring, habitat restoration, biosecurity measures, collaborative management, and public awareness initiatives. By understanding the complex interactions between pathogens, fish hosts, and their environment, the stakeholders can develop targeted strategies to mitigate the impacts of transboundary diseases and promote the health and resilience of fish populations and their habitats. Through collaboration, innovation, and a commitment to stewardship, we can work towards sustainable solutions that protect and conserve wild fish populations for posterity. This chapter aims to provide an overview on transboundary diseases as part of broader conservation efforts to safeguard the health and integrity of the aquatic ecosystems.
... The partially characterized ISKNV strain identified in native cichlids in this study was identical to previous strains identified in cultured tilapia and carp in Brazil, suggesting that viral transmission between these species may be possible (Swaminathan et al., 2022). Our findings underscore the importance of implementing proper biosecurity measures in Brazilian aquaculture to both control the impact and spread of endemic diseases as well as to prevent the entry of exotic pathogens. ...
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Peacock bass (syn.: tucunaré, Cichla ocellaris) and the pearl cichlids (syn.: acará, Geophagus brasiliensis) are South American cichlids that are highly valued in both the ornamental and sport fish industries. Since 2017, a number of outbreaks of infectious spleen and kidney necrosis virus (ISKNV) have been reported on Brazilian food and ornamental fish farms. In this study, we detected ISKNV in farmed peacock bass and pearl cichlid by PCR and sequence analysis of the partial major capsid protein (MCP) gene. Moribund peacock bass (n=10) and pearl cichlids (2) from a farm experiencing elevated mortality among juveniles and adults of these species, were submitted for bacteriological and molecular diagnostics. Spleen, liver, brain, and kidney tissues were cultured on 5% sheep blood agar and cystine heart agar with 1% glucose and bovine haemoglobin. No bacteria were isolated from the 12 fish. Additionally, DNA extracts from the liver and spleen of all animals were tested for ISKNV using two conventional polymerase chain reaction (cPCR) assays and two nested PCR (nPCR) assays. ISKNV DNA was amplified in all 12 fish DNA extracts tested, in two or more of the PCR assays. Selected ISKNV amplicons were confirmed by Sanger sequencing. The nucleotide sequences derived from these animals were identical to ISKNV strains previously detected in food (e.g., tilapia and carp) and ornamental species, including strains previously detected in fish from Brazil. To the authors’ knowledge, this is the first report of ISKNV in these native Brazilian cichlids.
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Mass mortality events of common carp (Cyprinus carpio, carp) associated with carp edema virus (CEV) alone or in coinfections with koi herpesvirus (KHV), is an emerging issue. Despite recent outbreaks of CEV in wild carp populations, the host range of North American species has not been well studied. To that end, we intensively sampled carp (n = 106) and co-habiting native fish species (n = 5 species; n = 156 total fish) from a CEV-suspect mass-mortality event of carp in a small Minnesota lake (Lake Swartout). Additionally, fecal and regurgitant samples (n = 73 each) from double-crested cormorants (Phalacrocorax auritus, DCCO) were sampled to test the potential of DCCO to act as a vector for virus transmission. CEV was confirmed to be widespread in the Lake Swartout carp population during the outbreak with high viral loads and histological confirmation, suggesting that CEV was the cause of the mortality event. There were no detections of CEV in any native fish species; however, DCCO regurgitant and fecal samples were positive for CEV DNA. In addition, three CEV-positive and one CEV + KHV-positive mortality events were confirmed with no observed mortality or morbidity of non-carp species in other lakes. This study provides evidence that CEV infection and disease may be specific to carp during mortality events with mixed-species populations, identifies DCCO as a potential vector for CEV, and further expands the known range of CEV, as well as coinfections with KHV, in North America.
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In late 2018, unusual patterns of very high mortality (>50% production) were reported in intensive tilapia cage culture systems across Lake Volta in Ghana. Samples of fish and fry were collected and analysed from two affected farms between October 2018 and February 2019. Affected fish showed darkening, erratic swimming and abdominal distension with associated ascites. Histopathological observations of tissues taken from moribund fish at different farms revealed lesions indicative of viral infection. These included haematopoietic cell nuclear and cytoplasmic pleomorphism with marginalisation of chromatin and fine granulation. Transmission electron microscopy showed cells containing conspicuous virions with typical Iridovirus morphology i.e. enveloped, with icosahedral and or polyhedral geometries and with a diameter c.160 nm. PCR confirmation and DNA sequencing identified the virions as Infectious Spleen and Kidney Necrosis Virus (ISKNV). Samples of fry and older animals were all strongly positive for the presence of the virus by qPCR. All samples tested negative for TiLV and Nodavirus by qPCR. All samples collected from farms prior to the mortality event were negative for ISKNV. Follow up testing of fish and fry sampled from 5 additional sites in July 2019 showed all farms had fish that were PCR positive for ISKNV, whether there was active disease on the farm or not, demonstrating the disease was endemic to farms all over Lake Volta by that point. The results suggest that ISKNV was the cause of disease on the investigated farms and likely had a primary role in the mortality events. A common observation of coinfections with Streptococcus agalactiae and other tilapia bacterial pathogens further suggests that these may interact to cause severe pathology, particularly in larger fish. Results demonstrate that there are a range of potential threats to the sustainability of tilapia aquaculture that need to be guarded against.
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Infectious spleen and kidney necrosis virus (ISKNV), a type species of the genus Megalocytivirus, poses a threat to ornamental fish trade as most cases show nonspecific symptoms, thus making timely diagnosis challenging. Apparently health molly (Poecilia sphenops) and angelfish (Pterophyllum scalare) collected from two distinct geographic localities of India were screened for four genera under Iridoviridae, Megalocytivirus {ISKNV, turbot reddish body iridovirus (TRBIV) and red seabream iridovirus (RSIV); ranaviruses and Singapore grouper iridovirus; and Lymphocystivirus} through molecular approach. In total five numbers out of 17 samples (29.4%), ISKNV genome fragments were detected. A PCR assay using major capsid protein (MCP) gene was standardised to detect and differentiate infections within the Megalocytivirus genus, even without aid of sequencing. This forms the first report of ISKNV infection in ornamental fish from India. Sequence analysis of MCP gene showed that Indian isolate being 100% similar to the complete genome or reference strain of ISKNV. Phylogenetic reconstruction demonstrated the present strain belonging to ISKNV genotype I. Furthermore, structural stability of the MCP revealed this strain was more stable than ISKNV genotype II, RSIV and TRBIV at 25 degree C and pH 7.0. Thus a strong pan-India surveillance is recommended to reduce trade risk.
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Megalocytivirus of family Iridoviridae has been identified as a pathogen that caused the fatal systemic infection to lead to massive death of numerous fish species in both ornamental and food fish industries. It caused significant economic losses due to severe mortality of infected fish. Since 2011 in Indonesia, megalocytivirus in giant gourami (Osphronemus goramy) had been found as the main pathogen in some cases of giant gourami outbreak, especially in West Java, Central Java, and Bali. The aim of this study is to identify and characterize the pathogen known as a megalocytivirus infection in freshwater-cultured giant gourami. We identified the virus using universal and specific primers for megalocytivirus and infectious spleen and kidney necrosis virus (ISKNV). Sequencing and BLAST analysis were used to develop a phylogenetic tree. The result showed that phylogenetic analysis of major capsid protein (MCP) gene unveiled a new member of megalocytivirus, designated as giant gourami iridovirus (GGIV). GGIV-formed cluster belonged to ISKNV and has 100% homology to ISKNV complete genome. Artificial infection by intraperitoneal injection with supernatant homogenate from spleen and kidney of naturally infected fish showed 93% cumulative mortality in 12 days. Fish showed a clinical sign of infection as lethargic, loss of appetite, pale or darken body color, and hemorrhages. Internal organ on dead fish showed swollen spleen and kidney and also a pale liver. Quantitative PCR analysis on internal organs showed spleen had the highest viral DNA copy number followed by kidney, gill, and liver. Histopathological analysis showed many abnormally hypertrophied cells in spleen which is typical histopathological characteristic of megalocytivirus infection. In conclusion, GGIV belonged to ISKNV from genus megalocytivirus.
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Mortality in wild fish populations represents a challenging issue for public fish health inspectors. When a single fish species is involved, an infective aetiology is frequently suspected, with focus on viral notifiable diseases. However, other viral agents not subjected to regulation and causing mortality in common carp have been reported such as carp edema virus (CEV). In mid-June 2020, a severe common carp mortality was observed in an artificial lake in north-east of Italy. Sleepy fish were noted some days before the beginning of the mortality itself, which lasted several days and involved over 340 adult specimens. During the outbreak, water temperature was around 15°C, water quality was normal, and no adverse meteorological events were reported in the area. Four specimens, which showed severe cutaneous hyperaemia and increased mucus production on skin and gills, were tested by bacteriological methods and virological analysis targeting the main carp pathogens. Molecular analysis performed on gills, kidney and brains from all the fish analysed resulted positive for CEV, which, based on anamnestic information and laboratory findings, was considered the responsible for the mortality event herein described.
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Megalocytivirus of family Iridoviridae was considered as one of the most alarming causative agents which caused fatally systemic infection and lead to mass mortalities to world-widely distributed freshwater and marine fish. In recent years, severe diseases with typical symptoms of body blackened, swims slowly to the water surface or surrounding the ponds were observed frequently in cultured Asian seabass, Lates calcarifer, in Zhuhai city of Guangdong province, where is the most important area for Asian seabass culture in mainland China. The causative agents were highly suspected as megalocytiviruses, however, the detailed and systemic studies about these diseases are still lacking. In this study, two cases of high morbidity and mortality of Asian seabass were investigated by systemic isolation and identification of pathogen. Virus isolation by MFF-1 cells and molecular detection confirmed that the causative agents were ISKNV genotype-II (ISKNV-II) of megalocytivirus. The mortalities from natural and artificial infected fish were 85.89% and 83.30%, respectively. A total of 21 single-nucleotide differences, including 20 same-sense mutations, were observed on the ISKNV-II compared with that of ISKNV-I. Histopathology examination showed that numerous abnormal enlarged cells, the most well-known ISKNV-specific clinicopathological features, were observed in spleen, kidney and liver. Results of immunohistochemistry (IHC) and immunofluorescence assay (IFA) examination confirmed that numerous abnormal enlarged cells with abundant viruses existed in these tissues. Moreover, large number of viral particles of diameter 150 nm were also observed both in infected tissues and MFF-1 cells by transmission electron microscope (TEM). Taken together, this is the first study with detailed and robust evidence confirming that ISKNV-II is the causative agent of the severe diseases in farmed Asian seabass in mainland China. This study contributes to the pathogenic and epidemiological studies of Asian seabass iridovirus disease in mainland China and other Asian countries and regions.
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Infectious spleen and kidney necrosis virus (ISKNV), a member of family iridoviridae, reported for the first time in a wide range of ornamental fish species in India. Significant mortalities during the year 2018‐19 were reported from a number of retailers in the region with various clinical signs. The samples of moribund, dead and apparently healthy ornamental fishes were collected from retailers, located in three districts of Karnataka, India. Out of 140 fish samples, 16 samples (11.42 %) representing 10 different fish species were found positive to ISKNV by OIE listed primers and same samples were reported to amplify the major capsid protein (MCP) gene of ISKNV. Further, sequence analysis of MCP gene showed that all strains detected in this study were closely related to other documented isolates from different countries with an identity ranging from 98.76 to 100 %. Further, they clustered in the clade of ISKNV, during the phylogenetic analysis. The sequence similarity was high (99.94%) to ISKNV strains from Japan, Australia, and Malaysia. This is the first report of an ISKNV infection in India. Moreover, out of 10 ISKNV positive fish species; three species were reported positive to ISKNV for the first time in the world. Further, the in‐vitro experiment showed the growth of virus in Asian seabass cell line, which is a natural host of ISKNV. Therefore, considering the lethal nature of megalocytiviruses to infect a vast range of species, proper biosecurity measures needs to be taken to control these emerging pathogens.
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We conducted a trial for PCR detection of carp edema virus, the causative agent of viral edema of carp. Two pairs of primers for nested PCR were designed from the sequence data of carp edema virus; F2 (5'-GCTGCTGCACTTTTAGGAGG-3'), R2 (5'-TGCAAGTTATTTCGATGCCA-3'), F1 (5'-GCTGTTGCAACCATTTGAGA-3'), R1 (5'-TGCAGGTTGCTCCTAATCCT-3') . In the case of nucleic acid extracted from gills of naturally diseased fish and artificially infected fish, expected sizes of the products of carp edema virus were specifically amplified in each primer pair and nested PCR. Amplification from the fish in natural outbreaks succeeded in all the cases of which existence of the virus was confirmed by infectivity trials. On the other hand, none of the product was found in normal fish. These results indicate that the PCR primers developed in this study are useful for a rapid detection of carp edema virus and the confirmatory diagnosis of the disease.
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Using cultures of the SKF-9 cell line, megalocytivirus AFIV-16 was isolated from imported angelfish Pterophyllum scalare held in quarantine at the Australian border. The cytopathic effect caused by isolate AFIV-16 presented as cell rounding and enlargement, but complete destruction of the infected cell cultures did not occur. The infected cells demonstrated immunocytochemical reactivity with monoclonal antibody M10, which is used for diagnosis of OIE-listed red sea bream iridoviral disease. Using electron microscopy, the virus particles, consisting of hexagonal nucleocapsids, were observed in the cytoplasm of SKF-9 cells. The replication of AFIV-16 in cultured SKF-9 cells was significantly greater at 28°C incubation than at 22 and 25°C incubation, whereas no difference in growth characteristics was observed for red sea bream iridovirus (RSIV) isolate KagYT-96 across this temperature range. Whole genome sequencing demonstrated that AFIV-16 has a 99.96% similarity to infectious spleen and kidney necrosis virus (ISKNV), the type species in the genus Megalocytivirus. AFIV-16 was classified into ISKNV genotype Clade 1 by phylogenetic analysis of the major capsid protein gene nucleotide sequence. This is the first report of whole genome sequencing of an ISKNV genotype megalocytivirus isolated from ornamental fish.