Fish Disease in Taiwan: Iridovirus-like particle in grouper

Fish Diseases inTaiwan

v i r u s

I r i d o v i r u s

Iridoviridae: Brief Introduction
Fish Iridoviruses
Grouper Iridovirus in Taiwan (TGIV)
Iridoviruses in Giant Seaperch and Largemouth Bass
Recent Study in this Laboratory

Iridoviridae: Brief Introduction
Iridovirus is a group of double strain DNA virus, spherical, enveloped or non-enveloped and icosahedral. Virions 160-350 nm in diameter.
The majority of the studies on iridovirus concentrated on insect iridovirus. It has been found in many insect species. Infected insect larvae showed iridescent color, and that's how this group of virus got its name. Most of the fish iridoviruses belong to the Genus Ranavirus, and Frog Virus 3 is the most studied.
Currently iridoviruses are classified into four genuses according to 6th ICTV Report
    Genus Iridovirus: insect iridescent viruses
    Genus Chloriridovirus: insect iridescent viruses
    Genus Ranavirus: frog viruses, some fish iridoviruses
    Genus Lymphocystivirus: lymphocystis virus
    unclassified viruses : goldfish virus 1; goldfish virus 2

Fish Iridoviruses other than lymphocystis virus
Here is a list of iridoviruses isolated from fish.
Isolated
Epizootic hematopoietic necrosis virus (EHNV), Perca fluviatilia, Australia, Langdon et al. 1986
Sheetfish iridovirus, Silurus glanis, Germany, Ahne et al. 1989
Red sea bream iridovirus (RSIV), Pagrus major, Japan, Inouye et al. 1992
European catfish iridovirus, Ictalurus melas, France, Pozet et al. 1992
Doctor fish iridovirus, Labroides dimidatus, US (SE Asia?), Hedrick and McDowell 1995
Guppy iridovirus, Poecilia reticulata, US (SE Asia?), Hedrick and McDowell 1995
Largemouth bass virus (LMBV), Micropterus salmoides, USA, Plumb et al. 1996
Seabass iridovirus, Latelabrax sp., Hongkong, Miyata et al. 1997
Grouper iridovirus, Epinephelus malabaricus, Thailand, Miyata et al. 1997
Grouper iridovirus in Taiwan (TGIV), Epinephelus sp., Taiwan, Chao and Pang 1997
Pike-perch iridovirus, Stizostedion lucioperca, Finland, Tapiovaara et al. 1998
White sturgen iridovirus, Acipenser transmontanus,
Observed but not isolated
Angelfish Pterophyllum scalare, UK, Rodger et al. 1997
Tilapia Oreochromis mossambicus, Australia, Ariel and Owens 1997
Tilapia Oreochromis niloticus, Canada (US?), McGrogan et al. 1998
Tilapia Oreochromis aureus, Avtalion and Shlapobersky 1994
,O. niloticus, Sarotherodon galilaeus, red tilapia,
Cichlid (Ram) Apistogramma ramirezi, US, Leibovitz and Riis 1980
Flounder Scophthalmus maximus, Denmark, Bloch and Larsen 1993
Chromide cichlid Etroplus maculatus, Singapore?, Armstrong and Ferguson 1989
Striped beakperch Oplegnathus fasciatus, Korea, Jung and Oh 2000
Mandarinfish Siniperca chuatsi, China, He et al. 2000

Iridovirus in Taiwan
We have seen iridivirus infection in three different fish species: grouper (Epinephelus spp.), giant seaperch (Lates calcarifer) and largemouth bass (Micropterus salmoides).
Grouper Iridovirus in Taiwan (TGIV)
This disease is a major killer in grouper culture, only next to white spot disease. It affects fish all year round except for winter. The outbreak usually cpntinues 1 to 2 months. In juvernile fish (5-10cm), nearly all fish will be infected and the mortality could reach 60%. In adult fish (over 25 cm) there's only 5-30 % mortality. Fish recovered from the disease can be infected again after several months but the mortality will be below 5%. The causative agent is named as TGIV (Grouper Iridovirus in Taiwan).
Affected fish shows darkening in color. Elevated frequency of opercular movement is noted. One of the quick identification of this disease is large epitheloid cells in gills. We often see this kind of cells appear in gill filament, block the circulation. Gill shows hyperemia and hemorrhage, then it turns pink or pale with the progress of infection.
Internally, we usually find swollen spleens and head kidneys. They increase the size by 2-10 folds. Infiltration by inflammatory cells can be seen in liver, cardiac muscle, spleen and kidney. The liver shows serious fatty change. In spleen, the number of lymphoid and hematopoietic tissue reduce due to extensive infiltration by inflammatory cells.
The most characteristic feature in histopathology is the universal distribution of giant cells. This big round cell appear in gill, spleen, head kidney, trunk kidney, heart and liver. They vary in size. The nucleus is pushed to side. The cytoplasm is either eosinophilic or basophilic, homogenous or granular. If we put giant cells under electron microscope, there is a lot of viral particles in their cytoplasms.
This is a double strand DNA virus, icosahedral, with double membranes. The side to side diameter is 120-130 nm and apex to apex is 160-170 nm.
Giant Seaperch (Lates calcarifer) and Largemouth Bass Iridoviruses
Seaperch culture in Taiwan also experienced Iridovirus infection for the past few years. But the mortality is not as high as in grouper. Until now there still no in depth study on this disease.

Relation to other Iridovirus and PCR Detection of TGIV
Similar diseases have been described in U.S., Japan and southeast asia. It caused serious economic loss in red sea bream culture in Japan. The causative agent is named RSIV (Red Sea bream Iridovirus). Primers designed for RSIV detection fail to amplify any fragment from TGIV sample in our lab. This indicates they are not the same virus although they are morphologically similar. Primers capable of amplifying other iridoviruses isolated in US, Europe and Australia (example, for Major Capsid Protein gene) also failed to yeild any product in TGIV. These result might indicate that TGIV is different from all other iridoviruses described before. Furhter study is needed to clarify this.
Our recent data on iridovirus infection in seaperch and largemouth bass showed that these causative agents are closely related but different viruses. They share very high sequence similarity in several genes we sequenced. Currently we are preparing this result for publication.
We also developed a PCR-based protocol to detect TGIV in grouper (Chao et al., submitted). This primer set can detect iridovirus in grouper, seaperch and largemouth bass, and did not amplify anything in lymphocystis sample.

Figure 1
Gill of infected grouper. Note pale spot in gill filaments.

Figure 2
Wet mount of gill. Note the characteristic enlarged cells inside gill filament.

Figure 3
Wet mount of gill. Note several enlarged cells inside gill filament.

Figure 4
Spleen of infected grouper is usually enlarged.

Figure 5
Enlarged cells can be easily seen in almost all internal organs.

Figure 6
Transmission electron micrograph of a infected spleen cell, reveal numerous viruses.

Figure 7
Depends on stages of infection, sometimes only a few virus can be found in cells.

Figure 8
Enlarged view of a single virus.

Last Update: 30 October 2001

	Figure 1 Gill of infected grouper. Note pale spot in gill filaments.
	Figure 2 Wet mount of gill. Note the characteristic enlarged cells inside gill filament.
	Figure 3 Wet mount of gill. Note several enlarged cells inside gill filament.
	Figure 4 Spleen of infected grouper is usually enlarged.
	Figure 5 Enlarged cells can be easily seen in almost all internal organs.
	Figure 6 Transmission electron micrograph of a infected spleen cell, reveal numerous viruses.
	Figure 7 Depends on stages of infection, sometimes only a few virus can be found in cells.
	Figure 8 Enlarged view of a single virus.