Lymphocystis Disease Virus Infection Research Articles

Identification of a 27.8 kDa protein from flounder gill cells involved in lymphocystis disease virus binding and infection

In vitro, lymphocystis disease virus (LCDV) infection of flounder gill (FG) cell cultures causes obvious cytopathic effect (CPE). We describe attempts to isolate and characterize the LCDV-binding molecule(s) on the plasma membrane of FG cells that were responsible for virus entry. The results showed that the co-immunoprecipitation assay detected a 27.8 kDa molecule from FG cells that bound to LCDV. In a blocking ELISA, pre-incubation of FG cell membrane proteins with the specific antiserum developed against the 27.8 kDa protein could block LCDV binding. Similarly, antiserum against 27.8 kDa protein could also inhibit LCDV infection of FG cells in vitro. Mass spectrometric analysis established that the 27.8 kDa protein and beta-actin had a strong association. These results strongly supported the possibility that the 27.8 kDa protein was the putative receptor specific for LCDV infection of FG cells.

Immune enhancement of chemotherapeutants on lymphocystis disease virus (LDV) infected Paralichthys olivaceus

In olive flounder, Paralichthys olivaceus infected with lymphocystis disease virus (LDV) bath treatment with formalin, hydrogen peroxide or Jenoclean at 50, 100, and 200 ppm daily 10 min for 60 days enhanced the innate immune response and disease resistance. Jenoclean enhanced the immune parameters at the lowest concentration of 50 ppm; on the other hand, hydrogen peroxide bath treatment enhanced the immunity level at 100 ppm, while formalin was effective only at 200 ppm. A low cumulative mortality and high relative percent survival was noted in Jenoclean treated group followed by formalin and hydrogen peroxide treated groups. In olive flounder at low concentrations of 50 ppm or 100 ppm hydrogen peroxide and Jenoclean effectively prevent LDV infection.

Effect of Punica granatum solvent extracts on immune system and disease resistance in Paralichthys olivaceus against lymphocystis disease virus (LDV)

We report the effect of aqueous, ethanol, and methanol solvent leaf extracts of Punica granatum on innate immune mechanisms, such as phagocytosis activity, respiratory burst activity, alternative complement activity, lysozyme activity and functional immunity in terms of percentage cumulative mortality and Relative Percent Survival (RPS) in olive flounder Paralichthys olivaceus naturally infected with lymphocystis disease virus (LDV) after 8 weeks. Infected fish were intraperitoneally administered with 0, 5, 50, and 100 mg kg −1 body weight of solvent extracts. In groups treated with 50 and 100 mg kg −1 body weight, the chosen innate immune parameters significantly increased after 8 weeks when compared to 0 mg kg −1 dose, but not with 5 mg kg −1. Administration of P. granatum solvent extracts for 8 weeks significantly reduced the percentage mortality with the consequent increase in RPS. The results suggest that intraperitoneal administration of the leaf extracts of P. granatum at 50 or 100 mg kg −1 dose clearly enhance the innate immune responses and disease resistance after 8 weeks in P. olivaceus against natural LDV infection.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 °C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection.

Lymphocystis disease virus persists in the epidermal tissues of olive flounder, Paralichthys olivaceus (Temminch & Schlegel), at low temperatures

Olive flounder artificially infected with lymphocystis disease virus (LCDV) were reared at 10, 20 and 30 degrees C for 60 days, to compare LCD-incidence. In the fish reared at 20 degrees C, lymphocystis cells appeared on the skin and fins at 35 days post-challenge, and the cumulative LCD-incidence was 80% at 60 days. High levels of LCDV, with a mean polymerase chain reaction (PCR) titre of 10(6) PCR-U mg(-1) tissue, were detected in the fins and skin of LCD-affected fish at 20 degrees C, but were not detected in the spleen, kidney, brain and intestinal tissues of these fish. No LCD clinical signs were observed in the fish reared at 10 degrees C and 30 degrees C; however, a low level of LCDV (10(3) PCR-U mg(-1) tissue) was detected in the fins and skin of these fish. By increasing the rearing temperature from 10 to 20 degrees C, lymphocystis clusters appeared on the skin and fins of the fish with no previous LCD clinical signs within 33 days after the temperature change. It was shown that permissive cells for LCDV infection exist in the epidermis of olive flounder. At low temperatures, small amounts of LCDV were able to persist over a period extended for a further 45 days in the fish epidermis, even though the fish showed no LCD clinical signs. The optimum growth temperature of LCDV is near 20 degrees C.

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt‐head seabream, Sparus aurata L.

Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease.

Whitespotted puffer Arothron hispidus, a new host for lymphocystis in Qingdao Aquarium of China

In April 2004 white nodular lesions were found on the fins of whitespotted puffer Arothron hispidus (Linnaeus). Diagnostic studies were carried out to confirm the disease using light and electron microscopy, histochemical methods and PCR. The results revealed that the nodules were composed of giant cells up to 400 microm in diameter. These cells were surrounded by a periodic acid-Schiff (PAS)-positive hyaline capsule containing dot-shaped, Feulgen-positive inclusion bodies in the cytoplasm and an irregular nucleus. Numerous virus particles 200 nm in diameter and with hexagonal profiles were observed in the cytoplasm. These features were consistent with those of lymphocystis disease. Additionally, based on the gene sequences of major capsid protein (MCP) of lymphocystis disease virus (LCDV) from Japanese flounder Paralichthys olivaceus, 2 pair primers were designed; after a nested PCR was performed for detection of LCDV in A. hispidus, a positive amplified product was obtained showing the presence of LCDV. Therefore, the white nodules were the lymphocystis lesions caused by LCDV infection and A. hispidus was demonstrated to be a new host for LCDV.

Induction of apoptosis in a flounder gill cell line by lymphocystis disease virus infection

Lymphocystis disease virus (LCDV), a large icosahedral DNA virus classified to the iridovirus family, is the causative agent of lymphocystis, a disease which occurs in marine and freshwater fish species and is characterized by formation of papilloma-like lesions on the surface of the skin. In vitro, LCDV infection causes flounder gill cells, an adherent cell line, to exhibit an obvious cytopathic effect (CPE). In order to test whether apoptosis is responsible for the observed CPE, cells infected with LCDV at a multiplicity of infection (m.o.i.) of 5 PFU per cell were examined at various time intervals for the appearance of apoptotic signs. Nuclear fragmentation, DNA laddering and caspase activation were observed in the infected cells at the time (i.e. 10 days post-infection) when an intensive CPE was observed. These findings demonstrate that LCDV is capable of inducing apoptosis in vitro, which is different from the result of LCDV infection in vivo, and consequently suggest an intricate LCDV-host interaction.

Molecular anatomy of lymphocystis disease virus.

Lymphocystis disease (LD) has been reported to occur in over one hundred different species of fish worldwide. The disease is caused by lymphocystis disease virus (LCDV), a member of the iridovirus family. Numerous fish species that play an important role in fishery and fish farming are highly susceptible to LCDV infection. The infected animals develop disseminated clusters of aberrant hypertrophied cells within their connective tissue, the so-called lymphocystis cells. In the cytoplasm of these cells a massive accumulation of virions can be observed. As a first step towards understanding the mechanisms of viral infection and pathogenesis the complete genomic nucleotide sequence of lymphocystis disease virus type 1 (LCDV-1; flounder isolate) was determined. LCDV-1 is the type species of the genus Lymphocystivirus within the family Iridoviridae. The virions contain a single linear double-stranded DNA molecule that is circularly permuted, terminally redundant and heavily methylated. Since there is no convenient cell system for virus replication we determined the complete nucleotide sequence of the viral genome (102,653 base pairs). Computer assisted analyses of 195 potential open reading frames resulted in the identification of a number of putative gene products with significant homology to functionally characterized proteins of other species.