Purification and antibacterial characterization of a novel isoform of the Manila clam lectin (MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum.

In many bivalve molluscs, lectins are present in the hemolymph and are thought to be important for internal host defense mechanisms. For this study, we purified a novel isoform of the Manila clam lectin (designated MCL-4) from the plasma of the Manila clam, Ruditapes philippinarum, using affinity chromatography and gel filtration. Native PAGE results showed that the MCL-4 consisted of 70 kDa protein. MCL-4 was found to be composed of 58-kDa and 43-kDa bands when examined using SDS-PAGE under reducing and non-reducing conditions. The native MCL-4 was revealed as a 147 kDa molecular mass protein by gel filtration. The purified MCL-4 agglutinates calcium-dependently in the erythrocytes of sheep and rabbit, but not in cells of the three species of marine bacteria tested. However, the phagocytic ability of the R. philippinarum hemocytes for the MCL-4-opsonized Vibrio tubiashii cells was significantly greater than that for the BSS-treated bacterial cells. Addition of purified MCL-4 markedly suppressed Alteromonas haloplanktis growth. These results suggest that MCL-4, because of its opsonizing and bacteriostatic properties, might contribute to the host defense mechanisms against invading microorganisms in R. philippinarum.

In vivo and in vitro analysis of the resistance against viral haemorrhagic septicaemia virus in Japanese flounder (Paralichthys olivaceus) precedingly infected with aquabirnavirus.

The resistance of Japanese flounder (Paralichthys olivaceus Temminck et Schlegel) against a viral haemorrhagic septicaemia virus (VHSV) challenge induced by a preceding non-lethal aquabirnavirus (ABV) challenge was investigated through experimental dual-infections with different intervals between the two challenges. The non-specific protection conferred by the primary ABV infection against the secondary VHSV infection commenced at Day 3 and persisted up to Day 14 but vanished at Day 21 post-ABV challenge. The in vitro assay using HINAE (hirame natural embryo) cells demonstrated anti-VHSV activity in the serum of ABV-challenged flounder from Day 1 to Day 14 but not at Day 21 post-ABV challenge. A high expression of a Mx gene, a molecular marker of type I interferon(s) (IFN) occurred in the head kidneys of ABV-challenged flounder from Day 1 to Day 7. These results suggest that the non-specific protection against the secondary VHSV infection in flounder was due to IFN(s) induced by the primary ABV infection.

Mapping a neutralizing epitope on the coat protein of striped jack nervous necrosis virus.

Striped jack nervous necrosis virus (SJNNV), a fish nodavirus, is the causative agent of viral nervous necrosis in marine fishes. The fish nodaviruses are divided into four different genotypes based on the nucleotide sequence of the coat protein gene. In the present study, partial coat protein genes of fish nodaviruses were expressed. This allowed the serological relationship among the different virus genotypes to be analysed and neutralizing epitopes on the coat protein to be mapped. Western blot analysis revealed that SJNNV and other fish nodavirus genotypes shared a significant number of common antigenic determinants, although SJNNV was serologically distinguishable. The results suggested that the SJNNV determinant for neutralizing MAbs was a linear epitope, which consisted of a repeated amino acid sequence within the coat protein. One of the neutralizing epitopes of SJNNV was deduced to be PAN at aa 254-256 in the coat protein.