| Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the important pathogens causing farmed and wild shrimp diseases. It is the knowned smallest shrimp virus and belongs to the Parvoviridae family and most closely related to the genus brevidensovirus. Though IHHNV has not been associated with mass mortalities in recent years, it is widespread and results in a disease called runt-deformity syndrome in Litopenaeus vannamei cultured by a lot of countries. At present, the complete or partial IHHNV genomic sequences, which are isolated from Hawaii, California, Thailand and Taiwan in China, have been deposited in GenBank. Although the IHHNV-infected penaeid shrimps were detected frequently in Chinese mainland, heretofore no IHHNV genome sequence from Chinese isolate was reported. In this paper, the IHHNV genome ends were cloned and sequenced by means of the addition ofpoly (A) to the 3' end of the DNA and nested PCR. According to sequences of the 5' and 3' ends termini, the full-length IHHNV genome was amplified by PCR with specific primers and sequenced. The full-length IHHNV genome isolated from Fujian contains 3833nt and shows high similarity to Hawaii and California isolation except for the main difference at the 5' terminal of the DNA. The viral genome analysis revealed three large open reading frames (ORFs), encoding two nonstructural proteins NS1 and NS2, as well as a capsid protein CP by analogy to other parvoviruses. In this work, the full-length IHHNV capsid protein(ICP) with the 6×His-Tag at its N terminal was expressed in Escherichia coli and purified by Ni-NTA agarose affinity matrix under native or denatured condition. The negative stain electron micrography analysis revealed that both the cell lysate supernatant and purified soluble ICP contained spherical VLPs with homogeneous size and shape. IHHNV VLPs with average 20 nm in diameter were similar to the native IHHNV particles and were specifically immunolabelled with a mouse polyclonal antibody against recombinant ICP (rICP). The denatured structural proteins failed to spontaneously renature and assemble into polymorphologic aggregations in vitro aider removal of the denaturant. Results presented in this study revealed that the full-length ICP alone was sufficient to self-assemble into VLPs in the absence of other IHHNV protein or its nucleic acid. In addition, the VLPs could be purified by Ni-NTA affinity chromatography, which strongly indicated that the N-terminus of ICP is located on the outer surface of VLP. Furthermore, immunofluorescence microscope showed that rICP had the capacity of binding and entry into the hemocytes of Penaeus wannamei. Based on the following facts: (i) IHHNV has similar host range to WSSV. (ii) Co-infection with WSSV has been reported. (iii) L. stylirostris that are highly infected with IHHNV show resistance to WSSV infection. We consider that IHHNV VLP may be a very promising vehicle for delivering drug or anti-virus material (such as siRNA) into shrimp cells against WSSV.
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