ORIGINAL ARTICLE Expression and self-assembly of virus-like particles of infectious hypodermal and hematopoietic necrosis virus in Escherichia coli Luhong Hou Æ Hao Wu Æ Limei Xu Æ Feng Yang Received: 12 December 2008 / Accepted: 9 February 2009 / Published online: 28 February 2009 Ó Springer-Verlag 2009 Abstract Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the highly pathogenic shrimp viruses. In this study, IHHNV capsid protein (CP) was recombinantly expressed in Escherichia coli and found to self-assemble into virus-like particles (VLPs) with homogeneous size and shape similar to native IHHNV par- ticles. Furthermore, we found that IHHNV-VLPs encapsidate RNA and DNA with a predominant size of 0.5 kb. Stability experiments revealed that the VLP could not be disassembled by EDTA, but its structure was completely disrupted by dithiothreitol (DTT). Non-reducing SDS/PAGE showed that no intermolecular disulfide bonds were formed between the CP molecules, suggesting that intra-CP disulfide bonds are essential for maintaining the structural integrity of the capsid. In addition, indirect immunofluorescence microscopy anal- ysis showed that the VLPs could efficiently enter primary hemocytes of the shrimp Litopenaeus vannamei, which implied that IHHNV-VLPs may be promising vehicles for the delivery of antiviral agents. Introduction Since 1970s, more than 20 shrimp viruses have been reported [21]. Viral diseases have become a major problem in shrimp culture and are believed to be responsible for approximately $1 billion loss per year in the world since 1994 [5]. The viruses with the most impact on shrimp culture are white spot syndrome virus (WSSV), Taura syndrome virus (TSV), infectious hypodermal and hemato- poietic necrosis virus (IHHNV) and yellowhead virus (YHV). IHHNV, a small non-enveloped virus, was first detected in 1981 from farmed penaeid shrimps [19, 20]. However, little was known about its molecular properties until sufficient IHHNV virions were purified from infected shrimp [2]. The purified virion contains a *4 kb single strand of DNA packaged in an icosahedral capsid approx- imately 22 nm in diameter. In 2000, the IHHNV genome was sequenced (GenBank accession no. AF273215) and showed three open reading frames, which encode two nonstructural proteins (NS1, NS2) and one structural capsid protein (CP) [33]. Its genomic organization is similar to that of mosquito viruses of the genus Brevidensovirus, which belong to the family Parvoviridae [7]. Over the past 20 years, the structural protein(s) of many viruses have been demonstrated to be able to self-assemble into virus-like particles (VLPs) when expressed in eukaryotic or prokaryotic expression systems. VLPs are generally similar to natural viral particles in size, shape, cellular uptake and intracellular trafficking, but non- infectious due to the lack of a viral genome, so they could act as a vehicle system for therapeutic purposes, such as delivery of nucleic acids, proteins, drugs, or small mole- cules [4, 8, 16, 17, 23, 26, 27, 31]. Although no relevant studies have been reported on expression and assembly of IHHNV-CP, certain CPs from other parvoviruses have Luhong Hou and Hao Wu contributed equally to this paper. L. Hou School of Life Sciences, Xiamen University, Xiamen 361005, People’s Republic of China H. Wu Á L. Xu Á F. Yang (&) Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, SOA, 178 Daxue Rd., Xiamen 361005, People’s Republic of China e-mail: mbiotech@public.xm.fj.cn F. Yang College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, People’s Republic of China 123 Arch Virol (2009) 154:547–553 DOI 10.1007/s00705-009-0336-6