A novel lectin domain-containing protein (LvCTLD) associated with response of the whiteleg shrimp Penaeus (Litopenaeus) vannamei to yellow head virus (YHV) Kingkamon Junkunlo a,b , Anuphap Prachumwat a,c , Amornrat Tangprasittipap a,c , Saengchan Senapin a,b,d , Suparerk Borwornpinyo c , Timothy W. Flegel a,b,d , Kallaya Sritunyalucksana a,b,c,⇑ a Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand b Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand c Shrimp–Virus Interaction Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Klong Luang, Pathumthani 12120, Thailand d National Science and Technology Development Agency (NSTDA), Thailand Science Park, Klong Luang, Pathumthani 12120, Thailand article info Article history: Received 24 October 2011 Revised 19 December 2011 Accepted 19 December 2011 Available online 27 December 2011 Keywords: C-type lectin LDL receptor Penaeus (Litopenaeus) vannamei Yellow head virus Recognition The proPO system abstract When using mRNA from gills of normal whiteleg shrimp Penaeus (Litopenaeus) vannamei as the tester and mRNA from yellow head virus (YHV)-infected shrimp as the driver, subtractive suppression hybridization (SSH) revealed that a novel EST clone of 198 bp with a putative C-type lectin-like domain (CTLD) was downregulated in YHV-infected shrimp. The clone nucleotide sequence had 99% identity with one contig MGID1052359 (1,380 bp) reported in an EST database of P. vannamei, and the presence of this target in normal shrimp was confirmed by RT-PCR using primers designed from the MGID1052359 sequence. Analysis of the primary structure of the deduced amino acid (a.a.) sequence of the contig revealed a short portion (40 a.a. residues) at its N-terminus with high similarity to a low density lipoprotein receptor (LDLR) class A domain and another 152 a.a. residues at its C-terminus with high similarity to a C-type lectin domain. Thus, the clone was named LvCTLD and three recombinant proteins (LvCTLD, the LDLR domain and the CTLD domain) were synthesized in a bacterial system based on its sequence. An in vitro encapsulation assay revealed that Sepharose 4B beads coated with rLvCTLD were encapsulated by shrimp hemocytes and that melanization followed by 24 h post-encapsulation. The encapsulation activity of rLvCTLD was inhibited by 100 mM galactose, but not mannose or EDTA. In vivo injection of rLvCTLD or rLvCTLD plus YHV resulted in a significant elevation of PO activity in the hemolymph of the challenged shrimp when compared to shrimp injected with buffer, suggesting that rLvCTLD could activate the proPO system. An ELISA test revealed that rLvCTLD could bind to YHV particles in the pres- ence of shrimp hemolymph. Phylogenetic analysis suggested that the LvCTLD sequence was more closely related to an antiviral gene found in Penaeus monodon (PmAV) than to other reported shrimp lectins. Taken together, we conclude that a novel shrimp LvCTLD is a host recognition molecule involved in the shrimp defense mechanism against YHV via recruitment of hemocytes, probably at the site of viral infection, and via activation of the proPO system. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Shrimp aquaculture is an important economic activity of many countries around the world and shrimp viral diseases are considered a major constraint for development of a sustainable culture indus- try. It is generally believed that a better understanding of how shrimp respond to viral infection at the molecular level will lead to new methods for disease prevention and control (Flegel and Sritu- nyalucksana, 2011; Liu et al., 2009). A number of proteins have been proposed to be involved in shrimp antiviral defense. These include PmRab7 (Sritunyalucksana et al., 2006), Pm-fortilin (Tonganunt et al., 2008), anti-lipopolysaccharide factor (ALF) (Liu et al., 2006) and lectins (Luo et al., 2003; Ma et al., 2007, 2008). Lectins have been reported to be involved in several cellular immune responses includ- ing apoptosis, encapsulation, phagocytosis and nodule formation. In invertebrates, encapsulation is a process by which hemocytes attach to non-self materials and lead to their melanization and destruction (Schmit and Ratcliffe, 1977). Recently, Penaeus (Penaeus) monodon lectin (PmLT) has been shown to stimulate hemocyte encapsulation when coated on agarose beads, suggesting a role in the shrimp innate immune response (Ma et al., 2008). Expression of many pen- aeid shrimp C-type lectin genes have been shown to be modulated by viral infection in shrimp. For example, the levels of LvLT from Penaeus (Litopenaeus) vannamei (Ma et al., 2007), FcLectin (Liu et al., 2007), Fc-hsl (Sun et al., 2008), FcLec2 (Zhang et al., 2009) 0145-305X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.12.010 ⇑ Corresponding author at: Shrimp–Virus Interaction Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Klong Luang, Pathumthani 12120, Thailand. Tel.: +66 26448150x449; fax: +66 23547344. E-mail address: kallaya@biotec.or.th (K. Sritunyalucksana). Developmental and Comparative Immunology 37 (2012) 334–341 Contents lists available at SciVerse ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci