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Suppression subtractive hybridization (SSH) was employed to identify yellow head virus (YHV)-responsive genes from the hemocytes of the black tiger shrimp, Penaeus monodon. Two SSH cDNA libraries were constructed to identify viral responsive genes in the early (24I) and late (48/72I) phases of YHV infection. From 240 randomly selected clones from each library, 155 and 30 non-redundant transcripts were obtained for the early and late libraries, respectively. From these clones, 72 and 16, respectively, corresponded to known genes (E-values < 1 x 10(-4)) that could be categorized according to their putative functions. The upregulated genes identified as likely to be associated with cell defense and homeostasis were found at a high proportion in the 24I SSH library, but not in 48/72I SSH library implying that these immune molecules participate in viral defense immunity in the early phase of YHV infection whereas their expressions were suppressed in the late phase of infection. Novel YHV-responsive genes were uncovered from these SSH libraries including caspases, histidine triad nucleotide-binding protein 2, Rab11, beta-integrin, tetraspanin, prostaglandin E synthase, transglutaminase, Kazal-type serine proteinase inhibitor and antimicrobial peptides. Among these YHV-responsive genes, several have been previously reported to participate in defense against white-spot syndrome virus (WSSV) implying that YHV infection in shrimp induces similar host immune responses as observed during WSSV infection. The expression of four apparently upregulated immune-related genes identified from the two SSH libraries, anti-lipopolysaccharide factor isoform 6 (ALFPm6), crustin isoform 1 (crustinPm1), transglutaminase and Kazal-type serine proteinase inhibitor isoform 2 (SPIPm2), was evaluated by real-time RT-PCR to reveal differential expression in response to YHV infection at 6, 24, 48 and 72 h post-infection. The results confirmed their differential expression and upregulation, and thus verified the success of the SSHs and the likely involvement of these genes in shrimp antiviral mechanisms., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Yellow head virus (YHV) is an invertebrate nidovirus that has caused mass mortality in penaeid shrimp since 1990. Several YHV types are known, but only the original type (YHV-type 1 or YHV-1) is highly virulent. Most studies have focused on acute YHV-1 infections and there is limited work on YHV-1 survivors. We compared moribund and surviving (14%) whiteleg shrimp Penaeus (Litopenaeus) vannamei from an experimental challenge with YHV-1. Although grossly normal, all survivors were positive for YHV-1 by specific, reverse transcriptase polymerase chain reaction (RT-PCR) assays, histological analysis or transmission electron microscopy (TEM), indicating that they were not resistant but tolerant to YHV-1. On the other hand, real-time PCR analysis revealed that mean YHV-1 copies/ng total RNA for survivors (2.8x10(4) +/- 6.9x10(4)) were approximately 40 times lower (P<0.05) than those in moribund shrimp (1.2x10(6) +/- 6.7x10(5)copies/ng total RNA). This was confirmed by strong positive immunohistochemical and in situ hybridization (ISH) reactions for YHV-1 in lymphoid organ tubules (LOT) of moribund shrimp and weak positive reaction only in lymphoid organ spheroids (LOS) of survivors. TEM revealed morphologically complete YHV virions in both groups. Furthermore, immuno-TEM and Western blot analysis revealed that YHV-1 structural proteins gp116 and p20 were present at comparable reactive levels in each group. Thus, YHV-1 tolerance was not associated with absence of gp116 as previously reported for palaemonid shrimp. Instead, it was associated with the presence of YHV-positive LOS and a relatively low viral load.