White spot syndrome virus (WSSV) was assigned to the genus Whispovirus belonging to new family Nimaviridae in the universal database of ICTV (International Committee of Taxonomy of Viruses, http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm). WSSV is probably the most important pathogen of the cultured penaeid shrimp resulting in high mortalities 1. Even though WSSV represents one of the largest known animal viruses with a 305 kb double-stranded circular DNA genome, most of the putative 185 ORFs bear no homology to known genes in the GenBank 23. The technical difficulty in characterization of the WSSV ORFs lies mainly in the lack of established shrimp cell lines for in vitro reproduction of the virus 4. During viral lytic infection, just as other DNA viruses, the genes encoded by WSSV can be classified as immediately early, delayed early, late and very late genes. Most, if not all, immediate-early genes encode transcriptional regulation proteins. They are distinguished from other viral genes by the fact that their transcription is independent of prior viral gene product expressed in transient assays 5. Although during the last decade, intensive efforts have been undertaken for characterization of the structural protein genes and a few non-structural protein genes that show homology to known sequences in the databases, little is known about the molecular mechanisms underlying the WSSV life cycle and mode of infection.

Recently, three viral transcripts (Orf427, Orf151 and Orf366) and their corresponding DNA sequence have been detected in both specific-pathogen-free (SPF) shrimps and WSSV-infected shrimps through a WSSV-specific DNA microarray study. From this study, Orf427, Orf151 and Orf366 were determined to be latency-associated genes of WSSV 6. These data suggest that WSSV remains latent in healthy shrimps. In a similar global analysis, three immediately early (IE) genes (ie1, ie2, and ie3) of WSSV were identified in infected shrimps 7. Identification of the IE genes and latency-associated genes can lead to better understanding of the life cycle of WSSV, shedding light on the molecular mechanisms in WSSV-induced mortality. In a previous study, we have found that latency-related ORF427 interacted with a shrimp protein phosphatase (PPs) 8. To further characterize the orf427 gene, we were interested to analyze the promoter of orf427 and its expression during viral pathogenesis.

To investigate whether promoter of orf427 is active without the existence of other viral proteins in the host cells, we tried to establish in vitro culture of fragments from lymphoid organ as reported previously 9. However, the primary shrimp cells were very sensitive to standard liposome-based transfection reagents. Thus, for the promoter analysis, we employed a transduction method mediated by baculovirus 10. Recombinant baculoviruses bearing EGFP-expressing cassettes were produced according to pFASTBac1 manufacturer instructions (Invitrogen) (Fig. 1A). Budded virus from insect cell culture medium was concentrated by ultrafiltration and infectious titers of both stock viruses were determined by plaque assay and adjusted to be 10¹⁰ plaque-forming units (PFU)/ml.

Infection of SF9 cells and transduction of shrimp primary cells with the recombinant baculovirses were carried out at a MOI of 10 and 100, respectively. As expected, the ie1 promoter drove the expression of the egfp reporter gene in both insect SF9 and the primary shrimp cells, as demonstrated by direct light and fluorescence microscopy; while the orf427 promoter didn't express egfp to a detectable level in either cell type (Fig. 1B and 1C). The expression of GFP could be confirmed in both cells through immunoblot assay using monoclonal anti-GFP antibody (Fig. 1D). We also noticed that the primary shrimp cells could only be transduced at a low percentage of about 5% (Fig. 1C).

In most cases, viruses establish latency in specific tissue(s). To test whether orf427 is transcribed only in specific latency sites or in all the tissues that support viral infection, in situ hybridization was performed on paraffin embedded tissue sections from shrimps at late infection (4 days after viral inoculation) using DIG-labeled antisense RNA probes specific for orf427. Results shown in fig. 2 indicated that in contrast to the control shrimp sections, orf427 was extensively transcribed in all the WSSV infected tissue sections including subcuticular epithelium cells (Fig. 2I), hemocytes lodged in the connective tissues (Fig. 2II), and stomach chamber lining cells (Fig. 2III). Also, we expressed and purified partial fragment of ORF427 in a GST-fusion form. Protein purity of the purified protein was more than 90% as judged by SDS-PAGE (figure not shown). Polyclonal antibody was developed by injection of the protein into Guinea pigs. ORF427 can be detected from homogenized infected shrimps through immunoblot assay using the anti-ORF427 antibody (Fig. 3).

In order to determine whether orf427 is transcribed in the early phase during viral lytic infection, we employed a RT-PCR approach to detect the transcriptional products of orf427. The sequences of the primers used are shown in Fig. 4A. P. monodon shrimps challenged through intramuscular injection with WSSV were sampled at different time points after viral inoculation, and total RNAs were extracted from the shrimp heads for RT-PCR analysis. As controls, fragments corresponding to the WSSV immediately early gene ie1 7, delayed early gene dnapol 11, and late gene vp28 12, were also amplified from the same RNA samples. A shrimp β-actin primer set was used as an internal control for RNA quality and amplification efficiency. Our results show that orf427 is only transcribed after 6 h post infection (Fig. 4B), which is at the late phase during viral lytic infection. As expected, ie1 can be detected from 3 h p.i., while dnapol and vp28 can be detected from 6 h p.i. (Fig. 4B).

Establishment and maintenance of latency in the host after primary infection have been investigated in some well-studied DNA viruses such as: herpes simplex virus (HSV) 13, human herpesvirus (HHV) 14, cytomegalovirus (CMV) 15, and Epstein-Barr virus 16. However, the molecular mechanisms that control virus latency and reactivation remain to be elucidated. Because of problems associated with conducting molecular studies in animals, it has proven difficult for investigators to move beyond phenomenal description and identification of latency-associated transcripts (LATs). Most of the characterized LATs were expressed at low levels during lytic replication but were major transcripts during latent infection, and their functions were not understood. These include a set of latency-associated transcripts from the HHV-6 IE-A region 17, a set of genes controlled by the Qp promoter of Epstein-Barr virus 16, and latency-associated transcripts from both DNA strands in the ie1/ie2 region of CMV 15. U94 gene of HHV-6 is one of the better-characterized LATs. U94 protein acts as a transactivator by binding to a transcription factor and enables the establishment and/or maintenance of latent infection at the primary infection site like monocytes and early bone marrow progenitor cells 18. Our data indicate that orf427 is a very late gene during viral lytic infection, and this correlates with the finding that ORF427 is not a transcriptional regulator, but a protein phosphatase-interacting protein 8.

Most recently, nuclear protein phosphatase-1 was reported to regulate HIV-1 transcription both in vitro and in vivo 19. Primary functional dissection of Orf427 suggests that orf427 most likely encodes a calcium-binding regulator of shrimp protein phosphatase, with the C terminus responsible for the binding of PPs (data not shown). This suggests that orf427 is not necessary for viral reactivation and only contributes to maintaining viral latency by affecting the function of shrimp protein phosphatase. Similarly, the LAT gene of HSV-1 has been shown to be dispensable for viral reactivation from latently infected mouse dorsal root ganglia cultured in vitro 20.

The development of a continuous shrimp cell line in vitro is urgently required for further characterization of WSSV infection at the molecular and cellular levels. In recent years, encouraging progress has been made in shrimp cell culture using conventional primary culture techniques. Several investigators have reported that WSSV infects the primary cultures of lymphoid organs from the black tiger shrimp, P. monodon; however, recent findings suggest that the replication of WSSV in lymphoid organ primary cell is low 4921. Besides this, the primary cell couldn't be transfected with common liposome methods. We thus took alternative approach to monitor the gene expression in the primary shrimp cells. Recently AcMNPV (Autographa californica multiple nucleopolyhedrovirus), containing an appropriate eukaryotic promoter, was used to efficiently transfer and express foreign genes in a variety of mammalian cells and several animal models 22. Considering that shrimp is more phylogenically related to arthropods, the natural host of AcMNPV, we employed recombinant baculovirus-mediated transduction to introduce foreign genes into the primary shrimp cells. As expected, the primary shrimp cells were transduced in our experiments; and the low transduction efficiency might be due to the possible inhibition effect of L15 medium on the attachment of baculovirus to the cell membrane (for example, the pH value of medium for insect cells to amplify baculovirus is 6.8, while the pH value of L15 medium is above 7.0). The transduction efficiency might be significantly increased by using VSV-G-containing baculovirus as gene delivery vehicle 10. The successful transduction of cultured shrimp cells with recombinant baculovirus may pave the way for the development of baculovirus-based vaccines for the shrimp farming industry.

The data presented here demonstrates that latency-associated Orf427 is only transcribed in the very late phase during viral lytic infection. In contrast to immediately early promoters, the promoter of orf427 couldn't drive the expression of an egfp reporter gene independently. Our data suggest that as a very late protein during viral lytic infection, ORF427 might only function in maintaining WSSV in the latent phase but is not required for virus reactivation.

The author(s) declare that they have no competing interests.

Jimmy Kwang designed the study and critically reviewed the manuscript. Liqun Lu performed all the experiments and wrote the manuscript. Wang Hai helped perform in situ hybridization. Ivanus Manopo helped prepare shrimp primary cells and critically review the manuscript. Yu Li constructed and tested the plasmid containing WSSV ie1 promoter.