Identification Of A Novel White Spot Syndrome Virus (WSSV)-induced Gene (MjVIG1) In Marsupenaeus Japonicus

Shrimp aquaculture production has rapidly expanded worldwide over the last few decades,and shrimp is considered the most economical commodity in fishery production.However,invasive pathogens are a critical risk factor with the rapid development of shrimp aquaculture industry.Marsupenaeus japonicus,an important shrimp species in Asia,has ever provided good economic benefits to farmers.Since various shrimp diseases outbreak in 1990 s,M.japonicus has also suffered a great loss.White spot disease is one of the most serious shrimp viral diseases,and its pathogen is white spot syndrome virus(WSSV).WSSV is a rod-shaped,double-stranded enveloped DNA virus that belongs to family Nimaviridae,genus Whispovirus,which is highly lethal to penaeid shrimp.Its wide spread has caused tremendous economic losses to the world shrimp farming.Until now,there is still no effective precautions.In addition,another yellow head virus(YHV)has also been reported to cause mass mortality in shrimp aquaculture.YHV is a bacilliform,enveloped,positive-sense single-stranded RNA virus classified in the family Roniviridae.It is reported that so far no drugs or chemicals have been able to control the virus.In this study,we will also mention one additional shrimp disease named as acute hepatopancreatic necrosis disease(AHPND),which caused by bacterial pathogen Vibrio parahaemolyticus.Information regarding the viral denfense system in penaeid shrimp is still limited.As invertebrates,crustaceans including penaeid shrimp solely depend on their innate immune systems for defense against pathogenic microorganisms.In general,the innate immunity contains two important defense systems,humoral and cellular responses.The shrimp hemolymph doesn’t have immunoglobulin,but contains such as lysozymes,lectins and superoxide dismutase,especially the humoral immune factors of prophenoloxidase(ProPO)activated system which released by the hemocytes.All of these immune factors play essential roles in the humoral immune responses.In crustaceans,they mainly function as recognizing the non-self materials and againsting the invasive pathogens.Meanwhile,cellular responses are closely related to humoral responses,which have the activities performed by hemocytes directly such as phagocytosis,encapsulation and nodule formation.Based on morphological classification,shrimp hemocytes were divided into three types,namely hyaline cells(HCs),semi-granular cells(SGCs)and granular cells(GCs).Different cell types have different functions but with some overlapping such as HCs performing phagocytosis;SGCs working in encapsulation,phagocytosis,prophenoloxidase(ProPO)system and cytotoxicity;and GCs playing an important role in the ProPO system.A newly reported study separated shrimp hemocytes into upper and lower two different density sub-popμlations by Percoll gradient centrifugation,and conducted transcriptome analysis.Several highly expressed transcripts were homologous to immune-related genes from upper layer,suggesting this layer’s hemocytes may play as the regulator of immune system and help to eliminate pathogens.Some contigs were differentially expressed among hemocyte sub-populations;among them,contig c15785_g1 was strongly expressed in the upper layer.Although c15785_g1 is an unknown gene,it may play an important immune regulatory role in hemocytes.In this study,I aimed to clone and characterize the novel contig c15785_g1.This new gene was found to be the first WSSV induced gene in M.japonicus hemocytes after performing different assays,and is hereafter referred to as MjVIG1.According to the EST sequence of MjVIG1 in the transcriptome sequencing results,the full MjVIG1 cDNA sequence was obtained by primer design and RACE PCR(rapid amplification of cDNA ends PCR).Sequence analysis showed that it had no similarity to known characterized protein.Furthermore,the result showed that MjVIG1 mRNA expression can be detected in many tissues,while expressed highest in hemocytes.During WSSV infection,MjVIG1 transcript level significantly increased.In contrast,MjVIG1 transcript level did not change after YHV and V.parahaemolyticus infections,suggesting MjVIG1 may have a specific immune response with WSSV.Moreover,immunostaining results revealed that MjVIG1 was expressed by a small portion in granule-containing hemocytes.This,together with flow cytometry results,showed that the ratio and number of MjVIG1 positive hemocytes was significantly increased after WSSV infection.In this study,knock down of MjVIG1 gene expression through specific dsRNA was detected,but it still need more information to verify its function.In summary,MjVIG1 might be a new gene which was induced by WSSV in M.japonicus hemocytes.In response to viral infections,a major step in the initation of the host defense system is the induction of host cell genes.Viruses induce the up-regμlation of many genes,some of which encode antiviral proteins that have antiviral acitivity.Therefore,functional analysis of MjVIG1 is needed to be further investigated and it may provide additional information in understanding shrimp’s innate immune system especially under threat by WSSV.