Antiviral responses and biological targets of a kazal-type serine proteinase inhibitor spipm2 from the black tiger shrimp Penaeus monodon

White spot syndrome (WSS) is a severe viral disease in Penaeus monodon which causes high mortality. The understanding of shrimp immunity will lead to the knowledge for prevention and solving the disease. In black tiger shrimp, several proteins are expressed in response to viral infection. Among others, a Kazal-type serine proteinase SPIPm2 is abundantly expressed in the hemocytes and shown to be involved in innate immune response against white spot syndrome virus (WSSV). In the healthy hemocyte, the immunofluorescent and immunogold labeling techniques showed that the SPIPm2 was expressed and stored in the granule of semi-granular and granular hemocytes. Immunocytochemical study showed that, after WSSV infection, the percentage of SPIPm2-producing hemocytes was reduced by about half. Expectably, the SPIPm2 was secreted readily from these hemocytes after WSSV challenge. The recombinant SPIPm2 (rSPIPm2) was used to investigate the effect of SPIPm2 on viral infection in shrimp. Injection of rSPIPm2 prior to WSSV injection prolonged the mortality rate of WSSV-infected shrimp. By using shrimp primary hemocyte cell culture, the antiviral activity was neutralization and protection. The rSPIPm2 temporarily and dose-dependently neutralizes the WSSV and protects the hemocytes from viral infection judging from the substantially less expression of WSSV late gene VP28. The antiviral activity was very likely due to the binding of SPIPm2 to the components of viral particle and hemocyte cell membrane. The yeast two-hybrid screening had identified a viral target protein of SPIPm2 in the WSSV cDNA library, namely WSV477. The WSV477 was reported to have ATP/GTPase activity. In vitro pull down assay confirmed the protein-protein interaction between rSPIPm2 and rWSV477. In WSSV-infected culture, silencing of WSV477 by dsRNA-WSV477 reduced the expression of viral late gene VP28 and, hence, the reduction of viral infectivity.