A sensitive and specific SYBR Green-based qPCR assay for detecting scale drop disease virus (SDDV) in Asian sea bass.

Scale drop disease virus (SDDV) is a megalocytivirus known to cause disease in Asian sea bass in Southeast Asia. To support SDDV diagnosis and surveillance, we report on a sensitive and specific SYBR Green qPCR assay. The qPCR primers were designed to target a 135 bp fragment of the SDDV ATPase gene. The optimized SDDV qPCR assay reliably detected 2 copies of a plasmid dsDNA control and did not cross-amplify DNA to any of 12 viral or bacterial pathogens commonly found in aquatic animals. When assessed with 86 field samples, the assay detected SDDV in DNA extracted from each of 34 scale drop disease-affected fish collected from 5 affected farms. The qPCR also detected SDDV in DNA from 30 of 52 overtly healthy fish collected from 9 farms where SDDV had not been detected previously, using a semi-nested conventional PCR. The higher sensitivity of our SDDV qPCR assay can thus be useful in detecting fish with subclinical/chronic infections. However, the qPCR showed that SDDV DNA loads varied from 8.0 × 102 to 6.8 × 104 viral DNA copies per 200 ng DNA template among the 8 organ tissue types sampled from 3 diseased fish. In circumstances requiring SDDV to be detected unequivocally in subclinical carriers with lower-level infection, qPCR testing of more than one type of tissue is advisable.

C-terminal domain of WSSV VP37 is responsible for shrimp haemocytes binding which can be inhibited by sulfated galactan.

Viral envelope proteins play an important role in facilitating the attachment of viruses to the surface of host cells. Here, we investigated the binding of White Spot Syndrome Virus (WSSV) VP37 to haemocytes of whiteleg shrimp, Litopenaeus vannamei. Three versions of recombinant VP37 proteins, including full length VP37 (VP37(1-281)), C-terminal domain VP37 (VP37(111-281)) and C-terminal domain disrupted VP37 (VP37(1-250)) were individually expressed and tested for their haemocytes binding ability. Through an ELISA-based binding assay, we found that VP37(111-281) bound to shrimp haemocytes in a similar way to VP37(1-281), while VP37(1-250) exhibited a significantly weaker binding. This suggests that the C-terminal domain of VP37 is required for the binding of VP37 to shrimp haemocytes. Furthermore, we found that the binding of VP37 to shrimp haemocytes was impaired by pre-incubation of VP37 with sulfated galactan (SG), a sulfated polysaccharide derived from red seaweed (Gracilaria fisheri). Previously, it has been shown that a type of sulfated polysaccharide, heparin, is also present in L. vannamei. To investigate the role of heparin as a receptor for VP37, the binding of VP37 to porcine heparin, whose structure is similar to that found in L.vannamei, was investigated in a Surface Plasmon Resonance (SPR) system. The results showed that VP37 bound strongly to heparin with binding affinity (KD) of 1.0 µM and the binding was significantly blocked by SG. These findings have lead us to propose that the attachment of WSSV might be mediated by the interaction between VP37 and a heparin-like molecule presented on the shrimp cells.