Detection and Simultaneous Differentiation of Three Co-infected Viruses in Zanthoxylum armatum.

Green Sichuan pepper (Zanthoxylum armatum) is an important economic fruit crop, which is widely planted in the southwest region of China. Recently, a serious disease, namely flower yellowing disease (FYD), broke out, and the virus of green Sichuan pepper nepovirus (GSPNeV) was identified to be highly correlated with the viral symptoms. Meanwhile, green Sichuan pepper idaeovirus (GSPIV) and green Sichuan pepper enamovirus (GSPEV) were also common viruses infecting green pepper. In our research, specific primers were designed according to the reported sequences of the three viruses, and a multiplex reverse transcription-polymerase chain reaction (RT-PCR) method for the simultaneous detection of GSPNeV, GSPIV, and GSPEV was established. The annealing temperature, extension time, and cycle number affecting the multiplex RT-PCR reaction were adjusted and optimized. Sensitivity analysis showed that the system could detect the three viruses simultaneously from the complementary deoxyribonucleic acid (cDNA) samples diluted by 10-3. The results of the ten samples detected by the multiplex RT-PCR system were consistent with the results of a single PCR, indicating that the method can be successfully used for rapid detection of field samples.

PCR Detection and Phylogenetic Analysis of Megalocytivirus Isolates in Farmed Giant Sea Perch Lates calcarifer in Southern Taiwan.

The Megalocytivirus genus includes three genotypes, red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV), and has caused mass mortalities in various marine and freshwater fish species in East and Southeast Asia. Of the three genotypes, TRBIV-like megalocytivirus is not included in the World Organization for Animal Health (OIE)-reportable virus list because of its geographic restriction and narrow host range. In 2017, 39 cases of suspected iridovirus infection were isolated from fingerlings of giant sea perch (Lates calcarifer) cultured in southern Taiwan during megalocytivirus epizootics. Polymerase chain reaction (PCR) with different specific primer sets was undertaken to identify the causative agent. Our results revealed that 35 out of the 39 giant sea perch iridovirus (GSPIV) isolates were TRBIV-like megalocytiviruses. To further evaluate the genetic variation, the nucleotide sequences of major capsid protein (MCP) gene (1348 bp) from 12 of the 35 TRBIV-like megalocytivirus isolates were compared to those of other known. High nucleotide sequence identity showed that these 12 TRBIV-like GSPIV isolates are the same species. Phylogenetic analysis based on the MCP gene demonstrated that these 12 isolates belong to the clade II of TRBIV megalocytiviruses, and are distinct from RSIV and ISKNV. In conclusion, the GSPIV isolates belonging to TRBIV clade II megalocytiviruses have been introduced into Taiwan and caused a severe impact on the giant sea perch aquaculture industry.