Detection of lymphocystis disease virus in Japanese flounder Paralichthys olivaceus and other marine teleosts from northern China

We isolated a strain of lymphocystis disease virus （LCDV） from Japanese flounder （Paralichthys olivaceus） cultured in northern China. Based on published sequences of major capsid protein （MCP） gene of LCDV-cn （GenBank： AF126405）, we designed two primer sets P1/P2 and P3/P4. We then used one-step or nested PCR and in-situ hybridization （ISFI） to detect LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral supematant, skin nodules, gut, gill, kidney, spleen, stomach, heart, and liver of Japanese flounder. We compared the DNA sequence with 14 MCP nucleotide sequences from GenBank, including Megalocytivirus （OFIV and RSIV）, lridovirus （CzlV and W/V）, Ranavirus （TFV and FV3）, and Lymphocystivirus （8 LCDV）. Based on the alignment, we confirmed the PCR product was from Lymphocystivirus （GenBank accession number DQ279090 （LCDV-HD））. Using ISH, we noted the presence of LCDV in the skin nodules, gut, gill, spleen, stomach, and heart of spontaneously infected Japanese flounders. We successfully amplified LCDV fragments from Schlegel＇s black rockfish （Sebastes schlegeli Higendorf）, redwing sea robin （Lepidotrigla microptera Gtinther） and turbot （Scophthalmus maximus） using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method.

We isolated a strain of lymphocystis disease virus(LCDV) from Japanese flounder(Paralichthys olivaceus) cultured in northern China.Based on published sequences of major capsid protein(MCP) gene of LCDV-cn(GenBank:AF126405),we designed two primer sets P1/P2 and P3/P4.We then used one-step or nested PCR and in-situ hybridization(ISH) to detect LCDV and identify the target tissues or cells in infected Japanese flounder.The PCR products were positive in purified viral supernatant,skin nodules,gut,gill,kidney,spleen,stomach,heart,and liver of Japanese flounder.We compared the DNA sequence with 14 MCP nucleotide sequences from GenBank,including Megalocytivirus(OFIV and RSIV),Iridovirus(CzIV and WIV),Ranavirus(TFV and FV3),and Lymphocystivirus(8 LCDV).Based on the alignment,we confirmed the PCR product was from Lymphocystivirus(GenBank accession number DQ279090(LCDV-HD)).Using ISH,we noted the presence of LCDV in the skin nodules,gut,gill,spleen,stomach,and heart of spontaneously infected Japanese flounders.We successfully amplified LCDV fragments from Schlegel's black rockfish(Sebastes schlegeli Higendorf),redwing sea robin(Lepidotrigla microptera Günther) and turbot(Scophthalmus maximus) using the one-step and nested PCR,suggesting the target genes can be widely detected in fish using this method.