Virus Research 118 (2006) 31–38 A new RNA virus found in black tiger shrimp Penaeus monodon from Thailand Kallaya Sritunyalucksana a , Somjai Apisawetakan a , Anutara Boon-nat b , Boonsirm Withyachumnarnkul a,c , Timothy W. Flegel a,∗ a Centex shrimp, Faculty of Science, Mahidol University and National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Klong Luang, Pathumthani 12120, Thailand b Aquatic Disease Center, Charoen Pokaphan, Mahachai, Samutsakorn, Thailand c Department of Anatomy, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand Abstract A new, apparently innocuous virus was found while investigating the cause of monodon slow growth syndrome (MSGS) in cultured black tiger shrimp (Penaeus monodon). It was identified via plasmid vector clones of E. coli containing randomly amplified cDNA fragments produced from total nucleic acid extracts of hemolymph from MSGS shrimp. Of 421 clones, 30 that failed to give positive dot blot hybridization with a digoxigenin (DIG)-labeled shrimp DNA probe were sequenced and compared to GenBank records. Of these, 22 corresponded to known shrimp DNA records. Of eight that did not, one (20A) showed significant deduced amino acid sequence similarity to RNA-dependent RNA polymerases (RdRp) of the viruses in the family Luteoviridae and alignment revealed commonly conserved amino acids including a GDD motif believed to be at the enzyme active site. However, phylogenetic analysis showed that the virus sequence did not cluster with the Luteoviridae or other known RNA virus sequences. Thus, in accordance with frequent practice, it was named according to the area where it was first collected as Laem-Singh virus (LSNV). In situ hybridization with a DIG-labeled 20A insert revealed strong cytoplasmic staining confined to the lymphoid organ (LO), the heart and hepatopancreatic connective tissue in both normal and MSGS shrimp. RT-PCR assays based on the 20A clone sequence also gave positive results with both normal and MSGS shrimp. Transmission electron microscopy (TEM) of LO tissue revealed viral-like particles of approximately 27 nm diameter (within the Luteoviridae size range) in locations that matched those of positive in situ hybridization reactions in parallel samples. Although not directly associated with MSGS in Penaeus monodon, the presence or effect of this virus with other crustacean species is presently unknown. © 2005 Elsevier B.V. All rights reserved. Keywords: RNA-dependent RNA polymerase (RdRp); Monodon slow growth syndrome (MSGS); Penaeus monodon; Laem-Sing virus (LSNV) 1. Introduction Viruses are the most common biological agents in the marine environment and it is known that marine crustaceans can be simultaneously infected by more than one type (Flegel, 2001; Flegel et al., 2004). Approximately 20 viruses are currently known for penaeid shrimp (Lightner, 1996; Loh et al., 1997; Lightner and Redman, 1998) and often cause no gross signs of disease, especially in the natural environment (Flegel, 2001). In stressful environments such as culture systems, some of these ∗ Corresponding author. Tel.: +66 2 201 5876; fax. 66 2 354 7344 E-mail address: sctwf@mahidol.ac.th (T.W. Flegel). viruses can become more virulent and cause significant eco- nomic loss by mortality or retarded growth. Monodon slow growth syndrome (MSGS) was first observed by shrimp farm- ers in cultured black tiger shrimp in 2002 and considered to a possible result of infection by an unknown infectious agent (Chayaburakul et al., 2004) since injection of 0.45 m-filtered lymphoid extracts from MSGS shrimp caused similar slow- growth symptoms in normal growing shrimp (Withyachum- narnkul et al., unpublished data). In addition, no correlation was found between MSGS and infection by known shrimp pathogens (Chayaburakul et al., 2004). For epidemiological purposes, a case definition for an MSGS pond was agreed by Thai researc- hes (http://library.enaca.org/Health/Publications/Slow growth syndrome.pdf) to be a coefficient of size variation ≥35%