Outbreaks of infectious myonecrosis virus (IMNV) in Indonesia confirmed by genome sequencing and use of an alternative RT-PCR detection method

Abstract

Outbreaks of disease due to infectious myonecrosis virus (IMNV) were first reported from Brazil in 2004 in Pacific white shrimp Penaeus (Litopenaeus) vannamei, an exotic species to Brazil that was relatively recently imported for cultivation in earthen ponds. In Indonesia, this non-native species has also been imported and successfully cultivated on a large scale since 2003. Starting in early 2006, there were anecdotal reports from Indonesian shrimp farmers of high mortality in cultivated P. vannamei with gross signs of white muscle, similar to those reported for IMNV outbreaks in Brazil. We obtained a sample of shrimp from one of these outbreak ponds and it gave a positive result for the presence of IMNV using a standard commercial detection kit. After sequencing the PCR fragment to confirm the presence of IMNV, additional primers were designed for cloning and sequencing the full 7.5 kb IMNV genome. Subsequent analysis (GenBank accession no.EF061744) revealed that the Indonesian IMNV sample had 99.6% nucleic acid sequence identity (a total of 29 differences in 7.5 kb) to that of Brazilian IMNV reported at GenBank. It is interesting that one of these differences was a single base insertion at nucleotide 7431 leading to the creation of a delayed termination (stop) codon that led to 13 additional amino acid residues in the deduced RdRp (RNA-dependent RNA polymerase) protein product. Due to some difficulty with very weak false positive results obtained using the commercial detection kit with some samples, we designed an alternative, nested RT-PCR detection method with specific primers to target the viral RdRp region instead of the capsid gene targeted by the commercial kit. In our hands, this protocol gave more consistent results and higher sensitivity that did the kit. © 2007 Elsevier B.V. All rights reserved.