Detection of infectious myonecrosis virus in penaeid shrimps using immunoassays: usefulness of monoclonal antibodies directed to the viral major capsid protein

Abstract

Despite the economic impact of the infectious myonecrosis virus (IMNV) on shrimp farms in several countries, no method for immunological detection is currently available. With the aim of developing immunodiagnostic methods for IMNV detection in infected shrimps, a recombinant fragment of the IMNV major capsid protein gene encoding amino acids 105-297 (rIMNV₁₀₅₋₂₉₇ was heterologously expressed in Escherichia coli and used to immunize Balb/c mice, generating monoclonal antibodies (MAbs). Six hybridomas were obtained, and four of these recognized the presence of IMNV in tissue homogenates from naturally infected shrimps by immunodot blot assay. Among these MAbs, three were able to detect a ~100-kDa protein, which corresponds to the predicted mass of the IMNV major capsid protein, as well as viral inclusion bodies in muscle fibroses by western blot and immunohistochemistry. Two MAbs showed high specificity and sensitivity, showing no cross-reaction with healthy shrimp tissues in any assays, indicating their usefulness for IMNV detection.

Studies on the occurrence of infectious myonecrosis virus in pond‐reared Litopenaeus vannamei (Boone, 1931) in India

“…IMNV‐positive samples were further confirmed by RT‐PCR using the primer sets designed by various workers (Tang et al. ; Seibert et al., ; Borsa et al., ; Kunanopparat et al., ). Further, the RT‐PCR product obtained from IMNV‐infected shrimp was sequenced and subjected to a general BLASTN search, and the results revealed 98% identity with the sequence of IMNV submitted by Naim et al.…”

“…Agarose gels showing RT ‐ PCR amplicons for detection of IMNV in infected Litopenaeus vannamei collected from shrimp ponds from West Bengal using different sets of primers designed by various workers. Lane M—100‐bp ladder marker; Lane M1—3‐Kbp ladder marker; Lane 1—139 bp (Poulos & Lightner, ); Lane 2—328 bp (Poulos & Lightner, ); Lane 3—600 bp (Borsa et al., ); Lane 4—686 bp (our laboratory primer); Lane 5—700 bp (Seibert et al., ); Lane 6—1014 bp (Kunanopparat et al., )…”

“…As the IMNV was reported for the first time in India, the primer sets designed by different workers were used to detect the IMNV in the infected shrimp (Tang et al. ; Seibert et al., ; Borsa et al., ; Kunanopparat et al., ). One set of primers was designed in our laboratory based on the whole‐genome sequence available in the GenBank (Accession No.…”

“…() have reported the susceptibility of wild Fafantepenaeus subtilis (Perez Farfante 1967) to IMNV. Various diagnostic tools such as in situ hybridization method (Tang et al., ), RT‐PCR (Poulos & Lightner, ), real‐time RT‐PCR (Andrade, Srisuvan, Tang, & Lightner, ), RT‐loop‐mediated isothermal amplification combined with a lateral flow dipstick (Puthawibool, Senapin, Kiatpathomchai, & Flegel, ) and immunological‐based diagnostics (Borsa et al., ; Chaivisuthangkura, Senapin, Wangman, Longyant, & Sithigorngul, ; Seibert et al., ) are available to detect the IMNV. This study reports the occurrence of IMNV infection for the first time in pond‐reared L. vannamei in India, experimental infection, River's postulates and sequence analysis.…”

Investigation of possible presence of infectious myonecrosis virus in shrimp in China

“…; Borsa et al . ; Kunanopparat et al . ), in situ hybridization using a specific cDNA probe (Tang et al .…”