Detection of major capsid protein of infectious myonecrosis virus in shrimps using monoclonal antibodies

Abstract

Infectious myonecrosis virus (IMNV) has been causing a progressive disease in farm-reared shrimps in Brazil and Indonesia. Immunodiagnostic methods for IMNV detection, although reliable, are not employed currently because monoclonal antibodies (MAbs) against this virus are not available. In this study, a fragment of the IMNV major capsid protein gene, comprising amino acids 300-527 (IMNV(300-527)), was cloned and expressed in Escherichia coli. The nucleotide sequence of the recombinant IMNV(300-527) fragment displayed a high degree of identity to the major capsid protein of IMNV isolates from Brazil (99%) and Indonesia (98%). Ten MAbs were generated against the expressed fragment, and eight of these, mostly IgG(2a) or IgG(2b), were able to bind to IMNV in tissue extracts from shrimps infected naturally in immunodot-blot assays. Six of these MAbs recognized a approximately 100 kDa protein in a Western-blot, which is the predicted mass of IMNV major capsid protein, and also bound to viral inclusions present in muscle fibroses and in coagulative myonecrosis, as demonstrated by immunohistochemistry. Among all those MAbs created, four did not cross-react with non-infected shrimp tissues; this observation supports their applicability as a sensitive and specific immunodiagnosis of IMNV infection in shrimps.

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

“…Several methods have been developed for IMNV detection, including immunostaining using specific antibodies (Seibert et al . ; Borsa et al . ; Kunanopparat et al .…”