Circulatory white spot syndrome virus in South-West region of Bangladesh from 2014 to 2017: molecular characterization and genetic variation

Abstract

White Spot Syndrome Virus (WSSV), the etiological agent of White Spot Disease (WSD) is a major impediment for shrimp aquaculture in the worldwide. A critical threshold level of WSSV load in infected shrimp is an important trait for disease manifestation and WSSV transmission in cultured shrimp and subsequently make outbreaks. The present study investigated 120 naturally infected cultured shrimp samples by SYBR Green based qPCR assay for WSD diagnosis and quantification of WSSV load. Among them, 94 samples resulted a variable count of WSSV load ranging from 2.1 × 10⁸ to 2.64 × 10¹⁴ copies/g of shrimp tissue. The severity of WSSV infection was assessed based on the established critical threshold load of WSSV in shrimp tissue. Compared to the established critical threshold value of WSSV load in shrimp tissue, our findings showed the horrifying scenario of the severity of WSSV infection in cultured shrimps of Bangladesh that was found to be above the critical limit to initiate an outbreak in the Bangladeshi shrimp aquaculture industry. The latest phylogenetic pattern was altered from the former monophyletic history among WSSVs of Bangladesh due to a variation at 500th nucleotide of VP28 coding gene. Viruses characterized from recent outbreaks in 2015 and 2017 displayed amino acid substitution at position 167 (G→E) on the surface of VP28 protein which has demonstrated the probable replacement of indigenous virus pool. Therefore, it is imperative to take initiative for the management and prevention of WSSV outbreak to sustain shrimp aquaculture in South-West region of Bangladesh.

Keywords: Cultured shrimp; Mutation; Phylogeny; Real-time qPCR; Severity; VP28.

Fig. 1

Amplification plots of WSSV positive samples and no template control (NTC). The amplification of positive samples was confirmed by significant fluorescent signal overhead the base line. The significant fluorescent signal above the baseline was not spotted for the negative control (NTC). The C_T (cycle threshold) value of NTC was beyond the determination index

Fig. 2

Specific dissociation peak of WSSV positive samples by melt curve analysis with corresponding conventional PCR amplicon (148 bp) targeting VP28 gene of WSSV. The ethidium bromide-stained agarose gel (1.5%) was used to visualized 148 bp PCR amplicon generated by qVP28F and qVP28R primers. The Tm value (81.1 ± 17 °C) of real-time PCR specified the unified WSSV amplicon in the samples

Fig. 3

Molecular phylogenetic analysis (codon based) of WSSV isolated from Bangladesh between 2014 to 2017. Nucleotide sequences of VP28 coding region were used to construct the tree based on the Tamura-Nei model by Maximum Likelihood method. The analysis included 42 nucleotide sequences among which 18 were from Bangladesh. The sequences generated from isolates of Bangladesh are shown in bold letter, which are placed in three distinct clusters among four. Sequences generated exclusively for this study and submitted from previous study (Hossain et al. 2015) are marked with (filled triangle) and (filled diamond) symbols, respectively. Monodon Baculovirus (Accession No. HQ222840) was taken as outgroup in the tree

Fig. 4

Three-dimensional structure of aligned WSSV VP28 envelope protein of BAN_SH_BG-1C_2014 that was taken as representative of isolates collection in 2014 in Bangladesh and PDB id 2ED6. The side chain of Glutamic acid (E) in the reference protein protruded out of the surface portion where the BD isolate contained Glycine (G). The amino acid positioned between the beta-strands seven and eight. The red color in the figure showed the protruded side chain. Paste color and gray color represented the target region (160–170 amino acid) and the rest of the protein. Surface and line style was used to visualize the protein and side chain of glutamic acid