Design and evaluation of reverse transcription nested PCR primers for the detection of betanodavirus in finfish

Abstract

Viral encephalopathy and retinopathy otherwise known as viral nervous necrosis (VNN) is a neuropathological condition affecting more than 50 fish species worldwide, mostly marine. Different PCR protocols with specific primers were reported from many countries for confirmation of VNN in fishes. In the present study, two pairs of primers were designed and evaluated for the diagnosis of clinical and subclinical cases of infections from field. These primers designated as BARL-F1/BARL-R1 amplified a 902 bp product in the variable region (T4) of the coat protein gene by first step PCR. Nested PCR primers BARL-F2/BARL-R2 amplified a fragment of 313 bp. The results were comparable with other commonly used primer sets such as F2/R3 and RG668f/RG919r primers. These new primers could detect betanodavirus in standard reference samples containing low, moderate and high viral load. Known positive and negative control samples of fish also revealed a predictive value of 100 % by RT-PCR diagnosis.

Keywords: Betanodavirus; Diagnosis; Mortality; Nested RT-PCR; Viral encephalopathy and retinopathy; Viral nervous necrosis.

Fig. 1

Histological section of brain of VNN affected Asian seabass larvae (code 01), H&E stain. a Peripheral vacuolation in the brain (×10); b severe vacuolations with coalescence of the vacuoles seen scattered in the brain tissue (arrow), H&E ×40

Fig. 2

Evaluation of the primers using VNN affected seabass larvae from clinical outbreak of disease (lanes 1–6 first step PCR using primer set BARL-F1/BARL-R1 and lanes 7–12 nested PCR using primer set BARL-F2/BARL-R2)—these samples were tested positive by OIE primer (F2/R3). M DNA ladder, 1 Asian seabass larvae—code 01, 2 Asian seabass larvae—code 02, 3 Asian seabass larvae—code 03, 4 reference strain (CSIRO), 5 negative control, 6 positive control, 7 Asian seabass larvae—code 01, 8 Asian seabass larvae—code 02, 9 Asian seabass larvae—code 03, 10 reference strain (CSIRO), 11 negative control, 12 positive control

Fig. 3

Evaluation of the primers using sub-clinically affected fishes (lanes 1–7 first step PCR using primer set BARL-F1/BARL-R1 and lanes 8–14 nested PCR using primer set BARL-F2/BARL-R2)—these samples were tested positive by OIE primer (F2/R3). M DNA ladder, 1 M. cephalus (code-15), 2 L. calcarifer (code-27), 3 L. calcarifer (code-28), 4 C. chanos (code-07), 5 negative control, 6 positive control, 7 M. cephalus (code-15), 8 L. calcarifer (code-27), 9 L. calcarifer (code-28), 10 C. chanos (code-07), 11 negative control, 12 positive control

Fig. 4

Detection of betanodavirus positive reference strain (CSIRO, Australia) containing low, moderate and heavy infection using the newly designed primers (lane M molecular marker (100 bp), lanes 1–3 Positive control (reference sample) containing low, moderate and high level of betanodavirus by first step PCR using primer set BARL-F1/BARL-R1, lanes 4–6 same samples by nested PCR using primer set BARL-F2/BARL-R2

Fig. 5

a Betanodavirus detection using published primers F2/R3 for first step (top) and RG668f/RG919r for nested PCR (bottom); b newly designed primers BARL-F1/BARL-R1 for first step (top) and BARL-F2/BARL-R2 for nested PCR (bottom) showing specific products; lane M DNA ladder (100 bp), lane N negative control, lanes 1–3 cell culture samples C1–C3 (positive control), lanes 4–7 healthy Asian seabass larval samples S1–S4 (known VNN-negative control)