Development of a SYBR Green quantitative PCR assay for detection of Lates calcarifer herpesvirus (LCHV) in farmed barramundi

Abstract

Lates calcarifer herpes virus (LCHV) is a new virus of farmed barramundi in Southeast Asia.2 However, a rapid detection method is yet to be available for LCHV. This study, therefore, 2 aimed to develop a rapid quantitative PCR (qPCR) detection method for LCHV and made it 2 timely available to public for disease diagnostics and surveillance in barramundi farming 3 countries. A newly designed primer set targeting a 93-bp fragment of the LCHV putative 3 major envelope protein encoding gene (MEP) was used for developing and optimizing a 3 SYBR Green based qPCR assay. The established protocol could detect as low as 10 viral 3 copies per µl of DNA template in a reaction containing spiked host DNA. No cross-3 amplification with genomic DNA extracted from host as well as common aquatic pathogens 3 (12 bacteria and 3 viruses) were observed. Validation test of the method with clinical samples 3 revealed that the virus was detected in multiple organs of the clinically sick fish but not in the 3 healthy fish. We thus recommend that barramundi farming countries should promptly initiate 3 active surveillance for LCHV in order to understand their circulation for preventing possibly 3 negative impact to the industry.

Scale Drop Disease Virus (SDDV) and Lates calcarifer Herpes Virus (LCHV) Coinfection Downregulate Immune-Relevant Pathways and Cause Splenic and Kidney Necrosis in Barramundi Under Commercial Farming Conditions

“…In Singapore, where this particular study has taken place, farmers have reported mass mortalities of juveniles during the transition between nursery phases (∼50 to 500 g) and the final grow out period as a recurring phenomenon over the last few years. Although several novel viral pathogens (SDDV, LCHV, LCBV) affecting barramundi have been first reported and identified in Singaporean farms (Gibson-Kueh et al, 2012;de Groof et al, 2015;Chang et al, 2018;Chen et al, 2019), their occurrence is now known to extend throughout Southeast Asia (Senapin et al, 2019;Nurliyana et al, 2020;Meemetta et al, 2020), affecting the industry as a whole. To better understand what is happening to the biology of barramundi in commercial farming conditions during one such disease outbreak, the presence of five putative pathogens was investigated using a comparative RNAseq and histological approach targeting multiple organs of affected and non-affected juveniles.…”

“…Copy numbers of SDDV and LCHV were calculated from respective standard graphs generated by qPCR amplifications of serially diluted plasmid containing corresponding viral insert target as previously described (Meemetta et al, 2020;Sriisan et al, 2020).…”

“…Other emerging pathogens in cultured barramundi include L. calcarifer herpes virus (LCHV) (Chang et al, 2018;Meemetta et al, 2020), infectious spleen and kidney necrosis virus (ISKNV) (Dong et al, 2017a), Vibrio harveyi causing scale drop and muscle necrosis (Vh-SDMND) (Dong et al, 2017b), L. calcarifer birnavirus (LCBV) (Chen et al, 2019), and red sea bream iridovirus (RSIV) (Girisha et al, 2020). SDDV and LCHV infections in barramundi reportedly caused similar gross signs of scale loss (Chang et al, 2018), reiterating the need to use more specific diagnostic tests.…”

“…SDDV and LCHV infections in barramundi reportedly caused similar gross signs of scale loss (Chang et al, 2018), reiterating the need to use more specific diagnostic tests. PCR-based diagnostic methods have been established for these pathogens (Gias et al, 2011;de Groof et al, 2015;Meemetta et al, 2020;Sriisan et al, 2020). However, epidemiological studies characterizing the prevalence of these pathogens in farming sites have not yet been carried out.…”

Pre-treatment of Nile tilapia (Oreochromis niloticus) with ozone nanobubbles improve efficacy of heat-killed Streptococcus agalactiae immersion vaccine

“…Serial dilutions of the pSNB1 plasmid spiked with 200 ng of tilapia DNA were used to construct a standard curve for quantifying S. agalactiae . Fish gill DNA was isolated using the conventional phenol-chloroform method [31,32] and 200 ng of each DNA sample was subjected to qPCR assays using the CFX Connect™ Real-time System (Bio-Rad, USA). The resulting C q value was used to compute bacterial DNA in the fish gills using the equation: copy number = 10 (Ct – Intercept)/Slope .…”