Phiwsaiya, K. | bioRxiv Science

bioRxiv2022

Detection of nervous necrosis virus RGNNV genotype in pearl gentian grouper (Epinephelus lanceolatus male x E. fuscoguttatus female) fry imported to Thailand

Nervous necrosis virus (NNV) is a deadly virus that affects more than 120 fish species worldwide, but there is little information about it in Thailand. In August 2019, a population of pearl gentian grouper fry imported to Thailand experienced mass mortality. The diseased fish exhibited darkening, floating on the water surface, sleepy behaviour, and erratic swimming. We received a set of samples for disease diagnosis. PCR analysis revealed that these samples were positive for NNV but negative for Megalocytivirus ISKNV and Ranavirus. Sequencing the viruss genome and phylogenetic analysis revealed that it is a member of the red-spotted grouper nervous necrosis virus (RGNNV) genotype. In situ hybridization using an NNV-specific probe revealed localization of the virus in the vacuolation lesions of the central nervous system (brain and spinal cord) and the retina of infected fish. The virus was successfully isolated from tissue homogenate from diseased fish using the E-11 cell line. This study identified NNV (RGNNV genotype) as a virus associated with massive die-offs in imported pearl grouper fry in Thailand. Considering the infectious nature of the virus and its broad host range, appropriate biosecurity measures are needed to prevent any loss to the marine aquaculture industry. HighlightsO_LIThis study reports detection of NNV in grouper larvae imported to Thailand C_LIO_LIThe virus was assigned to the RGNNV genotype, which is the most extensively distributed genotype C_LIO_LIHistopathology revealed a pathognomonic lesion of NNV C_LIO_LIISH using an NNV-specific probe revealed localization of NNV in the CNS and retina C_LIO_LIThe virus was successfully propagated from diseased fish using the E-11 cell line C_LI

pathology↗

bioRxiv2019

Genetic diversity of tilapia lake virus genome segment 1 from 2011 to 2019 and a newly validated semi-nested RT-PCR method

The gene of RNA viruses, encoding RNA-directed RNA polymerase (RdRp) is relatively conserved due to its crucial function in viral genome replication and transcription making it a useful target for genetic diversity study and PCR detection. In this study, we investigated the genetic diversity of 21 tilapia lake virus (TiLV) genome segment 1 sequences predictively coding for RdRp subunit P1. Those sequences were obtained from infected fish samples collected in Ecuador, Israel, Peru, and Thailand between 2011 and 2019 (nine sequences from this study and 12 sequences from GenBank). Primers were then designed from the highly conserved regions among all 21 TiLV segment 1 sequences and used in semi-nested RT-PCR condition optimization. The result revealed that all 21 TiLV segment 1 sequences showed 95.00-99.94 and 99.00-100% nucleotide and amino acid sequence identity, respectively. These isolates were phylogenically clustered into three separate genetic clades, called i) Israeli-2011 clade (containing of TiLV isolates from Israel collected in 2011, Ecuador, and Peru isolates), ii) monophyletic Israel-2012 clade (containing only TiLV isolates collected from Israel in 2012), and iii) Thai clade (containing only sequences obtained from Thailand isolates). The newly established PCR protocol was 100 times more sensitive than our previous segment 3-based protocol when comparatively assayed with RNA extracted from infected fish. The assay was also shown to be specific when tested against negative control samples, i.e. RNA extracted from clinical healthy tilapia and from bacterial and viral pathogens (other than TiLV) commonly found in aquatic animals. Validation experiment with RNA extracted from naturally infected fish specimens collected in 2013-2019 yielded positive test results for all samples tested, confirming that our newly designed primers and detection protocol against TiLV segment 1, have a potential application for detection of all current genetic variants of TiLV.

genetics↗