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Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV).

ABSTRACT: The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in freshwater and marine fishes. Its diagnosis officially relies on the isolation of the virus in cell culture and its identification by serological or polymerase chain reaction (PCR) methodologies. Nowadays, reverse transcription real-time quantitative PCR (RT-qPCR) is the most widely employed technique for the detection of this virus and some studies have reported the validation of RT-qPCR procedures for the detection, typing, and quantification of VHSV isolates. However, although the efficacy of this technique is not in doubt, it can be cumbersome and even impractical when it comes to processing large numbers of samples, a situation in which cross-contamination problems cannot be ruled out. In the present study, we have designed and validated a macroarray for the simultaneous detection, typing, and quantification of VHSV strains. Its analytical sensitivity (5-50 TCID₅₀/_mL), analytical specificity (intra and intergroup), efficiency (E = 100.0-101.1) and reliability (repeatability and reproducibility with CV < 5%, and standard curves with R² < 0.95) with strains from any VHSV genotype have been widely demonstrated. The procedure is based on the 'binary multiplex RT-qPCR system (bmRT-qPCR)' previously reported by the same team, applied to arrays of 96-well PCR strip tubes plates, which can be stored at -25 °C for three months and up to one year before their use, without significant loss of efficiency.

SUBMITTER: Lopez-Vazquez C

PROVIDER: S-EPMC8002285 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundViral hemorrhagic septicemia virus (VHSV) is a highly contagious viral disease of fresh and saltwater fish worldwide. VHSV caused several large scale fish kills in the Great Lakes area and has been found in 28 different host species. The emergence of VHS in the Great Lakes began with the isolation of VHSV from a diseased muskellunge (Esox masquinongy) caught from Lake St. Clair in 2003. VHSV is a member of the genus Novirhabdovirus, within the family Rhabdoviridae. It has a linear single-stranded, negative-sense RNA genome of approximately 11 kbp, with six genes. VHSV replicates in the cytoplasm and produces six monocistronic mRNAs. The gene order of VHSV is 3'-N-P-M-G-NV-L-5'. This study describes molecular characterization of the Great Lakes VHSV strain (MI03GL), and its phylogenetic relationships with selected European and North American isolates.ResultsThe complete genomic sequences of VHSV-MI03GL strain was determined from cloned cDNA of six overlapping fragments, obtained by RT-PCR amplification of genomic RNA. The complete genome sequence of MI03GL comprises 11,184 nucleotides (GenBank GQ385941) with the gene order of 3'-N-P-M-G-NV-L-5'. These genes are separated by conserved gene junctions, with di-nucleotide gene spacers. The first 4 nucleotides at the termini of the VHSV genome are complementary and identical to other novirhadoviruses genomic termini. Sequence homology and phylogenetic analysis show that the Great Lakes virus is closely related to the Japanese strains JF00Ehi1 (96%) and KRRV9822 (95%). Among other novirhabdoviruses, VHSV shares highest sequence homology (62%) with snakehead rhabdovirus.ConclusionPhylogenetic tree obtained by comparing 48 glycoprotein gene sequences of different VHSV strains demonstrate that the Great Lakes VHSV is closely related to the North American and Japanese genotype IVa, but forms a distinct genotype IVb, which is clearly different from the three European genotypes. Molecular characterization of the Great Lakes isolate will be helpful in studying the pathogenesis of VHSV using a reverse genetics approach and developing efficient control strategies.

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Design and Evaluation of a Macroarray for Detection, Identification, and Typing of Viral Hemorrhagic Septicemia Virus (VHSV).

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