Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/µl while the LOD for the RT-qPCR was 0.2 PFU/µl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Determination of the complete genome sequence of infectious hematopoietic necrosis virus (IHNV) Ch20101008 and viral molecular evolution in China.

This study determined the complete genomic sequence of the infectious hematopoietic necrosis virus (IHNV) strain Ch20101008 isolated from farmed brook trout (Salvelinus fontinalis) that died from a disease caused by the virus in northeast China. The sequence was determined from 10 overlapping clones obtained through RT-PCR amplification. The whole genome length of Ch20101008 comprised 11,129 nucleotides (nt), and the overall organization was typical of that observed for all other IHNV strains. The phylogenetic analysis results of the 65 IHNV glycoprotein genes and 47 IHNV partial nucleoprotein genes presented five major genogroups (J, U, L, E and M). The J genogroup included the J Nagano and J Shizuoka subgroups. The IHNV Ch20101008 strain belonged to the J Nagano subgroup of the J genogroup and was significantly related to other Chinese IHNV strains. All Chinese IHNV isolates are monophyletic, with a recent common ancestor, except for the BjLL strain. The N, P, M, G, NV and L genes of Ch20101008 were compared with the available IHNV sequences in GenBank. The results indicated that 198 nt were substituted, 53 of which exhibited amino acid change in the Ch20101008 genome. An adenine nucleotide deletion was found at position 4959 of the 5' UTR of the L gene. In the G gene, specific nucleotides and amino acid variations of the Chinese IHNV strains were observed when compared with 23 isolates from other countries. Of the 15 nucleotide sites that changed, seven resulted in amino acid substitution. The data further demonstrated that the J genogroup IHNV was introduced to and evolved in salmon farm environments in China.

A rapid assay for detecting antibody against Bluetongue virus with a latex agglutination test using recombinant VP7 antigen.

A latex agglutination test (LAT) for detecting antibody against Bluetongue virus (BTV) in ruminants was developed using latex beads coupled with recombinant VP7 protein. Compared with competitive enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of the LAT were 99.0% and 93.0%, respectively. There was excellent agreement between the results obtained by competitive ELISA and the LAT (kappa = 0.930). Because it is rapid and easy to use, the LAT could be used for BTV antibody detection, especially for screening many serum samples.