Phylogenetic Analyses And Diagnostic Assays For Chinese Infectious Haematopoietic Necrosis Virus(IHNV)

The product of salmon was 39,164 tons in 2014, but the disease, infectious haematopoietic necrosis(IHN), always made a serious loss since the coldwater fish culture was developmented in China. IHNV was reported in China in 1985 and has since undergone considerable spread in China. IHN has been reported in Gansu, Liaoning, Beijin, Heilongjiang, Jilin, Hebei and Shandong, etc. The positive rate of IHN was about 36.7% in China in 2014. IHN has became a threat to Chinese coldwater fish culture. The 10 infectious haematopoietic necrosis virus(IHNV) strains has been isolated from different reigions in China. In this study, phylogenetic analyses of the mid G sequences of 60 strains isolated from 9 provinces in China enable determination of the evolution and spread of the IHNV in China since the first report, including the 50 samples that were isolated from 7 provinces in this study. Sequence comparisons revealed 11 different sequence types, m G801J~m G810 J and m G8011 M. The results of phylogenetic analyses showed that most of Chinese IHNV isolates are belong to Nagano subgenotype, J genotype. The results suggest that there are co-circulating lineages of IHNV present within specific areas of China. More important, we first report the MA and MN subgenotype, M genotype in China. Further, the database allows us to analyze the pathway of distribution in China over time. In most of the cases, spread of IHNV was related to movement of infected egg or fish. The eggs from Heilongjiang maybe is the source of IHNV in Jilin, Liaoning, Gansu, Yunnan.This study determined the complete genomic sequence of the IHNV strain Ch20101008 isolated from farmed brook trout(Salvelinus fontinalis) that died from a disease caused by the virus in Jilin, China in 2010. 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. All Chinese IHNV isolates are VI monophyletic, with a recent common ancestor, except for the Bj LL strain. The N, P, M, G, NV and L genes of Ch20101008 were compared with the available IHNV sequences in Gen Bank. 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 4,959 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.We established two molecular biology detection assays and monoclonal antibody to detecte IHN, including reverse transcriptase loop-mediated isothermal amplification(RT-LAMP) and reverse transcriptase digital droplet PCR(RT-dd PCR). We reports development of RT-dd PCR for absolute IHNV quantification base on the RT-q PCR assay. Our comparison of standard material and clinic samples quantification by RT-dd PCR and RT-q PCR indicated that both of two assays showed great specificity, sestivity and precision. The slightly lower sensitivity was observed in RT-dd PCR comparing to RT-q PCR for detecting IHNV standard material, but the RT-dd PCR showed a better detectability while was applied to measure the IHNV copy in tissues of golden rainbow trout and arctic char. This absolute quantitation assay may be useful to standardize quantification of IHNV in fish sample. The aim of the study reported here was to examine the IHNV titres in the spleen, kidney and liver, etc 9 organs of golden rainbow trout and arctic char in early infected period. We try to explain the question “why the arctic Char is not sensitive to IHNV?”.The sensitivity of RT-LAMP was 3.64×10-7 μg/μL, which is 100 times higher than RT-PCR, but same as RT-q PCR. The Diagnostic sensitivity(DSe) and Diagnostic specificity(DSp) were calculated based on the result of 100 samples. The RT-LAMP, RT-q PCR and RT-PCR have the same DSp. The DSe of RT-LAMP was slightly lower than RT-q PCR, but much higher than RT-PCR. More important, the programme of RT-LAMP completed in 15~20 min, and can avoid the contamination comparing to tranditional LAMP. Additionally, we screemed two monoclonal antibody for detecting IHNV, and try to establish Fluorescent nanometer microspheres test card. The methods established in this study were applied to detect IHNV from the 100 samples, and the detectability was compared to OIE methods, the results indicated that the RT-dd PCR showed the best detectability(44%), the positive rate of RT-q PCR, RT-LAMP, virus isolation and fluorescent nanometer microspheres test card were 43%, 42%, 23% and 15%.The standard of Quarantine Protocol for Infectious Haematopoietic Necrosis(SN/T1474-2014) and The Diagnostic Assay of RT-LAMP for Infectious Haematopoietic Necrosis Virus were drafted base on the OIE Manual and the result in this study.