Molecular Epidemiology Of A New Yellow Head Virus Strain In China

In recent years, the shrimp farming industry as an important industry in our country is developing rapidly, with the promotion of intensive aquaculture model and the expand of the farming scale, the prevalence rate of shrimp increased year by year in China, the loss of up to billions of dollars due to shrimp diseases in our country every year. Yellow head virus is a major disease in shrimp aquaculture, it has caused serious losses for world shrimp farming in twenty years. The virus with strong pathogenicity, once infected, the mortality rate is very high, and it is difficult to control. The disease occurs in Southeast Asia and Australia, had never caused a loss of cultured shrimp in China.In 2012 our laboratory first detected YHV positive samples from Hebei shrimp farm according to the standards, and made a futher research.In order to find out the YHV presence and the variation situation of our country, 299 samples were collected during 2012-2014 and detected by nested RT-PCR method recommended by the world organization for animal health(OIE) manual, and some of the YHV positive samples gene were sequenced and phylogenetic tree analysis. Epidemiology survery showed that YHV positive rate was 11%. L. vannamei, F. chinensis, M. japonicus and Macrobrachium rosenbergii cultured in China were all found YHV positive samples, and F. chinensis have the highest positive rate, F. chinensis and Macrobrachium rosenbergii. were newly discovered natural host of YHV, which is important to supplement the host range of YHV. Then 1002 bp fragments located in the ORF 1b region were amplified, alignment results showed that six samples with the six known YHV genetypes displayed 81.0%—90.5% nucleic acid sequence identify. The phylogenetic tree showed that six samples were in the same branch, but were not in the same branch with the six known YHV genotypes, with YHV Type 1(YHV-1) is near. A sequence of 509 bp located in the ORF 3 region encoding the structural gene for gp 116 were amplified, it different with 545 bp for YHV Type 1a(YHV-1a), 383 bp for YHV-1b, 476 bp for YHV-2. The phylogenetic tree showed that six samples were in the same branch, and different with the six known YHV genotypes, sequences short than YHV-1a, and similarity is greater than YHV-1b. Comparison two samples of ORF 2 sequence, sequence similarity of 2 samples was 99.8% and had the same protein sequence, sequence similarity with YHV-1 was 85.9% and YHV-2 was 80.9%. The above gene sequences alignment results suggests that the YHV detected in China was a new pathogenic genotype. This provides important information for the study of China’s shrimp new etiology, the results also show that our country is facing the threat of new shrimp diseases.Based on the complete genome sequences of YHV China strain, by comparison of the whole genome of YHV China strain and the known YHV strains, we found the similarity is 75.4%-82.3%. A fragment about 200 bp in ORF2 is conserved sequence with the known YHV genetypes. A fragment about 250 bp sequences in CR2 of ORF1 are more conserved. LAMP primers were designed according to the conserved sequence in CR2 for detection of yellow head complex genotypes.YHV-1 is the main pathogenic genetype of yellow head complex of virues. For research the new YHV virus pathogenicity we found, samples of 20120706(Hebei Tanghai) and 20131018(Qingdao Jimo) were selected for L. vannamei artificial infection experiment. The results showed that the cumulative mortality rate of 20120706 sample was 75%, the cumulative mortality of 20131018 sample was 93.33%, and the high mortality rate is similar to the YHV-1 type, YHV China strains is a pathogenic strain.Previously, researchers have been separated 6 genotypes of YHV from diseased shrimp in all over the world, more genetypes are discovered. According to partial gene sequences have been published by our laboratory, scholars in Thailand named YHV China strain as YHV-9. How the relationship of YHV China strain with these known genetypes in the evolution, infected species and distribution were never reported. Based on the epidemiological investigation and the whole genome sequencing of YHV China strain, samples collect from different geographic regions of China were analysis of the homology of YHV ORF1, ORF2 and ORF3 sequences with known YHV strains. in order to clarify the phylogenetic relationship of YHV China strain with known YHV strains, enrich the knowledge of YHV genomic variation and evolution, to provide basic theory for the effective prevention and treatment of YHV.