Detection Methods, Sequence Of The Complete Genome Of Acute Viral Necrobiotic Virus Isolated From Scallop Chlamys Farreri

The scallop Chlamys farreri is one of the major species cultured in North China and its culture in commercial scale has been performed more than 20 years. However, the great expansion and intensification have induced the occurrence of disease since 1990's, especially the disease called "Acute Viral Necrobioitic Disease" (AVND) has been becoming epizootics since 1997 in north coast of China. The cumulative mortality could be more than 90% and the disease caused by a virus called "Acute Viral Necrobiotic Virus" (AVNV) has been becoming the major limiting factor in the development of the scallop industry, striking the economic process in the north coastal region of China.The virus particles take the shape of sphere and have a bilaminal envelope with spike outside it. The diameter of virions is approximately 130-170nm, while the nucleocapsids are 90-140nm. The space between the envelope and capsid is about 10-16nm. The virions could be found in the connective tissue cells and interstitial cells of digestive gland, kidney, mantle, and intestine. These morphological characteristics of AVNV are similar to Ostreid herpesvirus 1 (OsHV-1) which was reported to infect other bivalve mollusk such as oyster, clam and scallop.Firstly, we obtained some nucleic acid sequences of AVNV by using the primers (A3/A4, C2/C6 and Gp3/Gp4) for detection of OsHV-1 and AVNV DNA as DNA template. Analysis of these sequences revealed that the similarity on the A3/A4, C2/C6 and Gp3/Gp4 fragments arrived at 99%, 96% and 99% respectively between AVNV and OsHV-1. It is believed that great similarity is possible at the level of nucleotide between AVNV and OsHV-1. Then, primers for amplification of AVNV nucleotide sequence were designed according to the sequences of AVNV and OsHV-1 complete genome sequence which had been published (AY509253). PCR and molecular cloning techniques were used to set up a series of overlapping clones at the ends, covering the whole genomic sequence of AVNV. The entire genome of AVNV was determined, following the sequencing and splicing (GeneBank accession number: GQ153938). The genome was a linear double-stranded DNA of 210,993bp in length with a base composition of 61.5% A+T. There were two large inverted repeats at the end of AVNV genome (1-7,638 and 178,052-185,689, 187,200-197,411 and 200,782-210,993), which is the typical characteristic of herpesvirus. 123 open reading frames (ORFs) were identified with coding capacity for polypeptides ranging from 41 to 1878 amino acids. The percent coding density of AVNV accounted for 82% of the genetic information in the AVNV genome and the average length of each ORF in the AVNV genome was l,254bp. Computer-assisted analyses of the deduced amino acid sequences revealed that 44 putative gene products showed significant homology to functionally characterized proteins of other species in the Gene Bank/EMBL/GGBJ database. These proteins included enzymes and structural proteins involved in virus replication, nucleotide metabolism and modification and virus-host interaction, such as DNA polymerase (ORF99), ATPase subunit of DNA-packaging terminase (ORF108), SF2 helicase (ORF66), two subunits of ribonucleotide reductase (ORF20 and ORF50), a tentative primase (ORF24), deoxyuridine triphosphatase(ORF74), and dUTPase (ORF27, ORF33 and ORF74). Moreover, an analysis of codon preference, putative conserved domains or signatures, homology and possible function was done on these important genes. In comparison with the ORFs between AVNV and OsHV-1, the two viruses shared 29, 75, 3 and 8 genes with 100%, 91-99%, 81-90%, 40-80% amino acid identity, respectively. Although AVNV is closely related to OsHV-1, there are some divergences between their genomes in size, identity of gene products, host specificity and geological distribution. These differences suggest that AVNV and OsHV-1 shouldn't belong to the same species and AVNV should be considered a separate species belonging to Malacoherpesviridae in herpesvirales.With the acquisition of AVNV genomic DNA sequence, studies on detection and diagnosis methods of nucleic acid for AVNV were done. These methods included PCR, nested PCR, loop-mediated isothermal amplification (LAMP), and fluorescence quantitative polymerase chain reaction (FQ-PCR). These methods would be very critical for further research on AVNV, such as rapid detection of AVNV suspected case, pathogenic mechanism of AVNV infection, investigation of molecular epidemiology and the relationship between AVNV and outbreak of the disease.The primer sets for one-step PCR and nested PCR were designed from the DNA sequence of AVNV. The sizes of amplified products were found in each methods coincided with the predicted sizes of 706bp and 314bp respectively, whereas the uninfected scallop DNA, other DNA viruses and control reaction (no DNA template) were negative. The one-step PCR assay was able to detect 100fg AVNV DNA. The sensitivity of nested PCR amplification was 100 times higher than that of the one-step PCR, which corresponded to about 1fg AVNV DNA. These methods will be very useful for sensitive and specific detection of AVNV in the laboratory.LAMP primers were designed from AVNV genomic DNA sequence following the method of Notomi et al. (2000) and using the LAMP primer designing software PrimerExplorer V4 (http://primerexplorer.jp/e/). A set of four primers recognizing six distinct regions in the target sequence (F1c, F2, F3, B1c, B2, and B3) were designed. Forward inner primer for AVNV (AVNV-FIP) consisted of F1c, a TTTT linker and F2, and backward inner primer (AVNV-BIP) consisted of B1c, a TTTT linker and B2. The two outer primers, AVNV-F3 and AVNV-B3, were located outside the F2-B2 region, respectively. Reaction was carried out in a water bath and reaction temperature and time were optimized at 64°C for 60 min. The lower detection limit of this assay was 1fg AVNV DNA, which was demonstrated 100 times higher than that of one-strp PCR assay. This assay is a simple, rapid, sensitive and specific LAMP technique for detection of AVNV. The visual inspections of LAMP products by the naked-eye require very simple equipments and may facilitate the field application of the assay.A pair of specific primers which could amplify a 90bp fragment and a fluorescent TaqMan probe was designed, basing on the DNA sequences of AVNV. Fluorescence based quantitative polymerase chain reaction (FQ-PCR) assay for detection of AVNV was developed and optimized. It could obtain excellent linearity when the AVNV genome was between 10~8 and 10~2 copies, lgX = -0.29Ct + 13.28 (correlation coefficient R = 0.998). The detection limit of this assay was about 10 copies. It was confirmed that the FQ-PCR assay was a powerful tool for the detection of AVNV with rapidity, sensitivity, specificity, and quantification. It would be used for further research on AVNV, such as pathogenic mechanism of AVNV infection, dynamic distribution of AVNV, and the relationship between AVNV and outbreak of the disease.