Detection Of Norwalk-like Viruses In Aquaculture Oysters Of China And Part Gene Sequences Analysis

Norwalk-like viruses (NLVs) in the family Caliciviridae are important agents of human gastroenteritis. These small (20 ~ 35nm) round and nonenveloped viruses are positive single-stranded RNA viruses. According to the nucleotide sequence of the RNA polymerase region of NLVs genomes, NLVs are divided into three genogroups: genogroup â…  (Gâ…  ), genogroup â…¡ (Gâ…¡ ) and Sapporo virus. From 1976 to 1980, about 42% gastroenteritis was related to NLVs, according to the report of Centers for Disease Control and Prevention (CDC). NLVs are acid-stable, cold-stable and Cl-stable. The infectious dose can probably be as low as 10-100 virus particles. In 1995, the first NLVs case of China was found in the children' s stool specimens. Then there was report about AGE caused by eating fresh crab contaminated with NLVs. As a large aquaculture producers, China provide amount of shellfish every year. Especially oyster is the top one. In the south of China, especially in Zhejiang province, people are used to eat fresh seafood, once the seafood would have contaminated NLVs, gastroenteritis will break. On the other hand, large amount of oysters will be output from China to other countries. Thus it is necessary to set up a simple fast method to detect the NLVs in shellfish.Since 1980s several methods had been used to detect NLVs, such as electron microscopy, immune assay, enzyme immunoassays (EIAs) and western blot assay. However related viruses in cell culture also prevented the production of animal hyperimmune sera and restrict these methods. Public health controls are hampered by the absence of methods for the detection of these viruses in shellfish, as they cannot grow in tissue culture. SoPCR was widely used to detect the exit of NLVs, which take good advantage of sensitivity, specification and quickness.37 experimental oysters came from fishery of Shandong province were deal with Glycine buffer (pH9.5) and PEG 6000 orderly, then RNA was extracted to perform RT-PCR. 6 of 37 samples were detected that were contaminated with NLVs. Most of positive isolates were from Dec, 2002 to April, 2003 which shows that oysters are easier to be contaminated in winter. Purified, cloned and sequenced the RT-PCR products of positive samples. The results of Blastn of 6 isolates in this study indicate they are belong to genotype group II and are with high homology percent to Japan strains in GeneBank. This shows that NLVs in closer geographical areas, their genetic diversity are low.The results show the homology percent of these 6 isolates are 79. 2%. Fecal isolate is 77.0% homological to Japan oyster isolate while only 56. 7%^ 56. 1%^ 57. 3% and 65. 3% homological to other Chinese oysters isolates. The homology percent between Japan oyster isolate and Chinese isolates are 72.49k 73.6%^ 73. 0% and 79.4%.The homology percent among Chinese isolates is 95.5%, especially sample 1, 3, 5 are 99. 0% homological. According to these data we concluded that Chinese isolates belong to the same genotype. The difference between fecal isolate, Japan oyster isolate and Chinese isolates shows the genetic diversity. The alignment of the 6 isolates with strains listed on ICTV shows that Japan oyster isolate is closer to Lordsdale virus (X86557) while Chinese isolates are close to Mexico virus (U22498).To verify the result of RT-PCR, we designed a blot hybridization assay. Based on sequence anal ysis, a biotin label ed probe was designed. This blot hybridization assay provides a simple and quick method to confirm the result of PCR without sequencing of the PCR product. Comparing thesensitivity of RT-PCR and blot hybridization, the result demonstrated that this blot hybridization is a quick and sensitive and specific method for detection of Norwalk-like viruses.Since NLVs are unable to be cultured in cells or tissue organs. It' s hard to estimate the original copies of viruses, especially when the original amount is very low. In order to make quantitative analysis for NLVs, we have developed an assay based on real-time RT-PCR. New primers and probes for NLVs Gil are designed within conserved genome regions. And through this assay, we set up two standard curves of NLVs G I and NLVs G II respectively. According to the standard curve of NLVs Gil, we counted the original amounts (copies) of 3 viruses isolated from oyster: 2. 8X 10\ 3. 9X108fn 1.6X102. The detect limits of NLVs GI and NLVs Gllboth are 10X102 copies.