Basic Research On Screening Model And Document Study On Effective Herbs And Formulae For Norovirus Gastroenteritis

Noroviruses (NoV) are a leading cause of epidemics of acute nonbacterial gastroenteritis, affecting people of all ages worldwide. NoV infection with high infection rates exist in populations both in developing and developed countries. The viruses are highly contagious, usually resulting in large outbreaks in crowded communities or institutions such as schools, restaurants, hospitals, child care centers, nursing homes for the elderly, cruise ships, and military settings. It was estimated that 10% of the population of United States experienced NoV gastroenteritis each year (23 million cases); and NoV accounts for a substantial extent of disease and potential economic loss (35-75 million dollars), particularly to the health service where a large proportion of outbreaks occur. In China, acute gastroenteritis (AGE) is one of the most common illnesses of children, as well as in other developing countries. In family settings gastroenteritis is second in frequency only to the common cold. However, there are only few reports on the prevalence of NOV in China. So far, AGE caused by NoV infections were not a routine surveillance project, just classified into C grade infectious diseases as other infectious diarrhea, in China. And the role of NoV infections was over underestimated. NOV are small (～38 nm in diameter), nonenveloped, singlestranded, positive-sense RNA viruses belonging to the family Caliciviridae. The NOV genome encodes three open reading frames (ORF), one of which, ORF-2, encodes one major structural protein of～60 kDa that spontaneously forms virus-like particles (VLPs) when expressed in baculovirus or in other expression systems. These VLPs are morphologically and antigenically indistinguishable from the native forms of viruses found in human stools, providing valuable materials for the development of immunological assays, for the study of virus-receptor host interaction.NoV are difficult to study, because there is no cell culture or animal model available for them, and the disease is difficult to control because of a lack of vaccines or effective antivirals against NoV. It is therefore a public health priority to develop an effective strategy for the prevention and treatment of NoV infection. Despite the difficulty in cultivating noroviruses, significant advances in understanding the genomic structure, individual viral proteins, RNA replication strategy, and virus-host interaction of the virus have been made. These advancements provide new strategies in the development of antiviral agents against norovirus, including the inhibition of viral attachment to host cells through carbohydrate receptors, inhibition of viral protease and polymerase functions, and interference in viral replication.GII-4 norovirus strain now was recognized as the predominant strain circulating around the world in last 20 years. In this study, VLPs from a GⅡ-4 NoV, which was identified as the prevalence strain in China in our former studies, were expressed in insect cells. EIA methods for VLPs binding to HBGAs receptor were also established. These works will provide basis for future drug screening.Chinese medicine had a long history for viral diarrhea treatment. More and more prescriptions were explored in these years with good effect. In the present study, herb and prescriptions were screened by document study as future Chinese medicine candidate.1. Clone and Sequence Analysis of the ORF2 Gene of GⅡ-4 norovirusRNA was extracted from fecal sample. After transcription and PCR, the ORF2 gene was cloned in sample T-vector. Sequences were analyzed by using DNAstar software and compared with prototype sequences retrieved from the NCBI database by Clustal X and Treeview software. Exact whole ORF2 gene of norovirus GⅡ-4 strain was obtained which code 54 KD amino acid. The capsid region consists of two major domains, the shell (S) and the protrusion (P) domains, which are linked by a short hinge. The S domain forms the interior shell of the capsid, while the P domain builds up arch-like structures extending from the shell. Three ORF2 gene of norovirus GⅡ-4 strain was obtained and strains in this study showed 92-93% homology with VA387 strains. And there existed 3 conserved regions in ORF2 region:a short hinge (FLVPPTVE) between P and S domain; the trypsin cleavage sites (KT or RT amino acid) and a highly conserved arginine (R) cluster at the C terminus of the P domain, which is critical for receptor binding.2. Expression of NoV capsid protein (VLPs) in insect cellsGZ121 NoV ORF2 gene amplified by PCR by using PMD18-T plasmid, was inserted into baculovirus expression system, constructed a recombinant vector, pFastBac-GZ121 ORF2. After the transposition in DH10Bac, recombinant baculovirus rBacmid-GZ121 ORF2 was gained. After the transfection to Sf9 cells, rBac-GZ121 ORF2 was obtained in supernatant culture. VLPs expression were confirmed by using SDS-PAGE and Western blot and showed an expected molecular mass about 58KDa. And the expressed VLPs had a 30nm diameter under Electronic Microscope. It indicated the successful expression VLPs in insect cells, which will provide material basis for further studying the characteristic of NoV VLPs. 3. GZ121 VLPs bind to Saliva HBGAs receptor and its significanceSaliva specimens were collected from 63 volunteers; HBGAs antigens types were identified by using agglutination inhibition assays; the binding patterns of NoV to HBGAs antigens were identified by EIA. Among 63 volunteers, O-antigen accounted for 33.3%, B for 22.2%, A and non-secretor for 20.6% and AB for 3.2%, respectively. GZ121 VLPs can bind to all secretors (OD>2.5), but not to all nonsecretors (OD≦0.3). These results also confirmed our successful expression VLPs in insect cells. We established experimental methods on NoV binding to saliva HBGAs antigen; it will help us to explore the host adaptation character, virus evolution and epidemiology of NoV; it will also help us to establish technique platform of anti-NoV drugs screening, and lay the basis for NoV prevention and therapy in Chinese population.4. Screening and evaluation of effective herbs and formulae for viral diarrheaWe used words (viral diarrhea, autumn diarrhea, acute gastroenteritis, and Chinese medicine, Chinese herb, Chinese medicine product, integrated traditional chinese and western medicine and experience report) as key words and did search documents in CBMdisc and CNKI from Jan 1999 to Dec 2008; and we obtained 374 documents. After eliminating unqualified documents,158 documents were selected to our study. The herbs or formulae from one document saved as a record.After the frequency study of the herbs and formulae in these documents, some characteristics of herbs and formulae were found:(1) emphasizing on the use of classic traditional formulae. (2) Emphasizing on some herbs; (3) equally focusing on invigorating the spleen for eliminating dampness; (4) also focusing on the cause of the disease.Combining with the screening and analysis of the effective herbs for viral diarrhea, we make the essence prescription:baked white atractylodes rhizome (10g), Poria from Yunnan (10g), Agastache rugosus(10g), roasted Radx Puerariae(10g), plantago seed(10g),dark plum(5g).