Potential infections of H5N1 and H9N2 avian influenza do exist in Guangdong populations of China.

BACKGROUND: Southeast China is one of the sites of influenza origin. During 2003--2004, nine avian influenza outbreaks took place in Guangdong Province. But no human case was reported. To examine the status of potential human infection by human influenza (H1N1, H3N2) and avian influenza (H5N1, H7N7, H9N2) in the avian influenza epidemic area of Guangdong Province, China, we conducted a seroepidemiologic survey in the people of this area from April to June of 2004. METHODS: Three out of 9 H5N1 avian influenza affected poultry areas in Guangdong were randomly selected, and the population living within 3 kilometers of the affected poultries were chosen as the survey subjects. One thousand two hundred and fourteen people were selected from 3 villages at random. Human and avian influenza antibody titers were determined by hemagglutination-inhibition (HI) test and microneutralization test (MNT). RESULTS: The positive rate of antibody to H5N1 was 3.03% in the occupational exposure group and 2.34% in general citizens group; that of H9N2 was 9.52% in the occupational exposure group and 3.76% in the general citizens group. Moreover one case in the occupational exposure group was positive for H7N7. One year later, all previously positive cases had become negative except for one H5N1-positive case. CONCLUSION: The observations imply that H5N1 and H9N2 avian influenza silent infections exist in Guangdong populations.

Sequence analysis and structural prediction of the severe acute respiratory syndrome coronavirus nsp5.

The non-structural proteins (nsp or replicase proteins) of coronaviruses are relatively conserved and can be effective targets for drugs. Few studies have been conducted into the function of the severe acute respiratory syndrome coronavirus (SARS-CoV) nsp5. In this study, bioinformatics methods were employed to predict the secondary structure and construct 3-D models of the SARS-CoV GD strain nsp5. Sequencing and sequential comparison was performed to analyze the mutation trend of the polymerase nsp5 gene during the epidemic process using a nucleotide-nucleotide basic local alignment search tool (BLASTN) and a protein-protein basic local alignment search tool (BLASTP). The results indicated that the nsp5 gene was steady during the epidemic process and the protein was homologous with other coronavirus nsp5 proteins. The protein encoded by the nsp5 gene was expressed in COS-7 cells and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study provided the foundation for further exploration of the protein's biological function, and contributed to the search for anti-SARS-CoV drugs.

Variation analysis of the severe acute respiratory syndrome coronavirus putative non-structural protein 2 gene and construction of three-dimensional model.

BACKGROUND: The rapid transmission and high mortality rate made severe acute respiratory syndrome (SARS) a global threat for which no efficacious therapy is available now. Without sufficient knowledge about the SARS coronavirus (SARS-CoV), it is impossible to define the candidate for the anti-SARS targets. The putative non-structural protein 2 (nsp2) (3CL(pro), following the nomenclature by Gao et al, also known as nsp5 in Snidjer et al) of SARS-CoV plays an important role in viral transcription and replication, and is an attractive target for anti-SARS drug development, so we carried on this study to have an insight into putative polymerase nsp2 of SARS-CoV Guangdong (GD) strain. METHODS: The SARS-CoV strain was isolated from a SARS patient in Guangdong, China, and cultured in Vero E6 cells. The nsp2 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into eukaryotic expression vector pCI-neo (pCI-neo/nsp2). Then the recombinant eukaryotic expression vector pCI-neo/nsp2 was transfected into COS-7 cells using lipofectin reagent to express the nsp2 protein. The expressive protein of SARS-CoV nsp2 was analyzed by 7% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The nucleotide sequence and protein sequence of GD nsp2 were compared with that of other SARS-CoV strains by nucleotide-nucleotide basic local alignment search tool (BLASTN) and protein-protein basic local alignment search tool (BLASTP) to investigate its variance trend during the transmission. The secondary structure of GD strain and that of other strains were predicted by Garnier-Osguthorpe-Robson (GOR) Secondary Structure Prediction. Three-dimensional-PSSM Protein Fold Recognition (Threading) Server was employed to construct the three-dimensional model of the nsp2 protein. RESULTS: The putative polymerase nsp2 gene of GD strain was amplified by RT-PCR. The eukaryotic expression vector (pCI-neo/nsp2) was constructed and expressed the protein in COS-7 cells successfully. The result of sequencing and sequence comparison with other SARS-CoV strains showed that nsp2 gene was relatively conservative during the transmission and total five base sites mutated in about 100 strains investigated, three of which in the early and middle phases caused synonymous mutation, and another two base sites variation in the late phase resulted in the amino acid substitutions and secondary structure changes. The three-dimensional structure of the nsp2 protein was successfully constructed. CONCLUSIONS: The results suggest that polymerase nsp2 is relatively stable during the phase of epidemic. The amino acid and secondary structure change may be important for viral infection. The fact that majority of single nucleotide variations (SNVs) are predicted to cause synonymous, as well as the result of low mutation rate of nsp2 gene in the epidemic variations, indicates that the nsp2 is conservative and could be a target for anti-SARS drugs. The three-dimensional structure result indicates that the nsp2 protein of GD strain is high homologous with 3CL(pro) of SARS-CoV urbani strain, 3CL(pro) of transmissible gastroenteritis virus and 3CL(pro) of human coronavirus 229E strain, which further suggests that nsp2 protein of GD strain possesses the activity of 3CL(pro).

[Effect of dietary n-6/n-3 polyunsaturated fatty acid ratio on spleen lymphocyte's function and fatty acid composition in mice].

OBJECTIVE: To study the effect of dietary n-6/n-3 PUFA ratio on spleen lymphocyte's function and fatty acid composition in mice. METHODS: Male BALb/c mice were divided into 5 groups: the dietary S : m : p was 1 : 1.5 : 1 n-6/ n-3 PUFA ratio was 1, 7.5, 15, 30 respectively, and in the control group, dietary S : M : P was 1 : 1.5 : 3.7, which was followed the AIN-93G formulation. All groups consumed purified diet based on the AIN-93G formulation. The different n-6/ n-3 PUFA ratios in diets were varied by mixing of oils. After 12 wks breeding, all mice were sacrificed, and the function and fatty acid composition of Lymphocyte, IL-2 and PGE2 concentration were measured. RESULTS: When dietary n-6/n-3 PUFA ratio approximated 1, the lymphocyte proliferation, proportions of CD4+ and CD8+ T cell, IL-2 and PGE2 concentration decreased significantly. The concentration of C18 : 2, C20 : 4, n-6 PUFA in lymphocyte decreased significantly; Meanwhile, the concentration of C22 : 6, C16 : 1, C18 : 1 and total MUFA in lymphocyte significantly higher than other groups. The concentration of C2 : 6 in lymphocytes were significantly negative correlated with the proliferation of spleen lymphocytes. The concentration of C20 : 5 in lymphocyte was significantly negative correlated with the proportions of CD4+ T cell and IL-2 level. The concentration of C16 : 1 in lymphocyte was significantly negative correlated with the proportions of CD4+ and CD8+ T cell. CONCLUSION: The fatty acid composition of diet affected that of lymphocyte in mice. Compared with the T lymphocyte function in diet with n-6/n-3 ratio approximated 30, the T lymphocyte function in mice was suppressed when dietary n-6/n-3 ratio approximated 1.