ABSTRACT
<p><b>OBJECTIVE</b>To study the severe acute respiratory syndrome (SARS)-associated coronavirus genotype and its characteristics.</p><p><b>METHODS</b>A SARS-associated coronavirus isolate named ZJ01 was obtained from throat swab samples taken from a patient in Hangzhou, Zhejing province. The complete genome sequence of ZJ01 consisted of 29,715 bp (GenBank accession: AY297028, version: gi: 30910859). Seventeen SARS-associated coronavirus genome sequences in GenBank were compared to analyze the common sequence variations and the probability of co-occurrence of multiple polymorphisms or mutations. Phylogenetic analysis of those sequences was done.</p><p><b>RESULTS</b>By bioinformatics processing and analysis, the 5 loci nucleotides at ZJ01 genome were found being T, T, G, T and T, respectively. Compared with other SARS-associated coronavirus genomes in the GenBank database, an A/G mutation was detected besides the other 4 mutation loci (C:G:C:C/T:T:T:T) involved in this genetic signature. Therefore a new definition was put forward according to the 5 mutation loci. SARS-associated coronavirus strains would be grouped into two genotypes (C:G:A:C:C/T:T:G:T:T), and abbreviated as SARS coronavirus C genotype and T genotype. On the basis of this new definition, the ZJ01 isolate belongs to SARS-associated coronavirus T genotype, first discovered and reported in mainland China. Phylogenetic analysis of the spike protein gene fragments of these SARS-associated coronavirus strains showed that the GZ01 isolate was phylogenetically distinct from other isolates, and compared with groups F1 and F2 of the T genotype, the isolates of BJ01 and CUHK-W1 were more closely related to the GZ01 isolate. It was interesting to find that two (A/G and C/T) of the five mutation loci occurred in the spike protein gene, which caused changes of Asp to Gly and Thr to Ile in the protein, respectively.</p><p><b>CONCLUSION</b>Attention should be paid to whether these genotype and mutation patterns are related to the virus's biological activities,epidemic characteristics and host clinical symptoms.</p>
Subject(s)
Humans , Middle Aged , Genotype , Mutation , Severe acute respiratory syndrome-related coronavirus , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate a 272 base pair section of the 5'-non-coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV)-negative human subjects (not patients).</p><p><b>METHODS</b>This sequence section bears interest because (1) it harbors several potential methylation (Cp-rich) sites, and (2) it represents the largest part of its internal ribosomal entry site. A pre-PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: SmaI, XmaCI, MspI, and HpaII are inhibited if methylation(s) are present at certain cytosines within their cutting sequences.</p><p><b>RESULTS</b>The suspected HCV-specific sequence was found in the DNA of each subject tested. The pre-PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena.</p><p><b>CONCLUSIONS</b>The results provide formal proof that these HCV-specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences.</p>
Subject(s)
Humans , DNA Methylation , Hepacivirus , Genetics , Restriction Mapping , Sequence Homology, Nucleic AcidABSTRACT
To seek the optimum experiment methods of animal immunization with HCV gene and to explore the effect on antibody responses in mice immunized by pCD-HCV1 recombinant in different administration, recombinant pCD-HCV1 was constructed by technique of molecular biology and was injected into muscles of Balb/c of mice with different times, routes and dosage of inoculations as well as different treatment. The results showed that the serum antibody level reached 0.183±0.06,0.428±0.05,0.707±0.08 and 0.773±0.07(OD410 value) respectively after recombinant pCD-HCV1(100μg/mouse) were injected into mice once, twice, three times and four times. The antibody level of mice (n＝12) with four times inoculation was the highest; pCD-HCV1 was perfused into stomach orally in mice or were into mice by i.p, s.c and i.m(100μg/mouse, three times) in different routes (n＝6), and the antibody levels were 0.138±0.05, 0.178±0.07, 0.233±0.08 and 0.691±0.05 respectively; after the mice (n＝8) were inoculated with the pCD-HCV1 of different dosage(10μg, 50μg and 100μg) the antibody levels of three groups were 0.11±0.09, 0.33±0.04, and 0.700±0.07, and the results showed a significant difference (P＜0.01); Mice was injected with procaine (100μl, 0.4mg) by i.m or s.c. Then pCD-HCV1 was injected into mice and antibody levels were higher than that of mice immunized directly with recombinant pCD-HCV1 of same dosage. The results may provide a reference data deserved for screening the optimum immunization method of development HCV-DNA-based vaccine in mice model.