Influence of miR-30b regulating humoral immune response by genetic difference.

Investigation of genetic difference will be beneficial to researchers to understand the origins and nature of diseases. Previous studies have revealed that L-kynurenine (L-Kyn) level was changed significantly in patient with cancer and that miR-30b play different role in tumor cells and immune cells. Moreover, it has been also conformed that miR-30b involved in the process of L-Kyn-mediated suppression of humoral immune responses induced by lipopolysaccharide (LPS) in human normal B cells separated from volunteers' peripheral blood. Nevertheless, the miR-30b role regulating humoral immune response in B lymphoma cells has been still unclear due to the genetic difference between normal cells and tumor cells. The current study demonstrated that the selected concentration of L-Kyn (100, 1000 µM) significantly reduced the immunoglobulin M secretion induced by LPS when compared with the control group in B lymphoma, CH12.LX, and BCL-1 cells, which had, at least, incomplete dependence on Aryl hydrocarbon receptor, the receptor of L-Kyn. In addition, although L-Kyn (100 µM) significantly attenuated the expression of miR-30b in BCL-1 cells rather than in CH12.LX cells, no significant differences in the strength of L-Kyn-mediated suppression of humoral immune responses induced by LPS were detected by enzyme-linked immunosorbent assay between the LPS (10 µg/ml) + L-Kyn (100 µM) group and the LPS (10 µg/ml) + L-Kyn (100 µM) + miR-30b mimics/miR-30b inhibitor group in CH12.LX and BCL-1 cells, respectively. Further data also showed that mouse Bach2 mRNA was a novel target of miR-30b. These results suggest that genetic difference among cells has a great influence on the miR-30b role in the process of L-Kyn-mediated suppression of humoral immune responses induced by LPS.

Full-length genome of wild-type hepatitis A virus (DL3) isolated in China.

AIM: To characterize the genome of an wild-type HAV isolate (DL3) in China. METHODS: A stool specimen was collected from hepatitis A patient from Dalian, China. HAV (DL3) was isolated and viral RNA was extracted. The genome of DL3 was amplified by reverse transcription and polymerase chain reaction (RT-PCR), followed by cloning into pGEM-T vector. The positive colonies were selected and sequenced. The full-length genome of DL3 was analyzed and compared with other wild-type HAV isolates. RESULTS: The genome of DL3 was 7 476 nucleotides (nt) in size, containing 732-nt 5'untranslated region (UTR), 6 681-nt open reading frame (ORF) which encoded a polyprotein of 2 227 amino acids (aa), and 63-nt 3'UTR. The base composition was 28.96 % A (2 165), 16.08 % C (1 202), 22.11 % G(1 653) and 32.85% U (2 456). Genomic comparisons with wild-type HAV isolates revealed that DL3 had the highest identity of 97.5 % for nt (185 differences) with AH1, the lowest identity of 85.7 % (1 066 differences) with SLF88. The highest identity of 99.2 % for amino acid (18 differences) appeared among DL3, AH2 and FH3, and the lowest identity of 96.8 % (72 differences) between DL3 and SLF88. Based upon comparisons of the VP1/2A junction and the VP1 amino terminus, DL3 was classified as subgenotype IA. Phylogenetic analysis showed that DL3 was closest to the isolates in Japan. CONCLUSION: The sequence comparison and phylogenetic analysis revealed that DL3 is most similar to the isolates in Japan, suggesting the epidemiological link of hepatitis A happened in China and Japan.

Mutational characteristics in consecutive passage of rapidly replicating variants of hepatitis A virus strain H2 during cell culture adaptation.

AIM: To investigate the molecular mechanism of cell adaptation and rapid replication of hepatitis A virus strain H2 in KBM17 cells. METHODS: Virus of strain H2 at passage 7 was consecutively passaged in KBM17 cells for 22 passages, every passage was incubated for 14 days. Antigenic and infectious titers of every passage and one-step growth dynamics of passage 22 were determined with ELISA. Genomes of passage 6, passage 12, passage 18 and passage 22 were sequenced and compared with H2K7. RESULTS: During continuous passage of vaccine strain H2 at passage K7 in KMB17 cells, infectious and antigenic titers increased with the increase of passages, infectious titers at day 14 reached 6.77LgCCID(50)ml(-1) for passage 6 (P6), 7.0 LgCCID(50)ml(-1) for passage 12 (P12), 7.33 LgCCID(50)ml(-1) for passage 18 (P18) and 7.83 LgCCID(50)ml(-1) for passage 22 (P22), respectively. The one-step growth dynamics showed that replicating peak of P22 appeared at day 14 with infectious titers of 7.83 LgCCID(50)ml(-1) and antigenic titer of 1:1024. After passage 22 a new cell-adapted variant (P22) of H2K7 with rapid and shortened replication cycle from 28 days to 14 days was obtained. Sequencing and comparisons of genomes of P6, P12, P18 and P22 showed that mutational numbers in genomes of different passages increased with adaptive passages, and mutations scattered over the genome. In comparison with that of K7, P6 had only 6 nucleotides (nt) mutations, P12 had 7 mutational changes, in addition to 6 same mutations with P6, there appeared a new mutation in 5'NTR at nucleotide position 591 resulting in a nucleotide exchange from A to G. P18 had 10 nt mutations, among the 10 mutations, 7 mutational changes were same as with P12, three new mutational changes appeared in the genome, one in 5'NTR, one in 3C coding region, one in 3D coding region, at P22 there appeared 18 nucleotide changes in the genome, on the basis of P18,there occurred additional 8 nucleotide mutations, two in 5'NTR, three in 2C, one in 3A, one in 3C and one in 3D. The results suggested that although H2K7 was already an attenuated strain, the mutations of genome is not sufficient to completely adapt the KMB17, further mutations caused rapid replication adaptation. CONCLUSION: 18-nt changes scattering over the genome are cooperatively responsible for further adaptation characterized by rapid and shortened replication cycle from 28 days to 14 days in KMB17 cells. The mutations in 2C coding region play more important role in increase of infectious titer than other mutations, the mutations in 2B coding region show less important role than it usually does in cell adaptation, nucleotide changes in 5' NTR seem to be not relevant to cell adaptation during initial stages (before P6), but do in late stages.