A natural DNMT1 mutation elevates the fetal hemoglobin level via epigenetic derepression of the γ-globin gene in ß-thalassemia.

DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator of the formation of large macromolecular complexes that repress human γ-globin expression by maintaining DNA methylation. However, very little is known about the association of DNMT1 variants with ß-thalassemia phenotypes. We systematically investigated associations between variants in DNMT1 and phenotypes in 1142 ß-thalassemia subjects and identified a novel missense mutation (c.2633G>A, S878F) in the DNMT1 bromo-adjacent homology-1 (BAH1) domain. We functionally characterized this mutation in CD34+ cells from patients and engineered HuDEP-2 mutant cells. Our results demonstrate that DNMT1 phosphorylation is abrogated by substituting serine with phenylalanine at position 878, resulting in lower stability and catalytic activity loss. S878F mutation also attenuated DNMT1 interactions with BCL11A, GATA1, and HDAC1/2, and reduced recruitment of DNMT1 to the γ-globin (HBG) promoters, leading to epigenetic derepression of γ-globin expression. By analyzing the F-cell pattern, we demonstrated that the effect of DNMT1 mutation on increased fetal hemoglobin (HbF) is heterocellular. Furthermore, introduction of S878F mutation into erythroid cells by clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) recapitulated γ-globin reactivation. Thus, the natural S878F DNMT1 mutation is a novel modulator of HbF synthesis and represents a potential new therapeutic target for ß-hemoglobinopathies.

Immunogenicity and protective potency of Norovirus GII.17 virus-like particle-based vaccine.

OBJECTIVES: Noroviruses (NoVs) are major cause of acute viral gastroenteritis in worldwide, and the lack of a cell culture system that must be considered the virus like particles (VLPs) are used as an effective vaccine development. MATERIALS AND METHODS: In the present study, we investigated the expression of the major capsid protein (VP1) of the Genogroup II, genotype 17 (GII.17) NoV, using recombinant baculovirus system in insect cells, as well as a saliva binding blockade assay to detect their protective potency. RESULTS: Our results showed that GII.17 VLPs could be successfully generated in sf9 insect cells, and electron microscopic revealed that GII.17 VLPs appeared as spherical particles with a - 35 nm diameter. Immunized mice with purified VLPs produced GII.17 specific sera and could efficiently block GII.17 VLPs binding to the saliva histo-blood group antigens (HBGAs). CONCLUSIONS: Together, these results suggested that GII.17 VLPs represent a promising vaccine candidate against NoV GII.17 infection and strongly support further preclinical and clinical studies.

Functional analysis of transcriptional regulation of herpes simplex virus type 1 tegument protein VP22.

The herpes simplex virus type 1 (HSV-1) tegument proteins have important functions in the viral replication process. In order to investigate the role of the HSV-1 tegument protein VP22 in viral replication, its transcriptional regulation of viral promoters was investigated using the chloramphenicol acetyltransferase (CAT) assay. The results indicate that VP22 exerts a dose-dependent transcriptional inhibitory effect on the HSV-1 alpha4, TK, and gC gene promoters. VP22 had the capacity to repress transcriptional activation of promoters via different viral transcription regulatory factors such as VP16 and ICP0, as evidenced by the specific repression of the TK and gC gene promoters by ICP0. In addition, VP22 was capable of inhibiting the promotion of ICP0 transcriptional activation in the presence of HAT PCAF, which is even more remarkable than the VP22 repression of ICP0 transcriptional activation. Finally, the transcriptional inhibitory effect of VP22 on other viral promoters was demonstrated by the analysis of beta-galactosidase activities in internal controls.

[Genomic characteristics of an echovirus 20 strain (KM/EV20/2010) isolated in Kunming, Yunnan, China].

OBJECTIVE: To identify the complete genome sequence of an echovirus 20 isolate (KM/EV20/2010) and understand its genetic variation and evolution characteristics. METHODS: Seven overlapping clones covering the whole viral genome (excluding the poly-A tail) were obtained by RT-PCR and sequenced, and their nucleotide and amino acid sequences were aligned with other echovirus 20 isolates. Phylogenetic and pairwise alignment analyses based on genome and complete VP1 regions were conducted with software Mega 4.1, RDP3 and SimPlot 3.5.1. RESULTS: The genome of the echovirus strain was 7 395 nucleotides in length, and contained a 744-nt non-translated region (NTR) at the 5' end and a 96-nt NTR at the 3' end. The entire open reading frame contained 6 549 nt, encoding a 2 183-aa polyprotein. In the coding region, there was no nucleotide deletion or insertion. Based on the complete genome sequence alignments, the echovirus strain showed 80.1% nucleotide and 96.7% amino acid homology to echovirus 20 prototype JV-1 strain. The phylogenetic trees constructed on the genome and complete VP1 regions all indicated that the echovirus strain was not in one cluster with echovirus 20 prototype JV-1 strain, while had a closer relationship with echovirus 30 prototype Bastianni. Genotyping results from phylogenetic analysis showed that echovirus 20 has six genotypes. The strain used in this study belonged to genotype IV. The nucleotide divergence was 9.4%-21.7% among the 6 genotypes. The possible putative recombination was detected in its non-coding sequence of the echovirus 20 strain used in this study. CONCLUSION: Based on the bioinformatics analysis. The echovirus 20 strains isolated in China could be divided into six genotypes, and the echovirus 20 in this study belonged to genotype IV.

[Intragastric administration of interferon-α-transformed Bifidobacterium promotes lymphocyte proliferation and maturation in mice].

OBJECTIVE: To investigate the effects of intragastric administration of human interferon-α (hIFN-α)-transformed Bifidobacterium on immune functions of mice. METHODS: The E.coli-Bifidobacterium shuttle expression vector containing hIFN-α gene was constructed and transformed into Bifidobacterium. The hIFN-α-transformed Bifidobacterium suspension (1010 /ml) was prepared after induction with 0.2% L-arabinose for hIFN-α expression and administered intragastrically in male Balb/C mice at the dose of 0.1 ml every other day for 2 weeks, with the mice receiving empty vector-transformed Bifidobacteria as the negative control and those having an equal volume of saline as the blank control. The percentages of mononuclear cell subsets in the thymus, spleen and blood were detected in the mice by flow cytometry, and the serum levels of IL-4, IL-12, IFN-γ and TNF-α were assayed using mouse cytokine FlowCytomix Kit. RESULTS: The percentages of CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the thymus, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺CD8⁺ cells in the spleen, and CD3⁺CD8⁺ cells in the blood all increased significantly in IFN group as compared with those in the negative and blank control groups (P<0.01 or 0.05). The serum level of IFN-γ also increased significantly (P<0.05) while IL-4 level remained unchanged in IFN group compared with those in the two groups. CONCLUSION: Intragastric administration of hIFN-α-transformed Bifidobacterium promotes lymphocyte proliferation and maturation and increases the serum levels of Th1 cytokines in mice.

[Biochemical and pathological analysis of mice with type 2 diabetes mellitus induced by high-fat diet and low-dose streptozotocin injections].

OBJECTIVE: To analyze the biochemical and pathological changes in mice with type 2 diabetes mellitus (T2DM) induced by high-fat diet combined with low-dose streptozotocin (STZ) injections. METHODS: C57BL/6J mice were divided randomly into normal control group (NC group), high-fat diet group (HC group) and high-fat diet plus STZ group (HC+STZ group). The mice were fed on normal chow or a high-fat diet for 1 month before two introperitoneal injections of STZ (40 mg/kg) or citrate buffer with an interval of 24 h as appropriate. Fasting blood glucose (FBG) was detected every week for 4 weeks, and oral glucose tolerance test (OGTT) was performed one month after the injections, after which the biochemical profiles, islet and liver were evaluated by immunohistochemical and pathological analysis. RESULTS: In HC+STZ group, FBG was above the cutoff value (13.89 mmol/L) in 75% of the mice at 1 week after STZ injections and in all the mice at two weeks except for the death of 1 mouse, with a success rate of modeling of 91.3%. FBG in HC group, though slightly higher than that in NC group, remained normal (6.8 mmol/L). The body weight in HC+STZ and HC groups was significantly higher than that in NC group after feeding but without obvious increases after the injections (P<0.01). Blood glucose in HC+STZ group at 0.5 to 2 h after OGTT and the area under curve (AUC) were higher than those in NC and HC groups (P<0.01); the AUC in HC group was a also higher than that in NC group (P<0.05). Plasma creatinine was significantly higher in HC+STZ group than in NC (P<0.01) and HC (P<0.05) groups. Insulin secretion by the islets decreased obviously in HC+STZ and HC group. The mice in HC+STZ group showed atrophy, fibrosis, and vacuolization in the islets with mild fatty liver but no visible renal pathologies. CONCLUSION: High-fat diet and low-dose STZ injections can induce T2DM in mice with very similar biochemical and pathological changes to human T2DM and with such complications as fatty liver.

[Genomic characteristics of coxsackievirus B5 A210/KM/09 strain isolated in Yunnan, China].

OBJECTIVE: To characterize the complete genome sequence of coxsackievirus B5 (CVB5)A210/KM/09 strain which was isolated from Yunnan, China, 2009. METHODS: Eight overlapping clones covering the whole viral genome (excluding the poly-A tail)were obtained by RT-PCR and sequenced, with their nucleotide and amino acid sequences compared with other known CVB5 strains. RESULTS: The genome of the CVB5 A210/KM/09 strain had 7 372 nucleotides in length, and containing a 742-nt non-translated region (NTR) at the 5' end and a 98-nt NTR at the 3' end. The entire open reading frame contained 6 555 nt, encoding a 2 185-aa polyprotein. In the coding region, there appeared no nucleotide deletion or insertion, but some changes of amino acid seemed unique. Based on the complete genome sequence alignments, CVB5 isolate A210/KM/09 strain showed the highest nucleotide (92.5%) and amino acid (97.3%) identities to the CVB5/CC10/10. It also shared nucleotide (80.1%-92.5%) and amino acid (95.0%-97.3%) homology with other CVB5 strains: 17Y, 19CSF, 20CSF, 1954/85/US, 2000/CSF/KOR, 03001N, CoxB5/Henan/2010, VB5/SD/09 and Faulkner. Blast between genome fragments, A210/KM/09 showed similarity on nucleotide (80.1%-92.5%) and amino acid (95.0%-97.3%) identities with other CVB5 strains. The phylogenetic tree, constructed on the complete VP1 regions, indicated that CVB5 could be divided into genotype A, B, C and D. while Genotype C and D could be further divided into C1-C4 and D1-D4 subgenotypes. CONCLUSION: A210/KM/09 and other CVB5 predominant strains isolated in China belonged to CVB5 subgenotype C4.

Comparative study of the immunogenicity in mice and monkeys of an inactivated CA16 vaccine made from a human diploid cell line.

The coxsackie A16 virus (CA16), along with enterovirus 71 (EV71), is a primary pathogen that causes hand, foot, and mouth disease (HFMD). To control HFMD, CA16, and EV71 vaccines are needed. In this study, an experimental inactivated CA16 vaccine was prepared using human diploid cells, and the vaccine's immunogenicity was analyzed in mice and rhesus monkeys. The results showed that the neutralizing antibody was developed in a dose-dependent manner, and was sustained for 70 days with an average GMT (geometric mean titer) level of 80 to 90 in immunized mouse and for 56 days with GMT of higher than 300 in monkeys. The neutralizing antibody had a cross-neutralizing activity against different viral strains (genotype A and B), and the specific IFN-γ-secreting cell response was activated by these virus strains in an ELISPOT assay. This study provides evidence for the potential use of inactivated CA16 as a candidate for use in vaccines.

Thymosin alpha-1-transformed Bifidobacterium promotes T cell proliferation and maturation in mice by oral administration.

Thymosin alpha-1 (Tα1) has been used as an immune potentiator for treatment of immune deficiency diseases by injection administration. However, injection is inconvenient and may cause many side effects. In order to improve the administration convenience of Tα1, a human Tα1 gene transformed Bifidobacterium longum (BL-Tα1) was prepared and its effects on mice immunity by oral administration were investigated. The Balb/c mice were treated with BL-Tα1, which was pre-induced with 0.2% l-arabinose, every other day for 2 weeks. The B. longum transformed with empty vector (BL-0) was used as the negative control, and normal saline (NS, 0.9% saline) was used as the blank control. The results shown that (1) the CD3(+)CD4(+) and CD3(+)CD8(+) T-cells in blood, spleen and thymus, and the CD4(+)CD8(+) cells in thymus and spleen of BL-Tα1 group were all significantly increased than that of negative control BL-0 group respectively; (2) the interferon-γ (IFN-γ) and interleukin-12 (IL-12) in serum of BL-Tα1 group were significantly increased. No significant differences were found in the levels of tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) between BL-Tα1 group and BL-0 group; (3) thymic hyperplasia and lymphadenectasis were observed in BL-Tα1 group after three-month treatment. In conclusion, the Tα1-transformed B. longum promotes thymus and lymph nodes growth, stimulates T cell proliferation and maturation, and enhances cellular immunity through Th1 pathway by oral administration.

[Genomic characteristics of coxsackievirus B1 MSH/KM9/2009 strain isolated in Yunnan, China].

To characterize the complete genome sequence of coxsackievirus B1 (CVB1) MSH/KM9/2009 strain isolated from Yunnan, China,2009. Eight overlapping clones covering the whole viral genome (excluding the poly-A tail) were obtained by RT-PCR and sequenced, and their nucleotide and amino acid sequences were compared with other known CVB1 strains. The genome of the CVB1 MSH/KM9/2009 strain had 7384 nucleotides in length, and contained a 741nt non-translated region (NTR) at the 5' end and a 94nt NTR at the 3' end. The entire open reading frame contained 6 549 nt, encoding a 2 183-aa polyprotein. In the coding region, there was no nucleotide deletion or insertion, but some changes of amino acid were unique. The complete genome sequence alignments showed that the CVB1 isolate MSH/KM9/2009 strain shared the highest nucleotide (80.9%, 81.6%, 80.5% and 80%) and amino acid (95.6%, 95.8%, 96.2% and 95.6%) identities to the CVB1 M16560, pmMC, Tucson B1 and CVB1Nm strain, respectively. Phylogenetic tree analysis showed that the MSH/KM9/2009, CVB1 M16560, pmMC, Tucson B1 and CVB1Nm strain clustered into same group. The newly isolated CVB1 strain MSH/KM9/2009 from Yunnan Province belonged to genotype CVB1.

[Complete nucleotide sequence of a human coxsackievirus B3 strain A103/KM/09 isolated in Yunnan province, 2009].

OBJECTIVE: To analyze the genetic characterization of the complete genome from a human coxsackievirus B3 strain A103/KM/09 isolated in Yunnan province, 2009. METHODS: By using RT-PCR, all the eight fragments which containing about 1000 nucleotides and covering full viral genome, were sequenced. By using Mega 5.05,Geneious, RDP 3 and SimPlot 3.5.1 software, sequences were aligned with other enterovirus reference sequences. Phylogenetic and recombination analysis were also carried out. RESULTS: The A103/KM/09 isolate genome showed 7389 nucleotides in length , encoding for 2185 amino acids. In the complete genome, the homology of nucleotide and amino acid among the seven coxsackievirus B3 isolates were 81.0%-88.0% and 95.7%-98.0%, respectively. There appeared 81.0% and 95.7% homology when compared with that of Nancy prototype strain. Results from the Phylogenetic analysis showed that the coxsackievirus B3 formed five distinct clades, I-V. Nucleotide divergence rates between clades were 16.2%-24.3% . The A103/KM/09 strain belonged to clade V. Clade V was further divided into four sub-clades,A-D. The nucleotide divergence between sub-clades was 4.3%-11.4%. Putative recombinant event for A103/ KM/09 was detected. CONCLUSION: All coxsackievirus B3 isolates could be divided into five clades, with A103/KM/09 strain belonged to Clade V-D. Evolution of coxsackievirus B3 had occurred in China.

[Complete genome sequence characteristics of human enterovirus 71 strain isolated in Yunnan, China].

The complete nucleotide sequence of two human enterovirus 71 strains (KMM09 and KM186-09) isolated in Yunnan,China, were determined by RT-PCR and sequencing. As with other human enteroviruses, the genomes were 7 409 nucleotides (nts) in length and encoded 2 193 aa. Phylogenetic analysis based on VP1 regions revealed that the two isolates belonged to subgenotype C4a. In structural genomic regions, subgenotype C4 was most homologous to other strains of C genotype when compared to other genotypes. In non-structural genomic regions, subgenotype C4 was more homologous to CA16/G10 and other strains of B genotype than to other strains of C genotype. RDP3 and Blast analysis displayed evidence of recombination in non-structural genomic regions between subgenotype B3 and C4, C4 and CA16/G10. The full-length genome of the human enterovirus 71 strains provided an overview of the diversity of genetic characteristics of a circulatinghuman enterovirus 71.

A comparative study of the characteristics of two Coxsackie A virus type 16 strains (genotype B).

Coxsackie A virus is one of the major pathogens associated with hand, foot and mouth disease (HFMD). The etiological characteristics of Coxsackie A virus type 16 (CA16) are thought to correlate with the pathological process of its infection. Two CA16 strains that were isolated from a severe HFMD patient presented with different plaque forms. This observation, along with biological analysis, indicated that the differences in the strains' biological characteristics, such as proliferation kinetics and immunogenicity, correlate with differences in their pathogenicity toward neonatal mice. Furthermore, these differences are thought to be associated with the sequence of the 5' non-coding region of the viral genome and the VP1 structural region sequence. The results suggest that the biological and genetic characteristics of the CA16 viral strains are relevant to their pathogenicity.

[Feasibility study of using Sabin-IPV immunization in post-eradication era in China].

Based upon the World Health Organization (WHO)'s perspective, the post-eradication era is coming with a priority of global certifying the wild poliovirus eradication, by which then the Oral Poliomyelitis Attenuated Live Vaccine (OPV) will be ceased and Inactivated Poliovirus Vaccine (IPV) will be synchronized used, in particular the affordable Sabin-IPV (sIPV) is recommended to be used continuously in developing countries till the final eradication of poliomyelitis. However, no any sIPV has successfully been developed in the world. In this paper, the sIPV immunization strategy, current development status and its usage in industrialized countries are preliminarily analyzed. Feasibility analysis of potential sIPV usage in China, including immunization strategy, schedule, production technology and supply capacity is analysed as well. sIPV development in China should be accelerated in order to prepare production technology to the post-eradication era.