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.

[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.

[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.

[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.

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.

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.

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.