The purpose of the current study is to assess the effect of removing human cytomegalovirus ( HCMV) by leukocyte reduction filtration ( LRF) in blood center. Peripheral blood mononuclear cells ( PBMCs) from blood
samples negative for HCMV-IgG and pp65 were separated and co-cultured with HCMV-infected human fibroblasts in order to establish infection. The infected PBMCs were purified from fibroblasts by double selection using magnetic beads coated with anti-human fibroblast antibodies and anti-CD45 and then spiked into whole blood units prior to LRF. After LRF, HCMV load in whole blood and in fibroblasts was assessed using real-time quantitative polymerase chain reaction( PCR) for HCMV DNA and quantitative reverse transcriptase PCR( RT-PCR) for mRNA encoding the
HCMV DNA and fibroblast-specific splice variant of prolyl-4-hydroxylase, respectively. After LRF, the rate of leukocyte depletion in whole blood units ( 200 ml) was 99. 98%. After correcting for fibroblast-associated HCMV, the mean HCMV load in whole blood was reduced from3 742 to 22. 57 copies per microliter ( 2. 50 log10 reduction) by LRF. These results suggest that LRF reduces viral load but does not completely remove HCMV from blood components.

To understand the relationship between variation of enterovirus 71 ( EV 71) and its virulence, the sequence of the VP4 gene of EV71 virus was analyzed. Eight strains of EV71 isolated from clinical samples collected from infants and children with hand, foot and mouth disease in the Children’s Hospital Affiliated to Capital Institute of Pediatrics in Beijing during 2007 to 2008 were used for comparison. Full length VP4 genes from these eight EV71 strains were amplified by reverse-transcriptase PCR ( RT-PCR) and sequenced after the amplicons were cloned into pUCm-T. The sequences were compared with VP4 genes from EV71 in GenBank. Sequence analysis and type identification were performed by bioinformatics ( DNAStar) . The full length of VP4 gene has 207 bp coding for a protein of 69 amino acids with estimated molecular weight of 7 ×10³ . The nucleotide homology of VP4 genes among the eight strains were 94% ～100% and homology of the deduced amino acids were 100% . The nucleotide sequence homology between these eight strains isolated in Beijing and those isolated in Fuyang, Shenzhen and Taiwan was higher than those isolated elsewhere. The homology of deduced amino acid sequences encoded by VP4 between these eight isolates in Beijing and those in GenBank was 100% except one strain isolated from India. The amino acids at the 7th and 54th position of VP4 of the strain from India were different from others. There were many differences between the nucleotide sequences of VP4 from the eight Beijing strains and those from severe cases ( BrCr, MS and NCKU9822) , as reported in the literature, whereas only a few nucleotide differences were observed between severe
cases ( BJ97, BJ110B, BJ110Y and BJ4243) and mild cases ( BJ25, BJ47, BJ65 and BJ67) among these eight Beijing strains. Phylogenetic analysis of the VP4 sequences from these eight strains indicates that the EV71 viruses that circulated in Beijing during 2007 to 2008 should be classified as subtype C4. The VP4 genes from the isolates in Beijing from2007 and 2008 were highly conserved. There was no consistent divergence in the sequences of VP4 between strains isolated fromsevere cases and those frommild cases, suggesting that the virulence of the EV71 virus does not seem to be related to the sequence of VP4.

The genes encoding 19kD lipoprotein and early secreting antigenic target-6( ESAT-6) were amplified from genome of the standard Mycobacterium tuberculosis H37Rv using polymerase chain reaction, and then cloned into the vector pMD18-T followed by subcloning into the expression vector pET-21a, respectively. Recombinant 19kD-ESAT6 was expressed in E. coli, and then purified by Ni-NTA. The antigenicity of the fusion protein was analyzed by Western blot analysis. The recombinant 19kD-ESAT6 plasmid was constructed successfully, and could be expressed efficiently in E. coli BL21 ( DE3) . The relative molecular mass of the fusion protein was approximately 29 ×10³ by SDS-PAGE. The recombinant 19kD-ESAT6 protein showed specific antigenicity as determined by Western blot, and can be used for the development of serodiagnostic reagents.

Varicella is a highly contagious disease caused by varicella-zoster virus ( VZV) . Vaccination has provided the best means to control infection. A number of countries maintain a universal childhood vaccination policy since 1995, resulting in a dramatic decrease in the incidence, morbidity and mortality related to varicella. In this article, we review the development of varicella Oka vaccine, the research in vaccine efficiency and safety, the molecular mechanism of attenuation, as well as newideas in immunization strategy, summarizing the current advances and limits
of the varicella vaccine since its worldwide application.