ABSTRACT
Objective To construct a chimeric infectious clone of the fatal virulent strain SDLY 107, containing the gene fragments encoding 2A and 3B proteins of the mild virulent strain SDLY 1, and to establish a reverse genetic system platform for further investigation on virulence of enterovirus 71 strains. Methods The overlap PCR analysis was performed to obtain the gene fragments encoding 2A and 3B pro-teins of the mild virulent strain SDLY 1.The obtained gene fragments were digested and then cloned into a plasmid pMD19-T containing the full-length gene of SDLY 107 strain by using gene replacement strategy. The recombinant RNA was transfected into Vero cells for the preparation of recombinant virus particles.Sev-eral assays including the PCR, indirect immunofluorescence ( IFA) , Western blot and sequencing were per-formed for virus identification.Virus titers were measured by 50%cell culture infective dose ( CCID50 ) and plaque assay.Results The infectious clones of SDLY 107-2A-1 and SDLY 107-3B-1 chimeric virus strains were constructed successfully.Typical cytopathic effect was observed in Vero cells after viral transfection. Identification of the rescued viruses by PCR, IFA, Western blot and sequencing further confirmed the suc-cessful construction of infectious virus strains.The virus titers of SDLY 107-2A-1 and SDLY 107-3B-1 strains detected by CCID50 and plaque assay were 1.25 ×105 PFU/ml and 0.7 ×105 PFU/ml, respectively. Conclusion The chimeric viruses SDLY 107-2A-1 and SDLY 107-3B-1 were rescued successfully, causing cytopathic effects similar to those by using the parental virus strain SDLY 107.This study might pave the way for further investigation on in vitro and in vivo virulence of enterovirus 71 strains.
ABSTRACT
Objective To determine the function of conserved amino acids in the fusion-promoting domain of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein,clearly understanding mechanism of cell fusion.MethodsUsing a PCR-based site-directed mutagenesis method and the method of homology recombination occurred in vivo to change six conservative amino acids into alanine respectively.Wild type (WT) and all mutant HN proteins were exepressed in BHK-21 cells by the vacciniaT7 RNA polymerase expression system.The amount of each HN protein at the cell surface was determined by fluorescence-activated cell sorter (FACS).Cell fusion efficiency,hemadsorption activity (or receptor binding activity) and neuraminidase activity were determined.Results There was no statistic difference of cell surface expression among WT and each mutant HN protein ( P＜0.05 ).Cell fusion efficiency of each mutant protein decreased to some extent,especially 1103A decreased to 14.2％ in head.Hemadsorption activity of mutant proteins were reduced in different extent,the maximum reduction of which was also 1103A,28.2％ of wt NDV HN.There was different neuraminidase activity among each mutant HN protein.L74A increased slightly to 118.6％.L110A decreased most to 5.2％.I103A decreased second most to 5.7％.Conclusion Conserved amino acids in fusion-promoting domain of NDV HN played an important role in cell fusion.I103 was identified as a key amino acid in this domain.