Bing du xue bao = Chinese journal of virology最新文献

In this study, we examined the complete genome of coxsackievirus A6 (CVA6) from hand, foot, and mouth disease in Guangdong Province from 2013,and explored the genetic similarities and differences in epidemic and non-epidemic stains of CVA6.Eighteen strains of CVA6 were included in complete genome sequencing, and the sequences were subject to phylogenetic analysis,sequence alignment analysis and genetic recombination analysis using the software DNASTAR6.0,MEGA5.2and SimPlot3.5.1.The results showed that the complete genome of 18 Guangdong CVA6strains ranged from 7390bp to 7392bp.No insertions or deletions were detected in the coding region. There were several insertions and deletions in 5′UTR and 3′UTR.Phylogenetic analysis indicated that the nucleotide and amino acid sequence identity between the 18 complete genomes were 90.5%-99.6% and 97.5%-99.9%,respectively.The strains isolated in2013 could be further divided into two clusters, III and IV, while the strains isolated in 2011 were only present in the IV cluster. Genetic recombination analysis revealed that the Guangdong representative strain of CVA6,GD870/2013,had gene recombination in the P2 and P3regions,while the GD839/2013 strain did not show obvious genetic recombination. Genome-wide analysis of CVA6 revealed that there are two possible transmitted chains, III and IV, in epidemic strains from Guangdong Province in 2013.The transmitted chain Ⅲ originated from the strain with genetic recombination in the P2 and P3regions,whichwas completely different from the chain IV. Transmission of chain IV of CVA6 was only observed in the nonepidemic 2011 strain.

In order to develop a novel effective biological insecticide for controlling oleander hawk moth, a new pathogen was isolated from naturally diseased Daphnis nerii. Based on scanning electron microscopy, full-length amplification of cDNAs (FLAC), and phylogenetic analysis of genome segments 2and 10,the virus was identified as a new type of cypovirus (Da phnis nerii cypovirus [DnCPV]). Electrophoresis analysis showed that DnCPV had a genome comprising 10double-stranded RNA (dsRNA) segments, ranging from 892 to 4160bp.Using FLAC, the cDNAs from the 10 dsRNA segments of the new CPV were cloned and genome segments 2and 10 were sequenced. Sequencing results showed that segment 2 encoded RNA-dependent-RNA-polymerases (RdRps) and segment 10 encoded polyhedrin. These two segments shared conserved terminal sequences of AGUCAAA and AGC at the 5’and 3’ends,respectively.These conserved terminal sequences were not consistent with any of the known CPV types.Phylogenetic analysis of the RdRp and polyhedrin indicated that this CPV was more closely related to CPV type 19 and type 5than other CPV types. Based on the unique conserved terminal sequences and the electrophoresis pattern of the new virus, we tentatively named it DnCPV Nanchang isolate: DnCPV-NC.

As direct antiviral effector proteins, the innate antiviral proteins induced by pathogens and interferon can restrict intracellular viral infection at an early stage, establishing the state of the host antiviral immune response. Mx, PKR, OAS, IFITM, ZAP, and other proteins have all been shown to play important roles in the antiviral response. The host innate immune factors Mx and IFITM are both key antiviral effectors against influenza, while the antiviral mechanism of viperin remains to be completely elucidated. Research on the avian innate immune system is still at an early stage, resulting in fragmented knowledge in this area. Moreover, mechanisms of intrinsic and innate immunity in birds remain unclear.

The purpose of this study was to examine polymorphisms in the human cytomegalovirus(HCMV)UL144gene in children with asymptomatic HCMV infection. PCR was performed to amplify the UL144 open reading frame(ORF)from urine specimens of asymptomatic HCMV-DNA positive children and both strands of the amplicon were sequenced. Sequence analysis was performed with software including BioEdit,DNAstar,Mega5.0and GeneDoc. Twenty-one of 50 clinical strains were successfully amplified and sequenced, giving a positive rate of detection of 42%.Nucleotide sequence homology ranged from 80.2%to100% and amino acid sequence homology ranged from 77.8%to 100%.The UL144 sequences were distributed among two genotypes, type A(47.61%)and type B(52.38%).The Expasy database was used to analyze the important functional motifs of the UL144 protein. These results revealed that there was a high level of conservation of post-translational modification sites including ASN, PKC, TNFR, and NCD3 G.UL144type B added a PROKAR-LIPOPROTEIN site and ZF-CTCHY site between amino acid residues 1 and 16 and between amino acid residues 30 and 96,respectively,as compared with type A.Compared to the UL144 gene from the Toledo strain, there was a high level of conservation in the CRD1 and CRD2of UL144 type A, while significantly more variability was observed in CRD1 and CRD2of UL144 type B. The transmembrane and cytoplasmic domains were highly conserved in both UL144 type A and type B. Variation in nucleotide sequences of UL144 type A and type B did not cause major changes to the predicted isoelectric point or secondary structure of the UL144 protein. The UL144 genotype of children with asymptomatic HCMV infection was divided into type A and type B, which was different from children with symptomatic HCMV infection.

Enterovirus 71（EV71）is one of the major pathogens of hand, foot and mouth disease (HFMD). The EV71 genome encodes an RNA-dependent RNA polymerase（RdRp）,3D(pol),which is critical for genome transcription and translation. However, how the 3D(pol) interacts with the host remains unclear. Yeast two-hybrid systems provide an effective approach for detecting protein-protein interactions. In this report, we inserted the DNA sequence of 3D(pol) into the pGBKT7 vector as the bait plasmid for the yeast two-hybrid experiment and transformed the plasmid into the yeast AH109 strain. We detected the expression,cytotoxicity and self-activity of 3D(pol).The 3D(pol) expressed well without affecting cell growth but exhibited strong transcriptional activation in yeast cells. We further constructed a series of pGBKT7-3D(pol) deletion mutants and identified the shortest transcriptional activation domain（1-94aa）using a self-activation assay. The results provide a molecular basis for screening the host proteins that interact with 3D(pol) using the yeast two hybrid system.

Loop mediated isothermal amplification(LAMP)technology is a newly developed isothermal amplification technology for in vitro detection of nucleic acids. Although the LAMP assay is rapid, specific, sensitive, simple and has been widely applied for rapid detection of nucleic acids, it continues to improve and develop. In this paper, we summarize approaches to addressing amplification product contamination, primer design to avoid false positives, and the development of related techniques based on LAMP technology. This paper could serve as a reference for the application of the assay at the grassroots level.

Baculoviruses are a diverse group of viruses with double-stranded circular DNA genomes. They have certain advantageous properties for biotechnology applications, including high host specificity and environmental friendliness.Baculoviruses could play more important roles in sustainable agriculture as potential microbial insecticides. However, the popularization and application of baculovirus-based insecticides were seriously restricted due to deficiencies such as low virulence and slow rates of action. The infectivity of baculoviruses can be improved by synergistic factors. The present review summarizes characteristics of seven types of synergistic factors including baculovirus enhancin, entomopoxvirus fusolin and calcofluor. The mechanisms of these seven synergistic factors were analyzed. The information presented in this review can serve as a reference to aid in the development and application of baculovirus-based insecticides.

Influenza A H1N1pdm09 Virus; Chicken embryo; Phylogenetic tree; Egg-adaptation; Antigenic site; Drug-resistance site; Abstract: To investigate egg-adapted mutation in influenza A H1N1pdm09 viruses isolated from the Hubei influenza surveillance network, a comparative analysis was performed of three influenza A H1N1pdm09 viruses isolated in chicken embryo and the corresponding MDCK cell-derived viruses. Analyses included examination of the phylogenetic tree, evolutionary rates, amino acid substitutions, egg-adapted mutation and homology modeling. We found differences between the egg-adapted viruses and MDCK cell-derived viruses based on phylogenetic trees and evolutionary rates; the viruses showed a trend of " NA>HA>MP". Four amino acid substitutions(Q223R,V527 I,M19Iand H275Y)were found in three egg-adapted viruses.Q223 Rand V527Iwere present in the haemagglutinin protein, while M19I and H275Y were detected in neuraminidase.The Q223R mutation changed the structure of antigenic sites between Sb and Ca2. H275Y is a classic neuraminidase resistance mutation. The results suggest that the egg-adapted mutations were introduced when influenza viruses were isolated in chicken embryos from the Hubei influenza surveillance network. These mutations may affect the selection of vaccine candidates and vaccine efficacy; therefore, monitoring of egg-adapted mutations should be strengthened in the influenza surveillance network.

The purpose of this study was to construct recombinant full-length hepatitis E virus(HEV)fused with enhanced green fluorescent protein(EGFP),and assess its infectivity in A549 cells. Two fragments from the full-length HEV genome and the EGFP gene were amplified by PCR. The EGFP gene was inserted downstream of the HEV ORF2 and then cloned into the pGEM® -7Zf(+)vector containing the T7 and SP6RNA polymerase promoters, producing pGEM-HEV-EGFP. The construction of the pGEM-HEV-EGFP recombinant plasmid was confirmed by restriction enzyme digest and sequencing. The pGEM-HEV-EGFP recombinant plasmid was transfected into A549 cells to assess infectivity using Lipofectamine. EGFP expression was observed at 24hpost-transfection,and expression of the HEV ORF2 was detected by immunofluorescence, confirming the presence of the HEV ORF2 and EGFP fusion protein. Cytopathic effects were observed at day seven post-transfection. The infectivity of pGEM-HEV-EGFP was confirmed by the presence of fluorescence after three continuous passages. The recombinant pGEM-HEV-EGFP vector was successfully constructed and effectively infected A549 cells, which will facilitate future studies on the mechanisms of HEV infection and pathogenesis.

The purpose of this study was to explore the potential of the GB virus C(GBV-C)genotype 7E2 protein as a detection antigen for ELISA kit development. In this study, analyses of antigen epitopes, space structures and the linear B cell epitopes from the GBV-C genotype 7E2 protein were performed using an online analysis program. To establish a more reliable detection method for GBV-C studies, a 945bp gene fragment from GBV-C E2 was amplified by RT-PCR and ligated into the pET-32 a prokaryotic expression vector, which was then transformed into E. coli BL21 cells for protein expression. A protein with a molecular weight of 55 kDa was detected by 12% SDS-PAGE. The protein was found in inclusion bodies, and the His-tagged protein was detected by western blotting. The results showed that the cloned E2 gene sequence was 945 bp, and that the GBV-C E2 protein sequence had multiple antigenic epitopes. The recombinant protein formed inclusion bodies, which was consistent with expectations. These findings may provide the foundation for the development of a GBV-C detection kit.