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Characterization of a novel podoviridae-phage infecting Serratia marcescens isolated in China.

Feng-Yu XU ; Yong-Jie LIU ; Hong-Xia MA ; Yan ZHANG ; Sheng-Bing SU ; Chan-Juan SHEN ; Cheng-Ping LU

Chinese Journal of Virology.2012;28(4):439-443.

Serratia marcescens jn01 was employed as the host for the isolation of phages from environmental sewage. One strain of phage named SmPjn was purified by picking transparent plaque with 2mm diameter and clear edge on the double-layer agar repeatedly. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of (58 +/- 2.16) x (55 +/- 0.47) nm and (7 +/- 1.25) nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage was capable of infecting the other two isolates of S. marcescens, P25 and CMCC41002. The optimal multiplicity of infection was 1. A one-step growth curve of SmPjn indicated that the latent period and burst size were estimated at 50 min and 1,125 pfu/cell, respectively . Genomic DNA of SmPjn was above 27kb in size and could be digested by Hind Ill and EcoR I into 11 and 9 visible fragments after electrophoresis, respectively. A novel Podoviridae-phage infecting S. marcescens was firstly reported in China.
China ; DNA, Viral ; genetics ; isolation & purification ; metabolism ; Host Specificity ; Podoviridae ; genetics ; growth & development ; isolation & purification ; Restriction Mapping ; Serratia marcescens ; physiology

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The application of single-genome amplification and sequencing in genomic analysis of an attenuated EIAV vaccine.

Hua-Mian WEI ; Xue-Feng WANG ; Shan-Shan WANG ; Cheng DU ; Hai-Fang LIU ; Qiang LIU ; Jian-Hu ZHOU

Chinese Journal of Virology.2012;28(4):431-438.

Our previous studies found that the Chinese attenuated EIAV vaccine was composed of a pool of quasispecies, which showed a complicated diversity called "multi-species". Further determining the viral composition of these species in the vaccine should improve the identification of predominant viruses in the vaccine and facilitate the analysis of in vivo evolution of EIAV and the vaccine. In this study, the comparison of fidelities in amplifying and sequencing the V3 to V5 fragment of EIAV envelope gp90 gene by either a single-genome amplification (SGA) approach or the traditional RT-PCR (bulk PCR) was performed. Results revealed that the diversities were 1.84% and 1.88% for SGA- and bulk PCR-derived sequences, respectively. Futher analysis revealed that beside the sequences highly homologous to those derived by the bulk PCR, nine of 73 sequences derived by SGA contained a deduced amino acid domain that was identical to the corresponding domain in the virulent strain LN40. In addition, sequences with deletion of one predicted amino acid residual was detected by using SGA The presence of these less populated sequences provided additional evidence for the "multi-species" hypothesis for the action mechanism of the EIAV vaccine. Furthermore, based on the analysis of sampling bias, Our results that the difference in copy number of each viral specie in the pool of quasispecies resulted in the inefficiency to amplify viral sequences that were in low population by bulk PCR. Therefore, the sequences amplified by bulk PCR could not correctly represent the composition of quasispecies. As an approach based on the amplification and sequencing single isolated genome, SGA significantly improved the weakness of bulk PCR and appeared its advantage in analysis of EIAV genome composition with high variety.
Calibration ; Cloning, Molecular ; DNA, Complementary ; genetics ; Genome, Viral ; genetics ; Infectious Anemia Virus, Equine ; immunology ; Nucleic Acid Amplification Techniques ; methods ; Polymerase Chain Reaction ; Sequence Analysis ; methods ; Vaccines, Attenuated ; genetics ; Viral Envelope Proteins ; genetics ; Viral Vaccines ; genetics

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The development and application of a SYBR Green I real-time PCR assay for detection of infectious bursal disease virus.

Xin ZHOU ; Xia YANG ; Jun ZHAO ; Hong-Tao CHANG ; Xin-Wei WANG ; Lu CHEN ; Chuan-Qing WANG

Chinese Journal of Virology.2012;28(4):424-430.

To meet the needs of detection of infectious bursal disease virus (IBDV) under high efficient culture, a SYBR Green I real-time RT-PCR (qRT-PCR) was developed using a pair of primers specific to the conserved region of VP4 gene of IBDV and compared with TCID50 method by monitoring the proliferation dynamics of IBDV in DF-1 cell line adherent to micro carrier in tubular reactor. The results showed that the RT-PCRassay was linear in the range of 4. 03 X 10(1)-10(9) copies/microL. The IBDV RNA detection limit was 40 copies/microL, which was 1 000 times more sensitive than conventional PCR. No cross-reactions with other viruses was observed. The intra-assay coefficient of variation was less than 0.05%. There was a parallel correlation of IBDV proliferation dynamics in DF-1 cell under Micro carrier suspension and static adherent culture by the qRT-PCR assay and TCID50 method. The detection results of the IBDV samples from tubular and flask culture showed the differences of the micro carrier and adherent culture by both methods. In conclusion, the qRT-PCR assay is more rapid and sensitive than the TCID50 method, which is more appropriate for the real time detection of IBDV.
Animals ; Calibration ; Cell Line ; Conserved Sequence ; DNA Primers ; genetics ; Infectious bursal disease virus ; genetics ; isolation & purification ; Organic Chemicals ; chemistry ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; Reproducibility of Results ; Spectrometry, Fluorescence ; Viral Proteins ; genetics ; Virus Replication

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Identification and characterization of porcine bocavirus episomes.

Wan-Zhu YANG ; Can-Ping HUANG ; Zhao-Jun DUAN

Chinese Journal of Virology.2012;28(4):418-423.

To verify that the circular forms of bocavirus genome exist in their host, bocavirus episomes were detected in fecal samples of healthy piglets using a semi-nested PCR method. Two species of porcine bocaviruses (PBoVG2-episome and PBoVG3-episome) were identified for the first time. The relevant terminal sequences of the noncoding region (405 and 511 nt, respectively) were also obtained. Sequence analyses and secondary structure prediction indicated that the PBoVG2-episome was more similar to that of human bocavirus 3 (HBoV3) but the PBoVG3-episome was quite different from that of other members of the genus Bocavirus. Discovery of episomal forms of porcine bocaviruses (PBoV) suggested that PBoV, like HBoV, used a different replication mechanism from other parvoviruses. The sequencing of episome Inverted Terminal Repeats (ITRs) also contributes to a possible alternative strategy for constructing infectious molecular clones of bocavirus in a future study.
Animals ; Base Sequence ; Bocavirus ; genetics ; physiology ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Genome, Viral ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; Swine ; virology

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Degradation of 14-3-3beta appeared in apoptosis cell induced by PrP106-126 polypeptide.

Peng SUN ; Juan SONG ; Jin ZHANG ; Qin-Qin SONG ; Xing GAN ; Yu CUI ; Chen GAO ; Xiao-Zhen BO ; Jun HAN

Chinese Journal of Virology.2012;28(4):414-417.

To investigate changes of 14-3-3beta from apoptosis induced by PrP106-126 polypeptide, HeLa cell was incubated with PrP106-126 for 4h or 8h. Nucleus changes and the expression of PARP were detected differently by Hoechst staining and Western blotting. Expressing of protein and mRNA from 14-3-3beta was determined by Western blotting and Real-time PCR. The results show that typical nucleus pyknosis and chip of apoptosis and degradation of PARP were induced by PrP106-126 peptide in HeLa cells. Degradation of 14-3-3beta appeared in apoptosis groups induced by PrP106-126 peptide. However, 14-3-3beta mRNA did not display any changes in apoptosis groups. This study indicated that degradation of antiapoptosis protein 143-3beta induced by PrP106-126 peptide may be one of pathogenesis mechanism of prion disease.
14-3-3 Proteins ; metabolism ; Apoptosis ; drug effects ; HeLa Cells ; Humans ; Peptide Fragments ; pharmacology ; Prions ; pharmacology ; Proteolysis ; drug effects

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A redox process of ethanethiol group increases formation of beta-sheet structure and amyloid fibrils of wild type human prion protein.

Han SUN ; Qi SHI ; Shao-Bin WANG ; Fei GUO ; Wu-Ling XIE ; Cao CHEN ; Cun-Qi LIU ; Xiao-Ping DONG

Chinese Journal of Virology.2012;28(4):409-413.

In order to explore the potential influences of the disulfide bridge on the physical and chemical properties of PrP protein, the expressed recombinant human wild-type PrP protein was purified for using in an established redox process for the reduction and oxidation of the ethanethiol group within PrP. Sedimentation tests illustrated that redox process remarkably promoted the aggregation of recombinant PrP. Thioflavin T binding assay revealed an enhanced fibrillization of the recombinant human PrP after redox process. Far-UV circular dichroism demonstrated that the PrP treated with redox process showed a significant p-sheet rich structure. Furthermore, PrP-specific Western blot identified that the recombinant PrP after redox possessed stronger proteinase K-resistance. Those data indicates that the formation of the disulfide bridge induces the alteration of the secondary structure and enhances the progresses of aggregation and fibrillization of PrP protein.
Amyloid ; chemistry ; Endopeptidase K ; metabolism ; Humans ; Oxidation-Reduction ; Prions ; chemistry ; metabolism ; Protein Multimerization ; Protein Structure, Secondary ; Proteolysis ; Sulfhydryl Compounds ; chemistry

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Genetic diversity of adenoviruses in bats of China.

Li-Hong CHEN ; Zhi-Qiang WU ; Yong-Feng HU ; Fan YANG ; Jian YANG ; Qi JIN

Chinese Journal of Virology.2012;28(4):403-408.

Adenovirus remains a significant threat to public health. Recent studies showed that bats can harbor diverse adenoviruses. To further investigate the distribution and genetic diversity of bat adenoviruses in China, we collected throat and anal swab samples of 11 bat species from 6 provinces of China, including Beijing, Hunan, Jiangxi, Yunnan, Guizhou and Hainan. Nested PCR was used to identify potential bat adenoviruses from the samples, and positive results were cloned and sequenced for genetic diversity study. In addition, nucleotide sequence alignments based on corresponding amino acid sequence similarities were used for phylogenetic analyses. Our results showed that about 20% of bat species in China are positive to adenoviruses, and Myotis ricketti is likely to be the most important host of bat adenoviruses in all locations. Moreover, we identified two diverse sequences of bat adenoviruses from the same sample of Ia io in Guizhou province of China. In general, the average nucleotide and amino acid sequence similarities of the conserved region of DNA polymerases of bat adenoviruses are 66.6% and 74.7%, respectively. The differences between bat species and their residences environments may have driven the adaptive evolution of the viruses, leading to the genetic diversity of the bat adenoviruses.
Adenoviridae ; classification ; genetics ; isolation & purification ; physiology ; Animals ; China ; Chiroptera ; virology ; Genetic Variation ; Host Specificity ; Phylogeny

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Full-length genome analysis of four genotype 3 letogenic Newcastle disease viruses isolated from different hosts.

Shi-Qiang JIN ; Chun-Chun MENG ; Jun-Liang DENG ; Xiang-Le ZHANG ; Xv-Sheng QIU ; Lei TAN ; Sheng-Qing YU ; Zhi-Cai ZUO ; Chan DING

Chinese Journal of Virology.2012;28(4):394-402.

The purpose of this study is trying to analysis the homology between four lentogenic Class I genotype 3 Newcastle disease virus isolates from different hosts with NDV strain NDV 08-004, which was the first obtained complete genome sequence virus of class I genotype 3. The full-length genome of NDV isolates, JS/3/09/Ch, ZJ/3/10/Ch, AH/2/10/Du and JS/9/08/Go,were determined by RT-PCR and then an alyzed. All the genomes are 15 198 nucleotides (nt) in length. Compared with the full genome sequences of Class II NDV stains (genotype IV-IX),four isolates has a 6-nt deletion in the non-coding region of nuclear phosphoprotein gene between nucleotides 1 640-1 641 and 12-nt insertion in the coding region of phospho protein gene between nucleotides 2 381-2 382. All the isolates have the motifs 112EQ/RQE/GRL117 at the cleavage site of the fusion protein, which is typical of lenogenic NDV strains, and it is in agreement with the result of pathogenic tests. The full-length genome of 4 genotype 3 NDV isolates shared 93% nucleotide identity with NDV08-004. The results of alignment of 6 viral genes showed that NP gene shared the highest identity (98.3%-96.4%) and P gene shared the lowest identity (96.1%-91.9%). The results show the following two points. First, it is concluded that the isolates from different hosts share the same genotype has the insignificant divergence in the genetic information. Second, it is proposed that the mutation rates of NP/F/L genes are lower than P/M/HN genes.
Animals ; Genome, Viral ; genetics ; Genomics ; Genotype ; Host Specificity ; genetics ; Newcastle disease virus ; classification ; genetics ; isolation & purification ; Phylogeny

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Genetic characteristics of EV71 in Yunnan Province from 2009 to 2010.

Guo-Jun LIU ; Yan WU ; Bing-Jun TIAN ; Jian-Ping CUN ; Jie YIN ; Li-Li JIANG ; Yue MA ; Lin LU ; Wen XU

Chinese Journal of Virology.2012;28(4):389-393.

In order to learn about the genetic characteristic of human enterovirus type71 (HEV71) isolated from cases of Hand, Foot and Mouth Disease (HFMD) in Yunnan Province from 2009 to 2010. 50 isolates form HFMD cases were performed entire VP1 coding region amplification by reverse transcription-polymerase chain reaction and sequencing the nucleotide sequences; then the phylogenetic tree was constructed. The complete nucleotide sequences of region VP1 of the 50 strains were all 891nt length coding 297 amino acids. The result of molecular identification of the 50 strains is HEV71. Phylogenetic analysis indicated that 48 EV71 isolates belonged to subgenotype C4a and 2 EV71 isolates belonged to genotype A. From 2009 to 2010, the pathogen of HFMD cases were EV71 strains in Yunnan province, which were co-evolved with isolates from other provinces in mainland of China. There was no significant difference found in the whole sequence of VP1 gene of the strains isolated from different regions or under different diseases occurred, but the spread of genetype A appared in Yunnan Province in 2009.
Capsid Proteins ; genetics ; Child, Preschool ; China ; Enterovirus A, Human ; classification ; genetics ; isolation & purification ; pathogenicity ; Female ; Genotyping Techniques ; Hand, Foot and Mouth Disease ; virology ; Humans ; Male ; Phylogeny ; Sequence Analysis, DNA

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Detection and genotyping of human bocavirus 2 in children with acute diarrhea.

Hui ZHAO ; Lin-Qing ZHAO ; Yu SUN ; Yuan QIAN ; Li-Ying LIU ; Li-Ping JIA ; You ZHANG ; Hui-Jin DONG

Chinese Journal of Virology.2012;28(4):382-388.

To investigate the prevalence of HBoV2 in pediatric patients with acute diarrhea in Beijing and the characteristic of the genome of the virus, 553 stool specimens were collected from pediatric outpatients with acute diarrhea in Affiliated Children's Hospital of Capital Institute of Pediatrics during Nov. 2010 to Oct. 2011. TaqMan-based Real-time polymerase chain reaction was performed to detect HBoV2 DNA from these specimens. Two positive specimens with high viral loads were selected for segmented amplification and then the amplified fragments were cloned into the plasmid vector pGEM-T, transformed into Escherichia coli DH5alpha and sequenced. Then genomic sequences assembled from those DNA fragments were compared with other parvovirus genomic sequences in the GenBank. Among these 553 specimens tested, 15 (2.7%) were HBoV2 DNA positive. The highest positive rate was shown in July (7.0%) through the whole year and in 3-6 month age group (4.1%) among different age groups. All these 15 specimens positive for HBoV2 DNA were collected from patients younger than 2 years old, including 4 simultaneously positive for norovirus, 3 positive for rotavirus and 1 positive for adenovirus. By sequence analysis, 2 almost complete HBoV2 genomic sequences assembled from gene fragments amplified from specimens BJQ19 and BJQ390 were typical HBoV2. And they shared high homology with each other (99.2%), while they shared the highest homology with FJ375129 from Shanghai China (99.1% and 99.2%) among other parvoviruses. These data suggest that some of acute diarrhea in pediatric patients in Beijing were associated with HBoV2, and infants and young children aged from 3 months to 2 years, are more likely to be infected by HBoV2.
Acute Disease ; Adolescent ; Child ; Child, Preschool ; DNA, Viral ; analysis ; genetics ; Diarrhea ; virology ; Female ; Genome, Viral ; genetics ; Genotyping Techniques ; Human bocavirus ; genetics ; isolation & purification ; pathogenicity ; Humans ; Infant ; Male ; Phylogeny ; Sequence Alignment ; Viral Proteins ; genetics

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