pHA2 plasmid sequence,with Bacterial Artificial Chromosome(BAC) vector and the GFP expression cassette, was introduced into the UL23(TK) gene of Pseudorabies virus(PRV)strain ZJ by homologous recombination,and the recombinant PRV (rPRV-HA2) was confirmed and isolated by plaque purification. The circular genome of rPRV-HA2 was electroporated into Escherichia coli strain DH10B and then the PRV BAC (pPRV) was recovered. The transfection of pPRV into VeroE6 cells resulted in productive infection. The rescued virus isolated following transfection was indistinguishable from rPRV-HA2 in cytopathic effects (CPE) and replication curve in vitro. The growth kinetics of the viruses indicated that partial deletion of TK gene and BAC vector insertion had no effect on the viral titre and plaque size in vitro. The PRV BAC system will enable quick and reliable manipulation of the viral genome for the functional investigation on the PRV genes and the development of PRV vector in vaccine.
Animals ; Cercopithecus aethiops ; Chromosomes, Artificial, Bacterial ; genetics ; Genome, Viral ; Herpesvirus 1, Suid ; genetics ; physiology ; Pseudorabies ; virology ; Recombination, Genetic ; Swine ; Swine Diseases ; virology ; Vero Cells ; Virus Replication

Peste des petits ruminants virus is a member of Morbillivirus Paramyxoviridae. The complete genome of a Peste des petits ruminants virus (PPRV) isolate, China/Tib/07 was sequenced and molecular characteristics was analyzed. The internal sequences of the virus genome were amplified by RT-PCR with primers designed according to the published data in GenBank, while the sequences of the 3' and 5' ends of the genome were determined by RACE. Amplification products were directly sequenced,assembled and analyzed with DNAStar4.0. Results showed that China/Tib/07 genome consisted of 15 948 nucleotides in length, encoding six structural proteins and two non-structural proteins just like other known PPRV genomes. Phylogenetically, the virus genome shared homology of 91.6%-98.1% with Southwest Asian isolates among PPRV strains and the highest homology of 64.3% with rinderpest virus among morbillivirus members.
Animals ; Base Sequence ; Cercopithecus aethiops ; China ; Genome, Viral ; Molecular Sequence Data ; Peste-des-Petits-Ruminants ; veterinary ; virology ; Peste-des-petits-ruminants virus ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Alignment ; Sequence Analysis, DNA ; Sheep ; Sheep Diseases ; virology ; Vero Cells ; Viral Proteins ; genetics

To develop a reverse genetics system of Peste des petits ruminants virus(PPRV), five pairs of oligonucleotide primers were designed on the basis of the full-length genomic sequence of PPRV Nigeria 75/ 1 strain. Using RT-PCR technique, five over-lapping cDNA fragments, designated as JF1, JF2, JF3, JF4 and JF5, respectively, were amplified, followed by cloning into pcDNA3.1(+)vector. An AscI restriction enzyme site and a T7 promoter sequence were introduced immediately upstream of 5'-end, while a PacI restriction enzyme site was engineered downstream of 3'-end. Using pok12 as a plasmid vector, the full-length cDNA clone pok12-PPRV of Nigeria 75/1 was assembled by connecting the five cDNA fragments via the unique restriction endonuclease site of PPRV genome. The resultant nucleotide sequence of the PPRV Nigeria 75/1 strain in the study was compared with other members of genus morbillivirus, and phylogenetic analysis was used to examine the evolutionary relationships. The results showed that PPRV Nigeria 75/ 1 was antigenically closely related to Rinderpest virus and Measles virus. Successful construction of full-length cDNA clone of PPRV Nigeria 75/1 strain lays the basis rescuing PPRV effectively and enables further research of PPRV at molecular level.
Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Genome, Viral ; Molecular Sequence Data ; Peste-des-Petits-Ruminants ; virology ; Peste-des-petits-ruminants virus ; classification ; genetics ; Phylogeny ; Sequence Analysis, DNA

The nucleotide sequences of M and F genes from a field strain of peste des petits ruminants virus (PPRV) ("China/Tib/Gej/07-30") was firstly determined. The M gene was 1 483 nucleotides in length with a single open reading frame (ORF), encoding a protein of 335 amino acids. The F gene was 2411 nucleotides in length, encoding a protein of 546 amino acids. The resulting nucleotide sequence and the deduced amino acid sequences were compared with the homologous regions of other PPRV isolates. The nucleotide sequences of M and F genes of the "China/Tib/Gej/07-30" was 92.4%-97.7% and 85.5%-96.1% identical to other PPRV isolates, respectively, while a homology of 97.0%-98.2% and 94.3%-98.2% could be observed at the amino acids level respectively. Several sequence motifs in the M and F genes had been identified on the basis of conservation in the PPRVs and the morbilliviruses. The 3' untranslated region of M gene was 443 nucleotides in length with 82.4%-93.5% identical to other PPRV isolates. The 5' untranslated region of F gene was 634 nucleotides in length with 76.2%-91.7% identical to other PPRV isolates.
Amino Acid Sequence ; Animals ; Base Sequence ; Molecular Sequence Data ; Peste-des-Petits-Ruminants ; veterinary ; virology ; Peste-des-petits-ruminants virus ; chemistry ; classification ; genetics ; isolation & purification ; Phylogeny ; Sequence Homology, Amino Acid ; Sheep ; Sheep Diseases ; virology ; Tibet ; Viral Fusion Proteins ; chemistry ; genetics ; Viral Matrix Proteins ; chemistry ; genetics

Abstract:One H5N1 subtype avian influenza virus, A/duck/Shandong/009/2008 (Dk/SD/009/08), was isolated from apparently healthy domestic ducks in some live bird market in East China during our epidemiological surveillance. To investigate the genetic composition, Dk/SD/009/08 was subjected to genome sequencing. The amino acid motif of cleavage site was "PLRERRRK-R/GL", which was consistent with the characterization of the HPAIV. According to the newest unified nomenclature system of H5N1, Dk/SD/ 009/08 was classified into Clade 2.3.4. The BLAST results showed that four gene segments (HA, NA, NP and NS) had the highest nucleotide identities with H5N1 subtype AIVs whereas the remaining four (PB2, PB1, PA and M) displayed the closest relationship with H9N2 subtype. Therefore, Dk/SD/009/08 might be a natural reassortant virus. The phylogenetic analysis further indicated that G1-like H9N2 subtype AIVs which was prevalent mainly in quails of Southern China might provide the internal genes for Dk/ SD/009/08.
Animals ; Chick Embryo ; Genome, Viral ; Humans ; Influenza A Virus, H5N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza A Virus, H9N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; virology ; Molecular Sequence Data ; Phylogeny ; Reassortant Viruses ; classification ; genetics ; isolation & purification ; Recombination, Genetic ; Viral Proteins ; genetics

To study IgG antibody persistence and temporal change in SARS coronavirus (SARS-CoV) infected patients, 22 patients recovered from SARS in Beijing were recruited and followed-up from 2004 to 2008, serum samples from patients were collected every year. We checked and analyzed the SARS-CoV IgG antibody (Ab) for five consecutive years using the commercial ELISA test kit. The results showed that: all of the serum were SARS-IgG antibody-positive the first year after recovery, the titer of most serum remained at high levels at the 2ed and 3rd year post infection. The Ab titers significantly declined at 4th year post infection. The IgG Ab was almost undetectable after 5 years post infection. In conclusion, SARS-CoV IgG Ab can be maintained for more than 3 years post infection, however, the titer of IgG Ab has declined markedly 4 years later. These data provide a useful reference for diagnosis and control of SARS infection, the evaluation of immune response and vaccine efficacy.
Adult ; Antibodies, Viral ; blood ; immunology ; Female ; Follow-Up Studies ; Humans ; Immunoglobulin G ; blood ; immunology ; Male ; Middle Aged ; SARS Virus ; immunology ; Severe Acute Respiratory Syndrome ; blood ; immunology ; virology

To investigate HIV-1 subtype distribution and prevalence of HIV-1 drug resistance in Liuzhou and Nanning, a total of 304 HIV-infected subjects or AIDS patients from Liuzhou and Nanning were recruited. Whole blood was withdrawn from a peripheral vein of each subject. HIV RNA were extracted from plasma, and subjected to PCR amplification targeting HIV pol gene fragment and DNA sequencing. Sequences obtained were subtyped by phylogenetic analysis. Two subtypes, CRF01_AE and CRF07_BC, were found in subjects from Liuzhou, accounting for 75.2% and 24.8%, respectively. Subtype CRF01 AE, CRFO8_BC, B, and C were found in subjects from Nanning. CRF01_AE and CRF08 BC were still the dominant strains in Nanning, accounting for 85.8% and 11.5%, respectively. Sequences were also analyzed for drug resistance mutations, and rates of drug resistance were calculated. The rate of drug resistance was 3.3% in ART-naive subjects from Liuzhou, and 8.7% in the ART-experienced. For patients from Nanning, the rate was 1.4% in ART-naive subjects, and 27.5% in ART-experienced subjects.
Anti-HIV Agents ; pharmacology ; China ; epidemiology ; Drug Resistance, Viral ; Genotype ; HIV Infections ; epidemiology ; virology ; HIV-1 ; classification ; drug effects ; genetics ; isolation & purification ; Humans ; Molecular Sequence Data ; Phylogeny

An outbreak of hand foot and mouth disease occurred in Shang dong, China in 2009. Almost 20% of patient's swabs was positive for Coxsackie virus B5 (CVB5) identified by RT-PCR and sequencing. It was suggested that CVB5 may be another important pathogen for HFMD. Fifteen pairs of overlapping primers were designed and the genome sequence was sequenced. The genome of CVB5 was 7 399 nt in length, coding for 2 185aa. The genome displayed 80.6%-85.3% nucleotide sequence identity and 96.1%-96.9% amino acid sequence identity with another three CVB5 respectively. Phylogenetic analysis showed that different segment of genome underwent a distinct evolutionary and selective pressure. Simplot analysis displayed no evident recombination between genome of CVB5 and other HEV B viruses. The complete and characterized genome of CVB5/09 provides further insight into the genetics of CVB5 and other HEV B viruses, aiding in the surveillance and control of HFMD.
Base Sequence ; China ; Coxsackievirus Infections ; virology ; Enterovirus B, Human ; classification ; genetics ; isolation & purification ; Genome, Viral ; Humans ; Molecular Sequence Data ; Phylogeny ; Viral Proteins ; genetics

A novel dual luciferase expression vector was designed and its expression characteristics were studied in vitro and in vivo. Firstly, the Gluc and Fluc genes were connected via the TaV 2A sequence by overlaping PCR, and inserted into the expression vector pAAV2neoCAG, obtaining the recombinant plasmid pAAV2neoCAG-Gluc-2A-Fluc. Then pAAV2neoCAG-Gluc-2A-Fluc was transfected into BHK21 cells and the activity of Gluc and Fluc in the supernatant and cell lysates were assayed respectively. Results showed that both Gluc and Fluc were expressed successfully. The Gluc was mainly detected in the culture media while the Fluc was mostly within cells. The activity of Gluc in the supernatant increased gradually with time while the Fluc activity in cells almost kept stable. To investigate the expression of pAAV2neoCAG-Gluc-2A-Fluc in vivo, the plasmid was hydrodynamically injected into BALB/c mice through tail vein. The Gluc activity was assayed in a small volume of blood taken by tail vein at different time points. Results showed that Gluc was expressed stably at least 7 days. Live bioluminescence imaging technology was used to compare the expression characteristics of Gluc and Fluc. Whole body imaging was seen when coelenterazine, a specific substrate for Gluc, was injected, and the imaging signals decreased rapidly within 10 minutes. Liver imaging was showed when Flue specific substrate named D-Luciferin was injected, and the imaging remained stable at least for half an hour. The dual luciferase expression vector pAAV2neoCAG-Gluc-2A-Fluc combines the advantages of the secreted report gene Gluc and the non-secreted report gene Fluc, and will provide a new tool for cell labeling and tracing.
Animals ; Base Sequence ; Cricetinae ; Crustacea ; enzymology ; Fireflies ; enzymology ; Gene Expression ; Genes, Reporter ; Genetic Vectors ; genetics ; metabolism ; Luciferases ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data

A combinatorial human Fab library to the rabies virus was constructed using antibody genes derived from the blood of vaccinated donors. The library were panned and selected on purified rabies virus particles of aG or CTN strain with phage display. Eleven unique human Fab antibodies specific for the rabies virus glycoprotein were obtained by ELISA, IFA and DNA sequences analysis of these antibodies. Among these Fab antibodies, five human Fab antibodies were converted to full-length human IgG antibodies with recombinant baculovirus system. The five full-length human IgG antibodies were tested in vitro for rabies virus neutralization, resulting in all specificities to neutralize the virus. The obtained human anti-rabies antibodies lay the basis for the production of cocktail of anti-rabies monoclonal antibody with chinese intellectual property.
Amino Acid Sequence ; Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Cell Line ; Cricetinae ; Humans ; Immunoglobulin Fab Fragments ; genetics ; immunology ; Immunoglobulin G ; genetics ; immunology ; Molecular Sequence Data ; Neutralization Tests ; Rabies ; immunology ; virology ; Rabies virus ; genetics ; immunology