OBJECTIVEReverse genetics was used to construct the platform of flu pandemic strain A/California/07/2009 (H1N1).

METHODSEight genes fragements were amplified and ligated with bidirectional vector, recombinant plasmids were co transfected to the 293 T cells and rescued the virus. Gene sequencing, antigenic analysis and growth property were used to evaluate the rescued virus.

RESULTSRescued virus show the genes sequence correct, keep the same antigenicity and similar growth property compared with wild type virus.

CONCLUSIONThe pandemic virus reverse genetics platform of A/California/07/2009 (H1N1) were built. Based on this platform, rescued virus hold the similarity of antigenicity and growth ability with wild type virus.

Orthomyxoviridae ; genetics ; growth & development ; immunology ; Pandemics ; Plasmids ; Reverse Genetics

BACKGROUNDAlthough it was widely accepted that full-length HIV genome sequences is important in studying virus genetic evolution and variation as well as developing vaccine candidate, to directly sequencing HIV-1 genome of virion RNA remains as a challenge worldwide. Up to date, no published genomic sequences from virion RNA are available for Chinese prevalent HIV-1 strains due to the absence of specialized protocol and appropriate lab equipments. In this study we developed a straightforward approach for amplifying and sequencing HIV virion RNA from plasma by modifying published protocols and further confirmed it is suitable to process Chinese samples.

METHODSThe methods for viral RNA extraction and gene amplification was modified and optimized as could be widely used in most Chinese labs. Gene alignment of Chinese HIV-1 strains was employed for designing specialized primer sets for Thai-B and BC recombinant strains. Based on comprehensively consideration of high variable gene region and recombinant breakpoints in BC recombinant strains, a three-amplicon strategy (including 4.3-kb gag-pol, 2.9-kb pol-env and 2.7-kb env-nef) was developed. In addition, one amplicon (9 kb near full-length genome) was also used in 32 samples with varied viral loads. All amplicons were directly sequenced by DNA automated sequencer.

RESULTSTwenty-five percent (8/32) amplification efficiency was achieved by the one-amplicon strategy and 65.6% (21/32) by three-amplicon strategy. For one amplicon strategy, none of complete near full-length genome sequences was obtained by DNA sequencing. For three-amplicon strategy, 75% sequences were achieved in DNA sequencing. Amplification efficiency but not sequencing efficiency was closely associated with viral loads.

CONCLUSIONThree-amplicon strategy covering all encoding regions of HIV-1 is suitable for Thai-B and BC recombinant strains and could be potentially employed in less-well equipped Chinese labs.

Genome, Viral ; genetics ; HIV-1 ; genetics ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Virion ; genetics

Human immunodeficiency virus (HIV) is a retrovirus, belongs to Lentiviridae family. As long as viral genetic material entering into host cytoplasm, double-strand DNAs synthesis occurs which is catalyzed by reverse transcriptase (RT) with viral plus-strand RNA as template. This reverse transcription is a key link of HIV-1 life cycle and an important target for anti-HIV drug development. The process of reverse transcription can be divided into several steps: formation of minus-strand strong-stop DNA; the first translocation; initiation of plus-strand DNA synthesis; and, the second translocation and the completion of both strands. These steps can be detected individually by using polymerase chain reaction (PCR) according to the amplified products on the region of R/U5, U3, U5/PBS and the sequence between LTR and Gag. In this review, we summarize the principle for detecting stages of HIV-1 reverse transcription by using PCR.
DNA Replication ; genetics ; DNA, Viral ; biosynthesis ; genetics ; HIV Reverse Transcriptase ; genetics ; metabolism ; HIV-1 ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcription

This study was purposed to investigate the expression of the growth-factor independence 1 (GFI1) in patients with leukemia and its clinical significance. Bone marrow mononuclear cells were obtained from 65 newly diagnosed leukemia patients including 24 acute myeloid leukemia (AML), 18 chronic myelogenous leukemia (CML), 6 acute lymphoblastic leukemia (ALL), 17 blast crisis of chronic myelogenous leukemia and 13 patients with iron deficiency anemia (IDA) were used as controls. The relative expression of gene gfi1 was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and taqman quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). The results showed that gene expression of gene gfi1 in leukemia patients was obviously higher than that in controls and the difference was statistically significant (p < 0.01), in which the expression of gene gfi1 in newly diagnosed CML patients was higher than that in newly diagnosed AML, newly diagnosed ALL, CML-BCP patients and the difference was significant (p < 0.01). Expression of gene gfi1 in lymphocytic blast crisis of CML was higher than that in nonlymphocytic blast crisis of CML, and the difference was significant. It is concluded that gene gfi1 may play an important role in leukemia, especially in CML incidence and progression. The high level expression of gene gfi1 may be participate in the development of lymphoma.
DNA-Binding Proteins ; genetics ; Gene Expression ; Humans ; Leukemia ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factors ; genetics ; Transcription, Genetic ; genetics

Infectious bursal disease virus (IBDV) is an important member of the Birnaviridae family. IBUV mainly targets the bursa of Fabricius, the central immune organ of chicken, resulting in chicken infectious bursal disease (IBD). IBD represents one of the great challenges for ongoing development of the poultry industry. Reverse genetics for IBDV emerged over twenty years ago. Since then, the technologies behind virus rescue have continually improved leading to a deep understanding of IBDV gene function and tailored vaccine development. Our lab has also been instrumental in the field of IBDV research. Here we review studies on the pathogenic mechanism and the effective prevention and control of IBD.
Animals ; Birnaviridae Infections ; virology ; Chickens ; Infectious bursal disease virus ; genetics ; physiology ; Poultry Products ; virology ; Reverse Genetics

Animals ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; virology ; Humans ; Reverse Genetics ; methods

OBJECTIVEGenetic evolution of VP1 of enterovirus type 71 in Shenzhen were analyzed.

METHODSAll samples were tested by RT-PCR using EV71 specific primer. The VP1 of EV71 were amplified and sequenced. A phylogenetic tree was constructed by comparison of the sequences with subgenotype A, B and C using DNAStar, BioEdit and Mega 3.1 software.

RESULTSAmong 35 strains, the homogeneity of the VP1 nucleotide sequence was between 92.1%-100%. The homogeneity of the VP1 nucleotide sequence with subgenotype A and B was between 81.4% -91.1%. The VP1 nucleotide sequence of 35 strains of Shenzhen shared between 93% -97.4% identity with cluster C4. The prevalence strains of EV71 were cluster C4b from 1998 to 2004, and gradually moved to C4a since 2003. All of EV71 were C4b from 2006 to 2008. Also, the homogeneity of the VP1 nucleotide sequence with Anhui FY23 EV71 strain were 94.5% -94.7%, 95.7% -95.8%, 96.2%, 95.4% -97.5%, 96.3% -99.2% from 2003 to 2008. It shows that the homogeneity was increased year by year. There was a mutation (A --> C) at No. 66 nucleotide of VP1 of EV71 that two strains were isolated in 2003 and 8 strains in 2008, that caused amino acid mutation (Q --> H) at No. 22 of VP1.

CONCLUSIONEV71 C4b was gradually moved to C4a from 1998 to 2008. There was a missense mutation at No. 66 nucleotide of VP1.

Enterovirus ; classification ; genetics ; Humans ; Mutation, Missense ; genetics ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Viral Structural Proteins ; classification ; genetics

OBJECTIVETo explore PTEN gene expression and its clinical significance in acute leukemia.

METHODSThe expression levels of PTEN mRNA in 5 leukemia cell lines, 87 patients with acute leukemias (AL), including 59 acute myeloid leukemia (AML), 26 acute lymphoblastic leukemia (ALL), and 2 acute hybrid leukemia, 21 AL in complete remission (AL-CR), 31 chronic myelogenous leukemia (CML) and 14 normal controls were assayed by RT-PCR.

RESULTSPTEN mRNA was detected in K562 cell line, but not in Kasumi-1, HL-60, U937, Nalm-6 cell lines. The expression ratio of PTEN mRNA between CML (61.29%) and normal control (78.57%) had no statistical difference (P > 0.05). The expression ratios of PTEN mRNA in AL (18.39%) and AL-CR (42.86%) were significantly lower than that in normal control (P < 0.01 and P < 0.05, respectively), AL also has a lower expression ratio than that of AL-CR (P < 0.05). The decreased level of PTEN mRNA had a positive correlation with poor-prognostic factors (high white blood cell count of > or = 20 x 10(9)/L and chromosome abnormality).

CONCLUSIONThere is down-regulated expression of PTEN gene in AL. PTEN gene may play a role in leukemogenesis.

Cell Line, Tumor ; Humans ; Leukemia ; genetics ; metabolism ; PTEN Phosphohydrolase ; genetics ; metabolism ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo prepare the armored RNA containing M gene of influenza H3N2.

METHODSThe vector pAR-1 was constructed from expression vector pET30b in which the bacteriophage MS2 DNA fragment, containing the genes for maturase and coat protein and the pac site, was inserted. The M gene fragment of influenza A was inserted into the HindIII site downstream of the pac site on the pAR-1, which formed a new recombinant plasmid pAR-2. After the prokaryotic expression was carried out, armored RNA AR-2 containing M gene was obtained. AR-2 was purified, and then was quantified by real time RT-PCR. Moreover, the stability of AR-2 was checked.

RESULTSAR-2 was expressed successfully. AR-2 remained stable under various storage environments. Approximately 8.9 x 10(11) copies of AR-2 particles can be purified from one milliliter of culture.

CONCLUSIONIt showed that AR-2 was stable and RNase-resistant, which, as a virus surrogate, would be used as RT-PCR standards, controls and training or proficiency samples.

Influenza A Virus, H3N2 Subtype ; genetics ; Plasmids ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; standards ; Viral Matrix Proteins ; genetics

OBJECTIVETo evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform.

METHODSVirus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance.

RESULTSThe M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus.

CONCLUSIONThe M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

Genetic Variation ; Genome, Viral ; genetics ; Influenza A virus ; genetics ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods