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.

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

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

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

To construct a expression plasmid containing the full-length cDNA of rabies virus, four overlapped fragments covering full length cDNA of rabies virus street stain HN10 were cloned into pVAX1 sequentially in the genome except for the G-L noncoding region which was replaced with GFP gene. The plasmid containing the full-length viral cDNA was flanked by hammerhead ribozyme (HamRz) and hepatitis delta ribozyme (HdvRz) sequences and arranged under the control of the cytomegalovirus (CMV) promoter. The constructed plasmid could be directly used for the following procedure of producing the recombinant rabies virus street HN10.
Cloning, Molecular ; DNA, Complementary ; genetics ; Models, Genetic ; Plasmids ; genetics ; Rabies virus ; classification ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

To improve the reliability and credibility of genotyping hepatitis E virus (HEV) and to explore the possibility of unifying standards of HEV genotyping by designing HEV universal primers for amplification of a long genomic fragment of different HEV genotypes. A set of universal primers (HEVuPrimer) was designed based on conserved regions determined by alignment analysis of 82 HEV strains with complete genome in GenBank. HEVuPrimer was compared with a set of previously used primers (MXJ primers) for their sequence-matching to different HEV strains and applied to amplify HEV genomic fragments from HEV reference strains with known different genotypes and clinical serum samples with anti-HEV-IgM by RT-nPCR. HEV genotyping based on the fragments amplified with HEVuPrimer was compared and validated with that based on HEV full genome and fragments obtained with MXJ primers. HEV genotyping by the phylogenetic analysis supplemented with the percent of nucleotide identity of the HEVuPrimer-determined fragments showed good correspondence with that based on HEV full-length genome. In addition, HEVuPrimer was much better than MXJ primers in matching sequences of HEV strains available from GenBank, and was able to amplify all the reference HEV strains with different genotypes. Among 124 samples with anti-HEV-IgM, 60 were positive for HEV RNA determined by a 644bp amplicon of RT-nPCR with the HEVuPrimenr. All the positive isolates belonged to HEV genotype 4 with nucleotide homology of 80.0%-99.9%, and could be further divided into 4 subgenotypes. Moreover, a novel subtype was identified with 6 HEV strains isolated very recently. The RT-nPCR using the HEVuPrimer and phylogenetic analysis of the amplified region provided strong evidences for its feasibility in HEV genetic classification. Our data have new implication for the consensus of genotype classification of HEV.
DNA Primers ; genetics ; Genome, Viral ; genetics ; Genotype ; Hepatitis E virus ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods

The goal of this study is to research the genetic characteristics and relationship between HN and P genes of NDV. The nucleotide sequence and deduced amino acid sequence were analyzed for the Hemagglutinin-neuramindase (HN) and Phosphoprotein (P) gene of twelve field isolates of Newcastle disease virus (NDV) during 1997-2005 in China. The HN and P gene sequences of fifteen NDV reference strains from GenBank were also used in this study. The molecular evolution distance of nucleotides and amino acids were calculated by MEGA 4.0 software, and analysis of variance and correlations were analyzed by SPSS11.0 software among different length sequences of the HN gene or P gene. The nucleotide and amino acids correlation of HN and P gene were analyzed respectively. The correlation of evolution distance and isolation year were also calculated. The results indicated that there were difference and good correlation of nucleotide and amino acid among different length sequences of the HN gene or P gene. These results revealed that the HN and P gene of NDV have the different response to selective pressure to adopt to landscape and closely relationship on heredity mutations. Nucleotide variations of HN and P gene have relationship with isolation year of strains.
Evolution, Molecular ; Genetic Variation ; HN Protein ; genetics ; Newcastle disease virus ; classification ; genetics ; Phosphoproteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Group A rotavirus (RV) is the most important etiologic agent of severe gastroenteritis among children and the development of an effective vaccine becomes the top public health priority. Since survey of RV serotypes circulating in local community is important for introduction or development of RV vaccine, RV serotype G3 had proved as the predominant strain in Changchun from 2001 to 2005. Stool specimens collected from children with acute diarrhea were tested for group A rotavirus by enzyme-linked immunosorbent assay (ELISA) and RV isolates were typed by reverse transcription-polymerase chain reaction (RT-PCR) using serotype-specific primers. The complete VP7 gene segments of 31 rotavirus strains selected in Changchun from 1999 to 2005 were amplified with RT-PCR. Amplicons were cloned and sequenced. Comparative analysis of the VP7 sequences showed that there were no obvious differences among 31 RV strains. There was similar genetic variation among VP7 genes during the same RV season. The nucleotide sequence of VP7 gene of six G3 RV strains had one base deletion at nt1038 in 2003 RV season. The nucleotide mutations in regions A, B and C of VP7 gene took place at the same position or position near-by. Increase of nucleotide mutation in non- high variation region may benefit maintenance of serotype G3 as pre dominant strain after 2002. Increase of non continuous variation in non-high variation regions was notable.
Antigens, Viral ; genetics ; Capsid Proteins ; genetics ; Genetic Variation ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Rotavirus ; classification ; genetics ; Serotyping

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.