COVID-19/genetics* ; Genome, Viral/genetics* ; Humans ; RNA, Viral/genetics* ; RNA-Seq ; SARS-CoV-2/genetics* ; Whole Genome Sequencing

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

Cloning, Molecular ; DNA, Viral ; genetics ; Genome, Viral ; genetics ; Hepatitis B ; virology ; Hepatitis B virus ; genetics ; Humans

Total RNA was extracted from infected papaya (Carica papaya L.) leaves in Hainan Province, and the full-length sequences of papaya ringspot virus were amplified by RT-PCR and RACE, and its complete genomic sequence was assembled, named Hainan-P isolate. The RNA genome sequence of Hainan-P isolate was 10323 nucleotides (nts)in length,excluding the 3'-terminal poly(A) tail. And it was composed of a single open reading frame encoding a polyprotein of 3343 amino acids.. The result of homology analysis with twelve GenBank PRSV isolates showed that the polyprotein identity of Hainan-P ranged from 89. 8% to 93.2%, that was higher than the complete nt homology of 82.3% to 89.1%. The P1 amino acid was the least conserved (sharing homology only between 65.4% and 80.1%), whereas HC-Pro, CI and CP were the most conserved. Phylogenetic tree were constructed by the Neighbor-joining method in MEGA 3.1, which showed that PRSV isolates were obviously relevant to geographical origin, and it was impossible to delineate host-specific (P type and W type)evolution.
Carica ; virology ; Genome, Viral ; Phylogeny ; Potyvirus ; classification ; genetics

Base Pairing ; Dimerization ; Genome, Viral ; RNA, Viral ; chemistry ; genetics ; Retroviridae ; chemistry ; genetics

Genome, Viral ; Hepacivirus ; genetics ; physiology ; Models, Biological ; RNA, Viral ; biosynthesis ; genetics ; Replicon ; Transformation, Genetic ; Virus Replication

OBJECTIVETo probe the primary characteristic of 0507JS11 virus isolated from Culex sp. and determine the classification of 0507JS11 virus in taxonomy.

METHODS0507JS11 virus was cultured in Aedes albopictus C6/36 cells and cytopathic effects (CPEs) were recorded. Electro-microscopic morphology of 0507JS11 virus was observed. Total DNA extract of 0507JS11 virus was detected by 1% Agarose Gel Electrophoresis. Complete genomic sequence of 0507JS11 virus was sequenced and then made phylogenetic analysis.

RESULTS0507JS11 virus could cause CPEs in Aedes albopictus C6/36 cells. Viral particles have no envelope and appear icosahedron symmetry with diameter of 20 nm. The genome of 0507JS11 virus was positive single strand DNA (ssDNA) with full length of 3977 nt. However, a DNA band about 4 kbp was observed in the electrophoresis of total DNA extract of 0507JS11 virus. The coding region of the genome included three ORFs, ORF1 and ORF2 code NSP1 and NSP2, ORF3 codes VP. Phylogenetic analysis of the complete genomic sequence of 0507JS11 virus indicated an independent linear in Brevidensovirus.

CONCLUSION0507JS11 virus is a new member in Brevidensovirus.

Animals ; Culex ; virology ; DNA, Viral ; genetics ; Densovirinae ; classification ; genetics ; isolation & purification ; Genome, Viral ; Sequence Analysis, DNA

Chikungunya virus ; genetics ; isolation & purification ; China ; Genome, Viral ; RNA, Viral ; genetics ; Sequence Analysis, RNA

The Rana grylio virus (RGV) is a member of the genus Ranavirus. It belongs to the family Iridoviridae, and contains the gene 67R encoding dUTPase. In order to investigate the function of 67R in the replication and infection of RGV, we constructed Δ67R-RGV, a recombinant virus with deletion of 67R. First, we constructed the plasmid pGL3-67RL-p50-EGFP-67RR which carried an enhanced green fluorescence gene (EGFP) as a selectable marker. After homologous recombination between pGL3-67RL-p50-EG- FP-67RR and the RGV genome, Epithelioma papulosum cyprini (EPC) cells were infected with the resulting mixture. Through ten successive rounds of plaque isolation via EGFP selection, all plaques emitted green fluorescence, and finally Δ67R-RGV was generated. Total DNA of Δ67R-RGV infected cells was extracted for PCR analyses. Simulateously, mock infected and wild-type RGV (wt-RGV) infected cells were used as a comparison. Results showed that 67R could be detected in wt-RGV infected cells, but that only the EGFP gene was detected in Δ67R-RGV infected cells. Furthermore, one-step growth curves of wt-RGV and Δ67R-RGV were similar. Therefore, 67R and its encoding product dUTPase might not be essential for the growth of RGV. These results suggest that, homologous recombination and recombinant rana- virus could be used to study the gene function of viruses in aquatic animals.
Genes, Viral ; physiology ; Genome, Viral ; Polymerase Chain Reaction ; Pyrophosphatases ; genetics ; Ranavirus ; genetics ; Recombination, Genetic

Animals ; Genome, Viral ; Humans ; Viral Proteins ; genetics ; metabolism ; Yellow Fever ; virology ; Yellow fever virus ; genetics ; metabolism