A porcine parvovirus, designated as VRI-1, was isolated from a 30-day-old piglet. Replicative form of viral DNA from ST cells infected with VRI-1 was directly cloned into pUC19. The cloned DNA fragment contained the entire nonstructural and structural protein genes, covering approximately 85% of the viral genome. The nucleotide sequence of VRI-1 showed 99.4~99.5% identity in the nonstructural protein (NS) and 99.0~99.2% identity in the structural protein with previously reported PPV strains, respectively. Among the cloned genes, two types of defective genomes with deletion of 100 and 247 nucleotides at almost similar location of 3' region within NS gene were also identified in this study.
Base Sequence* ; Clone Cells ; DNA ; DNA, Viral ; Genome* ; Genome, Viral ; Nucleotides ; Parvovirus, Porcine*

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

OBJECTIVETo develop a vector inserted with complete genome of poliovirus strain Sabin I.

METHODSThe 3 fragments of the complete genome of Sabin I was amplified and cloned to pEASY-T3 by molecular biological technology. These cloned pEASY-T3 were then digested by Restriction enzymes and ligated to pWSK29 step by step and identified.

RESULTSThe complete genome of poliovirus strain Sabin I was successfully cloned into vector pWSK29 with 9 nucleotide mutations.

CONCLUSIONThe complete genome plasmid was constructed and it provided a basis for further research of the function of Sabin I.

Cloning, Molecular ; Genetic Vectors ; genetics ; Genome, Viral ; Mutation ; Poliovirus ; genetics

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. Previously, we have made the recombinant TYMV construct containing a 0.7 kb eGFP gene or a 1.8 kb GUS gene. The genomic RNAs from these constructs were efficiently encapsidated. To examine in more detail whether size constraint exists for replication and packaging of TYMV, we have inserted into the TY-GUS an extra sequence derived from either eGFP or GUS. We also made a recombinant containing RNA1 sequence of Flock house virus. These TYMV recombinants were introduced into Nicotiana benthamiana leaves by agroinfiltration. Northern blot analysis of the viral RNAs in the agroinfiltrated leaves showed that the genomic RNA band from the recombinant TYMV became weaker as longer sequence was inserted. The result also showed that the efficiency of genomic RNA encapsidation decreased sharply when an extra sequence of 2.2 kb or more was inserted. In contrast, the recombinant subgenomic RNA containing an extra sequence of up to 3.2 kb was efficiently encapsidated. Overall, these results show that size constraint exists for replication and encapsidation of TYMV RNA.
Blotting, Northern ; Genome ; Genome Size* ; Plant Viruses ; Product Packaging* ; RNA ; RNA, Viral ; Tobacco ; Tymovirus*

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

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

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

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

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