China ; Genotype ; Measles virus ; genetics ; isolation & purification ; Molecular Epidemiology ; methods ; Nucleoproteins ; genetics ; Polymerase Chain Reaction

OBJECTIVETo study the relationship between the distribution of rabies virus and genetic variation, the genetic characterization and variation of rabies virus strains in China were analyzed.

METHODSThe downstream 720 nucleotides of Nucleoprotein (N) gene coding region of the rabies specimens from different areas and host animals were sequenced, and then homology and phylogenesis were analyzed.

RESULTSNucleotide similarities of 34 N gene sequences were 87.5%-100%, and the deduced amino acid similarities were 93.3%-99.6%. Most of the nucleotide variations were synonymous mutations.

CONCLUSIONThe 34 rabies specimens all belong to genotype I and are of regional characteristic. The rabies viruses in high-incidence areas in China are of various origins and present the transmission tendency from high-incidence areas to surrounding regions. There may be cross-infection and mutual spread of rabies virus between wildlife and domestic animals as well as native and foreign animals.

Animals ; China ; Humans ; Molecular Epidemiology ; Nucleoproteins ; genetics ; Phylogeny ; RNA, Viral ; genetics ; Rabies ; virology ; Rabies virus ; classification ; genetics

OBJECTIVETo investigate the impact of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) on sperm DNA fragmentation and nucleoprotein transition.

METHODSBased on the recommended methods in the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th ed), we conducted routine semen analysis for 65 CP/CPPS patients and 30 healthy men. We also analyzed the results of papanicolaou staining, sperm DNA fragmentation and sperm nucleoprotein transition.

RESULTSCompared with the healthy control males, the CP/CPPS patients showed significant decreases in sperm concentration ([134.05 +/- 99.80] vs [94.75 +/- 92.07]) x 10(6)/ml, P <0.05), the percentage of morphologically normal sperm ([7.26 +/- 2.28] vs [5.61 +/- 3.40]%, P <0.05) and sperm progressive motility ([59.18 +/- 16.06] vs [47.68 +/- 17.62]%, P<0.05), but dramatic increases in sperm DNA fragmentation ([22.92 +/- 11.51] vs [43.58 +/- 17.07%, P<0.01) and sperm nucleoprotein transition ([23.26 +/- 5.97] vs [32.14 +/- 8.79]%, P<0.01).

CONCLUSIONCP/CPPS significantly reduces sperm quality and male fertility.

Adult ; Case-Control Studies ; DNA Fragmentation ; Humans ; Male ; Nucleoproteins ; genetics ; Prostatitis ; genetics ; Semen Analysis ; Sperm Count ; Sperm Motility ; Young Adult

OBJECTIVETo Construction of P and NP genes eukaryotic expression vectors of Newcastle Disease Virus LaSota strain,study its reverse genetics and functional genome of NDV.

METHODSP, NP genes were amplified and cloned into pGEM-T easy vector and then subcloned into pcDNA3.1 (+) expression vector respectively, the recombinant plasmids were named pcDNA3.1 (+)-P and pcDNA3.1 (+)-NP, Recombinant plasmids were transfected into 293 and BHK-21 cells respectively and were detected using IE and Western blot analysis.

RESULTSExpression of P, NP genes were detected and confirmed by the IE and WB analysis.

CONCLUSIONThe recombinant eukaryotic plasmids pcDNA3. 1(+)-P, pcDNA3.1 (+)-NP were expressed in 293 and BHK-21 cells successfully. This research may be helpful for further study of reverse genetics and functional genome of NDV.

Animals ; Cell Line ; Cricetinae ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Humans ; Newcastle disease virus ; genetics ; metabolism ; Nucleoproteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism

This study aims to investigate whether the nucleoprotein (NP) of severe fever with thrombocytopenia syndrome virus (SFTSV) can impact the cellular immunity of host cells. Gene segments that encode the NP and non-structural protein (NSs) of SFTSV were inserted into eukaryotic expression vector VR1012. Host proteins that interact with NP and affect immunity were identified with co-immunoprecipitation (IP), SDS-PAGE, mass spectrometry (MS), and Western blot. Co-localization of NP and the identified host proteins was confirmed by confocal microscopy. A 60kD SSA/Ro, a protein related to immunity, interacted with NP, as found by IP and MS. Confocal microscopy showed that NP and SSA/Ro were co-localized in cytoplasm. These results indicated that SFTSV NP may specifically bind to 60kD SSA/Ro and cause a series of immune responses and clinical symptoms.
Bunyaviridae Infections ; genetics ; metabolism ; virology ; HEK293 Cells ; Humans ; Nucleoproteins ; genetics ; metabolism ; Phlebovirus ; genetics ; metabolism ; Protein Binding ; Ribonucleoproteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism

Ebola virus (EBOV) is a key member of Filoviridae family and causes severe human infectious diseases with high morbidity and mortality. As a typical negative-sense single-stranded RNA (-ssRNA) viruses, EBOV possess a nucleocapsid protein (NP) to facilitate genomic RNA encapsidation to form viral ribonucleoprotein complex (RNP) together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. However, the mechanism of EBOV RNP formation remains unclear. In this work, we solved the high resolution structure of core domain of EBOV NP. The polypeptide of EBOV NP core domain (NP(core)) possesses an N-lobe and C-lobe to clamp a RNA binding groove, presenting similarities with the structures of the other reported viral NPs encoded by the members from Mononegavirales order. Most strikingly, a hydrophobic pocket at the surface of the C-lobe is occupied by an α-helix of EBOV NP(core) itself, which is highly conserved among filoviridae family. Combined with other biochemical and biophysical evidences, our results provides great potential for understanding the mechanism underlying EBOV RNP formation via the mobility of EBOV NP element and enables the development of antiviral therapies targeting EBOV RNP formation.
Crystallography, X-Ray ; Ebolavirus ; physiology ; Humans ; Nucleoproteins ; chemistry ; genetics ; metabolism ; Protein Structure, Tertiary ; Structure-Activity Relationship ; Virus Assembly ; physiology

OBJECTIVEThis study was to explore the differences in the nucleoprotein gene between rabies virus (RABV) and its vaccine strains in Guizhou province from year 2005 to 2010.

METHODSSamples from 4 rabies patients and cerebral tissue samples of 28 rabies infected dogs were collected from different districts in Guizhou province between year 2005 and 2010. Direct Immunofluorescence Assay (DFA) and RT-nested PCR assay were applied to detect the overall length of N gene sequence. Meanwhile, based on the comparison between the homology and phylogenetic tree, the differences in N gene sequence between the prevalent RABV and the RABV vaccine strains collected from NCBI database in these years.

RESULTSAccording to DFA and RT-nested PCR assay, the antigen and nucleic acid of the 21 dogs and 4 human samples were both confirmed positive; whose full length of N gene sequences were both 1353 bp. The homological analysis showed that the 25 strains of RABV virus and the RABV type I virus stored by GenBank database shared a high homology in N gene nucleotide and amino acid sequences, which were 89%-100% and 98%-100%, respectively. Besides, the homology between the 25 strains of RABV virus and its vaccines in nucleotide and amino acid sequences were separately 86%-95% and 96%-100%. The N gene of vaccines for livestock shared the highest homology with HEP-Flury strain in the nucleotide and amino acid, which were 88%-89% and 98%-99%, respectively. The vaccines for human use showed its greatest homology with the CTN strain in nucleotide (86%-100%) and amino acid (96%-100%). The phylogenetic tree analysis indicated that the 25 strains of RABV virus, RABV type I virus and the CTN vaccine strains constituted one individual cluster, which was least different from the CTN vaccine for human use.

CONCLUSIONThe prevalent RABV virus, the vaccine HEP-Flury for livestock and the vaccine CTN for human use were found to be highly similar in N gene expression in Guizhou province from 2005 to 2010.

Amino Acid Sequence ; Animals ; Dogs ; Genotype ; Humans ; Molecular Sequence Data ; Nucleoproteins ; genetics ; RNA, Viral ; genetics ; Rabies ; veterinary ; virology ; Rabies Vaccines ; genetics ; Rabies virus ; classification ; genetics ; isolation & purification

OBJECTIVETo study the genetic characterization of wild-type measles viruses isolated in Xinjiang in 2003 and 2004.

METHODSPeripheral blood mononuclear cells (PBMC) from 19 suspected measles cases collected between June 2003 and April 2004 were used to isolate measles virus by cocultivation with phytohemagglutinin (PHA)-stimulated cord blood mononuclear cells (CBMC). For positive samples, 676 nucleotides of the C-terminus of the nucleoprotein (N) gene of the measles virus genome were amplified by reverse transcription-polymerase chain reaction and then sequenced. These sequences were compared with those of other measles reference strains available in GenBank or measles isolates elsewhere in China using BLAST searches and phylogenetic analyses.

RESULTS6 measles virus strains were isolated with 3 strains (XJ03-26, XJ03-27, XJ03-74) from 2003 and 3 (XJ04-146, XJ04-150, XJ04-152) from 2004. The strain XJ03-26, differed from the Chinese measles vaccine strain S-191 (genotype A) by less than 1% at nucleotide level, and therefore appeared a vaccine-associated strain. The other 5 strains as XJ03-27, XJ03-74, XJ04-146, XJ04-150 and XJ04-152 were proved to be genotype H1 strains,among which XJ03-27, XJ03-74, XJ04-150 and XJ04-152, showing their nucleotide diversity were varied from 0.5% to 1.6%, when compared to the H1a reference strain China9322, and identified as H1a strains. XJ04-146 showed a nucleotide similarity of 98.7% when compared to H1b reference strain China9475, and was identified as H1b strain. Additionally, we found that there were two sets of strain (XJ03-27 and XJ04-150; XJ03-74 and XJ04-152), with almost identical nucleotide sequences, circulating in 2003 and 2004 and both having more nucleotide variability (up to 6.1%, 27 nucleotides).

CONCLUSIONGenotype H1 measles virus had been proven to have been circulated in Xinjiang, China during 2003 and 2004. H1a was the predominant epidemic strain while H1b strain stood the next.

China ; epidemiology ; Disease Outbreaks ; Genotype ; Humans ; Measles ; epidemiology ; Measles virus ; classification ; genetics ; isolation & purification ; Nucleoproteins ; genetics ; Phylogeny ; RNA, Viral ; genetics ; Sequence Analysis, RNA ; Viral Proteins ; genetics

OBJECTIVETo investigate the correlation of sperm DNA damage and sperm-nucleoprotein transition with acrosin activity and seminal parameters.

METHODSWe collected 535 semen samples, assessed sperm DNA damage by sperm chromatin dispersion test, and analyzed the correlation of sperm DNA damage and sperm-nucleoprotein transition with acrosin activity and seminal parameters according to the WHO criteria.

RESULTSStatistically significant differences were observed in sperm DNA damage among sperm-nucleoprotein transition, acrosin activity, sperm concentration and the percentage of grade a + b sperm (P < 0.01). Sperm DNA damage was positively correlated with age, sperm-nucleoprotein transition, sperm concentration and the percentage of grade d sperm (P < 0.01 or P < 0.05), but negatively correlated with acrosin activity (P < 0.001). Stepwise linear regression analysis demonstrated that age, sperm concentration, the percentage of grade d sperm, sperm-nucleoprotein transition and acrosin activity were independent variables related to the DNA fragmentation index (DFI). The abnormality rates of sperm-nucleoprotein transition, acrosin activity, sperm concentration and graded a + b sperm were significantly higher in the sperm DNA damage group (DFI > or = 30%) than in the normal control (DFI < 30%) (P < 0.01).

CONCLUSIONSperm DNA damage is closely related with sperm-nucleoprotein transition, acrosin activity and seminal parameters, which may become another important independent parameter for the evaluation of sperm quality.

Acrosin ; genetics ; Adult ; Chromatin ; DNA Damage ; DNA Fragmentation ; Humans ; Infertility, Male ; genetics ; Male ; Nucleoproteins ; genetics ; metabolism ; Sperm Count ; Sperm Motility ; Spermatozoa

OBJECTIVETo analyze the genetic characterizations of wild type measles viruses caused the measles outbreak in Xinjiang.

METHODSVero/Slam cell were used for measles viruses isolation from the specimens collected from measles outbreaks patients. Fragment of 676 nucleotide acids of the carboxylend of nucleoprotein gene were amplified using reverse transcription-polymerase chain reaction (RT-PCR) method and then the PCR products were directly sequenced and analyzed. Phylogenetic tree was constructed based on 450 nucleotide acids of the N-terminus of nucleoprotein gene, and homological analysis was performed at nucleotide acid level.

RESULTS11 measles viruses were sequenced and all belonged to H1a subgenotype. The nucleotide difference was 0-0.2% between 11 Xinjiang isolates. And the nucleotide difference was 2.2%-2.4% between Xinjiang isolates and H1 genotype reference strain.

CONCLUSIONThe Measles viruses causing the measles outbreak in Xinjiang were H1a subgenotype.

China ; epidemiology ; Disease Outbreaks ; Humans ; Measles ; epidemiology ; virology ; Measles virus ; classification ; genetics ; isolation & purification ; Molecular Sequence Data ; Nucleoproteins ; genetics ; Phylogeny ; Viral Proteins ; genetics