ABSTRACT
To study the B cell linear epitopes of rabies virus CVS-11 nucleoprotein, peptides were synthesized according to the amino acid sequences of B cell linear epitopes. Linear epitopes predicted by bioinformatics analysis were evaluated with immunological techniques. Indirect enzyme-linked immunosorbent assay showed that titers of antibodies to peptides (355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein) were above 1:12 800 in mouse sera. The antibodies recognized denatured rabies virus CVS-11 nucleoprotein in Western blot analysis. Purified anti-peptide antibodies recognized natural rabies virus CVS-11 nucleoprotein in BHK-21 cells in indirect fluorescent antibody test. The 355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein were validated as B cell linear epitopes.
Subject(s)
Animals , Female , Humans , Male , Mice , Amino Acid Sequence , Antibodies, Viral , Allergy and Immunology , Epitope Mapping , Epitopes, B-Lymphocyte , Chemistry , Genetics , Allergy and Immunology , Mice, Inbred BALB C , Molecular Sequence Data , Nucleoproteins , Chemistry , Genetics , Allergy and Immunology , Rabies , Allergy and Immunology , Virology , Rabies virus , Chemistry , Genetics , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To prepare monoclonal antibodies against a newly discovered and conserved linear epitope of Rabies virus nucleoprotein and to use them in a rabies diagnostic test.</p><p><b>METHODS</b>Synthetic peptide containing the epitope was used as immunogen to prepare hybridoma cell lines by classical hybridoma technology. Anti-peptide monoclonal antibodies produced in ascites of inoculated Balb/c mice were labeled with fluorescein isothiocyanate (FITC) after purification and used in fluorescent antibody test (FAT).</p><p><b>RESULTS</b>Two positive hybridoma cell lines, RVNP-mAb1-CL and RVNP-mAb2-CL, were obtained. RVNP- mAb1-CL produced a higher concentration of monoclonal antibody RVNP-mAb1 in Balb/c ascites. FITC-labeled RVNP-mAb1 showed correct results on certain Rabies virus-positive canine brain tissue samples and cells of a small subclone of baby hamster kidney 21 cell line (BSR).</p><p><b>CONCLUSION</b>FITC-labeled RVNP-mAb1 has potential application for laboratory diagnosis of rabies.</p>
Subject(s)
Animals , Cricetinae , Dogs , Mice , Antibodies, Monoclonal , Cell Line , Epitopes , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Hybridomas , Mice, Inbred BALB C , Nucleoproteins , Allergy and Immunology , Rabies virus , Allergy and Immunology , Viral Proteins , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>Compare the difference of the results referred to the WHO standard rabies immunoglobulin and the national standard human rabies immunoglobulin used in the rapid fluorescent focus inhibition test (RFFIT).</p><p><b>METHODS</b>Setting the WHO standard immunoglobulin and the national standard immunoglobulin in the same system and testing 12 human serum at the same time. Compare the fluorescence percentage of the two different standard immunoglobulin; compare the 12 serum results calculated from the two different standard immunoglobulin used the calculation formula of neutralization antibody titer.</p><p><b>RESULTS</b>The Results display that the 50% percent of the two standard immunoglobulin are all between the fifth and the sixth well, but the percentage of the national standard immunoglobulin is lower than the WHO one. The same testserum result calculated from the WHO standard immunoglobulin is little higher than the national one.</p><p><b>CONCLUSION</b>There is difference in the WHO standard immunoglobulin and the national one, but there is no influence in the results.</p>
Subject(s)
Humans , Fluorescent Antibody Technique , Methods , Reference Standards , Immunoglobulins , Allergy and Immunology , Neutralization Tests , Methods , Reference Standards , Rabies , Diagnosis , Allergy and Immunology , Rabies virus , Allergy and Immunology , Reference Standards , World Health OrganizationABSTRACT
Objective To establish a rapid fluorescent inhibition test (RFFIT) for testing rabies virus neutralizing antibody and the titer of rabies virus neutralizing antibody.CVS-11 was used as the standard challenge virus,and three generations prepared for the establishment of the virus library.Methods International standard for rabies immunoglobulin was used as the reference serum.RFFIT test was established under consulting the protocol of Institute of Pasteur,and its specificity,stability and reproducibility were validated.Results We established the RFFIT which showed both good specificity ( 100% ) and reproducibility (P＞0.5).Conclusion The establishment of RFFIT test perfected the rabies laboratory techniques and would enhance the overall ability in detecting rabies in China.
ABSTRACT
A combinatorial human Fab library to the rabies virus was constructed using antibody genes derived from the blood of vaccinated donors. The library were panned and selected on purified rabies virus particles of aG or CTN strain with phage display. Eleven unique human Fab antibodies specific for the rabies virus glycoprotein were obtained by ELISA, IFA and DNA sequences analysis of these antibodies. Among these Fab antibodies, five human Fab antibodies were converted to full-length human IgG antibodies with recombinant baculovirus system. The five full-length human IgG antibodies were tested in vitro for rabies virus neutralization, resulting in all specificities to neutralize the virus. The obtained human anti-rabies antibodies lay the basis for the production of cocktail of anti-rabies monoclonal antibody with chinese intellectual property.
Subject(s)
Animals , Cricetinae , Humans , Amino Acid Sequence , Antibodies, Neutralizing , Allergy and Immunology , Antibodies, Viral , Allergy and Immunology , Cell Line , Immunoglobulin Fab Fragments , Genetics , Allergy and Immunology , Immunoglobulin G , Genetics , Allergy and Immunology , Molecular Sequence Data , Neutralization Tests , Rabies , Allergy and Immunology , Virology , Rabies virus , Genetics , Allergy and ImmunologyABSTRACT
5 strains of virus isolated from Culex tritaeniorhynchus, Anopheles sinensis and Armigeres subalbatus, which caused cytopathic effect in C6/36 cells, had been obtained in the survey of arboviruses in Northwestern Yunnan Province. China. The virus particles displayed 70 nanometers diameter (n=7) with no envelope but spikes on the surfaces. RNA-PAGE of the genomes of the isolates showed 6-5-1 profile. A fragment of the 12th segment sequence was amplified by a pair of specific primers for Kadipiro virus strain JKT-7075 in RT-PCR. The full length of the 12th segment was 758 nucleotides, BLAST analysis revealed the highest identity was 90% to JKT-7075. Phylogenetic analysis demonstrated that the isolates appeared to be Kadipiro viruses (Family Reoviridae). It was the first report of kadipiro virus isolation in China.
Subject(s)
Animals , Amino Acid Sequence , Anopheles , Virology , Cell Line , China , Coltivirus , Classification , Genetics , Culex , Virology , Molecular Sequence Data , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
<p><b>OBJECTIVE</b>To study the clinical and laboratory characteristics of adult Japanese encephalitis (JE) patients in a JE outbreak in Yuncheng, Shanxi Province in 2006.</p><p><b>METHOD</b>All the clinical data from the Second People's Hospital in Yuncheng city were analyzed, part of patients' sera and cerebrospinal fluid were tested by serology and RT-PCR.</p><p><b>RESULTS</b>The majority of patients were middle-aged and elderly, 77.8% (35/45) of the total cases were more than 40 years old. Severe and fulminating type cases accounted for 60.0% (27/45). Most patients had underlying diseases. IgM antibody to JE virus (JEV) in serum was positive in each of the 45 patients analyzed and 4-fold or greater rise in sera neutralization antibody titer were found in convalescent serum. JEV nucleic acid was positive in part of cerebrospinal fluid specimens.</p><p><b>CONCLUSION</b>Viral encephalitis emerged in Yuncheng city, Shanxi Province was Japanese encephalitis B, and most of the cases belonged to elderly group.</p>
Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Male , Middle Aged , Young Adult , Antibodies, Viral , Blood , China , Epidemiology , Disease Outbreaks , Encephalitis Virus, Japanese , Genetics , Allergy and Immunology , Encephalitis, Japanese , Blood , Cerebrospinal Fluid , Epidemiology , Immunoglobulin M , Blood , Neutralization Tests , RNA, Viral , Genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
0507BS3 virus was isolated from a mixed pool of Culex sp. and Anopheles sp. collected in Kashi, Xinjiang, China. 0507BS3 virus could cause cytopathic effects on C6/36 cells but not on Vero and BHK-21 cells. Viral particles had no envelope and appeared round with diameter of about 60 nm (n = 20). Viral capsid was composed of a single layer and a central core. Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 10 double stranded RNA (dsRNA) segments. Sequencing of the tenth segment revealed the length of 964bp (GenBank ID: FJ150869). A single open reading frame (ORF) was found and encoded a protein of 275 amino acids with a molecular mass of 30.8kDa. The nucleotide sequence had no similarity with any other viral genomic sequences, but the deduced amino acid sequence significantly matched the polyhedrin genes of cytoplasmic polyhedrosis virus (CPV) in some sections. A phylogenetic tree was constructed to compare the polyhedrin gene sequences of all CPV types in GenBank. The tree demonstrated that 0507BS3 virus was only distantly related to the other CPV types and belonged to a new CPV type.
Subject(s)
Animals , Cell Line , China , Culicidae , Virology , Phylogeny , Reoviridae , Classification , GeneticsABSTRACT
0507JS60 virus was isolated from a pool of Culex sp. collected in Kashi, Xinjiang, which could be propagated stably on C6/36 cells and caused cytopathic effects continuously. Viral particles had no envelope and appeared round with diameter of about 55nm (n = 10). Capsomeres on the surface of capsid were clearly visible. Electrophoresis of viral genome showed a profile of 12 double stranded RNA (dsRNA) segments. Sequencing of the twelfth segment revealed the length of 760bp (GenBank ID: FJ157354). A single open reading frame (ORF) was found and encoded a protein of 174 amino acids with a molecular mass of 18.9kD. The nucleotide sequence had similarity over 89% with that of LNV, but the deduced amino acid sequence had similarity over 91% with that of LNV. A phylogenetic tree was constructed to compare the corresponding genetic sequences in Seadornavirus. The tree demonstrated that 0507JS60 virus lied in the same branch with LNV and more closely related to LNV-NE9712. 0507JS60 virus was identified as LNV, which was firstly isolated outside the Northeast of China.
Subject(s)
Animals , Cell Line , China , Culex , Virology , Molecular Sequence Data , Phylogeny , Reoviridae , Classification , GeneticsABSTRACT
<p><b>BACKGROUND</b>To study the arboviruses carried by mosquitoes collected in Hebei Province.</p><p><b>METHODS</b>Samples were collected from mosquito active sites and stored in liquid nitrogen till use. Pools of 20 to 30 mosquitoes were ground after sterilization, centrifugal supernant was inoculated onto C6/36 cell, cytopathic effect was observed for three sequential passages. Positive isolates were identified by IFA and RT-PCR.</p><p><b>RESULTS</b>Totally 1310 mosquitoes were collected from two villages of She county, Hebei province. They were divided into 46 pools and ground respectively. Thirteen positive isolates were obtained. Two isolates reacted with alphaviral antibodies and were amplified by alphaviral primers, nucleotide sequence showed the highest homology (98%) to Getah virus (AY702913.1), so the two isolates were identified as Getah virus.</p><p><b>CONCLUSION</b>Getah virus was isolated from mosquitoes in Hebei Province. This is the first report of isolating Getah virus from inland of China.</p>
Subject(s)
Animals , Arboviruses , Classification , Genetics , Cell Line , Cluster Analysis , Culicidae , Virology , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
<p><b>BACKGROUND</b>To disclose the species and distribution of tick-borne arboviruses in the southern part of Xinjiang.</p><p><b>METHOD</b>Totally 5045 ticks were collected from 36 collecting sites of 23 places in the southern Xinjiang, which were made into cDNA pools with pd(N)6 primer through RT-PCR method. Then PCR was used to detect viral nucleotide sequence from cDNA.</p><p><b>RESULTS</b>All 34 cDNAs showed negative to flavivirus and California serogroup virus primers; but nairovirus and primers derived from Xinjiang hemorrhagic fever virus had amplified and yielded some obvious bands corresponding to the nucleotide sequences of Xinjiang hemorrhagic fever virus. A phylogenetic analysis was done to the obtained partial sequences of L and S segments.</p><p><b>CONCLUSION</b>Nucleotide sequences of Neither flaviviruses nor California serogroup viruses were detected from the samples. However partial L segment sequence was first reported in China. Phylogenetic analysis of partial L and S segments disclosed the molecular characteristic of Xinjiang hemorrhagic fever virus.</p>