The N protein of the rinderpest virus (RPV) was analyzed topologically and antigenically by using anti-N monoclonal antibodies (Mabs). Ten Mabs were raised against the N protein of the RPV. At least six non-overlapping antigenic sites (sites A-F) were delineated by competitive binding assays using biotinylated Mabs. Of them 5 sites (A, C, D, E and F) on the N protein were recognized by RPV-specific Mabs in ELISA and IFA while site B was recognized by Mabs reacting with both RPV and PPRV. Non- reciprocal competition was found among sites C, D and E. Recombinant RPV N protein after exposure to 0.2% SDS exhibited higher ELISA titers in all Mabs recognizing 6 sites. Four sites (A, B, E and F) on 2% SDS-treated N protein lost completely reactivity with Mabs while the remaining sites (C and D) on the protein retained their antigenicity to some degree. It indicates that two sites (C and D) were sequential. Six representative Mabs bound to each site exhibited competition with rinderpest antibodies in a blocking ELISA, indicating that the sites were actively involved in antigenicity in cattle.
Antibodies, Monoclonal/*immunology ; Antigens, Viral/chemistry/*immunology ; Binding, Competitive ; Enzyme-Linked Immunosorbent Assay ; Epitopes/immunology ; Nucleocapsid Proteins/chemistry/*immunology ; Rinderpest virus/*immunology

Human noroviruses (huNoVs) recognize histo-blood group antigens (HBGAs) as attachment factors, in which genogroup (G) I and GII huNoVs use distinct binding interfaces. The genetic and evolutionary relationships of GII huNoVs under selection by the host HBGAs have been well elucidated via a number of structural studies; however, such relationships among GI NoVs remain less clear due to the fact that the structures of HBGA-binding interfaces of only three GI NoVs with similar binding profiles are known. In this study the crystal structures of the P dimers of a Lewis-binding strain, the GI.8 Boxer virus (BV) that does not bind the A and H antigens, in complex with the Lewis b (Le(b)) and Le(y) antigens, respectively, were determined and compared with those of the three previously known GI huNoVs, i.e. GI.1 Norwalk virus (NV), GI.2 FUV258 (FUV) and GI.7 TCH060 (TCH) that bind the A/H/Le antigens. The HBGA binding interface of BV is composed of a conserved central binding pocket (CBP) that interacts with the β-galactose of the precursor, and a well-developed Le epitope-binding site formed by five amino acids, including three consecutive residues from the long P-loop and one from the S-loop of the P1 subdomain, a feature that was not seen in the other GI NoVs. On the other hand, the H epitope/acetamido binding site observed in the other GI NoVs is greatly degenerated in BV. These data explain the evolutionary path of GI NoVs selected by the polymorphic human HBGAs. While the CBP is conserved, the regions surrounding the CBP are flexible, providing freedom for changes. The loss or degeneration of the H epitope/acetamido binding site and the reinforcement of the Le binding site of the GI.8 BV is a typical example of such change selected by the host Lewis epitope.
Binding Sites ; Blood Group Antigens ; chemistry ; immunology ; Caliciviridae Infections ; immunology ; virology ; Crystallography, X-Ray ; Epitopes ; chemistry ; immunology ; Evolution, Molecular ; Humans ; Lewis Blood-Group System ; chemistry ; immunology ; Norovirus ; chemistry ; immunology ; pathogenicity ; Protein Binding ; Viral Proteins ; chemistry ; immunology

BACKGROUNDTo study the composition and significance of the inclusion bodies of human cytomegalovirus (HCMV).

METHODSMicrodissection of inclusion bodies, PCR and Southern blot were adopted to detect DNA, and immunohistochemistry method and catalyzed signal amplification (CSA) were used to detect the different antigens of HCMV.

RESULTSThe inclusion bodies of HCMV were separated from the tissue section of human salivary gland. The fragments amplified by PCR from these dissected inclusion bodies were confirmed to be the DNA of HCMV. With the immunohistochemical method CSA, the immediately early and early antigens of HCMV were detected with monoclonal antibodies DDG9/CCH2, while matrix protein AAC10 was negative in the inclusion bodies.

CONCLUSIONThe ingredient of inclusion bodies of HCMV included the DNA and the antigens expressed in specific stage of the virus.

Antigens, Viral ; analysis ; immunology ; Cytomegalovirus ; genetics ; immunology ; Cytomegalovirus Infections ; diagnosis ; immunology ; virology ; DNA, Viral ; analysis ; genetics ; Humans ; Immunohistochemistry ; Inclusion Bodies ; chemistry ; immunology ; virology ; Microdissection ; Salivary Glands ; chemistry ; immunology ; virology

Antibodies, Monoclonal ; chemistry ; immunology ; Antigens, Viral ; chemistry ; genetics ; immunology ; Binding Sites ; Capsid Proteins ; chemistry ; genetics ; immunology ; Epitopes ; chemistry ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Hepatitis E ; immunology ; prevention & control ; virology ; Hepatitis E virus ; chemistry ; immunology ; Humans ; Molecular Docking Simulation ; Mutagenesis, Site-Directed ; Peptide Mapping ; Protein Binding ; Recombinant Proteins ; chemistry ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; biosynthesis

Human antibodies generated by phage antibody technology have been widely used in the immunotherapy of various diseases. Among the characteristics of these therapeutic antibodies, affinity is one of the most important determinants of their biological efficacy. The binding of an antibody and its corresponding antigen could be disrupted by thiocyanate solution of different concentrations, depend upon the affinity of the antibody. This mechanism has been adopted to determine the relative affinity of monoclonal or polyclonal antibodies in routine immunological practice. Correlation between the elution method and other techniques that measure the affinity such as equilibrium dialysis and biospecific interaction analysis (BIA) has been established. Here we describe the applications of the thiocyanate elution method in the determination of the relative affinity index (RAI) of phage antibodies (Phabs). Five clone antibodies, including 3 clones of anti-keratin antibodies (AK1, AK2 and AK3) and 2 clones of anti-HBsAg antibodies (HB1 and HB2) were selected to express Phabs and Fabs, and the RAI were determined by ELISA after thiocyanate elution. A HRP-conjugated anti-M13 was used as secondary antibody for Phabs and HRP-goat-anti-human Fab was used for Fabs. The affinity ranks of the Phabs were compared with that of the Fab fragments. The results showed that all the Phabs tested were tolerant to thiocyanate treatment. The relative affinity rank of 5 Phabs coincided well with that of their corresponding Fabs. We conclude that the thiocyanate elution can be used as an easy and rapid method to measure and compare the relative affinity of Phabs.
Antibodies ; immunology ; Antibodies, Monoclonal ; immunology ; Antibodies, Viral ; immunology ; Antibody Affinity ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; methods ; Hepatitis B Antibodies ; immunology ; Hepatitis B Surface Antigens ; immunology ; Keratins ; immunology ; Peptide Library ; Proteomics ; methods ; Thiocyanates ; chemistry ; Transfection

BACKGROUNDTo study the antigenic and genetic characteristics of influenza (H3N2) virus circulated in China in 2004.

METHODSSingle-way and cross-way hemagglutination inhibition (HI) tests were firstly used to determine the reactivity with the reference serum of virus isolates. Based on the serological results, virus isolates were selected according to the different time and location in China in 2004. The HA1 domain of HA gene of those virus isolates were then sequenced in order to analyze the gene characterization.

RESULTSSingle-way HI test results showed that 52.3% of isolates showed 4 folds or more HI titer difference compared to A/Fujian/411/2002 (H3N2) itself (international reference strain in 2004). Cross-way HI test results showed that the antigenic ratio was 4. The nucleic acid and amino acid sequence data of HA1 domain showed that the mutated virus appeared in early February of 2004, and became the dominant circulating strain gradually. There were four important mutant positions, they were 159 Y>F, 189 S>N, 145 K>N, 226 V>I, respectively. The results also indicated that the mutated viruses originated from southern China, then transmitted to northern China, according to the analysis of time and location distribution.

CONCLUSIONThe HA1 domain of HA gene of influenza virus (H3N2) isolated from 2004 in China showed mutation and antigenic drift, and the mutated viruses were becoming the dominant circulating strain in China, and showed amino acid sequence difference compared to A/Fujian/411/2002 (H3N2) A/Wellington/1/2004 (H3N2), the vaccine components pronounced by WHO for 2004-2005 northern hemisphere and 2005 southern hemisphere respectively, which suggested that further surveillance should be conducted to monitor the virus mutation in circulation.

Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; immunology ; Cell Line ; China ; DNA, Complementary ; chemistry ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; immunology ; Phylogeny ; RNA, Viral ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

In this study, Recombinant baculoviruses rBac-NF and rBac-NG were generated for expressing F and G proteins Nipah virus (NiV) . The expression of recommbinant G (rNG) and F (rNF) protein in rBac-NF and rBac-NG infected cells were confirmed by western-blot. Both rNG and rNF showed sensitive and specific antigenic reaction to rabbit serum anti-Nipah virus in indirect immunofluorescence detection and indirect ELISA. Immunization with rBac-NF and rBac-NG infected insect cells elicited G ad F protein specific antibody responses in mice. Furthermore, the G ad F specific antibodies could neutralize the infectivity of the VSVdeltaG* F/G, the NiV F and G envelope glycoproteins psudotyped recombinant Vesicular Stomatitis Virus expressing green fluorescence protein. The results demonstrated F and G protein expressed by the recombinant baculoviruses could be safe economic diagnostic antigens for the surveillance and monitoring of NiV and promising subunit vaccines for the prevention of NiV.
Animals ; Antigens, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Nipah Virus ; chemistry ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Recombination, Genetic ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology

Group-A rotaviruses are recognized as the most common cause of acute diarrhea. Phylogenetic analyses of the VP7 genes of rotaviruses circulating in Nanjing (China) could aid in the development of rotavirus vaccines. A total of 908 stool specimens were collected from patients suffering from acute diarrhea in Nanjing between October 2012 and December 2013, and were tested further for rotaviruses. Fifty rotavirus isolates selected randomly were typed by reverse transcription-polymerase chain reaction using serotype-specific primers for G genotyping. VP7 genes of 19 G9 strains were sequenced for further genetic characterization. Among the 908 stool specimens examined during the surveillance period, 103 (11.34%) were rotavirus-positive. G9 was the most predominant genotype (78.0%), followed by G2, G1 and G3. Sequence and phylogenetic analyses of the VP7 genes of serotype G9 rotaviruses revealed these strains to comprise two lineages (G9-VI, G9-III) and to be dominated by the G9-VI lineage (which belonged to a unique subcluster of Japanese and Chinese G9 strains). Amino-acid sequences of the four antigenic regions (A, B, C or F) were variant among a portion of strains, which may have contributed to the prevalence of G9 rotaviruses in this area.
Adult ; Amino Acid Sequence ; Antigens, Viral ; chemistry ; genetics ; Capsid Proteins ; chemistry ; genetics ; China ; Evolution, Molecular ; Humans ; Infant ; Molecular Sequence Data ; Mutation ; Phylogeny ; Rotavirus ; genetics ; immunology ; physiology ; Serogroup

To understand the HA1 genetic variation characterization of influenza H3N2 virus isolates in Zhu-hai during 2008-2009, we selected 20 of H3N2 Influenza strains cultured in MDCK cell. Viral RNAs were extracted and amplified by using RT-PCR. The amplified products were purified after identified by gel electrophoresis and then the nucleotide sequences of the amplicons were determined. The results were analyzed by the software ClustalX and MEGA4. 1. When compared with the amino acid sequences of the epitopes of HA1 district of H3N2 influenza vaccine recommended by WHO in 2008, changes were found in those of H3N2 influenza strains in Zhuhai in 2008: K140I in all of H3N2 influenza strains, L157S in 08-0343 and 08-0677, K158R in 08-0466, 08-0620 and 08-0667, K173E in 08-0466 and 08-0620, K173N in 08-0667, and I192T in 08-0667. The epitopes of HA1 district of H3N2 influenza strains in Zhuhai in 2009 are different from that of H3N2 influenza vaccine during the same time: K173Q and P194L occur in all of H3N2 influenza strains, N144K, K158N, and N189K occur in the strains except the strain 09-0056. HA1 domain of H3N2 influenza strains in 2009 has lost a glycosylation site at amino acid position 144 while the glycosylation sites of HA1 domain of H3N2 influenza stains isolated in 2008 remained. This study suggested that H3N2 influenza virus in Zhuhai in 2008 was not evolved a novel variant and H3N2 influenza variant in 2009 was attributed to antigenic drift in HA1 district.
Animals ; Antigens, Viral ; immunology ; Cell Line ; China ; Dogs ; Epitopes ; immunology ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; immunology ; metabolism ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; immunology ; isolation & purification ; Mutation ; Phylogeny ; Sequence Analysis, DNA

To develop a sensitive and specific ELISA for detection of antibodies to the nonstructural protein of FMDV. We cloned and expressed FMDV nonstructural protein 3AB in Escherichia coli expression system. The recombinant protein 3AB was purified with Ni-NTA HisBind Resins and characterized by Western blotting. An indirect ELISA based on purified protein 3AB as a coating antigen was established. The specificity and sensitivity of this assay were evaluated by comparison with a commercial 3ABC-ELISA kit in detecion of serum samples. The results showed that the recombinant protein 3AB was expressed as a formation of inclusion bodies in Escherichia coli. The purified protein could specificially react with FMDV infection antibodies in Western blotting assay, but no reaction with the immune antibodies induced with vaccine. Two assays were no significant differences in specificity and sensitivity for detection of field samples (P>0.05). Therefore, we speculated that the recombinant protein 3AB is a promising molecular marker, which may effectively differentiate FMD-infected from vaccinated animals in a herd.
Animals ; Antibodies, Viral ; analysis ; Antigens, Viral ; biosynthesis ; genetics ; immunology ; Cattle ; Cattle Diseases ; diagnosis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease ; diagnosis ; immunology ; Foot-and-Mouth Disease Virus ; chemistry ; genetics ; isolation & purification ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology