Monoclonal antibodies (MAbs) are widely applied in disease diagnoses. Herein, we report a MAb, WF-4, against Influenza A virus nucleoprotein (NP), its broad response with Influenza A virus, and its application in an immunohistochemistry (IHC) assay. WF-4 was screened by immunofluorescence assay (IFA). The results showed that its reactivity with baculovirus-expressed full-length recombinant NP (rNP) in Western blot (WB), indicating its IHC applicability. Fifteen Influenza A virus (reference subtypes H1 to H15) infected chicken embryonated chorioallantoic membranes (CAM), fixed by formalin, were all detectable in the WF-4-based IHC assay. Also, the reactivity of the IHC test with NP from experimentally inoculated H6N1 and from all recent outbreaks of H5 subtype avian Influenza A virus (AIV) field cases in Taiwan showed positive results. Our data indicate that CAM, a by-product of Influenza A virus preparation, is helpful for Influenza A virus-specific MAb characterization, and that the WF-4 MAb recognizes conserved and linear epitopes of Influenza A virus NP. Therefore, WF-4 is capable of detecting NP antigens via IHC and may be suitable for developing various tests for diagnosis of Influenza A virus and, especially, AIV infection.
Animals ; Antibodies, Monoclonal ; Blotting, Western ; Chickens ; Chorioallantoic Membrane ; Diagnosis ; Disease Outbreaks ; Epitopes ; Fluorescent Antibody Technique ; Formaldehyde ; Immunohistochemistry ; Influenza A virus ; Influenza in Birds ; Influenza, Human ; Nucleoproteins ; Taiwan

PURPOSE: The influenza B virus diverges into two antigenically distinct lineages: B/Yamagata and B/Victoria. Influenza B is the dominant circulating virus during some influenza seasons, and recent data demonstrated that influenza A and B infection similarly cause severe clinical symptoms in hospitalized patients. Nucleoprotein (NP) is a good target for a universal influenza vaccine. This study investigated whether NP epitope variation within two lineages affects the dominant cytotoxic T lymphocyte (CTL) responses induced by vaccination and the resultant protective immunity. MATERIALS AND METHODS: The NP of B/Yamagata/16/1988, the representative strain of the Yamagata lineage, includes a dominant CTL epitope, FSPIRITFL, while B/Shangdong/7/1997 from the Victoria lineage has one amino acid difference in this sequence, FSPIRVTFL. Two recombinant replication-deficient adenovirus (rAd)-vectored vaccines expressing either NP were prepared (rAd/B-NP(I) and rAd/B-NP(V), respectively) and administered to BALB/c mice intranasally. To examine the efficacy of vaccination, antibody responses, CTL responses, and morbidity/mortality after challenge were measured. RESULTS: Both vaccines induce similar antibody and CD8 T-cell responses cross-reacting to both epitopes, and also confer cross-protection against both lineages regardless of amino acid difference. CONCLUSION: The rAd-vectored vaccine expressing the NP could be developed as universal influenza B vaccine which provides broader protection.
Adenoviridae ; Animals ; Antibody Formation ; Epitopes ; Humans ; Influenza B virus* ; Influenza Vaccines ; Influenza, Human* ; Lymphocytes ; Mice ; Nucleoproteins* ; Seasons ; T-Lymphocytes ; T-Lymphocytes, Cytotoxic* ; Vaccination ; Vaccines ; Victoria

PURPOSE: The aim of the present study was to develop a serodiagnostic test for differentiation infected from vaccinated animal (DIVA) strategy accompanying the marker vaccine lacking an immunodominant epitope (IDE) of nucleoprotein of Newcastle disease virus (NDV). MATERIALS AND METHODS: Recombinant epitope-repeat protein (rERP) gene encoding eight repeats of the IDE sequence (ETQFLDLMRAVANSMR) by tetra-glycine linker was synthesized. Recombinant baculovirus carrying the rERP gene was generated to express the rERP in insect cells. Specificity and sensitivity of an indirect enzyme-linked immunosorbent assay (ELISA) employing the rERP was evaluated. RESULTS: The rERP with molecular weight of 20 kDa was successfully expressed by the recombinant baculovirus in an insect-baculovirus system. The rERP was antigenically functional as demonstrated by Western blotting. An indirect ELISA employing the rERP was developed and its specificity and sensitivity was determined. The ELISA test allowed discrimination of NDV infected sera from epitope deletion virus vaccinated sera. CONCLUSION: The preliminary results represent rERP ELISA as a promising DIVA diagnostic tool.
Animals ; Baculoviridae ; Blotting, Western ; Discrimination (Psychology) ; Enzyme-Linked Immunosorbent Assay ; Insects ; Molecular Weight ; Newcastle disease virus* ; Newcastle Disease* ; Nucleoproteins ; Sensitivity and Specificity

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.
Animals ; Antibodies* ; Asian Continental Ancestry Group ; Baculoviridae ; Cell Culture Techniques ; Chromatography ; Dogs* ; Enzyme-Linked Immunosorbent Assay* ; Humans ; Nucleoproteins ; Rabies virus* ; Rabies* ; Sensitivity and Specificity ; Vaccination

Rabies is known as the most fatal disease in all warm-blooded animals, including dogs. Among animals that transmit rabies, dogs are mainly responsible for transmitting animal rabies in Asian countries. Detection of rabies virus (RABV) antibodies in dogs is performed by fluorescent antibody virus neutralization (FAVN) test or rapid fluorescent focus inhibition test. These standard assays are difficult to carry out in diagnostic laboratories without sufficient instruments, designated RABV, and cell culture systems. An alternative assay that is easy to conduct and time efficient is required for rapid sero-surveillance following vaccination. Recombinant baculovirus expressing RABV nucleoprotein (RVN) was constructed and the recombinant protein was purified using Ni-NTA and fast protein liquid column chromatography. We developed and evaluated an indirect enzyme-linked immunosorbent assay (I-ELISA) with recombinant RVN for the detection of RABV antibodies in 122 dog serum samples. The I-ELISA results obtained from these samples were compared with FAVN results. The sensitivity, specificity, and accuracy of I-ELISA were 88.1%, 92.5%, and 91.0%, respectively, compared with FAVN. Results of I-ELISA were significantly correlated with that of FAVN (r = 0.81). These results suggest that I-ELISA with recombinant RVN is useful for sero-surveillance of RABV in dog sera.
Animals ; Antibodies* ; Asian Continental Ancestry Group ; Baculoviridae ; Cell Culture Techniques ; Chromatography ; Dogs* ; Enzyme-Linked Immunosorbent Assay* ; Humans ; Nucleoproteins ; Rabies virus* ; Rabies* ; Sensitivity and Specificity ; Vaccination

Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.
Animals ; Antibodies ; Flow Cytometry* ; Humans ; Lymphocytic choriomeningitis virus* ; Lymphocytic Choriomeningitis* ; Methods ; Mice ; Nucleoproteins ; Polymerase Chain Reaction

OBJECTIVETo correlate sperm nucleoprotein transition (SNT) with sperm morphology, DNA damage and embryo development, and assess its value for assisted reproductive technology (ART).

METHODSThe SNT of 437 infertile men underwent ART were assayed, and its correlation with sperm morphology, DNA damage, fertilization rate, normal fertilization rate, cleavage rate, available embryo rate, D3 high quality embryo rate, blastocyst formation rate and high quality blastocyst rate were analyzed.

RESULTSThe normal morphology rate of sperms, DNA damage, fertilization rate, normal fertilization rate, cleavage rate, embryo transfer rate (ETR), D3 high quality embryo rate, blastocyst formation rate (BFR) and high quality blastocyst in normal males (Group A, abnormal rate≤30%, 135 subjects) did not significantly differ from those with an abnormal rate between 30% and 60% (Group B, 170 subjects) (P>0.05). For those with an abnormal rate of above 60% (Group C, 132 subjects), the sperm normal morphology rate, DNA damage, normal fertilization rate, ETR, D3 high quality embryo rate, high quality blastocyst rate were significantly lower compared with Group A (P<0.01), while no significant difference was found in fertilization rate, cleavage rate and BFR between groups A and C (P>0.05).

CONCLUSIONSNT is related with sperm morphology rate, DNA damage and embryo development, and should be assessed before ART.

Adult ; Blastocyst ; metabolism ; DNA Damage ; Embryo Transfer ; Embryonic Development ; Female ; Fertilization in Vitro ; Humans ; Infertility, Male ; genetics ; metabolism ; Male ; Nucleoproteins ; genetics ; metabolism ; Spermatozoa ; metabolism

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.
Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Cloning, Molecular ; Ebolavirus ; genetics ; immunology ; Hemorrhagic Fever, Ebola ; immunology ; virology ; Humans ; Immunoglobulin Heavy Chains ; genetics ; immunology ; Mice ; Nucleoproteins ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV), a phlebovirus in the family Bunyaviridae, is an emerging tick-borne infectious disease that impacts humans. This disease manifests as a decreased blood cell count and multi-organ failure, with a case-fatality rate of more than 12% in China. Because vaccines or antiviral drugs for the treatment of this disease are not available, monitoring the SFTS circulation in animals and controlling the tick-mammal cycle are important for preventing SFTS. Monoclonal antibodies against the recombinant nucleoprotein of SFTSV were generated to develop a competitive enzyme-linked immunosorbent assay (cELISA) for the detection of antibodies against SFTSV infection in cattle. The specificity and sensitivity of cELISA was assessed by comparing the results of this assay to those of an immunofluorescence assay (IFA). The results of the cELISA using 416 field bovine serum samples and laboratory-immunized positive sera showed 98.1% consistency with those of the IFA. The cELISA used in this study did not show cross-reactivity with antisera against other viral cattle diseases. The cELISA presented in this study can be applied to detect antibodies against SFTSV in cattle.
Animals ; Antibodies ; Antibodies, Monoclonal ; Antiviral Agents ; Blood Cell Count ; Bunyaviridae ; Cattle ; Cattle Diseases ; China ; Communicable Diseases ; Diagnosis* ; Enzyme-Linked Immunosorbent Assay* ; Fever* ; Fluorescent Antibody Technique ; Humans ; Immune Sera ; Nucleoproteins ; Phlebovirus ; Sensitivity and Specificity ; Thrombocytopenia* ; Vaccines

The influenza A is an acute respiratory infection persistently threatening human health and social stability, and has caused high morbidity and mortality. The development of novel anti-influenza drugs based on new targets is very significant because of high mutation and drug resistance of influenza virus. The nucleoprotein of influenza A virus identified high conservation, provides cross immune protection as a potential target of anti-influenza drugs and reports on relevant studies have been published at home and a- board. Herbal drug as a traditional Chinese medicine shows the distinct advantages in the aspect of prevention and treatment of influenza A. This paper analyzes the structure and function of influenza a virus, and reviews the advances in the research on anti-influenza targets based on the nucleoprotein of the influenza A virus.
Animals ; Drug Discovery ; methods ; Humans ; Influenza A virus ; drug effects ; metabolism ; physiology ; Influenza, Human ; drug therapy ; Molecular Targeted Therapy ; methods ; Nucleoproteins ; chemistry ; metabolism ; Virus Replication ; drug effects