Nucleoprotein-based indirect enzyme-linked immunosorbent assay(indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays(ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in antiEbola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G(Ig G) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

AgNOR and rasp21 expression in gastric mucosal lesions with Helicobacter pylori infection

IM To study the number of AgNOR and rasp21 expression in different gastric mucosal lesions with Helicobacter pylori (Hp) infection to evaluate their biological behaviour and possible mechanism of Hp. METHODS Hp (using CLO test combined with WathinStarry staining), AgNOR (silver colloid technique) and rasp21 (monoclonal antibody and immunohistochemical staining—ABC method) were detected in 278 patients with endoscopically and pathologically confirmed gastric mucosal lesions, including chronic superficial gastritis (CSG), chronic atrophic gastritis (CAG), intestinal metaplasia (IM), dysplasia (Dys), and gastric cancer (GC). Among them, 146 cases were Hp positive, and 132 cases Hp negative. RESULTS The mean number of AgNOR in Hp positive group was significantly higher than that in Hp negative group in the gastric mucosal lesions except for CSG (P<005 or P<001). The positive rate of rasp21 expression in Hp positive group was also significantly higher than that in Hp negative group in gastric mucosal lesions except for CSG and CAG (P<005).CONCLUSION Hp+ gastric mucosal lesions have more biological behaviour of tumors. Hp may act as a promoter to activate ras gene and to stimulate cell over proliferation.

Rapid Detection of Filoviruses by Real-time TaqMan Polymerase Chain Reaction Assays

Ebola virus （EBOV） and Marburg virus （MARV） are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates and there is currently no licensed vaccine or therapeutics. To date, there is no specific laboratory diagnostic test in China, while there is a national need to provide differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. In this study, the TaqMan RT-PCR assays targeting the nucleoprotein genes of the Zaire Ebolavirus （ZEBOV） and MARV were developed and their sensitivities and specificities were investigated. Our results indicated that the assays were able to make reliable diagnosis over a wide range of virus copies from 103 to 109, corresponding to the threshold of a standard RNA transcript. The results showed that there were about 101 RNA copies per milliliter of virus culture supernatant, equivalent to 10,000 RNA molecules per infectious virion, suggesting the presence of many non-infectious particles. These data indicated that the TaqMan RT-PCR assays developed in this study will be suitable

Preparation and Initial Application of a Monoclonal Antibody Specific for a Newly Discovered Conserved Linear Epitope of Rabies Virus Nucleoprotein

Objective 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. Methods 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）. Results Two positive hybridoma cell lines, RVNP-mAbl-CL and RVNP-mAb2-CL, were obtained. RVNP- mAbl-CL produced a higher concentration of monoclonal antibody RVNP-mAbl in Balb/c ascites. FITC-labeled RVNP-mAbl 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）. Conclusion FITC-labeled RVNP-mAbl has potential application for laboratory diagnosis of rabies

Generation and application of two monoclonal antibodies targeting conserved linear epitopes in the NP protein of influenza A virus

Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

Expression of Nucleoprotein Gene of CTN Strain Rabies Virus from China in <i>E. coli </i>with Antigenicity

The nucleoprotein (NP) gene of rabies CTN strain isolated from China was recombined into pMal-c2x. The antigenicity of the recombined MBP-NP fusion proteins was examined by western blotting and by enzyme linked immunosorbent assay (ELISA). The results demonstrated that the recombined protein possesses predominant antigenicity.

Evaluation of Benzamide Derivatives as New Influenza A Nucleoprotein Inhibitors

Virus nucleoprotein (NP) is an emerging target for drug development for Influenza. We designed benzamide derivatives as new inhibitors of NP that demonstrate good potency in blocking influenza A. Screening revealed that compound 39 was the most potent molecule in the series, exhibiting IC50 values of 0.46 and 0.27 μM in blocking the replication of H3N2 (A/HK/8/68) and (A/WSN/33) influenza A viral strains. The observed inhibition of viral replication correlated well with cytopathic protection. Furthermore, based on computational analysis and fluorescence microscopy, it was determined that compound 39 inhibited nuclear accumulation by targeting influenza A viral nucleoproteins. Finally, the rodent pharmacokinetic profile of compound 32 displayed half-life of greater than 4 hours and bioavailability greater than 20%, suggesting this class of molecules had drug-like properties.

Phosphorylation sites within Ebola virus nucleoprotein

To understand the infection process, the viral multiplication and entry to the cell is widely studied. The Ebola virus nucleoprotein is the important problem for the pathological process. Focusing on the specific biological process, the post translational modification is needed. Here, the authors used the bioinformatics study to find the phosphorylation sites within the Ebola virus nucleoprotein and could identify many new sites.

Physiological Effects of Salmon Milt Nucleoprotein on Movement, Stress Tolerance and Lifespan of <i>C. elegans</i>

In recent years, various physiological functions of salmon milt extract, which consists of nucleic acid and nucleoprotein, have been reported. The objective of this study is to analyze the physiological function and its mechanism of salmon milt extract (NG) on nematodes (C. elegans). The wild type nematode N2 strain was bred on the plate containing of NG for four days, and its body length increased depending on NG concentration. When nematodes were bred with NG for a longer period, average lifespan was increased, and survival rate was increased by up to 20%. Generally, the movement of nematodes decreases with longer breeding period (i.e. aging). Analysis of movement (both gross thrashing movement and local pumping movement) showed that NG suppressed this decrease f movement with aging. Furthermore, the deease of survival rate by heat stress and oxidative stress was suppressed by NG administration. Nile Red staining analysis showed that fat accumulation varied depending on the concentration of NG. RT-PCR analysis revealed that the mRNA expression levels of the stress resistance genes sod-3 and sod-4 were increased. These results indicated that NG administration increased the expression of stress-tolerance-related genes, promoted stress tolerance, increased movement and prolonged lifespan in nematode.

SSINCC: Simple separation of interacting nucleoprotein complex components

Protein-DNA binding assays have been used in a va-riety of applications from fundamental studies re-garding the binding process itself to serve as probes for the detection, quantification and separation of target analytes. Here we describe a novel method of analyzing and identifying intermolecular DNA interactions that allows for the simple separation of interacting nucleoprotein complex components (SSINCC), focusing specifically on DNA-DNA interactions using P1 plasmid active partition system nucleoprotein complexes as a model to demonstrate DNA sequence specificity and tolerance of composite factor complexity. Traditional and recent assays of protein-DNA interaction are summarized and compared with SSINC. Although SSINC is examined here employing P1 partition nucleoprotein complex as an example of DNA-DNA intermolecular association, universal applications of this methodology to nucleo-protein complex studies can be envisioned.

Immunoglobulin G Specific Antibody Level against Ebola Viral Glycoprotein and Nucleoprotein in Ebola Virus Disease Survivors and Their Relatives

Ebola virus disease is a complex zoonosis that is highly virulent in humans. Despite its sorely pathogenic and lethal nature, survivors of this infection and even asymptomatic cases are able to develop both humoral and cellular immunity against several Ebola virus (EBOV) proteins. We aimed at determining immunoglobulin G (IgG) antibodies level against two Ebola viral antigens, the glycoprotein and the nucleoprotein in Ebola survivors and their relatives. Anti-EBOV glycoprotein (GP) and nucleoprotein (NP) IgG antibodies were quantified using ELISA. We enrolled 199 participants in two different sites as follow: 91 survivors at the Loreto clinic and 70 survivors with 38 relatives of Sierra Leone Association of Ebola Survivors Bombali Branch (SLAESB) tested for anti-EBOV NP and anti-EBOV GP IgG antibodies. Our findings revealed that the median anti-EBOV IgG level among survivors was 5.7128 U/ml [IQR: 2.793 - 7.783] for anti-EBOV GP IgG and 4.431 U/ml [IQR: 2.083 - 7.696] for anti-EBOV NP IgG. Survivors relatives had a median anti-EBOV GP IgG level of ?0.7128 U/ml [IQR: -0.903 to -0.04327] and -2.711 U/ml [IQR: -4.01 to -1.918] for anti-EBOV NP IgG. We observed that IgG levels in survivors were higher than in relatives with a significant difference of about 0.0001. The median value of anti-EBOV IgG level among seropositive relatives was 0.7043 U/ml [IQR: 0.5686 to 3.716] for anti-EBOV GP IgG and 4.05 U/ml [IQR: 0.2765 to 7.759] for anti-EBOV NP IgG respectively. Interestingly, we observed that 3.30% of Loreto clinic survivors did not developed anti-EBOV NP IgG antibodies;also about 10% survivors of the SLAESB were not reactive to anti-EBOV NP IgG and 1.43% of these survivors did not express antibodies against the Ebola viral glycoprotein. Our work is consistent with previous published studies showing heterogeneity in both survivors and asymptomatic cases of Ebola infection developing adaptive immunity against EBOV proteins.

Sequencing, Expression and Diagnostic Application of the Nucleoprotein Gene of Xinjiang Hemorrhagic Fever Virus

In order to analyze the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV), viral RNA was amplified by RT-PCR by using the proof-reading DNA polymerase to produce the complete NP gene. The PCR product was sequenced, analyzed for phylogenesis and cloned into the expression vector pET32a and the recombinant plasmid expressed in E.coli BL-21 with high yield. The primarily purified fused protein was used to coat ELISA plates for the detect antibodies. It was found the similarities between NP gene of BA88166 and other XHFVs in nucleotide level and amino acid contents were very significant, and the NP gene of BA88166 encoded a nucleoprotein with 482 amino acid and a deduced molecular weight (MW) of 54?kDa. Western blot assay showed that the fusion protein expressed in bacteria possessed good antigenicity. The results with ELISA for the detection of the human and animal sera collected in endemic areas were found to be in good accordance to the clinical diagnosis. It concluded that the relations of NP genes of XHFV BA88166 and other XHFVs appeared to be evolutionally close. The methodologies established in this study were accurate, specific, rapid and reproducible for the clinical examinations and epidemiological survey.

CLINICAL SIGNIFICANCE OF DETECTING SERUM ANTIBODIES TO NUCLEOPROTEIN AND GLYCOPROTEIN G_2 OF HANTAAN VIRUS IN PATIENTS WITH HEMORRHAGIC FEVER WITH RENAL SYNDROME

Antibody blocking enzyme linked immunosorbent assays, respectively detecting antibodies to Hantaan virus nucleoprotein (NPAb) and glycoprotein GZ (G,Ab), were developed using monoclonal antibody L133, L13r3, LV48A and LVZB28B NPAb and GZAb in 291 serum samples from 65 patientswith kemorrkagic fever with renal syudrome (HFRS) were detfrmlned by these methods. The positive rates or NPAb were 90N on day 2-3 and 100 % on day 8-9 arter onset of disease, respectively.NPAb titers Increased during fever period and reached Peak levels during kypotensive and oliguric periods of HFRS. It was suggested that NPAb might be an important component Involved in the immunopathogenlc lin'alrmeut of HFRS and the detection of NPAb might be useful for the early diagnosis or HFRS. The I,osltlve rates and titers of GZAh were very low during the rirst three periods,namely rever, hypoteuslve and ollgurlc periods, and reached high levels during the convalescent period. GRAb titers were negatively related to the I,rotelnurla levels during the course of HFRS. It wasIndicated that GZAb might be the main component or neutralizing autlhodles to Hantaan virus Infection and the efrlclent production or GZAb was a good marker ror predicting the recovery and betterprognosis of HFRS.

Structure and sequence analysis of influenza A virus nucleoprotein

Influenza A virus nucleoprotein (NP) forms homo-oligomers and multiple copies of NP wrap around genomic RNA, along with a trimeric polymerase making up ribonucleoprotein (RNP) complex. Sequence comparison of more than 2500 influenza A NP showed that this protein contains 30.1 % of polymorphic residues. NP is composed of a head and a body domain and a tail loop/ linker region. The head domain is more conserved than the body domain, as revealed from the structure-based sequence alignment. NP oligomerization is mediated by the insertion of the non-polymorphic and structurally conserved tail loop of one NP molecule to a groove of another NP. The different form of NP oligomers is due to the flexibility of the polymorphic linkers that join the tail loop to the rest of the protein. The RNA binding property of NP is known to involve the protruding element and the flexible basic loop between the head and body domains, both having high degree of primary sequence conservation. To bind RNA, NP may first capture the RNA by the flexible basic loop and then the RNA is clamped by the protruding element.

Insight into the Ebola virus nucleocapsid assembly mechanism： crystal structure of Ebola virus nucleoprotein core domain at 1.8 A resolution

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 （NPcore） pos- sesses 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 a- helix of EBOV NPcore itself, which is highly conserved among filoviridae family. Combined with other bio- chemical 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.

The Structure Analysis and Antigenicity Study of the N Protein of SARS-CoV

The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with other viral structural proteins, the low variation rate of the N protein with regards to its size suggests its importance to the survival of the virus.

The nucleoprotein of severe fever with thrombocytopenia syndrome virus processes a stable hexameric ring to facilitate RNA encapsidation

Severe fever with thrombocytopenia syndrome virus(SFTSV),a member of the Phlebovirus genus from the Bunyaviridae family endemic to China,is the causative agent of life-threatening severe fever with thrombocyto-penia syndrome(SFTS),which features high fever and hemorrhage.Similar to other negative-sense RNA viruses,SFTSV encodes a nucleocapsid protein(NP)that is essen-tial for viral replication.NP facilitates viral RNA encapsida-tion and is responsible for the formation of ribonucleopro-tein complex.However,recent studies have indicated that NP from Phlebovirus members behaves in inhomogene-ous oligomerization states.In the present study,we report the crystal structure of SFTSV NP at 2.8Åresolution and demonstrate the mechanism by which it processes a ring-shaped hexameric form to accomplish RNA encapsida-tion.Key residues essential for oligomerization are identi-fi ed through mutational analysis and identifi ed to have a signifi cant impact on RNA binding,which suggests that correct formation of highly ordered oligomers is a criti-cal step in RNA encapsidation.The fi ndings of this work provide new insights into the discovery of new antiviral reagents for Phlebovirus infection.

Extracellular nucleoprotein exacerbates influenza viruspathogenesis by activating Toll-like receptor 4 and the NLRP3inflammasome

Host immune responses, such as those initiated by pattern recognition receptor (PRR) activation, are important for viralclearance and pathogenesis. However, little is known about the interactions of viral proteins with surface PRRs or, moreimportantly, the association of innate immune activation with viral pathogenesis. In this study, we showed that internalinfluenza virus proteins were released from infected cells. Among these proteins, nucleoprotein (NP) played a critical role inviral pathogenesis by stimulating neighboring cells through toll-like receptor (TLR)2, TLR4, and the NLR family pyrin domaincontaining 3 (NLRP3) inflammasome. Through the activation of these PRRs, NP induced the production of interleukin (IL)-1β andIL-6, which subsequently led to the induction of trypsin. Trypsin induced by NP increased the infectivity of influenza virus,leading to increases in viral replication and pathology upon subsequent viral infection. These results reveal the role of releasedNP in influenza pathogenesis and highlight the importance of the interactions of internal viral proteins with PRRs in theextracellular compartment during viral pathogenesis.

Nucleoprotein phosphorylation site(Y385)mutation confers temperature sensitivity to influenza A virus due to impaired nucleoprotein oligomerization at a lower temperature

Mutations in viral proteins can lead to the cold adaption of influenza A virus and the cold-adapted virus is an important vaccination instrument.Here,we identify a novel strain of influenza A virus with cold sensitivity conferred by a mutation at a phosphorylation site within the nucleoprotein(NP).The highly conserved tyrosine 385 residue(Y385)of NP was identified as a phosphorylation site by mass spectrometry.The constructive NP phosphorylation mimicked by Y385 E mutation was fatal for virus replication,while the continuous Y385 dephosphorylation mimicked by Y385 F mutation had little impact on virus replication in vitro.Notably,the Y385 F virus showed much lower replicative capacity in turbinates of mice compared with the wild type virus.Moreover,the replication of Y385 F virus was significantly reduced in both A549 and MDCK cells grown at 33℃,when compared to that at 37℃.These results indicated that the Y385 F mutation led to cold sensitivity of virus.We further found that the cold sensitivity of Y385 F virus could be attributed to diminished NP oligomerization rather than any changes in intracellular localization.Taken together,these findings suggest that the phosphorylation of NP may be a critical factor that regulates the temperature sensitivity of influenza A virus.

Structure-based Design, Synthesis and Anti-influenza A Virus Activities of Substituted Phenyl-coupled Heterocyclic Ethylamide Derivatives

A series of new substituted phenyl-coupled heterocyclic ethylamide derivatives was designed and synthe- sized as anti-influenza agents. In vitro anti-influenza A（A/PR/8/34 H1N1 strain） activities of these compounds were investigated and compared to those of the commercial antiviral drugs（Arbidol and Ribavirin） against the influenza. Specifically, among these twelve compounds exhibiting moderate levels of antiviral activity against influenza A, compounds 30c and 30d are the most effective ones, and as efficacious as the positive control Ribavirin and much more effective than Ingavirin and Arbidol, indicating that they are prospective candidates for further exploration. These results are also consistent with the docking study results in terms of the design of compounds targeting in- fluenza A via viral nucleoprotein.