rN Protein Research Articles

Electrochemical biosensor for the evaluation of monoclonal antibodies targeting the N protein of SARS-CoV-2 virus

The emergence of COVID-19 caused by the coronavirus SARS-CoV-2 has prompted a global pandemic that requires continuous research and monitoring. This study presents a design of an electrochemical biosensing platform suitable for the evaluation of monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid (N) protein. Screen-printed carbon electrodes (SPCE) modified with gold nanostructures (AuNS) were applied to design a versatile and sensitive sensing platform. Electrochemical techniques, including electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), were used to investigate the interactions between immobilised recombinant N (rN) protein and several monoclonal antibodies (mAbs). The electrochemical characterisation of SPCE/AuNS/rN demonstrated a successful immobilisation of rN, enhancing the electron transfer kinetics. Affinity interactions between immobilised rN and four mAbs (mAb-4B3, mAb-4G6, mAb-12B2, and mAb-1G5) were explored. Although mAb-4B3 showed some non-linearity, the other monoclonal antibodies exhibited specific and well-defined interactions followed by the formation of an immune complex. The biosensing platform demonstrated high sensitivity in the linear range (LR) from 0.2 nM to 1 nM with limits of detection (LOD) ranging from 0.012 nM to 0.016 nM for mAb-4G6, mAb-12B2, and mAb-1G5 and limits of quantification (LOQ) values ranging from 0.035 nM to 0.139 nM, as determined by both EIS and SWV methods. These results highlight the system's potential for precise and selective detection of monoclonal antibodies specific to the rN. This electrochemical biosensing platform provides a promising route for the sensitive and accurate detection of monoclonal antibodies specific to the rN protein.

Development and validation of an enzyme-linked immunoassay kit for diagnosis and surveillance of COVID-19

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

DUPLICATE: DEVELOPMENT AND VALIDATION OF AN ENZYME-LINKED IMMUNOASSAY KIT FOR DIAGNOSIS AND SURVEILLANCE OF COVID-19

There is a massive demand to identify alternative methods to detect new cases of COVID-19 as well as to investigate the epidemiology of the disease. In many countries, importation of commercial kits poses a significant impact on their testing capacity and increases the costs for the public health system. We have developed an ELISA to detect IgG antibodies against SARS-CoV-2 using a recombinant viral nucleocapsid (rN) protein expressed in E. coli. Using a total of 894 clinical samples we showed that the rN-ELISA was able to detect IgG antibodies against SARS-CoV-2 with high sensitivity (97.5%) and specificity (96.3%) when compared to a commercial antibody test. After three external validation studies, we showed that the test accuracy was higher than 90%. The rN-ELISA IgG kit constitutes a convenient and specific method for the large-scale determination of SARS-CoV-2 antibodies in human sera with high reliability.

Serological methods for detection of infection with shrew-borne hantaviruses: Thottapalayam, Seewis, Altai, and Asama viruses

The infectivity of shrew-borne hantaviruses to humans is still unclear because of the lack of a serodiagnosis method for these viruses. In this study, we prepared recombinant nucleocapsid (rN) proteins of Seewis orthohantavirus, Altai orthohantavirus (ALTV), Thottapalayam thottimvirus (TPMV), and Asama orthohantavirus. Using monospecific rabbit sera, no antigenic cross-reactivity was observed. In a serosurvey of 104 samples from renal patients and 271 samples from heathy controls from Sri Lanka, one patient serum and two healthy control sera reacted with rN proteins of ALTV and TPMV, respectively. The novel assays should be applied to investigate potential infectivity of shrew-borne hantaviruses to humans.

Development of recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay for sero-survey of porcine reproductive and respiratory syndrome.

Background and Aim:Porcine reproductive and respiratory syndrome (PRRS) is a disease endemic in many countries and is of economic importance. India was free from PRRS until the first outbreak was reported from a North-East Indian state in 2013. Since then, disease outbreaks have been reported from North-East India and the pilot study conducted earlier showed that it is gradually spreading to the rest of India. Considering there are no locally developed population screening tests available for PRRS and imported diagnostic/screening tests are expensive, the present study was aimed at developing recombinant nucleocapsid (rN) protein-based indirect enzyme-linked immunosorbent assay (iELISA).Materials and Methods:The rN protein of PRRS virus (PRRSV) was produced following standard cloning, expression, and purification procedures. Using this antigen, iELISA was optimized for the detection of serum antibodies to PRRSV. The sensitivity and specificity of the test were assessed by comparing it with a commercial PRRSV antibody detection kit.Result:A total of 745 serum samples from ten different states of India were screened using the developed iELISA. The iELISA had a relative specificity of 76.18% and sensitivity of 82.61% compared to the commercial ELISA (Priocheck PRRSV ELISA kit, Thermo Fisher Scientific, USA).Conclusion:The iELISA, which deployed rN protein from Indian PRRSV, was found to be suitable in the serological survey and may be a useful tool in future disease surveillance programs.

Association of IL1RN VNTR polymorphism with chikungunya infection: A study from Western India.

Chikungunya, caused by the chikungunya virus (CHIKV) mostly presents as acute and chronic articular inflammatory manifestations. Interleukin 1 receptor antagonist (IL-1RN) is a potent endogenous competitive inhibitor of IL-1α and 1β and has an anti-inflammatory role. The present study evaluated the possible association of IL1RN variable number tandem-repeat (VNTR) alleles and genotypes, and CHIKV stimulated IL-1RN cytokine production with resistance and/or susceptibility to chikungunya infection and disease state in 224 patients withchikungunya (61 patients withacute chikungunya and 163 patients with chronic chikungunya) and 355 healthy controls. Polymerase chain reaction, CHIKV stimulated cytokine assay and luminex platform were used for assessing polymorphism and protein levels respectively. The study revealed a significant association of IL1RN*1/*1 genotype under recessive genetic model with the risk of developing chikungunya infection. Our findings also indicated that IL1RN *2 allele under dominant mode was associated with protection to chronic chikungunya. The results also revealed a higher production of IL-1 RN protein in patients with chronic chikungunya. To conclude, the results suggest the association of ILRN VNTR polymorphism and IL-RN protein levels with chronic chikungunya.

Cover Feature: Computational Design and Study of Artificial Selenoenzyme with Controllable Activity Based on an Allosteric Protein Scaffold (Chem. Eur. J. 44/2019)

A computational protocol that combines docking, molecular dynamics, and MM-PBSA was described to guide the design of an artificial selenoenzyme based on a Ca2+-responsive recoverin (Rn) protein. The catalytically active site of glutathione peroxidase (GPx) can be precisely incorporated into the allosteric domain of Rn, leading to a high GPx activity and tightly controlled function for the catalyzed reduction of H2O2 by glutathione (GSH). These results provide important insights into how to create an enzyme-like site in non-homologous protein scaffolds. More information can be found in the Full Paper by Q. Luo et al. on page 10350.

Computational Design and Study of Artificial Selenoenzyme with Controllable Activity Based on an Allosteric Protein Scaffold.

The establishment of new enzymatic function in an existing scaffold is a great challenge for protein engineers. In previous work, a highly efficient artificial selenoenzyme with controllable activity was constructed, based on a Ca2+ -responsive recoverin (Rn) protein. In this study, a design strategy combining docking, molecular dynamics, and MM-PBSA is presented, to predict the catalytically active site of glutathione peroxidase (GPx) on the allosteric domain of Rn. The energy contributions of the binding hot spot residues are evaluated further by energy decomposition analysis to determine the detailed substrate recognition mechanism of Rn, which provides clear guidance for artificial enzyme design for improved substrate binding (Michaelis-Menten constant, Km ).

Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis

BackgroundThe avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50 kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis.MethodsConstructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1 mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200 ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA).ResultsThe expected cDNA fragment of approximately 1240 bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10 mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot.ConclusionsThe association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.

Development of an indirect ELISA for detection of antibody to wobbly possum disease virus in archival sera of Australian brushtail possums (Trichosurus vulpecula) in New Zealand

AIMS: To develop an indirect ELISA based on recombinant nucleocapsid (rN) protein of wobbly possum disease (WPD) virus for investigation of the presence of WPD virus in Australian brushtail possums (Trichosurus vulpecula) in New Zealand.METHODS: Pre- and post-infection sera (n=15 and 16, respectively) obtained from a previous experimental challenge study were used for ELISA development. Sera were characterised as positive or negative for antibody to WPD virus based on western-blot using WPD virus rN protein as antigen. An additional 215 archival serum samples, collected between 2000–2016 from five different regions of New Zealand, were also tested using the ELISA. Bayesian modelling of corrected optical density at 450 nm (OD450) results from the ELISA was used to obtain estimates of receiver operating characteristic (ROC) curves to establish cut-off values for the ELISA, and to estimate the prevalence of antibody to WPD virus.RESULTS: Western blot analysis showed 5/14 (36%) pre-infection sera and 11/11 (100%) post-infection sera from experimentally infected possums were positive for antibodies to WPD virus. Bayesian estimates of the ROC curves established cut-off values of OD450≥0.41 for samples positive, and OD450<0.28 for samples negative for antibody to WPD virus, for sera diluted 1:100 for the ELISA. Based on the model, the estimated proportion of samples with antibodies to WPD virus was 0.30 (95% probability interval=0.196–0.418). Of the 230 archival serum samples tested using the ELISA, 48 (20.9%) were positive for antibody to WPD virus, 155 (67.4%) were negative and 27 (11.7%) equivocal, using the established cut-off values. The proportion of samples positive for WPD virus antibody differed between geographical regions (p<0.001).CONCLUSION: The results suggested that WPD virus or a related virus has circulated among possums in New Zealand with differences in the proportion of antibody-positive samples from different geographical regions. Antibodies to WPD virus did not seem to protect possums from disease following experimental infection, as one third of possums from the previous challenge study showed evidence of pre-existing antibody at the time of challenge. These results provide further support for existence of different pathotypes of WPD virus, but the exact determinants of protection against WPD and epidemiology of infection in various regions of New Zealand remain to be established.CLINICAL RELEVANCE: Availability of the indirect ELISA for detection of WPD virus antibody will facilitate prospective epidemiological investigation of WPD virus circulation in wild possum populations in New Zealand.

Development of Recombinant Nucleocapsid Protein-based Enzyme-linked Immunosorbent Assay for Serological Detection of Winter Dysentery Disease

Winter Dysentery Disease is an acute and contagious viral disease that adversely affects dairy cattle on farms in Thailand. An indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant nucleocapsid (rN) protein which has been developed for antibodies detection against Winter Dysentery Disease was produced by an Escherichia coli protein expression system. The epitope antigen N protein was designed using almost total N gene fragments (8-430 aa, N gene). It was identified to be approximately 48 kDa as the rN protein and can bind bovine coronavirus dairy cattle positive serum by western blot analysis. The conditions of the ELISA method were optimized. The rN protein was standardized with a coating antigen concentration of 5 μg/well. The rN protein was tested with sera in both infected and uninfected dairy cattle. The dilution of the primary antibodies was identified as 1:50 using a checkerboard titration. The intra- and inter-assays were repeatable. The cut-off of the corrected OD450 value from mean ± 2SD (standard deviations) was established at 0.049. The percentage of specificity, sensitivity and accuracy between the developed rNELISA and a SVANOVIR® BCV-Ab ELISA kit was 96.3, 84.8 and 86.1%, respectively. Cohen’s kappa value of the developed ELISA compared with the commercial test kit was 0.71. The correlation coefficient of absorbance values from these two tests was 0.68. The recombinant nucleocapsid protein ELISA method might be helpful for bovine coronavirus diagnosis and surveillance.

Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.

The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.

Immune Responses to Mucosal Vaccination by the Recombinant S1 and N Proteins of Infectious Bronchitis Virus

Infectious bronchitis virus (IBV) is prevented primarily by the use of live attenuated vaccines, which are known to have a limited strain range of protection. Alternative vaccines against the emerging new virus strains can improve control of the disease. The aim of this study was to evaluate the immunogenic potential of two recombinant viral proteins, when administered by eyedrop, without the assistance of a vector. The recombinant S1 (rS1) and N (rN) proteins of the M41 strain expressed in E. coli were tested, and the live attenuated vaccine H120 was used as a positive control. Protection was evaluated by re-isolation of virus from tracheas of vaccinated chickens after challenge with strain M41. After three immunizations, rS1 glycoprotein induced 40% protection, while vaccination with rN provided no protection. Vaccination with rS1, rN, or H120 induced a cellular immune response as demonstrated by in vitro ChIFN-γ production by splenocytes of vaccinated birds. Vaccination with H120, and to a lesser extent rS1, induced HI and virus-specific IgG antibody production. These findings indicate that recombinant viral proteins administered through the mucosal route can evoke an immune response without the assistance of a vector.

Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Araraquara Virus Recombinant Nucleocapsid Protein

Laboratory diagnosis of hantavirus cardiopulmonary syndrome (HCPS) in Brazil has been performed mostly by detection of IgM antibodies to recombinant antigen purified from Sin Nombre virus and Andes virus (ANDV). Recently, a recombinant nucleocapsid (rN) protein of Araraquara virus (ARAV), a Brazilian hantavirus, was obtained in Escherichia coli. To evaluate ARAV rN as antigen for antibody detection, serum samples from 30 patients from Argentina seropositive for hantavirus were tested. All samples were positive for IgG and IgM by enzyme-linked immunosorbent assay (ELISA) using either ARAV rN or ANDV rN antigens. In Brazil, six of 60 serum samples from patients with suspected HCPS (10%) were positive for IgM by ELISA using ARAV rN antigen and 7 were positive using ANDV rN antigen. For results obtained with 90 serum samples analyzed by IgM ELISA with ANDV rN antigen, the sensitivity of the IgM ELISA using ARAV rN antigen was 97.2%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 98.1%. The results show that ARAV rN is a suitable antigen for diagnosis of hantavirus infection in Brazil and Argentina.

Boosted expression of the SARS-CoV nucleocapsid protein in tobacco and its immunogenicity in mice

Vaccines produced in plant systems are safe and economical; however, the extensive application of plant-based vaccines is mainly hindered by low expression levels of heterologous proteins in plant systems. Here, we demonstrated that the post-transcriptional gene silencing suppressor p19 protein from tomato bushy stunt virus substantially enhanced the transient expression of recombinant SARS-CoV nucleocapsid (rN) protein in Nicotiana benthamiana. The rN protein in the agrobacteria-infiltrated plant leaf accumulated up to a concentration of 79μg per g fresh leaf weight at 3 days post infiltration. BALB/c mice were intraperitoneally vaccinated with pre-treated plant extract emulsified in Freund's adjuvant. The rN protein-specific IgG in the mouse sera attained a titer about 1:1,800 following three doses of immunization, which suggested effective B-cell maturation and differentiation in mice. Antibodies of the subclasses IgG1 and IgG2a were abundantly present in the mouse sera. During vaccination of rN protein, the expression of IFN-γ and IL-10 was evidently up-regulated in splenocytes at different time points, while the expression of IL-2 and IL-4 was not. Up to now, this is the first study that plant-expressed recombinant SARS-CoV N protein can induce strong humoral and cellular responses in mice.

Immunological characterizations of the nucleocapsid protein based SARS vaccine candidates

The recombinant nucleocapsid (rN) protein of the coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS) was cloned and expressed in Escherichia coli, extracted from cell lysates containing 6 M urea, then purified by Ni 2+-affinity chromatography. In animal immunogenicity studies, we found that most anti-rN protein antibodies were IgG2a in BALB/c mice vaccinated with rN emulsified in Montanide ISA-51 containing the synthetic oligodeoxynucleotide, CpG. In contrast, anti-rN protein antibodies of mice immunized with rN protein in PBS were found to mainly be IgG1. These results indicated that ISA-51/CpG-formulated rN protein was dramatically biased toward a Th1 immune response. To identify the B-cell immunodominant epitopes of the rN protein in the mouse and monkey, the reactivities of antisera raised against purified rN proteins formulated in ISA-51/CpG were tested with a panel of overlapping synthetic peptides covering the entire N protein sequence. Three immunodominant linear B-cell epitope regions were mapped to residues 166–180, 356–375, and 396–410 of the rN protein. When the reactivities of these peptides were screened with human sera from five SARS patients, peptides corresponding to residues 156–175 reacted strongly with sera from two of the SARS patients. These results indicated that the region around residues 156–175 of the N protein is immunogenic in the mouse, monkey, and human. We found that peptides corresponding to residues 1–30, 86–100, 306–320, and 351–365 contained murine immunodominant T-cell epitopes. To identify functional CTL epitopes of the N protein, BALB/c mice were immunized with peptides containing the H-2K d CTL motif emulsified in adjuvant ISA-51/CpG. Using an IFN-γ secretion cell assay and analysis by flow cytometry, peptides containing residues 81–95 were found to be capable of stimulating both CD4 + and CD8 + cell proliferation in vitro. We also only observed that peptides corresponding to residues 336–350 were capable of stimulating IFN-γ production in T-cell cultures derived from peripheral blood mononuclear cells (PBMCs) of macaques immunized with the rN protein emulsified in ISA/CpG adjuvant. Our current results together with those of others suggest that some immunodominant B-cell and T-cell epitopes are conserved in the mouse, monkey, and human. This information is very important for the development SARS diagnostic kits and a vaccine.

Serological Assays for the Detection of Human Andes Hantavirus Infections Based on Its Yeast-Expressed Nucleocapsid Protein

Background: The objective of the study was to develop and evaluate IgM and IgG ELISAs and an IgG Western blot test for the serological detection of human infections with Andes virus (ANDV), the major cause of hantavirus cardiopulmonary syndrome (HCPS) in South America. Methods: The entire nucleocapsid (N) protein-encoding sequence of ANDV (strain AH-1) was cloned and expressed in the yeast Saccharomyces cerevisiae. The polyhistidine-tagged recombinant N (rN) protein of ANDV was purified by nickel-chelation chromatography and characterized by its reactivity with different N-specific monoclonal antibodies. To detect an antibody response directed against ANDV in humans, indirect IgM and IgG ELISAs and an IgG Western blot test based on ANDV rN antigen were developed. The evaluation of the tests was performed using a negative serum panel and 63 blinded sera from Argentina and Chile, containing acute-phase and convalescent sera from HCPS patients. Results: The specificities and sensitivities for the IgM and IgG ELISAs were demonstrated to be very high. The IgG ELISA data were confirmed by the IgG Western blot assay based on the same rN antigen. Almost all anti-ANDV-positive sera reacted to higher endpoint titers with N protein of ANDV than with those of Sin Nombre, Laguna Negra or Puumala virus. The cross-reactivity of anti-ANDV-N IgG-positive sera to rN proteins of other hantaviruses was found to be increased with time after the onset of HCPS. Conclusion: The high sensitivity of the novel assays should facilitate early diagnosis of ANDV infections and might contribute to a successful treatment of HCPS patients.

Detection of Human Hantavirus Infections in Lithuania

In Europe certain hantaviruses are known to cause hemorrhagic fever with renal syndrome of different severity. The objective of the present investigation was to study the presence of hantavirus infections in Lithuania. Two different serum panels from cancer patients (n = 438) and blood donors (n = 299) from Lithuania were tested by monoclonal antibody capture IgG ELISA using yeast-expressed recombinant nucleocapsid (rN) proteins of Puumala virus (PUUV), Hantaan virus (HTNV) and Dobrava virus (DOBV). The reactivity of ELISA-positive sera was proven in Western blot tests using various hantavirus rN proteins. Selected serum samples were further analyzed by focus reduction neutralization assays. In the IgG ELISA 39 sera from the cancer patients and four sera from blood donors were found to be reactive with at least one of the rN proteins. By immunoblot using the three yeast-expressed rN proteins, the ELISA reactivity of 36 of 39 and two of four serum samples from cancer patients and blood donors, respectively, was confirmed; this corresponds to a seroprevalence of 8.2% and 0.7%, respectively. In ELISA, the majority of the samples reacted exclusively with rN proteins of HTNV and DOBV (31 of 36 and one of two in the two groups). In the group of sera selected for serotyping by focus reduction neutralization assay, this dominance was confirmed by the identification of eight DOBV but only four PUUV infections. No infection by HTNV or another hantavirus besides DOBV and PUUV was verified. Anti-hantavirus-positive human sera were detected in all seven investigated counties of Lithuania. In Lithuania at least two hantaviruses, DOBV and PUUV, circulate and cause human infections. Additional investigations are needed to study the seroprevalence more precisely and to search for clinical cases of hantavirus infections.

Development and evaluation of serological assays for detection of human hantavirus infections caused by Sin Nombre virus

The hantavirus cardiopulmonary syndrome (HCPS) was first recognized in 1993 after a cluster of acute respiratory distress syndrome deaths in the southwestern of the United States. The major causative agent of HCPS in North America is the Sin Nombre virus (SNV) carried by the deer mouse Peromyscus maniculatus. The first HCPS case imported to Europe was reported in 2002. The objective of the study was to develop and evaluate ELISA and Western blot tests for the serological detection of human infections caused by SNV including those imported to Europe. A polyhistidine (His)-tagged recombinant nucleocapsid (rN) protein of SNV was expressed in Saccharomyces cerevisiae and purified by nickel chelation chromatography. On the basis of the purified SNV rN protein mu-capture and indirect IgM and IgG ELISAs and an IgG Western blot were developed. The evaluation of the tests was performed using a negative serum panel and a blinded serum panel from the US containing acute-phase sera from HCPS patients. Based upon the results obtained using a panel of negative control sera the specificity for SNV mu-capture and indirect IgM and IgG ELISAs were found to be 100%. All 33 sera from SNV-infected HCPS patients included in the blinded panel were detected by the SNV mu-capture and indirect IgM ELISAs. Twenty-nine out of the 33 SNV-IgM positive sera reacted also in the SNV-IgG ELISA. An SNV-IgG Western blot confirmed the data of the SNV-IgG ELISA. Although the majority of anti-SNV positive sera cross-reacted with rN proteins of Puumala virus and Dobrava virus, the lacking reactivity of a few sera with these heterologous rN antigens in the corresponding IgM and IgG ELISAs demonstrates the value of virus-specific test formats for acute-phase sera. The novel SNV ELISA and Western blot tests represent a useful tool for the serological detection of SNV infections.

Yeast-expressed hantavirus Dobrava nucleocapsid protein induces a strong, long-lasting, and highly cross-reactive immune response in mice.

In Europe, Dobrava virus (DOBV) carried by the yellow-necked field mouse Apodemus flavicollis is one of the hantaviruses that can cause severe hemorrhagic fever with renal syndrome in humans. For several hantaviruses, the nucleocapsid (N) protein has proven to be very immunogenic in humans and rodents and even can protect rodents against a virus challenge. To investigate the immunogenicity of DOBV N protein, BALB/c and C57BL/6 mice were immunized three times with a DOBV recombinant N (rN) protein expressed in yeast Saccharomyces cerevisiae together with complete Freund's, with incomplete Freund's, and without adjuvant, respectively. Mice of both strains elicited N-specific antibodies with end-point titers being as high as 1:1,000,000 in C57BL/6 mice. The antibodies induced by DOBV rN protein were highly cross-reactive to the rN proteins of hantaviruses Puumala and Hantaan. In both mice strains, DOBV rN protein induced N-specific antibodies of all IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3), suggesting a mixed Th1/Th2 immune response. Taken together, yeast-expressed DOBV rN protein represents a promising vaccine candidate.