Development of an immunochromatography strip test based on truncated nucleocapsid antigens of three representative hantaviruses (original) (raw)
PLoS Neglected Tropical Diseases, 2013
In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in .90% of PUUV-and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV).
Rapid, whole blood diagnostic test for detecting anti-hantavirus antibody in rats
Journal of Virological Methods, 2013
Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibodydetecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats.
Competitive Homogeneous Immunoassay for Rapid Serodiagnosis of Hantavirus Disease
Journal of Clinical Microbiology, 2015
In this study we describe a competitive homogenous immunoassay making use of Förster 1 Resonance Energy Transfer (FRET) in rapid detection of pathogen-specific antibodies. The assay 2 principle is based on competition between a monoclonal antibody (MAb) and serum antibodies to a 3 given antigen. In the assay named as Competitive FRET Immuno Assay (CFRET-IA), FRET signal 4 is induced if MAb carrying a donor-label binds to an acceptor-labeled antigen. Specific antibodies 5 in serum compete for antigen binding, resulting in reduced FRET signal. 6 7
Journal of Virological Methods, 2003
Hantaviruses infecting humans in Eurasia include Hantaan, Seoul, Puumala and the closely related Dobrava and Saaremaa viruses. These viruses are causative agents of hemorrhagic fever with renal syndrome (HFRS), which is recognized as a severe health care problem in several countries. Diagnostics of hantavirus infections relies on serology, performed principally with enzyme immunoassay (EIA) or immunofluorescence assay (IFA). We developed four 5-min immunochromatographic IgM-antibody tests for diagnostics of acute Puumala, Dobrava and Hantaan virus infections and a similar combination test to detect all Eurasian pathogenic hantavirus infections. We evaluated the assays using 100 fingertip blood samples collected randomly from Finnish volunteers, 28 confirmed hantavirus IgM-negative sera, and 77 sera from patients with acute infections of various hantaviruses. The specificities and sensitivities of the Puumala-, Dobrava-and Hantaan virus-specific tests varied from 96 to 100%, whereas, the combination test showed 96% specificity and 80 to 93% sensitivity. Cross-reactions were observed commonly between the Dobrava and the Hantaan virus tests, but only rarely between the Puumala and the Hantaan virus, or the Puumala and the Dobrava virus, tests. Altogether, the rapid tests showed less cross-reactivity than the respective EIA tests. According to the results, the performance of these tests meets well the requirements for diagnostic use. Nevertheless, the specific one-antigen tests were markedly more sensitive than the combination test. However, if optimized, a combination test would be suitable for regions where several hantaviruses circulate. #
Laboratory diagnosis of human hantavirus infection: novel insights and future potential
Infections by Hantavirus (Bunyaviridae) can cause severe human diseases, such as hemorrhagic fever with renal syndrome in Eurasia and cardiopulmonary syndrome in the Americas. These diseases are emergent and became a serious public health problem worldwide. Thus, rapid, sensitive and reliable methods for diagnosis of hantavirus infection are necessary in order to manage patients and control this rodent-borne virosis. Serological methods, such as neutralization tests, immunoblots and enzyme immunoassays using hantavirus-recombinant proteins as antigens, are discussed in this article, as well new methods such as immunochromatographic test. Hantavirus genome detection, by different kinds of reverse transcription-PCR, including the real-time variant, is also discussed here.
Journal of Virological Methods, 2008
Indirect and capture enzyme-linked immunosorbent assays (ELISAs) for detection of Hantaan virus (HTNV)-specific immunoglobulins G (IgG) and M (IgM) in human serum samples were developed on the basis of recombinant yeast-expressed nucleocapsid (N) protein of HTNV. The sensitivities and specificities of the indirect and capture ELISAs were evaluated by comparing the reactivity of sera from patients with hemorrhagic fever with renal syndrome (HFRS) from China with that of a commercial IgG/IgM kit. The sensitivity of the indirect IgG and IgM ELISA tests was both 100% and the specificity of the indirect IgM and IgG ELISA test was 98% and 99%, respectively. The sensitivity and specificity of the capture IgM ELISA was 100% and 97%, respectively. The novel assays were found to detect HTNV-specific antibodies in acute phase sera from suspected HFRS patients in China. The results indicate that these novel ELISAs are suitable for the diagnosis of HTNV and for sero-epidemiological studies.
Quality control measures for the serological diagnosis of hantavirus infections
Journal of Clinical Virology, 2003
Background: With society's rapidly increasing mobility, patients infected with severe viral infections can become seriously ill at any place in Europe and elsewhere. Improving the diagnostics of these infections is the most important step in detecting the pathogens and dealing with them, and for this purpose, quality control measures are essential tools. Objectives: To assess the diagnostic reality for rare hantavirus infections in Europe by (1) running a pre-evaluation panel (four samples, sent out in 1999) to optimise sample preparation and shipping procedure and afterwards (2) starting an External Quality Assurance (EQA) program (20 samples, sent out in 2001). Study design: All samples sent out had to be tested for the presence of specific IgG and IgM antibodies against hantavirus. For the pre-evaluation panel, four samples were distributed (two samples IgG'//IgM(/, one sample IgG-borderline/IgM(/, one sample IgG(// IgM(/), for the EQA 20 samples (six samples IgG'//IgM'/, eight samples IgG'//IgM(/, one sample IgG(/borderline/ IgM(/, five samples IgG(//IgM(/). Thirteen laboratories took part in the pre-evaluation panel, 18 laboratories participated in the first EQA run. Results: For the pre-evaluation panel, the participants reported correct results for 64% of the IgG-positive samples (85% excluding borderline-positive sample), and 92% for the IgG-negative sample. IgM testing was correctly negative in all laboratories. For the EQA, the participants reported correct results for 76% of the IgG-positive samples, and 97% correct results for the IgG-negative samples. For the IgM-positive samples, 53% correct results were reported, and 98% correct results for the IgM-negative samples. Conclusions: The results presented here prove the importance of quality measures also for viruses only rarely suspected, like hantavirus, and they clearly demonstrate the need for improvement of the existing test systems.
Diagnostic Microbiology and Infectious Disease, 2007
The symptoms of hantavirus pulmonary syndrome (HPS) may resemble those of other febrile illnesses. The development of an accurate diagnostic test should therefore improve clinical prognosis and be useful in epidemiological studies. We evaluated the use of a recombinant antigen (rN∆ 85 ) based on the S-segment sequences of a Brazilian hantavirus for detecting IgM and IgG antibodies against hantavirus in an indirect enzyme immunoassay (EIA). We assayed 613 serum samples (570 from humans and 43 from rodents). IgM EIA had a sensitivity of 94.1% and a specificity of 99.1%. IgG EIA had a sensitivity of 95.2% and a specificity of 98.4%. This evaluation confirms that rN∆ 85 IgM and IgG EIA tests are potentially useful rapid, sensitive and cost-effective tools for detecting antibodies against hantaviruses indigenous to Brazil and other South American countries, in patients with acute or convalescent hantavirus infection, and in rodent reservoirs.
Journal of Clinical Virology, 2005
Background: 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. Objectives: 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. Study design: 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-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. Results: Based upon the results obtained using a panel of negative control sera the specificity for SNV-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-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. Conclusions: The novel SNV ELISA and Western blot tests represent a useful tool for the serological detection of SNV infections.
Journal of Virological Methods, 1989
Three enzyme-linked immunosorbent assay (ELISA) systems based upon different principles were developed for the serology of Hantaan virus infections and compared with an indirect immunofluorescence assay (IFA). The indirect IFA was carried out with y-irradiated Hantaan virus-infected and uninfected Vero E6 cells fixed with ethanol (-70°C) or acetone (20°C) on drop slides and a FITC-coupled sheep anti-human Ig preparation. Atypical staining in the IFA was avoided by using ethanol (-70°C) instead of acetone (20°C) fixation. In the first ELISA ('cellassay'), Hantaan virus-infected or uninfected Vero E6 cells were used as antigens, which after y-irradiation were seeded into microtiter ELISA strips. Serial dilutions of human sera were incubated and specific antibodies were demonstrated with a horseradish peroxidase (HRPO)-conjugated sheep anti-human Ig preparation. In the second ELISA ('competition-assay') an affinity-purified human Ig preparation was used as a capture antibody for Hantaan virus antigen. After incubation of serial dilutions of human sera with this coat, the reactivity of the affinity purified anti-Hantaan virus Ig coupled to HRPO was determined. In the third ELISA ('complex trapping blocking [CTB]-assay') the same capture antibody was used to react with a mixture of the antigen and serial dilutions of human sera. The reactivity with the same HRPO conjugate was then determined. The results obtained in the respective assay systems with sera from people at risk or suspected of Hantaan virus infection coincided well. The CTB-ELISA proved to be faster and more sensitive than both the other ELISA systems, without giving more non-specific re-
Journal of clinical microbiology, 1985
Antisera prepared against 16 strains of hantaviruses isolated from patients with hemorrhagic fever with renal syndrome (HFRS) or from rodents captured in HFRS-endemic and nonendemic regions were titrated against Hantaan virus strain 76-118, Puumala virus strain Sotkamo, and Prospect Hill virus strain Prospect Hill-I by using the indirect immunofluorescent antibody and plaque reduction neutralization tests. Isolates fell into one of four distinct groups or serotypes. Serotype 1 included Apodemus-derived strains, serotype 2 included Rattus-derived strains, serotype 3 included Clethrionomys-derived strains, and serotype 4 included Microtus-derived strains. Serotypic classification of hantavirus infections was possible for humans and rodents in widely varied geographical areas, but in a few instances, sera from patients with HFRS did not conform to any of the four serotypes, suggesting the existence of as yet unidentified serotypes. A definitive serological classification of hantaviruse...
Serum antibodies to structural proteins of hantavirus arise at different times after infection
Journal of Medical Virology, 1992
An enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of serum antibodies against group-specific epitopes of the glycoproteins (GI, G2) and nucleoprotein (NP) of the genus Hantavirus. This assay was used to study the kinetics of the development of serum antibodies after natural infection with Puumala-like virus in humans. To this end a panel of 34 serum samples collected from individuals at different times after natural infection was tested by the ELISA. The samples were also tested for specific IgM and IgG levels against Puumala-like virus, which provided confirmatory data about the presumed timing of infection. It was shown that serum antibodies against the zyxwvuts GI epitope were present in the acute and early convalescent period just before antibodies to the NP epitope could be demonstrated. In contrast, antibodies to two G2 epitopes were present not earlier than in the convalescent and late convalescent period. Since all these categories of antibodies seem to persist for long periods, antibodies against the GI epitope and the NP epitope may be of specific diagnostic value. Furthermore, levels of G1-specific antibodies and antibodies to either NP or G2 may allow estimation of the time elapsed following initial infection. zyxwvutsrq 0 1992 Wilev-Liss, Inc.
Expression of a hantavirus N protein and its efficacy as antigen in immune assays
Brazilian Journal of Medical and Biological Research, 2008
Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.
Fast amperometric immunoassay for hantavirus infection
Electroanalysis, 1996
The determination of specific antibodies against Hantavirus in blood plasma has been developed. The assay technique is based on a flow through amperometric immunoelectrode. Highly dispersed carbon material serves as an immunosorbent and at the same time as an electrode material. The analysis employed the 'sandwich' assay scheme. Recombinant protein of Hantavirus was immobilized on the surface of the immunosorbent. Target analyte captured by the immunosorbent at the first stage of incubation interacts with the peroxidase labeled antihuman antibodies at the second stage of incubation. The detection of peroxidase label was conducted amperometrically by electroreduction of iodine formed as a product of peroxidase catalyzed reaction. The technique permits determination of specific antibodies against Hantavirus in blood plasma using a 22min assay time.
Rapid field test for detection of hantavirus antibodies in rodents
Epidemiology and Infection, 2004
Puumala virus (PUUV) is the causative agent of nephropathia epidemica, a mild form of haemorrhagic fever with renal syndrome. PUUV is transmitted to humans via aerosolized excreta of the infected bank vole (Clethrionomys glareolus). Current methods for screening of the PUUV prevalence among bank vole populations are laborious, combining sampling in the field and subsequent analyses in the laboratory. In order to facilitate animal testing, a new serological immunochromatographic rapid test was developed. The test uses PUUV nucleocapsid protein as antigen, and it detects anti-PUUV IgG antibodies in rodents. With fresh and undiluted bank-vole blood samples (n=105) the efficacy of the test was 100 %, and with frozen and diluted samples (n=78) the efficacy was 91%. The test was also shown to detect related hantavirus infections in Norway lemmings and sibling voles (n=31) with 99% efficacy. The test provides an applicable tool for studying PUUV and related hantavirus infections in arvicoline rodents.
Journal of Virological Methods, 2009
Hantaviruses, which are mainly rodent-borne viruses, cause hemorrhagic fever with renal syndrome in the Old World, and hantavirus pulmonary syndrome in the New World. A neutralization test based on quantitative RT-PCR, the replication reduction neutralization test (RRNT), was developed for efficient detection of hantavirus-neutralizing antibodies. The effectiveness of the RRNT was evaluated by examining several hantaviruses and hantavirus-specific convalescent human serum samples. All convalescent serum samples tested by RRNT caused significant decreases in hantavirus genomes with only one specific hantavirus species, which allowed a straightforward identification of the related hantavirus. The results obtained by RRNT were completely comparable with the results obtained by focus reduction neutralization test (FRNT). The RRNT approach is a reliable and rapid alternative for FRNT, hitherto considered as the gold standard for hantavirus serology.
Archives of Virology, 1988
Consecutive serum samples collected from 235 patients with Haemorrhagic Fever with Renal Syndrome (HFRS), between two days and two years after onset of disease, have been analysed for the presence of IgG and IgM type of antibodies specific for Hanta-viruses. The sera were screened in parallel by a newly developed indirect Immuno Enzyme Assay (EIA) in parallel with Indirect Immunofluorescent Antibody Assay (IFA). In both tests the Hantaan virus strain 76-118 was used as the antigen. The EIA was much more sensitive than the IFA test for the detection of IgM type antibodies. With the indirect EIA IgM type antibodies against Hantaan virus 76-118 have been detected in HFRS patient's sera from the second day of illness indicating the usefulness of this test for the early serological diagnosis of this disease.
Journal of Clinical Virology, 2003
Background: Hantaviruses are rodent borne viruses in the family Bunyaviridae that cause significant morbidity in large areas of Europe. There are only a few reports available on hantavirus infections from Spain. Although the results of these earlier studies indicated the presence of hantavirus infections, no confirmative or serotype-specific analyses have been performed. Objectives: To investigate whether hantaviruses cause human infection/disease in Spain. Study design: Ten thousand, four hundred and eighteen serum samples from the general population and 599 sera from 492 patients with potential hantavirus infections (renal disease, pneumonia or hepatitis) were initially screened by immunofluorescence assay (IFA) using Hantaan, Seoul and Puumala hantavirus antigens. Altogether 193 suspicious samples (165 from healthy people and 28 from patients) were selected for confirmation by quality-assured assays. Results and conclusions: Of the 165 pre-screened serum samples from healthy individuals, only five could be confirmed by IFA for hantavirus-reactive antibodies (using Dobrava, Saaremaa, Hantaan or Puumala virus antigens). In addition, one serum was found weakly positive for hantavirus-reactive IgG by ELISA using recombinant Saaremaa virus (SAAV) nucleocapsid (N) antigen, and subsequently confirmed by immunoblotting. Thus, the results indicated a low (0.06%) total antibody prevalence to hantaviruses in Spain. Of 28 pre-screened serum samples from hospitalized patients, eight reacted as positive or showed borderline reactivities for hantavirus-specific IgM by ELISA using recombinant Saaremaa and Puumala virus N antigens. The IFA/ELISA reactive/border-line samples were subsequently analyzed by a focus reduction neutralization test, which revealed low titers (1:80) against SAAV in two samples from a patient with hepatic disease. The nature of the hantavirus(es) potentially involved remain, however, unknown, since none of the positive samples showed neutralizing titers of the expected range to any of the known European hantaviruses.