Am. J. Trop. Med. Hyg., 81(2), 2009, pp. 273–276 Copyright © 2009 by The American Society of Tropical Medicine and Hygiene
Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Araraquara Virus Recombinant Nucleocapsid Protein Luiz Tadeu Moraes Figueiredo,* Marcos Lazaro Moreli, Alessandra Abel Borges, Glauciane Garcia de Figueiredo, Soraya Jabur Badra, Ivani Bisordi, Akemi Suzuki, Silvana Capria, and Paula Padula Virology Research Center, School of Medicine of the University of São Paulo in Ribeirão Preto, Ribeirão Preto, Brazil; Adolfo Lutz Institute, São Paulo, Brazil; National Institute for Infectious Diseases Dr. Carlos G. Malbrán, Buenos Aires, Argentina
Abstract. 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.
virus came from Bolivia) virus (LNV), Castelo dos Sonhos virus (CASV) and Anajatuba virus (ANAV). The diagnosis of HCPS in Brazil has been made mostly on the basis of clinical presentation, history of exposure and positivity of serum IgM or IgG antibodies to hantavirus. Performance of hantavirus serologic testing has been confined to a few government public laboratories that use an enzyme-linked immunosorbent assay (ELISA) with the N protein of Sin Nombre virus (SNV) provided by the Centers for Disease Control and Prevention (Atlanta, GA) and Andes virus (ANDV) provided by the Carlos Malbrán Institute (Buenos Aires, Argentina) as antigens. Therefore, to supply laboratories in Brazil with a native antigen for hantavirus serologic testing, we have recently produced a recombinant homologous N protein based on the nucleotide sequence of ARAV obtained from a HCPS patient. In the present study, this N recombinant protein of ARAV was tested as antigen in an IgG ELISA and an IgM ELISA for diagnosis of human infection by hantavirus. These results were compared with those obtained with similar tests using the recombinant N proteins of either SNV or ANDV as antigens.
INTRODUCTION The genus Hantavirus of the family Bunyaviridae includes a large number of rodent-borne viruses that are distributed worldwide. These agents are enveloped, 80–120 nm in diameter, with a genome consisting of three segments of negativestranded RNA named small (S), medium (M), and large (L). The RNA segments are complexed with nucleocapsid protein (N) to form individual L, M, and S nucleocapsids. The S segment encodes the N protein, the M segment encodes a glycoprotein precursor that is processed into Gn and Gc proteins, and the L segment encodes the viral RNA polymerase.1 The N protein is synthesized soon after infection and is the most abundant protein. The encapsidation of viral RNA by the N protein is recognized as central in virus replication, but its details are largely unknown. It is believed that by encapsidation the N protein could protect new synthesized viral RNA from degradation by nucleases. In addition, the N protein interacts directly with the cytoplasmic tail of glycoprotein G1 to initiate virion assembly.2 The N protein induces strong humoral immune response, both in infected patients and rodents, based on three major epitopes located near the amino terminus of the N protein, which are cross-reactive among members of the genus Hantavirus.2 Hantavirus cardiopulmonary syndrome (HCPS), a newly discovered human disease, emerged in North America in 1993 in association with exposure to aerosols of urine and feces of wild rodents of the subfamily Sigmodontinae. Soon after HCPS was described in other American countries, it became a worrisome emerging health problem in Brazil with a case-fatality rate of 39% (884 deaths as of May 2007; Brazilian Ministry of Health, unpublished data). Five types of hantavirus are known to cause HCPS in Brazil: Juquitiba virus (JUQV), Araraquara virus (ARAV), Laguna Negra-like (the original Laguna Negra
MATERIALS AND METHODS Production of N recombinant protein of ARAV. The N recombinant protein of ARAV was prepared in the Virology Research Center, School of Medicine of the University of São Paulo in Ribeirão Preto, Brazil. Briefly, the N gene of ARAV was purified from the blood of a patient with HCPS, cloned into a pET200D vector, and used to transform expressioncompetent Escherichia coli BL21 star™ (Invitrogen, Carlsbad, CA). Previously, the complete 1,287-nucleotide sequence of this N gene was obtained and it showed 97% homology with a previously known sequence of ARAV. The recombinant N (rN) protein, which contained 429 amino acids and had a size of 52 kD, was purified under denaturing conditions.3 Purified plasmid pET200D with no insert was used as negative control antigen.3 Human sera and laboratory analysis. To assess the performance of IgM and IgG ELISAs developed with ARAV rN, studies were carried out at the Adolfo Lutz Institute in
* Address correspondence to Luiz Tadeu Moraes Figueiredo, Virology Research Center, School of Medicine of the University of São Paulo in Ribeirão Preto, Av. Bandeirantes 3900, Ribeirão Preto, SP, 14049-900, Brazil. E-mail:
[email protected]
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MORAES FIGUEIREDO AND OTHERS
São Paulo, Brazil. Comparisons were made with the rN antigens of SNV (National Institutes of Health, Bethesda, MD) and ANDV (National Institute for Infectious Diseases, Buenos Aires, Argentina).4 The tests using ARAV rN antigen were performed in the same manner with ANDV antigen at the National Institute for Infectious Diseases, and with SNV and ANDV antigens at the Adolfo Lutz Institute.5 In Brazil, 18 serum samples from persons with dengue or another acute febrile illness who were seronegative for hantavirus were tested by IgG ELISA with SNV and ARAV rN antigens.6 In addition, serum samples from 60 patients with a clinically suspected diagnosis of HCPS routinely submitted to the laboratory for serologic diagnosis of HCPS by IgM ELISA using ANDV rN were tested using ARAV rN antigen.4 In Argentina, samples from the bank of serum samples ANLIS (National Institute for Infectious Diseases Dr. C. G. Malbrán) were used for testing by IgM and IgG ELISAs with ARAV rN. Thirty human serum samples from HCPS patients with a diagnosis confirmed either by reverse transcription–polymerase chain reaction or seropositivity for IgM or IgG by ELISA for ANDV were re-analyzed for IgM and IgG by ELISA using ANDV and ARAV rN antigens (Table 1). IgG ELISA using ANDV, SNV, and ARAV rN antigens. All commercial reagents used for the IgM and IgG ELISAs were obtained from Kierkegaard and Perry Laboratories (Gaithersburg, MD). Tests were performed as described.4,5 Briefly, polystyrene microtiter plates (Polysorp, Nunc,
Denmark) were coated overnight in a wet chamber at 4°C with 4 µg/mL of ANDV rN, SNV rN, or control antigen, or with 2 µg/mL of ARAV rN antigen and the respective control. All incubations were conducted at 37°C for 1 hour and plates were washed six times with wash buffer (phosphate-buffered saline [PBS]–0.1% Tween 20) between each step. All reagents were added at volumes of 0.1 mL. All serum samples were diluted 1:400 in dilution buffer (5% skimmed milk powder in PBSTween-20) and 4-fold dilutions up to 1:25,600 were added to the antigen-coated wells. Peroxidase-labeled affinity-purified goat anti-human IgG Fc antibody was added and specific antibody binding was detected by the addition of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substrate with absorbance measured at 405 nm. Values were expressed as the optical density (OD) obtained with ANDV antigen or ARA rN antigen minus the OD values for the control antigens. The cut-off value of the test was determined by the mean plus 3 standard deviations of the ODs obtained for at least 4 negativecontrol serum samples plus 3 standard deviations of the mean. IgM ELISA using ANDV and ARAV rN antigens. A capture IgM ELISA using ANDV and ARAV rN antigens was performed as described.4 Briefly, U-bottom polyvinylchloride microtitration plates (Falcon, Irvine, CA) were coated with a 1:1,000 dilution of an affinity-purified goat antihuman IgM (µ chain) antibody.All reagents were added at volumes of 0.1 mL. All incubations were conducted at 37°C for 1 hour and the plates were washed six times with wash buffer (PBS–0.1% Tween 20)
Table 1 ELISA cross-reactivity of 30 serum samples from ANDV-infected HCPS patients in Argentina tested by ELISA with ANDV and ARAV rN antigens* IgM OD‡
IgG OD‡
Serum ID
Origin of serum
Sex
Age†
Days
ANDV
ARAV
ANDV
ARAV
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
South Center Center South South North Center South Center Center North West South Center Center South Center Center South Center North Center Center North Center Center South South South North North
M F F M M M M M F M M F F M M F F M M M M F F M M F F M F M
67 8 4 40 58 19 23 31 11 40 14 70 35 47 10 77 20 29 41 19 20 10 48 42 45 NA NA 37 24 23
4 6 1 17 6 3 NA 6 3 11 12 4 4 NA 6 NA 3 15 NA 5 6 9 3 11 9 NA NA 10 4 7
0.839 2.6 1.81 1.982 3.475 2.593 2.906 3.461 2.025 1.,863 1.743 1.085 3.51 2.767 3.377 1.599 3.498 2.71 3.497 2.092 2.194 3.02 1.982 1.3 2.192 2.648 2.488 3.03 3.478 0.839
0.923 3.056 2.445 3.238 0.881 1.906 3.113 1.719 2.331 1.967 3.505 0.787 3.493 1.734 3.497 1.809 2.69 2.516 2.567 3.314 3.048 2.759 2.477 2.014 1.515 1.62 2.299 1.374 1.587 2.055
3.111 2.08 2.69 1.67 3.43 0.87 2.25 1.92 2.91 3.02 3.345 2.321 2.87 2.59 1.784 2.34 2.08 3.12 0.723 1.667 0.8 2.919 2.418 2.098 3.111 2.08 2.69 1.67 3.43 1.784
2.982 2.93 3.04 1.41 3.5 1.28 3.05 2.18 3.07 2.65 3.432 3.072 3.44 3.09 2.39 2.99 3.508 3.441 1.412 2.132 2.29 3.485 3.394 1.853 1.845 1.638 2.918 3.495 3.503 2.493
* Optical density (OD) values of the unspecific reactions were subtracted from the OD of the corresponding antibody-antigen reactions. ELISA = enzyme-linked immunosorbent assay; ANDV, Andes virus; HCPS = hantavirus cardiopulmonary syndrome; ARAV, Araraquara virus; rN = recombinant nucleocapsid; ID identification; Days = number of days between serum collection and initial clinical symptoms; NA = not available. † Age is in years, except for serum 3, which was from a patient 4 months of age. ‡ The IgM serum dilution was 1:1,600 and the IgG serum dilution was 1:400.
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between each step. Serum samples were diluted 1:100 and 4-fold dilutions up to 1:6,400 in dilution buffer (5% skimmed milk powder in PBS-Tween-20) were made and dispensed into human IgM antibody–coated wells. The ANDV and ARAV rN, as well as control antigens, were diluted to a final concentration of 2 µg/mL in dilution buffer and dispensed into appropriate
wells. ANDV rabbit hyperimmune serum diluted 1:25,000 in dilution buffer, peroxidase-labeled goat anti-rabbit serum diluted 1:2,000, and ABTS substrate were also added. The cutoff value was determined as described for the IgG ELISA. Results obtained for 90 serum samples, including 60 samples from patients in Brazil with a suspected diagnosis of HCPS,
Table 2 IgM ELISA cross-reactivity of 60 serum samples from suspected patients with HCPS in Brazil using ANDV and ARAV recombinant N antigens* Patient/state of origin
1/SC 2/SC 3/SC 4/SC 5/SC 6/SC 7/SC 8/SC 9/SC 10/SC 11/DF 12/DF 13/SC 14/ES 15/SP 16/ES 17/SP 18/SP 19/SC 20/SC 21/SC 22/SC 23/SC 24/SC 25/MG 26/SC 27/MG 28/SP 29/MG 30/PR
Serum dilution
ARAV rN OD
ANDV rN OD
Result
Patient/state of origin
1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1/400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400
0.037 0.029 0.013 −0.027 0.014 0.051 0.085 0.009 0.009 0.020 0.015 0.018 0.047 0.023 0.011 0.086 0.025 0.018 0.015 0.033 0.630 0.549 0.018 0.038 0.087 0.088 0.016 0.008 0.062 0.040 0.009 0.060 0.025 0.021 0.027 0.008 0.030 0.041 0.041 0.092 0.076 0.061 0.001 0.027 0.023 0.079 0.698 0.585 0.093 0.091 0.020 0.019 0.082 0.099 0.649 0.571 0.056 0.129 0.039 0.045
0.027 0.038 0.069 0.076 0.034 0.044 0.056 0.092 0.019 0.046 0.012 0.012 0.036 0.039 0.021 0.025 0.041 0.045 0.050 0.081 0.626 0.481 0.001 0.033 0.318 0.292 0.003 0.036 0.058 0.032 0.012 0.018 0.020 0.032 0.002 0.001 0.020 0.016 0.022 0.001 0.059 0.051 0.039 0.089 0.054 0.071 1.118 1.019 0.039 0.039 0.092 0.034 0.060 0.062 0.964 0.814 0.056 0.062 0.045 0.016
–
31/MG
–
32/PR
–
33/MG
–
34/PR
–
35/MG
–
36/MG
–
37/MG
–
38/SP
–
39/SC
–
40/SC
+
41/SP
–
42/MG
+ for ANDV and – for ARAV –
43/SP
–
45/SP
–
46/MG
–
47/SP
–
48/MG
–
49/SP
–
50/PR
–
51/GO
–
52/PR
–
53/GO
+
54/SC
–
55/SP
–
56/SP
–
57/MG
+
58/MG
–
59/MG
–
60/MG
44/MG
Serum dilution
ARAV rN OD
1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400 1:100 1:400
0.085 0.091 0.023 0.029 0.041 0.091 0.111 0.004 0.724 0.925 0.817 0.585 0.032 0.108 0.558 0.595 0.074 0.081 0.055 0.092 0.006 0.013 0.052 0.030 0.059 0.036 0.228 0.189 0.001 0.004 0.106 0.057 0.003 0.029 0.060 0.037 0.055 0.023 0.059 0.049 0.052 0.013 0.041 0.117 0.038 0.018 0.033 0.046 0.015 0.016 0.260 0.085 0.028 0.006 0.033 0.020 0.043 0.012 0.009 0.033
ANDV RN OD
0.056 0.063 0.124 0.036 0.069 0.042 0.058 0.034 0.739 0.702 0.770 0.662 0.048 0.048 0.410 0.371 0.057 0.063 0.053 0.059 0.060 0.039 0.024 0.028 0.071 0.013 0.042 0.073 0.070 0.047 0.024 0.024 0.017 0.005 0.031 0.003 0.032 0.054 0.027 0.029 0.078 0.014 0.008 0.014 0.047 0.063 −0.003 0.017 0.053 0.025 0.160 0.019 0.017 0.029 0.030 0.006 0.069 0.029 0.046 0.018
Result
– – – – + + – + – – – – – – – – – – – – – – – – – – – – – –
* Optical density (OD) values of the unspecific reactions were subtracted from the OD of the corresponding antibody-antigen reactions. ELISA = enzyme-linked immunosorbent assay; HCPS = hantavirus cardiopulmonary syndrome; ANDV = Andes virus; ARAV = Araraquara virus; rN = recombinant nucleocapsid; SC = Santa Catarina Southern Region; MG = Minas Gerais Southeastern Region; PR = Parana; SP = São Paulo Southeastern Region; DF = Distrito Federal Middle West Region; ES = Espírito Santo Southeastern Region; GO = Goiás.
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MORAES FIGUEIREDO AND OTHERS
and 30 samples from patients in Argentina with a confirmed diagnosis of HCPS were used for determination of sensitivity, specificity, positive predictive value, and negative predictive value of the IgM ELISA with ARAV rN antigen. Comparisons were made with results obtained by IgM ELISA with ANDV rN antigen. RESULTS Serum samples from 18 persons in Brazil seronegative for hantaviruses were negative at a dilution of 1:400 by the IgG ELISA with either ARAV or SNV rN antigens. Conversely, serum samples from 30 persons from Argentina seropositive for hantavirus were positive at a dilution of 1:400 by the IgG ELISA with either ARAV or ANDV rN antigens. All serum samples from Argentina reacted with ARAV rN antigen, although when differences in OD values greater than 0.5 between the 2 antigens were considered, approximately the same proportion was found. Thus, 6 (10%) of 60 serum samples from patients with suspected HCPS in Brazil were positive at a dilution 1:400 by IgM ELISA with ARAV rN antigen, and 7 (11.6%) were positive by IgM ELISA for ANDV rN antigen (Table 2). All serum samples from 30 persons in Argentina previously infected by hantaviruses were positive at a dilution of 1,600 by the IgM ELISA with either ARAV or ANDV rN antigens (Table 1). OD values for both antigens varied between each other in approximately the same proportion. Results obtained for 90 serum samples analyzed by IgM ELISA with ANDV rN antigen as the gold-standard showed that the IgM ELISA with ARAV rN antigen had a sensitivity of 97.2%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 98.1%. However, one of the patients (patient 13 in Table 2) was seropositive by IgM ELISA with ANDV rN antigen, but not with ARAV rN and was later confirmed to have acute Chagas disease.
DISCUSSION The diagnosis of hantavirus infections has been routinely confirmed by the presence of antibodies to these viruses detected by ELISA. For that purpose, ideally, native hantavirus antigens should be used for higher sensitivity.4,5 However, the biosafety conditions necessary to propagate hantaviruses and their slow replication and variable yield in cell cultures make it difficult to produce large quantities of antigen for an ELISA. Therefore, recombinant protein antigens are a convenient alternative for hantavirus serologic analysis.4–6 Considering that hantavirus nucleocapsid protein elicits strong humoral immune response in infected patients, we produced a recombinant ARAV N protein in E. coli that was used for detection of IgG and IgM against hantaviruses by ELISA in serum samples from patients with suspected hantavirus infection from Brazil and Argentina. Virtually the same results were observed with 48 serum samples tested by IgG ELISA using SNV or ARAV rN antigens in Brazil and ANDV or ARAV rN antigens in Argentina. The cross-reactivity observed for ANDV and ARAV in the 30 serum samples from Argentina has been reported for many hantaviruses1,4 and is supported by the phylogenetic relationship between these two viruses.1 All American hantaviruses
are related to rodents of the sub-family Sigmodontinae, and ARAV and ANDV are similar to each other because of their geographic proximity.1 Testing of 90 serum samples from patients with suspected or confirmed HCPS enabled us to determine that the IgM ELISA using ARAV rN antigen has a high sensitivity (97.2%), specificity (100%), positive predictive value (100%), and negative predictive value (98.1%). The only divergent result occurred with a sample positive by IgM ELISA for ANDV rN antigen, which was negative for ARAV rN antigen. However, this result may have been caused by the high specificity of the ARAV rN antigen because it was later confirmed that this serum was from a patient with acute Chagas disease. It is likely that the high levels of IgM present in the serum of this patient caused the false-positive result. Our results show that SNV and ARAV rN antigens for the IgG ELISA and ANDV rN and ARAV rN antigens for the IgM ELISA are similar in sensitivity and specificity for the serologic diagnosis of hantavirus infections. These results should encourage the use of the rN of ARAV as antigen in ELISAs for detection of antibodies to hantaviruses in Brazil. Considering that little is known about the seroprevalence of hantavirus infections in different regions of Brazil, the rN protein of ARAV could also be used in serologic surveys for human and rodent samples. Our results show that ARAV rN antigen could also be used for diagnosis of hantavirus infections in Argentina. Received November 3, 2008. Accepted for publication April 28, 2009. Acknowledgment: We thank Dr. Eurico Arruda for reviewing the manuscript. Authors’ addresses: Luiz Tadeu Moraes Figueiredo, Marcos Lazaro Moreli, Alessandra Abel Borges, Glauciane Garcia de Figueiredo, and Soraya Jabur Badra, Virology Research Center, School of Medicine of the University of São Paulo in Ribeirão Preto, Av. Bandeirantes 3900, Ribeirão Preto, SP, 14049-900, Brazil. Ivani Bisordi and Akemi Suzuki, Adolfo Lutz Institute, São Paulo, SP, Brazil. Silvana Capria and Paula Padula, National Institute for Infectious Diseases Dr. Carlos G. Malbrán, Buenos Aires, Argentina.
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