Laboratory Diagnosis of Human Hantavirus Infection: Novel Insights and Future Potential

Alex Martins Machado; Glauciane Garcia de Figueiredo; Gilberto Sabino dos Santos Jr; Luiz Tadeu Moraes Figueiredo

Disclosures

Future Virology. 2009;4(4):383-389. 

In This Article

Serological Diagnosis

The detection of antihantavirus IgM and/or IgG antibodies in serum is the most common approach used for laboratory diagnosis of HCPS and HFRS. Thus, one of the first serologic tests used for diagnoses of HFRS in Europe and Asia was indirect immunofluorescence assay (IFA) using hantavirus-infected cells fixed as an antigen in glass plates. The preparation of hantavirus antigens for serologic tests is usually difficult because cell-culture infections result in low virus yield. In addition, there are biosafety precautions with regard to handling hantaviruses for concentration and purification procedures, which can only be performed in biosafety level 3 laboratories. Thus, most of the hantavirus antigens used in serologic tests have been obtained by avoiding handling of the microorganism and using rDNA methods. These antigens are mostly N-recombinant proteins (rNs), however, Gn and Gc proteins have also been produced. All the three hantavirus structural proteins (Gn, Gc and N) can induce a high level of IgM detectable at the onset of symptoms in the infected patient.[20–24] N, the most abundant viral protein, induces a strong humoral immune response in humans and rodents, based on three major epitopes of cross-reactive antigens for the Hantavirus genus. Thus, N is suitable for use as antigen in enzyme immunoassays (EIAs) for the diagnosis of hantavirus infection.[22–24] The N antigenic sites, conserved among those of the Hantavirus genus, are located in the aminoproximal region of the protein, based on studies with PUUV, Hantaan virus (HTNV) and Sin Nombre virus (SNV).[21,25–27] Furthermore, an important benefit of the application of the N antigen in immunoassays is that these tests are not difficult to standardize.

The most commonly used serological tests for hantavirus are indirect IgG and IgM ELISAs, as well as M antibody-capture ELISAs. These tests take approximately 4–6 h to be performed by trained personnel in the Brazilian public health laboratories. Recently, a rN of ARAV was obtained in Escherichia coli. To test ARAV rN as an antigen for antibody detection, sera from 30 patients from Argentina, previously known as seropositive for hantavirus, were tested and were all found to be positive for IgG and IgM by ELISA, using either ARAV or Andes vírus (ANDV) rN antigens. A total of six out of 60 serum samples from Brazilian patients with suspected HCPS (10%) were positive for IgM by ELISA using ARAV rN antigen and seven were positive using ANDV rN antigen. Considering the results obtained with these 90 sera, in terms of hantavirus antibody detection, 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 demonstrate that ARAV rN is a suitable antigen for the diagnosis of hantavirus infection in Brazil and Argentina.[23,24]

Immunoblot and neutralization tests have also been used for the serologic diagnosis of hantavirus infections.[25–28] Valdivieso et al. performed a neutralization test on ANDV and SNV in plasma samples from 20 HCPS patients from Chile and the USA using a focus-reduction neutralization assay in Vero E6 cells and found high titers of neutralizing antibodies to these viruses.[29] The authors suggest that this neutralization method could be used as a routine test for the serologic diagnosis of hantavirus infections. However, finding high titers in neutralization tests is not enough to pinpoint the infecting serotype. In theory, only a fourfold difference in neutralization test titers can make the difference. Furthermore, neutralization tests are known to be laborious and could only be performed by trained personal.

Considering that HCPS is a severe disease with a high case-fatality rate, there is a clear need for rapid diagnostic tests that are ideally simple to carry out and can be used in field conditions.[23,30,31]

Shountz et al. developed a 1 h EIA for detecting antibodies to SNV in deer mice and the test demonstrated a high specificity and sensitivity when results were compared with those of a standard EIA.[32] Navarrete et al. applied for the POC-PUUMALA™, a new immunochromatographic assay for diagnosis of the nephropathia epidemica, a mild form of HFRS that occurs in Europe.[33] This test for the detection of acute-phase infection by PUUV used a highly purified baculovirus-expressed PUUV N protein antigen, which was immobilized on the nitrocellulose membrane. Following the addition of sample (5 µl of serum, plasma or fingertip blood) and buffer, PUUV-specific IgM antibodies, if present, together with the gold-conjugated antihuman IgM goat antibody, formed a specific colored line in 5 min. POC-PUUMALA sensitivity and specificity were 97 and 90%, respectively, when compared with another commercial assay (Reagena, Finland). A similar technique, reported by Hujakka et al. demonstrated a 100% sensitivity and 99% specificity when testing unfrozen serum samples, in comparison with microcapture EIA for IgM and an IFA for IgG antibodies.[34] The same technique testing freeze-thawed serum samples demonstrated a 97.1% sensitivity and specificity. This test also has been commercialized by the Reagena company. An immunochromatographic test for the fast diagnosis of HCPS (Nanocore, Brazil) using the rN protein of ARAV produced in E. coli BL21 strain as the antigen has been tested.[23] Preliminary results with this test are encouraging since it allows the detection of, in 10–15 min, antihantavirus IgM and IgG antibodies in patient sera with high sensitivity and specificity [Moreli ML, Figueiredo LTM, Unpublished Data].

Research on the specificity of hantavirus protein epitopes is important in order to develop virus-specific serologic tests. Distinguishing between distinct hantavirus infections is not always possible with the available serologic tests. The neutralization test, the most specific of these tests, is laborious and expensive. Tischler et al. analyzed epitopes involved in serotype-specific and cross-reactive recognition of the N protein of hantavirus using monoclonal antibodies against N proteins of ANDV and SNV.[35] Schilling and colleagues have clearly shown the limits of serotyping by nonfocus reduction neutralization test methods.[36] In an outbreak of hantavirus that occurred in Germany, Schilling observed that, in most cases, serological testing identified PUUV as the causative agent. In contrast to the common view, standard methods such as ELISA, IFA and immunoblot analysis were not always able to determine the causative hantavirus species. In these cases, the testing of consecutive sera or typing of neutralizing antibodies by a focus reduction neutralization test was required to confirm the virus-specific diagnosis of PUUV infection.

In 2001, Araki et al. expressed truncated rNs of HTNV, SEOV and Dobrava virus (DOBV) in the baculovirus system.[37] The truncated rNs, which lacked 49 (rN50) or 154 (rN155) N-terminal amino acids of the N proteins of HTNV, SEOV and DOBV, were able to differentiate HTNV-, SEOV- and DOBV-specific immune sera. The cross-reactivity of these monoclonal antibodies with distinct hantavirus N proteins indicated that epitopes located within amino acids 244–286 were related to serotype specificity. Thus, rapid, sensitive and low cross-reactivity immunoassays using special epitopes of ANDV N protein have been recently developed. Lindkvist et al. identified residues involved in serospecific and cross-reactive recognition of the N proteins of PUUV, SEOV and SNV using serum samples from 17 patients having nephropathia epidemica.[25] The authors found considerable individual differences in cross-reactivity in the sera of PUUV-infected patients and also found that these reactions were in relation to four amino acid regions in the N protein: aa 14–17, 22–24, 26 and 35–38.

The immunoblot has also been used for the serologic diagnosis of hantavirus infection. This technique is more sensitive and specific than ELISA and has been used to confirm the presence of antihantavirus antibodies when ELISA results are dubious.[26] Schubert et al. evaluated two methods for diagnosis of hantavirus infection, an EIA and an immunoblot, concluding that both assays were highly sensitive for diagnosis of acute nephropathia epidemica.[27]

A new diagnostic method for hantavirus infection has been developed based on a highly dispersed immunoelectrode prototype amperometric immunosensor and has been used, in field conditions, for testing rodent sera. The test consists of a sandwich immunoassay where the naphthol, formed as result of the enzymatic reduction of naphthyl phosphate in the presence of alkaline phosphatase, is detected amperometrically. This approach combines the advantages of both a highly dispersed immunosorbent flow-through scheme of immunoassay and a highly sensitive electrochemical determination of enzyme labeling. The test does not require any pretreatment of the samples and can be performed in a short time interval (25 min). The results obtained with this new technique were compared with those obtained using the strip immunoblot assay from US CDC, demonstrating a high sensitivity.[30,31] For this analysis, the results of the CDC's ELISA were the gold standards and those samples that agreed or disagreed with the results obtained were labeled as the ELISA ‘true positives’ and ‘true negatives’, or ‘false positives’ or ‘false negatives’, respectively. The strip immunoblot assay and ELISA tests were in agreement in all cases. Out of the 96 tested samples, five were true positive, 83 were true negatives and eight were false positives.

These important tools were recently reported for the serologic diagnosis of hantavirus. However, it is necessary to further investigate these new methods in order to better evaluate their real usefulness with regard to diagnosis of hantavirus infection.

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