Detection and typing of human pathogenic hantaviruses by real-time reverse transcription-PCR and pyrosequencing.
Infection of humans with hantaviruses induces 2 severe diseases: hemorrhagic fever with renal syndrome (HFRS)  and hantavirus cardiopulmonary syndrome (HCPS). The Old World hantaviruses Dobrava (DOBV), Puumala (PUUV), and Tula (TULV) cause HFRS in Europe (6-8), whereas Hantaan virus (HNTV) and Seoul virus (SEOV) are the most prominent HFRS pathogens in Asia (9). The New World hantaviruses Andes (ANDV) and Sin Nombre (SNV) cause HCPS in the Americas (10-12).
Infection with PUUV, the Central European DOBV lineage DOBV-Aa, and TULV varies from moderate disease to subclinical courses, and infection with the hantavirus DOBV lineage from the Balkans, DOBV-Af, or the hantavirus species SEOV, HNTV, ANDV, and SNV affects various organs and can lead to more severe and even fatal disease (6, 11, 13, 14).
Standard diagnostic methods for hantaviruses are based on serologic and immunologic techniques [for a review, see Refs. (1, 15, 16)]. These serologic assays do not take into account the viremic status of the patient, and serologic cross-reactivity between different hantaviruses hinders species differentiation. Other serotyping approaches, such as the characterization of neutralizing antibodies by focus-reduction neutralization test (17), are labor intensive, time-consuming, and require BSL3/BSL4 safety conditions.
Consequently, a specific and sensitive diagnostic method for the detection, differentiation, and quantification of hantaviruses is required to detect hantavirus in an early state of infection, before the onset of clinical symptoms. Today, real-time PCR is considered the gold standard for rapid pathogen detection (18), and several assays were recently reported to detect individual hantavirus species such as PUUV (19) and DOBV (20). A recently published real-time reverse transcription (RT)-PCR assay based on degenerated primers amplified RNA of DOBV, PUUV, HNTV, and SEOV but differentiated these hantaviruses by species-specific 5' nuclease probes (21).
Based on real-time RT-PCR, this study describes the development of 3 real-time RT-PCR assays for the specific detection of the European hantaviruses DOBV, PUUV, and TULV. In addition, 2 real-time RT-PCR assays were established for the simultaneous detection of the Asian species HNTV and SEOV or the American species ANDV and SNV in 1 reaction tube each. For each real-time RT-PCR, a pyrosequencing assay was established to enable virus typing.
Pyrosequencing is a new technique in which the enzymatic incorporation of different nucleotides that are added separately and are complementary to the sequenced DNA strand is monitored online. For each incorporated nucleotide, a light signal is generated and presented as a peak histogram, called a pyrogram. Because of the loss of signal intensity with an increased number of incorporated nucleotides, pyrosequencing has its strength in the detection of sequences of up to 80 bases and the identification of single nucleotide polymorphisms. Usually this maximal length is sufficient for the identification and typing of pathogens, as shown for the highly diverse group of papillomaviruses (22). Recently, pyrosequencing was also used for determination of HIV resistance mutations (23) or influenza virus resistance to adamantane (24). In this study, we applied pyrosequencing to generate short sequence stretches of the respective real-time RT-PCR products, allowing hantavirus species differentiation.
Materials and Methods
In total, 552 clinical specimens from suspected hantavirus infections in humans and mice sent to the German Consultant Laboratory for Hantavirus Infections (Institute of Virology, Charit6 Berlin), including serum and urine, were treated for RNA preparation as described below for cell culture supernatant. We routinely tested specimens for hantavirus-specific antibodies by ELISA and hantavirus RNA by nested RT-PCR (25). Two human acute sera of HFRS patients from Greece were kindly provided by Anna Papa (Thessaloniki, Greece).
PCR TEMPLATES: ISOLATION OF VIRAL RNA, REVERSE TRANSCRIPTION, AND PLASMID CLONING
We performed real-time RT-PCR with the hantaviruses DOBV [DOBV-Aa, strain Slovakia (DOBV/SK) and DOBV-Af, strain Slovenia (DOBV/Slo)], PUUV (strain Vranica-Hallnas), TULV (strain Moravia), HNTV (strain 76-118), and SEOV (strain 80-39). Because propagation of ANDV and SNV was not possible due to BSL4 restrictions, we used yeast cells producing recombinant nucleocapsid proteins of ANDV (strain AH-1) (26) and SNV (strain 3H226) (27) to produce virus-specific RT-PCR templates. All other hantaviruses were propagated in Vero E6 cells, and the viral titers were determined by focus assay as described (17).
Total virus RNA was isolated from 140 [micro]L virus-containing cell culture supernatant, using the QIAamp Viral Mini Kit according to the manufacturer's instructions (Qiagen). cDNA was produced by reverse transcription in a total volume of 20 [micro]L containing 4 [micro]L 1O X buffer, 0.1 [micro]L 0.1 mol/L dithiothreitol, 3 [micro]L 2.5 mmol/L deoxynucleotide triphosphate, 5.0 pmol random hexamers, 0.5 [micro]L 40 units/[micro]L RNase inhibitor, 0.5 [micro]L 200 units/[micro]L Moloney murine leukemia virus reverse transcriptase, and 10 [micro]L purified RNA. Samples were incubated at 25 [degrees]C for 10 min, 42 [degrees]C for 10 min, and 96 [degrees]C for 6 min.
For calibration of all assays, we cloned the respective PCR amplicons into a TOPO TA cloning vector according to the manufacturer's instructions (Invitrogen). After plasmid preparation, we determined the DNA concentration by use of a spectrophotometer at 260 run and calculated the corresponding copy number. Calibration curves were generated by amplification of 10-fold serial dilutions of [10.sup.5] to [10.sup.1] plasmid copies per reaction in a constant background of 1 ng/[micro]L [lambda] DNA or mouse DNA.
Degenerated oligonucleotide primers and the minor groove binder 5' nuclease probes were designed to bind to a highly conserved region within the S-segment of the hantavirus genome. The sequences, genome locations, and annealing temperatures of oligonucleotide primers and minor groove binder probes are listed in Supplemental Data Table 1, which accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue11.
Real-time RT-PCR conditions for 1-step and 2-step RT-PCR were established for the ABI 7900/7500 series (Applied Biosystems) and the LightCycler (Roche Applied Science). All reaction conditions are listed in detail in Supplemental Data Table 2.
In addition to nucleic acids of various hantavirus species, the specificity of the assays developed was evaluated with nucleic acids of several other pathogens, including influenza A and B virus, yellow fever virus, dengue fever virus, tick-borne encephalitis virus, rift valley fever virus, Marburg virus, Ebola virus, adenovirus, poxviruses, and Bacillus anthracis, as well as cDNA of mice and humans (Table 1). All of these nucleic acids had previously tested positive with specific assays (data not shown), indicating target concentrations of > [10.sup.5] per reaction.
We determined the detection limits and imprecision of each assay by use of 2-step RT-PCR, measuring 3 different concentrations of viral cDNA, reflecting high, medium, and low viral load, and in addition serial dilutions of plasmids ([10.sup.5] to [10.sup.1]), each measured in quadruplicate on the TagMan 7900 and the LightCycler. All experiments were repeated on 3 consecutive days to determine interassay variability. Linearity and calibration curves were calculated with Microsoft Excel 2003.
To determine the sensitivity of pyrosequencing,10-fold serial dilutions of the corresponding plasmids were amplified by real-time PCR and directly subjected to pyrosequencing.
We performed pyrosequencing analysis by use of a PyroMark ID System (Biotage) directly after real-time RT-PCR was finished. Real-time RT-PCR was performed with 1 biotinylated primer per assay to allow efficient single-strand DNA preparation by using streptavidin-coated Sepharose beads. The single-stranded amplicon was annealed to a specific sequencing primer that is usually different from the amplification primers. For all pyrosequencing assays, the biotinylated real-time RT-PCR primers and sequencing primers are given in Supplemental Data Table 1. In the case of ANDV, a non-extendable "blocking primer" had to be designed and included to the sequencing mix to inhibit 3'-end hairpin formation of the target by unwanted sequence generation (28). Pyrosequencing reactions were performed using Pyrogold SQA reagents (Biotage) with the complete real-time RT-PCR product according to the manufacturer's instructions.
The aim of this study was to design real-time RT-PCR assays that can specifically detect various hantavirus species but display no additional cross-reactivity among the hantavirus genus. As proven by specificity testing including triplicate measurements, none of the 5 individual assays developed for the identification of DOBV, PUUV, TULV, HNTV/SEOV, and ANDV/SNV showed nonspecific cross-reactions to other hantaviruses.
Moreover, additional specificity tests performed with further pathogens and mouse and human nucleic acids showed no cross-reaction to these targets (Table 2).
The PCR efficiency for each assay is given in Table 3. All uniplex assays displayed a dynamic detection range from [10.sup.5] to [10.sup.1] target copies, with a correlation between [r.sup.2] = 0.98 and [r.sup.2] = 0.99 when performed as 2-step real-time RT-PCR for both platforms and a detection limit of [less than or equal to] 10 copies of a plasmid containing the RT-PCR target region (see Table 3). Representative amplification curves are shown in Supplemental Data Fig. 1.
For the determination of the reproducibility (intra- and interassay variability), cDNA from clinical specimens with [C.sub.T] values between 29 and 33 were measured in quadruplicate on 3 consecutive days. For all assays (except for the HNTV/SEOV interassay variation with HNTV cDNA as template) the range of the threshold cycle ([C.sub.T]) values was <1 [C.sub.T]. The combination of the uniplex PUUV and the uniplex DOBV assay into a multiplex reaction caused no adverse effects on the reproducibility (data not shown). Table 3 summarizes typical results of the variability and efficiency testing.
To shorten the hands-on time, all reactions were also established as 1-step real-time RT-PCR. For the respective reaction conditions, see Supplemental Data Table 2.
To confirm the real-time RT-PCR results of hantavirus species-specific assays or to identify the hantavirus species in specimens that came up positive in the multiplex assays (for the detection of DOBV/PUUV, HNTV/SEOV, or ANDV/SNV), we applied the pyrosequencing technique subsequently to real-time RT-PCR. Using the pyrosequencing primers (see Supplemental Data Table 1), virus-specific sequences between 24 and 67 bases for respective hantavirus species could be generated in < 1 h after the real-time RT-PCR run. Sequence comparison allowed the explicit classification of the virus species amplified by the 3 single (DOBV, PUUV, and TULV) or the 3 multiplex (DOBV/PUUV, HNTV/SEOV, and ANDV/SNV) real-time RT-PCR assays. Fig. 1 shows a representative pyrogram of the HNTV/SEOV assay performed with cDNA of HNTV or SEOV, respectively.
We determined the sensitivity of the additional pyrosequencing step by analyzing 10-fold dilutions of the corresponding plasmids by pyrosequencing directly after amplification by real-time PCR. The best sensitivity was obtained for PUUV and SEOV, with a sequencing detection limit of 10 amplified plasmid copies per reaction. For all other viruses, sequences analyzable by comparison to sequence databases could be obtained when [greater than or equal to] 100 copies of the corresponding plasmid were amplified and subjected to pyrosequencing.
Finally, 228 clinical specimens from humans suspected to be hantavirus positive and 324 mice specimens were tested by nested RT-PCR and the new real-time RT-PCR assays in duplicate. The results are summarized in Table 2. In total, 7 of 228 (3.1%) of the human specimens tested positive in nested and real-time RT-PCR. Of the mice specimens, 10 of 324 (3.1%) tested positive in nested RT-PCR and 9 of 324 (2.8%) in real-time RT-PCR assays. Two human acute sera of HFRS patients from Greece, kindly provided by Anna Papa (Thessaloniki, Greece), were detected to be positive for DOBV, and 5 human acute sera of HFRS patients and 5 bank vole (Clethrionomys glareolus) specimens from Germany were positive for PUUV. Of the mice specimens from Slovakia, 2 striped field mice (Apodemus agrarius) and 1 yellow-necked mouse (Apodemus flavicollis) were positive for DOBV, and 1 common vole (Microtus arvalis) was positive for TULV. Quantification in human serum revealed virus loads of 320 to 5200 genome equivalents/mL, and in mouse serum, 1000 to [10.sup.7] genome equivalents/mL. All real-time RT-PCR results could be confirmed by pyrosequencing yielding analyzable sequences of up to 30 bases. Although nested PCR assays are usually expected to have the best sensitivity, our data indicate nearly the same sensitivity for the newly developed real-time RT-PCR assays. Clinical specimens infected with other hantaviruses, like ANDV or SNV, were not available.
Hantavirus diagnostic was recently based on serologic and immunologic assays such as the focus-reduction assay, Western blot, ELISA, and immunofluorescence test, which are in part commercially available. However, they can be applied only after seroconversion, can be limited in sensitivity to detect infection, and, due to the high similarity of hantavirus proteins, also can lack in the specificity of hantavirus typing (15, 29). Moreover, IgG response may take weeks to develop, and cases with a general delayed antibody response have been reported. Galeno et al. (30) reported a fatal ANDV HCPS infection with viremia before the onset of symptoms at a time when neither IgM nor IgG was detectable. Although the viremic phase seems to be short-termed, its evaluation is important for further treatments, and virus serotyping might also be important for the differentiation of HFRS from HCPS in these early stages of infection.
[FIGURE 1 OMITTED]
In contrast to the recently published hantavirus-specific RT-PCR assays, our assay is suitable for a fast screening of the major hantaviruses in Europe, Asia, and the Americas and enables rapid and exact virus typing in clinical specimens. Three specific single real-time RT-PCR assays were developed for the European hantaviruses DOBV, PUUV, and TULV and 2 real-time RT-PCR assays for the detection of (a) the Asian hantaviruses HNTV and SEOV and (b) the American hantaviruses ANDV and SNV in 1 reaction tube each.
All assays featured the major benefits of real-time PCR-speed, a very low detection limit of 10 genome equivalents per reaction, and a linear quantification range from [10.sup.5] to [10.sup.1]. Despite the pronounced diversity of the RNA genome of hantaviruses that resulted in the design of highly degenerated primers, the detection limits and the assay imprecision were nearly identical for all assays under optimized reaction conditions. In addition, the uniplex assays for the European hantaviruses PUUV and DOBV could be combined in a multiplex RT-PCR with no significant loss in sensitivity and specificity, making it a feasible tool for the rapid screening of clinical specimens in Europe. To reduce specimen handling, all real-time RT-PCR assays could be performed as 1-step RT-PCR with common reagents, resulting in comparable specificity, sensitivity, and reproducibility.
For hantavirus typing or confirmation of the real-time RT-PCR results, pyrosequencing could be performed directly after RT-PCR amplification, including the differentiation of HNTV from SEOV as well as ANDV from SNV, which is not possible by real-time RT-PCR only.
To demonstrate the usefulness of the new developed assays, we tested 228 human clinical specimens and 324 mouse specimens. Altogether 16 of 552 (2.5%) of the specimens were positive for hantavirus RNA. Two human sera specimens were positive for DOBV, 5 human sera specimens as well as 5 mouse specimens were positive for PUUV, 3 mouse specimens were positive for DOBV, and 1 mouse specimen was positive for TULV. As expected, no clinical specimens were positive for HNTV/SEOV or ANDV /SNV.
Due to the fact that species typing in hantavirus infection is not possible during the early stage of infection by serologic methods, these assays offer a detection limit that promises the identification of the respective hantavirus directly after onset of clinical symptoms. However, it should be mentioned that hantavirus RNaemia is usually short-lasting and even undetectable in certain patients (1), which is reflected by the small number of PCR-positive specimens. Therefore the real-time assays for the major hantaviruses from Europe, Asia, and America should be understood as a supplemental technique to serologic diagnostic procedures for hantaviruses.
The real-time RT-PCR assays developed are specific for the desired hantavirus species even in concentrations close to the detection limit of [less than or equal to] 10 copies per reaction. By combining the specific assays for the clinically most relevant hantaviruses in Europe, DOBV and PUUV (31, 32), an expeditious screening of clinical specimens is possible. If necessary for confirmation of the real-time RT-PCR results or for differentiation of ANDV and SNV, as well as HNTV and SEOV, rapid pyrosequencing can be applied.
Grant/funding support: This study was supported by Deutsche Forschungsgemeinschaft (Grant KR1293/2).
Financial disclosures: None declared.
Acknowledgments: We are grateful to Heike Lerch, Brita Auste, and Julia Tesch for excellent technical assistance and to Ursula Erikli for critically reading the manuscript.
Received June 12, 2007; accepted August 8, 2007. Previously published online at DOI: 10.1373/clinchem.2007.093245
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 Nonstandard abbreviations: HFRS, hemorrhagic fever with renal syndrome; HCPS, hantavirus cardiopulmonary syndrome; DOBV, Dobrava virus; PUUV, Puumala virus; TULV, Tula virus; HTNV, Hantaan virus; SEOV, Seoul virus; ANDV, Andes virus; SNV, Sin Nombre virus; RT-PCR, reverse transcription-PCR; CT, threshold cycle.
MARIT KRAMSKI, [1,2] HELGA MEISEL,  BORIS KLEMPA, [1,3] DETLEV H. KRUGER,  GEORG PAULI  and ANDREAS NITSCHE  *
 Institute of Virology, Helm ut-Ruska-Haus, Charite Campus Mitte, Berlin, Germany.
 Robert Koch-Institut, Zentrum fur Biologische Sicherheit 1, Berlin, Germany.
 Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava, Slovakia.
* Address correspondence to this author at: Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany. Fax 49-(0)30-18754-2605; e-mail firstname.lastname@example.org.
Table 1. Results of the specificity testing of the hantavirus real-time RT-PCR assays. DOBV PUUV TULV DOBV SK (a) + [empty set] [empty set] DOBV SLO (a) + [empty set] [empty set] PUUV (a) [empty set] + [empty set] TULV (a) [empty set] [empty set] + HNTV (a) [empty set] [empty set] [empty set] SEOV (a) [empty set] [empty set] [empty set] ANDV (b) [empty set] [empty set] [empty set] SNV (b) [empty set] [empty set] [empty set] HNTV/SEOV ANDV/SNV DOBV SK (a) [empty set] [empty set] DOBV SLO (a) [empty set] [empty set] PUUV (a) [empty set] [empty set] TULV (a) [empty set] [empty set] HNTV (a) + [empty set] SEOV (a) + [empty set] ANDV (b) [empty set] + SNV (b) [empty set] + No cross-reactions were observed to FLU A, (a) FLU B, (c) YFV, (a) TBEV, (a) DNV, (a) RVFV, (a) MBV, (a) EBOV, (a) VEEV, (a) ADV, (a) VAC, (a) B. anthracis, (d) human DNA/cDNA, or mouse DNA/cDNA. (a) Cell culture supernatant; (b) recombinant nucleocapsid protein produced in yeast cells; (c) clinical specimen tested positive by specific PCR; (d) bacterial colony. DOBV SK, Dobrava virus-Aa, strain Slovakia; DOBV SLO, Dobrava virus-Af, strain Slovenia; PUUV, Puumala virus, strain Vranica; TULV, Tula virus, strain Moravia; HNTV, Hantaan virus, strain 76-118; SEOV, Seoul virus, strain 80-39; FLU, influenza; YFV, yellow fever virus; TBEV, tick-borne encephalitis virus; DNV, dengue virus types 1-4; RVFV, Rift Valley fevervirus; MBV, Marburg virus; EBOV, Ebola virus; VEEV, Venezuelan equine encephalitis virus; ADV, adenovirus; VAC, vaccinia virus. Table 2. Summary of the clinical specimens. No. of samplese (a) Total Germany Slovakia Greece Human 91 135 2 228 Mouse 96 228 0 324 Nested PCR Real-time RT-PCR Positive Negative Positive Negative Human 7 221 7 221 Mouse 10 314 9 315 (a) Specimens from suspected hantavirus cases between 2004 and 2007 sent to the Consultant Laboratory for Hantaviruses, Charite Campus Mitte, Institute of Virology, and specimens from mice trapped in hantavirus-conspicuous regions. Table 3. Variability and imprecision of the hantavirus real-time RT-PCR assays. (a) Variability (b) [C.sub.T] Assay Virus Intraassay Interassay DOBV DOBV (SK) 29.98 (0.28) 30.20 (0.30) DOBV (SLO) 33.51 (0.44) 33.82 (0.33) PUUV PUUV 28.03 (0.26) 28.30 (0.50) TULV TULV 29.22 (0.20) 29.13 (0.20) HNTV/SEOV HNTV 30.49 (0.14) 31.88 (1.63) SEOV 30.37 (0.14) 30.20 (0.23) ANDV/SNV ANDV 27.03 (0.86) 26.95 (0.62) SNV 29.12 (0.45) 29.83 (0.50) Efficiency (%) RNA cDNA (c) cDNA (c) 1-step 2-step 2-step Assay RT-PCR TM RT-PCR TM RT-PCR LC DOBV 92.7 89.8 56.9 98.4 89.0 66.1 PUUV 103.9 93.5 74.4 TULV 85.1 93.9 84.8 HNTV/SEOV 85.1 89.1 126.9 57.9 94.8 97.8 ANDV/SNV 100.1 89.8 107.2 82.4 90.6 78.3 Efficiency (%) Plasmid (d) Plasmid (d) 2-step 2-step Assay RT-PCR TM RT-PCR LC DOBV 78.9 110.9 118.4 120.5 PUUV 87.6 121.7 TULV 93.9 73.2 HNTV/SEOV 90.6 126.9 94.8 96.5 ANDV/SNV 86.8 89.1 96.7 92.2 (a) Variability is stated by means of the CT, and efficiency is specified for the TagMan (TM) and the LightCycler (LC). (b) For the determination of intraassay variability, cDNA with medium viral load was measured in quadruplicate, and for interassay variability, measurements were repeated on 3 consecutive days using the TagMan platform. (c) Calculations using the slope of the calibration curves for cDNA with 3 10-fold dilution steps. (d) Calculations using the slope of the calibration curves for plasmid standard (105 to 101 plasmid copies).
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|Title Annotation:||Molecular Diagnostics and Genetics|
|Author:||Kramski, Marit; Meisel, Helga; Klempa, Boris; Kruger, Detlev H.; Pauli, Georg; Nitsche, Andreas|
|Date:||Nov 1, 2007|
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