Department of Virology, Haartman Institute, University of Helsinki, Helsinki
Department of Virology and Medicity Research Laboratory, University of Turku, Turku
Department of Medical Microbiology, University of Oulu, Oulu, Finland
A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5' untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with genus-specific digoxigenin-labeled oligonucleotide probes. The sensitivity of the multiplex RT-PCR and liquid hybridization assay was 10 to 100 picornavirus genome equivalents for representatives of each picornavirus genus. The hepatitis A virus assay exhibited a sensitivity of 10 genome copies. Both the uniplex and the multiplex tests were highly specific for the target viruses. Twenty-three clinical samples, including cerebrospinal fluid, serum, and nasopharyngeal swab specimens, were used for clinical evaluation of the multiplex RT-PCR assay. The results obtained were consistent with the results of routine virus diagnostic assays. Furthermore, the assay was used to screen 68 stool specimens for the presence of parechoviruses and Aichi virus. One sample was found to contain parechovirus RNA, whereas no Aichi virus was detected. The assay described here can be applied for the efficient identification of human enteroviruses and rhinoviruses in clinical specimens and simultaneously enables the collection of information on the epidemiology and clinical outcomes of infections caused by the currently poorly known human parechoviruses and Aichi virus.
Picornaviruses include several important human and veterinary pathogens. The large genus of human enteroviruses (HEVs; 64 serotypes) includes polioviruses, coxsackie A and B viruses, echoviruses, and enterovirus types 68 to 71. Although polioviruses will be eradicated in the near future, other HEVs remain a significant cause of morbidity and are responsible for a wide spectrum of clinical symptoms, ranging from mild respiratory infections to severe disease conditions such as myocarditis, meningitis, and encephalitis (6). Human rhinoviruses (HRVs; more than 100 serotypes) are the major cause of the common cold, and they have also been described to have a role in lower respiratory tract infections and exacerbations of asthma (11, 16). Despite the availability of vaccines, hepatitis A virus (HAV; a hepatovirus) infections still occur widely. Human parechoviruses (HPEVs), earlier included in the HEVs, currently form their own genus. They appear to cause mainly gastroenteritis and respiratory tract infections, but central nervous system infections have also been associated with these viruses (22). The antibody prevalence of HPEV type 1 (HPEV1) in the Finnish population is greater than 90%, which suggests that the infections may be mild or asymptomatic and underdiagnosed (12). The only known member of the recently established kobuvirus genus, Aichi virus (AV), has been detected in patients with gastroenteritis in Japan (25).
Traditional assays for the diagnosis of HEV, HRV, and HPEV infections rely on virus isolation, followed by neutralization typing for HEVs and HPEVs and the acid lability test for HRVs. Due to the poor growth of HAV in cell culture, diagnosis of the infection is based on the detection of virus-specific immunoglobulin M (IgM) antibodies. The laborious and time-consuming nature of virus propagation in cell culture and the increasing knowledge of the molecular biology of picornaviruses have led to development of a number of reverse transcription (RT)-PCR-based assays that enable the rapid and specific detection of small amounts of viral nucleic acids in clinical specimens (3, 5, 9, 10, 12, 13, 19, 26). There are, however, limitations to the use of PCR in diagnostic virology, such as the relatively high costs, the lack of availability of adequate samples, as well as the narrow diagnostic coverage of the assays. To overcome these limitations, multiplex RT-PCR methods have been developed in which the addition of several primer pairs enables the simultaneous amplification of multiple viral targets in one PCR test (1, 2, 7, 18).
We describe here a qualitative test based on a nonnested multiplex RT-PCR and liquid hybridization reactions for the simultaneous identification of HEVs, HRVs, HPEVs, and AV. In addition, a separate assay for the detection of HAV was developed, thus complementing the test pattern so that all human picornaviruses are covered. The multiplex RT-PCR assay enables the combination of routine diagnostics for HEVs and HRVs with the collection of data on the rates of appearance and the clinical outcomes of HPEV and AV infections.
MATERIALS AND METHODS
cDNA clones and production of viral RNAs. The AV cDNA clone (strain A844/88) was a gift from T. Yamashita (Aichi Prefectural Institute of Public Health, Aichi, Japan), HAV cDNA (strain HM-175/7) was kindly provided by R. H. Purcell (Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Md.), the HPEV1 Harris strain and HRV1B cDNA clones were from G. Stanway (Department of Biological Sciences, University of Essex, Colchester, United Kingdom), and the echovirus 11 (EV11; Gregory strain) cDNA has been described by Dahllund et al. (4).
For production of viral RNAs, recombinant plasmids containing the full-length cDNA copies of the viral genomes were linearized with appropriate restriction endonucleases (New England Biolabs) and transcribed with RNA polymerase (T7 polymerase for AV, HPEV1, HRV1B, and EV11 and SP6 polymerase for HAV; Promega), according to the instructions of the manufacturer, for 60 min at 37°C. The template plasmids were degraded by incubation with DNase (Promega) for 15 min at 37°C, and the RNAs were purified with an RNeasy Mini kit (Qiagen) and analyzed by agarose gel electrophoresis. The RNA concentrations were determined by measuring the optical density at 260 nm.
Oligonucleotides. The primers used for RT and PCR amplification were designed from conserved regions of the 5' untranslated regions of the viral genomes. The sequences, map positions, and orientations of the primers are shown in Table 1. The sense primer of each pair was biotinylated at the 5' end during synthesis, and the probes used in liquid hybridization were labeled at the 5' end with a Digoxigenin Oligonucleotide Tailing kit (Roche).
Separate RT reactions. The reaction mixture (total volume, 20 μl) contained the following components: 1.25 μM primer, 1 mM deoxynucleoside triphosphates (dNTPs; Amersham Biosciences), 50 mM Tris-HCl (pH 8.4), 40 mM KCl, 5 mM MgCl2, 0.5% Tween, 20 U of RNasin RNase inhibitor (Promega), and 50 U of Expand reverse transcriptase (Roche). Prior to the reaction, template RNA, dNTPs, and the negative-strand primer were denatured at 85°C for 3 min and cooled on ice, and the other reaction components were added. RT was carried out at 42°C for 60 min.
Multiplex RT reactions. The reaction mixture consisted of 1.25 μM each primer, 1 mM dNTPs (Amersham Biosciences), 50 mM Tris-acetate (pH 8.4), 75 mM potassium acetate, 8 mM magnesium acetate, 10 mM dithiothreitol, 20 U of RNase OUT (Invitrogen), and 15 U of ThermoScript RT (Invitrogen) in a total volume of 20 μl. Template RNA, dNTPs, and negative-strand primers were treated as described above. The reaction took place at 65°C for 60 min, followed by a 5-min termination step at 85°C.
PCR amplification. The PCR mixture contained single or multiple primer pairs at concentrations of 0.5 μM, 200 μM dNTPs (Amersham Biosciences), 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 7.5 U of Ampli Taq Gold polymerase (Roche). Ten microliters of the RT reaction product was added to the PCR mixture (total volume, 100 μl). PCR was performed by using a touchdown format. After primary denaturation at 95°C for 7 min, the cycling conditions were the following: denaturation at 94°C for 40 s; annealing for 40 s, starting from 63°C followed by a 1°C decrease in temperature per cycle; and elongation at 72°C for 40 s. These steps were repeated eight times, and thereafter, 45 additional cycles were conducted by using the latter cycling conditions, albeit with annealing at 54°C. The amplification products were analyzed by agarose gel electrophoresis.
Liquid hybridization. The liquid hybridization procedure was performed in streptavidin-coated wells (Labsystems) with one probe at a time. Ten microliters of the amplification product was added to the wells containing 40 μl of binding buffer (25 mM Tris-HCl [pH 7.5], 125 mM NaCl, 5 mM EDTA, 0.1% Tween 20, 0.5x Denhardt's solution). The plates were incubated at 22°C for 30 min with agitation (650 rpm), 50 μl of elution buffer (100 mM NaOH, 300 mM NaCl) was added, and incubation was continued for 1 min. After the plate was washed with buffer 1 (0.25 M Tris-HCl [pH 7.5], 1.25 M NaCl, 20 mM MgCl2, 3% Tween 20), 50 μl of hybridization buffer (0.1% sodium dodecyl sulfate, 5x SSC [1x is 0.15 M NaCl plus 0.015 M sodium citrate], 1x Denhardt's solution) containing 6.7 fmol of the appropriate probe was added. Hybridization was performed at 42°C for 30 min with agitation (650 rpm). Unbound probe was removed by washing six times with buffer 2 (0.05x SSC, 0.3% Tween 20), and then 50 μl of conjugation buffer (25 mM Tris-HCl [pH 7.5], 125 mM NaCl, 2 mM MgCl2, 0.3% Tween 20, 1% bovine serum albumin) containing 5 mU of anti-digoxigenin-alkaline phosphatase conjugate (Roche) was added and the plates were incubated at 22°C for 30 min. After the plates were washed six times with buffer 1, 50 μl of Lumiphos 538 substrate (Lumigen Inc.) was added and the plates were incubated for 35 min at room temperature with protection from light. Luminescence was measured with a Labsystems Luminoskan luminometer. The liquid hybridization assay was done in separate wells for each probe and in duplicate for all amplification products.
Clinical specimens. Sixty-eight stool specimens sent to the Department of Virology at the University of Turku for routine virus diagnostic assays during a 12-month period (from January to December 2001) were screened for the presence of HPEVs and AV. In addition, five cerebrospinal fluid (CSF) samples and one serum sample found to be positive for HEVs by routine virus diagnostic assays at the Helsinki University Hospital between January and December 2001 were analyzed, and five nasopharyngeal samples shown to be positive for HRVs were also tested. Furthermore, three serum samples and nine CSF samples negative for HEVs and HRVs were included in the analysis. Nucleic acids from all the samples were isolated by phenol-chloroform extraction and were stored at –70°C until they were tested. Analysis of CSF and nasopharyngeal samples was repeated one to two times in separate RT-PCR assays, depending on the volume of the sample, and each amplification product was detected in two parallel liquid hybridization reactions.
Separate RT-PCR assays. The pair of RT-PCR primers (primer 3B+ and primer 4–; Table 1) used has previously been described by Lnnrot et al. (15) to detect at least 30 different enterovirus serotypes and 9 different rhinovirus serotypes. The sensitivity of the amplification reaction (amplicon length, 116 bp) was 10 genome equivalents of both EV11 RNA and HRV1B RNA (Fig. 1A). A sensitivity of 10 genome equivalents was also obtained for the HAV RT-PCR with primers Hav1B+ and Hav1–, which produced a 206-bp amplicon (data not shown). For HPEVs, several RT-PCR primers were designed from different parts of the 5' untranslated region and tested in different combinations to obtain optimal sensitivity. The highest sensitivity, 100 genome equivalents, was achieved by using primers Hpev1B+ and Hpev1–, which gave rise to a 253-bp amplicon (Fig. 1A). Of the four primers designed for AV RNA amplification, the combination of primers Aichi1B+ and Aichi1- was shown to be the most sensitive one, producing a 260-bp amplicon when 100 genome equivalents of RNA was used as the template. All these uniplex RT-PCRs resulted in the specific amplification products with the expected sizes.
Multiplex RT-PCR. Combining of the separate RT-PCR tests to obtain a multiplex assay was complicated due to apparent interactions of the primers at 42°C, which is the optimal temperature for the reverse transcriptases commonly used in RT-reactions. Therefore, use of a thermostable reverse transcriptase was necessary to minimize the cross-hybridization of the antisense primers in the multiplex RT reaction. An increase of the temperature to 65°C enabled simultaneous RT-PCR amplification of all the other viral target RNAs but not HAV RNA, and successful performance of the multiplex RT reaction therefore required exclusion of the HAV-specific primer. In contrast to the RT reaction, the conditions of the PCR did not require additional optimization when the separate amplification reactions for HEVs, HRVs, HPEVs, and AV were combined.
For HEVs and HRVs, the multiplex RT-PCR resulted in specific amplification products in agarose gel electrophoresis when 100 genome equivalents of RNA was used as the template (Fig. 1B). Also, amplicons corresponding to 100 HPEV and AV genomes were visible on the agarose gels. Although the intensities of the multiplex RT-PCR amplicons obtained agarose gel electrophoresis were lower than those obtained by the uniplex RT-PCRs, 100 genome equivalents of target RNAs were detected in repeated experiments. Simultaneous amplification of HEVs, HRVs, and HPEVs, picornaviruses found in respiratory samples, by multiplex RT-PCR with EV11, HRV1B, and HPEV1 RNAs as templates, resulted in sensitivities of 100 to 1,000 genome equivalents (data not shown). Amplification of HEVs, HPEVs, and AV, viruses present in stool specimens, simultaneously in a multiplex RT-PCR enabled detection of amplicons corresponding to 1,000 genome copies of HEVs and HPEVs and 100 genome copies of AV. When several virus RNAs were amplified simultaneously in a multiplex RT-PCR, the sensitivity of the assay could be improved by raising the MgCl2 concentration in the PCR mixture to 3.5 mM (data not shown). However, the elevated MgCl2 concentration had an inhibitory effect on the amplification of individual RNAs, and therefore, a concentration of 1.5 mM was used in all reaction mixtures.
Liquid hybridization. Of the several probes designed for the detection of HEVs, HRVs, HPEVs, AV, and HAV, the ones shown in Table 1 were specific in the liquid hybridization reactions following both the uniplex RT-PCR and the multiplex RT-PCRs. When the other probes were evaluated, problems concerning the specificity and/or the sensitivity of liquid hybridization occurred. In the hybridization reactions for the detection of HEV and HRV amplicons from uniplex RT-PCRs, the products representing 10 genome equivalents of RNA template gave a luminescence signal (Fig. 2). Although the luminescence signals from the hybridization reactions decreased when amplicons from the multiplex RT-PCR were used, HEV and HRV amplicons corresponding to 10 genome equivalents of viral RNA could be detected (Fig. 3A). In repeated experiments, the sensitivity of HPEV and AV amplicon detection varied from 10 to 100 genome equivalents by using both the uniplex and the multiplex RT-PCRs. However, the luminescence signals were decreased when the amplification products from the multiplex RT-PCR were detected (Fig. 2 and 3A). For HAV detection, only amplicons from the uniplex RT-PCRs were used in liquid hybridization. The sensitivity of the assay was 10 genome equivalents (Fig. 2). When the multiplex RT-PCR followed by the liquid hybridization reaction was used to simultaneously detect the representatives of respiratory picornaviruses, HEVs, HRVs, and HPEVs, the sensitivity of liquid hybridization decreased, allowing detection of 1,000, 10, and 1,000 genome equivalents of viral RNA, respectively (Fig. 3B). In the case of simultaneous amplification of the picornaviruses present in stool samples, HEVs, HPEVs, and AV, the hybridization assay had sensitivities of 1,000, 1,000, and 10 genome equivalents, respectively. The luminescence signal in duplicate liquid hybridization reactions, performed for all the amplification products, varied from 0.5 to 9.7% of the mean value when 10 to 1,000 target molecules were detected (data not shown).
Analysis of clinical specimens. All five CSF samples found to be positive by routine diagnostic assays for HEVs were also positive by the multiplex RT-PCR and the liquid hybridization assay (Table 2). Similarly, one serum sample known to be positive for HEVs gave a positive result by the multiplex assay. Furthermore, the five HRV-positive nasopharyngeal samples were positive by the multiplex assay. The three serum samples and nine CSF samples previously negative for HEVs and HRVs remained negative when they were analyzed by the multiplex RT-PCR and liquid hybridization assay (Table 2). The luminescence signals obtained with the positive clinical samples by the multiplex assay varied from 630 to 2,350 relative luminescence units (RLU), whereas the values obtained for the negative specimens were less than 4 RLU. Variations of 0.2 to 23.6% of the mean value were observed in the signals for the clinical samples when amplicons from one RT-PCR were tested in two parallel hybridization reactions (data not shown).
Of the 68 stool specimens screened for the presence of HPEVs and AV, one sample from a 1-month-old baby was found to be positive for HPEV by the uniplex RT-PCR and liquid hybridization. The sample was also positive when it was analyzed by the multiplex RT-PCR followed by liquid hybridization. No AV was found in any of the stool specimens. The multiplex RT-PCR was specific for the analysis of clinical specimens, as unspecific amplification was observed by agarose gel electrophoresis only in the case of stool specimens. Furthermore, the luminescence signals from liquid hybridization were also specific, with the mean background of luminescence with the different probes ranging from 1.0 to 9.7 RLU for all except three samples, which were positive for either HEVs (one CSF sample) or HRVs (two respiratory samples) and for which signals in the range of 45 to 63 RLU with the HPEV-specific probe were observed. Whether these findings represent real double infections or unspecific reactivity is unknown.
Identification of picornaviruses by laborious and time-consuming virus propagation in cell culture has largely been replaced by detection of viral nucleic acids by RT-PCR assays. The use of RT-PCR for the detection of HEVs and HRVs from clinical samples has been described in several studies, and the superior sensitivity of RT-PCR compared to that of virus cultivation is well documented (5, 8, 9, 13, 19, 23, 24). RT-PCR tests have also been developed for the detection of HAV in clinical, food, and environmental samples (3, 17, 21). More recently, RT-PCR has also been applied for the detection of HPEVs (12, 14) and AV (26) in clinical specimens. Multiplex RT-PCR assays for the detection of picornaviruses have also been reported; and the simultaneous detection of HEVs, HPEVs, and human herpesviruses in CSF samples has been of particular interest (2, 18). Multiplex RT-PCR assays that detect HRVs and other respiratory pathogens in clinical samples have also been described (1, 7).
At present, the major need in the clinical diagnosis of human picornavirus infections is the ability to detect HEVs (and possibly also HPEVs) in CSF samples and HEVs, HPEVs, and HRVs in respiratory specimens. Moreover, epidemiological studies also require assays for the identification of HEVs, HPEVs, HAV, and AV in stool samples. The need for the specific diagnosis of picornavirus infection will probably increase in the near future due to the development of agents with activities against picornaviruses (20). Optimally, detection of picornavirus RNA from CSF and nasopharyngeal specimens and collection of epidemiological data on the infections could be performed by one multiplex RT-PCR assay.
The qualitative multiplex RT-PCR designed in this study enables the simultaneous amplification of sequences from the HEV, HRV, HPEV, and AV genomes. Identification of the amplicons is performed by liquid hybridization reactions with genus-specific oligonucleotide probes. The capability of this assay to replace the currently used RT-PCR test for the detection of HEVs and HRVs in clinical samples was considered throughout the development process, and the assay design was based on the currently available laboratory facilities. The sensitivities of the multiplex test in the case of HEVs and HRVs corresponded to those of the uniplex RT-PCRs, as 10 genome equivalents of RNA could be detected when liquid hybridization was performed after multiplex RT-PCR. The sensitivity of HPEV and AV detection remained at the level of 10 to 100 genome equivalents when the amplicons from the multiplex RT-PCR were used. This can be considered satisfactory, especially when these viruses are mostly detected in stool specimens, in which the number of viral particles tends to be high. Combining the separate RT-PCRs seemed to have no significant effect on the sensitivity of the hybridization, as amplification products from the multiplex RT-PCRs barely perceptible to the eye by agarose gel electrophoresis could be reliably detected by liquid hybridization. Therefore, the decrease in the luminescence signal from the multiplex RT-PCR amplicons may be considered to result from the lower amplification efficiencies of the target sequences in the multiplex RT-PCRs. When the effect of coinfection was studied, amplification of three viral target sequences (HEV, HRV, and HPEV or HEV, HPEV, and AV) simultaneously in the multiplex RT-PCR was shown to decrease the sensitivity, as would be expected. Only relatively small variations in the luminescence signals were observed in duplicate liquid hybridization reactions performed with the amplification products. Furthermore, no cross-reactions of the primer pairs and probes used for the identification of different human picornavirus genera were detected, indicating the high degree of specificity of the assay. Although amplification of HAV RNA was not possible by the multiplex RT-PCR due to the apparent interactions of the primers, the separate HAV RT-PCR and liquid hybridization assay exhibited a high sensitivity and a high specificity for the detection of HAV RNA.
The assay was used to analyze clinical specimens, including 14 CSF specimens, 4 serum specimens, 5 nasopharyngeal specimens, and 68 stool specimens. For the CSF, serum, and nasopharyngeal specimens, the results obtained by our multiplex assay were consistent with the results obtained when the samples were previously tested by routine diagnostic assays. Variations in the luminescence signals were observed when the amplification products from separate RT-PCRs were tested by hybridization. As no internal controls are included in the assay, it is not known whether the variation in the luminescence signal results from sample handling, RT-PCR, or liquid hybridization. When stool specimens from 68 patients, aged 3 days to 86 years, were analyzed for the presence of HPEVs and AV, a sample from a 1-month-old baby was found to be positive for HPEVs, whereas no AV was detected. The method developed in the present study meets the current need to detect HEVs and HRVs in clinical specimens while simultaneously enabling the collection of information on the epidemiology and clinical outcomes of infections caused by the currently poorly known human picornaviruses, HPEVs and AV. Moreover, the collection of assays described here covers the genus-specific molecular biology-based diagnosis of all picornaviruses known to cause infections in humans.
We thank R. Vainionp and H. Piiparinen for providing us with the clinical specimens. Birgitta Grekula is acknowledged for technical assistance; and R. H. Purcell, G. Stanway, and T. Yamashita are acknowledged for providing us with the viral cDNAs.
The study was supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, the Instrumentarium Research Foundation, and the Finnish Cultural Foundation.
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