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Journal of Clinical Microbiology, February 2008, p. 759-762, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.02009-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Human Parechovirus Type 1, 3, 4, 5, and 6 Detection in Picornavirus Cultures{triangledown}

Michel de Vries,1 Krzysztof Pyrc,1,{dagger} Ron Berkhout,2 Wilma Vermeulen-Oost,2 Ronald Dijkman,1 Maarten F. Jebbink,1 Sylvia Bruisten,2 Ben Berkhout,1 and Lia van der Hoek1*

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands,1 Public Health Laboratory, Municipal Health Service, Amsterdam, The Netherlands2

Received 12 October 2007/ Returned for modification 26 November 2007/ Accepted 4 December 2007


   ABSTRACT
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Picornavirus cultures that could not be typed in neutralization assays were analyzed by VP1 reverse transcription-PCR (RT-PCR) and a virus discovery tool (VIDISCA). Human parechoviruses (HPeVs) were frequently identified, among which were the uncommon isolates HPeV-4, HPeV-5, and HPeV-6. The HPeV-5 isolate could be amplified only by VIDISCA and not by VP1 RT-PCR.


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Enteroviruses and human parechoviruses (HPeVs) belong to the family Picornaviridae. Infections can cause relatively mild symptoms such as diarrhea but also life-threatening diseases such as encephalitis (14). HPeVs were formerly known as echovirus 22 (HPeV-1) and echovirus 23 (HPeV-2; two strains) but were reclassified by phylogenetic analysis as a new genus (6, 8, 10, 13, 15). Additional genotypes have been identified recently (1-3, 7, 17), and one of the two strains of HPeV-2 (strain CT86-6760) was reclassified as HPeV-5 (2). The VP1 protein of enteroviruses and HPeV is the most variable and immunogenic protein. Serotyping is performed with specific antisera directed to the different enteroviruses and HPeV types. Nowadays, genotyping is used as an alternative method for typing. In genotyping, a clinical isolate with a VP1 nucleotide identity to known genotypes of higher then 75% or with an amino acid identity to known genotypes of 85% or higher is considered the same enterovirus type (11, 12).

In the study presented here, we analyzed 25 virus isolates that showed an enterovirus-like cytopathic effect (CPE) in cell culture. We used a universal enterovirus VP1 reverse transcription-PCR (RT-PCR) and a universal HPeV VP1 RT-PCR that should theoretically amplify all enteroviruses and all HPeVs (3, 9). Furthermore, we employed the VIDISCA method. VIDISCA is a virus discovery tool that can identify any RNA or DNA virus regardless of the virus family. The method uses restriction enzyme recognition sites and subsequent PCR amplification and was successfully used to identify a novel human coronavirus (16).

At the Municipal Health Service of Amsterdam, clinical samples that are sent in for virus diagnostics are routinely incubated on various cell cultures. If CPE is observed, a chloroform sensitivity test is performed to check whether the virus contains an envelope. For a nonenveloped virus, an acid stability test is performed to distinguish between rhinoviruses and other picornaviruses such as enteroviruses and HPeV. When viruses are acid stable, the isolates are tested with neutralization assays using antisera directed against poliovirus types 1 to 3; echoviruses 1 to 7, 9, 11 to 14, 20, 21, 25, 27, 29, 30, and 33; coxsackievirus B types 1 to 6; and HPeV-1 (RIVM, Bilthoven, The Netherlands). For some virus cultures these neutralization assays remain negative, although the CPE indicates the presence of a virus.

The 25 virus isolates that we analyzed could not be typed by the antiserum panel (Table 1). We first analyzed the virus isolates with the universal enterovirus and parechovirus VP1 RT-PCRs (3, 9). Twenty-three isolates were amplified with these PCRs, whereas two samples remained negative. Subsequent sequence analysis identified one echovirus 9, one echovirus 15, one echovirus 16, four echovirus 18, one echovirus 21, and five coxsackievirus type A2, A6, A7, A10, and A16 isolates. The parechoviruses included four HPeV-1 isolates, four HPeV-3 isolates, one HPeV-4 isolate, and one HPeV-6 isolate (Table 1).


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  		TABLE 1. Virus isolates typed by VP1 RT-PCR or VIDISCA

 
To determine the efficacy of VIDISCA in detecting and identifying picornaviruses, we also tested all of the isolated viruses with this virus discovery method. The VIDISCA method was performed exactly as described previously (16). Twenty-four of 25 samples yielded virus-specific DNA fragments. Cloning and sequencing confirmed the presence of viral sequences. The VIDISCA confirmed the typing by VP1 sequencing for all 23 cases (Table 1). Interestingly, there was one sample that remained negative in the VP1 RT-PCR but for which viral fragments were obtained by VIDISCA. The VIDISCA sequences displayed a high similarity with HPeV-5. Via genome walking the complete VP1 sequence was obtained, which confirmed that isolate 2000-1108 is indeed an HPeV-5 isolate (88% nucleotide identity) (GenBank accession numbers EU077510 to EU077513). We then compared the VP1 sequences of isolate 2000-1108 with those of the primers used for the VP1 RT-PCR. A mismatch close to the 3' end of one of the primers may explain the negative result in the VP1 RT-PCR (target sequence in strain 2000-1108, 5'-TCAGAATTCTTGGGGATC-3'; primer sequence, 5'-CCAAAATTCRTGGGGTTC-3').

For one sample (sample 2005-830), neither the VIDISCA method nor the VP1 RT-PCR supplied viral amplification products. We therefore tested this sample in a universal 5' untranslated region (5'UTR) RT-PCR for enterovirus and HPeV, but this assay also remained negative (data not shown).

Another sample (2005-823) was identified as HPeV-6. Since only a single full genome sequence of an HPeV-6 isolate from Japan (17) is available, full genome sequencing was performed with VIDISCA, combined with degenerate primers designed to anneal at conserved regions, and genome walking. The complete genome sequence except for the first 70 nucleotides of the 5'UTR was obtained (GenBank accession number EU077518). The HPeV-6 strain NII561-2000 and our HPeV-6 variant have identities in the coding region of 96.4% and 99.1% for nucleotides and amino acids, respectively.

The genome of HPeV-6 strain 2005-823 has the characteristic picornavirus organization, putatively encoding a single polyprotein of 2,182 amino acids that is predicted to be cleaved into 10 mature proteins by viral proteases. HPeV-6 strain 2005-823 contains a cis-acting replication element that is located in VP0, as described recently (2). Furthermore, HPeV-6 strain 2005-823 has a 28-amino-acid extension in the N terminus of VP3 and conserved motifs in the 2A protein that share homology with the H-rev-107 family of cellular proteins (5, 15). An imperfect repeat in the 3'UTR region is present, as described previously (2), which contains some nucleotide changes. The first repeat has the AUUAGACACUAAUCUG sequence, and the second has AUUGGAACACUAAUUCG.

HPeV-6 strain 2005-823 contains an RGD motif at the end of VP1 (Fig. 1). This RGD motif is critical for infectivity of HPeV-1 (2, 4, 15), and it has been suggested that this motif facilitates entry into the cell by binding to integrins. Such an RGD motif is present in all HPeVs except HPeV-3 isolates. Interestingly, an insertion of 3 amino acids is present 7 amino acids upstream of the RGD motif. This insertion is not observed in HPeV types 1, 2, 3, 4, and 5 but is present in our isolate and in the HPeV-6 isolate from Japan, which makes it unique for type 6 (Fig. 1).


Figure 1
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  		FIG. 1. Alignment of predicted amino acid sequence in the C terminus of the VP1 protein. The RGD motif is indicated by a black background. The insertion of 3 amino acids, located 7 amino acids upstream of the RGD motif, is marked with gray.

 
HPeV-6 was described recently by Watanabe et al. (17). The virus was isolated from a cerebrospinal fluid specimen from a 1-year old child with Reye syndrome with a fatal outcome. HPeV-6 was only observed in Japan in the years 2000 and 2001 and not in the years 1991 to 1999 and 2002 to 2005. Our sample was isolated in 2005 from a stool sample from a 2.5-month-old girl with a history of 9 days of fever, dehydration, bilateral otitis media, and anemia.

For all samples a neutralization assay was performed with a panel of antibodies before molecular analysis. This panel contains antibodies against the most prevalent enteroviruses and HPeV-1. For six of the viruses that we identified, antisera were present in this panel but neutralization assays remained negative. The antisera used in this assay were generated several years ago and possibly, due to evolutionary changes, do not recognize the currently circulating strains.

Since the assignment of parechoviruses as a separate genus, additional genotypes have been identified with the use of molecular biological techniques. Furthermore, phylogenetic analysis with these novel genotypes allowed the reclassification of HPeV-2 (strain CT86-6760) as HPeV-5 (2). Most of the novel genotypes have been identified in a short time period by independent groups in different continents, as with HPeV-6, described by Watanabe et al., and our variant (1-3, 7, 17). It is tempting to speculate that in addition to HPeV types 1 to 6, more HPeV types will be discovered in the near future. Within 2 years two new genotypes (HPeV-4 and HPeV-6) have been discovered (2, 3, 17). These viruses probably are not emerging viruses but were previously unrecognized. Universal RT-PCRs facilitated identification of these previously unknown genotypes. However, the sequence variation that we noticed in the primer-binding site of HPeV-5 isolate 2000-1108 illustrates that the degenerative primers that are currently in use have their limitations. With the discovery of the additional genotypes and monitoring of the sequence variation, the universal HPeV VP1 primers can be improved and redesigned, which might lead to detection of additional previously unrecognized HPeVs.

In conclusion, we successfully typed picornaviruses in 24 out of 25 cell cultures, which were previously untypeable using serology. Most interesting is the identification of HPeV-4, HPeV-5, and HPeV-6 in clinical samples. Infection with HPeV-5 and HPeV-6 has not been described previously in The Netherlands, whereas infection with HPeV-4 has been described only once (3).

Nucleotide sequence accession numbers. The sequences determined in this study have been deposited in GenBank under accession numbers EF155423, EF155422, EU077500 to EU077509, and EU077514 to EU077524.


   ACKNOWLEDGMENTS
 
We thank A. H. Brandenburg of the Public Health Laboratory, Leeuwarden, The Netherlands, for supplying three virus cultures (06-5030513, 06-5030740, and 06-5031010) and K. S. M. Benschop for HPeV VP1 primers. We also thank M. Damen of the Municipal Health Service, Amsterdam, for careful inspection of the clinical symptoms.

L.V.D.H., M.D.V., and R.D. are supported by VIDI grant 016.066.318 from the Netherlands Organization for Scientific Research.


   FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Experimental Virology, Academic Medical Center of the University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. Phone: 31-20-5667510. Fax: 31-20-6916531. E-mail: c.m.vanderhoek{at}amc.uva.nl Back

{triangledown} Published ahead of print on 12 December 2007. Back

{dagger} Current address: Microbiology Department, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland. Back


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Journal of Clinical Microbiology, February 2008, p. 759-762, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.02009-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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