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1 Enterovirus Laboratory, Department of Viral Diseases and Immunology, National Public Health Institute (KTL), Mannerheimintie 166, FIN-00300 Helsinki, Finland
2 Swedish Institute for Infectious Disease Control, SE-17182 Solna, Sweden
Correspondence
Merja Roivainen
Merja.Roivainen{at}ktl.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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v
3 receptor and decay accelerating factor) were not successful. Seroprevalence studies in the Finnish population revealed a high prevalence of this virus over the past two decades. The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are DQ916376–DQ916379 and EF107097–EF107098.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The primary site of enterovirus infection is the mucosal tissue of the respiratory or gastrointestinal tract. After spreading through the lymphatic system and circulation, the virus can infect secondary target tissues. The secondary replication sites largely define the clinical manifestations of a given enterovirus strain. In the intestinal mucosa, virus replication can continue for several weeks, during which time progeny virus is shed into faeces.
The enterovirus genome is a single-stranded RNA molecule of approximately 7500 nt consisting of a single open reading frame flanked by non-coding 5' and 3' regions. The 5' UTR contains an internal ribosome-binding site, which is essential for translation initiation (Pelletier & Sonenberg, 1988; Molla et al., 1992; Chen & Sarnow, 1995). The 3' UTR forms highly conserved secondary and tertiary structures that are thought to be important in replication initiation (Pilipenko et al., 1992, 1996; Mirmomeni et al., 1997). The open reading frame is translated into a single, large polypeptide, which is subsequently cleaved by viral proteases (reviewed by Palmenberg, 1990). The polypeptide is divided into three domains, P1 to P3, consisting of three to four proteins each. The P1 region contains viral capsid proteins VP1 to VP4, whilst P2 and P3 contain the non-structural proteins.
Enteroviruses were originally classified by their antigenic and pathogenic properties in humans and mice. As phylogenetic methods to classify enteroviruses became available, it became apparent that pathogenic properties were not sufficient to classify the evolutionarily related viruses into correct groups. Using molecular properties, human enteroviruses have been classified into five species, Human enterovirus A (HEV-A), HEV-B, HEV-C, HEV-D and Poliovirus (Stanway et al., 2005). Polioviruses are genetically related to HEV-C and will probably be reclassified into this species (Brown et al., 2003).
Within an enterovirus species, sequence divergence is greatest in the capsid protein (VP1)-coding region of the virus genome, and classifications based on sequence variation in this region correlate completely with the classification made using antigenic properties (Oberste et al., 1999, 2000). It has been suggested that enteroviruses should be classified in the same serotype if they have >75 % nucleotide similarity in the VP1-coding sequence (>85 % amino acid sequence similarity) and into different serotypes if they have <70 % nucleotide similarity (<85 % amino acid similarity). Molecular typing of serologically non-typable strains has led to the discovery of a large number of new enterovirus types (Oberste et al., 2001, 2004c, 2005; Norder et al., 2003).
Detection of circulating wild-type poliovirus strains has become more and more important as the World Health Organization (WHO)-coordinated global poliomyelitis eradication initiative is approaching its goal (Hovi, 2006). Poliomyelitis typically presents itself as a rapidly progressing, usually unilateral AFP, often with fever and residual paralysis at day 60 after onset. Along with standard clinical case-driven surveillance of AFP patients, environmental surveillance of sewage waters has been adopted as a supplementary method for the detection of poliovirus circulation in some countries, including Egypt. Samples are collected from water bodies contaminated with human faeces, concentrated and analysed using standard methods (El-Bassioni et al., 2003; Hovi et al., 2005). During environmental surveillance, non-polio enteroviruses are also found regularly.
In this study, we have reported the isolation and characterization of several strains of a proposed new serotype strain of HEV-D from an AFP case in the Democratic Republic of the Congo and from waste water in Egypt. Only two other serotypes have been found previously in the HEV-D species, EV68 and EV70. The new serotype was found to have an unusually wide tissue tropism and host species range in vitro. Seroprevalence studies in the Finnish population revealed a high prevalence of this virus over past two decades.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were provided by the WHO Polio Labnet. The Ohio strain of HeLa cells was kindly provided by Eurico Arruda (University of Virginia, Charlottesville, USA). The green monkey kidney cell lines GMK and Vero have been maintained in our laboratory since the 1960s. Human colorectal adenocarcinoma (CaCo-2), human lung carcinoma (A549), human larynx epidermoid carcinoma (Hep-2C), mouse embryo fibroblast (3T3), hamster kidney (BHK21), rabbit kidney (RK13), canine kidney (MDCK) and bovine kidney (MDBK) cell lines were purchased from ATCC. Human neuroblastoma (SK-N-SH) cells were kindly provided by Antti Vaheri (University of Helsinki, Finland). Human hepatocellular carcinoma (HUH-7) cells were kindly provided by Darius Moradpou (University Hospital Freiberg, Germany). Chinese hamster ovary (CHO) cells expressing human coxsackie- and adenovirus receptor (HCAR) were kindly provided by Jeffrey Bergelson (Children's Hospital of Philadelphia, USA). Mouse fibroblast M4 (LPR–/–, LDLR–/–) cells (Reithmayer et al., 2002) and intracellular adhesion molecule (ICAM-1)-expressing M4 cells were kindly provided by Dieter Blaas (Medical University of Vienna, Austria).
Isolation of viruses.
Collection of a 1 l sewage sample and concentration using a two-phase separation method were performed as described previously (El Bassioni et al., 2003; Hovi et al., 2005). Virus isolation was carried out in RD and L20B cell lines as described by Hovi et al. (2005). Virus strains producing cytopathic effect (CPE) in both cell lines were subjected to neutralization with polyclonal antisera specific for poliovirus types PV1, PV2 and PV3 (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) according to the protocol recommended by WHO. Virus strains escaping neutralization with PV-specific antisera were passaged once in L20B cells before molecular typing. The isolation and sequencing of viruses from stool samples has been described elsewhere (Junttila et al., 2007).
Partial VP1 RT-PCR and sequencing.
Viral RNA was extracted from 100 µl infected cell cultures with an RNeasy Total RNA kit (Qiagen) according to the manufacturer's instructions. RT-PCR using primers 292 and 222 (nt 2612–2627 and 2969–2951 relative to the genome of PV1-Mahoney) was carried out as described by Oberste et al. (2003). PCR amplicons were purified with the QIAquick gel extraction kit (Qiagen) and used as templates in cycle sequencing (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit, version 1.1; Applied Biosystems) in an automated sequencer (model 310; Applied Biosystems). Electropherograms were analysed with Vector NTI Advance 10.1 (Invitrogen) and the partial VP1 sequences obtained were compared with sequences available in GenBank using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). Virus isolates showing <70 % nucleotide sequence identity with known enterovirus serotypes were subjected to further analysis.
Plaque purification of environmental virus strains.
Viruses were purified using a plaque assay in monolayer cultures of RD cells and passaged once in RD cells at 36 °C, freeze–thawed three times and clarified by centrifugation at 250 g for 10 min. All subsequent experiments were performed using these plaque-purified virus preparations.
Full-length genome sequencing.
Total RNA was extracted from cells infected with virus strains using Trizol reagent (Gibco-BRL Life Technologies) according to the manufacturer's instructions. A SuperScript First-strand Synthesis System for RT-PCR kit (Gibco-BRL Life Technologies) was used for cDNA synthesis and a SuperScript One-step RT-PCR for Long Templates kit was used for PCRs. PCR products were purified using a QIAEXII agarose gel extraction kit (Qiagen). Complete or partial genomes of the virus isolates were sequenced using a primer-walking strategy.
Phylogenetic analysis.
Nucleotide and amino acid sequences were assembled and compared using the programs CONTIGEXPRESS and ALIGNX (Vector NTI Advance 10.1; Invitrogen). The sequences were aligned using CLUSTAL X (version 1.81) (Thompson et al., 1997). Phylogenetic trees were produced using the maximum-likelihood method implemented in TREE-PUZZLE version 5.2 (Schmidt et al., 2002) and the neighbour-joining method implemented in MEGA version 3.1 (Kumar et al., 2004). Bootstrap analysis was performed using 1000 replicates. The transition–transversion rate was estimated from the data and the TN93 model of substitution (Tamura & Nei, 1993) was used to calculate distances. Phylogenetic trees were visualized using MEGA version 3.1. The SIMPLOT 2.5 program was used for similarity plot and bootscanning analyses (Lole et al., 1999). For similarity plot analysis, a 200 nt window was moved in 20 nt steps and Jukes–Cantor correction was used. Bootscanning analysis (Salminen et al., 1995) was run with the neighbour-joining algorithm and 100 pseudoreplicates.
GenBank accession numbers.
The following sequences were obtained from GenBank: HEV-D serotype EV68 strains EV68-Fermon (AY426531
[GenBank]
), EV68-TX03 (AY426500
[GenBank]
), EV68-MD99 (AY426499
[GenBank]
), EV68-TX99 (AY426498
[GenBank]
), EV68-MN98 (AY426497
[GenBank]
), EV68-TX02-2 (AY426496
[GenBank]
), EV68-TX02-1 (AY426495
[GenBank]
), EV68-WI00 (AY426494
[GenBank]
), EV68-MO00 (AY426493
[GenBank]
), EV68-MD02-2 (AY426492
[GenBank]
), EV68-MD02-1 (AY426491
[GenBank]
), EV68-NY93 (AY426490
[GenBank]
), EV68-MN89 (AY426489
[GenBank]
), EV68-CA62-3 (AY426488
[GenBank]
), EV68-CA62-2 (AY426487
[GenBank]
) and EV68-CA62-1 (AY426486
[GenBank]
); HRV87-Corn (AY355268
[GenBank]
); serotype EV70 strains EV70-J670/71 (NC_001430
[GenBank]
), EV70-ENG/71 (D17595
[GenBank]
), EV70-FB/73 (D17596
[GenBank]
), EV70-G10/72 (D17597
[GenBank]
), EV70-HP185/81 (D17598
[GenBank]
), EV70-HP85/78 (D17599
[GenBank]
), EV70-I/72 (D17600
[GenBank]
), EV70-M51/76 (D17603
[GenBank]
), EV70-M8/72 (D17604
[GenBank]
), EV70-R20/71 (D17605
[GenBank]
), EV70-R6/71 (D17606
[GenBank]
), EV70-SEC32/71 (D17607
[GenBank]
), EV70-T260/74 (D17608
[GenBank]
), EV70-T62/73 (D17609
[GenBank]
), EV70-TW266/81 (D17610
[GenBank]
) and EV70-V1250/81 (D17611
[GenBank]
); HEV-A serotypes CVA8 strain Donovan (AY421766
[GenBank]
), CVA16 strain G-10 (U05876
[GenBank]
) and enterovirus 71 strain BrCr (U22521
[GenBank]
); HEV-B serotypes ECHO5 strain Noyce (AF083069
[GenBank]
), CVB3 strain Nancy (M16572
[GenBank]
) and CVA9 strain Griggs (D00627
[GenBank]
); HEV-C serotypes CVA11 strain Belgium-1 (AF499636
[GenBank]
) and CVA20 strain IH35 (AF499642
[GenBank]
), poliovirus 2 strain Lansing (M12197
[GenBank]
) and porcine enterovirus 8 (PEV8) strain V13 (AF406813
[GenBank]
).
Virus replication in different cell lines.
Confluent cell cultures in 24-well plates were infected with EV94-E210 at an apparently high m.o.i. After 30 min of adsorption at room temperature and a 30 min internalization period at 36 °C in 5 % CO2, the remaining inoculated virus was removed and cells were washed twice with Hanks' balanced salts solution supplemented with 20 mM HEPES (pH 7.4), and growth medium (Eagle's minimum essential medium supplemented with 20 mM HEPES, 20 mM MgCl, 1 % FBS and penicillin/streptomycin solution) was added to all cultures. Incubation was continued at 36 °C in a 5 % CO2 atmosphere.
For virus titration, cell cultures were harvested at different intervals and freeze-thawed three times. The total infectivity of each sample was subsequently determined by end-point titration in microwell cultures of the corresponding cell line or in RD cells for detection of possible replication in non-human cell lines. CPE was determined on day 6 after infection by microscopy and TCID50 titres were calculated using the Kärber formula (Lennette, 1969).
Human sera.
The study group for the serum neutralization assay consisted of 181 women at the end of the first trimester of pregnancy. The sera were derived from the Finnish Maternity Cohort and had been collected in 1983 (n=86) and 2002 (n=95).
Neutralization assay.
Aliquots of sera were inactivated at 56 °C for 20 min and stored at 4 °C. Serial 4-fold dilutions of sera were mixed with an equal volume of virus (100 TCID50). The mixtures were neutralized at 36 °C for 1 h and then overnight at 4 °C. The mixtures were transferred into 96-well cell culture plates with RD cells (
3x105 cells ml–1) and incubated at 36 °C in 5 % CO2 for 6 days. The highest dilution that completely inhibited viral CPE was taken as the end-point titre of the serum.
CPE protection assay.
Monoclonal antibodies to the second and third short consensus repeats of decay accelerating factor (DAF) were kindly provided by Douglas Lublin (Washington University School of Medicine, St Louis, USA). Polyclonal rabbit antisera to the vitronectin v3 receptor and to the poliovirus receptor (PVR) were produced in house (Ylipaasto et al., 2004). The antibodies were added to confluent 96-well cell culture plate monolayers in volumes of 30 µl and incubated for 2 h at 36 °C in 5 % CO2, after which pre-titrated virus (10 000 TCID50 per well) was administered. The mixture was incubated at 36 °C in 5 % CO2 for 20 h and the level of protection was examined by light microscopy.
Assay for acid sensitivity.
The acid sensitivities of environmental EV94 strains were tested using a standard protocol (Couch, 1992). Human rhinovirus 2 (HRV2) and PV1 Sabin strain were included as acid-sensitive and acid-insensitive control viruses, respectively. Briefly, an equal volume of buffer A [0.1 M citric acid buffer (pH 4.0) or 0.1 M phosphate buffer (pH 7.0)] was added to a virus-infected cell culture supernatant. The mixtures were incubated for 1 h at 36 °C and then neutralized using 0.5 M phosphate buffer (pH 7.2). The infectivities of the acid-treated and untreated viruses were assayed in RD cells in 96-well microplates at 36 °C in 5 % CO2. CPE was examined daily and end-point titres were calculated using the Kärber formula on day 7.
Immunofluorescence assay of virus-infected cells.
Infected cells were fixed with cold methanol for 15 min at –20 °C, washed three times in PBS without calcium or magnesium (PBS–) and incubated for 1 h at 36 °C in 5 % CO2 with enterovirus-specific polyclonal rabbit antiserum (KTL-482) produced against the peptide EAIPALTAVETGHTSQVC, which was designed according to the VP1 region of the enterovirus genome (Härkönen et al., 2002). Unbound antibody was removed by washing three times with PBS– and once with PBS– supplemented with 0.1 % BSA. The conjugate for virus antiserum (anti-rabbit FITC, cat. no. 711-095-152; Jackson ImmunoReseach Laboratories) was incubated for 30 min at 36 °C. After staining, slides were washed three times with PBS– and once with water. Slides were analysed under a confocal microscope (Leica TGS NT).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The complete VP1 sequence of one of the virus strains (E210) was submitted to the Picornavirus Study Group (University of Essex, Colchester, UK) for comparison with those of previously proposed new enterovirus serotypes, and consequently was registered as a new candidate enterovirus serotype, EV94. According to this proposal, from here on, the virus strains characterized in this study are referred to as strains of EV94.
Possible cross-reactions of EV94 with EV68 and EV70 were studied using a neutralization assay with virus-specific rabbit antisera raised against EV68 and EV70. The antiserum against EV68 was not able to protect RD cells from EV70 or EV94 infection. Likewise, the antiserum against EV70 was not able to protect GMK cells from EV68 infection or RD cells from EV94 infection, indicating that EV94 differed antigenically from these two previously determined HEV-D serotypes.
Further genetic characterization of EV94
The genome of EV94-E210 sequenced in this study was 7364 nt. The non-coding 5' end was 714 nt, followed by an open reading frame encoding a 2190 aa polypeptide. The length of the non-coding 3' region was 80 nt. The overall base composition of the genome was 30.1 % A, 22.2 % G, 21.5 % C and 26.2 % U.
Nucleotide similarities in the complete VP1 region ranged from 96.3 to 98.3 % among the four environmental EV94 isolates and from 85.7 to 86.3 % among clinical isolate 19-04 and the environmental isolates, confirming clustering of all six strains in the same serotype. The environmental isolate EV94-E210 and the clinical isolate EV94-19-04 had 81.5–89.9 % nucleotide similarity and 92.9–100 % amino acid similarity in the coding region (Tables 1 and 2).
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). In the 5' region, HEV-D and HEV-C serotypes were about equally close to EV94. As expected, the sequence similarities were lowest in the capsid-coding region and highest in the non-structural protein-coding regions. The complete genome nucleotide similarities between EV94-E210 and the prototype strains of EV70 (EV70-J670/71) and EV68 (EV68-Fermon) were 75.8 and 73.7 %, respectively. In the VP1 region, the nucleotide identities were 67.7 and 67.4 %, respectively. The nucleotide similarities between the EV94 isolates and the EV68 and EV70 isolates obtained from GenBank ranged from 66.1 to 69.6 % and from 66.6 to 69.4 %, respectively, in the complete VP1 regions (for EV68 vs EV94) or in the overlapping 912 nt in the VP1 region (for EV70 vs EV94).
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). The VP1 amino acid sequences of the EV68 and EV70 strains obtained from GenBank had 69.1–72.0 % and 74.7–76.6 % sequence similarity with EV94-E210 in the overlapping regions.
Phylogenetic analysis
Phylogenetic trees were generated from HEV-D 5' UTR, VP1 and 3D sequences (Fig. 2). On the basis of the VP1 and 3D regions, the EV94 isolates were closely related and formed a monophyletic group within the HEV-D species. In the 5' UTR region, EV94 grouped together with other HEV-D strains and HEV-C serotypes. The phylogenetic trees of the VP1 and 3D regions were essentially congruent, providing no evidence of recombination within HEV-D species. In order to verify the apparent lack of recombination between HEV-D serotypes, we analysed the nucleotide sequences of EV94-E210, EV94-19-04, EV70-J670/71 and EV68-Fermon using similarity plot and bootscanning methods (data not shown). No evidence of recombination was found.
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). Acid treatment did not affect the infectivities of the environmental EV94 isolates (Table 3), whereas the titre of the acid-sensitive control, HRV2, was reduced 10 000-fold. The titre of the acid-insensitive control, PV1-Sabin, was not affected by acid treatment.
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).
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). Virus replication without a strong CPE was found in rabbit RK13 cells. No signs of replication or altered morphology were found in mouse fibroblastic 3T3 cell line. In bovine kidney (MDBK) and canine kidney (MDCK) cells, the virus infection altered cell morphology and induced cell death, but no viable virus progeny formation was detected in the replication assay using RD cells. However, an enterovirus-specific antibody produced a positive signal in an immunofluorescence assay in MDBK cells, suggesting that EV94 is able to internalize into these cells and that there is at least limited gene expression (Fig. 3c). No viral proteins were detected in MDCK cells (not shown).
Receptor usage
The ability of viruses to use cell-surface molecules as receptors is considered to be an important determinant of tissue tropism, host range and pathogenesis. The role of DAF (CD55), vitronectin receptor (v3) and PVR (CD155) in cell attachment and virus internalization was studied using CPE protection assays (Nobis et al., 1985). None of the blocking antibodies was able to prevent infection in RD cells.
We used CHO cell lines expressing either the 2 subunit of 21 integrin or HCAR, and M4 cells expressing (ICAM-1) to assess the role of 21, HCAR and ICAM-1, respectively, in EV94 infection. Equal amounts of progeny formation were found in both 2- and HCAR-expressing CHO cells. No progeny virus production was found in M4 or M4–ICAM cells, but mild CPE was detected in M4–ICAM cells.
Neutralization assays with human sera
As EV94 is a previously unknown enterovirus, we assessed the prevalence of antibodies against EV94 in Finland using a serum neutralization assay. Serum samples of 181 pregnant women, collected in 1983 (n=86) and 2002 (n=95), were studied. Neutralizing antibodies against EV94 were found in 80 % of subjects in the year 1983 and 78.9 % in the year 2002 (Fig. 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Enterovirus strains should be classified in the same serotype if they have >75 % nucleotide similarity in the VP1-coding sequence (>85 % amino acid sequence similarity) and into different serotypes if they have <70 % nucleotide similarity (<85 % amino acid similarity) (Oberste et al., 2000, 2001, 2003). The sequence similarities and phylogenetic analyses suggested that the virus isolates described in this study belonged to the HEV-D species and formed a new type, preliminarily designated EV94. We suggest that E210 should be considered as the prototype strain of this new enterovirus type. The natural host range and the cellular receptor for this virus are not known, but the similarities between EV70 and EV94 in cellular host range in vitro suggest that these serotypes might share some biological characteristics. The genomic G+C content of EV94 and EV68 varied by 2.6 mol%, thus only just failing to meet the species demarcation criterion. We did not study proteolytic processing or encapsidation of EV94, but given the overall genomic similarity of EV94, EV70 and EV68, it is unlikely that there would be any major differences in these properties among HEV-D serotypes.
Strain EV94-19-04 was isolated from a patient with AFP. Moreover, the environmental strain EV94-E210 was able to induce cytolytic infection in neuroblastic SK-N-SH cells, suggesting that this new enterovirus serotype might also be neurovirulent in vivo. However, based on this data, we cannot conclude that EV94 is necessarily the causative agent of these AFP cases, as the observed association may be coincidental.
The EV94 isolates originating from Egypt and the Democratic Republic of the Congo had substantial divergence in nucleotide sequences but only a few amino acid substitutions were detected. The nucleotide substitutions were distributed evenly throughout the genome. This suggests that these are geographically divergent, independently circulating strains that probably share antigenic and other biological properties but have accumulated mostly neutral mutations over time. In phylogenetic analysis, the EV94 isolates were monophyletic with other HEV-D serotypes in all genome regions except the 5' UTR. In this region, all enteroviruses cluster into two major groups of which HEV-A and HEV-B species constitute the enterovirus 5' UTR cluster II and HEV-C and HEV-D species cluster I (Hyypiä et al., 1997). The phylogenetic trees constructed on the basis of amino acid sequences were essentially similar to nucleotide alignment-based trees (data not shown).
Intra- and intertypic recombination is common within enterovirus species HEV-A, HEV-B and HEV-C (Santti et al., 1999; Oprisan et al., 2002; Brown et al., 2003; Lindberg et al., 2003; Lukashev et al., 2003, 2005; Chevaliez et al., 2004; Oberste et al., 2004a; reviewed by Lukashev, 2005). However, no evidence of recombination between HEV-D serotypes has been found in this study or by others (Oberste et al., 2004b). Although bootscanning analysis showed some possible recombination sites, the similarity plot analysis suggested that EV68 and EV70 are almost equally distant from EV94, and any evidence of recombination may be a product of convergent evolution between lineages rather than a definite recombination event. Moreover, the phylogenetic trees of VP1 and 3D were essentially congruent. Apparently, any possible recombination event must have taken place a relatively long time ago. Our seroprevalence studies suggested that EV94 is a common virus; hence, co-infection of this virus with other HEV-D strains is possible and the apparent lack of recombination may be due to different tissue tropisms, as suggested previously by Oberste et al. (2004b). More complete genome sequences of field isolates of HEV-D serotypes are needed to elucidate the role of recombination within this species.
Most enteroviruses use the faecal–oral route of transmission. Interestingly, the other serotypes of HEV-D, EV68 and EV70, differ from most enteroviruses by infecting their primary target tissue directly. EV68 is associated with respiratory disease and EV70 is a causative agent of acute haemorrhagic conjunctivitis. In order to access primary replication sites in the alimentary channel mucosa, the virus has to get through the acidic environment of the stomach. It has been shown previously that EV68 (Blomqvist et al., 2002) and some strains of EV70 (Oberste et al., 2004b) are acid sensitive, and EV68 has a lower optimum growing temperature than most other enteroviruses. In this study, we showed that EV94-E210 is acid stable and thus is likely also to be able to access the small intestine in vivo. Moreover, EV94 was able to replicate in a cell line of alimentary channel origin, the colon epithelial CaCo-2 cell line, and the strains described in this study were originally isolated from sewage and faecal specimens, suggesting that this virus might use the faecal–oral route of transmission.
Most enteroviruses have a restricted cell tropism in vitro. To characterize the in vitro cell tropism of EV94, we studied its replication in various human and other mammalian cell lines. EV94 was cytolytic in a wide variety of human cell lines including muscle, glandular, lung epithelial, epithelioid and neuroblastic cells. Moreover, clear virus progeny production was observed in all of the human cell lines studied. EV94 was also able to replicate in some non-human cell lines including primate (GMK and Vero), hamster (BHK21), rabbit (RK13), mouse (L20B) and bovine (MDBK) cell lines, suggesting that EV94 resembles EV70 in having a wider host range in vitro than most other enteroviruses (Yoshii et al., 1977). The ability of the virus to infect and replicate in mouse L cells expressing human PVR presents an important practical issue. L20B cells are used in poliovirus diagnostics and thus EV94 may cause false-positive results if the serotype is not confirmed by other means. Continuous cell lines differ from cells in vivo in many respects; for example, virus receptor expression levels may be different in cultured cells and the corresponding cells in vivo. Therefore, further studies are needed to determine the tissue tropism and possible pathogenesis of EV94 in vivo.
Both virus and host determinants affect the tissue tropism of a virus. These determinants include receptor specificity, host-cell factors participating in translation and replication initiation (reviewed by Whitton et al., 2005) and the local cytokine (alpha/beta interferon) milieu at the site of infection (Ida-Hosonuma et al., 2005; Yoshikawa et al., 2006). The ability of EV94 to replicate in a wide variety of cell lines in vitro suggests that its receptor is expressed widely. Alternatively, the virus may use various receptors. As receptor specificity is considered to be an important determinant of tissue tropism, host range and pathogenesis, we studied the receptor usage of this serotype. In this study, we were not able to obtain any conclusive evidence of the receptor usage of EV94. The other serotypes of HEV-D (EV68 and EV70) use DAF as a receptor in HeLa cells (Karnauchow et al., 1996; Blomqvist et al., 2002). EV70 also uses sialic acid-containing receptors in a variety of other human cell lines (Alexander & Dimock, 2002; Haddad et al., 2004; Nokhbeh et al., 2005). Our results suggest that EV94 does not need DAF to infect RD cells.
The closest relative of EV94, EV70, replicates in cells derived from a wide range of mammalian species (Yoshii et al., 1977), and neutralizing antibodies against EV70 have been detected in the sera of cattle, sheep, swine, chickens, goats, dogs and monkeys (Kono et al., 1981; Sasagawa et al., 1982), suggesting that EV70 might have a wider host range than most other enteroviruses. However, EV70 isolates have not been found in animals. EV70 was discovered in Ghana in 1969 during an acute haemorrhagic conjunctivitis epidemic (Mirkovic et al., 1973). Miyamura et al. (1986) and Takeda et al. (1994) estimated that all EV70 isolates have a common ancestor that emerged in one place in 1967±15 months. The wide host range and recent origin of EV70 suggests that this serotype may have originated from an animal picornavirus. Our results show that EV94 is able to replicate in various mammalian cell lines and suggest that the wide host range may be a common characteristic of the HEV-D species. It is tempting to speculate that this new enterovirus serotype could be of animal origin or have an animal reservoir.
In this study, neutralizing antibodies against EV94 were found in a surprisingly large number of individuals since 1983, indicating that EV94 is not a new virus but that infections caused by this or an antigenically closely related virus strain have been common in Finland for at least two decades. The absence of clinical isolates of EV94 prior to this study might be due to lack of efficient methods to identify untypable enteroviruses before molecular methods became widely available. Alternatively, the clinical manifestations of EV94 might be rare or very mild, or the infection might be asymptomatic, and thus it may remain undetected in virological surveillance studies. EV94 replicates well in many cell lines, and it would be surprising if cell culture-based isolation methods failed to detect this virus.
In conclusion, our results suggest that EV94 is a new serotype of the HEV-D species and has an unusually wide tissue tropism and host species range in vitro. EV94 is probably able to use the faecal–oral route of transmission, is potentially neuropathogenic and has an unexpectedly high prevalence in the Finnish population. Further studies are needed to elucidate the pathogenic and epidemiological properties of EV94.
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ACKNOWLEDGEMENTS |
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Received 29 August 2006;
accepted 22 October 2006.
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