Download Enterovirus 94, a proposed new serotype in human enterovirus

Journal of General Virology (2007), 88, 849–858
DOI 10.1099/vir.0.82510-0
Enterovirus 94, a proposed new serotype in
human enterovirus species D
Teemu P. Smura,1 Nina Junttila,2 Soile Blomqvist,1 Helene Norder,2
Svetlana Kaijalainen,1 Anja Paananen,1 Lars O. Magnius,2 Tapani Hovi1
and Merja Roivainen1
Correspondence
Merja Roivainen
[email protected]
Received 29 August 2006
Accepted 22 October 2006
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
The genus Enterovirus (family Picornaviridae) contains five species with strains isolated from
humans: Human enterovirus A (HEV-A), HEV-B, HEV-C, HEV-D and Poliovirus. In this study, a
proposed new serotype of HEV-D was characterized. Four virus strains were isolated from sewage
in Egypt and one strain from acute flaccid paralysis cases in the Democratic Republic of the Congo.
The complete genome of one environmental isolate, the complete coding sequence of one clinical
isolate and complete VP1 regions from the other isolates were sequenced. These isolates had
66.6–69.4 % nucleotide similarity and 74.7–76.6 % amino acid sequence similarity in the VP1
region with the closest enterovirus serotype, enterovirus 70 (EV70), suggesting that the isolates
form a new enterovirus type, tentatively designated enterovirus 94 (EV94). Phylogenetic analyses
including sequences of the 59 UTR, VP1 and 3D regions demonstrated that EV94 isolates formed a
monophyletic group within the species HEV-D. No evidence of recombination was found between
EV94 and the other HEV-D serotypes, EV68 and EV70. Further biological characterization showed
that EV94 was acid stable and had a wide cell tropism in vitro. Attempts to prevent replication with
protective antibodies to known enterovirus receptors (poliovirus receptor, vitronectin avb3 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.
INTRODUCTION
The genus Enterovirus (family Picornaviridae) contains a
large group of human pathogens. Although the majority of
enterovirus infections are subclinical, enterovirus infection
can lead to a variety of acute and chronic diseases including
mild upper respiratory illness, febrile rash, aseptic meningitis, encephalitis, acute haemorrhagic conjunctivitis, pleurodynia, acute flaccid paralysis (AFP), diabetes, myocarditis
and neonatal sepsis-like disease (Pallansch & Roos, 2001).
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
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are DQ916376–DQ916379 and EF107097–
EF107098.
0008-2510 G 2007 SGM
frame flanked by non-coding 59 and 39 regions. The 59 UTR
contains an internal ribosome-binding site, which is
essential for translation initiation (Pelletier & Sonenberg,
1988; Molla et al., 1992; Chen & Sarnow, 1995). The 39 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
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849
T. Smura and others
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 nontypable 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 HEVD 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.
METHODS
Cell lines. A human rhabdomyosarcoma (RD) cell line and recombinant mouse L cells expressing human poliovirus receptor (L20B)
(Pipkin et al., 1993) 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
850
(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 (LPR2/2,
LDLR2/2) 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 ml 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 uC, 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 (GibcoBRL 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
(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
ALIGNX
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Journal of General Virology 88
EV94, a proposed new serotype of HEV-D
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 EV68Fermon (AY426531), EV68-TX03 (AY426500), EV68-MD99
(AY426499), EV68-TX99 (AY426498), EV68-MN98 (AY426497),
EV68-TX02-2 (AY426496), EV68-TX02-1 (AY426495), EV68-WI00
(AY426494), EV68-MO00 (AY426493), EV68-MD02-2 (AY426492),
EV68-MD02-1 (AY426491), EV68-NY93 (AY426490), EV68-MN89
(AY426489), EV68-CA62-3 (AY426488), EV68-CA62-2 (AY426487)
and EV68-CA62-1 (AY426486); HRV87-Corn (AY355268); serotype
EV70 strains EV70-J670/71 (NC_001430), EV70-ENG/71 (D17595),
EV70-FB/73 (D17596), EV70-G10/72 (D17597), EV70-HP185/81
(D17598), EV70-HP85/78 (D17599), EV70-I/72 (D17600), EV70M51/76 (D17603), EV70-M8/72 (D17604), EV70-R20/71 (D17605),
EV70-R6/71 (D17606), EV70-SEC32/71 (D17607), EV70-T260/74
(D17608), EV70-T62/73 (D17609), EV70-TW266/81 (D17610) and
EV70-V1250/81 (D17611); HEV-A serotypes CVA8 strain Donovan
(AY421766), CVA16 strain G-10 (U05876) and enterovirus 71 strain
BrCr (U22521); HEV-B serotypes ECHO5 strain Noyce (AF083069),
CVB3 strain Nancy (M16572) and CVA9 strain Griggs (D00627);
HEV-C serotypes CVA11 strain Belgium-1 (AF499636) and CVA20
strain IH35 (AF499642), poliovirus 2 strain Lansing (M12197) and
porcine enterovirus 8 (PEV8) strain V13 (AF406813).
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 uC 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 uC 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 uC
for 20 min and stored at 4 uC. Serial 4-fold dilutions of sera were
mixed with an equal volume of virus (100 TCID50). The mixtures
were neutralized at 36 uC for 1 h and then overnight at 4 uC. The
mixtures were transferred into 96-well cell culture plates with RD
cells (~36105 cells ml21) and incubated at 36 uC 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 avb3 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
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volumes of 30 ml and incubated for 2 h at 36 uC in 5 % CO2, after
which pre-titrated virus (~10 000 TCID50 per well) was administered. The mixture was incubated at 36 uC 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 virusinfected cell culture supernatant. The mixtures were incubated for
1 h at 36 uC 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 uC 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 220 uC, washed
three times in PBS without calcium or magnesium (PBS2) and incubated for 1 h at 36 uC 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 PBS2 and once
with PBS2 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 uC. After staining, slides were washed three times with PBS2 and once with water.
Slides were analysed under a confocal microscope (Leica TGS NT).
RESULTS
Identification of a new serotype of HEV-D
Sewage specimens collected in Egypt during environmental
surveillance for wild-type polioviruses were examined for
cytopathic viruses in both RD and L20B cells. Virus strains
that produced CPE in L20B cells and escaped neutralization
with poliovirus-specific antisera were characterized using
molecular methods in Helsinki. Several non-serotypable
enterovirus strains were isolated. Initial characterization was
performed by partially sequencing the VP1-coding region.
Four strains clustered close to EV70 and were subjected to a
more detailed study. Meanwhile, three non-serotypable
virus strains isolated from faecal specimens of AFP patients
in the Democratic Republic of the Congo were analysed in
Stockholm. One of these isolates turned out to be EV68,
whilst the other two grouped closer to the above-mentioned
Egyptian isolates.
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
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T. Smura and others
Table 1. Nucleotide sequence similarities (%) between
EV94-E210 and strains EV94-19-04, EV70-J670/71 and
EV68-Fermon
Table 2. Amino acid sequence similarities (%) between
EV94-E210 and strains EV94-19-04, EV70-J670/71 and
EV68-Fermon
EV94-19-04 EV70-J670/71 EV68-Fermon
Complete genome
59 UTR
VP4
VP2
VP3
VP1
2A
2B
2C
3A
3B
3C
3D
39 UTR
ND,
ND
ND
85.0
86.0
84.3
86.3
84.1
81.5
83.5
86.1
86.4
84.5
89.9
ND
75.8
83.5
79.7
74.8
70.9
67.8
73.6
75.1
77.8
78.3
66.7
76.3
82.7
80.7
73.7
76.2
77.3
74.2
70.1
67.7
69.6
76.4
77.4
78.3
75.8
72.9
77.0
68.8
Not determined.
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 59 end was 714 nt, followed by an
open reading frame encoding a 2190 aa polypeptide. The
length of the non-coding 39 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.
Polyprotein
VP4
VP2
VP3
VP1
2A
2B
2C
3A
3B
3C
3D
EV94-19-04
EV70-J670/71
EV68-Fermon
97.0
97.1
98.8
99.1
97.1
93.9
92.9
96.1
96.6
100
96.7
97.8
86.9
95.7
84.4
80.4
73.6
85.2
94.9
90.0
92.1
95.5
88.0
95.2
84.8
89.9
84.7
82.6
71.4
82.3
85.9
88.2
89.9
86.4
88.0
90.6
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).
A similarity plot analysis with prototype sequences of other
enterovirus serotypes suggested that EV94 was most closely
related to the HEV-D species in all genome regions except
in 59 UTR (Fig. 1). In the 59 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
Fig. 1. Similarity plot of complete enterovirus genomes using a sliding window of
200 nt moving in 20 nt steps. The sequence
of EV94-E210 was compared with representatives of HEV-A (CVA16), HEV-B (CVB3),
HEV-C (CVA20), poliovirus (PV2) and HEV-D
(EV68 and EV70) species.
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Journal of General Virology 88
EV94, a proposed new serotype of HEV-D
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).
We used the complete genome sequence of EV68 prototype
strain Fermon (GenBank accession no. AY426531) for
tentative estimation of the EV94 polypeptide cleavage sites.
The amino acid sequences of individual estimated EV94
proteins had 71.4–90.6 % similarity with those of EV68Fermon and 73.6–95.7 % similarity with EV70-J670/71
proteins (Table 2). 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 59 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 59 UTR region, EV94 grouped together with other HEVD 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.
Acid sensitivity
Sensitivity to acid treatment and optimum growth temperature infer the natural target tissue and route of
transmission of the virus. Rhinoviruses and EV68, which
multiply mainly in the respiratory tract epithelia, are sensitive to low pH and have a lower optimal growth temperature
than most enteroviruses, which are enteric pathogens. In
order to assess the possible transmission route of EV94,
we tested the acid sensitivity of environmental strain EV94E210 using a standard assay (Couch, 1992). 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.
Infectivity and virus replication
We infected a variety of human continuous cell lines with
EV94-E210. Virus replication was studied by light microscopy and by measuring the infectivity of samples collected
at different time points. The virus was able to induce CPE
and clear progeny virus production was detected in all of the
human cell lines studied (Fig. 3a).
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Fig. 2. Phylogenetic trees constructed from complete 59 UTR (a),
VP1 (b) and 3D (c) nucleotide sequences. Phylogenetic trees
were construction with TREE-PUZZLE 5.2 using the Tamura–
Nei model of substitution; the transition–transversion rate was
estimated from the data. Six representatives of both human enterovirus 59 UTR clusters were used to construct the tree in (a).
The viruses sequenced in this study and representatives of
each human enterovirus species were used to construct the trees
in (b) and (c). PEV8 was used as an outgroup to root the trees in
(b) and (c).
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T. Smura and others
Table 3. Stability of environmental EV94 isolates under
acidic conditions
Isolate
EV94-E210
EV94-E430
EV94-E435
EV94-E438
HRV2
PV1-Sabin
”Log TCID50
pH 4
pH 7
7.1
6.6
7.1
6.6
<1.6
7.1
6.6
6.6
7.1
6.6
5.6
7.1
Most human enteroviruses have strict host specificity and
can infect only human or other primate cells. To determine
the host species specificity of EV94 in vitro, we studied
infection and replication of EV94-E210 in a variety of
mammalian cell lines, as described above. EV94 was able to
induce CPE in green monkey kidney cells (GMK and Vero),
recombinant mouse L cells expressing human PVR (L20B)
and hamster kidney BHK21 cells (Fig. 3b). 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 (avb3) 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 a2 subunit of
a2b1 integrin or HCAR, and M4 cells expressing (ICAM-1)
to assess the role of a2b1, HCAR and ICAM-1, respectively,
in EV94 infection. Equal amounts of progeny formation
were found in both a2- 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.
Fig. 3. (a, b) Replication of EV94-E210 in human (a) and
other mammalian (b) cell lines. The cells were infected with an
apparently high m.o.i. The infectivity of each sample was determined by end-point titration in the corresponding cell line (a) or
in RD cells (b). (c) Infected and uninfected GMK and MDBK
cells were stained with enterovirus-specific polyclonal rabbit
antiserum (KTL-482, green staining, diluted 1 : 400 for GMK
and 1 : 800 for MDBK cells) 6 h after infection. Upper panels,
infected cells; lower panels, mock-infected cells. Magnification:
2006 (GMK cells); 4006 (MDBK 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
854
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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Journal of General Virology 88
EV94, a proposed new serotype of HEV-D
of these AFP cases, as the observed association may be
coincidental.
Fig. 4. Antibody prevalence and end-point titres of neutralizing
antibodies against EV94 in the sera of 181 Finnish women at
the end of the first trimester of pregnancy.
DISCUSSION
Members of an enterovirus species are considered to share
>70 % amino acid similarity in regions coding for the
capsid (P1) and non-structural proteins 2C and 3CD, to
share a limited number of host-cell receptors and natural
host range, to have a genomic G+C content that varies by
no more than 2.5 mol% and to share a significant degree of
compatibility in proteolytic processing, replication, encapsidation and genetic recombination (Stanway et al., 2005).
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
http://vir.sgmjournals.org
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 59 UTR. In this
region, all enteroviruses cluster into two major groups of
which HEV-A and HEV-B species constitute the enterovirus
59 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, coinfection 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,
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T. Smura and others
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
856
(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.
ACKNOWLEDGEMENTS
Strains isolated from sewage specimens were derived from an
environmental poliovirus surveillance project supported by WHO
(TSA18/181/256).
REFERENCES
Alexander, D. A. & Dimock, K. (2002). Sialic acid functions in
enterovirus 70 binding and infection. J Virol 76, 11265–11272.
Blomqvist, S., Savolainen, C., Raman, L., Roivainen, M. & Hovi, T.
(2002). Human rhinovirus 87 and enterovirus 68 represent a unique
serotype with rhinovirus and enterovirus features. J Clin Microbiol
40, 4218–4223.
Brown, B., Oberste, M. S., Maher, K. & Pallansch, M. A. (2003).
Complete genomic sequencing shows that polioviruses and members
of human enterovirus species C are closely related in the noncapsid
coding region. J Virol 77, 8973–8984.
Chen, C. Y. & Sarnow, P. (1995). Initiation of protein synthesis by
the eukaryotic translational apparatus on circular RNAs. Science 268,
415–417.
Chevaliez, S., Szendroi, A., Caro, V., Balanant, J., Guillot, S.,
Berencsi, G. & Delpeyroux, F. (2004). Molecular comparison of
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 15:48:41
Journal of General Virology 88
EV94, a proposed new serotype of HEV-D
echovirus 11 strains circulating in Europe during an epidemic of
multisystem hemorrhagic disease of infants indicates that evolution
generally occurs by recombination. Virology 325, 56–70.
Couch, R. (1992). Rhinoviruses. In Laboratory Diagnosis of Viral
Infections, pp. 709–729. Edited by E. H. Lennette. New York: Marcel
Dekker.
El Bassioni, L., Barakat, I., Nasr, E., de Gourville, E. M., Hovi, T.,
Blomqvist, S., Burns, C., Stenvik, M., Gary, H. & other authors
(2003). Prolonged detection of indigenous wild polioviruses in
sewage from communities in Egypt. Am J Epidemiol 158, 807–815.
Haddad, A., Nokhbeh, M. R., Alexander, D. A., Dawe, S. J., Grise, C.,
Gulzar, N. & Dimock, K. (2004). Binding to decay-accelerating factor
is not required for infection of human leukocyte cell lines by
enterovirus 70. J Virol 78, 2674–2681.
Härkönen, T., Lankinen, H., Davydova, B., Hovi, T. & Roivainen, M.
(2002). Enterovirus infection can induce immune responses that
cross-react with b-cell autoantigen tyrosine phosphatase IA-2/IAR.
Lukashev, A. N. (2005). Role of recombination in evolution of
enteroviruses. Rev Med Virol 15, 157–167.
Lukashev, A. N., Lashkevich, V. A., Ivanova, O. E., Koroleva, G. A.,
Hinkkanen, A. E. & Ilonen, J. (2003). Recombination in circulating
enteroviruses. J Virol 77, 10423–10431.
Lukashev, A. N., Lashkevich, V. A., Ivanova, O. E., Koroleva, G. A.,
Hinkkanen, A. E. & Ilonen, J. (2005). Recombination in circulating
Human enterovirus B: independent evolution of structural and nonstructural genome regions. J Gen Virol 86, 3281–3290.
Mirkovic, R. R., Kono, R., Yin-Murphy, M., Sohier, R., Schmidt, N. J.
& Melnick, J. L. (1973). Enterovirus type 70: the etiologic agent of
pandemic acute haemorrhagic conjunctivitis. Bull World Health
Organ 49, 341–346.
Mirmomeni, M. H., Hughes, P. J. & Stanway, G. (1997). An RNA
tertiary structure in the 39 untranslated region of enteroviruses is
necessary for efficient replication. J Virol 71, 2363–2370.
J Med Virol 66, 340–350.
Miyamura, K., Tanimura, M., Takeda, N., Kono, R. & Yamazaki, S.
(1986). Evolution of enterovirus 70 in nature: all isolates were
Hovi, T. (2006). Surveillance for polioviruses. Biologicals 34,
recently derived from a common ancestor. Arch Virol 89, 1–14.
123–126.
Molla, A., Jang, S. K., Paul, A. V., Reuer, Q. & Wimmer, E. (1992).
Hovi, T., Blomqvist, S., Nasr, E., Burns, C. C., Sarjakoski, T., Ahmed,
N., Savolainen, C., Roivainen, M., Stenvik, M. & other authors
(2005). Environmental surveillance of wild poliovirus circulation in
Cardioviral internal ribosomal entry site is functional in a genetically
engineered dicistronic poliovirus. Nature 356, 255–257.
Egypt – balancing between detection sensitivity and workload. J Virol
Methods 126, 127–134.
Hyypiä, T., Hovi, T., Knowles, N. J. & Stanway, G. (1997). Classifi-
cation of enteroviruses based on molecular and biological properties.
J Gen Virol 78, 1–11.
Ida-Hosonuma, M., Iwasaki, T., Yoshikawa, T., Nagata, N., Sato, Y.,
Sata, T., Yoneyama, M., Fujita, T., Taya, C. & other authors (2005).
Nobis, P., Zibirre, R., Meyer, G., Kuhne, J., Warnecke, G. & Koch, G.
(1985). Production of a monoclonal antibody against an epitope on
HeLa cells that is the functional poliovirus binding site. J Gen Virol
66, 2563–2569.
Nokhbeh, M. R., Hazra, S., Alexander, D. A., Khan, A., McAllister, M.,
Suuronen, E. J., Griffith, M. & Dimock, K. (2005). Enterovirus 70
binds to different glycoconjugates containing a2,3-linked sialic acid
on different cell lines. J Virol 79, 7087–7094.
The alpha/beta interferon response controls tissue tropism and
pathogenicity of poliovirus. J Virol 79, 4460–4469.
Norder, H., Bjerregaard, L., Magnius, L., Lina, B., Aymard, M. &
Chomel, J. J. (2003). Sequencing of ‘untypable’ enteroviruses reveals
Junttila, N., Leveque, N., Kabue, J. P., Cartet, G., Mushiya, F.,
Muyembe-Tamfum, J.-J., Trompette, A., Magnius, L., Lina, B. &
other authors (2007). New enteroviruses, EV-93 and EV-94,
two new types, EV-77 and EV-78, within human enterovirus type B
and substitutions in the BC loop of the VP1 protein for known
types. J Gen Virol 84, 827–836.
associated with acute flaccid paralysis in the Democratic Republic
of the Congo. J Med Virol (in press).
Oberste, M. S., Maher, K., Kilpatrick, D. R. & Pallansch, M. A. (1999).
Karnauchow, T. M., Tolson, D. L., Harrison, B. A., Altman, E., Lublin,
D. M. & Dimock, K. (1996). The HeLa cell receptor for enterovirus 70
is decay-accelerating factor (CD55). J Virol 70, 5143–5152.
Kono, R., Sasagawa, A., Yamazaki, S., Nakazono, N., Minami, K.,
Otatsume, S., Robin, Y., Renaudet, J., Cornet, M. & other authors
(1981). Seroepidemiologic studies of acute hemorrhagic conjuncti-
Molecular evolution of the human enteroviruses: correlation of
serotype with VP1 sequence and application to picornavirus
classification. J Virol 73, 1941–1948.
Oberste, M. S., Maher, K., Flemister, M. R., Marchetti, G., Kilpatrick,
D. R. & Pallansch, M. A. (2000). Comparison of classic and molecular
approaches for the identification of untypeable enteroviruses. J Clin
Microbiol 38, 1170–1174.
vitis virus (enterovirus type 70) in West Africa. III. Studies with
animal sera from Ghana and Senegal. Am J Epidemiol 114, 362–368.
Oberste, M., Schnurr, D., Maher, K., al-Busaidy, S. & Pallansch, M.
(2001). Molecular identification of new picornaviruses and character-
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: Integrated software
ization of a proposed enterovirus 73 serotype. J Gen Virol 82, 409–416.
for molecular evolutionary genetics analysis and sequence alignment.
Brief Bioinform 5, 150–163.
Oberste, M. S., Nix, W. A., Maher, K. & Pallansch, M. A. (2003).
Lennette, E. H. (1969). General principles underlying laboratory
Improved molecular identification of enteroviruses by RT-PCR and
amplicon sequencing. J Clin Virol 26, 375–377.
diagnosis of viral and rickettsial infections. In Diagnostic Procedures
for Viral and Rickettsial Infections, pp. 1–63. Edited by E. H. Lennette
& N. J. Schmidt. New York: American Public Health Association.
Oberste, M. S., Penaranda, S. & Pallansch, M. A. (2004a). RNA
Lindberg, A. M., Andersson, P., Savolainen, C., Mulders, M. N. &
Hovi, T. (2003). Evolution of the genome of Human enterovirus B:
Oberste, M. S., Maher, K., Schnurr, D., Flemister, M. R., Lovchik, J.
C., Peters, H., Sessions, W., Kirk, C., Chatterjee, N. & other authors
(2004b). Enterovirus 68 is associated with respiratory illness and
recombination plays a major role in genomic change during
circulation of coxsackie B viruses. J Virol 78, 2948–2955.
incongruence between phylogenies of the VP1 and 3CD regions
indicates frequent recombination within the species. J Gen Virol 84,
1223–1235.
shares biological features with both the enteroviruses and the
rhinoviruses. J Gen Virol 85, 2577–2584.
Lole, K. S., Bollinger, R. C., Paranjape, R. S., Gadkari, D., Kulkarni,
S. S., Novak, N. G., Ingersoll, R., Sheppard, H. W. & Ray, S. C.
(1999). Full-length human immunodeficiency virus type 1 genomes
Oberste, M. S., Michele, S. M., Maher, K., Schnurr, D., Cisterna, D.,
Junttila, N., Uddin, M., Chomel, J.-J., Lau, C.-S. & other authors
(2004c). Molecular identification and characterization of two
from subtype C-infected seroconverters in India, with evidence of
intersubtype recombination. J Virol 73, 152–160.
proposed new enterovirus serotypes, EV74 and EV75. J Gen Virol
85, 3205–3212.
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 13 May 2017 15:48:41
857
T. Smura and others
Oberste, M. S., Maher, K., Michele, S. M., Belliot, G., Uddin, M. &
Pallansch, M. A. (2005). Enteroviruses 76, 89, 90 and 91 represent a
Santti, J., Hyypiä, T., Kinnunen, L. & Salminen, M. (1999). Evidence
novel group within the species Human enterovirus A. J Gen Virol 86,
445–451.
Sasagawa, A., Miyamura, K. & Kono, R. (1982). Enterovirus type
Oprisan, G., Combiescu, M., Guillot, S., Caro, V., Combiescu, A.,
Delpeyroux, F. & Crainic, R. (2002). Natural genetic recombination
Schmidt, H. A., Strimmer, K., Vingron, M. & von Haeseler, A. (2002).
between co-circulating heterotypic enteroviruses. J Gen Virol 83,
2193–2200.
Pallansch, M. A. & Roos, R. P. (2001). Enteroviruses: polioviruses,
coxsackieviruses, echoviruses and newer enteroviruses. In Fields
Virology, 4th edn, pp. 723–775. Edited by D. M. Knipe, P. M.
Howley, D. E. Griffin, R. A. Lamb, M. A. Martin, B. Roizman & S. E.
Straus. Philadelphia: Lippincott Williams & Wilkins.
Palmenberg, A. C. (1990). Proteolytic processing of picornaviral
polyprotein. Annu Rev Microbiol 44, 603–623.
Pelletier, J. & Sonenberg, N. (1988). Internal initiation of translation
of eukaryotic mRNA directed by a sequence derived from poliovirus
RNA. Nature 334, 320–325.
Pilipenko, E. V., Maslova, S. V., Sinyakov, A. N. & Agol, V. I. (1992).
Towards identification of cis-acting elements involved in the replication
of enterovirus and rhinovirus RNAs: a proposal for the existence of
tRNA-like terminal structures. Nucleic Acids Res 20, 1739–1745.
Pilipenko, E. V., Poperechny, K. V., Maslova, S. V., Melchers, W. J.,
Slot, H. J. & Agol, V. I. (1996). Cis-element, oriR, involved in the
of recombination among enteroviruses. J Virol 73, 8741–8749.
70-neutralizing IgM in animal sera. Jpn J Med Sci Biol 35, 63–73.
TREE-PUZZLE:
maximum likelihood phylogenetic analysis using
quartets and parallel computing. Bioinformatics 18, 502–504.
Stanway, G., Brown, F., Christian, P., Hovi, T., Hyypiä, T., King,
A. M. Q., Knowles, N. J., Lemon, S. M., Minor, P. D. & other authors
(2005). Family Picornaviridae. In Virus Taxonomy. Eighth Report of
the International Committee on Taxonomy of Viruses, pp. 757–778.
Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff, U. Desselberger &
L. A. Ball. London: Elsevier/Academic Press.
Takeda, N., Tanimura, M. & Miyamura, K. (1994). Molecular
evolution of the major capsid protein VP1 of enterovirus 70.
J Virol 68, 854–862.
Tamura, K. & Nei, M. (1993). Estimation of the number of nucleotide
substitutions in the control region of mitochondrial DNA in humans
and chimpanzees. Mol Biol Evol 10, 512–526.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &
Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res 25, 4876–4882.
initiation of (-) strand poliovirus RNA: a quasi-globular multidomain RNA structure maintained by tertiary (‘kissing’) interactions. EMBO J 15, 5428–5436.
Whitton, J. L., Cornell, C. T. & Feuer, R. (2005). Host and virus
Pipkin, P. A., Wood, D. J., Racaniello, V. R. & Minor, P. D. (1993).
Ylipaasto, P., Klingel, K., Lindberg, A. M., Otonkoski, T., Kandolf, R.,
Hovi, T. & Roivainen, M. (2004). Enterovirus infection in human
Characterisation of L cells expressing the human poliovirus receptor
for the specific detection of polioviruses in vitro. J Virol Methods 41,
333–340.
Reithmayer, M., Reischl, A., Snyers, L. & Blaas, D. (2002). Species-
specific receptor recognition by a minor-group human rhinovirus
(HRV): HRV serotype 1A distinguishes between the murine and the
human low-density lipoprotein receptor. J Virol 76, 6957–6965.
determinants of picornavirus pathogenesis and tropism. Nat Rev
Microbiol 3, 765–776.
pancreatic islet cells, islet tropism in vivo and receptor involvement
in cultured islet beta cells. Diabetologia 47, 225–239.
Yoshii, T., Natori, K. & Kono, R. (1977). Replication of enterovirus 70
in non-primate cell cultures. J Gen Virol 36, 377–384.
Salminen, M. O., Carr, J. K., Burke, D. S. & McCutchan, F. E. (1995).
Yoshikawa, T., Iwasaki, T., Ida-Hosonuma, M., Yoneyama, M.,
Fujita, T., Horie, H., Miyazawa, M., Abe, S., Simizu, B. & Koike, S.
(2006). Role of the alpha/beta interferon response in the acquisition
Identification of breakpoints in intergenotypic recombinants of HIV
type 1 by bootscanning. AIDS Res Hum Retroviruses 11, 1423–1425.
of susceptibility to poliovirus by kidney cells in culture. J Virol 80,
4313–4325.
858
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IP: 88.99.165.207
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