Fourth human parechovirus serotype.We identified a novel human parechovirus (HPeV) type (K251176-02) from a neonate neonate /neo·nate/ (ne´o-nat) newborn infant. ne·o·nate n. A neonatal infant. neonate a newborn animal. with fever. Analysis of the complete genome showed K251176-02 to be a new HPeV genotype. Since K251176-02 could not be neutralized with antibodies against known HPeV serotypes 1-3, it should be classified as a fourth HPeV serotype serotype /se·ro·type/ (ser´o-tip) the type of a microorganism determined by its constituent antigens; a taxonomic subdivision based thereon. se·ro·type n. See serovar. v. . ********** Infections with human parechoviruses (HPeVs) are commonly associated with mild gastrointestinal and respiratory symptoms in young children (1-3), but more severe conditions, such as flaccid paralysis Flaccid paralysis Paralysis characterized by limp, unresponsive muscles. Mentioned in: Botulism flaccid paralysis Neurology Paralysis characterized by complete loss of muscle tone and tendon reflexes. Cf Spastic paralysis. (4) and encephalitis encephalitis (ĕnsĕf'əlī`təs), general term used to describe a diffuse inflammation of the brain and spinal cord, usually of viral origin, often transmitted by mosquitoes, in contrast to a bacterial infection of the meninges (5), have also been described. Recently, a new serotype (HPeV3) has been isolated, which has been associated with transient paralysis (6) and neonatal sepsis neonatal sepsis Sepsis of newborn, septicemia of newborn Pediatrics A severe systemic infection of the newborn caused primarily by group B streptococcus, a bacterium found in the GI and GU tracts, which causes ±3/4 (7). HPeV1 and HPeV2 were previously known as the enteroviruses Enteroviruses Viruses which live in the gastrointestinal tract. Coxsackie viruses, viruses that cause hand-foot-mouth disease, are an enterovirus. Mentioned in: Hand-Foot-and-Mouth Disease echovirus echovirus /echo·vi·rus/ (ek´o-vi?rus) an enterovirus isolated from humans, separable into many serotypes, certain of which are associated with human disease, especially aseptic meningitis. 22 and 23 but were reclassified into a new genus within the family Picornaviridae after phylogenetic phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. analysis showed that parechoviruses were distinct from other picornaviruses (1-3,8-11). HPeVs have predominantly been isolated from young children, and increasing evidence shows that HPeV can cause serious illness in these patients. We recently showed that infection with HPeV3 is associated with younger age and more severe disease than is infection with HPeV1 (12). During the screening of patient samples, we identified 1 aberrant HPeV type. Phylogenetic analysis of the full-length sequence and viral neutralization neutralization, chemical reaction, according to the Arrhenius theory of acids and bases, in which a water solution of acid is mixed with a water solution of base to form a salt and water; this reaction is complete only if the resulting solution has neither acidic nor assays showed that the isolate designated K251176-02 is a new HPeV genotype and serotype. The Study Viral culture viral culture A test in which a specimen–eg, throat swab, sputum, stool, CSF, urine, from a Pt is placed in live cells; various viruses–eg, adenovirus, enterovirus, herpes simplex, measles, mumps, myxovirus, paramyxovirus, rhinovirus, rubella, of the stool of a 6-day-old patient with a 2-day history of high fever and poor feeding and no history of gastrointestinal or respiratory symptoms showed enterovirus enterovirus /en·tero·vi·rus/ (en´ter-o-vi?rus) any virus of the genus Enterovirus. enterovi´ral Enterovirus /En·tero·vi·rus/ (en´ter-o-vi?rus cytopathic effects. However, PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) targeted at the 5" untranslated region (UTR UTR Untranslated Region (genetics) UTR Unicode Technical Report UTR Unique Taxpayer Reference (UK Inland Revenue) UTR Unable to Reach UTR Unable to Reproduce UTR University Technical Representative ) of enterovirus (13) was negative, whereas a 5' UTR PCR specific for HPeV (12) was positive. Results of sequencing the VP1 region (12) suggested that K251176-02 was a novel HPeV genotype. Therefore, the full-length sequence was determined. Combinations of consensus primers were used to generate partially overlapping amplicons that covered the complete genome. Amplicons were sequenced according to according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. a primer walking Primer walking is a sequencing method of choice for sequencing DNA fragments between 1.3 and 7 kilobases. Such fragments are too long to be sequenced in a single sequence read using the cyclic dideoxy terminator method (also known as PCR based dideoxy sequencing or Automated Sanger strategy. The 5" UTR was amplified by using the 5" RACE System (Invitrogen, Carlsbad, CA, USA). Because a primer composed of the first 22 nucleotides (nt) of published consensus parechovirus sequences was used to amplify the 5" UTR proximal end, these 22 nt could not be determined with absolute certainty (8). The 3' UTR end was amplified with a tagged oligo-dT primer. The complete genome of K251176-02 was 7,348 nt long, containing a 5' UTR of 708 nt, a large single open reading frame (ORF) of 6,549 nt, and a 3' UTR of 91 nt followed by a poly(A) tract. The full-length sequence of K251176-02 has been deposited in GenBank under accession no. DQ315670. We found a best-match nucleotide identity (14) of 72.2% in the VP1 gene with HPeV2 CT86-6760, which suggests that K251176-02 is most closely related to HPeV2 CT86-6760. Indeed, phylogenetic analysis of the capsid capsid /cap·sid/ (kap´sid) the shell of protein that protects the nucleic acid of a virus; it is composed of structural units, or capsomers. cap·sid n. nucleotide sequence based on Jukes Jukes: see Dugdale, Richard Louis. and Cantor distances showed K251176-02 to cluster with HPeV2 CT86-6760 (Figure 1A). However, the genetic distance was considerable (0.327) and comparable to the genetic distance between HPeV1 Harris and HPeV2 Williamson (0.332). Phylogenetic analysis of the nonstructural region showed that K251176-02 clustered with the HPeV3 prototypes A308-99 and Can82853-01 (Figure 1B). [FIGURE 1 OMITTED] To identify recombination recombination, process of "shuffling" of genes by which new combinations can be generated. In recombination through sexual reproduction, the offspring's complete set of genes differs from that of either parent, being rather a combination of genes from both parents. events between the different HPeV prototypes, a SimPlot analysis was performed on the known full-length nucleotide HPeV genomes against K251176-02. The SimPlot analysis (Figure 2) showed the differential similarity of K251176-02 with HPeV2 CT86-6760 in the highly variable P1 region and with HPeV3 in the more conserved P2-P3 region. This finding may be the result of a recombination event. [FIGURE 2 OMITTED] The secondary structure of the 5' UTR of K251176-02, determined by the MfoM program of Zuker and Turner (http://mfold2.wustl.edu), was predicted to be highly structured and was characterized by a stable hairpin hairpin a secondary structure that occurs in single-strand RNA during protein synthesis in which the strand turns back on itself. The structure is the result of base pairing and hydrogen bond formation. at the proximal end that was also found in known HPeV prototypes (8,11, data not shown). The predicted secondary structure of the 3' UTR of K251176-02 contained the same 1-stem loop organization as the HPeV prototypes and was similar to the secondary structure of HPeV1 Harris and HPeV2 Williamson and CT86-6760 (15). A comparison of the complete ORF of K251176-02 with the HPeV prototypes showed an amino acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins. identity of 86.9% to 90.1% (Table l). This amount is in the same range of amino acid identity as observed between known HPeV protoypes. For the VP1 gene, the greatest amino acid identity was observed with HPeV2 CT86-6760 (80.4%). In the nonstructural region, identity was greater to HPeV3, with 98.1% identity in the polymerase gene (3[D.sup.pol]). Comparison of the deduced amino acid sequence in the capsid region of K251176-02 with the HPeV prototypes showed that the sequences that are predicted to be part of the [beta]-barrel structure (6,10,11) are well conserved in K251176-02. Like HPeV1 and HPeV2, K251176-02 also contained an RGD RGD Rijksgebouwendienst RGD Rat Genome Database RGD Registered Graphic Designer (Canada) RGD Arginine-Glycine-Aspartic Acid RGD Rapid Gas Decompression RGD Reacting Gas Dynamics RGD Range Gate Deception RGD Returned Goods Damaged motif at the C-terminal end of the VP1 gene, which was absent in HPeV3 (6,7,15). K251176-02 also contained the common motifs [X.sub.2]GXGK(S/T S/T Such That S/T Self-Titled (also seen as ST) S/T Short Ton(s) ) and DDLXQ (2C gene), which are predicted to have a helicase function. The active-site cysteine cysteine (sĭs`tēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of mammalian protein. of the protease protease /pro·te·ase/ (pro´te-as) endopeptidase. pro·te·ase n. Any of various enzymes, including the proteinases and peptidases, that catalyze the hydrolytic breakdown of proteins. 3C is in the context of GXCG, and the active site of polymerase 3[D.sup.pol] contains the conserved sequence In biology, conserved sequences are similar or identical sequences that may occur within nucleic acids, proteins or polymeric carbohydrates within multiple species of organism or within different molecules produced by the same organism. YGDD. The well-conserved motifs within the 3[D.sup.pol] gene (KDELR, PSG PSG, n polysomnograph; polygraph performed during sleep. Physiological variables such as pulse, blood pressure, and respiration are monitored and charted. , and FLKR) were also found in K251176-02 (6,9,11). In summary, K251176-02 represents a new genotype in the genus Parechovirus. To confirm that K251176-02 is also a new serotype, a neutralization assay was performed. Table 2 shows that K251176-02 could not be neutralized by antisera directed against HPeV1 Harris, HPeV2 Williamson, and HPeV3 A308-99, which confirms that K251176-02 is a new genotype that can be classified as a fourth HPeV serotype. Conclusions HPeVs are classified in the genus Parechovirus in the family Picornaviridae. The recently identified HPeV3 has been associated with severe illness in young children in several studies (6,7,12). This association has increased the awareness of HPeVs as relevant pathogens in young children. We identified a new HPeV genotype in a stool specimen from a neonate with high fever. Since classification criteria based on genotyping have not been defined for HPeVs, we used the criteria proposed by Oberste et al. (14) for the classification of new enteroviral genotypes. According to these criteria, a new genotype is defined when a best-match nucleotide identity of <70% is found in the VP1 gene. A 70%-75% best-match nucleotide identity indicates further characterization is needed. Therefore, neutralization assays were conducted; these assays showed that K251176-02 did not neutralize with antisera directed against the 3 known HPeV serotypes. This finding indicates that K251176-02 is a new genotype that can be classified as a fourth HPeV serotype. The patient from whom K251176-02 was isolated had high fever but no signs of neonatal sepsis, as has been found in infections with HPeV3 (6,7,12). Previous data suggest differences in severity of disease between the different HPeV serotypes (12); however, more data are needed to elucidate epidemiologic and pathogenic features of the different HPeV serotypes, including K251176-02. HPeV2 CT86-6760 was genotypically as distinct from HPeV2 Williamson as from other HPeV types (Table 1). The existence of 2 genotypically divergent HPeV serotypes 2 is surprising and needs to be elucidated further. This finding, however, argues in favor of a universal typing method that is based on molecular characteristics (genotyping) instead of serotyping, provided classification criteria are well defined. Acknowledgments We thank Georgios Pollakis for his assistance in the phylogenetic analysis and for technical support in sequencing the full-length genome, Rene Minnaar for further technical support, and Hiroyuki Shimizu for antisera used in the neutralization assay. This study was supported by the Department of Medical Microbiology Medical microbiology is a branch of microbiology which deals with the study of microorganisms including bacteria, viruses, fungi and parasites which are of medical importance and are capable of causing diseases in human beings. at the Academic Medical Center, Amsterdam. References (1.) Stanway G, Joki-Korpela P, Hyypia T. Human parechoviruses--biology and clinical significance. Rev Med Virol. 2000; 10:57-69. (2.) Joki-Korpela P, Hyypia T. Parechoviruses, a novel group of human picornaviruses. Ann Med. 2001;33:466-71. (3.) Stanway G, Hyypia T. Parechoviruses. J Virol. 1999;73:5249-54. (4.) Figueroa JP, Ashley D, King D, Hull B. An outbreak of acute flaccid paralysis in Jamaica associated with echovirus type 22. J Med Virol. 1989;29:315-9. (5.) Koskiniemi M, Paetau R, Linnavuori K. Severe encephalitis associated with disseminated echovirus 22 infection. Scand J Infect Dis. 1989;21:463-6. (6.) Ito M, Yamashita T, Tsuzuki H, Takeda N, Sakae K. Isolation and identification of a novel human parechovirus. J Gen Virol. 2004;85:391-8. (7.) Boivin G. Human parechovirus 3 and neonatal infections. Emerg Infect Dis. 2005;11:103-5. (8.) Oberste MS, Maher K, Pallansch MA. Complete sequence of echovirus 23 and its relationship to echovirus 22 and other human enteroviruses. Virus Res. 1998;56:217-23. (9.) Hyypia T, Horsnell C, Maaronen M, Khan M, Kalkkinen N, Auvinen P, et al. A distinct picornavirus picornavirus Any of a group of the smallest known animal viruses. (Pico refers to their small size, rna to their core of RNA.) This group of spheroidal viruses includes viruses that attack the vertebrate intestinal tract and often invade the central nervous system as well group identified by sequence analysis. Proc Natl Acad Sci U S A. 1992;89:8847-51. (10.) Stanway G, Kalkkinen N, Roivainen M, Ghazi gha·zi n. pl. gha·zies Islam 1. A man who has fought successfully against infidels. 2. Often used as a title for such a warrior. F, Khan M, Smyth M, et al. Molecular and biological characteristics of echovirus 22, a representative of a new picornavirus group. J Virol. 1994;68:8232-8. (11.) Ghazi F, Hughes PJ, Hyypia T, Stanway G. Molecular analysis of human parechovirus type 2 (formerly echovirus 23). J Gen Virol. 1998;79:2641-50. (12.) Benschop KSM KSM Kellogg School of Management KSM Korean Service Medal KSM St. Mary's, Alaska (Airport Code) KSM Key Service Message (FIPS) KSM Khalid Shaik Mohammed KSM Knowledge Structure Map , Schinkel J, Minnaar RP, Pajkrt D, Spanjerberg L, Kraakman HC, et al. Human parechovirus infections in Dutch children and the association between serotype and disease severity. Clin Infect Dis. 2006;42:204-10. (13.) Beld M, Minnaar R, Weel a. & adv. 1. Well. n. 1. A whirlpool. 1. A kind of trap or snare for fish, made of twigs. J, Sol C, damen M, van der Avoort H, et al. Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription--PCR with an armored RNA RNA: see nucleic acid. RNA in full ribonucleic acid One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic internal control. J Clin Microbiol. 2004;42:3059-64. (14.) Oberste MS, Michele SM, Maher K, Schnurr D, Cisterna D, Junttila N, et al. Molecular identification and characterization of two proposed new enterovirus serotypes, EV74 and EV75. J Gen Virol. 2004;85:3205-12. (15.) Abed Y, Boivin G. Molecular characterization of a Canadian human parechovirus (HPeV)-3 isolate and its relationship to other HPeVs. J Med Virol. 2005;77:566-70. Address for correspondence: Kimberley S.M. Benschop, Department of Clinical Virology virology, study of viruses and their role in disease. Many viruses, such as animal RNA viruses and viruses that infect bacteria, or bacteriophages, have become useful laboratory tools in genetic studies and in work on the cellular metabolic control of gene expression , Meibergdreef 15, 1105 AZ Amsterdam, the Netherlands; email: k.s.benschop@amc.uva.nl Kimberley S.M. Benschop, * Janke Schinkel, * Manon E. Luken, ([dagger]) Peter J.M. van den Broek, ([dagger]) Matthias F.C. Beersma, ([double dagger]) Negassi Menelik, ([section]) Hetty W.M. van Eijk, * Hans L. Zaaijer, * Christina M.J.E. VandenBroucke-Grauls, * Marcel G.H.M. Beld, * and Katja C. Wolthers * * Academic Medical Center, Amsterdam, the Netherlands; ([dagger]) Primagen, Amsterdam, the Netherlands; ([double dagger]) Leiden University Medical Center The Leiden University Medical Center (Dutch: Leids Universitair Medisch Centrum) or LUMC, is the university hospital affiliated with Leiden University, of which it forms the medical faculty. , Leiden, the Netherlands; and ([section]) BovenlJ Ziekenhuis, Amsterdam, the Netherlands Ms Benschop is a PhD candidate who works at the Academic Medical Center, Amsterdam. Her primary research interests are the clinical and molecular epidemiology molecular epidemiology Molecular medicine An evolving field that combines the tools of standard epidemiology–case studies, questionnaires and monitoring of exposure to external factors with the tools of molecular biology–eg, restriction endonucleases, and pathogenesis of enteroviruses and human parechoviruses.
Table 1. Amino acid identity matrix of all known human parechovirus
(HPeV) full-length sequences *
Target HPeV1 (H) HPeV2 (W) HPeV2 (CT) HPeV3 (A308)
ORF
K251176-02 88.7 86.9 90.1 89.1
HPeV1 (H) -- 88.6 88.1 87.2
HPeV2 (W) -- -- 85.8 85.1
HPeV2 (CT) -- -- -- 86.7
HPeV3 (A308) -- -- -- --
HPeV3 (Can) -- -- --
P1
K251176-02 78.3 78.6 81.3 74.7
HPeV1 (H) -- 81.6 76.4 74.9
HPeV2 (W) -- -- 74.2 73.9
HPeV2 (CT) -- -- -- 73.0
HPeV3 (A308) -- -- -- --
HPeV3 (Can) -- -- -- --
P2-P3
K251176-02 94.3 91.5 94.9 97.0
HPeV1 (H) -- 92.4 94.5 93.8
HPeV2 (W) -- -- 92.2 91.2
HPeV2 (CT) -- -- -- 94.2
HPeV3 (A308) -- -- -- --
HPeV3 (Can) -- -- _ --
Target HPeV3 (Can)
ORF
K251176-02 89.3
HPeV1 (H) 87.3
HPeV2 (W) 85.0
HPeV2 (CT) 86.9
HPeV3 (A308) 98.2
HPeV3 (Can)
P1
K251176-02 75.1
HPeV1 (H) 75.3
HPeV2 (W) 73.9
HPeV2 (CT) 73.5
HPeV3 (A308) 97.4
HPeV3 (Can) --
P2-P3
K251176-02 97.1
HPeV1 (H) 93.9
HPeV2 (W) 91.1
HPeV2 (CT) 94.2
HPeV3 (A308) 98.6
HPeV3 (Can) --
* Amino acid identities for the open reading frame (ORF), capsid region
(P1), and nonstructural region (P2-P3) are based on p-distance models
between K251176-02 (DQ315670) and the HPeV prototypes, HPeV1 (H)
(Harris, S45208), HPeV2 (W) (Williamson, AJ005695), HPeV2 (CT)
(CT86-6760, AF055846), HPeV3 (A308) (A308-99, AB084913), and HPeV3
(Can) (Can82853-01, AJ889918). The full-length sequence of K251176-02
was aligned with the HPeV prototypes by using ClustalW, included in the
Vector NTI Advance 10 software package (Invitrogen, Carlsbad, CA, USA).
Alignment was edited by using GeneDoc software (version 2.6.02). The
matrix was constructed by using MEGA version 3.1. The 5' untranslated
region (UTR) and 3' UTR are excluded from the analysis because the
regions are noncoding.
Table 2. Neutralization assay with LLcMk2 cells *
Antiserum
[alpha]-
Virus [alpha]-HPeV1 (Harris) HPeV2 (Williamson)
HPeV1 Harris -- ++++
HPeV2 Williamson ++++ --
K251181-02 (HPeV3) ++++ ++++
K251176-02 ++++ ++++
Antiserum
Virus [alpha]-HPeV3 (A308-99) Viral controls
HPeV1 Harris ++++ ++++
HPeV2 Williamson ++++ ++++
K251181-02 (HPeV3) -- ++++
K251176-02 ++++ ++++
* Culture isolates of K251176-02, human parechovirus 1 (HPeV1,
echovirus 22) and HPeV2 (echovirus 23) from a reference panel (National
Institute for Public Health and the Environment, Bilthoven, the
Netherlands) and K251181-02 that was previously genotyped as HPeV3 (12)
were incubated with antisera (20 U/mL in Eagle minimal essential
medium) directed against HPeV1 Harris, HPeV2 Williamson, and HPeV3
A308-99. The antisera to HPeV1 and HPeV2 were raised in horses. The
antiserum to HPeV3 was raised in guinea pigs. Neutralization is done on
a 96-microtiter plate containing a monolayer of LLcMk2 cells that have
been incubated for 3 days. The assay was determined after viral
controls (no antisera used) of the 4 culture isolates showed cytopathic
effects >50% (++++).
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