Identification and Characterization of Two Strains of Human Parechovirus 4 Isolated from Two Clinical Cases in Fukuoka City, Japan

Abstract

Reverse transcription-PCR targeting the VP0 gene of human parechoviruses (HPeVs) was used to identify two isolates from two Japanese children's stool specimens. Molecular analysis revealed that these isolates belonged to HPeV type 4, and their nucleotide identity in the P1 region was 85.0%.Human parechoviruses (HPeVs), members of the Parechovirus genus of the Picornaviridae family, are genetically classified into six types. HPeVs have been associated with gastrointestinal and respiratory symptoms and also with severe symptoms such as transient paralysis and neonatal sepsis in young children (3,5). HPeV type 1 (HPeV1) and HPeV3 infections are common worldwide (5, 6); however, other HPeV infections, especially HPeV4 infections, are less common. HPeV4 strains have been isolated from young children with fever, TORCH syndrome, and lymphadenitis in a few instances (2, 10, 11).This paper describes the identification and characterization of HPeV4 strains using molecular and immunological techniques. In 2001 and 2005, we isolated viruses from stool specimens from a 1-year-old girl with herpangina and from a 3-year-old boy with acute gastroenteritis, respectively, in Fukuoka City, Japan. The stool specimens were cultured weekly at 37°C for three passages on Caco-2, RD-18S, VeroE6, and HEp-2 cells. Picornaviridae-like cytopathic effects were observed in the Caco-2, RD-18S, and VeroE6 cell lines. To identify these isolates, a molecular typing method based on reverse transcription-PCR (RT-PCR) and direct sequencing was carried out. Viral RNAs were extracted from cell culture supernatants by using a QIAamp viral RNA mini kit (Qiagen, Germany). RT-PCRs were performed with specific primers (187/011, 188/011, and 189/011 for the enterovirus VP1 gene and E23P1/HPV-N1 for the HPeV VP0 gene) (5, 9). Table 1 shows the molecular typing results from the cell culture samples. In case 1, the Caco-2 and RD-18S cell culture samples were positive by RT-PCR for enterovirus, and human coxsackievirus group A type 2 was identified by direct sequencing of the VP1 amplicon. In both case 1 and 2, all of the Caco-2, RD-18S, and VeroE6 culture samples were positive for HPeV by RT-PCR. The VeroE6 culture sample from case 1 and the Caco-2 culture sample from case 2 were designated Fuk2001-282 and Fuk2005-123, respectively. These amplicon sequences were compared with sequences from HPeV reference strains HPeV1 Harris, HPeV2 Williamson, HPeV3 A308-99, HPeV4 K251176-02, HPeV4 T75-4077, HPeV5 T92-15, and HPeV6 NII561-2000.Both Fuk2001-282 and Fuk2005-123 showed high similarities to the VP0 gene of the HPeV4 reference strains (positions 710 to 1,339 on HPeV1 Harris); the nucleotide identities of Fuk2001-282 to HPeV4 K251176-02 and HPeV4 T75-4077 were 84.9% and 89.5%, respectively, and those of Fuk2005-123 to K251176-02 and T75-4077 were 85.4% and 85.2%, respec-

Detection of human parechovirus in stool samples collected from children with acute gastroenteritis in Japan during 2007–2008

“…There have been four genotypes of HPeVs found in Japan up to date. They are HPeV1, HPeV3, HPeV4, and HPeV6 [Takao et al, 2001; Ito et al, 2004; Watanabe et al, 2007; Wakatsuki et al, 2008]. By this study, two different types of HPeVs, HPeV1 and HPeV3, were identified in Japanese infants and children with acute gastroenteritis, and the majority of the strains studied belonged to HPeV1.…”

Evolution and conservation in human parechovirus genomes

“…Several diagnostic laboratories now screen for HPeV infection and together with the retrospective analysis of archived material, a growing number of HPeV associations with disease have been reported recently (Baumgarte et al, 2008;Benschop et al, 2006aBenschop et al, , 2008bBoivin et al, 2005;De Souza Luna et al, 2008;De Vries et al, 2007;Drexler et al, 2009;Harvala et al, 2008Harvala et al, , 2009Li et al, 2009;Tauriainen et al, 2008;Wakatsuki et al, 2008;Wolthers et al, 2008). A greater understanding of the biology and molecular biology of these viruses is therefore required.…”

Emergence of Parechovirus A4 Central Nervous System Infections among Infants in Kansas City, Missouri, USA

“…The fourth human parechovirus type (PeV-A4) was identified by virus neutralization assays in 2006 (11) in a stool sample of a 6-day-old infant with fever and feeding problems (12,13). PeV-A4 is primarily associated with relatively mild infections of the gastrointestinal and respiratory tracts (13).…”