Genomic Characterization of Novel Human Parechovirus Type

Using a simple metagenomic approach, we identified a divergent human parechovirus (HPeV) in the stool of a child in Pakistan. Genomic characterization showed this virus was distinct enough from reported HPeV types to qualify as candidate prototype for the seventh HPeV type.

Using a simple metagenomic approach, we identifi ed a divergent human parechovirus (HPeV) in the stool of a child in Pakistan. Genomic characterization showed this virus was distinct enough from reported HPeV types to qualify as candidate prototype for the seventh HPeV type.
H uman parechoviruses (HPeVs) belong to the recently identifi ed genus Parechovirus of the family Picornaviridae. Serologic and molecular studies show that HPeV comprises 6 neutralization serotypes or corresponding types based on capsid protein similarities, HPeV1-6. HPeV1 and HPeV2, originally known as enterovirus echoviruses 22 and 23, were isolated in 1956 (1). Because echoviruses 22 and 23 had serologic, molecular, and biologic properties highly distinct from other enteroviruses, they were reclassifi ed in 1999 as members of the genus Parechovirus (2). The other 4 parechovirus types were identifi ed more recently from young children with clinical manifestations similar to those caused by human enteroviruses: HPeV3 in 2002 (3), HPeV4 in 2005 (4), and HPeV5 (5) and HPeV6 (6) in 2006. HPeV infections occur commonly in the general population and mostly cause mild gastrointestinal and respiratory symptoms in young children (7). More severe consequences also have been ascribed to HPeV infections, including acute fl accid paralysis (AFP) (3), encephalitis (8), aseptic meningitis (9), myocarditis (10), neonatal sepsis (11), and Reye syndrome (6), Nonpolio AFP may be caused by many viruses, including nonpolio enteroviruses, human adenoviruses, herpes simplex virus, Epstein-Barr virus, and West Nile virus (12). HPeV1 was associated with an AFP outbreak in Jamaica in 1986. In 2 of 3 AFP patients with HPeV1 detected in stool samples, antibody titer also increased signifi cantly (13). HPeV6 was isolated from the stool specimen of an AFP patient in Japan in 2001 (6). HPeV3 has not been reported from AFP cases but was identifi ed in 1 transient paralysis case and believed to cause serious central nervous system symptoms more frequently than HPeV1 (3,7). HPeV types 2, 4, and 5 have been less often observed in clinical studies.
Using sequence-independent PCR amplifi cation and sequence similarity searches, we recently investigated virus sequences in stool samples from children in Pakistan who had nonpolio AFP and from healthy children who had close contacts with persons who had AFP. Sequences of human parechoviruses were identifi ed in samples from 6 of 65 persons. Analysis showed 5 HPeV infections in 56 samples from persons who had nonpolio AFP, 1 HPeV1, 1 HPeV5, and 2 HPeV6; in 1 sample, HPeV type could not be determined because the sequenced fragment was located in a phylogenetically uninformative region. A highly divergent HPeV type also was identifi ed in 1 contact sample, and the full genome of this virus was sequenced. Phylogenetic analysis indicated that this virus, designated PAK5045, has the genetic characteristics expected of a new HPeV type.

The Study
HPeV PAK5045 was found in 1 stool sample from a healthy 2-year-old boy who had close contact with a person who had nonpolio AFP, using a previously described method applied here to stool samples (14). Briefl y, virus nucleic acids were purifi ed from stool samples, randomly amplifi ed by reverse transcription (RT)-PCR using 3′ randomized RT and PCR primers, subcloned, and sequenced. HPeV sequences were abundant in 1 sample with 24/48 plasmid subclones identifi ed as PAK5045. Assembly of these HPeV sequences produced 5 fragments covering ≈75% of the genome. Specifi c PCR primers were used to link these genome fragments, and rapid amplifi cation of cDNA ends was carried out to acquire the 5′ and 3′ ends.
The nucleotide sequence of PAK5045 virus was 7,127 nt, excluding a poly (A) tail. PAK5045 contained a partial 5′ untranslated region (UTR) of 511 nt, an open reading frame (ORF) encoding a putative polyprotein precursor of 2,175 aa, and a 3′ UTR of 88 nt. The nucleotide sequence of PAK5045 was generated with at least 2× coverage except for the 5′ UTR. The full-length sequence of PAK5045 has been deposited in GenBank under accession no. EU556224.
The  (Table 1). Of the 6 known HPeV types, the intertype amino acid identities ranged from 84.9% to 91.1%, and the intertype nucleotide identities of the ORF sequence ranged from 76.1% to 83.4% (data not shown), a range similar to their identities relative to PAK5045.
Phylogenetic analysis with the complete P1 amino acid sequences of fully sequenced HPeVs confi rmed the existence of the 6 types defi ned by previous studies (Figure 1, panel A) (15). PAK5045 virus was most similar to HPeV3 strains. The identity of P1 amino acid sequences between PAK5045 virus and both HPeV3 strains analyzed was 77.5%, which  was lower than some HPeVs intertype amino acid identities (e.g., average of 82.4% between HPeV1 and HPeV2, 78.3% between HPeV1 and HPeV4, 80.4% between HPeV1 and HPeV6, 78.4% between HPeV2 and HPeV6, and 80.5% between HPeV4 and HPeV5) (data not shown). Consistent with the P1 amino acid tree, phylogenetic analysis with VP1 capsid proteins also showed 6 established types (Figure 1, panel B) (5). PAK5045 VP1 was slightly closer to type 3 strains, with the greatest amino acid identity being 78.8% (Table 2), and more divergent from the other established HPeV types. We retrieved from Gen-Bank, and then analyzed, genetic relationships among 92 full-length VP1 amino acid sequences and with PAK5045. None clustered with PAK5045 as a close genetic lineage. The amino acid identities between PAK5045 and HPeV3 strains ranged from 69.9% to 78.8%, outside the HPeV3 intratype range of 85.8%-100% ( Table 2).
To identify recombination events between the different HPeV types, we performed SimPlot analysis (http:// sray.med.som.jhmi.edu/SCRoftware/simplot) of the 6 complete nucleotide HPeV prototype genomes against PAK5045 (Figure 2). In general, PAK5045 was closer to HPeV3 and HPeV4 than to the other viruses. PAK5045 showed a relatively higher degree of nucleotide similarity to HPeV3 A308/99 in the P1 region consistent with the P1 phylogenetic tree. Downstream of nucleotide position 3600, HPeV4 K251176-02 became the closest relative of PAK5045 in most of the nonstructural (P2/3) region, which suggests an ancient recombination event.

Conclusions
We identifi ed and characterized a novel HPeV type from the stool sample of a healthy child who had been in close contact with a person who had nonpolio AFP. The genome sequence diverged suffi ciently from the 6 known HPeVs to qualify as a candidate for the prototype of HPeV7. Using only a low-level shotgun sequencing method, we detected HPeVs in 9% (6/65) of stool samples from patients with nonpolio AFP, including HPeV types 1, 5, 6, and 7. A more sensitive method, such as HPeV-directed RTnested PCR, is likely to have detected a higher prevalence. The median age of sampled patients was 3 years (range 1 month-15 years), and all HPeV-positive patients were <3 years of age.
In a previous study, HPeVs were isolated from 0.3% of 13,656 various clinical samples collected in Japan (14 HPeV1, 16 HPeV3, 10 HPeV6, and 1 HPeV4) (6). In Germany, the detection rate of HPeVs did not differ significantly between patients with acute diarrhea and controls, with 11.6% (7/60) of children <2 years of age being HPeV positive (15). In a Dutch study of 303 isolates showing cytopathic effects consistent with enterovirus infection, 12% were HPeV positive, with 27 HPeV1 and 10 HPeV3, all in children <3 years of age (7). HPeV infection, therefore, seems to be associated with young children (<3 years). More studies are needed to associate HPeV infection (with any genotypes) with development of neurologic disease, such as AFP.