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Human parechovirus 1 (HPEV-1) is a prototype member of parechoviruses, a recently established picornavirus genus. Although there is preliminary evidence that HPEV-1 recognizes ␣ V integrins as cellular receptors, our understanding of early events during HPEV-1 infection is still very limited. The aim of this study was to clarify the entry mechanisms of HPEV-1, including the attachment of the virus onto the host cell surface and subsequent internalization. In blocking experiments with monoclonal antibodies against different receptor candidates, antibodies against ␣ V and ␤ 3 integrin subunits, in particular in combination, appeared to be the most efficient ones in preventing the HPEV-1 infection. To find out whether HPEV-1 uses clathrin-coated vesicles or other routes for the entry into the host cell, we carried out double-labeling experiments of virus-infected cells with anti-HPEV-1 antibodies and antibodies against known markers of the clathrin and the caveolin routes. At the early phase of infection (5 min postinfection [p.i.]) HPEV-1 colocalized with EEA1 (early endosomes), and later, after 30 min p.i., it colocalized with mannose-6-phosphate receptor (late endosomes), whereas no colocalization with caveolin-1 was observed. The data indicate that HPEV-1 utilizes the clathrin-dependent endocytic pathway for entry into the host cells. Interestingly, endocytosed HPEV-1 capsid proteins were observed in the endoplasmic reticulum and cis-Golgi network 30 to 60 min p.i. Depolymerization of microtubules with nocodazole inhibited translocation of the virus to the late endosomes but did not block HPEV-1 replication, suggesting that the RNA genome may be released early during the entry process.Early events in viral infection include specific attachment of the virion onto the cell surface receptor(s) followed by entry into the cell and subsequent release of the genome. Successful completion of this process is a prerequisite for the initiation of the infection cycle, and these events play an important role in tissue tropism and pathogenesis. Recently, numerous cell surface molecules, with wide variation in their structures and normal physiological functions, have been identified as virus receptors.The routes by which extracellular ligands, including viruses, are internalized into the cell include clathrin-mediated endocytosis, uptake via caveolae, macropinocytosis, phagocytosis, and other pathways that presently are poorly characterized. For some virus systems, the entry events have been described in detail. For instance, both adenovirus type 2, a nonenveloped DNA virus, and Semliki Forest virus, an enveloped RNA virus, enter the host cells through a clathrin-mediated pathway (7,8,19,39,42). Different members of the polyomavirus family are known to enter the host cell by distinct mechanisms; simian virus 40 uses the caveola-dependent endocytic route, while the human polyomavirus JC virus enters the cells through clathrinmediated endocytosis (24, 32).Picornaviruses include several important human pathogens which belong to ...

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Parechoviruses are a recently established group of human viral pathogens. At the time of their first isolation, parechoviruses were classified among the enterovirus genus in the picornavirus family, but based on their different biological properties they were separated into their own genus. The type member is human parechovirus 1 (HPEV1), which frequently infects humans, in particular small children. The parechovirus genus also includes HPEV2 and the Ljungan virus, which was recently isolated from rodents, is a candidate for the group. Seroepidemiological studies have shown that the prevalence of HPEV1 antibodies is surprisingly high, exceeding 95% in adult populations. According to present data, HPEV1 causes mainly gastrointestinal and respiratory infections; however, severe disease conditions, such as myocarditis and encephalitis, have also been reported. HPEV2 infections appear to be rare, and it is currently not known whether the Ljungan virus can infect humans.

Human parechoviruses 1 and 2 (HPEV1 and HPEV2, respectively), formerly known as echoviruses 22 and 23, have been assigned to a novel picornavirus genus on the basis of their distinct molecular and biological properties. To study the immunological characteristics of HPEV1 capsid proteins, antigenic analysis was carried out by a peptide scanning technique, which can be used to identify the immunogenic peptide sequences of a protein. Partially overlapping peptides, representing the capsid of HPEV1, were synthesized using a 12 aa window in a three residue shift and reactivity of rabbit and murine HPEV1 antisera against these peptides were tested. Using this method, an antigenic site in the VP0 polypeptide, recognized by both rabbit and murine antisera, was identified. The sequence of this region was conserved among HPEV1 clinical isolates obtained from Finland and the United States. Antiserum against this peptide region showed neutralizing activity against HPEV1 in cell culture. Because the C-terminal region of HPEV1 VP1 contains a functional RGD motif, the antigenicity of this region was also tested. By using the corresponding peptide antiserum, neutralization of HPEV1 was observed. Cross-neutralization between HPEV1 and coxsackievirus A9, an enterovirus with a similar RGD motif in VP1, was also detected.

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A qualitative multiplex reverse transcription (RT)-PCR and liquid hybridization assay for the detection of human enteroviruses, rhinoviruses, parechoviruses, and Aichi virus was developed. Furthermore, a separate assay for the recognition of hepatitis A virus was established to complement the test pattern so that all human picornaviruses were covered. The amplicons, which represented the 5 untranslated regions of the viral RNA genomes, were identified in liquid hybridization reactions with genus-specific digoxigenin-labeled oligonucleotide probes. The sensitivity of the multiplex RT-PCR and liquid hybridization assay was 10 to 100 picornavirus genome equivalents for representatives of each picornavirus genus. The hepatitis A virus assay exhibited a sensitivity of 10 genome copies. Both the uniplex and the multiplex tests were highly specific for the target viruses. Twenty-three clinical samples, including cerebrospinal fluid, serum, and nasopharyngeal swab specimens, were used for clinical evaluation of the multiplex RT-PCR assay. The results obtained were consistent with the results of routine virus diagnostic assays. Furthermore, the assay was used to screen 68 stool specimens for the presence of parechoviruses and Aichi virus. One sample was found to contain parechovirus RNA, whereas no Aichi virus was detected. The assay described here can be applied for the efficient identification of human enteroviruses and rhinoviruses in clinical specimens and simultaneously enables the collection of information on the epidemiology and clinical outcomes of infections caused by the currently poorly known human parechoviruses and Aichi virus.Picornaviruses include several important human and veterinary pathogens. The large genus of human enteroviruses (HEVs; 64 serotypes) includes polioviruses, coxsackie A and B viruses, echoviruses, and enterovirus types 68 to 71. Although polioviruses will be eradicated in the near future, other HEVs remain a significant cause of morbidity and are responsible for a wide spectrum of clinical symptoms, ranging from mild respiratory infections to severe disease conditions such as myocarditis, meningitis, and encephalitis (6). Human rhinoviruses (HRVs; more than 100 serotypes) are the major cause of the common cold, and they have also been described to have a role in lower respiratory tract infections and exacerbations of asthma (11,16). Despite the availability of vaccines, hepatitis A virus (HAV; a hepatovirus) infections still occur widely. Human parechoviruses (HPEVs), earlier included in the HEVs, currently form their own genus. They appear to cause mainly gastroenteritis and respiratory tract infections, but central nervous system infections have also been associated with these viruses (22). The antibody prevalence of HPEV type 1 (HPEV1) in the Finnish population is greater than 90%, which suggests that the infections may be mild or asymptomatic and underdiagnosed (12). The only known member of the recently established kobuvirus genus, Aichi virus (AV), has been detected in patients wi...

Our findings confirm the association between previous exposure to C. trachomatis and ectopic pregnancy. We found no association between C. trachomatis seropositivity and miscarriage or preterm birth.