Human parechoviruses (HPeVs) are widespread pathogens belonging to the Picornavirus family. The aim of the present study was to assess the prevalence and genetic diversity of HPeV in Shanghai, China, during a HPeV screening program in 2008 and 2009. Of 300 stool samples from children under the age of 5 years with acute diarrhea seen at Children's Hospital, Fudan University, Shanghai, China, 165 (55%) were HPeV-positive. The median age of infected children was 3 months. The prevalence of HPeV was high (57%) in infants up to 2 years old but dropped to 30.4% in children between 2 and 5 years old. The prevalence did not differ by sex. Infections were present throughout the year but peaked in July and August. The most predominant genotype was HPeV1. Of the 139 strains, 4 were found in 9 samples: HPeV4 (n = 4), HPeV5 (n = 1), HPeV6 (n = 1), and HPeV8 (n= 3). This study provided useful data on the epidemiology of HPeV infection by documenting the distribution of genotypes, age of infection, and seasonal patterns in Shanghai, China.

Between April 1999 and March 2008, a total of 4,976 stool specimens collected from patients with suspected viral infection through infectious agent surveillance in Aichi, Japan, were tested for the presence of human parechoviruses (HPeVs). We detected HPeVs in 110 samples by either cell culture, reverse transcriptase PCR (RT-PCR), or both. Serotyping either by neutralization test or by nucleotide sequence determination and phylogenetic analysis of the VP1 region and 5' untranslated region (5'UTR) regions revealed that 63 were HPeV type 1 (HPeV-1), followed by 44 HPeV-3 strains, 2 HPeV-4 strains, and 1 HPeV-6 strain. The high nucleotide and amino acid sequence identities of the Japanese HPeV-3 isolates in 2006 to the strains previously reported from Canada and Netherlands confirmed the worldwide prevalence of HPeV-3 infection. Ninety-seven percent of the HPeV-positive patients were younger than 3 years, and 86.2% younger than 12 months. The clinical diagnoses of HPeV-positive patients were gastroenteritis, respiratory illness, febrile illness, exanthema,"hand, foot, and mouth disease," aseptic meningitis, and herpangina. Among 49 HPeV-positive patients with gastroenteritis, 35 were positive with HPeV-1 and 12 with HPeV-3, and out of 25 with respiratory illness, 11 were positive with HPeV-1 and 14 with HPeV-3. HPeV-3 seemed to be an important etiological agent of respiratory infection of children. While HPeV-1 was detected predominantly during fall and winter, the majority of the HPeV-3 cases were detected during summer and fall. A different pattern of clinical manifestations as well as seasonality suggested that there are different mechanisms of pathogenesis between HPeV-1 and HPeV-3 infections.

HASH(0x2b7088baf840)

BACKGROUND: From December 1987 through August 2004, lung tissue, nasopharyngeal swabs, and colon swab specimens obtained during 1263 autopsies of infants and young children were examined to assess the role of viruses in deaths of children aged <2 years. METHODS: Multiple cell cultures were used to isolate viruses. With 4 exceptions, virus isolates were identified by neutralization, immunofluorescence assay, or enzyme immunoassay. RNA extracted from these 4 isolates and associated autopsy specimens was tested using parechovirus-specific real-time polymerase chain reaction (RT-PCR) and sequencing assays. RESULTS: Specimens from 426 (34%) autopsies were positive for at least 1 virus; enteroviruses and adenoviruses were the most commonly identified. Human parechoviruses (HPeVs) were identified antigenically in isolates from 18 decedents (all HPeV type 1) and by RT-PCR in isolates and multiple autopsy specimens from 4 decedents with untypeable virus isolates. Sequencing of the VP1 region identified these 4 HPeVs as HPeV type 3 (n = 3) and HPeV type 6 (n = 1). Despite the detection of HPeV, the deaths of decedents 3 and 4 were determined to have been from noninfectious causes. CONCLUSIONS: These are the first confirmed HPeV type 3 and HPeV type 6 detections in the United States. This is also the initial report of fatal cases with associated HPeV type 3 infection. These results support prior findings associating HPeVs with serious disease in young children. Clinical testing for HPeVs and routine HPeV surveillance by public health laboratories will help determine the burden of disease caused by HPeVs.

BACKGROUND:: Human parechoviruses (HPeVs) and enteroviruses (EVs) belong to the family Picornaviridae. EVs are known to cause a wide range of disease such as meningitis, encephalitis, and sepsis. HPeV1 and 2 have been associated with mild gastrointestinal or respiratory symptoms in young children. HPeV3 is associated with more severe neonatal infection. Little is known about the epidemiology and pathology of HPeV4-6 in children. METHODS:: We evaluated the clinical symptoms of the children with an HPeV 4, 5, or 6 infection. The patients with positive HPeV4-6 in stool samples were selected and available plasma or cerebrospinal fluid samples from these patients were tested for HPeV. Data on clinical symptoms, diagnosis, presence and duration of fever, medical history, mean age, use of antibiotics of the children infected with HPeV4-6 were retrospectively documented. RESULTS:: HPeV4-6 were found in 31 of the 277 HPeV positive children (11%). Coinfection with EV was seen in 8 patients. Fever was seen in 13 (42%) patients. Of the HPeV4-6 positive patients, 20 of the 31 children (64%) presented with gastrointestinal complaints and 18 of 31 (58%) patients had respiratory symptoms. The mean age was 14 months, 58% of the patients had an underlying disorder such as bronchomalacia or a cardiac disorder. CONCLUSIONS:: Symptomatic HPeV4-6 infections are seen in relative young children and are associated with respiratory and/or gastrointestinal symptoms. HPeV type 4 was detected more frequently than HPeV types 5 and 6.

Background. Human parechoviruses (HPeVs), along with human enteroviruses (HEVs), are associated with neonatal sepsis and meningitis. We determined the relative importance of these viruses and the specific HPeV types involved in the development of central nervous system-associated disease. Methods. A total of 1575 cerebrospinal fluid (CSF) samples obtained during 2006-2008 were screened for HPeV by means of nested polymerase chain reaction. All samples for which results were positive were typed by sequencing of viral protein (VP) 3/VP1. Screening for HEV was performed in parallel, as was detection of HPeV in respiratory and fecal surveillance samples, to identify virus types circulating in the general population. Results. HPeV was detected in 14 CSF samples obtained exclusively from young infants (age,<3 months) with sepsis or pyrexia. The frequency of detection of HPeVs varied greatly by year, with the highest frequency (7.2%) noted in 2008 exceeding that of HEVs. Direct typing of CSF samples revealed that all infections were caused by HPeV type 3, a finding that is in contrast to the predominant circulation of HPeV1 in contemporary respiratory and fecal surveillance samples. Conclusion. HPeV was a significant cause of severe sepsis and fever with central nervous system involvement in young infants, rivaling enteroviruses. The specific targeting of young infants by HPeV type 3 may reflect a difference in tissue tropism between virus types or a lack of protection of young infants by maternal antibody consequent to the recent emergence of HPeV.

Human parechoviruses (HPeVs) are members of the large and growing family of Picornaviridae. Although originally described as echovirus 22 and 23 within human enteroviruses because of their clinical and morphological properties, they have since been shown to be distinct from this and other picornavirus groups in several features of their genome organisation, structure and replication. Human parechoviruses show genetic and antigenic heterogeneity and a number of distinct HPeV types are known to circulate widely in human populations throughout the world. Although the majority of HPeV infections occur early in life without specific symptoms, disease manifestations associated with many of the currently described types have been described, ranging from gastroenteritis and respiratory infections to neurological disease, particularly in neonates. Although HPeV diagnosis has historically been made by virus isolation, a new generation of sensitive and specific molecular tests for HPeV RNA promises to greatly improve the effectiveness of HPeV detection and type identification, as well as providing a greater understanding its molecular epidemiology. By this means, we will learn much more about the clinical relevance of HPeVs and the association of different HPeV types with specific disease presentations.

Human parechoviruses (HPeVs) are frequent pathogens with seroprevalance of over 90% in adults. Recent studies on these viruses have increased the number of HPeV types to 6. Here we analyse the complete genome of one clinical isolate, PicoBank/HPeV1/a, and VP1 and 3D protein sequences of PicoBank/HPeV6/a, isolated from the same individual 13 months later. The sequences indicate that this individual was first infected with an HPeV1 strain, and then an HPeV6 strain, showing that the first infection was not cross-protecting. PicoBank/HPeV1/a is closely related to another recent HPeV1 isolate, BNI-788St, but is distinct from the HPeV1 Harris prototype isolated 50 years ago. The availability of an increasing number of HPeV sequences has allowed a detailed analysis of these viruses. The results add weight to the observations that recombination plays a role in the generation of HPeV diversity. An important finding is the presence of unexpected conservation of codons utilised in part of the 3D-encoding region, some of which can be explained by the presence of a phylogenetically-conserved predicted secondary structure domain. This suggests that in addition to the cis-acting replication element, RNA secondary structure domains in coding regions may play a key role in picornavirus replication.

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.

Viruses are important pathogens causing respiratory tract infections both in the community and health-care facility settings. They are extremely common causes of morbidity in the competent hosts and some are associated with significant mortality in the compromised individuals. With wider application of molecular techniques, novel viruses are being described and old viruses are found to have new significance in different epidemiological and clinical settings. Some of these emerging pathogens may have the potential to cause pandemics or global spread of a severe disease, as exemplified by severe acute respiratory syndrome and avian influenza. Antiviral therapy of viral respiratory infections is often unnecessary in the competent hosts because most of them are self-limiting and effective agents are not always available. In the immunocompromised individuals or for infections caused by highly pathogenic viruses, such as avian influenza viruses (AIV), antiviral treatment is highly desirable, despite the fact that many of the agents may not have undergone stringent clinical trials. In immunocompetent hosts, antiviral therapy can be stopped early because adaptive immune response can usually be mounted within 5-14 days. However, the duration of antiviral therapy in immunosuppressed hosts depends on clinical and radiological resolution, the degree and duration of immunosuppression, and therefore maintenance therapy is sometimes needed after the initial response. Immunotherapy and immunoprophylaxis appear to be promising directions for future research. Appropriate and targeted immunomodulation may play an important adjunctive role in some of these infections by limiting the extent of end-organ damage and multi-organ failure in some fulminant infections.

Summary: Clinical laboratories historically diagnose seven or eight respiratory virus infections using a combination of techniques including enzyme immunoassay, direct fluorescent antibody staining, cell culture, and nucleic acid amplification tests. With the discovery of six new respiratory viruses since 2000, laboratories are faced with the challenge of detecting up to 19 different viruses that cause acute respiratory disease of both the upper and lower respiratory tracts. The application of nucleic acid amplification technology, particularly multiplex PCR coupled with fluidic or fixed microarrays, provides an important new approach for the detection of multiple respiratory viruses in a single test. These multiplex amplification tests provide a sensitive and comprehensive approach for the diagnosis of respiratory tract infections in individual hospitalized patients and the identification of the etiological agent in outbreaks of respiratory tract infection in the community. This review describes the molecular methods used to detect respiratory viruses and discusses the contribution that molecular testing, especially multiplex PCR, has made to our ability to detect respiratory viruses and to increase our understanding of the roles of various viral agents in acute respiratory disease.

Background.@nbsp; Enteroviruses (EVs) belong to the family Picornaviridae and are a well-known cause of neonatal sepsis and viral meningitis. Human parechoviruses (HPeVs) type 1 and 2, previously named echovirus 22 and 23, have been associated with mild gastrointestinal or respiratory symptoms in young children. Six HPeV genotypes are currently known, of which HPeV3 is associated with neonatal sepsis and meningitis. Methods.@nbsp; Cerebrospinal fluid samples from children aged <5 years previously tested by EV-specific polymerase chain reaction (PCR) during 2004-2006 were selected ([Formula: see text]). Samples from 716 of those children were available for retrospective testing by HPeV-specific real-time PCR. The prevalence of EV and HPeV in these samples was compared. Data on clinical presentation of children infected with HPeV were retrospectively documented. Results.@nbsp; HPeV was found in cerebrospinal fluid samples from 33 (4.6%) of the children. Yearly prevalence of HPeV in cerebrospinal fluid varied remarkably: 8.2% in 2004, 0.4% in 2005, and 5.7% in 2006. EV was detected in 14%(108 of 761 samples), with no variation in yearly prevalence. Children with HPeV in cerebrospinal fluid presented with clinical symptoms of sepsislike illness and meningitis, which led to hospitalization and antibiotic treatment. Conclusion.@nbsp; EV-specific PCRs do not detect HPeVs. The addition of an HPeV-specific PCR has led to a 31% increase in detection of a viral cause of neonatal sepsis or central nervous system symptoms in children aged <5 years. HPeV can be considered to be the second cause of viral sepsis and meningitis in young children, and rapid identification of HPeV by PCR could contribute to shorter duration of both antibiotic use and hospital stay.

Background.@nbsp; Human parechovirus (HPeV) 1 is a common virus that infects almost everyone during childhood. Because clinical symptoms are poorly documented, we evaluated the symptoms associated with HPeV1 infection in a cohort of children followed prospectively from birth at 3-month intervals. Methods.@nbsp; Symptoms such as fever, cough, those of the common cold, otitis media, and gastroenteritis were determined from hospital records and from questionnaires administered to the parents of 59 children during regular study visits. HPeV1 infections were diagnosed by measuring neutralizing antibodies in follow-up serum samples. Additionally, HPeV RNA was analyzed in middle ear fluid (MEF) and nasopharyngeal aspirate samples from 33 patients with otitis media by reverse-transcription polymerase chain reaction. Results.@nbsp; Otitis media showed a clear association with HPeV1 infection-it developed in 50% of the 3-month follow-up periods that yielded evidence for HPeV1 infection but in only 14% of the HPeV1-negative periods (odds ratio [OR], 6.14 [95% confidence interval {CI}, 2.75-13.77]). In children with recurring otitis media, MEF samples were positive for HPeV in 15% of episodes. Cough was also associated with HPeV1 infection, but this association was weaker (OR, 3.67 [95% CI, 1.66-8.09]). Other symptoms were not linked to HPeV1 infection. Conclusions.@nbsp; HPeV1 infections are common in childhood and may cause otitis media and cough.

BACKGROUND: Human parechoviruses (HPeVs) have been associated with severe conditions such as neonatal sepsis and meningitis in young children. Rapid identification of an infectious agent in such serious conditions in these patients is essential for adequate decision making regarding treatment and hospital stay. OBJECTIVES: We have developed an HPeV specific real-time PCR assay based on the conserved 5'untranslated region. STUDY DESIGN: To determine the detection limit of the assay, serial dilutions of HPeV in vitro RNA were tested in a background of HPeV and EV RNA-negative cerebrospinal fluid (CSF). The specificity was tested by analyzing culture isolates of HPeV 1-6, enterovirus (EV) types, human rhinoviruses (HRVs) and hepatitis A virus (HAV). To establish diagnostic relevance, 522 CSF samples from children <5 years were tested. RESULTS: The detection limit of the assay was 75 copies of HPeV cDNA per reaction. The assay was highly specific for HPeV, detecting all HPeV types. We identified HPeV infections in CSF of 20 children (3.8%), all with severe conditions such as sepsis and meningitis. CONCLUSIONS: These results suggest that HPeV screening of paediatric clinical samples should be included in viral diagnostics in suspected cases of neonatal sepsis and meningitis.

Human parechoviruses (HPeVs), members of the family Picornaviridae, are classified into six types. To investigate the dynamics and likelihood of recombination among HPeVs, we compared phylogenies of two distant regions (VP1 and 3Dpol) of 37 HPeV isolates (types 1 and 3-5) and prototype sequences (types 1-6). Evidence for frequent recombination between HPeV1, 4, 5 and 6 was found. The likelihood of recombination was correlated with the degree of VP1 divergence and differences in isolation dates, both indicative of evolutionary times of divergence. These temporal dynamics were found to be most similar to those of human enterovirus species B variants. In contrast, HPeV3 remained phylogenetically distinct from other types throughout the genome. As HPeV3 is equally divergent in nucleotide sequence from the other HPeV types, its genetic isolation may reflect different biology and changed cellular tropisms, arising from the deletion of the RGD motif, and likely use of a non-integrin receptor.

Parechovirus epidemiology and disease association are not fully understood. Real-time RT-PCR for all HPeV was applied on stool samples from two groups of patients. Both contained patients with acute enteritis of all age groups, seen during one full year. Patients with norovirus, adenovirus, enterovirus, astrovirus and rotavirus infection were excluded. In 118 patients from outbreak and hospital settings, no HPeV was detected. In a prospective study group of 499 non-hospitalized patients, detection rate was 1.6%. One virus-positive patient occurred in 39 control patients. Positive samples occurred only in summer and autumn. Only one patient had accompanying respiratory symptoms. Association with travel or animal contact was not found. All positive patients except one were <2 years of age, with neutral gender ratio. In children <2 years of age, detection rate was 11.6%(7 of 60 children). The range of viral loads was 3170 - 503,377,290 copies per gram or millilitre of stool. One of the highest viral loads occurred in a control child without symptoms at the time of testing. Phylogenetic analysis showed mainly contemporary HPeV1 strains in our patients, but also a separate new lineage of HPeV1 in evolutionary transition from the historical prototype strain. Moreover, a novel sixth HPeV type was identified. Full genome analysis of two viruses revealed recombination between HPeV1 and 3 in one, and HPeV6 and 1 in another. HPeV seems relevant in children <2 years and specific RT-PCR for HPeV should be included in enteritis screening.

A serological ELISA test was developed using a synthetic peptide from the VP0 protein of human parechoviruses (HPeVs). Seroprevalence for HPeVs was 70% in children </= 5 years and 95% in adults. In children with serial sera, seropositivity increased from 22% to 88% between 2 and 24 months of age.

ABSTRACT: BACKGROUND: Enteroviruses are among the most common viruses infecting humans worldwide and they are associated with diverse clinical syndromes. Acute flaccid paralysis (AFP) is a clinical manifestation of enteroviral neuropathy, transverse myelitis, Guillian-Barre Syndrome, Traumatic neuritis and many other nervous system disorders. The objective of this study was to understand the role of Non-Polio Enteroviruses (NPEV) towards this crippling disorder. METHODS: Stool specimens of 1775 children, aged less than 15 years, suffering from acute flaccid paralysis were collected after informed consent within 14 days of onset of symptoms during January 2003 to September 2003. The specimens were inoculated on RD and L20B cells using conventional tube cell culture while micro-neutralization test was used to identify the non-polio enterovirus (NPEV) serotypes. Detailed clinical information and 60-days follow-up reports were analyzed for NPEV-associated AFP cases. RESULTS: NPEV were isolated from 474 samples. The male to female ratio was 1.4:1. The isolation of NPEV decreased significantly with the increase in age. Cases associated with fever at the onset of NPEV-associated AFP were found to be 62%. The paralysis was found asymmetrical in 67% cases, the progression of paralysis to peak within 4 days was found in 72% cases and residual paralysis after 60 days of paralysis onset was observed in 39% cases associated with NPEV. A clinical diagnosis of Guillian-Barre syndrome was made in 32% cases. On Microneutralization assay, echo-6 (13%) and coxsackievirus B (13%) were the most commonly isolated serotypes of NPEV along with E-7, E-13, E-11, E-4 and E-30. The isolates (n=181) found untypable by the antiserum pools were confirmed as NPEV by PCR using Pan-Enterovirus primers. CONCLUSIONS: The present study suggests that NPEV are a dominant cause of AFP and different serotypes of NPEV are randomly distributed in Pakistan. The untypable isolates need further characterization and analysis in order to determine their association with clinical presentation of a case.