Isolation and Characterization of Hepatitis E Virus Subtype 4b Using a PLC/PRF/5 Cell-Derived Cell Line Resistant to Porcine Sapelovirus Infection.

The human hepatocarcinoma cell line PLC/PRF/5 is susceptible to hepatitis E virus (HEV) infection and is used for HEV isolation. It is difficult to use the cell line for this purpose directly from fecal specimens of swine or wild boar contaminated with porcine sapelovirus (PSV) because PSV infection results in rapid and extensive cytopathic effects in PLC/PRF/5 cells, interrupting the growth of HEV. Herein, we used a PSV infection-resistant cell line, N1380, derived from PLC/PRF/5 cells, and successfully isolated a HEV-4b strain from a PSV-positive swine fecal specimen. Our results indicated that N1380 cells are a useful tool for the isolation of HEV from swine or wild boar fecal specimens, even when the cells are co-infected with PSV.

Polymerase chain reaction primer sets for the detection of genetically diverse human sapoviruses.

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.

Broadly reactive real-time reverse transcription-polymerase chain reaction assay for the detection of human sapovirus genotypes.

Sapoviruses are associated with acute gastroenteritis. Human sapoviruses are classified into four distinct genogroups (GI, GII, GIV, and GV) based on their capsid gene sequences. A TaqMan probe-based real-time reverse transcription-polymerase chain reaction (RT-PCR) assay that detects the representative strains of these four genogroups is widely used for screening fecal specimens, shellfish, and environmental water samples. However, since the development of this test, more genetically diverse sapovirus strains have been reported, which are not detectable by the previously established assays. In this study, we report the development of a broader-range sapovirus real-time RT-PCR assay. The assay can detect 2.5 × 107 and 2.5 × 10 1 copies of sapovirus and therefore is as sensitive as the previous test. Analysis using clinical stool specimens or synthetic DNA revealed that the new system detected strains representative of all the 18 human sapovirus genotypes: GI.1-7, GII.1-8, GIV.1, and GV.1, 2. No cross-reactivity was observed against other representative common enteric viruses (norovirus, rotavirus, astrovirus, and adenovirus). This new assay will be useful as an improved, broadly reactive, and specific screening tool for human sapoviruses.

Clinical and Epidemiologic Features of Severe Viral Gastroenteritis in Children: A 3-Year Surveillance, Multicentered Study in Taiwan With Partial Rotavirus Immunization.

The global epidemiological landscape of childhood acute gastroenteritis (AGE) is changing after the introduction of 2 effective rotavirus vaccines in 2006. A comprehensive evaluation for viral etiology of childhood AGE in Taiwan, where rotavirus vaccination was provided by the private sector since 2006, is lacking.From 2009 to 2011, children younger than 5 years of age with AGE who were hospitalized at 3 sentinel hospitals were enrolled in this surveillance study. Stool specimens were tested for rotavirus, norovirus, enteric adenovirus, and astrovirus. The epidemiologic and clinical information was collected by questionnaire-based interviews and chart reviews.Viral agents were detected in 1055 (37.5%) of 2810 subjects, with rotavirus (21.2%) being the leading cause of disease, followed by norovirus (14.9%), enteric adenovirus (3.74%), astrovirus (2.10%), and a mixture of at least 2 of 4 above-mentioned viruses (4.06%). The majority (56%) of the viral AGE occurred in children <2 years of age. Rotavirus and norovirus were detected more frequently in cool seasons (P < 0.0001 for both), whereas no seasonal variation was observed for adenovirus and astrovirus. Adult households with diarrhea and a Vesikari score >10 were independent factors respectively associated with an increased risk of norovirus (adjusted odds ratio [aOR] 9.034, P = 0.0003) and rotavirus (aOR, 3.284, P < 0.0001) infections. Rotavirus immunization and female gender were protective factors against rotavirus (aOR, 0.198, P < 0.0001) and astrovirus (aOR, 0.382, P = 0.0299) infections, respectively.Rotavirus and norovirus are the 2 most important viral agents of childhood AGE in Taiwan with partial rotavirus immunization. In addition, different enteric viruses are associated with distinct epidemiologic and clinical features.

Epidemiology and clinical peculiarities of norovirus and rotavirus infection in hospitalized young children with acute diarrhea in Taiwan, 2009.

BACKGROUND/PURPOSE: Acute diarrhea is one of the most common morbidities in pediatrics worldwide. We conducted a study to investigate the incidence of norovirus in young children hospitalized with acute diarrhea in Taiwan and its clinical peculiarity compared with rotavirus gastroenteritis. METHODS: Between January and December, 2009, patients younger than 5 years and admitted to hospital with acute diarrhea were randomly selected; and their stool samples were collected and tested for presence of rotavirus and norovirus by enzyme immunoassay and reverse transcription-polymerase chain reaction, respectively. The clinical manifestations and laboratory findings of the enrolled patients were analyzed. RESULTS: A total of 989 cases were enrolled with a mean age of 21.6 ± 13.7 months and a male proportion of 56.0%. Rotavirus and norovirus was detected in 20.2% and 14.6% of all patients, respectively. Genogroup II was the predominant strain of norovirus (80.6%). Children aged 6-36 months accounted for the majority of patients positive for rotavirus and norovirus (73.0% and 81.3%, respectively). The incidences of norovirus and rotavirus infection were higher during winter and early spring. Most patients with rotavirus and norovirus diarrhea experienced vomiting (74.9%vs. 74.8%, respectively) and fever (94.7%vs. 71.3%, respectively). CONCLUSION: Most young diarrheal patients presenting with vomiting were likely to have norovirus or rotavirus infection. Patients with norovirus diarrhea experienced an absence of, or low-grade fever and longer duration of vomiting compared with those positive for rotavirus infection. A family history of current gastroenteritis may suggest the possibility of norovirus infection.

Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction.

Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI-GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5 x 10(7) to 2.5 x 10(1) copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses.