The mechanism and machinery of hepatitis C virus (HCV) RNA replication are still poorly characterized. Our previous study has shown that HCV RNA synthesis occurs on a lipid raft membrane structure [J. Virol. 77 (2003) 77 4160]. In this study, we further characterized these replication complexes (RCs) in Huh-7 cells that support active RNA replication of a subgenomic HCV replicon. Biochemical analysis showed that these membrane structures were resistant to Nonidet P-40 or Triton X-100 (TX-100) at 4 degrees C while solubilized by beta-octylglucoside at 4 degrees C or Triton TX-100 at 37 degrees C, characteristic of lipid rafts. Cholesterol sequestration assay further demonstrated the association between HCV nonstructural (NS) proteins and cholesterol-rich lipid rafts. The RCs contained both minus- and plus-strand HCV RNA, with the plus-stranded RNA being approximately 10-fold more abundant than the minus-strand. Furthermore, the HCV RNA and NS proteins were resistant to RNase and protease digestion, respectively, but became sensitive after treatment with the raft-disrupting agents. These results suggested that the HCV RCs are protected within lipid rafts. Detergent-resistant membrane (DRM) fractions containing NS proteins and viral RNA were capable of HCV RNA synthesis using the endogenous HCV RNA template. NS proteins were distributed in both the ER and the Golgi, but the majority of the active RCs were detected in the Golgi-derived membrane. Depletion of cellular cholesterol selectively reduced HCV RNA replication. These findings provide further insights into the mechanism of HCV replication in vivo.

Human bocavirus (HBoV), a newly cloned human virus of the genus Bocavirus, was detected by PCR from nasopharyngeal swab samples (8 of 318; 5.7%) collected from children with lower respiratory tract infections. HBoV may be one of the causative agents of lower respiratory tract infections in young children.The family Parvoviridae contains two subfamilies: Parvovirinae, which infects vertebrates, and Densovirinae, which infects insects. The subfamily Parvovirinae consists of five genera: Parvovirus, Erythrovirus, Dependovirus, Amdovirus, and Bocavirus (12). Parvovirus B19, which belongs to the genus Erythrovirus, is a well-known human pathogen (3,12). A new human virus of the genus Bocavirus, provisionally named human bocavirus (HBoV), was recently cloned from pooled human respiratory tract samples and is considered to be pathogenic to humans (1). In this study, nasopharyngeal swab samples obtained from children with lower respiratory tract infections were investigated for the presence of HBoV.

Human metapneumovirus (hMPV) has been recently discovered as an etiological agent of acute respiratory infections. Our purpose was to asses the virological and clinical features of children with respiratory infections caused by hMPV. We examined 658 nasopharyngeal swab samples obtained from 637 children with respiratory infections for hMPV by using reverse transcription-PCR (RT-PCR). A total of 268 samples from 637 children were inoculated onto tertiary monkey kidney cells. A total of 36 serum samples (26 in the acute phase and 10 in the convalescent phase) from the 26 hMPV-positive children were tested for immunoglobulin G (IgG) and IgM antibodies to hMPV by using an indirect immunofluorescence assay. We detected hMPV in 57 (8.9%) of the 637 samples by using RT-PCR and isolated 7 (2.6%) hMPV strains of the 268 samples in cell cultures. A total of 12 (46.2%) of 26 hMPV-positive children were suspected to have primary infection with hMPV as determined by an indirect immunofluorescence assay. The infected children were diagnosed as having wheezy bronchitis (36.8%), upper respiratory tract infection (26.3%), bronchitis (22.8%), and pneumonia (14.0%). We showed that two hMPV groups were circulating in different regions during the same period and that reinfection with hMPV frequently occurs in childhood. The RT-PCR test is the most sensitive test for detection of hMPV, and a serological test may be useful to differentiate between primary infection and reinfection with hMPV.

A new human virus, provisionally named human bocavirus (HBoV), was discovered by Swedish researchers in 2005. A new immunofluorescence assay using Trichoplusia ni insect cells infected with a recombinant baculovirus expressing the VP1 protein of HBoV was developed, and the levels of immunoglobulin G antibody to the VP1 protein of HBoV in serum samples were measured. The overall seroprevalence rate of antibodies against the VP1 protein of HBoV in a Japanese population aged from 0 months to 41 years was 71.1% (145 of 204). The seropositive rate was lowest in the age group of 6 to 8 months and gradually increased with age. All of the children had been exposed to HBoV by the age of 6 years. A rise in titers of antibody against the VP1 protein of HBoV during the convalescent phase was observed for four patients with lower respiratory tract infections, and HBoV DNA was detected in nasopharyngeal swab and serum samples from all four patients. These results suggest that HBoV is a ubiquitous virus acquired early in life and that HBoV might play a role in the course of lower respiratory tract infections.

Human enteroviruses (EVs) are the major cause of a variety of acute and chronic illnesses. Virus isolation and neutralization tests are usually done to identify the causative virus, but these tests are labor intensive, time consuming, and sometimes require suckling mice from which certain viruses have been isolated. This study investigated a rapid and reliable method based on reverse-transcription polymerase chain reaction and phylogenetic analysis. The phylogenetic tree constructed by neighbor-joining on the basis of the VP4 sequence from 66 prototypes grouped all human EVs into 5 distinct clusters. These clusters correspond closely to the 5 newly designated species-human EV A-D and poliovirus. The VP4 sequences of 89 isolates from 26 serotypes obtained over >30 years plus those of 66 prototype strains were analyzed. Each isolate formed a monophyletic cluster along with its respective prototype strain, allowing for serotype identification (with the exception of E-8). VP4-based classification appears to be an effective tool for the molecular epidemiology study of EVs.