Nobuhisa Ishiguro

Detection of Human Bocavirus in Japanese Children with Lower Respiratory Tract Infections

ABSTRACT 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.

Human Metapneumovirus Infection in Japanese Children

ABSTRACT 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.

Detection of human coronavirus NL63 in young children with bronchiolitis.

HCoV‐NL63, the fourth human coronavirus, has been isolated recently from children with respiratory tract infections, including upper respiratory infection, bronchiolitis, and pneumonia. The virus has been also detected in immunocompromised adults with respiratory tract infections. A total of 118 nasopharyngeal swab samples from 118 hospitalized young children aged less than 2 years with bronchiolitis who were not infected with human respiratory syncytial virus, influenza A or B, or human metaneumovirus were selected. Three (2.5%) of the 118 samples were positive for HCoV‐NL63 by reverse transcription‐polymerase chain reaction tests. HCoV‐NL63 may be one of the causative agents of bronchiolitis in young children. J. Med. Virol. 75:463–465, 2005.

High genetic diversity of the attachment (G) protein of human metapneumovirus.

ABSTRACT Complete genes encoding the predicted nucleoprotein (N), phosphoprotein (P), matrix protein (M), fusion protein (F), M2-1protein, M2-2protein, small hydrophobic protein (SH), and attachmentprotein (G) of seven newly isolated human metapneumoviruses (hMPVs) were analyzed and compared with previously published data for hMPV genes. Phylogenetic analysis of the nucleotide sequences indicated that there were two genetic groups, tentatively named groups 1 and 2, similar to the grouping of human respiratory syncytial virus. Although the predicted amino acid sequences of N, P, M, F, and M2 were highly conserved between the two groups (amino acid identities, 96% for N, 85% for P, 97% for M, 94% for F, 95% for M2-1, and 90% for M2-2), the amino acid identities of the SH and G proteins were low (SH, 58%; G, 33%). Furthermore, each group could be subdivided into two subgroups by phylogenetic analysis, tentatively named subgroups 1A and 1B and subgroups 2A and 2B. The predicted amino acid sequences of G within members of each subgroup were highly conserved (amino acid identities, 88% for group 1A, 93% for group 1B, and 96% for group 2B). The G of hMPV is thought to be the major antigenic determinant and to play an important role in the production of neutralizing antibodies. Clarification of the antigenic diversity of G is important for epidemiological analysis and for establishment of strategies to prevent hMPV infection.

Detection of Human Metapneumovirus Antigens in Nasopharyngeal Secretions by an Immunofluorescent-Antibody Test

ABSTRACT Human metapneumovirus (hMPV) is a recently discovered pathogen associated with respiratory tract infections, primarily in young children, immunocompromised individuals, and elderly individuals. Reverse transcription-PCR (RT-PCR) has been reported to be a more sensitive method for the diagnosis of hMPV infections than virus isolation by culture and serological study. However, there has been no report on rapid methods, such as an immunofluorescent-antibody test or an enzyme-linked immunosorbent assay, for the detection of hMPV antigens in nasopharyngeal secretions. In this study, we compared an indirect immunofluorescent-antibody test (IFA) with a monoclonal antibody with RT-PCR for detection of hMPV in nasal secretions from 48 hospitalized children with respiratory tract infections. Fifteen of the 48 children were positive for hMPV by RT-PCR. IFA results were positive for 11 of the 15 RT-PCR-positive children (sensitivity, 73.3%) and 1 of the 33 RT-PCR-negative children (specificity, 97.0%). Although the sensitivity of IFA is lower than that of RT-PCR, IFA is a rapid and useful test for the diagnosis of hMPV infections in children.

Early reinfection with human metapneumovirus in an infant

ABSTRACT Human metapneumovirus (hMPV) is one of the major pathogens of respiratory illness. Reinfection with hMPV occurs frequently throughout life. We describe an infant who was infected with two different hMPV strains during a period of only 1 month.

Lack of Association between New Haven Coronavirus and Kawasaki Disease

To the Editor—The new human coronavirus NL63 (HCoV-NL63) was discovered by van der Hoek et al. [1] and Fouchier et al. [2]. HCoV-NL63 has been shown to cause respiratory tract disease in young children [3, 4]. Esper et al. have reported a novel HCoV designated the “New Haven coronavirus” (HCoV-NH) that has been shown by sequence analysis to be very similar to HCoV-NL63 [5]. Esper et al. also reported that HCoV-NH was detected by reverse-transcription polymerase chain reaction (RT-PCR) in 8 (72.7%) of 11 respiratory tract samples from children with Kawasaki disease (KD) and in 1 (4.5%) of 22 age-matched samples from control subjects [6]. On the basis of these data, they suggested that HCoV-NH infection was associated with KD. To further investigate whether HCoV-NH disease is associated with KD, we performed a retrospective study. From October 2002 to May 2003, 19 nasopharyngeal swab samples were collected from 19 children who fulfilled the criteria for KD and who were treated at Tenshi Hospital in Sapporo, Japan. All of the samples were collected after informed consent was obtained from the children’s parents. All of the samples were obtained within 7 days of the onset of illness. The mean age of the children with KD was 22.6 months (range, 4 months–5 years). We used as controls 208 nasopharyngeal swab samples that were collected from children with diagnoses of respiratory tract disease who were admitted to hospitals in Sapporo, Japan, during the same period. All of these samples were examined after the possibility of infection with human respiratory syncytial virus or influenza A or B was excluded by rapid antigen-detection tests. The mean age of the children with respiratory tract disease was 21.6 months (range, 4 months–5 years). After extraction of total RNA and synthesis of cDNA, we performed RT-PCR to detect the HCoVNH genome, as described by Esper et al. [6]. The primer set and the PCR conditions in our PCR assay were the same as those used in their PCR assays. Sequencing of the PCR products was also performed to confirm the presence of HCoV-NH. Although RNA sequences of HCoV-NH were detected in samples from 5 (2.4%) of the 208 control children with respiratory tract disease, we could not detect any RNA sequences of HCoV-NH in 19 samples from children with KD (table 1). On the basis of these data, we have some reservations about the findings described by Esper et al. [6]. They collected respiratory tract swab samples from children with KD as part of an ongoing epidemiological investigation of respiratory tract viruses. We collected respiratory tract swab samples from all of the patients with KD, regardless of the presence of respiratory tract symptoms, who were treated at Tenshi Hospital from October 2002 to May 2003. Because no RNA sequences of HCoV-NH were detected in samples from 19 patients with KD in our study, there is a possibility that Esper et al. tested samples from patients with KD who had respiratory tract symptoms. Our results suggest that Esper et al.’s results may be coincidental and that HCoVNH does not play a dominant role in the etiology or pathogenesis of KD in Japan.

Microbial production of hydroxy and oxo fatty acids by several microoganisms as a model of adipocere formation

Some varieties of aerobic or anaerobic microorganisms from the human stool and adipocere were separated and identified. These separated microorganisms together with other authentic ones produced 10-hydroxystearic acid from oleic acid. Some bacteria could convert oleic acid to 10-oxostearic acid as well as 10-hydroxystearic acid. These findings indicate that the microbial enzyme(s) catalyzes the hydration of oleic acid and probably the oxidation of this hydrated product. Aerobic bacteria as well as anaerobic microorganisms were found to be involved in the formation of adipocere.

Heterogeneous, restricted patterns of Epstein-Barr virus (EBV) latent gene expression in patients with chronic active EBV infection

Epstein-Barr virus (EBV) has been shown to infect T lymphocytes and to be associated with a chronic active infection (CAEBV), which has been recognized as a mainly non-neoplastic T-cell lymphoproliferative disorder (T-cell LPD). The systemic distribution of EBV genomes was studied, by real-time PCR, in multiple tissues from six patients with CAEBV, including three patients with T-cell LPD, one patient with B-cell LPD and two patients with undetermined cell-type LPD. There were extremely high loads of EBV genomes in all tissues from the patients. This reflects an abundance of circulating and infiltrating EBV-infected cells and a wide variety of clinical symptoms in the affected tissues. We chose one sample from each patient that was shown by real-time PCR to contain a high load of EBV genomes and examined the expression of EBV latent genes by RT-PCR. EBER1 and EBNA1 transcripts were detected in all samples. Only one sample also expressed EBNA2, LMP1 and LMP2A transcripts in addition to EBER1 and EBNA1 transcripts. Two of the remaining five samples expressed LMP1 and LMP2A transcripts. One sample expressed LMP2A but not LMP1 and EBNA2 transcripts. Another sample expressed EBNA2 but not LMP1 and LMP2A transcripts. The other sample did not express transcripts of any of the other EBNAs or LMPs. None of the samples expressed the viral immediate-early gene BZLF1. These results showed that EBV latent gene expression in CAEBV is heterogeneous and that restricted forms of EBV latency might play a pathogenic role in the development of CAEBV.

Differential gene expression of S100 protein family in leukocytes from patients with Kawasaki disease

S100 family proteins are calcium-binding proteins, some of which have been shown to have intracellular and extracellular functions associated with inflammation. The serum concentration of S100A12 has been reported to increase in the acute phase of Kawasaki disease. The purpose of this study was to evaluate leukocyte gene expressions of S100 family proteins in the acute phase of Kawasaki disease. Ten paired blood samples were obtained from ten patients with Kawasaki disease in the acute phase and in the convalescent phase. We examined leukocyte expression levels of 18 S100 genes in the acute phase compared with those in the convalescent phase by using quantitative real-time polymerase chain reaction. Significantly elevated expression of seven S100 genes (S100A6, A8, A9, A11, A12, S100P, and S100Z) was observed in the acute phase. Conclusion:Of the upregulated S100 genes, calgranulin members of S100 genes (S100A8, S100A9, and S100A12) were most highly expressed in the acute phase. Only one S100 gene, the S100A13 gene, exhibited a significantly decreased expression level in the acute phase.