Hideaki Kikuta

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.

Seroepidemiology of Human Bocavirus in Hokkaido Prefecture, Japan

ABSTRACT 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.

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.

Efficient Multiplication of Human Metapneumovirus in Vero Cells Expressing the Transmembrane Serine Protease TMPRSS2

ABSTRACT Human metapneumovirus (HMPV) is a major causative agent of severe bronchiolitis and pneumonia. Its fusion (F) protein must be cleaved by host proteases to cause membrane fusion, a critical step for virus infection. By generating Vero cells constitutively expressing the transmembrane serine protease TMPRSS2 and green fluorescent protein-expressing recombinant HMPV, we show that TMPRSS2, which is expressed in the human lung epithelium, cleaves the HMPV F protein efficiently and supports HMPV multiplication. The results indicate that TMPRSS2 is a possible candidate protease involved in the development of lower respiratory tract illness in HMPV-infected patients.

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.

Relationship between gene polymorphisms of mannose-binding lectin (MBL) and two molecular forms of MBL

Mannose‐binding lectin (MBL) activates complement through MBL‐associated serine proteases (MASP). A deficiency in MBL due to mutations at exon 1 of the human MBL gene is reported to cause vulnerability to infection. We examined sera of known MBL genotype by gel filtration and assessed their elution patterns using an ELISA for MBL and identified two MBL forms, a high‐molecular‐mass form and a lower‐molecular‐mass form. By the identification of either or both forms in individual sera, three types of patterns emerged: type 1 consisted of a high‐molecular form; type 2, of a low‐molecular form; and type 3, of both forms. Types 1, 2 and 3 corresponded, respectively, to a wild type (A/A), a homozygous mutation at codon 54 (B/B) and their heterozygote (A/B). One exception was a heterozygous LXPA/LYPB phenotype that exhibited the type‐2 pattern. Binding to mannan and MASP‐1/3 occurred exclusively with the high‐molecular form. An apparent MBL deficiency does not in fact representa deficiency in MBL molecules but rather the presence of circulating oligomeric mutant MBL with impaired function.

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.

Unique character and metabolism of high density lipoprotein (HDL) in fetus

Lipid and lipoprotein profiles, and enzymes for the lipid metabolism were compared between cord and adult blood. Consistent with previous reports, the major lipoprotein in cord blood was high-density lipoprotein (HDL), and that in adult blood was low-density lipoprotein (LDL). The level of apolipoprotein E (apo E) in cord blood was almost equivalent to that in adult blood, while other apolipoproteins and lipids were all lower than the adult level. In cord blood, apo E-rich HDL cholesterol represented more than 30% of total HDL cholesterol (around 11% in adult), and the concentration was about twice of that in adult blood. This apo E-rich HDL cholesterol was poorly esterified (E/T 56%) compared with that in adults (93%). The lecithin:cholesterol acyltransferase (LCAT) activity in cord blood was extremely low, while the activity and mass of cholesteryl ester transfer protein (CETP) were higher than those in adult blood. The apo E genotype did not show influences on total cholesterol, LDL cholesterol, total HDL cholesterol, and apo E rich HDL cholesterol levels in cord blood, as opposed to those in adult blood. The association of D442G mutation of the CETP gene with the increased HDL cholesterol in adult blood was not seen in cord blood. Rather, the mutation was associated with low total cholesterol and LDL cholesterol levels in cord blood. These results indicate that, in fetus, the character and metabolism of HDL, especially of apo E-rich HDL cholesterol, are distinct from those in adults.

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.