Detection of human bocaviruses 1 to 4 from nasopharyngeal swab samples collected from patients with respiratory tract infections.

Human bocaviruses (HBoV) 1, 2, 3, and 4 (HBoV1-4) were detected in 132 (15.5%), 5 (0.6%), 3 (0.4%), and 5 (0.6%) of 850 nasopharyngeal swab samples collected from children with respiratory tract infections, respectively. Out of the 145 HBoV1-4-positive samples, 62 (42.8%) were codetected with other respiratory viruses.

Novel serological tools for detection of Thottapalayam virus, a Soricomorpha-borne hantavirus.

We developed serological tools for the detection of hantavirus-specific antibodies and hantavirus antigens in shrews. The work was focussed to generate Thottapalayam virus (TPMV)-specific monoclonal antibodies (mAbs) and anti-shrew immunoglobulin G (IgG) antibodies. The mAbs against TPMV nucleocapsid (N) protein were produced after immunization of BALB/c mice with recombinant TPMV N proteins expressed in Escherichia coli, baculovirus and Saccharomyces cerevisiae-mediated expression systems. In total, six TPMV N-protein-specific mAbs were generated that showed a characteristic fluorescent pattern in indirect immunofluorescence assay (IFA) using TPMV-infected Vero cells. Out of the six mAbs tested, five showed no cross-reaction to rodent-associated hantaviruses (Hantaan, Seoul, Puumala, Tula, Dobrava-Belgrade and Sin Nombre viruses) in IFA and enzyme-linked immunosorbent assay (ELISA), although one mAb reacted to Sin Nombre virus in IFA. None of the mAbs cross-reacted with an amino-terminal segment of the shrew-borne Asama virus N protein. Anti-shrew-IgG sera were prepared after immunization of rabbits and BALB/c-mice with protein-G-purified shrew IgG. TPMV-N-protein-specific sera were raised by immunisation of Asian house shrews (Suncus murinus) with purified yeast-expressed TPMV N protein. Using these tools, an indirect ELISA was developed to detect TPMV-N-protein-specific antibodies in the sera of shrews. Using an established serological assay, high TPMV N protein specific antibody titres were measured in the sera of TPMV-N-protein-immunized and experimentally TPMV-infected shrews, whereas no cross-reactivity to other hantavirus N proteins was found. Therefore, the generated mAbs and the established ELISA system represent useful serological tools to detect TPMV, TPMV-related virus antigens or hantavirus-specific antibodies in hantavirus-infected shrews.

Detection of KI polyomavirus and WU polyomavirus DNA by real-time polymerase chain reaction in nasopharyngeal swabs and in normal lung and lung adenocarcinoma tissues.

Polyomaviruses KI (KIPyV) and WU (WUPyV) were detected from 7 (3.0%) and 38 (16.4%) of 232 children with respiratory tract infections by real-time PCR. The rates of infection by KIPyV and WUPyV alone were 3 of 7 (42.9%) and 20 of 38 (52.6%), respectively. In the other samples, various viruses (human respiratory syncytial virus, human metapneumovirus, human rhinovirus, parainfluenza virus 1 and human bocavirus) were detected simultaneously. One case was positive for KIPyV, WUPyV and hMPV. There was no obvious difference in clinical symptoms between KIPyV-positive and WUPyV-positive patients with or without coinfection. KIPyV was detected in one of 30 specimens of lung tissue (3.3%). Neither of the viruses was detected in 30 samples of lung adenocarcinoma tissue.

Truncated hantavirus nucleocapsid proteins for serotyping Sin Nombre, Andes, and Laguna Negra hantavirus infections in humans and rodents.

Sin Nombre virus (SNV), Andes virus (ANDV), and Laguna Negra virus (LANV) have been known as the dominant causative agents of hantavirus pulmonary syndrome (HPS). ANDV and LANV, with different patterns of pathogenicity, exist in a sympatric relationship. Moreover, there is documented evidence of person-to-person transmission of ANDV. Therefore, it is important in clinical medicine and epidemiology to know the serotype of a hantavirus causing infection. Truncated SNV, ANDV, and LANV recombinant nucleocapsid proteins (trNs) missing 99 N-terminal amino acids (trN100) were expressed using a baculovirus system, and their applicability for serotyping SNV, ANDV, and LANV infection by the use of enzyme-linked immunosorbent assays (ELISA) was examined. HPS patient sera and natural-reservoir rodent sera infected with SNV, ANDV, and LANV showed the highest optical density (OD) values for homologous trN100 antigens. Since even patient sera with lower IgM and IgG antibody titers were serotyped, the trN100s are therefore considered useful for serotyping with early-acute-phase sera. In contrast, assays testing whole recombinant nucleocapsid protein antigens of SNV, ANDV, and LANV expressed in Escherichia coli detected homologous and heterologous antibodies equally. These results indicated that a screening ELISA using an E. coli-expressed antigen followed by a serotyping ELISA using trN100s is useful for epidemiological surveillance in regions where two or more hantavirus species cocirculate.

Molecular epidemiological and serological studies of hantavirus infection in northern Vietnam.

The distribution of anti-hantavirus antibodies in humans and rodents in northern Vietnam was examined. In total, 837 serum samples from healthy humans (617) and patients with fever (220), living in six different areas were screened for IgG antibodies against Hantaan or Seoul virus (SEOV) by ELISA, IFA, and Western blot analysis. Antibody-positive sera were identified in 7/617 (1.1%) healthy donors, 5/150 port workers in the port of Hai Phong, and 2/185 residents of Ha Nam Province. In comparison, positive sera were detected in 5/220 (2.3%) fever patients in the provinces of Ha Nam (1/58) and Thanh Hoa (4/146). Antibody-positive Rattus norvegicus were found in the provinces of Ha Nam (7/52) and Thanh Hoa (1/67), in Haibatrung District (7/43) in Hanoi, and in Hai Phong Port (21/62), while antibody-positive R. rattus (2/17) were found in Hai Phong Port. Part of the Gc region from the viral genome was amplified by RT-PCR using lung tissue samples from R. norvegicus in Haibatrung (2/7) and Hai Phong Port (7/9), but not from R. rattus (0/2). Viral sequences were located in the SEOV clade and formed a single lineage with Indonesian SEOV, suggesting that Vietnamese SEOV is part of a distinct lineage among Asian SEOVs.

Early-life lessons of the courtship dance in a dance-duetting songbird, the Java sparrow.

Vocal learners, such as songbirds, must practise singing in a developmentally sensitive period to master songs. Yet, knowledge remains limited about the development of visual displays in birds, even when courtship includes well-coordinated vocalizations (songs) and body motions. The Java sparrow (Lonchura oryzivora) is a species of songbird that exhibits a courtship duet dancing exchange between the sexes, with this behaviour driving mating success. In this study, juvenile male Java sparrows were observed in captivity, showing that they repeatedly practise the courtship dance in their early life. We called it 'practice', as juvenile birds frequently dance towards family members or other juveniles well before sexual maturation. Based on our observation that dance motor performance increased with age, we propose that the practice is needed for motor learning. In addition, it could also be important for establishing vocal-motional coordination or socialization. Older juveniles gradually became capable of singing and dancing simultaneously, and participated in duet dancing more often. We also found that repeated encounters with the same individual promote dance movement. Though our results do not show how much social experiences account for the development of dance communication, early-life dance practising might influence future reproductive success, like song practising does.

[Survey on patients' satisfaction with opioid rescue guidance by pharmacists].

To examine the influence of drug therapy guidance by pharmacists on the use of a rescue dose (RD) for opioid analgesics (opioids) and pain as well as drug therapy guidance in cancer pain treatment, we conducted a patient satisfaction survey. The subjects were 56 cancer patients undergoing opioid therapy in hospitals belonging to the Symptom Control Research Group (SCORE-G). The survey period was 2 months (from November 1 until December 31, 2006). Drug therapy guidance regarding the use of RD was performed twice in each patient to evaluate the patients' satisfaction. RD was prescribed in 87.8% of the patients in the first guidance and in 80.5% in the second guidance periods. The proportion of patients who used RD significantly increased from 63.8% to 87.5%. Five items significantly improved in the second guidance period: "marked analgesic effects," "satisfaction with current treatment," "correct understanding of RD usage," "relief through RD," and "appropriate use of RD." On comprehensive evaluation following the second round of guidance, 81% of the patients reported overall satisfaction, and 78% reported the usefulness of guidance in pain treatment. These results suggest that positive guidance by pharmacists increases patients' satisfaction. In providing guidance, it was important to confirm the characteristics and side effects of opioids as well as the necessity of RD to patients accurately and repeatedly.

[Usefulness of parenteral colistin in treating of lower respiratory infection due to multidrug-resistant Pseudomonas aeruginosa].

We report a case of cystic fibrosis in a 19-year-old woman who suffered from frequent exacerbations of lower respiratory infection due to multidrug-resistant Pseudomonas aeruginosa and who was successfully treated with parenteral colistin. Multidrug-resistant Pseudomonas aeruginosa isolated from sputum had become resistant to all parenteral antibiotics commercially available in Japan. She did not show clinical improvement despite treatment with several different combinations of available antibiotics. We therefore obtained parenteral colistin from a pharmacy outside Japan. She responded well to parenteral colistin without apparent side effects such as serious nephrotoxicity or neurotoxicity. Colistin is therefore an important alternative antibiotic for treating multidrug-resistant Pseudomonas aeruginosa and its use should be considered in severe infection. We hope that parenteral colistin will become available in Japan in the near future.

Clonal expansion of multiphenotypic Epstein-Barr virus-infected lymphocytes in chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus (EBV) infection has been recognized as clonal non-neoplastic lymphoproliferative diseases. However, some reports of cases with a multiphenotypic expansion of EBV-infected lymphocytes give rise to questions of how EBV infects multiphenotypic lymphocytes and whether chronic active EBV infection is a truly monoclonal lymphoproliferative disease. We report two patients with chronic active EBV infection who showed expansion of multiphenotypic EBV-infected lymphocytes. EBV DNA was detected in CD4+ and CD8+ T cells and in B cells from pleural fluid of one patient and in T and B cells from a cervical lymph node of the other patient by polymerase chain reaction (PCR). Although real-time PCR showed that there were equally high loads of EBV genomes in CD4+ and CD8+ T cells from the pleural fluid, Southern blot hybridization with terminal repeats of the EBV genome showed a single band of the same molecular weight in three tissue samples from the patient. The results indicated biphenotypic expansions of CD4+ and CD8+ T cells infected with the same clone of EBV. Furthermore, bisulfite PCR analysis showed hypermethylated status in the Cp region in the two patients regardless of their cell populations. There has been a discrepancy between clonality and expansion of multiphenotypic EBV-infected lymphocytes. We speculate that lymphoid progenitor cells that have not differentiated into T and B cell progenitors are infected with EBV, resulting in clonal expansion of EBV-infected multiphenotypic cells.

Epidemiology of hantavirus infection in Thousand Islands regency of Jakarta, Indonesia.

Hemorrhagic fever with renal syndrome (HFRS) is a rodent-borne zoonotic disease caused by hantavirus infection. Many HFRS cases have been reported in East Asia and North Europe, while the situation in Southeast Asia remains unclear. In this study, the prevalence of hantavirus infection in rodents and humans in Thousand Islands regency, which is close to the port of Jakarta, one of the largest historic ports in Indonesia, was investigated. A total of 170 rodents were captured in 2005, and 27 (15.9%) of the rodents were antibody-positive against Hantaan virus antigen in an immunofluorescence assay (IFA) and Western blotting. Despite the high prevalence in rodents, human sera collected from 31 patients with fever of unknown origin and 20 healthy volunteers in the islands in 2009 did not show positive reaction to the antigen in IFA. To identify the virus in rodents genetically, a total of 59 rodents were captured in 2009. Sera from the rodents were screened for antibody by ELISA, and lung tissues were subjected to RT-PCR. 20 (33.9%) of the 59 rodents were antibody-positive, and 3 of those 20 rodents were positive for S and M genome segments of hantaviruses. Genetic analysis showed that the viruses belonged to Seoul virus and formed a cluster with those in Vietnam and Singapore. These results suggest that a unique group of Seoul viruses has spread widely in Southeast Asia.

Application of truncated nucleocapsid protein (N) for serotyping ELISA of murinae-associated hantavirus infection in rats.

Truncated recombinant nucleocapsid proteins (trNs) that lack N-terminally located cross-reactive epitopes of four Murinae rodent-associated hantaviruses, Seoul virus (SEOV), Thailand virus, Hantaan virus (HTNV) and Dobrava-Belgrade virus, were produced by using a baculovirus expression system. ELISA with the trNs as antigens enabled serotyping of immune sera from rats experimentally inoculated with the corresponding hantaviruses with cut-off OD values of 60% of those of whole N of HTNV. The trN-based ELISA could serotype 12 out of 13 sera obtained from wild rodents (Rattus norvegicus) naturally infected with SEOV using the 60% cut-off value. These results indicate that screening with whole N followed by serotyping with trNs using a cut-off OD value of 60% of that of whole N is a useful method for serological surveillance of Murinae-associated hantavirus infection among rodents.

Development of a serotyping enzyme-linked immunosorbent assay system based on recombinant truncated hantavirus nucleocapsid proteins for New World hantavirus infection.

New World hantaviruses were divided into five groups based on the amino acid sequence variability of the internal variable region (around 230-302 amino acids) of hantavirus nucleocapsid protein (NP). Sin Nombre virus (SNV), Andes virus, Black Creek Canal virus (BCCV), Carrizal virus (CARV) and Cano Delgadito virus belong to groups 1, 2, 3, 4 and 5, respectively. Patient and rodent sera were serotyped successfully by an enzyme-linked immunosorbent assay (ELISA) with recombinant truncated NP lacking 99 N-terminal amino acids (trNP100) of SNV, CARV and BCCV. The trNP100 of BCCV showed lower reactivity to heterologous sera. In contrast, whole recombinant NP antigens detected both homologous and heterologous antibodies equally. The results together with results of a previous study suggest that trNP100 can distinguish infections among viruses in groups 1, 2, 3 and 4 of New World hantaviruses. The serotyping ELISA with trNP100 is useful for epidemiological surveillance in humans and rodents.

Studies on hantavirus infection in small mammals captured in southern and central highland area of Vietnam.

To investigate the distribution of hantaviruses among animals in Southern and Central Highland area of Vietnam, a total of 1311 serum samples were obtained from rats and Asian house shrews (Suncus murinus) captured at 11 locations between 2006 and 2009. A total of 1066 serum samples from rats were examined for IgG antibodies against Hantaan virus, and there were 30 antibody-positive serum samples from rats that had been captured mainly in a port area and urban area in Ho Chi Minh City (HCMC) (2.8%). All of the antibody-positive rats were Rattus norvegicus, and they had Seoul virus (SEOV) genome in their lungs. SEOV sequences detected from rats captured in Southern Vietnam belonged to the same lineage as those from rats captured at Haiphong Port and a market area in Hanoi City. SEOV strain CSG5 was isolated from a rat captured at Saigon Harbor. Strain CSG5 showed a cross-neutralization pattern almost the same as that of a representative strain of SEOV. A total of 245 Asian house shrews were captured in the Central Highland area and near HCMC. Sera were examined for IgG antibodies against Thottapalayam virus (TPMV), and 32 (13.1%) of the antibody-positive shrews were mainly from the Central Highland area and showed a neutralizing antibody against TPMV. These results indicated that SEOV is distributed among R. norvegicus inhabiting harbor and urban areas of Southern Vietnam and that TPMV or an antigenically related virus is distributed among Asian house shrews in Central Highland area.

Antibodies against structural and nonstructural proteins of human bocavirus in human sera.

Immunofluorescence assays (IFAs) for detection of human bocavirus (HBoV) proteins (VP1, VP2, NP-1, and NS1) were developed. The VP1 IFA was the most sensitive for detection of IgG antibody and suitable for screening. IgG antibodies in convalescent-phase sera from HBoV-positive patients were detected by VP1 and VP2 IFAs. Sensitivities of NP-1 and NS1 IFAs were low.

Different cross-reactivity of human and rodent sera to Tula virus and Puumala virus.

Tula virus (TULV) and Puumala virus (PUUV) are hantaviruses carried by the bank vole (Myodes glareolus) and European common vole (Microtus arvalis), respectively. PUUV is a causative agent of hemorrhagic fever with renal syndrome (HFRS), while TULV is thought to be apathogenic to humans. The N-terminal regions of the N proteins from TULV and PUUV were expressed and applied as enzyme-linked immunosorbent assay (ELISA) antigens. Colonized Japanese grass voles (Microtus montebelli) and BALB/c mice were used for experimental inoculation of the vole-borne hantaviruses TULV and PUUV. Voles and mice showed significant antibody production toward both viruses, but these antisera showed little cross-reactivity between TULV and PUUV in the immunofluorescence antibody assay and ELISA. In contrast, sera from patients with HFRS caused by PUUV exhibited high cross-reactivity against the TULV antigen, and sera from a natural rodent reservoir showed moderate cross-reactivity against the heterologous antigen, indicating that the antigenic cross-reactivity between TULV and PUUV differs in sera from rodents and humans.

Detection of four genetic subgroup-specific antibodies to human metapneumovirus attachment (G) protein in human serum.

Human metapneumovirus (hMPV) strains are classified into two genetic groups, A and B, each of which is further divided in two genetic subgroups, A1, A2, B1 and B2. hMPV encodes two major surface glycoproteins, the fusion (F) and attachment (G) proteins, which may be immunogenic and protective antigens. Although the amino acid sequences of hMPV F protein are highly conserved, those of the G protein are highly variable with low amino acid identity between the two groups. To address the antigenic variation between the genetic subgroups, we developed an immunofluorescence assay (IFA) method using Trichoplusia ni (Tn5) insect cells infected with each recombinant baculovirus-expressed hMPV G (Bac-G) protein of the four genetic subgroups. The titre of each antibody to the four Bac-G proteins was measured by the IFA in 12 paired serum samples obtained from children infected with hMPV of each genetic subgroup. Although 11 of the 12 acute-phase serum samples in paired samples were negative for the antibody to any Bac-G proteins, all of the convalescent-phase serum samples in those paired samples were positive for the antibody to only one of the four Bac-G proteins of the infecting genotype of hMPV. Since the antibody response to hMPV G protein was transient and genetic subgroup-specific without cross-reactivity, four genetic subgroups on the basis of hMPV G protein could be identified as different serotypes. This assay may be useful for the study of immune responses of humans to different hMPV strains, especially for clarifying the risk of reinfection with hMPV.

Seroepidemiology of human bocavirus in Hokkaido prefecture, Japan.

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.

Development of a rapid chromatographic immunoassay for detection of human metapneumovirus using monoclonal antibodies against nucleoprotein of hMPV.

Human metapneumovirus (hMPV) nucleocapsid (N) protein is a major structural protein that encapsidates the RNA genome and is essential for replication and transcription of the hMPV genome. We developed two mouse monoclonal antibodies (MAbs), designated 3D1 and 5B10, against N protein of hMPV and characterized them by an immunofluorescence assay and an immunoprecipitation assay using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV N protein. Both MAbs were found to be reactive to two groups of hMPV by an immunofluorescence assay using two groups of hMPV-infected cells. A chromatographic immunoassay (lateral flow assay) was developed using the MAbs. The assay is a sandwich immunoassay that uses a paper membrane with a gold colloid-conjugated MAb (5B10) in a liquid phase and an MAb (3D1) in a solid phase. We preliminarily examined the sensitivity and specificity using hMPV-infected cells, Tn5 insect cells infected with a recombinant baculovirus-expressing hMPV N protein, hMPV, and purified N protein. The assay had good specificity and sufficient sensitivity to detect hMPV. Therefore, the assay may be a rapid and useful test for diagnosis of hMPV infections.

Detection of antibodies against human metapneumovirus by Western blot using recombinant nucleocapsid and matrix proteins.

Detection of antibodies against individual proteins of human metapneumovirus (hMPV) is important in the analysis of immune responses to hMPV. Specific antibodies against nucleocapsid (N) and matrix (M) proteins in 97 serum samples were tested by Western blot using recombinant N and M proteins of hMPV expressed in Escherichia coli. The results were compared with those of immunofluorescence assays (IFAs) based on hMPV-infected LLC-MK2 cells, which expressed the whole hMPV proteins. Thirty (61.2%) and 31 (63.3%) of 49 serum samples with titers of > or = 1:160 by IFA reacted with N and M proteins, respectively. Only 2 (4.2%) of 11 serum samples with titers of 1:80 by IFA reacted with N and M proteins. Antibodies against N and M proteins were not detected in 37 serum samples with titers of < 1:40 by IFA. These results indicate that the antibodies against N and M proteins are highly specific (100%) but less sensitive (42.1%, N protein; 40.8%, M protein) than those against whole proteins of hMPV detected by IFA. The reactivity of sera with the recombinant N protein and that with the recombinant M protein correlated well (correlation coefficient of 0.79), and the concordance of reactivities was 91% (kappa = 0.79). In summary, both recombinant N and M proteins of hMPV were antigenic, and the responses to N and M protein varied among patients. Therefore, Western blot using N and M proteins provide a useful tool for analysis of immune responses to hMPV.

Detection of human bocavirus in Japanese children with lower respiratory tract infections.

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.