In Vitro Characterization of Multidrug-Resistant Influenza A(H1N1)pdm09 Viruses Carrying a Dual Neuraminidase Mutation Isolated from Immunocompromised Patients.

Influenza A(H1N1)pdm09 viruses carrying a dual neuraminidase (NA) substitution were isolated from immunocompromised patients after administration of one or more NA inhibitors. These mutant viruses possessed an H275Y/I223R, H275Y/I223K, or H275Y/G147R substitution in their NA and showed enhanced cross-resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir compared to single H275Y mutant viruses. Baloxavir could be a treatment option against the multidrug-resistant viruses because these dual H275Y mutant viruses showed susceptibility to this drug. The G147R substitution appears to stabilize the NA structure, with the fitness of the H275Y/G147R mutant virus being similar or somewhat better than that of the wild-type virus. Since the multidrug-resistant viruses may be able to transmit between humans, surveillance of these viruses must continue to improve clinical management and to protect public health.

Epidemiological Aspects of Escherichia albertii Outbreaks in Japan and Genetic Characteristics of the Causative Pathogen.

Zoonotic pathogen Escherichia albertii has been identified as the cause of several human disease outbreaks; however, factors such as the general symptoms and incubation period of E. albertii infection have yet to be defined. Therefore, we aimed to determine the unique aspects of E. albertii outbreaks in Japan and to examine the genetic characteristics of the causative pathogen. We studied all known E. albertii outbreaks that occurred in Japan up until 2015, which consisted of five confirmed outbreaks and one putative outbreak (Outbreaks 1-6). Outbreaks were re-examined based on personal communications between researchers in prefectural and municipal public health institutes, and through examination of any published study conducted at the time. Draft genome sequences of outbreak-associated E. albertii isolates were also generated. The most common symptom displayed by patients across the six episodes was watery diarrhea (>80%), followed by abdominal pain (50-84%) and fever (37.0-39.5°C) (26-44%). The estimated average incubation period of E. albertii infection was 12-24 h. We assumed that most of the outbreaks were foodborne or waterborne, with restaurant foods, restaurant water, and boxed lunches being the suspected transmission vehicles. Three of the six outbreak-associated E. albertii isolates possessed intact ETT2 regions, while the remaining isolates contained disrupted ETT2-encoding genes. Virulence gene screening revealed that more than half (44/70) of the tested genes were present in all 5 strains examined, and that each of the strains contained more than 1 gene from 14 out of the 21 groups of virulence genes examined in this study. The five E. albertii strains were classified into four of the five known phylogroups. Therefore, we determined that multiple E. albertii genotypes in Japan have the potential to cause outbreaks of diarrhea, abdominal pain, and/or fever following infection of a human host.

Five-year Study of Viral Etiology and Features of Febrile Respiratory Tract Infections With Prolonged Fever in Japanese Pediatric Outpatients.

Over 5 years, we prospectively collected nasopharyngeal aspirate samples from pediatric outpatients with prolonged fever (≥5 days, ≥38.0°C). Real-time polymerase chain reaction assays identifying 13 different respiratory viruses and Mycoplasma pneumoniae were performed on the test samples. Real-time polymerase chain reaction assays identified at least 1 pathogen in 273 (75.4%) of the 362 samples assessed (239 single and 34 multiple infections).

Detection of gastroenteritis viruses among pediatric patients in Hiroshima Prefecture, Japan, between 2006 and 2013 using multiplex reverse transcription PCR-based assays involving fluorescent dye-labeled primers.

Multiplex reverse transcription (RT)-polymerase chain reaction (PCR)-based assays involving fluorescent dye-labeled primers were modified to detect 10 types of gastroenteritis viruses by adding two further assays to a previously developed assay. Then, these assays were applied to clinical samples, which were collected between January 2006 and December 2013. All 10 types of viruses were effectively detected in the multiplex RT-PCR-based assays. In addition, various viral parameters, such as the detection rates and age distributions of each viral type, were examined. The frequency and types of mixed infections were also investigated. Among the 186 virus-positive samples, genogroup II noroviruses were found to be the most common type of virus (32.7%), followed by group A rotaviruses (10.6%) and parechoviruses (10.3%). Mixed infections were observed in 37 samples, and many of them were detected in patients who were less than 2 years old. These observations showed that the multiplex RT-PCR-based assays involving fluorescent dye-labeled primers were able to effectively detect the viruses circulating among pediatric acute gastroenteritis patients and contributed to the highly specific and sensitive diagnosis of gastroenteritis. J. Med. Virol. 89:791-800, 2017. © 2016 Wiley Periodicals, Inc.

Coronavirus Infections in Pediatric Outpatients with Febrile Respiratory Tract Infections in Hiroshima, Japan, over a 3-Year Period.

Previously, we conducted a 3-year prospective study to determine the viral causes of acute respiratory tract infections among 495 febrile pediatric outpatients. We collected 495 nasopharyngeal aspirate specimens, and used both real-time PCR assays and viral culture to test each for respiratory viruses other than coronavirus. Here, we used real-time PCR to test the 495 archival specimens for four human coronavirus strains. We identified 15 coronavirus-positive specimens: eight with OC43, 5 with NL63, 2 with HKU1, and none with 229E. Of the 15 children (5 boys) infected with human coronavirus, the mean age was 3.5 years, and the age range was 1.1 to 5.8 years; one child was diagnosed with lower respiratory infection; the other 14 were diagnosed with upper respiratory infection. Of these 15 patients, none were hospitalized, 5 were infected with coronavirus alone, 8 were co-infected with another virus, and 2 were co-infected with 2 other viruses. The multi-virus infections involved 6 adenoviruses, 3 respiratory syncytial viruses, 2 parainfluenza viruses, and 1 rhinovirus. In conclusion, the burden of human coronaviruses was relatively light among this cohort of 495 pediatric outpatients, and the incidence of these infections was low.

Comparison of throat swab and nasopharyngeal aspirate specimens for rapid detection of adenovirus.

Nasopharyngeal aspirate (NPA) and throat swab (TS) specimens from individual patients were compared with regard to usefulness for adenovirus detection. In 153 adenovirus-infected patients, rapid test sensitivities with NPAs (90.8%) were nearly equivalent to those with TSs (91.5%) based on real-time polymerase chain reaction standards, indicating that NPAs are equally useful.

[Clinical, epidemiological, and etiological studies of aseptic meningitis in adults].

From summer to autumn, we noted the occurrence of a small epidemic of aseptic meningitis in adults. Over the last 10 years, we have encountered 203 male (mean age, 34.6 ± 15.0 years) and 157 female (mean age, 35.6 ± 16.3 years) patients with aseptic meningitis. We could identify the causative virus in 17 (81%) of 21 cases during the abovementioned months in 2012. Identification rates of the virus in the stool, cerebrospinal fluid, throat swab, and serum samples were 71%, 67%, 42%, and 5%, respectively. The etiological viruses included enteroviruses in all cases, such as echovirus type 9 (E9) in 9 cases, echovirus type 6 (E6) in 4 cases, coxsackievirus type A9 in 1 case, and unknown type of enterovirus in 3 cases. No differences in the clinical manifestations and laboratory findings were noted between E9 meningitis and E6 meningitis. In addition, we countered 14 cases of mumps meningitis, 7 cases of varicella-zoster virus meningitis and 6 cases of herpes simplex meningitis during the last 10 years; these cases did not occur as an epidemic, but occurred sporadically. Cases of mumps meningitis were noted in all seasons, and cases of varicella-zoster virus meningitis were only noted from summer to winter. The etiology of epidemic aseptic meningitis in adults could be mainly due to enterovirus infection, and its prognosis was benign.

Three-year study of viral etiology and features of febrile respiratory tract infections in Japanese pediatric outpatients.

BACKGROUND: For most febrile respiratory tract infections (RTIs) in children, the causative pathogen is never identified. We sought to identify the causative pathogen in individual cases of pediatric outpatient with RTIs and to determine whether particular clinical features of RTIs are associated with particular viruses. METHODS: Over 3 years, we prospectively collected nasopharyngeal aspirate specimens from individual pediatric outpatients with an RTI accompanied by persistent fever (>3 days, ≥38.0°C) and peak temperature ≥39.0°C. Two methods-(1) viral culture for respiratory viruses and (2) real-time polymerase chain reaction (PCR) assays identifying 9 different respiratory viruses and 2 respiratory bacteria-were used to test specimens. RESULTS: For 495 specimens, viral culture and real-time PCR assays together identified at least 1 pathogen in 83.0% and ≥1 viruses alone in 79.4%. These 2 methods identified 138 children with respiratory syncytial virus, 66 with human metapneumovirus, 73 with parainfluenza viruses, 124 with adenovirus, 23 with rhinovirus, 38 with enterovirus, 11 with influenza type C virus, 15 with Mycoplasma pneumoniae and 3 with Chlamydophila pneumoniae; the coinfection rate was 19.7% among all infections. Among the patients with single-pathogen infections, the rate of lower RTI was 37.6% for respiratory syncytial virus, 40.7% for human metapneumovirus, 18.2% for parainfluenza viruses and 2.2% for adenovirus (P < 0.01). CONCLUSIONS: Viral culture and real-time PCR assays were used together to identify causative pathogens in 83% of febrile outpatient children with RTI; specific viruses were associated with particular clinical diagnoses.

Use of two rapid influenza diagnostic tests, QuickNavi-Flu and QuickVue Influenza A+B, for rapid detection of pandemic influenza A (H1N1) 2009 viruses in Japanese pediatric outpatients over two consecutive seasons.

A prospective study of outpatient children conducted during 2 consecutive seasons (2009 and 2011) of pandemic influenza A (H1N1) 2009 virus determined the sensitivity of a chromatographic immunoassay test; real-time reverse transcription-polymerase chain reaction was the standard, and the test was 87.2% (117 patients in 2009) and 97.4% (114 patients in 2011) sensitive.

Influenza viral load and rapid influenza diagnostic tests in children and adults.

We compared children and adults with regard to rapid influenza test sensitivity and viral load. Specimen volumes were measured, rapid tests were conducted, and viral load was determined. There was no difference between children and adults in test sensitivity or viral load, but children had higher specimen volumes.

Evaluation of influenza virus A/H3N2 and B vaccines on the basis of cross-reactivity of postvaccination human serum antibodies against influenza viruses A/H3N2 and B isolated in MDCK cells and embryonated hen eggs.

The vaccine strains against influenza virus A/H3N2 for the 2010-2011 season and influenza virus B for the 2009-2010 and 2010-2011 seasons in Japan are a high-growth reassortant A/Victoria/210/2009 (X-187) strain and an egg-adapted B/Brisbane/60/2008 (Victoria lineage) strain, respectively. Hemagglutination inhibition (HI) tests with postinfection ferret antisera indicated that the antisera raised against the X-187 and egg-adapted B/Brisbane/60/2008 vaccine production strains poorly inhibited recent epidemic isolates of MDCK-grown A/H3N2 and B/Victoria lineage viruses, respectively. The low reactivity of the ferret antisera may be attributable to changes in the hemagglutinin (HA) protein of production strains during egg adaptation. To evaluate the efficacy of A/H3N2 and B vaccines, the cross-reactivities of postvaccination human serum antibodies against A/H3N2 and B/Victoria lineage epidemic isolates were assessed by a comparison of the geometric mean titers (GMTs) of HI and neutralization (NT) tests. Serum antibodies elicited by the X-187 vaccine had low cross-reactivity to both MDCK- and egg-grown A/H3N2 isolates by HI test and narrow cross-reactivity by NT test in all age groups. On the other hand, the GMTs to B viruses detected by HI test were below the marginal level, so the cross-reactivity was assessed by NT test. The serum neutralizing antibodies elicited by the B/Brisbane/60/2008 vaccine reacted well with egg-grown B viruses but exhibited remarkably low reactivity to MDCK-grown B viruses. The results of these human serological studies suggest that the influenza A/H3N2 vaccine for the 2010-2011 season and B vaccine for the 2009-2010 and 2010-2011 seasons may possess insufficient efficacy and low efficacy, respectively.

Catalytic and biochemical features of a monoclonal antibody heavy chain, JN1-2, raised against a synthetic peptide with a hemagglutinin molecule of influenza virus.

It has long been an important issue to produce a catalytic antibody that possesses the ability to lose the infectivity of a bacteria or virus. The monoclonal antibody JN1-2 was generated using a synthetic peptide (TGLRNGITNKVNSVIEKAA) conjugated with human IgG. The peptide sequence includes the conserved region of the hemagglutinin molecule (HA(1) and HA(2) domains), which locates on the envelope of the influenza virus and plays an important role in influenza A virus infection. The monoclonal antibody specifically reacted with the HA2 domain, not only of H2 but also of an H1 strain of the H1N1 subtype (H1 strain). The heavy chain (JN1-2-H) isolated from the parent antibody showed catalytic activity cleaving the above antigenic peptide with very high turnover (kcat = 26 min(-1)), and it could slowly degrade the recombinant HA(2) domain by the catalytic function. Interestingly, the heavy chain exhibited the ability to reduce the infectivity of type A H1N1 but not type B, indicating specificity to type A. This characteristic monoclonal catalytic antibody heavy chain could suppress the infection of the influenza virus in vitro assays.

[Simultaneous multiple assay (Luminex xTAG respiratory viral panel FAST assay) efficacy in human respiratory virus detection].

Luminex xTAG respiratory viral panel FAST (RVP FAST) assay detects 17 human respiratory virus strains per measurement. Studying RVP FAST efficacy in detecting respiratory viruses in 67 aspirate samples from the nasal cavities of children with acute respiratory infection, we compared RVP FAST results to those of conventional nucleic acid amplification tests (NAT), e.g., real-time PCR, targeting 8 strains. RVP FAST assay detected 13 strains (98 isolates) in 59 of 67 samples. Of these, 8--influenza virus (Inf.V)-AH1, Inf. V-AH3, novel Inf.V-AH1, and Inf.V-B, and adenovirus, RS virus, metapneumovirus, and bocavirus--were compared to NAT results. RVP FAST showed higher sensitivity (83.3-100%) and specificity (98.2-100%) than NAT. RVP FAST also detected coronavirus (CoV) 229E, OC43, NL63, and HKU1 from 10 virus strain samples and enterovirus and/or rhinovirus from 35. RVP FAST assay thus comprehensively detects clinically important viruses in a single measurement, making RVP FAST assay useful in detecting causative respiratory tract viruses.

[Rapid detection of novel influenza A virus and seasonal influenza A (H1N1, H3N2) viruses by reverse transcription-loop-mediated isothermal amplification (RT-LAMP)].

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay we developed detects novel influenza A (H1N1) of swine origin and seasonal influenza A (H1N1 and H3N2) viruses. Individual primer sets targeting the HA gene for novel H1N1, H1N1, and H3N2 were newly designed to specifically detect these subtypes. No cross-reactions occurred among novel H1N1, H1N1, and H3N2, and 7 respiratory viruses-influenza B virus, influenza C virus, adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, and rhinovirus-had no reaction to 3 RT-LAMP assays. RT-LAMP is assayed at 63 degrees C for 40 min. In our RT-LAMP assay, Eriochrome Black T was added to the reaction mixture as an amplification indicator to detect virus genomes without using real-time turbidimetry. Positive reactions were indicated in blue and negative reactions remained purple. Of 139 samples from suspected novel H1N1 subjects tested by both RT-LAMP and real-time RT-PCR assay, 110 were positive in both assays. Two samples with low copy numbers were positive only in real-time RT-PCR assay. Of 27 novel negative H1N1 samples, 4 were positive for H3N2 on viral isolation and conventional RT-PCR assay. RT-LAMP assay for detecting H3N2 obtained the same findings. Our RT-LAMP assay is thus potentially useful in rapidly detecting influenza A virus such as novel H1N1, H1N1, and H3N2.

Rapid diagnosis of adenovirus respiratory tract infections using the chromatographic immunoassay test in the pediatric outpatient setting.

To assess the usefulness of a new rapid chromatographic immunoassay test for the detection of adenovirus, a prospective 3-year study was conducted in 587 febrile outpatient children suspected of adenovirus infection. A total of 332 children were diagnosed with this infection, using a viral culture. The sensitivity and specificity of the rapid test were 89.2% and 98.0%, respectively.

Transition of genotypes associated with norovirus gastroenteritis outbreaks in a limited area of Japan, Hiroshima Prefecture, during eight epidemic seasons.

The transition of genotypes implicated in 102 NoV gastroenteritis outbreaks in Hiroshima Prefecture, Japan, during eight epidemic seasons was investigated. Eighteen genotypes were implicated in the outbreaks, with the chronological characteristics as in GII.3, GII.4, GII.5 and GII.12. In GII.4 variants, amino acid changes and positively selected sites were of note and significantly concentrated in the surface-exposed P2 subdomain of the VP1 protein. Notably, variant-specific epitopes at which positively selected sites are located may be significant for distinguishing a new GII.4 variant. The interaction of these genetic changes with developing immunity seems to influence NoV epidemics.

Human metapneumovirus infection in febrile children with lower respiratory diseases in primary care settings in Hiroshima, Japan.

Human metapneumovirus (hMPV) has been shown to be a leading cause of viral lower respiratory tract infections in children. Nevertheless, few reports regarding hMPV infections over consecutive years in children in primary care settings are available. We carried out virologic and clinical studies to determine the role of hMPV in febrile lower respiratory infections in children at a primary care clinic over 3 years and 5 months. Nasopharyngeal aspirates obtained from children with acute respiratory tract infections accompanied by high-grade fever (> or = 39 degrees C) and productive cough were studied for hMPV by reverse transcription-polymerase chain reaction and for other respiratory viruses by viral cultures and immunoassays. Of 379 patients tested, 202 were positive for at least 1 virus, including 98 with hMPV, 69 with respiratory syncytial virus, 18 with adenovirus, 12 with enterovirus, 8 with parainfluenza virus, 3 with rhinovirus, 2 with influenza virus type C, and 1 with herpes simplex virus. The male:female ratio of hMPV-infected children was 0.96:1 with an overall mean age of 3.5 years (range, 2 months to 9 years). These infections occurred predominantly from February to July, and the hospitalization rate was 4%. Of 93 patients infected with hMPV alone, 52 (56%) showed evidence of a lower respiratory tract infection.