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.

Evaluation of Diagnostic Assay for Rickettsioses Using Duplex Real-Time PCR in Multiple Laboratories in Japan.

Tsutsugamushi disease and Japanese spotted fever are representative rickettsioses in Japan, and are caused by infection with Orientia tsutsugamushi and Rickettsia japonica, respectively. For molecular-based diagnosis, conventional PCR assays, which independently amplify respective rickettsial DNA, are usually used; however, this approach is time-consuming. Here, we describe a new duplex real-time PCR assay for the simultaneous detection of O. tsutsugamushi and spotted fever group rickettsiae, and its evaluation using several PCR conditions in 6 public health laboratories. The detection limit of the assay was estimated to be 102 copies and the sensitivity was almost identical to that of 3 conventional PCR methods. A total of 317 febrile patients were selected as clinically suspected or confirmed cases of rickettsioses. The detection efficiency of this assay for O. tsutsugamushi from blood or skin (eschar) specimens appeared to be almost the same as that of the conventional PCR method, even when performed in different laboratories, whereas the efficiency for spotted fever group rickettsiae tended to be higher than that of the 2 traditional double PCR assays. Our duplex real-time PCR is thus a powerful tool for the rapid diagnosis of rickettsioses, especially at the acute stage of infection.

Extremely Low Genomic Diversity of Rickettsia japonica Distributed in Japan.

Rickettsiae are obligate intracellular bacteria that have small genomes as a result of reductive evolution. Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fevers". The life cycle of SFG rickettsiae is closely associated with that of the tick, which is generally thought to act as a bacterial vector and reservoir that maintains the bacterium through transstadial and transovarial transmission. Each SFG member is thought to have adapted to a specific tick species, thus restricting the bacterial distribution to a relatively limited geographic region. These unique features of SFG rickettsiae allow investigation of how the genomes of such biologically and ecologically specialized bacteria evolve after genome reduction and the types of population structures that are generated. Here, we performed a nationwide, high-resolution phylogenetic analysis of Rickettsia japonica, an etiological agent of Japanese spotted fever that is distributed in Japan and Korea. The comparison of complete or nearly complete sequences obtained from 31 R. japonica strains isolated from various sources in Japan over the past 30 years demonstrated an extremely low level of genomic diversity. In particular, only 34 single nucleotide polymorphisms were identified among the 27 strains of the major lineage containing all clinical isolates and tick isolates from the three tick species. Our data provide novel insights into the biology and genome evolution of R. japonica, including the possibilities of recent clonal expansion and a long generation time in nature due to the long dormant phase associated with tick life cycles.

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.

Influenza A(H1N1)pdm09 virus exhibiting enhanced cross-resistance to oseltamivir and peramivir due to a dual H275Y/G147R substitution, Japan, March 2016.

An influenza A(H1N1)pdm09 virus carrying a G147R substitution in combination with an H275Y substitution in the neuraminidase protein, which confers cross-resistance to oseltamivir and peramivir, was detected from an immunocompromised inpatient in Japan, March 2016. This dual H275Y/G147R mutant virus exhibited enhanced cross-resistance to both drugs compared with the single H275Y mutant virus and reduced susceptibility to zanamivir, although it showed normal inhibition by laninamivir.

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.

Phylogenetic and Geographic Relationships of Severe Fever With Thrombocytopenia Syndrome Virus in China, South Korea, and Japan.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne acute infectious disease caused by the SFTS virus (SFTSV). SFTS has been reported in China, South Korea, and Japan as a novel Bunyavirus. Although several molecular epidemiology and phylogenetic studies have been performed, the information obtained was limited, because the analyses included no or only a small number of SFTSV strains from Japan. METHODS: The nucleotide sequences of 75 SFTSV samples in Japan were newly determined directly from the patients' serum samples. In addition, the sequences of 7 strains isolated in vitro were determined and compared with those in the patients' serum samples. More than 90 strains that were identified in China, 1 strain in South Korea, and 50 strains in Japan were phylogenetically analyzed. RESULTS: The viruses were clustered into 2 clades, which were consistent with the geographic distribution. Three strains identified in Japan were clustered in the Chinese clade, and 4 strains identified in China and 26 in South Korea were clustered in the Japanese clade. CONCLUSIONS: Two clades of SFTSV may have evolved separately over time. On rare occasions, the viruses were transmitted overseas to the region in which viruses of the other clade were prevalent.

Sensitive and specific PCR systems for detection of both Chinese and Japanese severe fever with thrombocytopenia syndrome virus strains and prediction of patient survival based on viral load.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV). A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.

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.

[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.

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.

Contribution of the leader sequence to homologous viral interference among Sendai virus strains.

Sendai viruses (SeV) derived from persistent infection have a capacity to interfere with co-infected wild-type virus. Here we showed that interference was also caused by the laboratory strains Z and Nagoya. The leader mutations A(20)U and A(24)U related to viral adaptation from mice to chicken eggs significantly affected the capacity for viral interference, especially through genome amplification. Furthermore, recombinant SeV that possessed the mutations A(34)G and G(47)A, which are commonly found in the leader sequence of persistent infection-derived SeV strains, had an increased capacity for interference. Viral replication of human parainfluenza viruses 1, 2, and 3, but not the mumps virus or Newcastle disease virus, was suppressed by co-infection of a persistent infection-derived SeV strain, suggesting suppression of closely related human paramyxoviruses. These results indicate that homologous interference is partly dependent on the promoter sequence and further suggest involvement of promoter activity for genome amplification related to host factors in viral interference.

The YLDL sequence within Sendai virus M protein is critical for budding of virus-like particles and interacts with Alix/AIP1 independently of C protein.

For many enveloped viruses, cellular multivesicular body (MVB) sorting machinery has been reported to be utilized for efficient viral budding. Matrix and Gag proteins have been shown to contain one or two L-domain motifs (PPxY, PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular proteins involved in MVB sorting, which are recruited to the viral budding site. However, for many enveloped viruses, L-domain motifs have not yet been identified and the involvement of MVB sorting machinery in viral budding is still unknown. Here we show that both Sendai virus (SeV) matrix protein M and accessory protein C contribute to virus budding by physically interacting with Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and its specific interaction with the N terminus of Alix/AIP1(1-211) was important for the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP budding was inhibited by the overexpression of some deletion mutant forms of Alix/AIP1 and depletion of endogenous Alix/AIP1 with specific small interfering RNAs. The YLDL sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, and even YPxL. C protein was also able to physically interact with the N terminus of Alix/AIP1(212-357) and enhanced M-VLP budding independently of M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may enhance the efficiency of the utilization of cellular MVB sorting machinery for efficient SeV budding.

[Evaluation of immunochromatography test for rapid detection of influenza A and B viruses using real-time PCR].

The sensitivity of rapid diagnostic kits to influenza B is lower than to influenza A. The cause-poor performance of the kit or the scarcity of viruses in type B specimens-has yet to be clarified. Using real-time PCR, we measured the amount of influenza viruses with nasopharyngeal aspirate fluid previously identified by virus isolation culture and passing the rapid diagnosis test by four types of kits, including the ESPLINE Influenza A&B-N (Fujirebio Corp., Japan). We classified the results of virus isolation and rapid diagnosis tests into three groups and examined them: group 1 (12 specimens, influenza B, all negative in tests using four types of kits); group 2 (57 specimens, influenza B, all positive in tests); and group 3 (36 specimens, AH3, all positive in tests). The average amount of viruses in group 1 (6.60 +/- 0.81 log10copies/mL) was significantly lower (p<0.0001) than that in group 2 (8.51 +/- 0.57 log10copies/mL) or group 3 (8.72 +/- 0.63 log10copies/mL). No significant difference was seen in the amount of viruses between groups 2 and 3. We concluded that the cause of low sensitivity in rapid diagnostic kits to influenza B are attributable to the scarcity of viruses in the specimen.

Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay.

In this study, we developed a one-step, single-tube genogroup-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NoV) genomes targeting from the C terminus of the RNA-dependent RNA polymerase gene to the capsid N-terminal/shell domain region. This is the first report on the development of an RT-LAMP assay for the detection of NoV genomes. Because of the diversity of NoV genotypes, we used 9 and 13 specially designed primers containing mixed bases for genogroup I (GI) and II (GII), respectively. The RT-LAMP assay had the advantages of rapidity, simplicity, specificity, and selectively and could obtain results within 90 min, generally even within 60 min, under isothermal conditions at 62 degrees C. The detection limits for NoV genomes were between 10(2) and 10(3) copies/tube for GI and GII with differentiation by genotype, and no cross-reactions among NoV GI and GII and other gastroenteritis viruses, such as sapovirus, human astrovirus, adenovirus type 40 and 41, and group A and C rotavirus, were found. In the evaluation tests with fecal specimens obtained from gastroenteritis outbreaks, the sensitivity and specificity of the RT-LAMP assay with regard to RT-PCR were 100 and 94% for GI and 100 and 100% for GII, respectively. These findings establish that the RT-LAMP assay is potentially useful for the rapid detection of NoV genomes from fecal specimens in outbreaks of food-borne and person-to-person-transmitted gastroenteritis.

[Eleven cases of co-infection with influenza type A and type B suspected by use of a rapid diagnostic kit and confirmed by RT-PCR and virus isolation].

In the 2004/05 influenza season there were epidemics of influenza caused by several types of viruses (type B and A (H3) viruses, and type B, A (H3), and A (H1) viruses) in many areas of Japan. In such epidemics a single individual could be co-infected with several influenza viruses. From February to March in 2005, we examined 15 patients who were positive for influenza type A and B viruses when tested with a rapid diagnostic kit. The type A (H3) and B influenza virus genes were successfully amplified by RT-PCR in 10 of the 15 patients, confirming that they were co-infected with type A (H3) and B viruses. The type A (H1) and B virus genes were successfully amplified in another patient, confirming that the patient was co-infected with type A (H1) and B viruses. By contrast, 2 patients were clearly positive for type A and B viruses according to the rapid diagnostic kit, but positive for type B virus alone by RT-PCR. No influenza virus genes were detected by RT-PCR in the remaining 2 patients. To isolate one type from a mixture of two different types of influenza viruses in a specimen, we neutralized one of the types with type-specific antiserum, and isolated the other with MDCK (+) cells. The results obtained by virus isolation were identical to those obtained by RT-PCR. Influenza viruses corresponding to the results of RT-PCR were isolated from 9 of the 11 patients in which isolation was attempted. No viruses were isolated from the 2 patients in whom no virus genes were detectable by RT-PCR. Based on these results we concluded that 11 of 15 patients who were positive for type A and B viruses according to the rapid diagnostic kit were co-infected with type A (H3) or A (H1) and B virus. When several types of influenza viruses are prevalent, as in the 2004/05 influenza season, the possibility of a patient being co-infected with more than one type of influenza virus should be considered.