[Show abstract][Hide abstract] ABSTRACT: Six influenza A(H1N1)pdm09 viruses were detected in Sapporo, Japan, between November and December 2013. All six viruses possessed an H275Y substitution in the neuraminidase protein, which confers cross-resistance to oseltamivir and peramivir. No epidemiological link among the six cases could be identified; none of them had received neuraminidase inhibitors before specimen collection. The haemagglutinin and neuraminidase genes of the six viruses were closely related to one another, suggesting clonal spread of a single resistant virus.
Full-text · Article · Jan 2014 · Eurosurveillance: bulletin europeen sur les maladies transmissibles = European communicable disease bulletin
[Show abstract][Hide abstract] ABSTRACT: Influenza virus RNA-dependent RNA polymerase is composed of three viral proteins, PB1, PB2, and PA. The host protein Ebp1 (ErbB3-binding protein1) interacts with PB1, and inhibits both in vitro RNA synthesis and virus replication. On Western blotting, the induction of Ebp1 was observed after influenza virus infection. To understand the induction of Ebp1 by influenza virus infection, we introduced a series of deletions within the 981-nucleotide long sequence located upstream of the Ebp1 gene (-664 to +317 nt from the transcription initiation site) and ligated them to the GFP gene. GFP expression assays indicated that the 981-nt upstream region was required for expression of GFP in not all cells but some cells. Microscopic analysis of the transformants showed that GFP expression was up-regulated by the influenza virus infection. Furthermore, quantitative real-time PCR indicated that influenza virus infection induced Ebp1 mRNA expression. Our data showed that (i) the newly synthesized vRNP of influenza virus induces Ebp1 expression; (ii) the Ebp1 promoter localizes between -664 nt and the initiation site of the Ebp1 gene, +317-nt long sequence in the noncoding region is required for regulation of Ebp1 gene expression; and (iii) Ebp1 expression level is correlated with virus protein expression level.
[Show abstract][Hide abstract] ABSTRACT: To monitor and characterize oseltamivir-resistant (OR) pandemic (H1N1) 2009 virus with the H275Y mutation, we analyzed 4,307 clinical specimens from Japan by neuraminidase (NA) sequencing or inhibition assay; 61 OR pandemic (H1N1) 2009 viruses were detected. NA inhibition assay and M2 sequencing indicated that OR pandemic (H1N1) 2009 virus was resistant to M2 inhibitors, but sensitive to zanamivir. Full-genome sequencing showed OR and oseltamivir-sensitive (OS) viruses had high sequence similarity, indicating that domestic OR virus was derived from OS pandemic (H1N1) 2009 virus. Hemagglutination inhibition test demonstrated that OR and OS pandemic (H1N1) 2009 viruses were antigenically similar to the A/California/7/2009 vaccine strain. Of 61 case-patients with OR viruses, 45 received oseltamivir as treatment, and 10 received it as prophylaxis, which suggests that most cases emerged sporadically from OS pandemic (H1N1) 2009, due to selective pressure. No evidence of sustained spread of OR pandemic (H1N1) 2009 was found in Japan; however, 2 suspected incidents of human-to-human transmission were reported.
Full-text · Article · Mar 2011 · Emerging Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: In March 2009, pandemic influenza A(H1N1) (A(H1N1)pdm) emerged in Mexico and the United States. In Japan, since the first outbreak of A(H1N1)pdm in Osaka and Hyogo Prefectures occurred in the middle of May 2009, the virus had spread over 16 of 47 prefectures as of June 4, 2009.
We analyzed all-segment concatenated genome sequences of 75 isolates of A(H1N1)pdm viruses in Japan, and compared them with 163 full-genome sequences in the world. Two analyzing methods, distance-based and Bayesian coalescent MCMC inferences were adopted to elucidate an evolutionary relationship of the viruses in the world and Japan. Regardless of the method, the viruses in the world were classified into four distinct clusters with a few exceptions. Cluster 1 was originated earlier than cluster 2, while cluster 2 was more widely spread around the world. The other two clusters (clusters 1.2 and 1.3) were suggested to be distinct reassortants with different types of segment assortments. The viruses in Japan seemed to be a multiple origin, which were derived from approximately 28 transported cases. Twelve cases were associated with monophyletic groups consisting of Japanese viruses, which were referred to as micro-clade. While most of the micro-clades belonged to the cluster 2, the clade of the first cases of infection in Japan originated from cluster 1.2. Micro-clades of Osaka/Kobe and the Fukuoka cases, both of which were school-wide outbreaks, were eradicated. Time of most recent common ancestor (tMRCA) for each micro-clade demonstrated that some distinct viruses were transmitted in Japan between late May and early June, 2009, and appeared to spread nation-wide throughout summer.
Our results suggest that many viruses were transmitted from abroad in late May 2009 irrespective of preventive actions against the pandemic influenza, and that the influenza A(H1N1)pdm had become a pandemic stage in June 2009 in Japan.
[Show abstract][Hide abstract] ABSTRACT: Nanomanipulation of a single influenza virus in a microfluidic chip for biomedical innovation was developed. We employed Robot on a Chip (RoC) to achieve stable manipulation of nano-scale biomaterials such as virus (size is about 100 nm). The microfluidic chip has independent virus chamber and cell chamber to make quantitative analysis such as single virus infection to a specific cell. Gel-microtool generated by local heating of thermosensitive hydrogel poly(N-isopropylacrylamide) (PNIPAAm) by near infrared laser was used for stable and high-speed virus manipulation. Single selected virus was transported to cell chamber using the gelmicrotool. To construct the stable experimental environment in a microfluidic chip, solution reservoir having air damper and leak port was fabricated and short response time of flow control within 1 second was achieved. To avoid the incursion of the extra virus to the cell chamber, isolation of the cell chamber to virus chamber was performed by stacking the microchannel between these chambers using in-situ photoplolymerization of photo-crosslinkable resin polyethylene glycol methacrylate (PEG-MA) (200). In this paper, we demonstrated single influenza virus manipulation and isolation of cell chamber and virus chamber using optical tweezers, quick flow control devices and in-situ photopolymerization in a microfluidic chip.
[Show abstract][Hide abstract] ABSTRACT: Influenza virus PB1 protein plays the key role for the assembly and catalytic function of viral RNA polymerase. We showed that Ebp1 interacted with PB1 and interfered both the viral RNA polymerase function in vitro and the virus growth. The Ebp1 expression was induced by influenza virus infection using western blotting and quantitative real time PCR assay. However, the quantitative determination has been requested of the relationship between influenza virus growth and Ebp1 level. To analyze the relationship between virus growth and Ebp1 level, we developed the assay system of single cell. Using this system, we confirmed the tight correlation between the Ebp1 level and the influenza virus growth.
[Show abstract][Hide abstract] ABSTRACT: Influenza virus attaches on the sialic acid on cellular membrane through it's HA protein and enters cytoplasm by receptor-mediated endocytosis, then move from endocytic vesicles to early endosomes and late endosome where the influenza viruses fuse with the endosomal membrane to release viral genome followed to transport the genome to nuclei where virus replicate their genome. Previously we found that host cell protein Ebpl (ErbB3 binding protein 1) interacted with the catalytic subunit PB1 of influenza virus RNA dependent RNA polymerase and interfered with its function. Ebpl expression is regulated by cell cycle, showing high level expression especially in Gl to S phase. Recently we identified another host protein (PA4c9) interacts with subunit PA of influenza virus RNA polymerase. Again PA4c9 is one of the cell cycle dependent proteins. These findings encouraged us to investigate the host cell cycle dependency of influenza infection. To identify the cell phase at which influenza virus attach most efficiently, the cells were treated with the fluorescent-labeled influenza viruses for 5-15 minutes and fixed with paraformaldehyde followed by observation under the confocal microscope. In parallel, attempts were made to manipulate a single particle of fluorescent-labeled virus using laser manipulator for infection onto a single cell at different phase. The results altogether indicated that influenza virus preferred the membrane of stable cell phase (G0/G1) to attach for infection.
[Show abstract][Hide abstract] ABSTRACT: We propose in situ formation of gel microbeads made of a thermoreversible hydrogel for indirect laser micromanipulation of microorganisms, DNA and viruses. Irradiation, using a 1064 nm laser, of an aqueous solution mixed with poly-(N-isopropylacrylamide) through a high magnification lens resulted in the formation of a gel microbead at the laser focus due to heating. The gel microbead was trapped by the laser, and was used for indirect laser micromanipulation of microscale and nanoscale samples. Laser tweezers can typically handle a microscale object with size ranging from several tens of nm to several hundreds of mum in a stable manner. However, a nanoscale object with a size of a few nm cannot be stably manipulated, and laser beam heating is a major problem. This paper shows a method of indirect manipulation of microorganisms, DNA and viruses using a gel microbead made from the poly-(N-isopropylacrylamide) aqueous solution. We succeeded in reducing the laser power for gel microbead formation, and in using the laser-trapped gel microbead for the manipulation of microorganisms, DNA and viruses
[Show abstract][Hide abstract] ABSTRACT: Influenza virus RNA polymerase is composed of three subunits, PB1, PB2 and PA, which is involved in both transcription and replication of viral genome RNA, negative strand RNA. Influenza virus gene product RNA dependent RNA polymerase synthesizes two different RNAs, one is mRNA and another is cRNA which become a template for virus genome RNA synthesis. To synthesize two different RNAs the RNA polymerase should be converted by some factor(s) in the virus infected cell. It was speculated that between polymerase structure and function could be some relations. It is started to screen the protein(s) which interact(s) with RNA polymerase using yeast two-hybrid screening system. Nine host proteins were identified, which interacted with different polymerase subunits. One of them, PB1c45, which interacted with PB1 subunit in polymerase and molecular weight was 45kDa, was analyzed the effect on polymerase activity and influenza virus infectivity. The PB1c45 inhibited the polymerase activity and also reduced the influenza virus infectivity