Antiviral susceptibility of influenza viruses isolated from patients pre- and post-administration of favipiravir.

Favipiravir, a viral RNA-dependent RNA polymerase inhibitor, has recently been approved in Japan for influenza pandemic preparedness. Here, we conducted a cell-based screening system to evaluate the susceptibility of influenza viruses to favipiravir. In this assay, the antiviral activity of favipiravir is determined by inhibition of virus-induced cytopathic effect, which can be measured by using a colorimetric cell proliferation assay. To demonstrate the robustness of the assay, we compared the favipiravir susceptibilities of neuraminidase (NA) inhibitor-resistant influenza A(H1N1)pdm09, A(H3N2), A(H7N9) and B viruses and their sensitive counterparts. No significant differences in the favipiravir susceptibilities were found between NA inhibitor-resistant and sensitive viruses. We, then, examined the antiviral susceptibility of 57 pairs of influenza viruses isolated from patients pre- and post-administration of favipiravir in phase 3 clinical trials. We found that there were no viruses with statistically significant reduced susceptibility to favipiravir or NA inhibitors, although two of 20 paired A(H1N1)pdm09, one of 17 paired A(H3N2) and one of 20 paired B viruses possessed amino acid substitutions in the RNA-dependent RNA polymerase subunits, PB1, PB2 and PA, after favipiravir administration. This is the first report on the antiviral susceptibility of influenza viruses isolated from patients after favipiravir treatment.

[Opioid induction using rapid release drugs and the shift to fentanyl patches].

SUBJECT AND METHODS: From April 2011 to March 2013, 20 patients with cancer pain that was not controlled by non-opioid analgesics were treated with a short-acting opioid for cancer pain management.The primary carcinoma sites were the stomach( n=5), colo-rectum(n=5), lungs(n=3), urinary bladder(n=2), breast(n=2), pancreas(n=2), and liver(n=1). The analgesic effects and adverse events were evaluated, and a shift to fentanyl patches was made for patients whose cancer pain was relieved.After the shift, the efficiency and safety were validated. RESULTS: All 6 patients with a numeric rating scale (NRS)less than 5 at the time of opioid induction had a good analgesic effect, and in only 1 patient, grade 2 constipation and grade 3 anorexia was observed.Of the 14 patients who had an NRS of 6 or greater, 11 had a good analgesic effect.However, 3 patients experienced no effect, and their survival periods after opioid induction were very short.In the 11 patients with good pain control, only 3 patients exhibited grade 2 adverse events.Nine patients out of 17 with a good analgesic effect caused by short-acting opioids were shifted to fentanyl patches, and 8 patients were under good analgesic control for 2 weeks or more. CONCLUSION: Opioid induction using rapid release drugs was effective and safe.However, these drugs should be clinically considered at an early stage.Furthermore, in patients where a shift to a fentanyl patch was possible, good long-term pain control was achieved.

Influenza virus infection induces cellular Ebp1 gene expression.

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.

Rapid discrimination of oseltamivir-resistant 275Y and -susceptible 275H substitutions in the neuraminidase gene of pandemic influenza A/H1N1 2009 virus by duplex one-step RT-PCR assay.

Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus caused significant outbreaks worldwide last year (2009). A number of oseltamivir-resistant A/H1N1pdm viruses possessing an H275Y substitution in the neuraminidase (NA) protein were reported sporadically in several countries, including Japan, but they were sensitive to zanamivir and did not spread in the community. In this study, to monitor rapidly and simply oseltamivir-resistant A/H1N1pdm viruses possessing H275Y, a duplex one-step RT-PCR assay (H275Y RT-PCR assay) was developed based on an endpoint genotyping analysis method. H275Y RT-PCR assay evaluated using several subtypes/types of influenza A and B viruses and other respiratory pathogenic viruses and shown to have high sensitivity and high specificity. Forty-four clinical specimens were tested after RNA purification using the H275Y RT-PCR assay, resulting in one clinical specimen being found to contain a virus possessing the H275Y mutation. Seventy-three clinical isolates were then tested with the H275Y assay by using clinical isolates in the cultured supernatants of cells directly, without RNA purification, and the results were consistent with the NA sequencing. Since the H275Y RT-PCR assay could detect the H275Y mutation in clinical isolates without RNA purification, as well as a H275Y mutated virus in clinical specimens after RNA purification, the assay was considered a powerful tool for surveillance screening of oseltamivir-resistant A/H1N1pdm virus activity.

Monitoring and characterization of oseltamivir-resistant pandemic (H1N1) 2009 virus, Japan, 2009-2010.

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.

[Clinical analysis of 12 cases of pneumothorax during intensive chemotherapy for malignant neoplasms].

Twelve cases of pneumothorax during intensive chemotherapy for malignant neoplasms were found in a retrospective study of patients being treated at our hospital in the period 2001-2009. All of the patients were men, and their diseases were lung cancer (9 cases), gastric cancer (2 cases) and esophageal cancer (one case). Operation for pneumothorax was performed 9 times in 7 patients(twice in two patients). Thoracoscopic surgery was done in 6 patients, and one patient with severe pulmonary emphysema was performed by open thoracotomy. Pleurodesis was performed 5 times using OK-432 only or OK-432 and minocyclin. Five patients died during the treatment for pneumothorax. The causes of death were interstitial pneumonia after pleurodesis (one case), and progression of cancer during interruption of chemotherapy (4 cases). Development of pneumothorax during intensive chemotherapy should be recognized and treated as soon as possible because it may hinder the treatment for malignant tumors and lead to severe pulmonary dysfunction.

Molecular evolutionary analysis of the influenza A(H1N1)pdm, May-September, 2009: temporal and spatial spreading profile of the viruses in Japan.

BACKGROUND: 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. METHODS/PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.