The N-terminal fragment of PA subunit of the influenza A virus effectively inhibits ribonucleoprotein (RNP) activity via suppression of its RNP expression.

The influenza RNP, which is formed from PB1, PB2, PA, NP subunits, and vRNA, is autonomously replicated and transcribed in the infected cell. The simplest method to inhibit RNP activity is to impair the formation of the RNP. Thereupon we confirmed whether the peptides/fragments mimicking one of RNP components can interfere with their formation. During the process of this inhibitory study we found interesting suppression of protein expression of the RNP components by the N-terminal fragment of PA subunit. Especially, we found two residues (D108 and K134) on the fragment that were critical for the suppression. Furthermore, we determined the combination of three amino acids (P28, M86 and E100) on the fragment that are important for the strong suppression, and identified the minimum essential region (residues from 1 to 188) of the PA subunit that allowed its suppression. Our findings indicate that the N-terminal fragment of PA subunit may become one of candidates for an effective inhibitor of influenza RNP activity.

The N-terminal fragment of a PB2 subunit from the influenza A virus (A/Hong Kong/156/1997 H5N1) effectively inhibits RNP activity and viral replication.

BACKGROUND: Influenza A virus has a RNA-dependent RNA polymerase (RdRp) that is composed of three subunits (PB1, PB2 and PA subunit), which assemble with nucleoproteins (NP) and a viral RNA (vRNA) to form a RNP complex in the host nucleus. Recently, we demonstrated that the combination of influenza ribonucleoprotein (RNP) components is important for both its assembly and activity. Therefore, we questioned whether the inhibition of the RNP combination via an incompatible component in the RNP complex could become a methodology for an anti-influenza drug. METHODOLOGY/PRINCIPAL FINDINGS: We found that a H5N1 PB2 subunit efficiently inhibits H1N1 RNP assembly and activity. Moreover, we determined the domains and important amino acids on the N-terminus of the PB2 subunit that are required for a strong inhibitory effect. The NP binding site of the PB2 subunit is important for the inhibition of RNP activity by another strain. A plaque assay also confirmed that a fragment of the PB2 subunit could inhibit viral replication. CONCLUSIONS/SIGNIFICANCE: Our results suggest that the N-terminal fragment of a PB2 subunit becomes an inhibitor that targets influenza RNP activity that is different from that targeted by current drugs such as M2 and NA inhibitors.

Comparison study of single and concurrent administrations of carbapenem, new quinolone, and macrolide against in vitro nontypeable Haemophilus influenzae mature biofilms.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen and a common cause of otitis media in children, chronic bronchitis, and pneumonia in patients with chronic obstructive pulmonary disease. Many studies have reported that NTHi is capable of producing biofilms, which may be one of the important factors involved in chronic diseases and accelerating antimicrobial resistance. Unfortunately, there is still no consensus about the elimination of biofilms. In this study, concurrent administrations of levofloxacin (LVFX)-imipenem (IPM) and clarithromycin (CAM)-IPM, as well as the single administration of IPM, LVFX, and CAM, were performed to treat the mature biofilms produced by NTHi, respectively. Biofilm inhibition was quantified using microtiter biofilm assay (MBA), and relative biomass was calculated as the ratio compared to that of untreated control biofilms. The relative biomasses of biofilms treated with IPM, LVFX-IPM, and CAM-IPM against a ß-lactamase-negative ampicillin-resistant strain was 1.10, 0.08, and 0.13 at 1× minimum inhibitory concentration (MIC), 0.90, 0.05, and 0.07 at 10× MIC, and 0.80, 0.06, and 0.07 at 100× MIC, respectively. Biofilms were also visually observed by scanning electron microscopy, and a focused ion-beam system showed that high concentrations of combined administration strongly inhibited the biofilms, which was consistent with the results of MBA. Our data demonstrated the antibiofilm effect of concurrent administration against mature NTHi biofilms, which indicated a rationale for the potential use of concurrent administrations in diseases involving chronic NTHi biofilms.

Infection control for a methicillin-resistant Staphylococcus aureus outbreak in an advanced emergency medical service center, as monitored by molecular analysis.

A methicillin-resistant Staphylococcus aureus (MRSA) outbreak occurred in an advanced emergency medical service center between 2010 and 2011. Our objective was to evaluate the status of the MRSA outbreak, as monitored by molecular analysis. Twenty-eight MRSA strains were isolated from blood samples from 11 patients, from other specimens (pharynx, nasal cavity, etc.) from 12 patients, from two environmental samples, and from the skin, middle nasal meatus, and urine of one patient each from other wards. Pulsed-field gel electrophoresis (PFGE) was performed to evaluate horizontal transmission. Molecular typing by PFGE showed that the 28 MRSA strains presented 7 patterns in total, and that 11 of the MRSA strains had the same PGFE pattern. Unselective use of intranasal mupirocin ointment, MRSA monitoring for new inpatients, and prevention of direct or indirect contact infection were performed. However, the number of inpatients with MRSA did not quickly decrease, and additional molecular typing by PFGE showed that 10 of 19 MRSA strains found (5 of 6 from blood, 5 of 13 from other specimens) were the same as those found previously. Lectures and ward rounds were performed repeatedly, and staff participation in ward rounds was suggested. Finally, the number of inpatients with MRSA significantly decreased more than 6 months after the intervention. Although the MRSA outbreak was thought to have ended, follow-up molecular typing by PFGE showed that horizontal transmission persisted. Our data suggest that various combinations of infection control measures are essential when dealing with an MRSA outbreak, and monitoring by molecular analysis using PFGE is useful to identify the status of the outbreak.