Community- and hospital-acquired infections with oseltamivir- and peramivir-resistant influenza A(H1N1)pdm09 viruses during the 2015–2016 season in Japan

We report five cases of community- and hospital-acquired infections with oseltamivir- and peramivir-resistant A(H1N1)pdm09 viruses possessing the neuraminidase (NA) H275Y mutation during January–February 2016 in Japan. One case was hospitalized and was receiving oseltamivir for prophylaxis. The remaining four cases were not taking antiviral drugs at the time of sampling. These cases were geographically distant and epidemiologically unrelated. The five viruses showed ~300-fold rise in IC₅₀ values against oseltamivir and peramivir, defined as highly reduced inhibition according to the WHO definition. Overall, the prevalence of the H275Y A(H1N1)pdm09 viruses was 1.8 % (5/282). The resistant viruses possessed the V241I, N369 K, and N386 K substitutions in the NA that have been previously reported among A(H1N1)pdm09 to alter transmission fitness. Analysis of Michaelis constant (Km) revealed that two of the isolates had reduced NA affinity to MUNANA, while the other three isolates displayed a slightly decreased affinity compared to the sensitive viruses. Further studies are needed to monitor the community spread of resistant viruses and to assess their transmissibility.     

Emergence of influenza A (H1N1) PDM09 in the remote Islands of India – A molecular approach

Background: A disease outbreak of A (H1N1) PDM09 was reported in Andaman and Nicobar islands in 2009 with an attack rate of 33.5% among settler population and 26.3% among the aboriginal Nicobarese tribe. During the ongoing outbreak of A (H1N1) PDM09 disease in different parts of the world, a subject working in Dubai city of Saudi Arabia, came to Port Blair, following which the pandemic triggered for the first time in these Islands. Materials and Methods: During the period August 2009 to January 2011, 30 confirmed cases of Influenza A (H1N1) PDM09 virus infection was detected. To understand the genetic relationship, the NA gene sequences of the viruses were phylogenetically analysed together along with the virus sequence isolated from other parts of the world. Result: Formation of multiple clusters were observed, with the sequences of Andaman Islands, mainland India, Mexico, Saudi Arabia and few other counties clustering together. The sequence analysis data revealed that there was no specific mutation conferring resistance to oseltamivir among the Andaman A (H1N1) PDM09 virus isolates. The result of phylogenetic analysis have also revealed that the A (H1N1) PDM09 virus might have spread in these remote Islands of India via the subject from Saudi Arabia/Dubai. Conclusion: A (H1N1) PDM09 Influenza outbreak have highlighted the need to strengthen the region-specific pandemic preparedness plans and surveillance strategies.     

Inhibition of PKR ameliorates lipopolysaccharide-induced acute lung injury by suppressing NF-κB pathway in mice

Acute lung injury (ALI) is characterized by dramatic lung inflammation and alveolar epithelial cell death. Although protein kinase R (PKR) (double-stranded RNA-activated serine/threonine kinase) has been implicated in inflammatory response to bacterial cell wall components, whether it plays roles in lipopolysaccharide (LPS)-induced ALI remains unclear. This study was aimed to reveal whether and how PKR was involved in LPS-induced ALI pathology and the potential effects of its specific inhibitor, C16 (C₁₃H₈N₄OS). During the experiment, mice received C16 (100 or 500 ug/kg) intraperitoneally 1?h before intratracheal LPS instillation. Then, whole lung lavage was collected for analysis of total protein levels and proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6. The lungs were tested for Western blot, transferase-mediated dUTP nick-end labeling (TUNEL) stain and immunohistochemistry. Results showed that PKR phosphorylation increased significantly after LPS instillation. Furthermore, PKR specific inhibition attenuated LPS-induced lung injury (hematoxylin and eosin stain), reduced lung protein permeability (total protein levels in whole lung lavage) and suppressed proinflammatory cytokines (TNF-α, IL-1β and IL-6) and lung apoptosis (TUNEL stain and caspase3 activation). Moreover, mechanism-study showed that C16 significantly suppressed I kappa B kinase (IKK)/I kappa B alpha (IκBα)/NF-κB signaling pathway after LPS challenge. These findings suggested that PKR inhibition ameliorated LPS-induced lung inflammation and apoptosis in mice by suppressing NF-κB signaling pathway.     

Seasonal H1N1 2007 influenza virus infection is associated with elevated pre‐exposure antibody titers to the 2009 pandemic influenza A (H1N1) virus

The new influenza strain detected in humans in April 2009 has caused the first influenza pandemic of the 21st century. A cross‐reactive antibody response, in which antibodies against seasonal H1N1 viruses neutralized the 2009 pandemic influenza A (H1N1) virus (2009 pH1N1), was detected among individuals aged >60 years. However, factors other than age associated with such a cross‐reactive antibody response are poorly documented. Our objective was to examine factors potentially associated with elevated pre‐exposure viro‐neutralization and hemagglutination‐inhibition antibody titers against the 2009 pH1N1. We also studied factors associated with antibody titers against the 2007 seasonal H1N1 virus. One hundred subjects participating in an influenza cohort were selected. Sera collected in 2008 were analysed using hemagglutination inhibition and viro‐neutralization assays for the 2009 pH1N1 virus and the 2007 seasonal H1N1 virus. Viro‐neutralization results were explored using a linear mixed‐effect model and hemagglutination‐inhibition results using linear‐regression models for interval‐censored data. Elevated antibody titers against 2009 pH1N1 were associated with seasonal 2007 H1N1 infection (viro‐neutralization, p 0.006; hemagglutination‐inhibition, p 0.018). Elevated antibody titers were also associated with age in the viro‐neutralization assay (p <0.0001). Seasonal 2007 H1N1 infection is an independent predictor of elevated pre‐exposure antibody titers against 2009 pH1N1 and may have contributed to lowering the burden of the 2009 pH1N1 pandemic.     

Oseltamivir-resistant 2009–2010 pandemic influenza A (H1N1) in an immunocompromised patient

Although neuraminidase inhibitors are active against most 2009–2010 pandemic influenza A (H1N1) swine-origin strains, sporadic cases of oseltamivir resistance have been described. Since April 2009, 54 cases of oseltamivir-resistant H1N1 swine-origin have been reported in the USA (http://www.cdc.gov/flu/weekly/; accessed 1 February 2010). Approximately 1.4% of tested isolates are oseltamivir resistant. We report a patient with an underlying hematological malignancy who was hospitalized with influenza A (H1N1) swine-origin and whose strain developed oseltamivir resistance during therapy.     

Influenza A(H1N1)pdm 2009 and influenza B virus co-infection in hospitalized and non-hospitalized patients during the 2015–2016 epidemic season in Israel

BackgroundInfluenza A and B viruses co-infections are rare events and mainly occurred in immunocompromised patients.ObjectivesIn this study we report an unusually high occurrence of influenza A (H1N1)pdm 2009 and influenza B virus co-infections during the epidemic year 2015–2016.Study designNasopharyngeal swabs were collected from 1919 patients visiting 26 outpatient clinics distributed throughout Israel and presenting with influenza-like illness. In addition, hospitalized patient tested for influenza viruses were also included in the study. Patients samples collected between October 2015 and April 2016 were tested for the presence of influenza viruses by real-time PCR.ResultsOf the 1919 patient samples tested, 11 (0.6%) were co-infected with both influenza A(H1N1)pdm 2009 and influenza B/Victoria viruses. Similar observation was noted in four hospitalized patients during the same period. Patients at ages 1–72 years, and their clinical symptoms were similar to that of patients infected with either influenza A or B viruses. Of all patients, only one hospitalized patient was immunocompromised.In conclusion: Co-infection of influenza A(H1N1)pdm 2009 and influenza B viruses is an increasingly recognized phenomenon. This co-infection can occur not only in immunocompromised individuals, but also in immunocompetent patients. Although co-infection appears to be a rare event, it may still play a role in the epidemiology, pathogenicity and evolution of influenza viruses.     

Epithelial-derived TGF-β1 acts as a pro-viral factor in the lung during influenza A infection

Mucosal surfaces are under constant bombardment from potentially antigenic particles and so must maintain a balance between homeostasis and inappropriate immune activation and consequent pathology. Epithelial cells have a vital role orchestrating pulmonary homeostasis and defense against pathogens. TGF-β regulates an array of immune responses—both inflammatory and regulatory—however, its function is highly location- and context-dependent. We demonstrate that epithelial-derived TGF-β acts as a pro-viral factor suppressing early immune responses during influenza A infection. Mice specifically lacking bronchial epithelial TGF-β1 (epTGFβKO) displayed marked protection from influenza-induced weight loss, airway inflammation, and pathology. However, protection from influenza-induced pathology was not associated with a heightened lymphocytic immune response. In contrast, the kinetics of interferon beta (IFNβ) release into the airways was significantly enhanced in epTGFβKO mice compared with control mice, with elevated IFNβ on day 1 in epTGFβKO compared with control mice. This induced a heighted antiviral state resulting in impaired viral replication in epTGFβKO mice. Thus, epithelial-derived TGF-β acts to suppress early IFNβ responses leading to increased viral burden and pathology. This study demonstrates the importance of the local epithelial microenvironmental niche in shaping initial immune responses to viral infection and controlling host disease.     

Emergence of H274Y oseltamivir-resistant A(H1N1) influenza viruses in Japan during the 2008–2009 season

BackgroundA substantial increase in oseltamivir-resistant A(H1N1) influenza viruses was reported in Europe in late 2007.ObjectivesTo monitor the antiviral susceptibility profile of human A(H1N1) influenza viruses in Japan during the 2007–2008 and 2008–2009 seasons.Study designViruses were obtained from respiratory samples of patients with influenza collected in Japan between December 2007 and April 2008 (n = 1046) and between December 2008 and April 2009 (n = 1789). Oseltamivir resistance was determined by an H274Y-specific real-time PCR cycling probe assay and a neuraminidase inhibition assay. Amantadine resistance was assessed by sequencing the M2 gene. Sequencing of the hemagglutinin and NA genes was performed to infer phylogenetic relationships between different strains.ResultsThree of 687 (0.4%) A(H1N1) viruses from the 2007–2008 season and 745 of 745 (100%) viruses from the 2008–2009 season carried the NA–H274Y substitution and demonstrated a >300-fold reduction in oseltamivir susceptibility. All oseltamivir-resistant viruses from the 2008–2009 season possessed an A193T substitution in the receptor-binding domain of the hemagglutinin. Amantadine resistance was detected in 431 of 687 (62.7%) and 0 of 745 (0.0%) of the A(H1N1) viruses from the 2007–2008 and 2008–2009 seasons, respectively.ConclusionsA dramatic surge in oseltamivir-resistant A(H1N1) viruses possessing the NA–H274Y substitution was detected in Japan during the 2008–2009 season. The emergence of oseltamivir-resistant viruses was facilitated by mutations in the viral genome. Intensified surveillance, including phenotypic assays and sequencing of the hemagglutinin, neuraminidase, and M2 gene would allow monitoring of the spread and evolution of drug-resistant influenza virus variants.     

Early production of IL-17A by γδ T cells in the trachea promotes viral clearance during influenza infection in mice

The innate immune response generated against influenza infection is critical for the inhibition of viral dissemination. The trachea contains different types of innate immune cells that protect the respiratory tract from pathogen invasion. Among them, γδ T cells have the ability to rapidly generate large amounts of pro-inflammatory cytokines to preserve mucosal barrier homeostasis during infection. However, little is known about their role during the early phase of influenza infection in the airways. In this study, we found that, early after infection, γδ T cells are recruited and activated in the trachea and outnumber αβ T cells during the course of the influenza infection that follows. We also showed that the majority of the recruited γδ T cells express the Vγ4 TCR chain and infiltrate in a process that involves the chemokine receptor CXCR3. In addition, we demonstrated that γδ T cells promote the recruitment of protective neutrophils and NK cells to the tracheal mucosa. Altogether, our results highlight the importance of the immune responses mediated by γδ T cells.     

A modified “double-hit” induced acute lung injury model in rats and protective effects of tetramethylpyrazine on the injury via Rho/ROCK pathway

We focused on the production and evaluation of a modified “double-hit” induced acute lung injury (ALI) model, which closely mimics the clinical situation. Further, tetramethylpyrazine (TMP), an alkaloid contained in ligustrazine was evaluated for its potent anti-inflammatory effects in this model. Rats were randomized into 4 groups: G1 (NS control group), G2 (“double-hit” group), G3 (low dosage TMP group) and G4 (high dosage TMP group). The rats in G2, G3 and G4 were intraperitoneally injected with a low dose of LPS followed by intratracheal injection with median dose of LPS to establish the “double-hit” model. The rats in G3, G4 were intraperitoneally injected with low (G3), high (G4) dosage TMP for the protection against ALI. Upon termination of the experiment, TMP attenuated the harmful changes in animal model reaction, breathing frequency, histological examination, lung W/D-weight ratio, BAL fluid PMNs percentage, MPO activity and ROCK2 mRNA expression. We found inhibiting RhoA/ROCK pathway might attribute to TMP-induced protection against ALI.