Research Items (12)
Influenza viruses can cause severe life threatening infections in high-risk patients, including young children, the elderly and patients with compromised immunity due to underlying medical conditions or immunosuppressive treatment. The impaired immunity of these patients causes prolonged virus infection and combined with antiviral treatment facilitates the emergence of viruses with resistance mutations. The diverse nature of their immune status makes them a challenging group to study the impact of influenza virus infection and the efficacy of antiviral therapy. Immunocompromised ferrets may represent a suitable animal model to assess influenza virus infection and antiviral treatment strategies in immunocompromised hosts. Here, ferrets were given a daily oral solution of mycophenolate mofetil, tacrolimus and prednisolone sodium phosphate to suppress their immune system. Groups of immunocompromised and immunocompetent ferrets were inoculated with an A/H3N2 influenza virus and were subsequently treated with Oseltamivir or left untreated. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed on the throat and nose specimens to study virus replication during the course of infection. All immunocompromised ferrets had prolonged presence of viral RNA and a higher total amount of virus shedding compared to the immunocompetent ferrets. Although Oseltamivir reduced the total amount of virus shedding from the nose and throat of treated ferrets, it also resulted in the emergence of the neuraminidase R292K resistance substitution in all these animals, as determined by mutation specific RT-PCR and next-generation sequencing. No additional mutations that could be associated with the emergence of the R292K resistance mutation were detected. The immunocompromised ferret model can be used to study A/H3N2 virus shedding and is a promising model to study new antiviral strategies and the emergence of antiviral resistance in immunocompromised hosts.
Scheme of time of treatment start and virus inoculation in ferrets of the 4 experimental groups. Four days before virus inoculation, all ferrets were given a cocktail of antibiotics (10 mg/kg amoxicillin and 2.5 mg/kg clavulanic acid) once daily till the end of the experiment. The ferrets assigned to the immunosuppressive groups (group 2 and 3) were given a mixture of immunosuppressant (20 mg/kg MMF, 0.5 mg/kg, tacrolimus and 8 mg/kg prednisolone) starting 3 days prior to virus inoculation twice daily till the end of the experiment. All ferrets were inoculated on day 0 with 104 TCID50 A/NL/16/98 (H3N2) influenza virus. One day after virus infection, OSP (10 mg/kg) was administered to ferrets from group 3 and 4. Ferrets from groups 1, 2 and 3 were euthanized 18 days post infection and the once from group 4 were euthanized 7 days post infection. (PDF)
Emergence of oseltamivir resistant viruses in ferrets. The emergence of oseltamivir resistant viruses was monitored in throat and nose samples from untreated immunocompetent ferrets (A), untreated immunocompromised ferrets (B), oseltamivir treated immunocompromised ferrets (C) and treated immunocompetent ferrets (D) by mutation-specific RT-PCR for the oseltamivir-resistance mutation R292K. The green bars depict the presence of the wildtype genotype (292R) and the magenta the presence of the resistant genotype (292K). If both genotypes were present in one sample, the proportions of the genotypes are stacked. (PDF)
Correspondence between the R292K resistance mutation as measured by mutation specific RT-PCR and Illumina next generation sequencing. The relative proportion of the R292K resistance mutation detected in samples of all oseltamivir treated immunocompromised (grey circles) and immunocompetent (open squares) ferrets inoculated with A/NL/16/98 (H3N2) as determined by mutation specific RT-PCR was compared to the relative proportion determined by Illumina NGS. (PDF)
The frequency of the R292K mutation as determine by Illumina next generation sequencing in oseltamivir treated immunocompetent ferrets. The percentage of the R292K variant was determined in pooled throat/nose samples collected at 2, 4, 6, 8 and 10 dpi from immunocompetent ferrets that received oseltamivir treatment. The graph depicts the frequency of the R292K mutation over time for each ferret separately, with circles, squares, triangles, down triangles, diamonds and open circles representing ferrets 1–6 respectively. (PDF)
Viral RNA load in ferrets inoculated with influenza virus A/NL/16/98. Immunocompetent (blue, purple) and immunocompromised (red, green) ferrets were inoculated with influenza virus A/NL/16/98 (H3N2) and subsequently treated with oseltamivir (green, purple) or left untreated (blue, red). Virus RNA load in samples of the throat (A) and nose (C) was determined by qRT-PCR daily for 7 days. The area under the curve of panels A and C was used to estimate the total amount of viral RNA shedding from the throat (B) and nose (D) of the inoculated ferrets. The line and bar graphs depict the mean ± S.E.M. The asterisks indicates a statistically significant P value (0.01<**P>0.001, ***P<0.001). (PDF)
Influenza antibody titers of immunocompetent and immunocompromised ferrets against influenza viruses A/NL/16/98 and A/NL/271/95. Serum influenza antibody titers of the untreated immunocompetent and immunocompromised ferrets against the influenza homologous strain (A) A/NL/16/98 and (B) the heterologous strain A/NL/271/95 of immunocompetent and immunocompromised ferrets were determined by hemagglutinin inhibition (HI) assay. The antibody titer of each animal are depicted as individuals points. The horizontal bars represent the mean ± S.E.M. (PDF)
In October 2016, a severe infection with swine influenza A(H1N1) virus of the Eurasian avian lineage occurred in a child with a previous history of eczema in the Netherlands, following contact to pigs. The patient’s condition deteriorated rapidly and required life support through extracorporeal membrane oxygenation. After start of oseltamivir treatment and removal of mucus plugs, the patient fully recovered. Monitoring of more than 80 close unprotected contacts revealed no secondary cases. © 2016, European Centre for Disease Prevention and Control (ECDC). All rights reserved.
Chikungunya virus (CHIKV) re-emerged and caused an outbreak in the Caribbean and the Americas. CHIKV can cause incapacitating arthralgia, which may be evolved in chronic arthritis that is similar to rheumatoid arthritis that lasts for months or years. This review provides an overview of known and hypothesized mechanisms that CHIKV uses to promote chronic arthritis. We hypothesized that the chronic inflammatory response that is stimulated by persisting CHIKV replication in the joints results in the arthritic symptoms seen in patients. Most hypotheses proposed in this review need to be tested or confirmed, which may help in the development of new specific treatments and vaccines against CHIKV that will not only combat viral persistence but also prevent tissue damage.