Date of Award:
Master of Science (MS)
Animal, Dairy, and Veterinary Sciences
E. Bart Tarbet
E. Bart Tarbet
Donald F. Smee
Influenza viruses cause between 3 and 5 million cases of respiratory infection each year and are responsible for between 250 and 500 thousand deaths. Two avenues for the treatment and prevention of influenza virus infections are vaccination and antiviral chemotherapy. Prevention is largely accomplished through vaccination. While vaccines remain the preferred method for controlling the spread of influenza, antiviral treatment is important for severe infections caused by new and emerging virus strains. Occasionally new viruses emerge to which the population has no previous immunity. Such was the case when the pandemic H1N1 influenza virus appeared in 2009. When new viruses such as this emerge, it can take months to formulate a vaccine to protect individuals from infection. In this case, antiviral drugs are used to treat influenza infections and thus decrease the spread of the disease.
One drug currently approved by the Food and Drug Administration (FDA) is Tamiflu® (oseltamivir). Treatment with in mice has shown that some viruses have a unique response to oseltamivir. A study performed at Utah State University showed that oseltamivir is unable to protect mice from a lethal infection even though the viruses remain sensitive to oseltamivir in vitro. In the previous study, oseltamivir was unable to protect mice from a lethal infection with two oseltamivir-sensitive influenza strains: A/Duck/MN/1525/81 (H5N1) and A/Victoria/3/75 (H3N2). This research was performed to verify differences in mortality observed in the Utah State study and to determine if there are significant differences between the viruses that allow them to overcome treatment with oseltamivir.
We used the A/Duck/MN/1525/81 (H5N1) virus, the A/Victoria/3/75 (H3N2) virus, and the A/NWS/33 (H1N1) virus. All three were used in the study completed at Utah State. We added the 2009 pandemic influenza virus, A/California/04/2009 (H1N1), for a more recent comparison. For each virus, we observed viral replication in cell culture, measured performance in a cell culture antiviral essay, and tested for oseltamivir resistance with a neuraminidase inhibition assay.
Groups of mice were infected with each virus to determine effects of virus strain on survival, mean body weight, mean lung virus titers, lung weights, and discoloration of the lungs due to hemorrhage. Lungs were also sent for histopathology. A complete blood count (CBC) was run on infected mice and 16 pro-inflammatory cytokines were measured to determine the host response to infection.
We then completed an animal experiment to observe the effects of oseltamivir treatment at 5 mg/kg/day and 40 mg/kg/day on mortality, mean body weight, lung virus titers, lung weights, and discoloration of the lungs. Following oseltamivir treatment, lungs were sent for histopathology as well.
We confirmed that the A/Duck/1525/81 (H5N1) and A/Victoria/3/75 (H3N2) viruses caused significantly higher mortality in mice than the A/NWS/33 (H1N1) and A/California/04/2009 (H1N1) viruses when treated with oseltamivir. We also discovered that the A/Duck/MN/1525/81 (H5N1) virus causes mortality more quickly in mice than the other viruses. This would make infection with that virus more difficult to treat with oseltamivir. The A/Victoria/3/75 (H3N2) virus did not cause early mortality but was still less susceptible to treatment with oseltamivir than the A/NWS/33 (H1N1) and the A/California/04/2009 (H1N1) virus.
Treatment with oseltamivir at either dose did not significantly lower the lung virus titers in mice infected with the A/Duck/MN/1525/81 (H5N1) and A/Vicotria/3/75 (H3N2) viruses. Both doses of oseltamivir were able to moderately reduce the lung virus titers in mice infected with the A/California/04/2009 (H1N1) virus. Treatment with either dose of oseltamivir was able to reduce the lung virus titers in mice infected with the A/NWS/33 (H1N1) virus significantly more than any of the other infected mice. This indicates that the A/NWS/33 (H1N1) virus is uniquely sensitive to oseltamivir treatment compared to the other three viruses.
Lung weights, discoloration of the lungs, and histopathology results all pointed to a more rapid onset of disease in mice infected with the A/Duck/MN/1525/81 (H5N1) and A/Victoria/3/75 (H3N2) viruses. Lung weights were the most solid evidence of a more severe infection with those two viruses. Lung weights increased more dramatically earlier in the infection with these two viruses. The infection progresses more rapidly in mice infected with the A/Duck/MN/1525/81 (h5N1) and A/Victoria/3/75 (H3N2) viruses.
Since oseltamivir is one of the primary drugs that would be used with an emerging pandemic, more research concerning its effectiveness should be done. This study showed that despite remaining sensitive to oseltamivir in vitro, some viruses are able to cause mortality in mice despite treatment with oseltamivir. Further studies can be done to optimize oseltamivir treatment by increasing concentration or lengthening the duration of treatment. If oseltamivir will be used in a future pandemic, knowledge of its strengths and weaknesses will maximize its effectiveness.
Hurst, Brett L., "In Vitro and In Vivo Comparison of the Pathogenicity of Four Influenza Virus Strains" (2012). All Graduate Theses and Dissertations, Spring 1920 to Summer 2023. 1220.
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