Pathogenetic Features of Inflammation in Mice with Avian Influenza and Its Prevention by Oxidized Dextran

The dissertation is devoted to the study of the pathogenetic features of the inflammatory process developing in mice upon infection with H5N1 subtype avian influenza virus strain A/goose/Krasnoozerskoye/627/05, isolated in the Russian Federation in 2005. The study was conducted on experimental animals with the aim of elucidating the mechanisms of pathogenesis and morphogenesis of the infectious disease at the tissue, cellular, and subcellular levels. Special attention is given to the study of viral topology, structural and ultrastructural changes in target organs — the lungs and liver — as well as the role of proinflammatory cytokines in the development of the infectious process.

The work also considers the possibility of avian influenza prevention using oxidized dextran, a nonspecific immunomodulatory agent. Its efficacy is evaluated in the context of reducing lethality, alleviating pathological changes in organs, and increasing the lifespan of infected mice. The work is significant for understanding the pathogenesis of highly pathogenic avian influenza and for the search for potentially promising means of its prevention.

Relevance of the problem

Influenza A viruses are among the most significant causative agents of infectious diseases for humanity. The emergence of highly pathogenic subtypes of the virus in the domestic poultry population and the increasing number of registered cases of direct transmission of avian influenza agents to humans are obviously associated with the threat of a new pandemic and the need to search for new means of prevention and treatment of the disease (Shortridge K.F. et al., 2000).

The major epidemics of influenza in the 20th century were caused by viruses that originated directly from avian viruses through genetic reassortment between strains of avian and human influenza (the pandemics of 1957 and 1968) or by adaptation of the avian strain to humans (the "Spanish flu" of 1918–1919) (World Health Organization [WHO], 2006; Suzuki, Y., 2005).

In recent times, direct transmission of influenza viruses circulating among birds has been gaining increasing significance. According to WHO data as of June 30, 2008, the total number of confirmed cases of human disease caused by H5N1 subtype influenza A viruses amounted to 385, of which 243 (63%) resulted in death (WHO).

Particular interest is aroused by the H5N1 subtype avian influenza agent, since certain parallels can be drawn between this virus and the "Spanish flu" virus (H1N1 subtype), which caused the death of up to 40 million people during the 1918–1919 pandemic. Like the "Spanish flu" virus, influenza A virus of H5N1 subtype can be transmitted directly from infected birds to humans, possesses unusually high pathogenicity, and the ability to cause generalized infection in the absence of previously formed specific immunity in the human population (To K.F. et al., 2001; De Jong M.D. et al., 2005; Pei F. et al., 2005; Suzuki Y., 2005; Uiprasertkul M., et al., 2005).

Studies of circulating strains of the agent demonstrate ongoing viral evolution and expansion of the host range. All these circumstances necessitate regarding influenza A virus of H5N1 subtype as the most likely cause of future influenza pandemics (WHO, 2006).

In 2005, in the village of Suzdalka, Novosibirsk Oblast, Russian Federation, an outbreak of domestic poultry was recorded, caused by H5N1 subtype avian influenza virus. In the shortest possible time, the viruses spread across Western Siberia, and subsequently to the central regions and the southwest of Russia. Epizootics among domestic and wild birds occurred in Novosibirsk, Omsk, Tyumen, Kurgan, and Chelyabinsk Oblasts and Altai Krai. By the end of August of the same year, the total number of avian influenza outbreaks in Russia reached 53. Avian influenza epizootics led to significant economic losses associated with the death and destruction of poultry. As a result of the spread of infection, more than 1 million head of domestic poultry died or were destroyed (Onishchenko G.G. et al., 2005).

On the territory of Russia, not a single case of human infection with avian influenza has been registered to date. However, it has been shown that H5N1 subtype viruses isolated in 2005 in Novosibirsk Oblast are highly pathogenic for mammals (Onishchenko G.G. et al., 2006; Evseenko V.A. et al., 2007).

Since these were the first cases of detection of highly pathogenic H5N1 subtype avian influenza viruses on the territory of Russia, it became relevant to obtain data on the phenotypic features of the isolated viruses, their pathogenicity for birds and mammals, phylogenetic position, and serological properties.

Furthermore, it has been proven that in recent years, H5N1 subtype avian influenza viruses have acquired resistance to the action of the main drugs used for the treatment and prevention of influenza. Strains isolated in Novosibirsk Oblast during the 2005 epizootic were no exception (Iatsyshina S.B., Shestopalov A.M. et al., 2008).

Alongside virological studies that make it possible to identify the subtype of the circulating virus and its molecular-genetic properties, it is necessary to study the features of pathological processes arising in the bodies of animals as a result of exposure to a new viral agent. Given the real danger of the spread of avian influenza virus in the human population, there is an acute need to identify the mechanisms of pathogenesis and morphogenesis of the infectious disease. For this purpose, it is necessary to investigate the features of the "virus-cell" relationship at both the light-optical and ultrastructural levels.

Analysis of structural and ultrastructural changes in target organs during infection makes it possible to identify ontogenetic features of the virus associated with the ways of its entry into cells, reproduction, and circulation in the animal body, and therefore the reasons for the development of pathogenetic events leading to severe consequences for the virus-affected organism. The use of microscopic methods is thus a necessary condition for understanding the basic mechanisms of development and the most important manifestations of avian influenza infection, and in particular the H5N1 subtype.

For the development of new approaches to combating viral infections, it is necessary to develop H5N1 subtype avian influenza models on experimental animals (mammals), to study the structure of virions and the features of their formation in mammalian cells, with the aim of searching for new prophylactic agents directed at reducing the level of risk of influenza infection in humans and its severe complications.

In light of the above, the goal and objectives of this research were formulated.

Goal of the research

To study the features of the manifestation of the inflammatory process in the lungs and liver of mice in avian influenza caused by the highly pathogenic H5N1 subtype avian influenza strain A/goose/Krasnoozerskoye/627/05, and its prevention by oxidized dextran.

Objectives of the research:

1. To investigate the pathogenicity of H5N1 subtype avian influenza virus A/goose/Krasnoozerskoye/627/05 for mice.

2. To investigate the efficacy of oxidized dextran in the prophylaxis of experimental viral infection caused by H5N1 subtype avian influenza virus A/goose/Krasnoozerskoye/627/05 in mice.

3. To study the features of the topology of H5N1 subtype avian influenza virus A/goose/Krasnoozerskoye/627/05 and subcellular changes in the lungs and liver of infected mice.

4. To study morphological changes in the lungs and liver of mice infected with H5N1 subtype avian influenza virus A/goose/Krasnoozerskoye/627/05.

5. To study the role of proinflammatory cytokines in the pathogenesis of the infectious process caused by H5N1 subtype avian influenza virus A/goose/Krasnoozerskoye/627/05.

6. To study the pathogenetic features of avian influenza manifestation in the lungs of experimentally infected mice under preventive administration of oxidized dextran.

Scientific novelty of the research results

During the experiment, data on the pathogenetic features of inflammation in avian influenza in mice caused by an avian influenza virus isolated on the territory of the Russian Federation were obtained for the first time. The features of the manifestation of the infectious process and its complications in the lungs and liver of mice were studied in detail at the tissue, cellular, and subcellular levels. By transmission electron microscopy, the features of the morphotypes of virions A/goose/Krasnoozerskoye/627/05, its topology, and its tropism to various cells of the lungs and liver were identified.

Data were obtained on the level of expression of proinflammatory cytokines and apoptosis factors in the lung and liver tissues of mice infected with H5N1 subtype avian influenza virus.

A means (oxidized dextran) for the prophylaxis of avian influenza in mammals was proposed and tested experimentally in mice. Its high efficacy was demonstrated.

Practical significance of the work

The pathogenetic features of avian influenza manifestations in mice caused by a new strain of H5N1 subtype avian influenza virus A/goose/Krasnoozerskoye/627/05, potentially dangerous to humans, were investigated. A means for the prophylaxis of the main complications of avian influenza in mice was proposed, which may be promising for the development of means for the prophylaxis of avian influenza in humans due to the nonspecific nature of its effects.

The following theses are submitted for defense:

1. H5N1 subtype avian influenza virus A/goose/Krasnoozerskoye/627/05 possesses high pathogenicity for mice, attributable to viral replication in lung and liver cells, the development of a generalized infectious process, and high lethality.

2. Avian influenza virus A/goose/Krasnoozerskoye/627/05 possesses high tropism to alveolar epithelial cells, lung and liver macrophages, hepatocytes, and hepatic sinusoidal endothelial cells, accompanied by the development of thrombotic complications, hemorrhages, interstitial inflammation in the lungs, edematous syndrome, dystrophic and necrotic changes in the liver and lungs, and early fibrosis in these organs.

3. Oxidized dextran exerts a pronounced prophylactic effect in experimental infection of outbred white mice with H5N1 subtype avian influenza virus A/goose/Krasnoozerskoye/627/05, which manifests as a reduction in lethality and an increase in the lifespan of the animals, depends on the timing and dose of drug administration, and is attributable to a significant reduction in the severity of the main pathological manifestations of inflammation.