As we again approach the regular worldwide flu season, questions remain on what impact H5N1 will have on our increasingly better but not sufficiently prepared health systems. Dr. Ryabchikova provides our ASA family of readers a comprehensive review of the pathology involved and terminology used across the professional sphere. She first presented this information at CBMTS-Industry V.

The Pathology of Avian Influenza in Birds and Animals:
An Analytical Review

Elena I. Ryabchikova, Tatyana N. Getmanova
State Research Center of Virology and Biotechnology "Vector".
Koltsovo, Novosibirsk region, 630559 Russia

ABSTRACT

          Analysis of the published scientific papers containing data on H5N1 avian influenza gross anatomy, histopathology and the virus nucleoprotein antigen presence has been performed. Common features of the gross anatomy in birds and susceptible mammalian species are presented. Main light macroscopic findings in chicks and other birds and immunohistochemical data on H5N1 influenza virus antigen presence in different cells in birds are given. Pathohistological features and data on H5N1 virus antigen occurence in mice having respiratory and neurological forms of the infection are presented; and, ferrets and non-human primate data are presented. Our analysis showed that infection of the endothelium is a common feature of H5N1 avian influenza in birds, but mammalian species do not show presence of the virus antigen in these cells. Infection of the endothelial cells may play a crucial role in pathogenesis of severe H5N1 influenza virus disease in birds.

INTRODUCTION

          All avian influenza (AI) viruses belong to the Influenzavirus A genus of the Orthomyxoviridae family, and have a negative-strand, segmented RNA genome. Influenza A viruses are divided into subtypes depending on presence of one of 15 antigenically distinct haemagglutinin (HA) antigens (H1 to H15), and one of nine neuraminidase (NA) antigens (N1 to N9). Influenza A viruses infecting poultry compose two distinct groups differing by their ability to cause disease. (1) Highly pathogenic avian influenza (HPAI), which usually results in 100% mortality, sometimes is called "avian plague". HPAI viruses have been restricted to subtypes H5 and H7, although not all viruses of these subtypes cause HPAI (2). Low pathogenicity avian influenza (LPAI) usually represents mild disease; however these viruses may change their virulence and cause severe disease (2). So, classification of avian influenza viruses as HPAI and LPAI is nominal. It has been shown that virulent properties of HPAI viruses are related to their ability to exploit a ubiquitous protease(s) for cleavage of HA (31), which permits reproduction in various organs and tissues. In contrast, LPAI viruses can be cleaved by trypsin and trypsin-like enzymes, and thus restricted to replication at sites in the host where such enzymes are present (i.e., the respiratory and intestinal tracts) (28).

          Infuenza A virions are pleomorphic, enveloped particles having diameter of 80±120 nm (Fig.1). The viral genome consists of eight negative-sense, single-stranded RNAs, and encodes ten polypeptides. The virus envelope contains three integral membrane proteins: haemagglutinin (HA), neuraminidase (NA) and the M2 ion channel protein, which play an important role in influenza pathogenesis. Replication of influenza virus is localized in the nucleus of the host cell. Viral RNAs are transported to the plasma membrane, where assembly of progeny particles takes place by budding (10).

          Influenza A virus subtypes are able to infect various animal and bird species, and show numerous clinical manifestations and features of the influenza disease. More than 1600 articles devoted to H5N1 AI virus subtype may be found via ProMed database representing research activity related to this agent during the 10 years after its discovery. The goal of our work was to collect and analyze data published in international scientific journals, and to attempt make a generalized view of H5N1 AI pathology in animal and bird hosts.

METHOD

          We analyzed more than 50 papers containing morphological data which came from the studies of more than 60 strains of H5N1 AI virus. Some papers were devoted to examination of natural infection in birds and mammals, and were aimed at diagnostics of H5N1 strains. The microscopy was included in these works as a necessary routine examination. In addition to Gallinaceous and duck species, it has been found that H5N1 AI virus can cause severe (sometimes deadly) disease in swans(34), flamingo, various duck species, sparrow, pigeon(6); gulls(3); magpies(14); and emus(25).

DISCUSSION

          Gross pathology in birds naturally infected with H5N1 AI was represented as varying combinations of the following changes: congestion of the intestines, trachea, brain, liver, kidneys, lung congestion and edema; swollen, mottled or congested spleen; increased pericardial fluid; petechial hemorrhages on cardiac fat; red-brown mottled pancreas; hemorrhagic duodenal contents; focal liver necrosis; friable liver; thickened air sacs; scanty contents in gizzard and proventriculus (3, 6, 24, 32, 34).

          Histopathological examination of the birds infected by H5N1 AI virus showed diverse combinations of the pathological changes. Marked congestion and lung edema, congestion and edema with loss of mucus gland and epithelial cells in the trachea were observed. In the brain, congestion with or without multiple small foci of necrosis and gliosis, or multifocal non-suppurative meningo-encephalitis were found. The spleen showed congestion and multifocal necrosis. Hepatic congestion, hepatocyte necroses and vacuolar degeneration; congestion and small foci of necrosis in small intestines and caeca, necrosis of lymphoid tissues in intestinal lamina propria were observed. Multifocal acute necrosis in the pancreas with prominent haemostatic alterations should be especially noted. Thymus congestion with small foci of necrosis in medulla, congestion and lymphoid depletion, and congestion and hemorrhage in the oviduct and the ovary were also detected (3, 6, 11, 17, 22, 24, 32, 34).

          Particular sensitivity of a chick to H5N1 influenza virus resulted in the grave pathological changes of all organs and tissues. Severity of gross and microscopic pathology was absolutely consistent with the heavy presence of virus nucleoprotein antigen in organs and tissues found by immunohistochemical examinations (1, 11, 14, 17, 22, 24, 25, 29, 32). Presence of H5N1 influenza virus antigens was detected in the lungs and other parts of respiratory tract; liver and spleen, heart, intestines, gizzard, proventriculus, and oviduct. The antigen also was found in the brain neurons and glia, kidney, pancreas, and ovary. Presence of the virus antigen in endothelium was noted in various tissues from the early infection period. This finding is evidence for the important role of endothelial damage in AI pathogenesis in chicks.

          Association of the fibrin thrombi in lung arterioles with the presence of viral antigen in endothelial cells of chickens has been shown. Hematological analyses revealed that coagulopathy was induced at the infection early stages when viral antigen was detected only in the endothelial cells and monocytes/macrophages. In addition, gene expression of the tissue factor (the main inducer of blood coagulation) was suppressed in the spleen, lung, and brain of H5N1 AI virus infected chickens. These data suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI H5N1 AI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens (21).

          Immunohistochemical examination of AI nucleoprotein antigen in ducks confirmed that AI in these birds has less severe nature: for most organs examined, antigen was detected in duck tissues at significantly lower proportions than in chicks (1, 14, 25). In swans H5N1 AI virus antigen has very similar to chicken distribution in tissues, however clear neurotropism was evident. Heavy immunostaining was observed in a large number of neurons and glial cells, especially Purkinje cells. In contrast, in the lungs, the antigen was detected in few endothelial cells but not within pneumocytes (34).

          Avian influenza studies in animals mostly represent two directions: development of experimental models, and examination of animal susceptibility to H5N1 AI virus and their possible role in the virus transmission. The mammalian species shown to be naturally or experimentally susceptible to H5N1 AI virus currently include man, non-human primates (cynomolgus and rhesus monkeys), mouse, hamster, pig, ferret, stone marten, dog, domestic cat, tiger, leopard and the civet (4, 7, 8, 13, 15, 16, 20, 26, 30, 38, 41).

THE MOUSE AS A MODEL

          Studies of H5N1 AI virus infection in mice are the most numerous, because the mouse is the most frequent experimental model for examination of viral pathogenesis and antiviral efficacy of vaccines and medicinal preparations. In contrast to other influenza viruses, the H5N1 strain is able to kill mice without adaptation (9, 20). Such a property was recently shown also in H1N1 (Spanish) influenza virus strain, recently reconstructed by molecular biological methods (37).

          Infection of mice by H5N1 AI virus cause generally two kinds of the disease: predominant damage of respiratory or nerve system. In the first case mice showed variable degrees of degeneration and necrosis of respiratory epithelium in the nasal cavity, trachea, bronchi, and bronchioles, associated with fibrin deposits and neutrophils. The lungs had peribronchial alveolitis characterized by intra-alveolar serofibrinous exudate, erythrocytes and neutrophils, and increased numbers of alveolar macrophages. This bronchointerstitial pattern of pneumonia was most frequent, but in the most severe cases, a diffuse interstitial pneumonia pattern affected entire lung lobes (5). Generalized infection was detected (7, 9) or was not detected (5).

          In the case of neuropathogenic form of the disease, foci of neuronal degeneration and neuronophagia associated with inflammatory infiltration were observed in the brain stem and spinal cord. Foci of mononuclear cell infiltrates were also seen in the meninges and in the perivascular spaces of the brain stem and spinal cord neuropil. In the spinal cord, there were occasional degenerating glial cells, neurons with marginated chromatin and an eosinophilic inclusion that partially or completely filled the nucleus. Necrotic ependymal cells were scattered among the ependyma lining the central canal of the spinal cord. Neuronal degeneration and inflammatory infiltrates were also evident in some dorsal root ganglia, and there were many foci of necrosis and inflammatory cells associated with the perivertebral adipose tissue (18).

          The evidences that H5N1 virus spread to the brain by intracranial nerves have been adduced in one article (12). Immunohistochemical examinations of intranasally infected mice revealed the presence of H5N1 AI virus nucleoprotein antigen in cells of respiratory tract: in nasal cavity epithelium (mostly degenerating cells), in trachea and bronchi epithelium, in debris inside bronchi, and in occasional alveolar macrophages (17, 19, 20, 29, 33, 39). The presence of the antigen in brain of intranasally infected mice was thought to be the evidence of the infection generalization and neurovirulence of the AI virus particular strains (5, 7, 20, 23, 33). Only one paper reported presence of H5N1 AI virus antigen (Hong Kong 483RG, Hong Kong 6PB2-627K strains) in liver, heart, skin, and fatty tissue, in addition to respiratory and nerve system (29). Intravenous infection causes the neurological form of H5N1 AI virus infection (33). Presence of nucleoprotein antigen means that a cell is infected and produces AI virus nucleoprotein. However, this fact is not strong evidence for the production of viral progeny by antigen-positive cells: only electron microscopy is able to show budding of influenza virus on the surface of particular cell type. There are no published electron microscopic studies of H5N1 influenza virus in mice, birds and other animals.

          A comparative study of human Hong Kong H5N1 AI virus isolates showed that they differ in virulence for mice. The isolates were obtained from humans who died and showed higher virulence than isolates obtained from recovered persons. The first replicated systemically, while the second replicated only in the respiratory tract (7). It is attractive to use this relationship and the mouse model for prediction of the AI virulence in humans. However, other studies are needed to be sure that such a relationship really exists. Thus, studies of H5N1 AI virus infection in mice revealed two forms of the disease, and dependence of the virulence and presence of the virus antigen on virus strain and inoculation route.

OTHER ANIMAL MODELS

          Data about the pathology in other animals susceptible for H5N1 AI virus are few. Main gross anatomy features in carnivorous animals (cat, tiger, and leopard) were necroses and focal hemorrhages in visceral organs and tonsils (15, 39, 13, 35, 27, 36). Microscopically, the lesions were characterized by inflammation and necroses. In large felids, thrombocytopenia, hemorrhagic lesions in the lung and encephalitis associated with multifocal infiltration by neutrophils and macrophages have been reported (13, 35). Presence of nucleoprotein antigen was shown in cat lungs in association with foci of tissue consolidation (15), and in various lung cells (39). The antigen was also detected in the liver, heart, brain, renal glomeruli, adrenal gland and sometimes the spleen and pancreas (27, 30); and it has also been found in the large intestine (40). Wide spreading of the antigen evidences for systemic infection however no virus has been isolated from blood (27). The most interesting finding was detection of the virus antigen in a liver of cat (27, 30) and tiger (35).

          Ferrets are known as a good laboratory model for studies of influenza A virus infection. Detailed study of H5N1 AI infection pathogenesis in ferrets has been published (41). Gross anatomy showed focal areas of redness in lungs on days 1-3 postinfection. Consolidation of the lungs was evident from day 3. Extrapulmonary lesions in ferrets included discoloration and petechiae on the liver, and lesions on the intestines and kidneys. Microscopic studies revealed acute bronchiolitis, bronchopneumonia, and interstitial pneumonia characterized by suppurative exudates in the bronchi, bronchioles, and adjacent alveolar spaces. Prominent epithelial necroses and intraalveolar edema, alveolar epithelial hyperplasia and various proportions of fibrin and mixed inflammatory cells (predominantly mononuclear cells) in alveoli were also present. Histopathologic features detected in the brains of ferrets included the presence of glial nodules, polymorphonuclear cells in the parenchyma, lymphocyte perivascular infiltration, neuronophagia, and increased lymphocyte infiltration in the choroid plexus (8, 41). Meningeal mononuclear infiltration, scattered foci of marked neuronal degeneration and neuronophagia associated with inflammatory cell infiltrates were noted in the neuropil of the olfactory bulb, cerebrum, and brain stem on day 6-7 after ferret infection (8).

          Immunohistochemical staining for the presence of viral antigen yielded rare positive cells in the ferret lungs (type II pneumocytes, alveolar macrophages, nonciliated cuboidal epithelial cells in terminal bronchioles) (39, 41).
Non-human primates (Rhesus and Cynomolgus monkeys) were experimentally infected with H5N1 AI virus, and developed respiratory disease. Gross anatomy showed prominent swelling of the lymph nodes; focal pulmonary consolidation, consolidated tissue was red-purple, firm, slightly depressed, with disseminated pulmonary hemorrhages (1-5 mm in diameter), mild subpleural emphysema and large area of necrosis, characterized by brown-red, dry tissue and a rough pleura. Trachea and bronchi contained foamy fluid (3, 15).

          Immunohictochemical study showed presence of H5N1 AI virus nucleoprotein antigen in monkey alveolar macrophages, type 1 and type 2 pneumocytes, bronchiolar and bronchial epithelium, neutrophils, and in unidentified mononuclear cells. In tonsils antigen-positive reaction was detected in sloughed epithelial cells in the crypts and in dendritic cells of some germinal centers. The reaction for the antigen was not observed in the spleen, heart, and brain (3, 15, 26, 39).

CONCLUSION

          The analysis of published works devoted to H5N1 AI virus infection showed that in chicks the virus caused extremely severe pathology resembling fast poisoning. Other bird species show less severe pathology, however common feature for birds was systemic infection. Early viral damage of endothelium and clotting system in chicks may be a key feature of the pathogenesis of fulminant H5N1 AI disease. Mammals in general showed less severe pathology than birds, however systemic infections were detected in various species and were associated with prominent lesions. Mammalian species were not examined carefully except for mice. A model testing virulent properties of a new H5N1 AI strains in mice and extrapolating the data to humans may be possible.

Key words: H5N1 influenza virus, analytical review, bird and animal pathology.

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Ed. Note: Our thanks to Elena Ryabchikova and Tatyana Getmanova for their efforts in bringing forward this excellent review. VECTOR has been at the forefront in the international efforts on avian influenza. It was at CBMTS almost 10 years ago when VECTOR's Sergey Netesov first recommended the world look at the flyways - starting in Mongolia and Western China to check on an emerging H5N1 that might prove very difficult to control.

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