PMC:2700745 / 7512-24909 JSONTXT

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Seasonal influenza\n\n2.1 Models of benign influenza\nInfluenza is usually an acute, self-limited respiratory tract infection that begins with the sudden onset of high fever, followed by inflammation of the upper respiratory tree and trachea, with coryza, cough, headache, prostration, malaise and other signs and symptoms that persist for 7–10 days (Taubenberger and Morens, 2008). The virus replicates in both the upper and lower respiratory tract. In experimental infections in healthy volunteers, influenza A viral replication peaks approximately 48 h after inoculation into the nasopharynx, declining thereafter, with usually little or no virus shed after 6 days. However, viral antigen can still be detected in cells from the respiratory tract and secretions of infected individuals by enzyme immunoassay for several days after infectious virus can no longer be recovered (Wright et al., 2007). A summary of current animal models available for studying benign seasonal influenza is presented in Table 2 .\nTable 2 Summary of current animal models available for studying benign disease associated with seasonal influenza.\nVirus Disease Model Animal Model References\nInfluenza A strains Mild Pathogenesis Mouse Gambaryan et al. (2005)\nAichi/2/68 (H3N2) BALB/cJCitMoise (B/c)\nA/NIB/26/90M (H3N2) A/SnJCitMoise\nA/NIB/23/89M (H1N1) CBA/CaLacSto\nA/NIB/23/89-MA (mouse adapted) C57BL/6LacSto\n\u2028\u2028\nInfluenza A/Aichi/2/68 (H3N2) Mild Pathogenesis Ferret Svitek et al. (2008)\nInfluenza A/Port/ Chalmers/173 (rat adapted after 11 passages in rats Mild Pathogenesis Rat (Rattus) Brown Norway Fischer-344 Sprague–Dawley Daniels et al. (2003)\n\u2028\u2028\nSwine influenza A strains Mild Pathogenesis Pig Vincent et al. (2007)\nX98 H3N2\nA/SW/CO/23619/ 99 H3N2\nA/SW/IA/00239/2004 rH1N1\n\u2028\u2028\nInfluenza A H1N1 Mild Pathogenesis/Pneumonia Pigtailed macaque Baas et al. (2006)\nA/Texas/36/91\n\n2.1.1 Mice\nIn mice infected with influenza viruses of low pathogenicity, a mild illness occurs with few of the signs and symptoms that are seen in humans. The major parameters of mild to moderate disease are weight loss and the detection of virus, primarily in the lungs (Sidwell and Smee, 2004). In such a model, a polymer-bound 6′sialyl-N-acetyllactosamine was found to ameliorate weight loss in treated animals (Gambaryan et al., 2005).\n\n2.1.2 Ferrets\nLess virulent strains of influenza virus cause mild influenza-like symptoms in ferrets that rarely end in death. For example ferrets infected with A/Aichi/2/68 (H3N2) did not lose weight (Svitek et al., 2008). Only mild respiratory disease occurred in Aichi-infected animals with occasional sneezing and little serous nose exudate expressed. Virus infection elicits a rapid and strong upregulation of IFN-α, IFN-γ, and TNF-α, while more virulent strains induced significantly lower levels of IFN-α during the first 2 days after infection. During the first 4 days after infection, only IL-8 was detected in samples from animals inoculated with A/Aichi/2/68, whereas IL-6 expression was associated with the more virulent viruses (Svitek et al., 2008).\nThe utility of a ferret model in which a benign infection was induced is illustrated by the results from the following vaccine study. Ferrets immunized with virosome-based intranasal influenza vaccine consisting of hemagglutinins from influenza A/Beijing/262/95like (H1N1), A/Sydney/5/97 (H3N2), and influenza B/Harbin/7/94 and then challenged with the A/Sydney/5/97 (H3N2) strain had less viral shedding in nasal secretions and the vaccine elicited a vaccine-specific antibody response (Lambkin et al., 2004). In addition, the vaccine regimen protected against fever, weight loss, and infiltration of inflammatory cells.\n\n2.1.3 Rats\n“True” rat species have been evaluated to determine their suitability as models for influenza disease and to ascertain whether genetic background impacts their susceptibility to infection. In one study, Brown Norway (BN), Fischer-344 (F344) and Sprague–Dawley (SD) rats were challenged with a rat-adapted influenzaA/Port/Chalmers/173 (H3N2) virus (Daniels et al., 2003). The virus was adapted to rats by 11 successive passages thorough infected lung homogenate. The F344 and SD rats were most sensitive to the infection, with 100-fold higher lung virus titers than seen in the BN rats. Alveolar macrophages, lactate dehydrogenase activity, and total lung protein concentrations (an indicator of pulmonary edema) were higher in the BN rats. Neutrophil numbers, interleukin 6 levels, and TNF-α activity were greatest in the bronchoalveolar lavage fluids from F344 and SD rats. Nevertheless, the infection was not lethal and few pathologic abnormalities were noted in the lungs. Although the study provided insights into factors of importance in protecting the host from influenza virus infections, rat models probably have not been characterized well enough to be recommended for use in evaluating anti-influenza therapies.\nRecently Alarcon et al. (2007) demonstrated the use of the Brown Norway rat in evaluating influenza vaccines. Using microneedle technology for i.d. administration of three different types of influenza vaccines (Fluzone® 2003–2004 formulations and DNA plasmid-based vaccine) the investigators demonstrated in a rat model, that a whole inactivated virus elicited antibody responses to the corresponding wild type parent H3N2 and B strains. Animals treated with multiple doses of DNA plasmid vaccine also responded with antibody response to the parent strains.\n\n2.1.4 Pigs\nBecause influenza A viruses frequently adapt to efficient transmission among pigs, these animals have occasionally been used as a model for testing vaccines (van der Laan et al., 2008). Signs of illness include fever, loss of appetite, labored breathing and coughing. However, the animals rarely die from the disease unless virus is directly inoculated into the trachea, in which case they exhibit signs of pneumonia. Pigs have been found to have low susceptibility to recently emerged H5N1 strains (Lipatov et al., 2008). To date, the main use of the pig has been in the development of vaccines against swine influenza (reviewed by van der Laan et al., 2008).\nProblems that may preclude the use of pigs for influenza studies include caging, the complexities of animal husbandry and waste management. Thus, the recent development of a model in Ellegaard Göttingen minipigs appears to offer an alternative, if these animals can be shown to develop a fulminant pneumonia using a less intrusive route than intratracheal inoculation. However, as in the case of common pigs, minipigs were not susceptible to infection with H5N1 strains such as A/chicken/Yamaguchi/7/04 (Isoda et al., 2006). As with normal pigs one must always be cautious to obtain animals with no previous exposure to influenza viruses, since minipigs can be hosts for swine influenza A viruses (Hansen et al., 1997).\n\n2.1.5 Nonhuman primates\nBecause nonhuman primates are much more closely related to humans than small animals typically used to study influenza, they have been used as models for human disease. In particular, rhesus macaques have been used to study pathogenesis and evaluate therapeutic and prophylactic strategies (see Baas et al., 2006 for review). In addition, the suitability of pigtailed macaques as models of influenza in the context of transcriptional studies has been evaluated (Baskin et al., 2004). A recent study examined the innate immune response in affected lung tissue with viral genetic material present (Baas et al., 2006). The authors used histopathological analysis of lung tissue, immunohistochemistry, viral and host gene expression by microarray analysis, proteomics, gene expression in circulating blood cells, and quantitative real-time RT-PCR to study individual animal responses until the end of the experiment. The infections were mild, without pneumonia or significant lung pathology. The investigators were able to demonstrate significant differences in gene expression within regions in influenza virus-induced lesions, based on the presence or absence of viral mRNA, and correlated them with transcriptional markers of early disease in peripheral white blood cells (Baas et al., 2006).\nThe use of viruses causing benign infections in macaques has primarily been used for vaccine studies. For example, Rimmelzwaan et al. (2001) demonstrated that protective immunity induced by immune stimulating complex (ISCOM)-based vaccines consisting of the membrane glycoproteins of A/Philippines/2/82 did not protect macaques from a challenge infection with A/Netherlands/18/94. However, vaccination of monkeys, which had had a prior infection with an influenza A/Philippines/2/82-like virus with a single dose of ISCOM vaccine, induced long-lasting protective antibody immunity against challenge infection with the homologous virus A/Netherlands/18/94.\n\n2.2 Models of severe seasonal influenza and pneumonia\nOccasionally in healthy individuals, but much more often in the very young, the elderly, and the immunocompromised, acute seasonal influenza develops into hemorrhagic bronchitis and viral pneumonia, characterized by dyspnea, hemoptysis, pulmonary edema and cyanosis. Death can occur within 48 h after the onset of symptoms. A summary of animal models available to study such infections is presented in Table 3 .\nTable 3 Summary of currently available animal models for studying severe seasonal influenza pneumonia.\nVirus Animal Model References\nInfluenza A H3N2 strains Mouse (BALB/c) Reviewed by Sidwell and Smee (2000)\nA/Shangdong/09/93\nA/Victoria/3/79\n\u2028\u2028\nInfluenza B strains\nB/HongKong/5/72\nB/Lee/40\n\u2028\u2028\nB/Sichuan/379/99 BALB/c Smee et al. (2006)\nInfluenza A H1N1 strains BALB/c Smee et al. (2008)\nA/WSN/33 (H1N1) virus containing the HA gene of A/New Caledonia/20/99\n\u2028\u2028\nA/New Caledonia/20/99 BALB/c Smee et al. (2006)\n\u2028\u2028\nInfluenza A strains\nA/Charlottesville/31/95 (H1N1) Ferret Smee et al. (2008)\n\u2028\u2028\nA/USSR/90/77 (H1N1) Ferret Svitek et al. (2008)\nA/Port Chalmers/1/73 (H3N2)\n\u2028\u2028\nInfluenza A H3N2 A/Wuhan/359/95 Cotton Rat Ottolini et al. (2005), Review by Eichelberger (2007)\nIn cases of severe influenza pneumonia in humans, histological changes in the lungs include capillary and small vessel thromboses, necrotizing bronchitis and bronchiolitis, interstitial edema and inflammatory infiltrates, the formation of hyaline membranes in alveoli and alveolar ducts, varying degrees of acute edema between the alveoli with or without hemorrhaging, and diffuse alveolar damage. The later stages of the disease are characterized by organizing diffuse alveolar damage, fibrosis, epithelial regeneration, and squamous metaplasia. If concomitant bacterial or fungal infection is present, there may be pronounced neutrophil infiltration into alveolar air spaces, with a lesser degree of alveolar hemorrhage and edema than in primary influenza pneumonia (Taubenberger and Morens, 2008).\n\n2.2.1 Mice\nThe mouse remains the primary model for evaluating the antiviral therapy of influenza pneumonia, due to the inexpensiveness of the animals and their caging and the general fidelity of the illness in mice to the human disease (Sidwell and Smee, 2004). In addition, many reagents are available to study the effects of virus replication and treatment on the mouse immune system. The laboratory mouse can be experimentally infected with both seasonal influenza A viruses and influenza B viruses, but this usually requires some adaptation by multiple lung passage. The adapted virus can then infect murine lung cells, probably as a result of amino acid changes in the surface hemagglutinins that enable it to bind to cell-surface alpha 2,3-linked sialic acid molecules (Ibricevic et al., 2006). A recent advance in influenza A modeling has been the finding that the reconstructed 1918 pandemic influenza virus is lethal for mice (Jeffery et al., 2001, Taubenberger, 2006).\nA number of parameters may be used to monitor influenza virus infection in mice, including change in body weight, decline in arterial oxygen saturation, increase in serum alpha-1-acid glycoprotein, mean time to death, and lung weight, viral titer and pathology scores (Sidwell and Smee, 2000). Disease manifestations often depend on the infectivity and challenge dose of the virus and (for seasonal influenza virus strains) how well the virus has adapted to the host. If a virus replicates in mice without causing apparent illness, the effects of therapy can be monitored using parameters such as lung viral titers, increase in lung weight and increase in 1-acid glycoprotein (1-AG), all of which increase in nonlethally infected mice (Ottolini et al., 2005, Sidwell and Smee, 2000).\nMost of the histopathological features of influenza viral pneumonia listed above are also seen in mice, but some signs and symptoms of human influenza are rarely observed. Mice do not show outward signs of fever and do not have increased rectal temperatures, nor is dyspnea, cyanosis or hemoptysis easily, if ever detected in mice infected with strains other than the H5N1 virus. However, as in humans, reduced blood oxygen saturation levels, a measure of lung function, can be measured in mice, and these levels are dramatically lower as pneumonia progresses and the mice approach death (Barnard et al., 2007). Weight loss is also a good marker of disease severity (Sidwell and Smee, 2000). The reader is referred to the following recent papers that show the utility of the seasonal influenza mouse model for evaluating vaccines (reviewed by van der Laan et al., 2008, Hagenaars et al., 2008, Hai et al., 2008) and antiviral drugs (Dimmock et al., 2008, Gilbert and McLeay, 2008, Reading et al., 2008, Smee et al., 2008, Wang et al., 2008).\n\n2.2.2 Ferrets\nBecause influenza-virus-infected ferrets develop many of the typical signs of infection in humans, including nasal discharge, anorexia, watery eyes, otologic symptoms and fever, they are now being used as an animal model for influenza-like pneumonia (Sidwell and Smee, 2000, Govorkova et al., 2007, Smee et al., 2008, Svitek et al., 2008). Virus in high titers can be recovered from the respiratory tract (Smith and Sweet, 1988, Potter et al., 1976). Although ferrets mimic seasonal influenza A infections in humans, they appear to be less responsive to infections by influenza B viruses (Pinto et al., 1969).\nA limitation of ferret studies is the lack of specific reagents for studying the ferret immune system, compared with similar resources for mice. However, this issue will probably be resolved in the near future with the development of reagents such as cross-reactive monoclonal antibodies (mAb). For example, CD8+ cells in mouse bronchoalveolar lavages from pneumonic ferrets were identified with mAb to human CD8 (Rutigliano et al., 2008). Caging can also be a problem, because most animal facilities require that 1–3 ferrets be housed in rabbit-style cage systems. Proper caging for work with H5N1 influenza virus is at the final stages of development, but not yet available for use. Access to specific pathogen-free (influenza virus seronegative) ferrets is also an issue, and will become increasingly so as the demand for ferret studies grows. Finally, although ferrets are relatively passive in temperament relative to their mink cousins, they can become fairly aggressive after being exposed to invasive procedures.\nAs a result of the studies described above, a number of studies have been done to show the adequacy of ferrets as models for testing vaccine efficacy (reviewed by van der Laan et al., 2008; (Huber et al., 2008; Parks et al., 2007) and the effectiveness of antiviral drug therapy (Smee et al., 2008, Oxford et al., 2007, Malakhov et al., 2006).\n\n2.2.3 Cotton rats\nCotton rats (Sigmodon hispidus) have several advantages over mice and ferrets as a model for human influenza, including the availability of reagents to study immunological responses. When compared to mice of any strain, cotton rats appear to have all the innate and adaptive immune responses seen in humans, such as Mx gene-mediated responses. Viruses isolated from humans do not have to be adapted to cotton rats to cause disease (Eichelberger, 2007). The disadvantages of the cotton rat are primarily animal availability and the aggressiveness of species, regardless of gender.\nThe influenza A cotton rat model is a nasal and pulmonary infection in adult inbred cotton rats. Animals infected intranasally with doses of a recent H3N2 influenza strain had increased breathing rates accompanied by weight loss and decreased temperature (Ottolini et al., 2005). Virus replication peaked within 24 h in the lung, with peak titers proportional to the infecting dose. Although virus was cleared from the lung by day 3, replication persisted in nasal tissues for 6 days. Pulmonary pathology included early bronchiolar epithelial cell damage, followed by extensive alveolar and interstitial pneumonia detectable in animals for up to about three weeks. Cytokine levels were typical of an inflammatory response to a lung infection; their upregulation appeared to coincide with increased virus replication. Since this model has not been gained wide acceptance due to the limitations described above, there are a paucity of papers using cotton rats to vaccine efficacy (Straight et al., 2008) and antiviral efficacy (Eichelberger et al., 2004, Stertz et al., 2007) against influenza virus infections."}