4 H5N1 avian influenza In contrast to the human-adapted influenza A viruses that cause benign seasonal influenza in the majority of patients, the recently emerged Southeast Asian H5N1 virus causes acute viral pneumonia aggravated by ARDS, toxic shock and multiple organ failure in previously healthy, immunocompetent individuals (Peiris et al., 2007, Beigel et al., 2005). Its markedly enhanced virulence for humans appears at least in part to reflect a difference in viral tropism for cells of the lower respiratory tract, but until recently, those target cells were unknown for both humans and other mammals (Rimmelzwaan et al., 2006, van Riel et al., 2006). In a recent study (van Riel et al., 2006), an H5N1 virus was found to preferentially bind to type II pneumocytes, alveolar macrophages, and nonciliated bronchiolar cells in the lower respiratory tract of cats, similar to the clinical scenario in humans (Matrosovich et al., 2004). How well various animal models resemble human disease may thus reflect the difference in distribution of infection in the respiratory tract. Because the H5N1 virus infects a wide range of mammalian species without prior adaptation, a number of animal models are available for study (Table 5 ). Table 5 Summary of current animal models available for studying human disease caused by highly pathogenic H5N1 avian viruses. Virus Disease Model Animal Model References A/duck/Tuva/01/06 (H5N1) Interstitial pneumonia Mouse (BALB/c) Evseenko et al. (2007) 

 A/Vietnam/1203/2004 (BALB/c) Hatta et al. (2007) VN1204PB2-627Lys (mutant) A/Vietnam/1204/2004 VN1203PB2-627Glu (mutant) A/chicken/Vietnam/NCVD5/2003 A/muscovy/duck/Vietnam/NCVD18/2003 

 A/Vietnam/1194/04 (BALB/c) A/Vietnam/1203/04 (VN1203) Hatta and Kawaoka (2005) A/Vietnam/1204/04 (VN1204) A/duck/Vietnam/NCVD-5/03 A/duck/Vietnam/NCVD-18/03 A/duck/Vietnam/NCVD-25/03 A/chicken/Vietnam/NCVD-8/03 A/chicken/Vietnam/NCVD-15/03 A/chicken/Vietnam/NCVD-30/03 

 A/chicken/Yamaguchi/7/04 (BALB/c) Isoda et al. (2006) 

 H5N1 viruses A/Hong Kong/481/97 A/HongKong/483/97 (BALB/c) Katz et al. (2000b) A/Hong Kong/485197 A/Hong Kong1486197 A/Hong Kong/507/97 

 H5N1 viruses A/ck/HK/220/97 BALB/c Dybing et al. (2000) A/ck/HK/728/97 A/HK/156/97 A/ck/Scotland/59 A/tk/England/91 

 A/HongKong/156/97 (BALB/c) Gubareva et al. (1998a) 

 Influenza A H5N1 Pathogenesis/Pneumonia Ferret Zitzow et al. (2002) A/Hong Kong/486/97 A/Hong Kong/483/97 

 Influenza B clinical isolate related to B/Beijing/184/93 and B/Guangdong/8/93 Gubareva et al. (1998b) Influenza A H5N1 Pathogenesis/Pneumonia Cat Rimmelzwaan et al. (2006) A/Vietnam/1194/04 Influenza A H5N1 Pathogenesis/Pneumonia Cynomolgus macaque Kuiken et al. (2003) A/HongKong/156/97 Rimmelzwaan et al. (2003) 4.1 Mice Isolates of H5N1 virus recovered from human patients can cause lethal disease in BALB/c mice without prior adaptation (Evseenko et al., 2007, Hatta et al., 2007, Hatta and Kawaoka, 2005, Isoda et al., 2006, Katz et al., 2000b, Gubareva et al., 1998a). Although the infection is primarily pulmonary, there is strong evidence of systemic spread to solid organs, including the brain (Gubareva et al., 1998a, Katz et al., 2000a). Most of these viruses cause necrosis in respiratory epithelium of the nasal cavity (Hatta et al., 2007), trachea (Hatta et al., 2007), bronchi, and bronchioles (Katz et al., 2000b, Hatta and Kawaoka, 2005) with accompanying inflammation and accumulation of fibrin and neutrophils (Katz et al., 2000b). The pathology in the lungs can be characterized as a peribronchial alveolitis with intra-alveolar serofibrinous exudate, erythrocytes and neutrophils, and increased numbers of alveolar macrophages (Katz et al., 2000b). Often the infection results in a bronchointerstitial pneumonia and alveolar edema or in severe cases it may become a diffuse interstitial pneumonia affecting entire lung lobes (Dybing et al., 2000). Body temperatures usually decrease by four of five degrees and animals lose at least 20% or more body when they succumb to the effects of the infection. In another study, a recent H5N1 isolate was shown to promote significantly higher levels of pro-inflammatory cytokines in whole lungs and primary human macrophages probably resulting in early and excessive infiltration of macrophages and neutrophils in the lungs of mice (Perrone et al., 2008). Another study has suggested that in addition to the typical proinflammatory cytokines, TNF-α may also contribute to the morbidity during H5N1 influenza virus infection (Szretter et al., 2007). Because of the severity of H5N1 infections in humans caused by highly pathogenic avian influenza viruses, numerous vaccine and antiviral efficacy studies have recently been done in mice. See the following recent references for vaccine studies (reviewed by van der Laan et al., 2008, Chen et al., 2008, Chen et al., 2009, Hickman et al., 2008, de Vries et al., 2008) and for antiviral efficacy studies (Barnard et al., 2007, Boltz et al., 2008a, Ilyushina et al., 2008, Zheng et al., 2008). 4.2 Ferrets Two H5N1 viruses isolated in Hong Kong during the 1997 outbreak were found to readily infect ferrets (Zitzow et al., 2002). Since then, more H5N1 isolates that are highly pathogenic for birds have been found to replicate in ferret lungs without prior host adaptation. The infections generally have been characterized by severe lethargy, fever, weight loss, transient lymphopenia, and virus replication in the upper and lower respiratory tract and in multiple organs including the brain. More importantly, the illness induced by these agents was more severe than that induced by recently isolated human H3N2 viruses. The lungs of H5N1-infected ferrets showed diffuse inflammation of interalveolar septa with infiltrates of mononuclear cells and intra-alveolar edema, regardless of the time post infection; in contrast to the scattered infiltrates seen with other influenza A viruses, these changes were observed throughout the lungs (Maines et al., 2005). Viral antigens were detected in alveolar bronchial cells or bronchioles in the majority of animals by day 3 post virus exposure. Brain tissues showed mononuclear cell infiltrates in the meninges, choroid plexus, and brain parenchyma, and viral antigens were detected in neurons in the same areas. In an effort to determine why the H5N1 virus is more virulent for ferrets than seasonal influenza A isolates, Cameron et al. (2008) analyzed the expression of host innate immune response genes during the course of lethal infection. The authors found that many interferon response genes, including IFI44, ISG15 (G1P2), MX2, OAS1, OAS2, STAT1, TAP1, and UBE1L, were significantly upregulated ferrets infected with the H5N1 virus, in comparison to an H3N2 isolate. CXCL10, a chemoattractant of activated Th1 lymphocytes and natural killer cells, was also upregulated to a much greater extent in an H5N1 infection than with the H3N2 virus. CXCL10 and its receptor CXCR3 are thought to play a role in the temporal development of innate and adaptive immunity in concert with type I and II interferons (Neville et al., 1997). In support of that hypothesis, Cameron et al. (2008) found that treatment of H5N1-infected ferrets with AMG487, a CXCR3 antagonist, markedly reduced the severity of symptoms and delayed death, compared to untreated animals. Vaccines (reviewed by Subbarao and Luke, 2007, Chen et al., 2008, Lalor et al., 2008, Mahmood et al., 2008) and a number antiviral compounds (Malakhov et al., 2006, Govorkova et al., 2007, Boltz et al., 2008b, Yun et al., 2008) have been tested in ferrets infected with H5N1 viruses very often have verified findings in mice (Malakhov et al., 2006, Govorkova et al., 2007, Boltz et al., 2008b, Yun et al., 2008). 4.3 Cats Rimmelzwaan et al. (2006) assessed the virulence of a H5N1 virus in cats by infecting them by intratracheal inoculation or through feeding on virus-infected chicks. Within 2 days, most animals developed fever, conjunctivitis, lethargy, and labored breathing. Virus was detected in the throat, nasal, and rectum, regardless of the original site of infection. The virus spread systemically and was detected in the respiratory and digestive tracts, liver, kidney, heart, brain, and lymph nodes. Cellular damage in infected tissues correlated with the detection of viral proteins. Histopathological examination of the lungs revealed multiple or coalescing foci of inflammation and necrosis in the bronchioles. The alveolar and bronchiolar lumina were infiltrated with alveolar macrophages, neutrophils, and erythrocytes, mixed with fibrin, edema fluid, and cellular debris. More importantly, some alveoli were covered by hyaline membranes, which are also seen human lungs upon autopsy. Hyaline membrane formation has not been observed in mice or other animal models. The epithelium of bronchiolar and alveolar walls, which were moderately thickened, also had evidence of both necrosis and hyperplasia. There was also edema and moderate accumulation of mononuclear cells around pulmonary artery branches. Although there has been some work in defining the cat influenza model for evaluating pathogenesis of H5N1 infections, few if any studies have been published which describe the suitability of the cat for doing vaccine (Karaca et al., 2005, Vahlenkamp et al., 2008) and antiviral efficacy studies. 4.4 Dogs Dogs can also be infected with the H5N1 avian influenza virus. Because these animals are often in close contact with both wild and domestic birds and with humans, this raises the possibility that the virus could adapt to dogs and be transmitted to humans while retaining virulence. To date it has been found that dogs are susceptible to experimental infection but are not capable of transmitting the virus to other mammals (Giese et al., 2008). The disease in dogs was characterized by the development of conjunctivitis and fever within 2 days after virus exposure, which resolved with no other adverse events by day 7. Dogs may be useful more as sentinel for human disease than as a model for human influenza disease (Cleaveland et al., 2006). 4.5 Nonhuman primates A cynomolgus macaque (Macaca fascicularis) model of H5N1 (A/HongKong/156/97) virus infection with pneumonia and ARDS has been developed (Kuiken et al., 2003, Rimmelzwaan et al., 2003). The principal change in the lung was a necrotizing bronchial interstitial pneumonia, similar to that described for primary human influenza pneumonia. In contrast to human H5N1 cases, detection of influenza virus antigen was limited to pulmonary tissue and tonsils. The model was used to evaluate the efficacy of intravenous zanamivir, which has a longer half-life than oseltamivir (Stittelaar et al., 2008). Drug levels in the epithelial lining fluids of the lungs were equivalent to those in plasma. Treated macaques had lower gross pathology scores and less lung pathology than untreated animals. However, there was considerable variability in viral lung titers and gross pathology within groups of macaques, and scattering of foci of lung infection from animal to animal, making it difficult to establish statistically significant differences. The authors pointed out that such variability probably reflects what happens within the human population. Some vaccine studies with macaques have also been done (de Vries et al., 2008, Kreijtz et al., 2008, Ruat et al., 2008 among others).