Archives of Medical Research 40 (2009) 655e661

REVIEW ARTICLE

Comparison of the Pathology Caused by H1N1, H5N1,and H3N2 Influenza Viruses

Jeannette Guarnera and Reynaldo Falcon-Escobedob

aDepartment of Pathology and Laboratory Medicine, Emory University Hospital, Atlanta, GeorgiabDepartment of Pathology, Hospital Central Dr. Ignacio Morones Prieto, San Luis Potosi, Mexico

Received for publication September 8, 2009; accepted September 21, 2009 (ARCMED-D-09-00424).

Address reprint re

Hospital, 1634 Clifton

712-2631; E-mail: jgu

0188-4409/09 $eseedoi: 10.1016/j.arcm

The spectrum of morbidity and mortality of H1N1, H5N1, and H3N2 influenza A virusesrelates to the pathology they produce. In this review, we describe and compare thepathology of these viruses in human cases and animal models. The 1918 H1N1, the novel2009 H1N1 pandemic virus, and H5N1 show inflammation, congestion, and epithelialnecrosis of the larger airways (trachea, bronchi and bronchioles) with extension intothe alveoli causing diffuse alveolar damage. Seasonal influenza A viruses (H3N2 andH1N1) have primarily caused inflammation, congestion and epithelial necrosis of thelarger airways with lesser extension of the inflammatory process to alveoli. Localizationof the inflammation and cellular damage relate to the presence of virus in different celltypes. Infections with 1918 H1N1, the novel 2009 H1N1 pandemic virus, and H5N1 showvirus in mucosal epithelial cells of the airways (from the nasopharynx to the bronchioles),alveolar macrophages, and pneumocytes, whereas infections with seasonal influenzaviruses show viral antigens primarily in mucosal epithelial cells of the larger airways.The increased morbidity that has been encountered with the 2009 H1N1 virus is relatedto infection of cells in the upper and lower airways. The 2009 H1N1 virus shows similarpathology to that encountered with other highly virulent influenza A viruses such as the1918 H1N1 and H5N1 viruses. � 2009 IMSS. Published by Elsevier Inc.

Key Words: Pathology, Influenza A virus, Novel H1N1, avian H5N1, Seasonal H3N2, Human cases,

Animal models.

Introduction

Preparedness for an influenza pandemic has includedsurveillance for all influenza viruses but attention has beenprimarily focused on transmission of avian H5N1 virus tohumans and further transmission of H5N1 virus amonghumans. Attention to the H5N1 virus started in 1997 whenhumans became infected during a poultry outbreak of thishighly pathogenic avian influenza virus in Hong Kong(1e3). Animal studies and studies of autopsies of patientsinfected with the different viruses (avian H5N1, swineH1N1, and seasonal H3N2) have revealed that the cells inthe respiratory tract these viruses infect are different result-ing in different pathologic pictures and morbidity andmortality rates (4e11). In addition, further molecularstudies of autopsy material from the 1918 pandemic lead

quests to: Jeannette Guarner, MD, Emory University

Rd, Room C179A, Atlanta, GA, 30322; Phone 404-

arne@emory.edu

front matter. Copyright � 2009 IMSS. Published by Elseved.2009.10.001

to recognition that the virus causing disease was of swineorigin (H1N1) (12,13). Reconstruction and successfulanimal infections with the 1918 H1N1 virus have addedto the spectrum of clinicopathological outcomes that thedifferent influenza A viruses can cause (14,17). Beforethe 2009e2010 season begins, a comparison of thepathology between the recent 2009 H1N1 pandemic virus(18,19) and the other influenza A viruses may help to betterprepare healthcare professionals to understand themorbidity and mortality that may be encountered.

The signs and symptoms of H1N1, H5N1 and H3N2influenza A viruses is primarily in the respiratory tractand the pathological damage encountered mirrors the clin-ical presentation with the most frequent pathology observedin the airways and lungs. Clinical symptoms of myocarditisand encephalopathy have been described less frequently inpatients with influenza A infections, and pathology in theheart has been observed in some of these cases. It shouldbe noted that the pathological descriptions in humans come

ier Inc.

656 Guarner and Falcon-Escobedo/ Archives of Medical Research 40 (2009) 655e661

primarily from autopsies; thus, our knowledge relates to themost severe cases with the poorest outcomes (20). Study ofthe pathology of confirmed cases is important to be able tobetter understand the differences in presenting signs andsymptoms, duration of the disease, complications, andmortality that these viruses can produce. In addition,knowing the pathology these viruses cause will enablepathologists to perform presumptive diagnosis and seekother diagnostic techniques (PCR, viral culture) that willallow confirmation of the virus involved for specific cases.

This paper will describe and compare the pathologyencountered in autopsies and animal models infected withH1N1, H5N1 and H3N2 influenza viruses.

Pathology of H1N1

Detailed descriptions of the pathology of cases infectedwith the pandemic 2009 H1N1 virus have not yet appearedin the literature. The descriptions presented here are basedon the authors’ observations of four cases and a publishedreport of one case (18). The lung pathology of deceasedpatients infected with the pandemic 2009 H1N1 haveshown primarily diffuse alveolar damage with extensivehyaline membrane formation and intraalveolar edema(Figure 1A). The vessels in alveolar walls show thrombosis,which has lead to necrosis of the alveolar walls (Figure 1B)and varying degrees of mixed inflammatory infiltrate.Alveolar epithelial cells and pneumocytes show reactivechanges, and their nuclei appear enlarged. Desquamatedmacrophages and pneumocytes are observed within thealveolar spaces. Prominent proliferation of fibroblasts isobserved in patients with longer duration of disease. Intra-alveolar hemorrhage and erythrophagocytosis have alsobeen observed. Immunohistochemical assays have demon-strated viral antigens in pneumocytes type I and II andintraalveolar macrophages (personal communication,Dr. Sherif Zaki, Centers for Disease Control and Preven-tion, Atlanta, GA, 2009). Bacterial pneumonias, definedas accumulation of neutrophils in the alveoli, have beenobserved in 30% of cases and may be either communityacquired or nosocomial (ventilator-associated) (18,19).

The trachea and major bronchi of deceased patients in-fected with the pandemic 2009 H1N1 have shown necroticdesquamation of the mucosa and edema and mixed inflam-matory infiltrate in the submucosa (Figure 1C). The glandsin these airways show loss of goblet cells and necrosis ofsurrounding submucosa (Figure 1D). Viral antigens havebeen noted in the epithelial cells and mucous glands oftrachea, bronchi, and bronchioles (personal communica-tion, Dr. Sherif Zaki, Centers for Disease Control andPrevention, Atlanta, GA, 2009). PCR demonstrating virusin tissues from the respiratory tract and lung has shownbetter sensitivity and specificity than immunohistochem-istry. Myocardial infarction has been documented in severalpatients infected with the 2009 H1N1 virus (18).

The1918 H1N1 pandemic virus is recorded in history ashaving caused the highest number of excess deaths (20).Multiple studies of the pathology were published at thetime and newer studies have reviewed the archivedpathology material to ascertain the role of viral pneumoniaand bacterial superinfections (17,21e24). From the patho-logical standpoint, H1N1 infections during the 1918pandemic can be divided into those patients with viralpneumonia and those with superimposed bacterial pneu-monia. Those patients who died early during the infectionshowed viral pneumonia with focal necrosis of the alveolarwalls associated with capillary thrombosis, prominenthyaline membrane formation, and intraalveolar edema. Inthe trachea and major bronchi, autopsies showed necroticmucosa with edema and inflammation in the submucosa.The loss of epithelium was more pronounced in the bron-chioles where the epithelial layer is thinner. In comparison,those patients with superimposed bacterial pneumoniasshowed abundant intraalveolar neutrophilic infiltrate asso-ciated with edema, fibrin and necrosis in a predominantlobular distribution. It is estimated that up to 90% ofpatients who died during the 1918 H1N1 pandemic hadbacterial superinfections with Streptococcus pneumoniae,S. hemolyticus, and mixed pathogens being the mostfrequently isolated bacteria (17). Myocarditis was reportedin patients with the 1918 H1N1 infections (22).

Several animal models have been used to study thepathology of H1N1 infections and the results vary depend-ing on the host animal and virus used for the infection(5,8,10,14e16,25). In guinea pigs and pigs, low pathogenicseasonal H1N1 virus (A/Puerto Rico/8/34 and A/swine/Thailand/HF6/05) showed viral antigens in the mucosalepithelium associated with epithelial cell damage and couldbe seen from the upper airways to the bronchioles (5,10).Damage of the epithelium resulted in increased mucousproduction and mild inflammatory infiltrate. Inflammationsurrounded bronchioles but did not spill into the lung inter-stitium. In ferrets, the seasonal H1N1 (A/Netherlands/26/2007) showed multifocal necrotizing rhinitis with abundantviral antigens in the nasal turbinates and focal consolidationof the lungs without evidence of viral antigens in the lowerrespiratory tract (25). When low pathogenic seasonal H1N1virus (A/Puerto Rico/8/34) was used to infect mice, thelungs showed complete consolidation due to bronchitisand interstitial pneumonia, the thymus was reduced in sizeby 30% due to depletion of cortical lymphocytes, and theheart showed inflammatory foci (8). Thus, for the low path-ogenic seasonal H1N1 viruses the extent of damage de-pended on the infected host.

Animal models using ferrets and pandemic 2009 H1N1have shown more extensive consolidation of the lungs thanthat encountered with seasonal H1N1 virus (25). Histopath-ologically, ferrets show multifocal mild to moderate necro-tizing rhinitis, tracheitis, bronchitis, and bronchiolitis, andviral antigens are observed from the nasal turbinates to

Figure 1. Histopathological characteristics of fatal cases with confirmed pandemic 2009 H1N1 virus infection showing diffuse alveolar damage in the early

exudative phase characterized by hyaline membranes (A,B), intraalveolar edema (A), and alveolar wall necrosis (B) accompanied by inflammatory infiltrate

(A). The trachea and bronchi showed denuded mucosa (C,D) with inflammation (C) and necrosis (D) of the submucosa. Hematoxylin and eosin stains; orig-

inal magnifications: �10 (A), �20 (B,D), and �5 (C). Color version of this figure available online at www.arcmedres.com.

657Comparison of Pathology of Influenza A Viruses

the bronchioles. The pandemic 2009 A (H1N1) influenzaviruses replicate to higher titers in lung tissue and can berecovered from the upper and lower airways as well asthe intestinal tract (26).

Infection of mice or monkeys with highly pathogenic re-constructed 1918 H1N1 viruses (1918 HA/NA:WSN or1918 HA/NA:Tx/91) produced large areas of pneumoniathat microscopically showed necrotizing bronchitis andinflammation of alveolar walls by neutrophils and macro-phages (14e16). Necrotic debris was noted in the lumenof bronchioles and alveoli. Viral antigens were localized

in bronchiolar epithelium and alveolar macrophages. Theseanimals have also shown increased expression of cytokinesand chemokines.

Pathology of H5N1

Even though the mortality from H5N1 infection is |60%,there have been !15 autopsies reported in the literature(2e4,27e29). The lung pathology of deceased patients in-fected with the H5N1 virus has shown diffuse alveolardamage in the lungs. Depending on the duration of illness,

658 Guarner and Falcon-Escobedo/ Archives of Medical Research 40 (2009) 655e661

diffuse alveolar damage may be in the exudative earlyphase and show hyaline membrane formation, congestionof vasculature, and lymphocytes infiltrating the interstitiumor in the later fibrous proliferative phase where fibroblastproliferation is added to the picture (Figure 2A) (3). Inflam-mation around bronchioles, alveolar interstitium, and in thepleura has been present. Cells involved in the pathologyinclude presence of T-lymphocytes in the alveolar septae,macrophages in the alveolar lumen, and hyperplasia anddesquamation of type II pneumocytes. Several cases have

Figure 2. Histopathological characteristics of fatal cases with confirmed H5N1

erative phase characterized by fibroblast proliferation (A) and deposition of coll

hemophagocytosis (C). Hematoxylin and eosin stains; original magnifications: �1

www.arcmedres.com.

shown intraalveolar hemorrhage (Figure 2B), whereasothers have shown hemophagocytosis. Hemophagocytosishas been noted not only in the lung but also in bonemarrow, lymph nodes (Figure 2C), spleen, and liver. Otherorgans with pathology have included the spleen with deple-tion and apoptosis of lymphocytes, kidneys with acutetubular necrosis, and liver with necrosis, fatty change, acti-vation of Kupffer cells, and cholestasis. Two cases haveshown demyelinated areas in the brain with necrosis andreactive histiocytes. Several techniques have demonstrated

virus infection showing diffuse alveolar damage in the later fibrous prolif-

agen (A,B). Intraalveolar hemorrhage is seen in some cases (B) as well as

0 (A), �5 (B), and �63 (C). Color version of this figure available online at

Figure 3. Histopathological characteristics of fatal cases with confirmed seasonal H3N2 virus infection showing mucosal desquamation and disorganization

and submucosal congestion, hemorrhage, and inflammation in the trachea (A). The lungs show intraalveolar hemorrhage and edema (B). The heart shows

focal areas of mononuclear inflammatory infiltrate (C). Hematoxylin and eosin stains; original magnifications: �10 (A), �5 (B), and �40 (C). Color version

of this figure available online at www.arcmedres.com.

659Comparison of Pathology of Influenza A Viruses

virus that can replicate in the lung, and immunohistochem-ical assays have localized the viral antigens in ciliated andnonciliated epithelial cells and in type II pneumocytes.Viral sequences and antigens have been found in thebrain, intestines, and placenta as well as lymphocytes andmacrophages.

Several animal models have been used to study thepathology of H5N1 infections and the results vary depend-ing on the pathogenicity of the virus used for the infection(11,14,15,30). In general, those viruses that have beenrecovered from patients with fatal infections (i.e., A/HongKong/483/97) tend to produce more severe disease in thedifferent animal models than those recovered from patientswith milder disease (i.e., A/HongKong/486/97) (11,30).

The animals that have been used include mice, ferrets,and primates.

Macroscopically, all animals showed various degrees ofconsolidation and discoloration in the lungs and hemor-rhages in adipose tissue surrounding liver, intestines,kidneys, and bladder (11,30). Macroscopic changesoccurred earlier in animals infected with the highly virulentviruses compared to those infected with low-virulenceviruses. Microscopically, the lungs showed inflammationand edema surrounding bronchioles and spilling into thealveolar septae (11,15,30). Histopathology and flow cytom-etry have shown that the inflammatory infiltrate consistedof neutrophils and macrophages (14). The extent of inflam-mation spilling into the alveoli appeared earlier (about

660 Guarner and Falcon-Escobedo/ Archives of Medical Research 40 (2009) 655e661

5e9 days postinfection) and was more pronounced in thoseanimals infected with highly virulent virus compared tothose infected with low-virulence virus. Similarly, the pres-ence of epithelial necrosis has been described for thoseanimals infected with highly virulent virus. At about 3 dayspostinoculation and before there are inflammatory infil-trates, viral antigens can be detected in type II pneumocytes(15). The brains of animals infected with the highly virulentviruses showed glial nodules, perivascular cuffing bylymphocytes and neutrophils, neuronophagia, and inflam-mation in the choroid plexus more frequently and exten-sively than those animals infected with low-virulenceviruses (11,30). Viral antigens have been detected inneurons.

Pathology of H3N2

Publications of the pathology observed in patients infectedwith seasonal or interpandemic influenza have included51 pediatric patients with fatal disease and 11 adults (six withfatal disease and five who survived the infection) (6,7,31,32).The pathological features described for H3N2 have a biastowards severe and complicated infections that have requiredopen lung biopsies or were fatal, and an autopsy was per-formed to understand the cause of death. The pathologicalchanges observed in surviving patients have ranged fromacute lung injury including patchy fibrinous alveolarexudates, hyaline membrane formation, interstitial edema,and necrosis of bronchiolar mucosa to reparative changessuch as proliferation of type II pneumocytes, interstitialchronic inflammatory infiltrates, and organization ofairspaces and interstitium (31,32).

In autopsies of patients infected with the H3N2 virus, themost frequent and sometimes the only pathology observedhas been mononuclear inflammation and congestion of thetrachea, major bronchi and bronchioles commonly accom-panied by necrosis of the epithelium and submucosalhemorrhage (Figure 3A) (6,7). Viral antigens may only bepresent in the bronchial epithelium. Changes in the lowerrespiratory tract include mononuclear inflammation in thealveolar septa, hyaline membrane formation and intraalveo-lar hemorrhage (Figure 3B). Viral antigens are rarely foundin the alveoli. In 50% of the cases there were focal areas ofpneumonia with Staphylococcus aureus and group A strep-tococci being the most frequently identified bacterial super-infections. Lymph nodes showed hemophagocytosis in halfthe cases. In the heart, patchy areas of mononuclear inflam-mation and necrosis (Figure 3C) were observed in 30% ofcases.

There are several descriptions of the pathology of animalsinfected with the H3N2 virus that have usually beencompared to animals that have been infected with seasonalH1N1 viruses. Mice, guinea pigs, pigs and ferrets have beenused in these models (5,8,10,30). In all of these animalmodels there is desquamation and disorganization of the

epithelium and inflammation of the mucosa of the airways(nasal, paranasal, sinuses, trachea, bronchus, and bronchi-oles) starting at 1 day postinfection. Changes in the respira-tory mucosa are accompanied by the presence of viralantigens that decline sharply 3 days postinfection. Macro-scopically the lungs appear consolidated, and microscopi-cally there is neutrophilic and mononuclear inflammationin the bronchioles and alveolar intestitium. Viral antigenswere detected in the epithelial cells and macrophages.Outside the lungs, viral antigens have been found in the heartand thymus. In pigs, lesions produced by seasonal H1N1were more extensive than those produced by infections withthe H3N2 virus (10).

Comparison of the Pathological Features Caused byInfluenza A Viruses

The pathology caused by these viruses in humans or animalmodels depends on the virulence of the infecting agent andhost response. All the viruses infect the respiratory epithe-lium from the nasal passages to bronchioles; however, high-virulence viruses (1918 H1N1, H5N1, and probably 2009H1N1) also tend to infect pneumocytes and intraalveolarmacrophages. The localization and amount of the inflam-matory reaction will depend on the infected cells. Thus,low-virulence viruses (seasonal H3N2 and H1N1) causeprimarily inflammation, congestion and epithelial necrosisof the larger airways (trachea, bronchi and bronchioles),whereas high-virulence viruses have equal inflammationin these localizations with additional, more extensive areasof inflammation in the alveoli. In susceptible individuals,inflammation of the alveolar walls will result in diffusealveolar damage. It needs to be remembered that influenzaA viruses do not cause inclusions or visible cytopathiceffects in vivo; however, infected cells show reactivechanges with enlargement of the nuclei. In addition, thepathological picture will change to intraalveolar neutro-philic infiltrate if bacterial superinfection is present. Thefrequency and type of bacterial superinfections that wecan expect during the 2009e2010 season will be differentfrom those encountered during the 1918 H1N1 pandemic.The use of antibiotics, supportive measures (ventilators),and vaccinations for S. pneumoniae and Haemophilus influ-enzae will probably reduce the frequency of these superin-fections but will bring community and nosocomial bacterialinfections that will have acquired antibiotic resistance.

References1. Claas E, Osterhaus A, van-Beek R, et al. Human influenza A H5N1

virus related to a highly pathogenic avian influenza virus. Lancet

1998;351:472e477.

2. To K, Chan P, Chan K, et al. Pathology of fatal human infection asso-

ciated with avian ifluenza A H5N1 virus. J Med Virol 2001;63:

242e246.

661Comparison of Pathology of Influenza A Viruses

3. Korteweg C, Gu J. Pathology, molecular biology, and pathogenesis of

avian influenza A (H5N1) infection in humans. Am J Pathol 2008;172:

1155e1170.

4. Ng W, To K. Pathology of human H5N1 infection: new findings.

Lancet 2007;370:1106e1108.

5. Tang X, Chong K. Histopathology and growth kinetics of influenza

viruses (H1N1 and H3N2) in the upper and lower airways of guinea

pigs. J Gen Virol 2009;90:386e389.

6. Guarner J, Paddock C, Shieh W, et al. Histopathologic and immuno-

histochemical features of fatal influenza virus infection in children

during the 2003e2004 season. Clin Infect Dis 2006;43:132e140.

7. Guarner J, Shieh W, Dawson J, et al. Immunohistochemical and in situ

hybridization studies of influenza A virus infection in human lungs.

Am J Clin Pathol 2000;114:227e233.

8. Fislova T, Gocnık M, Sladkova T, et al. Multiorgan distribution of

human influenza A virus strains observed in a mouse model. Arch

Virol 2009;154:409e419.

9. Kuikena T, Taubenberger J. Pathology of human influenza revisited.

Vaccine 2008;26S:D59eD66.

10. Sreta D, Kedkovid R, Tuamsang S, et al. Pathogenesis of swine influ-

enza virus (Thai isolates) in weanling pigs: an experimental trial. Virol

J 2009;6:34e44.

11. Maines T, Lu X, Erb S, et al. Avian influenza (H5N1) viruses isolated

from humans in Asia in 2004 exhibit increased virulence in mammals.

J Virol 2005;79:11788e11800.

12. Taubenberger J, Reid A, Krafft A, et al. Initial genetic characterization

of the 1918 ‘‘Spanish’’ influenza virus. Science 1997;275:1793e1796.

13. Tumpey T, Basler C, Aguilar P, et al. Characterization of the reconstructed

1918 Spanish influenza pandemic virus. Science 2005;310:77e80.

14. Perrone L, Plowden J, Garcıa-Sastre A, et al. H5N1 and 1918

pandemic influenza virus infection results in early and excessive infil-

tration of macrophages and neutrophils in the lungs of mice. PLoS

Pathog 2008;4:e1000115.

15. Baskin C, Bielefeldt-Ohmann H, Tumpey T, et al. Early and sustained

innate immune response defines pathology and death in nonhuman

primates infected by highly pathogenic influenza virus. Proc Natl

Acad Sci 2009;106:3455e3460.

16. Tumpey T, Garcia-Sastre A, Taubenberger J, et al. Pathogenicity

of influenza viruses with genes from the 1918 pandemic virus:

functional roles of alveolar macrophages and neutrophils in

limiting virus replication and mortality in mice. J Virol 2005;79:

14933e14944.

17. Morens D, Taubenberger J, Fauci A. Predominant role of bacterial

pneumonia as a cause of death in pandemic influenza: implications

for pandemic influenza preparedness. J Infect Dis 2008;198:

962e970.

18. Perez-Padilla R, de-la-Rosa-Zamboni D, Ponce-de-Leon S, et al.

Pneumonia and respiratory failure from swine-origin influenza A

(H1N1) in Mexico. N Engl J Med 2009;361:680e689.

19. Centers for Disease Control and Prevention (CDC). Bacterial coin-

fections in lung tissue specimens from fatal cases of 2009 pandemic

influenza A (H1N1)- United States, May-August 2009. MMWR Morb

Mortal Wkly Rep 2009;583:1071e1074.

20. Novel-Swine-Origin-Influenza-A-(H1N1)-Virus-Investigation-Team.

Emergence of a novel swine-origin influenza A (H1N1) virus in

humans. N Engl J Med 2009;360:2605e2615.

21. Taubenberger J, Morens D. The pathology of influenza virus infec-

tions. Annu Rev Pathol Mech Dis 2008;3:499e522.

22. Goodpasture E. The significance of certain pulmonary lesions in relation

to the etiology of influenza. Am J Med Sci 1919;158:863e867.

23. Lucke B, Wight T, Kime E. Pathologic anatomy and bacteriology of influ-

enza: epidemic of autumn, 1918. Arch Intern Med 1919;24:154e237.

24. MacCallum W. Pathology of epidemic pneumonia in camps and

cantonments in 1918. Med Rec 1919;95:776e784.

25. Bakwin H. Gross pathology of influenzal pneumonia in France. Am J

Med Sci 1920;159:435e442.

26. Munster V, de-Wit E, van-den-Brand J, et al. Pathogenesis and trans-

mission of swine-origin 2009 A(H1N1) influenza virus in ferrets.

Science 2009;325:481e483.

27. Maines T, Jayaraman A, Belser J, et al. Transmission and pathogenesis

of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice.

Science 2009;325:484e487.

28. Uiprasertkul M, Kitphati R, Puthavathana P, et al. Apoptosis and path-

ogenesis of avian influenza A (H5N1) virus in humans. Emerg Infect

Dis 2007;13:708e712.

29. Chotpitayasunondh T, Ungchusak K, Hanshaoworakul W, et al.

Human disease from influenza A (H5N1), Thailand, 2004. Emerg

Infect Dis 2005;11:201e209.

30. Gu J, Xie Z, Gao Z, et al. H5N1 infection of the respiratory tract and

beyond. Lancet 2007;370:1137e1145.

31. Zitzow L, Rowe T, Morken T, et al. Pathogenesis of avian influenza A

(H5N1) viruses in ferrets. J Virol 2002;76:4420e4429.

32. Noble R, Lillington G, Kempson R. Fatal diffuse influenzal pneumonia:

premortem diagnosis by lung biopsy. Chest 1973;63:644e666.