The International Journal of Biochemistry & Cell Biology 40 (2008) 2671–2674
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Medicine in focus
Human H5N1 inﬂuenza: Current insight into pathogenesis Tran Tan Thanh a , H. Rogier van Doorn a,b , Menno D. de Jong a,b,c,∗ a
Oxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, District 5, Ho Chi Minh City, Viet Nam Centre for Tropical Medicine, Nufﬁeld Department of Clinical Medicine, University of Oxford, Centre for Clinical Vaccinology and Tropical Medicine, Oxford, UK c Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands b
a r t i c l e
i n f o
Article history: Received 12 May 2008 Received in revised form 30 May 2008 Accepted 30 May 2008 Available online 6 June 2008 Keywords: H5N1 Inﬂuenza Emerging infections Zoonosis Pathogenesis
a b s t r a c t Since their emergence as avian (1996) and zoonotic human pathogens (1997), H5N1 inﬂuenza viruses have become endemic among poultry in large parts of Asia, but outbreaks have also been seen in Africa and Europe. Transmission from animals to humans remains sporadic, but mortality of human infection is high (63%). To date, reported cases of human to human transmission have been rare. Patient and laboratory data suggest that highly efﬁcient viral replication and the resulting intensiﬁed immune response of the human host are the determining factors in H5N1 pathogenesis and case fatality rate. Therefore, in the management of H5N1 disease (early) suppression of viral replication is key. The underlying biochemistry and cell biology of H5N1 pathogenesis and treatment are brieﬂy discussed in this review. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction Inﬂuenza A viruses belong to the family of Orthomoyxoviridae. Their genome consists of 8 single-stranded negative sense RNA segments, encoding eleven proteins. Inﬂuenza A viruses are classiﬁed according to the antigenicity of their two surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). To date, 16 HA (H1-16) and 9 NA (N1-9) subtypes have been described, all of which are found in the virus’ natural host: waterfowl. A limited number of subtypes have established species-speciﬁc lineages in humans (H1N1, H2N2, and H3N2). Besides the two surface glycoproteins, which are essential for virus binding to (HA) and release from (NA) the host cell, other proteins known to be involved in pathogenicity and transmissibility of inﬂuenza viruses are the proteins of the replication complex (Polymerase acidic [PA], and basic 1 [PB1] and 2
∗ Corresponding author at: Oxford University Clinical Research Unit, Hospital for Tropical Diseases, 190 Ben Ham Tu, District 5, Ho Chi Minh City, Viet Nam. Tel.: +84 8 8384009; fax: +84 8 9238904. E-mail address: [email protected]
(M.D. de Jong). 1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2008.05.019
[PB2], and the nucleoprotein [NP]) and the non-structural protein 1 (NS1) (Peiris et al., 2007) (Fig. 1). Since their emergence as zoonotic human pathogens in 1997, highly pathogenic avian inﬂuenza H5N1 viruses have spread to large parts of the world. Human infections remain sporadic, but are associated with severe disease and high mortality (63% [241/382], per 30-4-08 (WHO, 2008)). High viral replication and an increased inﬂammatory response with cytokine dysregulation are thought to play central roles in the pathogenesis of human disease. 2. Pathogenesis 2.1. Clinical syndrome Most human cases present as severe pneumonia with rapid progression to acute respiratory distress syndrome (ARDS). Patients show radiological evidence of pneumonia, sometimes with bilateral inﬁltration and collapse or consolidation. Complications associated with fatal outcome include ARDS and multi-organ failure. Occasionally, encephalitis has been observed. In recovered patients,
T.T. Thanh et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2671–2674
Fig. 1. Genes and proteins involved in H5N1 pathogenesis, virulence and treatment. PB = Polymerase Basic, PA = Polymerase Acidic, HA = Hemagglutinin, NP = Nucleoprotein, NA = Neuraminidase, M = Matrix, NS = Nonstructural.
radiological evidence of lung damage may still be observed after several months (de Jong and Hien, 2006). High nasopharyngeal viral loads were associated with fatal outcome (de Jong et al., 2006). Evidence of actively replicating H5N1 virus was found in the trachea and lower respiratory tract. In addition, viral RNA was demonstrated in autopsy samples of multiple non-respiratory organs including intestines, liver, spleen and brain, suggesting widespread viral dissemination (Abdel-Ghafar et al., 2008). In a pregnant woman, viral RNA and antigen was also detected in fetus and placenta (Gu et al., 2007). The isolation of H5N1 viruses from plasma suggests that the blood is a route of dissemination from primary infected (respiratory tract) sites to other organs. It remains unknown how H5N1 virus reaches the central nervous system: hematogenously through crossing of the blood–brain barrier or continuously by spread from peripheral nerve endings, as suggested by studies in cats and mice (Rimmelzwaan et al., 2006; Tanaka et al., 2003). Patients with severe H5N1 disease often have lymphopenia, thrombocytopenia, and increased levels of serum aminotransferases (de Jong and Hien, 2006). Most examined patients have increased levels of chemokines (interferon [IFN]-inducible protein 10 [IP-10], monokine induced by IFN-␥ [MIG], monocyte chemotactic protein-1 [MCP-1], Interleukin [IL]-8) and pro- and anti-inﬂammatory cytokines (IL6, IFN-␥, IL-10), the levels of most of which were positively correlated with both nasopharyngeal virus loads and fatal outcome (de Jong et al., 2006). In vitro experiments in macrophages and respiratory epithelial cells have shown that H5N1 viruses may induce cytokine expression to a larger extent than human seasonal inﬂuenza viruses (Chan et al., 2005; Cheung et al., 2002). This observed high production of cytokines may be explained by strong activation of the p38 mitogen-activated protein kinase
(p38 MAPK) signaling pathway by H5N1 (Lee et al., 2005). 2.2. Hemagglutinin (HA) The hemagglutinin (HA) plays a role in viral entry into host cells and determines host speciﬁcity of inﬂuenza viruses. HA binds to glycan receptors terminated by a sialic acid (SA) linked to a galactose residue. Traditionally it was thought that avian hosts have ␣2,3 linked sialic acids (SA ␣2,3-Gal) in their respiratory and gastrointestinal tract, while humans express SA ␣2,6-Gal. Likewise, avian and human viruses preferentially bind to the corresponding sialic acids of their hosts. Receptor speciﬁcity may change by a small number of amino acid changes in the receptorbinding pocket of HA (Naeve et al., 1984). Recent studies revealed expression of SA ␣2,3-Gal in human respiratory epithelial cells: cells of the upper respiratory tract (nasal mucosa, paranasal sinuses, pharynx, and trachea) express mainly SA ␣2,6-Gal, whereas cells in the lower respiratory tract (type II pneumocytes and nonciliated cuboidal epithelial cells) express both SA ␣2,6- and ␣2,3-Gal. SA ␣2,3-Gal bearing cells in the lower respiratory tract are believed to act as primary target cells for H5N1 virus infections. The predominance of these receptors in the lung may explain the propensity of H5N1 viruses to cause pneumonia in humans as well as the difﬁculty in transmission of this virus from birds and between humans (Shinya et al., 2006; van Riel et al., 2006). Compared to adults, respiratory cells from children seem to express more SA ␣2,3-Gal, suggesting – in accordance with epidemiological data – that they may be more susceptible to H5N1 infection (Nicholls et al., 2007a,b). However, data from glycan arrays show that the structural topology of the glycan also plays an important part besides the linkage of the terminal sialic acid (Chandrasekaran et al., 2008) and that viruses
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may bind to a whole range of other glycans apart from SA ␣2,3 and ␣2,6-Gal (Stevens et al., 2006), demonstrating that inﬂuenza receptor speciﬁcity is (far) more complex than previously recognized. 2.3. The polymerases Changes in the viral polymerase complex, particularly in PB2, seem essential for adaptation of avian inﬂuenza viruses to efﬁcient replication and pathogenicity in mammalian hosts. Speciﬁcally, the presence of a lysine (Lys) instead of a glutamic acid (Glu) at position 627 of PB2 has been identiﬁed as an important determinant of host range (Subbarao et al., 1993). Furthermore, in vitro studies have shown that viruses with Lys627 replicate efﬁciently at lower temperatures (33 ◦ C), providing opportunities for efﬁcient growth in the upper respiratory tract of mammalian hosts. Accordingly, viruses containing Lys627 replicate to higher titres in nasal turbinates of mice than viruses with Glu627 (Neumann et al., 2007). While mouse models suggest that Lys627 in H5N1 PB2 is a major determinant of high pathogenicity in mammals, a clear association with poor clinical outcome in humans has not been found. Interestingly, in a series of Vietnamese H5N1 patients, most viruses lacking Lys627 harboured an alternative change at position 701 (Asp to Asn) which has also been linked to adaptation of avian viruses to mammalian hosts (de Jong et al., 2006). In mice, H5N1 viruses lacking an Asn701 residue were unable to replicate and cause disease (Li et al., 2005). In H7N7 viruses, the presence of Asn at position 701 was shown to be involved in binding to mammalian importin 1␣, thereby enabling more efﬁcient transport of the replication machinery to the nucleus of the host cell (Gabriel et al., 2008). While speciﬁc changes in PB2 thus seem important for efﬁcient replication and pathogenicity in mammals, changes in other polymerase genes likely also play a role. This is illustrated by reverse genetics experiments showing more pronounced virulence changes when all three polymerase genes from a highly virulent virus, rather than single genes, were introduced in a non-pathogenic virus (Salomon et al., 2006). 2.4. The PB1-F2 protein The recently discovered PB1-F2 protein is translated from an alternate reading frame of the PB1 gene segment. PB1-F2 causes apoptosis in macrophages, reducing their ability to induce an immune response and therefore delaying viral clearance (Coleman, 2007). This reading frame is also present in H5N1, and mutations cause virulence changes (Conenello et al., 2007). The exact role in pathogenicity has yet to be determined. 2.5. The NS1 protein The NS1 protein is the only nonstructural protein of inﬂuenza viruses. This protein plays an important role in the pathogenicity of inﬂuenza viruses by protecting the virus from the antiviral effects of the host IFN responses. NS1 exerts its IFN antagonism by binding to dsRNA and
to the cellular RNA helicase retinoic acid inducible gene 1 (RIG-1), an upstream regulatory component of the IFN production cascade (Mibayashi et al., 2007). In highly pathogenic H5N1 viruses, the NS1 gene is also implicated in causing cytokine dysregulation, which is thought to play an important role in H5N1 disease pathogenesis in mammals, including humans. Compared to human H1N1 and H3N2 viruses, the NS1 gene of H5N1 viruses or its product is a potent inducer of proinﬂammatory cytokines in vitro, particularly TNF-␣ (Cheung et al., 2002). Contemporary human H1N1 virus carrying the NS1 of a highly pathogenic H5N1 virus isolated during the 1997 Hong Kong outbreak induced high pulmonary concentrations of pro-inﬂammatory cytokines and prolonged viral shedding in pigs (Seo et al., 2002). While this effect was linked to a speciﬁc amino acid change in NS1 (Asp92Glu), this change has not been observed in current H5N1 strains. Whole genome sequencing revealed a PDZ domain ligand (PL) in the NS1 carboxy terminus of inﬂuenza virus. Proteins that contain PDZ domains play important roles in many key signaling pathways. Avian PL signatures from H5N1 and H1N1 were shown in vitro to bind human PDZ domains more efﬁciently than human H2N2 and H3N2 viruses, possibly disrupting more cellular processes (Obenauer et al., 2006). The exact consequences of these ﬁndings on the virulence of inﬂuenza viruses in general and of H5N1 viruses in particular have yet to be determined. 3. Treatment At present, two classes of drugs are in use for the treatment of inﬂuenza virus infections: the adamantanes (amantadine and rimantadine), targeting the M2 ion channel of inﬂuenza A viruses, and the neuraminidase inhibitors (zanamivir and oseltamivir). Susceptibility to adamantanes is varying in current H5N1 viruses. Adamantane resistance rates are high in certain genetic clades of H5N1 viruses, which may be explained by the use of amantadine in poultry farming (Abdel-Ghafar et al., 2008; Deyde et al., 2007; He et al., 2008). H5N1 viruses are usually susceptible to neuraminidase inhibitors, although up to 30-fold differences in in vitro 50% inhibitory concentrations of oseltamivir are observed between different genetic clades of current H5N1 viruses (McKimm-Breschkin et al., 2007). Development of high levels of resistance of H5N1 virus can occur during treatment with oseltamivir and is associated with treatment failure (de Jong et al., 2005). While oseltamivir resistant viruses remain susceptible to zanamivir (Wetherall et al., 2003), the currently available locally acting inhaled formulation of this drug limits its potential use in human H5N1 infections in view of the systemic nature of this disease. Parenteral formulations of neuraminidase inhibitors as well as novel drugs with alternative targets, such as the viral polymerase, are desirable and currently under clinical development. The potential use of combination therapy to enhance antiviral efﬁcacy and prevent the development of drug resistance may need consideration. The beneﬁts of antiviral treatment are highly dependent on the timing of treatment. Early installment of treatment clearly seems associated with improved outcome of
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human H5N1 infections (Abdel-Ghafar et al., 2008), likely due to prevention of irreversible tissue damage by the virus and the ensuing host immune response. At present there is insufﬁcient evidence suggesting a role of speciﬁc immunomodulatory agents in the treatment of H5N1 infections. Studies in knock-out mice revealed conﬂicting results showing milder disease courses in TNF receptordeﬁcient mice in one study and unchanged outcomes of TNF or TNF receptor-deﬁcient mice in another (Salomon et al., 2007; Szretter et al., 2007). The latter study also showed no beneﬁcial effects of glucocorticoid treatment of mice but combined antiviral and steroid treatment was not evaluated (Salomon et al., 2007). Observational data in humans showed higher, rather than lower mortality rates in H5N1-infected patients receiving steroids in addition to oseltamivir suggesting there is no role of steroids in the treatment of human H5N1 diseases (Abdel-Ghafar et al., 2008). 4. Conclusion Knowledge gained from clinical observations and experimental research suggest that high viral replication efﬁciency, broad tissue tropism and an intense inﬂammatory response play critical roles in the pathogenesis of H5N1 inﬂuenza. Clinical management should be focused on early and effective suppression of viral replication. The potential role of immunomodulatory intervention remains unclear and requires further unraveling of the molecular and cellular mechanisms underlying H5N1 pathogenesis to afford a rational approach. References Abdel-Ghafar AN, Chotpitayasunondh T, Gao Z, Hayden FG, Nguyen DH, de Jong MD, et al. Update on avian inﬂuenza A (H5N1) virus infection in humans. N Engl J Med 2008;358:261–73. Chan MC, Cheung CY, Chui WH, Tsao SW, Nicholls JM, Chan YO, et al. Proinﬂammatory cytokine responses induced by inﬂuenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells. Respir Res 2005;6:135. Chandrasekaran A, Srinivasan A, Raman R, Viswanathan K, Raguram S, Tumpey TM, et al. Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin. Nat Biotechnol 2008;26:107– 13. Cheung CY, Poon LL, Lau AS, Luk W, Lau YL, Shortridge KF, et al. Induction of proinﬂammatory cytokines in human macrophages by inﬂuenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet 2002;360:1831–7. Coleman JR. The PB1-F2 protein of Inﬂuenza A virus: increasing pathogenicity by disrupting alveolar macrophages. Virol J 2007; 4:9. Conenello GM, Zamarin D, Perrone LA, Tumpey T, Palese P. A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 inﬂuenza A viruses contributes to increased virulence. PLoS Pathog 2007;3:1414– 21. de Jong MD, Hien TT. Avian inﬂuenza A (H5N1). J Clin Virol 2006;35: 2–13. de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, Chau TN, et al. Fatal outcome of human inﬂuenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 2006;12: 1203–7. de Jong MD, Tran TT, Truong HK, Vo MH, Smith GJ, Nguyen VC, et al. Oseltamivir resistance during treatment of inﬂuenza A (H5N1) infection. N Engl J Med 2005;353:2667–72. Deyde VM, Xu X, Bright RA, Shaw M, Smith CB, Zhang Y, et al. Surveillance of resistance to adamantanes among inﬂuenza A(H3N2) and A(H1N1) viruses isolated worldwide. J Infect Dis 2007;196:249–57.
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