THE ROLE OF AVIAN AND HUMAN INFLUENZA VIRUS RECEPTORS IN RELATION TO THE TRANSMISSABILITY OF THE H5N1 VIRUS
Virusul gripal aviar H5N1 este raspunzator de gripa aviara – o boala infectioasa fatala care poate determina potential o pandemie devastatoare, daca virusul H5N1 sufera o mutatie in forma diseminarii eficiente in populatia umana. Descoperirile recente au condus la intelegerea de baza a histopatologiei celulare si de organ cauzate de virusul H5N1. Aceasta lucrare este un review al datelor din literatura a patologiei virusului aviar H5N1 raportat postmortem si in studiile clinice raportate la mecanismele-cheie patogenice. Dereglarile in sinteza citokinelor si chemokinelor poate fi unul din mecanismele-cheie implicate in patogeneza gripei aviare cu H5N1. Se va face, de asemenea, trecerea in revista a diferitilor determinanti moleculari inalt patogeni care au fost identificati in ultimii ani si rolul receptorilor virusurilor gripale aviare si umane in relatie cu transmisibilitatea virusului H5N1. O apreciere comprehensiva a mecanismelor patogenice ale virusului aviar H5N1 este necesara in elaborarea strategiilor efective pentru prevenirea, diagnosticul si tratamentul acestei patologii cu grad de urgenta ridicat.
Cuvinte cheie: virusul gripal aviar H5N1, virusul uman gripal, mecanisme patogenice, receptori virali
H5N1 avian influenza is a highly fatal infectious disease that could cause a potentially devastating pandemic if the H5N1 virus mutates into a form that spreads efficiently among humans. Recent findings have led to a basic understanding of cell and organ
histopathology caused by the H5N1 virus.
This is a review of literature data of the pathology of H5N1 avian influenza reported in postmortem and clinical studies and discuss the key pathogenetic mechanisms. Dysregulation of cytokines and chemokines is likely to be one of the key mechanisms in the pathogenesis of H5N1 influenza.
We also review the various molecular determinants of increased pathogenicity
that have been identified in recent years and the role of avian and human influenza virus receptors in relation to the transmissibility of the H5N1 virus.
A comprehensive appreciation of H5N1 influenza pathogenetic mechanisms should aid in the design of effective strategies for prevention, diagnosis, and treatment of this emerging disease.
Key words: H5N1 avian influenza virus, human influenza virus, pathogenic mechanisms, virus receptors
Human and avian viruses bind to different receptors. The HA protein of avian influenza viruses preferentially binds to sialic acids linked to galactose by _-2,3 linkage (avian
influenza virus receptors), which are located on the intestinal epithelial cells of avians. In contrast, the HA protein of human influenza viruses primarily recognizes _-2,6-linked sialic acids (human influenza virus receptors),which are notably expressed on epithelial cells of the human trachea.[1–5]. Because of these differences in receptor specificity and distribution, as well as the limited replication in humans, avian influenza viruses were initially thought to be incapable of causing human infection.
However, this presumption was proved incorrect when in 1997 the H5N1 avian influenza virus infected and killed several humans. Since then a number of studies have been performed aiming to explain the ability of avian influenza viruses to infect humans.
Human influenza virus receptors are mainly expressed in the upper respiratory tract, whereas avian influenza virus receptors are primarily expressed in the lower respiratory tract (type II alveolar cells) [6,7,8] However, avian influenza virus
receptors have also been detected on epithelial cells in human tracheobronchial cell cultures and in human tissue sections of trachea and bronchi, albeit to a lesser
extent than human influenza virus receptors[.9]
This may explain the capability of the virus to infect humans. In addition, H5N1 viruses are capable of infecting ex vivo nasopharyngeal tissues, despite a limited number of avian influenza virus receptors detected.Therefore, it has been suggested that the H5N1 virus may also use alternative binding sites on the epithelium to enter target cells.
Conflicting results have been reported as to the cell type expressing avian influenza virus receptors in the trachea and bronchi. Some studies have found such receptors to be located mainly on basal cells or only on goblet cells, whereas others have detected their presence primarily on ciliated cells– and only on a small proportion of nonciliated cells, including basal cells. In tracheobronchial cell cultures avian influenza viruses have been found to infect mainly ciliated cells. Alveolar macrophages appear to have none or very few avian influenza virus receptors.
The receptor distribution in extra-pulmonary tissuesas been less extensively studied. Thus far neurons and the epithelial cells of the pancreatic and bile ducts have been found to express avian influenza receptors. [14,15] In addition, avian influenza virus receptors have been detected on endothelial cells in many organs throughoutthe body. Some studies have reported the presence of avian influenza virus receptors on the epithelial cells ofthe intestinal mucosa, [16,17] whereas others did not find their presence on such cells. 
With respect to immune cells, avian influenza virus receptors have been detected on T cells87 and Kupffer cells of the liver.  The receptor distribution pattern as detected by lectin histochemistry broadly resembles that of infected organs and cells as demonstrated by in situ hybridization.
However, the abundant expression of avian influenza virus receptors found on the endothelial cells of various organs contrasts with the absence of virus in these cells. In addition, the widespread and abundant expression of avian influenza virus receptors in the lungs is not in line with the limited number of infected pneumocytes, as detected by in situ hybridization/ IHC.7 At the same time the absence of avian influenza virus receptors on placental macrophages, alveolar macrophages, cytotrophoblasts, and intestinal epithelial cells is inconsistent with the detected infectionof such cells.
These discrepancies further support the assumption that other receptors, co-receptors, or mechanisms may play a role in the interaction between the virus and its target cells, thus warranting further investigation.
Receptor Switch and Human-to-Human Transmission
Most avian and human H5N1 isolates only bind to avian influenza virus receptors. Only a limited number of H5N1 human isolates have been identified that are capable of binding to human influenza virus receptors in vitro. It is thought that for efficient human-tohuman transmission the HA protein of influenza viruses should preferentially recognize human influenza virus receptors.
Previous studies with H1, H2, and H3 serotypes have demonstrated that minor mutations in the HA gene may cause receptor specificity to switch from recognizing avian influenza virus receptors to recognizing human influenza virus receptors. Similar mutations have been introduced on the framework of an A/Vietnam/2004 H5N1 virus.
Mutations enabling H1 serotypes to recognize human receptors applied to H5N1 virus affect its affinity for avian receptors but do not result in human receptor specificity. In contrast, mutations enabling H2 and H3 receptors to recognize human receptors applied to H5N1 virus resulted in significant binding of the mutant virus to a natural, branched _-
2,6-linked biantennary N-linked glycan and in a reduced binding to _-2,3-linked SA receptors. Viruses with these properties would be able to evade the virus-neutralizing effects of mucins containing _-2,3-linked SAs and bind more avidly to lung epithelial cells expressing _-2,6-linked biantennary N-linked glycans. Yamada and colleagues have recently provided further insight in possible mutations affecting the ability of the H5N1 virus to bind to human receptors.
By performing reverse genetics studies and crystal structure determination, they have identified two mutations at position 182 and 192 of HA that enhance binding of H5N1 viruses to human influenza virus receptors. Despite the capability of a number of human H5N1 isolates to bind to human influenza virus receptors in vitro, these isolates do not spread efficiently from human-to-human in vivo.
Animal experiments with reassortant viruses have also shown that the mere acquisition of human influenza surface proteins does not necessarily confer transmissibility of H5N1 virus.  Inoculation with a reassortant virus containing genes for internal proteins of H5N1 A/Hk/486 and for surface proteins of human H3N2 A/vic/75 did not result in efficient transmission among ferrets in a respiratory droplet experimental design, even though viral replication was not compromised. In addition, four H5N1 human isolates(A/Vn/1203/04, A/Vn/JP36-2/05, A/Hk/213/03, and A/Turkey/65-596/06), including isolates capable of binding to human influenza virus receptors (A/Hk/213/03 and A/Turkey/65-596/06), have been studied in a direct contact model of ferrets.35 No transmission of either H5N1 A/Vn/1203/04 or A/Turkey/65-596/06 virus was detected.
Although transmission of both H5N1 A/Hk/213/03 and A/Vn/JP36-2/05 viruses occurred, it appeared to be far from efficient. No secondary transmission from an infected contact ferret to a na?ve contact ferret was demonstrated.
On the basis hereof it appears that increased binding affinity for human influenza receptors alone is not sufficient for efficient transmission. Additional molecular determinants seem to be required. It has been suggested that certain biological properties such as the capacity to induce virus excretion from the upper respiratory tract (coughing and sneezing) may enhance efficient transmission.
As mentioned above, the presence of lysine at position 627 is a molecular determinant associated with such a capacity and may, therefore, contribute to efficient spread among humans. However, it seems likely that various additional amino acid changes are required to give avian influenza viruses the capacity to spread among humans..
In fact for the 1918 H1N1 influenza virus, a human influenza virus that is supposed to be derived solely from an avian source, various amino acid changes differentiate the human isolate from its avian consensus sequences.
Implications of Receptors
The widespread distribution of avian influenza virus receptors in various organs may explain the multiple organ involvement seen in H5N1-infected humans. However, there are a number of discrepancies between the cell types expressing avian influenza receptors and the cell types found to be infected by the H5N1 virus.
Despite the relative lack of avian influenza virus receptors, viral replication has been demonstrated in the upper respiratory tract. Therefore, further research is needed to investigate the possible role of other receptors or mechanisms involved in the interaction between the virus and its target cells. The identification of other receptors could help the
design of effective drugs treating H5N1 infection or preventing transmission. Recent studies have shown that not only the acquisition of the capacity to bind to human
influenza virus receptors but also other genetic changes may be necessary for efficient transmission among humans.
Continuous surveillance of the circulating H5N1 strains is of key importance as the emergence of amino acid substitutions similar to those demonstrated for the 1918 H1N1 virus might indicate that the virus could acquire pandemic potential in the near future.
Since 1997 several studies have contributed to fundamental insights into the pathology and pathogenesis of human H5N1 influenza. Aside from the respiratory tract,other organs such as the intestines, the brain, and the placenta appear to be infection targets of the virus. The H5N1 virus is also capable of transplacental transmission to the fetus.
Dysfunction of the immune system may be a key pathogenetic mechanism. At the molecular level,several viral genes and mutations in gene products have been suggested to be involved in increased virulence of H5N1 viruses.
At the same time, however, it becomes increasingly apparent that what is known today about the virus and its pathogenicity is only the tip of the iceberg and that there are likely several additional pathogenetic mechanisms and molecular determinants of pathogenicity in H5N1 influenza yet to be identified
In light of the subsisting threat of a potentially devastating influenza pandemic, further investigations in these respects are urgently required.
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