Dr. Emil Ionita
I. Cantacuzino Institute

Virusul gripal H5N1este cunoscut a avea o slaba capacitate de transmitere de la om la om si de a determina o pandemie. Factorii-cheie in determinarea daca virusul gripal A va deveni pandemic sunt: specificitatea de legare a receptorului si infectivitatea virala. Determinantii infectiei si susceptibilitatii umane la virusul H5N1 nu au fost pe deplin elucidati. Lucrarea de fata este o trecere in revista a datelor din literatura de specialitate privind aminoacizii importanti si domeniile proteinelor virale legate de specificitatea de legare a receptorului care au fost raportate atat pentru om, cat si pentru pasari, pe modelele animale.
Cuvinte-cheie: legarea de receptor, transmisia, H5N1, mamifer, Virusul gripal A

H5N1influenza virus is known to have a weak ability for human-human transmission and pandemic infection. The key factors in determining whether influenza A virus becames pandemic are receptor binding specificity and virus infectivity. The determinants of infection and susceptibility of humans to H5N1 virus have not been fully elucidated.
This paper is a review of literature data concerning the important amino acids and domains of viral proteins related to receptor binding specificity that have been reported both for humans and avians using animal models.

Keywords: receptor binding, transmission, H5N1, mammal, A virus influenza


In 1996 in China the highly pathogenic avian influenza (HPAI) H5N1 virus was first isolated from sick geese,[1] and in October 8th, 2013, outbreaks of H5N1 influenza across the globe have resulted in millions of deaths in birds and 377 deaths (mortality rate of 59.3%) in humans. [2]. In 1997 in Hong Kong were first reported cases of H5N1 virus infection in humans., which coincided with an outbreak of the virus in the territory’s chicken farms.[3,4] More than 15 years after this outbreak, other avian H5N1 virus strains have not acquired the level of transmissibility and replication required to cause a human pandemic. In anticipation of this possibility, much attention has been focused on transmission and adaptation of avian H5N1 virus in humans and the potential increase in virulence caused by amino acid mutations in viral proteins.
Recently, the geographic distribution of avian H5N1 virus infection has expanded to Kazakstan, Mongolia, Djibouti, Egypt, Turkey and Russia, indicating that more of the world’s population is at risk.[5] However, the determinants of infection and susceptibility of humans to H5N1 virus have not been fully elucidated. To become a pandemic virus, the viral strain should be able to transmit efficiently between humans, which is determined by factors such as whether the virus grows to high enough titers in the human lungs.[6,7] Fortunately, the current H5N1 virus strains have not acquired such abilities in humans.
However, two recent experimental studies have shown that reassortant H5N1 viruses with four mutations in hemagglutinin (HA) were capable of droplet transmission in a ferret model[8,9]
Therefore, the potential risk does exist for a natural H5N1 virus to evolve into a pandemic virus after continuous circulation between avian and human hosts, which will provide opportunities for the acquisition of the necessary amino acid mutations in viral proteins, particularly HA.
This review of the literature data is focused on studies reporting on the receptor binding of H5N1 virus in humans and other mammals, as well as the amino acid mutations in viral proteins related to transmission of the virus in humans and experimental animals.
In virus binding studies, the attachment of viruses can be directly measured by labeling viruses and then applying them to tissue sections in a method coined virus histochemistry, which has been used to study the pattern of virus attachment in different tissues.
By comparing virus binding and transmission in different animal models, this knowledge will help to elucidate potential transmission routes and will provide the genetic basis for predicting whether a mutated virus strain may evolve into a pandemic virus.
An important aspect of influenza virus infection is the interaction between the viral surface glycoprotein HA and the corresponding receptor on host cells. In order to infect host cells, influenza virus utilizes HA to bind to complex glycans on the host cell surface via a terminal sialic acid (SA).Influenza viruses have different preferences for SAs with different linkages.
For example, human influenza virus prefers sialic acid linked to galactose via an α-2,6 bond (SAα-2,6Gal), whereas avian influenza virus prefers the terminus with sialic acid linked to galactose via an α-2,3 bond (SAα-2,3Gal). SAα-2,6Gal is the major linkage for vicinal galactose in the human upper respiratory epithelium. Epithelial cells in the paranasal sinuses, pharynx, trachea and bronchi mainly express SAα-2,6Gal, which is also expressed in ciliated and goblet cells in the human lung.[10,11]
Apart from SAα-2,6Gal, the human respiratory tract also expresses SAα-2,3Gal on non-ciliated cuboidal bronchiolar cells, which are situated at the junction between the respiratory bronchiole and alveolus.[12,13] Highly pathogenic avian H5N1 virus labeled by fluorescein isothiocyanate (FITC) was shown to preferentially attach to type-II pneumocytes, alveolar macrophages, and non-ciliated cuboidal epithelial cells in the terminal bronchioles of the human lower respiratory tract.[14] The binding of H5N1 virus rarely occurs at the trachea and upper respiratory tract, which is consistent with pathological findings observed at autopsy, such as diffuse alveolar damage, interstitial pneumonia, focal hemorrhage, and bronchiolitis.[15,16]
Some strains of the highly pathogenic H5N1 virus, such as A/Hong Kong/486/97 (HK486) or A/Duck/Hong Kong/200/01, show high binding affinity to SAα-2,3Gal but not SAα-2,6Gal. However, some other strains isolated from patients, such as A/Hong Kong/212/03 and A/Hong Kong/213/03 (HK213), can recognize both SAα-2,3Gal and SAα-2,6Gal.[17,18] These findings implicate the ability of H5N1 virus, which preferentially binds to SAα-2,3Gal, to switch to SAα-2,6Gal if the virus is transmitted and begins to adapt to humans.
However, efficient replication of H5N1 virus can only occur in the lower, rather than upper, respiratory tract, implicating that the virus may not be readily spread by sneezing and coughing.[19] This may account for the inefficient human-to-human transmission of H5N1 virus. The H5N1 virus was also shown to infect nasopharyngeal and oropharyngeal epithelia, which do not express SAα-2,3Gal, suggesting that other binding sites on nasopharyngeal and oropharyngeal epithelia may be able to mediate virus entry.
Among the 16 HA subtypes, only the H1, H2 and H3 subtypes have become adapted to humans. These three virus subtypes caused the pandemics of 1918, 1957, 1968 and 2009. The particular mutations within the HA viral protein of these pandemic viruses, which contributed to the adaptation of avian influenza virus to humans, have been identified. H1 of the 1918 pandemic H1N1 virus switched its binding from SAα-2,3Gal to SAα-2,6Gal via the E190D and G225D mutations (H3 numbering) in HA.[20] The H2N2 and H3N2 pandemic viruses were avian-human reassortant viruses.[20] The 1957 and 1968 pandemic H2 and H3 viruses were able to switch their binding specificity from SAα-2,3Gal to SAα-2,6Gal via two amino acid changes of Q226L and G228S (H3 numbering) within HA. When these mutations were introduced into H5 HA of A/Vietnam/1203/04 (VN1203),22 the E190D and G225D mutations abolished H5 HA binding to all glycans, while the Q226L and G228S mutations afforded H5 HA binding to natural human SAα-2,6Gal. However, a complete shift from SAα-2,3Gal to SAα-2,6Gal binding preference was not observed.
Further studies showed that these two mutations (Q226L and G228S) combined with a loss of glycosylation at 158N26 and the K193R27 mutation could enhance the binding affinity to SAα-2,6Gal. Some other HA mutations in the rHK486 and rVN1203 viruses were shown to result in a binding shift from SAα-2,3Gal to SAα-2,6Gal[21] Although receptor preferences of the viral mutants were changed by HA mutations in this study, almost all virus mutants exhibited attenuated infection efficiency in vitro and in vivo. These studies indicated that switching the binding preference of H5N1 virus from SAα-2,3Gal to SAα-2,6Gal by mutation in HA was possible; however, a single mutation in HA was unable to benefit the H5N1 virus because these artificial virus mutants could not replicate and transmit efficiently, either in vitro or in vivo. In nature, some H5N1 isolates from human patients have acquired the ability to increase their binding to SAα-2,6Gal while maintaining a decreased binding capability for SAα-2,3Gal.[22] These viruses were highly pathogenic, even though efficient transmission was not observed. In addition, the A134V mutation emerged during the course of virus replication in a fatal case of human infection, and this mutation was shown to increase the binding ability to SAα-2,6Gal.[23] Thus, with continual circulation of H5N1 viruses between avian and human hosts, a new pandemic virus may emerge after the virus obtains suitable binding and efficient replication ability.
H5N1 virus can replicate robustly in BALB/c mouse lungs to reach high titers and cause lethal disease without prior adaptation. Studies of FITC-labeled H5N1 virus binding in the lung tissue of mice showed that H5N1 virus mainly binds to the tracheal epithelia and becomes progressively weaker in binding toward the alveoli, a pattern of binding opposite to that of the human respiratory tract. This is consistent with the observation that mouse tracheal epithelia preferentially express SAα-2,3Gal but not SAα-2,6Gal. Thus, the mouse model is suitable for studying the transmission and pathogenicity of influenza viruses that preferentially bind to the receptor via SAα-2,3Gal linkage [23] but is inappropriate for the investigation of H5N1 virus transmission in humans.
The HK213 virus exhibits binding affinity to both SAα-2,3Gal and SAα-2,6Gal. When the HA and NA of this virus were substituted with the HA and NA of VN1203, the recombinant virus demonstrated excellent binding affinity for synthetic α-2,3-linked SA receptor, resulting in expanded organ tropism and increased lethality in mice. In contrast, recombinant VN1203 virus, in which HA was substituted with the HA of HK213, showed reduced lethality and systemic spread in mice.[24] In addition, a human H5N1 virus isolate with the mutation HA222E was mainly confined to the mouse lung, although virus with the HA222K mutation could be isolated from the mouse brain. In particular, the HA222E variant showed a reduced binding affinity for synthetic α-2,3Gal-linked SA receptor analogues compared to that of HA222K.[25] These studies show that HA plays an important role in viral tropism, which can be attributed to the HA binding preference for SAα-2,3Gal- or SAα-2,6Gal-linked receptors as well as the virulence of avian influenza virus in mice. However, because it has been demonstrated that other viral proteins also play a role in viral organ tropism in mice, HA is not the only element affecting viral organ tropism.
Pigs have been traditionally been considered efficient mixing vessels for avian and human influenza A viruses, and both SAα-2,6Gal and SAα-2,3Gal can be detected in the porcine trachea by lectin histochemistry. Moreover, some avian-human reassortant viruses have been isolated from pigs. A recent study showed that only human influenza A virus strains have the ability to attach to the porcine trachea, whereas H5N1 virus cannot attach to the trachea of pigs. One possible reason for this distinction is that the lectin histochemistry technique is only an indirect measure of influenza A virus binding to host tissues and thus cannot account for other variables that may influence binding between the virus and host cells. Intranasal inoculation with H5N1 virus or the consumption of infected chicken meat was unable to cause severe influenza virus infection in pigs. Moreover, replication of H5N1 virus was restricted to the lower respiratory tract, mainly to the bronchiole and alveoli. These results are consistent with the binding study of H5N1 virus in pigs, which showed that H5N1 virus can only bind to the alveolus and not the trachea, bronchus or bronchiole.[26]
Macaques have been studied as a non-human primate for H5N1 virus infection in some experiments, and the pattern of H5N1 virus binding in cynomolgus macaques is similar to the viral binding pattern in humans. However, no H5N1 virus can be detected in the trachea of macaques, essentially because the virus preferentially binds to type I, but not type II, pneumocytes in macaques, which is different from the binding of H5N1 virus in humans. Moreover, the precise cell types to which H5N1 virus can bind in the respiratory tract remain controversial. For instance, it was reported that H5N1 virus could also be detected in the epithelium of the trachea and bronchi of macaques,[27] and these differences may have resulted from the different techniques used in these studies. Indeed, although macaques may provide a model for H5N1 receptor binding and transmission studies, ethical constraints, availability and cost may limit the utility of these animals for infection studies of H5N1 virus.



The currently available literature data show that mutations in HA of H5N1 virus can increase the binding capacity of this viral protein for SAα-2,6Gal. Furthermore, these binding and transmission studies have shown that the binding properties of HA can affect virulence, organ tropism and transmission in mammals. Receptor binding studies of H5N1 virus have suggested that the H5N1 virus preferentially binds to SA via the linkage of SAα-2,3Gal, which is mainly located on type II alveolar epithelial cells and alveolar macrophages in the human lower airway. Therefore, the distribution of SAα-2,3Gal may account for the limited transmission between humans. Due to the circulation of H5N1 virus strains such as HK212, HK213 and DKGX/35, these viruses can recognize both SAs in α-2,3Gal and α-2,6Gal linkages. In addition, certain amino acid substitutions in HA can switch the binding specificity of a virus from SAα-2,3Gal to SAα-2,6Gal, which can affect organ tropism and even enable transmission between ferrets.
In contrast to H5N1 virus, the 2009 H1N1 pandemic virus preferentially binds to the SAα-2,6Gal receptor, although viruses with a D222G mutation in HA switch the viral receptor binding preference from SAα-2,6Gal to SAα-2,3Gal and were frequently observed in the lower respiratory tracts of patients with severe clinical outcomes. Therefore, the binding properties of influenza virus HA to glycan receptors affect interspecies transmission, organ tropism and virulence in the host.
When regions with H5N1 in circulation are continually surveyed, it should be noted if amino acid mutations exist that are related to binding affinity, as this may provide early evidence for the genesis of a pandemic virus and should contribute to future pandemic prevention efforts.



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