Molecular and Genetic Medicine

Almost all influenza virus proteins are found to contain caspase cleavage motifs. Two caspase cleavage consensus sequences, EXDBY and D/EXXDBY (caspase motifs) were identified in N- and C-terminal regions of influenza virus proteins nucleocapsid NP (positions D16 and D497) and ionic channel M2 (positions D23 and D87). Using reverse genetics with the highly-virulent avian influenza virus A/FPV/Rostock (H7N1), as a vector precursor, these NP and M2 caspase motifs were artificially altered by site-directed mutagenesis and pathogenicity of the generated caspase mutant viruses was tested in chickens. Three main groups of virus mutants were identified. The first group of mutants was characterized by high replication in cells and low virulence in chickens. These virus mutants possessed the altered N-terminal NP and C-terminal M2 caspase motifs. The second group of virus mutants, possessing the altered N-terminal caspase motif of M2, was characterized by attenuated replication in cultured cells and reduced pathogenic properties in chickens. Third, mutations generated in the C-terminus of NP were lethal and restricted virus rescue by reverse genetics, implying a critical role of this caspase site in virus replication. Thus, these data suggested that, (i) caspase motifs in virus proteins play a significant role in virus pathogenicity; (ii) the lack of direct correlation between replication potential and pathogenicity, observed in caspase mutants of the first virus group, implied that virus caspase motifs could affect immunopathogenesis during the infection process, rather than simply controlling virus production in target cells in the chicken host.

Mutations in avian influenza A viral hemagglutinin HA1 domain may alter the binding specificity of HA for F-sialosaccharide receptors, shifting the virus’s host range from birds to humans. The amino acid mutations can occur at the sialoside binding site, as well as the antigenic site, far from the binding site. Thus, a theoretical study involving the in silico prediction of HA-sialosaccharide binding may require quantum chemical analysis of HA1 full domain complexed with sialosides, balancing a computational cost with model size of HA1-sialoside complex. In addition, there is no insight to relationship between the model size of HA1-sialoside complex and its binding energy. In this study, H3 subtype HA1 full domains complexed with avian- and human-type Neu5AcF(2-3 and 2-6)Gal receptor analogs was investigated by ab initio based fragment molecular orbital (FMO) method at the level of second-order Møller–Plesset perturbation (MP2)/6- 31G. Using this approach, we found avian H3 HA1 to bind to avian F2-3 receptor more strongly than to human F2-6 receptor in gas phase, by a value of 15.3-16.5 kcal/mol. This binding benefit was larger than that in the small model complex. Analysis of the interfragment interaction energies (IFIEs) between Neu5Ac-Gal receptor and amino acid residues on the full domain of H3 HA1 also confirmed the higher avian H3-avian F2-3 binding specificity. It was particularly important to evaluate the IFIEs of amino acid residues in a 13Å radius around Neu5Ac-Gal to take account of long-range electrostatic interactions in the larger HA1- sialoside complex model. These results suggest suitable size of HA1-sialoside complex is significant to estimate HA1-sialoside binding energy and IFIE analysis with FMO method.

Avian influenza viruses are considered to be key contributors to the emergence of human influenza pandemics. A major determinant of infection is the presence of virus receptors on susceptible cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid E2,3-galactose (SAE2,3-Gal) linked receptors, whereas human strains bind to sialic acid E2,6-galactose (SAE2,6-Gal) linked receptors. While ducks are the major reservoir for influenza viruses, they are typically resistant to the effects of viral infection, in contrast to the frequently severe disease observed in chickens. In order to understand whether differences in receptors might contribute to this observation, we studied the distribution of influenza receptors in organs of ducks and chickens using lectin histochemistry with linkage specific lectins and receptor binding assays with swine and avian influenza viruses. Although the intestinal epithelial cells of both species expressed only SAE2,3-Gal receptors, we found widespread presence of both SAE2,6-Gal and SAE2,3-Gal receptors in many organs of both chickens and ducks. Co-expression of both receptors may allow infection of cells with both avian and human viruses and so present a route to genetic reassortment. There was a marked difference in the primary receptor type in the trachea of chickens and ducks. In chicken trachea, SAE2,6-Gal was the dominant receptor type whereas in ducks SAE2,3-Gal receptors were most abundant. This suggests that chickens could be more important as an intermediate host for the generation of influenza viruses with increased ability to bind to SAE2,6-Gal receptors and thus greater potential for infection of humans. Chicken tracheal and intestinal epithelial cells also expressed a broader range of SAE2,3-Gal receptors (both G(1-4)GlcNAc and G(1-3)GalNAc subtypes) in contrast to ducks, which suggests that they may be able to support infection with a broader range of avian influenza viruses.

During the past decade, H9N2 low pathogenic avian influenza virus (LPAI) has caused considerable economic loss due to decreased production, increased mortality and the cost of vaccination in Iranian poultry industry. Because of widespread occurrence of this disease and the virus potential to mutate to highlypathogenic (HP) form and transmission to humans, it is, therefore, imperative to understand the pathogenesis and properties of these viruses. In this study, a two step TaqMan real time PCR assay was performed for the quantitation of A/chicken/Iran/772/1998(H9N2) virus in various organs of broiler chickens at different days post inoculation (DPI). Forty 5-week-old commercial broiler chickens were inoculated with the virus. Five chickens were randomly selected on days 1, 3, 6 and 9 PI. Their trachea, lungs, spleen, kidneys, pancreas, blood and faeces were collected for virus detection. A PCR test was performed and the positive samples were used for quantitative real time PCR assay. The result of RT-PCR assay showed the presence of the virus in trachea (40%, 33%), lungs (20%, 66.6%) and spleen (20%, 50%) of infected chickens on days 3 and 6 PI, respectively. The virus was also detected in the kidneys of inoculated chickens on 3 (40%), 6 (60%) and 9 (100%) DPI. In faecal samples the virus was only detected on day 6 PI (83.3%). The molecular quantitation of AIV showed that the AIV titre in the trachea, lungs and spleen of chickens at 3 DPI is lower than the AIV titre at 6 DPI in these organs. The highest titre was observed in the faeces. The AIV titre in all organs of the birds which died at 6 DPI was higher than those of the same organs in the other experimental birds.

Influenza A viruses are highly infectious respiratory pathogens that can infect many species. Birds are the reservoir for all known influenza A subtypes; and novel influenza viruses can emerge from birds and infect mammalian species including humans. Because swine are susceptible to infection with both avian and human influenza viruses, novel reassortant influenza viruses can be generated in this mammalian species by reassortment of influenza viral segments leading to the “mixing vessel” theory. There is no direct evidence that the reassortment events culminating in the 1918, 1957 or 1968 pandemic influenza viruses originated from pigs. Genetic reassortment among avian, human and/or swine influenza virus gene segments has occurred in pigs and some novel reassortant swine viruses have been transmitted to humans. Notably, novel reassortant H2N3 influenza viruses isolated from the US pigs, most likely infected with avian influenza viruses through surface water collected in ponds for cleaning barns and watering animals, had a similar genetic make-up to early isolates (1957) of the H2N2 human pandemic. These novel H2N3 swine viruses were able to cause disease in swine and mice and were infectious and highly transmissible in swine and ferrets without prior adaptation. The preceding example shows that pigs could transmit novel viruses from an avian reservoir to other mammalian species. Importantly, H2 viruses pose a substantial risk to humans because they have been absent from mammalian species since 1968 and people born after 1968 have little preexisting immunity to the H2 subtype. It is difficult to predict which virus will cause the next human pandemic and when that pandemic might begin. Importantly, the establishment and spread of a reassorted mammalian-adapted virus from pigs to humans could happen anywhere in the world. Therefore, both human and veterinary research needs to give more attention to potential cross-species transmission capacity of influenza A viruses.

Influenza A virus (IAV) is one of the most common infectious pathogens in humans and causes considerable morbidity and mortality. The recent spread of highly-pathogenic avian IAV H5N1 viruses has reinforced the importance of pandemic preparedness. In the pathogenesis of IAV infection, cellular proteases play critical roles in the process of viral entry into cells that subsequently leads to tissue damage in the infected organs. Since there are no processing protease for the viral membrane fusion glycoprotein hemagglutinin precursor (HA0) in IAV, entry of the virus into cells is determined primarily by the host cellular HA0 processing proteases that proteolytically activate membrane fusion activity. HA0 of seasonal human IAV has the consensus cleavage site motif Q(E)-T/X-R and is selectively processed by at least seven different trypsintype processing proteases identified to-date in animal model experiments using mouse-adapted IAV or gene expression system in MDCK cells. As is the case for the highly pathogenic avian influenza (HPAI) A virus, endoproteolytic processing of the HA0 occurs through ubiquitous cellular processing proteases, which selectively recognize the multi-basic consensus cleavage site motifs, such as R-X-K/R-R, and K-X-K/R-R. The cleavage enzymes for the R-X-K/R-R motif, but not K-X-K/R-R motif, have been reported to be furin and pro-protein convertase (PC)5/6 in the trans-Golgi network. Here we report new members of type II transmembrane serine proteases of the cell membrane, mosaic serine protease large form (MSPL) and its splice variant TMPRSS13, which recognize and cleave both R-X-K/R-R and K-X-K/R-R motifs without calcium. Furthermore, IAV infection significantly up-regulates a latent ectopic pancreatic trypsin, one of the potent HA processing proteases, and pro-matrix metalloprotease-9, in various organs. These proteases may synergistically damage the blood-brain barrier in the brain and basement membrane of blood vessels in various organs, resulting in severe edema and multiple organ failure. In this review, we discuss these proteases as new drug target molecules for IAV treatment acting by inhibition of IAV multiplication and prevention of multiple organ failure, other than anti-viral agents, viral neuraminidase inhibitors.