Pathogenic assessment of avian influenza viruses in migratory birds.

ABSTRACTSeveral subtypes of avian influenza (AI) viruses have caused human infections in recent years; however, there is a severe knowledge gap regarding the capacity of wild bird viruses to infect mammals. To assess the risk of mammalian infection by AI viruses from their natural reservoirs, a panel of isolates from 34 wild birds was examined in animal models. All selected AI virus subtypes were found to predominantly possess Eurasian lineage, although reassortment with North American lineage AI viruses was also noted in some isolates. When used to infect chickens, 20 AI isolates could be recovered from oropharyngeal swabs at 5 days post-infection (dpi) without causing significant morbidity. Similarly, mild to no observable disease was observed in mice infected with these viruses although the majority replicated efficiently in murine lungs. As expected, wild bird AI isolates were found to recognize avian-like receptors, while a few strains also exhibited detectable human-like receptor binding. Selected strains were further tested in ferrets, and 15 out of 20 were found to shed the virus in the upper respiratory tract until 5 dpi. Overall, we demonstrate that a diversity of low-pathogenic AI viruses carried by wild migratory birds have the capacity to infect land-based poultry and mammalian hosts while causing minimal signs of clinical disease. This study reiterates that there is a significant capacity for interspecies transmission of AI viruses harboured by wild aquatic birds. Thus, these viruses pose a significant threat to human health underscoring the need for continued surveillance.

Influenza Virus with Increased pH of Hemagglutinin Activation Has Improved Replication in Cell Culture but at the Cost of Infectivity in Human Airway Epithelium.

Pandemic H1N1 (pH1N1) influenza virus emerged from swine in 2009 with an adequate capability to infect and transmit between people. In subsequent years, it has circulated as a seasonal virus and evolved further human-adapting mutations. Mutations in the hemagglutinin (HA) stalk that increase pH stability have been associated with human adaptation and airborne transmission of pH1N1 virus. Yet, our understanding of how pH stability impacts virus-host interactions is incomplete. Here, using recombinant viruses with point mutations that alter the pH stability of pH1N1 HA, we found distinct effects on virus phenotypes in different experimental models. Increased pH sensitivity enabled viruses to uncoat in endosomes more efficiently, manifesting as increased replication rate in typical continuous cell cultures under single-cycle conditions. A more acid-labile HA also conferred a small reduction in sensitivity to antiviral therapeutics that act at the pH-sensitive HA fusion step. Conversely, in primary human airway epithelium cultured at the air-liquid interface, increased pH sensitivity attenuated multicycle viral replication by compromising virus survival in the extracellular microenvironment. In a mouse model of influenza pathogenicity, there was an optimum HA activation pH, and viruses with either more- or less-pH-stable HA were less virulent. Opposing pressures inside and outside the host cell that determine pH stability may influence zoonotic potential. The distinct effects that changes in pH stability exert on viral phenotypes underscore the importance of using the most appropriate systems for assessing virus titer and fitness, which has implications for vaccine manufacture, antiviral drug development, and pandemic risk assessment.IMPORTANCE The pH stability of the hemagglutinin surface protein varies between different influenza strains and subtypes and can affect the virus' ability to replicate and transmit. Here, we demonstrate a delicate balance that the virus strikes within and without the target cell. We show that a pH-stable hemagglutinin enables a human influenza virus to replicate more effectively in human airway cells and mouse lungs by facilitating virus survival in the extracellular environment of the upper respiratory tract. Conversely, after entering target cells, being more pH stable confers a relative disadvantage, resulting in less efficient delivery of the viral genome to the host cell nucleus. Since the balance we describe will be affected differently in different host environments, it may restrict a virus' ability to cross species. In addition, our findings imply that different influenza viruses may show variation in how well they are controlled by antiviral strategies targeting pH-dependent steps in the virus replication cycle.

Assessment of Molecular, Antigenic, and Pathological Features of Canine Influenza A(H3N2) Viruses That Emerged in the United States.

Background: A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods: In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results: A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions: Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health.

Risk assessment of recent Egyptian H5N1 influenza viruses.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are enzootic in poultry populations in different parts of the world, and have caused numerous human infections in recent years, particularly in Egypt. However, no sustained human-to-human transmission of these viruses has yet been reported. We tested nine naturally occurring Egyptian H5N1 viruses (isolated in 2014-2015) in ferrets and found that three of them transmitted via respiratory droplets, causing a fatal infection in one of the exposed animals. All isolates were sensitive to neuraminidase inhibitors. However, these viruses were not transmitted via respiratory droplets in three additional transmission experiments in ferrets. Currently, we do not know if the efficiency of transmission is very low or if subtle differences in experimental parameters contributed to these inconsistent results. Nonetheless, our findings heighten concern regarding the pandemic potential of recent Egyptian H5N1 influenza viruses.

Assessment of Antiviral Properties of Peramivir against H7N9 Avian Influenza Virus in an Experimental Mouse Model.

The H7N9 influenza virus causes a severe form of disease in humans. Neuraminidase inhibitors, including oral oseltamivir and injectable peramivir, are the first choices of antiviral treatment for such cases; however, the clinical efficacy of these drugs is questionable. Animal experimental models are essential for understanding the viral replication kinetics under the selective pressure of antiviral agents. This study demonstrates the antiviral activity of peramivir in a mouse model of H7N9 avian influenza virus infection. The data show that repeated administration of peramivir at 30 mg/kg of body weight successfully eradicated the virus from the respiratory tract and extrapulmonary tissues during the acute response, prevented clinical signs of the disease, including neuropathy, and eventually protected mice against lethal H7N9 influenza virus infection. Early treatment with peramivir was found to be associated with better disease outcomes.

Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems.

BACKGROUND: Each year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus' fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen's eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers. METHODS: In this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles. RESULTS: For the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles. CONCLUSIONS: This study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.

Combined quantum mechanics/molecular mechanics (QM/MM) simulations for protein-ligand complexes: free energies of binding of water molecules in influenza neuraminidase.

The applicability of combined quantum mechanics/molecular mechanics (QM/MM) methods for the calculation of absolute binding free energies of conserved water molecules in protein/ligand complexes is demonstrated. Here, we apply QM/MM Monte Carlo simulations to investigate binding of water molecules to influenza neuraminidase. We investigate five different complexes, including those with the drugs oseltamivir and peramivir. We investigate water molecules in two different environments, one more hydrophobic and one hydrophilic. We calculate the free-energy change for perturbation of a QM to MM representation of the bound water molecule. The calculations are performed at the BLYP/aVDZ (QM) and TIP4P (MM) levels of theory, which we have previously demonstrated to be consistent with one another for QM/MM modeling. The results show that the QM to MM perturbation is significant in both environments (greater than 1 kcal mol(-1)) and larger in the more hydrophilic site. Comparison with the same perturbation in bulk water shows that this makes a contribution to binding. The results quantify how electronic polarization differences in different environments affect binding affinity and also demonstrate that extensive, converged QM/MM free-energy simulations, with good levels of QM theory, are now practical for protein/ligand complexes.

Strategies to calculate water binding free energies in protein-ligand complexes.

Water molecules are commonplace in protein binding pockets, where they can typically form a complex between the protein and a ligand or become displaced upon ligand binding. As a result, it is often of great interest to establish both the binding free energy and location of such molecules. Several approaches to predicting the location and affinity of water molecules to proteins have been proposed and utilized in the literature, although it is often unclear which method should be used under what circumstances. We report here a comparison between three such methodologies, Just Add Water Molecules (JAWS), Grand Canonical Monte Carlo (GCMC), and double-decoupling, in the hope of understanding the advantages and limitations of each method when applied to enclosed binding sites. As a result, we have adapted the JAWS scoring procedure, allowing the binding free energies of strongly bound water molecules to be calculated to a high degree of accuracy, requiring significantly less computational effort than more rigorous approaches. The combination of JAWS and GCMC offers a route to a rapid scheme capable of both locating and scoring water molecules for rational drug design.

The effect of the MDCK cell selected neuraminidase D151G mutation on the drug susceptibility assessment of influenza A(H3N2) viruses.

Propagation of influenza A(H3N2) viruses in MDCK cells has been associated with the emergence of neuraminidase (NA) variants carrying a change at residue 151. In this study, the pyrosequencing assay revealed that ∼90% of A(H3N2) virus isolates analyzed (n=150) contained more than one amino acid variant (D/G/N) at position 151. Susceptibilities of the virus isolates to zanamivir and oseltamivir were assessed using the chemiluminescent and fluorescent NA inhibition (NI) assays. In the chemiluminescent assay, which utilizes NA-Star® substrate, up to 13-fold increase in zanamivir-IC50 was detected for isolates containing a high proportion (>50%) of the G151 NA variant. However, an increase in zanamivir-IC50s was not seen in the fluorescent assay, which uses MUNANA as substrate. To investigate this discrepancy, recombinant NAs (rNAs) were prepared and tested in both NI assays. Regardless of the assay used, the zanamivir-IC50 for the rNA G151 was much greater (>1500-fold) than that for rNA D151 wild-type. However, zanamivir resistance conferred by the G151 substitution was masked in preparations containing the D151 NA which had much greater activity, especially against MUNANA. In conclusion, the presence of NA D151G variants in cell culture-grown viruses interferes with drug susceptibility assessment and therefore measures need to be implemented to prevent their emergence.

Discover binding pathways using the sliding binding-box docking approach: application to binding pathways of oseltamivir to avian influenza H5N1 neuraminidase.

Drug binding and unbinding are transient processes which are hardly observed by experiment and difficult to analyze by computational techniques. In this paper, we employed a cost-effective method called "pathway docking" in which molecular docking was used to screen ligand-receptor binding free energy surface to reveal possible paths of ligand approaching protein binding pocket. A case study was applied on oseltamivir, the key drug against influenza a virus. The equilibrium pathways identified by this method are found to be similar to those identified in prior studies using highly expensive computational approaches.

Assessment of the antiviral properties of recombinant porcine SP-D against various influenza A viruses in vitro.

The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.

Assessment of Streptococcus pneumoniae capsule in conjunctivitis and keratitis in vivo neuraminidase activity increases in nonencapsulated pneumococci following conjunctival infection.

PURPOSE: The pneumococcal capsule is required for pathogenesis in systemic infections, yet reports show most conjunctivitis outbreaks are caused by nonencapsulated pneumococci, while keratitis infections are caused by encapsulated strains. This study aims to determine the effect of capsule in pneumococcal keratitis and conjunctivitis in rabbit models of infection. METHODS: A capsule-deficient isogenic mutant was created using homologous transformation. Parent and mutant strains were injected within the upper bulbar conjunctiva (conjunctivitis) or into the corneal stroma (keratitis) of New Zealand white rabbits. Clinical examinations were performed 24 and 48 hr post-infection at which time corneas or conjunctivae were removed, homogenized, and plated to determine the recovered bacterial load. Whole eyes were removed for histological examination. The neuraminidase activity was determined following in vitro and in vivo growth. RESULTS: There were no significant differences in clinical scores between the eyes infected with the parent or mutant for either infection, nor was there a difference in the amount of bacteria recovered from the cornea. In the conjunctivae, however, the mutant strain was cleared by the host faster than the parent strain. Histological examination showed slightly more infiltrating polymorphonuclear leukocytes (PMN) and macrophages in the conjunctivae infected with the parent strain. The neuraminidase activity of both strains was not significantly different when the strains were grown in vitro. However, the neuraminidase activity of the parent was significantly less than that of the mutant at 3 and 12 hr post conjunctival infection. CONCLUSIONS: Although more outbreaks of pneumococcal conjunctivitis are tied to nonencapsulated S. pneumoniae strains, this study showed that an encapsulated strain was capable of establishing conjunctivitis in a rabbit injection model and survive attack by the host immune system longer than its nonencapsulated isogenic mutant. Nonetheless, the nonencapsulated pneumococci had an increased neuraminidase activity level in vivo when compared to the parent strain.

In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity.

The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, alpha2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to alpha2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with alpha2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.

Five novel mutations in the lysosomal sialidase gene (NEU1) in type II sialidosis patients and assessment of their impact on enzyme activity and intracellular targeting using adenovirus-mediated expression.

Sialidosis is an autosomal recessive disease resulting from a deficiency of lysosomal sialidase. Type II sialidosis is a rare disease characterized clinically by hydrops fetalis, hepatosplenomegaly, and severe psychomotor retardation. Genomic DNA from four unrelated sialidosis patients was screened for mutations within the sialidase gene NEU1. Five novel mutations were identified. Four are missense and one is nonsense: c.674G>C (p.R225P), c.893C>T (p.A298V), c.3G>A (p.M1?), c.941C>G (p.R341G), and c.69G>A (p.W23X). We have used our findings and diagnostic tools to confirm the presence of a homozygous null allele in a neonate sibling. Recombinant adenoviruses expressing the mutant sialidase alleles in primary cell cultures were utilized to assess the impact of each mutation on enzyme activity and intracellular localization. None of the mutant alleles expressed significant enzymatic activity. The p.R341G mutation exerts its pathological effect by perturbing substrate binding, while the p.A298V and p.R225P mutations appear to impair the folding of the sialidase enzyme. Our findings point to mutation-sensitive amino acids involved in catalytic function or structural stability and indicate the potential utility of these mutations for molecular diagnosis of this rare disease.

Exoglycosidase purity and linkage specificity: assessment using oligosaccharide substrates and high-pH anion-exchange chromatography with pulsed amperometric detection.

Simplified HPLC protocols to determine the activity and linkage specificity and to detect the most commonly-encountered contaminants in available exoglycosidase preparations (Jacob and Scudder, Methods Enzymol., 230, 280-300, 1994) were developed. Monosaccharides and oligosaccharides were analyzed in a single chromatographic step using high-pH anion-exchange chromatography with pulsed amperometric detection. All analyses were performed with underivatized oligosaccharide substrates and by direct injection of unprocessed, diluted enzyme digests into the chromatograph. The sialidase from Newcastle disease virus was found to release both alpha (2-->3)- and alpha (2-->6)-linked Neu5Ac from a triantennary, lactosamine-type oligosaccharide. The activity of alpha-galactosidase from green coffee beans was assayed using Gal alpha(1-->3)[Fuc-alpha(1ar2)]Gal by detection of Gal and Fuc alpha(1-->3)Gal. The linkage specificities of beta-galactosidases from Streptococcus pneumoniae and bovine testis were assessed using Gal beta(1-->3 or 4)GlcNAc beta(1-->3)beta(1-->4)Glc as substrates. Contaminating beta-N-acetylhexosaminidase activity in the beta-galactosidase preparation was assayed using an agalactobiantennary oligosaccharide. The alpha(1-->3 or 4) linkage specificity of fucosidase III from almond meal was confirmed (Scudder et al., J. Biol. Chem. 265, 16472-16477, 1990) by its inactivity against a biantennary oligosaccharide with all Fuc residues linked alpha(1-->6). An alpha-fucosidase from chicken liver was found to cleave alpha(1-->2,3 or 6)-linked Fuc residues from oligosaccharides. The activity of jack bean (Canavalia ensiformis) alpha-mannosidase was assayed with a relatively resistant substrate, Man alpha(1-->3)- Man beta(1-->4)GlcNAc. A GlcNAc beta(1-->4)-terminated triantennary oligosaccharide was used to assay for contaminating beta-N-acetylhexosaminidase activity in alpha-mannosidase preparations and to determine the linkage and branch specificity of beta-N-acetylhexosaminidase at different enzyme concentrations.

Epididymal and testicular enzymes as monitors for assessment of male antifertility drugs.

PIP: Since the synthesis and maturational processes of sperm are associated with characteristic alterations in different marker enzyme activities in testes and epididymis, it is possible to monitor these enzymes to investigate whether 3 antispermatogenic agents, WIN 18 446, alpha-chlorohydrin (AC), and cyproterone acetate (CA), have any characteristic effects on biochemical events associated with spermatogenesis and maturation of sperm; acid, neutral, and alkaline proteinases, particulate and soluble arylamidases, and sialidase were studied after treatment with 1 of the 3 agents in male albino rats (CIBA strain) by measuring these enzyme levels in rat testicular and epididymal tissues. After WIN treatment, sialidase activity as well as sialic acid content increased in the testis, whereas no such effect occurred in the epididymis at any dose. At low dose, AC (10 mg/kg/day for 7 days) and CA (50 mg/kg/day for 10 days) decreased the sialic acid content and the sialidase activity in the epididymis, whereas the sialic acid and sialidase activity in the testis remained unchanged. At higher dose levels, AC (25 mg) and CA (50 mg) both affected the tissues significantly, i.e., enhancing sialidase activity and lowering sialic acid content. Therefore, the effect of CA and AC is more prominent on the maturational phenomena than the testicular spermatogenesis. AC and CA deserve further investigation for use as a male contraceptive. The relationship between proteinase, sialidase, and arylamidase activities and different phases of spermatogenesis and maturation was established by these test results.^ieng