Assessment of Pandemic and Seasonal Influenza A (H1N1) Virus Susceptibility to Neuraminidase Inhibitors in Three Enzyme Activity Inhibition Assays

The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC₅₀s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC₅₀s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC₅₀s between the wild type and the variants, whereas the IC₅₀s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.For the treatment and chemoprophylaxis of infections caused by influenza A viruses, the U.S. Food and Drug Administration (FDA) has approved four drugs: amantadine and rimantadine as well as zanamivir and oseltamivir. These drugs belong to two classes, adamantanes (i.e., M2 ion-channel blockers) and neuraminidase (NA) inhibitors (NAIs), respectively. In recent years, the effectiveness of M2 blockers has been greatly compromised, which limits their usefulness in clinical practice. This is largely due to the rapid emergence and widespread circulation of adamantane-resistant influenza viruses (1, 5, 6, 7, 14, 17). More recently, the emergence and worldwide spread of seasonal H1N1 viruses resistant to oseltamivir, currently the most widely used drug against influenza infections, became a considerable public health concern (15, 21, 25, 32). Monitoring the NAI resistance of influenza viruses is an ongoing public health issue since the emergence in 2009 of pandemic viruses that are resistant to M2 blockers.Cell culture-based assays are typically not used for assessment of virus sensitivity to NAIs because of the unpredictable effect of hemagglutinin (HA) receptor binding (2, 34). Instead, drug susceptibility can be monitored by functional (biochemical) NA inhibition (NI) assays, and subsequent genotypic methods are generally required to identify the molecular marker(s) of resistance in the NA. The principle underlying the functional methods relies on the enzymatic nature of the NA, a viral surface glycoprotein and antigen. NA acts by cleaving the terminal neuraminic acid (also called sialic acid) from receptors recognized by influenza viral HA, thus facilitating the release of progeny virions from infected cells and preventing self-aggregation (29). Structurally, NAIs mimic the natural substrate, neuraminic acid, and produce tight interactions, with conserved residues of the NA active site competing with neuraminic acid for binding (11, 23). Preincubation of virus with NAIs leads to the inhibition of enzyme activity, which is detected after the addition of enzyme substrate. Most NI assays commonly used for virus surveillance utilize as substrates small synthetic conjugates that produce either a luminescent or a fluorescent signal upon cleavage by the NA enzyme. The chemiluminescent (CL) assay uses the 1,2-dioxetane derivative of neuraminic acid substrate in the influenza neuraminidase inhibitor resistance detection (NA-Star) kit (8), while the fluorescent (FL) assay employs 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid substrate (MUNANA) (30). The results of the NI assays are expressed as the 50% inhibitory concentration (IC₅₀), which represents the NAI concentration that inhibits 50% of the enzyme activity of the virus. As the NA activity of clinical specimens is usually insufficient for determining the IC₅₀ due to a low viral content, NI assays, using either the substrate provided with the NA-Star kit or the MUNANA substrate, require virus propagation in cell cultures or embryonated chicken eggs. It is noteworthy that IC₅₀s are specific to the virus type/subtype and to the individual NAI tested (8, 19, 20, 24, 32, 37). The IC₅₀s obtained can be used for assessment of virus susceptibility to NAIs, including detection of resistant viruses, as well as for comparing the potencies of antiviral drugs belonging to the NAI class. Although both the CL and FL assays allow reliable detection of NAI resistance, the more recently developed CL assay was reported to be about 70 times more sensitive in detecting NA activity and has a greater linear range than the FL assay (8). The CL assay was also selected for use in the global drug susceptibility surveillance program by the Neuraminidase Inhibitor Susceptibility Network (NISN) (37, 39) and by other surveillance laboratories (28, 32). It should also be noted that IC₅₀s may vary even for the same virus when the NI assay is done using the NA-Star substrate (CL assay) and the MUNANA substrate (FL assay), according to reports on seasonal viruses (37). Whether one of the two assays, the CL or FL assay, more reliably predicts the level of resistance and the drug concentration required for the NA activity inhibition in vivo are key points of interest and remain to be elucidated.A third assay, the colorimetric (CM) assay, which utilizes fetuin as the substrate of the NA, is typically used to determine the titer of anti-NA antibodies because small substrates do not effectively compete with antibodies (3, 31). This assay is not widely used for antiviral susceptibility testing. Unlike the NA-Star and MUNANA synthetic substrates, fetuin is a large, natural, and soluble bovine glycoprotein that contains abundant neuraminic acids at the ends of its oligosaccharide moiety (which include the presence of two residues of α2,3-linked sialic acid and one residue of α2,6-linked sialic acid) (4, 33) and has been used as a substrate in NA-catalyzed reactions (3). Given that NAIs compete with the enzyme substrate for binding to the active site, the structure of the substrate can potentially influence the outcome of the competition and, as a result, the IC₅₀. In this respect, fetuin may represent a better natural substrate for the enzyme-neuraminic acid attached via an α2,3 or α2,6 linkage to oligosaccharide chains on the cell surface. Furthermore, since the cleavage of each neuraminic acid is chemically converted, the CM assay can be a quantifiable method from which the resulting IC₅₀s would correlate more closely to the NA activity of the virus tested. Despite these apparent advantages to the use of fetuin, the CM method relies on chemical reactions that are time-consuming, cumbersome, and impractical for high-throughput use. In addition, the assay requires concentrated virus stocks for testing. Thus, fetuin is still considered an undefined substrate that does not confer sufficient sensitivity or specificity for use in routine NAI susceptibility assays (34). The potential usefulness of a large substrate such as fetuin for assessment of the NAI susceptibilities of novel H1N1 viruses or novel inhibitors remains largely unexplored.Resistance to NAIs is not defined as clearly as that to adamantanes. In NI assays, a drug-resistant virus should have IC₅₀s consistently greater than the threshold value that is determined for each viral type/subtype and drug tested (27, 32, 37). Since the 2007-2008 influenza season, about a decade after the introduction of NAIs into clinical use, an NA framework mutation, H275Y (H274Y in N2 numbering), was consistently and most commonly detected in oseltamivir-resistant H1N1 viruses isolated worldwide (15, 21, 25, 32). Although the H275Y substitution represents the most-defined oseltamivir resistance marker of influenza viruses carrying the NA of the N1 subtype (35), novel NAI resistance-associated mutations—determined by elevated IC₅₀s in NI assays—continue to be revealed (21, 22, 32). Importantly, oseltamivir-resistant viruses from the ongoing H1N1 pandemic have been detected and reported around the world (9, 10, 26, 38). Seasonal and 2009 pandemic H1N1 viruses have the same phylogenetically distant NA gene ancestors (16), which necessitates the comprehensive assessment of the drug susceptibilities of the new pandemic viruses. Therefore, it is necessary to evaluate existing NI assays in order to better understand which assay may be the most sensitive for the detection of NAI resistance and/or the most predictive of virus susceptibility to NAIs in vivo.In the present study, we assessed the susceptibilities of a panel of seasonal and pandemic H1N1 influenza viruses, including virus variants bearing the established oseltamivir resistance mutation, H275Y in the NA, against five NAIs: two FDA-approved NAIs, zanamivir and oseltamivir, and three investigational NAIs, peramivir, R-125489 (the bioactive metabolite of the prodrug CS-8958 [laninamivir]), and A-315675 (a bioactive form of the prodrug A-322278). In order to better characterize and assess the consistency of IC₅₀s and levels of susceptibility, these viruses were tested in the widely used CL and FL assays, as well as with the CM method.     

Computerized hydrodynamic system "Flow" for testing artificial heart valves

The computerized mock circulation system is described for analyzing the hydrodynamic characteristics of artificial heart valves. Flow studies have been carried out on 6 mechanical valves of various types. The volumetric flow, pressure upstream and downstream were measured and the mean systolic pressure drop and energy losses were calculated. The commercial model of the EMIKS valve shows much lower values of pressure drop and one of the smallest value of energy losses among the tested artificial heart valves.     

TREK-1 channels do not mediate nitrergic neurotransmission in circular smooth muscle from the lower oesophageal sphincter

Background and purpose:

The ionic mechanisms underlying nitrergic inhibitory junction potentials (IJPs) in gut smooth muscle remain a matter of debate. Recently, it has been reported that opening of TWIK-related K⁺ channel 1 (TREK-1) K⁺ channels contributes to the nitrergic IJP in colonic smooth muscle. We investigated the effects of TREK-1 channel blockers on nitrergic neurotransmission in mouse and opossum lower oesophageal sphincter (LOS) circular smooth muscle (CSM).

Experimental approach:

The effects of TREK-1 channel blockers were characterized pharmacologically in murine and opossum gut smooth muscle using conventional intracellular and tension recordings.

Key results:

In LOS, L-methionine depolarized the resting membrane potential (RMP) but did not inhibit the nitrergic IJP. Cumulative application of theophylline hyperpolarized the RMP and inhibited the nitrergic IJP concentration dependently. The induced membrane hyperpolarization was prevented by pre-application of caffeine, but not by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. 8-Br-cAMP significantly hyperpolarized membrane potential and increased the amplitude of the nitrergic IJP. In opossum LOS muscle strips, L-methionine increased resting tone but had no effect on nerve-mediated LOS relaxation. On the other hand, theophylline markedly inhibited tone. In CSM from mouse proximal colon, L-methionine caused modest inhibition of nitrergic IJPs.

Conclusions and implications:

TREK-1 channels were not involved in the nitrergic IJP in LOS CSM. Not only does L-methionine have no effect on the nitrergic IJP or LOS relaxation, but the effect of theophylline appears to be due to interruption of Ca²⁺-releasing pathways (i.e. caffeine-like effect) rather than via blockade of TREK-1 channels.     

In Vitro Antiviral Activity of Favipiravir (T-705) against Drug-Resistant Influenza and 2009 A(H1N1) Viruses

Favipiravir (T-705) has previously been shown to have a potent antiviral effect against influenza virus and some other RNA viruses in both cell culture and in animal models. Currently, favipiravir is undergoing clinical evaluation for the treatment of influenza A and B virus infections. In this study, favipiravir was evaluated in vitro for its ability to inhibit the replication of a representative panel of seasonal influenza viruses, the 2009 A(H1N1) strains, and animal viruses with pandemic (pdm) potential (swine triple reassortants, H2N2, H4N2, avian H7N2, and avian H5N1), including viruses which are resistant to the currently licensed anti-influenza drugs. All viruses were tested in a plaque reduction assay with MDCK cells, and a subset was also tested in both yield reduction and focus inhibition (FI) assays. For the majority of viruses tested, favipiravir significantly inhibited plaque formation at 3.2 μM (0.5 μg/ml) (50% effective concentrations [EC₅₀s] of 0.19 to 22.48 μM and 0.03 to 3.53 μg/ml), and for all viruses, with the exception of a single dually resistant 2009 A(H1N1) virus, complete inhibition of plaque formation was seen at 3.2 μM (0.5 μg/ml). Due to the 2009 pandemic and increased drug resistance in circulating seasonal influenza viruses, there is an urgent need for new drugs which target influenza. This study demonstrates that favipiravir inhibits in vitro replication of a wide range of influenza viruses, including those resistant to currently available drugs.In the United States alone, seasonal influenza is responsible annually for infecting between 5 and 20% of the American population, resulting in more than 200,000 hospitalizations and 36,000 deaths (8). Globally, seasonal influenza causes between 250,000 and 500,000 deaths every year (60). Influenza is not only a disease of great medical importance but also of economic importance. Despite available vaccines, a recent study predicted that in the United States influenza results in direct medical costs of the order of $10.4 billion each year, with the total economic burden for the United States being projected at $87.1 billion each year (44). It is widely accepted that vaccination remains the most effective approach for the prevention of viral infections (48). Although there is a safe and effective annual trivalent influenza vaccine, a large proportion of the global population does not receive the yearly influenza vaccine. This can be due to a variety of reasons, including the lack of access to adequate health care, unavailability of vaccine supply, allergies, and adverse reactions. During the 2009 pandemic (pdm), in addition to the vaccination and epidemiological control measures being exerted by health care officials, antivirals targeting influenza offer an essential tool in treating infected patients, in addition to protecting those at high risk of infection, such as the young, elderly, and health care workers.Currently, there are two classes of anti-influenza drugs licensed in the United States for use in the treatment and management of influenza infections in humans: M2 ion channel blockers (also known as adamantanes) and neuraminidase (NA) inhibitors (NAIs) (30). Influenza antivirals are highly effective in the treatment of influenza infections if used promptly following the onset of symptoms or following exposure (45, 46). Both the M2 blockers amantadine and rimantadine are taken by the patient orally (45). However, of the two available NAIs, only oseltamivir is available as an oral formulation (zanamivir has to be inhaled [14, 53]), although other routes of administration have been investigated (31). The use of the M2 blockers amantadine and rimantadine is limited due to the rapid emergence of transmissible drug-resistant mutant viruses and the fact that they offer protection only against influenza A virus infections (32). The high prevalence of adamantane resistance in seasonal A(H3N2) viruses and oseltamivir resistance in seasonal A(H1N1) viruses is reflected in the CDC recommendations for the use of influenza antivirals (6).The majority of adamantane-resistant A(H3N2) and A(H1N1) viruses circulating globally in recent years share the same mutation, S31N, in the M2 protein (20), although other resistance-conferring mutations have been detected also (including A30T, L26F, and V27A) (20, 49). The globally spread oseltamivir-resistant seasonal A(H1N1) viruses share the same mutation, H275Y (H274Y in N2 subtype amino acid numbering), in the drug-targeted enzyme neuraminidase, although other mutations are known to cause reduced susceptibility in vitro (19, 47, 50).Seasonal A(H1N1) viruses resistant to both the adamantanes and the NAI oseltamivir have previously been reported, without an apparent link to treatment (12, 50). Currently, zanamivir is the only drug effective against both adamantane-resistant and/or oseltamivir-resistant influenza viruses, but due to the fact that it has to be inhaled, it is less suitable for use with several high-risk groups, including the severely ill (41), infants (33), and the elderly (22). Furthermore, zanamivir may decrease pulmonary function, so it is not recommended for the treatment of infections in individuals with chronic underlying lung and heart disease conditions (23).Since 1997, there have been several outbreaks of highly pathogenic avian influenza A(H5N1) infections in poultry, with a substantial number of infections occurring in humans (1). The overall case fatality of A(H5N1) infections in humans is over 60% and, unlike seasonal influenza, is most deadly in the young and healthy (ages 10 to 19 years) (59). Oseltamivir is the medication of choice for treating individuals infected with A(H5N1) (17). However, resistance in A(H5N1) viruses has been detected following the treatment of patients with oseltamivir (18, 38). In addition, naturally occurring reduced susceptibility to oseltamivir (35, 40) and possibly to zanamivir (29) has been documented for circulating A(H5N1) viruses, including novel mutations in the NA (29, 35). Adamantane resistance is widely spread among A(H5N1) viruses that carry mutations at amino acid residues 26, 27, and 31 in the M2 protein (13, 35) and among swine viruses circulating in Eurasia (27).In April 2009, a novel reassortant A(H1N1) virus was first identified as circulating in humans in both Mexico and the United States (7, 9). Since April, the virus has continued to transmit among humans, and on 11 June 2009 the World Health Organization classified the outbreak as the first influenza pandemic of the 21st century (58). The 2009 A(H1N1) pandemic viruses consist of a unique combination of gene segments, including those of the North American (triple reassortants) and Eurasian swine lineages (27, 54). The 2009 A(H1N1) pandemic viruses are resistant to the adamantanes and sensitive to the NAIs (3, 16). Yet, concerns exist about the possibility of acquisition of resistance to the NAI oseltamivir, since the majority of A(H1N1) viruses which have been circulating predominantly worldwide during the 2008-2009 influenza season are oseltamivir resistant due to the resistance-conferring H275Y mutation in the NA. Such an acquisition of resistance by the 2009 A(H1N1) pandemic viruses would be a major setback and would further limit the already sparse therapeutic options (15, 57). There have been laboratory-confirmed cases of oseltamivir-resistant 2009 A(H1N1) pandemic viruses (each carrying the H275Y resistance-conferring mutation in the NA) in the United States (5).Collectively, these recent findings emphasize not only the need for new effective antivirals to control and treat influenza infections but also the need to identify new molecular targets (47).One such compound which is currently being investigated and undergoing clinical trials for the treatment of influenza infections is favipiravir (T-705), a pyrazine derivative (2, 26, 31). Favipiravir targets the RNA-dependent RNA polymerase (RdRp), a component of influenza virus different from that of currently licensed influenza antivirals (24, 25). It was shown that favipiravir can inhibit the viral replication of influenza type A, B, and C viruses (24, 25, 55). Favipiravir reduces influenza virus replication by selectively inhibiting the viral RdRp, since it does not affect the synthesis of host cellular DNA and RNA (25). Favipiravir has also shown great potential to act as a broad-spectrum antiviral against many RNA viruses, as reviewed by Furuta and coworkers (26).The purpose of this study was to evaluate the ability, in vitro, of favipiravir to inhibit the viral replication of contemporary influenza viruses as well as viruses with pandemic potential, including viruses resistant to the currently available and licensed anti-influenza drugs. In this report we demonstrate that favipiravir is a potent inhibitor of seasonal influenza A and B virus replication, including that of drug-resistant and drug-sensitive viruses. In addition, favipiravir was shown to effectively inhibit influenza A viruses of other antigenic subtypes, including A(H2N2), viruses of avian origin [A(H4N2), A(H7N2), and A(H5N1)], and viruses of swine origin [A(H1N1) and A(H1N2)], as well as the 2009 A(H1N1) pandemic viruses.     

Critical ovarian hyperstimulation syndrome in a coasted in-vitro fertilization patient

We report an instance of critical ovarian hyperstimulation syndrome in a highly responsive in-vitro fertilization patient despite the preventive measure of a 4 day 'coast' interval during which no gonadotrophins were administered while gonadotrophin-releasing hormone agonist therapy continued until serum oestradiol concentrations fell below 3000 pg/ml.      

Relationship between oxygen anion-radical generation and lipid peroxidation in liver microsomes

Determination of the O₂ consumption and accumulation of malondialdehyde, induced by phenobarbital and 3-methylcholanthrene in rat or rabbit liver microsomes revealed inhibition of lipid peroxidation but a relatively high level of O₂ ^?-radical generation. It is postulated that the absence of direct correlation between lipid peroxidation activity and O₂ ^? generation in microsomes depends on the antioxidant level in the microsomal membrane.     

Prevalence and risk of migraine headaches in adult fragile X premutation carriers

FMR1 premutation carriers are common in the general population (1/130–260 females and 1/250–810 males) and can be affected by fragile X‐associated tremor ataxia syndrome, fragile X‐associated primary ovarian insufficiency, anxiety, depression, hypertension, sleep apnea, fibromyalgia, and hypothyroidism. Here we report the results of a pilot study to assess the prevalence and risk of migraine in FMR1 premutation carriers. Three hundred fifteen carriers (203 females; 112 males) and 154 controls (83 females; 71 males) were seen sequentially as part of a family study. A standardized medical history, physical examination and confirmation of diagnosis of migraine headaches were performed by a physician. The prevalence of migraine was 54.2% in female carriers (mean age/SD: 49.60/13.73) and 26.79% in male carriers (mean age/SD: 59.94/14.27). This prevalence was higher compared to female (25.3%; mean age/SD: 47.60/15.21; p = 0.0001) and male controls (15.5%; mean age/SD; 53.88/13.31; p = 0.0406) who underwent the same protocol and were confirmed to be negative for the FMR1 mutation by DNA testing. We hypothesize that the increased prevalence of migraine headaches in FMR1 premutation carriers is likely related to the mitochondrial abnormalities that have recently been reported. Screening for migraine should be considered when evaluating FMR1 premutation carriers in the future.