Assessing the Viral Fitness of Oseltamivir-Resistant Influenza Viruses in Ferrets,Using a Competitive-Mixtures Model

To determine the relative fitness of oseltamivir-resistant strains compared to susceptible wild-type viruses, we combined mathematical modeling and statistical techniques with a novel in vivo “competitive-mixtures” experimental model. Ferrets were coinfected with either pure populations (100% susceptible wild-type or 100% oseltamivir-resistant mutant virus) or mixed populations of wild-type and oseltamivir-resistant influenza viruses (80%:20%, 50%:50%, and 20%:80%) at equivalent infectivity titers, and the changes in the relative proportions of those two viruses were monitored over the course of the infection during within-host and over host-to-host transmission events in a ferret contact model. Coinfection of ferrets with mixtures of an oseltamivir-resistant R292K mutant A(H3N2) virus and a R292 oseltamivir-susceptible wild-type virus demonstrated that the R292K mutant virus was rapidly outgrown by the R292 wild-type virus in artificially infected donor ferrets and did not transmit to any of the recipient ferrets. The competitive-mixtures model was also used to investigate the fitness of the seasonal A(H1N1) oseltamivir-resistant H274Y mutant and showed that within infected ferrets the H274Y mutant virus was marginally outgrown by the wild-type strain but demonstrated equivalent transmissibility between ferrets. This novel in vivo experimental method and accompanying mathematical analysis provide greater insight into the relative fitness, both within the host and between hosts, of two different influenza virus strains compared to more traditional methods that infect ferrets with only pure populations of viruses. Our statistical inferences are essential for the development of the next generation of mathematical models of the emergence and spread of oseltamivir-resistant influenza in human populations.The neuraminidase (NA) inhibitors are a class of influenza antiviral drugs that are specifically designed to inhibit the enzymatic function of the NA, thereby preventing normal viral replication. Since 1999, two NA inhibitors (NAIs), oseltamivir (Tamiflu) and zanamivir (Relenza), have been shown to be effective for the treatment and prophylaxis of patients infected with not only seasonal influenza, but also highly pathogenic A(H5N1) and the newly emerged A(H1N1) pandemic virus. Prior to 2007, resistance to this class of drugs was considered relatively uncommon, particularly in comparison with the other class of influenza antivirals, the adamantanes, which readily select for viral resistance in treated patients. During early clinical trials, oseltamivir resistance was detected in only 1 to 2% of adults (14) and 5 to 6% of children (33) under treatment, although later studies detected resistance in up to 18% of oseltamivir-treated children (16). In contrast, resistance following zanamivir treatment is rare, with only one reported case observed in an immunocompromised patient (6). Influenza viruses that develop resistance to these drugs typically contain mutations in the NA which, either directly or indirectly, alter the shape of the NA enzymatic site, thereby reducing the ability of the drugs to bind to this specific pocket. One of the most commonly observed mutations in oseltamivir-resistant A(H3N2) viruses is an arginine-to-lysine mutation at residue 292 (R292K) of the NA, while the predominant NA mutation in oseltamivir-resistant A(H1N1) viruses is a histidine-to-tyrosine mutation at residue 274 (H274Y) (N2 NA amino acid numbering, equivalent to residue 275 based on N1 numbering). Both of these mutations have an indirect impact on drug binding, as they affect the ability of the glutamic acid residue at position 276 to reorientate, as required for slow binding by oseltamivir (3). Many mutations that cause NAI resistance also cause reduced NA enzyme activity and, consequently, can compromise viral fitness.Previous studies have demonstrated that viruses with an R292K NA mutation demonstrated compromised growth in vitro (36) and in ferrets were significantly less infectious and did not transmit (9). The replication and transmission fitness of the H274Y mutation has also been studied previously. An H274Y mutant A(H1N1) strain isolated from a patient under oseltamivir treatment demonstrated compromised growth in cell culture compared to a wild-type (WT) virus (13), although a strain carrying the same mutation selected in vitro was found to replicate as well as the wild type (32). The infectivity and transmissibility of an H274Y mutant were found to be restricted in ferrets (13), although a second study demonstrated that transmission of the mutant virus between ferrets was possible, but required a greater viral dose of the mutant compared to the wild type (10). These results suggest that resistant virus variants with the same NA mutation may differ in replication or transmission fitness depending on other viral components. Nevertheless, based on these data and the viral fitness of other resistant mutants, it was believed that NAI-resistant viruses were unlikely to spread throughout the community due to their compromised viral fitness in the absence of drug selective pressure. This was proven incorrect during the Northern Hemisphere 2007-2008 influenza season, when large numbers of oseltamivir-resistant seasonal A(H1N1) viruses with an H274Y mutation were detected in patients who had not been treated with oseltamivir (4, 24). The mutant strain continued to spread to the Southern Hemisphere, such that by late 2008 virtually all circulating seasonal A(H1N1) viruses were oseltamivir resistant (11). The rapid global spread of this strain clearly suggested that the oseltamivir-resistant seasonal A(H1N1) virus had fitness equivalent to or greater than that of the previous oseltamivir-sensitive A(H1N1) strain. The reasons for enhanced viral fitness in this strain, when previous studies demonstrated that the acquisition of an H274Y mutation led to reduced viral fitness, remain unclear but probably involve compensatory mutations or reassortment events which may have improved the hemagglutinin (HA)/NA balance, allowing efficient transmission (5, 26).Experimental methods have been developed to assess the relative fitness of NAI-resistant strains compared with respective wild-type viruses, both in vitro and in vivo. Ferrets have been considered the most appropriate model animal for influenza research, and fitness studies have assessed variables such as minimum dose required to achieve infection, duration of viral shedding, and levels of viral load to allow comparisons between viruses. The guinea pig model has also been previously used to assess the viral fitness of influenza viruses, particularly in comparing the transmissibility of strains via either the contact or aerosol route (2). As an alternative to these traditional approaches, we have investigated a methodology that involves coinfection of ferrets with a mixture of two influenza viruses. Daily monitoring of changes in the relative proportion of those viruses over the course of the infection allows determination of the relative replication fitness of the viruses. Monitoring of recipient ferrets exposed to the infected ferrets enables the relative transmissibility of the viruses (henceforth, the relative transmission fitness) to be determined. In this study, the “competitive-mixtures” methodology was used to assess the relative replication and transmission fitness of an oseltamivir-resistant R292K mutant A(H3N2) virus compared with an oseltamivir-sensitive A(H3N2) wild-type strain and also to asses the relative replication and transmission fitness of an oseltamivir-resistant H274Y seasonal A(H1N1) mutant compared with an oseltamivir-sensitive A(H1N1) wild-type strain. Quantitative estimates for the replication fitness of mutant viruses were determined using a simple mathematical model of within-host viral replication and mixed-effects statistical tests. Transmission fitness was evaluated by application of a graphical technique that demonstrated the relationship between the proportion of mutant virus in the infectee ferrets as a function of the proportion of mutant virus in the infector ferrets.Inferences drawn from the statistical analyses presented here are essential for the refinement of existing mathematical models that simulate the spread of influenza in the human population and model the deployment of antiviral agents. These models are designed to assess the likely impact of different antiviral agent deployment strategies to control pandemic influenza (18, 21, 35). At present, data on the probability of emergence of NAI-resistant strains, the relative transmission fitness of these strains, and the probability of an individual''s infection reverting to an NAI-sensitive strain in the absence of ongoing selective pressure are severely limited. In consequence, human population-level models of influenza spread must make gross assumptions on the likely characteristics of NAI-resistant strains. Data such as those presented here will be used to inform new models of drug deployment and result in improved pandemic policy advice (20, 23).     

Patients with ovarian carcinoma excrete different altered levels of urine CD59, kininogen-1 and fragments of inter-alpha-trypsin inhibitor heavy chain H4 and albumin

Background  

Diagnosis of ovarian carcinoma is in urgent need for new complementary biomarkers for early stage detection. Proteins that are aberrantly excreted in the urine of cancer patients are excellent biomarker candidates for development of new noninvasive protocol for early diagnosis and screening purposes. In the present study, urine samples from patients with ovarian carcinoma were analysed by two-dimensional gel electrophoresis and the profiles generated were compared to those similarly obtained from age-matched cancer negative women.     

Inhibition of Na+-Taurocholate Co-transporting Polypeptide-mediated Bile Acid Transport by Cholestatic Sulfated Progesterone Metabolites

Sulfated progesterone metabolite (P4-S) levels are raised in normal pregnancy and elevated further in intrahepatic cholestasis of pregnancy (ICP), a bile acid-liver disorder of pregnancy. ICP can be complicated by preterm labor and intrauterine death. The impact of P4-S on bile acid uptake was studied using two experimental models of hepatic uptake of bile acids, namely cultured primary human hepatocytes (PHH) and Na⁺-taurocholate co-transporting polypeptide (NTCP)-expressing Xenopus laevis oocytes. Two P4-S compounds, allopregnanolone-sulfate (PM4-S) and epiallopregnanolone-sulfate (PM5-S), reduced [³H]taurocholate (TC) uptake in a dose-dependent manner in PHH, with both Na⁺-dependent and -independent bile acid uptake systems significantly inhibited. PM5-S-mediated inhibition of TC uptake could be reversed by increasing the TC concentration against a fixed PM5-S dose indicating competitive inhibition. Experiments using NTCP-expressing Xenopus oocytes confirmed that PM4-S/PM5-S are capable of competitively inhibiting NTCP-mediated uptake of [³H]TC. Total serum PM4-S + PM5-S levels were measured in non-pregnant and third trimester pregnant women using liquid chromatography-electrospray tandem mass spectrometry and were increased in pregnant women, at levels capable of inhibiting TC uptake. In conclusion, pregnancy levels of P4-S can inhibit Na⁺-dependent and -independent influx of taurocholate in PHH and cause competitive inhibition of NTCP-mediated uptake of taurocholate in Xenopus oocytes.     

Temporal distribution of larval Chironomidae (Diptera) in experimental rice fields in Penang,Malaysia

The distribution and abundance of chironomid larvae were investigated in experimental rice fields at the Bukit Merah Agricultural Experimental Research Station, Penang, Northern Malaysia. The mean density of chironomid larvae during the period of the study (including two cultivation seasons) was 121 larvae m^?2. Chironomus kiiensis (48.6%) was the most abundant species, while four other species, Polypedilum trigonus (4.8%), Tanytarsus formosanus (22.6%), Tanypus punctipennis (13.9%), and Clinotanypus sp. (10.1%) occurred less frequently, although they were present during all wet phases of rice cultivation. Generally, water depth, rice plant height, conductivity, and nitrate–nitrogen content of the water only weakly influenced the abundance of the larvae. Other environmental parameters such as pH, dissolved oxygen, daytime water temperature, total suspended solids, phosphate, and sediment total organic matter did not significantly affect larval populations. The community structure of chironomids in this rice agroecosystem followed the dynamic changes of the field including agronomic practices, patterns of water availability, and phases of rice plant growth.     

Contribution of charged and polar residues for the formation of the E1–E2 heterodimer from Hepatitis C Virus

The transmembrane domains of the envelope glycoprotein E1 and E2 have crucial multifunctional roles in the biogenesis of hepatitis C virus. We have performed molecular dynamics simulations to investigate a structural model of the transmembrane segments of the E1–E2 heterodimer. The simulations support the key role of the Lys370–Asp728 ion pair for mediating the E1–E2 heterodimerization. In comparison to these two residues, the simulation results also reveal the differential effect of the conserved Arg730 residue that has been observed in experimental studies. Furthermore, we discovered the formation of inter-helical hydrogen bonds via Asn367 that stabilize dimer formation. Simulations of single and double mutants further demonstrate the importance of the ion-pair and polar interactions between the interacting helix monomers. The conformation of the E1 fragment in the simulation of the E1–E2 heterodimer is in close agreement with an NMR structure of the E1 transmembrane segment. The proposed model of the E1–E2 heterodimer supports the postulated cooperative insertion of both helices by the translocon complex into the bilayer.     

Bile Acid-Induced Arrhythmia Is Mediated by Muscarinic M2 Receptors in Neonatal Rat Cardiomyocytes

Background

Intrahepatic cholestasis of pregnancy (ICP) is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC), which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM). Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart.

Methods and Results

Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M₂ receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M₂ receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters.

Conclusion

We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M₂ receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.     

Allergenic proteins of natural rubber latex

As the living cytoplasm of laticiferous cells, Hevea brasiliensis latex is a rich blend of organic substances that include a mélange of proteins. A small number of these proteins have given rise to the problem of latex allergy. The salient characteristics of H. brasiliensis latex allergens that are recognized by the International Union of Immunological Societies (IUIS) are reviewed. These are the proteins associated with the rubber particles, the cytosolic C-serum proteins and the B-serum proteins that originate mainly from the lutoids. Procedures for the isolation and purification of latex allergens are discussed, from latex collection in the field to various preparative approaches adopted in the laboratory. As interest in recombinant latex allergens increases, there is a need to validate recombinant proteins to ascertain equivalence with their native counterparts when used in immunological studies, diagnostics, and immunotherapy.     

Complex combination of seed dormancy and seedling development determine emergence of Viburnum tinus (Caprifoliaceae)

BACKGROUND AND AIMS: The shrub Viburnum tinus is widely distributed in mattoral vegetation of the Mediterranean basin. The purpose of the present study was to classify the seed dormancy type and examine the requirements for embryo growth, root protrusion and shoot emergence. METHODS: Overwintered fruits were collected in western Spain in April 2001 and prepared in three ways: entire pericarp was removed, exocarp and mesocarp were removed or fruits were left intact. Fruits treated in these three ways were subjected to artificial annual temperature cycles or to constant temperature regimes for 1.5 years. KEY RESULTS: Removal of exocarp and mesocarp was necessary for embryo growth and germination. High temperature favoured dormancy alleviation and embryo growth, intermediate to low temperatures favoured root protrusion, and intermediate temperature shoot emergence. There was substantial germination at constant temperature regimes, indicating an overlap between temperature intervals suitable for the different stages of embryo and seedling development. Functionally, V. tinus has the same root and shoot emergence pattern that is described for other Viburnum species considered to have epicotyl dormancy. However, the requirement for high and low temperatures for radicle protrusion and epicotyl emergence, respectively, was missing in V. tinus; these characters are the foundation for the epicotyl dormancy classification. CONCLUSIONS: It is concluded that V. tinus does not have epicotyl dormancy. Instead, there is a combination of a weak morphophysiological dormancy and a slow germination process, where different temperatures during an annual cycle favour different development stages. The present study suggests that the first complete seedlings would emerge in the field 1.5 years after fruit maturation in October, i.e. seed dispersal during winter, embryo growth during the first summer, root protrusion and establishment during the second autumn and winter, and cotyledon emergence during the second spring.     

Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage

The nucleocapsids of hepatitis B virus (HBV) are made of 180 or 240 subunits of core proteins or known as core antigens (HBcAg). A fusion bacteriophage bearing the WSFFSNI sequence that interacts tightly to HBcAg was employed as a diagnostic reagent for the detection of the antigen using the phage-enzyme-linked immunosorbent (phage-ELISA), dot blot and immunoprecipitation assays. The results from phage-ELISA and dot blot assay showed that as low as 10 ng of HBcAg can be detected optimally by 1.0x10(12) pfu/ml fusion M13 bacteriophage. The sensitivity of the dot blot assay corresponds with that of the phage-ELISA. HBcAg in HBV positive serum samples can also be detected using the fusion phage via the phage-ELISA and phage-dot blot assay. The phage cross-linked to cyanogen bromide (CNBr) activated agarose can also be used to precipitate HBcAg in bacterial lysate. The optimum amount of phage needed for cross-linking to 1 g of agarose is about 7.0x10(6) pfu/ml which could also precipitate purified and unpurified HBcAg in bacterial lysate. This study demonstrates the potential of fusion bacteriophage bearing the sequence WSFFSNI as a diagnostic reagent and a ligand for the detection and purification of HBcAg respectively.     

African sharptooth catfish Clarias gariepinus does not detoxify ammonia to urea or amino acids but actively excretes ammonia during exposure to environmental ammonia

The African sharptooth catfish Clarias gariepinus lives in freshwater, is an obligatory air breather, and exhibits high tolerance of environmental ammonia. This study aimed at elucidating the strategies adopted by C. gariepinus to defend against ammonia toxicity during ammonia exposure. No carbamoyl phosphate synthetase (CPS) I or III activities were detected in the liver or muscle of the adult C. gariepinus. In addition, activities of other ornithine-urea cycle (OUC) enzymes, especially ornithine transcarbamylase, were low in the liver, indicating that adult C. gariepinus does not have a "functional" hepatic OUC. After being exposed to 50 or 100 mM NH4Cl for 5 d, there was no induction of hepatic OUC enzymes and no accumulation of urea in tissues of the experimental animals. In addition, the rate of urea excretion remained low and unchanged. Hence, ammonia exposure did not induce ureogenesis or ureotely in C. gariepinus as suggested elsewhere for another obligatory air-breathing catfish of the same genus, Clarias batrachus, from India. Surprisingly, the local C. batrachus did not possess any detectable CPS I or III activities in the liver or muscle as had been reported for the Indian counterpart. There were no changes in levels of alanine in the muscle, liver, and plasma of C. gariepinus exposed to 50 or 100 mM NH4Cl for 5 d; neither were there any changes in the glutamine levels in these tissues. Yet even after being exposed to 100 mM NH4Cl for 5 d, there was no significant increase in the level of ammonia in the muscle, which constitutes the bulk of the specimen. In addition, the level of ammonia accumulated in the plasma was relatively low compared to other tropical air-breathing fishes. More importantly, for all NH4Cl concentrations tested (10, 50, or 100 mM), the plasma ammonia level was maintained relatively constant (2.2-2.4 mM). These results suggest that C. gariepinus was able to excrete endogenous ammonia and infiltrated exogenous ammonia against a very steep ammonia gradient. When exposed to freshwater (pH 7.0) with or without 10 mM NH4Cl, C. gariepinus was able to excrete ammonia continuously to the external medium for at least 72 h. This was achieved while the plasma NH4+ and NH3 concentrations were significantly lower than those of the external medium. Diffusion trapping of NH3 through boundary layer acidification can be eliminated as the pH of the external medium became more alkaline instead. These results represent the first report on a freshwater fish (C. gariepinus) adopting active excretion of ammonia (probably NH4+) as a major strategy to defend against ammonia toxicity when exposed to environmental ammonia.