Environmental Stability of Swine and Human Pandemic Influenza Viruses in Water under Variable Conditions of Temperature, Salinity, and pH.

UNLABELLED: The movement of influenza A viruses (IAVs) from wild bird reservoirs to domestic animals and humans is well established, but the transmission mechanisms that facilitate efficient movement across and within these host populations are not fully defined. Although predominant routes of transmission vary between host populations, the extent of environmental stability needed for efficient IAV transmission also may vary. Because of this, we hypothesized that virus stability would differ in response to varied host-related transmission mechanisms; if correct, such phenotypic variation might represent a potential marker for the emergence of novel animal or human influenza viruses. Here, the objective was to evaluate the ability of eight swine and six human IAV isolates to remain infective under various pH, temperature, and salinity conditions using a preestablished distilled water system. Swine and human viruses persisted longest at near-neutral pH, at cold temperatures, or under "freshwater" conditions. Additionally, no significant differences in persistence were observed between pandemic and nonpandemic IAVs. Our results indicate that there have been no apparent changes in the environmental stability of the viruses related to host adaptation. IMPORTANCE: This study assessed the environmental stability of eight swine and six human influenza A viruses (IAVs), including viruses associated with the 2009 H1N1 pandemic, in a distilled water system. The important findings of this work are that IAV persistence can be affected by environmental variables and that no marked changes were noted between human and swine IAVs or between pandemic and nonpandemic IAVs.

Optimizing Surveillance for South American Origin Influenza A Viruses Along the United States Gulf Coast Through Genomic Characterization of Isolates from Blue-winged Teal (Anas discors).

Relative to research focused on inter-continental viral exchange between Eurasia and North America, less attention has been directed towards understanding the redistribution of influenza A viruses (IAVs) by wild birds between North America and South America. In this study, we genomically characterized 45 viruses isolated from blue-winged teal (Anas discors) along the Texas and Louisiana Gulf Coast during March of 2012 and 2013, coincident with northward migration of this species from Neotropical wintering areas to breeding grounds in the United States and Canada. No evidence of South American lineage genes was detected in IAVs isolated from blue-winged teal supporting restricted viral gene flow between the United States and southern South America. However, it is plausible that blue-winged teal redistribute IAVs between North American breeding grounds and wintering areas throughout the Neotropics, including northern South America, and that viral gene flow is limited by geographical barriers further south (e.g., the Amazon Basin). Surveillance for the introduction of IAVs from Central America and northern South America into the United States may be further optimized through genomic characterization of viruses resulting from coordinated, concurrent sampling efforts targeting blue-winged teal and sympatric species throughout the Neotropics and along the United States Gulf Coast.

Co-infection of mallards with low-virulence Newcastle disease virus and low-pathogenic avian influenza virus.

Waterfowl are considered the natural reservoir of low-virulence Newcastle disease viruses (loNDVs) and low-pathogenic avian influenza viruses (LPAIVs). The objective of this study was to investigate the effect of co-infections with loNDV and LPAIV on the infectivity and excretion of these viruses in mallards. One-month-old mallards were inoculated intranasally with 10(6) median embryo infectious doses of a wild-bird-origin loNDV and A/Mallard/MN/199106/99 (H3N8) LPAIV on the same day or received the LPAIV 2 or 5 days after loNDV inoculation. All mallards became infected with both viruses based on detection of seroconversion and viral shedding. Co-infection resulted in a higher number of cloacal swabs detected positive for LPAIV and a lower number of cloacal swabs detected positive for loNDV in some groups, although differences between groups were not statistically significant. Co-infection did not affect replication of LPAIV in epithelial cells of the lower intestine and bursa of Fabricius. In summary, the results of this study indicate that co-infection with LPAIV and loNDV does not affect the ability of mallards to be infected with either virus although it may have minimal effects on patterns (source and timing) of viral shedding.

Detection of avian influenza viruses from shorebirds: evaluation of surveillance and testing approaches.

Although influenza A viruses have been isolated from numerous shorebird species (Family: Scolopacidae) worldwide, our understanding of natural history of these viruses in this diverse group is incomplete. Gaining this information can be complicated by sampling difficulties related to live capture, the need for large sample sizes related to a potentially low prevalence of infection, and the need to maintain flexibility in diagnostic approaches related to varied capabilities and resources. To provide information relevant to improving sampling and testing of shorebirds for influenza A viruses, we retrospectively evaluated a combined data set from Delaware Bay, USA, collected from 2000 to 2009. Our results indicate that prevalence trends and subtype diversity can be effectively determined by either direct sampling of birds or indirect sampling of feces; however, the extent of detected subtype diversity is a function of the number of viruses recovered during that year. Even in cases where a large number of viruses are identified, an underestimate of true subtype diversity is likely. Influenza A virus isolation from Ruddy Turnstones can be enhanced by testing both cloacal and tracheal samples, and matrix real-time PCR can be used as an effective screening tool. Serologic testing to target species of interest also has application to shorebird surveillance. Overall, all of the sampling and diagnostic approaches have utility as applied to shorebird surveillance, but all are associated with inherent biases that need to be considered when comparing results from independent studies.

The pathogenesis of low pathogenic avian influenza in mallards.

Mallards are important natural hosts involved in the epidemiology of low pathogenic avian influenza viruses (LPAIVs). LPAIVs are mainly transmitted by a fecal-oral route and are excreted in high concentrations in the feces. We investigated the pathology, viral antigen distribution, and the expression of alpha2,3 sialic acid (SA) influenza virus receptors in mallards after intranasal inoculation with A/Mallard/MN/199106/99 (H3N8) or A/Mallard/MN/355779/00 (H5N2). Gross lesions were not observed. Avian influenza virus (AIV) nucleoprotein (NP) antigen was detected in rare epithelial cells of the larynx and trachea only at 1-day postinoculation (dpi) in the birds infected with H3N8 LPAIV, but infection with either virus was associated with lymphocytic tracheitis and laryngitis on 1 and 2 dpi. AIV NP antigen was detected in enterocytes of the lower intestine from 1 to 4 dpi and in epithelial cells of the bursa of Fabricius from 2 to 3 dpi in birds infected with either virus. Oropharyngeal and cloacal viral shedding was detected from 1 dpi, with higher cloacal viral shedding detected at 2 and 3 dpi with both viruses. Mallards abundantly expressed alpha2,3 sialic acid receptors in epithelial cells of the respiratory tract, lower intestine, and bursa of Fabricius. Some infected birds had decreased alpha2,3 sialic acid expression in epithelial cells of the bursa of Fabricius and in enterocytes of the ceca and colon. In conclusion, the main sites of LPAIV replication in mallards are the enterocytes of the lower intestinal tract and epithelial cells of the bursa of Fabricius in the first days after infection, when these birds are shedding AIV in high titers in the feces.

Effect of different routes of inoculation on infectivity and viral shedding of LPAI viruses in mallards.

We studied the effect of different routes of inoculation on the infectivity and duration of viral shedding in mallards (Anas platyrhynchos) infected with wild bird-origin low pathogenic avian influenza viruses (LPAIVs). One-month-old mallards were inoculated with 10(6) median embryo infectious doses of either A/mallard/MN/199106/99 (H3N8) or A/mallard/MN/355779/00 (H5N2) via 1 of 5 different routes: intranasal (IN), intratracheal (IT), intraocular (IO), intracloacal (IC), or intra-ingluvial (II). Birds in all routes of inoculation groups became infected with LPAIV as detected by virus isolation, real time reverse transcription polymerase chain reaction, and serology. Mallards in different route of inoculation groups had similar viral shedding through oropharynx and cloaca from 1 day postinoculation (dpi). The peak of oropharyngeal (OP) viral shedding was reached between 2 and 3 dpi in all routes of inoculation groups infected with either virus. The peak of cloacal (CL) viral excretion was reached between 2 and 3 dpi in all routes of inoculation groups infected with H3N8 LPAIV and in the IO-, IC-, and II-inoculated groups infected with H5N2 LPAIV, with a delayed and shorter peak for the IN- and IT-inoculated groups. The birds inoculated via the II route had more productive OP and CL viral shedding after infection with either LPAIV, as evidenced by higher number of swabs testing positive over the study period. In conclusion, mallards can be infected with LPAIV by various routes of inoculation, and this corroborates their high susceptibility to infection by these viruses.

The mouse Aire gene: comparative genomic sequencing, gene organization, and expression.

Mutations in the human AIRE gene (hAIRE) result in the development of an autoimmune disease named APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy; OMIM 240300). Previously, we have cloned hAIRE and shown that it codes for a putative transcription-associated factor. Here we report the cloning and characterization of Aire, the murine ortholog of hAIRE. Comparative genomic sequencing revealed that the structure of the AIRE gene is highly conserved between human and mouse. The conceptual proteins share 73% homology and feature the same typical functional domains in both species. RT-PCR analysis detected three splice variant isoforms in various mouse tissues, and interestingly one isoform was conserved in human, suggesting potential biological relevance of this product. In situ hybridization on mouse and human histological sections showed that AIRE expression pattern was mainly restricted to a few cells in the thymus, calling for a tissue-specific function of the gene product.

Relations among mock jurors' attitudes, trial evidence, and their selections of an insanity defense verdict: a path analytic approach.

This study examined an important question relevant to the domain of the insanity defense: What are the interrelationships among important evidential and attitudinal factors which influence how jurors decide their final verdicts? To answer this question, a mock trial in which the insanity defense was argued was presented to 224 college undergraduates by means of an audiotape and slide show. Following the presentation, participants were asked to answer a series of questions regarding the trial. A path model was specified with four evidential factors as endogenous variables, i.e., evaluation of the defendant's mental status, belief that the defendant could be rehabilitated, beliefs regarding the accuracy of the expert witnesses, and mock-jurors' predeliberation verdicts. In addition, three attitudinal factors were specified as exogenous variables, i.e., attitudes toward the insanity defense, attitudes towards due process vs crime control, and attitudes towards the death penalty. The path model was consistent with previous literature, suggesting that jurors' attitudes toward the death penalty and the insanity defense had a direct effect on how they evaluated the accuracy of the expert testimony and their evaluation of the defendant's over-all mental status. In turn, mock jurors' evaluations of the defendant's mental status had a direct effect on their selections of verdict. Importantly, mock jurors' evaluations of the evidential factors, particularly the mental status of the defendant, were a stronger predictor of their selections of verdict than were their initial attitudes.

Alcohol consumption, strength of religious beliefs, and risky sexual behavior in college students.

Relationships among alcohol use, strength of religious convictions, and unsafe sexual practices of 210 students at a large public university in the "bible belt" were examined. The women with strong religious beliefs consumed less alcohol and were less likely to engage in risky sexual behavior than were female participants with weaker religious convictions. Among the men, religious conviction was not significantly correlated with alcohol consumption or risky sexual behavior, but alcohol consumption and inconsistent use of condoms and multiple sexual partners were significantly correlated. Men had higher rates of alcohol consumption and unprotected sexual activity than women did, yet the two groups did not differ in overall frequency of sexual activity. Future research is needed to (a) provide greater understanding of gender differences in alcohol use, risky sexual behavior, and religious beliefs of college students in the region and (b) determine whether similar correlations exist in other areas of the country.

PIP: Relationships between alcohol consumption, strength of religious beliefs, and risky sexual behavior were examined among 210 students at East Carolina University, North Carolina, a large public university in the US's "bible belt." The study sample largely reflected the overall composition of the student body: 61% of the respondents were women and 39% were men; 9% were Black, 86% were White, and 4% were other; and they were aged 18-36 years, of mean age 21 years. 84% reported having had sexual intercourse, with 34% of the entire sample reporting a frequency of 1-3 times per week, and 27% reporting a frequency of 1-2 times per month. 27% reported the consistent use of condoms, 60% reported inconsistent use, and 13% reported never using condoms. 48% of respondents reported having sexual intercourse with multiple partners during the past year. 60% of respondents believed in attending church or attended church on a regular basis, 78% believed that God operated in their daily lives, and 80% believed that they would go to heaven when they died. 66% did not believe that premarital sex was a sin and 77% did not believe that alcohol drinking was a sin. 35% reported being intoxicated more than 5 times in the past month and 33% reported drinking so much alcohol that they passed out at least once during the past month. The women with strong religious beliefs consumed less alcohol and were less likely to engage in risky sexual behavior than were female participants with weaker religious convictions. Among the men, religious conviction was not significantly related to alcohol consumption or risky sex behavior, but the inconsistent use of condoms and having multiple sex partners were significantly positively correlated with alcohol consumption. Men had higher rates of alcohol consumption and unprotected sexual activity than women did, although the two groups did not differ in the overall frequency of sexual activity.

Coreless DC motors for sterilizable medical devices.

Coreless dc motors provide certain advantages when used in medical devices that require repeated sterilization. This article discusses the various sterilization methods used for medical devices, the effects that each method has on coreless dc motors, and ways to prevent damage to the motors during sterilization.

The effect of an in situ-produced oil shale processing water on metabolism.

The effects of in situ-produced oil shale retort water on the metabolism of various substrates was studied both in vivo and in vitro. The induction observed in rats was classified as Type I due to an increase in metabolism of hexobarbital and ethylmorphine without subsequent increases in zoxazolamine metabolism. The maximal absorption of the cytochrome-P450-CO complex was observed to be 450 millimicron, also consistent with Type I inducers. Cytochrome P-450 levels were also significantly increased over controls.

Effects of lipids on the activity of ferrochelatase.

Removal of lipids from submitochondrial particles or detergent-solubilized mitochondrial preparations of rat liver resulted in a 90% loss of ferrochelatase (protochemeferro-lyase, EC 4.99.1.1) activity. The addition of either a fatty acid or phospholipid restored enzyme activity; the extent of reactivation being correlated with the degree of unsaturation of the fatty acid or acyl chain and independent of the polar head group of the phospholipid, Arrhenius plots of the ferrochelatase activities of submitochondrial particles and detergent-solubilized mitochondrial preparations showed transition temperatures of 37 and 28.5 degrees C, respectively. Ferrochelatase of submitochondrial particles or detergent-solubilized preparations had an absolute requirement for Ca2+. The ferrous salt of oxalic acid, a Ca2+ chelator, was a very poor substrate for these preparations. In contrast, ferrochelatase activities of fatty acid- or lipid-supplemented acetone extracts of these preparations were not dependent on the presence of Ca2+ and ferrous oxalate served as substrate for these extracts.20

The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX in mammalian mitochondria.

Protoporphyrinogen oxidase, an enzyme which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells, has been found in several mammalian tissues. It has been extracted from rat liver mitochondria by sonication in the presence of salt and detergent and partially purified. The enzyme is similar in many respects to yeast protoporphyrinogen oxidase. Based on its behavior on Sephadex G-200 the molecular weight of the enzyme is approximately 35,000. Catalysis by protoporphyrinogen oxidase was specific for proteoporphyrinogen IX (apparent Km of 11 muM) and proceeded maximally at pH 8.6 to 8.7. The effect of temperature on enzyme activity plotted according to Arrhenius gave a value of E of 9,100 calories per mol. Enzyme activity was inhibited in the presence of high salt concentrations and temperatures above 45 degrees. Oxygen was essential for protoporphyrinogen oxidase activity and an alternative elevtron acceptor has not yet been found. No requirement for a metal or other cofactor could be demonstrated. The presence of monothiol groups was indicated; however, it is not known whether the thiol groups are involved directly in the binding of substrate to the enzyme.

The regulation of heme biosynthesis.

Changes in the levels of the heme biosynthetic enzymes were studied in cells and protoplasts of Saccharomyces cerevisiae during glucose derepression and respiratory adaptation. On aerobic or anaerobic glucose derepression or in the presence of 3', 5' cyclic AMP the levels of beta-aminolevulinic acid dehydratase, protoporphyrinogen oxidase and ferrochelatase increased whereas the levels of the enzymes catalysing the reactions from porphobilinogen to protoporphyrinogen IX remained constant. In contrast, the level of beta-aminolevulinic acid synthetase decreased during aerobic glucose derepression and in the presence of 3', 5' cyclic AMP, but it remained unchanged during anaerobic derepression. The heme content of the cells increased during aerobic glucose derepression but no increase was observed during anaerobic derepression of the cells. It is concluded that, in yeast, the inhibitory effect of anaerobiosis on heme synthesis is due, at least in part, to the requirement for oxygen in the oxidation of protoporphyrinogen IX, and that the effect of glucose on heme biosynthesis is mediated via glucose repression of protoporphyrinogen oxidase and possibly ferrochelatase.

The enzymic conversion of protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidase activity in mitochondrial extracts of Saccharomyces cerevisiae.

The oxidation of protoporphyrinogen IX to protoporphyrin IX in yeast cells is enzyme-dependent. The enzyme, protoporphyrinogen oxidase, associated with purified mitochondria isolated from Saccharomyces cerevisiae was solubilized by sonic treatment in the presence of detergent and partially purified. The molecular weight of the enzyme was 180,000 plus or minus 18,000. The purified preparation could be stored at -20 degrees in the presence of 20% glycerol for several months without loss of activity. Enzyme activity was destroyed by heating above 40 degrees and by proteolytic digestion and irreversible inactivation occurred outside the pH range of 4.0 to 9.5. The pH optimum of the enzymic reaction was 7.45 and the value of the Michaelis constant was approximately 4.8 muM. Protoporphyrinogen oxidase did not catalyse the oxidation of coproporphyrinogen I or III or uroporphyrinogen I or III and the rate of enzymic oxidation of mesoporphyrinogen IX was less than 20% of that observed with protoporphyrinogen IX. The presence of thiol groups in the enzyme system was indicated but no metal ion or other cofactor requirement was demonstrated. Enzyme activity was insensitive to cyanide, 2,4-dinitrophenol, and azide whereas it was inhibited in the presence of Cu-2+ or Co-2+ ions, high ionic strength, heme, or hemin.