Role of Permissive Neuraminidase Mutations in Influenza A/Brisbane/59/2007-like (H1N1) Viruses

Neuraminidase (NA) mutations conferring resistance to NA inhibitors were believed to compromise influenza virus fitness. Unexpectedly, an oseltamivir-resistant A/Brisbane/59/2007 (Bris07)-like H1N1 H275Y NA variant emerged in 2007 and completely replaced the wild-type (WT) strain in 2008–2009. The NA of such variant contained additional NA changes (R222Q, V234M and D344N) that potentially counteracted the detrimental effect of the H275Y mutation on viral fitness. Here, we rescued a recombinant Bris07-like WT virus and 4 NA mutants/revertants (H275Y, H275Y/Q222R, H275Y/M234V and H275Y/N344D) and characterized them in vitro and in ferrets. A fluorometric-based NA assay was used to determine Vmax and Km values. Replicative capacities were evaluated by yield assays in ST6Gal1-MDCK cells. Recombinant NA proteins were expressed in 293T cells and surface NA activity was determined. Infectivity and contact transmission experiments were evaluated for the WT, H275Y and H275Y/Q222R recombinants in ferrets. The H275Y mutation did not significantly alter Km and Vmax values compared to WT. The H275Y/N344D mutant had a reduced affinity (Km of 50 vs 12 µM) whereas the H275Y/M234V mutant had a reduced activity (22 vs 28 U/sec). In contrast, the H275Y/Q222R mutant showed a significant decrease of both affinity (40 µM) and activity (7 U/sec). The WT, H275Y, H275Y/M234V and H275Y/N344D recombinants had comparable replicative capacities contrasting with H275Y/Q222R mutant whose viral titers were significantly reduced. All studied mutations reduced the cell surface NA activity compared to WT with the maximum reduction being obtained for the H275Y/Q222R mutant. Comparable infectivity and transmissibility were seen between the WT and the H275Y mutant in ferrets whereas the H275Y/Q222R mutant was associated with significantly lower lung viral titers. In conclusion, the Q222R reversion mutation compromised Bris07-like H1N1 virus in vitro and in vivo. Thus, the R222Q NA mutation present in the WT virus may have facilitated the emergence of NAI-resistant Bris07 variants

Are soil carbon credits empty promises? Shortcomings of current soil carbon quantification methodologies and improvement avenues

As the consequences of climate change are looming large, agricultural soil carbon credits have emerged as an increasingly advocated lever to incentivize the reduction of greenhouse gas emissions and promote carbon storing farming practices. These credits are exchanged on self-regulated voluntary carbon markets, each of them using distinct protocols to assess the changes in soil carbon stocks and convert them into carbon credits. Although serious discrepancies between protocols have already been noted regarding general carbon credit accounting principles, an in-depth evaluation of how changes in soil organic carbon stocks are calculated is still lacking. In this context, the primary objective of our study was to investigate how changes in soil organic carbon stock are estimated by the major carbon credit protocols worldwide. We evaluated the requirements of each protocol regarding the estimation of the initial SOC stock as well as the modelling and/or measurement of changes in stock with time. We found that existing protocols vary greatly in their scientific rigour. We showed in particular that some protocols do not require in situ soil analyses to estimate initial soil carbon stocks but rely on regional values, leading them to potentially overestimate these stocks by up to 2.5 times. Our study also found that the protocols relying on models require different farming practices and different levels of information for each practice to estimate SOC stock changes. The protocols relying, at least partly, on soil sampling also displayed different requirements for the sampling design, sampling tools, SOC analysis methods and SOC stock calculation methods. On this basis, we suggest reforms designed to improve and standardize the quantification of carbon stock changes in soils and to improve the reliability of soil carbon credits

E119D Neuraminidase Mutation Conferring Pan-Resistance to Neuraminidase Inhibitors in an A(H1N1)pdm09 Isolate From a Stem-Cell Transplant Recipient

Background. An influenza A(H1N1)pdm09 infection was diagnosed in a hematopoietic stem cell transplant recipient during conditioning regimen. He was treated with oral oseltamivir, later combined with intravenous zanamivir. The H275Y neuraminidase (NA) mutation was first detected, and an E119D NA mutation was identified during zanamivir therapy. Methods. Recombinant wild-type (WT) E119D and E119D/H275Y A(H1N1)pdm09 NA variants were generated by reverse genetics. Susceptibility to NA inhibitors (NAIs) was evaluated with a fluorometric assay using the 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) substrate. Susceptibility to favipiravir (T-705) was assessed using plaque reduction assays. The NA affinity and velocity values were determined with NA enzymatic studies. Results. We identified an influenza A(H1N1)pdm09 E119D mutant that exhibited a marked increase in the 50% inhibitory concentrations against all tested NAIs (827-, 25-, 286-, and 702-fold for zanamivir, oseltamivir, peramivir, and laninamivir, respectively). The double E119D/H275Y mutation further increased oseltamivir and peramivir 50% inhibitory concentrations by 790- and >5000-fold, respectively, compared with the WT. The mutant viruses remained susceptible to favipiravir. The NA affinity and velocity values of the E119D variant decreased by 8.1-fold and 4.5-fold, respectively, compared with the WT. Conclusions. The actual emergence of a single NA mutation conferring pan-NAI resistance in the clinical setting reinforces the pressing need to develop new anti-influenza strategie

Coastal Subsistence, Maritime Trade, and the Colonization of Small Offshore Islands in Eastern African Prehistory

Recent archaeological research has firmly established eastern Africa's offshore islands as important localities for understanding the region's pre-Swahili maritime adaptations and early Indian Ocean trade connections. While the importance of the sea and small offshore islands to the development of urbanized and mercantile Swahili societies has long been recognized, the formative stages of island colonization—and in particular the processes by which migrating Iron Age groups essentially became “maritime”—are still relatively poorly understood. Here we present the results of recent archaeological fieldwork in the Mafia Archipelago, which aims to understand these early adaptations and situate them within a longer-term trajectory of island settlement and pre-Swahili cultural developments. We focus on the results of zooarchaeological, archaeobotanical, and material culture studies relating to early subsistence and trade on this island to explore the changing significance of marine resources to the local economy. We also discuss the implications of these maritime adaptations for the development of local and long-distance Indian Ocean trade networks

Randomized Controlled Ferret Study to Assess the Direct Impact of 2008–09 Trivalent Inactivated Influenza Vaccine on A(H1N1)pdm09 Disease Risk

During spring-summer 2009, several observational studies from Canada showed increased risk of medically-attended, laboratory-confirmed A(H1N1)pdm09 illness among prior recipients of 2008–09 trivalent inactivated influenza vaccine (TIV). Explanatory hypotheses included direct and indirect vaccine effects. In a randomized placebo-controlled ferret study, we tested whether prior receipt of 2008–09 TIV may have directly influenced A(H1N1)pdm09 illness. Thirty-two ferrets (16/group) received 0.5 mL intra-muscular injections of the Canadian-manufactured, commercially-available, non-adjuvanted, split 2008–09 Fluviral or PBS placebo on days 0 and 28. On day 49 all animals were challenged (Ch0) with A(H1N1)pdm09. Four ferrets per group were randomly selected for sacrifice at day 5 post-challenge (Ch+5) and the rest followed until Ch+14. Sera were tested for antibody to vaccine antigens and A(H1N1)pdm09 by hemagglutination inhibition (HI), microneutralization (MN), nucleoprotein-based ELISA and HA1-based microarray assays. Clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. Baseline characteristics were similar between the two groups of influenza-naïve animals. Antibody rise to vaccine antigens was evident by ELISA and HA1-based microarray but not by HI or MN assays; virus challenge raised antibody to A(H1N1)pdm09 by all assays in both groups. Beginning at Ch+2, vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at Ch+5 (7.4% vs. 5.2%; p = 0.01). At Ch+5 vaccinated animals had higher lung virus titers (log-mean 4.96 vs. 4.23pfu/mL, respectively; p = 0.01), lung inflammatory scores (5.8 vs. 2.1, respectively; p = 0.051) and cytokine levels (p>0.05). At Ch+14, both groups had recovered. Findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. While they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior 2008–09 TIV receipt on A(H1N1)pdm09 illness. As such, they warrant further in-depth investigation and search for possible mechanistic explanations

Randomized controlled ferret study to assess the direct impact of 2008-09 trivalent inactivated influenza vaccine on A(H1N1)pdm09 disease risk

During spring-summer 2009, several observational studies from Canada showed increased risk of medically-attended, laboratory-confirmed A(H1N1)pdm09 illness among prior recipients of 2008-09 trivalent inactivated influenza vaccine (TIV). Explanatory hypotheses included direct and indirect vaccine effects. In a randomized placebo-controlled ferret study, we tested whether prior receipt of 2008-09 TIV may have directly influenced A(H1N1)pdm09 illness. Thirty-two ferrets (16/group) received 0.5 mL intra-muscular injections of the Canadian-manufactured, commercially-available, non-adjuvanted, split 2008-09 Fluviral or PBS placebo on days 0 and 28. On day 49 all animals were challenged (Ch0) with A(H1N1)pdm09. Four ferrets per group were randomly selected for sacrifice at day 5 post-challenge (Ch+5) and the rest followed until Ch+14. Sera were tested for antibody to vaccine antigens and A(H1N1)pdm09 by hemagglutination inhibition (HI), microneutralization (MN), nucleoprotein-based ELISA and HA1-based microarray assays. Clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. Baseline characteristics were similar between the two groups of influenza-naïve animals. Antibody rise to vaccine antigens was evident by ELISA and HA1-based microarray but not by HI or MN assays; virus challenge raised antibody to A(H1N1)pdm09 by all assays in both groups. Beginning at Ch+2, vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at Ch+5 (7.4% vs. 5.2%; p = 0.01). At Ch+ 5 vaccinated animals had higher lung virus titers (log-mean 4.96 vs. 4.23pfu/mL, respectively; p = 0.01), lung inflammatory scores (5.8 vs. 2.1, respectively; p = 0.051) and cytokine levels (p.0.05). At Ch+14, both groups had recovered. Findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. While they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior 2008-09 TIV receipt on A(H1N1)pdm09 illness. As such, they warrant further in-depth investigation and search for possible mechanistic explanations

Do lambs perceive regular human stroking as pleasant? Behavior and heart rate variability analyses

Stroking by humans is beneficial to the human-animal relationship and improves welfare in many species that express intraspecific allogrooming, but very few studies have looked at species like sheep that do not express such contact except around parturition. This study investigated the way lambs perceive regular human tactile contact using behavioral and physiological responses. Twenty-four lambs were reared and bucket-fed in groups of four. All were stroked daily by their familiar caregiver. At 8 weeks of age, the lambs were individually tested in their home pen but in a 1×1m open-barred pen after a 15h period of habituation to physical separation from peers while remaining in visual and auditory contact. Half of the lambs received stroking by their caregiver for 8min and half were exposed to their caregiver’s immobile presence. Heart rate and heart rate variability were recorded and analyzed by 2-min slots over the same interval based on three measures: mean heart rate value (HR), root mean square of successive differences (RMSSD) and standard deviation of all intervals measured between consecutive sinus beats (SDNN). Behavioral responses (ear postures of the lamb and time spent in contact with the familiar caregiver, on the knees of the familiar caregiver, and moving) were recorded throughout the test. Lamb HR decreased continuously while in the presence of their caregiver. Lambs being stroked showed slower HR and higher RMSSD which reflected positive emotional states compared to lambs left unstroked. All behavioral variables were highly correlated with the main component axis of the PCA analyses: the more the animals stayed in contact with their caregiver, the less they moved and the more their ears were hanging. This first component clearly differentiates lambs being stroked or not. Behavioral and physiological observations support the hypothesis that gentle physical contact with the caregiver is perceived positively by lambs

Short- and mid-term effects on performance, health and qualitative behavioural assessment of Romane lambs in different milk feeding conditions

The common practice of artificially rearing lambs from prolific meat breeds of sheep constitutes a welfare issue due to increased mortality rates and negative health issues. In this multidisciplinary study, we investigated the possible short- and mid-term advantages of artificially feeding fresh ewe's milk instead of commercial milk replacer on lambs' growth, health and welfare. Romane lambs were either separated from their mothers on D3 and fed with Lacaune ewes' milk (LAC, n?=?13) or milk replacer (REP, n?=?15), or they were reared by their mothers (MOT, n =?15). On D45, they were weaned, gathered in single-sex groups until the end of the study on D150. Lamb performance and biomarkers of overall health were assessed by measuring: growth, dirtiness of the perianal area, enteric pathogens in the faeces, total antioxidant status and redox status assessed by plasma reduced glutathione/oxidised glutathione ratio, and immune response after vaccination against chlamydiosis. As an exploratory approach, blood cell transcriptomic profiles were also investigated. Last, qualitative behaviour assessment (QBA) was performed as an integrated welfare criterion. Lacaune ewes' milk and REP never differed in their average daily gain but grew less than MOT lambs in the early suckling period and just after weaning. No effect was detected afterwards. On D30, LAC and REP lambs had lower total antioxidant and higher redox status than MOT lambs but did not differ among themselves. Lacaune ewes' milk and MOT had a cleaner perianal area than REP lambs on D21, while faecal pathogen infection did not vary between the treatment groups. After vaccination, LAC also had a stronger immune response on D90 compared to REP lambs. Transcriptome analysis performed on D150 showed differential gene expression, mainly in relation to inflammatory, immune and cell cycle response, between male lambs of the LAC group and those of the MOT and REP groups. Based on QBA, LAC lambs never differed from MOT lambs in their general activity and varied from REP only on D21; REP lambs were always more agitated than MOT lambs. In conclusion, artificial milk feeding impaired early growth rate, health and emotional state mainly during the milk feeding period and at weaning. Feeding artificially reared lambs with fresh ewe's milk partly mitigated some of the negative effects induced by milk replacer but without achieving the full benefit of being reared by the mother

Coastal subsistence, maritime trade, and the colonization of small offshore islands in eastern African prehistory

Recent archaeological research has firmly established eastern Africa’s offshore islands as important localities for understanding the region’s pre-Swahili maritime adaptations and early Indian Ocean trade connections. While the importance of the sea and small offshore islands to the development of urbanized and mercantile Swahili societies has long been recognized, the formative stages of island colonization—and in particular the processes by which migrating Iron Age groups essentially became “maritime”—are still relatively poorly understood. Here we present the results of recent archaeological fieldwork in the Mafia Archipelago, which aims to understand these early adaptations and situate them within a longer-term trajectory of island settlement and pre-Swahili cultural developments. We focus on the results of zooarchaeological, archaeobotanical, and material culture studies relating to early subsistence and trade on this island to explore the changing significance of marine resources to the local economy. We also discuss the implications of these maritime adaptations for the development of local and long-distance Indian Ocean trade networks.The Sealinks Project is funded through a grant toNicole Boivin from the European Research Council (StarterGrant 206148) under the “Ideas” specific Programme of the 7th Framework Programme (FP7). Additional funding includes: postdoctoral fellowships from the British Academy (2010–2013) and the University of Queensland (2015–2017) to Alison Crowther; a Fondation Fyssen Postdoctoral Grant toEr´endira Quintana Morales; and a postdoctoral fellowship from the Government of the Balearic Islands (Conselleria d’Educaci´o) and the European Social Fund to Llorenc¸ Picornell-Gelabert. Fieldwork was carried out under COSTECH Research Permit no. 2012-303-ER-2011-85 and the Division of Antiquities (Ministry of Natural Resources and Tourism, Tanzania) excavation license no. EA.402/605/01 issued to Alison Crowther, and with the support and permission of theRegionalAdministrative Secretary (Dar es Salaam andCoast/Pwani), the District Administrative Secretary (Mafia), and the Village Executive Officer (Mtendaji wa Kijiji, Juani).http://www.tandfonline.com/loi/uica20hb2016Anthropology and Archaeolog