Genetic and Pathobiologic Characterization of Pandemic H1N1 2009 Influenza Viruses from a Naturally Infected Swine Herd

Since its initial identification in Mexico and the United States, concerns have been raised that the novel H1N1 influenza virus might cause a pandemic of severity comparable to that of the 1918 pandemic. In late April 2009, viruses phylogenetically related to pandemic H1N1 influenza virus were isolated from an outbreak on a Canadian pig farm. This outbreak also had epidemiological links to a suspected human case. Experimental infections carried out in pigs using one of the swine isolates from this outbreak and the human isolate A/Mexico/InDRE4487/2009 showed differences in virus recovery from the lower respiratory tract. Virus was consistently isolated from the lungs of pigs infected with A/Mexico/InDRE4487/2009, while only one pig infected with A/swine/Alberta/OTH-33-8/2008 yielded live virus from the lung, despite comparable amounts of viral RNA and antigen in both groups of pigs. Clinical disease resembled other influenza virus infections in swine, albeit with somewhat prolonged virus antigen detection and delayed viral-RNA clearance from the lungs. There was also a noteworthy amount of genotypic variability among the viruses isolated from the pigs on the farm. This, along with the somewhat irregular pathobiological characteristics observed in experimentally infected animals, suggests that although the virus may be of swine origin, significant viral evolution may still be ongoing.The zoonotic potential of swine influenza viruses is well recognized (18), and pigs have been considered a leading candidate for the role of intermediate host in the generation of reassortant influenza A viruses with pandemic potential. This has been largely based on genomic analysis of influenza A viruses isolated from swine and the fact that α2,3-linked sialic acid (avian-like) and α2,6-linked sialic acid (human-like) receptors are both abundant in the swine respiratory tract (12). Despite this, there is no direct evidence that the reassortment of the 1957 and the 1968 human pandemic viruses occurred in pigs (28). Furthermore, it is very likely that the 1918 pandemic virus was introduced to pigs from humans (8, 31). The origins of influenza A viruses that have been isolated from pigs include those that are wholly human or avian, as well as reassortants containing swine, human, and avian genes (2, 20, 29). Although there have been several instances of swine-to-human transmission, for example, that of triple-reassortant swine influenza (H1) viruses (rH1N1), which appeared after 1998, they did not lead to establishment of sustained transmission in the human population (23).In the early spring of 2009, Mexico and the United States reported clusters of human pneumonia cases caused by a novel H1N1 influenza A virus. This virus subsequently spread across the globe at an unprecedented rate, prompting the WHO to declare a pandemic in June 2009. Phylogenetic analysis has inferred that the virus is likely a reassortant between a North American triple-reassortant swine H1N1 or H1N2 virus and a Eurasian lineage H1N1 swine influenza virus (7, 19). Bayesian molecular-clock analysis of each gene of this novel H1N1 virus (24) concluded that the mean evolutionary rate is typical of that of swine influenza viruses but that the duration of unsampled diversity for each gene segment had means that ranged from 9.24 to 17.15 years, suggesting that the proposed ancestors of this virus may have been circulating undetected for nearly a decade. Inadequate surveillance and characterization of influenza A viruses that circulate in swine have been blamed for this evolutionary gap.On 28 April 2009 the Canadian Food Inspection Agency (CFIA) became involved in a suspected outbreak of swine influenza on a pig farm in Leslieville, Alberta, Canada. The farm was a 220-sow farrow-to-finish operation consisting of approximately 2,200 animals that ranged from newborn piglets to market weight pigs. The animals were not vaccinated against swine influenza, and although there had been prior problems with porcine reproductive and respiratory syndrome virus and Mycoplasma hypopneumoniae, two etiologic agents of the swine respiratory disease complex, the herd had been stable with respect to respiratory disease. Beginning 20 April, approximately 25% of the pregrower and grower pigs in two of the barns exhibited respiratory problems with clinical signs that included an acute onset of coughing, lethargy, and loss of appetite. These clinical signs were preceded by the hiring of a carpenter on 14 April to work on the ventilation system in the same two barns. This individual had been ill for 2 days after his return from Mexico on 12 April (10). Given the evolving situation in Mexico and the United States, the CFIA and Alberta Agriculture and Rural Development decided to place the herd under quarantine and to carry out a full epidemiological and laboratory investigation.Here, we report on the characterization of the first pandemic H1N1 2009 viruses to be isolated from a naturally infected pig herd. Genetic sequence data from several viruses isolated from this outbreak have provided a glimpse into the mutation frequencies associated with replication of the virus in the swine host. Experimental infections of pigs comparing one of these swine isolates with the human isolate A/Mexico/InDRE4487/2009(H1N1) were also carried out and have provided insights into the pathobiological behavior of these viruses in pigs.     

Cooperation of C1q receptors and integrins in C1q-mediated endothelial cell adhesion and spreading

The interaction of C1q with endothelial cells elicits a multiplicity of biologic responses. Although these responses are presumed to be mediated by the interaction of C1q with endothelial cell surface proteins, the identity of the participants is not known. In this study we examined the roles of two C1q binding proteins, cC1q-R/calreticulin and gC1q-R/p33, in C1q-mediated adhesion and spreading of human dermal microvascular endothelial cells (HDMVEC). When HDMVEC were cultured in microtiter plate wells coated with concentrations of C1q ranging from 0 to 50 microg/ml, a specific and dose-dependent adhesion and spreading was observed. The extent of adhesion and spreading was similar to the adhesion seen on collagen-coated wells. Spreading (68 +/- 12%) and to a moderate extent adhesion (47 +/- 9%) were inhibited by anti-gC1q-R mAb 60.11. Similar effects were noted with polyclonal anti-cC1q-R but not with control nonimmune IgG. The two Abs had a slight additive effect (75 +/- 13% inhibition) when mixed together in the proportion of 100 microg/ml anti-gC1q-R and 30 microg/ml anti-cC1q-R. More importantly, a 100% inhibition of spreading, but not adhesion, to C1q-coated wells was observed when HDMVEC were cultured in the presence of 30 microM of the peptide GRRGDSP but not GRRGESP. Furthermore, while anti-beta(1) integrin Ab blocked both adhesion and spreading, anti-alpha(5) integrin blocked only spreading and not adhesion. Ag capture ELISA of endothelial cell membrane proteins using polyclonal anti-gC1q-R showed the presence of not only beta(1) and alpha(5) integrins but also CD44. Taken together these results suggest that endothelial cell adhesion and spreading require the cooperation of both C1qRs and beta(1) integrins and possibly other membrane-spanning molecules.     

Reduced knee joint moment in ACL deficient patients at a cost of dynamic stability during landing

The current study aimed to examine the effect of anterior cruciate ligament deficiency (ACLd) on joint kinetics and dynamic stability control after a single leg hop test (SLHT). Twelve unilateral ACLd patients and a control subject group (n=13) performed a SLHT over a given distance with both legs. The calculation of joint kinetics was done by means of a soft-tissue artifact optimized rigid full-body model. Margin of stability (MoS) was quantified by the difference between the base of support and the extrapolated center of mass. During landing, the ACLd leg showed lower external knee flexion moments but demonstrated higher moments at the ankle and hip compared to controls (p<0.05). The main reason for the joint moment redistribution in the ACLd leg was a more anterior position of the ground reaction force (GRF) vector, which affected the moment arms of the GRF acting about the joints (p<0.05). For the ACLd leg, trunk angle was more flexed over the entire landing phase compared to controls (p<0.05) and we found a significant correlation between moment arms at the knee joint and trunk angle (r2 = 0.48;p<0.01). The consequence of this altered landing strategy in ACLd legs was a more anterior position of the center of mass reducing the MoS (p<0.05). The results illustrate the interaction between trunk angle, joint kinetics and dynamic stability during landing maneuvers and provide evidence of a feedforward adaptive adjustment in ACLd patients (i.e. more flexed trunk angle) aimed at reducing knee joint moments at the cost of dynamic stability control.     

Halobacteriovorax,an underestimated predator on bacteria: potential impact relative to viruses on bacterial mortality

Predation on bacteria and accompanying mortality are important mechanisms in controlling bacterial populations and recycling of nutrients through the microbial loop. The agents most investigated and seen as responsible for bacterial mortality are viruses and protists. However, a body of evidence suggests that predatory bacteria such as the Halobacteriovorax (formerly Bacteriovorax), a Bdellovibrio-like organism, contribute substantially to bacterial death. Until now, conclusive evidence has been lacking. The goal of this study was to better understand the contributors to bacterial mortality by addressing the poorly understood role of Halobacteriovorax and how their role compares with that of viruses. The results revealed that when a concentrated suspension of Vibrio parahaemolyticus was added into microcosms of estuarine waters, the native Halobacteriovorax were the predators that responded first and most rapidly. Their numbers increased by four orders of magnitude, whereas V. parahaemolyticus prey numbers decreased by three orders of magnitude. In contrast, the extant virus population showed little increase and produced little change in the prey density. An independent experiment with stable isotope probing confirmed that Halobacteriovorax were the predators primarily responsible for the mortality of the V. parahaemolyticus. The results show that Halobacteriovorax have the potential to be significant contributors to bacterial mortality, and in such cases, predation by Halobacteriovorax may be an important mechanism of nutrient recycling. These conclusions add another dimension to bacterial mortality and the recycling of nutrients.     

Visual and proprioceptive contributions to postural control of upright stance in unilateral vestibulopathy

Preserving upright stance requires central integration of the sensory systems and appropriate motor output from the neuromuscular system to keep the centre of pressure (COP) within the base of support. Unilateral peripheral vestibular disorder (UPVD) causes diminished stance stability. The aim of this study was to determine the limits of stability and to examine the contribution of multiple sensory systems to upright standing in UPVD patients and healthy subjects. We hypothesized that closure of the eyes and Achilles tendon vibration during upright stance will augment the postural sway in UPVD patients more than in healthy subjects. Seventeen UPVD patients and 17 healthy subjects performed six tasks on a force plate: forwards and backwards leaning, to determine limits of stability, and upright standing with and without Achilles tendon vibration, each with eyes open and closed (with blackout glasses). The COP displacement of the patients was significantly greater in the vibration tasks than the controls and came closer to the posterior base of support boundary than the controls in all tasks. Achilles tendon vibration led to a distinctly more backward sway in both subject groups. Five of the patients could not complete the eyes closed with vibration task. Due to the greater reduction in stance stability when the proprioceptive, compared with the visual, sensory system was disturbed, we suggest that proprioception may be more important for maintaining upright stance than vision. UPVD patients, in particular, showed more difficulty in controlling postural stability in the posterior direction with visual and proprioceptive sensory disturbance.     

Estimation of Parameters in the Two-Compartment Model for Exhaled Nitric Oxide

The fractional concentration of exhaled nitric oxide (FeNO) is a biomarker of airway inflammation that is being increasingly considered in clinical, occupational, and epidemiological applications ranging from asthma management to the detection of air pollution health effects. FeNO depends strongly on exhalation flow rate. This dependency has allowed for the development of mathematical models whose parameters quantify airway and alveolar compartment contributions to FeNO. Numerous methods have been proposed to estimate these parameters using FeNO measured at multiple flow rates. These methods—which allow for non-invasive assessment of localized airway inflammation—have the potential to provide important insights on inflammatory mechanisms. However, different estimation methods produce different results and a serious barrier to progress in this field is the lack of a single recommended method. With the goal of resolving this methodological problem, we have developed a unifying framework in which to present a comprehensive set of existing and novel statistical methods for estimating parameters in the simple two-compartment model. We compared statistical properties of the estimators in simulation studies and investigated model fit and parameter estimate sensitivity across methods using data from 1507 schoolchildren from the Southern California Children''s Health Study, one of the largest multiple flow FeNO studies to date. We recommend a novel nonlinear least squares model with natural log transformation on both sides that produced estimators with good properties, satisfied model assumptions, and fit the Children''s Health Study data well.     

Khat Chewing and Restrictive Dietary Behaviors Are Associated with Anemia among Pregnant Women in High Prevalence Rural Communities in Eastern Ethiopia

Background

Anemia affects a high proportion of pregnant women in the developing countries. Factors associated with it vary in context. This study aimed to determine the prevalence and predictors of anemia among pregnant women in the rural eastern Ethiopia.

Methods

A community-based cross-sectional study was done on 1678 pregnant women who were selected by a cluster random sampling technique. A pregnant woman was identified as anemic if her hemoglobin concentration was <11 g/dl. Data were collected in a community-based setting. Multilevel mixed effect logistic regression was used to determine the adjusted odds ratios (AOR) with 95% confidence intervals (CI) for the predictors of anemia.

Results

Anemia was observed among 737(43.9%) of the 1678 pregnant women studied (95% CI 41.5%–46.3%). After controlling for the confounders, the risk of anemia was 29% higher in the women who chewed khat daily than those who sometimes or never did so (AOR, 1.29; 95% CI, 1.02–1.62). The study subjects with restrictive dietary behavior (reduced either meal size or frequency) had a 39% higher risk of anemia compared to those without restrictive dietary behavior (AOR, 1.39; 95% CI, 1.02–1.88). The risk of anemia was increased by 68% (AOR, 1.68; 95% CI, 1.15–2.47), and 60% (AOR, 1.60; 95% CI, 1.08–2.37) in parity levels of 2 births and 3 births, respectively. Compared to the first trimester, the risk of anemia was higher by two-fold (AOR, 2.09; 95% CI, 1.46–3.00) in the second trimester and by four-fold (AOR, 4.23; 95% CI, 2.97–6.02) in the third trimester.

Conclusion

In this study, two out of five women were anemic. Chewing khat and restrictive dietary habits that are associated with anemia in the setting should be addressed through public education programs. Interventions should also focus on the women at higher parity levels and those who are in advanced stages of pregnancy.     

Cytokine Profile in a Cohort of Healthy Blood Donors Carrying Polymorphisms in Genes Encoding the NLRP3 Inflammasome

Background

The NLRP3 inflammasome has been recognized as one of the key components of the innate immunity by sensing a diversity of insults. Inflammasome activation results in the maturation of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Increased production of IL-1β is found in patients with gain-of-function polymorphisms in genes encoding the NLRP3 inflammasome. Since approximately 5% of the Swedish population are heterozygote carriers of these combined gene variants, their impact on inflammasome status and a relationship on disease development is therefore highly relevant to study. The present study investigates levels of inflammasome-produced cytokines as a measure of inflammasome activation in healthy individuals carrying Q705K polymorphism in the NLRP3 gene combined with C10X in the CARD8 gene.

Materials and Methods

Genotyping of 1006 healthy blood donors was performed for the polymorphisms Q705K in the NLRP3 and C10X in the CARD8 genes. IL-1β, IL-18, IL-33, as well as a number of other pro-inflammatory cytokines, were analyzed by Luminex or ELISA in plasma from individuals carrying the polymorphisms and in age and gender matched non-carrier controls.

Results & Discussion

The prevalence of the polymorphisms was in line with previous studies. Plasma levels of IL-1β and IL-33 were elevated among carriers of combined Q705K+C10X polymorphisms compared to controls, whereas no difference was found for IL-18 and the other cytokines measured. Moreover, carriers of C10X or Q705K per se had similar plasma levels of IL-1β as non-carriers. These data suggest that the combined polymorphisms create inflammasomes with increased basal activation state, which might provide a more favourable innate immune response. In spite of this, it could also represent the mechanisms by which the inflammatory loop is triggered into a long-term inflammatory phenotype.