Coinfection with SARS-CoV-2 and Influenza A Virus Increases Disease Severity and Impairs Neutralizing Antibody and CD4+ T Cell Responses.

Given the current coronavirus disease 2019 (COVID-19) pandemic, coinfection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) is a major concern for public health. However, the immunopathogenic events occurring with coinfections of SARS-CoV-2 and IAV remain unclear. Here, we report the pathogenic and immunological consequences of SARS-CoV-2 and IAV H1N1 coinfection in the K18-hACE2 transgenic mouse model. Compared with a single infection with SARS-CoV-2 or IAV, coinfections not only prolonged the primary virus infection period but also increased immune cell infiltration and inflammatory cytokine levels in bronchoalveolar lavage fluid leading to severe pneumonia and lung damage. Moreover, coinfections caused severe lymphopenia in peripheral blood, resulting in reduced total IgG, neutralizing antibody titers, and CD4+ T cell responses against each virus. This study sheds light on the immunopathogenesis of SARS-CoV-2 and IAV coinfection, which may guide the development of effective therapeutic strategies for the treatment of patients coinfected with these viruses. IMPORTANCE The cocirculation of influenza virus merging with the COVID-19 pandemic raises a potentially severe threat to public health. Recently, increasing numbers of SARS-CoV-2 and influenza virus coinfection have been reported from many countries. It is a worrisome issue that SARS-CoV-2 coinfection with other pathogens may worsen the clinical outcome and severity of COVID-19 and increase fatality. Here, we evaluated SARS-CoV-2 and IAV coinfection using the K18-hACE2 mouse model. Coinfected mice exhibited increased mortality with prolonged IAV shedding. Furthermore, coinfected mice showed a higher level of cytokines and chemokines than a single infection condition. Interestingly, our data show that coinfected mice showed significantly fewer virus-specific and neutralizing antibodies than the mice with a single infection. Overall, this study suggests that coinfection aggravates viral pathology by impaired neutralizing antibody response.

Fibrillin2 in chloroplast plastoglobules participates in photoprotection and jasmonate-induced senescence.

Fibrillins (FBNs) are the major structural proteins of plastoglobules (PGs) in chloroplasts. PGs are associated with defense against abiotic and biotic stresses, as well as lipid storage. Although FBN2 is abundant in PGs, its independent function under abiotic stress has not yet been identified. In this study, the targeting of FBN2 to PGs was clearly demonstrated using an FBN2-YFP fusion protein. FBN2 showed higher expression in green photosynthetic tissues and was upregulated at the transcriptional level under high-light stress. The photosynthetic capacity of fbn2 knockout mutants generated using CRISPR/Cas9 technology decreased rapidly compared with that of wild-type (WT) plants under high-light stress. In addition to the photoprotective function of FBN2, fbn2 mutants had lower levels of plastoquinone-9 and plastochromanol-8. The fbn2 mutants were highly sensitive to methyl jasmonate (MeJA) and exhibited root growth inhibition and a pale-green phenotype due to reduced chlorophyll content. Consistently, upon MeJA treatment, the fbn2 mutants showed faster leaf senescence and more rapid chlorophyll degradation with decreased photosynthetic ability compared with the WT plants. The results of this study suggest that FBN2 is involved in protection against high-light stress and acts as an inhibitor of jasmonate-induced senescence in Arabidopsis (Arabidopsis thaliana).

Comparison of outcomes of EUS-guided ablation and surveillance only for pancreatic cystic lesions: a propensity score-matching study (with videos).

BACKGROUND AND AIMS: EUS-guided ethanol ablation is a recently introduced treatment approach for pancreatic cystic lesions (PCLs), including branch-duct intraductal papillary mucinous neoplasms (BD-IPMNs). However, the utility of this procedure is limited because of its relatively low efficacy in treating PCLs. METHODS: We retrospectively reviewed patients with PCLs, including those with enlarging suspected BD-IPMNs or those with PCLs measuring >3 cm, who were suboptimal candidates for surgery and had been managed using EUS-guided rapid ethanol lavage (EUS-REL; immediate ethanol lavage performed 4 times, 2015-2022) or surveillance only (SO; 2007- 2022). Propensity score matching (PSM) was performed to minimize bias. The primary outcome was the cumulative incidence rate of BD-IPMN progression. Secondary outcomes were the efficacy and safety of EUS-REL, surgical resection rate (SR), overall survival (OS), and disease-specific survival (DSS) in both groups. RESULTS: Overall, 169 and 610 patients were included in the EUS-REL and SO groups, respectively. PSM created 159 matched pairs. The radiologic complete resolution rate after EUS-REL was 74%. Procedure-related pancreatitis in the EUS-REL group was 13.0% (n = 22; 19 mild and 3 moderate grade); no severe adverse events were reported. The 10-year cumulative incidence rate of BD-IPMN progression was significantly lower in the EUS-REL group than in the SO group (1.6% vs 21.2%; hazard ratio, 12.35; P = .003). EUS-REL showed a lower tendency of SR compared with that associated with SO. The rates of 10-year OS and 10-year DSS were comparable in both groups. CONCLUSIONS: EUS-REL was associated with a significantly lower 10-year cumulative incidence rate of BD-IPMN progression and a lower tendency of SR, whereas its 10-year OS and DSS rates were similar to those of SO for PCLs. EUS-REL may be a viable alternative to SO for managing patients with enlarging suspected BD-IPMNs or those with PCLs >3 cm who are suboptimal candidates for surgery.

Comparison of EUS-guided ablation and surgical resection for nonfunctioning small pancreatic neuroendocrine tumors: a propensity score-matching study.

BACKGROUND AND AIMS: Treatment strategies for small pancreatic neuroendocrine tumors (PNETs) <2 cm in size are still under debate. The feasibility and safety of EUS-guided ethanol ablation (EUS-EA) have been demonstrated. However, sample sizes in previous studies were small with no comparative studies on surgery. Therefore, we aimed to compare the safety and long-term outcomes of EUS-EA with those of surgery for the management of nonfunctioning small PNETs. METHODS: We retrospectively reviewed patients with PNETs who were managed by EUS-EA (from 2011 to 2018) and surgery (from 2000 to 2018) at Asan Medical Center. Propensity score matching (PSM) was performed to increase comparability. The primary outcome was early and late major adverse events (Clavien-Dindo grade ≥III) after treatment. Secondary outcomes were 10-year overall (OS) and disease-specific survival (DSS) rates, length of hospital stay, and development of endocrine pancreatic insufficiency. RESULTS: Of all patients, 97 and 188 patients were included in the EUS-EA and surgery groups, respectively. PSM created 89 matched pairs. EUS-EA was associated with a significantly lower rate of early major adverse events (0% vs 11.2%, P = .003). Late major adverse events occurred more frequently after surgery, with no significant difference between groups (3.4% vs 10.1%, P = .07). Both treatment modalities showed comparable 10-year OS and DSS rates. The length of hospital stay was significantly shorter in the EUS-EA group (4 days vs 14.1 days, P < .001), and endocrine pancreatic insufficiency was less common after EUS-EA than after surgery (33.3% vs 48.6%, P = .121). CONCLUSIONS: EUS-EA had fewer adverse events and a shorter hospital stay with similar OS and DSS rates compared with surgery, suggesting that EUS-EA may be a preferred alternative to surgical resection in selected patients with nonfunctioning small PNETs.

Associations among workplace incivility, stress coping, and nursing performance in hospital nurses: A path analysis.

PURPOSE: This study aimed to identify the associations among workplace incivility, stress-coping strategy, and nursing performance and confirm how workplace incivility influences nursing performance through the stress-coping strategy. DESIGN: This is a descriptive, cross-sectional study. Data were collected using a self-reported questionnaire from 245 nurses working at seven hospitals in Korea between December 2019 and January 2020. METHODS: The associations among the study variables (workplace incivility, stress coping, and nursing performance) were analyzed using path analysis with bootstrapping. RESULTS: Supervisors' incivility affected directly and negatively nursing performance although problem-focused coping was mediating between them. By contrast, coworkers' and doctors' incivility was not directly associated with nursing performance through stress-coping strategies. CONCLUSION: Problem-focused coping enhanced nursing performance and was a more effective stress-coping strategy than emotion-focused coping for nurses affected by coworkers' and doctors' incivilities. Supervisors' incivility may be considered a threatening factor to nursing performance even though problem-focused coping partially mediates between supervisors' incivility and nursing performance. CLINICAL RELEVANCE: Organizations must prevent all kinds of workplace incivilities from occurring. Nursing managers should periodically monitor the relationship between the supervisors and nurses and be aware of nurses' stress-coping strategies under stressful situations.

Chloroplast-localized PITP7 is essential for plant growth and photosynthetic function in Arabidopsis.

Recent studies of chloroplast-localized Sec14-like protein (CPSFL1, also known as phosphatidylinositol transfer protein 7, PITP7) showed that CPSFL1 is necessary for photoautotropic growth and chloroplast vesicle formation in Arabidopsis (Arabidopsis thaliana). Here, we investigated the functional roles of CPSFL1/PITP7 using two A. thaliana mutants carrying a putative null allele (pitp7-1) and a weak allele (pitp7-2), respectively. PITP7 transcripts were undetectable in pitp7-1 and less abundant in pitp7-2 than in the wild-type (WT). The severity of mutant phenotypes, such as plant developmental abnormalities, levels of plastoquinone-9 (PQ-9) and chlorophylls, photosynthetic protein complexes, and photosynthetic performance, were well related to PITP7 transcript levels. The pitp7-1 mutation was seedling lethal and was associated with significantly lower levels of PQ-9 and major photosynthetic proteins. pitp7-2 plants showed greater susceptibility to high-intensity light stress than the WT, attributable to defects in nonphotochemical quenching and photosynthetic electron transport. PITP7 is specifically bound to phosphatidylinositol phosphates (PIPs) in lipid-binding assays in vitro, and the point mutations R82, H125, E162, or K233 reduced the binding affinity of PITP7 to PIPs. Further, constitutive expression of PITP7H125Q or PITP7E162K in pitp7-1 homozygous plants restored autotrophic growth in soil but without fully complementing the mutant phenotypes. Consistent with a previous study, our results demonstrate that PITP7 is essential for plant development, particularly the accumulation of PQ-9 and photosynthetic complexes. We propose a possible role for PITP7 in membrane trafficking of hydrophobic ligands such as PQ-9 and carotenoids through chloroplast vesicle formation or direct binding involving PIPs.

Co-Development of Adolescents' Cooperative and Competitive Attitudes: How it Predicts Mental Health and Academic Achievement.

This study used nationally representative longitudinal data in South Korea to examine how joint changes in adolescents' (N = 7324; Mage ≈ 11 years) cooperative and competitive attitudes from sixth to ninth grade relate to mental health and achievement in 10th grade. The parallel process model showed that both cooperative and competitive attitudes declined over time. Higher cooperative attitudes at baseline indicated higher competitive attitudes, and a faster decline in cooperative attitudes indicated a faster decline in competitive attitudes. The intercept of cooperative attitudes was positively related to mental health but negatively related to achievement. Opposite patterns were found for the intercept of competitive attitudes. These findings highlight the usefulness of considering the co-development of cooperative and competitive attitudes.

High Light Acclimation Mechanisms Deficient in a PsbS-Knockout Arabidopsis Mutant.

The photosystem II PsbS protein of thylakoid membranes is responsible for regulating the energy-dependent, non-photochemical quenching of excess chlorophyll excited states as a short-term mechanism for protection against high light (HL) stress. However, the role of PsbS protein in long-term HL acclimation processes remains poorly understood. Here we investigate the role of PsbS protein during long-term HL acclimation processes in wild-type (WT) and npq4-1 mutants of Arabidopsis which lack the PsbS protein. During long-term HL illumination, photosystem II photochemical efficiency initially dropped, followed by a recovery of electron transport and photochemical quenching (qL) in WT, but not in npq4-1 mutants. In addition, we observed a reduction in light-harvesting antenna size during HL treatment that ceased after HL treatment in WT, but not in npq4-1 mutants. When plants were adapted to HL, more reactive oxygen species (ROS) were accumulated in npq4-1 mutants compared to WT. Gene expression studies indicated that npq4-1 mutants failed to express genes involved in plastoquinone biosynthesis. These results suggest that the PsbS protein regulates recovery processes such as electron transport and qL during long-term HL acclimation by maintaining plastoquinone biosynthetic gene expression and enhancing ROS homeostasis.

Comparison of the seed nutritional composition between conventional varieties and transgenic soybean overexpressing Physaria FAD3-1.

BACKGROUND: PfFAD3 transgenic soybean expressing omega-3 fatty acid desaturase 3 of Physaria produces increased level of α-linolenic acid in seed. Composition data of non-transgenic conventional varieties is important in the safety assessment of the genetically-modified (GM) crops in the context of the natural variation. RESULTS: The natural variation was characterized in seed composition of 13 Korean soybean varieties grown in three locations in South Korea for 2 years. Univariate analysis of combined data showed significant differences by variety and cultivation environment for proximates, minerals, anti-nutrients, and fatty acids. Percent variability analysis demonstrated that genotype, environment and the interaction of environment with genotype contributed to soybean seed compositions. Principal component analysis and orthogonal projections to latent structure discriminant analysis indicated that significant variance in compositions was attributable to location and cultivation year. The composition of three PfFAD3 soybean lines for proximates, minerals, anti-nutrients, and fatty acids was compared to a non-transgenic commercial comparator (Kwangankong, KA), and three non-transgenic commercial varieties grown at two sites in South Korea. Only linoleic and linolenic acids significantly differed in PfFAD3-1 lines compared to KA, which were expected changes by the introduction of the PfFAD3-1 trait in KA. CONCLUSION: Genotype, environment, and the interaction of environment with genotype contributed to compositional variability in soybean. PfFAD3-1 soybean is equivalent to the conventional varieties with respect to these components. © 2020 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform.

BACKGROUND: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. METHODS: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). RESULTS: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). CONCLUSIONS: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.

An I436N substitution confers resistance of influenza A(H1N1)pdm09 viruses to multiple neuraminidase inhibitors without affecting viral fitness.

The resistance of influenza viruses to neuraminidase (NA) inhibitors (NAIs; i.e. oseltamivir, zanamivir, peramivir and laninamivir) can be associated with several NA substitutions, with differing effects on viral fitness. To identify novel molecular markers conferring multi-NAI resistance, the NA gene of oseltamivir-resistant (H275Y, N1 numbering) 2009 pandemic influenza [A(H1N1)pdm09] virus was enriched with random mutations. This randomly mutated viral library was propagated in Madin-Darby canine kidney (MDCK) cells under zanamivir pressure and gave rise to additional changes within NA, including an I436N substitution located outside the NA enzyme active site. We generated four recombinant A(H1N1)pdm09 viruses containing either wild-type NA or NA with single (I436N or H275Y) or double (H275Y-I436N) substitutions. The double H275Y-I436N mutation significantly reduced inhibition by oseltamivir and peramivir and reduced inhibition by zanamivir and laninamivir. I436N alone reduced inhibition by all NAIs, suggesting that it is a multi-NAI resistance marker. I436N did not affect viral fitness in vitro or in a murine model; however, H275Y and I436N together had a negative impact on viral fitness. Further, I436N alone did not have an appreciable impact on viral replication in the upper respiratory tract or transmissibility in ferrets. However, the rg-H275Y-I436N double mutant transmitted less efficiently than either single mutant via the direct contact and respiratory droplet routes in ferrets. Overall, these results highlight the usefulness of a random mutagenesis approach for identifying potential molecular markers of resistance and the importance of I436N NA substitution in A(H1N1)pdm09 virus as a marker for multi-NAI resistance.

Vaccine Efficacy of Inactivated, Chimeric Hemagglutinin H9/H5N2 Avian Influenza Virus and Its Suitability for the Marker Vaccine Strategy.

In order to produce a dually effective vaccine against H9 and H5 avian influenza viruses that aligns with the DIVA (differentiating infected from vaccinated animals) strategy, we generated a chimeric H9/H5N2 recombinant vaccine that expressed the whole HA1 region of A/CK/Korea/04163/04 (H9N2) and the HA2 region of recent highly pathogenic avian influenza (HPAI) A/MD/Korea/W452/14 (H5N8) viruses. The chimeric H9/H5N2 virus showed in vitro and in vivo growth properties and virulence that were similar to those of the low-pathogenic avian influenza (LPAI) H9 virus. An inactivated vaccine based on this chimeric virus induced serum neutralizing (SN) antibodies against both H9 and H5 viruses but induced cross-reactive hemagglutination inhibition (HI) antibody only against H9 viruses. Thus, this suggests its compatibility for use in the DIVA strategy against H5 strains. Furthermore, the chimeric H9/H5N2 recombinant vaccine protected immunized chickens against lethal challenge by HPAI H5N8 viruses and significantly attenuated virus shedding after infection by both H9N2 and HPAI H5N8 viruses. In mice, serological analyses confirmed that HA1- and HA2 stalk-specific antibody responses were induced by vaccination and that the DIVA principle could be employed through the use of an HI assay against H5 viruses. Furthermore, each HA1- and HA2 stalk-specific antibody response was sufficient to inhibit viral replication and protect the chimeric virus-immunized mice from lethal challenge with both mouse-adapted H9N2 and wild-type HPAI H5N1 viruses, although differences in vaccine efficacy against a homologous H9 virus (HA1 head domain immune-mediated protection) and a heterosubtypic H5 virus (HA2 stalk domain immune-mediated protection) were observed. Taken together, these results demonstrate that the novel chimeric H9/H5N2 recombinant virus is a low-pathogenic virus, and this chimeric vaccine is suitable for a DIVA vaccine with broad-spectrum neutralizing antibody against H5 avian influenza viruses.IMPORTANCE Current influenza virus killed vaccines predominantly induce antihemagglutinin (anti-HA) antibodies that are commonly strain specific in that the antibodies have potent neutralizing activity against homologous strains but do not cross-react with HAs of other influenza virus subtypes. In contrast, the HA2 stalk domain is relatively well conserved among subtypes, and recently, broadly neutralizing antibodies against this domain have been isolated. Therefore, in light of the need for a vaccine strain that applies the DIVA strategy utilizing an HI assay and induces broad cross-protection against H5N1 and H9N2 viruses, we generated a novel chimeric H9/H5N1 virus that expresses the entire HA1 portion from the H9N2 virus and the HA2 region of the heterosubtypic H5N8 virus. The chimeric H9/H5N2 recombinant vaccine protected immunized hosts against lethal challenge with H9N2 and HPAI H5N1 viruses with significantly attenuated virus shedding in immunized hosts. Therefore, this chimeric vaccine is suitable as a DIVA vaccine against H5 avian influenza viruses.

Evaluation of the Immune Responses to and Cross-Protective Efficacy of Eurasian H7 Avian Influenza Viruses.

Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%). In vivo immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses.IMPORTANCE Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses.

Fibrillin 5 Is Essential for Plastoquinone-9 Biosynthesis by Binding to Solanesyl Diphosphate Synthases in Arabidopsis.

Fibrillins are lipid-associated proteins in plastids and are ubiquitous in plants. They accumulate in chromoplasts and sequester carotenoids during the development of flowers and fruits. However, little is known about the functions of fibrillins in leaf tissues. Here, we identified fibrillin 5 (FBN5), which is essential for plastoquinone-9 (PQ-9) biosynthesis in Arabidopsis thaliana. Homozygous fbn5-1 mutations were seedling-lethal, and XVE:FBN5-B transgenic plants expressing low levels of FBN5-B had a slower growth rate and were smaller than wild-type plants. In chloroplasts, FBN5-B specifically interacted with solanesyl diphosphate synthases (SPSs) 1 and 2, which biosynthesize the solanesyl moiety of PQ-9. Plants containing defective FBN5-B accumulated less PQ-9 and its cyclized product, plastochromanol-8, but the levels of tocopherols were not affected. The reduced PQ-9 content of XVE:FBN5-B transgenic plants was consistent with their lower photosynthetic performance and higher levels of hydrogen peroxide under cold stress. These results indicate that FBN5-B is required for PQ-9 biosynthesis through its interaction with SPS. Our study adds FBN5 as a structural component involved in the biosynthesis of PQ-9. FBN5 binding to the hydrophobic solanesyl moiety, which is generated by SPS1 and SPS2, in FBN5-B/SPS homodimeric complexes stimulates the enzyme activity of SPS1 and SPS2.

Pathogenicity and genetic characterisation of a novel reassortant, highly pathogenic avian influenza (HPAI) H5N6 virus isolated in Korea, 2017.

We investigated influenza A(H5N6) viruses from migratory birds in Chungnam and Gyeonggi Provinces, South Korea following a reported die-off of poultry in nearby provinces in November 2017. Genetic analysis and virulence studies in chickens and ducks identified our isolate from December 2017 as a novel highly pathogenic avian influenza virus. It resulted from reassortment between the highly virulent H5N8 strain from Korea with the N6 gene from a low-pathogenic H3N6 virus from the Netherlands.

Growth and Pathogenic Potential of Naturally Selected Reassortants after Coinfection with Pandemic H1N1 and Highly Pathogenic Avian Influenza H5N1 Viruses.

Coinfection of ferrets with H5N1 and pH1N1 viruses resulted in two predominate genotypes in the lungs containing surface genes of highly pathogenic avian influenza H5N1 virus in the backbone of pandemic H1N1 2009 (pH1N1). Compared to parental strains, these reassortants exhibited increased growth and virulence in vitro and in mice but failed to be transmitted indirectly to naive contact ferrets. Thus, this demonstrates a possible natural reassortment following coinfection as well as the pathogenicity of the potential reassortants.

Genetic characterisation of novel, highly pathogenic avian influenza (HPAI) H5N6 viruses isolated in birds, South Korea, November 2016.

A novel genotype of H5N6 influenza viruses was isolated from migratory birds in South Korea during November 2016. Domestic outbreaks of this virus were associated with die-offs of wild birds near reported poultry cases in Chungbuk province, central South Korea. Genetic analysis and animal studies demonstrated that the Korean H5N6 viruses are highly pathogenic avian influenza (HPAI) viruses and that these viruses are novel reassortants of at least three different subtypes (H5N6, H4N2 and H1N1).

A microcomputed tomography evaluation of the marginal fit of cobalt-chromium alloy copings fabricated by new manufacturing techniques and alloy systems.

STATEMENT OF PROBLEM: Although new digital manufacturing techniques are attracting interest in dentistry, few studies have comprehensively investigated the marginal fit of fixed dental prostheses fabricated with such techniques. PURPOSE: The purpose of this in vitro microcomputed tomography (µCT) study was to evaluate the marginal fit of cobalt-chromium (Co-Cr) alloy copings fabricated by casting and 3 different computer-aided design and computer-aided manufacturing (CAD-CAM)-based processing techniques and alloy systems. MATERIAL AND METHODS: Single Co-Cr metal crowns were fabricated using 4 different manufacturing techniques: casting (control), milling, selective laser melting, and milling/sintering. Two different commercial alloy systems were used for each fabrication technique (a total of 8 groups; n=10 for each group). The marginal discrepancy and absolute marginal discrepancy of the crowns were determined with µCT. For each specimen, the values were determined from 4 different regions (sagittal buccal, sagittal lingual, coronal mesial, and coronal distal) by using imaging software and recorded as the average of the 4 readings. For each parameter, the results were statistically compared with 2-way analysis of variance and appropriate post hoc analysis (using Tukey or Student t test) (α=.05). RESULTS: The milling and selective laser melting groups showed significantly larger marginal discrepancies than the control groups (70.4 ±12.0 and 65.3 ±10.1 µm, respectively; P<.001), whereas the milling/sintering groups exhibited significantly smaller values than the controls (P=.004). The milling groups showed significantly larger absolute marginal discrepancy than the control groups (137.4 ±29.0 and 139.2 ±18.9 µm, respectively; P<.05). In the selective laser melting and milling/sintering groups, the absolute marginal discrepancy values were material-specific (P<.05). Nonetheless, the milling/sintering groups yielded statistically comparable (P=.935) or smaller (P<.001) absolute marginal discrepancies to the control groups. CONCLUSIONS: The findings of this in vitro µCT study showed that the marginal fit values of the Co-Cr alloy greatly depended on the fabrication methods and, occasionally, the alloy systems. Fixed dental prostheses produced by using the milling/sintering technique can be considered clinically acceptable in terms of marginal fit.

Combinatorial mutagenesis of the voltage-sensing domain enables the optical resolution of action potentials firing at 60 Hz by a genetically encoded fluorescent sensor of membrane potential.

ArcLight is a genetically encoded fluorescent voltage sensor using the voltage-sensing domain of the voltage-sensing phosphatase from Ciona intestinalis that gives a large but slow-responding optical signal in response to changes in membrane potential (Jin et al., 2012). Fluorescent voltage sensors using the voltage-sensing domain from other species give faster yet weaker optical signals (Baker et al., 2012; Han et al., 2013). Sequence alignment of voltage-sensing phosphatases from different species revealed conserved polar and charged residues at 7 aa intervals in the S1-S3 transmembrane segments of the voltage-sensing domain, suggesting potential coil-coil interactions. The contribution of these residues to the voltage-induced optical signal was tested using a cassette mutagenesis screen by flanking each transmembrane segment with unique restriction sites to allow for the testing of individual mutations in each transmembrane segment, as well as combinations in all four transmembrane segments. Addition of a counter charge in S2 improved the kinetics of the optical response. A double mutation in the S4 domain dramatically reduced the slow component of the optical signal seen in ArcLight. Combining that double S4 mutant with the mutation in the S2 domain yielded a probe with kinetics <10 ms. Optimization of the linker sequence between S4 and the fluorescent protein resulted in a new ArcLight-derived probe, Bongwoori, capable of resolving action potentials in a hippocampal neuron firing at 60 Hz. Additional manipulation of the voltage-sensing domain could potentially lead to fluorescent sensors capable of optically resolving neuronal inhibition and subthreshold synaptic activity.

Environmental Contamination and Viral Shedding in MERS Patients During MERS-CoV Outbreak in South Korea.

BACKGROUND: Although Middle East Respiratory Syndrome coronavirus (MERS-CoV) is characterized by a risk of nosocomial transmission, the detailed mode of transmission and period of virus shedding from infected patients are poorly understood. The aims of this study were to investigate the potential role of environmental contamination by MERS-CoV in healthcare settings and to define the period of viable virus shedding from MERS patients. METHODS: We investigated environmental contamination from 4 patients in MERS-CoV units of 2 hospitals. MERS-CoV was detected by reverse transcription polymerase chain reaction (PCR) and viable virus was isolated by cultures. RESULTS: Many environmental surfaces of MERS patient rooms, including points frequently touched by patients or healthcare workers, were contaminated by MERS-CoV. Viral RNA was detected up to five days from environmental surfaces following the last positive PCR from patients' respiratory specimens. MERS-CoV RNA was detected in samples from anterooms, medical devices, and air-ventilating equipment. In addition, MERS-CoV was isolated from environmental objects such as bed sheets, bedrails, IV fluid hangers, and X-ray devices. During the late clinical phase of MERS, viable virus could be isolated in 3 of the 4 enrolled patients on day 18 to day 25 after symptom onset. CONCLUSIONS: Most of touchable surfaces in MERS units were contaminated by patients and health care workers and the viable virus could shed through respiratory secretion from clinically fully recovered patients. These results emphasize the need for strict environmental surface hygiene practices, and sufficient isolation period based on laboratory results rather than solely on clinical symptoms.