Role of CIV NS1 Protein in Innate Immunity and Viral Replication.

The innate immune pathway serves as the first line of defense against viral infections and plays a crucial role in the host's immune response in clearing viruses. Prior research has indicated that the influenza A virus has developed various strategies to avoid host immune responses. Nevertheless, the role of the NS1 protein of the canine influenza virus (CIV) in the innate immune pathway remains unclear. In this study, eukaryotic plasmids of NS1, NP, PA, PB1, and PB2 were constructed, and it was found that these proteins interact with melanoma differentiation-associated gene 5 (MDA5) and antagonize the activation of IFN-ß promoters by MDA5. We selected the NS1 protein for further study and found that NS1 does not affect the interaction between the viral ribonucleoprotein (RNP) subunit and MDA5, but that it downregulates the expression of the laboratory of genetics and physiology 2 (LGP2) and retinoic acid-inducible gene-I (RIG-I) receptors in the RIG-I pathway. Additionally, NS1 was found to inhibit the expression of several antiviral proteins and cytokines, including MX dynamin like GTPase 1 (MX1), 2'-5'oligoadenylate synthetase (OAS), Signal Transducers and Activators of Transcription (STAT1), tripartite motif 25 (TRIM25), interleukin-2 (IL-2), IFN, IL-8, and IL-1ß. To further investigate the role of NS1, a recombinant H3N2 virus strain (rH3N2) and an NS1-null virus (rH3N2ΔNS1) were rescued using reverse-genetic technology. The rH3N2ΔNS1 virus exhibited lower viral titers compared to rH3N2, but had a stronger activation effect on the receptors LGP2 and RIG-I. Furthermore, when compared to rH3N2, rH3N2ΔNS1 exhibited a more pronounced activation of antiviral proteins such as MX1, OAS, STAT1, and TRIM25, as well as antiviral cytokines such as IL-6, IFN-ß, and IL-1ß. These findings suggest a new mechanism by which NS1, a nonstructural protein of CIV, facilitates innate immune signaling and provides new avenues for the development of antiviral strategies.

Psychometric evaluation of critical incident video instruments for nursing education.

The Critical Incident Video (CIV) Project is a mixed method longitudinal study that uses CIVs to prepare nursing faculty members to address common teaching challenges. CIVs are short videos that present unresolved teaching challenges. Given the lack of specific instruments for evaluating the usefulness of CIVs, the aim of this phase of the project was to develop and test two instruments measuring the pedagogical effects of CIVs. The CIV Preparation and Confidence Scale (CIVPCS©) and the CIV Simulation Experience Scale (CIVSES©) were assessed for validity and reliability. Using a Delphi method, a convenience sample of 23 nurse educators provided feedback enhancing the validity and clarity of the CIVPCS©. Reliability of the CIVPCS© was determined using Cronbach's alpha and test-retest method. No changes were recommended for the CIVSES©. Findings from the assessment of these newly developed CIV instruments are reported and implications for faculty development are discussed.

Proteomic Analysis Reveals That Mitochondria Dominate the Hippocampal Hypoxic Response in Mice.

Hypoxic stress occurs in various physiological and pathological states, such as aging, disease, or high-altitude exposure, all of which pose a challenge to many organs in the body, necessitating adaptation. However, the exact mechanisms by which hypoxia affects advanced brain function (learning and memory skills in particular) remain unclear. In this study, we investigated the effects of hypoxic stress on hippocampal function. Specifically, we studied the effects of the dysfunction of mitochondrial oxidative phosphorylation using global proteomics. First, we found that hypoxic stress impaired cognitive and motor abilities, whereas it caused no substantial changes in the brain morphology or structure of mice. Second, bioinformatics analysis indicated that hypoxia affected the expression of 516 proteins, of which 71.1% were upregulated and 28.5% were downregulated. We demonstrated that mitochondrial function was altered and manifested as a decrease in NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 expression, accompanied by increased reactive oxygen species generation, resulting in further neuronal injury. These results may provide some new insights into how hypoxic stress alters hippocampal function via the dysfunction of mitochondrial oxidative phosphorylation.

Phylogenetic analysis and evolutionary dynamics of H3N2 canine and feline influenza virus strains from 2006 to 2019.

H3N2 feline influenza virus (FIV) and canine influenza virus (CIV) are very common in cats and dogs. Due to the ability of the influenza virus to spread across hosts and frequent contact between pets and people, there exist huge public health problems. In this study, we collected H3N2 CIV and FIV genomes from 2006 to 2019 from NCBI and analyzed the evolutionary dynamics and molecular variation using a series of phylogenetic analysis methods. Results indicated that H3N2 FIVs were closely related to CIVs with high posterior probability and CIVs and FIVs have certain regional characteristics. However, compared with previous studies, the significance of geographical structure correlation decreased. Furthermore, we also found that the intrasubtypic reassortment between FIVs and CIVs were common during epidemics. The integrated analysis was also performed for different selection pressure acting on HA (566 codons), NA (469 codons), M1 (252 codons), and M2 (97 codons) proteins. One HA, two NA, three M1, and two M2 sites were found under positive selection. We subsequently performed the evolutionary dynamics of H3N2 CIV. The results indicated that the time of the most recent common ancestor of CIV H3N2 may have occurred earlier than indicated in a previous study. The Bayesian skyline plot analysis in this study showed the period of divergence of major H3N2 CIVs segments occurred between 2008 and 2010. Notably, according to our research, the PB1 has experienced two divergence periods (2006-2008 and 2009-2011).

Epidemiological survey and genetic evolution of H3N2 subtype influenza viruses from stray dogs in Shanghai, China.

An avian-origin canine influenza virus (CIV) has recently emerged in dogs and is spreading in China. Given that humans have frequent contact with dogs, this has prompted an increased emphasis on biosafety. In this study, we collected 693 nasal swab samples and 800 blood samples from stray dogs in animal shelters to survey canine influenza epidemiology and characterize the evolution of CIV H3N2 in Shanghai. We tested samples for canine influenza antibodies and canine influenza RNA in January-May, 2019, and the results showed that the positive rate was 17.62% by ELISA, 15.75% by microneutralization (MN) assay, and 18.51% by real time RT-PCR, respectively. We also performed phylogenetic and genomic analysis on six H3N2 CIV isolates. The H3N2 viruses which prevailed in Shanghai originated from Beijing and Jiangsu isolates. Phylogenetic analysis showed that the sequences of CIV isolates have multiple amino acid antigenic drifts, deletions, and substitutions. The time of the most recent common ancestor (TMRCA) of HA and NA was 2004 and 2005, respectively. Notably, the substitution, 146S, in hemagglutinin and the deletion in the neuraminidase (NA) stalk region we found in this study warrant attention because they have frequently been identified in human influenza viruses. The potential adaptation of this CIV H3N2 clade to mammals and its public health threat should be further evaluated.

May-Thurner syndrome: an uncommon and incidental finding in a postpartum female.

May-Thurner syndrome or Cockett syndrome is a pathological condition that arises due to extrinsic compression on iliocaval venous territory, leading to venous outflow obstruction. Here, author presents an incidental finding of left common iliac vein extrinsic compression by right common iliac artery with collateral vessels in the pelvis in a postpartum female.

Assessment of Molecular, Antigenic, and Pathological Features of Canine Influenza A(H3N2) Viruses That Emerged in the United States.

Background: A single subtype of canine influenza virus (CIV), A(H3N8), was circulating in the United States until a new subtype, A(H3N2), was detected in Illinois in spring 2015. Since then, this CIV has caused thousands of infections in dogs in multiple states. Methods: In this study, genetic and antigenic properties of the new CIV were evaluated. In addition, structural and glycan array binding features of the recombinant hemagglutinin were determined. Replication kinetics in human airway cells and pathogenesis and transmissibility in animal models were also assessed. Results: A(H3N2) CIVs maintained molecular and antigenic features related to low pathogenicity avian influenza A(H3N2) viruses and were distinct from A(H3N8) CIVs. The structural and glycan array binding profile confirmed these findings and revealed avian-like receptor-binding specificity. While replication kinetics in human airway epithelial cells was on par with that of seasonal influenza viruses, mild-to-moderate disease was observed in infected mice and ferrets, and the virus was inefficiently transmitted among cohoused ferrets. Conclusions: Further adaptation is needed for A(H3N2) CIVs to present a likely threat to humans. However, the potential for coinfection of dogs and possible reassortment of human and other animal influenza A viruses presents an ongoing risk to public health.

Spread of Canine Influenza A(H3N2) Virus, United States.

A canine influenza A(H3N2) virus emerged in the United States in February-March 2015, causing respiratory disease in dogs. The virus had previously been circulating among dogs in Asia, where it originated through the transfer of an avian-origin influenza virus around 2005 and continues to circulate. Sequence analysis suggests the US outbreak was initiated by a single introduction, in Chicago, of an H3N2 canine influenza virus circulating among dogs in South Korea in 2015. Despite local control measures, the virus has continued circulating among dogs in and around Chicago and has spread to several other areas of the country, particularly Georgia and North Carolina, although these secondary outbreaks appear to have ended within a few months. Some genetic variation has accumulated among the US viruses, with the appearance of regional-temporal lineages. The potential for interspecies transmission and zoonotic events involving this newly emerged influenza A virus is currently unknown.

Identification and function analysis of canine stimulator of interferon gene (STING).

Stimulator of interferon gene (STING) plays an important role in the cyclic GMP-AMP synthase (cGAS)-mediated activation of type I IFN responses. In this study, we identified and cloned canine STING gene. Full-length STING encodes a 375 amino acid product that shares the highest similarity with feline STING. Highest levels of mRNA of canine STING were detected in the spleen and lungs while the lowest levels in the heart and muscle. Analysis of its cellular localization showed that STING is localizes to the endoplasmic reticulum. STING overexpression induced the IFN response via the IRF3 and NF-κB pathways and up-regulated the expression of ISG15 and viperin. However, knockdown of STING did not inhibit the IFN-ß response triggered by poly(dA:dT), poly(I:C), or SeV. Finally, overexpression of STING significantly inhibited the replication of canine influenza virus H3N2. Collectively, our findings indicate that STING is involved in the regulation of the IFN-ß pathway in canine.

Multiplex RT-PCR detection of H3N2 influenza A virus in dogs.

A multiplex RT-PCR (mRT-PCR) assay to detect H3N2 CIV genomic segments was developed as a rapid and cost-effective method. Its performance was evaluated with forty-six influenza A viruses from different hosts using three primer sets which amplify four segments of H3N2 CIV simultaneously. The mRT-PCR has been successful in detecting the viral segments, indicating that it can improve the speed of diagnosis for H3N2 CIV and its reassortants.

Molecular analyses of H3N2 canine influenza viruses isolated from Korea during 2013-2014.

Canine influenza A virus (CIV) causes a respiratory disease among dog populations and is prevalent in North America and Asia. Recently, Asian H3N2 CIV infection has been of particular concern, with recent reports related to reassortants with pandemic 2009 strains, direct transmission from a human H3N2, a possibility of H3N2 CIV transmission to other mammals, and even the first outbreak of H3N2 CIVs in North America in April 2015. However, despite these global concerns, our understanding of how influenza A virus transmission impacts the overall populations of H3N2 CIVs remains incomplete. Hence, we investigated the evolutionary history of the most recent two Korean CIV isolates, A/canine/Korea/BD-1/2013 and A/canine/Korea/DG1/2014, along with 57 worldwide CIVs, using comprehensive molecular analyses based on genomic genotyping. This study presents that the new Korean CIV isolates are closely related to the predominantly circulating H3N2 CIVs with genotypes K, G, E, 3B, F, 2D, F, and 1E, carrying several mutations in antigenic and host determinant sites. Also, our findings show that the genome-wide genetic variations within the H3N2 CIVs are low; however, two antigenic protein (HA and NA) analysis demonstrates genetic diversification of the H3N2 CIVs, which evolves independently between Korea and China.

Enhanced insecticidal activity of Chilo iridescent virus expressing an insect specific neurotoxin.

Previously we have generated a recombinant Chilo iridescent virus (CIV) by inserting the green fluorescent protein gene (gfp) into the CIV 157L open reading frame (ORF) locus and showed that this recombinant (rCIV-Δ157L-gfp) was fully infectious both in cell culture as well as in insect larvae. This study opened up a new avenue for increasing the speed of kill of CIV and other iridoviruses by inserting virulence or toxin genes into the viral genome. In the current study we constructed a recombinant CIV (rCIV-Δ157L/gfp-AaIT) where the 157L ORF was replaced with both the AaIT neurotoxin gene from the scorpion Androctonus australis and the gfp gene, each under control of the viral major capsid protein (mcp) gene promoter. Recombinant virus was purified by successive rounds of plaque purification using Spodoptera frugiperda (Sf-9) cells. One-step growth curves for the recombinant viruses, rCIV-Δ157L/gfp-AaIT and rCIV-Δ157L-gfp, and wild-type CIVs in Sf-9 cells showed similar profiles. AaIT toxin expression in infected third instar Galleria mellonella larvae was confirmed by western blot analysis using an antibody against the AaIT protein. rCIV-Δ157L/gfp-AaIT infection at a concentration that kills 100% of the larvae caused paralysis in infected third instar G. mellonella larvae from two days after injection, whereas infection with non-AaIT containing viruses showed mortality starting much later (>10days). Bioassays on these larvae demonstrated that the speed of kill of CIV carrying AaIT was strikingly enhanced as compared to wild-type CIV. These results suggest that insertion of a toxin gene into CIV provides further opportunities to control a wide range of pest insects, such as weevils, using an iridovirus.

Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study.

In this mini review, we briefly survey the molecular processes that lead to reactive oxygen species (ROS) production by the respiratory complex III (CIII or cytochrome bc1). In particular, we discuss the "forward" and "reverse" electron transfer pathways that lead to superoxide generation at the quinol oxidation (Qo) site of CIII, and the components that affect these reactions. We then describe and compare the properties of a bacterial (Rhodobacter capsulatus) mutant enzyme producing ROS with its mitochondrial (human cybrids) counterpart associated with a disease. The mutation under study is located at a highly conserved tyrosine residue of cytochrome b (Y302C in R. capsulatus and Y278C in human mitochondria) that is at the heart of the quinol oxidation (Qo) site of CIII. Similarities of the major findings of bacterial and human mitochondrial cases, including decreased catalytic activity of CIII, enhanced ROS production and ensuing cellular responses and damages, are remarkable. This case illustrates the usefulness of undertaking parallel and complementary studies using biologically different yet evolutionarily related systems, such as α-proteobacteria and human mitochondria. It progresses our understanding of CIII mechanism of function and ROS production, and underlines the possible importance of supra-molecular organization of bacterial and mitochondrial respiratory chains (i.e., respirasomes) and their potential disease-associated protective roles. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Respiratory complex III dysfunction in humans and the use of yeast as a model organism to study mitochondrial myopathy and associated diseases.

The bc1 complex or complex III is a central component of the aerobic respiratory chain in prokaryotic and eukaryotic organisms. It catalyzes the oxidation of quinols and the reduction of cytochrome c, establishing a proton motive force used to synthesize adenosine triphosphate (ATP) by the F1Fo ATP synthase. In eukaryotes, the complex III is located in the inner mitochondrial membrane. The genes coding for the complex III have a dual origin. While cytochrome b is encoded by the mitochondrial genome, all the other subunits are encoded by the nuclear genome. In this review, we compile an exhaustive list of the known human mutations and associated pathologies found in the mitochondrially-encoded cytochrome b gene as well as the fewer mutations in the nuclear genes coding for the complex III structural subunits and accessory proteins such as BCS1L involved in the assembly of the complex III. Due to the inherent difficulties of studying human biopsy material associated with complex III dysfunction, we also review the work that has been conducted to study the pathologies with the easy to handle eukaryotic microorganism, the yeast Saccharomyces cerevisiae. Phenotypes, biochemical data and possible effects due to the mutations are also discussed in the context of the known three-dimensional structure of the eukaryotic complex III. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system.

Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in 'broken mitochondria' (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.

Rapid diagnosis of canine respiratory coronavirus, canine influenza virus, canine distemper virus and canine parainfluenza virus with a Taqman probe-based multiplex real-time PCR.

Canine Infectious Respiratory Disease Complex (CIRDC) is a highly infectious diseases. Canine respiratory coronavirus (CRCoV), Canine influenza virus (CIV), Canine distemper virus (CDV), and Canine parainfluenza virus (CPiV) are crucial pathogens causing CIRDC. Due to the similar clinical symptoms induced by these viruses, differential diagnosis based solely on symptoms can be challenging. In this study, a multiplex real-time PCR assay was developed for detecting the four RNA viruses of CIRDC. Specific primers and probes were designed to target M gene of CRCoV, M gene of CIV, N gene of CDV and NP gene of CPiV. The detection limit is 10 copies/µL for CIV or CRCoV, while the detection limit of CDV or CPiV is 100 copies/µL. Intra-group and inter-group repeatability coefficient of variation (CV) were both less than 2 %. A total of 341 clinical canine samples were analyzed, and the results indicated that the method developed in our study owns a good consistency and better specificity compared with the conventional reverse transcription PCR. This study provides a new method to enable the simultaneous detection of all four pathogens in a single reaction, improving the efficiency for monitoring the prevalence of four viruses in CIRDC, which benefits the control of CIRDC.

Identification of Two Isoforms of Canine Tetherin in Domestic Dogs and Characterization of Their Antiviral Activity against Canine Influenza Virus.

Canine influenza virus (CIV) significantly threatens the canine population and public health. Tetherin, an innate immune factor, plays an important role in the defense against pathogen invasion and has been discovered to restrict the release of various enveloped viruses. Two isoforms of canine tetherin (tetherin-X1 and tetherin-X2) were identified in peripheral blood leukocytes of mixed-breed dogs using reverse transcription polymerase chain reaction (RT-PCR). Amino acid alignment revealed that relative to full-length tetherin (tetherin-X1) and truncated canine tetherin (tetherin-X2) exhibited deletion of 34 amino acids. The deletion occurred at the C-terminus of the coiled-coiled ectodomain and the N-terminus of the glycosylphosphatidylinositol (GPI)-anchor domain. Tetherin-X2 was localized subcellularly at the cell membrane, which was consistent with the localization of tetherin-X1. In addition, canine tetherin-X1 and tetherin-X2 restricted the release of H3N2 CIV. However, canine tetherin-X1 had higher antiviral activity than canine tetherin-X2, indicating that the C-terminus of the coiled-coiled ectodomain and the N-terminus of the GPI-anchor domain of canine tetherin (containing the amino acids deleted in tetherin-X2) are critical for its ability to restrict H3N2 CIV release. This study provides insights for understanding the key functional domains of tetherin that restrict CIV release.

[Tetralogy of Fallot with absent pulmonary valve syndrome diagnosed in adulthood by infective endocarditis : Case of report]. / Agénésie de la valve pulmonaire avec communication interventriculaire découverte à l'âge adulte suite à une endocardite infectieuse : à propos d'un cas.

Absent pulmonary valve syndrome is a rare congenital heart disease. Associated with ventricular septal defect, it is considered a rare variant of Tetralogy of Fallot "Tetralogy of Fallot with absent pulmonary valve syndrome". It is characterized by its association with aneurysmal pulmonary arteries responsible for airways compression. Survival to adulthood of this unrepaired congenital heart disease is very rare, and the case of the patient we report in this article is added to the rare cases reported in the literature. Clinical tolerance depends on the degree of severity of the malformation and in particular on the importance of the aneurysmal dilation of the pulmonary arteries, thus determining the age of the diagnosis, the severity of symptoms, and the mode of evolution. Diagnosis of Tetralogy of Fallot with absent pulmonary valve syndrome must be established by transthoracic echography. Other investigations can be of capital contribution, such as thoracic computed tomography angiography and cardiac catheterization. The treatment is surgical and includes closure of the ventricular septal defect, relieve right ventricular outflow tract obstruction, and surgical reduction of the aneurysmal pulmonary arteries.

The accelerated evolution of human cytochrome c oxidase - Selection for reduced rate and proton pumping efficiency?

The cytochrome c oxidase complex, complex VI (CIV), catalyzes the terminal step of the mitochondrial electron transport chain where the reduction of oxygen to water by cytochrome c is coupled to the generation of a protonmotive force that drive the synthesis of ATP. CIV evolution was greatly accelerated in humans and other anthropoid primates and appears to be driven by adaptive selection. However, it is not known if there are significant functional differences between the anthropoid primates CIV, and other mammals. Comparison of the high-resolution structures of bovine CIV, mouse CIV and human CIV shows structural differences that are associated with anthropoid-specific substitutions. Here I examine the possible effects of these substitutions in four CIV peptides that are known to affect proton pumping: the mtDNA-coded subunits I, II and III, and the nuclear-encoded subunit VIa2. I conclude that many of the anthropoid-specific substitutions could be expected to modulate the rate and/or the efficiency of proton pumping. These results are compatible with the previously proposed hypothesis that the accelerated evolution of CIV in anthropoid primates is driven by selection pressure to lower the mitochondrial protonmotive force and thus decrease the rate of superoxide generation by mitochondria.

H3N2 canine influenza virus NS1 protein inhibits canine NLRP3 inflammasome activation.

Inflammation is an innate immune response of the body against pathogens and other irritants. The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome is a major player in the inflammatory response against pathogenic microorganisms. In this study, we analyzed the relationship between the NLRP3 inflammasome and the influenza virus NS1 protein, which is involved in host immune escape. The canine influenza virus NS1 protein transcriptionally attenuated proinflammatory cytokines by inhibiting the nuclear factor-κB (NF-κB) activator. NS1 also directly interacted with NLRP3 and blocked ASC (Apoptosis-associated speck-like protein containing CARD) oligomerization, which deactivated the NLRP3 inflammasome. In addition, NS1 inhibited pro-caspase 1 cleavage into caspase-1, which prevents maturation of IL-1ß and IL-18 from their respective precursors, eventually reducing the inflammatory response. Taken together, the influenza NS1 protein evades host immunity, and our findings provide a theoretical basis for the prevention and treatment of canine influenza.