An Optimized Real-Time PCR to Avoid Species-/Tissue-Associated Inhibition for H5N1 Detection in Ferret and Monkey Tissues

The real-time PCR diagnostics for avian influenza virus H5N1 in tissue specimens are often suboptimal, since naturally occurring PCR inhibitors present in samples, or unanticipated match of primer to unsequenced species' genome. With the principal aim of optimizing the SYBR Green real-time PCR method for detecting H5N1 in ferret and monkey (Chinese rhesus macaque) tissue specimens, we screened various H5N1 gene-specific primer pairs and tested their ability to sensitively and specifically detect H5N1 transcripts in the infected animal tissues, then we assessed RNA yield and quality by comparing Ct values obtained from the standard Trizol method, and four commonly used RNA isolation kits with small modifications, including Roche High Pure, Ambion RNAqueous, BioMIGA EZgene, and Qiagen RNeasy. The results indicated that a single primer pair exhibited high specificity and sensitivity for H5N1 transcripts in ferret and monkey tissues. Each of the four kits and Trizol reagent produced high-quality RNA and removed all or nearly all PCR inhibitors. No statistically significant differences were found between the Ct values from the isolation methods. So the optimized SYBR Green real-time PCR could avoid species- or tissue-associated PCR inhibition in detecting H5N1 in ferret and monkey tissues, including lung and small intestine

Transmission of H7N9 influenza virus in mice by different infective routes.

BackgroundOn 19 February 2013, the first patient infected with a novel influenza A H7N9 virus from an avian source showed symptoms of sickness. More than 349 laboratory-confirmed cases and 109 deaths have been reported in mainland China since then. Laboratory-confirmed, human-to-human H7N9 virus transmission has not been documented between individuals having close contact; however, this transmission route could not be excluded for three families. To control the spread of the avian influenza H7N9 virus, we must better understand its pathogenesis, transmissibility, and transmission routes in mammals. Studies have shown that this particular virus is transmitted by aerosols among ferrets.MethodsTo study potential transmission routes in animals with direct or close contact to other animals, we investigated these factors in a murine model.ResultsViable H7N9 avian influenza virus was detected in the upper and lower respiratory tracts, intestine, and brain of model mice. The virus was transmissible between mice in close contact, with a higher concentration of virus found in pharyngeal and ocular secretions, and feces. All these biological materials were contagious for naïve mice.ConclusionsOur results suggest that the possible transmission routes for the H7N9 influenza virus were through mucosal secretions and feces

The mouse and ferret models for studying the novel avian-origin human influenza A (H7N9) virus.

BackgroundThe current study was conducted to establish animal models (including mouse and ferret) for the novel avian-origin H7N9 influenza virus.FindingsA/Anhui/1/2013 (H7N9) virus was administered by intranasal instillation to groups of mice and ferrets, and animals developed typical clinical signs including body weight loss (mice and ferrets), ruffled fur (mice), sneezing (ferrets), and death (mice). Peak virus shedding from respiratory tract was observed on 2 days post inoculation (d.p.i.) for mice and 3-5 d.p.i. for ferrets. Virus could also be detected in brain, liver, spleen, kidney, and intestine from inoculated mice, and in heart, liver, and olfactory bulb from inoculated ferrets. The inoculation of H7N9 could elicit seroconversion titers up to 1280 in ferrets and 160 in mice. Leukopenia, significantly reduced lymphocytes but increased neutrophils were also observed in mouse and ferret models.ConclusionsThe mouse and ferret model enables detailed studies of the pathogenesis of this illness and lay the foundation for drug or vaccine evaluation

A single-amino-acid substitution in the HA protein changes the replication and pathogenicity of the 2009 pandemic A (H1N1) influenza viruses in vitro and in vivo

BACKGROUND: The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and since then it spread world wide over a short period of time. Although the virus infection is generally associated with mild disease and a relatively low mortality, it is projected that mutations in specific regions of the viral genome, especially within the receptor binding domain of the hemagglutinin (HA) protein could result in more virulent virus stains, leading to a more severe pandemic. RESULTS: Here, we found that a single amino acid substitution of Asp-to-Gly at position 222 in the HA protein of the A (H1N1) virus occurred after two passage propagation in the allantoic cavities of chicken embryonated eggs, and this single residue variation dramatically increased the viral replication ability in MDCK cells and pathogenicity in BALB/c mice. CONCLUSIONS: A substitution of Asp-to-Gly at position 222 in the HA protein was prone to occur under positive selection pressures, and this single amino acid mutation could dramatically increase the virus replication ability in vitro and pathogenicity in vivo. Our finding offers a better understanding of the transmission and evolution of the 2009 pandemic A (H1N1) virus and brings attention to further potentially severe influenza pandemic that may result from cross-host evolution of the influenza viruses

Identification of acetyltransferase genes (HAT1 and KAT8) regulating HBV replication by RNAi screening

Background: The initiation of hepatitis B virus (HBV) replication involves the formation of covalently closed circular DNA (cccDNA) and its transcription into pregenomic RNA (pgRNA) in hepatocyte nuclei. The regulatory mechanism of HBV replication by acetyltransferase is thus far not well understood, but human acetyltransferase has been reported as being involved in the regulation of HBV replication. Results: Depletion of KAT8 or HAT1 via RNA interference (RNAi) markedly down-regulated HBV-DNA and pgRNA levels in HepG2.2.15 cells, with KAT8 knockdown reducing both HBsAg and HBeAg more than HAT1 knockdown. Con-sistent with these observations, HBV replication regulators hepatocyte nuclear factor-4-α (HNF4α) and peroxisome proliferator-activated receptor gamma coactivator- (PPARGC-) 1-α were decreased following knockdown of HAT1 or KAT8. Conclusions: These data suggest that KAT8 or HAT1 regulate HBV replication and may be potential drug targets of anti-HBV therapy

A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms

We describe a genetic variation map for the chicken genome containing 2.8 million single-nucleotide polymorphisms ( SNPs). This map is based on a comparison of the sequences of three domestic chicken breeds ( a broiler, a layer and a Chinese silkie) with that of their wild ancestor, red jungle fowl. Subsequent experiments indicate that at least 90% of the variant sites are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about five SNPs per kilobase for almost every possible comparison between red jungle fowl and domestic lines, between two different domestic lines, and within domestic lines - in contrast to the notion that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated before domestication, and there is little evidence of selective sweeps for adaptive alleles on length scales greater than 100 kilobases

Adaption of Seasonal H1N1 Influenza Virus in Mice

The experimental infection of a mouse lung with influenza A virus has proven to be an invaluable model for studying the mechanisms of viral adaptation and virulence. The mouse adaption of human influenza A virus can result in mutations in the HA and other proteins, which is associated with increased virulence in mouse lungs. In this study, a mouse-adapted seasonal H1N1 virus was obtained through serial lung-to-lung passages and had significantly increased virulence and pathogenicity in mice. Genetic analysis indicated that the increased virulence of the mouse-adapted virus was attributed to incremental acquisition of three mutations in the HA protein (T89I, N125T, and D221G). However, the mouse adaption of influenza A virus did not change the specificity and affinity of receptor binding and the pH-dependent membrane fusion of HA, as well as the in vitro replication in MDCK cells. Notably, infection with the mouse adapted virus induced severe lymphopenia and modulated cytokine and chemokine responses in mice. Apparently, mouse adaption of human influenza A virus may change the ability to replicate in mouse lungs, which induces strong immune responses and inflammation in mice. Therefore, our findings may provide new insights into understanding the mechanisms underlying the mouse adaption and pathogenicity of highly virulent influenza viruses

Genomic Polymorphism of the Pandemic A (H1N1) Influenza Viruses Correlates with Viral Replication, Virulence, and Pathogenicity In Vitro and In Vivo

The novel pandemic A (H1N1) virus was first identified in Mexico in April 2009 and quickly spread worldwide. Like all influenzas, the H1N1 strain-specific properties of replication, virulence, and pathogenicity are a result of the particular genomic sequence and concerted expression of multiple genes. Thus, specific mutations may support increased virulence and may be useful as biomarkers of potential threat to human health. We performed comparative genomic analysis of ten strains of the 2009 pandemic A (H1N1) influenza viruses to determine whether genotypes associated with clinical phenotypes, which ranged from mild to severe illness and up to lethal. Virus replication capacity was tested for each strain in vitro using cultured epithelial cells, while virulence and pathogenicity were investigated in vivo using the BALB/c mouse model. The results indicated that A/Sichuan/1/2009 strain had significantly higher replication ability and virulence than the other strains, and five unique non-synonymous mutations were identified in important gene-encoding sequences. These mutations led to amino acid substitutions in HA (L32I), PA (A343T), PB1 (K353R and T566A), and PB2 (T471M), and may be critical molecular determinants for replication, virulence, and pathogenicity. Our results suggested that the replication capacity in vitro and virulence in vivo of the 2009 pandemic A (H1N1) viruses were not associated with the clinical phenotypes. This study offers new insights into the transmission and evolution of the 2009 pandemic A (H1N1) virus

Ionogel and polymeric ionic liquid vitrimers as healable ionically conducting coatings for electroactive smart textiles

Le développement de textiles électroactifs actionneurs et de vêtements haptiques nécessite l'élaboration de revêtements conducteurs ioniques, en tant que source d'ions nécessaire à leur fonctionnement à l'air libre. Les travaux de cette thèse visent donc à concevoir des matériaux conducteurs ioniques adaptés à l'enduction de fibres textiles avec une conductivité ionique élevée, de bonnes propriétés mécaniques et une capacité de cicatrisation grâce à la chimie des ionogels, des polymères liquides ioniques (PIL) et des vitrimères.Après un état de l'art sur les vitrimères et leurs applications comme conducteurs ioniques cicatrisables, deux mécanismes d'échange associatif dynamique ne nécessitant pas de catalyseur ont été sélectionnées :la transalkylation entre thioéther et sulfonium et l'échange associatif dynamiques des esters boroniques.Des ionogels dynamiques à base de polythioéther ont été synthétisés par addition de Michael entre un PEG diacrylate et des précurseurs di- et trithiol, en présence de 50 % en poids de liquide ionique : le 1-éthyl-3-méthylimidazolium bis(trifluorométhylsulfonyl)imide ou le 1-éthyl-3-méthylimidazolium trifluorométhanesulfonate. Les propriétés dynamiques de vitrimère ont été introduites au sein des ionogels soit par l'alkylation des groupes thioéther présents dans le réseau de polymère, soit par l'utilisation d'un monomère dithiol contenant un groupement ester boronique dynamique. Ces deux séries de ionogels vitrimères ont présenté à température ambiante des modules d'Young inférieurs à 0,9 MPa, des allongements à la rupture de l'ordre de 30 ~ 65 % et des conductivités ioniques de l'ordre de 10-4 S·cm-1. Les deux matériaux présentent les caractéristiques dynamiques attendues et se sont avérés prometteurs pour le développement de fibres actionneurs.Des réseaux dynamiques de polymères liquides ioniques, ne contenant aucune phase liquide, ont ensuite été développés. Tout d'abord, un PIL cationique linéaire présentant des cations imidazolium fixes, des groupes fonctionnels allyle pendants et des contre-ions mobiles bis(trifluoroméhylsulfonyl)imide a été synthétisé. Ce PIL linéaire a ensuite été réticulé à l'aide d'un dithiol contenant la fonction dynamique ester boronique. Des chaînes pendantes de PEG ont également été introduites pour augmenter le volume libre et pour augmenter la mobilité ionique. L'ajustement des quantités de réticulant et de chaîne pendante a permis l'obtention de matériaux étirables (E = 0,2 MPa et ε = 300 %) et hautement conducteur ioniques (σ ≈ 10-5 S·cm-1). La réaction d'échange entre les groupements esters boroniques leur a conféré des propriétés vitrimères telles que la cicatrisation, la soudure et la recyclabilité. Le processus d'enduction de ces matériaux conducteur ionique sur des fibres textiles et le développement d'actionneurs fibres fonctionnant à l'air libre ont également été démontrés.En conclusion, des ionogels vitrimères et des réseaux vitrimères de polymères liquides ioniques, peuvent être fabriqués en combinant judicieusement les avancées récentes en science des matériaux (PIL, ionogel et vitrimère) et une chimie des polymères adéquate. Leur conception ouvre des perspectives prometteuses dans différentes technologies émergentes telles que les électrolytes pour les supercondensateurs, les batteries, les piles à combustible et les revêtements ioniques pour le textile électronique.The development of smart electroactive textile actuators and haptic garments requires the elaboration of ionic coatings, acting as the ion source enabling their open-air actuation. The work of this thesis aimed at conceiving ionically conducting materials suitable for textile coating application with high ionic conductivity, good mechanical properties, and healing ability thanks to the chemistry of ionogels, polymeric ionic liquid (PIL)-based materials and vitrimers.After a state-of-the-art reviewing vitrimers and their applications as healable ion conductors, two catalyst-free vitrimer chemistries were selected to endow materials with dynamic properties, i.e., the transalkylation exchange of thioether/sulfonium and the associative exchange of boronic esters.Dynamic polythioether ionogels were obtained from thiol-ene Michael addition between diacrylate and di- and trithiol groups, in the presence of 50wt% of ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide or 1-ethyl-3-methylimidazolium trifluoromethanesulfonate. The dynamic vitrimer properties were introduced either by the alkylation of the thioether groups present along the polymer backbone thanks to an alkylating agent or by introducing a dithiol-containing boronic ester dynamic bonds. These two series of vitrimer ionogels showed at room temperature Young's modulus below 0.9 MPa, elongations at break in the range of 30 ~ 65 %, ionic conductivities in the order of 10-4 S·cm-1. Both materials show dynamic features, and they were demonstrated to be promising for developing yarn actuators.Then, liquid-free dynamic ionically conducting materials based on PILs were developed. First, a PIL with a backbone containing imidazolium cations and pendant allyl functional groups with bis(trifluoromehylsulfonyl)imide counter ions was synthesized. This linear PIL was subsequently crosslinked with a dithiol-containing boronic ester crosslinker. PEG dangling chains were also introduced to increase the free volume and to increase ionic mobility. Adjusting the crosslinker and dangling chain quantities yielded soft (E = 0.2MPa), stretchable (ε = 300%), and highly conducting ionic membranes (σ ≈ 10-5 S·cm-1). The exchange reaction between boronic ester groups endowed these materials with vitrimer properties such as healing, welding and recyclability. The dip-coating process on yarns and the development of dual-electrodes actuators with these materials were also demonstrated.In conclusion, dynamic ionogels and liquid-free ionically conducting polymers can be fabricated by combining wisely recent achievements in material science (PIL, ionogel and vitrimer) and adequate polymer chemistry. Their design opens promising perspectives in different emerging technologies as electrolytes for supercapacitors, batteries, fuel cells and ionic coatings for electroactive e-textile