Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion

H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the ‘Eastern’ G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages

Poultry trading behaviours in Vietnamese live bird markets as risk factors for avian influenza infection in chickens

Vietnamese poultry are host to co‐circulating subtypes of avian influenza viruses, including H5N1 and H9N2, which pose a great risk to poultry productivity and to human health. AIVs circulate throughout the poultry trade network in Vietnam, with live bird markets being an integral component to this network. Traders at LBMs exhibit a variety of trading practices, which may influence the transmission of AIVs. We identified trading practices that impacted on AIV prevalence in chickens marketed in northern Vietnamese LBMs. We generated sequencing data for 31 H9N2 and 2 H5N6 viruses. Viruses isolated in the same LBM or from chickens sourced from the same province were genetically closer than viruses isolated in different LBMs or from chickens sourced in different provinces. The position of a vendor in the trading network impacted on their odds of having AIV infected chickens. Being a retailer and purchasing chickens from middlemen was associated with increased odds of infection, whereas odds decreased if vendors purchased chickens directly from large farms. Odds of infection were also higher for vendors having a greater volume of ducks unsold per day. These results indicate how the spread of AIVs is influenced by the structure of the live poultry trading network

Immune Escape Variants of H9N2 Influenza Viruses Containing Deletions at the Hemagglutinin Receptor Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic Characteristics

H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence

Inhibition of NF-kB 1 (NF-kBp50) by RNA interference in chicken macrophage HD11 cell line challenged with Salmonellaenteritidis

The NF-kB pathway plays an important role in regulating the immunity response in animals. In this study, small interfering RNAs (siRNA) were used to specifically inhibit NF-kB 1 expression and to elucidate the role of NF-kB in the signal transduction pathway of the Salmonella challenge in the chicken HD11 cell line. The cells were transfected with either NF-kB 1 siRNA, glyceraldehyde 3-phosphate dehydrogenase siRNA (positive control) or the negative control siRNA for 24 h, followed by Salmonella enteritidis (SE) challenge or non-challenge for 1 h and 4 h. Eight candidate genes related to the signal pathway of SE challenge were selected to examine the effect of NF-kB 1 inhibition on their expressions by mRNA quantification. The results showed that, with a 36% inhibition of NF-kB 1 expression, gene expression of both Toll-like receptor (TLR) 4 and interleukin (IL)-6 was consistently and significantly increased at both 1 h and 4 h following SE challenge, whereas the gene expression of MyD88 and IL-1β was increased at 1 h and 4 h, respectively. These findings suggest a likely inhibitory regulation by NF-kB 1, and could lay the foundation for studying the gene network of the innate immune response of SE infection in chickens

Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

The immunogenicity and protective efficacy of vaccines can be enhanced by the selective delivery of antigens to the antigen-presenting cells (APCs). In this study, H9N2 avian influenza virus haemagglutinin (HA) antigen, was targeted by fusing it to single-chain fragment variable (scFv) antibodies specific to CD83 receptor expressed on chicken APCs. We observed an increased level of IFNγ, IL6, IL1β, IL4, and CxCLi2 mRNA upon stimulation of chicken splenocytes ex vivo by CD83 scFv targeted H9HA. In addition, CD83 scFv targeted H9HA induced higher serum haemagglutinin inhibition activity and virus neutralising antibodies compared to untargeted H9HA, with induction of antibodies as early as day 6 post primary vaccination. Furthermore, chickens vaccinated with CD83 scFv targeted H9HA showed reduced H9N2 challenge virus shedding compared to untargeted H9HA. These results suggest that targeting antigens to CD83 receptors could improve the efficacy of poultry vaccines

Kuntosalin laitekannan kehittämissuunnitelma : case: Fitness Club Nurmijärvi

Yritysten kehittäminen on yksi kilpailukeinoista, joten tästä johtuen yritysten tulee tehdä jatkuvasti muutoksia. Muutoksilla pyritään lisäämään kannattavuutta ja samalla myös ylläpitää nykyistä tilannetta, jotta nykyiset asiakkaat saavat sen mitä he haluavat. Fitness Club Nurmijärvellä muutoksilla on vaikutusta nykyisiin asiakkaisiin ja potentiaalisiin asiakkaisiin. Muutoksilla on tarkoitus täyttää käyttäjien tarpeet ja sitä kautta sitten luoda tuottavaa arvoa asiakkaille. Tämä opinnäytetyö on tehty Fitness Club Nurmijärven toimeksiannosta. Yrityksen toiminta on kuntosaliliiketoimintaa. Se on perustettu vanhan toimijan jatkumoksi vuonna 2016. Laitekanta on siirtynyt oston mukana ja osa on jo uusia laitteita. Laitekanta on merkittävässä roolissa kuntosaliliiketoiminnassa, joten tästä johtuen siihen on tarvittu uusi kehittämissuunnitelma. Kehittämissuunnitelmalla on tarkoitus ennakoida tulevia investointeja ja samalla kehittää palvelua paremmaksi. Suunnittelulla pyritään ohjaamaan tulevaa ja samalla tutustua paremmin nykyisen asiakaskunnan tarpeisiin ja toiveisiin. Kehittämistyön avulla olen pyrkinyt löytämään keinot, joilla palvelutuotteesta saataisiin parempi. Menetelminä ovat olleet on ollut yhteiskehittely, koekäyttäminen erilaisilla harjoitusohjelmilla, vertaisanalysoiminen ja eri henkilöiden haastatteleminen. Näillä keinoilla kuntosalista pyritään luomaan parempi. Kerättyjen tietojen avulla, tarkoituksena on täyttää asiakkaiden tarpeet, jotta he tulevaisuudessakin pysyisivät asiakkaina ja samalla vaikuttaa hyvillä valinnoilla potentiaalisiin asiakkaisiin. Tavoitteena on löytää vaihtoehdot, jotka ovat taloudellisesti kannattavia investointeja. Keskeisimpänä tuloksena oli muutos käytettävyyteen ja säädettävyyteen. Käytettävyyteen vaikuttavat erilaiset pinnoitteet ja käytettävyyteen säädöt mm. ristitaljassa, jolloin eri kokoiset ihmiset pystyvät harjoittamaan lihaksia tarkoituksen mukaisilla kulmilla ja säädöillä. Suunnitelmassa on pyritty ajattelemaan kokonaisvaltaisesti, jotta muutokset eivät vaikuttaisi negatiivisesti toisiin asiakassegmentteihin.The development of companies is one of their competition tools, so that is why companies must make changes to their business. By changes companies try to make more profitability and at the same moment maintain the situation, so that current customers also get what they want. When Fitness Club Nurmijärvi makes changes, it has an effect on the current customers and on potential customers. Changes have a goal to fill user needs and that way to give productive value to customers. This Bachelor’s thesis has been made as an assignment of Fitness Club Nurmijärvi. The company´s activity is a fitness business. The company was founded in 2016 and it´s based on the continuation of the old entrepreneur´s business. Gym equipments have became new owners property by the acquisition and there are already a few new equipment. The equipment base has a remarkable role at a fitness club business, so that is the reason why Fitness Club Nurmijärvi Ltd needs a new development plan. It´s meaning is to forecast costs which are coming and at the same time develop the service. By planning, company also has a reason to control the future and the company also wants to also understand the customers´ needs and wishes. By the research, there has been tried to find ways how the service would become better. Research methods have been co-creation, testing with different variations, benchmarking and discussions with other people who are not customers. With these methods, the gym should get better. With the information company tries to fill customers needs in a way that they would stay longer time as a customers and at the same to affect potential customers by changes. The target is to find options which are good investments financially. The most remarkable result was the change to usability and adjustability. Different surfaces effects usability and adjustments for example in cross pulley, when different sized people can work their muscles with right angels which affects to adjustability. Results of thesis are based on research methods which have been chosen. There has been thought a bigger picture when I was doing a plan, so the changes wouldn´t effect negatively to other customer segments

Poultry trading behaviours in Vietnamese live bird markets as risk factors for avian influenza infection in chickens

Vietnamese poultry are host to co-circulating subtypes of avian influenza viruses, including H5N1 and H9N2, which pose a great risk to poultry productivity and to human health. AIVs circulate throughout the poultry trade network in Vietnam, with live bird markets being an integral component to this network. Traders at LBMs exhibit a variety of trading practices, which may influence the transmission of AIVs. We identified trading practices that impacted on AIV prevalence in chickens marketed in northern Vietnamese LBMs. We generated sequencing data for 31 H9N2 and two H5N6 viruses. Viruses isolated in the same LBM or from chickens sourced from the same province were genetically closer than viruses isolated in different LBMs or from chickens sourced in different provinces. The position of a vendor in the trading network impacted on their odds of having AIV-infected chickens. Being a retailer and purchasing chickens from middlemen was associated with increased odds of infection, whereas odds decreased if vendors purchased chickens directly from large farms. Odds of infection were also higher for vendors having a greater volume of ducks unsold per day. These results indicate how the spread of AIVs is influenced by the structure of the live poultry trading network

Towards the selection of chickens resistant to Salmonella and Campylobacter infections

Resistance to infection with enteric pathogens such as Salmonella and Campylobacter can be at many levels and include both non-immune and immune mechanisms. Immune resistance mechanisms can be specific, at the level of the adaptive immune response, or non-specific, at the level of the innate immune response. Whilst we can extrapolate to some degree in birds from what is known about immune responses to these pathogens in mammals, chickens are not "feathered mice", but have a different repertoire of genes, molecules, cells and organs involved in their immune response compared to mammals. Fundamental work on the chicken's immune response to enteric pathogens is therefore still required. Our studies focus particularly on the innate immune response, as responses of heterophils (the avian neutrophil equivalent) from commercial birds, and macrophages from inbred lines of chickens, correlate with resistance or susceptibility to Salmonella infection with a variety of Salmonella serovars and infection models. We work on two basic resistance mechanisms - resistance to colonization with Salmonella or Campylobacter, and resistance to systemic salmonellosis (or fowl typhoid). To map genes involved in resistance to colonization with Salmonella and Campylobacter, we are using a combination of expression quantitative trait loci (eQTLs) from microarray studies, allied with whole genome SNP arrays (WGA), a candidate gene approach and analysis of copy number variation across the genome. For resistance to systemic salmonellosis, we have refined the location ofa novel resistance locus on Chromosome 5, designated SAL1, using high density SNP panels, combined with advanced back-crossing of resistant and susceptible lines. Using a 6th generation backcross mapping population we have confirmed and refined the SAL1 locus to 8-00 kb of Chromosome 5. This region spans 14 genes, including two very striking functional candidates; CD27-binding protein (Siva) and the RAC-alpha serine/threonine protein kinase homologue, AKT1