However, a direct comparison of the contribution of these residues to viral attenuation, immunogenicity and protection efficacy has not been conducted. Here, we compared the In vitro and in vivo phenotype of recombinant influenza A/Puerto Rico/8/34 H1N1 (PR8) viruses containing the ts, ca and att mutations of the US (PR8/AA) and the Russian (PR8/Len) MDVs. Our results show that PR8/Len is more attenuated in vivo than PR8/AA, although both viruses induced similar levels of humoral and cellular responses, and protection against homologous and heterologous viral challenges. Our findings support the feasibility of using a different virus backbone as MDV for the development of improved LAIVs for the prevention of IAV infections.Influenza viruses cause annual, seasonal infection across the globe. Vaccination represents the most effective strategy to prevent such infections and/or to reduce viral disease. Two major types of influenza vaccines are approved for human use: inactivated influenza vaccines (IIVs) and live attenuated influenza vaccines (LAIVs). Two Master Donor Virus (MDV) backbones have been used to create LAIVs against influenza A virus (IAV): the United States (US) A/Ann Arbor/6/60 (AA) and the Russian A/Leningrad/134/17/57 (Len) H2N2 viruses. The mutations responsible for the temperature sensitive ( In the United States (US), the Centers for Disease Control and Prevention (CDC) has estimated that influenza has caused between 9.3 and 49.0 million illnesses each year, since 2010- along with between 12,000 and 79,000 deaths annually [Vaccination is the most cost-effective approach to prevent influenza infections . Currentts) viruses that replicate efficiently at low temperatures  but not at elevated temperatures ; such viruses also possess an attenuated phenotype (att) in vivo [LAIVs have been generated by passaging influenza viruses at gradually reduced temperatures in eggs or cultured cells to generate temperature sensitive .The traditional methods for generating seasonal LAIVs is based on the co-infection of embryonated chicken eggs with a Master Donor Virus (MDV) and the seasonal virus recommended by the CDC and international health agencies to be part of the vaccine . The virts, ca and att phenotype have been mapped to PB2 (N265S), PB1  and NP (D34G) [ts, ca and att phenotype have been mapped to PB2 (V478L), PB1 (K265N and V591I) and NEP (M100I) [ts, ca and att phenotypes as that of the original US AA MDV. However, introduction of the mutations of the US AA MDV into the pandemic A/California/04/09 H1N1 virus  resulted in reduced ts and ca in vitro and limited attenuation in vivo [ts, ca and att phenotypes induced by the mutations of the US AA MDV are influenced by the virus backbone into which they are introduced. Although some groups have generated recombinant viruses by using the internal genes of AA or the Len (with the LAIV mutations) as MDVs with different HA and NA combinations [Two approaches have been used to develop seasonal IAV LAIVs that differ in the MDV: (1) the US A/Ann Arbor/6/60 H2N2 LAIV (AA) that is commercialized under the names of FluMist in the US and Canada, and Fluenz in the European Union; and (2) the Russian A/Leningrad/134/17/57 H2N2 LAIV (Len) that is commercialized under the names Ultravac (Russia) and Nasovac-S (India)  and alsoP (D34G) ,33. In t (M100I) ,36, A/ca (M100I) ,12,21 or in vivo ,38,39. Tinations ,41,42, aHere, we compared the contribution of the mutations present in the US AA and the Russian Len MDVs using the backbone of influenza PR8 In vitro and in vivo. Our results show that PR8 containing the mutations of the Russian Len MDV (PR8/Len) is more attenuated in vivo than the PR8 containing the mutations of the US AA MVD (PR8/AA). However, both PR8/AA and PR8/Len induced similar levels of humoral and cellular responses and both induce protection against homologous  and partially heterologous  viral challenges. Collectively, these findings support the feasibility of using the mutations of the US AA or the Russian Len MDVs in the PR8 virus backbone for the development of novel and improved LAIVs for the prevention of IAV infections. The use of circulating IAVs as MDVs in new LAIVs could grant a higher level of protection by the induction of more robust cellular immune responses against internal viral proteins . In addition, our findings have an important impact on the development and implementation of LAIVs with high levels of attenuation.l-glutamine, 2 mM) at 37 °C with 5% CO2 as described [Human embryonic kidney 293T  and Madin-Darby canine kidney  cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS), and 1% PSG  was generated using previously described plasmid-based reverse techniques [ts, ca, and att mutations of the USA/Ann Arbor/6/60 H2N2 (PR8/AA) are described elsewhere [The recombinant A/Puerto Rico/8/34 H1N1 (PR8) virus containing the chniques . The reclsewhere ,44. All lsewhere ,46.N-tosyl-l-phenylalanine chloromethyl ketone (TPCK)-treated trypsin (Sigma). Viral titers were determined by immunofocus assay  in MDCK cells at 33 °C using a monoclonal antibody (mAb) against influenza virus nucleoprotein, NP ; as described [For viral infections, viral stocks were diluted in phosphate buffered saline (PBS) containing 0.3% bovine albumin (BA) and 1% penicillin and streptomycin (PS) (PBS/BA/PS). After 1 h viral adsorption at room temperature (RT), MDCK cells were maintained in post-infection (p.i.) DMEM media supplemented with 0.3% BA, 1% PSG, and 1 μg/mL of escribed .ts, ca and att phenotypes of the MDV A/Leningrad/134/17/57 H2N2 LAIV: PB2 V478L; PB1 K265N and V591I; and NEP M100I. Mutations were confirmed by sequencing (ACGT). Mutated PB2, PB1 and NS PR8/Len viral segments were subcloned from the shuttle pUC19 vector into the ambisense pDZ plasmid for virus rescue with the rest of the PR8 viral genes [ts, ca and att mutations of the MDV A/Ann Arbor/6/60 H2N2 LAIV: PB2 N265S; PB1 K391E, E581G and A661T were previously described [To generate PR8/Len, the PR8/WT PB2, PB1 and NS viral segments were subcloned into a pUC19 plasmid (New England BioLabs) to introduce, using site-directed mutagenesis, the mutations responsible for the al genes ,47. The escribed .5 cells/well, triplicates). Cells were co-transfected in suspension, using Lipofectamine 2000 , with 250 ng of the indicated combinations of ambisense pDZ-PB2 , pDZ-PB1 , pDZ-PA and pDZ-NP plasmids, together with 500 ng of two reporter viral (v)RNA-like expression plasmids encoding the GFP and Gluc driven by a human RNA polymerase I promoter , and 100 ng of a reporter plasmid expressing Cypridina luciferase (Cluc) under the control of the constitutively active simian virus 40 (SV40) promoter (pSV40-Cluc) to normalize transfection efficiencies. Cells transfected in the absence of pDZ-NP were included as a negative control. Empty pDZ plasmid was used to keep the amount of transfected plasmid DNA constant in the negative control. At 24 h post-transfection (p.t.), cells were analyzed for GFP expression and photographed using a fluorescent microscope (Olympus IX81) and camera ; Gluc and Cluc expression levels were determined in tissue culture supernatants (TCS) using the BioluxGaussia and Cypridina Luciferase Assay kits (New England BioLabs) and quantified with a Lumicount luminometer (Packard). Reporter Gluc gene activation was normalized to that of Cluc and shown as fold induction over the level of induction of the negative control (no pDZ-NP). Mean values and standard deviations (SDs) were calculated and statistical analysis was performed using a two-tailed Student t-test with Microsoft Excel software. Data are represented as relative activity considering the activity of each polymerase complex combination at 33 °C as 100%.The ability of PR8/WT, PR8/AA and PR8/Len viral polymerase complexes to replicate and transcribe at different temperatures  were evaluated in HEK293T cells  were co-transfected in suspension, using Lipofectamine 2000, with 1 μg of the eight-ambisense PR8 pDZ-PB2-Len, -PB1-Len, -PA, -HA, -NP, -NA, -M, and -NS-Len plasmids. At 12 h p.t., medium was replaced with p.i. medium and TCS were collected at three days p.t., clarified, and used to infect fresh monolayers of MDCK cells. After three days p.i., PR8-Len virus was plaque purified and scaled up using MDCK cells at 33 °C, as described [PR8/Len virus rescue was performed as described for PR8/WT and PR8/AA viruses ,12,43,465 cells/well, triplicates) with PR8/WT, PR8/AA and PR8/Len at a multiplicity of infection (MOI) of 0.001. After 1 h of viral adsorption at RT, infection medium was replaced by p.i. medium and plates were incubated at different temperatures . TCS were collected at the indicated times p.i. and viral titers were determined by immunofocus assay (FFU/mL) in MDCK cells as indicated before [Multicycle growth kinetics were carried out by infecting MDCK cells  were infected with PR8/WT, PR8/AA and PR8/Len for 1 h at RT, overlaid with agar, and incubated at 33 °C, 37 °C, or 39 °C [Monolayers of MDCK cells  were anesthetized intraperitoneally (i.p.) with 2,2,2-tribromoethanol  and inoculated intranasally (i.n.) with 30 μL of a virus preparation containing the indicated doses of virus diluted in PBS [50) was determined using the Reed and Muench method [N = 6) were anesthetized and infected as described and viral titers in homogenized lungs at 2 (N = 3) and 4 (N = 3) days p.i. were determined by immunofocus assay (FFU/mL) as previously described in MDCK cells at 33 °C [Six-to-eight-week-old female C57BL/6 mice were purchased from the National Cancer Institute (NCI) and maintained in the animal care facility at the University of Rochester under specific pathogen-free conditions. All animal protocols were approved by the University Committee of Animal Resources  and complied with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Research Council . To evald in PBS . Mice wed in PBS . The 50%at 33 °C ,13,21,49N = 5) were anesthetized and vaccinated i.n. with the indicated doses and viruses. A group of mice (N = 5) was also mock vaccinated with PBS as negative control. At fourteen days post-vaccination, mouse sera were collected by submandibular bleeding to evaluate the presence of total influenza antibodies (Abs) by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibiting antibodies by hemagglutination inhibition (HAI) assay [50) of WT PR8. For the heterologous challenge, since the majority of the neutralizing antibodies induced after LAIV vaccination are directed against the viral surface HA and NA glycoproteins that are different between our vaccine PR8 viruses and the challenge X31 virus, a low viral dose (50 MLD50) was used in these experiments.For the vaccination and challenge experiments, 6–8-week-old female C57BL/6 mice (I) assay . Twenty-We evaluated the levels of total virus-specific Abs in the sera of vaccinated mice by ELISA as described ,46,49,51N = 5) were calculated from the last well where hemagglutination was inhibited [To evaluate the presence of hemagglutination inhibiting antibodies, mouse sera were treated with receptor-destroying enzyme  for 16 h at 37 °C and heat inactivated for 30 min at 56 °C. Then, sera were serially 2-fold diluted (starting dilution of 1:50) in 96-well V-bottom plates and mixed 1:1 with 4 hemagglutinating units (HAU) of the indicated viruses during 30 min at RT. HAI titers were determined by adding 0.5% turkey red blood cells to the virus-Ab mixtures followed by a 30 min incubation on ice . The geonhibited .3 FFU of PR8/AA or with 103 and 104 FFU of PR8/Len, or mock- vaccinated with PBS. At 10 days p.i., lungs and spleens were collected and processed to obtain cellular preparations. Lungs were perfused with PBS before being surgically removed and dissociated in Gentle MACS (MiltenyiBiotek) C tubes using the Lung01 program. Samples were incubated in 5 mL (2 μg/mL) of Collagenase II in RPMI-1640 medium containing 8% FBS for 30 min at 37 °C with gentle agitation every 10 min. After digestion, samples were further dissociated using the Heart01 program. Cell suspensions were filtered through 70 μm filters prior to 75:40 Percoll  discontinuous gradient separation. The top layer (containing fat and other debris) was eliminated by aspiration. The cell layer was then harvested and washed, prior to counting and staining. Single-cell suspensions were prepared from collected spleens by disruption in RPMI +8% FBS; yields were quantitated by viable cell counts using Trypan blue exclusion on a hemocytometer.Six-to-eight-week-old female C57BL/6 mice (N = 5) were anesthetized and immunized with 10Single cell suspensions were prepared for Flow cytometry analysis by staining in PBS containing 1% FBS, purified CD16/32 (clone 2.4G2), NP and PA tetramers, and the following antibodies: TCRβ-PerCPCy5.5, CD8α-FITC, CD4-BV421, CD44-APCCy7, and CD62L-PECy7. Cells were subsequently stained for viability using Live Dead Aqua (Invitrogen). All antibodies were obtained from eBioscience, BD Biosciences, or Biolegend. PA and NP tetramers ,12,25,52ts, ca and att phenotypes of the US AA MDV [In a previous work , we intrS AA MDV ,33 into S AA MDV .ts, ca and att mutations of the Russian Len MDV [To evaluate if the  Len MDV  resultedts phenotype effect than the AA PB2 mutations. In the case of PR8/Len, both the PB1 and PB2 segments induced a similar decrease in GFP and Gluc expression at 37 °C and 39 °C  have the aforementioned mutations.Both of the commercial/licensed lineages of LAIV (AA and Len) have mutations in the PB2 and PB1 viral segments. Thus, we next evaluated which of the viral segments has a more significant impact on the temperature sensitive phenotype in PR8. Using the same minigenome approach and different combinations of PB2 and PB1 viral segments of PR8/WT, PR8/AA or PR8/Len, GFP A and Glund 39 °C A,B. RemaBased on the minigenome results using transfected cells in which Len MDV mutations induced a stronger reduction in the PR8 polymerase activity at high temperatures (37 °C and 39 °C) than those induced by the AA MDV mutations , we nextN = 5) were infected with 103, 104, 105 and 106 fluorescent forming units (FFU) of PR8/AA  of ~1.5 × 104 FFU, as previously described [6 FFU died at day 6p.i., 40% of mice survived infection with 105 FFU and those infected with 104 and 103 FFU did not show body weight loss and all survived viral infection, resulting in a calculated MLD50 of ~6.8 × 104 FFU  [In our next set of experiments, we compared the virulence of both PR8 LAIVs in vivo . To thatf PR8/AA A,B or PRf PR8/AA C,D and mf PR8/AA A,C and sf PR8/AA ,D. As prmes p.i. A, resultmes p.i. B. Two miN = 6) were infected with 103 and 104 FFU of PR8/Len or 103 FFU of PR8/AA  and 4 (N = 3) p.i.  p.i. A. No staN = 5) were immunized with the same non-lethal doses of PR8/Len (103/104 FFU) and PR8/AA (103 FFU)  , or mock PR8/Len B. Humora PR8/Len C or puri PR8/Len D. Sera fN = 5) were immunized with 103 or 104 FFU of PR8/Len, 103 FFU PR8/AA, or mock-vaccinated (PBS). At 10 days p.i., lungs and spleens were recovered and processed to collect CD8 T cells. The results showed that vaccination with both PR8 LAIVs induced NP- and PA-specific CD8 T cells in the lungs  were vaccinated with the same PR8 LAIVs doses used in the immunogenicity studies, or mock (PBS) vaccinated (50 of PR8/WT and monitored over 10 days for body weight loss (3 FFU). Since all mice survived the challenge with PR8/WT, we cannot conclude which one of the two LAIVs (PR8/AA or PR8/Len) is more protective.Having confirmed the immunogenicity of both LAIVs, we proceeded with challenge experiments, initially focusing on a homologous viral challenge . Groups ccinated . At day ght loss A and surght loss B. All might loss , it is aN = 5) were vaccinated with the same PR8/Len and PR8/AA viral doses used in the homologous challenge studies  contain their respective H1N1 glycoproteins and, therefore, the antibodies detected in the ELISA are mainly directed against internal PR8 viral proteins, which are the same in all viruses.We next examined the humoral immune responses and the protection efficacy of PR8/Len and PR8/AA against a heterologous challenge with X31 (H3N2) . Groups  studies . Fourtee50 of X31 H3N2 and monitored daily over 10 days for body weight loss  of PR8/Len than PR8/AA to induce the same levels of protective immunity against a heterologous challenge.Then, fifteen days post-vaccination, mice were challenged with 50× MLDght loss A and surght loss B. Mock-vhallenge A,B. Micet weight A but 60%t weight B. These ts, ca and att phenotypes of the two commercially available MDVs (US (AA) and Russian (Len)) have been previously described [ts, ca and att phenotype of US AA MDV. Mutations located in the PB2 (V478L), PB1 (K265N and V591I) and in the NEP (M100I) are responsible for the ts, ca and att phenotype of the Russian Len MDV  than PR8/AA (103 FFU) to reach similar levels of humoral  seasonal circulating strain. However, it will be important to demonstrate that introduction of these ts, ca and att mutations results in the desired attenuated phenotype since we have previously shown that introduction of the US AA MDV mutations into the backbone of a recent pandemic Cal/09 did not resulted in an attenuated phenotype in vivo [Collectively, these results demonstrate that the mutations responsible for the ng human , canine ng human ,12,21 anng human  IAVs. Li in vivo ,38,39.


Baloxavir showed broad-spectrum in vitro replication inhibition of 4 types of influenza viruses (90% effective concentration range 1.2–98.3 nmol/L); susceptibility pattern was influenza A ˃ B ˃ C ˃ D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neuraminidase inhibitors. Influenza viruses are classified into 4 types: A, B, C, and D  2 blockers and neuraminidase inhibitors (NAIs) were approved to control influenza. M2 blockers are effective only against influenza A viruses and are not recommended because of widespread resistance. NAIs are used for treatment of influenza A and B virus infections, but NAI-resistant viruses have emerged  is nearly identical in all 4 influenza virus types  showed that all influenza D viruses have valine at position 38, whereas influenza C viruses have isoleucine, similar to most influenza A and B viruses. Nevertheless, valine at 38 in influenza A and B viruses had little or no effect (<3-fold) on baloxavir susceptibility  values in a low nanomolar range (50 values were much higher than those for baloxavir (50 values comparable to avian and seasonal influenza A viruses (,First, we tested 25 influenza viruses of avian origin, representing H5, H6, H7, H9, and H10 subtypes, by using FRA or HINT as described  Table 1.ar range  Table 1.aloxavir  Table 1. viruses  Table 2 It is prudent to analyze PA sequences of emerging influenza A viruses for markers previously associated with reduced baloxavir susceptibility (Baloxavir displayed broad antiviral activity against diverse influenza viruses, including all 4 types and animal-origin influenza A viruses with pandemic potential. Our findings suggest that baloxavir might offer the first therapeutic option against influenza C virus infections. Further studies are needed to provide comprehensive assessment of baloxavir susceptibility by using a large panel of representative influenza C viruses. Ongoing monitoring of baloxavir susceptibility of emerging avian and swine influenza A viruses with pandemic potential is needed to inform clinical management and public health preparedness efforts.Additional information on susceptibility of influenza A, B, C, and D viruses to baloxavir.


Primary human osteoclasts were differentiated from CD14+ monocytes and exposed to hypoxia (2% O2) and A2B receptor inhibitors . The hypoxic increase in bone resorption was prevented by the inhibition of the A2B receptor, at least partly by the attenuation of glycolytic and mitochondrial metabolism via inhibition of HIF. A2B receptor inhibition also reduced osteoclastogenesis in hypoxia by inhibiting early cell fusion . The A2B receptor is only functional in hypoxic or inflammatory environments when the extracellular concentrations of adenosine  are sufficient to activate the receptor. Inhibition of the A2B receptor under normoxic conditions therefore did not affect any parameter tested. Reciprocal positive regulation of HIF and the A2B receptor in a hypoxic microenvironment thus enhances glycolytic and mitochondrial metabolism in osteoclasts to drive increased bone resorption. A2B receptor inhibition could potentially prevent the pathological osteolysis associated with hypoxic diseases such as rheumatoid arthritis.Osteoclast-mediated bone destruction is amplified in the hypoxic synovial microenvironment of rheumatoid arthritis (RA). This increased bone resorption is driven by the hypoxia-inducible transcription factor HIF. We identified hypoxic induction of the HIF-regulated adenosine A Rheumatoid arthritis (RA) is a chronic inflammatory disease characterised by the progressive destruction of bone and articular cartilage. Bone resorption in RA is mediated by multi-nucleated osteoclasts and contributes significantly to disability and poor prognosis ,2,3. RA 1, P2Y12 and P2X7 receptors [1, A2A, A2B, A3), and the majority of studies have focused on the A1 and A2A receptors.Increasing attention has been paid to the role of purinergic signaling, a form of extracellular signaling mediated by purine nucleotides and nucleosides, in osteoclast differentiation and function. This has mainly focused on the effect of extracellular ATP to increase the formation and activity of osteoclasts, acting predominantly via the P2Yeceptors . There ieceptors ,10,11,12eceptors ,14 or haeceptors ,16 osteo2B receptor  as hypoxia-inducible in primary human osteoclasts [2B receptor is hypoxia-inducible in many tissues but is also only activated in hypoxia; because of its low affinity for adenosine (EC50 = 24 μM), it is only under hypoxic or inflammatory conditions that sufficient extracellular adenosine is available to activate the A2B receptor [Extracellular adenosine is formed by hydrolysis of extracellular ATP and ADP by ectonucleotidases located in the plasma membrane. ATP release into the extracellular space is increased under conditions of hypoxia or inflammation, via increased vesicular exocytosis from osteoblasts  for exameoclasts . The A2Breceptor .2B receptor [2B receptor expression is reported within both rheumatoid synovium [RA is both inflammatory and hypoxic, and therefore adenosine levels would be expected to be elevated in RA patients. Adenosine is very unstable, however, and is hydrolysed by the adenosine deaminase present in synovial fluid, making its actual levels hard to measure clinically, although elevation of adenosine has been observed in murine models of RA ,22. Highreceptor . Clinicasynovium  and rheusynovium . This su2B receptor mediates the hypoxia-induced increase of osteoclast-mediated bone destruction. It describes how A2B receptor inhibitors specifically reduce hypoxic bone resorption in osteoclasts, which is driven by a hypoxia-induced increase in A2B receptor expression as well as its activation by accumulation of extracellular adenosine. In turn, A2B receptor activation further stabilises HIF-1α to promote bone resorption via an increase in cellular metabolic rate.This work aims to determine whether the AReagents were obtained as follows: human M-CSF , human soluble RANKL , MRS1754 and PSB603 . Elephant dentine was obtained from HM Revenue & Customs, Heathrow Airport, UK. Unless stated, other reagents were from Sigma-Aldrich . The study was conducted in accordance with the Declaration of Helsinki; the use of leucocyte cones for osteoclast differentiation was approved by the London–Fulham Research Ethics Committee (11/H0711/7).6 cells/ml onto dentine discs or plastic dishes in α-MEM (without ribonucleosides/deoxyribonucleosides) containing 10% heat-inactivated foetal bovine serum (FBS), 2 mM L-glutamine, 50 IU/ml penicillin and 50 μg/ml streptomycin sulphate. Osteoclastogenesis was induced by treatment with 25 ng/ml M-CSF and 30 ng/ml RANKL every 3–4 days for 9 days. Monocytes were maintained in 25 ng/ml M-CSF. Primary human osteoblasts were purchased from Sigma-Aldrich. Hypoxic exposure was conducted at 2% O2, 5% CO2, balance N2 in a MiniGalaxy incubator .CD14+ monocytes were positively selected from the peripheral blood mononuclear cell component of leucocyte cones  using magnetic CD14+ microbeads . Monocytes were seeded at 1–1.25 × 10Tartrate-resistant acid phosphatase (TRAP) staining of formalin-fixed cells used naphthol AS-BI phosphate as a substrate, with reaction of the product with Fast Violet B salt. Multi-nucleated cells containing three or more nuclei were considered osteoclasts. Vitronectin receptor (VNR) was detected by immunocytochemistry for CD51/61 . Resorption tracks produced by mature osteoclasts on dentine discs were visualised by staining with 0.5% toluidine blue following the removal of cells by sonication. Dentines were photographed, resorption tracks highlighted, and the resorbed area quantified using ImageJ .HIF1α, HIF2A or a HIF1α scrambled control using RNAiMAX . Duplexes were removed after 4 h, and osteoclasts were cultured for a further 48 h prior to assay. Cells were transfected with a phosphoglycerate kinase (PGK) hypoxia response element (HRE)–firefly luciferase plasmid , A2B receptor-coding sequence (Hs_ADORA2B_1_SG), HIF1A (Hs_HIF1A_1_SG) and HIF2A (Hs_EPAS1_1_SG) or designed in-house against CD39 , CD73  and PER2 . Relative quantification (comparative CT) of target gene mRNA was normalised to β-actin (ACTB) mRNA.RNA was extracted in TRI reagent  and reverse-transcribed, and quantitative PCR was performed using Fast SYBR Green Master Mix in a Viia7 Real-Time PCR system . Human primers were either pre-validated Quantitect primers  against 3VO4, 1 mM NaF in PBS). Gels were evenly loaded with 40–60 μg protein/lane, and molecular weights were compared against the full-range Rainbow molecular weight marker . Primary antibodies were against the A2B receptor , HIF-1α , HIF-2α , GLUT1 , NFκB p65 and phospho NFκB p65  and β-tubulin . Densitometric quantification of Western blots was performed in ImageJ , normalizing experimental bands to the corresponding β-tubulin control.Cells were homogenized in HIF lysis buffer  or phospho-lysis buffer , 1 mM NaGlucose, adenosine and ATP were measured in the media using the Glucose (GO) Assay Kit, the Adenosine Assay Kit  and the ATP Determination Kit . Lactate was assayed in heat-inactivated medium by the increase in absorbance (340 nm) as NAD+ was converted to NADH in the presence of 0.32 M glycine, 0.32 M hydrazine, 9.6 mM NAD+ and 3 U/ml lactate dehydrogenase. Mitochondrial dehydrogenase activity within the electron transport chain was assessed by adding Alamar blue  to cells in culture for 4 h. The results were normalized to the number of osteoclasts.The concentrations of intracellular cyclic AMP (cAMP) and secreted interleukin-6 (IL-6) were measured using the cAMP Parameter and IL-6 Quantikine ELISA kits , according to the manufacturer’s instructions.2B receptor was visualized using the rabbit polyclonal H-200 antibody  and a PBS control and either the VECTASTAIN Elite Universal ABC Kit and DAB  or an AlexaFluor594 fluorescent secondary antibody. For F-actin ring staining, the cells were permeabilised with 0.5% Triton X-100 and stained with FITC-conjugated phalloidin (0.5 μg/mL). Osteoclasts were fixed in 4% formalin. The Ap < 0.05.The results were derived from three or more independent experiments. Data are presented as mean ± standard error of mean (SEM) of data transformed to the mean of the control group. Data were analysed using Prism . Statistical analysis comprised one-way or two-way ANOVA using Dunnett’s or Tukey’s multiple comparison as a post-hoc test. For experiments with only two conditions, a Students’ t-test was applied. The results were considered significant at 2B receptor mRNA (p < 0.002) [p < 0.01). A2B receptor protein was also induced by hypoxia, with hypoxic conditions verified by stabilisation of HIF-1α   that we f HIF-1α B,C. CoClectively D,E and ator mRNA F and protor mRNA G, indica2B receptor might be a HIF-regulated mediator of these processes. The A2B receptor inhibitors MRS1754 and PSB603 were first tested for their effects on the survival and bone resorption capacity of mature osteoclasts. No concentration tested affected osteoclast number . Exposure to MRS1754 or PSB603 during this 24 h period had no effect under normoxic culture conditions but delayed fusion under hypoxia at the 3–4-day timepoint . Extracellular adenosine is formed by the hydrolysis of extracellular ATP by the ectonucleotidases CD39 and CD73, restricting its actions to cells close to the release site. Expression of CD39 and CD73 was also increased in hypoxic osteoclasts (p > 0.01) and their CD14+ mononuclear precursors  [2B receptor inhibition  . IL-6 sthibition .2B receptor also increases the glycolytic rate via an intracellular feedback loop whereby hypoxic activation of the A2B receptor further activates HIF-1α signaling, which in turn stimulates glycolysis as part of the cellular metabolic adaptation to hypoxia [2B receptor reduced the stabilisation of HIF-1α . A2B receptor signaling commonly involves adenylyl cyclase and leads to an increase in intracellular cAMP [2B receptor inhibition under hypoxia was evident in either case  of this process. The Aer cells ,38,39,40ack loop ,33 is st2B receptor expression in osteoclasts increases during their differentiation from mononuclear precursors [2B receptor in both normoxic and hypoxic osteoclasts was predominantly cytoplasmic as described (www.proteinatlas.org), with a peri-nuclear bias and some nuclear involvement similar to that observed in human sclera fibroblasts and retinal pigment epithelial cells [2B receptor is only activated at the elevated concentrations of extracellular adenosine achieved in hypoxic conditions, despite the protein being present in normoxia. Osteoclast cultures exhibited increased concentrations of ATP and adenosine in the media following acute and extended periods of hypoxic exposure, respectively. Extracellular adenosine is formed by hydrolysis of extracellular ATP and ADP by the ectonucleotidases CD39 and CD73 located in the plasma membrane, whose mRNA levels also increased following hypoxic exposure. The controlled release of ATP from osteoclasts via the P2X7 receptor results in the accumulation of extracellular adenosine [2B receptor activation in adjacent hypoxic osteoclasts in vivo.Aecursors  and subsal cells ,43. Due denosine ,28, althdenosine  could al2B receptor inhibition with either MRS1754 or PSB603 only suppressed osteoclast fusion and bone resorption in hypoxic culture conditions, when elevated extracellular concentrations of adenosine were sufficient to activate the A2B receptor in the hypoxic cells. Fusogenic actions of extracellular adenosine have been described in both murine and human osteoclast cultures and attributed to the A1 or A2A receptors [2B receptor, adding an additional fusogenic role for this receptor to the list. There are potential A2B receptor-independent effects of both inhibitors. MRS1754 is less selective than PSB603 (200-fold versus >17000-fold selectivity) and therefore might partially inhibit other adenosine receptors, although the fact that the effects were only observed in hypoxia would indicate predominant inhibition of the A2B receptor. Despite high selectivity for the A2B receptor, PSB603 increased the rate of oxygen consumption of colorectal cancer cells in an A2B receptor-independent manner [2B receptor-driven pathway. For the reasons above, Aeceptors ,11,12,44t manner . However2B receptor inhibitors suggests that adenosine also stimulates processes involved in bone resorption via the A2B receptor. No effect of A2B receptor inhibitors on bone resorption was seen under normoxic conditions. Previous reports regarding the effects of the A2B receptor on osteoclast activity are opposing. Bone marrow-derived osteoclast formation and bone resorption ex vivo was enhanced in cells from A2B receptor knockout mice [2B receptor should be functionally inactive, suggestive of potential off-target effects in the knock-out animals. The A2B receptor partial agonist BAY60-6583 inhibited precursor fusion and the expression of osteoclast marker genes during differentiation of murine and human bone marrow-derived mononuclear cells into osteoclasts, as well as reduced F-actin ring formation and bone resorption [2B receptor activates alternative intracellular signaling pathways to those activated by physiological stimulation of the receptor, resulting in cellular effects that are not related to the normal cellular functions of the A2B receptor. It has, for example, been noted that in the presence of high levels of adenosine, BAY60-6583 can instead act as an antagonist and block the effects of adenosine at A2B receptors [Prevention of the hypoxic increase in osteoclast-mediated bone resorption by Aout mice . Howeversorption ,47. Thiseceptors .2B receptor–HIF signaling pathway is one intracellular pathway that would likely not be activated by A2B receptor agonists administered in normoxic culture, due to the requirement for hypoxia to cause inactivation of the HIF-regulating prolyl hydroxylase (PHD) enzymes and subsequent stabilization of HIF-α proteins [2B receptor amplify HIF signaling under hypoxic conditions, contributing to further stabilisation of HIF protein and activation of HIF-mediated transcription in neuroendocrine enterochromaffin cells of the gut [2B receptor- and HIF-dependent increase in glycolytic metabolism [The adenosine Aproteins ,49. Aden the gut , smooth  the gut , oral sq the gut , cardiom the gut  and micr the gut , as well the gut . Activattabolism ,52. Incrtabolism ,35. The 2B receptor-dependent manner [2B receptor is expressed within both the rheumatoid synovium [There is evidence that similar pathways are active in osteoclasts in vivo. Plasma adenosine levels are markedly elevated in rats with adjuvant-induced arthritis, a murine model of RA. The time of elevation of adenosine corresponds to the time-point at which the administration of MTX fails to suppress inflammatory bone destruction . In vitrt manner . Clinicasynovium  and rheusynovium , suggestsynovium , which m2B receptor in a hypoxic microenvironment enhances glycolytic and mitochondrial metabolism in osteoclasts to promote osteoclast differentiation and drive increased bone resorption. The adenosine A2B receptor–HIF signaling pathway is only active in hypoxic microenvironments, meaning that the inhibition of the A2B receptor only affects these cellular processes under hypoxia. As tissue hypoxia is a disease-specific environment, the A2B receptor could represent a potential therapeutic target, with specificity for hypoxic diseased tissue, which could prevent the pathological osteolysis associated with diseases such as RA.This study demonstrates that the reciprocal positive regulation of HIF and the A


Low pathogenic A(H9N2) subtype avian influenza viruses (AIVs) were originally detected in Cambodian poultry in 2013, and now circulate endemically. We sequenced and characterised 64 A(H9N2) AIVs detected in Cambodian poultry (chickens and ducks) from January 2015 to May 2016. All A(H9) viruses collected in 2015 and 2016 belonged to a new BJ/94-like h9-4.2.5 sub-lineage that emerged in the region during or after 2013, and was distinct to previously detected Cambodian viruses. Overall, there was a reduction of genetic diversity of H9N2 since 2013, however two genotypes were detected in circulation, P and V, with extensive reassortment between the viruses. Phylogenetic analysis showed a close relationship between A(H9N2) AIVs detected in Cambodian and Vietnamese poultry, highlighting cross-border trade/movement of live, domestic poultry between the countries. Wild birds may also play a role in A(H9N2) transmission in the region. Some genes of the Cambodian isolates frequently clustered with zoonotic A(H7N9), A(H9N2) and A(H10N8) viruses, suggesting a common ecology. Molecular analysis showed 100% of viruses contained the hemagglutinin (HA) Q226L substitution, which favours mammalian receptor type binding. All viruses were susceptible to the neuraminidase inhibitor antivirals; however, 41% contained the matrix (M2) S31N substitution associated with resistance to adamantanes. Overall, Cambodian A(H9N2) viruses possessed factors known to increase zoonotic potential, and therefore their evolution should be continually monitored. Subtype AH9) avian influenza viruses (AIVs) circulate globally in wild avian species and are endemic in domestic poultry in many Asian, Middle Eastern and African countries ,2. Despi avian inA(H9) AIVs in poultry pose a zoonotic risk to humans . Since tCambodian AIV surveillance started in response to the detection of highly pathogenic avian influenza (HPAI) A(H5N1) in Cambodian poultry in 2004. These viruses now circulate endemically in domestic poultry species ,25. In 2https://www.internationalreagentresource.org/Home.aspx) as described previously . In. In33]. eviously ,28.A subset of A(H9) AIV positive samples, with matrix gene qRT-PCR Ct values <30, were isolated in embryonated chicken eggs (ECEs) . Briefly™ Plus Fragment Library Kit (Life Technologies). Samples were barcoded with unique Ion Xpress barcode adapters (Life Technologies), pooled and purified using AMPure XP reagent (Agencourt). The final libraries were quantified using the Ion Library Quantitation Kit (Life Technologies) and 20 pM was used to perform an emulsion PCR to enrich Ion Sphere Particles™ (ISPs) on the Ion OneTouch 2 instrument (Life Technologies). The ISPs were then loaded onto a 318™ Chip v2 (Life Technologies) and run on the Ion Torrent PGM.A(H9N2) AIV positive isolates were sent to the WHO Collaborating Centre for Reference and Research on Influenza, Melbourne, Australia for whole genome sequencing (WGS). Prior to sequencing, RNA was extracted from samples with the NucleoMag® VET kit (Macherey-Nagel) and all genomic segments were amplified in a single reverse transcription PCR (RT-PCR) reaction . The staQuality control of the NGS reads was assessed using CLC Genomic Workbench v10 (QIAGEN). Low quality reads less than 50 nucleotides in length were removed and a minimum base call quality Phred score of 20 was set. Remaining sequences were aligned to a reference genome . Genes containing gaps or areas of low sequence coverage were completed using Sanger sequencing. Briefly, segment specific primers were used to amplify appropriate regions using conventional PCR . SequencSequences included in the phylogenetic analysis were obtained from GISAID , GenBankTime to the most recent common ancestor (TMRCA) for each node of the HA and NA phylogenies was estimated using a Bayesian Markov Chain Monte Carlo (MCMC) approach with BEAST v1.8.4 run on the CIPRES Science Gateway web portal . For eacReassortment was visualised by producing a phylogenetic congruency map, whereby segments of the individual viruses were linked across the ML phylogenies. The ML trees were created using IQ-tree as described above with a subset of viruses that had sequencing data available for all eight genomic segments . IncongrFor the purpose of this study, the genotyping system used follows conventions initially described by Li et al., 2005 . Using tThe H3 HA and N2 NA numbering systems were used throughout the text, unless otherwise stated. Molecular markers associated with changes in viral fitness or resistance to antivirals were investigated using the molecular inventory matrix produced by Suttie et al., 2019 . N-linkeSelection pressures acting on each gene of the Cambodian A(H9N2) AIVs was analysed by calculating the ratio of non-synonymous to synonymous mutations  using the HyPhy software package  accessed50) of viruses. This protocol has been described in detail by Leang et al., 2017 . . 59]. GeScreens for mutations known to affect AIV pathogenicity , are sumCambodian A(H9N2) viruses contained numerous substitutions associated with increased polymerase activity of AIVs in mammalian cell lines Tables  and S3E.Only a small number of mutations in MP and NS1 were detected that increase AIV virulence in mammalian or avian models Tables . CambodiGenetic analysis of known A(H9) HA antigenic sites  showed h11NSTE, 280NTTL, 287NVSK/R, 295NCSK and 474NGTY in the HA stalk domain, as well as 123NVSY and 200NRTF in the HA globular domain (In the Cambodian A(H9N2) viruses, seven conserved HA glycosylation sites were predicted, including: r domain . More vaSelection pressure analysis showed that the majority of sites in each gene of Cambodian A(H9N2) viruses were under neutral or negative selection. A small number of codons in HA , NA , PB2 389, 758), M2  and NS2 (119) were under positive selection . Substit8, M2  viruses showed 41% (n = 26) had the S31N marker associated with resistance to adamantanes . Mappingeramivir .Since integration of detection protocols in 2013, it is clear that subtype A(H9) AIVs now circulate endemically in Cambodian domestic poultry and they are currently the most prevalent subtype detected . BetweenTransmission by migration of wild birds may also play a minor role in viral diversity in Cambodia. A single A(H9N2) isolate, designated A/chicken/Cambodia/a77W22M1/2016, clustered separately from other Cambodian viruses, possibly representing a separate A(H9) introduction via migration of wild birds along the Eastern Asia/Australasian flyway. This virus clustered with A(H9N2) AIVs detected in poultry from Vietnam between 2014 and 2018, Japan in 2016 and with Indonesian A(H9N2) AIVs detected from 2016 to 2018. The first detection of A(H9N2) in Indonesia occurred during AIV outbreaks in 2016 and was associated with increased flock mortality and an 18% reduction in the egg production of layers . FurtherSince 2013, A(H9) genotype S viruses have been the most prevalent AIV lineage detected in Chinese chickens . The enhThe internal genomic cassette of genotype S viruses have been repeatedly donated to zoonotic AIVs such as A(H5N6), A(H7N9) and AH10N8) –21. In C0N8 –21. Molecular analyses indicate the Cambodian A(H9N2) AIVs were LPAI viruses with multiple molecular markers associated with adaptation to mammalian species. Cambodian A(H9N2) viruses contained multiple HA markers associated with increased binding to “human-like” α2,6-linked sialic acid residues . One welOther substitutions identified in internal proteins of Cambodian A(H9N2) viruses have also been associated with increased AIV replication and virulence in mammals . While mCandidate A(H9) vaccine viruses are selected as part of pandemic preparedness plans . The CamAssessing the effectiveness of available antivirals is vital for combating zoonotic AIVs as part of influenza pandemic preparedness strategies . MoleculIn summary, Cambodian A(H9N2) AIVs isolated from domestic poultry in LBMs between 2015 and 2016 belonged to the BJ/94 H9-4.2.5 lineage. They were closely related to viruses identified in surrounding countries, suggesting frequent circulation of these viruses possibly through cross-border movement of live poultry. Wild birds may also play a role in A(H9) transmission. The Cambodian A(H9N2) viruses contained multiple genomic segments clustering with genotype S lineage AIVs, with a small number close to zoonotic A(H7N9), A(H9N2) and A(H10N8) viruses. Additionally, all Cambodian A(H9N2) viruses had molecular markers associated with viral adaptation to mammalian species, raising further concerns about their increased zoonotic potential. However, these viruses remained susceptible to therapeutic and prophylactic prevention strategies utilizing NAIs. Overall, the continued endemicity and evolution of A(H9N2) AIVs in Cambodian domestic poultry underlines the need for continued, vigilant surveillance analysis of AIVs within the country.S1 Fighttp://www.naturalearthdata.com/).Samples were collected from two LBMs during this period, Orussey market (Phnom Penh) and Takeo market (Takeo), indicated by red dots. The map was produced with QGIS v2.18.4 using public domain data obtained from Natural Earth ((TIF)Click here for additional data file.S2 FigCambodian AIVs are coloured based on the year of detection: 2013 is shown in orange, 2015 in green and 2016 in blue. Trees were produced using BEAST v1.84 with GTR + Γ and SRD06 nucleotide substitution model. The nodes are labelled with the proposed TMRCA and node bars show corresponding 95% HPD ranges. Branch lengths are time-proportional and the time scale is shown on the x-axis.(PDF)Click here for additional data file.S3 FigGenetic diversity of the HA and NA A(H9N2) genes was estimated using the Gaussian Markov Random Field (GMRF) model. The x-axis measures time in years and the y-axis is an estimate of genetic diversity calculated from Neτ (effective population size and the generation length in years) shown in log scale. The median estimate of genetic diversity over time is shown as a solid black line and the purple shading represents the 95% HPD intervals.(TIF)Click here for additional data file.S4 Figa) PB2 b) PB1 c) PA d) NP e) MP and f) NS. Trees were generated with IQ-Tree using the GTR+ I + Γ model with 1,000 ultrafast boostrap replicates. Cambodian viruses are coloured based on the year of detection: 2015 is light blue and 2016 dark blue. AIVs identified in humans are coloured red, and G57 lineage viruses  are pink. Candidate vaccine viruses are indicated by an asterisks (*) next to the taxa name. Viruses from A(H9) reference lineages are shown in bold and the lineage Cambodian A(H9N2) viruses fall under is indicated on the right hand side of the tree. Bootstrap values of 70 or greater are displayed on branches. The scale bar indicates number of nucleotide substitutions per site.(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Tablea-h Molecular analysis of the eight genomic segments for the Cambodian A(H9N2) isolates identified between 2015 and 2016. Data for each segment, as well as any associated accessory proteins is listed: a) PB2, b) PB1, c) PA, d) HA, e) NP, f) NA, g) MP and h) NS(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.


After publication of this article , concernThere were errors in the preparation of the left panel of In The original real time PCR data files and raw flow cytometry data are no longer available. The authors have provided the available original underlying data for Figs 1-8 as Supporting Information files –S9 Data.S1 File(PDF)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.S2 Data(ZIP)Click here for additional data file.S3 Data(PPTX)Click here for additional data file.S4 Data(PPTX)Click here for additional data file.S5 Data(ZIP)Click here for additional data file.S6 Data(ZIP)Click here for additional data file.S7 Data(ZIP)Click here for additional data file.S8 Data(ZIP)Click here for additional data file.S9 Data(ZIP)Click here for additional data file.


The genotypes of the H9N2 avian influenza viruses have changed since 2013 when almost all H9N2 viruses circulating in chickens in China were genotype 57 (G57) with the fittest lineage of each gene. To characterize the H9N2 variant viruses from 2011 to 2014, 28 H9N2 influenza viruses were isolated from live poultry markets in China from 2011–2014 and were analyzed by genetic and biological characterization. Our findings showed that 16 residues that changed antigenicity, two potential N-linked glycosylation sites, and one amino acid in the receptor binding site of the HA protein changed significantly from 2011–2014. Moreover, the HA and NA genes in the phylogenetic tree were mainly clustered into two independent branches, A and B, based on the year of isolation. H9N2 virus internal genes were related to those from the human-infected avian influenza viruses H5N1, H7N9, and H10N8. In particular, the NS gene in the phylogenetic tree revealed genetic divergence of the virus gene into three branches labeled A, B, and C, which were related to the H9N2, H10N8, and H7N9 viruses, respectively. Additionally, the isolates also showed varying levels of infection and airborne transmission. These results indicated that the H9N2 virus had undergone an adaptive evolution and variation from 2011–2014. Escherichia coli, Chlamydia psittaci, Ornithobacterium rhinotracheale, Staphylococcus aureus, Haemophilus paragallinarum and others [The subtype H9N2 low-pathogenic avian influenza viruses have been prevalent in China since 1994. H9N2 viruses can cause great economic losses to the domestic poultry industry when co-infected with H5 or H7 influenza viruses or other pathogens including d others –9. Additd others –14. TherHemagglutinin (HA) is an important surface protein that plays a vital role when influenza viruses are introduced into host cells. According to epidemiological and genetic studies, the HA genes in H9N2 viruses can be divided into three distinct lineages in China including A/chicken/Beijing/1/94-like , A/quail/Hong Kong/G1/97-like , and A/duck/Hong Kong/Y439/97-like  . Since 2To understand the evolutionary characteristics of the H9N2 avian influenza viruses from 2010–2014, molecular changes, the antigenic residues, potential glycosylation sites, and receptor-binding sites in HA from 28 H9N2 strains isolated from 2011 to 2014 were analyzed. Moreover, the infection and airborne transmission from eight H9N2 isolates in this study were identified.The specific-pathogen-free (SPF) chickens and chicken embryos used in this study were purchased from Beijing Center for Laboratory Animals. Procedures involving the care and use of animals were approved by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXK-SU-2007-0005) and complied with the Jiangsu Laboratory Animal Welfare and Ethics guidelines of the Jiangsu Administrative Committee of Laboratory Animals.50) for the isolates were determined by propagating serial dilutions of viruses in eggs (0.2 mL per embryo) and calculated as described by Reed & Muench [From 2011 to 2014, tracheal or cloacal swabs collected from live poultry markets in China, including Jiangsu, Anhui, Heilongjiang, and Fujian, were placed into phosphate buffered saline (PBS) containing 2000 units/mL penicillin, 2 mg/mL streptomycin, 50 μg/mL gentamycin, and 1000 units/mL mycostatin. After freezing, the supernatants were collected through a 0.22 μm pore size filter after centrifugation at 1000 g for 10 min at 4°C. Next, 200 μL of the supernatant was inoculated in the allantoic cavities of 10-day-old SPF embryonated chicken eggs. After 72 h of incubation at 37°C, the viruses were tested for HA activity. The isolated viruses were identified as H9 subtype by hemagglutination inhibition (HI) assay using antisera against H9 subtype, H5 subtype avian influenza viruses (AIV), and Newcastle disease viruses (NDV). The 50% egg infective doses  into cDNA using 12-base universal primers for influenza A viruses (U12 A/G: AGCG/AAAAGCAGG). The gene fragments were amplified by polymerase chain reaction  after reverse transcription using specific primers, sequenced, and assembled by Takara Biotechnology  [Total viral RNA was extracted from allantoic fluids infected with purified isolates using the TRIZol Reagent . Viral RNA was reverse-transcribed  , 26. TheTo investigate the antigenic drift, the mutation levels for each of 18 reported antigenic residues from 28 isolates were quantified by comparison with 1081 full-length H9N2 HA sequences deposited in GenBank from 2010 to 2015 . The three commercial vaccine strains SH/F/98, SD/6/96, and GD/SS/94 were used as controls.http://www.cbs.dtu.dk/services/NetNGlyc/). Some key residues from all gene fragments were also investigated.The potential N-linked glycosylation sites (PNLGSs) in HA were predicted using the NetNGlyc 1.0 Server online software (v/v) for 24h at 37°C. The antisera were collected from vaccinated SPF chickens in two weeks after the booster vaccination and used to characterize the antigenicity of the 28 H9N2 isolates and the two commercial vaccine strains. The HI assay was expressed as the reciprocal of the highest serum dilution in which HA was inhibited. Then, antigenic cartography was performed with the Antigen Map program (http://sysbio.cvm.msstate.edu/AntigenMap), which uses matrix completion multidimensional scaling to map HI titers in two dimensions [To investigate the antigenic relationship between the H9N2 isolates and two commonly used vaccine strains (SH/F/98 and GD/SS/94), the nine H9N2 isolates  and two commercial vaccine strains (SH/F/98 and GD/SS/94) were selected according to the genotype of the isolates and the year of isolation. The antiserum of each strain was made, then the HI titer of each straun were detected as following description. Briefly, 3-week-old SPF chickens were immunized twice by subcutaneous injection of 0.3 mL of oil-emulsion of inactive whole virus vaccine of the indicated virus, which was inactivated by adding 0.2% formalin (mensions .6 EID50 of each virus or a PBS control. Tissue samples (trachea and lung) from the inoculated chickens were collected at 3 days and 5 days post-inoculation (dpi). Briefly, three SPF chickens were euthanatized using CO2 asphyxiation at designated times, and half of the tissues were harvested, washed, and ground into 20% (w/v) suspension in 1 mL PBS. Virus titers in the trachea and lung were determined in 10-day-old SPF embryonated chicken eggs. The other half of the tissues were fixed with 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 mm, and processed for staining with hematoxylin and eosin for histopathological examination. These experiments were repeated three times.To study the viral replication and pathogenicity of the H9N2 influenza virus in chicken, groups of six 3-week-old SPF chickens were orally, intranasally, or intratracheally inoculated with 0.2 mL of 106 EID50 of the indicated virus. The airborne contact group was placed in a poultry isolator adjacent to the infected group with a distance of 50 cm between poultry isolators. At day 3 and 5 post-inoculation, tracheal and cloacal swabs from chickens were collected in 1 mL of PBS containing antibiotics and, following one freeze-thaw cycle, were centrifuged at 3000 rpm for 10 min. Of the resulting supernatant, 0.2 mL were taken, and the EID50 titers of the tracheal and cloacal swabs collected from the indicated passaged virus were determined by serial titration of viruses in SPF embryonated chicken eggs using the Reed & Muench method [For studying the viral transmission, fifteen 3-week-old SPF chickens were divided into five groups: (i) inoculated group (three chickens), (ii) direct contact group (three chickens), (iii) airborne contact group (three chickens), (iv) PBS control group (three chickens), (v) inoculated group for sera (three chickens). To prevent cross-contamination, the chickens from the experimental groups (inoculated group and direct contact group were in the same poultry isolator) were housed in a separated poultry isolator after fumigation with the formaldehyde-potassium permanganate. The infected group was inoculated orally, intranasally, and intratracheally with 10h method . Sera coh method . The expAllantoic fluids infected with tracheal or cloacal swabs taken from chickens were harvested from embryonated SPF chicken eggs. The viruses were identified as H9 subtype by HI assay using chicken H9 antiserum and were identified as N2 via PCR using specific primers. The isolated viruses did not react with H5 or Newcastle disease virus antiserum. Twenty-eight H9N2 strains from chickens in live poultry markets were isolated in this study. HA genes from the 28 H9N2 isolates and seven other genes (genes neuraminidase (NA), polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase acidic (PA), nucleoprotein (NP), matrix (M), nonstructural (NS)) from the 9 representative isolates for the 28 H9N2 isolates were sequenced and submitted to GenBank under the accession numbers MG280611-MG280630 and MG280639-MG280709 .The phylogenetic analysis included the HA nucleotide sequences from the 28 field isolates and the other 42 reference strains, which include 18 reference strains from different lineages, three vaccine strains, three G57 reference strains, and 18 field strains isolated from 2010–2015. The results showed that HA genes from all 28 H9N2 isolates were clustered into the BJ/94-like lineage. According to the year of isolation, the viruses in the phylogenetic tree could mainly be clustered into two independent branches labeled A  and B . The viruses in branch A which were isolated from 2011–2012 shared a 92%-93.5% sequence identity with the three vaccine strains: SH/F/98, SD/6/96, and GD/SS/94. However, the strains in branch B which were collected from 2013–2015 only had a 90%-92% nucleotide identity with the three vaccine strains .Based on the HA phylogenetic analysis, nine isolates  were selected, and a phylogenetic analysis of these was performed on the seven genes of interest . The phylogenetic analysis of the NA nucleotide sequences showed that the viruses in the phylogenetic tree could be mainly grouped into two independent branches labeled A  and B , which were closely related to the year of isolation . The strResults from a reciprocal HI assay demonstrated that antisera against the SD/C9QH/11, JS/TM58/13, JS/JT95/13, or AH/WB/14 strains had low reactivity with most of the isolates and the two vaccine strains, SH/F/98 and GD/SS/94. The two vaccine strains and most of the isolates reacted well with the antisera against JS/TM71/14 or JS/TM59/13 . The antThe mutations for each of the 18 reported antigenic residues were analyzed by comparison with 1081 full-length H9N2 HA sequences deposited in GenBank . In comparison to three commercial vaccine strains , the 18 antigenic residues in the HA protein of the isolates were changed significantly from 2011–2014, including H66Q, G90E, S127R, S145N, D153G, Q164K/R, N167G/S/K, A168D/N, E181G, A198T/V, T200R, N201D, D216E, T220I, Q235M, R254K, N256D, and S283R . In addiCompared with the three vaccine strains, the receptor-binding sites (RBS) with amino acids Y109, W161, T163, A191, L202, and Y203 were conserved among the 28 isolates, though a mutation occurred at position A198. Among the 28 isolates, 11 strains possessed an A198V mutation, and 8 strains possessed an A198T mutation. The 198T position began to be dominant in 2013, and the proportion of 198V or 198A decreased yearly since 2012 or 2011, respectively . The amiThe hemadsorbing sites , active center (140–157), and antigenic determinants  in the NA protein from 9 representative isolates in the two branches were also analyzed . All theSome critical residues in internal genes contributing pathogenicity or infectivity were analyzed, and the results showed that 627E and 701D in PB2 were observed among nine isolates (701N in PB2 was observed only in the virus JS/TM59/13), which are characteristics of low-pathogenic AIVs. In contrast, D253N and Q591K in PB2 were not observed, which could increase AIV pathogenicity . 588V in50 counts from oropharyngeal and cloacal swabs, tracheal and lung homogenates, and tracheal and lung histological specimens were conducted on 3 or 5 dpi. The EID50 results from the tracheal and lung homogenates on 3 and 5 dpi showed that all isolates replicated better in tracheae than in lungs. In particular, the JS/JT12/11, ZJ/618/12, JS/YZ640/12, JS/TM58/13, and JS/JT95/13 strains reached higher EID50 on 3 and 5 dpi than other strains in the tracheae. The AH/WB/14 and JS/TM71/14 strains reached lower EID50 on 5 dpi in the tracheas than on 3 dpi, while no SD/C9QH/11 virus was detected on 5 dpi in the trachea. Only the ZJ/618/12 strain was detected in lung homogenates on 3 and 5 dpi, and the JS/TM58/13 or JS/JT95/13 viruses were detected in lung homogenates on 3 dpi or 5 dpi, respectively, while little or no virus was detected in the lung homogenates on 3 and 5 dpi in the groups infected with the rest of the isolates  and some field strains isolated in recent years were selected for phylogenetic analysis. The HA segment of the 28 isolates belonged to G57-like viruses. Additionally, the locations of all the strains in the phylogenetic tree are closely related to the year of isolation, and the later the isolation, the farther they are located from the original G57 ZJ/HJ/07 strain in the phylogenetic tree. Specifically, the NA gene has gradually evolved into an undefined lineage. In comparison to other lineages of each gene, the fittest and most advantaged one of each gene may help H9N2 viruses to survive easily in hosts. Additionally, our findings also showed that 16 antigenic residues, two PNLGSs, and one RBS residue changed significantly from 2011–2013, which might be related to the emergence of the G57 lineage. It is generally believed that the RSSR sequence at the HA cleavage site is a characteristic of low-pathogenic AIVs . In thisThe virulence, infectivity, and transmission of the H9N2 virus are affected by multiple factors, particularly genetic factors. In this study, most isolates possessed residues in critical sites that increase the infectivity and transmission. Subsequently, animal experiments showed that the isolates caused different degrees of histopathological changes concerning host tissue tropism and better replication in the upper respiratory tract. Better airborne transmission and replication in the host can create an opportunity for H9N2 viruses to survive easily.Currently, H9N2 viruses are circulating in China without an effective vaccination strategy. H9N2 viruses escape selection pressure from virus-host interactions by evolution, which may have directly resulted in the emergence of an epidemic G57 virus. To understand the evolution of the H9N2 virus from 2011–2013 when the G57 virus became predominant, we performed a molecular analysis of H9N2 isolates in this study. From 2011–2013 changes in antigenic residues, PNLGSs, and some critical residues might have contributed to the increased H9N2 virus survival and evolution. These changes might help the G57 virus to become the predominant strain and could potentially cause enhanced infectivity and transmission by the circulating H9N2 virus.In summary, our findings suggest that the H9N2 virus surface glycoproteins have undergone evolution and variation from 2011–2014, which might be related to the G57 virus outbreaks and emergence since 2013. The H9N2 virus internal genes were closely related to those from the human-infected avian influenza viruses H5N1, H7N9, and the emerging influenza virus H10N8. In particular, some NS genes from H9N2 viruses were closely related to the H10N8 and H7N9 viruses, respectively.


Kaposi's sarcoma (KS) is a kind of cancer that causes flat or raised lesions containing Human herpes virus 8 (HHV8). The KS lesions are common among immunosuppressed HIV patients. Highly Active Antiretroviral (HHART) treats and prevents the development of KS. The objective of this study was to determine the presence of K1 and K15  genes in Kaposi's sarcoma-associated herpes virus (KSHV) among immunosuppressed patients due to HIV-1.This was a cross-sectional descriptive study where consecutive sampling technique was adopted to pick archived tissue blocks from the Thematic Unit of Anatomic Pathology, Department of Human Pathology, College of Health Sciences, University of Nairobi and Department of Laboratory Medicine, Histology Section, Kenyatta National Hospital.Upon staining 81 tissue blocks with H & E, 84% (68/81) were diagnosed as KS and 16% (13/81) as KS-like. The K1 and K15 (P) genes were both detected at 88.9% (72/81) in the tissue blocks, with 95.8% (69/72) detection from KS and 4.2% (3/72) from the KS-like.The K1 and K15 (P) genes of KSHV were present among the immunosuppressed patients with Human Immunodeficiency Virus (HIV)-1. It is important to carry out K1 and K15 (P) genes detection on tissues that are diagnosed as KS or KS-like by histology technique. Current studies indicate that all types of Kaposi's sarcoma (KS) are caused by Human Herpes Virus (HHV)-8 infections, and the difference in them is attributed by the contributions of many cofactors . GeneticHypothesis: the existence KSHV gene K1 and K15 (P) gene is associated with KS among patients who had HIV-1 and AIDS.4 cell count, tumor location, number of tumors, and antiretroviral treatment status of the patients whose tissue blocks had been collected and used were obtained from the registry records in the cytology and human pathology department. For this study, the inclusion criteria were tissue blocks that were previously diagnosed as KS or KS-like with a comprehensive bio-data information linking the blocks and registry records. Tissue blocks previously diagnosed as KS or KS-like that had missing or incomplete bio-data information from the registry records were excluded from the study. Consecutive sampling technique was used to pick a total of 81 tissue blocks from the 104 FFPE because they had met the inclusion criteria. A rotary microtome was used to section FFPE blocks.This was a cross-sectional descriptive study where 104 formalin-fixed paraffin-embedded (FFPE) tissues blocks were retrieved from the Thematic Unit of Anatomic Pathology, Department of Human Pathology, College of Health Sciences, University of Nairobi and Department of Laboratory Medicine, Histology Section, Kenyatta National Hospital between the year 2013 and 2016. The demographics, HIV-1 status, CDTissue sectioning: different blades per FFPE block were used to prevent carry-over of DNA. After each section, the surface of the microtome was sterilized using DNAZapTM PCR DNA (Thermo Fisher Scientific Company Cat No./ID: AM9890) degradation solutions. One tissue section was cut up to 10 µm thick. The tissue sections were processed for Hematoxylin and Eosin (H and E) staining and the results reported by a qualified human pathologist.DNA extraction: DNA extraction was carried out from the sectioned tissues using Qiagen kit GeneRead DNA FFPE (Qiagen®company Cat No./ID: 180134).The extraction kit removes paraffin and reverses formalin cross-links from the DNA tissues before it is bound to the QIAampMinElute column. The eluted DNA was then ready to be used for nested PCR.DNA detection: taq PCR Core Kit-Qiagen (Qiagen® company Cat No./ID: 201223) was used to detect K1 and K5 (P) gene. The two (K1 & K15 (P)) selected genes of KSHV were detected using two set of primers. The set of primers used were; K1 product size 868 bpK1a-f ATGTTCCTGTATGTTGTCTGC; K1a-r AGTACCAATCCACTGGTTGCG K1 product size 840 bp K1b-f GTCTGCAGTCTGGCGGTTTGC; K1b-r CTGGTTGCGTATAGTCTTCCG, K15 (P) product size 365 bp K15P-OF TGCAGGCTTGGTCATGGGTTAC; K15P-OR GGGACCACGCTGCAATTAAATG; K15 (P) product size 285 bpK15-3C ACGCATACATGTACTGCCAC; K15-4C CTTTGATATTGCCAGTGGTG. The PCR cycling condition of all the two targeted KS regions were similar and it consisted of 30 number of cycles which entailed; initial denaturation at 94°C for 3 minutes, denaturation at 94°C for 1 minute, annealing at 63°C for 1 minute, extension at 72°C for 1 minute and final extension at 72°C for 10 minutes. The amplified PCR products were analyzed by electrophoresis on a 1% agarose gel containing ethidiumbromide (1µl/ml of agarose solution) and were visualized under ultraviolet light alongside 1Kb DNA ladder. A known case of KS was used as positive control and RNase free water used as a negative control.Ethical clearance: authors of this manuscript obtained ethical clearance for the study from Kenyatta National Hospital and University of Nairobi .Statistical analysis: sample size of 81 tissue blocks was calculated using the standard statistical formula [ formula t-test. A P value < 0.05 was considered statistically significant.where Z^2 = 1.96, p = 5.6% based on prevalence HIV infection, trends, and risk factors among persons aged 15-64 years in Kenya: results from a nationally representative study , q = 0.94 counts of the subjects whose tissue blocks were retrieved from the records demonstrated that none of the recruited subjects had a CD4 count of above 350 cell/ mm3. The number of CD4 count was grouped into two groups, 250 being the cut-off point. Among the tissue blocks that had the K1 and K15 (P) genes detected 51.4% (37/72) had CD4 count above 250 cells/ mm3 and 48.6% (35/72) had CD4 count below 250 cells/ mm3. The distribution of age and CD4 cell count were significantly skewed and were bimodal among those who had the K1 and K15 (P) genes detected at a P value of 0.039 and 0.004 respectively . The records also indicated the distribution and the number of KS lesions among these subjects whose tissue samples were archived as FFPE blocks. The number of KS lesions in these subjects was grouped into two, above 10 and below 10. The tissue blocks that the K1 and K15 (P) genes were detected and had > 10 KS lesions was 55.6% (40/72) and < 10 KS lesion 44.4% (32/72). The distribution of KS was grouped as either localized or generalized, with 37.5% (27/72) localized and 62.5 (45/72) generalized among those that the K1 and K15 (P) genes were detected. The distribution of the KS lesions among the tissue blocks that had the two targeted genes detected were as follows: skin 31.6% (23/72), lower limbs 27.8% (20/72), lymphedema 16.7% (12/72), upper limbs 13.9% (10/72), palate 5.6% (4/72), trunk 1.4% (1/72), genitals 1.4% (1/72) and eye 1.4% (1/72). The study did histology screening on the retrieved tissue blocks to describe the morphology of the KS lesions. Tissue blocks were processed, sectioned, stained by H & E, results read by qualified pathologist and were described as patches, plaques and/or nodules (The K1 and K15 (P) genes detected as positive were 88.9% (72/81) and negative was 11.1% (9/81). Among the 88.9% tissue bocks that were positive for the targeted HHV-8 genes, 41.7% (30/72) were from female and 58.3% (30/72) male. All the subjects whose tissue blocks were retrieved for this study were found to be HIV positive. The antiretroviral status of the subjects was also determined, and it showed that out of the 88.9% that had K1 and K15 (P) genes detected, 95.8% (69/72) were on antiretroviral treatment and 4.2 (3/72) were HAART naïve. The CD nodules . In this nodules  was done4 cell count, treatment and distribution of KS lesions showed a strength in the association: gender for example, we noticed that compared to females, males were twice represented among those with K1 and K5 (P) genes cases.Genotyping analysis in this study revealed that the Kaposi's sarcoma-associated herpes virus (KSHV) genes, K1 and K15 (P) were detected among the selected tissue blocks. These findings affirm the earlier hypothesis that K1 and K15 (P) genes of Kaposi's sarcoma-associated herpes virus (KSHV) were present in the tissue blocks retrieved from patients with HIV-1 and AIDS. A previous study  conducte3. Other studies [3 are linked to high risk of developing KS whether one is on HIV treatment or not. However, recommendations have been made on the need to lookout on KS cases in subjects with high CD4 cell count [4 cell count is elevated and clinical presentation of KS tumors either stabilize or resolve [These findings were in agreement with another study  where it studies , 25 havell count . For tre resolve . However resolve , althoug resolve . The mos resolve . Typical resolve . For thi resolve  and Zamb resolve  where it resolve  where woIn conclusion, Kaposi's sarcoma-associated herpes virus (KSHV) genes, K1 and K15 (P) were detected among tissue blocks collected from patients infected with HIV-1 and AIDs. The morphology of KS lesions upon histological diagnosis was distributed as patches, plaques and nodules. East Africa is traditionally known to have a high prevalence of KS . The preIn resources limited health facilities, Kaposi's sarcoma is routinely diagnosed by clinical presentations and cytological identification using H & E staining technique.There is need to detect HHV-8 using a robust diagnostic techniques to aid a precise patient management because the patients in this study had a dark skin pigmentation and in another study conducted on dark-skinned patients, KS had been confirmed to mimic a number of non-KS like dermatological conditions;It further stated that the patients who presented with violaceous skin lesions were likely to be considered a suspected case of KS which is difficult to demonstrate in dark-skinned individuals.The authors declare no competing interests.


Mycobacterium leprae, the intracellular bacterium that causes leprosy, the addition of exogenous innate or adaptive immune ligands to the infected monocytes/macrophages was required to detect a vitamin D-dependent antimicrobial activity. We investigated whether there is an intrinsic immune response to M. leprae in macrophages that is inhibited by the pathogen. Upon infection of monocytes with M. leprae, there was no upregulation of CYP27B1 nor its enzymatic activity converting the inactive prohormone form of vitamin D (25-hydroxyvitamin D) to the bioactive form . Given that M. leprae-induced type I interferon (IFN) inhibited monocyte activation, we blocked the type I IFN receptor (IFNAR), revealing the intrinsic capacity of monocytes to recognize M. leprae and upregulate CYP27B1. Consistent with these in vitro studies, an inverse relationship between expression of CYP27B1 vs. type I IFN downstream gene OAS1 was detected in leprosy patient lesions, leading us to study cytokine-derived macrophages (MΦ) to model cellular responses at the site of disease. Infection of IL-15-derived MΦ, similar to MΦ in lesions from the self-limited form of leprosy, with M. leprae did not inhibit induction of the vitamin D antimicrobial pathway. In contrast, infection of IL-10-derived MΦ, similar to MΦ in lesions from patients with the progressive form of leprosy, resulted in induction of type I IFN and suppression of the vitamin D directed pathway. Importantly, blockade of the type I IFN response in infected IL-10 MΦ decreased M. leprae viability. These results indicate that M. leprae evades the intrinsic capacity of human monocytes/MΦ to activate the vitamin D-mediated antimicrobial pathway via the induction of type I IFN.Following infection, virulent mycobacteria persist and grow within the macrophage, suggesting that the intrinsic activation of an innate antimicrobial response is subverted by the intracellular pathogen. For  Mycobacterium leprae, the etiological agent of leprosy, we describe an immune evasion mechanism whereby this bacterial pathogen utilizes our own antiviral immune response against the macrophage. Type I interferons (IFN) are a major part of our immune response to viral infections; however, this response will also suppress our ability to fight opportunistic bacterial infection. During infection of our macrophages, M. leprae induces an aberrant type I IFN response that subsequently suppresses our macrophage’s ability to activate the vitamin D-mediated antimicrobial pathway, a critical antimicrobial response for containment of mycobacterium. Thus, understanding how these pathogens can evade our immune response will be important for the development of new therapies against these chronic infections.Our macrophages are equipped with the ability to detect and kill invading pathogens, and yet, these cells of the innate immune system are still subject to infection by intracellular bacterium. In particular, mycobacterium, the type of intracellular bacteria responsible for diseases such as tuberculosis and leprosy, are very successful at establishing infection within macrophages. By studying  The ability of macrophages (MΦ) to kill intracellular pathogens is critical to the outcome of infection. Addition of exogenous ligands derived from the pathogen such as a Toll-like receptor 2 ligand (TLR2L) or from human immune cells, such as IFN-γ, provides an extrinsic signal to activate a vitamin D-dependent antimicrobial activity against mycobacteria in infected MΦ , 2. In tMycobacterium leprae, presents as a spectrum of disease in which the clinical manifestation correlates with the immunological state of the patient. At one end is the self-limiting tuberculoid form (T-lep) and the other end is the disseminated lepromatous form (L-lep) -25D3  substrate for 5 hours in serum-free RPMI media. [3H]-metabolites were purified using a C18 column and separated using a Zorbax-sil column . Radioactivity was measured in each sample by scintillation counting. The amount of each metabolite present was quantified from counts per minute plotted against elution profiles of standards for 25D3, 1,25D3, and 24,25D3 [Following treatments as indicated in the text, monocytes or MΦ were incubated with radiolabeled  24,25D3 .Total RNA was harvested using TRIzol  and cDNA synthesized using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). mRNA expression levels for CYP27B1, CYP24A1, VDR, CAMP, and IFNB were assessed using the Taqman system and pre-verified primer and probe sets (Thermo Fisher Scientific). Relative expression was quantified by comparing to the housekeeping gene 18S rRNA and using the ΔΔCt method , 14. OASSecreted IFN-β protein in the supernatant were measured using VeriKine Human Interferon-Beta ELISA Kit (PBL Interferon Source) following the manufacturer’s protocols.http://www.ncbi.nlm.nih.gov/geo/) accession number GSE17763.The microarray experiment was previously published . Gene ex3 U/mL IL-4 , 10μg/mL IL-10 , or 200ng/mL IL-15 (R&D Systems) for 48 hours immediately following plastic adherence [in vitro by the addition of 25-hydroxyvitamin D3  to the extracellular medium of MΦ cultures.MΦ were differentiated using our previously described rapid protocols; briefly, monocytes were directly treated with 10dherence , 13. ComM. leprae at MOI of 10 in antibiotic free RPMI1640 supplemented with 10% FBS for 16 hours; then washed vigorously to remove extracellular bacterium and cultured in RPMI 1640 supplemented with 10% FBS, penicillin and streptomycin.MΦ were infected with single cell suspensions of M. leprae as indicated above. Following washing the infected MΦ were cultured with 10% 25D sufficient human serum. Total RNA and DNA were harvested from M. leprae-infected macrophages as indicated in the text and cDNA was synthesized from the RNA fraction. Bacterial 16S rRNA and M. leprae repetitive genomic element DNA (RLEP) were measured using quantitative PCR. Bacterial viability was calculated by comparing 16S rRNA to RLEP DNA as previously published [MΦ were infected with ublished .Our previous study indicated that components of the vitamin D-mediated antimicrobial pathway, including CYP27B1 mRNA, were more highly expressed in T-lep lesions as compared to L-lep lesions . ExaminiM. leprae bacilli are more abundant in L-lep lesions, we determined the effect of M. leprae on CYP27B1 expression and function. Cultures of primary human monocytes were infected overnight with live M. leprae at a multiplicity of infection (MOI) of 10. Following infection of monocytes, total RNA was isolated, then CYP27B1 mRNA expression was assessed by qPCR. M. leprae infection did not significantly induce CYP27B1 mRNA expression in monocytes, whereas stimulation with exogenous Toll-like receptor 2/1 ligand (TLR2L) did . While monocytes treated with the exogenous TLR2L were able to significantly convert 25D3 into 1,25D3, consistent with previous studies [M. leprae-infected monocytes did not  did . Given t did not . Conversnd TLR2L . The rednd TLR2L . These ring TLR2 , it doesM. leprae induces monocytes to release a broad array of cytokines [M. leprae infection, IFN-β [M. leprae infected cells [M. leprae induction of type I IFN inhibits the intrinsic upregulation of CYP27B1 in the infected cells. Mining unpublished data from our previous study [M. leprae infection significantly induced mRNA expression of IFN-β and IFN-β protein; however, TLR2L had no effect  and two way ANOVA analysis confirms that leprosy type of the lesion is significantly (P = 0.0003) associated with differential gene expression of CYP27B1, CD163 and OAS1 . Indeed, when exogenous 25D3 was added to M. leprae infected IL-15 MΦ at various doses, CAMP gene expression was induced in a dose-dependent fashion , treatment with the IFNAR neutralizing monoclonal antibody significantly reduced M. leprae viability as compared to the isotype control  derived from the pathogen provides an extrinsic mechanism to activate a vitamin D-dependent antimicrobial response in infected MΦ, the intrinsic activation of innate antimicrobial responses by the pathogen is not sustained . Here weM. tuberculosis suggests that subversion of the initial host response is critical to establishing infection [M. leprae infection in the absence of exogenous triggers (TLR2L or IFN-γ), which is inhibited by the aberrant infection-induced expression of type I IFN. M. leprae may activate the intrinsic immune response in MΦ through a variety of pattern recognition receptors including TLR4, TLR9 and NOD2, but most importantly, TLR2 via the 19kDa [There is evidence for the existence of intrinsic anti-mycobacterial responses, although these are not usually sustained, and escaping the initial antimicrobial response is key for the pathogen to establish long term infection . The idenfection . Therefonfection  or supprhe 19kDa  and mLEPhe 19kDa , since TM. leprae showed little intrinsic induction of CYP27B1 expression or enzyme activity. When the type I IFN receptor (IFNAR) was neutralized during M. leprae infection, the intrinsic induction of CYP27B1 during M. leprae infection was uncovered. This suppression of CYP27B1 has in vivo relevance, as we found that CYP27B1 is more highly expressed in T-lep vs. L-lep lesions, the self-limited vs. progressive forms of leprosy, respectively, and was inversely correlated with type I IFN signaling. Using our previously characterized and described in vitro models of the L-lep and T-lep resident MΦ (M2-like IL-10 MΦ and M1-like IL-15 MΦ) [M. leprae infection of IL-10 MΦ induces a type I IFN response that inhibits CYP27B1 expression, similar to observations in monocytes. In contrast, IL-15 MΦ maintain their CYP27B1 expression and function in the presence of M. leprae infection. These data indicate that the regulation of CYP27B1 in infected MΦ at the site of disease is critical to activation of the intrinsic antimicrobial response.The intrinsic activation of the vitamin D antimicrobial pathway requires the metabolic conversion by the CYP27B1 enzyme of 25D, the inactive circulating prohormone (25D) and requires into the bioactive form  to transactivate its cognate receptor, the vitamin D receptor (VDR). Activation of human monocytes and MΦ by exogenous innate and adaptive immune signals  have been shown to induce expression and function of CYP27B1 [Streptococcus pneumonia [M. leprae infection in vitro, the induction of type I IFN provides an escape mechanism by which the bacterium subverts the vitamin D-mediated antibacterial response. The ability of type I IFN to inhibit CYP27B1 expression is likely related to the production of the immunosuppressive cytokine IL-10, which has been shown to be induced by type I IFN during M. leprae infection and inhibit CYP27B1 expression [http://motifmap.ics.uci.edu/), which is a type I IFN inducible transcription factor with known gene suppression functions [M. leprae blocks host defenses, some mediated through the mycobacterial cell wall component phenolic glycolipid 1 (PGL-1). Several studies have shown that PGL-1 manipulates host defense mechanisms such as complement activation, phagocytosis as well as cytokine release to inhibit maturation of dendritic cells and modulate T cell responses [M. leprae infection and not induced by purified TLR2 ligands [M. leprae infected IL-10 MΦ resulted in decreased bacterial viability by uncovering the intrinsic vitamin D-mediated antimicrobial response. Indeed, further understanding of the pathways by which M. leprae initiates immune inhibitory mechanisms such as induction of the type I IFN pathway will provide novel therapeutic targets for mycobacterial diseases.The induction of type I IFN is a well-studied host defense mechanism against viral infection; however, their role in the immune response against intracellular infection with mycobacteria and bacteria is less defined . While tneumonia . The facneumonia , suggestpression , 17. Altunctions . Other sesponses –33; all esponses . Similar ligands . ImportaThere is a well characterized genetic and functional association of the vitamin D pathway with leprosy. Single nucleotide polymorphisms in the VDR gene are associated with the different forms of leprosy , 34, as S1 Data(XLSX)Click here for additional data file.


ATPase is the most studied 2C protein in the Picornaviridae family. It is involved in RNA replication, encapsidation and uncoating and many inhibitors have been found that target PV 2CATPase. Despite numerous investigations to characterize its functions, a high-resolution structure of PV 2C has not yet been determined. We report here the crystal structure of a soluble fragment of PV 2CATPase to 2.55Å, containing an ATPase domain, a zinc finger and a C-terminal helical domain but missing the N-terminal domain. The ATPase domain shares the common structural features with EV71 2C and other Superfamily 3 helicases. The C-terminal cysteine-rich motif folds into a CCCC type zinc finger in which four cysteine ligands and several auxiliary residues assist in zinc binding. By comparing with the known zinc finger fold groups, we found the zinc finger of 2C proteins belong to a new fold group, which we denote the “Enterovirus 2C-like” group. The C-terminus of PV 2CATPase forms an amphipathic helix that occupies a hydrophobic pocket located on an adjacent PV 2CATPase in the crystal lattice. The C-terminus mediated PV 2C-2C interaction promotes self-oligomerization, most likely hexamerization, which is fundamental to the ATPase activity of 2C. The zinc finger is the most structurally diverse feature in 2C proteins. Available structural and virological data suggest that the zinc finger of 2C might confer the specificity of interaction with other proteins. We built a hexameric ring model of PV 2CATPase and visualized the previously identified functional motifs and drug-resistant sites, thus providing a structure framework for antiviral drug development.Poliovirus (PV) 2C ATPase encoded by poliovirus is one of the most important drug targets, and many inhibitors of 2C have been found. We report here the crystal structure of a soluble portion of PV 2CATPase, containing an ATPase domain, a zinc finger and a C-terminal helical domain. Our findings not only revealed common and individual structural features in the picornaviral 2C protein family, but also allowed us to visualize a large collection of functional motifs and drug-resistant sites identified from the decades-long investigations of poliovirus 2CATPase. Our findings are invaluable for understanding the function of picornavirus 2C proteins and the development of antiviral drugs.Since the launch of the Global Polio Eradication Initiative, the number of poliomyelitis cases has significantly reduced but obstacles to disease eradication remain. In the endgame phase, anti-poliovirus drugs will be critical in controlling transmission of vaccine-derived polioviruses and in treating patients with chronic infection. However, no effective anti-poliovirus drugs are yet available. The 2C The incidence of paralytic polio in 1988 was 1,000 children per day, and this number decreased to 400 per day in 2013 labelled ATP. The mixtures were incubated at 30°C and the reactions were initiated by adding enzyme. At the given time point, 10μL of the reaction mixture was removed and mixed with EDTA  to quench the reaction. At least three time points were recorded for each reaction. The mixtures were resolved by thin-layer chromatography using PEI (Polyethylenimine) Cellulose Plates (Sigma-Aldrich) with a buffer containing 0.8M acetic acid and 0.8M Lithium chloride [ATPase assays were performed as previously described ,36,54. Tchloride . The PEI2 in DMEM, supplemented with 10% Fetal Bovine serum. EV71 infectious clone contained the full-length cDNA of WT virus. The mutations were introduced by the site-directed mutagenesis. The plasmids were linearized by HindIII digestion and were in vitro transcribed to RNAs using the MEGA script T7 Kit (Ambion). Subsequently, the RNAs were transfected in Vero cells with Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. The Vero cells were cultured at 37°C. 72 hours post-transfection, the supernatants of cell culture were collected to infect RD cells seeded in a 24-well plate at the given temperatures. The RD cells were fixed 24 hours post-infection. Immunofluorescence assays were performed to probe the production of EV71 virus using mouse anti-EV71 monoclonal antibody and Donkey anti-mouse immunoglobulin G secondary antibody. At least eight different visual fields from each well were photographed randomly under the microscope. The positive dots were counted using the software ImageJ v1.2.0 [Vero and RD cells were grown with 5% COJ v1.2.0 .The Superdex200 10/300 GL column  was equilibrated with a buffer containing 20mM HEPES pH 7.5 and 100mM NaCl and 5mM DTT. The column was calibrated with Gel Filtration Standard (BIO-RAD) containing thyroglobulin (670kDa), γ-globulin (158kDa), ovalbumin (45kDa), myoglobin (17kDa), and vitamin B12 (1.35kDa). The purified proteins were loaded and eluted with a flow rate of 0.5ml/min. The Log of the molecular mass (kDa) of the standards was plotted as the function of the elution volume. Four standard proteins: thyroglobulin, γ-globulin, ovalbumin and myoglobin were used for a linear-fitting to generate a standard curve. The molecular mass of PV-2C-ΔN variants was then calculated using the standard curve.S1 FigThe ribbon models of chain A (red), chain B (orange), chain C (yellow), chain D (green), chain E (cyan) and chain H (blue) were aligned using Dali pairwise structure comparison server. The structural comparison between chains A and B gave Z-score = 33.1, rmsd = 1.3Å with 204 residues aligned. The largest structural deviation between A and B chains is contributed by a small loop region 180-184aa of B chain between β3 and α2, which is resulted from the crystal packing. The structural comparison between chains A and C gave Z-score = 31.5, rmsd = 0.5Å with 188 residues aligned. The structural comparison between chains A and D gave Z-score = 34.6, rmsd = 0.4 Å with 202 residues aligned. The structural comparison between chains A and E gave Z-score = 32.8, rmsd = 0.8Å with 201 residues aligned. The structural comparison between chains A and H gave Z-score = 10, rmsd = 1.2Å with 89 residues aligned. The C-terminal helix of different chains exhibits only slightly different conformation, but this is not comparable with the large conformational changes observed at the C-terminal helix in EV71 2C structure (PDB entries: 5GQ1 and 5GRB).(DOCX)Click here for additional data file.S2 FigThe CD spectrum of the MBP-tagged PV 2C with the wild-type sequence (red) is overlaid with the CD spectrum of the mutant  used for crystallization. The experiment shows that the triple mutations does not affect the overall folding of the protein.(DOCX)Click here for additional data file.S3 Figepresentative structures of different zinc fingers. The structures are shown with ribbon models and colored by secondary structure elements; α-helices are red, β-sheets are yellow, loops are green. The zinc ions are shown with gray spheres. Ligands for zincs, histidine or cysteine, are shown with stick models. (A) Left, structure of PV 2C zinc finger with 4 cysteine ligands (Cys4 type); right structure of EV71 2C zinc finger with 3 cysteine ligands (Cys3 type). These zinc fingers cannot be placed into any of the eight known zinc finger fold groups (illustrated in panel B-I), therefore, we classify them into a new fold group, denoted, Enterovirus 2C-like group. B to I. Representative structures from eight zinc finger fold groups defined by Krishna and colleagues. (B) Fold group 1: C2H2-like, PDB code: 1T6D chain D residues 42–69. (C) Fold group 2: Gag knuckle, PDB code: 1A1T chain A residues 12–30. (D) Fold group 3: treble clef, PDB code: 1HCQ chain A residues 5–36. (E) Fold group 4: zinc ribbon, PDB code: 1TFI chain A residues 1–50. (F) Fold group 5: Zn2/Cys6 like, PDB code: 2HAP chain C residues 62–95. (G) Fold group 6: TAZ2 domain like, PDB code: 1F81 chain A residues 37–65. (H) Fold group 7: zinc binding loops, PDB code: 1I3Q chain C residues 84–96. (I) Fold group 8: metallothioneins, PDB code: 4MT2 chain A residues 1–61.(DOCX)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 File(PDF)Click here for additional data file.