The name Influenza was given during Renaissance when unknown infection hit Florence, Italy, and people have correlated the disease with the stars alliance: Influence of the Stars ( it.
Influenza ) . Once an avian virus, it was transmitted to worlds around 10,000 old ages ago during the Ice Age ( Collier and Oxford, 2000 ) .I.1.1. Epidemiology of Influenza A VirusRemembering the pandemic Spanish grippe in 1918 and the avian grippe in 1997, 2004, and the pandemic H1N1 Swine beginning grippe virus ( SOIV ) from 2009 ( Medina and Garcia-Sastre, 2011 ) we realise that Influenza A virus viral pathogens have the possible to take to new pandemic viruses ( Cheung and Poan, 2007 ) .Harmonizing to World Health Organisation ( WHO ) , influenza causes yearly around three to five million instances of terrible unwellnesss. Almost 250,000 to 500,000 instances where Influenza A virus is responsible terminal up in deceases.
Most vulnerable to influenza are people aged 65 and over and babies ( WHO, 2012 ) .Influenza A virus is frequently associated with seasonal morbidity and mortality. Influenza A viruses are subtyped harmonizing to their antigenic and familial nature of the glycoproteins found at the surface: 15 hemagglutinin and 9 neuraminidase. So far lone viruses of H1N1, H2N2 and H3N2 subtypes are associated with human epidemics. The epidemiology of these viruses is due to the antigenic fluctuations events that are present at the surface of the two surface glycoproteins of the virus: HA and NA. These new fluctuations have the ability to infect people despite anterior inoculation.
Mutants of HA and NA cistrons are taking to new fluctuations of grippe A virus and these mutants are go oning as a consequence of RNA polymerase unfunctional activity during the transcribing of the grippe A virus genome. The new strains appear as response to familial alterations that encode the amino-acids in the HA and NA. Antigenic displacement is a type of fluctuation which occurs in grippe A viruses when a new IAV is developed by the alteration of HA or sometimes NA that is introduced in human population.
This event might go on when IAV is transmitted from an animate being or bird to worlds. Or when because of familial reassortment between the animate being and human a new offspring virus is arised. Some say that the avian grippe A viruses should get at least one cistron from worlds when it crosses the species barrier. Worlds can besides be infected with swine influenza A viruses and these sporadic infections are really unsafe for pregnant adult females of individuals with weak immune system ( Cox and Subbarao, 2000 ) .
I.1.2. Pathogenesis of Influenza A virusInfluenza A virus is considered to be the most infective member of its household. It can infect worlds of all age taking to sporadic infections in population to epidemics and pandemics ( Julkunen et al. , 2001 ) .
The Orthomyxoviridae household members cause grippe in mammals and birds as it is illustrated in Table 1. The Orthomyxoviridae household consist of Influenza A, B and C virus and 2 other household members.Virus typeNumber of cistron sectionSurface glycoproteinsHostInfluenza A8HA+NAWorlds, hogs, giants, birds, and othersInfluenza B8HA+NAWorlds and sealsInfluenza C7Hemagglutinin Esterase-Fusion ( HEF )Largely worlds, besides found in swineTable 1. Features of different types of grippe virus ( reupdated after Cheung and Poan, 2007 ) .Influenza viruses replicate in the epithelial cells of the upper respiratory piece of land.I.1.3.
Influenza A virus morphology and constructionAs morphology, the virions are spherical and are about 100-200 nanometer in diameter and filiform and step about 100 nanometers in diameter and 20Aµ long. HA is known to be really of import in the fond regard and entry of virus to the host ‘s cell, whereas NA is associated with the release of the new virus from cells. Following fond regard to specific receptors, virions are endocytosed and the acid pH in the endosomes facilitate a alteration of the HA. Protons facilitate the release of M1 protein from the ribonucleoprotein complex, following RNA to come in the cell karyon for written text and reproduction ( Collier and Oxford, 2000 ) .Influenza A Virus is an Orthomyxoviridae household member, with an enveloped negative sense RNA genome. It has 8 RNA sections that encode 11 proteins, and the late discovered N40 protein that is expressed in the PB1 section ( Medina and Garcia-Sastre, 2011 ) . Eight of the proteins are packaged in enveloped virions and the viral envelope is made out of two envelope glycoproteins: hemagglutin ( HA ) which is the receptor adhering membrane merger protein, and the enzyme neuraminidase ( NA ) , ( Enami et al, 1991 ) .The undermentioned elements are found in grippe A viruses: 3 polymerases ( PB1, PB2, and PA ) , matrix protein M1, ion channel protein M2 and non structural proteins NS1 and NS2 ( Julkunen et al.
, 2001 )Influenza A virus is an enveloped virus with pleumorphic virions. The forms differ from little spherical to long filiform. HA, NA, M2 viral proteins are impacting the morphology of the grippe virus atoms. The lipid envelope feature for grippe A virus is hosts cell derived during budding. HA, NA and M2 are found in the lipid envelope. The spike glycoproteins are rod molded and mushroom shaped harmonizing to electron micrographs surveies. Homotrimer HA involved in membrane merger and it ‘s a receptor binding, NA homotetramer hydrolyses sialic acid gropus in order to let go of the new viral atoms. M2 is an built-in membrane homotetramer and maps as an ion channel ( Cheung and Poon, 2007 ) .
In 1980 ‘s it was discovered that the Influenza A virus written text was located in the karyon. This major determination has led to the decision that influenza viruses uses the atomic compartment to transcribe and retroflex its genome utilizing the host ‘s RNA Pol II written text and splicing machinery ( Amorim and Digard, 2006 ) .I.1.4. Influenza A virus constituentsInfluenza A virus has 8 sections that are encoding for 11 viral cistrons: hemagglutinin ( HA ) , neuraminidase ( NA ) , matrix 1 ( M1 ) , matrix 2 ( M2 ) , nucleoprotein ( NP ) , non-structural proteins ( NS1, NS2 ) , polymerase acidic protein ( PA ) , polymerase basic protein ( PB1, PB2, PB1-F2 ) . Largely all the grippe A virus virions have been shown to be spherical, and HA, NA and M ” are the three viral transmembrane proteins that are doing the viral envelope lipid bilayer. The host ‘s lipid bilayer is rich in cholesterin enriched lipid tonss.
HA is found in copiousness as an envelope protein and a really little per centum for NA and M2. HA and NA are affiliated with the lipid tonss. M1 is found underneath the viral lipid membrane and is called matrix proteins because that where it holds the vRNPs.
The vRNPs are created out of negative stranded RNAs that are packaged around NP. The polymerase proteins PB1, PB2 and PA are found at the border of vRNPs and together are bring forthing the viral RNA polymerase composite ( Tasleem Samji, 2009 ) .I.1.4. Influenza A virus life rhythmThe grippe A virus has the undermentioned life rhythm phases: host cell entry, vRNPs entry into the karyon, written text and reproduction of the viral genome, vRNPs export from the karyon, assembly and budding at the host ‘s cell plasma membrane ( Tasleem Samji, 2009 ) .The sialic acid found at the cell surface glycoproteins are working as receptors for grippe A virus.
The viral atom is so endocytosed thru clarithrin – dependent endocytotic tract. The merger is induced by the low pH in endosomes, and as a response the viral ribonucleoprotein ( vRNP ) is released into the cytol. Viral nucleocapsid is introduced into the karyon and mRNA synthesis is induced by viral RNA polymerase which contains PB1, PB2 and PA polymerases. M1 and NS2 proteins are besides migrating to the karyon and as a consequence of their interaction with the vRNPs the conveyance of the freshly synthesised vRNPs is controlled. vRNP-M1 protein complex collaborates with HA and Na molecules and as a consequence the budding of the virions takes topographic point. Once the IAV production is dynamic it destroys the hosts cell pre-mRNAs, inhibits interlingual rendition of messenger RNA and the hosts cell is killed by programmed cell death or by cytolitic mechanisms ( Julkunen et al. , 2001 ) .
Host cell entryHA plays the most of import function in this first phase. HA is a homotrimer and signifiers spikes at the surface of the viral lipid membrane. These spikes are sialic acid edge at the host ‘s cell. After attaching of the viral atoms they are incorporated into the host ‘s cell utilizing the clathrin – dependent endocytosis. The pH in the endosome is low and that leads to merger between the viral and endosomal membrane. The acidic environment in the endosomal compartment leads to the activation of the M2 ion channel. M2 is a transmembrane protein that forms a proton selective ion channel.
Because of the M2 ion channel acidification the vRNPs are released ( Tasleem Samji, 2009 )Harmonizing to Pielak and Chou, 2010 the M2 protein is encoded on the 7th RNA section along with M1 and has three sections: an extracellular N-terminal section, a transmembrane section and an intracellular C terminus section. M2 protein is activated proton channel that mediates the uncoating of the viral atoms after their merger in endosomes by commanding the acidification of the interior parts of the endocytosed atoms ( Stouffer et al. , 2008 ) . M2 specific 97 amino acid membrane protein is indispensable in the successful release of the viral genome during grippe A virus entry.
With the interaction of M1 the viral genome is released and the viral RNA sections will go to the host ‘s karyon. This dynamic procedure is followed by grippe by influenza virus RNA reproduction and written text ( McCown and Pekosz, 2006 ) .Research on spherical virions has shown that grippe virus enters the cell by receptor mediated endocytosis, although the mechanism on how the endocytosis is induced is still non known. A intuition is that spherical virus binding atoms are triping a receptor tyrosine kinase ensuing in cellular signalling that triggers the incorporation of influenza virions. It is believed that the filiform influenza virions may come in the cells by utilizing the macropinocytosis. In septic cells, both signifiers of grippe virus usage lipid raft spheres located at the plasma membrane.
These lipid tonss are used as the sites of virus assembly and budding ( Rossman and Lamb, 2011 ) .Lipid tonss are located at the plasma membrane and are specific microdomains that are involved in cellular traffic and signal transduction tract. In some surveies the tonss have been identified as drifting elements within the cytoplasmatic fluid. Because of their little size, mobility and instability tonss are acting as transporters of proteins from the Golgi setup to the cell surface ( Chazal and Gerlier, 2003 ) .The viral merger requires that the responsible viral protein to trip a hydrophobic merger peptide. This alteration is favourised by impersonal pH, where the viral envelope ‘s glycoproteins are adhering to the cellular receptors or by an acidic pH merger protein.
As a response the viral atoms are foremost induced in endocytosis, that being the instance of grippe viruses. Experimental information has shown that lipid tonss located at the membrane have function in the virus entry. First there is the aggregation of the viral glycoproteins in the tonss and their interaction and recrution of the cellular receptor in tonss.
Finally the virus suppression is resulted as a response to a low degree of cholesterin ( Chazal and Gerlier, 2003 ) .A important function in virus entry, assembly and budding has the matrix protein M2 a proton selective, ion channel sphere that together with M1 provide the virion ‘s construction and intercede the viral lipid membrane – ribonucleoproptein ( RNP ) interaction ( Rossman and Lamb, 2011 ) . It was shown that low pH in the endosome has effects in triping the HA mediated merger of the viral endosomal membranes and besides for the M2 activation channel. HA and NA in grippe A virus have an intrinsic relation with the lipid raft spheres compared with the M2 protein that is outside of these spheres. M2 is responsible for interceding the proton conductivity to the virion nucleus.
This will hold effects on the RNP of M1 that will trip its conveyance to the karyon and marks the start of the viral reproduction ( Rossman et al. , 2010 ) .vRNPs entry into the karyonThe viral written text and entry of grippe A virus is effectuated in the karyon. NP, PA, PB1, PB2, are viral proteins that create the vRNPs. Because of the atomic localization of function signals ( NLS ) vRNPs can adhere to the nucleus import machinery, therefore they enter the karyon ( Tasleem Samji, 2009 ) .Transcription and reproductionInfluenza a virus has a negative strand RNA and it needs a positive sense RNA strand for its genome to be transcribed.
The positive strand RNA serves a templet for the vRNAs production. Viral RNA dependent RNA polymerase ( RdRp ) initiates RNA synthesis and many of the host ‘s cell machinery are used, such as host written text machinery. The hosts cellular molecules that are involved in the RNA synthesis are: BAT1, Heat daze protein 90 ( Hsp90 ) , the mini-chromosome care composite ( MCM ) , Tat-SF1 and DNA dependent polymerase II. The vRNA are transcribed into messenger RNA and replicated. Cellular RNA polymerase II binds to DNA and initiates written text. RdRp is known to adhere to Pol II. Splicing factor BAT1 for messenger RNA interacts with NP and leads to the formation of NP-RNA complex. Tat-SF1 facilitates the NP-vRNP formation composite.
Hsp90 interacts with PB2 and stimulates viral RNA synthesis ( Watanabe et al. , 2010 ) .Export of vRNPs from the karyonNegative sense vRNPs are exported from the karyon utilizing the CRM1 dependant tract through the atomic pores.
NP is interacting with CRM1, M1 protein is interacting with the vRNPs because of its C-terminal and because if it ‘s N-terminal of M1 binds to NEP. After the binding of M1 to the RNP composite, NEP attaches to M1-RNP complex utilizing its C-terminal sphere. Heat daze blood relation ( Hsc ) 70 protein and MAP kinase cascade are believed to play of import functions in the atomic conveyance of the vRNP composites ( Tasleem Samji, 2009 ) .Assembly and buddingOnce the vRNAs are outside of the karyon, the virus prepares its viral atoms to be released outside of the cell.
Influenza A virus uses the host ‘s plasma membrane to organize the viral atoms. All the viral proteins that are found in the lipid bilayer HA, NA and M2 are found at the viral atom forming. Virus particles bud from the apical side of polarised cells, triping the conveyance of HA, NA, and M2 to the apical plasma membrane. Surveies have shown that M2 cytoplasmic tail ( CT ) plays a really of import function in viral atoms formation. The omission of M2 CT led to the production of extended viral atoms.
M1 is involved in concluding stairss of budding. A really of import phase happening right before the release of the new viral atom is the cleavage of sialic acid residue from glycoproteins. Prior to bud release, NA removes these sialic acids and the freshly made viral bud is released ( Tasleem Samji, 2009 ) .Viral morphogenesis and budding need four stairss: viral constituents assembly, bud induction, bud developing and incorporation in the plasma membrane. The most of import events of the viral budding happen during assembly, when the viral constituents are brought to the budding site where they initiate budding.
Influenza virus atoms require three elements: the viral envelope incorporating a lipid bilayer and the three viral transmembrane proteins: HA, NA and M2. The protein that forms a span between the envelope and the viral nucleus is M1 protein, located underneath the lipid bilayer. The viral nucleocapsid or viral nucleus consists chiefly of vRNP ( viral nucleocapsid ) ( Nayak et al. , 2009 ) .HA associated with the lipid raft is important for viral reproduction. Nevertheless surveies have shown that the activation of the antiviral protein Viperin causes an instability of the lipid raft spheres that has effects in grippe A virus reproduction decreasing.
In surveies were virus like atoms ( VLP ) have been used, the HA protein buds from cells in cysts and it does non necessitate any viral protein look. VLPs deficiency of protein synthesis that normal viral proteins have and as a consequence the VLPs show aberrant assembly and budding. M1 viral protein is a good campaigner as a budding completion component. M1 interacts in the virion with the plasma membrane and the NP and it was shown that M1 is polymerised at the sites of budding therefore it may supply the viral elongation mechanism. When used in VLP system it was noticed that M1 together with HA induce increased efficiency of the viral budding. But what was more surprising was that the add-on of M2 protein in the HA+M1+M2 composite that was used in the VLP system resulted in important budding increasing. Thus M2 is indispensable for the completion of the viral budding procedure ( Rossman and Lamb, 2011 ) .I.
2. M2 viral proteinM2 viral protein is a proton selective ion channel incorporated within the viral envelope and allows protons to come in virus atoms in endosomes. M2 is a tetramer which consists of four polypeptide ironss of 97 residues.
The 24th residue is an N – terminal extracellular sphere that acts as a transmembrane sphere ( Rossman et al. , 2010 ) .M2 is pH activated and controls the degrees of acid inside the viral atoms. Rossman et al.
, have shown that M2 amphipathic spiral is conserved when studied in grippe A virus. The amphipathic spiral is used by the viral proteins to modify the membrane ‘s form, M2 is able to modify the form of the membrane as the cholesterin is able to modify the amphipathic spiral membrane curvature. When examined the localization of function of M2 by immunogold labelling and negatron microscopy in septic cells it was revealed that M2 is localised preponderantly at the base of budding virions. The most of import determination in this survey was that mutated M2 amphipathic spiral blocks membrane scission bespeaking a major function of M2 during the concluding phase of viral budding ( Rossman et al. , sept 2010 ) .I.
3. Annexin A6Derived from Grecian “ annexin ” which means “ bring/hold together ” proposing the belongings of the annexins to keep precise biological constructions such as membranes. Annexin proteins are adhering Ca2+ spheres and incorporate a conserved annexin repetition of 70 amino acid sequence ( Gerke and Moss, 2002 ) .Annexin A6 is a member of the Annexin household of calcium/ phospholipid binding proteins.
Annexin A6 is expressed in mammalian cells largely in specialised cell types such as endocrine cells, and different types of Annexin are regulated in different ways ( Smith et al. , 1994 ) .Annexin A6 contains two spheres: a Ca2+ dependant conserved nucleus and phospholipid binding ; and a variable N terminus tail ( Enrich et al.
, 2010 ) .The known maps of annexin A6 associated with the maps of the annexin household member are: phopholipase – A2 suppression, anticoagulation, ordinance of the sarcoplasmic Reticulum Ca channel, and Annexin A6 entirely is involved in growing ordinance, vesicular trafficking mechanisms such as endocytosis and exocytosis. Annexin A6 interacts with the cytoskeleton and plasma membrane and stray Annexin A6 from sciatic nervus binds to actin. The human Annexin A6 is localised on chromosome 5q ( Edwards and Moss, 1995 ) .Annexin A6 localization of function surveies have revealed that the Annexin A6 is present in chondriosome ‘s interior membrane of different tissues by utilizing immunogold negatron microscopy. The localization of function is associated with the Ca2+ high degrees found in chondriosome ( Rainteau et al. , 1995 ) .Annexin A6 lowers the degrees of cholesterin in the Golgi setup at the plasma membrane due to the fact that Annexin A6 sequesters cholesterol in endosomes.
Annexin A6 besides is aiming proteins for some signalling tracts, the most of import being p120GAP, that downregulates Ras. It is believed that the regulators enrolling potency of Annexin A6 involved in the EGFR/Ras tract is promoted by the Annexin A6 actin remodelling. There are features that lead us to another property: Annexin A6 organizer of the membrane spheres. Annexin A6 is a modulator of cholesterin homeostasis, scaffold formation by the signalling composites and membrane – actin adjustor in the endocytic and exocytic conveyance ( Enrich et al. , 2010 ) .
It was besides demonstrated that Annexin A6 had no effects in the virus entry phases ( Ma et al. , 2012 ) .The group of Dr. Beatrice Nal has hypothesised in 2011 that M2 viral protein may interact with host factors that will assist take to suppression or have an heightening consequence in the grippe A virus infection procedure. A good campaigner for this hypothesis is Annexin A6 human protein that was identified as fresh cellular factor that interacts with M2.
As hypothesised, the consequences showed that M2CT interacts with human Annexin A6 utilizing yeast two intercrossed screen as seen in table 2.FeaturesDescriptionBaitM2 CT ( cytoplamic tail )LibraryRandom primed human placenta complementary DNANumbers of interactions tested63.89 millionPreyAnnexin A6Numbers of positive ringers for Annexin A615Table2. Yeast two – loanblend showing, which have led to the designation of the Annexin A6 – M2 CT interaction ( Reupdated after Ma et al. , 2011 ) .
M2 was used as mark ( come-on ) was used to test a random primed cDNA library from human placenta in a yeast-two-hybrid check. This experiment has tested 63.89 million interactions and 15 positive consequences were identified that have shown interaction between M2 CT and Annexin A6. Further experiments have showed that M2 binds to Annexin A6 in human cells HEK293T infected with grippe a virus A/WSN/33.
A549 cells were used to analyze the comparative subcellular localization of function of M2 and Annexin A6 interaction, A549 cells were transfected with an Annexin A6 encoding plasmid and infected with A/WSN/33. In non infected samples Annexin A6 was distributed in the same proportion in the cytosol, the infected but non transfected samples showed M2 localised at the endoplasmic Reticulum and Golgi setup and besides at the plasma membrane. The transfected septic cells showed same M2 degree as the non transfected 1s, but after utilizing a confocal microscope it was revealed colocalisation of M2 and Annexin A6 at the plasma membrane in the septic cells.
Hushing Annexin A6 led to important progeny virus titre increasing, therefore Annexin A6 negatively modulated grippe A virus infection. Nevertheless farther experiments showed that negative transition induced by the absence of Annexin A6 happens merely in late phases of virus life rhythm. And the most of import find was that human protein Annexin A6 negatively modulates influenza A virus reproduction by non leting the virus budding procedure to finish. Following Dr. Beatrice Nal group ‘s determination that Annexin A6 is a negative modulator of grippe A virus infection at the budding phase, indicated by the figure bead of the virus offspring PFU titre in supernatants and the released virions in A431-Anx A6 cells that were demoing an broad extended form alternatively of the spherical form which is characteristic for grippe A virus. Annexin A6 is involved in biological development of the host ‘s mechanisms, and these mechanisms are besides used by the grippe virus in of import viral stairss such as assembly or budding, for illustration cholesterin and cortical actin ( Ma et al. , 2012 ) .In another survey, the cytoplasmatic portion of M2 influenza A virus was found to interact with caveolin-1, that is cholesterol adhering hosts protein and it is found copiously in the lipid raft ( Wang et al.
, 2011 ) .The current intervention used against grippe A virus infection is Amantadine drug that targets M2. Drug opposition and side effects of this drug are increasing so scientific discipline is in demand of a new antiviral drug. As ion channels are indispensable for the viral life rhythm, surveies are concentrating on these membrane proteins with purpose to happen new possible interactions that are taking topographic point between the viral protein channels and the host ‘s protein channels.
Cellular factors that are interacting with M2 are possible campaigners for negatively modulating its activity. As it was demonstrated that Annexin A6 is curtailing influenza a virus infection at the budding phase new radical interaction finds will take to better drug and intervention to be developed ( Nieva et al. , 2012 ) .Annexin A6 known as the member of the Annexin household and a Ca2+ dependant membrane adhering protein, known to “ convey together ” Ca signalling and membrane maps. Annexin A6 interacts with AP-1-clathrin composite at the Aµ1 and Aµ2 fractional monetary units that are involved in endocytosis ( Ma et al. , 2011 ) .The molecular mechanism affecting Annexin A6 and responsible for the negative transition of virions ‘ release from septic cells and/or morphogenesis defects is non known.
Having as get downing point the survey made by Dr. Nal ‘s group in Hong Kong, there are still pending inquiries that need to be answered. Further surveies should clear up whether or non Annexin A6 is a linker to stabilized cortical F-actin. This linkage could be related to M2-mediated late phase of virus budding and be a possible factor of the virus budding completion hold or defect that was found in A431 cells Annexin A6 overexpression. Annexin A6 induced alterations in cortical actin cytoskeleton which may besides impact other actin dependent stairss of virus assembly and budding ( Ma et al. , 2011 ) . In order to happen an reply to this inquiry, farther surveies should concentrate on viral life rhythm phases alterations in F-actin depleted cells.
Knowing that Anx A6 is besides a regulator of cholesterin and Anx A6 is besides a regulator of cholesterin and Anx A6 overexpression led to reduced degrees of condensed membrane spheres, a new survey should clear up if the viral budding is depleted as a consequence of Anx A6 look or because of low degrees of cholesterin and reduced degrees of condensed membrane spheres induced by the Anx A6 depletion or overexpression.And at last but non least new experiments should clear up the molecular mechanism that underlies the Anx A6 mediated depletion of influenza virus budding.I.4. HypothesisIn this undertaking we have used MDCK cells as a control and A431- human vulval squamous epithelial and A549- human dental consonant basal epithelial stable cells as experimental stuffs.The hypothesys of my work is that Annexin A6 localises with M2 at the cervix of budding in grippe A virus at the surface of septic epithelial cells.
Following this hypothesys a farther aim of the survey is to demo that annexin A6 affects virus morphogenesis through its interaction with grippe A virus M2.The chief aim of this undertaking is to optimize the conditions for the Immunofluorescence check by labelling M2 viral protein and Annexin A6 human protein in grippe A virus A/WSN/33 ( H1N1 ) infected cells. The optimised conditions for the Immunofluorescence check will be applied in farther checks that will look into the Anx A6 – M2 interaction and the faulty viral budding phenotype.
Another aim of this survey is to happen the optimum conditions for scanning negatron microscopy ( SEM ) protocol. The SEM experiment was done on MDCK influenza A virus A/WSN/33 ( H1N1 ) infected cells to detect the viral buds morphology. Further SEM experiments should hold the optimised conditions for elaborate probe by labelling M2 and Annexin A6. Besides these conditions could be applied for a future negatron microscopy experiment on grippe A virus septic cells.Chapter II. Materials and MethodsII.1. CellsHuman alveolar basal epithelial ( A549 ) , human vulval squamous epithelial ( A431 ) and Madin-Darby Canine Kydney ( MDCK ) cells were sent by our confederates Francois Kien from the Pasteur Institute in Hong Kong, and grown in Dulbecco ‘s modified Eagle ‘s medium ( DMEM ) ( Thermo Scientific, Rockford, USA ) supplemented with 10 % foetal bovine serum ( FBS ) ( Invitrogen, New-York, USA ) , 100Aµg/ml penicillin and 100 Aµg/ml streptomycin.
II.2. VirusII.2.1. IAV elaborationMDCK cells were grown in 150 cm2 at a concentration of 1 ten 105. Before infection, the cells were washed twice with PBS and 10 milliliter of pure DMEM was added to the flasks.
Consecutive virus dilutions were prepared get downing from a virus titre of 1.8 ten 106 pfu/ml to 1.8 tens 105 pfu/ml and eventually the needed viral dilution per flask of 1.8 ten 104 pfu/ml. We added 1ml of virus MOI: 0.001 into the flask and left it to be absorbed by the cells for 1 H at 37oC. After 1 H we removed the unabsorbed virus and rinsed the cells with PBS and added 25 milliliter of infection medium: DMEM with concluding concentration of 1 % P/S, 1 Aµg/ml TPCK – Trypsin, 0.
3 % BSA ( Invitrogen, California, USA ) . After 3 yearss at 37oC the medium of the cell civilization became partially cloudy due to cell decease.II.2.2 IAV Titration Hemagglutination assayAn IAV titration was performed by utilizing haemagglutination check in order to an accurate virus titre.
The trial was performed utilizing 4ml of Chicken ruddy blood cells ( TCS Biosciences, Buckingham, UK ) and Sheep red blood cells that have been topped up to 9 milliliter with PBS and so centrifuged at 2000 revolutions per minute, 4oC for 10 min. The cell pellet is diluted 1:10 in PBS utilizing a 96 good home base we will add to each good 50 Aµl PBS and 50Aµl RBC resuspended at a concentration of 0.5 % . Get downing from left to compensate we added 50Aµl of IAV A/WSN/33 virus to the first good mixed good so transferred 50Aµl to the following well and so on. After 30 proceedingss we could read the positive consequences that should hold formed unvarying ruddy coloring material and the negative 1s that will look as points in the Centre of the Wellss.II.2.3 IAV Plaque AssaySeveral plaque checks have been performed to happen the accurate virus titre of the stock.
Different conditions have been tested, in the first topographic point the MDCK cells have been seeded on 6 good home bases at a concentration of 3 ten 105, so we prepared the viral dilution ( 0 to 8 ) in the 0 well was added merely virus and in the remainder of the Wellss was added infection media every bit good. After 1 h incubation the unabsorbed virus was removed and the 2 % Agar sheathing in PBS was assorted with the infection medium ( DMEM, 2 % PS, 1Aµg/ml Trypsin-TPCK ( ) and 0.6 % BSA ) , to ensue a 1:1 dilution. We added 2-3 milliliter of sheathing medium to each well and allowed to solidify at room temperature.
The home bases were so turned over and left 3 yearss for incubation. To see any plaques we had to take the agar with the forceps and stain the cells with 0.1 % Cristal violet in PBS.II.
1. Primary antibodiesPrimary antibodies used in this survey were monoclonal mouse anti-M2 ( Santa Cruz Biothechnology, California, USA ) ; polyclonal coney anti-annexin antibody ( Abnova, Taipei, Taiwan ) ; monoclonal mouse anti-annexin antibody ( Abcam, Cambridge, UK ) ; rabbit anti-myc antibody ( Sigma, Dorset, UK ) .II.3.2.
Secondary antibodiesThe secondary antibodies used in this survey were: caprine animal anti-mouse FITC ( Jackson Immunosearch, Pennsylvania, USA ) ; Phalloidin ( Life Technologies, California, USA ) ; goat anti-rabbit Texas-Red ( Invitrogen, California, USA ) .II.4. Immunofluorescence checkII.4.1.
Immunofluorescence assay on non permeabilised septic cells was done to analyze the co localisation of viral protein M2 on MDCK and A431, A431 Annexin A6 cells. The cells were seeded on coverslips prior to infection at 3×105 was on coverslips and infected with influenza A/WSN/33 virus, utilizing different MOI of 1, 0.1, 0.01. The cells were fixed in 4 % paraformaldehyde ( PFA ) ( VWR International, Lutterworth, UK ) in PBS for 15 proceedingss.
Quenching in 50mM NH4Cl ( Fisher, Leicestershire, UK ) in PBS for 15 proceedingss and so the cells were washed 2 times in PBS. For barricading the broad binding of antibodies the cells were incubated for 30 min in 10 % normal caprine animal serum ( NGS ) ( Thermo Scientific, Rockford, USA ) in PBS. The cells were incubated in monoclonal mouse anti-M2 ( Santa Cruz Biotechnology, California, USA ) diluted in 5 % NGS in PBS and so conjugated with secondary antibody Goat anti-mouse – FITC ( Jackson Immunosearch, Pennsylvania, USA ) diluted in 5 % NGS in PBS for 1 hr at room temperature.
DAPI was used to stain the karyon and the images were captured with an Olympus microscope BX 41.II.4.
2. Immunofluorescence assay on non permeabilised, permeabilised A/WSN/33 influenza A virus infected MDCK was done to analyze the carbon monoxide localisation on M2 viral protein in MDCK cells. The cells have been grown on coverslips at a concentration of 3 ten 105 infected with influenza A/WSN/33 virus at MOI.
After 8h incubation the cells have been fixed in 4 % paraformaldehyde in PBS pH 7.4 nightlong. To seek different conditions in different phases of infection some coverslips have been fixed after 24 hours post infection for 10 proceedingss in 50mM NH4CL. Some coverslips have been permeabilised in 0.08 % Triton ( Fisher Scientific, Leicestershire, UK ) x 100 in PBS for 5 min, or in Tween20 ( Fisher, New Jersey, USA ) 0.1 % in PBS. The non permeabilised samples were labelled for M2 viral protein with monoclonal mouse anti-M2 ( Santa Cruz Biotechnology, California, USA ) followed by incubation with goat anti-mouse FITC ( Jackson Immunosearch, Pennsylvania, USA ) , the staining of F-Actin was done utilizing Phalloidin ( Life Technologies, California, USA ) that straight stains actin in blue without adding a primary antibody. Staining after permealisation was done for 5 min in 0.
08 % Triton in PBS at 4oC. Nuclei were stained for non-permeabilised cells with DAPI and the images were captured with Olympus microscope BX 41.II.4.3. Immunofluorescence assay on permeabilised non infected A431 Annexin A6-myc, A431, A549 Annexin A6-myc and MDCK cells was done to analyze different antibodies on order to co localise Annexin A6. The cells were foremost grown on coverslips at a concentration of 3 ten 105. Then they were fixed in 4 % PFA in PBS for 15 proceedingss and quenched for 15 min in 50mM NH4CL.
The cells were permeabilised in 0.08 % Triton in PBS for 5 min at 4oC. After barricading for 30 min in 10 % NGS in PBS the cells were incubated utilizing the polyclonal coney anti annexin antibody ( Abnova, Taipei, Taiwan ) , and the monoclonal mouse anti annexin antibody ( Abcam, Cambridge, UK ) diluted in 5 % NGS and so conjugated with the secondary antibodies Goat anti-rabbit Texas Red ( Invitrogen, California, USA ) and Goat anti-mouse FITC ( Jackson Immunosearch, Pennsylvania, USA ) . To place myc we incubated the coverslips in coney anti myc antibody ( Sigma, Dorset, UK ) diluted in 5 % NGS. The karyon were stained with DAPI and the images were captured with Zeiss – Axioplan 2 microscope.
II.5. SEM assay on WSN infected MDCK cellsScaning negatron microscopy was performed on MDCK cells infected with A/WSN/33 virus to analyze the virus budding. Cells were primary fixed at 8 and 24 hours with 2.5 % glutaraldehyde ( Agar Scientific, Stansted, UK ) in 0.1 Phosphate buffer ( Fisher Scientific, Leicestershire, UK ) pH 7.
2 for 2 hours. Cells were washed in 0.1M Phosphate buffer pH 7.
2 twice for 10 min following a secondary arrested development with 1 % Osmium tetroxide ( Agar Scientific, Essex, UK ) in 0.1 Phosphate buffer pH 7.2 for 1h at 4oC. The cells were washed twice with distilled H2O and so they were dehydrated in increasing concentration of ethyl alcohol. Critical point dry phase followed in CO2 utilizing drier Quorum K 859. After drying the coverslips were mounted onto aluminum holders and so coated with gold to increase their electrical conduction. The samples were examined with Zeiss Supra 35 field emanation scanning electron microscope.Chapter III.
ConsequencesIn this survey we are seeking to corroborate that Annexin A6 is a host cellular regulator of grippe A virus by negatively act uponing the viral budding. However this transition will non be taking topographic point without the interaction of M2 with Annexin A6. The molecular mechanism that underlies the Annexin A6 – M2 interaction still needs to be investigated.III.
1.Following our aim to find the best conditions of infectivity for A431 and A431 Annexin A6, an immunofluorescence check was performed on non permeabilised septic cells.Analysis of M2 look at the surface of septic cells was undertaken and the aim of this experiment was to find the best conditions for the infection of A431 and A431 annexin A6 cells with A/WSN/33 ( H1N1 ) grippe virus. Another aim was the visual image of M2 viral protein at the surface of septic cells. To seek different conditions the cells were infected with grippe A virus at different MOI, several 1, 0.1, 0.
01 ( merely for MDCK cells ) . After 8h p.i. the cells were fixed and labelled without any permeabilisation measure. MDCK cells were used as positive control. Monoclonal mouse anti-M2 conjugated with caprine animal anti-mouse FITC were used as primary and secondary antibody.In A431 cells M2 is distributed in the cytol at MOI 0.1 ( Figure 1, panel A ) , at MOI 1, M2 is largely found at the plasma membrane noticeable in Figure 1, panel B.
In A431 Annexin A6, M2 is seeable at the plasma membrane ( Figure 2, panel A ) but can be noticed in intracellular compartments as good, near to the perinuclear part. This part is expressive for the M2 localization of function at the endoplasmic Reticulum and the Golgi setup. Panel B confirms that M2 is copiously distributed in the intracellular compartments.
These consequences are confirmed by the MDCK control cells in Figure 3, that at 0.01 MOI ( panel A ) there is seeable staining of M2 at the plasma membrane. At higher MOI 0.
1 ( panel B ) and MOI 1 ( panel C ) M2 is seeable at the plasma membrane but it is besides seeable in the cytosol.These findings indicate that M2 viral protein and Annexin A6 human protein are modulating the infectivity of grippe A virus as shown that at the cells infected at the same grippe A virus MOI have resulted in M2 overexpression in the A431 Annexin A6 cells.III.2. Following our nonsubjective to corroborate that M2 has an of import function during virus budding and release at late phases we performed an immunofluorescence check on non permeabilised, permeabilised A/WSN/33 influenza A virus infected MDCK. This experiment has has confirmed that influenza A virus M2 viroporin plays a major function in virus atom release.
This experiment was done to analyze the grippe A virus M2 look at the surface of MDCK cells. The aim of this experiment was to colocalise and depict the grippe A virus M2 look at different station infection times 8h and 24 H p.i. in different conditions: permeabilised and non permeabilised. For better colocalisation of M2 we stained F-actin in MDCK influenza A virus septic cells utilizing Phalloidin. Another aim of this experiment was the optimization of the infection conditions in MDCK cells utilizing A/WSN/33 ( H1N1 ) grippe virus.
MDCK cells were infected with A/WSN/33 ( H1N1 ) and fixed at 8h p.i. and 24h p.i.
M2 was labelled with monoclonal mouse anti-M2 and conjugated with caprine animal anti-mouse FITC in non permeabilised cells. Two types of permeabilisation have been tried before labelling with 0.08 % Triton x100 and 0.1 % Tween 20. after permeabilisation the samples were labelled for M2 utilizing the same antibodies mentioned above, and F-actin was labelled utilizing Phalloidin.
As control we used not septic MDCK cells that have been tested for all the conditions.When permeabilised with 0.08 % Triton the M2 protein staining done on grippe A virus septic cells was stronger. As seen in Figure 4, panel Angstrom, at 24h p.i. with grippe A virus MOI 0.5 – 5, M2 viral protein is copiously present at the plasma membrane where some filiform formations can be seen.
In panel B the MDCK 24h p.i. with grippe A virus cells were non permeabilised and the image analysis reveals that M2 is seeable at the plasma membrane, but M2 is copiously found in the fiolopodia formed constructions.At 8h p.i with grippe A virus the MDCK cells seeable in Figure 5, panel A show besides M2 distribution at the plasma membrane but in add-on M2 is found in the cytosol every bit good. Distinct punta-like countries are seeable in the cytosol, where M2 and F-actin are expressed. In panel B, M2 was labelled before permeabilisation and the strength of the labelling is non as strong.
As observed in panel A, M2 labelling has the same form and in add-on there are some rich F-actin countries that coincide with with the M2 rich countries.Again the permeabilisation with 0.08 % Triton resulted in better labelling as seen in Figure 6, panel A. Apart from the same distribution model some rich M2 countries can be observed. These punta-like forms correspond with the punta-like F-actin rich countries.
Panel B does non demo a strong labelling, this might be due to the fact that Tween20 was used to permeabilise the cell, the image reveals some filopodia projections.The consequences of this experiment leads us to reason that permeabilisation with 0.08 % Triton leads to better consequences, however farther probe is required.III.3. The immunofluorescence check on permeabilised non infected A431 Annexin A6-myc, A431, A549 Annexin A6-myc and MDCK cells has revealed that Annexin A6 is distributed in the cytol.
Analysis of Annexin A6 was done at the surface of A431 Annexin A6, A431, A549 Annexin A6 and MDCK cells. The intent of this experiment was to analyze the colocalisation of Annexin A6 – myc utilizing different antibodies against annexin A6 and myc. After arrested development the cells were permeabilised in 0.08 % Triton x100. the cells were incubated in polyclonal coney anti-annexin antibody or monoclonal mouse anti-annexin antibody in order to label annexin A6, and they were conjugated with goat anti-rabbit Texas Red and goat anti-mouse FITC.
To label myc we incubated the cells in coney anti-myc antibody conjugated with goat anti-rabbit Texas Red. As control we used A431 Annexin A6, A431, A549 Annexin A6 and MDCK labelled merely with the secondary antibodies.In Figure 7, panel Angstrom it is seeable that for the conditions used the mouse anti-annexin works better that the coney anti-annexin. The image analysis revealed that Annexin A6 is equally distributed in the cytol. Panel B shows that Annexin A6 is largely found at the plasma membrane.When Annexin A6 is labelled in Figure 8, panel A utilizing coney anti-annexin, it was revealed that Annexin A6 is distributed within the cytol and some punta-like Annexin A6 rich parts are seeable.
In panel B myc and Annexin A6 was labelled utilizing both mouse anti-annexin and coney anti-myc, and the consequences showed that Annexin A6 is rich at the plasma membrane and myc is distributed in the cytol.When used a different type of cell A549 Annexin A6 in Figure 9, Annxexin A6 distribution turned out to be throught the cytol ( panel A ) and dinstict Annexin A6 rich zones seen around the atomic envelope. In the panel B the II merely labelled control was used to demo that the signal revealed in the other cells have specific signal. In panel C we can see that sneak anti-annexin gave better consequences than the coney anti-annexin. Annexin is found distributed in the cytol.
Figure 10, panel A coney anti-myc seems to work better than the rabit anti-annexin ( Pannel B ) . Myc is found in the cytol and distinguishable puncta-like myc zone rich are seeable. Annexin A6 is found in the cytol and once more form of puncta-like zone rich Annexin A6 is seeable.
Reasoning these consequences we noticed that mouse anti-annexin antibody gave a more intense labelling than the coney anti-annexin antibody and when used coney anti-myc antibody resulted in nice specific labelling. Further probes need to be done to uncover if the consequence of my experiment is accurate.III.4. SEM assay on WSN infected MDCK cellsAnalysis of the grippe A virus A/WSN/33 ( H1N1 ) morphology at the surface of MDCK infected cells was determined by scanning negatron microscopy ( SEM ) . The intent of this experiment was to depict SEM observations on the surface extremist construction of grippe A virus A/WSN/33 ( H1N1 ) and analyze the morphology of the virus at different phases of infection. MDCK cells were infected with A/WSN/33 ( H1N1 ) and repairing was done at 8h p.
i and 24h p.i in glutaraldehyde followed by 2nd arrested development in 1 % Os tetroxide. After critical point drying the coverslips were mounted onto aluminum holders and coated with gold.
As control we used not septic MDCK cells.In Figure 11, panel A, the most obvious difference after analising the non infected, the 8h p.i and 24h p.i.
influenza A virus MDCK septic cells is the cell count. In the non infected we can see legion interrelated cells. In the 8h p.i. the cells lost their morphology and some dusts are seeable. The cells loose their cellular connexion. In panel B under x1000 magnification does non uncover more than the panel A, and the image 12 panel confirms the observations made. In the panel B under the x5000 magnification in the non infected image legion dusts can be observed, and filopodia and lamellipodia constructions are revealed.
In the 8h p.i dust are abundant, no more lamellipodia is present merely some filopodia spike fibrils. In the 24h p.i cells, the cytoskeleton of the cells is still seeable and some projections are get downing to take signifier.
Figure 13, panel A reveals cytoplasmatic projections or filopodia in the non septic sample. In the 8h p.i dust and filopodia are present but less that in the non infected. The 24h p.
i. reveals under the x10000 magnification little projections at the plasma membrane. The morphological alterations of the cells cytoskeleton are besides seeable. In panel B the 24h p.i reveals viral atoms under x20000 magnification. Under the x50000 magnification there are seeable viral atoms and their sizes coincide with the grippe A virus general size of approximate 100nm. These viral atoms besides show the same morphology as the grippe A virus: spherical. Some viral atoms are demoing extended morphology.
Most all of the viral atoms are released at the plasma membrane and some spherical budding observed that is connected to the cell surface by a cervix.Reasoning these findings, it can be said that the conditions used for scanning negatron microscopy can be applied in future experiments where M2 viral protein and Annexin A6 human protein will be labelled to analyze further their look.Chapter IV. DiscussionThe distribution of Annexin A6 at low intracellular Ca2+ is diffuse, but as the Ca2+ degrees are increased Annexin A6 is found at the plasma membrane, endosomes and in the secretory cysts.
This leads us to believe that because of the Ca2+ degrees dysregulation after grippe A virus infection, Annexin A6 is merely found in different subcellular membrane countries.Overexpressing Annexin A6 might consequences in stabilization of the cortical cytoskeleton as Annexin A6 interacts and rearranges the F-actin.