Major capside protein of Polyomaviridae family viruses is able to selfassemble into virus-like particle (VLP) even without the presence of minor proteins, bind exogenous DNA non-specifically and recognise the receptor on the cellular surface. These characteristics determine its use as vector in gene therapy or immunotherapy. It was discovered before that MPyV VLPs significantly stimulate immune system and have strong adjuvant effect. Chimeric VLP derived from mouse polyomavirus carrying exogenous antigene or epitop is supposed to elicit specifically targeted immune response after immunisation. The main obstacle is choice of immunogene that is strong enough to cause adequate immune response. The goal of this thesis was to construct chimeric particles carrying epitop of malignant melanoma, one of the most immunogenic tumours, on their surface, using methods of genetic engineering. For future research of particle's immunogenic properties three types of particles were developed - particles with human and mouse melanoma epitopes, respectively and control particles with ovalbumine epitop. For the purpose of production of chimeric protein was used baculovirus expression system. It was verified then, with the use of electron microscopy, that introduction of tumour antigen into one of surface loops of VP1...

In this study, we focused on interactions of structural proteins of mouse polyomavirus (MPyV) and BK virus (BKV) with the nuclear lamina. Our goal was to examine whether and how can the virus, hence viral structural proteins, interact with the nuclear lamina and how would these interactions affect its properties. We supposed, that the expression of viral proteins would induce disintegration of the structure of nuclear lamina, thus enabling nuclear egress of virions in the late phase of infection. Viral structural proteins were expressed transiently in cells transfected with an expression vector pMPyV LATE. In these cells, VP1 was localized in a likewise manner as it shows in infected cells - mostly in a perinuclear area. Concurrently, defects in staining of nuclear lamina were observed in these cells, similarly to infected cells. Also, another expression vector was used in our experiments, the pMPyV mut3 VP1 encoding for a mutated protein VP1. When transiently expressed in cells, the mutated VP1 protein showed mostly diffuse nuclear localization. However, we observed significant morphological deformations and defective staining of the nuclear lamina. These observations imply an important role of VP1 in mechanical and biochemical properties alterations of the nuclear lamina in transfected and...

In 2014 SÖRAB constructed a continuous biological treatment system (KBR) to handle leachate waterfrom the landfill at the facility in Löt, north of Stockholm. The KBR is mainly focused on removal ofammonium nitrogen which would otherwise be released in to the recipient and contribute toeutrophication and damage to the environment. This project has focused on replacing the currentcarbon source in the process Brenntaplus VP1 and evaluating the efficiency of denitrification andeconomy of transitioning to a new carbon source. The carbon sources glycerol and ethanol wereevaluated and compared to Brenntaplus VP1 for the denitrification efficiency and microbial profile.The experiments were performed in laboratory conditions and in pilot scale using leachate water fromLöt. The reduction of ammonia was evaluated by chemical precipitation, addition of carbon sources bymeasuring ammonia-N and nitrate-N under aerobic (nitrification) and anaerobic (denitrification)conditions. The combination of ethanol and glycerol showed an enhanced denitrification and increasedmicrobial community both in lab and pilot scale studies with reduced hydraulic retention time. Therate of nitrate reduction was 0.23 mgNO3-N 1 -1 h -1 for ethanol/glycerol compared to 0.12-0.17mgNO 3- -N 1 -1 h -1 for Brenntaplus VP1 in pilot scale. The results indicate that using ethanol, glycerolor a mix of the two as a substitute for Brenntaplus VP1 is viable. This has been based on laboratoryand pilot scale studies. Each of the carbon sources examined during this project have showed a uniqueimpact on the process and its parameters such as: denitrification rate, microbial density and microbialcomposition. The carbon sources had an impact with temperature fluctuation and faster denitrificationcompared to the conventional KBR system. This implies that the carbon sources tested in this projectcan be advantageous and beneficial for Sörab depending on the carbon source availability and theseasonal variations. / Under 2014 konstruerade SÖRAB ett kontinuerligt biologiskt reningsverk (KBR) för att hanteralakvatten från deponin för ickefarligt avfall vid anläggningen i Löt, norr om Stockholm. KBR ärfrämst konstruerad för rening av ammoniumkväve som annars skulle släppas ut till recipienten ochbidra till övergödning och skador på miljön i området. Detta projekt har fokuserat på att ersätta dennuvarande kolkällan Brenntaplus VP1 som används i processen och utvärdera effektiviteten idenitrifieringen samt ekonomin vid övergång till en ny kolkälla. Kolkällorna glycerol och etanol varde kolkällor som valdes för utvärdering i detta projekt, dessa jämfördes med Brenntaplus VP1 i desseffekt på denitrifikationseffektivitet och mikrobiell sammansättning under laboratorieförhållanden ochi pilotskala. Möjligheten att reducera ammoniumkoncentrationen i lakvattnet utvärderades genomkemisk fällning och genom mätning av ammoniumkväve och nitratkväve under aeroba (nitrifikation)och anaeroba (denitrifikation) förhållanden. Kombinationen av etanol och glycerol indikerade enförbättrad denitrifikation och ökad mikrobiell densitet både i laboratorie- och pilotskala med reduceradhydraulisk retentionstid. Nitratreduktionshastigheten var 0,23 mgNO 3- -N 1 -1 h -1 för blandningen avetanol/glycerol jämfört med 0,12 - 0,17 mgNO 3- -N 1 -1 h -1 för Brenntaplus VP1 i pilotskala. Resultatenindikerar att användning av etanol, glycerol eller en blandning av de två har goda förutsättningar föratt ersätta Brenntaplus VP1. Var och en av de tre kolkällorna som undersöktes under detta projekt harvisat en unik inverkan på processen och dess parametrar såsom: denitrifikationshastighet, mikrobielldensitet och mikrobiell sammansättning. Genom att byta kolkälla i KBR kan prestandan ökas genomatt minska den hydrauliska retentionstiden samtidigt som systemet tycks bli mindre känsligt förtemperatursvängningar. Kolkällorna som utvärderats i detta projekt kan därför vara fördelaktiga för SÖRAB beroende på dess tillgänglighet och pris.

Orientadores: Clarice Weis Arns, Isabela Cristina Simoni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T00:22:52Z (GMT). No. of bitstreams: 1 Fernandes_MariaJuditeBittencourt_D.pdf: 1711625 bytes, checksum: d2876be51222b1ba7526b13ab7a72795 (MD5) Previous issue date: 2010 / Resumo: A doença infecciosa da bursa (IBD), denominada também doença de Gumboro, é uma doença aguda, imunossupressora, altamente contagiosa de aves jovens e de grande importância econômica para a avicultura. O vírus da doença infecciosa da bursa (IBDV), sorotipo 1, pode ser classificado de acordo com sua antigenicidade e patogenicidade em amostras clássicas virulentas (cv), atenuadas, variantes antigênicas ou muito virulentas (vv). Estas diferenças antigênicas são encontradas na região hipervariável do gene VP2, que é responsável pela indução de anticorpos neutralizantes e também dos possíveis marcadores de virulência que ainda não estão bem estabelecidos. O gene VP1 parece também apresentar um papel na virulência do vírus. Primeiramente, o objetivo do presente trabalho foi a identificação e caracterização molecular de 66 amostras brasileiras de IBDV através da RT-PCR de um fragmento do gene VP2 seguida pela digestão por enzimas de restrição (RE) e posterior confirmação pelo sequenciamento. A análise da RT-PCR/RE classificou 25 isolados como cepas vv e 16 como cepas cv além da classificação de 6 grupo moleculares. O sequenciamento também confirmou esta classificação com a presnça dos aminoácidos (aa) típicos das amostras vv (222A, 242I, 256I e 294I). Em 3 destes amostras vv também se observou mutações únicas que mostram pequenas, mas contínuas alterações dos vvIBDV circulantes nas granjas brasileiras. A arvore filogenética confirmou a origem comum das nossas amostras vv com os isolados de outros países assim como a origem monofilética destas amostras. Posteriormente foi feito a RT-PCR de um fragmento representativo do gene VP1 das amostras positivas para IBDV e a análise das sequências e filogenética. Quatorze amostras vv e três cv tiveram êxito nas sequências analisadas. Treze amostras vv apresentaram as substituições de aa comuns para as amostras vv (145T, 146D, 147N e 242E), exceto um que apresentou a sequência das amostras cv e na filogenia agrupou-se com estas amostras. A árvore a partir da VP1 pressupõe um rearranjo genético deste gene. Esta amostra com perfil do segmento A de amostra vv e do segmento B de cv seria o primeiro relato no Brasil de um rearranjo genético natural. Estes rearranjos de segmentos que também foram observados em amostras de outros países ou que podem ser produzidos em laboratório (quimeras) mostram que o segmento B pode estar contribuindo para a patogênese deste vírus. A origem destes rearranjos pode ser de troca genética com o uso de vacinas vivas ou se aceita a hipótese de que o segmento VP1 dos vvIBDV se originaram de um rearranjo genético de fonte desconhecida, estes rearranjos com segmento vvVP2 e cvVP1, seriam descendentes dos ancentrais dos vvVP1. Apenas um seqüenciamento completo das duas sequências e estudos in vivo poderão caracterizar o papel da VP1 na virulência desta amostra. Assim, o monitoramento contínuo das amostras de IBDV através da caracterização molecular pela análise das sequências dos genes e a detecção de alterações genéticas que possam influenciar a patogenicidade do vírus são de extrema importância, pois geram informações fundamentais que possibilitam e subsidiam o controle desta doença no Brasil / Abstract: Infectious bursal disease virus (IBDV) causes a disease among young chickens of great economic importance to the poultry industry worldwide both for the both mortality as the immunosuppression. Two distinct serotypes, 1 and 2, of IBDV are recognized. Only the serotype 1 is pathogenic for chickens and classified according to the antigenicity and/or pathogenicity in classical virulent (cv) strains, very virulent (vv) strains, antigenic variant strains, and attenuated strains. This classification has been based mainly on the VP2 gene sequence, more specifically on the hypervariable region corresponding to the induction of neutralizing antibodies and the serotype specificity. However, the fundamental molecular basis for pathogenicity is not yet clear. Studies with the VP1 gene have also shown its possible role in this virulence and pathogenicity. Firstly, the aim of the present paper was the molecular characterization of sixty-six Brazilian IBDV isolates from broiler and layers flocks during the period from 1997 to 2005 by RT-PCR followed by restriction enzyme analysis of a fragment from VP2 gene variable region. Sequence and phylogenetic analysis of the positive isolates were also carried out. Twenty-five of the isolates were identified as very virulent (vv) and sixteen as classic virulent (cv). All of vv isolates had the typical amino acid (aa) residues and clustered in a phylogenetic tree with the vvIBDV strains. Three vv isolates presented four common aa substitutions and differed from other vv strains indicating that the vvIBDVs circulating on Brazilian farms are undergoing slight but continuous exchanges. Furthermore, the Brazilian IBDV isolates characterized by the VP2 sequence in cv and vv strains were analyzed by the sequence and phylogeny of the VP1 gene fragment. Our vv isolates maintained clustered with the other vvIBDVs in phylogenetic tree obtained from the VP1 gene and presented the common aa too. The same occurred with the cv isolates. However, one isolate vv showed both characters, cv and vv into VP1 sequence and clustered with the ours and other cv isolates in the tree. This isolate has similar type of a reassortment / Doutorado / Microbiologia / Doutor em Genetica e Biologia Molecular

Polyomaviruses are small unenvelope DNA viruses, whose replication take place in cell nucleus. Despite its small genome size, these viruses can cause significant changes in the host cell, one of the most significant is cell transformation. Most studies of human pathogens from this family is the focus of clinical research, but do not provide enough information about the individual events of the life cycle of viruses. This thesis mainly aims to determining the exact time when the creation of the individual viral products and generate a timeline of events during natural infection in cells that are targets for BKV in the human body. It was found that the time course of the life cycle of BKV is very similar to those for model polyomaviruses MPyV and SV40 and in permissive cells takes about 40 - 50 hours.

Title from PDF of title page. / Document formatted into pages; contains 86 pages. / Thesis (M.S.P.H.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: The Enterovirus genus within the family Picornaviridae contains over 100 serotypes, of which sixty-four are known to be human pathogens. Infection with this group of RNA viruses produces a myriad of clinical conditions including poliomyelitis, meningitis, encephalitis, respiratory illnesses, and hand-foot-and-mouth disease. Outbreaks have been documented worldwide; significant morbidity and mortality exist to warrant laboratory surveillance. Traditionally, enteroviruses have been identified to the level of serotype by the serum neutralization assay. However, numerous problems are associated with this assay. The serum neutralization assay is labor intensive, results are often ambiguous, and reagents are becoming difficult to obtain. Recently, molecular-based typing protocols have been described that are cost effective and produce results that are more reliable. / ABSTRACT: The overall objective of this thesis was to implement a molecular-based typing protocol to replace the serum neutralization method currently used. Three specific aims were identified to reach this objective. First, a database cataloging all enteroviruses isolated at the Florida Department of Health - Tampa Branch Laboratory from 1981 through 2002 was created. Serotype prevalence, specimen submission rates, and temporal trends were analyzed to demonstrate the public health importance of enterovirus surveillance. Next, five oligonucleotide primer sets were compared with respect to sensitivity, specificity, and overall utility in molecular typing protocols developed to accurately determine enterovirus type. Finally, the most effective molecular assay was used to conduct two basic molecular epidemiological analyses of intratypic variation of Coxsackievirus B5 isolates, and of intratypic variation of successive Echovirus 9 passages. / ABSTRACT: The results from this study show that implementation of a molecular-based typing system for enteroviruses would be an improvement over current enterovirus serotyping methods. Results are obtained more rapidly and are more reliable. The implementation of such a system would improve the surveillance capabilities of the State of Florida Department of Health. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

Merkel cell polyomavirus (MCPyV) is a recently discovered human virus, having it's genome often integrated in a genome of Merkel carcinoma cells. Although this type of carcinoma is not so usual, it is very aggressive and it's incidence has been rising in last few years. It is not surprising that this virus is nowadays in the centre of scientific interest, as well as other pathogens and mechanisms affecting human life. Because the virus was discovered not so long ago, its research has been at the whole beginning. This diploma thesisaims to contribute to the study of this virus from the molecular-virology point of view. A neutralizing monoclonal antibody, type IgG2a, targeted against the main capsid protein of MCPyV, VP1, and recognizing its conformational epitote was prepared. This antibody was then used for a pilot study of VP1 VLPs MCPyV movement in mammalian cells. Results showed that the studied virus, at least particularly, utilizes caveolin-1-carrying vesicles for its movement in cells (colocalisation of VP1 VLPs and caveolin-1 was observedColocalisation with EEA1 marker of early endosomes, LamP2 marker of endolysosomal compartments or with BiP marker of endoplasmic reticulum was sporadic but significant. These preliminary results suggest that MCPyV might utilise an endocytic pathway leading...

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I takt med att Uppsala växer behöver kapaciteten för avloppsvattenrening på Kungsängsverket byggas ut. För att möta en framtida ökad belastning bedömer Uppsala Vatten & Avfall AB att en kolkälla kommer behöva tillsättas i den biologiska kvävereningen för att effektivisera den heterotrofa denitrifikationen på Kungsängsverket, som idag sker utan tillsats av kolkälla. Potentialen till förbättrad denitrifikation med olika kolkällor utvärderades genom satsvisa laboratorieförsök och litteraturstudier. Syftet var att bättre förstå de studerade kolkällornas funktion och prestanda i denitrifikation för att ge underlag inför en framtida fullskalig implementering av kolkälla. Det övergripande målet var att identifiera vilken eller vilka kolkällor som är mest fördelaktiga med avseende på reningseffektivitet, processpåverkan, doseringsbehov, ekonomi och miljöpåverkan. Triplikata försök genomfördes som denitrifikationstester med aktivt slam vid en genomsnittlig slamtemperatur på 14 ℃ och pH 7-8, där fem externa kolkällor (etanol, Brenntaplus VP1 och tre industriella restprodukter) samt försedimenterat avloppsvatten testades. I litteraturstudien inkluderades även metanol. Från försöksdata bestämdes specifika denitrifikationshastigheter, COD/N-kvoter och utbyteskoefficienter. Även bieffekter såsom nitritackumulering och fosforsläpp studerades. Därefter uppskattades doseringsbehov och kostnader baserat på erhållna resultat och antaganden om framtida produktionsmål för nitratkväve. En likartad prestanda erhölls med en av restprodukterna (RTP-vätska) och etanol som uppnådde högst denitrifikationshastigheter och reduktionsgrader (98 % respektive 97 %). Doseringsbehovet uppskattades vara 4 gånger högre med RTP-vätska jämfört med etanol. Med de två andra restprodukterna (dextrandrank och sackaroslösning) uppnåddes lägst denitrifikationseffektivitet och reduktionsgraderna uppgick till 79 % respektive 47 %. Vid test av sackaroslösning observerades dessutom ofullständig denitrifikation samt höga fosforsläpp. Dextrandranken uppträdde på liknande sätt. I egenskap av restprodukt är RTP-vätskan intressant för fortsatt utvärdering. Fullskalig implementering av RTP-vätska förutsätter att doseringsbehoven kan tillgodoses samt att lämplig distribuering och lagerhållning kan ordnas på Kungsängsverket. / The city of Uppsala is expanding and consequently enhanced capacity at the wastewater treatment plant of Kungsängen will be required in the future. As for the biological nitrogen removal process, Uppsala Vatten & Avfall AB expects an additional carbon source to be necessary in the future denitrification process. Currently, the nitrogen removal is employed without the addition of a carbon source. The potential of enhancing denitrification with different carbon sources was evaluated by conducting lab-scale batch tests and compiling literature data. The objective of this work was to better understand the performance of the chosen carbon sources as electron donors in heterotrophic denitrification and thereby provide groundwork for a future full-scale implementation of a carbon source. Based on information drawn from batch tests and literature, the carbon sources were evaluated with respect to removal efficiency, process compliance, quantitative dosing requirements, costs, and environmental sustainability. Lab-scale trials were conducted as denitrification tests (triplicate) at a mean sludge temperature of 14 ℃ and pH 7-8 with five external carbon sources (ethanol, Brenntaplus VP1, and three industrial waste products) and pretreated wastewater. In the literature review, methanol was included as well. Results obtained from the batch tests were used to determine kinetic parameters, mainly specific denitrification rates, COD/N ratios, and anoxic yield coefficients. Moreover, unwanted side effects due to addition of carbon sources were examined. Dosing requirements and costs were assessed based on previously determined kinetic parameters and supposed future production guidelines for effluent quality with respect to nitrate concentration. Similar performance was observed with one of the waste products (RTP liquid) and ethanol which achieved the highest denitrification rates and degree of removal (98 and 97 %, respectively). The estimated dosing requirement was 4 times higher with the RTP liquid compared to ethanol. The other two waste products, solutions of fructose (dextran) and sucrose, reached the lowest denitrification efficiency and removal degrees were 79 and 47 %, respectively. During tests with sucrose solution, incomplete denitrification and release of phosphorous were observed. The fructose solution showed somewhat similar behavior but to a lesser degree. Being a waste product, the RTP liquid is interesting for future evaluation. Full-scale implementation needs further considerations regarding dosing requirements, distribution, and storage conditions at the site of Kungsängsverket.

Rotavirus is one of the leading etiological agents of gastroenteritis in young of many species including humans worldwide and is responsible for about 600,000 infant deaths per annum. Rotavirus belongs to the Reoviridae family, and its genome is composed of 11 double-stranded RNA segments that encode six structural proteins and six nonstructural proteins. Rotavirus replication is fully cytoplasmic and occurs within highly specialized regions called viroplasms. NSP2 and NSP5 have been shown to be essential for viroplasm formation and, when co-expressed in uninfected cells, to form viroplasm¬like structures. A recent study suggest a key role for NSP5 in architectural assembly of viroplasms and in recruitment of viroplasmic proteins, containing four structural (VP1, VP2, VP3 and VP6) and two nonstructural (NSP2 and NSP5) proteins. NSP5, the translation product of gene segment 11 has a predicted molecular eight of 21 kDa. NSP5 has been reported to exist in multiple isoforms ranging in size from 28-and 32-35 kDa from a 26-kDa precursor has been attributed to O-glycosylation and hyperphosphorylation. To study different properties of the protein, recombinant NSP5 containing an N-terminal hisidine tag was expressed in bacteria and purified by affinity chromatography. A significant observation was the similarity in phosphorylation property of the bacterially expressed and that expressed in mammalian cells. While the untagged recombinant protein failed to undergo phosphorylation in vitro, addition of His tag or deletions at the N-terminus promoted phosphorylation of the protein in vitro, which is very similar to the reported properties exhibited by the corresponding proteins expressed in mammalian cells. Phosphorylation of NSP5 in vitro is independent of the cell type from which the extract is derived suggesting that the kinases that phosphorylate NSP5 are distributed in all cell types. Among the C-terminal deletion mutants studied, NH-∆C5 and NH-∆C10 were phosphorylated better than full-length NSP5, but NH-∆C25 and NH¬∆C35 showed substantial reduction in the level of phosphorylation compared to full-length NSP5. These results indicate that the C-terminal 30 residues spanning the predicted α-helical domain of NSP5 are critical for its phosphorylation in vitro which is in correspondence with previous findings that C-terminal 21 amino acids of NSP5 direct its insolubility, hyperphosphorylation, and VLS formation. The results revealed that though the tagged full-length and some of the mutants could be phosphorylated in vitro, they are not suitable substrates for hyperphosphorylation unlike the similar proteins expressed in mammalian cells or infected cells. Analysis by western blot and mass spectrometry revealed that the bacterially expressed NH-NSP5 is indeed phosphorylated. It appears that prior phosphorylation in bacteria renders the protein conformationally not amendable for hyperphosphorylation by cellular kinases in vitro. Mutation of the highly conserved proline marginally enhanced its phosphorylation in vitro but the stability of protein is affected. Notably, mutation of S67A, identified as a critical residue for the putative caesin kinase-I and-II pathways of NSP5 phosphorylation, affected neither the phosphorylation nor the ATPase activity of NSP5. These results suggest that bacterially expressed NSP5 by itself has undectable auto-kinase activity and it is hypophosphorylated. Purified recombinant NSP5 has been reported to possess an Mg¬ 2+-dependent ATP-specific triphosphatase activity. The results indicated that deletion of either C-terminal 48 amino acids or N-terminal 33 residues severely affected the ATPase activity of recombinant NSP5, underlying the importance of both N-and C-terminal domains for NSP5 ATP hydrolysis function. NSP5 expressed in rotavirus infected cells exists as inter-molecular disulfide-linked dimeric forms and it appears that the 46 kDa isoforms, that are phosphorylated, corresponds to dimer as revealed by western blotting. Analytical gel filtration analysis of NH-NSP5, NH-ΔN43 and NH-ΔN33-ΔC25 showed most of the proteins in void volume, but an additional peak corresponding to the mass of dimeric species further supports that NSP5 is basically a dimer that undergoes oligomerization. Analysis by sucrose-gradient fractionation revealed that NH-NSP5 and NH-ΔN43 proteins were mainly distributed in the lower fraction of the gradient suggesting the existence of high molecular weight complexes or higher oligomers. The multimeric nature of NSP5 and its mutants was further confirmed by dynamic light scattering which suggests that high molecular weight complexes are of homogenous species. The correlation curves showed a low polydispersity distribution and a globular nature of recombinant NH-NSP5 proteins. The present results clearly demonstrate that dimer is the basic structural unit of NSP5 which undergoes oligomerization to form a complex consisting of about 20-21 dimers. The nonstructural protein 5 is hyperphosphorylated in infected cells and cellular kinases have been implicated to be involved in its phosphorylation. NSP5 contains multiple consensus sites for phosphorylation by several kinases, but the cellular kinases that specifically phosphorylate NSP5 in infected cells are yet to be identified. Previous studies from our laboratory using signaling pathway inhibitors revealed that recombinant NH¬NSP5 and its deletion mutants can be phosphorylated in vitro by purified cellular kinases and by mammalian cell extracts. These studies also showed the involvement of PI3K-Akt and MAPK signaling pathways in NSP5 phosphorylation and a negative role for GSK3β in the phosphorylation of bacterially expressed recombinant NSP5 in vitro. In the present work, using phospho-specific anti-Ser9 GSK3β antibody, we observed that GSK3β is inactivated in a rotavirus infected MA104 cells in a strain-independent manner. GSK3β¬specific small interfering RNA (siRNA-GSK3β) reduced GSK3β levels leading to increased level of synthesis of the structural rotavirus protein VP6 and NSP5 hyperphosphorylation compared to control siRNA. The pharmacological kinase inhibitors (LY294002, Genistein, PD98059, and Rapamycin) studies at the concentrations tested did not significantly affect rotavirus infection as seen from the number foci, while U0126 severely affected rotavirus replication. The results clearly demonstrated the importance of the MEK1/2 signaling pathway in the successful replication of rotavirus and NSP5 hyperphosphorylation in rotavirus-infected cells. In contrast inhibition of GSK3β activity by LiCl, increased in general, the number of foci by greater than 2-fold for all viral strains studied. These results suggest that MEK1/2 pathway majorly contributes to GSK3β inactivation in rotavirus infected cells. Thus, our results reveal that rotavirus activates both the PI3K/Akt and FAK/ERK1/2 MAPK pathways and appears to utilize them as a strategy to activate mTOR, and inhibit GSK3β through phosphorylation on serine 9, the negative regulator of rotavirus NSP5 phosphorylation, and thus facilitate translational competence of rotaviral mRNAs during virus replication cycle. It was shown previously in the laboratory by co-immunoprecipitation assay that Hsp70 interacts with rotaviral proteins VP1 and VP4 in rotavirus-infected mammalian cells. In this study, the interactions between Hsp70 with VP1 and VP4 were further evaluated in vitro by GST-pull down assay. It was observed that the N-terminal ATPase and C-terminal peptide-binding domain of Hsp70 is necessary for its direct interaction with VP1 and VP4. The presence of Hsp70 in purified double-and triple-layered virus particles further supported the observed interactions of rotaviral proteins VP1 and VP4 with Hsp70. However, the specific interaction observed between Hsp70 and rotaviral capsid proteins, VP1 and VP4 in viral particles suggests that Hsp70 has an important role during rotavirus assembly which requires further investigation.