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1.
Funct Integr Genomics ; 17(5): 607-620, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28361233

RESUMO

In present investigation, differential expression of transcriptome after classical swine fever (CSF) vaccination has been explored at the cellular level in crossbred and indigenous (desi) piglets. RNA Sequencing by Expectation-Maximization (RSEM) package was used to quantify gene expression from RNA Sequencing data, and differentially expressed genes (DEGs) were identified using EBSeq, DESeq2, and edgeR softwares. After analysis, 5222, 6037, and 6210 common DEGs were identified in indigenous post-vaccinated verses pre-vaccinated, crossbred post-vaccinated verses pre-vaccinated, and post-vaccinated crossbred verses indigenous pigs, respectively. Functional annotation of these DEGs showed enrichment of antigen processing-cross presentation, B cell receptor signaling, T cell receptor signaling, NF-κB signaling, and TNF signaling pathways. The interaction network among the immune genes included more number of genes with greater connectivity in vaccinated crossbred than the indigenous piglets. Higher expression of IRF3, IL1ß, TAP1, CSK, SLA2, SLADM, and NF-kB in crossbred piglets in comparison to indigenous explains the better humoral response observed in crossbred piglets. Here, we predicted that the processed CSFV antigen through the T cell receptor signaling cascade triggers the B cell receptor-signaling pathway to finally activate MAPK kinase and NF-κB signaling pathways in B cell. This activation results in expression of genes/transcription factors that lead to B cell ontogeny, auto immunity and immune response through antibody production. Further, immunologically important genes were validated by qRT-PCR.


Assuntos
Peste Suína Clássica/imunologia , Imunogenicidade da Vacina/genética , Suínos/genética , Transcriptoma , Animais , Peste Suína Clássica/genética , Peste Suína Clássica/prevenção & controle , NF-kappa B/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Suínos/imunologia , Fator de Necrose Tumoral alfa/genética , Vacinação/veterinária
2.
Arch Virol ; 161(9): 2395-405, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27294845

RESUMO

Hemagglutinin neuraminidase (HN) is a membrane protein of Newcastle disease virus (NDV) with the ability to induce apoptosis in many transformed cell lines. TNF-α is a multi-factorial protein that regulates cell survival, differentiation and apoptosis. In a previous study, we reported that HN protein induces apoptosis by downregulating NF-κB expression. Further, we speculated that downregulation of NF-κB expression might sensitize HeLa cells to TNF-α-mediated apoptosis. Therefore, the present study was undertaken to investigate if HN protein could sensitize HeLa cells to TNF-α and to examine the apoptotic potential of the HN protein and TNF-α in combination. The results revealed that the pro-apoptotic effects were more pronounced with the combination of HN and TNF-α than with HN or TNF-α alone, which indicates that the HN protein indeed sensitized the HeLa cells to TNF-α-induced cell death. The results of the study provide a mechanistic insight into the apoptotic action of HN protein along with TNF-α, which could be valuable in treating tumor types that are naturally resistant to TNF-α.


Assuntos
Apoptose/fisiologia , Proteína HN/metabolismo , NF-kappa B/metabolismo , Vírus da Doença de Newcastle/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Regulação para Baixo , Regulação da Expressão Gênica , Proteína HN/genética , Células HeLa , Humanos , NF-kappa B/genética , Regulação para Cima , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia
3.
Indian J Exp Biol ; 54(3): 180-6, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27145631

RESUMO

Newcastle Disease (ND) is one of the major causes of economic loss in the poultry industry. Newcastle Disease Virus (NDV) is a single-stranded, negative-sense enveloped RNA virus (Fam. Paramyxoviridae; Order Mononegavirales). In the present study three monoclonal antibodies (MAbs) were produced by polyethyleneglycol (PEG)-mediated fusion of lymphocytes sensitized to NDV Bareilly strain and myeloma cells. NDV possesses ability to agglutinate erythrocytes of avian species. All the three MAbs designated as 2H7, 3E9 and 3G6 caused hemagglutination inhibition of NDV by specifically binding to NDV. The reactivity for all the 3 MAbs on indirect ELISA was found to be significantly higher than the antibody and antigen controls. On flowcytometry of HeLa cells infected with NDV using the MAbs as primary antibodies, there was a significant difference in the percentage of cells showing positive fluorescence compared to the mock control. One of the MAbs (3E9) was found to react with hemagglutinin-neuraminidase (HN) protein on western blot.


Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Antivirais/biossíntese , Vírus da Doença de Newcastle/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular
4.
Tumour Biol ; 37(9): 12089-12102, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27209409

RESUMO

The canine parvovirus NS1 (CPV2.NS1) protein selectively induces apoptosis in the malignant cells. However, for an effective in vivo tumor treatment strategy, an oncolytic agent also needs to induce a potent anti-tumor immune response. In the present study, we used poly (I:C), a TLR3 ligand, as an adjuvant along with CPV2.NS1 to find out if the combination can enhance the oncolytic activity by inducing a potent anti-tumor immune response. The 4T1 mammary carcinoma cells were used to induce mammary tumor in Balb/c mice. The results suggested that poly (I:C), when given along with CPV2.NS1, not only significantly reduced the tumor growth but also augmented the immune response against tumor antigen(s) as indicated by the increase in blood CD4+ and CD8+ counts and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed that Th1 cytokines (IFN-γ and IL-2) were significantly upregulated in the treatment group indicating activation of cell-mediated immune response. The present study reports the efficacy of CPV2.NS1 along with poly (I:C) not only in inhibiting the mammary tumor growth but also in generating an active anti-tumor immune response without any visible toxicity. The results of our study may help in developing CPV2.NS1 and poly (I: C) combination as a cancer therapeutic regime to treat various malignancies.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Parvovirus Canino/química , Poli I-C/farmacologia , Proteínas não Estruturais Virais/farmacologia , Animais , Apoptose , Citocinas/sangue , Feminino , Humanos , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia
5.
Int Immunopharmacol ; 35: 163-173, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27064544

RESUMO

BACKGROUND: Many viral proteins exhibit selective cytotoxicity for tumor cells without affecting the normal diploid cells. The apoptin protein of chicken infectious anemia virus is one of such proteins, which has been shown to kill tumor cells specifically. However, an effective cancer treatment strategy also requires assistance from the immune system. Recently, poly (I:C) has been shown to be an effective cancer vaccine adjuvant. AIM: In this study, we assessed the anti-tumor potential of apoptin gene transfer alone and in combination with poly (I:C) in a 4T1 mouse mammary tumor model. METHODS: 4T1 cells were used to induce mammary tumor in Balb/c mice. Mice bearing tumors were divided into 6 groups, and each group received six intratumoral injections during a period of one month. After the last immunization, the animals were sacrificed, and peripheral blood, spleen, lungs, liver, heart, kidney and tumor tissues were collected for immunological, molecular and pathological analysis. RESULTS: We report that intratumoral administration of apoptin plasmid along with poly (I:C) not only significantly inhibited the growth of mammary tumor, but also induced a potent anti-tumor immune response as indicated by the increase in blood CD4+, CD8+ cells and infiltration of immune cells in the tumor tissue. Further, blood serum analysis of the cytokines revealed increased secretion of Th1 cytokines (IFN-γ and IL-2). CONCLUSIONS: The results of our study demonstrate that the inclusion of poly (I:C) significantly enhanced the anti-tumor activity of apoptin mainly by inducing a potent anti-tumor immune response. Therefore, we report the use of apoptin and poly (I:C) combination as a novel and powerful strategy for cancer immunotherapy.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Vacinas Anticâncer , Proteínas do Capsídeo/genética , Neoplasias Mamárias Animais/terapia , Poli I-C/administração & dosagem , Células Th1/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Feminino , Terapia Genética , Interferon gama/sangue , Interleucina-2/sangue , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética
6.
Virus Res ; 213: 289-298, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26739427

RESUMO

Many viral proteins have the ability to kill tumor cells specifically without harming the normal cells. These proteins, on ectopic expression, cause lysis or induction of apoptosis in the target tumor cells. Parvovirus NS1 is one of such proteins, which is known to kill high proliferating tumor cells. In the present study, we assessed the apoptosis inducing ability of canine parvovirus type 2 NS1 protein (CPV2.NS1) in vitro in 4T1 cells, and found it to cause significant cell death due to induction of apoptosis through intrinsic or mitochondrial pathway. Further, we also evaluated the oncolytic activity of CPV2.NS1 protein in a mouse mammary tumor model. The results suggested that CPV2.NS1 was able to inhibit the growth of 4T1 induced mouse mammary tumor as indicated by significantly reduced tumor volume, mitotic, AgNOR and PCNA indices. Further, inhibition of tumor growth was found to be because of induction of apoptosis in the tumor cells, which was evident by a significant increase in the number of TUNEL positive cells. Further, CPV2.NS1 was also able to stimulate the immune cells against the tumor antigens as indicated by the increased CD4+ and CD8+ counts in the blood of CVP2.NS1 treated mice. Further optimization of the delivery of NS1 protein and use of an adjuvant may further enhance its anti-tumor activity.


Assuntos
Antineoplásicos/imunologia , Antineoplásicos/metabolismo , Vacinas Anticâncer/administração & dosagem , Neoplasias Mamárias Experimentais/terapia , Parvovirus Canino/imunologia , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/isolamento & purificação , Apoptose , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Expressão Gênica , Neoplasias Mamárias Experimentais/patologia , Camundongos , Parvovirus Canino/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resultado do Tratamento , Proteínas não Estruturais Virais/isolamento & purificação
7.
Appl Biochem Biotechnol ; 177(4): 940-56, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26306526

RESUMO

Many viral proteins are responsible for causing induction of apoptosis in the target cells. Hemagglutinin neuraminidase (HN), a multifunctional protein of Newcastle disease virus (NDV), is one of such proteins. The present study was undertaken to determine the apoptotic potential of the HN gene in cultured human cervical cancer cell line (HeLa cell) and to elucidate the molecular mechanisms involved. The results of the study indicate that HN protein causes apoptosis in HeLa cells, as observed by the translocation of Phosphatidylserine, activation of caspases, cleavage of poly (ADP-ribose) polymerase (PARP), and DNA fragmentation. Further, we report that expression of HN protein upregulates the SAPK/JNK pathway leading to transactivation of c-Jun which in turn activates apoptosis signaling. The results of our study provide an insight into the mechanism through which HN induces apoptosis.


Assuntos
Apoptose , Proteína HN/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Vírus da Doença de Newcastle/genética , Proteína HN/genética , Células HeLa , Humanos , Interferon-alfa/metabolismo , Regulação para Cima
8.
Indian J Exp Biol ; 53(5): 249-55, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-26040021

RESUMO

Viral gene oncotherapy, targeted killing of cancer cells by viral genes, is an emerging non-infectious therapeutic cancer treatment modality. Chemo and radiotherapy in cancer treatment is limited due to their genotoxic side effects on healthy cells and need of functional p53, which is mutated in most of the cancers. VP3 (apoptin) of chicken infectious anaemia (CIA) and NS1 (Non structural protein 1) of Canine Parvovirus-2 (CPV-2) have been proven to have oncolytic potential in our laboratory. To evaluate oncolytic potential of VP3 and NS1 together these genes needed to be cloned in a bicistronic vector. In this study, both these genes were cloned and characterized for expression of their gene products and its apoptotic potential. The expression of VP3 and NS1 was studied by confocal microscopy and flowcytometry. Expression of VP3 and NS1 in pVIVO.VP3.NS1 transfected HeLa cells in comparison to mock transfected cells indicated that the double gene construct expresses both the products. This was further confirmed by flowcytometry where there was increase in cells expressing VP3 and NS1 in pVIVO.VP3.NS1 transfected group in comparison with the mock control group. The apoptotic inducing potential of this characterized pVIVO.VP3.NS1 was evaluated in human cervical cancer cell line (HeLa) by DNA fragmentation assay, TUNEL assay and Hoechst staning. This double construct was observed to induce apoptosis in HeLa cells.


Assuntos
Apoptose/genética , Proteínas do Capsídeo/genética , Vírus Oncogênicos/genética , Terapia Viral Oncolítica , Proteínas não Estruturais Virais/genética , Animais , Proteínas do Capsídeo/uso terapêutico , Galinhas/virologia , Cães , Vetores Genéticos , Células HeLa , Humanos , Parvovirus Canino/genética , Proteínas não Estruturais Virais/uso terapêutico
9.
Cell Mol Life Sci ; 72(6): 1073-94, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25408521

RESUMO

Many viruses have the ability to modulate the apoptosis, and to accomplish it; viruses encode proteins which specifically interact with the cellular signaling pathways. While some viruses encode proteins, which inhibit the apoptosis or death of the infected cells, there are viruses whose encoded proteins can kill the infected cells by multiple mechanisms, including apoptosis. A particular class of these viruses has specific gene(s) in their genomes which, upon ectopic expression, can kill the tumor cells selectively without affecting the normal cells. These genes and their encoded products have demonstrated great potential to be developed as novel anticancer therapeutic agents which can specifically target and kill the cancer cells leaving the normal cells unharmed. In this review, we will discuss about the viral genes having specific cancer cell killing properties, what is known about their functioning, signaling pathways and their therapeutic applications as anticancer agents.


Assuntos
Genes Virais , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genética , Adenoviridae/genética , Animais , Apoptose , Proteínas do Capsídeo/análise , Proteínas do Capsídeo/genética , Vírus da Anemia da Galinha/genética , Humanos , Parvovirus/genética
10.
Genome Announc ; 2(1)2014 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-24526640

RESUMO

We report the complete genome sequence of the Sungri/96 vaccine strain of peste des petits ruminants virus (PPRV). The whole-genome nucleotide sequence has 89 to 99% identity with the available PPRV genome sequences in the NCBI database. This study helps to understand the epidemiological and molecular characteristics of the Sungri/96 strain.

11.
Indian J Exp Biol ; 51(2): 116-23, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23923604

RESUMO

Viral gene oncotherapy is emerging as a biotherapeutic cancer treatment modality based on targeted killing of cancer cells by viral genes. Newcastle disease virus (NDV) has the property to cause selective oncolysis of tumor cells sparing normal cells. NDV has a single stranded negative sense RNA genome, which is 15,186 nucleotide long and consists of six genes, which codes for eight proteins. NDV like other paramyxoviruses has the ability to generate multiple proteins from the P gene. P protein is encoded by an unedited transcript of the P gene, whereas the V and W protein are the results of RNA editing event in which one and two G residues are inserted at a conserved editing site within the P gene mRNA resulting in V and W transcripts, respectively. Although NDV is known to cause oncolysis by triggering apoptosis, the role of different viral proteins in selective oncolysis is still unclear. P gene edited products are known for its anti-apoptotic property in homologous host. In the present study, NDV P gene and its RNA edited products were amplified, cloned, sequenced and in vitro expression was done in HeLa cells. Further constructs were assayed for their apoptosis inducing ability in HeLa cells. Preliminary study suggested that P, V and W proteins are not apoptotic to HeLa cells.


Assuntos
Genes Virais/genética , Vírus da Doença de Newcastle/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Anexina A5/metabolismo , Sequência de Bases , Galinhas , Clonagem Molecular , Regulação Viral da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Fosfoproteínas/química , Reprodutibilidade dos Testes , Proteínas Virais/química
12.
Indian J Med Res ; 136(4): 571-84, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23168697

RESUMO

Cancer is one of the major causes of death worldwide. In spite of achieving significant successes in medical sciences in the past few decades, the number of deaths due to cancer remains unchecked. The conventional chemotherapy and radiotherapy have limited therapeutic index and a plethora of treatment related side effects. This situation has provided an impetus for search of novel therapeutic strategies that can selectively destroy the tumour cells, leaving the normal cells unharmed. Viral oncotherapy is such a promising treatment modality that offers unique opportunity for tumour targeting. Numerous viruses with inherent anti-cancer activity have been identified and are in different phases of clinical trials. In the era of modern biotechnology and with better understanding of cancer biology and virology, it has become feasible to engineer the oncolytic viruses (OVs) to increase their tumour selectivity and enhance their oncolytic activity. In this review, the mechanisms by which oncolytic viruses kill the tumour cells have been discussed as also the development made in virotherapy for cancer treatment with emphasis on their tumour specific targeting.


Assuntos
Apoptose/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Engenharia Genética/métodos , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/metabolismo , Humanos , Terapia Viral Oncolítica/tendências
13.
Indian J Exp Biol ; 49(9): 654-9, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21941937

RESUMO

Parvoviruses are small, 260-A-diameter, icosahedral, non-enveloped, single-stranded DNA viruses with a genome of approximately 5 kb. Non structural protein, (NS-1) is especially relevant, being both essential for virus replication and the main factor responsible for virus pathogenicity and cytotoxicity. This protein has also been reported to possess the property of killing of transformed cells. The present study was carried out to clone, characterize and express the NS-1 gene of canine parvovirus. NS-1 complete CDS 2020bp was amplified, cloned into eukaryotic expression vector pcDNA 3.1(+), sequenced and characterized by in vitro expression analysis. Functional activity of recombinant construct, pcDNA.cpv.NS-1, was evaluated by RT-PCR and flow cytometry for the expression of NS-1 specific mRNA and NS-1 protein, respectively, in transfected HeLa cells. This recombinant plasmid may serve as an important tool to evaluate the apoptotic potential of NS-1 protein of canine parvovirus in cultured HeLa cells.


Assuntos
Parvovirus Canino/genética , Proteínas não Estruturais Virais/biossíntese , Sequência de Bases , Clonagem Molecular , DNA Recombinante/genética , DNA Viral/genética , Citometria de Fluxo , Células HeLa , Humanos , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/fisiologia
14.
Indian J Exp Biol ; 49(2): 140-5, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21428216

RESUMO

Newcastle disease (ND) is highly contagious, economically important viral disease affecting most of avian species worldwide. Newcastle disease virus (NDV) has single stranded negative sense RNA genome which encodes for six structural and two non-structural proteins. Envelope glycoproteins i.e. hemagglutinin-neuraminidase (HN) and the fusion (F), elicit protective immune response. In this study, HN and F genes of velogenic (virulent) strain were amplified and cloned at multiple cloning sites A and B, respectively into pIRES bicistronic vector for use as bivalent DNA vaccine against ND. The recombinant plasmid was characterized for its orientation by restriction enzyme digestion and PCR. Expression of HN and F genes was assessed in transfected Vero cells at RNA level using RT-PCR in total RNA as well as protein level using IFAT, IPT and western blot using NDV specific antiserum. All these experiments confirmed that HN and F genes cloned in recombinant pIRES.nd.hn.f are functionally active. The recombinant construct is being evaluated as DNA vaccine against ND.


Assuntos
Clonagem Molecular , Proteína HN/genética , Vírus da Doença de Newcastle/genética , Vacinas de DNA/genética , Proteínas Virais de Fusão/genética , Animais , Chlorocebus aethiops , DNA Viral/genética , Vírus da Doença de Newcastle/imunologia , Proteínas Recombinantes de Fusão/genética , Transfecção , Vacinas de DNA/imunologia , Células Vero , Proteínas Virais de Fusão/imunologia
15.
Indian J Exp Biol ; 48(12): 1175-80, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21250598

RESUMO

Granulocyte-macrophage colony stimulating factor (GMCSF), a multifunctional cytokine can enhance immune responses when administered along with DNA vaccine. Aim of the present study was to clone and express the chicken GMCSF cytokine for use as 'genetic adjuvant'. Chicken GMCSF gene 435bp was amplified using specific primers in which restriction sites of BamHI and HindIII were at forward and reverse primers respectively. The PCR product was cloned into eukaryotic expression vector pcDNA 3.1(+) and clones were confirmed by restriction digestion and nucleotide sequencing. Functional activity of recombinant GMCSF was checked by expression of GMCSF specific mRNA in transfected Vero cells by RT-PCR of total RNA isolated from transfected Vero cells. The recombinant plasmid can be used as genetic adjuvant in chicken.


Assuntos
Clonagem Molecular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Sequência de Bases , Galinhas , Chlorocebus aethiops , Vetores Genéticos , Dados de Sequência Molecular , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Células Vero
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