Aditya Prasad Sahoo

Apoptosis induced by NS1 gene of Canine Parvovirus-2 is caspase dependent and p53 independent

Apoptosis is programmed cell death that normally occurs during development and aging in multicellular animals. Apoptosis also occurs as a defense mechanism against disease or harmful external agents. It can be initiated by a variety of stimuli including viruses and viral proteins. Canine parvovirus type 2 (CPV-2) that causes acute disease in dogs has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. Though non structural protein 1 (NS1) of many parvoviruses has been found to be apoptotic, no report on the apoptotic potential of NS1 of CPV-2 (CPV-2.NS1) exists. In this study, we evaluated the apoptotic potential of CPV-2.NS1 in HeLa cells. CPV-2.NS1 has been found to induce apoptosis which was evident through characteristic DNA fragmentation, increase in hypodiploid cell count, phosphatidyl serine translocation and activation of caspase-3. Increase in caspase-3 activity and no change in p53 activity with time in CPV-2.NS1 expressing HeLa cells showed the induction of apoptosis to be caspase dependent and p53 independent.

Rapid label-free visual assay for the detection and quantification of viral RNA using peptide nucleic acid (PNA) and gold nanoparticles (AuNPs).

A rapid label-free visual assay for the detection of viral RNA using peptide nucleic acid (PNA) probes and gold nanoparticles (AuNPs) is presented in this study. Diagnosis is a crucial step for the molecular surveillance of diseases, and a rapid visual test with high specificity could play a vital role in the management of viral diseases. In this assay, the specific agglomerative behavior of PNA with gold nanoparticles was manipulated by its complementation with viral RNA. The assay was able to detect 5-10 ng of viral RNA from various biological samples, such as allantoic fluids, cell culture fluids and vaccines, in 100 μl of test solution. The developed assay was more sensitive than a hemagglutination (HA) test, a routine platform test for the detection of Newcastle disease virus (NDV), and the developed assay was able to visually detect NDV with as little as 0.25 HA units of virus. In terms of the specificity, the test could discriminate single nucleotide differences in the target RNA and hence could provide visual viral genotyping/pathotyping. This observation was confirmed by pathotyping different known isolates of NDV. Further, the PNA-induced colorimetric changes in the presence of the target RNA at different RNA to PNA ratios yielded a standard curve with a linear coefficient of R(2)=0.990, which was comparable to the value of R(2)=0.995 from real-time PCR experiments with the same viral RNA. Therefore, the viral RNA in a given samples could be quantified using a simple visual spectrophotometer available in any clinical laboratory. This assay may find application in diagnostic assays for other RNA viruses, which are well known to undergo mutations, thus presenting challenges for their molecular surveillance, genotyping and quantification.

Canine parvovirus NS1 induced apoptosis involves mitochondria, accumulation of reactive oxygen species and activation of caspases

The non-structural protein (NS1) of parvoviruses plays an important role in viral replication and is thought to be responsible for inducing cell death. However, the detailed mechanism and the pathways involved in canine parvovirus type 2 NS1 (CPV2.NS1) induced apoptosis are not yet known. In the present study, we report that expression of CPV2.NS1 in HeLa cells arrests cells in G1 phase of the cell cycle and the apoptosis is mitochondria mediated as indicated by mitochondrial depolarization, release of cytochrome-c and activation of caspase 9. Treatment of cells with caspase 9 inhibitor Z-LEHD-FMK reduced the induction of apoptosis significantly. We also report that expression of CPV2.NS1 causes accumulation of reactive oxygen species (ROS) and treatment with an antioxidant reduces the ROS levels and the extent of apoptosis. Our results provide an insight into the mechanism of CPV2.NS1 induced apoptosis, which might prove valuable in developing NS1 protein as an oncolytic agent.

Canine parvovirus type 2a (CPV-2a)-induced apoptosis in MDCK involves both extrinsic and intrinsic pathways.

The canine parvovirus type 2 (CPV-2) causes an acute disease in dogs. It has been found to induce cell cycle arrest and DNA damage leading to cellular lysis. In this paper, we evaluated the apoptotic potential of the “new CPV-2a” in MDCK cells and elucidated the mechanism of the induction of apoptosis. The exposure of MDCK cells to the virus was found to trigger apoptotic response. Apoptosis was confirmed by phosphatidylserine translocation, DNA fragmentation assays, and cell cycle analysis. Activation of caspases-3, -8, -9, and -12 and decrease in mitochondrial potential in CPV-2a-infected MDCK cells suggested that the CPV-2a-induced apoptosis is caspase dependent involving extrinsic, intrinsic, and endoplasmic reticulum pathways. Increase in p53 and Bax/Bcl2 ratio was also observed in CPV-2a-infected cells.

Genomic analysis of host - Peste des petits ruminants vaccine viral transcriptome uncovers transcription factors modulating immune regulatory pathways

Peste des petits ruminants (PPR), is an acute transboundary viral disease of economic importance, affecting goats and sheep. Mass vaccination programs around the world resulted in the decline of PPR outbreaks. Sungri 96 is a live attenuated vaccine, widely used in Northern India against PPR. This vaccine virus, isolated from goat works efficiently both in sheep and goat. Global gene expression changes under PPR vaccine virus infection are not yet well defined. Therefore, in this study we investigated the host-vaccine virus interactions by infecting the peripheral blood mononuclear cells isolated from goat with PPRV (Sungri 96 vaccine virus), to quantify the global changes in the transcriptomic signature by RNA-sequencing. Viral genome of Sungri 96 vaccine virus was assembled from the PPRV infected transcriptome confirming the infection and demonstrating the feasibility of building a complete non-host genome from the blood transcriptome. Comparison of infected transcriptome with control transcriptome revealed 985 differentially expressed genes. Functional analysis showed enrichment of immune regulatory pathways under PPRV infection. Key genes involved in immune system regulation, spliceosomal and apoptotic pathways were identified to be dysregulated. Network analysis revealed that the protein - protein interaction network among differentially expressed genes is significantly disrupted in infected state. Several genes encoding TFs that govern immune regulatory pathways were identified to co-regulate the differentially expressed genes. These data provide insights into the host - PPRV vaccine virus interactome for the first time. Our findings suggested dysregulation of immune regulatory pathways and genes encoding Transcription Factors (TFs) that govern these pathways in response to viral infection.

Whole-Genome Sequence of Sungri/96 Vaccine Strain of Peste des Petits Ruminants Virus

ABSTRACT 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.

In-vitro Characterization and Evaluation of Apoptotic Potential of Bicistronic Plasmid Encoding HN Gene of Newcastle Disease Virus and Human TNF-α

The viral gene oncotherapy in combination with cytokines emerges as an exciting strategy for cancer therapy due to its minimal side effects and tumor specificity. HN is the surface protein of NDV which is involved in virus infectivity and is known to kill many cancerous cell types. TNF-α, a multifactorial cytokine has direct anti-tumor activity by activating the extrinsic pathways of apoptosis. In the present study, HN gene of NDV and TNF-α of human were cloned at multiple cloning sites (MCS) 1 and 2 of bicistronic expression vector pVIVO2. Expression pattern of recombinant clone was checked on transcriptional and translational level by RT-PCR, Immunofluorescence assay and flow cytometry. On flow cytometric analysis HN gene expression was found to be 28.30 ± 1.21; 5.22 ± 0.60%, and TNF-α gene expression was found to be 15.44 ± 0.42; 6.51 ± 0.757%, in HeLa cells transfected with pVIVO.nd.hn.hu.tnf and pVIVO2 empty vector control, respectively. These assays confirm that HN and TNF-α act synergistically in the induction of apoptosis in HeLa cells.

Viral genes as oncolytic agents for cancer therapy.

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.

Non-Structural protein 1 (NS1) gene of Canine Parvovirus-2 regresses chemically induced skin tumors in Wistar rats

The Non-Structural protein 1 of Canine Parvovirus-2 (CPV2.NS1) plays a major role in viral cytotoxicity and pathogenicity. CPV2.NS1 has been proven to cause apoptosis in HeLa cells in vitro in our laboratory. Here we report that CPV2.NS1 has no toxic side effects on healthy cells but regresses skin tumors in Wistar rats. Histopathological examination of tumor tissue from CPV2.NS1 treated group revealed infiltration of mononuclear and polymorphonuclear cells with increased extra cellular matrix, indicating signs of regression. Tumor regression was also evidenced by significant decrease in mitotic index, AgNOR count and PCNA index, and increase in TUNEL positive apoptotic cells in CPV2.NS1 treated group. Further, CPV2.NS1 induced anti-tumor immune response through significant increase in CD8(+) and NK cell population in CPV2.NS1 treated group. These findings suggest that CPV2.NS1 can be a possible therapeutic candidate as an alternative to chemotherapy for the treatment of cancer.

HN Protein of Newcastle Disease Virus Induces Apoptosis Through SAPK/JNK Pathway.

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.