Characterisation of genotype VII Newcastle disease virus (NDV) isolated from NDV vaccinated chickens, and the efficacy of LaSota and recombinant genotype VII vaccines against challenge with velogenic NDV

A Newcastle disease virus (NDV) isolate designated IBS002 was isolated from a commercial broiler farm in Malaysia. The virus was characterised as a virulent strain based on the multiple basic amino acid motif of the fusion (F) cleavage site 112RRRKGF117 and length of the C-terminus extension of the hemagglutinin-neuraminidase (HN) gene. Furthermore, IBS002 was classified as a velogenic NDV with mean death time (MDT) of 51.2 h and intracerebral pathogenicity index (ICPI) of 1.76. A genetic distance analysis based on the full-length F and HN genes showed that both velogenic viruses used in this study, genotype VII NDV isolate IBS002 and genotype VIII NDV isolate AF2240-I, had high genetic variations with genotype II LaSota vaccine. In this study, the protection efficacy of the recombinant genotype VII NDV inactivated vaccine was also evaluated when added to an existing commercial vaccination program against challenge with velogenic NDV IBS002 and NDV AF2240-I in commercial broilers. The results indicated that both LaSota and recombinant genotype VII vaccines offered full protection against challenge with AF2240-I. However, the LaSota vaccine only conferred partial protection against IBS002. In addition, significantly reduced viral shedding was observed in the recombinant genotype VII-vaccinated chickens compared to LaSota-vaccinated chickens

Differential modulation of immune response and cytokine profiles in the bursae and spleen of chickens infected with very virulent infectious bursal disease virus

Background: Very virulent infectious bursal disease virus (vvIBDV) induces immunosuppression and inflammation in young birds, which subsequently leads to high mortality. In addition, infectious bursal disease (IBD) is one of the leading causes of vaccine failure on farms. Therefore, understanding the immunopathogenesis of IBDV in both the spleen and the bursae could help effective vaccine development. However, previous studies only profiled the differential expression of a limited number of cytokines, in either the spleen or the bursae of Fabricius of IBDV-infected chickens. Thus, this study aims to evaluate the in vitro and in vivo immunoregulatory effects of vvIBDV infection on macrophage-like cells, spleen and bursae of Fabricius. Results: The viral load was increased during the progression of the in vitro infection in the HD11 macrophage cell line and in vivo, but no significant difference was observed between the spleen and the bursae tissue. vvIBDV infection induced the expression of pro-inflammatory and Th1 cytokines, and chemokines from HD11 cells in a time- and dosage-dependent manner. Furthermore, alterations in the lymphocyte populations, cytokine and chemokine expression, were observed in the vvIBDV-infected spleens and bursae. A drastic rise was detected in numbers of macrophages and pro-inflammatory cytokine expression in the spleen, as early as 2 days post-infection (dpi). On 4 dpi, macrophage and T lymphocyte infiltration, associated with the peak expression of pro-inflammatory cytokines in the bursae tissues of infected chickens were observed. The majority of the significantly regulated pro-inflammatory cytokines and chemokines, in vvIBDV-infected spleens and bursae, were also detected in vvIBDV-infected HD11 cells. This cellular infiltration subsequently resulted in a sharp rise in nitric oxide (NO) and lipid peroxidation levels. Conclusion: This study suggests that macrophage may play an important role in regulating the early expression of pro-inflammatory cytokines, first in the spleen and then in the bursae, the latter tissue undergoing macrophage infiltration at 4 dpi

Characterization of Newcastle disease virus (NDV) isolated from NDV vaccinated broiler farms and investigation of vaccine efficacy against challenge with Velogenic genotype VII NDV

Vaccines to control Newcastle disease (ND) were introduced more than 60 years ago. Despite that, ND is still one of the most significant avian diseases affecting major poultry farms in various countries. Class II of Newcastle disease viruses (NDV) can be divided into 10 different genotypes based on the F gene. However, since 1990s genotype VII NDV is the predominant velogenic NDV circulating in South-East Asia causing outbreaks even in well vaccinated flocks. Various factors such as inappropriate vaccination scheme,concurrent infections, immunosuppression and presence of variant NDV have been implicated as the probable causes of the outbreak. The current NDV vaccines comprised of genotype I and/or II viruses. Through the use of reverse genetic technology, genotype–matched NDV vaccine has been used in certain countries. However, the efficacy of this vaccine against the circulating velogenic genotype VII NDV is not well characterized. This study focused on the isolation and characterization of velogenic NDV from NDV vaccinated chickens in Malaysia. Sequencing and phylogenetic analysis based on the F gene of five NDV isolates (IBS001 to IBS005) showed that the viruses belong to genotype VII and sub-genotype VIId of NDV, with F cleavage site motif of 112RRRKRF117. In addition, sequencing of the Cterminus of the HN gene revealed that, viruses lack extension and encoded a typical amino acid sequence length of virulent NDV. Hence, molecular characterization based on the F and HN genes indicated the viruses (IBS001 to IBS005) belong to velogenic genotype VII NDV. One of the isolates, IBS002, was further characterized based on sequencing of the complete length of F and HN genes and pairwise comparisons between different genotypes. A maximum distance was detected between IBS002 and LaSota with nucleotide/amino acids variation between 17.71% to 18.67% for F gene and 20.89% to 23.37% for HN gene. Nucleotide/amino acids variations of 8.79% to 9.77% for F gene and 9.17% to 11.60% for HN genes were detected between isolate IBS002 and genotype VII Dalguban N+ vaccine. In addition, IBS002 has a mean death time (MDT) of 51.2 hours and intracerebral pathogenicity index (ICPI) of 1.76, further confirming that the virus is a velogenic strain. Both genotype matched (Dalguban N+) and mismatched (LaSota and Avinew) vaccines induced 100% protection against mortality and severe clinical symptoms following challenge with 105 ELD50 of IBS002. Vaccinated chickens also showed significant (P<0.05) lower pathogenicity scores although there was no significant (P<0.05) difference among the vaccinated groups. However, sentinel birds of Avinew and Dalguban N+ groups showed lower pathogenicity score compared to sentinels in LaSota group (P<0.05). Furthermore, Avinew and Dalguban N+ vaccinated chickens shed significantly (P<0.05) less virus after challenge and the viral load decreased faster than LaSota group. Moreover, sentinel birds mortality in LaSota vaccinated and non-vaccinated groups were significantly (P<0.05) higher than Avinew and Dalguban N+ vaccinated groups suggesting the importance of genotype matched vaccine (Dalguban N+) and enteric based NDV vaccine (Avinew) in inducing vaccine induced immunity. Vaccine that matched with the hemagglutination-inhibition (HI) test’s antigen induced significantly (P<0.05) higher antibody compared to vaccine from other genotypes where a 2 Log2 difference were detected when genotype VII and genotype II NDV antigens were used to detect homologous and heterologous HI titters. Immunophenotyping study showed significant increased (P<0.01) in KUL-1+ macrophages in PBMCs and splenocytes of control challenged birds. On the other hand, CD3+/CD4+ and CD3+/CD8+ T cells in spleen of different vaccinated groups were increased upon challenge suggesting the possible involvement of these cells in curtailing virus replication. In conclusion, isolated NDVs were classified as velogenic strains and belonged to genotype VIId of class II of NDV. Both genotype matched and mismatched NDV vaccines were able to confer protection against challenge with velogenic genotype VII NDV. However, genotype matched and enteric based NDV vaccines seems to be able to confer a more complete protection against virus shedding and transmission to susceptible chickens following challenged with velogenic genotype VII NDV

Synthesis of an Anthraquinone Derivative (DHAQC) and its Effect on Induction of G2/M Arrest and Apoptosis in Breast Cancer MCF-7 Cell Line

Anthraquinones are an important class of naturally occurring biologically active compounds. In this study, anthraquinone derivative 1,3-dihydroxy-9,10-anthraquinone-2- carboxylic acid (DHAQC) (2) was synthesized with 32% yield through the Friedel–Crafts condensation reaction. The mechanisms of cytotoxicity of DHAQC (2) in human breast cancer MCF-7 cells were further investigated. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that DHAQC (2) exhibited potential cytotoxicity and selectivity in the MCF-7 cell line, comparable with the naturally occurring anthraquinone damnacanthal. DHAQC (2) showed a slightly higher IC50 (inhibitory concentration with 50% cell viability) value in the MCF-7 cell line compared to damnacanthal, but it is more selective in terms of the ratio of IC50 on MCF-7 cells and normal MCF-10A cells. (selective index for DHAQC (2) was 2.3 and 1.7 for damnacanthal). The flow cytometry cell cycle analysis on the MCF-7 cell line treated with the IC50 dose of DHAQC (2) for 48 hours showed that DHAQC (2) arrested MCF-7 cell line at the G2/M phase in association with an inhibited expression of PLK1 genes. Western blot analysis also indicated that the DHAQC (2) increased BAX, p53, and cytochrome c levels in MCF-7 cells, which subsequently activated apoptosis as observed in annexin V/propidium iodide and cell cycle analyses. These results indicate that DHAQC (2) is a synthetic, cytotoxic, and selective anthraquinone, which is less toxic than the natural product damnacanthal, and which demonstrates potential in the induction of apoptosis in the breast cancer MCF-7 cell line

Molecular characterisation and pathotyping of recently isolated Newcastle disease virus isolates based on F protein’s cleavage site

Intensive vaccine programs have been implemented in many countries including Malaysia, but Newcastle disease virus (NDV) outbreaks have occurred, even in well-vaccinated farms. Hence, the present study was aimed to characterize five NDV isolates obtained from NDV vaccinated broiler farms in 2011 based on sequence and phylogenetic analysis of partial fusion gene. All the isolated NDV strains showed that they are categorized as velogenic NDV based on the presence of multi-basic protease cleavage sites, 112RRRKRF117. In addition, phylogenetic analysis showed that the isolates can classified under the genotype VII, subgenotype VIId

Molecular characterization of fusion gene of recently isolated Newcastle disease virus isolates

Intensive vaccine programs have been implemented in many countries including Malaysia, but Newcastle disease virus (NDV) outbreaks have occurred, even in well-vaccinated farms. Hence, the present study was aimed to characterize five NDV isolates obtained from NDV vaccinated broiler farms in 2011 based on sequence and phylogenetic analysis of partial fusion gene. All the isolated NDV strains showed that they are categorized as velogenic NDV based on the presence of multi-basic protease cleavage sites, 112RRRKRF117. In addition, phylogenetic analysis showed that the isolates can classified under the genotype VII, subgenotype VIId

Synthesis of anthraquinone derivative (DHAQC) and its effect on induction of G2/M arrest and apoptosis in breast cancer MCF-7 cell lines

Anthraquinones are an important class of naturally occurring biologically active compounds. In this study, anthraquinone derivative 1,3-dihydroxy-9,10-anthraquinone-2- carboxylic acid (DHAQC) (2) was synthesized with 32% yield through the Friedel-Crafts condensation reaction. The mechanisms of cytotoxicity of DHAQC (2) in human breast cancer MCF-7 cells were further investigated. Results from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that DHAQC (2) exhibited potential cytotoxicity and selectivity in the MCF-7 cell line, comparable with the naturally occurring anthraquinone damnacanthal. DHAQC (2) showed a slightly higher IC50 (inhibitory concentration with 50% cell viability) value in the MCF-7 cell line compared to damnacanthal, but it is more selective in terms of the ratio of IC50 on MCF-7 cells and normal MCF-10A cells. (selective index for DHAQC (2) was 2.3 and 1.7 for damnacanthal). The flow cytometry cell cycle analysis on the MCF-7 cell line treated with the IC50 dose of DHAQC (2) for 48 hours showed that DHAQC (2) arrested MCF-7 cell line at the G2/M phase in association with an inhibited expression of PLK1 genes. Western blot analysis also indicated that the DHAQC (2) increased BAX, p53, and cytochrome c levels in MCF-7 cells, which subsequently activated apoptosis as observed in annexin V/propidium iodide and cell cycle analyses. These results indicate that DHAQC (2) is a synthetic, cytotoxic, and selective anthraquinone, which is less toxic than the natural product damnacanthal, and which demonstrates potential in the induction of apoptosis in the breast cancer MCF-7 cell line