A study was conducted to isolate and identify chicken anaemia virus (CAV) from field samples of clinically infected broiler chickens in Malaysia. A total of 125 samples were collected from chickens aged 2-6 weeks with clinically depressed and retarded growth, of which five samples were found positive to CAV directly by polymerase chain reaction (PCR). Later, five isolates of CAV from the respective five PCR positive samples were isolated in MDCC-MSB1 cells at passage 4 based on cytopathic effects, PCR and indirect immunofluorescent antibody test. The isolates were identified as BL-1, BL-2, BL-3, BL-4 and BL-5. These CAV isolates were found to resist treatment with chloroform and heat at 37 degrees C for 2 h, 56 degrees C for 30 min and 70 degrees C for 5 min. One of the isolates, BL-5 produced significant reduction (p < 0.001) of hematocrit values (9-19%), pale bone marrow, thymus atrophy and haemorrhages in skin/muscle when inoculated into 1-day old SPF chickens. Restriction enzyme digestion of 926 bp genomic fragments of all the isolates including Cux-1 isolate with HindIII exhibited a similar pattern of bands in 2% agarose gel. The present findings confirmed the presence of CAV in Malaysia.
Two areas in the chicken anemia virus (CAV) genome have high G:C content with secondary structures. These two G:C rich areas could not be sequenced with Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit. Several modifications were carried out to solve the problem. Finally, a package of modified method was developed to sequence the high G:C areas. The result showed that the Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit with the normal procedures are not suitable for sequencing the high G:C regions of the CAV genome. The present developed method made the Perkin Elmer's Kit useful for the first time to sequence the G:C rich hairpin structures of the CAV genome. The system may be useful to sequence all other G:C rich DNA templates.
Specific-pathogen-free (SPF) chickens inoculated with low passage Chicken anaemia virus (CAV), SMSC-1 and 3-1 isolates produced lesions suggestive of CAV infection. Repeated passages of the isolates in cell culture until passage 60 (P60) and passage 123 produced viruses that showed a significantly reduced level of pathogenicity in SPF chickens compared to the low passage isolates. Sequence comparison indicated that nucleotide changes in only the coding region of the P60 passage isolates were thought to contribute to virus attenuation. Phylogenetic analysis indicated that SMSC-1 and 3-1 were highly divergent, but their P60 passage derivatives shared significant homology to a Japanese isolate A2.
Pasteurella multocida a Gram-negative bacterium has been identified as the causative agent of many economically important diseases in a wide range of hosts. Hemorrhagic septicemia is a disease caused by P. multocida serotype B:2 and E:2. The organism causes acute, a highly fatal septicemic disease with high morbidity and mortality in cattle and more susceptible in buffaloes. Therefore, the aim of this study was to investigate the clinical signs, blood parameters, post mortem and histopathology changes caused by P. multocida Type B:2 infections initiated through the oral and subcutaneous routes.
Sudden death is usually the main finding in field animals during haemorrhagic septicaemia outbreaks caused by Pasteurella multocida type B:2 that causes acute, fatal and septicaemic disease in cattle and buffaloes. This situation may be due to failure in early detection of the disease where early treatment of antibiotics may improve the prognosis of the animal and other surviving animals. Thus, there is a grey area on the knowledge on the potential usage of pro-inflammatory cytokines and acute phase proteins as early biomarkers in the diagnosis of haemorrhagic septicaemia. In addition, exploration of the cerebrospinal fluid during infection has never been studied before. Therefore, this study was designed to fill up the grey areas in haemorrhagic septicaemia research. Twenty-one buffalo calves were divided into seven treatment groups where group 1 was inoculated orally with 10 mL of sterile phosphate-buffered saline pH 7 which act as a negative control group. Groups 2 and 3 were inoculated orally and subcutaneously with 10 mL of 1012 colony-forming unit of P. multocida type B:2. Group 4 and 5 buffaloes were inoculated orally and intravenously with 10 mL of lipopolysaccharide broth. Groups 6 and 7 were administered orally and subcutaneously with 10 mL of outer membrane protein broth. During the post-infection period of 21 days, blood and cerebrospinal fluid were sampled for the analyses of pro-inflammatory cytokines, acute phase proteins and cytological examination. Buffalo calves infected with P. multocida and its immunogens via different routes of inoculation showed significant changes (p
West Nile virus (WNV) is a zoonotic single-strand RNA arbovirus (family Flaviviridae: Flavivirus), transmitted among avian hosts in enzootic cycles by a mosquito vector. The virus has a significant disease effect on humans and equines when it bridges into a cycle with various sequelae with epidemic potential. This study was carried out to identify the potential spectrum of WNV hosts in three geographic areas with climatologically distinct features: Malaysia, Qatar, and the United States of America (U.S.). Serum samples were collected from avian and mammal species suspected to be reservoirs for the virus at these areas in a cross-sectional epidemiologic study. The samples were tested for the presence of antibodies against the virus using an enzyme-linked immunosorbent assay. Data on putative risk factors were also collected and analyzed for significance of association with seropositivity using the logistic regression analysis. Among the tested avian and mammalian species, raccoons had the highest seroconversion rate (54%) followed by crows (30%), horses (27%), camels (10%), other avian species (7%), and canine species (3%). It was almost twice as likely to detect seroconversion among these mammalian and avian species in the fall in comparison to other seasons of the year. Only mammalian and avian species and seasons of the year were significantly associated with the likelihood of seroconversion to WNV when we controlled for other factors in the multivariate analysis. Our data from the U.S. showed that raccoons and camels are susceptible to infection by the virus and may play a role in the perpetuation of endemic foci for the disease.
Marine microalgae have been prominently featured in cancer research. Here, we examined cytotoxic effect and apoptosis mechanism of crude ethanol extracts of an indigenous microalga, Chaetoceros calcitrans (UPMAAHU10) on human breast cell lines. MCF-7 was more sensitive than MCF-10A with IC50 value of 3.00 ± 0.65, whilst the IC50 value of Tamoxifen against MCF-7 was 12.00 ± 0.52 μg/mL after 24 hour incubation. Based on Annexin V/Propidium iodide and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells was through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increased compared to extract treated MCF-10A cells which showed only 6 and 7 folds increased at the same time points, respectively. Based on GeXP study, EEC induced apoptosis on MCF-7 cells via modulation of CDK2, MDM2, p21Cip1, Cyclin A2, Bax and Bcl-2. The EEC treated MCF-7 cells also showed an increase in Bax/Bcl-2 ratio that in turn activated the caspase-dependent pathways by activating caspase 7. Thus, marine microalga, Chaetoceros calcitrans may be considered a good candidate to be developed as a new anti-breast cancer drug.
Very virulent infectious bursal disease virus (vvIBDV) targets B lymphocytes in the bursa of Fabricius (BF), causing immunosuppression and increased mortality rates in young birds. There have been few studies on the host immune response following vvIBDV infection at different inoculum doses in chickens with different genetic backgrounds. In this study, we characterized the immune responses of specific-pathogen-free (SPF) chickens and Malaysian red jungle fowl following infection with vvIBDV strain UPM0081 at 103.8 and 106.8 times the 50% embryo infectious dose (EID50). The viral burden, histopathological changes, immune cell populations, and expression of immune-related genes were measured and compared between infected and uninfected bursa at specific intervals. The populations of KUL1+, CD3+CD4+ and CD3+CD8+ cells were significantly increased in both types of chickens at 3 dpi, and there was significant early depletion of IgM+ B cells at 1 dpi in the red jungle fowl. vvIBDV infection also induced differential expression of genes that are involved in Th1 and pro-inflammatory responses, with groups receiving the higher dose (106.8 EID50) showing earlier expression of IFNG, IL12B, IL15, IL6, CXCLi2, IL28B, and TLR3 at 1 dpi. Although both chicken types showed equal susceptibility to infection, the red jungle fowl were clinically healthier than the SPF chickens despite showing more depletion of IgM+ B cells and failure to induce IFNB activation. In conclusion, high-dose vvIBDV infection caused an intense early host immune response in the infected bursa, with depletion of IgM+ B cells, bursal lesions, and cytokine expression as a response to mitigate the severity of the infection.
Infectious bursal disease is a highly contagious disease in the poultry industry and causes immunosuppression in chickens. Genome-wide regulations of immune response genes of inbred chickens with different genetic backgrounds, following very virulent infectious bursal disease virus (vvIBDV) infection are poorly characterized. Therefore, this study aims to analyse the bursal tissue transcriptome of six inbred chicken lines 6, 7, 15, N, O and P following infection with vvIBDV strain UK661 using strand-specific next-generation sequencing, by highlighting important genes and pathways involved in the infected chicken during peak infection at 3 days post-infection. All infected chickens succumbed to the infection without major variations among the different lines. However, based on the viral loads and bursal lesion scoring, lines P and 6 can be considered as the most susceptible lines, while lines 15 and N were regarded as the least affected lines. Transcriptome profiling of the bursa identified 4588 genes to be differentially expressed, with 2985 upregulated and 1642 downregulated genes, in which these genes were commonly or uniquely detected in all or several infected lines. Genes that were upregulated are primarily pro-inflammatory cytokines, chemokines and IFN-related. Various genes that are associated with B-cell functions and genes related to apoptosis were downregulated, together with the genes involved in p53 signalling. In conclusion, bursal transcriptome profiles of different inbred lines showed differential expressions of pro-inflammatory cytokines and chemokines, Th1 cytokines, JAK-STAT signalling genes, MAPK signalling genes, and their related pathways following vvIBDV infection.
The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.
Plasmid DNA (pDNA)-based vaccines have emerged as effective subunit vaccines against viral and bacterial pathogens. In this study, a DNA vaccine, namely plasmid internal ribosome entry site-HN/F, was applied in ovo against Newcastle disease (ND). Vaccination was carried out using the DNA vaccine alone or as a mixture of the pDNA and dextran-spermine (D-SPM), a nanoparticle used for pDNA delivery. The results showed that in ovo vaccination with 40 μg pDNA/egg alone induced high levels of antibody titer (P<0.05) in specific pathogen-free (SPF) chickens at 3 and 4 weeks postvaccination compared to 2 weeks postvaccination. Hemagglutination inhibition (HI) titer was not significantly different between groups injected with 40 μg pDNA + 64 μg D-SPM and 40 μg pDNA at 4 weeks postvaccination (P>0.05). Higher antibody titer was observed in the group immunized with 40 μg pDNA/egg at 4 weeks postvaccination. The findings also showed that vaccination with 40 μg pDNA/egg alone was able to confer protection against Newcastle disease virus strain NDIBS002 in two out of seven SPF chickens. Although the chickens produced antibody titers 3 weeks after in ovo vaccination, it was not sufficient to provide complete protection to the chickens from lethal viral challenge. In addition, vaccination with pDNA/D-SPM complex did not induce high antibody titer when compared with naked pDNA. Therefore, it was concluded that DNA vaccination with plasmid internal ribosome entry site-HN/F can be suitable for in ovo application against ND, whereas D-SPM is not recommended for in ovo gene delivery.
Skin cancer or cutaneous carcinoma, is a pre-eminent global public health problem with no signs of plateauing in its incidence. As the most common treatments for skin cancer, surgical resection inevitably damages a patient's appearance, and chemotherapy has many side effects. Thus, the main aim of this study was to screen for a cell penetrating peptide (CPP) for the development of a targeting vector for skin cancer. In this study, we identified a CPP with the sequence NRPDSAQFWLHH from a phage displayed peptide library. This CPP targeted the human squamous carcinoma A431 cells through an interaction with the epidermal growth factor receptor (EGFr). Methyl-β-cyclodextrin (MβCD) and chlorpromazine hydrochloride (CPZ) inhibited the internalisation of the CPP into the A431 cells, suggesting the peptide entered the cells via clathrin-dependent endocytosis. The CPP displayed on hepatitis B virus-like nanoparticles (VLNPs) via the nanoglue successfully delivered the nanoparticles into A431 cells. The present study demonstrated that the novel CPP can serve as a ligand to target and deliver VLNPs into skin cancer cells.
Chemotherapy is widely used in cancer treatments. However, non-specific distribution of chemotherapeutic agents to healthy tissues and normal cells in the human body always leads to adverse side effects and disappointing therapeutic outcomes. Therefore, the main aim of this study was to develop a targeted drug delivery system based on the hepatitis B virus-like nanoparticle (VLNP) for specific delivery of 5-fluorouracil-1-acetic acid (5-FA) to cancer cells expressing epithelial growth factor receptor (EGFR). 5-FA was synthesized from 5-fluorouracil (5-FU), and it was found to be less toxic than the latter in cancer cells expressing different levels of EGFR. The cytotoxicity of 5-FA increased significantly after being conjugated on the VLNP. A cell penetrating peptide (CPP) of EGFR was displayed on the VLNP via the nanoglue concept, for targeted delivery of 5-FA to A431, HT29 and HeLa cells. The results showed that the VLNP displaying the CPP and harboring 5-FA internalized the cancer cells and killed them in an EGFR-dependent manner. This study demonstrated that the VLNP can be used to deliver chemically modified 5-FU derivatives to cancer cells overexpressing EGFR, expanding the applications of the VLNP in targeted delivery of chemotherapeutic agents to cancer cells overexpressing this transmembrane receptor.
Bacteriophage EC1-UPM is an N4-like bacteriophage which specifically infects Escherichia coli O78:K80, an avian pathogenic strain that causes colibacillosis in poultry. The complete genome sequence of bacteriophage EC1-UPM was analysed and compared with other closely related N4-like phage groups to assess their genetic similarities and differences.
Neoplastic transformation appears to be a multi-step process in which the normal controls of cell proliferation and cell-cell interaction are lost, thus transforming normal cells into cancer. The tumorigenic process involves the interplay between oncogenes and tumour suppressor genes. In this study, we have selected the ras family, c-myc and epidermal growth factor receptor (EGFR) genes to detect whether their abnormalities are associated with the expression and progression of glioma cases in Malay patients. We have used the polymerase chain reaction-single stranded conformation polymorphism followed by direct sequencing for the study. For the ras gene family, we screened the point mutations in codons 12 and 61 of the H-, K-, and N-ras gene; for EGFR and c-myc, we analyzed only the exon 1 in glioma samples. In mutational screening analyses of the ras family, c-myc and EGFR gene, there was no mobility shift observed in any tumour analyzed. All patterns of single stranded conformation polymorphism (SSCP) band observed in tumour samples were normal compared to those in normal samples. The DNA sequencing results in all high-grade tumours showed that all base sequences were normal. All 48 patients survived after five years of treatment. In simple logistic regression analysis, variables which were found to be significant were hemiplegia (p=0.047) and response radiotherapy (p=0.003). Hemiplegics were 25 times more likely to have high pathological grade compared to those without. Patients with vascular involvement were 5.5 times more likely to have higher pathological grade. However, these findings were not significant in multivariate analysis. Patients who had radiotherapy were nearly 14 times more likely to have higher pathological grade. Multivariate analysis revealed that patients with hemiplegia were more likely to have higher pathological grade (p= 0.008). Those with higher pathological grading were 80 times more likely to have radiotherapy (p=0.004).
Newcastle disease virus (NDV) exerts its naturally occurring oncolysis possibly through the induction of apoptosis. We hypothesized that the binding of the virus to the cell via the hemagglutinin-neuraminidase (HN) glycoprotein may be sufficient to not only induce apoptosis but to induce a higher apoptosis level than the parental NDV AF2240 virus. NDV AF2240 induction of apoptosis in MCF-7 human breast cancer cells was analyzed and quantified. In addition, the complete HN gene of NDV strain AF2240 was amplified, sequenced and cloned into the pDisplay eukaryotic expression vector. HN gene expression was first detected at the cell surface membrane of the transfected MCF-7 cells. HN induction of apoptosis in transfected MCF-7 cells was analyzed and quantified. The expression of the HN gene alone was able to induce apoptosis in MCF-7 cells but it was a less potent apoptosis inducer compared to the parental NDV AF2240 strain. In conclusion, the NDV AF2240 strain is a more suitable antitumor candidate agent than its recombinant HN gene unless the latter is further improved by additional modifications.
Newcastle disease virus (NDV) AF2240 Malaysian strain is a very virulent avian virus. NDV strain AF2240 was previously demonstrated to induce apoptosis in human breast carcinoma MCF-7 cells. However, at which stage of the NDV life cycle apoptosis is induced and whether NDV replication and protein synthesis are involved in apoptosis induction have yet to be determined. In the present study, we investigated the time course of NDV strain AF2240 nucleoprotein (NP) gene expression and the early apoptotic signs in the form of activation of caspase-8 and mitochondrial transition pore opening. In addition, the induction of apoptosis by both ultraviolet-inactivated and cycloheximide-treated NDV-infected MCF-7 cells were examined. Our findings showed that NDV strain AF2240 induced apoptosis at 1 h post-infection (pi) through activation of mitochondrial transition pore opening and at 2 h through activation of caspase-8, while the NP gene was expressed at 6 h pi. The induced apoptosis was independent of both virus replication and protein synthesis. In conclusion, NDV strain AF2240 induces apoptosis at an early stage of its life cycle, possibly during virus binding or fusion with the cell membrane. The mitochondrial-related pathway may be the central activator in NDV strain AF2240-induced apoptosis.
For centuries, Edible Bird Nest (EBN) has been used in treatment of variety of respiratory diseases such as flu and cough as a Chinese natural medicine.
Edible Bird's Nest (EBN) as a popular traditional Chinese medicine is believed to have health enhancing and antiviral activities against influenza A virus (IAV); however, the molecular mechanism behind therapeutic effects of EBN is not well characterized.