Detection and molecular identification of infectious bronchitis virus isolated in Malaysia

Infectious bronchitis is a disease of economic importance in the poultry industry in Malaysia. Currently, natural outbreaks of IB are controlled through the use of vaccine. However, outbreaks still occur in vaccinated flocks. One of the major factors speculated for these outbreaks is the heterologous protection afforded by the current standard IB vaccines against different IB virus (IBV) serotypes and variants present in the country. Rapid and sensitive methods for the detection and identification of serotypes causing these outbreaks would therefore be valuable to the understanding and control of IB. In this study, reverse-transcription polymerase chain reaction (RT-PCR) using the published primers (C2U/C3L, S1oligo3'/S1oligo5', S1oligo3'/S1Newoligo5', IBP1+IIBRP2-, IBVN2+IIBVN1- and UTR2+/UTR1-) were used to detect 14 Malaysian IBV isolates. Only primers UTR2+/uTR1- and IBP l +IIBRP2- were found to be able to detect all of the 14 Malaysian isolates. These primers could therefore be used as universal primers for the detection of Malaysian IBV isolates. The Sl gene of a Malaysian nephropathogenic IBV (MH5365/95) and the variable region in the S l gene of the remaining 13 IBV isolates were RT-PCR using primers Slolig05-1/Sl olig03' and TM897FffM1328R respectively. The PCR products were cloned, sequenced and compared the sequences with published sequences of the S1 gene of other IBV strains. Based on this sequence comparison, MH5365/95 was found to be different from the other IBV strains. The remaining 13 isolates were classified into 2 general groups. The first group, that was designated as Group A, consists of three isolates and was closely related (more than 95% homology) to Massachusetts type (M41), the most commonly used vaccine strain in this country. The second group which consists of 10 isolates was genetically different from the published IBVs showing not more than 82% homology with the published sequence. These 10 isolates were further subdivided into Groups B and C, comprising 7 and 3 isolates respectively. The 3 isolates of Group C belong to the same group as MH5365/95. The Group B isolates, however, were more closely related to the M41 serotype (about 82% in homology) compared with that of Group C isolates (less than 80%). There was about 78% nucleic acid homology when Groups B and C were compared. It could therefore be concluded that the outbreaks in this country were caused by the new strains of IBV, which were different from the vaccine strains, thereby resulting in the vaccines becoming less efficacious to protect the chickens against the local IBVs

Cellular transcripts regulated during infectinos with highly pathogenic H5N1 avian influenza virus in 3 host systems.

BACKGROUND: Highly pathogenic Avian Influenza (HPAI) virus is able to infect many hosts and the virus replicates in high levels in the respiratory tract inducing severe lung lesions. The pathogenesis of the disease is actually the outcome of the infection as determined by complex host-virus interactions involving the functional kinetics of large numbers of participating genes. Understanding the genes and proteins involved in host cellular responses are therefore, critical for the elucidation of the mechanisms of infection. METHODS: Differentially expressed transcripts regulated in a H5N1 infections of whole lung organ of chicken, in-vitro chick embryo lung primary cell culture (CeLu) and a continuous Madin Darby Canine Kidney cell line was undertaken. An improved mRNA differential display technique (Gene Fishing™) using annealing control primers that generates reproducible, authentic and long PCR products that are detectable on agarose gels was used for the identification of differentially expressed genes (DEGs). Seven of the genes have been selected for validation using a TaqMan® based real time quantitative PCR assay. RESULTS: Thirty seven known and unique differentially expressed genes from lungs of chickens, CeLu and MDCK cells were isolated. Among the genes isolated and identified include heat shock proteins, Cyclin D2, Prenyl (decaprenyl) diphosphate synthase, IL-8 and many other unknown genes. The quantitative real time RT-PCR assay data showed that the transcription kinetics of the selected genes were clearly altered during infection by the Highly Pathogenic Avian Influenza virus. CONCLUSION: The Gene Fishing™ technique has allowed for the first time, the isolation and identification of sequences of host cellular genes regulated during H5N1 virus infection. In this limited study, the differentially expressed genes in the three host systems were not identical, thus suggesting that their responses to the H5N1 infection may not share similar mechanisms and pathways

Cellular transcripts regulated during infections with Highly Pathogenic H5N1 Avian Influenza virus in 3 host systems

<p>Abstract</p> <p>Background</p> <p>Highly pathogenic Avian Influenza (HPAI) virus is able to infect many hosts and the virus replicates in high levels in the respiratory tract inducing severe lung lesions. The pathogenesis of the disease is actually the outcome of the infection as determined by complex host-virus interactions involving the functional kinetics of large numbers of participating genes. Understanding the genes and proteins involved in host cellular responses are therefore, critical for the elucidation of the mechanisms of infection.</p> <p>Methods</p> <p>Differentially expressed transcripts regulated in a H5N1 infections of whole lung organ of chicken, <it>in-vitro </it>chick embryo lung primary cell culture (CeLu) and a continuous Madin Darby Canine Kidney cell line was undertaken. An improved mRNA differential display technique (Gene Fishing™) using annealing control primers that generates reproducible, authentic and long PCR products that are detectable on agarose gels was used for the identification of differentially expressed genes (DEGs). Seven of the genes have been selected for validation using a TaqMan^®based real time quantitative PCR assay.</p> <p>Results</p> <p>Thirty seven known and unique differentially expressed genes from lungs of chickens, CeLu and MDCK cells were isolated. Among the genes isolated and identified include heat shock proteins, Cyclin D2, Prenyl (decaprenyl) diphosphate synthase, IL-8 and many other unknown genes. The quantitative real time RT-PCR assay data showed that the transcription kinetics of the selected genes were clearly altered during infection by the Highly Pathogenic Avian Influenza virus.</p> <p>Conclusion</p> <p>The Gene Fishing™ technique has allowed for the first time, the isolation and identification of sequences of host cellular genes regulated during H5N1 virus infection. In this limited study, the differentially expressed genes in the three host systems were not identical, thus suggesting that their responses to the H5N1 infection may not share similar mechanisms and pathways.</p

The study of records management policies and procedures in Safeguards Corporation Sdn. Bhd. / Nur Mahirah Maizan... [et al.]

Records management should be systematic and organize system in dealing with records. It is a process starting from the creation of records until disposal which requires skills and knowledge towards it. The purpose of this research is to analyze the behavioural of employee towards the policies and procedures thus the Safeguard Records Management Department was chosen to determine and identify policies and procedures used and investigate their staff expertise and qualification in this field. The questionnaire was designated and distribution of questionnaires was distributed to the all level of staff. Data were gathered from 57 respondents who came from selected branches and based on the study there are some findings arise in dealing with records management thus the recommendation towards the issues were suggested in this research

Acoustic evaluation of HEMA polymer gel dosimeter phantoms

A new method for the evaluation of radiotherapy 3D polymer gel dosimeters has been developed using ultrasound to assess the significant structural changes that occur following irradiation of the dosimeters. Polymer gel dosimeters were being fabricated using a monomer named 2-Hydroxyl-Ethyl-Meta-Acrylate (HEMA) with the presence of gelatine as a gelling agent. The readymade gel which is the concentration for HEMA fixed at 2, 4 and 5% would then undergo an ultrasonic evaluation to test for the propagation of sound speed through it. In the observation of relationship between the ultrasound propagation speeds as the doses increase (focusing at the cross region from overlapped beams) and concentration of monomer, the propagation speed for all the three polymer gel dosimeter phantoms still varies between 1460 to 1570 m/s which is still in the range of speed of sound for human tissue [3]. The ultrasonic absorption attenuation coefficient dose sensitivity for polymer gel dosimeters for 2, 4 and 5% of monomer are in the range of 0.02 to 0.6 dB which is equivalent to human tissue. As a comparison, it can be seen that gel phantoms with high concentration of monomer (5%) is more sensitive to the radiation compared to the lower (2% and 4%) concentrations. Regarding the absolute results of mechanical and acoustic properties; the copolymer-in-oil phantom is equivalent with soft tissue

Potency and efficacy of a low pathogenic H5N2 inactivated vaccine against challenge with a Malaysian H5N1 highly pathogenic avian influenza virus

The potency and efficacy of an avian influenza (AI) H5N2 inactivated vaccine that was developed at Veterinary Research Institute, Ipoh was tested. The percentage sequence identity of the HA gene of the H5N2 vaccine virus to the challenge virus [A/chicken/Malaysia/5858/04 (H5N1)] was 88.2% by nucleotide and 90% by amino acid sequences similarities, respectively. As for the HAI segment, the nucleotide sequence similarities were 88.3 % and by amino acid sequence 87.7%.For potency testing, the heterologous killed H5N2 AI vaccine, formulated as an oil emulsion was administered only once subcutaneously in twenty five two-week old commercial broiler chickens. The HI antibodies were not detectable at week 1 post vaccination. The HI GMT attained was 30, 63, 200, 54 and 32 by week 2, 3, 4, 5, and 6 post vaccinations. Efficacy study was conducted on ten SPF chickens at week 3 post vaccination. 60% of the birds (6/10) with HI titres ≥ 64 - 128 survived the challenged. H5N1 challenge virus was reisolated from all the birds with HI titre ≤ 32 that died, and each of the birds that survived with HI titres of 64 and 128, from the oropharynx and cloaca at day 3 post challenge. This vaccine protected 60% of chickens against mortality and did not prevent shedding after challenged with a HPAI H5N1 virus

Cloning and expression of truncated Spike (S-f200) Glycoprotein of Infectious Bronchitis Virus (IBV) in Escherichia coli, and its immunogenicity to mice

Complete S1 gene of the Infectious Bronchitis Virus (IBV) was amplified and cloned into transfer vector. Truncated S1 gene designated as Sf200 (containing five antigenic sites located at 24–61, 291–398 and 497–543 amino acid residues of S1 glycoprotein) were amplified by overlap PCR, cloned into prokaryotic expression vector resulting pET-Sf200 and confirmed the construct by sequencing. The recombinant plasmid was identified by restriction enzyme and sequencing analysis. The in vitro expression of the truncated protein was analyzed in E. coli with a molecular weight of 38kDa determined through SDSPAGE and confirmed by Western blotting. The recombinant truncated protein was then purified from the culture media. The immunogenicity of the protein was studied in an animal experiment on mice, in which mice were injected subcutaneously. These findings suggest that the truncated Sf200 expressed in the pET- 32a (+) prokaryotic vector can be used as antigen to detect antibodies against IBV

Detection of equine herpesvirus infection: conventional versus molecular approaches

Equineherpes virus(EHV)are the highly contagious pathogens that infect both domestic and wild equine populations causing a major impact on equine industry worldwide. The methods for diagnosis of EHV have shown a vast improvement in the last decade. Although some conventional techniques are still applicable in certain cases, most of the clinical testing now focusing on rapid diagnosis by using the nucleic acid amplification-based techniquesas major advances for the detection of EHV. The diagnosis of EHV does not only depend on clinical situation alone, but the suitability of diagnostic test is also vital for equine clinicians to make a decision regarding the specific treatments and control measures to be taken. Therefore, crucial understanding of the strengths and limitations of each assay are needed in order to interpret the results. Realizing the issue, this review intends to outline the clinical application of conventional approaches and the progress of the new molecular approaches. Relative advantages and limitations of each method have also been discussed

Contemporary strategies and current trends in designing antiviral drugs against dengue fever via targeting host-based approaches

Dengue virus (DENV) is an arboviral human pathogen transmitted through mosquito bite that infects an estimated ~400 million humans (~5% of the global population) annually. To date, no specific therapeutics have been developed that can prevent or treat infections resulting from this pathogen. DENV utilizes numerous host molecules and factors for transcribing the single-stranded ~11 kb positive-sense RNA genome. For example, the glycosylation machinery of the host is required for viral particles to assemble in the endoplasmic reticulum. Since a variety of host factors seem to be utilized by the pathogens, targeting these factors may result in DENV inhibitors, and will play an important role in attenuating the rapid emergence of other flaviviruses. Many experimental studies have yielded findings indicating that host factors facilitate infection, indicating that the focus should be given to targeting the processes contributing to pathogenesis along with many other immune responses. Here, we provide an extensive literature review in order to elucidate the progress made in the development of host-based approaches for DENV viral infections, focusing on host cellular mechanisms and factors responsible for viral replication, aiming to aid the potential development of host-dependent antiviral therapeutics