Thermostability and Protein Studies of Newcastle Disease Virus

The heat stability of four strains of Newcastle disease virus (NDV) belonging to three different pathotypes were studied. The strains were the lentogenic V4 and its heat stable variant V4-UPM, the mesogenic S strain and the velogenic strain AF2240. Analyses of their haemagglutination and neuraminidase activities (which are the functions of the HN protein) and the hemolytic activities at various temperatures showed that strains AF2240, V4 and V4 -UPM were heat stable compared to strain S. There were no differences observed in the mobilities of the various NDV proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) studies. However, analysis of the various peptides with Staphylococcus aureus protease showed that the digested HN proteins of strain V4-UPM was different from the strains V4, AF2240 and S. The peptide analysis was repeated using Pseudomonas fragi Endoproteinase Asp-N and Lysobacter enzymogenes Lys-C and found to be similar except in strain V4-UPM. These proteins were further analysed by the two dimensional polyacrylamide gel electrophoresis (2DPAGE). The gels were then Western blotted and protein spots were identified using HN,NP and Fi Mabs and then analysed by the UVP GDS Gel Documentation System (United Kingdom). It was observed that in the heat stable V4 strain the isoelectric point (pI) of the HN protein was in the acidic region, strains V4-UPM and AF2240 in the neutral/weak basic regions and in the thermolabile strain s, the HN protein was shifted to the basic end of the isoelectric focussing run. The pI changes in the NP protein was seen in strain S only. The F protein was at the basic region for all strain except strain V4-UPM. In strain S it was seen that the HN, NP and F proteins were in the basic region and this basic pI could be responsible for the different biological characteristics seen in the thermostable strains compared to the thermosensitive strain

Epstein-barr virus and its association with breast cancer / Prof Madya Dr Hjh Zuridah Hj Hassan and Dr Zeenathul Nazariah Allauddin

Epstein-barr virus (EBV) is widely distributed with 90-95% of adults are seropositive. This virus is spread by intimate contact between susceptible persons and asymptomatic shedders. EBV can be the causative agent of some B and T cell lymphomas, Hodgkin disease, nasopharyngeal carcinoma and also as one of the cofactor in the development of many types of carcinomas including breast cancer. The role of Epstein-barr virus (EBV) in breast cancer is not well documented and reports are very controversial. On the other hand, current belief holds that chromosomal deficiency and translocation, deletion, insertion and point mutation in the p53 tumor suppressor gene (p53 gene) which plays an important role in the regulation of cell growth, differentiation and DNA repair can play a role. Abnormal cell proliferation is thought to be induced by p53 gene deficiency. In human carcinogenesis, p53 mutations are reported in esophageal carcinoma with documented changes in protein expression. There is also evidence that mechanisms other than point mutation may result in p53 protein accumulation and inactivation in a subset of breast cancer. Epidemiologic studies have implicated several lifestyle risk factors: tobacco exposure, alcohol consumption, diet, obesity in esophageal carcinoma. However, the clinical significance and potential applications of these observations in breast cancer remains unclear

Molecular Characterization Of Human Group A Rotavirus Isolates From Malaysia And Development Of A Colorimetric Pcr-Based Test For The Detection Of P[8] Genotype

Rotavirus has been recognized as a leading cause of the diarrhoeal illness in children under 5 years of age in the developing world. Latex agglutination test was used to detect group A rotavirus from 157 in-patients from different hospitals in Malaysia during 2000 to 2001. Diarrhoea was detected in 31 (19.7%) children and majority were under two years of age. When viewed under electron microscope by negative staining, rotavirus was seen as both double-shelled and single-shelled particles. Thirty one rotavirus antigen positive samples with typical group A electropherotype were further characterized into their G or P types by polymerase chain reaction (PCR) assay. The two common electropherotypes were IIC (51.6%) and IIG (35.5%). The most prevalent VP4 genotype was 25 (80.6%) P[8] and 1 (3.2 %) P[6]. Genotype P[4] and P[9] were not isolated and 5 (16.1 %) were P untypable (PUT). Regarding the VP7 genotype, G4 was the most prevalent (64.5 %), followed by G1 (6.45%), G2 (6.45%) and G3 (3.2 %). Neither G8 nor G9 was found and 6 (19.4 %) were G untypable (GUT). Studies in many countries found that G1P[8], G4P[8], G2P[4] and G3P[8] are the group A rotavirus strains more commonly seen in children. However from this present study, the common strains in Malaysia were G4P[8], G1P[8] and G3P[8]. One GUTP[6] strain (designated as 7W) was identified for the first time in Kuala Lumpur. Restriction endonuclease HaeIII and Sau96I were also used to characterize the VP7 gene of the local 7W strain. However a restriction profile could not be assigned. The P[8] and P[6] local strains (represented by 67F and 7W, respectively) were also characterized by nucleotide sequence analysis. Phylogenetic analysis revealed that the VP4 genes of the 67F and 7W formed a distinct lineage. The P[8] and P[6] are encoded by distinct VP4 gene alleles. The main diagnostic problem is the genetic diversity of these alleles among different rotavirus strains. To overcome this problem, a method that employs non-radioactive dot hybridization was successfully developed for P[8] and P[6]. VP4 cDNA rotavirus-specific probes were prepared and labelled with digoxigenin (DIG). Anti-DIG-alkaline phosphatase and the substrate NBT/BCIP were used to detect the binding of the probe to target sequence. A simple, practical, sensitive and specific assay based on polymerase chain reaction (PCR) and a colorimetric detection method (ELISA) for the typing of rotavirus in infected faeces has been developed succesfully. A set of oligonucleotides was employed for a single-tube reverse-transcription nested PCR (RT-nPCR). Upon synthesis of the first strand cDNA, a first stage of 10 cycles of PCR amplification was run to generate an 876-bp dsDNA from the 5’ terminal third of gene 4. The process was completed in the same tube by performing another 35 cycles of second stage amplification incorporating a biotinylated and digoxigenin 5’-end labelled primers. The RT-nPCR produced a 180-bp amplicon representing the VP4 P[8] type. The sensitivity of the RT-nPCR method was compared to non-nested PCR method and nested PCR was found to be 100 times more sensitive. To further increase the sensitivity, the enzyme-linked immunoassay (ELISA) was incorporated into the system. Streptavidin-coated microtitre plate was used to capture the biotinylated PCR-amplified products. This RT-nPCR ELISA was able to detect RNA as low as 4 pg nucleic acid. It was designed to type the single most epidemiologically important human rotavirus VP4 P[8] type which is often associated with rotavirus G1, G3 and G4 types. Monoclonal antibodies (Mabs) were used for G serotyping, but no Mabs were availabe for P serotyping. Therefore, the RT-nPCR ELISA method is a very useful technique to detect rotavirus

Effect of glycerol feed in methanol induction phase for hepatitis B surface antigen expression in Pichia pastoris strain KM71

This study describes expression of HBs Ag in methylotrophic yeast, Pichia Pastoris under alcohol oxidase promoter. A single copy number of HBs Ag gene was transformed into pichia strain of KM 71, a Mutˢ type, by using pA0815 pichia expression vector. The recombinant was cultivated in a shake flask either using methanol or a mixed feed of glycerol -methanol for induction. The HBs Ag gene integrity was justified using direct PCR method. The expressed products in the soluble cell extracts were analyzed by Western blot, SDS page, Bradford assay and ELISA tests. The recombinant HBs Ag was expressed successfully in Pichia pastoris strain KM71 at a high level of HBs Ag protein expression. Thus, an addition of glycerol in the ratio of glycerol per methanol 1/1 (g g-1) consistently produced 2-fold increment in both biomass accumulation and HBs Ag productivity

Establishment of rat brain endothelial cells susceptible to rat cytomegalovirus ALL-03 infection

Endothelial cells have been implicated as key cells in promoting the pathogenesis and spread of cytomegalovirus (CMV) infection. This study describes the isolation and culture of rat brain endothelial cells (RBEC) and further evaluates the infectious potential of a Malaysian rat CMV (RCMV ALL-03) in these cultured cells. Brain tissues were mechanically fragmented, exposed to enzymatic digestion, purified by gradient density centrifugation, and cultured in vitro. Morphological characteristics and expression of von Willebrand factor (factor VIII-related antigen) verified the cells were of endothelial origin. RBEC were found to be permissive to the virus by cytopathic effects with detectable plaques formed within 7 d of infection. This was confirmed by electron microscopy examination which proved the existence of the viral particles in the infected cells. The susceptibility of the virus to these target cells under the experimental conditions described in this report provides a platform for developing a cell-culture-based experimental model for studies of RCMV pathogenesis and allows stimulation of further studies on host cell responses imposed by congenital viral infections

The relevance of viral culture based methods for the detection of rat cytomegalovirus (RCMV all-03) in the modern era of technology

In spite of the unequivocal advantages of traditional virological methods, its utilization seems to have taken a back seat coincident with the concurrent advances in science and technology. This study examined the impact of such techniques in the groundwork identification of rat cytomegalovirus ALL-03 strain. Virus was validated in terms of its intrauterine novelty, cytopathogenicity and was identified based on morphologic criteria classical of herpesvirus. Our findings demonstrate the suitability of primary brain endothelial cells for the propagation of the virus resulting in concomitant production of cytopathic effects and formation of plaques comparable to the ones produced in established secondary cell lines. In the absence of any known sequences for molecular based detection method, such findings provided critical clues crucial for the preliminary identification of the virus. This paper concludes that conventional methods such as tissue culture and electron microscopy are still in the forefront in the field of virology and remains relevant, but should ideally be used in conjunction with more advanced methodologies for a more comprehensive evaluation of novel viruses

Cross-reactivity of Malaysian rat cytomegalovirus strains with its human counterpart

This study probes into the prospect of cross-reactivity of HCMV with RCMV which has not been acknowledged to date. We describe the uncovering of a protein with an estimated size of between 61-68 kDa from local RCMV strains which reacted with HCMV positive sera. Our findings are a first disclosure of a plausible immunological cross-reactivity between RCMV with its human counterpart which grounds substantial interest implying existence of conserved determinants between rat and human CMV polypeptides. The cross-reactive protein most likely represents an enveloped glycoprotein, though the precise identification and its degree of similarity needs to be evidently defined and further elucidated in forthcoming experiments

Plasma cholinesterase assay among Malaysian vector control operators occupationally exposed to insecticides

Increase in insecticide space spraying (fogging) activities in Selangor, Malaysia for management of dengue outbreaks submits vector control operators towards occupational low-level exposure towards hazardous organophosphate and pyrethroid compounds. Hence, this study was aimed to determine insecticide poisoning among these workers occupationally exposed to insecticides. Plasma cholinesterase level was assayed on 106 vector control operators (subject group) and 74 blood donor samples (control group) to identify insecticide poisoning. Information regarding the workers recent exposure, type of insecticides used and compliance to personal protective equipment was obtained through questionnaire. Statistical analysis was then applied to determine differences in cholinesterase mean level between the subject and control group. Comparison of mean cholinesterase level between the subjects (7879.70 ±227.94 IU/L) and control group (7067.86 ±1609.71 IU/L) showed significantly higher (p<0.05) mean cholinesterase level in the subject group. The study indicated absence of inhibition of cholinesterase level among subject group compared to the control group thus reflects free from organophosphate poisoning in the vector control operators. Majority of the vector control operators were Malays. Strict implementation of personal protective equipment use proved to prevent pesticide poisoning among these workers. These mean cholinesterase levels for both different cohorts is therefore recommended to be used as reference and need to be updated from time to time. However, it is better if each individual cholinesterase level is made available as compared to mean population valu

Plasma cholinesterase assay among Malaysian vector control operators occupationally exposed to insecticides

Increase in insecticide space spraying (fogging) activities in Selangor, Malaysia for management of dengue outbreaks submits vector control operators towards occupational low-level exposure towards hazardous organophosphate and pyrethroid compounds. Hence, this study was aimed to determine insecticide poisoning among these workers occupationally exposed to insecticides. Plasma cholinesterase level was assayed on 106 vector control operators (subject group) and 74 blood donor samples (control group) to identify insecticide poisoning. Information regarding the workers recent exposure, type of insecticides used and compliance to personal protective equipment was obtained through questionnaire. Statistical analysis was then applied to determine differences in cholinesterase mean level between the subject and control group. Comparison of mean cholinesterase level between the subjects (7879.70 ±227.94 IU/L) and control group (7067.86 ±1609.71 IU/L) showed significantly higher (p<0.05) mean cholinesterase level in the subject group. The study indicated absence of inhibition of cholinesterase level among subject group compared to the control group thus reflects free from organophosphate poisoning in the vector control operators. Majority of the vector control operators were Malays. Strict implementation of personal protective equipment use proved to prevent pesticide poisoning among these workers. These mean cholinesterase levels for both different cohorts is therefore recommended to be used as reference and need to be updated from time to time. However, it is better if each individual cholinesterase level is made available as compared to mean population valu

Assessment of insecticide poisoning among vector control operators using a new biomarker, the enyzme β-glucuronidase

The increase of insecticide space spraying to control Aedes mosquitoes submits vector control operators to low-level exposure to toxic organophosphates. Plasma β-glucuronidase level is found to increase in most organophosphate poisoning cases. This study aims to identify presence of organophosphate poisoning using β-glucuronidase assay as an alternative biomarker. A total of 180 blood samples (74 controls and 106 subjects) were examined for this enzyme using a spectrophotometer. These results were then compared to the routine screening assay, butyrylcholinesterase (BChE) assay using a chemical analyzer. Both results showed absence of organophosphate poisoning indicated by no increased in β-glucuronidase level with no reduced in BChE level. Results of BChE level in the subject group (7879.70 ±227.935 IU/L), was significantly higher than the control group (7067.86 ±1609.712 IU/L) (p<0.05). Meanwhile, β- glucuronidase level between the subject (194.48 ±114.711 U/dL) and control group (192.60 ±122.685 U/dL) showed no significant difference (p>0.05). The findings of this evaluation show that assaying for the β-glucuronidase enzyme can be an alternative method to determine organophosphate poisoning. Furthermore, β-glucuronidase assay using spectrophotometer is recommended due to its ease of use and suitability for small laboratories with limited facilities