Molecular characterisation of rice tungro bacilliform virus isolated from Bario, Sarawak

Rice tungro disease is one of the most damaging and destructive diseases of rice in South and Southeast Asia. The disease is caused by the co-infection of two viruses, the Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). The symptoms and severity of the disease depend on these two viral agents, if rice is coinfected by both viruses, it will show the typical severe symptoms of yellow-orange leaf discoloration, plant stunting and reduced in yield. On the other hand, if rice is infected only with RTBV, it shows milder symptoms and in contrast, rice plants will show no symptoms if they are infected only with RTSV. The disease had been detected in Malaysia since the 1930s. However, the first incursion of the disease was only reported in Sarawak in 2012. Since the disease was not seen in the Sarawak until recently, very little information on local virus isolate is available. This study was conducted to obtain and record the nucleotide sequence of partial coat protein gene of two primary isolates of RTBV collected from Bario, Sarawak in 2012 and 2013. Methodology and results: Based on the phylogenetic analysis, the isolates cluster with the Southeast Asia group with sequence identity at nucleotide and amino acid level of 91.1 to 95.1% and 98.6 to 99.5% respectively. Conclusion, significance and impact of study: This study provide the first genetic information on RTBV isolates from Sarawak. This data is important for future reference of the virus variants and diversity for epidemiological and diagnosis purposes

Interaction of antigenic recombinant coat proteins against the affinity purified antibody of rice tungro viruses: A preliminary study

Rice tungro disease (RTD) is a major destructive rice viral disease. To date, the common practice of detecting RTD is still based on symptoms visualisation. This is because methods like polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) which are conducted in the lab are limited for many farmers. As paddy planting is more dynamic in the rural region, sending samples is inconvenient as facilities providing the services are located far from the farms. Therefore, an easy and rapid method that can be utilized at point-of-need is advantageous to help manage RTD. This study reports on the use of antigenic recombinant coat proteins of the tungro viruses; Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) to develop a rapid serological for the detection of RTD. The aim is to produce affinity purified antibody against the coat proteins (CPs) of the tungro viruses. In our previous study, we have successfully cloned the RTBV CP and RTSV CP3. Here we report the result of the reactivity of the IgG purified polyclonal antibodies of the RTBV CP and RTSV CP3 tested in Western Blot and indirect ELISA. The binding affinity of the antibody to the antigen was evaluated in these two assays. The result showed similar pattern in reactivity between the two IgG antibodies of the RTSV CP3 and RTBV CP sera to the corresponding recombinant coat proteins (rCPs) in the immunoblots. The indirect ELISA result shows that the RTSV CP3 protein reacted strongly to both IgG antibodies, however it was noticed that the binding affinity started to decrease at higher concentration of the antigenic protein. This differs with the reactivity of the RTBV CP protein as binding of the IgG antibodies for the protein gradually increased with protein concentration. In conclusion, the IgG purified polyclonal antibodies against the rCPs of RTSV and RTBV show potential binding ability to be used in a rapid serological assay. However, the sensitive immunoassay can be further developed by optimizing the conditions of the rCPs and the affinity purified antibodies. Keywords: Coat protein, rice tungro viruse

Antioxidant and toxicity studies of 50% methanolic extract of Orthosiphon stamineus benth.

The present study evaluated the antioxidant activity and potential toxicity of 50% methanolic extract of Orthosiphon stamineus (Lamiaceae) leaves (MEOS) after acute and subchronic administration in rats. Superoxide radical scavenging, hydroxyl radical scavenging, and ferrous ion chelating methods were used to evaluate the antioxidant properties of the extract. In acute toxicity study, single dose of MEOS, 5000 mg/kg, was administered to rats by oral gavage, and the treated rats were monitored for 14 days. While in the subchronic toxicity study, MEOS was administered orally, at doses of 1250, 2500, and 5000 mg/kg/day for 28 days. From the results, MEOS showed good superoxide radical scavenging, hydroxyl radical scavenging, ferrous ion chelating, and antilipid peroxidation activities. There was no mortality detected or any signs of toxicity in acute and subchronic toxicity studies. Furthermore, there was no significant difference in bodyweight, relative organ weight, and haematological and biochemical parameters between both male and female treated rats in any doses tested. No abnormality of internal organs was observed between treatment and control groups. The oral lethal dose determined was more than 5000 mg/kg and the no-observed-adverse-effect level (NOAEL) of MEOS for both male and female rats is considered to be 5000 mg/kg per day

Antioxidant and Toxicity Studies of 50% Methanolic Extract of Orthosiphon stamineus Benth

The present study evaluated the antioxidant activity and potential toxicity of 50% methanolic extract of Orthosiphon stamineus (Lamiaceae) leaves (MEOS) after acute and subchronic administration in rats. Superoxide radical scavenging, hydroxyl radical scavenging, and ferrous ion chelating methods were used to evaluate the antioxidant properties of the extract. In acute toxicity study, single dose of MEOS, 5000mg/kg, was administered to rats by oral gavage, and the treated rats were monitored for 14 days. While in the subchronic toxicity study, MEOS was administered orally, at doses of 1250, 2500, and 5000mg/kg/day for 28 days. From the results, MEOS showed good superoxide radical scavenging, hydroxyl radical scavenging, ferrous ion chelating, and antilipid peroxidation activities. There was no mortality detected or any signs of toxicity in acute and subchronic toxicity studies. Furthermore, there was no significant difference in bodyweight, relative organ weight, and haematological and biochemical parameters between bothmale and female treated rats in any doses tested.No abnormality of internal organs was observed between treatment and control groups.Theoral lethal dose determined wasmore than 5000mg/kg and the no-observed-adverse-effect level (NOAEL) of MEOS for both male and female rats is considered to be 5000mg/kg per day

Antioxidant and Toxicity Studies of 50% Methanolic Extract of Orthosiphon stamineus Benth

The present study evaluated the antioxidant activity and potential toxicity of 50% methanolic extract of Orthosiphon stamineus (Lamiaceae) leaves (MEOS) after acute and subchronic administration in rats. Superoxide radical scavenging, hydroxyl radical scavenging, and ferrous ion chelating methods were used to evaluate the antioxidant properties of the extract. In acute toxicity study, single dose of MEOS, 5000 mg/kg, was administered to rats by oral gavage, and the treated rats were monitored for 14 days. While in the subchronic toxicity study, MEOS was administered orally, at doses of 1250, 2500, and 5000 mg/kg/day for 28 days. From the results, MEOS showed good superoxide radical scavenging, hydroxyl radical scavenging, ferrous ion chelating, and antilipid peroxidation activities. There was no mortality detected or any signs of toxicity in acute and subchronic toxicity studies. Furthermore, there was no significant difference in bodyweight, relative organ weight, and haematological and biochemical parameters between both male and female treated rats in any doses tested. No abnormality of internal organs was observed between treatment and control groups. The oral lethal dose determined was more than 5000 mg/kg and the no-observed-adverse-effect level (NOAEL) of MEOS for both male and female rats is considered to be 5000 mg/kg per day

Sentinel surveillance for human enterovirus 71 in Sarawak, Malaysia: lessons from the first 7 years

BACKGROUND: A major outbreak of human enterovirus 71-associated hand, foot and mouth disease in Sarawak in 1997 marked the beginning of a series of outbreaks in the Asia Pacific region. Some of these outbreaks had unusually high numbers of fatalities and this generated much fear and anxiety in the region. METHODS: We established a sentinel surveillance programme for hand, foot and mouth disease in Sarawak, Malaysia, in March 1998, and the observations of the first 7 years are described here. Virus isolation, serotyping and genotyping were performed on throat, rectal, vesicle and other swabs. RESULTS: During this period Sarawak had two outbreaks of human enterovirus 71, in 2000 and 2003. The predominant strains circulating in the outbreaks of 1997, 2000 and 2003 were all from genogroup B, but the strains isolated during each outbreak were genetically distinct from each other. Human enterovirus 71 outbreaks occurred in a cyclical pattern every three years and Coxsackievirus A16 co-circulated with human enterovirus 71. Although vesicles were most likely to yield an isolate, this sample was not generally available from most cases and obtaining throat swabs was thus found to be the most efficient way to obtain virological information. CONCLUSION: Knowledge of the epidemiology of human enterovirus 71 transmission will allow public health personnel to predict when outbreaks might occur and to plan interventions in an effective manner in order to reduce the burden of disease

MRI Study of Minor Physical Anomaly in Childhood Autism Implicates Aberrant Neurodevelopment in Infancy

Background: MPAs (minor physical anomalies) frequently occur in neurodevelopmental disorders because both face and brain are derived from neuroectoderm in the first trimester. Conventionally, MPAs are measured by evaluation of external appearance. Using MRI can help overcome inherent observer bias, facilitate multi-centre data acquisition, and explore how MPAs relate to brain dysmorphology in the same individual. Optical MPAs exhibit a tightly synchronized trajectory through fetal, postnatal and adult life. As head size enlarges with age, inter-orbital distance increases, and is mostly completed before age 3 years. We hypothesized that optical MPAs might afford a retrospective 'window' to early neurodevelopment; specifically, inter-orbital distance increase may represent a biomarker for early brain dysmaturation in autism. Methods: We recruited 91 children aged 7-16; 36 with an autism spectrum disorder and 55 age- and gender-matched typically developing controls. All children had normal IQ. Inter-orbital distance was measured on T1-weighted MRI scans. This value was entered into a voxel-by-voxel linear regression analysis with grey matter segmented from a bimodal MRI data-set. Age and total brain tissue volume were entered as covariates. Results: Intra-class coefficient for measurement of the inter-orbital distance was 0.95. Inter-orbital distance was significantly increased in the autism group (p = 0.03, 2-tailed). The autism group showed a significant relationship between inter-orbital distance grey matter volume of bilateral amygdalae extending to the unci and inferior temporal poles. Conclusions: Greater inter-orbital distance in the autism group compared with healthy controls is consistent with infant head size expansion in autism. Inter-orbital distance positively correlated with volume of medial temporal lobe structures, suggesting a link to "social brain" dysmorphology in the autism group. We suggest these data support the role of optical MPAs as a "fossil record" of early aberrant neurodevelopment, and potential biomarker for brain dysmaturation in autism. © 2011 Cheung et al.published_or_final_versio

Molecular Epidemiology of Tungro Viruses in East Malaysia and Development of Serological Detection Tools for Diagnosis of Rice Tungro Disease

Malaysia aspires to be a rice self-sufficient nation as rice is the main food staple for her citizens. Thus, a rice disease outbreak can cause major losses for the rice granaries in the country. One of the viral rice diseases of concern in Malaysia is the rice tungro disease (RTD). RTD is caused by two viruses, namely the Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). The disease was detected in West Malaysia and Sabah in East Malaysia since the early twentieth century, and only recently in Sarawak in East Malaysia. RTD is very well-studied in West Malaysia, where the nucleotide sequences of both tungro viruses isolated from West Malaysia were published. In contrast, relatively little is known of RTD and the tungro viruses circulating in East Malaysia. Therefore, the aims of this study were to characterise the nucleotide sequences of tungro viruses isolated from East Malaysia and develop a RTD diagnostic method using in-house generated reagents such as sera and antigens in the form of recombinant proteins. The sequences of coat protein 1 (CP1), CP2 and CP3 of RTSV, and the open reading frame 1 (ORF1), ORF2, ORF3 (CP gene) and ORF4 of RTBV isolated from East Malaysia were characterized and compared with sequences of other published isolates by phylogenetic analysis. The identities and nucleotide sequences of fourteen RTBV and five RTSV isolates from East Malaysia determined from this study could be the first record of tungro viruses from East Malaysia. Then, recombinant proteins were generated by cloning and expressing CP genes of RTSV and RTBV isolated from Sabah using the SUMO fusion expression system. The tungro isolates selected for cloning and protein expression had high similarities in the nucleotide and deduced amino acid sequences with all tungro isolates from East Malaysia. This was to ensure that the antibodies generated from the iv selected tungro isolates are able to detect both tungro viruses from East Malaysia. Lastly, in-house polyclonal antibodies against tungro viruses were generated using the purified tungro viruses and the produced recombinant proteins. Two recombinant proteins that have potential to replace purified tungro virions as antigens in antibody production, and a reactive anti-serum for use in serological detection of RTD were finally obtained from the cloning and expression of recombinant CP of tungro viruses and production of antibodies against tungro viruses, respectively

Identification of mimotopes of human enterovirus 71

A random phage-displayed peptide library was used to screen a raised rabbit serum antibodies against recombinant viral protien (VP)1 of B4 strain of human enterovirus (HEV)71 (R410 serum) for mimotopes of VP1 protein of HEV71

Over expression of Recombinant CP3 Protein of Rice Tungro Spherical Virus in Prokaryotic Expression System

Currently, serological based diagnosis of rice tungro disease (RTD) is being limited by the availability of antisera. This study aims to produce recombinant protein from the coat protein (CP)3 of rice tungro spherical virus (RTSV) as a potential antigen in producing high titre antibodies for use in detection of RTD. Amplified RTSV CP3 gene of approximately 900 bp was cloned into pETSUMO vector. The recombinant plasmid was transformed into cloning host Mach1™-T1® E.coli cells and screened for recombinant gene in the correct orientation. Plasmid DNA from positive transformants was isolated and transformed into expression host BL21 (DE3) E.coli cells. One positive clone was selected for large scale expression where the expressed protein was purified and analysed by SDS-PAGE and Western blot. This recombinant RTSV CP3 protein, 46 kDa in molecular mass with 6x His-tag and SUMO protein fused to its N-terminal, was found to be antigenic when it reacted with our in-house generated polyclonal antibodies against tungro viruses