Analytical and Clinical Evaluation of a TaqMan Real-Time PCR Assay for the Detection of Chikungunya Virus.

Due to the general symptoms presented by the Chikungunya virus (CHIKV)-infected patients, a laboratory test is needed to differentiate CHIKV from other viral infections. The reverse transcription-quantitative real-time PCR (RT-qPCR) is a rapid and sensitive diagnostic tool, and several assays have been developed for detecting and quantifying CHIKV. Since real-time amplification efficiency varies within and between laboratories, an assay must be validated before being used on patient samples. In this study, the diagnostic performance of a TaqMan RT-qPCR assay was evaluated using synthetic RNA and archived patient samples. The cutoff quantification cycle (Cq) value for the assay was determined by experimental evidence. We found the in-house assay was highly sensitive, with a detection limit of 3.95 RNA copies/reaction. The analytical specificity of the assay was 100%. The analytical cutoff Cq value was 37, corresponding to the mean Cq value of the detection limit. Using archived samples characterized previously, the sensitivity and specificity of the assay were 76% and 100%, respectively. The in-house assay was also compared with a commercial assay, and we found that the in-house assay had higher sensitivity. Although further evaluation with prospective patient samples is needed in the future, this validated RT-qPCR was sensitive and specific, which shows its potential to detect CHIKV in clinical samples. IMPORTANCE Chikungunya virus causes chikungunya fever, a disease characterized by fever, rash, and joint pain. In the early phase of infection, chikungunya fever is always misdiagnosed as other arbovirus infections, such as dengue. Laboratory tests such as RT-qPCR are therefore necessary to confirm CHIKV infection. We evaluated the performance of an in-house RT-qPCR assay, and our study shows that the assay could detect CHIKV in clinical samples. We also show the cutoff determination of the assay, which provides important guidance to scientists or researchers when implementing a new RT-qPCR assay in a laboratory.

Partial characterization of bacteriocin-like compound (BLIS) produced by Burkholderia stagnalis strain K23/3 against Burkholderia pseudomallei

Aims@#Burkholderia pseudomallei, the human pathogen that causes melioidosis, is intrinsically resistant towards a wide range of antibiotics and there have been reports of acquired resistance towards antibiotics used for melioidosis treatments. Antimicrobial peptides (AMP) such as bacteriocins are gaining the interests of researchers as alternative for treating infections caused by multidrug resistant bacteria. In this study, we aimed to identify Burkholderia spp. isolated from soil in Sarawak that possess the potential in inhibiting the growth of B. pseudomallei and to further characterize the antagonistic compound produced.@*Methodology and results@#A total of 50 Burkholderia spp. isolates of environmental origin and two isolates of Ralstonia solanacearum were screened against five clinical isolates of B. pseudomallei using spot-on-lawn assay and flip streak method. Burkholderia stagnalis isolate K23/3 showed clear zones of inhibition (ZOI) in both preliminary tests. Cell-free supernatant (CFS) was obtained from B. stagnalis K23/3 broth culture and was tested via agar well diffusion assay (AWDA). The antagonistic compound secreted at the early log phase of the bacterial growth was shown to be stable in a wide range of temperatures and pH. Treatment with different enzymes revealed that it was sensitive towards proteinase K, suggesting that it is proteinaceous. The bacteriocin-like-substance (BLIS) was subjected to ammonium sulfate precipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE gel was overlaid with indicator B. pseudomallei isolates where the active protein was shown to be less than 7.1 kDa.@*Conclusion, significance and impact of study@#Burkholderia stagnalis isolate K23/3 was able to secrete bacteriocin-like-substance (BLIS) that has the potential in biocontrol of B. pseudomallei in the environment or as potential treatment for melioidosis.

Overexpression of recombinant domain III envelope protein of Zika virus

Aims@#Zika virus (ZIKV) is a member of the Flaviviridae family and is transmitted to humans by mosquitoes. In humans, it causes disease known as Zika fever. The severity of the infection ranged from asymptomatic to mild disease and to infection associated with neurological disorders and congenital anomaly. The common symptoms are maculopapular rash, fever, arthralgia, myalgia, headache and conjunctivitis. The flavivirus genome consists of structural and nonstructural proteins. The envelope (E) glycoprotein is the major structural protein which is responsible for virus entry and represents a major target for neutralizing antibodies. The E protein consists of three distinct domains: domain I, domain II and domain III. The domain III (DIII) of the E protein has shown to be useful as antigen for flavivirus serologic diagnosis and immunization in animal model. Hence, the aim of this work is to express the DIII of E protein (EDIII) of ZIKV for immunoreactivity study@* Methodology and results@#The EDIII of ZIKV was cloned into pET SUMO cloning vector and transformed into Mach-T1 competent E. coli cells. Positive clone was selected, verified and transformed into BL21 (DE3) competent E. coli for protein expression. The expression of the recombinant protein was analysed on SDS-PAGE and western blot. The recombinant fusion protein of EDIII/SUMOHIS (rEDIII) was successfully expressed at a molecular weight of approximately 38.2 kDa.@*Conclusion, significance and impact of study@#The expression of the protein was confirmed by detection with antihistidine and a flavivirus antiserum, HPR.

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

Aims@#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.