Displaying publications 1 - 20 of 267 in total

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  1. Roslan ND, Sundram S, Hong LW, Ling KL, Vadamalai G
    Mol Biol Rep, 2023 Nov;50(11):9699-9705.
    PMID: 37676433 DOI: 10.1007/s11033-023-08771-0
    BACKGROUND: Sequence variation has been attributed to symptom variations but has not been investigated in Orange Spotting-Coconut cadang-cadang viroid (OS-CCCVd) infected palms. Likewise, the relationship between Coconut cadang-cadang viroid (CCCVd) variants, Orange Spotting (OS) severity and the accumulation of the viroid in the palms have not been elucidated. This paper describes the characterization of CCCVd variants by cloning and sequencing, followed by correlation with symptom expression.

    METHODS AND RESULTS: Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura × Pisifera (D × P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21 years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279.

    CONCLUSION: The OS symptoms observed in the field were characterized into three categories based on the size and morphology of the orange spots on the affected fronds. In addition, there was no direct correlation between disease severity and the accumulation of CCCVd variants in oil palm. This finding is the first report describing the sequence variation of the CCCVd RNA and symptom variation in OS oil palm field samples.

    Matched MeSH terms: RNA, Viral/genetics
  2. Andrew A, Citartan M, Wong KA, Tang TH, Magdline Sia Henry S, Ch'ng ES
    Microbiol Spectr, 2023 Aug 17;11(4):e0008823.
    PMID: 37272795 DOI: 10.1128/spectrum.00088-23
    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.
    Matched MeSH terms: RNA, Viral/genetics
  3. Chin KL, Teoh BT, Sam SS, Loong SK, Tan KK, Azizan NS, et al.
    Trop Biomed, 2022 Dec 01;39(4):518-523.
    PMID: 36602210 DOI: 10.47665/tb.39.4.005
    Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.
    Matched MeSH terms: RNA, Viral/analysis; RNA, Viral/genetics
  4. Drexler JF, Corman VM, Gloza-Rausch F, Seebens A, Annan A, Ipsen A, et al.
    PLoS One, 2009;4(7):e6367.
    PMID: 19636378 DOI: 10.1371/journal.pone.0006367
    Henipaviruses (Hendra and Nipah virus) are highly pathogenic members of the family Paramyxoviridae. Fruit-eating bats of the Pteropus genus have been suggested as their natural reservoir. Human Henipavirus infections have been reported in a region extending from Australia via Malaysia into Bangladesh, compatible with the geographic range of Pteropus. These bats do not occur in continental Africa, but a whole range of other fruit bats is encountered. One of the most abundant is Eidolon helvum, the African Straw-coloured fruit bat.
    Matched MeSH terms: RNA, Viral/genetics*
  5. Aziz MA, Norman S, Mohamed Zaid S, Simarani K, Sulaiman R, Mohd Aris A, et al.
    Arch Microbiol, 2023 Jan 28;205(2):76.
    PMID: 36708390 DOI: 10.1007/s00203-023-03417-y
    Wastewater monitoring for SARS-CoV-2 has attracted considerable attention worldwide to complement the existing clinical-based surveillance system. In this study, we report our first successful attempt to prove the circulation of SARS-CoV-2 genes in Malaysian urban wastewater. A total of 18 wastewater samples were obtained from a regional sewage treatment plant that received municipal sewage between February 2021 and May 2021. Using the quantitative PCR assay targeting the E and RdRp genes of SARS-CoV-2, we confirmed that both genes were detected in the raw sewage, while no viral RNA was found in the treated sewage. We were also able to show that the trend of COVID-19 cases in Kuala Lumpur and Selangor was related to the changes in SARS-CoV-2 RNA levels in the wastewater samples. Overall, our study highlights that monitoring wastewater for SARS-CoV-2 should help local health professionals to obtain additional information on the rapid and silent circulation of infectious agents in communities at the regional level.
    Matched MeSH terms: RNA, Viral/genetics
  6. Lai MY, Bukhari FDM, Zulkefli NZ, Ismail I, Mustapa NI, Soh TST, et al.
    BMC Infect Dis, 2021 Nov 17;21(1):1162.
    PMID: 34789179 DOI: 10.1186/s12879-021-06876-0
    BACKGROUND: Current assays for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rely on time consuming, costly and laboratory based methods for virus isolation, purification and removing inhibitors. To address this limitation, we propose a simple method for testing RNA from nasopharyngeal swab samples that bypasses the RNA purification step.

    METHODS: In the current project, we have described two extraction-free reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for the detection of SARS-CoV-2 by using E gene and RdRp gene as the targets.

    RESULTS: Here, results showed that reverse transcription loop-mediated isothermal amplification assays with 88.4% sensitive (95% CI: 74.9-96.1%) and 67.4% sensitive (95% CI: 51.5-80.9%) for E gene and RdRp gene, respectively.

    CONCLUSION: Without the need of RNA purification, our developed RT-LAMP assays for direct detection of SARS-CoV-2 from nasopharyngeal swab samples could be turned into alternatives to qRT-PCR for rapid screening.

    Matched MeSH terms: RNA, Viral/genetics
  7. Babadi AA, Rahmati S, Fakhlaei R, Heidari R, Baradaran S, Akbariqomi M, et al.
    Sci Rep, 2022 Nov 12;12(1):19416.
    PMID: 36371566 DOI: 10.1038/s41598-022-23996-y
    The current COVID-19 pandemic outbreak poses a serious threat to public health, demonstrating the critical need for the development of effective and reproducible detection tests. Since the RT-qPCR primers are highly specific and can only be designed based on the known sequence, mutation sensitivity is its limitation. Moreover, the mutations in the severe acute respiratory syndrome β-coronavirus (SARS-CoV-2) genome led to new highly transmissible variants such as Delta and Omicron variants. In the case of mutation, RT-qPCR primers cannot recognize and attach to the target sequence. This research presents an accurate dual-platform DNA biosensor based on the colorimetric assay of gold nanoparticles and the surface-enhanced Raman scattering (SERS) technique. It simultaneously targets four different regions of the viral genome for detection of SARS-CoV-2 and its new variants prior to any sequencing. Hence, in the case of mutation in one of the target sequences, the other three probes could detect the SARS-CoV-2 genome. The method is based on visible biosensor color shift and a locally enhanced electromagnetic field and significantly amplified SERS signal due to the proximity of Sulfo-Cyanine 3 (Cy3) and AuNPs intensity peak at 1468 cm-1. The dual-platform DNA/GO/AuNP biosensor exhibits high sensitivity toward the viral genome with a LOD of 0.16 ng/µL. This is a safe point-of-care, naked-eye, equipment-free, and rapid (10 min) detection biosensor for diagnosing COVID-19 cases at home using a nasopharyngeal sample.
    Matched MeSH terms: RNA, Viral/genetics
  8. Marya A, Karobari MI, Selvaraj S, Adil AH, Assiry AA, Rabaan AA, et al.
    Int J Environ Res Public Health, 2021 May 29;18(11).
    PMID: 34072456 DOI: 10.3390/ijerph18115848
    OBJECTIVE: Healthcare workers in general are at a high risk of potential infections with COVID-19, especially those who work with aerosol generating procedures. Dentists fall in this category, as not only do they operate with aerosol generating procedures but also operate within a face-to-face contact area.

    METHODS: A structured self-administered questionnaire was developed at Najran University and provided to the participants for data collection. The data collected included information on risk perception and incorporation of measures for protection against COVID-19 to gauge the attitude of dentists during this period. Also, clinical implementation of various protective measures was reviewed.

    RESULTS: Of the n = 322 dentists that answered the questions, 50% were general dentists and 28.9% were dentists working at specialist clinics, while the remaining 21.1% of dentists were employed in academic institutions. Among the newer additions to the clinic, 36.3% of dentists answered that they had added atomizers to their practices, followed by 26.4% of dentists that had incorporated the use of UV lamps for sterilization. We found that 18.9% dentists were using HEPA filters in their clinics, while 9.9% of dentists were making use of fumigation devices to control the risk of infection. One-way ANOVA was also carried out to demonstrate that there was a statistically significant difference (p = 0.049) between groups of dentists utilizing HEPA filters, UV lamps, atomizers, and fumigation devices to prevent the spread of SARS-CoV2 across their workplaces.

    CONCLUSION: Dentists are aware of recently updated knowledge about the modes of transmission of COVID-19 and the recommended infection control measures in dental settings. A better understanding of the situation and methods to prevent it will ensure that the dental community is able to provide healthcare services to patients during the pandemic.

    Matched MeSH terms: RNA, Viral
  9. SHAHRUL HISHAM ZAINAL ARIFFIN, ROSLINA SHAMSUDIN, NURUL ATIKAH AHMAD, ZULKIFLIE ZAMROD
    Sains Malaysiana, 2012;41:423-430.
    Sistem minigenom telah digunakan untuk mengkaji replikasi dan transkripsi virus RNA tidak bersegmen. Objektif kajian ini adalah untuk membina sistem minigenom bagi virus NDV strain tempatan, AF2240 serta bagi mengkaji mekanisme transkripsi dan replikasi virus ini. Bagi tujuan ini lima plasmid digunakan iaitu pMGNDV, pCITENP, pCITEP, pTriEX-T7, dan pGEML. Kesemua plasmid diekstrak secara berskala besar dan dimendakkan menggunakan polietilina glikol. Hasil ekstrak ini digunakan untuk transfeksi ke dalam sel. Translasi in vitro dilakukan dengan menggunakan pCITENP, pCITEP, dan pTriEX-T7 untuk memastikan kesemua konstruk ini berfungsi. Hasil pemblotan western menunjukkan protein bersaiz ~100 kDa (T7), ~53 kDa (NP), ~53 dan 55 kDa (P) berjaya diekspreskan. Protein CAT diperoleh apabila plasmid yang mengekodkan minigenom NDV ditransfeksi bersama plasmid yang mengekodkan protein nukleokapsid (NP), fosfoprotein (P) dan subunit besar polimerase (L) ke dalam sel BHK-21. Dianggarkan 55 pg protein CAT berjaya diperoleh menggunakan kit CAT ELISA. Hasil pemblotan western turut menunjukkan protein CAT bersaiz 25 kDa dihasilkan. Kesimpulannnya, system minigenom ini berupaya untuk berfungsi dan mampu mengekspreskan gen asing di dalam sel mamalia BHK-21.
    Matched MeSH terms: RNA, Viral
  10. Anusha B, Philip R, Norain K, Harvinder S, Gurdeep SM
    Med J Malaysia, 2012 Dec;67(6):641-3.
    PMID: 23770968 MyJurnal
    Nasopharyngeal carcinoma (NPC) is rare among people of Indian ethnicity. A short retrospective case review of clinical records of Indian patients diagnosed with nasopharyngeal carcinoma in a period of 5 years was conducted. Their slides were further subjected to EBV encoded RNA (EBER) - In- situ Hybridization (ISH). The histologic subtype was nonkeratinizing carcinoma in all 4 patients. All were Epstein Barr Virus (EBV) positive. We believe that the crucial factor responsible for nasopharyngeal carcinoma is genetics; either a genetic susceptibility among high risk groups or genetic resistance/immunity in low risk groups. Further genetic studies are required to look for somatic or inherited chromosomal mutations among the various risk populations.
    Matched MeSH terms: RNA, Viral*
  11. Pritchard LI, Chua KB, Cummins D, Hyatt A, Crameri G, Eaton BT, et al.
    Arch Virol, 2006 Feb;151(2):229-39.
    PMID: 16205863
    After the outbreak of Nipah virus (NiV) in 1998-99, which resulted in 105 human deaths and the culling of more than one million pigs, a search was initiated for the natural host reservoir of NiV on Tioman Island off the east coast of Malaysia. Three different syncytia-forming viruses were isolated from fruit bats on the island. They were Nipah virus, Tioman virus (a novel paramyxovirus related to Menangle virus), and a reovirus, named Pulau virus (PuV), which is the subject of this study. PuV displayed the typical ultra structural morphology of a reovirus and was neutralised by serum against Nelson Bay reovirus (NBV), a reovirus isolated from a fruit bat (Pteropus poliocephalus) in Australia over 30 years ago. PuV was fusogenic and formed large syncytia in Vero cells. Comparison of dsRNA segments between PuV and NBV showed distinct mobility differences for the S1 and S2 segments. Complete sequence analysis of all four S segments revealed a close relationship between PuV and NBV, with nucleotide sequence identity varying from 88% for S3 segment to 56% for the S1 segment. Similarly phylogenetic analysis of deduced protein sequences confirmed that PuV is closely related to NBV. In this paper we discuss the similarities and differences between PuV and NBV which support the classification of PuV as a novel mammalian, fusogenic reovirus within the Nelson Bay orthoreovirus species, in the genus Orthoreovirus, family Reoviridae.
    Matched MeSH terms: RNA, Viral/genetics
  12. Tan KE, Lim YY
    FEBS J, 2021 08;288(15):4488-4502.
    PMID: 33236482 DOI: 10.1111/febs.15639
    Circular RNAs (circRNAs) are a recently discovered class of noncoding RNAs found in many species across the eukaryotic kingdom. These intriguing RNA species are formed through a unique mechanism that is known as back splicing in which the 5' and 3' termini are covalently joined. Recent research has revealed that viruses also encode a repertoire of circRNAs. Some of these viral circRNAs are abundantly expressed and are reported to play a role in disease pathogenesis. A growing number of studies also indicate that host circRNAs are involved in immune responses against virus infections with either an antiviral or proviral role. In this review, we briefly introduce circRNA, its biogenesis, and mechanism of action. We go on to summarize the latest research on the expression, regulation, and functions of viral and host-encoded circRNAs during the host-virus interaction, with the aim of highlighting the potential of viral and host circRNAs as a suitable target for diagnostic biomarker development and therapeutic treatment of viral-associated diseases. We conclude by discussing the current limitations in knowledge and significance of elucidating the roles of circRNAs in host-virus interactions, as well as future directions for this emerging field.
    Matched MeSH terms: RNA, Viral/genetics
  13. Tee KK, Chan PQ, Loh AM, Singh S, Teo CH, Iyadorai T, et al.
    J Med Virol, 2023 Feb;95(2):e28520.
    PMID: 36691929 DOI: 10.1002/jmv.28520
    Pteropine orthoreovirus (PRV), an emerging bat-borne virus, has been linked to cases of acute respiratory infections (ARI) in humans. The prevalence, epidemiology and genomic diversity of PRV among ARI of unknown origin were studied. Among 632 urban outpatients tested negative for all known respiratory viruses, 2.2% were PRV-positive. Patients mainly presented with moderate to severe forms of cough, sore throat and muscle ache, but rarely with fever. Phylogenetic analysis revealed that over 90% of patients infected with the Melaka virus (MelV)-like PRV, while one patient infected with the Pulau virus previously found only in fruit bats. Human oral keratinocytes and nasopharyngeal epithelial cells were susceptible to clinical isolates of PRV, including the newly isolated MelV-like 12MYKLU1034. Whole genome sequence of 12MYKLU1034 using Nanopore technique revealed a novel reassortant strain. Evolutionary analysis of the global PRV strains suggests the continuous evolution of PRV through genetic reassortment among PRV strains circulating in human, bats and non-human primate hosts, creating a spectrum of reassortant lineages with complex evolutionary characteristics. In summary, the role of PRV as a common etiologic agent of ARI is evident. Continuous monitoring of PRV prevalence, pathogenicity and diversity among human and animal hosts is important to trace the emergence of novel reassortants.
    Matched MeSH terms: RNA, Viral/genetics
  14. Chaibun T, Puenpa J, Ngamdee T, Boonapatcharoen N, Athamanolap P, O'Mullane AP, et al.
    Nat Commun, 2021 02 05;12(1):802.
    PMID: 33547323 DOI: 10.1038/s41467-021-21121-7
    Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.
    Matched MeSH terms: RNA, Viral/genetics
  15. Rashid SA, Nazakat R, Muhamad Robat R, Ismail R, Suppiah J, Rajendran K, et al.
    Front Public Health, 2023;11:1208348.
    PMID: 37965510 DOI: 10.3389/fpubh.2023.1208348
    Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may transmit through airborne route particularly when the aerosol particles remain in enclosed spaces with inadequate ventilation. There has been no standard recommended method of determining the virus in air due to limitations in pre-analytical and technical aspects. Furthermore, the presence of low virus loads in air samples could result in false negatives. Our study aims to explore the feasibility of detecting SARS-CoV-2 ribonucleic acid (RNA) in air samples using droplet digital polymerase chain reaction (ddPCR). Active and passive air sampling was conducted between December 2021 and February 2022 with the presence of COVID-19 confirmed cases in two hospitals and a quarantine center in Klang Valley, Malaysia. SARS-CoV-2 RNA in air was detected and quantified using ddPCR and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The comparability of two different digital PCR platforms (QX200 and QIAcuity) to RT-PCR were also investigated. Additionally negative staining transmission electron microscopy was performed to visualize virus ultrastructure. Detection rates of SARS-CoV-2 in air samples using ddPCR were higher compared to RT-PCR, which were 15.2% (22/145) and 3.4% (5/145), respectively. The sensitivity and specificity of ddPCR was 100 and 87%, respectively. After excluding 17 negative samples (50%) by both QX200 and QIAcuity, 15% samples (5/34) were found to be positive both ddPCR and dPCR. There were 23.5% (8/34) samples that were detected positive by ddPCR but negative by dPCR. In contrast, there were 11.7% (4/34) samples that were detected positive by dPCR but negative by ddPCR. The SARS-CoV-2 detection method by ddPCR is precise and has a high sensitivity for viral RNA detection. It could provide advances in determining low viral titter in air samples to reduce false negative reports, which could complement detection by RT-PCR.
    Matched MeSH terms: RNA, Viral/analysis
  16. Kalaycioglu AT, Baykal A, Guldemir D, Bakkaloglu Z, Korukluoglu G, Coskun A, et al.
    J Med Virol, 2013 Dec;85(12):2128-35.
    PMID: 23959542 DOI: 10.1002/jmv.23714
    Genetic characterization of measles viruses (MVs) combined with acquisition of epidemiologic information is essential for measles surveillance programs used in determining transmission pathways. This study describes the molecular characterization of 26 MV strains (3 from 2010, 23 from 2011) obtained from urine or throat swabs harvested from patients in Turkey. MV RNA samples (n = 26) were subjected to sequence analysis of 450 nucleotides comprising the most variable C-terminal region of the nucleoprotein (N) gene. Phylogenetic analysis revealed 20 strains from 2011 belonged to genotype D9, 3 to D4, 2 strains from 2010 to genotype D4 and 1 to genotype B3. This study represents the first report describing the involvement of MV genotype D9 in an outbreak in Turkey. The sequence of the majority of genotype D9 strains was identical to those identified in Russia, Malaysia, Japan, and the UK. Despite lack of sufficient epidemiologic information, the presence of variants observed following phylogenetic analysis suggested that exposure to genotype D9 might have occurred due to importation more than once. Phylogenetic analysis of five genotype D4 strains revealed the presence of four variants. Epidemiological information and phylogenetic analysis suggested that three genotype D4 strains and one genotype B3 strain were associated with importation. This study suggests the presence of pockets of unimmunized individuals making Turkey susceptible to outbreaks. Continuing molecular surveillance of measles strains in Turkey is essential as a means of acquiring epidemiologic information to define viral transmission patterns and determine the effectiveness of measles vaccination programs designed to eliminate this virus.
    Matched MeSH terms: RNA, Viral/blood; RNA, Viral/genetics; RNA, Viral/urine
  17. Wong HV, Vythilingam I, Sulaiman WY, Lulla A, Merits A, Chan YF, et al.
    Am J Trop Med Hyg, 2016 Jan;94(1):182-6.
    PMID: 26598564 DOI: 10.4269/ajtmh.15-0318
    Vertical transmission may contribute to the maintenance of arthropod-borne viruses, but its existence in chikungunya virus (CHIKV) is unclear. Experimental vertical transmission of infectious clones of CHIKV in Aedes aegypti mosquitoes from Malaysia was investigated. Eggs and adult progeny from the second gonotrophic cycles of infected parental mosquitoes were tested. Using polymerase chain reaction (PCR), 56.3% of pooled eggs and 10% of adult progeny had detectable CHIKV RNA, but no samples had detectable infectious virus by plaque assay. Transfected CHIKV RNA from PCR-positive eggs did not yield infectious virus in BHK-21 cells. Thus, vertical transmission of viable CHIKV was not demonstrated. Noninfectious CHIKV RNA persists in eggs and progeny of infected Ae. aegypti, but the mechanism and significance are unknown. There is insufficient evidence to conclude that vertical transmission exists in CHIKV, as positive results reported in previous studies were almost exclusively based only on viral RNA detection.
    Matched MeSH terms: RNA, Viral/genetics*; RNA, Viral/isolation & purification*
  18. Chow WZ, Al-Darraji H, Lee YM, Takebe Y, Kamarulzaman A, Tee KK
    J Virol, 2012 Oct;86(20):11398-9.
    PMID: 22997419
    A novel HIV-1 genotype designated CRF53_01B was recently characterized from three epidemiologically unrelated persons in Malaysia. Here we announced three recently isolated full-length genomes of CRF53_01B, which is likely to be phylogenetically linked to CRF33_01B, circulating widely in Southeast Asia. The genome sequences may contribute to HIV-1 molecular surveillance and future vaccine development in the region.
    Matched MeSH terms: RNA, Viral/analysis; RNA, Viral/genetics
  19. Mohammadi MR, Vadamalai G, Joseph H
    PMID: 21534490
    Coconut cadong-cadong viroid (CCCVd) causes the Lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroid RNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4 degrees C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After adding sodium acetate, pH 5.6 to 200 mM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4 degrees C and another centrifugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer (50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 degrees C for 90 min and 16 h, respectively followed by two low stringency washes (0.5 X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60 degrees C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result the blue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.
    Matched MeSH terms: RNA, Viral/genetics; RNA, Viral/isolation & purification*
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