Displaying publications 21 - 40 of 48 in total

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  1. Singh S, Chow VT, Chan KP, Ling AE, Poh CL
    J Virol Methods, 2000 Aug;88(2):193-204.
    PMID: 10960707
    A specific and sensitive method based on RT-PCR was developed to detect enterovirus 71 (EV71) from patients with hand, foot and mouth disease, myocarditis, aseptic meningitis and acute flaccid paralysis. RT-PCR primers from conserved parts of the VP1 capsid gene were designed on the basis of good correlation with sequences of EV71 strains. These primers successfully amplified 44 strains of EV71 including 34 strains isolated from Singapore in 1997 and 1998, eight strains from Malaysia isolated in 1997 and 1998, one Japanese strain and the neurovirulent strain EV71/7423/MS/87. RT-PCR of 30 strains of other enteroviruses including coxsackievirus A and B, and echoviruses failed to give any positive amplicons. Hence, RT-PCR with these primers showed 100% correlation with serotyping. Direct sequencing of the RT-PCR products of 20 EV71 strains revealed a distinct cluster with two major subgroups, thus enabling genetic typing of the viruses. The genetic heterogeneity of these strains culminated in amino acid substitutions within the VP1, VP2 and VP3 regions. The sequencing of a 2.9 kb fragment comprising the capsid region and the major part of 5' UTR of two Singapore strains revealed that they belonged to a group distinct from the prototype EV71/BrCr strain and the EV71/7423/MS/87 strain. The dendrogram generated from 341 bp fragments within the VP1 region revealed that the strains of Singapore, Malaysia and Taiwan belong to two entirely different EV71 genogroups, distinct from the three genogroups identified in another recent study.
  2. Kho CL, Mohd-Azmi ML, Arshad SS, Yusoff K
    J Virol Methods, 2000 Apr;86(1):71-83.
    PMID: 10713378
    A sensitive and specific RT-nested PCR coupled with an ELISA detection system for detecting Newcastle disease virus is described. Two nested pairs of primer which were highly specific to all the three different pathotypes of NDV were designed from the consensus fusion gene sequence. No cross-reactions with other avian infectious agents such as infectious bronchitis virus, infectious bursal disease virus, influenza virus, and fowl pox virus were observed. Based on agarose electrophoresis detection, the RT-nested PCR was about 100 times more sensitive compared to that of a non-nested RT-PCR. To facilitate the detection of the PCR product, an ELISA detection method was then developed to detect the amplified PCR products and it was shown to be ten times more sensitive than gel electrophoresis. The efficacy of the nested PCR-ELISA was also compared with the conventional NDV detection method (HA test) and non-nested RT-PCR by testing against a total of 35 tissue specimens collected from ND-symptomatic chickens. The RT-nested PCR ELISA found NDV positive in 21 (60%) tissue specimens, while only eight (22.9%) and two (5.7%) out of 35 tissue specimens were tested NDV positive by both the non-nested RT-PCR and conventional HA test, respectively. Due to its high sensitivity for the detection of NDV from tissue specimens, this PCR-ELISA based diagnostic test may be useful for screening large number of samples.
  3. Arivananthan M, Yadav M, Kumar S
    J Virol Methods, 1997 Jun;66(1):5-14.
    PMID: 9220385
    Human herpesvirus-6 exists in two forms, HHV-6A which has not been clearly associated with any disease, and HHV-6B, the causative agent of exanthem subitum. The two variants have been distinguished by techniques such as dot blotting and restriction fragment length polymorphism of PCR products. This study aims to establish the prevalence of HHV-6A and HHV-6B in carcinoma tissues using variant-specific oligonucleotide probes. A total of 73 archived carcinoma biopsies from the oral, salivary gland, larynx, breast and cervix were obtained with seven histologically normal controls. In situ hybridization was carried out with nonradioactively labelled variant-specific probes. Samples that hybridized with both variant A and B probes were subjected further to nested PCR and digested with HindIII to distinguish the variants. A hybridization signal was observed in 76.2% of oral carcinoma tissue and 75.0% of salivary gland carcinoma tissue. In contrast, only 33.3% of cervical carcinoma tissue were positive for HHV-6 DNA. A hybridization signal was noted in all 4 laryngeal carcinoma tissues studied. However, the 10 breast carcinoma tissues studied were negative, as was the histologically normal tissue. The virus possesses tumourigenic potential and demonstrates virus transactivating properties. The frequency of HHV-6 variants in certain tumours suggest a cofactorial role in multistep carcinogenesis. While PCR amplifies selectively the predominant variant in a sample, this was not seen by in situ hybridization. The in situ hybridization technique allowed the localization of both HHV-6A and HHV-6B in the nuclei of transformed regions.
  4. Chan SY, Kautner IM, Lam SK
    J Virol Methods, 1994 Oct;49(3):315-22.
    PMID: 7868649
    The potential of RT-PCR to rapidly diagnose dengue infections from both acute and convalescent phase patients' sera was evaluated. The RNA extraction method involved binding of the viral RNA to silica particles in the presence of high concentration of guanidine thiocyanate. The protocol that was established was sensitive enough to detect 40 plaque forming units per 100 microliter of serum and results could be obtained within one day. Results from this study indicate that clinical samples should be collected in the early acute phase of illness when anti-dengue antibodies were undetectable or of low titres to ensure a more reliable diagnosis.
  5. Yap KL, Lam SK
    J Virol Methods, 1994 Apr;47(1-2):217-26.
    PMID: 8051228
    A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) was developed for the fast-growing and cytopathic cell culture-adapted hepatitis A virus (HAV) strain HM175A.2. Infectivity titration by EIA correlated well with titration by cytopathic effects. The reliability of this assay was demonstrated by close agreement in virus infectivity titers among different assays of the same virus aliquot and between assays of different virus aliquots. HAV infected cell cultures after fixation could be stored for up to 1 week before testing without decline in virus titer.
  6. Cheng HM, Foong YT, Mathew A, Sam CK, Dillner J, Prasad U
    J Virol Methods, 1993 Apr;42(1):45-51.
    PMID: 7686558
    An ELISA using the Epstein-Barr virus nuclear antigen 1 (EBNA 1) was found to detect selectively specific IgA in sera from patients with nasopharyngeal carcinoma (NPC). The antigen, p107, was a 20-amino acid synthetic peptide, representing a major epitope of EBNA 1.267/294 (90.8%) of NPC patients had IgA antibodies to p107 but in normal individuals, only 41/577 (7.1%) had IgA/p107. In sera from patients with other cancers, 11/77 (14.3%) had IgA/p107 reactivity. 124 IgA/VCA positive and 86 IgA/VCA negative NPC sera were also tested for IgA/p107 binding in ELISA. The majority of IgA/VCA positive sera (117) also contained IgA/p107 antibodies. Of interest was the detection of 74/86 IgA/p107 reactive sera in the IgA/VCA negative group. The results suggest that the IgA/p107 ELISA could become a useful, complementary screening assay to the IgA/VCA immunofluorescence test for detection of NPC.
  7. Permeen AM, Sam CK, Pathmanathan R, Prasad U, Wolf H
    J Virol Methods, 1990 Mar;27(3):261-7.
    PMID: 2157729
    The presence of Epstein Barr virus (EBV) DNA in biopsies from the post-nasal space (PNS) of patients suspected of nasopharyngeal carcinoma (NPC) was detected by in situ cytohybridization with an EBV DNA probe labelled with the novel labelling compound digoxigenin. The digoxigenin probe was hybridised to cryostat sections of NPC biopsies and subsequently detected by an enzyme immunoassay procedure. It was found that in situ cytohybridization using the digoxigenin probe was much more rapid and sensitive (96 h compared to five weeks) than the current method of using 3H-labelled probe. Using the digoxigenin EBV probe, it was found that in all the eighteen NPC biopsies tested, EBV DNA was detected in malignant epithelial cells and infiltrating lymphocytes. EBV DNA was also detected in some normal epithelial cells in these NPC biopsies. EBV DNA was not detected in epithelial cells of non-malignant biopsies.
  8. Tang KH, Yusoff K, Tan WS
    J Virol Methods, 2009 Aug;159(2):194-9.
    PMID: 19490973 DOI: 10.1016/j.jviromet.2009.03.015
    Hepatitis B is a major public health problem worldwide which may lead to chronic liver diseases, cirrhosis and hepatocellular carcinoma. An interaction between hepatitis B virus (HBV) envelope protein, particularly the PreS1 region, and a specific cell surface receptor is believed to be the initial step of HBV infection through attachment to hepatocytes. In order to develop a gene delivery system, bacteriophage T7 was modified genetically to display polypeptides of the PreS1 region. A recombinant T7 phage displaying amino acids 60-108 of the PreS1 region (PreS1(60-108)) was demonstrated to be most effective in transfecting HepG2 cells in a dose- and time-dependant manner. The phage genome was recovered from the cell lysate and confirmed by PCR whereas the infectious form of the internalized phage was measured by a plaque-forming assay. The internalized phage exhibited the appearance of green fluorescent dots when examined by immunofluorescence microscopy. Surface modification, particularly by displaying the PreS1(60-108) enhanced phage uptake, resulting in more efficient in vitro gene transfer. The ability of the recombinant phage to transfect HepG2 cells demonstrates the potential of the phage display system as a gene therapy for liver cancer.
  9. Tan SW, Ideris A, Omar AR, Yusoff K, Hair-Bejo M
    J Virol Methods, 2009 Sep;160(1-2):149-56.
    PMID: 19447142 DOI: 10.1016/j.jviromet.2009.05.006
    SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C(t)) 18.19+/-3.63 and a melting temperature (T(m)) 86.0+/-0.28 degrees C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C(t) value 14.70+/-2.32 and T(m) 87.4+/-0.21 degrees C. The assay had a dynamic detection range which spans over a 5log(10) concentration range, 10(9)-10(5) copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45+/-0.31% and 1.30+/-0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.
  10. Yap WB, Tey BT, Ng MY, Ong ST, Tan WS
    J Virol Methods, 2009 Sep;160(1-2):125-31.
    PMID: 19433111 DOI: 10.1016/j.jviromet.2009.04.038
    The core antigen of the hepatitis B virus (HBcAg) has been used widely as a diagnostic reagent for the identification of the viral infection. However, purification using the conventional sucrose density gradient ultracentrifugation is time consuming and costly. To overcome this, HBcAg particles displaying His-tag on their surface were constructed and produced in Escherichia coli. The recombinant His-tagged HBcAgs were purified using immobilized metal affinity chromatography. Transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA) revealed that the displayed His-tag did not impair the formation of the core particles and the antigenicity of HBcAg.
  11. Zainah S, Wahab AH, Mariam M, Fauziah MK, Khairul AH, Roslina I, et al.
    J Virol Methods, 2009 Feb;155(2):157-60.
    PMID: 19022293 DOI: 10.1016/j.jviromet.2008.10.016
    The performance of a commercial immunochromatography test for rapid detection of dengue NS1 antigen present in serum or plasma of patients was evaluated against a commercial dengue NS1 antigen-capture ELISA. The rapid immunochromatography test gave an overall sensitivity of 90.4% with a specificity of 99.5%. The sensitivity was highest for serum samples from which virus was isolated (96.3%) and lowest for those from which virus was not isolated and RT-PCR was negative (76.4%). The sensitivity was significantly higher for serum samples from patients with acute primary dengue (92.3%) than those from patients with acute secondary dengue (79.1%). The positive predictive value and negative predictive value of this commercial immunochromatography test were 99.6% and 87.9% respectively.
  12. Opitz L, Zimmermann A, Lehmann S, Genzel Y, Lübben H, Reichl U, et al.
    J Virol Methods, 2008 Dec;154(1-2):61-8.
    PMID: 18840469 DOI: 10.1016/j.jviromet.2008.09.004
    Strategies to control influenza outbreaks are focused mainly on prophylactic vaccination. Human influenza vaccines are trivalent blends of different virus subtypes. Therefore and due to frequent antigenic drifts, strain independent manufacturing processes are required for vaccine production. This study verifies the strain independency of a capture method based on Euonymus europaeus lectin-affinity chromatography (EEL-AC) for downstream processing of influenza viruses under various culture conditions propagated in MDCK cells. A comprehensive lectin binding screening was conducted for two influenza virus types from the season 2007/2008 (A/Wisconsin/67/2005, B/Malaysia/2506/2004) including a comparison of virus-lectin interaction by surface plasmon resonance technology. EEL-AC resulted in a reproducible high product recovery rate and a high degree of contaminant removal in the case of both MDCK cell-derived influenza virus types demonstrating clearly the general applicability of EEL-AC. In addition, host cell dependency of EEL-AC was studied with two industrial relevant cell lines: Vero and MDCK cells. However, the choice of the host cell lines is known to lead to different product glycosylation profiles. Hence, altered lectin specificities have been observed between the two cell lines, requiring process adaptations between different influenza vaccine production systems.
  13. Lee KW, Tan WS
    J Virol Methods, 2008 Aug;151(2):172-180.
    PMID: 18584885 DOI: 10.1016/j.jviromet.2008.05.025
    The recombinant hepatitis B virus (HBV) core antigen (HBcAg) expressed in Escherichia coli self-assembles into icosahedral capsids of about 35 nm which can be exploited as gene or drug delivery vehicles. The association and dissociation properties of the C-terminally truncated HBcAg with urea and guanidine hydrochloride (GdnHCl) were studied. Transmission electron microscopy (TEM) revealed that the dissociated HBcAg was able to re-associate into particles when the applied denaturing agents were physically removed. In order to evaluate the potential of the particles in capturing molecules, purified green fluorescent protein (GFP) was applied to the dissociated HBcAg for encapsidation. The HBcAg particles harbouring the GFP molecules were purified using sucrose density gradient ultracentrifugation and analysed using native agarose gel electrophoresis and TEM. A method for the encapsidation of GFP in HBcAg particles which has the potential to capture drugs or nucleic acids was established.
  14. Ong WT, Omar AR, Ideris A, Hassan SS
    J Virol Methods, 2007 Sep;144(1-2):57-64.
    PMID: 17512062
    Avian influenza viruses are pathogens of economical and public health concerns. However, infections caused by low pathogenic avian influenza particularly H9N2 subtype are not associated with clear clinical features. Hence, rapid detection and subtyping of the virus will enable immediate measures to be implemented for preventing widespread transmission. This study highlights the development of a multiplex real-time reverse-transcriptase polymerase chain reaction (RRT-PCR) assay using SYBR Green 1 chemistry for universal detection of avian influenza viruses and specific subtyping of H9N2 isolates based on melting temperatures (T(m)) discriminations. Three melting peaks generated simultaneously at temperatures 85.2+/-1.0, 81.9+/-0.9 and 78.7+/-0.9 degrees C represent NP, H9 and N2 gene products, respectively. The RRT-PCR assay was about 10-100-fold more sensitive when compared to the conventional RT-PCR method using reference H9N2 isolate. In addition, the RRT-PCR assay was 100% sensitive as well as 92% specific according to the standard virus isolation method in detecting experimentally infected specific-pathogen-free (SPF) chickens.
  15. Yaiw KC, Ong KC, Chua KB, Bingham J, Wang L, Shamala D, et al.
    J Virol Methods, 2007 Aug;143(2):140-6.
    PMID: 17442409
    Tioman virus is a newly described bat-urine derived paramyxovirus isolated in Tioman Island, Malaysia in 2001. Hitherto, neither human nor animal infection by this virus has been reported. Nonetheless, its close relationship to another paramyxovirus, the Menangle virus which had caused diseases in humans and pigs [Philbey, A.W., Kirkland, P.D., Ross, A.D., Davis, R.J., Gleeson, A.B., Love, R.J., Daniels, P.W., Gould, A.R., Hyatt, A.D., 1998. An apparently new virus (family Paramyxoviridae) infectious for pigs, humans, and fruit bats. Emerg. Infect. Dis. 4, 269-271], raises the possibility that it may be potentially pathogenic. In this study, mice were experimentally infected with Tioman virus by intraperitoneal and intracerebral routes, and the cellular targets and topographical distribution of viral genome and antigens were examined using in situ hybridization and immunohistochemistry, respectively. The possible association between viral infection and apoptosis was also investigated using the TUNEL assay and immunohistochemistry to FasL, Caspase-3, Caspase-8, Caspase-9 and bcl-2. The results showed that Tioman virus inoculated intracerebrally was neurotropic causing plaque-like necrotic areas, and appeared to preferentially replicate in the neocortex and limbic system. Viral infection of inflammatory cells was also demonstrated. TUNEL and Caspase-3 positivity was found in inflammatory cells but not in neurons, while FasL, Caspase-8 and Caspase-9 were consistently negative. This suggests that neuronal infection was associated with necrosis rather than apoptosis. Moreover, the data suggest that there may be an association between viral infection and apoptosis in inflammatory cells, and that it could, at least in part, involve Caspase-independent pathways. Bcl-2 was expressed in some neurons and inflammatory cells indicating its possible role in anti-apoptosis. There was no evidence of central nervous system infection via the intraperitoneal route.
  16. Cardosa MJ, Hooi TP, Shaari NS
    J Virol Methods, 1988 Oct;22(1):81-8.
    PMID: 3058737
    Partially purified DEN3 virus was used as antigen in a sensitive dot enzyme immunoassay (DEIA) for the detection of antibodies to flavivirus antigens. We describe here the method used to prepare and optimise the antigen-bearing nitrocellulose membranes and present the results obtained from screening 20 acute phase sera from patients shown to have had recent dengue infections by the haemagglutination inhibition (HI) test. Sixteen pairs of acute and convalescent sera from dengue-negative patients had no detectable antibody to dengue virus by HI. These were shown to have no antibody detectable by DEIA. Sera positive for dengue antibodies by HI had DEIA titers ranging from 10 to several thousand times greater than the titers detected by HI.
  17. Tan le V, Tuyen NT, Thanh TT, Ngan TT, Van HM, Sabanathan S, et al.
    J Virol Methods, 2015 Apr;215-216:30-6.
    PMID: 25704598 DOI: 10.1016/j.jviromet.2015.02.011
    Enterovirus A71 (EV-A71) has emerged as the most important cause of large outbreaks of severe and sometimes fatal hand, foot and mouth disease (HFMD) across the Asia-Pacific region. EV-A71 outbreaks have been associated with (sub)genogroup switches, sometimes accompanied by recombination events. Understanding EV-A71 population dynamics is therefore essential for understanding this emerging infection, and may provide pivotal information for vaccine development. Despite the public health burden of EV-A71, relatively few EV-A71 complete-genome sequences are available for analysis and from limited geographical localities. The availability of an efficient procedure for whole-genome sequencing would stimulate effort to generate more viral sequence data. Herein, we report for the first time the development of a next-generation sequencing based protocol for whole-genome sequencing of EV-A71 directly from clinical specimens. We were able to sequence viruses of subgenogroup C4 and B5, while RNA from culture materials of diverse EV-A71 subgenogroups belonging to both genogroup B and C was successfully amplified. The nature of intra-host genetic diversity was explored in 22 clinical samples, revealing 107 positions carrying minor variants (ranging from 0 to 15 variants per sample). Our analysis of EV-A71 strains sampled in 2013 showed that they all belonged to subgenogroup B5, representing the first report of this subgenogroup in Vietnam. In conclusion, we have successfully developed a high-throughput next-generation sequencing-based assay for whole-genome sequencing of EV-A71 from clinical samples.
  18. Thanarajoo SS, Kong LL, Kadir J, Lau WH, Vadamalai G
    J Virol Methods, 2014 Jun;202:19-23.
    PMID: 24631346 DOI: 10.1016/j.jviromet.2014.02.024
    A reverse transcription loop-mediated isothermal amplification (RT-LAMP) detected Coconut cadang-cadang viroid (CCCVd) within 60 min at 60 °C in total nucleic acid extracted from oil palm leaves infected with CCCVd. Positive reactions showed colour change from orange to green in the reaction mix after the addition of fluorescent reagent, and a laddering pattern band on 2% agarose gel electrophoresis. Conventional RT-PCR with LAMP primers produced amplicons with a sequence identical to the 297-nt CCCVd oil palm variant with the primers being specific for CCCVd and not for other viroids such as PSTVd and CEVd. RT-LAMP was found to be rapid and specific for detecting oil palm CCCVd.
  19. Lazouskaya NV, Palombo EA, Poh CL, Barton PA
    J Virol Methods, 2014 Mar;197:67-76.
    PMID: 24361875 DOI: 10.1016/j.jviromet.2013.12.005
    Enterovirus 71 (EV 71) is a causative agent of mild Hand Foot and Mouth Disease but is capable of causing severe complications in the CNS in young children. Reverse genetics technology is currently widely used to study the pathogenesis of the virus. The aim of this work was to determine and evaluate the factors which can contribute to infectivity of EV 71 RNA transcripts in vitro. Two strategies, overlapping RT-PCR and long distance RT-PCR, were employed to obtain the full-length genome cDNA clones of the virus. The length of the poly(A) tail and the presence of non-viral 3'-terminal sequences were studied in regard to their effects on infectivity of the in vitro RNA transcripts of EV 71 in cell culture. The data revealed that only cDNA clones obtained after long distance RT-PCR were infectious. No differences were observed in virus titres after transfection with in vitro RNA harbouring a poly(A) tail of 18 or 30 adenines in length, irrespective of the non-viral sequences at the 3'-terminus.
  20. Chua CL, Chan YF, Sam IC
    J Virol Methods, 2014 Jan;195:126-33.
    PMID: 24134938 DOI: 10.1016/j.jviromet.2013.10.015
    Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which has recently re-emerged globally and poses a major threat to public health. Infection leads to severe arthralgia, and disease management remains supportive in the absence of vaccines and anti-viral interventions. The high specificities of monoclonal antibodies (mAbs) have been exploited in immunodiagnostics and immunotherapy in recent decades. In this study, eight different clones of mAbs were generated and characterised. These mAbs targeted the linear epitopes on the CHIKV E2 envelope glycoprotein, which is the major target antigen during infection. All the mAbs showed binding activity against the purified CHIKV virion or recombinant E2 when analysed by immunofluorescence, ELISA and Western blot. The epitopes of each mAb were mapped by overlapping synthetic peptide-based ELISA. The epitopes are distributed at different functional domains of E2 glycoprotein, namely at domain A, junctions of β-ribbons with domains A and B, and domain C. Alignment of mAb epitope sequences revealed that some are well-conserved within different genotypes of CHIKV, while some are identical to and likely to cross-react with the closely-related alphavirus O'nyong-nyong virus. These mAbs with their mapped epitopes are useful for the development of diagnostic or research tools, including immunofluorescence, ELISA and Western blot.
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