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Quantitation of hepatitis B virus DNA in serum of patients with hepatoma

Lie-Injo LE, Lopez CG, Latu J, Lim ML, Balasegaram M

Cytobios, 1987;50(201):101-6.
PMID: 3036422

Hepatitis B virus (HBV) DNA in the serum of 31 patients with histologically confirmed primary hepatocellular carcinoma (PHC) from Malaysia and Indonesia was quantitated by densitometric scanning of autoradiograms obtained by Southern blot DNA hybridization, after electrophoresis using a 32P DNA cloned into plasmid pBR325 as a probe. This quantitation after electrophoresis is more informative than the usual spot hybridization technique. Five of the 31 sera were positive for HBV DNA. Levels ranged between 1.36 pq and 143.18 pq per ml of serum, and the levels of HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe in the serum were serologically determined. All five sera positive for HBV DNA were also positive for HBsAg. Three of the five positive for HBV DNA were positive for HBeAg and negative for anti-HBe. Two of the sera positive for HBV DNA were negative for HBeAg but positive for anti-HBe. All sera negative for HBV DNA were also negative for HBeAg. Many sera which were negative for HBV DNA and HBeAg were positive for HBsAg. Of the 31 sera from PHC patients, 23 had at least one HBV marker positive (74.2%).

Matched MeSH terms: DNA, Viral/blood*
The frequency of pre-core gene mutations in chronic hepatitis B infection: a study of Malaysian subjects

Yap SF, Wong PW, Chen YC, Rosmawati M

Southeast Asian J Trop Med Public Health, 2002 Mar;33(1):102-9.
PMID: 12118437

A retrospective study was carried out to determine the frequency of the pre-core stop codon mutant virus in a group of chronic hepatitis B carriers: 81 cases were considered [33 hepatits B e antigen (HBe) positive and 48 HBe negative]. All of the HBe positive cases had detectable viral DNA by hybridization analysis; in the case of the HBe negative cases, one third had detectable viral DNA by hybridization analysis and two thirds had HBV DNA detectable by polymerase chain reaction (PCR) amplification. Pre-core stop codon mutant detection was carried out on all specimens using allele-specific oligonucleotide hybridization following PCR amplification of the target sequence. The pre-core mutant was detected in 13/33 (39.4%) of HBe positive cases and in 32/48 (66.7%) of HBe negative cases. Sequence analysis was carried out on 8 of the 16 HBe negative specimens that did not carry the pre-core mutant virus to determine the molecular basis for the HBe minus phenotype in these cases: the 1762/1764 TA paired mutation in the second AT rich region of the core promoter was detected in five cases; a start codon mutation was detected in one case. The predominant mutation resulting in the HBe minus phenotype in our isolates was the 1896A pre-core ("pre-core stop codon") mutation; other mutations responsible for the phenotype included the core promoter paired mutation and pre-core start codon mutation. In view of the high frequency of the pre-core mutant virus, sequence analysis was performed to determine the virus genotype on the basis of the nucleotide sequence of codon 15. The sequences of 21 wild type virus (14 HBe positive and 7 HBe negative cases) were examined: 15 were found to be codon 15 CCT variants (71.4%); the frequency in the HBe positive group was 12/14 (85.7%), while that in the HBe negative group was 3/7 (42.9%). The high frequency of the codon 15 CCT variant in association with the frequent occurrence of the pre-core mutant in our isolates concurs with the results of other studies.

Matched MeSH terms: DNA, Viral/genetics
Fulltext Simple and cost-effective restriction endonuclease analysis of human adenoviruses

Adhikary AK, Hanaoka N, Fujimoto T

Biomed Res Int, 2014;2014:363790.
PMID: 24734232 DOI: 10.1155/2014/363790

Restriction endonuclease analyses (REAs) constitute the only inexpensive molecular approach capable of typing and characterizing human adenovirus (HAdV) strains based on the entire genome. However, the application of this method is limited by the need for time-consuming and labor-intensive procedures. We herein developed a simple and cost-effective REA for assessing HAdV. The method consists of (1) simple and cost-effective DNA extraction, (2) fast restriction endonuclease (RE) digestion, and (3) speedy mini agarose gel electrophoresis. In this study, DNA was isolated according to the kit-based method and 21.0 to 28.0 μg of viral DNA was extracted from prototypes (HAdV-1, HAdV-3, HAdV-4, and HAdV-37) in each flask. The amount of DNA ranged from 11.4 to 57.0 μg among the HAdV-3 (n=73) isolates. The obtained viral DNA was found to be applicable to more than 10 types of REAs. Fast-cut restriction endonucleases (REs) were able to digest the DNA within 15 minutes, and restriction fragments were easily separated via horizontal mini agarose gel electrophoresis. The whole procedure for 10 samples can be completed within approximately six hours (the conventional method requires at least two days). These results show that our REA is potentially applicable in many laboratories in which HAdVs are isolated.

Matched MeSH terms: DNA, Viral/chemistry
Fulltext Comparison of PCR-based genotyping methods for hepatitis B virus

Lim CK, Tan JT, Ravichandran A, Chan YC, Ton SH

Malays J Pathol, 2007 Dec;29(2):79-90.
PMID: 19108399 MyJurnal

Hepatitis B virus (HBV) is classified into eight genotypes (A to H). In this study, three genotyping methods were compared for their sensitivity and accuracy, namely PCR-RFLP on the S region, PCR-RFLP on the pre-S region and nested PCR with type specific primers. Sixty HBV samples from infected sera were genotyped. The nested PCR with type specific primers was found to be the most sensitive and produced substantial numbers of co-infections by genotypes B and C. The sensitivities for both PCR-RFLP methods were lower and did not reveal co-infections. Generally, the three methods produced consistent genotyping results in samples infected by single genotypes but for co-infections by genotypes B and C, the two PCR-RFLP methods yielded only single genotypic results. For the cases of single genotypic infections, genotypes ascertained by sequencing were in concordance across all three methods. However, when co-infections occurred, PCR analysis on clones revealed only single genotypic infections.

Matched MeSH terms: DNA, Viral/analysis
Fulltext Dermatological features of an imported case of chikungunya in an infant

Lee CY, Ng LC, Koh TH

Singapore Med J, 2008 Nov;49(11):959-60.
PMID: 19037568
Matched MeSH terms: DNA, Viral/metabolism
Nuclear targeting of viral and non-viral DNA

Chowdhury EH

Expert Opin Drug Deliv, 2009 Jul;6(7):697-703.
PMID: 19552613 DOI: 10.1517/17425240903025744

The nuclear envelope presents a major barrier to transgene delivery and expression using a non-viral vector. Virus is capable of overcoming the barrier to deliver their genetic materials efficiently into the nucleus by virtue of the specialized protein components with the unique amino acid sequences recognizing cellular nuclear transport machinery. However, considering the safety issues in the clinical gene therapy for treating critical human diseases, non-viral systems are highly promising compared with their viral counterparts. This review summarizes the progress on exploring the nuclear traffic mechanisms for the prominent viral vectors and the technological innovations for the nuclear delivery of non-viral DNA by mimicking those natural processes evolved for the viruses as well as for many cellular proteins.

Matched MeSH terms: DNA, Viral/administration & dosage*
Multiple facial seborrheic keratosis-like lesions in a young woman with epidermodysplasia verruciformis

Foong HB, Ibrahimi OA, Elpern DJ, Tyring S, Rady P, Carlson JA

Int J Dermatol, 2008 May;47(5):476-8.
PMID: 18412865 DOI: 10.1111/j.1365-4632.2008.03559.x
Matched MeSH terms: DNA, Viral/analysis
Fulltext Real-time PCR detection of male-specific coliphages

Yong SF, Ngeow YF, Tong YK, Ong JT

Malays J Pathol, 2006 Dec;28(2):79-82.
PMID: 18376795 MyJurnal

Male-specific coliphages are often used as indicators of contamination by enteric viruses. These phages can be detected in water samples by plaque assays and by polymerase chain reaction. In this study, the M13 coliphage was used to develop a real-time PCR assay for the detection of male-specific DNA coliphages. The real-time PCR was found to have a reaction efficiency of 1.45 and detection limit of 10(-3) plaque forming units per reaction mix. Repeated amplification and melting curve analyses demonstrated high specificity and reproducibility of the real-time assay. Quantitative detection with the real-time PCR should allow rapid assessment of the level of viral contamination in water.

Matched MeSH terms: DNA, Viral/analysis*
Fulltext Chikungunya virus infection

Sam IC, Sulaiman AB

Med J Malaysia, 2006 Jun;61(2):264-9.
PMID: 16898330 MyJurnal

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus which causes epidemic fever, rash and polyarthralgia in Africa and Asia. Two outbreaks have been reported in Malaysia, in Klang, Selangor (1998) and Bagan Panchor, Perak (2006). It is not known if the outbreaks were caused by the recent introduction of CHIKV, or if the virus was already circulating in Malaysia. Seroprevalence studies from the 1960s suggested previous disease activity in certain parts of the country. In Asia, CHIKV is thought to be transmitted by the same mosquitoes as dengue, Aedes aegypti and Ae. albopictus. Due to similarities in clinical presentation with dengue, limited awareness, and a lack of laboratory diagnostic capability, CHIKV is probably often underdiagnosed or misdiagnosed as dengue. Treatment is supportive. The prognosis is generally good, although some patients experience chronic arthritis. With no vaccine or antiviral available, prevention and control depends on surveillance, early identification of outbreaks, and vector control. CHIKV should be borne in mind in sporadic cases, and in patients epidemiologically linked to ongoing local or international outbreaks or endemic areas.

Matched MeSH terms: DNA, Viral/analysis
Fulltext Re-emergence of Chikungunya virus in Malaysia

Kumarasamy V, Prathapa S, Zuridah H, Chem YK, Norizah I, Chua KB

Med J Malaysia, 2006 Jun;61(2):221-5.
PMID: 16898316 MyJurnal

An outbreak of Chikugunya (CHIK) fever occurred among the fishing community in Bagan Pancor, Perak. The outbreak was laboratory confirmed within 48 hours after the receipt of the specimens. Fifty-three patients' serum samples were submitted for laboratory investigation and 47 (88.7%) were confirmed to be positive for CHIK infection by RT-PCR, and/or virus isolation, and/or in-house immunoflourescent test. RT-PCR and virus isolation were the tests of choice for patients with illness of four days or less and detection of CHIK specific IgM for those with more than four days of fever. The nucleic acid sequence based on the 354- and 294-bp of the nsP1 and E1 genes of the CHIK virus detected from pools of adults Aedes aegypti mosquitoes were identical to those CHIKV virus isolated from humans in the same locality. Phylogenetic analysis of the CHIK virus based on the 257 nts partial E1 gene indicates that Bagan Panchor's strain was closely related to the first CHIK virus isolated during the outbreak in Klang in 1998.

Matched MeSH terms: DNA, Viral/analysis
Fulltext Duplex real-time PCR for detection and quantification of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon

Tang KF, Lightner DV

Dis Aquat Organ, 2011 Feb 22;93(3):191-8.
PMID: 21516971 DOI: 10.3354/dao02293

We describe a duplex real-time PCR assay using TaqMan probes for the simultaneous detection of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV). Both MBV and HPV are shrimp enteric viruses that infect intestinal and hepatopancreatic epithelial cells. Both viruses can cause significant mortalities and depressed growth in infected larval, postlarval, and early juvenile stages of shrimp, and thus present a risk to commercial aquaculture. In this duplex assay, we combined 2 single real-time PCRs, amplifying MBV and HPV, in a one-tube PCR reaction. The 2 viruses were distinguished by specific fluorescent labels at the 5' end of TaqMan probes: the MBV probe was labeled with dichlorodimethoxyfluorescein (JOE), and the HPV probe was labeled with 6-carboxyfluorescein (FAM). The duplex real-time PCR assay was performed in a multi-channel real-time PCR detection system, and MBV and HPV amplification signals were separately detected by the JOE and FAM channels. This duplex assay was validated to be specific to the target viruses and found to have a detection limit of single copies for each virus. The dynamic range was found to be from 1 to 1 x 10(8) copies per reaction. This assay was further applied to quantify MBV and HPV in samples of infected Penaeus monodon collected from Malaysia, Indonesia, and Thailand. The specificity and sensitivity of this duplex real-time PCR assay offer a valuable tool for routine diagnosis and quantification of MBV and HPV from both wild and farmed shrimp stocks.

Matched MeSH terms: DNA, Viral/genetics
In situ hybridization: detecting viral nucleic acid in formalin-fixed, paraffin-embedded tissue samples

Mabruk MJ

Expert Rev Mol Diagn, 2004 Sep;4(5):653-61.
PMID: 15347259

In situ hybridization is a method for detecting specific nucleic acid sequences within individual cells. This technique permits visualization of viral nucleic acid or gene expression in individual cells within their histologic context. In situ hybridization is based on the complementary binding of a labeled nucleic acid probe to complementary sequences in cells or tissue sections, followed by visualization of target sequences within the cells. It has been used widely for the detection of viral nucleic acid sequences within individual cells. This review will define the technical approaches of in situ hybridization and its current application to detect viral nucleic acids within formalin-fixed, paraffin-embedded tissue samples, with special reference to the Epstein-Barr virus.

Matched MeSH terms: DNA, Viral/analysis*
Nucleotide sequence of precore region of hepatitis B virus DNA in HBsAg-positive carriers in Malaysia

Ton SH, Iskandar K, Noriah R, Thanaletchimy N

Scand. J. Infect. Dis., 1996;28(6):543-8.
PMID: 9060053

As most published studies on precore mutants have been carried out on isolates from patients with liver diseases, and it is unclear whether HBsAg carriers with viraemia in the absence of HBeAg are also generally infected by such mutants, it was decided to sequence the precore region in some HBV-DNA isolated from HBsAg-positive carriers. Precore sequences of HBV-DNA from 43 HBsAg carriers in Malaysia were studied. Three HBV subtypes were identified according to the nucleotide sequence of the precore region. Most of the carriers were found to be infected by the subtype adr. Mutations were detected in the precore regions. The most common conserved mutation was a silent mutation involving conversion from T to C (CCT to CCC) at position 1858 at codon 15 (proline). It was found that 4/43 (9.3%) had a mutation at the penultimate codon where TGG was changed to TAG. All 4 isolates with the TAG mutation had nt T at position 1858. Of the 4 carriers who were infected by these mutant viruses, 2 were coinfected with the wild type, 1 was infected only by a variant with the mutation at position 1896, while another was infected by a variant with mutations at positions 1896 and 1899. Three of the 4 were anti-HBe positive while 1 was HBeAg positive. Alanine aminotransaminase activities in all 4 carriers were normal. This study therefore demonstrated that variants with stop codons at the penultimate codon could be found in asymptomatic carriers in Malaysia.

Matched MeSH terms: DNA, Viral/genetics*; DNA, Viral/isolation & purification
Fulltext Co-administration of avian influenza virus H5 plasmid DNA with chicken IL-15 and IL-18 enhanced chickens immune responses

Lim KL, Jazayeri SD, Yeap SK, Alitheen NB, Bejo MH, Ideris A, et al.

BMC Vet Res, 2012;8:132.
PMID: 22866758 DOI: 10.1186/1746-6148-8-132

DNA vaccines offer several advantages over conventional vaccines in the development of effective vaccines against avian influenza virus (AIV). However, one of the limitations of the DNA vaccine in poultry is that it induces poor immune responses. In this study, chicken interleukin (IL) -15 and IL-18 were used as genetic adjuvants to improve the immune responses induced from the H5 DNA vaccination in chickens. The immunogenicity of the recombinant plasmid DNA was analyzed based on the antibody production, T cell responses and cytokine production, following inoculation in 1-day-old (Trial 1) and 14-day-old (Trial 2) specific-pathogen-free chickens. Hence, the purpose of the present study was to explore the role of chicken IL-15 and IL-18 as adjuvants following the vaccination of chickens with the H5 DNA vaccine.

Matched MeSH terms: DNA, Viral/genetics; DNA, Viral/immunology*
Fulltext Identification of a novel Getah virus by Virus-Discovery-cDNA random amplified polymorphic DNA (RAPD)

Hu T, Zheng Y, Zhang Y, Li G, Qiu W, Yu J, et al.

BMC Microbiol, 2012;12:305.
PMID: 23268691 DOI: 10.1186/1471-2180-12-305

The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA--random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method.

Matched MeSH terms: DNA, Viral/genetics; DNA, Viral/chemistry
Combined use of wild-type HBV precore and high serum iron marker as a potential tool for the prediction of cirrhosis in chronic hepatitis B infection

Chook JB, Ngeow YF, Yap SF, Tan TC, Mohamed R

J Med Virol, 2011 Apr;83(4):594-601.
PMID: 21328372 DOI: 10.1002/jmv.22016

Hepatitis B virus (HBV) and high liver iron deposits have both been associated with the development of cirrhosis. Among HBV factors, genotype and mutations in the basal core promoter (BCP) and precore regions have been most frequently studied but the evidence for a positive association with cirrhosis has been inconsistent. In this study, sera from persons with chronic HBV infection with and without cirrhosis were used for whole HBV genome analysis and for the estimation of serum iron marker (serum iron or ferritin) levels. Single codon analysis showed that the precore wild-type, TGG (nt 1,895-1,897), gave the highest accuracy (77.5%) for the identification of cirrhosis compared to other codons. When TGG was analyzed together with the precore start codon wild-type, ATG (nt 1,814-1,816), the accuracy was improved to 80.0% (odds ratio=35.29; 95% confidence interval=3.87-321.93; Phi=0.629; P<0.001). When the serum iron marker was included for analysis, it was clear that a combination of a precore wild-type and high serum iron marker gave a better accuracy (90.0%) (odds ratio=107.67; 95% confidence interval=10.21-1,135.59; Phi=0.804; P<0.001) for the identification of cirrhosis than either biomarker alone. It appeared that a combined use of both these biomarkers might help to predict the development of cirrhosis in a person with chronic HBV infection, but longitudinal studies are required to test this hypothesis.

Matched MeSH terms: DNA, Viral/genetics; DNA, Viral/chemistry
Fulltext Genetic characterization of porcine circovirus 2 found in Malaysia

Jaganathan S, Toung OP, Yee PL, Yew TD, Yoon CP, Keong LB

Virol J, 2011;8:437.
PMID: 21914166 DOI: 10.1186/1743-422X-8-437

Porcine circovirus type 2 is the primary etiological agent associated with a group of complex multi-factorial diseases classified as Porcine Circovirus Associated Diseases (PCVAD). Sporadic cases reported in Malaysia in 2007 caused major economic losses to the 2.2 billion Malaysian ringgit (MYR) (approximately 0.7 billion US dollar) swine industry. The objective of the present study was to determine the association between the presence of PCV2 and occurrences of PCVAD.

Matched MeSH terms: DNA, Viral/genetics; DNA, Viral/chemistry
Fulltext Detection of human herpesvirus 6 (HHV-6) in saliva of healthy adults in Malaysia

Choo, H.L., Shoji, Y., Leong, C.O.

Malaysian Journal of Medicine and Health Sciences, 2012;8(2):55-64.
MyJurnal

Human herpesvirus-6 (HHV-6) levels have been considered as markers for various diseases. The aim of this study was to evaluate the prevalence of HHV-6 infection in healthy adults in Malaysia. Methods: The level of HHV-6 in saliva was investigated in 36 healthy adults, age 19 to 23 years, at Kuala Lumpur, Malaysia using variant-specific TaqmanTM quantitative real-time PCR (qPCR). Results: The amount of HHV-6 DNA in the saliva of healthy adults ranged from negative to 10,000 HHV-6 genomes/ml of saliva (median, 360 genomes/ml of saliva). Of the 36 samples tested, 30 (83%) contained HHV-6 DNA. HHV-6B was the only variant detected in the saliva of all the positive cases. Conclusions: The detection of HHV-6 DNA in saliva by real-time PCR assay provides a sensitive and specific quantitation of HHV-6. Our pilot study suggests the wide prevalence of HHV-6 in saliva from
healthy adults.

Matched MeSH terms: DNA, Viral
Acceptability of Women Self-Sampling versus Clinician-Collected Samples for HPV DNA Testing: A Systematic Review

Morgan K, Azzani M, Khaing SL, Wong YL, Su TT

J Low Genit Tract Dis, 2019 Jul;23(3):193-199.
PMID: 30933030 DOI: 10.1097/LGT.0000000000000476

OBJECTIVES: Female self-sampling for human papillomavirus (HPV) DNA testing is an alternative screening method that can potentially increase cervical cancer screening coverage. This review addresses the acceptability of HPV DNA testing using self-sampling compared with conventional clinician-collected sampling. Barriers to and others factors associated with acceptability of either method were also examined.
METHODS: The following electronic resources were searched: Medline @EBSCOHOST(Medline), Embase, PubMed, and CINAHL databases. Manual searches were also conducted. The main outcome of interest was the acceptability of HPV DNA testing by self-sampling in comparison with clinician-collected sampling.
RESULTS: In total, 23 articles were included in this systematic review. The majority (19 studies) were quantitative intervention studies and 4 studies were qualitative observational studies. Eleven studies reported a preference for self-sampling by women compared with clinician-collected sampling (64.7%-93%). The remaining studies found that women preferred clinician-collected sampling because mainly of respondents' lack of confidence in their ability to complete self-sampling correctly. In most articles reviewed, the studied associated factors, such as demographic factors (age, marital status, and ethnicity), socioeconomic factors (income, education level), reproductive factors (condom use, number of children, current use of contraception, and number of partners), and habits (smoking status) were not found to be significantly associated with preference.
CONCLUSIONS: Both methods of sampling were found to be acceptable to women. Self-sampling is cost-effective and could increase the screening coverage among underscreened populations. However, more information about the quality, reliability, and accuracy of self-sampling is needed to increase women's confidence about using to this method.

Matched MeSH terms: DNA, Viral/isolation & purification*
Fulltext First molecular detection and complete sequence analysis of porcine circovirus type 3 (PCV3) in Peninsular Malaysia

Tan CY, Opaskornkul K, Thanawongnuwech R, Arshad SS, Hassan L, Ooi PT

PLoS One, 2020;15(7):e0235832.
PMID: 32706778 DOI: 10.1371/journal.pone.0235832

Porcine circovirus type 3 (PCV3) is a newly emerging virus in the swine industry, first reported recently in 2016. PCV3 assembles into a 2000 bp circular genome; slightly larger than PCV1 (1758-1760 bp), PCV2 (1766-1769 bp) and PCV4 (1770 bp). Apart from being associated with porcine dermatitis and nephropathy syndrome (PDNS), PCV3 has been isolated from pigs with clinical signs of reproductive failures, myocarditis, porcine respiratory disease complex (PRDC) and neurologic disease. Given that PCV3 is increasingly reported in countries including Thailand and U.S. with whom Malaysia shares trade and geographical relationship; and that PCV3 is associated with several clinical presentations that affect productivity, there is a need to study the presence and molecular characteristics of PCV3 in Malaysian swine farms. Twenty-four commercial swine farms, three abattoirs and retail shops in Peninsular Malaysia were sampled using convenience sampling method. A total of 281 samples from 141 pigs, including 49 lung archive samples were tested for PCV3 by conventional PCR. Twenty-eight lung samples from wild boar population in Peninsular Malaysia were also included. Nucleotide sequences were analyzed for maximum likelihood phylogeny relationship and pairwise distances. Results revealed that PCV3 is present in Peninsular Malaysia at a molecular prevalence of 17.02%, with inguinal lymph nodes and lungs showing the highest molecular detection rates of 81.82% and 71.43% respectively. Despite wide reports of PCV3 in healthy animals and wild boars, no positive samples were detected in clinically healthy finishers and wild boar population of this study. PCV3 strain A1 and A2 were present in Malaysia, and Malaysian PCV3 strains were found to be phylogenetically related to Spanish, U.S. and Mexico strains.

Matched MeSH terms: DNA, Viral/genetics

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