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.
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.
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.
Centrifugal microfluidic systems utilize a conventional spindle motor to automate parallel biochemical assays on a single microfluidic disk. The integration of complex, sequential microfluidic procedures on these platforms relies on robust valving techniques that allow for the precise control and manipulation of fluid flow. The ability of valves to consistently return to their former conditions after each actuation plays a significant role in the real-time manipulation of fluidic operations. In this paper, we introduce an active valving technique that operates based on the deflection of a latex film with the potential for real-time flow manipulation in a wide range of operational spinning speeds. The reversible thermo-pneumatic valve (RTPV) seals or reopens an inlet when a trapped air volume is heated or cooled, respectively. The RTPV is a gas-impermeable valve composed of an air chamber enclosed by a latex membrane and a specially designed liquid transition chamber that enables the efficient usage of the applied thermal energy. Inputting thermo-pneumatic (TP) energy into the air chamber deflects the membrane into the liquid transition chamber against an inlet, sealing it and thus preventing fluid flow. From this point, a centrifugal pressure higher than the induced TP pressure in the air chamber reopens the fluid pathway. The behaviour of this newly introduced reversible valving system on a microfluidic disk is studied experimentally and theoretically over a range of rotational frequencies from 700 RPM to 2500 RPM. Furthermore, adding a physical component (e.g., a hemispherical rubber element) to induce initial flow resistance shifts the operational range of rotational frequencies of the RTPV to more than 6000 RPM. An analytical solution for the cooling of a heated RTPV on a spinning disk is also presented, which highlights the need for the future development of time-programmable RTPVs. Moreover, the reversibility and gas impermeability of the RTPV in the microfluidic networks are validated on a microfluidic disk designed for performing liquid circulation. Finally, an array of RTPVs is integrated into a microfluidic cartridge to enable sequential aliquoting for the conversion of dengue virus RNA to cDNA and the preparation of PCR reaction mixtures.
The aim of this study was to determine if knowledge of both the serum HCV RNA and serum anti core IgM antibody status enabled one to predict the histological severity in chronic hepatitis C. We studied 45 female patients with chronic hepatitis C infection. The presence or absence of IgM antibodies to HCV and HCV RNA by PCR in each patient's serum was determined. Liver biopsies performed were scored according to a modified Desmet's histological activity index. Negative HCV RNA patients had least histological change. HCV RNA positive patients who were also IgM antibody positive had lower scores than their IgM negative counterparts. The grade of histological severity is more accurately predictable from knowledge of both the HCV RNA and IgM anti HCV status of the patient.
A 12-month study was carried out on the molecular epidemiology of rotavirus in urban and suburban Malaysian children. Analysis of faecal samples from 973 hospitalized diarrhoeic children by polyacrylamide gel electrophoresis detected 268 rotaviruses (28%). All isolates were group A rotaviruses, which produced 22 electropherotypes: 16 (91.5%) with long RNA migration patterns and 6 (8.5%) with short patterns. One of the long-pattern electropherotypes was the predominant strain (71.1% of the total electropherotypes) isolated during this study. Although 3 other strains were detected sporadically over the study period, 16 others were present only during the first 7 months and 2 others were confined to the last 5 months. Long- and short-pattern electropherotypes were found to co-circulate extensively. There was a significant association of short-pattern electropherotypes with infection in older children. In addition, the prevalence of vomiting and mean duration of diarrhoea were significantly associated with different electropherotypes.
Dengue viral antigens have been demonstrated in several types of naturally infected human tissues, but little is known of whether these same tissues have detectable viral RNA. We studied tissue specimens from patients with serologically or virologically confirmed dengue infections by immunohistochemistry (IHC) and in situ hybridization (ISH), to localize viral antigen and RNA, respectively. IHC was performed on specimens obtained from 5 autopsies and 24 biopsies and on 20 blood-clot samples. For ISH, antisense riboprobes to the dengue E gene were applied to tissue specimens in which IHC was positive. Viral antigens were demonstrated in Kupffer and sinusoidal endothelial cells of the liver; macrophages, multinucleated cells, and reactive lymphoid cells in the spleen; macrophages and vascular endothelium in the lung; kidney tubules; and monocytes and lymphocytes in blood-clot samples. Positive-strand viral RNA was detected in the same IHC-positive cells found in the spleen and blood-clot samples. The strong, positive ISH signal in these cells indicated a high copy number of viral RNA, suggesting replication.
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.
An analysis of rotavirus electropherotypes circulating in Kuala Lumpur, Malaysia, over 7 years showed that all except 1 of the 360 electropherotypes encountered were characteristic of group A rotaviruses. The long electropherotype predominated annually, and there was a rarity of short electropherotypes. Extensive genome variability and cocirculation of different electropherotypes were observed annually. A sequential appearance of the predominant electropherotype was observed in all years of the study, except for 1985 and 1988, when one electropherotype predominated throughout the study periods. There was no shift in the predominant electropherotype over a 6-year period.
Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus that can result in severe pulmonary disease and fatal encephalitis in humans and is responsible for outbreaks in Bangladesh, Malaysia, Singapore, India and possibly the Philippines. NiV has a negative-sense RNA genome that contains six genes and serves as a template for production of viral mRNA transcripts. NiV mRNA transcripts are subsequently translated into viral proteins. Traditionally, NiV quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assays have relied on using primer sets that amplify a target (N that encodes the nucleocapsid) within the coding region of the viral gene that also amplifies viral mRNA. Here we describe a novel one-step qRT-PCR assay targeting the intergenic region separating the viral F and G proteins, thereby eliminating amplification of the viral mRNA. This assay is more accurate than the traditional qRT-PCR in quantifying concentrations of viral genomic RNA.
Nipah and Hendra viruses belong to the novel Henipavirus genus of the Paramyxoviridae family. Its zoonotic circulation in bats and recent emergence in Malaysia with fatal consequences for humans that were in close contact with infected pigs, has made the reinforcement of epidemiological and clinical surveillance systems a priority. In this study, TaqMan RT-PCR of the Nipah nucleoprotein has been developed so that Nipah virus RNA in field specimens or laboratory material can be characterized rapidly and specifically and quantitated. The linearity of the standard curve allowed quantification of 10(3) to 10(9) RNA transcripts. The sensitivity of the test was close to 1 pfu. The kinetics of Nipah virus production in Vero cells was monitored by the determination of infectious virus particles in the supernatant fluid and by quantitation of the viral RNA. Approximately, 1000 RNA molecules were detected per virion, suggesting the presence of many non-infectious particles, similar to other RNA viruses. TaqMan real-time RT-PCR failed to detect Hendra virus DNA. Importantly, the method was able to detect virus despite a similar ratio in viremic sera from hamsters infected with Nipah virus. This standardized technique is sensitive and reliable and allows rapid detection and quantitation of Nipah RNA in both field and experimental materials used for the surveillance and specific diagnosis of Nipah virus.
BACKGROUND & OBJECTIVESI: Transovarial transmission of dengue virus in the Aedes vectors is now a well-documented phenomenon reported from many parts of the endemic areas in the world, which played an important role in initiating and maintaining the outbreak in human populations. This study investigated the factors affecting breeding habitats and the relationship with transovarial dengue virus in larvae of Aedes aegypti and Ae. albopictus.
To investigate if two important epidemic viral encephalitis in children, Enterovirus 71 (EV71) encephalomyelitis and Japanese encephalitis (JE) whose clinical and pathological features may be nonspecific and overlapping, could be distinguished.
This retrospective study examined the G/P type of rotavirus in RNA samples that have previously been e-typed by RNA-PAGE in 1996. The results were then compared to 2007 samples to ascertain the extent of changes that may have occurred in this 11-years time interval. The G and P genotypes were determined by hemi-nested PCR and further analysed by phylogenetic study. In 1996, the G/P combination G1P[8], G(UT)P[8] and G1P(UT) prevalence rate were 81%, 9% and 7%, respectively. As expected, the G9 genotype which has already emerged worldwide was identified in 42% of the 2007 samples with the remaining 33% G1P[8] and 25% G1P(UT) Analysis of the RNA pattern showed that majority of the isolates were long e-type in both series, nevertheless minor differences within electropherotypes were observed. Genetic diversity in some strains of the human group A rotaviruses was analysed by phylogenetic methods. These findings will help in the decision to introduce rotavirus vaccines within the next decade.
Nipah virus is a zoonotic virus isolated from an outbreak in Malaysia in 1998. The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero) using the one-step SYBR Green I-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay.
The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV.
The objective of this study was to ascertain the extent changes have occurred in the epidemiology of human rotavirus electropherotypes from the same location 7 to 8 years after an earlier study. Genomic RNA profiles of rotaviruses from diarrhoeic children admitted to the Kuala Lumpur Hospital from April to December 1996 were determined by polyacrylamide gel electrophoresis and silver staining. A total of 179 group A rotaviruses were detected from 870 children: 175 with legible staining of all RNA segments were classified into 14 distinct electropherotypes (10 and 4 with long and short migration patterns respectively). In addition, the results revealed: high predominance of long pattern electropherotypes (94% of the total electropherotypes); most long electropherotypes with RNA profiles which all 11 RNAs migrated separately (8 of 10 electropherotypes); all short electropherotypes had segments 2 and 3 that co-migrated; presence of a very numerically dominant electropherotype (75% of all electropherotypes); frequent co-circulation of the dominant electropherotype-present throughout the study period--with other electropherotypes present for limited periods; sequential temporal appearances by similar electropherotypes. These observations were similar to that of an earlier study conducted in 1988/89. Nevertheless, the dominant electropherotype in the present study was different and not among the electropherotypes detected in the earlier study.
Thirty-four suspected rabid brain samples from 2 humans, 24 dogs, 4 cats, 2 mongooses, I jackal and I water buffalo were collected in 1995-1996 in Sri Lanka. Total RNA was extracted directly from brain suspensions and examined using a one-step reverse transcription-polymerase chain reaction (RT-PCR) for the rabies virus nucleoprotein (N) gene. Twenty-eight samples were found positive for the virus N gene by RT-PCR and also for the virus antigens by fluorescent antibody (FA) test. Rabies virus isolates obtained from different animal species in different regions of Sri Lanka were genetically homogenous. Sequences of 203 nucleotides (nt)-long RT-PCR products obtained from 16 of 27 samples were found identical. Sequences of 1350 nt of N genes of 14 RT-PCR products were determined. The Sri Lanka isolates under study formed a specific cluster that included also an earlier isolate from India but did not include the known isolates from China, Thailand, Malaysia, Israel, Iran, Oman, Saudi Arabia, Russia, Nepal, Philippines, Japan and from several other countries. These results suggest that one type of rabies virus is circulating among human, dog, cat, mongoose, jackal and water buffalo living near Colombo City and in other five remote regions in Sri Lanka.
This report extends the knowledge on the epizootical situation of foot-and-mouth disease in Asia. RNA from six samples of type A and five of type O virus, isolated between 1987 and 1997 in Bangladesh, Iran, Malaysia and Turkey, was subjected to reverse transcription-dependent polymerase chain reactions that amplify large parts of the capsid protein VP1 encoding genome region. The amplification products were sequenced, and the sequences aligned to each other and to published sequences. This showed the type O isolates of 1987-1997 from Bangladesh to be of same genotype and closely related to isolates of 1988 and later from Saudi Arabia, 1990 from India, 1996 from Greece and Bulgaria, and 1997 from Iran. Among the analyzed type A isolates, those of 1992 and 1996 from Turkey were of same genotype and related to previously described isolates of 1987 from Iran and of 1992 from Saudi Arabia. The isolate of 1997 from Malaysia was found to be related to isolates from Thailand of 1993 and 1996. The isolates of 1987 from Bangladesh and 1997 from Iran, however, represent different so far not described genotypes. Monoclonal antibodies, raised against the vaccine production strains A22 Iraq, Asial Shamir, O1 Kaufbeuren and O1 Manisa, and the recent type A field isolates Saudi Arabia/92 and Albania/96, were used in an ELISA to compare the reaction patterns of many of the field isolates. The monoclonal antibodies were further characterized for virus-neutralizing activity and binding to trypsinized homologous virus. The failure of neutralizing antibodies in binding to trypsinized homologous as well as to heterologous virus suggested the epitopes to reside at the major antigenic component of the virus, which is the capsid protein VP1. Two non-neutralizing antibodies that bind to trypsin-sensitive epitopes cross-reacted, however, with heterologous virus. This indicates the existence of a trypsin-sensitive antigenic site outside of VP1. In summary, the results obtained by ELISA confirm the observed sequence differences, but indicate further sequence differences at minor antigenic sites that do not reside on VP1.