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.
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.
Dengue viruses pose a considerable global public health problem with an estimated 100 million cases of illness every year. This illustrates the need for rapid and reliable diagnostic methods for proper patient management and disease control. Currently, laboratory diagnosis depends on serology or virus isolation, with both methods having certain drawbacks. Alternatively, reverse transcription and polymerase chain reaction (RT-PCR) offers the potential for the rapid, highly sensitive and specific detection of dengue viruses. Since we occasionally encounter the problem of insufficient amounts of patient serum for the direct detection of dengue viruses, a method was developed for the extraction of viral RNA after biological amplification in mosquito larvae. Using this method, 15 of 19 clinical samples tested were correctly identified using RT-PCR.
In recent years, a large amount of nucleotide sequence data for dengue viruses has been published. Most of it was derived by sequencing cDNA synthesized from highly purified genomic viral RNA. This paper presents a simple and rapid method for the isolation of total RNA from mosquito cells infected with dengue viruses. This RNA can be used for direct nucleotide sequencing with specific primers without the need for further purification.
Forty-six strains of Japanese encephalitis (JE) virus from a variety of geographic areas in Asia were examined by primer-extension sequencing of the RNA template. A 240 nucleotide sequence from the pre-M gene region was selected for study because it provided sufficient information for determining genetic relationships among the virus isolates. Using 12% divergence as a cutoff point for virus relationships, the 46 isolates fell into three distinct genotypic groups. One genotypic group consisted of JE virus isolates from northern Thailand and Cambodia. A second group was composed of isolates from southern Thailand, Malaysia, Sarawak and Indonesia. The remainder of the isolates, from Japan, China, Taiwan, the Philippines, Sri Lanka, India and Nepal, made up a third group. The implications of these findings in relation to the epidemiology of JE are discussed. Results of this study demonstrate that the comparison of short nucleotide sequences can provide insight into JE virus evolution, transmission and, possibly, pathogenesis.
Malaysia had 10,219 confirmed cases of COVID-19 as of September 20, 2020. About 33% were associated with a Tablighi Jamaat religious mass gathering held in Kuala Lumpur between February 27 and March 3, 2020, which drove community transmission during Malaysia's second wave. We analysed genome sequences of SARS-CoV-2 from Malaysia to better understand the molecular epidemiology and spread. We obtained 58 SARS-CoV-2 whole genome sequences from patients in Kuala Lumpur and performed phylogenetic analyses on these and a further 57 Malaysian sequences available in the GISAID database. Nine different SARS-CoV-2 lineages (A, B, B.1, B.1.1, B.1.1.1, B.1.36, B.2, B.3 and B.6) were detected in Malaysia. The B.6 lineage was first reported a week after the Tablighi mass gathering and became predominant (65.2%) despite being relatively rare (1.4%) globally. Direct epidemiological links between lineage B.6 viruses and the mass gathering were identified. Increases in reported total cases, Tablighi-associated cases, and community-acquired B.6 lineage strains were temporally linked. Non-B.6 lineages were mainly travel-associated and showed limited onward transmission. There were also temporally correlated increases in B.6 sequences in other Southeast Asian countries, India and Australia, linked to participants returning from this event. Over 95% of global B.6 sequences originated from Asia Pacific. We also report a nsp3-C6310A substitution found in 47.3% of global B.6 sequences which was associated with reduced sensitivity using a commercial diagnostic real-time PCR assay. Lineage B.6 became the predominant cause of community transmission in Malaysia after likely introduction during a religious mass gathering. This event also contributed to spikes of lineage B.6 in other countries in the Asia-Pacific. Mass gatherings can be significant causes of local and global spread of COVID-19. Shared genomic surveillance can be used to identify SARS-CoV-2 transmission chains to aid prevention and control, and to monitor diagnostic molecular assays. Clinical Trial Registration: COVID-19 paper.
In mid-January 2000, the reappearance of Japanese encephalitis (JE) virus activity in the Australasian region was first demonstrated by the isolation of JE virus from 3 sentinel pigs on Badu Island in the Torres Strait. Further evidence of JE virus activity was revealed through the isolation of JE virus from Culex gelidus mosquitoes collected on Badu Island and the detection of specific JE virus neutralizing antibodies in 3 pigs from Saint Pauls community on Moa Island. Nucleotide sequencing and phylogenetic analyses of the premembrane and envelope genes were performed which showed that both the pig and mosquito JE virus isolates (TS00 and TS4152, respectively) clustered in genotype I, along with northern Thai, Cambodian, and Korean isolates. All previous Australasian JE virus isolates belong to genotype II, along with Malaysian and Indonesian isolates. Therefore, for the first time, the appearance and transmission of a second genotype of JE virus in the Australasian region has been demonstrated.
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.
The coding region of the nucleocapsid (N) gene was amplified from the viral RNA and inserted into the bacterial expression vector, pTrcHis2, for intracellular expression in three Escherichia coli strains: TOP 10, BL 21 and SG 935. The N protein was expressed as a fusion protein containing the myc epitope and His-tag at its C-terminal end. The amount of the fusion protein expressed in strain SG 935 was significantly higher than the other two strains, and was detected by the anti-myc antibody, anti-His and swine anti-NiV serum. Hence, the N(fus) protein produced in E. coli could serve as an alternative antigen for the detection of anti-NiV in swine.
The VP4 genetic groups of 151 field strains of human rotaviruses obtained from infants and young children with diarrhea from four locations in Malaysia were analyzed. The strains were adapted to growth in tissue culture and studied further by molecular hybridization of northern blotted RNA to PCR-generated cDNA probes representing amino acids 84-180 of the KU strain VP4, 83-181 of the DS-1 strain VP4, and 83-180 of either the 1076 or K8 strain VP4, representing VP4 genetic groups 1-4 (P1A, P1B, P2, and P3), respectively. The majority (79% of the field strains hybridized with the KU VP4 genetic group 1 probe and were associated with G1, G3, G4, untypable, or mixed G serotypes. VP4 genetic group 1 (P1A) strains were the most common in all locations in Malaysia between 1978-1988. Three strains which exhibited G3 and subgroup I specificity hybridized with the K8 VP4 genetic group 4 probe. These three VP4 genetic group 4 (P3) strains were detected in two different years and locations, extending the initial detection of this VP4 genetic group (the K8 strain) in Japan to a larger geographical area of Asia.
Hepatitis E virus (HEV) has been frequently detected from pork liver and liver products, which can usually cause self-limiting diseases in healthy adults, yet may result in fatality in immunosuppressed groups. Nevertheless, there is so far no standardized method for HEV detection available from pork liver and/or liver products. The present study aimed to optimize the virus extraction method of HEV from raw pork liver, which is often consumed in Asia undercooked to avoid a grainy texture. By comparing different sample preparation protocols and by applying the selected protocol to 60 samples collected from Singapore retail markets, we demonstrated that homogenization of 0.25 g raw pork liver with FastPrep™ Lysing Matrix Y containing yttria-stabilized zircondium oxide beads in 2 ml tubes and with harsh mechanical force at 6 ms-1, 40 s/cycle, for 5 cycles with 300 s pause time after each cycle is promising in both releasing the potentially intracellular viruses and resulting in satisfactory virus recovery rates (> 1%). A high prevalence (52%) of HEV genome was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) from the 60 samples collected from Singapore retail markets imported from Indonesia, Australia and Malaysia. However, RNase treatment decreased the HEV prevalence to 33.3%, and all of the 20 positive samples were with high RT-qPCR Ct values above 35, suggesting that the positive RT-qPCR signals maybe largely due to the inactive viruses and/or exposed HEV RNA traces in raw pork liver products. Therefore, conscious care should be taken when interpreting molecular detection results of viruses from food samples to be correlated with public health risks.
An efficient electrochemical impedance genosensing platform has been constructed based on graphene/zinc oxide nanocomposite produced via a facile and green approach. Highly pristine graphene was synthesised from graphite through liquid phase sonication and then mixed with zinc acetate hexahydrate for the synthesis of graphene/zinc oxide nanocomposite by solvothermal growth. The as-synthesised graphene/zinc oxide nanocomposite was characterised with scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy and X-ray diffractometry (XRD) to evaluate its morphology, crystallinity, composition and purity. An amino-modified single stranded DNA oligonucleotide probe synthesised based on complementary Coconut Cadang-Cadang Viroid (CCCVd) RNA sequence, was covalently bonded onto the surface of graphene/zinc oxide nanocomposite by the bio-linker 1-pyrenebutyric acid N-hydroxysuccinimide ester. The hybridisation events were monitored by electrochemical impedance spectroscopy (EIS). Under optimised sensing conditions, the single stranded CCCVd RNA oligonucleotide target could be quantified in a wide range of 1.0×10-11M to 1.0×10-6 with good linearity (R =0.9927), high sensitivity with low detection limit of 4.3×10-12M. Differential pulse voltammetry (DPV) was also performed for the estimation of nucleic acid density on the graphene/zinc oxide nanocomposite-modified sensing platform. The current work demonstrates an important advancement towards the development of a sensitive detection assay for various diseases involving RNA agents such as CCCVd in the future.
The public health burden of dengue is most likely under reported. Current dengue control measures only considered symptomatic dengue transmission. Hence, there is a paucity of information on the epidemiology of inapparent dengue. This study reports that many people have been unknowingly exposed to dengue infection. Almost 10% and 70% of individuals without any history of dengue infection and living in a dengue hotspot, in Selangor, Malaysia, were dengue IgM and IgG positive respectively. When dengue-positive mosquitoes were detected in the hotspot, 11 (6.3%) of the 174 individuals tested were found to have dengue viremia, of which 10 were asymptomatic. Besides, upon detection of a dengue-infected mosquito, transmission was already widespread. In a clinical setting, it appears that people living with dengue patients have been exposed to dengue, whether asymptomatic or symptomatic. They can either have circulating viral RNA and/or presence of NS1 antigen. It is also possible that they are dengue seropositive. Collectively, the results indicate that actions taken to control dengue transmission after the first report of dengue cases may be already too late. The current study also revealed challenges in diagnosing clinically inapparent dengue in hyperendemic settings. There is no one best method for diagnosing inapparent dengue. This study demonstrates empirical evidence of inapparent dengue in different settings. Early dengue surveillance in the mosquito population and active serological/virological surveillance in humans can go hand in hand. More studies are required to investigate the epidemiology, seroprevalence, diagnostics, and control of inapparent dengue. It is also crucial to educate the public, health staff and medical professionals on asymptomatic dengue and to propagate awareness, which is important for controlling transmission.
Rice tungro disease (RTD) is one of the most destructive diseases of rice in South and Southeast Asia. RTD is routinely detected based on visual observation of the plant. However, it is not always easy to identify the disease in the field as it is often confused with other diseases or physiological disorders. Here we report the development of two serological based assays for ease of detection of RTD. In this study we had developed and optimized an indirect ELISA and dot-blot assay for detection of RTD. The efficiency of both assays was evaluated by comparing the specificity and sensitivity of the assays to PCR assay using established primer sets. The indirect ELISA showed 97.5% and 96.6%, while the dot-blot assay showed 97.5% and 86.4% sensitivity and specificity, respectively, when compared to established PCR method. The high sensitivity and specificity of the two assays merit the use of both assays as alternative methods to diagnose RTD. Furthermore, the dot-blot assay is a simple, robust, and rapid diagnostic assay that is suitable for field test for it does not require any specialized equipment. This is a great advantage for diagnosing RTD in paddy fields, especially in the rural areas.
Long-COVID is a postviral illness that can affect survivors of COVID-19, regardless of initial disease severity or age. Symptoms of long-COVID include fatigue, dyspnea, gastrointestinal and cardiac problems, cognitive impairments, myalgia, and others. While the possible causes of long-COVID include long-term tissue damage, viral persistence, and chronic inflammation, the review proposes, perhaps for the first time, that persistent brainstem dysfunction may also be involved. This hypothesis can be split into two parts. The first is the brainstem tropism and damage in COVID-19. As the brainstem has a relatively high expression of ACE2 receptor compared with other brain regions, SARS-CoV-2 may exhibit tropism therein. Evidence also exists that neuropilin-1, a co-receptor of SARS-CoV-2, may be expressed in the brainstem. Indeed, autopsy studies have found SARS-CoV-2 RNA and proteins in the brainstem. The brainstem is also highly prone to damage from pathological immune or vascular activation, which has also been observed in autopsy of COVID-19 cases. The second part concerns functions of the brainstem that overlap with symptoms of long-COVID. The brainstem contains numerous distinct nuclei and subparts that regulate the respiratory, cardiovascular, gastrointestinal, and neurological processes, which can be linked to long-COVID. As neurons do not readily regenerate, brainstem dysfunction may be long-lasting and, thus, is long-COVID. Indeed, brainstem dysfunction has been implicated in other similar disorders, such as chronic pain and migraine and myalgic encephalomyelitis or chronic fatigue syndrome.
Immunomagnetic beads-PCR (IM-PCR), positively-charged virosorb filters (F), or a combination of both methods (F-IM-PCR) were used to capture, concentrate and rapidly detect hepatitis A virus (HAV) in samples of lettuce and strawberries experimentally contaminated. Direct reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of the collected HAV-beads complex showed a detection limit of 0.5 plaque forming units (PFU) of the virus present in 1-ml of wash solution from the produce, which was several hundred-fold more sensitive than that demonstrated by RT-PCR. In separate trials, virus-containing wash solutions from the produce were passed through the filters and the captured virus was eluted with 10 ml volumes of 1% beef extract. Of the 62% filter-captured HAV, an average of 34.8% was eluted by the 1% beef extract. PCR amplification of 2 microl from this eluate failed to produce a clear positive band signal. As little as 10 PFU, present on each piece of the lettuce or strawberry, was detectable by the F-IM-PCR, which was almost 20 times less sensitive than the detection limit of 0.5 PFU by the IM-PCR. However, considering the large volumes (< or =50 ml) used in the F-IM-PCR, the sensitivity of detection could be much greater than that of the IM-PCR, which was restricted to < or =20 ml volumes. These data indicate that the F-IM-PCR method provides the potential for a greater sensitivity of detection than the IM-PCR, since low levels of virus could be detected from large volumes of sample than possible by the IM-PCR method. Although positively-charged filters captured a greater amount of virus than both the IM-PCR and F-IM-PCR methods, direct PCR amplification from beef extract eluates was not successful in detecting HAV from produce.
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.
Foot-and-mouth disease (FMD) is a major cause of endemic outbreaks in Vietnam in recent years. In this work, six serotype A foot-and-mouth disease viruses (FMDV), collected from endemic outbreaks during January and February of 2009 in four different provinces in Vietnam, were genetically characterized for their complete genome sequences. Genetic analysis based on the complete viral genome sequence indicated that they were closely related to each other and shared 99.0-99.8% amino acid (aa) identity. Genetic and deduced aa analysis of the capsid coding gene VP1 showed that the six Vietnamese strains were all classified into the genotype IX from a total of 10 major genotypes worldwide, sharing 98.1-100% aa identity each other. They were most closely related to the type A strains recently isolated in Laos (A/LAO/36/2003, A/LAO/1/2006, A/LAO/6/2006, A/LAO/7/2006, and A/LAO/8/2006), Thailand (A/TAI/2/1997 and A/TAI/118/1987), and Malaysia (A/MAY/2/2002), sharing 88.3-95.5% nucleotide (nt) identities. In contrast, Vietnamese type A strains showed low nt identities with the two old type A FMDVs, isolated in 1960 in Thailand (a15thailand iso43) and in 1975 in the Philippines (aphilippines iso50), ranging from 77.3 to 80.9% nt identity. A multiple alignment based on the deduced amino acid sequences of the capsid VP1 coding gene of type A FMDV revealed three amino acid substitutions between Vietnamese strains and the strains of other Southeast Asian countries (Laos, Thailand, Malaysia, and the Philippines). Alanine was replaced by valine at residue 24, asparagine by arginine at residue 85, and serine by threonine at residue 196. Furthermore, type A FMDV strains recently isolated in Vietnam, Laos, Thailand, and Malaysia all have one amino acid deletion at residue 140 of the capsid VP1 protein compared with the two old type A FMDV strains from Thailand and the Philippines as well as most other type A representatives worldwide. This article is the first to report on the comprehensive genetic characterization of type A FMDV circulating in Vietnam.