Storage of dengue virus (DENV) culture stocks in -80°C is a common laboratory practice to maintain the viability of the virus for long-term usage. However, the efficiency of this method could still be hindered by multiple factors. In our laboratory, we observed a constant and substantial deterioration in the titer of DENV in Vero culture supernatant stored in -80°C. Such incident had badly hampered the laboratory work and prompted an investigation to determine the cause. DENV isolates representing all four serotypes were propagated and the culture supernatants were harvested and stored in aliquots of original stock and 10 fold dilutions (10-1 -10-4). DENV titer in these stocks was determined prior to storage and reassessed on the third and sixth month of storage by focus forming unit assay (FFUA). The result demonstrated a constant preservation of titer ranging from 104 ffu/ml to 105 ffu/ml in the diluted DENV virus culture stocks of 10-1, and 10-2 of DENV1-4, a minor reduction of titer from 103 ffu/ml to 102 ffu/ml at dilution 10-3 for DENV4 only and complete deterioration in undiluted culture stock and lower dilution (10-4) within 6 months of storage in -80°C for all serotypes. It is recommended that propagated DENV in Vero cells are stored in 10 fold dilutions as compared to the original form to preserve the titer for long-term usage.
Dengue virus type 2 (DENV-2) infection induced apoptotic cellular DNA fragmentation in Vero cells within 8 days of infection. The addition of high concentrations of extracellular Zn(2+) but not Ca(2+), Mg(2+) or Mn(2+) to the cell culture medium hastened the detection of apoptosis to within 4 h after infection. No apoptotic cellular DNA fragmentation was detected in the cell culture treated with Zn(2+) alone or infected with heat- or ultraviolet light-inactivated DENV-2 in the presence of Zn(2+). These results suggest that (i) apoptosis is induced in African green monkey kidney cells infected with live DENV-2 and (ii) the addition of high extracellular Zn(2+) accelerates detection of apoptosis in the DENV-2-infected cells.
The number of dengue cases has been increasing on a global level in recent years, and particularly so in Malaysia, yet little is known about the effects of weather for identifying the short-term risk of dengue for the population. The aim of this paper is to estimate the weather effects on dengue disease accounting for non-linear temporal effects in Selangor, Kuala Lumpur and Putrajaya, Malaysia, from 2008 to 2010. We selected the weather parameters with a Poisson generalized additive model, and then assessed the effects of minimum temperature, bi-weekly accumulated rainfall and wind speed on dengue cases using a distributed non-linear lag model while adjusting for trend, day-of-week and week of the year. We found that the relative risk of dengue cases is positively associated with increased minimum temperature at a cumulative percentage change of 11.92% (95% CI: 4.41-32.19), from 25.4 °C to 26.5 °C, with the highest effect delayed by 51 days. Increasing bi-weekly accumulated rainfall had a positively strong effect on dengue cases at a cumulative percentage change of 21.45% (95% CI: 8.96, 51.37), from 215 mm to 302 mm, with the highest effect delayed by 26-28 days. The wind speed is negatively associated with dengue cases. The estimated lagged effects can be adapted in the dengue early warning system to assist in vector control and prevention plan.
We present a compact, cost-effective, label-free, real-time biosensor based on long-range surface plasmon polariton (LRSPP) gold (Au) waveguides for the detection of dengue-specific immunoglobulin M (IgM) antibody, and we demonstrate detection in actual patient blood plasma samples. Two surface functionalization approaches are proposed and demonstrated: a dengue virus serotype 2 (DENV-2) functionalized surface to capture dengue-specific IgM antibody in blood plasma and the reverse, a blood plasma functionalized surface to capture DENV-2. The results obtained via these two surface functionalization approaches are comparable to, or of greater quality, than those collected by conventional IgM antibody capture enzyme linked immunosorbent assay (MAC-ELISA). Our second functionalization approach was found to minimize nonspecific binding, thus improving the sensitivity and accuracy of the test. We also demonstrate reuse of the biosensors by regenerating the sensing surface down to the virus (or antibody) level or down to the bare Au.
Doxycycline is an antibiotic derived from tetracycline that possesses antimicrobial and anti-inflammatory activities. Antiviral activity of doxycycline against dengue virus has been reported previously; however, its anti-dengue properties need further investigation. This study was conducted to determine the potential activity of doxycycline against dengue virus replication in vitro. Doxycycline inhibited the dengue virus serine protease (DENV2 NS2B-NS3pro) with an IC50 value of 52.3 ± 6.2 μM at 37 °C (normal human temperature) and 26.7 ± 5.3 μM at 40 °C (high fever temperature). The antiviral activity of doxycycline was first tested at different concentrations against DENV2 using a plaque-formation assay. The virus titter decreased significantly after applying doxycycline at levels lower than its 50 % cytotoxic concentration (CC50, 100 μM), showing concentration-dependent inhibition with a 50 % effective concentration (EC50) of approximately 50 μM. Doxycycline significantly inhibited viral entry and post-infection replication of the four dengue serotypes, with serotype-specific inhibition (high activity against DENV2 and DENV4 compared to DENV1 and DENV3). Collectively, these findings underline the need for further experimental and clinical studies on doxycycline, utilizing its anti-dengue and anti-inflammatory activities to attenuate the clinical symptoms of dengue virus infection.
This study investigates the effects of 2-phenyl-1-benzopyran-4-one (flavone) on DENV-2 infectivity in Vero cells. Virus adsorption and attachment and intracellular virus replication were investigated using a foci forming unit assay (FFUA) and quantitative RT-PCR, respectively. Addition of flavone (100 μg/mL) significantly increased the number of DENV-2 foci by 35.66% ± 1.52 and 49.66% ± 2.51 when added during and after virus adsorption to the Vero cells, respectively. The average foci size after 4 days of infection increased by 33% ± 2.11 and 89% ± 2.13. The DENV-2 specific RNA copy number in the flavone-treated infected cells increased by 6.41- and 23.1-fold when compared to the mock-treated infected cells. Flavone (100 μg/mL) did not promote or inhibit Vero cell proliferation. The CC₅₀ value of flavone against Vero cells was 446 µg/mL. These results suggest that flavone might enhance dengue virus replication by acting antagonistically towards flavonoids known to inhibit dengue virus replication.
Dengue virus-host cell interaction initiates when the virus binds to the attachment receptors followed by endocytic internalization of the virus particle. Successful entry into the cell is necessary for infection initiation. Currently, there is no protective vaccine or antiviral treatment for dengue infection. Targeting the viral entry pathway has become an attractive therapeutic strategy to block infection. This study aimed to investigate the effect of silencing the GRP78 and clathrin-mediated endocytosis on dengue virus entry and multiplication into HepG2 cells.
Dengue Viral Infection (DVI) imperils an estimated 2.5 billion people living in tropical and subtropical regions. World Health Organization (2011) guidelines also classified dengue as 'Expanded Dengue Syndrome' to incorporate wide spectrum of unusual manifestations of dengue infection affecting various organ systems - including liver, kidney, heart and brain. Renal involvements are least appreciated area of dengue infection, therefore, we systematically reviewed studies describing renal disorders in dengue infection, with emphasis on Acute Kidney Injury (AKI). The purpose of current review is to underscore clinicians’attention to this neglected intricacy of DVI. It suggests that dengue induced renal involvements vary from glomerulonephritis, nephrotic range proteinuria and AKI. We observed great disparity in incidence of AKI among dengue patients, based upon criteria used to define AKI. AKI among dengue patients was found to be associated with significant morbidity, mortality and longer hospitalization, adding financial burden to patients and healthcare system. Additionally, we identified several predictors of AKI in dengue patients including old age, obesity, severe dengue infection and concurrent bacterial or viral infection. Direct viral injury and deposition of antigen-antibody complex in glomerulus were found to be possible causes of renal disorders in dengue infection. Prior knowledge of clinico-laboratory characteristics and risk factors with early detection of AKI by using appropriate criteria would not only reduce morbidity and mortality but also decrease burden to patients and healthcare system.
Our understanding of the proteolytic processing events at the NS1-2A junction in the flavivirus polyprotein has not markedly progressed since the early work conducted on dengue virus (DENV). This work identified an octapeptide sequence located immediately upstream of the cleavage site thought to be important in substrate recognition by an as yet unknown, endoplasmic reticulum-resident host protease. Of the eight amino acid recognition sequence, the highly conserved residues at positions P1, P3, P5, P7 and P8 (with respect to N-terminus of NS2A) are particularly sensitive to amino acid substitutions in terms of DENV NS1-NS2A cleavage efficiency; however, the role of the octapeptide in efficient NS1 and NS2A production of other flaviviruses has not been experimentally addressed.
Natural reservoir hosts can sustain infection of pathogens without succumbing to overt disease. Multiple bat species host a plethora of viruses, pathogenic to other mammals, without clinical symptoms. Here, we detail infection of bat primary cells, immune cells, and cell lines with Dengue virus. While antibodies and viral RNA were previously detected in wild bats, their ability to sustain infection is not conclusive. Old-world fruitbat cells can be infected, producing high titres of virus with limited cellular responses. In addition, there is minimal interferon (IFN) response in cells infected with MOIs leading to dengue production. The ability to support in vitro replication/production raises the possibility of bats as a transient host in the life cycle of dengue or similar flaviviruses. New antibody serology evidence from Asia/Pacific highlights the previous exposure and raises awareness that bats may be involved in flavivirus dynamics and infection of other hosts.
Aedes aegypti mosquitoes carrying the wAlbB Wolbachia strain show a reduced capacity to transmit dengue virus. wAlbB has been introduced into wild Ae. aegypti populations in several field sites in Kuala Lumpur, Malaysia, where it has persisted at high frequency for more than 2 years and significantly reduced dengue incidence. Although these encouraging results indicate that wAlbB releases can be an effective dengue control strategy, the long-term success depends on wAlbB maintaining high population frequencies and virus transmission inhibition, and both could be compromised by Wolbachia-host coevolution in the field. Here, wAlbB-carrying Ae. aegypti collected from the field 20 months after the cessation of releases showed no reduction in Wolbachia density or tissue distribution changes compared to a wAlbB laboratory colony. The wAlbB strain continued to induce complete unidirectional cytoplasmic incompatibility, showed perfect maternal transmission under laboratory conditions, and retained its capacity to inhibit dengue. Additionally, a field-collected wAlbB line was challenged with Malaysian dengue patient blood, and showed significant blocking of virus dissemination to the salivary glands. These results indicate that wAlbB continues to inhibit currently circulating strains of dengue in field populations of Ae. aegypti, and provides additional support for the continued scale-up of Wolbachia wAlbB releases for dengue control. This article is part of the theme issue 'Novel control strategies for mosquito-borne diseases'.
Dengue is an arthropod-borne infectious disease caused by dengue virus (DENV) infection and transmitted byAedesmosquitoes. Approximately 50-100 million people are infected with DENV each year, resulting in a high economic burden on both governments and individuals. Here, we conducted a systematic review and meta-analysis to summarize information regarding the epidemiology, clinical characteristics, and serotype distribution and risk factors for global dengue outbreaks occurring from 1990 to 2015. We searched the PubMed, Embase and Web of Science databases through December 2016 using the term "dengue outbreak." In total, 3,853 studies were identified, of which 243 studies describing 262 dengue outbreaks met our inclusion criteria. The majority of outbreak-associated dengue cases were reported in the Western Pacific Region, particularly after the year 2010; these cases were primarily identified in China, Singapore and Malaysia. The pooled mean age of dengue-infected individuals was 30.1 years; of the included patients, 54.5% were male, 23.2% had DHF, 62.0% had secondary infections, and 1.3% died. The mean age of dengue patients reported after 2010 was older than that of patients reported before 2010 (34.0 vs. 27.2 years); however, the proportions of patients who had DHF, had secondary infections and died significantly decreased after 2010. Fever, malaise, headache, and asthenia were the most frequently reported clinical symptoms and signs among dengue patients. In addition, among the identified clinical symptoms and signs, positive tourniquet test (OR= 4.86), ascites (OR= 13.91) and shock (OR= 308.09) were identified as the best predictors of dengue infection, DHF and mortality, respectively (bothP< 0.05). The main risk factors for dengue infection, DHF and mortality were living with uncovered water container (OR= 1.65), suffering from hypotension (OR= 6.18) and suffering from diabetes mellitus (OR= 2.53), respectively (allP< 0.05). The serotype distribution varied with time and across WHO regions. Overall, co-infections were reported in 47.7% of the evaluated outbreaks, and the highest pooled mortality rate (2.0%) was identified in DENV-2 dominated outbreaks. Our study emphasizes the necessity of implementing programs focused on targeted prevention, early identification, and effective treatment.
Dengue infection can cause various effects on the central and peripheral nervous systems. Direct neurotropism and immunological mechanisms are responsible for most such neurological manifestations. We present the case of a 64-year-old woman with rapidly progressive dementia with seizures following dengue infection.
INTRODUCTION: Dengue virus (DENV) is principally transmitted by the Aedes aegypti mosquito. To date, mosquito population control remains the key strategy for reducing the continuing spread of DENV. The focus on the development of new vector control strategies through an understanding of the mosquito-virus relationship is essential, especially targeting the midgut, which is the first mosquito organ exposed to DENV infection.
METHODOLOGY: A cDNA library derived from female adult A. aegypti mosquito midgut cells was established using the switching mechanism at the 5' end of the RNA transcript (SMART), in combination with a highly potent recombination machinery of Saccharomyces cerevisiae. Gal4-based yeast two-hybrid (Y2H) assays were performed against DENV-2 proteins (E, prM, M, and NS1). Mammalian two-hybrid (M2H) and double immunofluorescence assays (IFA) were conducted to validate the authenticity of the three selected interactions.
RESULTS: The cDNA library was of good quality based on its transformation efficiency, cell density, titer, and the percentage of insert size. A total of 36 midgut proteins interacting with DENV-2 proteins were identified, some involved in nucleic acid transcription, oxidoreductase activity, peptidase activity, and ion binding. Positive outcomes were obtained from the three selected interactions validated using M2H and double IFA assays.
CONCLUSIONS: The identified proteins have different biological activities that may aid in the virus replication pathway. Therefore, the midgut cDNA library is a valuable tool for identifying DENV-2 interacting proteins. The positive outcomes of the three selected proteins validated supported the quality of the cDNA library and the robustness of the Y2H mechanisms.
To identify a novel antiviral peptide against dengue virus serotype 2 (DENV-2) by screening a phage display peptide library and to evaluate its in vitro antiviral activity and mode of action.
It is important to obtain frequent measurements of the abundance, distribution, and seasonality of mosquito vectors to determine the risk of disease transmission. The number of cases of dengue infection has increased in recent years on Penang Island, Malaysia, with recurring epidemics. However, ongoing control attempts are being critically hampered by the lack of up-to-date information regarding the vectors. To overcome this problem, we examined the current situation and distribution of dengue vectors on the island. Residences throughout the urban, suburban, and rural areas were inspected through wet and dry seasons between February 2009 and February 2010. Two vectors were encountered in the survey, with Aedes aegypti present in especially high numbers mostly in urban areas. Similar observations were noted for Ae. albopictus in rural areas. The former species was more abundant in outdoor containers, while the latter showed almost equivalent abundance both outdoors and indoors. The dengue virus was active in both urban and rural areas, and the number of cases of infection was higher in areas where Ae. aegypti was predominant. The abundance of immature Ae. albopictus was positively correlated with rainfall (r2 = 0.461; P < 0.05), but this was not the case for Ae. aegypti. For both species, the size of immature populations tended to increase with increasing intensity of rain, but heavy rains resulted in population loss. In addition to updating data regarding the larval habitats and locations (outdoors and indoors), this study highlighted the importance of spatial vector control stratification, which has the potential to reduce costs in control programs.
A main challenge in parasitology is the development of reliable tools to prevent or treat mosquito-borne diseases. We investigated the toxicity of magnetic nanoparticles (MNP) produced by Magnetospirillum gryphiswaldense (strain MSR-1) on chloroquine-resistant (CQ-r) and sensitive (CQ-s) Plasmodium falciparum, dengue virus (DEN-2), and two of their main vectors, Anopheles stephensi and Aedes aegypti, respectively. MNP were studied by Fourier-transform infrared spectroscopy and transmission electron microscopy. They were toxic to larvae and pupae of An. stephensi, LC50 ranged from 2.563 ppm (1st instar larva) to 6.430 ppm (pupa), and Ae. aegypti, LC50 ranged from 3.231 ppm (1st instar larva) to 7.545 ppm (pupa). MNP IC50 on P. falciparum were 83.32 μg ml(-1) (CQ-s) and 87.47 μg ml(-1) (CQ-r). However, the in vivo efficacy of MNP on Plasmodium berghei was low if compared to CQ-based treatments. Moderate cytotoxicity was detected on Vero cells post-treatment with MNP doses lower than 4 μg ml(-1). MNP evaluated at 2-8 μg ml(-1) inhibited DEN-2 replication inhibiting the expression of the envelope (E) protein. In conclusion, our findings represent the first report about the use of MNP in medical and veterinary entomology, proposing them as suitable materials to develop reliable tools to combat mosquito-borne diseases.
In South East Asia, dengue epidemics have increased in size and geographical distribution in recent years. We examined the spatiotemporal distribution and epidemiological characteristics of reported dengue cases in the predominantly rural state of Sabah, in Malaysian Borneo-an area where sylvatic and urban circulation of pathogens are known to intersect. Using a public health data set of routinely notified dengue cases in Sabah between 2010 and 2016, we described demographic and entomological risk factors, both before and after a 2014 change in the clinical case definition for the disease. Annual dengue incidence rates were spatially variable over the 7-year study period from 2010-2016 (state-wide mean annual incidence of 21 cases/100,000 people; range 5-42/100,000), but were highest in rural localities in the western districts of the state (Kuala Penyu, Nabawan, Tenom and Kota Marudu). Eastern districts exhibited lower overall dengue rates, although a high proportion of severe (haemorrhagic) dengue cases (44%) were focused in Sandakan and Tawau. Dengue incidence was highest for those aged between 10 and 29 years (24/100,000), and was slightly higher for males compared to females. Available vector surveillance data indicated that during large outbreaks in 2015 and 2016 the mosquito Aedes albopictus was more prevalent in both urban and rural households (House Index of 64%) than Ae. aegypti (15%). Demographic patterns remained unchanged both before and after the dengue case definition was changed; however, in the years following the change, reported case numbers increased substantially. Overall, these findings suggest that dengue outbreaks in Sabah are increasing in both urban and rural settings. Future studies to better understand the drivers of risk in specific age groups, genders and geographic locations, and to test the potential role of Ae. albopictus in transmission, may help target dengue prevention and control efforts.