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Objectives: Plasma leakage, a hallmark of disease in Dengue virus (DENV) infection, is an important clinical manifestation and is often associated with numerous factors such as viral factors. The aim of this study is to investigate the association of virus serotype, viral load kinetics, history of infection, and NS1 protein with plasma leakage., Methods: Subjects with fever ≤ 48 hours and positive DENV infection were included. Serial laboratory tests, viral load measurements, and ultrasonography examination to assess plasma leakage were performed., Results: DENV-3 was the most common serotype found in the plasma leakage group (35%). Patients with plasma leakage demonstrated a trend of higher viral load and a longer duration of viremia compared to those without. This was significantly observed on the fourth day of fever (p = 0.037). We found higher viral loads on specific days in patients with plasma leakage in both primary and secondary infections compared to those without. In addition, we also observed more rapid viral clearance in patients with secondary infection. NS1 protein, especially after 4 days of fever, was associated with higher peak viral load level, even though it was not statistically significant (p = 0.470). However, pairwise comparison demonstrated that peak viral load level in the group of patients with circulating NS1 detected for 7 days was significantly higher than the 5-day group (p = 0.037)., Conclusion: DENV-3 was the most common serotype to cause plasma leakage. Patients with plasma leakage showed a trend of higher viral load and a longer duration of viremia. Higher level of viral load was observed significantly on day 5 in patients with primary infection and more rapid viral clearance was observed in patients with secondary infection. Longer duration of circulating NS1 protein was also seen to be positively correlated with higher peak viral load level although not statistically significant., Competing Interests: The authors affirm no conflict of interest in this study., (Copyright: © 2023 Nainggolan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Dengue virus (DENV) infection remains to be a serious health problem in Indonesia. Community-based dengue studies to determine circulating DENV serotypes based on the geography and season are limited owing to the expensive cost and significant effort required. Many patients with DENV infection are not hospitalized and many visit the hospital in the later phase of the disease. In this study, we performed active DENV surveillance in a community in Jakarta to study the circulating dengue serotypes; adult febrile patients with fever less than 48 hours were recruited. Disease severity was defined using the World Health Organization (WHO) 1997 guidelines. Rapid NS1 dengue antigen detection was used to screen patients with DENV in the community. Viral culture using the C6/36 cell line, an increased antibody titer on hemagglutination inhibition test and enzyme linked immunosorbent assay, or detection of the viral genome on reverse transcription-polymerase chain reaction was used to confirm DENV infection. Of the 102 patients, 68 (66.7%) were confirmed to have DENV infection, with DENV-2 being the most dominant serotype, followed by DENV-3, DENV-1, and DENV-4, in concordance with several reports of mixed DENV infection. Interestingly, in terms of disease severity, although DENV-3 infection was not the predominant circulating serotype, infection with it tended to cause a more severe disease than infection with DENV-2.

Infection with hepatitis C virus (HCV) results in hepatitis C, a disease characterized by chronic infection, cirrhosis, and hepatocellular carcinoma. Currently, the standard therapy is a combination of pegylated interferon-α plus ribavirin with NS3 protease inhibitors. Addition of NS3 protease inhibitors to the standard therapy improves response rates; however, use of NS3 protease inhibitors is also associated with significant adverse effects and an increase in the overall cost of treatment. Therefore, there is a need to develop safe and inexpensive drugs for the treatment of HCV infections. In this study, we examined the antiviral activity of a crude extract from Dimocarpus longan leaves against HCV (genotype 2a strain JFH1). The D. longan crude extract (DL-CE) exhibited anti-HCV activity with a 50% effective concentration (EC50) of 19.4 μg/ml without cytotoxicity. A time-of-addition study demonstrated that DL-CE has anti-HCV activity at both the entry and post-entry steps and markedly blocks the viral entry step through direct virucidal activity with marginal inhibition of virion assembly. Co-treatment of DL-CE with cyclosporine A, an immunosuppressant or telaprevir, an NS3 protease inhibitor, resulted in additive and synergistic antiviral effects, respectively. Our findings suggest that DL-CE may be useful as an add-on therapy candidate for treating HCV infections.

The development of a dengue virus vaccine is a major priority in efforts to control the diseases. Several researchers are currently using the Asian 1 and Asian 2 genotypes as vaccine candidates for dengue type 2 virus (DENV-2). However, in this study, we constructed a recombinant plasmid-based prM/E gene, from a DENV-2 Cosmopolitan genotype strain as a dengue DNA vaccine candidate. The protein expression of the recombinant plasmid in CHO cells was analyzed using an enzyme-linked immunosorbent assay, western blotting, and sucrose gradient sedimentation. After being used to immunize ddY mice three times at doses of 25 or 100 μg, the DNA vaccine induced humoral immune responses. There was no difference in the neutralizing antibody titer (focus reduction neutralization test 50% value) of mice immunized with 25 and 100 μg DNA vaccine doses. When challenged with 3 × 10(5) FFU DENV-2, immunized mice could raise anamnestic neutralizing antibody responses, which were observed at day 4 and day 8 post-challenge. Analysis of immunogenicity using BALB/c mice showed that their antibody neutralization titers were lower than those of ddY mice. In addition, the antibodies produced after immunization and challenge could also neutralize a DENV-2 Asian 2 genotype (New Guinea C) strain. Therefore, the DENV-2 Cosmopolitan genotype may be a DENV-2 vaccine candidate.

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection-enhancing activities at sub-neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM-E) gene-based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM-E gene backbone showed high neutralizing antibody titers with less DENV infection-enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy., (© 2014 The Societies and Wiley Publishing Asia Pty Ltd.)

Dengue hemorrhagic fever has become a worldwide health issue. Heterologous infection by different serotypes may lead to severe forms of dengue infection and even death. In a cohort study in Jakarta from 2009 to 2010 with inclusion criteria of adults with fever of less than 48 hours, 72% were confirmed dengue infection from a total of suspected 190 dengue patients. Using RT-PCR, 16 patients were infected with DENV-4 with or without co-infection with other serotype(s). Dengue fever (DF) patients (73%) were infected with DENV-4 alone, while mixed infections were present in more severe clinical manifestations of DHF grade I and II. The nucleotide sequences of envelope (E) protein gene of 3 isolates of DENV-4 showed that they belonged to genotype II. There were 50 nucleotide substitutions, but only 3 amino acid changes were found at positions 130, 233 and 455, present in E protein isolated from DHF grade II patient.

Sequence analysis was conducted on structural and non-structural genes of 7 strains of dengue virus type-3 (DENV-3 virus) isolated in Indonesia and Thailand in the year 1973, 1994, and 1998 from patients with different clinical manifestations. In general, sequence similarity among isolates was greater than 93%, indicating that the mutation rate of DENV-3 circulating in this region was not more than 7% in the last 3 decades and suggesting that sequences that may responsible for viral architectures and/or biological function were strictly conserved. Mutations unique to viral strains associated with specific clinical manifestations were not found. Alignment of PrM/M and E nucleic acid sequences followed by parsimony analysis of sequences obtained in this study and published elsewhere allowed generation of phylogenetic trees, demonstrating that DENV-3 strains isolated in Indonesia in 1998 belonged to a separate cluster (subtype 2) from those isolated between 1973-1985 (subtype 1).

There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

A reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed as a rapid diagnostic test of dengue viremia. To detect dengue viruses in serum or plasma specimens, a pair of universal primers was designed for use in the RT-PCR. Using these primers, the 3'-noncoding region of dengue virus types 1, 2, 3, and 4 could be amplified, but not those of other flaviviruses, such as West Nile virus, Japanese encephalitis virus, and yellow fever virus, or the alphavirus Sindbis virus. The sensitivity of the RT-PCR assay was similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture. Combining a silica method for RNA isolation and RT-PCR dengue virus could be detected in a 6-hr assay. In a preliminary study using this method, we detected dengue virus in 38 of 39 plasma specimens from which dengue virus had been isolated by mosquito inoculation. We then applied this method for detecting dengue viremia to 117 plasma samples from 62 children with acute febrile illnesses in a dengue-endemic area. We detected dengue viremia in 19 of 20 samples obtained on the day of presentation, which had been confirmed as acute dengue infection by mosquito inoculation and antibody responses. The overall sensitivity of this method was 91.4% (32 of 35; 95% confidence interval [CI] = 82.2-100%). The results from testing plasma samples from febrile nondengue patients showed a specificity of 95.4% (42 of 44; 95% CI = 89.3-100%).

To clarify the correlation between metabolic stabilization of p53 protein and cellular transformation, we transformed the normal rat cell line F2408 with various viral transforming genes and examined the expression and stability of p53 protein in these transformed cells by pulse-chase immunoprecipitation experiments. As expected, the level of p53 in the SV40-transformed rat cell line was higher than that in the normal cell line and its stability was also increased. In contrast, in cells transformed with the E7 and E6 genes of human papillomavirus type 16, the level and stability of p53 were similar to those in the normal cells. In cells transformed by the middle T or large T of polyomavirus, v-K-ras, and v-src, the levels and stabilities of p53 were also not elevated, although the level of p53 was increased in activated c-H-ras-transformed cells without an increase in its stability. These results show that increased stability and expression of the p53 protein are not common events in viral transformation of the rat cell line. In addition, we demonstrated with a monoclonal antibody specific for the mutant form of p53 that cellular transformation by viral transforming genes does not involve mutational activation of p53 to an oncogenic form.

Human papillomavirus type 16 (HPV 16) is often found in cervical carcinomas, while HPV 6 is frequently associated with benign genital lesions. We have compared the abilities of the E7 transforming proteins of HPV 6 and 16 to transform various established and primary rodent cells by using the same heterologous promoter system. HPV 16 E7 efficiently induced anchorage-independent growth of all the rodent cell lines tested and immortalized or cooperated with ras in transforming primary rat cells. On the other hand, the transforming activity of HPV 6 E7 was lower and was restricted. By construction of chimeras of HPV 6 and 16 E7, we found that the difference in transforming activity between the two E7 proteins was mainly determined by the difference in their 30 N-terminal amino acid residues, although some activities seem to be slightly affected by differences in their residual C-terminal portions.

Human papillomavirus (HPV) type 52 from a cervical carcinoma in Indonesia was molecularly cloned and characterized. By hybridization with cervical carcinoma DNAs from Indonesian patients, HPV 52 was detected in 3 of 52 cases (6%), whereas HPV 16 and 18 were detected in 8 and 7 cases, respectively (15% and 13%). Sequence analysis revealed that the E6-E7 ORFs contained several DNA binding motifs (Cys-X-X-Cys) like previously sequenced HPVs. The E6 ORF also contained splice donor and acceptor signals, which may allow the expression of E6* protein. The E7 ORF encoded an amino acid sequence that is conserved in some DNA tumor viruses and is involved in binding to Rb protein and in cellular transformation. Transfection of a subgenomic fragment of HPV 52 under the control of a heterologous promoter showed that the E7 ORF alone induced anchorage-independent growth of established rodent cells and immortalized primary rat embryo fibroblasts (REF), and that in cooperation with activated ras, it induced malignant transformation of REF. The E6 ORF also induced, less efficiently, anchorage-independent growth. These results strongly suggest that HPV 52, like HPV 16 and 18, has oncogenic potential.

Three revertants defective in the ability to form colonies in semisolid medium were isolated from a rat cell line transformed by the E6 and E7 genes of human papillomavirus type 16 (HPV16). These revertants appeared to be defective in a cellular factor(s) necessary for transformation by HPV16-E6E7 genes since they still expressed a comparable amount of HPV16-E6E7 mRNA and E7 protein to the parental cells, harbored rescuable transforming virus, and were resistant to retransformation by HPV16-E6E7 genes. All these reverted phenotypes of the three mutants were recessive on somatic cell hybridization with normal cells, because all the hybrids showed transformed phenotypes.