Timely diagnosis of respiratory tract infections: evaluation of the performance of the Respifinder assay compared to the xTAG respiratory viral panel assay

BackgroundDengue virus (DENV), which causes mosquito-borne disease dengue hemorrhagic fever (DHF), consists of four serotypes co-circulating in endemic areas. Currently, DENV serotypes can be identified by laborious virus isolation followed by immunofluorescent assay and sophisticated RT-PCR.ObjectiveTo establish a new assay designated as “serotyping-NS1-ELISA” to detect the NS1 protein and to identify DENV serotypes simultaneously.Study designThe monoclonal antibodies (Mabs) against NS1 of each DENV serotype were produced and characterized for their serotype-specificity. To develop serotyping-NS1-ELISA, the selected serotype-specific anti-NS1 Mabs were applied to detect the NS1 antigen, which was previously captured by a flavivirus cross-reactive anti-NS1 Mab. Serotyping accuracy of the developed assay was validated with NS1 from DENV-infected cell culture supernatants and from well-characterized clinical specimens.ResultsOf 30 anti-NS1 Mabs, 1 serotype-specific anti-NS1 Mab to each DENV serotype was selected based on NS1 capture ELISA results for developing the serotyping-NS1-ELISA. Using DENV-infected cell culture supernatants for validation, the selected antibodies were shown to be capable of differentiating four DENV serotypes. When acute phase plasma from DENV-infected patients was used for validation, 65 out of 85 specimens (76.5% overall sensitivity) were positive to one of the four serotypes developed in our assay. Interestingly, identification of DENV serotypes by our serotyping-NS1-ELISA was 100% accurate for DENV1, 3 and 4 and 82.4% for DENV2 as compared with standard RT-PCR. Assay specificity was 100% (90/90).ConclusionsThe developed serotyping-NS1-ELISA provides an alternative for simultaneous detection of DENV NS1 and identification of its serotype in acute patients’ specimens. The assay would be applicable for dengue diagnosis and epidemiological studies.     

Letter to the editor: molecular genotyping of Dengue Virus Types 2 and 4 from the Guatemalan and Honduran Epidemics of 2007 using the envelope glycoprotein gene

Eight serum specimens collected from dengue patients in Guatemala and Honduras during the Central American epidemic of 2007 were analyzed. Virus identification and serotyping performed by a nested RT-PCR assay revealed two DENV-1 isolates from Guatemala, four DENV-2 isolates, two each from Guatemala and Honduras, and two DENV-4 isolates from Honduras. Viral genotyping determined by phylogenetic analysis of the complete envelope gene sequences demonstrated that the DENV-2 isolates from Guatemala and Honduras fell into the American/Asian Genotype III, and were most closely related to DENV-2/NI/BID-V2683-1999 isolated from a dengue case in Nicaragua in 1999; and the DENV-4 F07-076 isolate from Honduras belonged to genotype II, and was most closely related to DENV-4/US/BID-V1093/1998 isolated from Puerto Rico in 1998. Our results suggest that the 2007 dengue outbreaks in Guatemala and Honduras were most likely caused by the re-emergence of earlier, indigenous DENV strains rather than by newly introduced strains and there were at least three serotypes of DENV co-circulating during the 2007 Central American epidemics.     

A retrospective survey of dengue virus infection in fatal cases from an epidemic in Brazil

Dengue virus can infect many cell types from the vascular, muscular and hematological systems causing diverse clinical and pathological signs. The purpose of the present study was to investigate by different diagnostic methods dengue virus in human tissue specimens obtained from fatal cases (n=29) during a large-scale dengue fever epidemic in 2002 in the State of Rio de Janeiro, Brazil. The combination of four procedures provided diagnostic confirmation of DENV-3 infection in 26 (89.6%) out of the 29 suspected fatal cases. Dengue virus (DENV) was isolated from 2/74 (2.7%) tissue samples, inoculated into C6/36 cells and identified as DENV-3, nested RT-PCR accusing 22/72 (30.5%) samples as DENV-3. Real-time RT-PCR yielded the highest positivity rate, detecting viral RNA in 45/77 (58.4%) clinical specimens, including the liver (n=18), lung (n=8), spleen (n=8), brain (n=6), kidney (n=3), bone marrow (n=1) and heart (n=1). Immunohistochemical tests recognized the DENV antigen in 26/59 (44%) specimens. Given the accuracy and effectiveness of real-time RT-PCR in this investigation, this approach may play an important role for rapid diagnosis of dengue infections.     

Antibody-dependent enhancement of dengue virus infection in vitro by undiluted sera from monkeys infected with heterotypic dengue virus

Two monkeys (Macaca fascicularis) each were infected with dengue virus type 1 (DENV-1) and type 2 (DENV-2). High levels of neutralizing antibody to homotypic serotype were detected from day 10 to week 58 after infection. Levels of cross-reactive neutralizing antibody to other serotypes were at lower levels or undetectable. Serum samples collected from day 10 to week 58 enhanced infection by homotypic and heterotypic serotypes of DENV when diluted, demonstrating antibody-dependent enhancement (ADE). The ADE activities to heterotypic and homotypic dengue virus infections peaked at dilutions of 1:10–1:100 and 1:100–1:1,000, respectively. Serum samples collected enhanced heterotypic dengue virus infection without any dilution. The results indicate that sera from infected monkeys have an ability to enhance heterotypic dengue virus infection in vitro without dilution, although some of these sera also possess neutralizing activity.     

Differential proinflammatory and angiogenesis-specific cytokine production in human pulmonary endothelial cells, HPMEC-ST1.6R infected with dengue-2 and dengue-3 virus

In this study, the ability of dengue virus serotypes 2 (DENV-2) and 3 (DENV-3) to infect and induce increased production of proinflammatory cytokines in a pulmonary endothelial cell line (HPMEC-ST1.6R) was investigated. This cell line exhibits the major constitutive and inducible endothelial cell characteristics, as well as angiogenic response. DENV-2 and DENV-3 infection was confirmed by an observed cytopathic effect (CPE), as well as RT-PCR and immunofluorescence assays. Increases in Th-1 and Th-2 cytokines IL-4, IL-8, IL-6, IL-10, GM-CSF, INF-gamma, and tumor necrosis factor (TNF-alpha) within DENV-2- and DENV-3-infected cells were demonstrated using a microbead-based Bio-plex assay. Proinflammatory cytokine increases and the expression of a potent angiogenic inducer protein, VEGF were confirmed by dot-blot analysis using the TranSignal Human Angiogenesis Antibody Array. Dengue virus-infected HPMEC-ST1.6R cells exhibited an elongated cytoplasmic morphology, possibly representing a response to VEGF and activation of angiogenesis. The increased levels of Th-1 cytokines and VEGF in DENV-2 virus infected-HPMEC-ST1.6R could be distinguished from those infected by DENV-3. This suggests that cytokine patterns associated with DENV infections may be serotype and strain-specific. The experimental approaches described here could be developed further into a useful diagnostic tool for the characterization of dengue hemorrhagic fever cases, leading to enhancement of treatment therapy.     

Phylogenetic and evolutionary analyses of dengue viruses isolated in Jakarta,Indonesia

Dengue has affected Indonesia for the last five decades and become a major health problem in many cities in the country. Jakarta, the capital of Indonesia, reports dengue cases annually, with several outbreaks documented. To gain information on the dynamic and evolutionary history of dengue virus (DENV) in Jakarta, we conducted phylogenetic and evolutionary analyses of DENV isolated in 2009. Three hundred thirty-three dengue-suspected patients were recruited. Our data revealed that dengue predominantly affected young adults, and the majority of cases were due to secondary infection. A total of 171 virus isolates were successfully serotyped. All four DENV serotypes were circulating in the city, and DENV-1 was the predominant serotype. The DENV genotyping of 17 isolates revealed the presence of Genotypes I and IV in DENV-1, while DENV-2 isolates were grouped into the Cosmopolitan genotype. The grouping of isolates into Genotype I and II was seen for DENV-3 and DENV-4, respectively. Evolutionary analysis revealed the relatedness of Jakarta isolates with other isolates from other cities in Indonesia and isolates from imported cases in other countries. We revealed the endemicity of DENV and the role of Jakarta as the potential source of imported dengue cases in other countries. Our study provides genetic information regarding DENV from Jakarta, which will be useful for upstream applications, such as the study of DENV epidemiology and evolution and transmission dynamics.     

Detection and serotyping of dengue virus in serum samples by multiplex reverse transcriptase PCR-ligase detection reaction assay

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.     

Molecular Studies on Dengue Viruses Detected in Patients from Central India

Purpose: Dengue viruses (DENVs), the causative agents of dengue (DEN), are classified into four serotypes and several genotypes. Identifying circulating serotypes and genotypes has clinical and epidemiological importance; however, limited information in this regard is available from Central India. This laboratory-based study was done to fill this lacuna. Materials and Methods: The samples collected in the acute phase of illness were subjected to DEN NS1 ELISA, and NS1-positive samples (n = 80) were subjected to serotyping; representative samples from each serotype were sequenced to identify genotypes. Results: Seventy-one (88.75%) samples could be serotyped. All the four DENV serotypes with dominance of DENV-3 (n = 33; 47%) were detected. DENV-4 was detected after a gap of 3 years. Cases with multiple DENV serotype infection were identified. Genotyping showed that DENV-1 belonging to genotype III, DENV-2 cosmopolitan (IV), DENV-3 genotype III lineage C and DENV-4 genotype I were in circulation in the year 2016. Conclusion: Our study documents the molecular characteristics of DENV circulating in the area. Detection of heterologous DENV serotype with dominance of DENV-3 emphasises the need for regular molecular monitoring.     

Antibodies play a greater role than immune cells in heterologous protection against secondary dengue virus infection in a mouse model

The four serotypes of dengue virus (DENV1–4) are causative agents of dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Previous DENV infection is a risk factor for DHF/DSS during subsequent infection by a different serotype. Nonetheless, most primary and secondary DENV infections are asymptomatic. To investigate the possible mechanisms of immune protection in vivo, 129/Pas mice lacking IFN-α/β and -γ receptors (AG129) were used to model secondary infection using both DENV1–DENV2 and DENV2–DENV4 sequences. At intervals between sequential infections of 4 to 52 weeks, protection against secondary heterologous DENV infection was observed. Passive transfer of DENV-immune serum was protective against replication of heterologous challenge virus in all tissues tested, whereas adoptive transfer of DENV-immune cells significantly protected mice from replication of the challenge virus only when a lower inoculum was administered. These findings are relevant for understanding both natural and vaccine-induced immunity to DENV.     

Comprehensive mapping of immunodominant and conserved serotype- and group-specific B-cell epitopes of nonstructural protein 1 from dengue virus type 1

The dengue virus (DENV) nonstructural protein 1 (NS1) is an immunogenic protein that holds potential for the development of vaccines and diagnostic reagents; however, the epitopes of NS1 have not been comprehensively mapped. We mapped B-cell linear epitopes on NS1 using 149 monoclonal antibodies with DENV serotype specificity and cross-reactivity as well as antisera from 27 mice immunized with the four DENV serotypes. Epitope recognition analysis was performed using a set of 15-mer sequential overlapping peptides that spanned the entire NS1 protein from DENV-1. This strategy identified three regions of NS1 that are DENV-1 serotype-specific epitopes, namely amino acid residues 1-15, 71-85, and 338-352. We also identified five group-specific B-cell epitopes that were highly conserved among isolates of the four DENV serotypes. These novel immunodominant serotype- and group-specific B-cell epitopes of DENV NS1 may aid the development of new dengue vaccines and diagnostic assays.     

Molecular Characterisation and Phylogenetic Analysis of Dengue Outbreak in Pasighat,Arunachal Pradesh,Northeast India

Background and Objectives: Dengue is one of the most prevalent arboviral diseases in the world with 390 million dengue infections per year. In this study, we report the molecular characterisation of dengue outbreak in Pasighat, Arunachal Pradesh, Northeast India during 2015. Subjects and Methods: A total of 613 dengue-suspected cases were screened for dengue virus by dengue NS1 Ag and anti-dengue IgM antibody depending on the duration of sample collection and onset of symptom. Further, molecular characterisation was done by amplifying the C-PrM region by real-time polymerase chain reaction followed by phylogenetic analysis. Results: Molecular characterisation revealed that the dengue outbreak was predominantly due to dengue virus serotype-1 (DENV-1) (90.9%) while DENV-2 was detected in 7.5% of samples. Co-infection of DENV-1 and DENV-2 was detected in one case. Phylogenetic analysis of the DENV-1 strains with the prototype revealed that the DENV-1 strains were grouped within genotype III. Similarly, DENV-2 strains were clustered within genotype IV. The study revealed a change in the predominant serotype in recent years with DENV-3 in 2012 to DENV-1, 2, 3 and 4 in 2014 to DENV-1 in 2015 in the study region. A unique L24M mutation was observed in the DENV-1 strains of Arunachal Pradesh which was absent in all the circulating strains in India except one strain from the state of Kerala in South India. Marked variation within the DENV-2 strains was observed at A102V and I163V in one strain similar to earlier circulating isolates in India. Conclusions: The present study reveals a shift in the serotype dominance in the study region. As serotype shifts and secondary infection with a heterologous DENV serotype are frequently associated with disease severity, there is an urgent need for sustained monitoring of the circulating serotypes and enhanced surveillance operations, especially in the monsoon and post-monsoon periods to prevent large-scale, severe dengue outbreaks in this region.     

Molecular genotyping of dengue viruses by phylogenetic analysis of the sequences of individual genes

The prevalence of four serotypes of dengue virus (DENV) has risen dramatically in recent years accompanied by an increase in viral genetic diversity. The evolution of DENV has had a major impact on their virulence for humans and on the epidemiology of dengue disease around the world. In order to perform disease surveillance and understand DENV evolution and its effects on virus transmission and disease, an efficient and accurate method for genotype identification is required. Phylogenetic analysis of viral gene sequences is the method used most commonly, with envelope (E) gene the most frequently selected target. To determine which gene might be suitable targets for genotyping DENV, phylogenetic analysis was performed on 10 individual coding genes plus the 3'-non-translated region (3'NTR) for 56 geographically divergent DENV strains representing all identified genotypes. These were reflected in eleven maximum likelihood phylogenetic trees. Based on the bootstrap values (over 90%) supporting the major nodes, the best target genes were identified for each serotype: for DENV-1, the sequences of all coding genes except non-structural gene 4A (NS4A), for DENV-2, PrM/M, E, NS1, NS3, NS4A and NS5, for DENV-3, all coding genes and the 3'NTR, and for DENV-4, C, PrM/M, E, NS1, NS2A, NS2B, NS4A and NS5.     

Evaluation of widely used diagnostic tests to detect West Nile virus infections in horses previously infected with St. Louis encephalitis virus or dengue virus type 2

Primary West Nile virus (WNV) infections can be diagnosed using a number of tests that detect infectious particles, nucleic acid, and specific IgM and/or IgG antibodies. However, serological identification of the infecting agent in secondary or subsequent flavivirus infections is problematic due to the extensive cross-reactivity of flavivirus antibodies. This is particularly difficult in the tropical Americas where multiple flaviviruses cocirculate. A study of sequential flavivirus infection in horses was undertaken using three medically important flaviviruses and five widely utilized diagnostic assays to determine if WNV infection in horses that had a previous St. Louis encephalitis virus (SLEV) or dengue virus type 2 (DENV-2) infection could be diagnosed. Following the primary inoculation, 25% (3/12) and 75% (3/4) of the horses mounted antibody responses against SLEV and DENV-2, respectively. Eighty-eight percent of horses subsequently inoculated with WNV had a WNV-specific antibody response that could be detected with one of these assays. The plaque reduction neutralization test (PRNT) was sensitive in detection but lacked specificity, especially following repeated flavivirus exposure. The WNV-specific IgM enzyme-linked immunosorbent assay (IgM ELISA) was able to detect an IgM antibody response and was not cross-reactive in a primary SLEV or DENV response. The WNV-specific blocking ELISA was specific, showing positives only following a WNV injection. Of great importance, we demonstrated that timing of sample collection and the need for multiple samples are important, as the infecting etiology could be misdiagnosed if only a single sample is tested.