Optimization of Flow-Cytometry Based Assay for Measuring Neutralizing Antibody Responses against Each of the Four Dengue Virus Serotypes

Dengue is an important public health problem worldwide, with India contributing nearly a third of global dengue disease burden. The measurement of neutralizing antibody responses is critical for understanding dengue pathophysiology, vaccine development and evaluation. Historically, dengue virus neutralization titers were measured using plaque reduction neutralization tests (PRNTs), which were later adapted to focus reduction neutralization tests (FRNTs). Given the slow and laborious nature of both these assays, there has been interest in adapting a high-throughput flow cytometry based neutralization assay. However, flow cytometry based assays typically underestimate neutralization titers, and in situations where the titers are low they can even fail to detect neutralization activity. In this study, by evaluating graded numbers of input Vero cell numbers and viral inoculum, we optimized the flow cytometry based neutralization assay in such a way that it is sensitive and scores titers that are in concordance with focus reduction neutralization tests for each of the four dengue virus serotypes (p < 0.0001). Given that dengue is a global public health concern, and several research groups are making efforts to understand its pathophysiology and accelerate vaccine development and evaluation both in India and worldwide, our findings have timely significance for facilitating these efforts.

Regional Variation in Dengue Virus Serotypes in Sri Lanka and Its Clinical and Epidemiological Relevance

Dengue is a significant health concern in Sri Lanka, but diagnosis of the infecting dengue virus (DENV) serotype has hitherto been largely restricted to the Colombo district in the western province. Salinity tolerant Aedes vectors are present in the island’s northern Jaffna peninsula, which is undergoing rapid groundwater salinization. Virus serotypes were determined by RT-qPCR in 107 and 112 patients diagnosed by NS1 antigen positivity from the Jaffna district in 2018 and 2019, respectively, and related to clinical characteristics. DENV1 and DENV2 were the most common serotypes in both years. Infections with multiple serotypes were not detected. DENV1 was significantly more prevalent in 2019 than 2018, while DENV3 was significantly more prevalent in 2018 than 2019 among the Jaffna patients. Limited genomic sequencing identified DENV1 genotype-I and DENV3 genotype-I in Jaffna patients in 2018. Dengue was more prevalent in working age persons and males among the serotyped Jaffna patients. DENV1 and DENV2 were the predominant serotypes in 2019 in the Colombo district. However, DENV1 and DENV3 were significantly more prevalent in Colombo compared with Jaffna in 2019. The differences in the prevalence of DENV1 and DENV3 between the Jaffna and Colombo districts in 2019 have implications for dengue epidemiology and vaccination. Salinity-tolerant Aedes vector strains, widespread in the Jaffna peninsula, may have contributed to differences in serotype prevalence compared with the Colombo district in 2019. Significant associations were not identified between virus serotypes and clinical characteristics among Jaffna patients.

Neutralization of Dengue Virus Serotypes by Sera from Dengue-Infected Individuals Is Preferentially Directed to Heterologous Serotypes and Not against the Autologous Serotype Present in Acute Infection

Sequential infections of humans by the four different dengue serotypes (DENV-1–4) lead to neutralizing antibodies with group, cross, and type specificity. Virus neutralization of serotypes showed monotypic but mostly multitypic neutralization profiles due to multiple virus exposures. We have studied neutralization to heterologous, reference DENV serotypes using paired sera collected between days 6 and 37 after onset of fever. The DENV-primed neutralization profile of the first serum sample, which was monitored by a foci reduction neutralization test (FRNT), was boosted but the neutralization profile stayed unchanged in the second serum sample. In 45 of 47 paired serum samples, the predominant neutralization was directed against DENV serotypes distinct from the infecting serotype. Homologous neutralization studies using sera and viruses from the same area, 33 secondary sera from DENV-1 infected Cambodian patients and eight virus isolates from Cambodia, showed that the FRNT assay accurately predicted the lack of a predominant antibody response against the infecting DENV-1 serotype in contrast to FRNT results using the WHO set of DENV viruses. This report provides evidence that DENV-primed multitypic neutralizing antibody profiles were mainly boosted and stayed unchanged after secondary infection and that DENV neutralization was predominantly directed to heterologous DENV but not against the infecting homologous serotype.

RANCANGAN PRIMER UNTUK DETEKSI VIRUS DENGUE SEROTIPE DENV-3 DAN DENV-4 DENGAN METODE NASBA DAN LFIA

Simple, fast, and accurate early detection is expected to reduce the mortality rate due to dengue infection. The dengue virus RNA detection method using Nucleic Acid Sequence-Based Amplification (NASBA) is an alternative method that can reduce the use of a thermocycler. The detection of NASBA amplicons was carried out using the Lateral Flow Assay (LFIA). This study was conducted to prove the effectiveness of the new primer design to detect dengue virus serotypes DENV-3 and DENV-4. In addition, this study was also conducted to measure the sensitivity and specificity of the LFIA method to detect dengue virus serotypes DENV-3 and DENV-4. The initial stage of this research was the isolation of dengue virus RNA from C6/36 cell cultures, then proceeded to design primers for NASBA and LFIA probes. NASBA reactions were performed on dengue virus serotypes DENV-3 and DENV-4 and DENV-4. The NASBA reaction products were then visualized on LFIA and agarose gel electrophoresis. The NASBA method with a new primary design can be used for the detection of dengue virus serotypes DENV-3 and DENV-4 as evidenced by electrophoresis results bands at 196 bp and 144 bp. The LFIA method with probe design can be used for the detection of dengue virus serotype DENV-3 and DENV-4 dengue virus serotype with a positive line on the test line on LFIA. The LFIA method for the detection of the dengue virus has a sensitivity up to a concentration of 10-3 (1 g/μl). The results of this study indicate that the newly designed primers are specific and sensitive for DENV-3 and DENV-4 dengue virus serotypes detection. Abstrak Deteksi dini yang sederhana, cepat dan akurat diharapkan dapat mengurangi tingkat kematian akibat infeksi dengue. Metode deteksi RNA virus dengue dengan Nucleic Acid Sequence-Based Amplification (NASBA) merupakan metode alternatif yang dapat mengurangi penggunaan thermocycler. Deteksi amplikon hasil NASBA dilakukan dengan Lateral Flow Assay (LFIA). Studi ini dilakukan untuk membuktikan efektivitas desain baru primer untuk mendeteksi virus dengue serotipe DENV-3 dan DENV-4. Di samping itu, studi ini juga dilakukan untuk mengukur sensitivitas dan spesifisitas metode LFIA untuk mendeteksi virus dengue serotype DENV-3 dan DENV-4. Tahap awal penelitian ini adalah isolasi RNA virus dengue dari kultur sel C6/36, kemudian dilanjutkan dengan merancang primer untuk NASBA dan probe LFIA. Reaksi NASBA dilakukan pada virus dengue serotipe DENV-3 dan DENV-4 dan DENV-4. Produk reaksi NASBA kemudian divisualisasikan pada LFIA dan elektroforesis gel agarosa. Metode NASBA dengan desain primer baru dapat digunakan untuk deteksi virus dengue serotipe DENV-3 dan DENV-4 yang dibuktikan oleh pita hasil elektroforesis pada 196 bp dan 144 bp. Metode LFIA dengan desain probe dapat digunakan untuk deteksi virus dengue serotipe DENV-3 dan DENV-4 serotipe virus dengue dengan garis positif pada garis uji pada LFIA. Metode LFIA untuk deteksi virus dengue memiliki sensitivitas hingga konsentrasi 10-3 (1 μg/μl). Hasil penelitian ini menunjukkan bahwa primer baru yang dirancang bersifat spesifik dan sensitif untuk deteksi serotipe virus dengue DENV-3 dan DENV-4.

Multifoci and multiserotypes circulation of dengue virus in Senegal between 2017 and 2018

Background Dengue fever is a mosquito born disease associated with self-limited to life threatening illness. First detected in Senegal in the nineteenth century, and despite its growing incidence this last decade, significant knowledge gaps exist in our knowledge of genetic diversity of circulating strains. This study highlights the circulating serotypes and genotypes between January 2017 and December 2018 and their spatial and temporal distribution throughout all regions of Senegal. Methods We used 56 dengue virus (DENV) strains for the analysis collected from 11 sampling areas: 39 from all regions of Senegal, and 17 isolates from Thiès, a particular area of the country. Two real time RT-qPCR systems were used to confirm dengue infection and corresponding serotypes. For molecular characterization, CprM gene was sequenced and submitted to phylogenetic analysis for serotypes and genotypes assignment. Results Three dengue virus serotypes (DENV-1–3) were detected by all used methods. DENV-3 was detected in 50% (28/56) of the isolates, followed by DENV-1 and DENV-2, each representing 25% (14/56) of the isolates. DENV-3 belongs to genotype III, DENV-1 to genotype V and DENV-2 to Cosmopolitan genotype. Serotype 3 was detected in 7 sampling locations and a co-circulation of different serotypes was observed in Thiès, Fatick and Richard-toll. Conclusions These results emphasize the need of continuous DENV surveillance in Senegal to detect DENV cases, to define circulating serotypes/genotypes and to prevent the spread and the occurrence of severe cases.