Development of Multiplex Molecular Assays for Simultaneous Detection of Dengue Serotypes and Chikungunya Virus for Arbovirus Surveillance.

The major arboviruses mainly belong to the Bunyaviridae, Togaviridae, and Flaviviridae families, among which the chikungunya virus and dengue virus have emerged as global public health problems. The main objective of this study was to develop specific, sensitive, and cost-effective molecular multiplex RT-PCR and RT-qPCR assays for the rapid and simultaneous detection of CHIKV and the four serotypes of DENV for arbovirus surveillance. Specific primers for all viruses were designed, and one-step multiplex RT-PCR (mRT-PCR) and RT-qPCR (mRT-qPCR) were developed using reference strains of the CHIKV and DENV serotypes. The specificity of the test for all the viruses was confirmed through sequencing. The standard curves showed a high correlation coefficient, R2 = 0.99, for DENV-2 and DENV-3; R2 = 0.98, for DENV-4; and CHIKV; R2 = 0.93, for DENV-1. The limits of detection were calculated to be 4.1 × 10-1 copies/reaction for DENV-1, DENV-3, and CHIKV and 4.1 × 101 for DENV-2 and DENV-4. The specificity and sensitivity of the newly developed mRT-PCR and mRT-qPCR were validated using positive serum samples collected from India and Burkina Faso. The sensitivity of mRT-PCR and mRT-qPCR are 91%, and 100%, respectively. The specificity of both assays was 100%. mRT-PCR and mRT-qPCR assays are low-cost, and a combination of both will be a useful tool for arbovirus surveillance.

Cytokine profile, neutralisation potential and viral replication dynamics in sera of chikungunya patients in India: a cross-sectional study.

Background: Chikungunya disease (CHIKD) is a threat to global health, as it impairs the quality of life of an infected individual ranging from months to years. A systematic evaluation of the serological, virological, and immunological aspects of the circulating viruses and their impact on the host response is imperative for better understanding of the evolving disease dynamics. Methods: Serum samples were collected from 196 acute CHIKD patients from ten tertiary care hospitals across India during 2016-2021. Out of 196 patients, paired convalescent samples were collected from 51 patients (one-month post-onset of symptoms). The serum samples were profiled for cytokines and neutralisation capacity. Further, chikungunya virus (CHIKV) was isolated from the acute sera and the replication kinetics of the clinical isolates was evaluated. Findings: Serological analysis indicated that neutralisation could be correlated to seroconversion in the convalescent phase but not found significant in acute phase. In the acute phase samples, there was a correlation between elevated serum levels of IFN-γ, IP-10, MCP-1 and MIG and disease severity. During convalescent phase, pro-inflammatory markers such as IL-6, IL-1ß, IL-9 and IP-10 were found to be elevated with a corresponding decline in the secretion of anti-inflammatory cytokines such as IL-4 and IL-10, which correlated with persistent arthralgia. Analysis of replication of the clinical isolates revealed that 68.4% of viruses were fast-growing in the Vero cells (cytopathic effect [CPE] observed within 24 h post-infection), and their corresponding acute serum samples showed an elevated secretion of IFN-α, IL-1RA, IL-17F, IL-9, MCP-1 and MIP-1α. Interpretation: This study provides an important overview of neutralisation capabilities and cytokine responses along with virus pathogenesis associated with CHIKV infections in India. Funding: Biotechnology Industry Research Assistance Council (BIRAC).

Short communication: Virological and B cell profiles of chikungunya and Dengue virus co-infections in Delhi during 2017-2019.

With explosive epidemics of chikungunya in India since 2004, chikungunya virus (CHIKV) now co-circulates in geographical areas where Dengue virus (DENV) is already endemic and thus provides opportunity for the same mosquito to be infected with both viruses. Although there are excellent studies that have addressed the clinical of mono and co-infection, we have little to no knowledge on the current viral sequences that pre-dominate co-infections, and the B cell response elicited. In this study, we analyzed febrile patients that were confirmed to have DENV-CHIKV co-infections and asked the following questions: 1) what is the frequency of co-infections found in a single cycle of transmission; 2) what are the viral sequences associated with them; 3) what does the antibody secreting cell / plasmablast response look like in patients that are co-infected with both viruses. We report those co-infections occur at a frequency of 6.7% in the transmission cycle, and while DENV-3 is now frequently detected, we do not see a serotype bias in the patients that are co-infected with ESCA strain of CHIKV. Moreover, the effector B cell response (plasmablasts) observed are specific to both infecting viruses indicating no overt bias. Further studies to associate whether any of these properties have a bearing on clinical disease manifestation will be both timely and important.

Detection of Dengue viruses among febrile patients in Lagos, Nigeria and phylogenetics of circulating Dengue serotypes in Africa.

Dengue fever, a mosquito borne viral disease, is caused by Dengue virus. This virus and its vector is endemic in most tropical countries including Nigeria. Dengue presents with febrile symptoms and is a major cause of morbidity and mortality in affected countries. The infection presently has no licensed drugs and vaccine is only available for previously exposed individuals. Despite the endemicity of Dengue in Nigeria, very few studies have identified circulating Dengue genotypes in the country. There is also sparse information on the occurrence, distribution and temporal patterns of circulating dengue virus serotypes as well as genotypes in Africa. This situation creates barriers to effective control of the infection in the continent. This study identified Dengue serotypes and genotypes among febrile patients in two health centers in Lagos, Nigeria. Phylogenetic analysis of Dengue sequences previously collected from African countries and submitted to GenBank database from 1944 till date was also performed. One hundred and thirty febrile persons were recruited for the study between April and August 2018. Eleven (8.5%) persons were Dengue virus positive. Dengue virus serotypes 1 (genotype I) and 3 (genotype I) were identified as actively circulating in Lagos, Nigeria. DENV 1 genotype V, DENV 2 cosmopolitan genotype and DENV 3 genotype III has over the years been the predominant circulating Dengue strains in Africa. Relative genotypic stability of circulating Dengue serotypes in Africa occurred over the past five decades. This may be due to limited investigations on circulating Dengue serotypes among asymptomatic individuals in the region as most studies focused on disease outbreaks and imported cases. There is the need to describe circulating Dengue genotypes in northern Africa, southern Africa as well as among asymptomatic individuals in other parts of Africa as this will provide further information on the diversity of Dengue genotypes circulating in the region.