Comparison of the Mosquito Inoculation Technique and Quantitative Real Time Polymerase Chain Reaction to Measure Dengue Virus Concentration

Abstract

Abstract. An accurate measure of infectious dengue virus in human and mosquito tissues is critical to fully understand virus-host relationships, disease severity, viral fitness, and pathogenesis. In recent years, RNA copy number measured by quantitative real time-polymerase chain reaction has been used to measure dengue virus concentration in vitro and in vivo. In this study, we detail important differences in the measurement of viral growth kinetics in Vero and C6/36 tissue cultures, in Aedes aegypti mosquitoes, and in viremic human sera using RNA genomic equivalents and mosquito infectious dose 50 (MID 50 ). Although there was reasonably good correlation between the two methods, RNA copy number was 2 to 5 logs greater than infectious virus titers. These differences varied significantly depending on virus strain, viral platform, infectious virus assay, and viral growth phase. The results have important implications for the correct interpretation of biological and epidemiological data from experimental and clinical studies, and show that genomic equivalents should be interpreted with caution when used as a proxy for infectious virus in such studies.Epidemic dengue/dengue hemorrhagic fever (DF/DHF) has emerged as the most important mosquito-borne viral disease of humans in the past 40 years with both the viruses and mosquito vectors spreading globally in the tropics.1 This spread has been closely linked to the global trends of urbanization and globalization, combined with a lack of effective mosquito control. Most urban centers of the tropics are now hyperendemic with multiple virus serotypes co-circulating. The result has been larger and more frequent epidemics associated with more severe disease. 1From the 1940s when dengue viruses (DENV) were first isolated 2,3 until the 1960s, scientists relied on suckling mice for isolation and assay of DENV. In the 1960s, mammalian cell cultures were used, 4 but both methods were highly insensitive to primary DENV isolates that had not been adapted by serial passage.5 Subsequent development of the mosquito inoculation technique and the C6/36 Aedes albopictus cell line in the 1970s provided significant improvement in sensitivity, and permitted work with unpassaged DENVs. [5][6][7] However, the relatively insensitive plaque assay that measures plaqueforming units (PFU) 8 has continued to be used to measure infectious DENV in experimental studies, and the C6/36 cell culture system has been primarily used for virus isolation [6][7][8][9] because most virology laboratories lacked the ability to work 7,8,10 with live mosquitoes. The efficacy of clinical diagnosis, surveillance, prevention, and control of dengue has been limited by the lack of easy to use and sensitive diagnostic tests. Currently, laboratory diagnosis in most dengue-endemic countries relies on detecting immunoglobulin M (IgM) antibody in acute serum samples. More recently, commercial tests combining NS1 antigen and IgM antibody detection have become increasingly popular. 11For DENV detection and quantitation, qu...

Asymptomatic humans transmit dengue virus to mosquitoes

“…It also suggests that the amount of virus might differ between venous blood (used to measure viremia) and capillary or venule blood (imbibed by mosquitoes) and/or between plasma and whole blood. As previously reported, it is also possible that the high sensitivity of the mosquito infection model may help to detect low levels of viremia that are below the limit of detection by qRT-PCR, but are actually sufficient to infect mosquitoes (21). In our multivariate analysis (Table 1), factors other than the amount of viral RNA in plasma (e.g., serotype and disease category) contributed to differences in participant infectiousness.…”

“…The number of infectious virus particles measured by the mosquito inoculation technique can be 2-5 logs lower, depending on the virus strain, cell type used, level of viremia, host immune response, and several other factors such as handling and storage conditions of viremic blood sample (21). RT-PCR detects RNA from infectious viruses, but also from immature virions and defective particles.…”

Evolutionary potential of the extrinsic incubation period of dengue virus inAedes aegypti

“…; Choy et al. ). Second, in the wild, mosquitoes are more likely to take multiple small blood meals (Scott et al.…”

“…In contrast, plaque assays, which require much larger volumes of starting material, quantify actual infectious particles. While there is a strong correlation between the two measures, the estimated RNA copy number is usually 2-5 logs higher than the number of infectious units due to the presence of immature and/or defective virus (Richardson et al 2006;Choy et al 2013). Second, in the wild, mosquitoes are more likely to take multiple small blood meals (Scott et al 2000).…”

The role of Culex pipiens mosquitoes in transmission of West Nile virus in Europe

“…Although there is a correlation between the number of viral RNA copies and infectious virus particles, the viral genome copies are often 100-5000 times higher than the number of truly infectious virus particles (Bae et al 2003, Choy et al 2013. Use of infectivity assays or proper validation of qRT-PCR results is, thus, essential to draw valid conclusions on outcomes of vector competence studies.…”