Development of a Real-Time qPCR Assay for Quantification of Covert Baculovirus Infections in a Major African Crop Pest

Abstract

Many pathogens and parasites are present in host individuals and populations without any obvious signs of disease. This is particularly true for baculoviruses infecting lepidopteran hosts, where studies have shown that covert persistent viral infections are almost ubiquitous in many species. To date, the infection intensity of covert viruses has rarely been quantified. In this study, we investigated the dynamics of a covert baculovirus infection within the lepidopteran crop pest Spodoptera exempta. A real-time quantitative polymerase chain reaction (qPCR) procedure using a 5' nuclease hydrolysis (TaqMan) probe was developed for specific detection and quantification of Spodoptera exempta nucleopolyhedrovirus (SpexNPV). The qPCR assay indicated that covert baculovirus dynamics varied considerably over the course of the host life-cycle, with infection load peaking in early larval instars and being lowest in adults and final-instar larvae. Adult dissections indicated that, contrary to expectation, viral load aggregation was highest in the head, wings and legs, and lowest in the thorax and abdomen. The data presented here have broad implications relating to our understanding of transmission patterns of baculoviruses and the role of covert infections in host-pathogen dynamics.

Keywords: Spodoptera exempta; TaqMan real-time qPCR; baculovirus; covert infections; nucleopolyhedrovirus.

Figure 1

Covert SpexNPV DNA as measured in asymptomatic insects sampled as adults, eggs, neonates, and 4, 6, 8, 10 and 12 days post egg hatch. SpexNPV load as a function of (A) age (days post-hatch); (B) larval instar (1st to 5th); and (C) head capsule width (mm). In (A) the curve shown is the smoothing spline through the raw data. In (B,C) the linear regression lines are shown.

Figure 2

Covert SpexNPV DNA levels in different body regions of adult S. exempta moths. Means ± S.E. are shown. Different letters above the error bars indicate means that are significantly different according to Fisher’s least significant difference test.

Figure 3

Dynamics of SpexNPV genome replication following ingestion of 10⁴ OBs (treated) and dH₂O (control). The lines are the fitted Gompertz curves (see text for details).