26 March 2013

3 minute read

At present these techniques are entirely reliant upon interpretation of semi-quantitative RT-PCR (sqPCR) data in the form of Cq values used to infer the presence of viral RNA in samples.

Introduction

Schmallenberg virus (SBV) is a pathogen of ruminants which was initially identified in late 2011 through metagenomic studies conducted by the Friedrich Loeffler Institute in Germany . Infection with SBV in adult sheep and cattle can result in a mild disease whose clinical signs include diarrhea and reduced milk yield. A major economic impact of SBV infection, however, lies in the occurrence of congenital defects in offspring of infected ruminants . Recent phylogenetic analyses have demonstrated that SBV is most closely related to viruses of the species Sathuperi virus and is not a reassortant with other species of the Simbu serogroup . Viruses of this serogroup, including Akabane (AKAV) and Aino virus, have previously been isolated from pools of livestock associated Culicoides biting midges in Japan where the vector most clearly implicated is Culicoides oxystoma . Vector competence studies for these arboviruses are most complete for AKAV, particularly in Australia where the role of Culicoides brevitarsis in transmitting and spreading the virus has been characterized in detail . Incidence of AKAV in field collected Culicoides has also been studied in Israel , the Oman and the Republic of South Africa , where the major Afro-tropical arbovirus vector Culicoides imicola is believed to play a primary role in transmission.

Methodology/Principal Findings

This study investigates the advantages and limitations of sqPCR in this role by comparing infection and dissemination rates of Schmallenberg virus (SBV) in two colony lines of Culicoides. Through the use of these behaviorally malleable lines we provide tools for demarcating arbovirus infection and dissemination rates in Culicoides which to date have prevented clear implication of primary vector species in northern Europe. The study demonstrates biological transmission of SBV in an arthropod vector, supporting the conclusions from field-caught Culicoides and provides a general framework for future assessment of vector competence of Culicoides for arboviruses using sqPCR.

Figure 1. Observed C _q values for Schmallenberg virus (SBV) in Culicoides biting midges infected via different routes and processed ten days post infection. (A) C. sonorensis infected via intrathoracic inoculation, incubated for ten days after which the abdomen/thorax, head and saliva of individual insects were processed separately using sqPCR. The box-and-whisker plot shows the median (horizontal line), interquartile range (box), 1.5 times the interquartile range (whiskers) and any outliers (crosses). (B) C. sonorensis fed orally on SBV-infected blood via a membrane-based system and processed separately as whole insects using sqPCR (C) C. nubeculosus fed orally on SBV-infected blood via a membrane-based system and processed separately as whole insects using sqPCR. doi:10.1371/journal.pone.0057747.g001

Conclusions/Significance

When adopting novel diagnostic technologies, correctly implicating vectors of arboviral pathogens requires a coherent laboratory framework to fully understand the implications of results produced in the field. This study illustrates these difficulties and provides a full examination of sqPCR in this role for the Culicoides-arbovirus system.

Citation: Veronesi E, Henstock M, Gubbins S, Batten C, Manley R, et al. (2013) Implicating Culicoides Biting Midges as Vectors of Schmallenberg Virus Using Semi-Quantitative RT-PCR. PLoS ONE 8(3): e57747. doi:10.1371/journal.pone.0057747

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March 2013