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Abstract: Since mid-December 2011, samples from malformed lambs and calves are sent to CODA-CERVA in Belgium for diagnosis of Schmallenberg virus (SBV), a novel Orthobunyavirus that was first detected by researchers of the Friedrich-Loeffler-Institut (FLI, Germany) in German cattle in autumn 2011 and was later shown to be involved in congenital malformations in lambs, goat kids and calves. Surprisingly, by making use of real time RT-PCR (rRT-PCR) assays developed by the FLI, presence of SBV RNA could only be confirmed in part of the SBV suspected newborns examined. To investigate possible causes for non-confirmation by rRT-PCR, a comparative analysis between different organs and tissues (cerebrum, cerebellum, brain stem, spinal cord, thymus, spleen, lymph nodes, meconium) originating from respectively 90 and 81 malformed lambs and calves was undertaken. Furthermore, thoracic fluids of respectively 55 malformed lambs and calves were examined by a virus neutralization test (VNT) to evaluate the presence of neutralizing anti-SBV antibodies in these animals. Our results show that among the different organs tested by rRT-PCR, brain stem material is the most appropriate tissue for SBV detection while it could also be detected in all other tissues but to a more variable degree. The VNT test showed that 95% of the malformed lambs were positive for anti-SBV neutralizing antibodies while this was only the case for 44% of malformed calves. These immunological data suggest that a humoral immune response could assist in the clearance of SBV from the fetus during gestation and that SBV specific antibody testing should be considered together with rRT-PCR analysis for confirmation of SBV infection. [Copyright &y& Elsevier]