The hemorrhagic disease of grass carp (Ctenopharyngodon idellus), caused by grass carp reovirus (GCRV), is the most severe disease of the fish that leads to huge economic losses. GCRV, belonging to the genus Aquareovirus of the family Reoviridae, has been classified into three genotypes based on their phylogenetic relationship. It is essential to develop an effective method to inhibit the replication of different genotypes of GCRV simultaneously. In this report, two multiple-shRNAs expression vectors, named pMultishVP2/2 and pMultishVP6/7, were generated and investigated. pMultishVP2/2 targeted the VP2 gene of GCRV-JX0901 (genotype I) and the VP2 gene of HGDRV (Hubei grass carp disease reovirus; genotype III). pMultishVP6/7 targeted the VP7 gene of GCRV-JX0901 and the VP6 gene of HGDRV. These two multiple-shRNAs expression vectors can simultaneously, significantly inhibit the replication of GCRV-JX0901 and HGDRV in vitro. Compared to the positive control, CPE induced by GCRV-JX0901 or HGDRV in cell transfected with shRNA transcribing vector was significantly delayed. The quantitative PCR analysis of the GCRV genomic RNA revealed that the pMultishVP2/2 could simultaneously inhibit the GCRV-JX0901 and HGDRV VP2 coding genes by 89.02% and 89.84%, respectively. The pMultishVP6/7 could simultaneously inhibit the GCRV-JX0901 VP7 coding gene and HGDRV VP6 coding gene by 80.63% and 86.78%, respectively. Furthermore, compared to the positive control, the indirect immunofluorescence assay and western blot demonstrated that the protein expression of the two genotypes of GCRV decreased significantly. The results in this study indicated that this multiple-shRNAs expression system could be used as a cross-reactive antiviral agent for treating the hemorrhagic disease of grass carp caused by multiple genotypes of GCRV.,Copyright ? 2014 Elsevier B.V. All rights reserved.